Experimental Medicine Reviews1

plumelia
ricerca
E xperimental M edicine R eviews

Morphophysiological Remarks
in english and italian
Edited by Aldo Gerbino Giovanni Zummo Giuseppe Crescimanno
plumelia
edizioni
Dipartimento di Medicina Sperimentale
Di.Me.S.
Universit degli Studi di Palermo Facolt di Medicina e Chirurgia Di.Me.S. Dipartimento di Medicina Sperimentale
Sezioni: Anatomia Umana Emerico Luna (via del Vespro, 129) Istologia ed Embriologia Arcangelo Pasqualino di Marineo (via del Vespro, 129) Fisiologia Umana G. Pagano (corso Tukory, 129)
Experimental Medicine Reviews Morphophysiological Remarks

in English and Italian
Palermo, Italy - vol 1, 2007 Edited by Aldo Gerbino, Full Professor of Histology and Embryology, Histology Section Giovanni Zummo, Full Professor of Human Anatomy, Human Anatomy Section Giuseppe Crescimanno, Full Professor of Physiology, Physiology Section Scientific editorial office Francesco Cappello, Laura Uzzo, Maurizio Casarrubea Editing Simona Corrao Graphic design Rosario Notaro Plumelia edizioni Collana Ricerca ISBN 978-88-89876-08-4 This volume can be found on line at the following address: www.unipa.it/dimes Printed by Officine Tipografiche Aiello & Provenzano, Bagheria (Palermo)
Preface Il Dipartimento di Medicina Sperimentale dellUniversit degli Studi di Palermo stato istituito con Decreto Rettorale n 1446 del 01.12.2000, con decorrenza dal 01 gennaio 2001, dalla volont dei Docenti di Anatomia Umana, Fisiologia Umana e di Istologia ed Embriologia generale di costituire un centro di ricerca coordinata tra discipline di base che si riconoscono in una matrice culturale comune, la conoscenza del corpo umano e delle sue funzioni, sia pure con approcci metodologici propri. Le attivit didattiche e scientifiche dei Docenti del Dipartimento di Medicina Sperimentale, DiMeS, sono rappresentate nel sito online www.unipa. it/dimes/ Dallepoca della sua costituzione il Dipartimento ha sperimentato molteplici modalit di cooperazione tra i settori disciplinari che lo costituiscono e con centri di ricerca italiani, europei ed extraeuropei tanto da includere ben due Dottorati di ricerca, quello di Fisiopatologia Neurosensoriale e quello di Scienze delle Attivit Motorie. Fin dallepoca della sua costituzione, il Dipartimento di Medicina Sperimentale ha svolto il suo compito istituzionale sia nellattivit di ricerca che in quella di didattica in tutte le Facolt in cui sono presenti gli insegnamenti che vi insistono. Lattivit didattica stata svolta in coerenza con gli ordinamenti didattici dei Corsi di Laurea delle Facolt di Medicina e Chirurgia, Scienze Matematiche, Fisiche e Naturali, Farmacia e Scienze Motorie. Lattivit di ricerca stata di interesse The Department of Experimental Medicine, University of Palermo was created on 01.12.2000 by the Deans note n 1446. This Department, operative from 01.01.2001, was shaped as a cross-disciplinary centre where Human Anatomy, Physiology and Histology and Embryology combine their cultural bases, their different side of knowledge on the human body and its functions. Both the scientific and didactic activieties of the DiMeS (standing for Dipartimento di Medicina Sperimentale), are found in the website www.unipa.it/dimes/ From its constitution the Department has been working cooperating with Italian and internation research centres, so that today it produces two Phd courses, in Neurosensorial Fisiopathology and in Movement Science. The Department has also run courses and research activities in any Faculty where its topics are taught, adapting the courses according to the main strain of the specific Faculty (Faculty of Medicine and Surgery, Mathematics Phisics and Natural Sciences and Movement Science). The scientific research has been in accordance with the CIVR evaluation standards and with the cultural mission given since the department origin. Conferences, Workshops, International congresses have collaborated to feed the cultural debate, born from the local and international collaborations led by the Department. After seven years since its constitution, the Scientific Board has therefore felt the need
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scientifico, conforme sia alla valutazione del CIVR sia agli obiettivi culturali che erano stati prefissati alla costituzione del Dipartimento. Il dibattito culturale stato intenso e stimolante tanto che, dallepoca della sua costituzione, sono state svolte numerose conferenze, workshop e simposi internazionali, frutto delle numerose collaborazioni locali, nazionali ed internazionali e dei diversi temi scientifici svolti nei laboratori del Dipartimento. Pertanto, dopo sette anni dalla sua fondazione, il Consiglio ha sentito la necessit di consolidare la decisione di mantenere stabili le fondamenta culturali del Dipartimento promuovendo il presente volume che vuole essere non solo lo strumento dellinformazione ma anche lavvio di un confronto sia interno tra i ricercatori del Dipartimento di Medicina Sperimentale sia tra le componenti dellAteneo palermitano, che attraverso il riconoscimento dei temi e delle metodiche possano stabilire interrelazioni e possibili collaborazioni. Come gi evidente dal sommario sono riportati la maggior parte dei temi di ricerca che vengono svolte nei laboratori scientifici del DiMeS e si evidenziano i gruppi e le collaborazioni consolidate. Sono convinto che sia impossibile parlare in modo compiuto e definito della ricerca. Di essa sono state date numerose definizioni, che tuttavia ne descrivono solo la soggettivit storica o gli interessi commerciali. In questultimo trentennio frequentemente abbiamo sentito distinguere la ricerca di base da quella applicata, la prima producendo mera conoscenza, la seconda, invece, tecnologia,
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of a volume representing not only a functional cultural instrument but also a virtual dialogue between researches to create new paths of cultural interrelations and collaborations. In the summary, the main themes, working teams and collaborations among scientific labs are highlighted. lm strongly convinced that a definite and pro per way to talk about research is possible. Many definitions have been given but they describe only commerciai aspects or historical monodisciplinary aspects. During these last thirty years two different kinds of research
D.i.Me.S - Anatomia Umana e Istologia ed Embriologia, Palermo
D.i.Me.S - Fisiologia Umana, Palermo
innovazione, ricchezza. In questi tempi di ristrettezze finanziarie, la ricerca di base o ricerca pura viene considerata un accessorio, un lusso da sacrificare perch la congiuntura economica poco favorevole agli sprechi. Questo concetto stato esasperato in modo strumentale in questi ultimi anni, in particolare considerando lo slittamento degli obiettivi della politica a favore dei valori economici, esaltati dallindustria. AI valore delle idee si contrappone quello del profitto. Permettetemi una provocazione: la ricerca applicata non esiste. Esiste solo la ricerca ed esistono poi le sue applicazioni che possono essere immediate o futuribili. A nessuno sfugge che quando qualcuno osserv che un cerchio ruotava su se stesso, ancora non erano noti n i raggi n gli assali n alcun sistema di ammortizzamento n alcun strumento da utilizzare per il trasporto. La ruota ed il carro sono nati non da un bisogno immediato ma da singole e conseguenti osservazioni (ricerche, conoscenze, competenze) cui sono conseguite le applicazione tecniche. Non vanno confuse la cultura e le sue applicazioni tecniche. Non va confusa la sperimentazione con le esigenze del commercio. Da qui il tentativo ancora limitato di dimostrare come ricerca ed applicazioni possano convivere e come la sperimentazione di laboratorio possa rappresentare obiettivi culturali attuali e non necessariamente indirizzati allimmediatezza del profitto. La ricerca non pu che essere anarchica. La ricerca certezza del dubbio e dellignoranza. So che non so quel che non so; invidio coloro che ne sa-
have been outlined, the first producing me re knowledge, the second producing innovations, technology and wealth through its applicability. We are living n an age where the pure knowledge is considered a waste of money because it cant produce money itself, an idea that has been pushed by the political and industriai world. The idea of culture is set against the value of wealth, and it looses. Let me share a provocation: the applied research does not exist. Reserch means pure cultural speculation, and only in a second moment its application in immediate or programmed technologies. It is infact undoubt that the wheel wasnt born because of an immediate desire of a means of transportation but because someone realised that something with a circle form could move for its own. I mean that the wheel, the carriage, and our car are originated by our observation and speculations, followed by their applications. Culture and its applications are not the same thing. Research must not be confused with commerciai needs. This volume intends to show how research and its applicability can both represent current cultural goals not necessarily addressed to an immediate economic advantage. Research must be anarchic, it is the knowledge of doubt and ones ignorance I know that I dont know what I know not. I envy those who will know more that I know that like me, they will need to measure, assume, hypothesize, mistrust their own deductions. They will need to tell the truth from the falsehood and consider there is always a part of falsehood in truth. (M.
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pranno di pi, ma so che anchessi: come me, avranno da misurare, pesare, dedurre e diffidare delle deduzioni ottenute, stabilire nellerrore qual la parte del vero e tener conto nel vero delleterna presenza del falso (M. Yourcenar: Lopera al nero). Noi speriamo che lo notte della ricerca finisca presto e che si torni a riconoscere alla ricerca scientifica il ruolo di motore indispensabile per lo crescita e il rilancio della cultura e dei valori etici che essa rappresenta. Palermo Dicembre 2007 Giovanni Zummo
Yourcenar: The Work in the Black Phase). We do hope it could be recognised the role of the scientific research as the cultural engine that is essential for the growth and the acceleration of those ethical and cultural values it represents.
Note The heterogeneity of scientific papers, a member of different schools, is divided into trasversal methodology that today spreads through way of research. This makes, on value editorial uniformity of single capitols, some problems about harmonization. It is necessary and perphas useful that rise some differences about impostation, that not seem damage editings quality. The Editors
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Guest contribution
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno) Plumelia Ricerca (ISBN 978-88-89876-08-4) - Vol. 1 - 2007
GASTRULATION IN SEA URCHIN AND AMPHIBIAN EMBRYOS

[Gastrulazione nel Riccio di mare e negli Anfibi] Giuseppina Turturici, Fabiana Geraci, Gabriella Sconzo and Giovanni Giudice
Dipartimento di Biologia Cellulare e dello Sviluppo Alberto Monroy, Facolt di Scienze Matematiche, Fisiche e Naturali, Universit degli Studi di Palermo (I)
Key words: Gastrulation, Paracentrotus lividus, Xenopus, Xbra, BENI Parole chiave: Gastrulazione, Paracentrotus lividus, Xenopus, Xbra, BENI Abstract. The molecular aspects of the sea urchin Paracentrotus lividus and Strongylocentrotus purpuratus and of the amphibian Xenopus are reviewed. Particular emphasis is given to the gene activity, as for example that of Xbra for Xenopus and genes active before gastrulation for P. lividus. Riassunto. In questa review sono trattati gli aspetti molecolari della gastrulazione del riccio di mare Paracentrotus lividus e dello Strongylocentrotus purpuratus e dellanfibio Xenopus. Si d speciale enfasi allattivit genica, cos come ad esempio quella di Xbra per lo Xenopus e a quella dei geni attivi prima della gastrulazione per il P. lividus.
Gastrulation in sea urchin embryos In this short review we will deal with three most significant examples of gastrulation i.e. that of sea urchins and that of amphibians, seen from a cytological and from a molecular bioological point of view (fig. 1,2,3). In sea urchins, as thorougly described by Hardin [1], the all process begins with the invagination of few cells at the vegetal pole of the blastula embryo, .i.e. the primary mesenchyme cells, followed by invagination of other neighbouring cells, so that a primary intestine starts to be formed. At the same time an active elongation of the intestinal cells occurs, so that gatrulation proceeds. This process involves many sulfate containing macromolecules and can be inhibited by a variety of means, as e.g. treatment with valproate, [2] treatment with Cadmium [3, 4] (or inhibition of RNA synthesis at selected times before blastula) [5]. The process of gastrulation advances with the aid of some philopodia emanating form the secondary mesenchyme cells and [6] explore the walls of the blastocoel, in a process of touching and detouching trough a selective adhesion, till they find a place where to selectively attacch, i.e. where the future mouth will beopened, and from where they pull the other cells to invaginate, thus completing sea urchin gastrulation.
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Giuseppina Turturici, Fabiana Geraci, Gabriella Sconzo and Giovanni Giudice
Gastrulation in amphibians In Xenopus different cell behaviours have been described by Wilson et al [7-10]. In any case, cells from different prospective germ layers are brought to their proper locations by concerted cell movements, so that at the end of the process the ectoderm forms the external part of the embryo, while mesoderm is the middle part of it and endoderm is inside. As described by Nie and Chang [11] several signals influence Xenopus gastrulation, all related to the Erb B tyrosine Kinase receptor. Homma et al. [12] found that a novel gene, which they called BENI (from Brachiury Expression Nuclear Inhibitor) is required for the phase of convergent extension of Xenopus gastrulation, as shown by experimental inhibition with morpholinos or by experiments of BENI [1] iperexpression followed by whole mounts observations. The conclusion of the authors is that the results suggest that BENI expression is regulated by activin-like signaling and that this regulation is crucial for Xbra expression. Ito et al. [13] suggest that SU(H)2 (CBF-1 Suppressor od Hairless, Lag1) is an essential factor for gene expression and morphogenesis of the Xenopus gastrula embryos. They base their conclusions a on experiments of inhibitions with morpholinos and of rescue with the appropriate mRNAs. Chung et al. [14] reported about the role of the protein ANR5, a target of FGF, in regulating Xenopus gastrulation, as shown by experiments of morholinos inhibition and of rescue, by its mRNA. This ankinin repeat domain, called xANR5 acts through Rho and interacts with PAPC, a paraxial protocadherin, to regulate cell protrusion formation and tissue separation in Xenopus gastrulaion. Yun et al. [15] showed that SRF, (Serum Responsive Factor) negatively regulates the Activin/Nodal signal, wich is important for Xenopus gastrulation, because it is crucial for ectoderm specification and for correct positioning of mesoderm and endoderm. Its transcription is restricted to the animal pole ectoderm of the early embryos. The experimental ectopic expression of SRF suppresses mesoderm induction in both the marginal zone in
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Gastrulation in sea urchin and amphibian embryosb
[2,3]
vivo and that caused by Activin/Nodal signal in the animal caps. The inhibition of SRF transcription by antisense or morpholino expands the expression of mesentoderma genes toward the ectodermal territory and enhances the activity id activin, SRFyntercts withh Smad-2 and Fast-1. The authors therefore suggest that XRF might act to ensure proper mesoderm induction in the appropriate region by inhibiting the mesoderm inducing signals during early embryogenesis. Suzawa et al. [16] said that the glucose transporter (xGLUT1) is required for Xenopus gastrulation. Actually the gene GLUT1 is the most important among the thirteen GLUT genes described in animal tissues. Loss of function of GLUT1 experimentally caused in Xenopus brings about microcefaly and axis elongation error. The authors show by whole mount analyses that this gene is expressed in the dorsal region of the embryos especialy in dorsal blastopore lip at the gastrula stage. The authors conclusion is that GLUT1 is an important player in Xenopus gastrulation. Nie and Chang [11] showed that PI3K and Erk MAPK mediate Erb signalings in Xenopus gastrulation. This conclusion is supported by data on rescue of gastrulation defects, by activation of MAPK or P13K in ErbB4 morphant embryos. The authors conclusion is that PI3K Erk and MAPK act downstream of ErbB to participate in gastrulaion morphogenesis. Finally Wallingford and Harland [17] comment on the importance of BMPs in vertebrate gastrulation, through their control of cell adhesion.
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Giuseppina Turturici, Fabiana Geraci, Gabriella Sconzo and Giovanni Giudice
References [1] [2] [3] [4] Hardin J. The cellular basis of sea urchin gastrulation. Current topics in developmental biology vol 33 159-262. Ed. Pedersen and Schatten. Academic press (1996). Sconzo G, Fasulo G, Romancino D, Cascino D, Giudice G. Effect of retinoic acid and valproate on sea urchin development. Pharmazie 1996 51: 175-80. Roccheri MC, Agnello M, Bonaventura R, Matranga V. Cadmium induces the expression of specific stress proteins in sea urchin embryos. Biochem Biophys Res Commun 2004 321: 80-7. Luparello C., Sirchia R., Paci L., Miceli V., Vella R., Scudiero R., Trinchella F. Response to cadmium stress by neoplastic and immortalized human breast cells: evidence for different modulation of gene expression. In Corvin A.J. (ed.) New Developments in Cell Apoptosis Research, pp. 213-239. Happauge, NY (USA): Nova Science Publishers, Inc. (2007). Giudice G, Mutolo V, Donatuti G. Gene expression in sea urchin development. Wilhelm Roux Archiv 1968 161: 118-128. Wu SY, McClay DR. The Snail repressor is required for PMC ingression in the sea urchin embryo. Development 2007 134: 1061-70. Wilson P, Keller R. Cell rearrangement during gastrulation of Xenopus: direct observation of cultured explants. Development 1991 112: 289-300. Winklbauer R,, Nagel M, Selchow A and Wacker S. Mesoderm migration in the Xenopus gastrula. Int J Dev Biol 1996 40: 305311. Keller R, Davidson LA, Shook DR. How we are shaped: the biomechanics of gastrulation. Differentiation 2003 71: 171-205. L. Solnica-Krezel, Conserved patterns of cell movements during vertebrate gastrulation, Curr. Biol 2005 15: R213R228. Nie S, Chang C. Regulation of Xenopus gastrulation by ErbB signaling. Dev Biol 2007 303: 93-107. Homma M, Inui M, Fukui A, Michiue T, Okabayashi K, Asashima M. A novel gene, BENI is required for the convergent extension during Xenopus laevis gastrulation. Dev Biol 2007 303: 270-80. Ito M, Katada T, Miyatani S, Kinoshita T. XSu (H)2 is an essential factor for gene expression and morphogenesis of the Xenopus gastrula embryo. Int J Dev Biol 2007 51: 27-36. Chung HA, Yamamoto TS, Ueno N. ANR5, an FGF target gene product, regulates gastrulation in Xenopus. Curr Biol 2007 17: 932-9. Yun CH, Choi SC, Park E, Kim SJ, Chung AS, Lee HK, Lee HJ, Han JK. Negative regulation of Activin/ Nodal signaling by SRF during Xenopus gastrulation. Development 2007 134: 769-77. Suzawa K, Yukita A, Hayata T, Goto T, Danno H, Michiue T, Cho KW, Asashima M. Xenopus glucose transporter 1 (xGLUT1) is required for gastrulation movement in Xenopus laevis. Int J Dev Biol 2007 51: 183-90. Wallingford JB, Harland RM. Vertebrate gastrulation: the BMP sticker shock. Curr Biol 2007 17: R206-9.
[5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17]
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Airway cells
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EXERCISE-INDUCED CHANGES IN AIRWAY CELLS

[Variazioni delle cellule delle vie aeree indotte dallesercizio fisico] Laura Chimenti1, Giuseppe Morici2,3, Alessandra Patern1, Anna Bonanno3, Vincenzo Bellia1, and Maria R. Bonsignore1,3
Department of Medicine, Pneumology, Physiology and Nutrition (DIMPEFINU), 2Department of Experimental Medicine (DIMES) and 3Institute of Biomedicine and Molecular Immunology (IBIM), National Council of Research (CNR), Palermo (I)
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Key words: Airway inflammation, Bronchial epithelial damage, Remodelling, Induced sputum, Endurance training Parole chiave: Infiammazione delle vie aeree, Danno dellepitelio bronchiale, Rimodellamento, Espettorato indotto, Allenamento di endurance Abstract. Background. Exercise-induced changes in airway cells are largely documented in athletes and animal models. Increased inflammatory cells have been reported in the airways of non-asthmatic endurance athletes independently of symptoms or spirometric alterations, but the functional significance of such findings is still uncertain. Aim. The purpose of this review is to summarize the current state of knowledge about the physiological changes in airway cells induced by acute exercise and training. Results. Runners and athletes of different endurance activities carried out under moderate environmental conditions showed increased airway neutrophils at rest, which tended to further increase after exercise. Skiers and swimmers also showed large increases in airway lymphocytes and eosinophils, possibly related to chronic exposure to cold and dry air or irritants, respectively. In endurance trained mice we found increased inflammatory cells and damaged bronchial epithelium in small airways. Nevertheless, the increase in airway inflammatory cells observed in athletes and mice was not associated with inflammatory activation, and the airway inflammation of athletes did not correlate with bronchial hyperreactivity or post exercise respiratory symptoms. Conclusions. Training-induced airway changes may represent adaptive responses to exercise hyperventilation. However, further studies are necessary to understand the mechanisms responsible for control of inflammatory activation, and assess the relationship between amount/intensity of training and morphologic/ functional changes in airways. Riassunto. Premessa. Variazioni delle cellule delle vie aeree indotte dallesercizio sono ampiamente documentate negli atleti e nei modelli animali. Nelle vie aeree di atleti di endurance non asmatici stato descritto un incremento delle cellule infiammatorie indipendente da sintomi o alterazioni spirometriche, ma il significato funzionale di queste osservazioni resta incerto. Scopo. Lo scopo di questa review di riassumere lo stato attuale delle conoscenze sulle variazioni fisiologiche delle cellule delle vie aeree indotte dallesercizio acuto e dallallenamento. Risultati. Podisti ed atleti di altre attivit dendurance praticate in condizioni ambientali moderate mostravano, a riposo, un aumento dei neutrofili nelle vie aeree che tendevano ad aumentare dopo esercizio. Sciatori
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Laura Chimenti, Giuseppe Morici, Alessandra Patern, Anna Bonanno, Vincenzo Bellia, and Maria R. Bonsignore
di fondo e nuotatori, inoltre, mostravano un notevole aumento di linfociti ed eosinofili, possibilmente correlati, rispettivamente, ad una esposizione cronica ad aria fredda e secca o irritanti. In topi sottoposti ad allenamento dendurance abbiamo trovato un aumento delle cellule infiammatorie e danno allepitelio bronchiale nelle piccole vie aeree. Tuttavia, laumento delle cellule infiammatorie delle vie aeree osservato negli atleti e nei topi non era necessariamente associato ad attivazione infiammatoria e linfiammazione delle vie aeree non era sempre correlata ad iperreattivit bronchiale o a sintomi respiratori post-esercizio. Conclusioni. I cambiamenti delle vie aeree indotte dallallenamento potrebbero rappresentare risposte adattative alliperventilazione durante lesercizio. Ulteriori studi sono necessari, tuttavia, per capire i meccanismi responsabili del controllo dellattivazione infiammatoria, e per valutare le relazioni tra quantit/intensit dellallenamento e cambiamenti morfo/funzionali nelle vie aeree.
Exercise-induced bronchoconstriction and airway inflammation Paragraph 1: Exercise-induced changes in airways cells were initially studied in relation to occurrence of exercise-induced bronchoconstriction (EIB). It was hypothesized that athletes developing EIB after intense exercise might show a background of inflammatory activation in their airways. However, changes in airways cells were shown to commonly occur in athletes independent of concomitant symptoms or spirometric alterations. Paragraph 2: EIB is defined as a decrease in forced expiratory volume of 1s (FEV1) 10% from the baseline value after appropriate exercise provocation [1], and describes the acute, transient airway narrowing that usually occurs typically 5 to 15 min after cessation of exercise. In some instances, a late-phase response (LPR) can also occur 3 to 13 h after completing exercise [2]. Exercise is the most common trigger of bronchospasm in those who are known to be asthmatic, and 50% to 90% of all individuals with asthma have airways that are hyperreactive to exercise [3]. However, EIB also occurs in up to 10% of subjects who are not known to be atopic or asthmatic [4]. Paragraph 3: The pathogenesis of EIB is likely multifactorial and is not completely understood. The mechanism of EIB is believed to relate to the consequences of insufficient heating and humidification of large volumes of air during exercise. The thermal hypothesis proposed that cooling of the airways needed to be followed by rapid re-warming and that these two events caused vasocostriction followed by reactive hyperemia of the bronchial microcirculation, together with edema of the airway wall [5]. Nevertheless, neither airway cooling or re-warming appeared to be necessary for EIB to occur [6]. Paragraph 4: The main theory on EIB pathophysiology is that exercise hyperventilation causes drying of the epithelial surface, thus increasing osmolarity of the airway surface lining fluid [3, 6, 7]. As water evaporates, the airway surface liquid becomes hyperosmolar and provides an osmotic stimulus for water to move from any cell nearby, resulting in cell
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Exercise-induced changes in airway cells
volume loss. Therefore, the increase in regulatory volume after cell shrinkage is believed to be the key event, which results in release of inflammatory mediators that cause airway smooth muscle to contract and the airways to narrow [6]. Paragraph 5: The functional and cellular events triggered by exercise hyperventilation have been studied in experimental models. In anesthetized dogs challenged with high flows of air into a lung segment during bronchoscopy, hyperventilation with dry air caused hyperosmolarity of airway surface lining [8] and bronchoconstriction [9]. Repeated dry air challenges (DACs), mimicking chronic exposure such as in athletes during training, caused epithelial damage with eosinophil and neutrophil influx and increased peptidoleukotriene concentrations in bronchoalveolar lavage fluid (BALF) [10]. In cultured human bronchial epithelial cells, exposure to a hyperosmolar medium or cooling-rewarming triggered an inflammatory cascade by increasing the expression of IL-8 and RANTES partly through the activation of p38 MAP Kinase [11, 12]. Therefore both hyperventilation and airway hyperosmolarity appear capable to cause bronchoconstriction and inflammatory response. Paragraph 6: Athletes who compete in endurance sports such as cross-country skiing, swimming and long-distance running are more likely to experience symptoms of EIB as ventilation is increased for long periods of time during training and competitions, allowing for relatively more evaporative water loss and subsequent airway narrowing [13]. The increased prevalence of EIB in winter sports athletes (cross-country skiing, skating, hockey) is believed to be linked to enhanced exposure to dry and cold air and the relative increase in the reactive hyperemia in the pulmonary vasculature [14]. Finally, athletes who train under environmental conditions of pollutant exposure are at increased risk for the development of EIB. Chlorine compounds in swimming pools and chemicals related to ice-resurfacing machinery in ice rinks may be additional risks for certain populations of athletes [15, 16]. Particulate matter and gases such as carbon monoxide and nitrogen dioxide, which are abundant in indoor ice arenas, and chlorine from swimming pools may trigger and/or exacerbate bronchospasm in athletes who are predisposed to EIB. Paragraph 7: This hypothesis is supported by the finding that 78% of athletes who were EIB-positive after a sport- and environment-specific exercise test turned negative when the test was repeated in the laboratory [17]. However, clinical occurrence of EIB is variable in EIB-positive athletes, since exercise-induced respiratory symptoms poorly predicted EIB [14, 18]. Paragraph 8: In summary, both EIB and airway inflammation may result from exposure to different environmental factors. However, a causal association between inflammation and EIB is likely to occur only in a limited set of conditions. Moreover, inflammatory cells in the airways are increased in well-trained athletes independent of EIB.
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Airway cells after training Paragraph 9: Airway inflammation has been largely described in athletes who strenuously exercise at low temperatures as skaters and cross-country skiers [19, 20]. Crosscountry skiers studied at rest showed increased lymphocytes in BALF (19) and evidence of airway remodelling in endobronchial biopsies of proximal airways [21]. Airway damage was also observed in sled dogs after intense and prolonged exercise in extreme cold and dry environment [22]. Skiers showed neutrophils infiltration, which is not a typical finding in either atopic or non-atopic asthma, and relatively mild infiltration with eosinophils, mast cells, and macrophages. Moreover, bronchial biopsy findings did not correlate with bronchial hyperresponsiveness (BHR), atopy, or symptoms of asthma [21]. These results suggested the hypothesis that repeated cold weather hyperpnea can predispose these athletes to chronic airway disease different from classic asthma. The term ski-asthma, in fact, describes the syndrome of non-atopic airway inflammation and hyper-reactivity in elite winter sport athletes [19-22]. The peculiar features of ski asthma are further underlined by the lack of changes in airway cells after administration of inhaled steroids [23], a finding opposite to the response of classic asthma to steroid treatment [24]. Paragraph 10: In non-asthmatic amateur runners exercising in a temperate environment, the percentage of neutrophils in induced sputum at rest was higher than in sedentary controls, suggesting a chronic increase in neutrophils in the airways possibly related to habitual training [25]. Neutrophil counts in induced sputum further increased after a marathon race, in the absence of post-race respiratory symptoms or spirometric changes. Because the subjects were not asthmatic, these data suggest that endurance exercise may cause airway inflammation independent of associated asthma or BHR [21]. Paragraph 11: Similar findings were obtained in athletes of other sports. Well-trained young competitive rowers with normal bronchial reactivity to methacholine showed predominance of neutrophils in induced sputum both at rest and after exercise [26]. In swimmers, airway neutrophil differential counts at baseline were higher than in sedentary subjects but cell counts did not change significantly after a 5-km trial in an outdoor pool [27]. After a 5-km race in the sea (hypertonic airway exposure), the same swimmers showed slightly increased eosinophils and lymphocyte differential counts in induced sputum [27]. Paragraph 12: Data obtained in an experimental model in mice support the interpretation that exercise causes limited inflammation in the airways of athletes. Mice were kept sedentary or underwent mild intensity training for 6 weeks under standard laboratory conditions, in order to avoid the potential effect of environmental factors such as air temperature or humidity. Increased leukocyte infiltrate was observed in bronchiolar walls and lumen of endurance trained mice (Figure 1) [28]. Paragraph 13: Inflammatory cell recruitment into the airways could be triggered by da20
mage of airway epithelium, but data obtained in vivo are variable. In large airways of skiers, no clear evidence of epithelial damage was reported despite evidence of remodelling [20]. In marathon runners and swimmers, BEC counts in induced sputum were low at rest and after exercise [25, 26], but their apoptosis increased after a race. Only in rowers after an all-out test did BEC counts in induced sputum tend to increase [26]. Epithelial shear stress caused by very high ventilation observed during supramaximal exercise may account for this result [29]. Conversely, BEC damage occurred in horses after exercise while breathing cold air [30], and repeated dry and cold air challenges in dogs caused loss of ciliated epithelium and airway remodeling [31]. In endurance-trained mice bronchiolar epithelium showed progressive changes during training. After 45-days, the number of ciliated epithelial cells was four-fold lower in trained compared to sedentary mice, and apoptosis of bronchiolar epithelial cells almost doubled in trained mice (Figure 2, panels A and B). Epithelial thickness was 56% higher in trained than in sedentary mice, and bronchiolar epithelium showed a five-fold increase in the number of proliferating cells in trained mice. (Figure 2, panels C and D). The changes observed in trained mice were similar to the epithelial damage described in horses [30] and in dogs [31]. However, the evidence of active repair, indicated by the increase in epithelial thickness and proliferation, suggested that habitual exercise may increase epithelial turnover in bronchioles. In summary, the hypothesis that epithelial damage may trigger influx of inflammatory cells into the airways is supported by some pieces of experimental evidence, although it is hard to test in human studies. Markers of airway inflammation Paragraph 14: To assess whether the increased inflammatory cells in the airways were activated, markers of inflammation were analysed in endurance athletes at rest or after exercise. The available data agree that the increase of airway inflammatory cells in athletes is not usually associated with major evidence of activation, either in BALF and induced sputum in cross-country skiers studied at rest [23] or in induced sputum of runners studied at rest and after a marathon race [25]. Increased concentrations of inflammatory markers (eosinophil peroxidase, neutrophil lipocalin) in induced sputum were observed only in elite swimmers of the Finnish National team [15], while in amateur swimmers training outdoor throughout the year, there was no evidence of inflammatory cell activation at rest or after exercise in outdoor pool or sea [27]. Therefore, data from athletes studies do not provide clear evidence of significant inflammatory activation in the airways induced by acute exercise or prolonged training. Paragraph 15: Other data, obtained in a murine model of allergic asthma, suggest that inflammatory activation in the airways may actually be inhibited by exercise training. In
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ovalbumin-sensitized mice, nuclear translocation of nuclear-factor-kB (NF-kB) in airway cells was lower in trained than in sedentary animals [32]. Furthermore, in small airways of endurance trained nonasthmatic mice, NF-kB translocation and inhibitor-alpha of NF-kB (IkBa) phosphorylation were not affected and goblet cells in bronchioles were negative at Alcian-PAS staining, indicating that training did not cause excess mucus production [28]. Expression of adhesion molecules by inflammatory cells in the airways Paragraph 16: Expression of adhesion molecules by inflammatory cells provides information on their activation state. In runners or swimmers, airway neutrophils after exercise were shown to express low levels of adhesion molecules, suggesting a possible mechanism for the frustrated airway inflammation found in endurance athletes [7]. L-selectin and b2-integrins on the surface of leukocytes play an important role in leukocyte-endothelial cell interaction. These molecules undergo quantitative and qualitative changes in response to various stimuli. Exercise is known to mobilize neutrophils in peripheral blood, an event associated with shedding of L-selectin [33]. In both runners [25] and swimmers [27], expression of L-selectin by airway neutrophils decreased after exercise, while CD11b/CD18 decreased in runners but was unaffected in swimmers. Paragraph 17: A low level of expression of adhesion molecules, however, does not account for the mechanism of inflammatory cell recruitment at the airway levels in athletes, since a stimulus is necessary for chemoattraction of neutrophils from the bloodstream into the airways. As reported above, bronchial epithelial cells were shown to release IL-8 and RANTES [11, 12] upon exposure to a hyperosmolar medium or cooling-rewarming, suggesting a possible mechanism for exercise-induced leukocyte migration into airways. However, besides recruiting inflammatory cells, hyperosmolar exposure could also decrease their activation state [7]. Therefore, the same stimulus could account for the apparent discrepancy between presence of inflammatory cells in the airways of athletes and lack of inflammatory activation (Figure 3). Discussion Paragraph 18: Studies in trained athletes have shown increased inflammatory cells in the airways with some variability among sports possibly explained by different environmental exposures during exercise. A frustrated inflammatory process possibly related to modulation of adhesion molecules was also proposed. In animal models endurance training caused: decrease in the number of ciliated cells in bronchiolar epithelium; epithelial damage, increased epithelial thickness and proliferation suggesting active repair processes; leukocytes infiltrate in bronchiolar walls and lumen with blunted or absent activation. Paragraph 18: The present state of knowledge only allows to hypothesize the sequence
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of events leading to increased inflammatory cells in the airways (Figure 3). The airway influx of neutrophils is likely driven by chemoattractant mediators released by BECs. Trained mice showed increased leukocyte infiltrate in bronchiolar walls, in line with the findings obtained in samples from large airways in human athletes of different disciplines [19-23]. We speculate that exercise may cause influx of inflammatory cells into the airways secondary to epithelial changes. BECs might be affected by osmotic changes or cooling-rewarming during exercise, and trigger an inflammatory cascade [11, 12]. However, the increase in inflammatory cells in the lumen could also represent a mechanism limiting inflammation. During an inflammatory event, excessive accumulation of leukocytes can be prevented by a fine balance of immune cell recruitment and removal in the damaged tissue. Apoptosis is a mechanism potentially useful to remove granulocytes during inflammation [34], but apoptosis of leukocytes was similar in trained and sedentary mice [28]. Therefore, transepithelial migration might be considered as a highly efficient mechanism, alternative to apoptosis, for clearance of mucosal granulocytes [35]. Paragraph 19: As for the mechanism(s) involved in the pathogenesis of epithelial changes, intense exercise hyperpnea can affect airway epithelium by causing changes in viscosity, tonicity, or amount of the airway lining fluid [6]. It is also possible that high airflows during exercise may cause epithelial shear stress, although this hypothesis is little considered in the current literature on the effects of exercise on airway cells [29]. After all-out rowing, which requires a very high ventilation, BECs in induced sputum tended to increase, suggesting a potential link between maximal airflows during exercise, and epithelial damage [27]. Paragraph 20: Increased inflammatory cells in induced sputum of athletes showed no evidence of activation at rest or after exercise [7]. Markers of inflammation were not found to be increased in BALF or induced sputum in cross-country skiers at rest [23] and in runners at rest and after a marathon race [25]. In trained mice, goblet cells in bronchioles were negative at Alcian-PAS staining. In addition, training did not increase translocation of the NF-kB subunit p65 or IkBa phosphorylation, suggesting blunted or absent activation of inflammatory cells in small airways of trained mice [28]. Overall, an increase in the number of inflammatory cells without activation might represent an adaptive response to increased ventilation during exercise. Conclusions Paragraph 22: Long-term consequences of habitual training are unknown, but available evidence supports that exercise-induced changes do not necessarily mean that exercise is harmful to the lungs. The data collected through these studies suggest that inflammatory activation in the airways is blunted after exercise, and imply that athletes are not inevitably
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exposed to significant risk in terms of detrimental effects on respiratory health. Exercise per se might cause physiological adaptive responses rather than airway damage. However, further studies are necessary to ascertain the effects of acute exercise and regular training on airway cell pathophysiology in athletes.
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Figure 1. Immunohistochemistry for inflammatory (CD45+) cell infiltration in small airways of endurance-trained mice. In lung sections from sedentary (Panels A and C, original magnification 20x and 40x, respectively) and trained mice (Panels B and D, original magnification 20x and 40x, respectively), CD45+ cells can be identified in the bronchiolar walls (B, large arrow) and the luminal side (D, small arrows).
Figure 2. Immunohistochemical staining (original magnification: 40x) of bronchiolar epithelial cells for apoptosis (TUNEL, panels A and B) and proliferation (PCNA, panels C and D).
Figure 3. Cellular events possibly triggered by exercise hyperventilation.
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] Kyle JM, Walzer RB, Hanshaw SL, Leaman JR, Frobase JK. Exercise-induced bronchospasm in the young athlete: guidelines for routine screening and initial management. Med Sci Sports Exerc 1992; 24:856-859. Speelberg B, van der Berg NJ, Oosthoek CHA, Verhoeff NPLG, van der Brick WTJ. Immediate and late asthmatic response induced by exercise in patients with reversible airflow limitation. Eur Respir J 1989; 2: 402-408. Rundell KW, Jenkinson DM. Execise-induced bronchospasm in the elite atlete. Sports Med 2002; 32: 583-600. Gotshall RW. Exercise-induced bronchoconstriction. Drugs 2002; 62: 1725-1739. McFadden ER Jr. Hypothesis: exercise-induced asthma as a vascular phenomenon. Lancet 1990; 335: 880-3. Anderson SD, Daviskas E. The mechanism of exercise-induced asthma is. J Allergy Clin Immunol 2000; 106: 453-459. Bonsignore MR, Morici G, Vignola AM, Riccobono L, Bonanno A, Profita M, Abate P, Scichilone N, Amato G, Bellia V, Bonsignore G. Increased airway inflammatory cells in athletes: what do they mean? (Review) Clin Exper Allergy 2003; 33: 14-21. Freed AN, Davis MS. Hyperventilation with dry air increases airway surface fluid osmolarity in canine peripheral airways. Am J Respir Crit Care Med 1999; 159: 1101-7. Freed AN, Bromberger-Barnea B, Menkes HA. Dry air-induced constriction in lung periphery: a canine model of exercise-induced asthma. J Appl Physiol 1985; 59: 1986-90. Davis MS, Freed AN. Repeated hyperventilation causes peripheral airways inflammation, hyper-reactivity, and impaired bronchodilation in dogs. Am J Respir Crit Care Med 2001; 164: 785-9. Hashimoto S, Matsumoto K, Gon Y, Nakayama T, Takeshita I, Horie T. Hyperosmolarity-induced interleukin-8 expression in human bronchial epithelial cells through p38 mitogen-activated protein kinase. Am J Respir Crit Care Med 1999; 159: 634-640. Hashimoto S, Gon Y, Matsumoto K, Takeshita I, Maruoka S, Horie T. Inhalant corticosteroids inhibit hyperosmolarity-induced, and cooling and rewarming-induced interleukin-8 and RANTES production by human bronchial epithelial cells. Am J Respir Crit Care Med 2000; 162: 1075-1080.. Holzer K, Brukner P. Screening of athletes for exercise-induced bronchoconstriction. Clin J Sport Med 2004; 14: 134-138. Rundell KW, Im J, Mayers LB, Wilber RL, Szmedra L, Schimtz HR. Self-reported symptoms and exerciseinduced asthma in the elite athletes. Med Sci Sports Exerc 2001; 33:208-213. Helenius IJ, Rytila P, Metso T, Haahtela T, Venge P, Tikkanen HO. Respiratory symptoms, bronchial responsiveness, and cellular characteristics of induced sputum in elite swimmers. Allergy 53: 346-352, 1998. Brauer M, Spengler JD. Nitrogen dioxide exposures inside ice skating rinks. Am J Public Health 1994; 84: 429-433. Rundell KW, Wilber RL, Szmedra L, jenkinson DM, Mayers LB. Exercise-induced asthma screening of elite athletes: field versus laboratory exercise challenge. Med Sci Sports Exerc 2000; 32: 309-316. De Baets F, Bodart E, Dramaix-Wilmet M, Van Daele S, De Bilderling G, Masset S, Velmeire P, Michel O. Exercise-induced respiratory symptoms are poor predictors of bronchoconstriction. Pediatr Pulmonol 2005; 39: 301-305. Sue-Chu M, Larsson L, Moen T, Rennard SI, Bjermer L. Bronchoscopy and bronchoalveolar lavage find-
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ings in cross-country skiers with and without ski asthma. Eur Respir J 1999; 13: 626-32. [20] Provost-Craig MA, Arbour KS, Sestili DC, Chabalko JJ, Ekinci E. The incidence of exercise-induced bronchospasm in competitive figure skaters. J Asthma 1996; 33: 67-71. [21] Karjalainen EM, Laitinen A, Sue-Chu M, Altraja A, Bjermer L, Laitinen LA. Evidence of airway inflammation and remodeling in ski athletes with and without bronchial hyperresponsiveness to methacholine. Am J Respir Crit Care Med 2000; 161: 2086-2091. [22] Davis MS, McKiernan B, McCullough S, Nelson S Jr, Mandsager RE, Willard M, Dorsey K. Racing Alaskan sled dogs as a model of ski asthma. Am J Respir Crit Care Med 2002; 166: 878-882. [23] Sue-Chu M, Karjalainen EM, Laitinen A, Larsson L, Laitinen LA, Bjermer L. Placebo-controlled study of inhaled budesonide on indices of airway inflammation in bronchoalveolar fluid and bronchial biopsies in cross-country skiers. Respiration 2000; 67: 417-425. [24] National Asthma Education and Prevention Program. Expert panel report 2: guidelines for the diagnosis and management of asthma. Bethesda, MD: National Institute of Health, April 1997:97-4051. [25] Bonsignore MR, Morici G, Riccobono L, Insalaco G, Bonanno A, Profita M, Paterno A, Mirabella A, Vassalle C, Vignola AM. Airway inflammation in non-asthmatic amateur runners. Am J Physiol 2001; 281: 668-676. [26] Morici G, Bonsignore MR, Zangla D, Riccobono L, Profita M, Bonanno A, Patern A, Di Giorgi R, Mirabella F, Chimenti L, Benigno A, Vignola AM, Bellia V, Amato G, Bonsignore G. Airway cell composition at rest and after an all-out test in competitive rowers. Med Sci Sports Exerc 2004; 1723-1729. [27] Bonsignore MR, Morici G, Riccobono L, Profita M, Bonanno A, Paterno A, Di Giorgi R, Chimenti L, Insalaco G, Cuttitta G, Abate P, Mirabella F, Vignola AM, Bonsignore G. Airway cells after swimming outdoors or in the sea in nonasthmatic athletes. Med Sci Sports Exerc 2003; 35: 1146-1152. [28] Chimenti L, Morici G, Paterno A, Bonanno A, Siena L, Licciardi A, Veca M, Guccione W, Macaluso F, Bonsignore G, Bonsignore MR. Endurance training damages small airway epithelium in mice. Am J Respir Crit Care Med 2007; 175(5): 442-449. [29] Nucci G, Suki B, Lutchen K. Modeling airflow-related shear stress during heterogeneous constriction and mechanical ventilation. J Appl Physiol 2003; 95: 348-356. [30] Davis MS, Lockard AJ, Marlin DJ, Freed AN. Airway cooling and mucosal injury during cold weather exercise. Equine Vet J Suppl 2002; 34: 413-416. [31] Davis MS, Schoefield B, Freed AN. Repeated peripheral airway hyperpnea causes inflammation and remodelling in dogs. Med Sci Sports Exerc 2003; 35: 608-616. [32] Pastva A, Estell K, Schoeb TR, Atkinson P, Schwiebert LM. Aerobic exercise attenuates airway inflammatory responses in a mouse model of atopic asthma. J Immunol 2004; 172: 4520-4526. [33] Van Eeden SF, Granton J, Hards JM, Moore B, Hogg JC. Expression of the cell adhesion molecules on leukocytes that demarginate during acute maximal exercise. J Appl Physiol 1999; 86: 970-976. [34] Bratton DL, Fadok VA. Theirs but to do and dye. J Allergy Clin Immunol 1999; 103: 555-558. [35] Erjefalt JS, Uller L, Malm-Erjefalt M, Persson CG. Rapid and efficient clearance of airway tissue granulocytes through transepithelial migration. Thorax 2004; 59: 136-143.
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NEW PERSPECTIvES ON THE ROLES Of PROTEINASES AND LUNG STRUCTURAL CELLS IN THE PATHOGENESIS Of CHRONIC OBSTRUCTIvE PULMONARY DISEASE
[Nuove prospettive sui ruoli delle proteasi e delle cellule strutturali polmonari nella patogenesi della bronchite cronica ostruttiva]
Giampiero La Rocca*, Rita Anzalone, Francesca Magno, Simona Corrao, Marco Carbone, Tiziana Loria, Marco Gervasi, Melania Lo Iacono, Giovanni Zummo and Felicia Farina
Sezione di Anatomia Umana, Dipartimento di Medicina Sperimentale, Universit degli Studi di Palermo, Italy * Corresponding author - The two authors contributed equally to this work
Key words: Metalloproteinases, Cigarette smoke, COPD, Fibroblasts, Proteinase inhibitors Parole chiave: Metalloproteasi, Fumo di sigaretta, BPCO, Fibroblasti, Inibitori delle proteasi Abstract. Cigarette smoke is among the major risk factors for the development of chronic lung diseases such as COPD. In the last years, a number of reports investigated the multiple roles of proteolytic enzymes in lung pathophysiology. These molecules are expressed at various levels since the beginnings of lung development, and their expression should be restored during injury repair as well as inflammatory processes. Recent literature reports have enlightened the role of fibroblasts as key cells in mediating not only the composition of the extracellular matrix in the lungs, but also as possible regulators of the inflammatory processes following the exposure to environmental toxic stimuli as cigarette smoke. These cells constitutively produce MMP-2 as well as other proteolytic enzymes, and the expression of these molecules is regulated by cigarette smoke ad different extents. Since inhibition of proteolytic enzymes is being viewed as a promising therapeutic strategy for lung diseases, it is important to know which enzymes should be inhibited for their pro-inflammatory action, and which ones should not, since it has been demonstrated their anti-inflammatory role. Riassunto. Il fumo di sigaretta uno dei principali fattori di rischio per lo sviluppo di malattie polmonari croniche come la BPCO. Negli ultimi anni, diversi gruppi hanno studiato i molteplici ruoli degli enzimi proteolitici nella fisiopatologia polmonare. Queste molecole sono espresse a vari livelli sin dalle fasi iniziali dello sviluppo polmonare, e possono essere riespresse durante i processi di riparo delle ferite e infiammazione. Diversi articoli recenti hanno messo in luce il ruolo centrale dei fibroblasti nel mediare non solo la composizione della matrice extracellulare polmonare, ma anche nella regolazione dei processi infiammatori innescati dallesposizione a stimoli ambientali tossici come il fumo di sigaretta. I fibroblasti producono costitutivamente la MMP-2, insieme ad altri enzimi litici, e lespressione di tali molecole regolata a diversi livelli dal fumo di sigaretta. Poich linibizione dellattivit degli enzimi proteolitici della matrice vista come una strategia terapeutica promettente per le malattie polmonari, importante conoscere quali enzimi debbano essere inibiti a causa del loro ruolo pro-infiammatorio, e quali invece debbano essere preservati per il loro dimostrato ruolo anti-infiammatorio.
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Giampiero La Rocca, Rita Anzalone, Francesca Magno, Simona Corrao, Marco Carbone, Tiziana Loria, Marco Gervasi, Melania Lo Iacono, Giovanni Zummo, Felicia Farina
1. Introduction Chronic lung inflammatory diseases constitute a broad group of pulmonary pathologies, with a growing number of affected subjects and an increase in the sanitary expense worldwide. New pharmacological tools are being developed to fight these diseases, which include anti-inflammatory drugs and molecule-aimed drugs to interfere with intracellular an extracellular mechanisms involved in the pathogenetic process. Chronic Obstructive Pulmonary Disease (COPD) is a progressive and death-causing disease, which progression from the mild to severe grade is little affected by drug administration, therefore being viewed as a leading cause of death in western countries. The disruption of the airway wall organisation is one of the key features of such pathologies, followed by an increase in collagen deposition which leads to a progressive loss of lung function [1]. Cigarette smoke is among the major risk factors for the development of chronic lung diseases such as COPD and emphysema [2]. This mixture of as many as 3000 different chemical species, most of which possess a high reactivity towards biological molecules, may interact with cellular populations of the upper as well as lower airways. Between the most striking phenomena caused by smoke exposure, there is the accumulation of macrophages and neutrophils, observed in pulmonary emphysema, and, as demonstrated for COPD, the inflammatory state is maintained, even following the removal of the initial causing stimulus (e.g. for smoke cessation after the diagnosis) [2,3]. 2. The role of extracellular matrix in lung pathophysiology The extracellular matrix represents a complex mixture of macromolecules which provide a stable substrate for cells and, as shown by different reports in recent years, is able to provide morphogenetic signals as well as serving as a reservoir of matrix-bound growth factors [4]. Therefore, it is now accepted that there is a delicate balance between growth factors production and their interaction with molecules bound to the extracellular matrix, therefore regulating growth factors function itself [5,6]. Apart from the known instructive role in lung morphogenesis and repair events and from serving as a molecular scaffold for tissue organisation, cell-extracellular matrix interactions are believed to play a role in lung pathology. For instance, in the development of inflammatory processes, one of the potential mechanisms for the perpetuation of the inflamed state in airways may involve the control of extracellular matrix (ECM) turnover [7]. In the case of COPD, the activation of inflammatory cells by cigarette smoke results also in the production of large amount of proteinases, as well as the decrease of their tissue inhibitors levels. Therefore, the global effect is an imbalance of tissue homeostasis which favours a pathogenetic remodelling of the matrix [8,9]. The production of matrix-degrading enzymes goes more far than the simple degradation of molecules to favour inflammatory cells migration in response to chemotactic stimuli. In
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New perspectives on the roles of proteinases and lung structural cells in the pathogenesis of chronic obstructive pulmonary disease
fact, the effects of inflammation may be prolonged also indirectly, for example by the generation of proteolytic fragments (matrikins) of ECM molecules by the proteolytic enzymes secreted by different cell types, even after the cessation of the causative stimulus (cigarette smoke, oxidative stress, air pollution). This process may take place by the recruiting activity of ECM fragments towards neutrophils and monocytes, but also by the activation of growth/survival factors (or their mobilisation from their extracellular matrix storage sites); all of these processes may contribute to trigger inflammation [10,11]. Therefore in airways, as well as in other body systems, the delicate balance between production, activation and inhibition of proteinases should stringently regulate the tissutal responses to external injuries. The alteration of this equilibrium should favour the development of pathologies which reside in the inability to repair properly the injuries. 3. Matrix metalloproteinases and their roles in lung pathophysiology Matrix degrading proteinases belong to different classes, grouped on the basis of their structural and catalytic features. In particular, matrix metalloproteinases (MMPs) constitute a broad family of 25 members, which share a significant structural homology and domain organisation and feature a zinc ion binding site into their catalytic domain [12,13]. Different subgroups of MMPs have been characterised, on the basis of their substrate specificity (e.g. collagenases, elastases and gelatinases). As a key feature of these molecules, the same substrate may be cleaved by different enzymatic species. This overlap of target molecules involves both ECM structural proteins and regulatory ones, therefore reflecting the intricate organisation of matrix microenvironmental regulation. Between MMPs, gelatinases, also named Type IV collagenases, are two enzymes (MMP-2 or gelatinase A and MMP-9 or gelatinase B) which play a key role in a number of physiological processes. These enzymes are able to degrade the type IV collagen, which is abundant in the basement membranes, as well as gelatin derived from collagenases-dependent cleavage of the fibrillar collagen types. It is well demonstrated that in lung ECM biology these molecules are involved in developmental processes, as well as in tissue repair and fibrosis [14]. An increased knowledge on the function and spatiotemporal regulation of these enzymes during pulmonary development will be of central importance to understand lung repairing processes. Various enzymes participate in the development of lung from the earliest stages of lung buds formation through the final development of the bronchial tree, with a strict regulation both in terms of places and length of expression, with some MMPs considered as key players in these processes. As reviewed recently by Greenlee and coworkers (15 and refs therein), there are multiple experimental works on the differential roles of MMPs during the development of mice lungs, showing that while some MMPs were absent (as demonstrated for MMP-3 and MMP-9), MMP-2 and its main activator, MMP-14, decreased
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with the progression of lung development. Therefore, two key enzymes are of central importance during development: MMP-2 and MMP-14. In particular, some reports indicated that gelatinase A expression persists during lung development [16]. Although not all members of the MMP family are found within the adult lung tissue, some enzymatic activities are upregulated during the acute and chronic phases of pulmonary diseases. In fact, their role has been highlighted in several pathological conditions, as asthma, COPD and lung cancer. Although small amounts of MMP2 and MMP14 are present in the lining fluid of the lungs under normal conditions, other MMPs such as MMP-9, and MMP-12 are significantly upregulated under such pathological conditions, as shown for macrophage-derived enzymes. Therefore, a wrong pathway of proteolytic signaling may involve, as suggested by different reports, the cleavage of a-1 proteinase inhibitor. The decrease of this molecule is associated with smoke-induced COPD development and the cleavage by MMP-9 is likely to enhance the activity of neutrophil elastases, therefore resulting in elastin degradation [17,18]. Furthermore, it must be considered that damaging external stimuli (as airborne pollutants or smoke components inhaled with respiration) can provide a variety of signals which can induce an inflammatory response. The presence of reactive oxygen species as well as other highly reactive chemical species, favours the interactions with cellular populations of the respiratory tract, in both upper and lower airways. In recent years, the attention of researchers has been focused on the contribution of structural cells of the lungs, as fibroblasts, to the development and the maintenance of chronic lung diseases following their exposure to noxious agents. The use of cigarette smoke (and in particular its soluble extracts, named CSE) as source of damage for lung cells allows mimicking the effects that may take place in vivo as a consequence of smoking and, as demonstrated in several studies on animal models, cigarette smoke exposure is currently considered the best model for COPD development [19]. Indeed, CSE-treated lung fibroblasts are able to secrete chemotactic molecules for both neutrophils and macrophages [20,21]. In addition, fibroblasts exposed to cigarette smoke may produce prostaglandin, thereby contributing to the creation of a proinflammatory microenvironment [22]. Moreover, gelatinases produced by structural cells may play a key role in the pathogenesis of COPD, as demonstrated for mouse lung fibroblasts. Indeed, this process appeared to be regulated by CSE exposure [23]. Therefore it is apparent that the pulmonary fibroblast should no more be viewed as a mere structural cell of the respiratory apparatus. In fact, fibroblasts regulate the composition of the ECM scaffold, by deposing new collagen and elastin molecules, both in the physiological turnover of matrix and in the reparative processes following injuries in the lungs. Instead, when a persistent inflammatory state is triggered by cigarette smoke, the general mechanisms of tissue repair fail, and matrix alterations may accumulate, leading to disease development.
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In this process, the correct balance between proteinases and inhibitors is critical for tissue repair and remodelling [24]. The potential contribution of fibroblasts as active players in chronic lung diseases has been further elucidated in a recent study from our group. By performing several experiments, we determined the variations in the gelatinolytic pattern of cultured human lung fibroblasts exposed to increasing concentrations of CSE. Interestingly, the most significant variations were observed for the extracellular MMP-2 levels, which were shown to decrease following exposure to growing doses of cigarette smoke. In addition, we demonstrated a decrease in MMP-2 mRNA and protein, which suggested a transcriptional modulation due to cigarette smoke exposure [25]. Moreover, the mRNA levels of TIMP-2, the main inhibitor of MMP-2, conversely increased following exposure to cigarette smoke, thus reinforcing the global negative effect on MMP-2 enzyme levels. The observed modulator effect of CSE on fibroblast-secreted gelatinases and inhibitors may in part explain the effects due to cigarette smoke exposure in vivo, and confirm the recent hypotheses on the central role of fibroblasts in the development of chronic lung diseases. Since the anti-inflammatory properties of enzymes as MMP-2, we argued that in this case, an inhibition of the enzyme should just worsen the clinical features of the patients. Recent experimental evidences from different groups support our hypothesis. First of all, some members of the metalloproteinases family, and in particular gelatinase A, have been reported to have a limiting role in inflammatory processes [26,27]. In fact, some chemokines may be inactivated by MMP-2, thereby restricting monocyte migration towards sites of injury. Therefore, it should be suggested that in a normal inflammatory process some enzymes (as macrophage-derived MMP-9) may be initially required to promote cell influx to damaged areas, while when inflammation must be dampened, other enzymatic activities belonging to the same family may participate degrading proinflammatory mediators to stop the process. Indeed, when the inflammatory process persists, as in COPD, the cellular interplay, as well as cell-matrix interactions are severely affected. In addition, in a more recent work which described a novel functional proteomics approach, the authors identified several proteins in the BAL fluid that should be cleaved by gelatinases A and B, and are essential for regulating inflammatory pathways in experimental asthma, as S100A8 and S100A9 [28]. These proteins all have chemotactic properties and are upregulated in allergic lung inflammation. 4. Therapeutic approaches of proteases inhibition: are they always beneficial? Given the importance of cell-ECM interactions for the development of inflammatory lung diseases, the possibility to use natural or synthetic inhibitors of metalloproteinases for the clinical management of COPD patients is being investigated by various groups [29]. One point in favour of this option is the clear relationship between increased expression of
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some enzymes (MMP-8, MMP-9, and MMP-12) and the progression of the disease. Therefore, the use of specific inhibitors designed to inhibit these enzymatic activities should be beneficial for the treatment of patients. On the other hand, given their high structural similarity, it is a very difficult task to obtain a molecule with single enzymatic specificity; therefore it is likely that an inhibitor should affect a number of enzymes, and therefore blocking several different extracellular signalling pathways. Indeed, currently it is not clear whether MMP inhibition should be beneficial or harmful for patients treatment, and in which situations it would really be of clinical usefulness. The complexity of the pattern is increased by the evidence that different activation pathways are known for the different forms of metalloproteinases, and these mechanisms should be varied under different pathological conditions. A second aspect to be considered is the possible but not sure redundancy in function between similar MMPs. In this case, the two gelatinases, even if they share a great number of substrate molecules between the metalloproteinase families, should play non-redundant roles in lung pathologies. For example, it has been demonstrated that MMP-2 and MMP-9 should play a nonredundant role in the pathogenesis of obliterative airway disease (OAD), a known complication of chronic lung allograft rejection. Given the experimental results obtained, it has been proposed that inhibition of MMP-9, rather than MMP-2, may represent a useful therapeutic opportunity for chronic lung allograft rejection [30]. In conclusion, despite the current efforts to use proteinase inhibitors in the clinical practice for the treatment of inflammatory lung diseases, a lot of work is still to be performed. Understanding the single roles of the different enzymes, in physiology and pathology, will give us the opportunity to design better drugs to improve the patients condition.
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References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] Di Stefano A, Caramori G, Ricciardolo FLM, Capelli A, Adcock IM, Donner CF. Cellular and molecular mechanisms in chronic obstructive pulmonary disease: an overview. Clin. Exp. Allergy 2004; 34: 1156-1167. Shapiro SD. End stage chronic obstructive pulmonary disease: The cigarette is burned out but inflammation rages on. Am J Respir Crit Care Med 2001; 164: 339-340. Shapiro SD. The macrophage in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999; 160: S29-S32. Ramirez F, Rifkin DB. Cell signalling events: a view from the matrix. Matrix Biol 2003; 22: 101-107. Corbel M, Belleguic C, Boichot E, Lagente V. Involvement of gelatinases (MMP-2 and MMP-9) in the development of airway inflammation and pulmonary fibrosis. Cell Biol Toxicol 2002; 18: 51-61. Aumailley M, Gayraud B. Structure and biological activity of the extracellular matrix. J Mol Med 1998; 76: 253-265. Kleinmann HK, Philp D, Hoffman MP. Role of the extracellular matrix in morphogenesis. Curr Opin Biotechnol 2003; 14: 526-532. Ten Hacken NHT, Postma DS, Tiemens W. (2003). Airway remodeling and long-term decline in lung function in asthma. Curr Opin Pulmon Med 2003; 9 :9-14. Shapiro SD. Proteolysis in the lung. Eur. Respir. J. 2003; 22 (Suppl. 44): 30s-32s. Fowlkes JL, Winkler MK. Exploring the interface between metallo-proteinase activity and growth factor and cytokine bioavailability. Cytokine Growth Factor Rev 2002; 13: 277-287. Schenk S, Quaranta V. (2003). Tales from the crypt[ic] sites of the extracellular matrix. TRENDS Cell Biol 2003; 13: 366-375. Sternlicht MD, Werb Z. How matrix metalloproteinases regulate cell behaviour. Annu Rev Cell Dev Biol 2001; 17: 463-516. Somerville RPT, Oblander SA, Apte SS. Matrix metalloproteinases: old dogs with new tricks. Genome Biol 2003; 4: 216. Chua F, Sly PD, Laurent GJ. Pediatric lung disease: from proteinases to pulmonary fibrosis. Pediatr Pulmonol 2005; 39: 392-401. Sato E, Koyama S, Takamizawa A, Masubuchi T, Kubo K, Robbins RA, Nagai S, IzumiT. Smoke extract stimulates lung fibroblasts to release neutrophil and monocyte chemotactic activities. A JP Lung 1999; 277: 1149-1157. Greenlee KJ, Werb Z, Kheradmand F. Matrix metalloproteinases in lung: multiple, multifarious and multifaceted. Physiol Rev 2007; 87: 69-98. Masumoto K, de Rooij JD, Suita S, Rottier R, Tibboel D, de Krijger RR. Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases during normal human pulmonary development. Histopathology 2005; 47: 410419. Liu Z, Zhou X, Shapiro SD, Shipley JM, Twining SS, Diaz LA, Senior RM, Werb Z. The serpin alpha1proteinase inhibitor is a critical substrate for gelatinase B/MMP-9 in vivo. Cell 2000; 102: 647655. Senior RM. Mechanisms of COPD: conference summary. Chest 2000; 117: 320S323S. Numanami H, Koyama S, Nelson DK, Hoyt JC, Freels JL; Habib MP, Amano J, Haniuda M, Sato E, Robbins RA. Serine protease inhibitors modulate smoke-induced chemokine release from human lung fibroblasts. Am J Respir Cell Mol Biol 2003; 29: 613-619. Martey CA, Pollock SJ, Turner CK, OReilly KM, Baglole CJ, Phipps RP, Sime PJ. Cigarette smoke induces cyclooxygenase-2 and microsomal prostaglandin E2 synthase in human lung fibroblasts: implications
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for lung inflammation and cancer. Am J Physiol Lung Cell Mol Physiol 2004; 287: L981-L991. [22] Ning W, Li C-J, Kaminski N, Feghali-Bostwick CA, Alber SM, Di YP, Otterbein SL, Song R, Hayashi S, Zhou Z, Pinsky DJ, Watkins SC, Pilewski JM, Sciurba FC, Peters DG, Hogg JC, Choi AM. Comprehensive gene expression profiles reveal pathways related to the pathogenesis of chronic obstructive pulmonary disease. PNAS 2004; 101: 14895-14900. [23] Mott JD, Werb Z. Regulation of matrix biology by matrix metalloproteinases. Curr Opin Cell Biol 2004; 16: 558-564. [24] La Rocca G, Anzalone R, Magno F, Farina F, Cappello F, Zummo G. Cigarette smoke exposure inhibits extracellular MMP-2 (gelatinase A) activity in human lung fibroblasts. Respir Res 2007; 8: 23. [25] McQuibban GA, Gong JH, Tam EM, McCulloch CA, Clark-Lewis I, Overall CM. Inflammation dampened by gelatinase A cleavage of monocyte chemoattractant protein-3. Science 2000; 289: 1202-1206. [26] McQuibban GA, Gong JH, Wong JP, Wallace JL, Clark-Lewis I, Overall CM. Matrix metalloproteinase processing of monocyte chemoattractant proteins generates CC chemokine receptor antagonists with anti-inflammatory properties in vivo. Blood 2002; 100: 1160-1167. [27] Greenlee KJ, Corry DB, Engler DA, Matsunami RK, Tessier P, Cook RG, Werb Z, Kheradmand F. Proteomic identification of in vivo substrates for matrix metalloproteinases 2 and 9 reveals a mechanism for resolution of inflammation. J Immunol 2006; 177: 7312-7321. [28] Belvisi MG, Bottomley KM. The role of matrix metalloproteinases (MMPs) in the pathophysiology of chronic obstructive pulmonary disease (COPD): a therapeutic role for inhibitors of MMPs? Inflamm Res 2003; 52: 95-100. [29] Fernandez FG, Campbell LG, Liu W, Shipley JM, Itohara S, Patterson GA, Senior RM, Mohanakumar T, Jaramillo A. Inhibition of obliterative airway disease development in murine tracheal allografts by matrix metalloproteinase-9 deficiency. Am J Transplant 2005; 5: 671683.
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AIRWAY CELLS IN SWIMMERS: A CASE REPORT AND A REvIEW Of THE LITERATURE

[Cellule delle vie aeree nei nuotatori: case report e rassegna della letteratura] Giuseppe Morici1,3, Laura Chimenti2, Anna Bonanno3, Loredana Riccobono3, Mirella Profita3, Alessandra Patern3 and Maria R. Bonsignore2,3
Department of Experimental Medicine (DIMES)1, and Department of Medicine, Pneumology, Physiology and Nutrition (DIMPEFINU)2; Institute of Biomedicine and Molecular Immunology (IBIM), National Council of Research (CNR)3, Palermo (I)
Key words: Endurance training, Marathon swimming, Induced sputum, Adhesion molecules Parole chiave: Allenamento dendurance, Nuoto di gran fondo, Espettorato indotto, Molecole di adesione Abstract. Background. Inflammatory cells are increased in the airways of non-asthmatic endurance athletes without clear evidence of activation. Their role and the mechanisms involved in their recruitment into the airways, however, are still poorly understood. Previous studies suggest that habitual training and exercise duration may be critical factors in determining airway cell counts and composition. Aim. To assess the effects of very prolonged endurance exercise on airway cells we studied a well-trained non-asthmatic amateur swimmer who covered the distance of 34.6 km between Lipari (Eolian Islands) and Capo dOrlando (Northern coast of Sicily) in 15 hours. Results. Induced sputum, collected at baseline and 12 hours after the trial, showed a very high percentage of neutrophils (98% and 82.5%, respectively). Moreover, eosinophils were slightly increased after the trial, similar to previous data collected in a group of swimmers after a competition in the sea. In peripheral blood, leukocytosis and neutrophilia, release of muscle enzymes and evidence of systemic inflammatory activation were also found. Conclusions. These data confirm that endurance exercise affects both airway and peripheral blood cells, and suggest a modulating role of exercise duration in neutrophil recruitment into the airways. Riassunto. Premessa. Le vie aeree di atleti endurance a riposo mostrano un aumento delle cellule infiammatorie, senza tuttavia evidenza di attivazione. Il significato fisiologico e i meccanismi responsabili del reclutamento delle cellule infiammatorie nelle vie aeree sono ancora poco chiari. Studi precedenti suggeriscono che lallenamento abituale e la durata dellesercizio possono modulare la composizione e la quantit di cellule presenti nelle vie aeree. Scopo. Gli effetti di un esercizio dendurance molto prolungato sulle cellule delle vie aeree sono stati valutati in un nuotatore amatoriale ben allenato, non-asmatico, che ha percorso la distanza di 34,6 km tra Lipari (Isole Eolie) e Capo DOrlando (Costa Nord della Sicilia) in 15 ore. Risultati. Lespettorato indotto mostrava una percentuale di neutrofili molto alta sia in condizioni di base che 12 ore dopo la prova (98% e 82.5%, rispettivamente). Inoltre, gli eosinofili erano leggermente aumentati dopo la prova, confermando dati precedenti ottenuti in un gruppo di nuotatori dopo una gara a mare. Il nuoto molto prolungato era anche associato a leucocitosi e neutrofilia del sangue periferico, rilascio di enzimi muscolari ed evidenza di attivazione infiammatoria sistemica.
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Giuseppe Morici, Laura Chimenti, Anna Bonanno, Loredana Riccobono, Mirella Profita, Alessandra Patern and Maria R. Bonsignore
Conclusioni. Questi dati confermano che lesercizio dendurance influenza le cellule delle vie aeree e del sangue periferico, e suggeriscono che la durata dellesercizio possa modulare il reclutamento dei neutrofili nelle vie aeree.
Background Paragraph 1: Several studies have shown increased inflammatory cells in the airways of endurance athletes exercising in a cold [1-6] or moderate [7-9] environment. The degree and type of airway inflammation under resting conditions was variable in athletes of different sports, and the role of inflammatory cells in the airways is currently unclear. In cross-country skiers studied at rest, lymphocytes were increased in bronchoalveolar lavage fluid [1], and endobronchial biopsies showed increased inflammatory cells (lymphocytes, but also neutrophils and eosinophils) and evidence of airway remodeling [3] suggesting chronic airway disease (ski asthma) [1-6]. Paragraph 2: We have previously reported that in non-asthmatic amateur runners exercising under moderate environmental conditions neutrophil counts in induced sputum were increased after a marathon race over the baseline level, in the absence of post-race respiratory symptoms or spirometric changes [7]. Under baseline conditions, the percentage of neutrophils in induced sputum of runners was higher than in sedentary controls, suggesting a chronic increase in neutrophils in the airways, possibly related to habitual training [7]. Similar to runners, well-trained, non-asthmatic young competitive rowers showed predominance of neutrophils in induced sputum both at rest and after exercise [9]. In swimmers, airway neutrophil differential counts at baseline were higher than in sedentary controls but cell counts did not change significantly after a 5-km trial in an outdoor pool [8]. After a 5-km race in the sea, a condition of hypertonic airway exposure during exercise, the same swimmers showed slightly increased eosinophils and lymphocyte differential counts in induced sputum [8]. Overall, 5-km swimming exerted smaller effects on airway cells than running a marathon. As for the mechanism of cell recruitment, it is possible that changes in epithelial cells associated with hyperosmolarity of the airway surface lining fluid or with cooling-rewarming of the airways, may drive inflammatory cells into the airways, as suggested by in vitro studies [10, 11]. Paragraph 3: The increase in inflammatory cells in the airways of athletes was not associated with evidence of inflammatory activation at rest or after exercise [7, 8, 12] as assessed by markers of inflammation in BALF or induced sputum in cross-country skiers studied at rest [13] and runners studied at rest and after a marathon race [7]. Furthermore, in both runners [7] and swimmers [8], expression of L-selectin by airway neutrophils decreased after exercise, while CD11b/CD18 decreased in runners but was unaffected in
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Airway cells in swimmers: a case report and a review of the literature
swimmers, confirming lack of inflammatory activation in the airways of endurance athletes. Similarly, intracellular markers of inflammation such as nuclear-factor-kB (NF-kB) translocation and inhibitor-alpha of NF-kB (IkBa) phosphorylation were not increased in endurance trained compared to sedentary mice [14]. Elite swimmers of the Finnish National team were the only athletes where increased concentrations of inflammatory markers (eosinophil peroxidase, neutrophil lipocalin) were found in induced sputum at rest [15]. Instead, in amateur swimmers training outdoor throughout the year, analysis of induced sputum showed no evidence of inflammatory cell activation at rest or after exercise either in outdoor pool or sea, in fact, neutrophil elastase concentration was low at all times [8]. It is likely that exposure to high chlorine compound concentrations in indoor pool may account for the inflammatory pattern found in the Finniah athletes, confirming the importance of environmental factors in exercise-induced airway changes. Paragraph 4: Estimating the respective role of endurance exercise and airway exposure to environmental factors in increasing airway inflammatory cells in athletes is difficult. Increased neutrophils in the airways may be a direct consequence of endurance training, as they were found in all athletes tested, irrespective of practised sport. Conversely, increased eosinophils and lymphocytes may depend on environmental exposure to factors such as chlorine compounds in swimmers, or dry and cold air in skiers. We report the results of a study on an amateur long-distance swimmer who completed a 35-km trial between Eolian Islands and Sicily. These data may shed some light on the effects of very prolonged endurance exercise on airway cells, extending our previous data collected in swimmers after 5-km races. Case report Paragraph 5: We studied an amateur non-asthmatic swimmer who covered the 35-Km distance between Lipari (Eolian Islands, Mediterranean Sea) and Capo dOrlando (Northern coast of Sicily) in 15 hours. Paragraph 6: The athlete (age: 30 yr; body weight: 67 kg; height: 169 cm) had been swimming at amateur level for ten years. To prepare for this trial, he swam 48 km/week. He was not taking any drug. Spirometric values at rest were: FEV1 4.15 L (104% predicted), FVC 5.32 L (113% predicted). The protocol was approved by the Ethical Committee, University of Palermo, and written informed consent was obtained before the study. Paragraph 7: The trial started at 07:00 a.m., at the Faraglioni of Lipari, Eolian Islands, Italy. Arrival at Capo dOrlando, on the Northern coast of Sicily (linear distance: 34.6 Km) was at 10:00 p.m. of the same day. The weather was good (barometric pressure: 1017-1019 mbar), with no or light wind. Air temperature increased from 25.5C at 07:00 a.m to 37.5C at 03:00 p.m; then, it decreased gradually to 26.5C at 10:00 p.m. The
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athlete did not stop or mount on boat; he only briefly interrupted swimming to drink or eat small snacks. Body weight at arrival was 64 kg. The trial was regular. The athlete referred no major complaint during or after the trial. Paragraph 8: Methods. Data were collected over four weeks: at rest, two weeks before the trial (baseline-intensive training); at rest, in the pre-trial week during progressive decrease in training intensity and duration (baseline-tapering); at the end of the trial; and 12 and 36 hours post-trial. Samples of induced sputum were obtained at baseline-tapering, and 12 hours post-trial. Samples were processed as previously reported [7, 8], and analyzed for cell counts and composition. Briefly, the subject underwent hypertonic saline (3%) aerosol for 20-min, and expectorated into sterile ampoules. After addition of an equal volume of 0.1% dithiothreitol saline solution (Sigma Chemical Co, St Louis, MO, USA), each sample was mixed (37C, 15 min), and centrifuged (800g, 10 min). The supernatant was frozen at 20C, for subsequent analysis of albumin and neutrophil elastase concentration. Cell pellets were resuspended in saline, and cell count (standard hemocytometer) and viability (Trypan blue exclusion) assessed in 10-l aliquots. Cells were cytocentrifuged (Cytospin 2, Shandon Instruments, Runcorn, UK) and stained (DiffQuick, Merz-Dade, Dudingen, Switzerland). Slides were blindly read (at least 400 cells/slide), and differential counts expressed as corrected percentage after subtracting squamous cells. Paragraph 9: Expression of adhesion molecules was analyzed in cells of induced sputum and peripheral blood neutrophils (Ficoll-Hypaque gradient separation), as previously reported [7,8]. Slides were prepared (acetone/methanol, 4C, 10 min), and incubated (37C, 1 h) with monoclonal antibodies (DAKO A/S, Denmark) anti-L-selectin, CD11a/CD18 (LFA-1) and CD11b/CD18 (MAC-1). Immunoreactivity was revealed by the labeled streptavidin biotin (LSAB) alkaline-phosphatase technique. Four hundred cells/slide were counted, and results were expressed as percentage of positive cells. Paragraph 10: Blood was drawn from the antecubital vein into sterile tubes containing EDTA (Vacutainer, Becton Dickinson) for complete blood cell and reticulocyte counts (ADVIA counter, Bayer). Aliquots of plasma and serum were collected and stored at 80C. Muscle enzymes (lactic dehydrogenase or LDH and creatine kinase or CK) and plasma neutrophil elastase concentrations were measured by enzymatic assays (Olympus Diagnostica, GmbH, and Ecoline, Kit Merck, Germany). Serum cortisol was assessed by radioimmunoassay (Immunotech, SA, Marseille, France). Paragraph 11: Results. In induced sputum (Table 1): total corrected cell counts were in the normal range, but increased at 12 hours post-trial compared to baseline; neutrophils accounted for >90% of cells in induced sputum at baseline, and for a slightly lower percentage (82.5%) after the trial; absolute neutrophil counts post-trial increased by 54% over baseline; neutrophil elastase concentration did not differ between samples; eosinophils
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were absent at baseline, and accounted for about 1% of total cells post-trial. Expression of adhesion molecules by neutrophils in induced sputum was low. L-selectin was expressed by 14.8 and 18.3% of neutrophils, before and after the trial respectively; corresponding figures were 27.6 and 19.0% for LFA-1, and 13.7 and 18.0% for MAC-1 expression. Paragraph 12: In peripheral blood (Table 2): red blood cell counts did not change, while reticulocytes increased slightly at 12h post-trial; prolonged swimming doubled WBC counts, largely due to increased neutrophil counts; peripheral blood leukocytosis and neutrophilia were still present at 12 hours, and returned to baseline by 36 h after the trial; the percentage of circulating neutrophils expressing adhesion molecules (L-selectin, LFA-1, MAC-1) did not differ among time points (data not shown). Paragraph 13: Markers of muscle damage, stress, and systemic inflammation (Table 3): after the trial, serum creatine phosphokinase (CK) and interleukin-6 (IL-6) levels showed a fifteen- and a seven-fold increase, respectively. Neutrophil elastase and TGF-1beta concentrations also increased, while lactic dehydrogenase (LDH) and cortisol levels did not show major changes compared to baseline values. Discussion Paragraph 14: Despite the obvious limitation of studying a single athlete, analysis of the effects of 35-km swimming may allow some insight into the effects of very prolonged exercise on airway cells. Based on previous work from our laboratory [7, 8], we hypothesized that exercise duration might be important in modulating airway cells. Neutrophil counts in induced sputum were much higher in runners after a marathon race (exercise lasting 3 to 4 h) than in swimmers after a 5-km race (lasting about 1 h). In the present study, the induced sputum sample obtained at rest after intensive training showed that almost all cells were neutrophils; a similar pattern was observed in the post-trial sample. Therefore, the data support the interpretation that intense endurance training and prolonged exercise increase airway neutrophils. However, no evidence of active inflammation in the airways was found after 35-km swimming. Neutrophil elastase concentration did not increase in induced sputum, and expression of adhesion molecules by airway neutrophils was low, in agreement with previous findings in runners and swimmers [7, 8]. Therefore, airway inflammation in athletes appears under tight control even after very prolonged exercise [12]. Paragraph 15: Our previous study in swimmers showed that eosinophils in induced sputum slightly increased after 5-km swimming in the sea [8]; because eosinophils did not increase after swimming in an outdoor pool, we interpreted their change as related to exposure to sea water. The development of hyperosmolarity of the airway surface layer during exercise is a highly debated issue [16, 17], and swimming in the sea might be a model of airway exposure to a hypertonic environment during exercise. After 35-km swimming,
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eosinophils in induced sputum increased very slightly, suggesting that very prolonged exposure to sea water did not exert deleterious effects. Swimming in the sea for prolonged time seems safe for healthy airways. Paragraph 16: Muscle enzymes were elevated after the trial, suggesting significant muscle damage. The changes in peripheral blood cells and mediators after 35-km swimming confirm that endurance exercise causes systemic inflammatory activation [18,19]. Plasma cortisol and IL-6 concentration, however, were much lower in this swimmer compared to values found after a marathon race [20], possibly because exercise intensity was lower than during a marathon race [21]. Conclusions Paragraph 17: Our athlete showed intense airway neutrophilia at rest and after prolonged swimming in the sea, but no active inflammation in the airways. Changes in peripheral blood cells and mediators were in line with the known systemic inflammatory activation induced by endurance exercise. Our findings suggest that exercise duration is involved in the modulation of the effects of endurance exercise at both airway and peripheral blood levels. However, very prolonged exposure to sea water did not appear to exert harmful effects to the airways. Acknowledgments We thank the athlete Mauro Giaconia who repeatedly came to the laboratory for data collection. We also thank the dedicated people who made this study possible, in particular Dr. Pietro Miraglia, Laboratory of Clinical Pathology Delta, Brolo (Messina), for his help in sample processing at night after end of the trial.
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References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] Sue-Chu M, Larsson L, Moen T, Rennard SI, Bjermer L. Bronchoscopy and bronchoalveolar lavage findings in cross-country skiers with and without ski asthma. Eur Respir J 1999; 13: 626-32. Provost-Craig MA, Arbour KS, Sestili DC, Chabalko JJ, Ekinci E. The incidence of exercise-induced bronchospasm in competitive figure skaters. J Asthma 1996; 33: 67-71. Karjalainen EM, Laitinen A, Sue-Chu M, Altraja A, Bjermer L, Laitinen LA. Evidence of airway inflammation and remodeling in ski athletes with and without bronchial hyperresponsiveness to methacholine. Am J Respir Crit Care Med 2000; 161: 2086-2091. Sue-Chu M, Karjalainen EM, Altraja A, Laitinen A, Laitinen LA, Noess AB, Larsson L, Bjermer L. Lymphoid aggregates in endobronchial biopsies from young elite cross-country skiers. Am J Respir Crit Care Med 1998; 158: 597-601. Larsson K, Ohlsen P, Larsson L, Malmberg P, Rydstrom PO, Ulriksen H. High prevalence of asthma in cross country skiers. BMJ 1993; 307: 1326-1329. Davis MS, McKiernan B, McCullough S, Nelson S Jr, Mandsager RE, Willard M, Dorsey K. Racing Alaskan sled dogs as a model of ski asthma. Am J Respir Crit Care Med 2002;166: 878-882. Bonsignore MR, Morici G, Riccobono L, Insalaco G, Bonanno A, Profita M, Paterno A, Mirabella A, Vassalle C, Vignola AM. Airway inflammation in non-asthmatic amateur runners. Am J Physiol 2001; 281: L668-L676. Bonsignore MR, Morici G, Riccobono L, Profita M, Bonanno A, Paterno A, Di Giorgi R, Chimenti L, Insalaco G, Cuttitta G, Abate P, Mirabella F, Vignola AM, Bonsignore G. Airway cells after swimming outdoors or in the sea in nonasthmatic athletes. Med Sci Sports Exerc 2003; 35: 1146-1152. Morici G, Bonsignore MR, Zangla D, Riccobono L, Profita M, Bonanno A, Patern A, Di Giorgi R, Mirabella F, Chimenti L, Benigno A, Vignola AM, Bellia V, Amato G, Bonsignore G. Airway cell composition at rest and after an all-out test in competitive rowers. Med Sci Sports Exerc 2004; 1723-1729. Hashimoto S, Matsumoto K, Gon Y, Nakayama T, Takeshita I, Horie T. Hyperosmolarity-induced interleukin-8 expression in human bronchial epithelial cells through p38 mitogen-activated protein kinase. Am J Respir Crit Care Med 1999; 159: 634-640. Hashimoto S, Gon Y, Matsumoto K, Takeshita I, Maruoka S, Horie T. Inhalant corticosteroids inhibit hyperosmolarity-induced, and cooling and rewarming-induced interleukin-8 and RANTES production by human bronchial epithelial cells. Am J Respir Crit Care Med 2000; 162: 1075-1080. Bonsignore MR, Morici G, Vignola AM, Riccobono L, Bonanno A, Profita M, Abate P, Scichilone N, Amato G, Bellia V, Bonsignore G. Increased airway inflammatory cells in athletes: what do they mean? Clin Exper Allergy 2003, 33: 14-21. Sue-Chu M, Karjalainen EM, Laitinen A, Larsson L, Laitinen LA, Bjermer L. Placebo-controlled study of inhaled budesonide on indices of airway inflammation in bronchoalveolar fluid and bronchial biopsies in cross-country skiers. Respiration 2000; 67: 417-425. Chimenti L, Morici G, Patern A, Bonanno A, Siena L, Licciardi A, Veca M, Guccione W, Macaluso F, Bonsignore G, Bonsignore MR. Endurance training damages small airway epithelium in mice. Am J Respir Crit Care Med 2007; 175(5): 442-449. Helenius IJ, Rytila P, Metso T, Haahtela T, Venge P, Tikkanen HO. Respiratory symptoms, bronchial responsiveness, and cellular characteristics of induced sputum in elite swimmers. Allergy 1998; 53: 346-352. Kotaru C, Hejal RB, Finigan JH, Coreno AJ, Skowronski ME, Brianas LJ, McFadden ER Jr. Influence of hyperpnea on airway surface fluid volume and osmolarity in normal humans. J Appl Physiol 2002;
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93:154-160. [17] Freed AN, Davis MS. Hyperventilation with dry air increases airway surface fluid osmolality in canine peripheral airways. Am J Respir Crit Care Med 1999; 159: 1101-1107. [18] Shek PN, Shephard RJ. Physical exercise as a human model of limited inflammatory response. Can J Phyiol Phramacol 1998; 76: 589-97. [19] Jordan J, Beneke R, Hutler M, Veith A, Luft FC, Haller H. Regulation of MAC-1 (CD11b/CD18) expression on circulating granulocytes in endurance runners. Med Sci Sports Exerc 1999; 31: 362-367. [20] Bonsignore MR, Morici G, Santoro A, Pagano M, Cascio L, Bonanno A, Insalaco G, Abate P, Mirabella F, Profita M, Gioia M, Vignola AM, Majolino I, Testa U, Hogg JC. Circulating hematopoietic precursor cells (HPCs) in well-trained runners. J Appl Physiol 2002, 93: 1691-97. [21] Dwyer J. Marathon swimmers: physiologic characteristics. J Sports Med Phys Fitness 1983; 23: 263-272.
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PRESENZA DEL fATTORE NATRIURETICO ATRIALE NELLAPPARATO RESPIRATORIO

[Presence of Atrial Natriuretic Peptides in the Respiratory System] Biagio Valentino, Diego Lipari, Giovanni Peri e Elvira Farina Lipari
Dipartimento di Medicina Sperimentale, Sezione Anatomia Umana, Facolt di Medicina e Chirurgia, Universit degli Studi di Palermo (I)
Parole chiave: ANP, Vie aerifere, Polmone Key words: ANP, Airways, Lung Riassunto. LANP un neuropeptide con propriet natriuretiche diuretiche e vasoattive. Tali propriet sono state indagate con metodiche immunoistochimiche e PCR analisi. Abbiamo indagato con tali metodiche la presenza di ANP nelle cavit nasali, trachea polmone e pleura per studiare il ruolo dellANP nei diversi organi deputati al trasporto dellaria ed alla ematosi area-sangue. Abstract. ANP is a neuropeptide that plays natriuretic, diuretic and vasoactive actions. These activities had been by immunohistochemical methods and PCR analysis in the nasal cavity, trachea, lung and pleura to study the ANP role in different organs that are involved in the air-transport and in the airblood haemostasis.
Introduzione Il fattore natriuretico atriale (ANP) un neuropeptide costituito da 28 amminoacidi ottenuto per attivit proteolitica da un precursore costituito da 126 amminoacidi C- terminale con attivit natriuretica, diuretica e vasoattiva primariamente trovato nel cuore di diversi mammiferi compreso luomo. In questi ultimi anni sono stati trovati altri in altri organi altri neuropeptidi BNP, CNP legati allANP con propriet natriuretiche diuretiche e vasoattive. Lespressione dei neuropeptidi appartenenti alla famiglia di ANP stata indagata con tecniche immunoistochimiche usando anticorpi anti-ANP o anticorpi anti-proANP e per mezzo di ibridizzazione in situ e PCR amplificazione.Lazione dellANP comporta il rilascio della muscolatura liscia dei vasi, lincremento della diuresi e natriuresi che ha come effetto la regolazione della distribuzione dei fluidi tra i compartimenti extra e intravasasali. Da diversi anni il nostro gruppo di lavoro si interessato allo studio e alla classificazione dellANP e degli altri neuropeptidi legati allANP in diversi organi ed apparati [2,3,4,5]. Fra gli apparati abbiamo preso in esame lapparato respiratorio che per la sua organizzazione morfologica e per la sua funzione dipende molto dal sistema nervoso e da diversi neurotrasmettitori. Sono stati indagati con metodiche immunoistochimiche e amplificazione c-DNA per ANP, le cavit nasali la trachea, il polmone e la pleura di diversi mammiferi e delluomo.
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Biagio Valentino, Diego Lipari, Giovanni Peri, Elvira Farina Lipari
Analisi immunotopografica Le cavit nasali Lorganizzazione strutturale e langioarchitettura dei turbinati delluomo sono atti a modificare alcune caratteristiche fisico-chimiche dellaria inspirata. stato osservato da diversi AA. che la termoregolazione dellaria inspirata dipende da diversi neurotrasmettitori quali VIP (vasoactive intestinal peptide) NPT (neuropeptide Y) somatostatina e ANP che producono effetti diversi sui vasi sanguiferi. Infatti il neurotrasmettitore ANP agendo sulla muscolatura liscia dei vasi sanguiferi e sulla attivit secretoria degli acini ghiandolari partecipa alla variazione di alcuni parametri fisici dellaria inspirata. I turbinati presentano una superficie molto estesa che agevola i contatti fra aria inspirata e mucosa e una tonaca propria infarcita di numerose ghiandole sieromucose e vasi sanguiferi. Le nostre indagini immunoistochimiche per ANP hanno messo in evidenza che lANP presente nelle cellule acinari delle ghiandole, nelle cellule epiteliali sierose ed in alcune cellule della tonaca propria poste in vicinanza ai sinusoidi e shunts artero-venosi. Tale disposizione spaziale dei siti reattivi per ANP permette di supporre che lANP, per azione paracrina, possa agire sulla muscolatura liscia dei sinusoidi inducendo vasodilatazione e aumentandone la capacitanza mentre agendo sugli shunts artero-venosi ne diminuisce la resistenza. Inoltre la presenza di ANP nelle cellule acinari modificherebbe la composizione e la viscosit del muco. Trachea e Polmone Nella trachea e nel polmone la distribuzione dellANP stato studiato con metodiche immunoistochimiche e PCR amplificazione. I risultati immunoistochimici su sezioni di trachea, confermati dai risultati di amplificazione PCR di ANP c-Dna, indicano che le cellule mucose,le cellule basali e le cellule ciliate sono immunopositive per ANP. Tali risultati concordano con quelli di Lofton (ottenuti con cellule isolate di trachea. Questi risultati indicano che nella trachea lepitelio della mucosa respiratoria sede di sintesi di ANP che potrebbe essere coinvolto nel regolare il battito ciliare e parteciperebbe alla stratificazione del muco sulla mucosa stessa. Nel polmone i risultati immunoistochimici mettono in evidenza pneumociti di 2 ordine risultano immunopositivi per ANP in accordo con i dati di Perrault e Gutkwoska [1]. La presenza di ANP nei pneumociti di 2 ordine interviene nel regolare la secrezione e la composizione del surfactant alveolare (fig. 1). Pleura La ricerca di ANP nelle cellule pleurali stata eseguita con metodiche immunoistochimiche e amplificazione PCR ANPc-DNA. I risultati hanno evidenziato cellule mesoteliali positive per ANP con granuli disposti nello spazio perinucleare (fig. 2). Le cellule mesoteliali svolgono
48
Presenza del fattore natriuretico atriale nellapparato respiratorio
un ruolo fondamentale nella produzione del liquido pleurico per cui lANP presente su tali cellule svolgerebbe un ruolo determinante sulla sintesi e omeostasi del fluido pleurico. Da queste osservazioni risulta che lapparato respiratorio sede di sintesi di ANP che regolando con gli altri neuropeptidi lattivit specifica dei diversi organi che lo compongono permette lo scambio gassoso fra laria inspirata e il sangue.
Fig. 1 - Polmone di coniglio. Cellule immunopositive per ANP.
Fig. 2 - Pleura parietale di coniglio. Cellule mesotelieli positive per ANP. 49
Biagio Valentino, Diego Lipari, Giovanni Peri, Elvira Farina Lipari
Bibliografia [1] [2] [3] [4] [5] Perrault T, Gutkowska J. Role of atrial natriuretic factor in lung physiology and pathology. Am. J. Resp. Crit. Care Med. 1995; 151:226-42. Valentino B, Farina Lipari E, Carini F, Valenza V. Immunohistochemical localization of atrial natriuretic factor (ANP) in the excretory of the rabbit parotid gland. Eur. J. Histochem. 1999; 43:241-5. Farina Lipari E, Valentino B, Lipari D, Dieli F. ANF-immunolocalization in the rabbit airways and lung. It. J. Anat. Embryol. 1999; 104 (suppl.1) :85. Valentino B, Lipari D, Dieli F, Leone A, Farina Lipari E. Distribution of Atrial Natriuretic Factor (ANF) in the Respiratory Tract and Lung showed by Immunohistochemical and PCR Methods. Dent. Med. Probl. 2003; 40:17-22. Farina Lipari E, Lipari D, Leone A, Dieli F, Valentino B. Localization of Atrial Natriuretic Factor (ANF) in the Normal Parietal Pleura in Rabbit. Immunohistochemical Studies and PCR Analysis. Dent. Med. Probl. 2004; 41:209-212.
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IMMUNOHISTOCHEMICAL EXPRESSION Of CB1 CANNABINOIDS RECEPTOR IN RAT LUNG

[Espressione immunoistochimica dei recettori CB1 cannabinoidi del polmone di ratto] Giuseppe Bonaventura, Daniela Cucco, Diego Lipari1, Giovanni Francesco Spatola, Maria Laura Uzzo e Vincenzo Tessitore
DI.ME.S. Dipartimento di medicina sperimentale - Sezione di Istologia ed Embriologia Arcangelo Pasqualino di Marineo. (1) e Sezione di Anatomia umana Emerico Luna, Facolt di Medicina e Chirurgia, Universit degli Studi di Palermo (I)
Key words: CB1 receptor, Endocannabinoids, Lung, Immunohistochemistry Parole chiave: Recettore CB1, Endocannabinoidi, Polmone, Immunoistochimica Abstract. Recent studies document that the CB1 receptor considered since its first identification as brain specific could be expressed by different pheripheral tissues (adipose organ, enteric nervous system, skeletal muscles, hepatocytes, salivary glands). Following this method we have considered interesting to carry out a study on the CB1 receptor immunohistochemical expression and distribution in rat lung. Our results indicate that CB1 receptor are expressed with different intensity in the various cytotypes of respiratory pseudostratified epithelium, in neuroepithelial bodies (NEBs) in nerve bundles distribute to peribronchial myocytes, and in the type II pneumocytes. The findings provide a morphohistochemical basis to speculate novel pulmonary peripheral role played by endocannabinoids with neurocrine, paracrine, endocrine mechanism. Riassunto. Studi recenti documentano che il recettore CB1, considerato in passato come brain specific, pu essere espresso in altri tessuti periferici (organo adiposo, sistema nervoso enterico, muscolo scheletrico, epatociti, ghiandole salivari). Ci sembrato interessante, pertanto, estendere lo studio dellespressione immunoistochimica e della distribuzione del recettore CB1 nel polmone di ratto. I nostri reperti documentano lespressione del CB1 nellepitelio respiratorio pseudostratificato, nei corpi neuroepiteliali (NEBs), nei tronchi nervosi distribuiti alla muscolatura peribronchiale e nei pneumociti di II tipo. I nostri risultati forniscono la base morfoistochimica per prospettare un nuovo ruolo periferico polmonare svolto dagli endocannabinoidi con meccanismo neurocrino, paracrino ed endocrino.
Introduction The toxicological study of the inhalation effects of the marijuana smoke on the lung functions has aroused great interest at the end of the 70s [1,2,3,4]; as regards, the bibliography of those years is full of contributions above all regarding the investigations done on the respiratory effects of the major psychoactive component in cannabis: the delta9-tetraidrocannabinol (THC). Pulmonary studies with inhaled delta-9-tetraidrocannabinol demonstrated a bronchodilating activity in normal human volunteers and reversal of exer-
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Giuseppe Bonaventura, Daniela Cucco, Daniela Lipari, Giovanni Francesco Spatola, Maria Laura Uzzo, Vincenzo Tessitore
cise-and methacholine-induced bronchospasm and hyperinflation in asthmatics [5,6]. The identification of the receptors CB1 and CB2 [7,8,9] for the cannabinoids and then of the respective endogenous ligands (endocannabinoids) [10,11,12,13,14] has risen a new interest in front of this theme in the 90s again; some outcome of previous investigations have been revised and the recent studies have acquired a more accurate scientific value concentrating the attention on receptorial molecules and on their endogenous ligands to cannibimimetic activity that together constitute the endocannabinoid system. The observations given by recent studies document that the CB1 receptor, considered since its first identification as brain specific, that is expressed exclusively by the central nervous system neurons, could be expressed by different peripheral tissues (adipose organ, enteric nervous system, striated muscles, hepatocytes, salivary glands) [15,16,17] and that the endocannabinoid system could have other perypheral targets outside of the central nervous system. Our previous studies document that CB1 receptor is widely distributed in many structures of the gastroenteropancreatic system (GEP) and in the rat salivary glands [18,19,20]; following this method on the exam of the immunohistochemical distribution of CB1 receptor in peripheral tissues, we have considered interesting to carry out a study on the CB1 receptor expression in the rat lung. Materials and methods Specimens of rat trachea, primary bronchi and lung were fixed in Bouins mixture and embedded in paraffin; obtained sections were processed with anti CB1 (Biosource Europe S.A.) by En-Vision + System HRP (AEC). Negative controls were performed by omission of primary antibody, and by absorption control on the primary antibody with the purified antigen. Other sections near by those processed have been submitted at the histological stain using the method hematoxylin-eosin. All the samples have been studied with microscope Leica DMLB. Results Intense immunohistochemical staining for CB1 receptor was found in the entire pseudostratified columnar epithelium of trachea, large and small bronchi, and bronchioles. Immunoreactivity appears more often diffuse in cytoplasm or distributed in thin and spread granula and was absent in the cell membranes. (Fig.1,2,3) The various epithelial cytotypes express the CB1 immunoreactivity not uniformly but with different intensity: it is absent in the goblet mucous cells, strong in ciliated cells and in Clara cells (Fig.4) very strong in the so-called neuroepithelial bodies (NEBs) (Fig. 5a-5b) that are corpuscular organoid structures within the bronchial epithelium, composed of pulmonary neuroendocrine cells (PNECS) which are part of the diffused neuroendocrine system (DNES), distributed through the body
52
Immunohistochemical expression of CB1 cannabinoids receptor in rat lung
(cardiovascular system, GEP system, genital male and female tracts). NEB cells appears as tall columnar cells extending from the basement membrane to the airway lumen (Fig.6). In tangential cut, they usually appear as distinct ovoid corpuscles which are predominantly innervated by afferent sensory fibres of vagal origin with cell bodies residing in the nodose ganglion [21, 22]. Remarkable CB1 immunoreactivity appears in the nerve bundles that pass in the bronchial submucosal layer (Fig. 7a-7b) whose axon terminals distribute themselves to peribronchial miocytes, that are poorly immunoreactive (Fig. 8). In the alveolus strong CB1 immunoreactivity, was observed exclusively in the type II pneumocytes (Fig. 9a-b). Conclusions Our findings justify some clarifications. The CB1 immunoreactivity expressed by citotypes of bronchial epithelium authorizes to believe that the endocannabinoids, via CB1 activation, influence the secretory function of Clara cells, the cleansing function of ciliated cells and the general epithelial trophism during the development. Another important aspect of pulmonary CB1 immunoreactivity, that our research put in evidence for the first time, its a very strong immunopositivity in all cells of NEBs, that demonstrate that the endocannabinoids, activating the CB1 receptors of the neuroepithelial bodies, may modulate the NEB functions. Physiologic studies have affirmed that the NEBs represent intrapulmonary hypoxia sensory chemioreceptors, regulating local ventilation/ perfusion, ratio in the lung lobule [23-24-25]. These function is particularly important during intrauterine life, and during the birth, for the control of pulmonary circulation, in which could be implicated the endocannabinoids. The big CB1 immunoreactivity that appears in the nerve fibres distributed among bronchial and bronchiolar smooth muscles, and with low intensity in the same miocytes surrouding the lamina mucosa, suggests that endocannabinoids influence the bronchial muscle contractility. Previous biochemical analyses indicate that anandamide is synthesized in lung tissues on calcium-ion stimulation and this suggests that locally generated anandamide partecipates in the intrinsic control of airway responsiveness, as well as a prejunctional mechanism, as directly interacting with CB1 receptors expressed by smooth muscles [26]. Our findings agrees with previous studies of Calignano et al. [26] that have studied the ultrastructural localization of CB1 receptor in rat lung, demonstrating that CB1 receptor are present in axon terminals in close proximity to smooth muscle cells. Previous study by Rice et al. demonstrate that CB1 receptor mRNA is expressed in alveolar type II cells [27]. Our findings document, for the first time, that the CB1 immu53
noreactivity in the alveoli is just present in the alveolar type II pneumocytes, and suggest that endocannabinoids regulate surfactant production from alveolar type II cells that is responsible of successful adaptation to air-breathing at birth in the perinatal period. Cerlet and Scott have recently investigated the potential health risks of marijuana use studying the effects of delta-9-tetraidrocannabinol on foetal lung development specifically related to surfactant production by alveolar type II cells isolated from foetal rabbit lungs. The result obtained suggests that THC significantly reduced formation of surfactant [28]. In conclusion, our findings demonstrated that CB1 receptor is expressed in selected structures of rat lung and provide the morphohistochemical basis to display that endocannabinoids, acting through the CB1 receptor, affect several lung function, carrying on new perypherical functions done with different mechanisms: neurocrine, autocrine-paracrine and endocrine. In addiction, the lung represents the birthplace of endocannabinoids (anandamide). This checking further emphasizes the endocannabinoids role in the control of the pulmonary functions. Consequently the lung can be now added rightfully to the list of sites of peripherical tissues that express the CB1 receptor outside the central nervous system.
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[1]
[2]
[3]
[4]
[5a]
[5b]
Figures 1-5b: [1] Intense but not uniform CB1 immunoreactivity in bronchiolar pseudostratified epithelium. Ob. 40x - [2] CB1 immunoreactivity diffuse in bronchiolar epithelium. Ob. 40x - [3] CB1 immunoreactiviy: microgranular staining in the bronchiolar epithelium. Ob. 20x - [4] Strong CB1 immunoreactivity in bronchiolar Clara cells. Ob. 20x - [5a-b] Very strong CB1 immunoreactivity in all neuroendocrine cells of NEB. Ob. 40x.
[6]
[7a]
[7b]
[8]
[9a]
[9b]
Figures 6-9b: [6] A neuroepithelial body stained with hematoxylin eosin . Ob. 40x - [7a-b] CB1 immunoreactivity in nerve bundle of submucosal layer Ob. 20x (a) Ob. 40x (b) - [8] CB1 immunoreactivity in bronchiolar mucosal epithelium, in peribronchial smooth muscle and in type 2 pneumocytes. Ob. 40x - [9a-b] Remarkable CB1 Immunoreactivity is observed exclusively in alveolar type 2 pneumocytes. Ob. 40x.
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] Rosenkrantz H, Fleischman RW. - Effects of cannabis on lungs. Adv Bio Sci 1978 Jul 22-23; 22-23: 279-99. Fleischman RW, Baker JR, Rosenkrantz H. Pulmonary pathologic changes in rats exposed to marihuana smoke for 1 year. Toxicol Appl Pharmacol 1979 Mar 15;47(3):557-66. Vachon L, Fitzgerald MX , Solliday NH, Gould IA & Gaensler EA. Single-dose effects of marihuana smoke. Bronchial dynamics and respiratory-center sensitivity in normal subjects. New Engl J Med 1973 288, 985 989. Tashkin DP, Shapiro BJ, Lee YE & Harper CE. Effects of smoked marijuana in experimentally induced asthma. Am Rev Respir Dis 1975 112, 377386. Tashkin DP, Reiss S, Shapiro BJ, Calvarese B, Olsen JL, Lodge JW. Bronchial effects of aerosolized delta 9-tetrahydrocannabinol in healthy and asthmatic subjects. Am Rev Respir Dis 1977 Jan;115(1):57-65. Abboud RT, Sanders HD. Effect of oral administration of delta-9-tetrahydrocannabinol on airway mechanics in normal and asthmatic subjects. Chest 1976 70, 480 485. Matsuda LA, Lolait SJ, Brownstein MJ, Young AC and Bonner TI. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature, 1990 346:561-564. Munro S, Thomas KL, Abu-Shaar M. Molecular characterization of a peripheral receptor for cannabinoids. Nature, 1993 365:61-65. Herkenham M. in: Cannabinoid Receptors (Pertwee RG, ed. Academic Press) 1995 145-166. Devane WA, Hanus L, Breuer A, Pertwee RG, Stevenson LA, Griffin G, Gibson D, Mandelbaum A, Etinger A, Mechoulam R. Isolation and structure of a brain costituent that binds to the cannabinoid receptor. Science, 1992 258:1946-1949. Hanus L, Gopher A, Almog S, Mechoulam R. Two new unsaturated fatty acid ethanolamides in brain that bind to the cannabinoid receptor. J Med Chem, 1993 36:3032-3034 Bayewitch M, Avidor-Reiss T, Levy R, Barg J, Mechoulam R, Vogel Z. The peripheral cannabinoid receptor: adenylate cyclase inhibition and G protein coupling. FEBS Lett, 1995 375:143-147. Sugiura T, Kondo S, Sukagawa A, Nakane S, Shinoda A, Itoh K, Yamashita A, Waku K. 2-Arachidonoylglycerol: a possible endogenous cannabinoid receptor ligand in brain. Biochem Biophys Res Comm, 1995 215:89-97. Mechoulam R, Ben-Shabat S, Hanus L, Ligumsky M, Kaminski NE, Schatz AR, Gopher A, Almog S, Martin BR, Compton DR. Identification of an endogenous 2-monoglyceride, present in canine gut, that binds to cannabinoid receptors. Biochem Pharmacol, 1995 50:83-90. Pertwee RG. Evidence for the presence of CB1 cannabinoid receptors on peripheral neurons and for the existence of neuronal non CB1 cannabinoid receptors. Life Sci 1999 Vol65, Nos. 6/7, pp597-605. Pinto L, Capasso R, Di Carlo G, Izzo AA. Endocannabinoids and the gut. Prostaglandins Leukot Essent Fatty Acids 2002 Feb-Mar; 66(2-3):333-41A. Storr M, Sibaev A, Marsicano G, Lutz B, Schusdziarra V, Timmermans JP, Allescher HD. Cannabinoid receptor type 1 modulates excitatory and inhibitory neurotransmission in mouse colon. Am J Physiol Gastrointest Liver Physiol 2004 Jan;286(1). Tessitore V, Uzzo ML, Spatola G, Cucco D, Bonaventura G. Immunohistochemical studies on leptin, grelin, orexin-A, orphanin FQ and endocannabinoids expression in the stomach of lean and obese (fa/fa) Zucker rats. First World Congress on Therapies against Obesity Paris 18-19 2006. Tessitore V, Uzzo ML, Spatola G, Cucco D, Bonaventura G. Immunhistochemical study and densitometric comparison on CB1 receptor expression in the gastrointestinal tract and pancreas of obese (fa/fa)and
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lean Zucker rats. It J Anat Embryol 2006 vol. 111 suppl.2, p. 256. [20] Bonaventura G, Mandracchia R, Spatola G, Uzzo ML. Immunohistochemical evidence for the presence of CB1 cannabinoid receptor in rat salivary glands. It J Anat Embryol 2006 vol. 112 (1), p. 43. [21] Cutz E. Neuro-endocrine (APUD-type) cells of the lung. In: Motta PM, Ultrastructure of endocrine cells and tissues, 1984. [22] Lauweryns JM, Cokelaere M, Theunynck P. Neuro-epithelial bodies in the respiratory mucosa of various mammals. Z Zellforsch 1972; 135: 569592. [23] Linnoila RI. Functional facets of the pulmonary neuroendocrine system. Lab Invest 2006 May; 86(5): 425-44. [24] Cutz E, Jackson A. Neuroepithelial bodies as airway oxygen sensors. Respir Physiol 1999; 115: 201214. [25] Adriaensen D, Brouns I, Van Genechten J, Timmermans JP. Functional morphology of pulmonary neuroepithelial bodies: extremely complex airway receptors. Anat Rec A Discov Mol Cell Evol Biol 2003 Jan; 270(1): 25-40. [26] Calignano A, Ktona I, Dsarnaud F, Giuffrida A, La Rana G, Mackie K, Freund TF, Piomelli D. Bidirectional control of airway responsiveness by endogenous cannabinoids. Nature 2000 Nov 2; 408(6808): 96-101. [27] Rice W, Shannon JM, Burton F, Fiedeldey D. Expression of a brain-type cannabinoid receptor (CB1) in alveolar Type II cells in the lung: regulation by hydrocortisone. Eur J Pharmacol 1997 May 30; 327(2-3): 227-32. [28] Cherlet T, Scott JE. Tetrahydrocannabinol (THC) alters synthesis and release of surfactant-related material in isolated fetal rabbit type II cells. Drug Chem Toxicol 2002 May; 25(2): 171-90.
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Heart, microcirculation
MORPHOLOGICAL fEATURES Of THE IDIOPATHIC DILATED CARDIOMYOPATHY

[Aspetti morfologici della Cardiomiopatia Dilatativa Idiopatica] Consolato Sergi, Francesco Cappello*, Hans-Joerg Steiner, Valentina Di Felice*, Giovanni Zummo* and Gregor Mikuz
Institute of Pathology, Medical University of Innsbruck, A-6020 Innsbruck, Austria, and *Dipartimento di Medicina Sperimentale, Sezione di Anatomia Umana, Universit di Palermo (I)
Key words: Cardiomyopathies, Idiopathic Dilated Cardiomyopathy, Genetic diseases Parole chiave: Cardiomiopatie, Cardiomiopatia Dilatativa Idiopatica, Malattie genetiche Abstract. Cardiomyopathies are a group of myocardial diseases that are characterized by not always defined etiologies, with a mixture of genetic and acquired contributing factors, by the frequent coexistence of distinctive pathologic traits and non-specific clinical and pathological features, and finally by an almost consistent overlap of natural history and outcome. In this paper, we review the main morphological features of Idiopathic Dilated Cardiomyopathy. Riassunto. Le cardiomiopatie sono un groppo di malattie del miocardio caratterizzate da una non sempre definibile e chiara eziologia, derivando da un insieme di fattori contribuenti genetici e acquisiti, e mostrando la coesistenza di caratteristiche patologiche specifiche e non, oltre a una storia naturale non patognomonica. In questo lavoro riportiamo le principali caratteristiche morfologiche della Cardiomiopatia Dilatativa Idiopatica.
Definition Dilated cardiomyopathy is defined as a heart muscle disorder defined by the presence of a dilated and poorly functioning left ventricle in the absence of abnormal loading conditions (hypertension, valve disease) or ischaemic heart disease sufficient to cause global systolic impairment [1]. It is important to stress that the presence of coronary atherosclerosis does not rule out a diagnosis of dilated cardiomyopathy. However, a conditio sine qua non is that the degree of myocardial dysfunction is not explained by the extent of ischemic damage. In a minority of cases with dilated cardiomyopathy genetic factors, viruses, toxins, catabolytes of an altered metabolism or immunological causes are found. However, the majority of cases with of dilated cardiomyopathy remains idiopathic (IDCM) and therefore it remains the reference standard in books and medical journals [2]. Clinical Diagnosis Diagnostic criteria for IDC are an ejection fraction < 0.45 and/or a fractional shorten61
Consolato Sergi, Francesco Cappello, Hans-Joerg Steiner, Valentina Di Felice, Giovanni Zummo and Gregor Mikuz
ing of < 25%, and a left ventricular end diastolic dimension of > 112% predicted value corrected for age and body surface area. Exclusion criteria include absence of systemic hypertension (> 160/100 mm Hg), coronary artery disease (> 50% in one or more major branches), chronic excess alcohol (> 40 g/day female, > 80 g/day male for more than five years after six month abstinence), systemic disease known to cause dilated cardiomyopathy, pericardial diseases, congenital heart disease, cor pulmonale. The criteria for left ventricular enlargement are based on data of Henry et al. [3]. Macroscopy Grossly, hearts harboring IDCM show a massive increase in mass in both pediatric and adult age. In adults, heart weight often exceeds 500 g and the heading of cor bovinum is used in these circumstances. The survival of patients affected with IDCM influences the heart weight. In fact, long-term survivors have significantly heavier hearts than those who die in the short term. In this case, the functionality plays a major role. Moreover, hearts of patients affected with IDCM show an apical rounding other than ventricular dilation (Roman arch). In the usual and complete configuration with four-chamber dilation, the heart shows an almost spherical appearance with disappearance of a proper apex. The epicardium is usually normal and coronary arteries do not usually show high-grade atherosclerosis. On cut surface, the myocardium is flaccid and there is eccentric hypertrophy. Heart hypertrophy is shown by cardiac weight increase, although this finding is not always evident because of dilation. The ratio of parietal thickness to cavity diameter drops from 0.48 (normal adult value) to 0.17-0.21, according to the survival time. Fibrosis is also seen with areas of replacement scarring. At this time, the pathologist needs to make a differential diagnosis with ischemic heart disease (coronary artery check) and myocarditis (virus real-time PCR). Other findings include endocardial fibrosis and mural thrombi. These latter are due to the reduced ejection fraction (a pattern of the cardiac apex that is also called: the change from caput vivum to caput mortuum). Cross-section planes may also show signs of secondary valvular incompetence with mitral valve leaflets harboring rolled edges. Light Microscopy Light microscopy plays an important role in the normal routine of a pathologist and IDCM is characterized by a particular complex of nonspecific features, that are probably due to the parietal stress rather than etiological features. IDCM change involves all myocardial components, including myocytes, interstitium, small vessels, and endocardium. Myocytes show cell hypertrophy defined by a global enlargement of hyperchromatic, often bizarrely shaped nuclei. Moreover, there are two other characteristics associated with dilation and increased lengthening, including thinning, waving, and side-to-side slippage of
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Morphological features of the Idiopathic Dilated Cardiomyopathy
myocytes within muscular bundles. Myocytes may assume the form of attenuated ones, showing an enlarged, rectangular and hyperchromatic nucleus that is transversely arranged and occupies the entire cross-sectional area. Thus, there is an increase of variability in the myocellular area. Another histologic feature is constituted by the myofibril loss, which causes hydropic change of the myocyte. Hydrops of the myocyte is often localized in the subendocardial layer and ranges from a perinuclear halo to a pattern of colliquative myocytolysis. A disordered arrangement of desmin intermediate filaments also characterizes a myocellular derangement of the myocytes as detected by immunohistochemistry. In particular, an immunohistochemical procedure is also needed to demonstrate an abnormal type I/type III fibrillar collagen ratio. In a recent study, atrial natriuretic protein (ANP) and CD34 (an endothelial and stem cell marker) were significantly over-expressed in IDCM compared to normal heart (NH). Patients with a difference of more than 20 myocardial fibers in the compared expression between CD34 and troponin T were associated with a quite less favorable survival although the difference was not significant [4]. The increase in ANP positive cells in IDCM could be a consequence of neuro-hormonal activation due to a decline of the impaired myocyte contractility. Further, since it was already shown that ANP could be important in the control of vascular remodeling, we postulated that the increment of CD34 positive cells could be functionally correlated to the increase of ANP production. Differential expression of CD34 and troponin T might be used for future studies to evaluate their prognostic value. Four types of fibrosis are commonly seen in IDCM hearts. Interstitial (interfiber) and perivascular fibroses prevail, whereas replacement and endocardial fibroses are evident focally. A number of inflammatory cells may be found in the myocardium, mostly close to areas of replacement fibrosis. These cells are almost exclusively lymphocytes, but no diagnosis of myocarditis should be made. The paucity of the infiltration and the topographic distribution help in distinguishing these two diseases. This can, however, become a challenge if limited sampling, e.g. endomyocardial biopsy, is the material to work on. Finally, intramyocardial branches of the coronary arteries may show a proliferation of smooth muscle cells of the tunica media that can evolve to a luminal narrowing. Morphometric analysis is an important help in light microscopy. The potential advantages of measurement in histopathology have been recognized for many years. The need for measurement arises from our realization that some of the diagnostic decisions that we make are poorly reproducible and the incorporation of computers in photo-microscopy and morphometry has opened the possibility of better evaluating remodeling structures during development [5]. A morphometric system has been used for the development of the biliary tract that has proved to be useful for cardiac morphometry as well [6]. We used a set-up that consisted of the following components: a light microscope (Zeiss, Jena, Germany),
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CCD grayscale video camera (Sony CCD-IRIS, Sony Corp., Cologne, Germany), BNC cable hookup, an Intel-based personal computer equipped with video frame grabber card, a Windows 3.11 operating system, Bioscan Optimas version 5.2 software (Bioscan, Edmonds, WA, U.S.A.) running in interlaced mode, a 200 color monitor (GDM, 2063 MS, Stimmler, Munich, Germany), and a drawing pad (WacomTM digitizer II, equipped with an induction mouse). The Optimas software was calibrated using a prefixed grid 2 of known dimensions (checkerboard pattern, square area of 2025 mm) using a 6.33 objective. Using a random number generator up to ten areas may be select for morphometry and quantitative analysis of the biliary epithelial cells or myocytes. Each freeze frame is saved on the hard disk (Tiff files, Tiff Image Archive System) or an external hard-drive permitting SATA files transfer. The outline shape may be drawn manually using the induction mouse on a drawing pad (WacomTM) or automatically using an open-source free-software Image J. Nuclear area and myofibril volume fraction are two morphometric variables, which have achieved an important status for their relationship to cardiac function and patients outcome. Both nuclear enlargement and reduced myofibril volume fraction (myofibril loss) are associated with a worse functional status and poorer prognosis. Electron Microscopy Even though the study of the ultrastructure is nonspecific, it may reveal characteristics associated with the myofibril loss. Electron microscopy studies show enlargement of nuclei with irregular shape and deep infoldings of the nuclear membrane, multiple nucleoli, nuclear inclusions of sarcoplasmic components, double nuclei, dilation and proliferation of T tubules, rough endoplasmic reticulum, and an increase of the number of sarcomeres and mitochondria (changes consistent with myocellular hypertrophy). Myofibril loss is characterized by a range from rarefaction to complete absence of sarcomeres. Conclusion Clinically, fatigue, exercise intolerance, and chest pain are the most frequent symptoms. Physical examination reveals a variable degree of cardiomegaly and aspects of congestive pump failure. Pulmonary vascular redistribution is detected by chest radiography and complex ventricular arrhythmias are common and have been linked to the degree of left ventricular functional impairment. In children, recurrent or incessant supraventricular tachycardias may be the cause, and not the effect of ventricular dysfunction, because arrhythmia therapy may induce improvement, or even regression of IDCM changes. In terms of risks and effectiveness the indications for endomyocardial biopsy are still controversial. However, it adds minimal risk to a cardiac catheterization in specialized centers and should be considered a useful complement in the diagnostic procedure in distinguishing IDCM
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from other common causes of cardiomyopathy, to exclude myocarditis (real-time PCR), to suspect a mitochondrial cardiomyopathy (electron microscopy), to identify abnormal deposition (myocardial toxicity), to address appropriate genetic counseling (clinical and molecular genetics), and to provide the basis for future research (autoimmunity?). Pathological anatomy of IDCM may show characteristics that taken singularly are not diagnostic, but together provide not only an important diagnostic help, but also the basis for the study of this fascinating chapter of cardiovascular pathology.
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Fig. 1. Cardiomyocytes of a patient with IDCM showing perinuclear halos, contraction bands, hyperchromatic enlarged nuclei and waving of the fibers shape (x 400, original magnification, hematoxylin and eosin staining).
Figure 2. Myocardial tissue for morphometric analysis of cardiomyocytes in an endomyocardial biopsy from a patient affected with idiopathic dilated cardiomyopathy. We use a particular thin section of 1-2 micrometer to study cardiomyocyte morphometry (x 200, original magnification, alcian-hematoxylin staining).
References [1] [2] [3] [4] [5] [6] Elliott P. Cardiomyopathy. Diagnosis and management of dilated cardiomyopathy. Heart 2000; 84:106112. Gallo P, dAmati G. Cardiomyopathies. In: Cardiovascular Pathology Silver, Gotlieb, Schoen (Eds). Third Edition, pp. 285-294. Henry WL, Gardin JM, and Ware JH. Echocardiographic measurements in normal subjects from infancy to old age. Circulation 1980; 62:10541061. Ardizzone N, Cappello F, Di Felice V, Rappa F, Minervini F, Maras S, Maras L, Rabl W, Zummo G and Sergi C. Atrial Natriuretic Peptide and CD34 Overexpression in Human Idiopathic Dilated Cardiomyopathies. AMPIS, in press 2007. Hamilton PW, Allen DC. Morphometry in histopathology. J Pathol 1995; 175:369379. Sergi C, Adam S, Kahl P, Otto HF. The remodeling of the primitive human biliary system. Early Hum Dev 2000; 58:167-178.
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HYDROGEN PEROXIDE IN ENDOTHELIUM: MULTIfACETED ROLES IN CELLULAR STRESS AND SIGNALLING

[Il perossido didrogeno nellendotelio: molteplici ruoli nello stress e nella segnalazione cellulare]
Rita Anzalone, Giampiero La Rocca*, Francesca Magno, Simona Corrao, Marco Carbone, Tiziana Loria, Francesca Pizzo, Giuseppina Maria Ciaccio, Antonino Di Stefano+, Pantaleo Giannuzzi, Felicia Farina and Giovanni Zummo
Sezione di Anatomia Umana, Dipartimento di Medicina Sperimentale, Universit degli Studi di Palermo, Palermo (I) - + Fondazione S. Maugeri IRCCS, Centro Medico di Veruno (NO) - The two authors contributed equally to this work - * Corresponding author
Key words: Hydrogen peroxide, Endothelial cells, Oxidative stress, Nitric oxide, Chronic heart failure Parole chiave: Perossido di idrogeno, Cellule endoteliali, Stress ossidativo, Ossido nitrico, Scompenso cardiaco cronico Abstract. The endothelial cells are a population of squamous cells that line the blood vessels throughout the body. These cells are being now viewed as key modulators of several physiological functions, and multiple cardiovascular diseases are due to impairment of their function. An increasing number of studies have shown the ability of a variety of vascular cells, not only endothelial cells, but also fibroblasts and smooth muscle cells, to produce reactive oxygen species (ROS). Moreover, as a distinctive feature, endothelial cells are able to manage with higher concentrations of ROS as well as reactive nitrogen species (RNS) with respect to the other cell types. The importance of hydrogen peroxide in endothelial biology is object of growing interest, and consequently the enzymes involved in both its generation and its clearance are now viewed as novel mediators of broad relevance. Historically, superoxide ion has been viewed as the most important ROS in vasculature. In fact, it can be produced by a number of intracellular enzymes, and it can directly influence nitric oxide bioavailability through its transformation in peroxynitrite. More recently, the dismutation product of superoxide, i.e. hydrogen peroxide, has been regarded as an important signaling agent; capable to transduce the initial stimulus longer and towards longer distances. Given the comprehension of the molecular mechanisms involving these molecules should be the key to better understanding the pathophysiology of cardiovascular diseases in which oxidative stress plays a key role. Riassunto. Le cellule endoteliali costituiscono una popolazione di elementi pavimentosi che rivestono I vasi ematici nel corpo. Queste cellule sono ormai considerate modulatrici di diverse funzioni fisiologiche, e diverse malattie cardiovascolari sono dovute ad uno sbilancio nella loro funzionalit. Un cresente numero di studi ha mostrato labilit delle cellule vascolari, non solo endoteliali, ma anche fibroblasti e cellule muscolari lisce, nella produzione di specie reattive dellossigeno (ROS). In particolare, le cellule endoteliali sono in grado di tollerare maggiori concentrazioni di ROS e di specie reattive dellazoto (RNS) rispetto ad altri citotipi. Limportanza del perossido di idrogeno nella biologia endoteliale oggetto di crescente interesse, e di conseguenza gli enzimi coinvolti nella generazione e nella eliminazione di questa molecola sono considerati mediatori di grande rilevanza. Storicamente, lo ione superossido stato considerato il pi importante ROS nella
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Rita Anzalone, Giampiero La Rocca, Francesca Magno, Simona Corrao, Marco Carbone, Tiziana Loria, Francesca Pizzo, Giuseppina Maria Ciaccio, Antonino Di Stefano, Pantaleo Giannuzzi, Felicia Farina, Giovanni Zummo
vascolatura. Infatti, esso pu essere prodotto da diversi enzimi cellulari, e pu rapidamente influenzare la biodisponibilit dellossido nitrico attraverso la sua trasformazione in perossinitrito. Pi recentemente, il prodotto di dismutazione del superossido, cio il perossido didrogeno, stato considerato come unimportante molecola di segnalazione, capace di trasdurre lo stimolo iniziale pi a lungo e a maggiore distanza. La comprensione dei meccanismi molecolari che coinvolgono tali molecole pu essere la chiave per meglio chiarire la fisiopatologia delle malattie cardiovascolari in cui lo stress ossidativo gioca un ruolo chiave.
1. Endothelium: origin, phenotype, in vitro models Endothelial cells (ECs) line the blood vessels in all our organs since the initial phases of development. They derive from mesoderm germ layer, and are characterized by a very early differentiation process, together with hematopoietic tissues. In fact, following angioblasts proliferation in the yolk sac, multiple foci of endothelial cells can be observed in the embryo. From the further proliferation of these cells, the heart as well as the embryonic vessels will be formed [1,2]. As a fundamental characteristic, these processes are finely regulated by a series of molecular switches between two endothelial phenotypes: migratory and non migratory, in order to extend the primitive vascular network following the developmental processes [3]. Multiple recent reports suggested that angioblasts (or cells classified as endothelial precursors) can be found also in adult tissues as well as in the bloodstream. The central importance of endothelial cells for the study of cardiovascular diseases has been highlighted in a growing number of experimental studies, both in vitro and in vivo. In particular, after the development of techniques to isolate and successfully propagate endothelial cells in vitro, with the use of appropriate substrates and supplements [4,5], in vitro studies increased by number giving a fundamental contribution to the definition of endothelial biology. In particular, HUVECs (human umbilical vein endothelial cells) have been widely used as a model system to investigate endothelial phenotype under physiologic and pathologic conditions (e.g. for experiments on angiogenesis, atherosclerosis, inflammation) [6]. The main counterpoint to the use of this model system was its derivation from an extraembryonic vascular structure, which lasts only some months and therefore, cannot develop significant vascular alterations in this period. Moreover, the need to obtain organ-specific endothelial cells has developed following the observations that vascular beds show a great heterogeneity between different organs and tissues and even inside the same organ [7]. To this regard, endothelial cells obtained from adult organs (also defined primary microvascular cells) can constitute a more valid model for the study of vascular diseases. Moreover, at the molecular level, the phenotypical characterization of endothelial cells should be based on more than one marker (the most common being CD31 and von Willebrand Factor, vWF) since not all endothelia in all types of vessels express uniformly
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Hydrogen peroxide in endothelium: multifaceted roles in cellular stress and signalling
these two markers, differences being present even inside the same organ [8]. More strikingly, other endothelial markers, as CD34, are used with caution due to their unconstant rate of distribution. In a classical work [9], it has been shown that while cells from the umbilical artery are highly positive to this molecule, HUVEC (which are extracted from the umbilical vein) present a lower number of CD34+ cells, therefore influencing experimental works based on the expression of this surface marker (i.e. homing of hematopoietic stem cells). In addition, the phenomenon of the lack of positivity to phenotypical as well as functional markers is accentuated in stable commercial endothelial cell lines, widely used in in vitro experiments. Most of the lines, in fact, even if capable to perform an higher number of population doublings with respect to primary cells, have been shown to lack one or more markers, adhesion molecules or receptors, therefore justifying the efforts of researchers in using primary cell lines as HUVEC or organ-specific microvascular cells as primary models for vascular pathophysiology [10]. Among the number of functions characterizing endothelial cells in vivo, they provide a structural barrier between the circulation and the surrounding tissues, with local variations in morphology and permeability, therefore regulating molecular trafficking via specific transport systems. In fact, ECs are able to transport specifically molecules from the luminal side to the basal one, by the process of transcytosis [11]. Moreover, the regulation of blood pressure and blood flow is influenced by endothelium-dependent release of molecular mediators: vasodilator molecules such as nitric oxide (NO) as well as vasoconstrictor ones. NO is receiving great attention for its multiple roles in vascular biology and cellular signaling processes. Endothelial cells elaborate NO constitutively, by the activity of one of the isoforms of NO synthase (NOS), eNOS or the NOS3 gene product, which is constitutively expressed in ECs. All isoforms of NOS require the formation of an active dimer to exert their enzymatic function, and the regulation of the enzyme dimeric state is an important regulatory process for the maintenance of its activity. Endothelial-cell derived NO has several important effects on the vasculature: first of all, NO maintains basal tone of vessels by relaxing vascular smooth muscle cells, therefore providing endothelium-dependent vasorelaxation. In addition, nitric oxide inhibits smooth muscle cell migration and proliferation, therefore providing signals which act limiting neointimal proliferation after vascular injury [12]. 2. Roles of ROS in endothelium The term endothelial dysfunction refers to a number of processes characteristic of several cardiovascular pathologies. While oxidative stress is a common denominator of endothelial dysfunction, the mechanisms underlying the impairment of endothelium-dependent responses can be pathology-specific and therefore markedly different [13]. Between the key factors that are associated to endothelial dysfunction, an important role is played by
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a disequilibrium in the cellular levels of ROS. Often this intracellular variation triggers a complex cascade of consequences, which may be accompanied by, or lead to, a reduced bioavailability of NO. As a final consequence, therefore, alterations in nitric oxide concentration should lead to the formation of other nitrogen-containing species, namely reactive nitrogen species (RNS), therefore linking together two of the fundamental outcomes of cellular stress events: oxidative stress and nitrosative stress [14]. A first way by which ROS can contribute to the development of cardiovascular pathologies should be by direct chemical interactions, e.g. by oxidation of the cellular constituents, or by influencing the bioavailability of NO, or even by influencing the signal transduction pathways [15]. Interestingly, the endothelium constitutes a particular example of tissue in which the basal levels of some ROS, as hydrogen peroxide, are more elevated than in other cell types. In addition, in vitro and in silico data have shown that the cellular permeability to molecules as hydrogen peroxide is enhanced in cultured endothelial cells. Moreover, several enzymes in these cells may constitute potential sources of ROS. One example may be the NADPH oxidase, together with uncoupled eNOS or xanthine oxidase (XO) [16]. These enzymes have been shown to be able to produce superoxide ion, as discussed further below. Finally, it is well established that the balance between ROS-producing and ROS-scavenging mechanisms is of key relevance for the maintenance of a proper endothelial function. Therefore, the reduction of ROS-scavenging processes, characteristic of chronic heart failure, may contribute to the development of oxidative stress conditions. More importantly, in heart failure additional processes influencing the delicate balance between ROS and RNS species should take place. In fact, xanthine oxidase, a superoxideproducing enzyme, is upregulated following heart failure [17]. Interestingly, it has been suggested that the unbalance between enzymes (NOS versus XO) in failing heart should reflect the disequilibrium of nitric oxide and superoxide ion levels. It has been hypothesized that, while in physiologic context the equilibrium is shifted towards an abundance of NO with respect to superoxide, favoring S-nitrosylation reactions, in heart failure superoxide is the prevalent molecular specie, and this should favor oxidative stress development due to oxidation reactions taking place [14]. 3. Hydrogen Peroxide in endothelium: sources and clearance Hydrogen peroxide is a reactive oxygen species which is capable to exert several physiological effects on endothelial cells. In vitro experiments have shown that endothelial cells isolated from different vascular beds increase their proliferative activity in presence of exogenous hydrogen peroxide in the low micromolar range (lower than 50 M, but higher in certain subtypes of endothelial cells) (reviewed in ref. 18). In fact, hydrogen peroxide action should lead to an increase of expression of VEGF, as well as other growth factors.
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On the other hand, excess levels of hydrogen peroxide should lead to an increase of cell death. Endogenous H2O2 is a ROS which is mainly generated by dismutation of superoxide ion. There are many intracellular sources of superoxide ion in endothelial cells, such as xanthine oxidase, uncoupled eNOS, or the mitochondrial respiratory chain. Therefore, even if this ion possesses a short half-life, it can trigger different responses in several pathways. For example, one of the most chemically-favored reactions involving superoxide is the production of peroxynitrite (ONOO-), thus lowering the intracellular levels of NO [15,18]. The second important reaction is the dismutation, which is carried out by SOD (superoxide dismutase) enzymes, in order to produce hydrogen peroxide, a molecule which is being viewed as the long-term effector of superoxide signaling. In fact, with respect to other ROS, it is not a free radical, since it does not possess an unpaired electron in its outer layer. The short half-life and potential of diffusion of superoxide constitute a severe limit to the role of this ROS as an important paracrine hormone in vascular biology. Therefore hydrogen peroxide has two advantages: it is more stable, and is also capable to diffuse into different cellular compartments. Regarding superoxide dismutation, several literature reports suggested a differential activity of the three SOD isoforms depending on the source of superoxide, i.e. the location of the enzymatic activity which is responsible for its production (reviewed in ref. 19). In fact, mitochondrial manganese SOD (Mn-SOD) is primarily involved in the processing of O2- derived from mitochondrial electron transport chain. Cytosolic copper/zinc (Cu/Zn)-SOD dismutes O2- derived from cytosolic oxidases. Moreover, extracellular superoxide produced by leukocytes (and probably other cell types) is determined by the extracellular form of Cu/Zn-SOD (ec-SOD) [20]. A so high number of cellular sources for hydrogen peroxide corresponds to several enzymatic system to properly manage the oxidant properties of this molecule. Therefore, hydrogen peroxide levels are maintained at a steady state by the actions of scavengers as catalase and glutathione peroxidase (Gpx1) [21]. Gpx1 is located in both cytoplasm and mitochondria, and is a key enzyme for the cellular defense against oxidative stress. It has been suggested that in cells exposed to doses of hydrogen peroxide lower than 100M, Gpx1 is the main enzyme involved in H2O2 reduction. Catalase is a peroxisomal enzyme and exclusively catalyzes the conversion of H2O2 to water, after the so-called catalase latency. More recently, a new class of antioxidants has been described. Peroxiredoxins are able to reduce hydrogen peroxide using thioredoxin as electron donor. Recent studies have characterized their involvement in the regulation of signaling processes by hydrogen peroxide [22]. As discussed earlier, the reduction of ROS-scavenging molecules, characteristic of chronic heart failure, may contribute to the development of oxidative stress conditions,
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therefore the proper maintenance of peroxide-producing as well as clearance mechanisms is essential for endothelial function. 4. The differential effects of hydrogen peroxide on endothelial biology In recent years, hydrogen peroxide emerged as a molecule possessing several effects on endothelium. First of all, in vitro experiments gave contrasting results on viability effects of exogenous hydrogen peroxide on ECs. In fact, while several reports indicated that doses of hydrogen peroxide over 50M in the culture medium should lead to an increase in apoptosis in HUVEC cells [23], which increased at higher doses, other studies have shown that artery-derived endothelial cells should overcome doses of H2O2 even four fold higher. Interestingly, clonogenic endothelial progenitor cells showed a high sensitivity to oxidative stress [24]. Moreover, similar doses of hydrogen peroxide may have differential effects on cellular viability with respect to the type of endothelial cells used for the experiments. In fact, in a very recent paper, La Rocca and collaborators [25] showed that exposure of HUVEC to 60M hydrogen peroxide for 3 and 6 hours resulted in a slight decrease of viability (4 to 7% less). When the same experiments were performed on endocardial endothelial cells, derived from the endocardium of CHF patients, these cells responded with a significant increase in proliferation. The authors therefore suggested that endothelial cells isolated from an organ primed for oxidative stress should be more resistant to the presence of stressor molecules [25]. Apart from these roles regarding endothelial proliferation, it has been shown a mutual interdependence between the concentrations of hydrogen peroxide and nitric oxide in endothelium: NO has been shown to possess a dual effect on H2O2 mediated toxicity, limiting or enhancing this process with respect to the concentrations of both species [26]. Endothelial hydrogen peroxide has been shown to exert endothelium-dependent vasorelaxation, a process due to the action of the endothelial isoform of NOS. It has been demonstrated that in vitro exogenous hydrogen peroxide upregulates eNOS transcription [18]. The increase of enzyme molecules leads to an increase in NO bioavailability, thereby causing vascular relaxation. Moreover, it has recently been suggested that hydrogen peroxide can function as a coupler of myocardial oxygen consumption to coronary blood flow increase. In fact, since superoxide ion is produced proportionally to the rate of cardiac metabolism, its dismutation leads to an increase in intracellular H2O2 concentration. Since one of the downstream effects is the upregulation of eNOS, the final point is a vasoactive effect [27]. On the contrary, in cardiovascular pathologies a number of factors should lead to the uncoupling of NOS. This process causes the arrest of NO production and the formation of superoxide ion. Self-maintaining circuits have been hypothesized for the generation of H2O2 in endothe74
lial cells. The ability of elevations in ROS to promote oxidase activation has been reported in some past experiments: Li et al. [28] showed that exogenous H2O2 was able to activate NAD(P)H oxidase to produce O2 independently of xanthine oxidase or eNOS. These findings were further confirmed by other groups, who proposed that even small concentrations of hydrogen peroxide derived from NAD(P)H oxidase can promote sustained activation of NAD(P)H oxidase itself [29]. Finally, as discussed earlier, H2O2 increase should lead to the reduced bioavailability of eNOS cofactors, therefore resulting in uncoupled eNOS and leading to further peroxide production. Enzymatic uncoupling should be caused by different processes: on one hand, it can be caused by the reduction of bioavailability of L-arginine, the NOS substrate, due to its chlorination by hypochlorous acid. On the other hand, the deficiency of tetrahydrobiopterine (BH4), an essential cofactor in NO synthesis, should be the result of the downregulation of dihydrofolate reductase (DHFR) by hydrogen peroxide. In fact, this enzyme is involved in recycling of oxidized BH4, a molecule which levels were linked to eNOS uncoupling by multiple reports [30,31]. Present data therefore reinforce the concept that the equilibrium of hydrogen peroxide levels, maintained by its intra- or extracellular sources on one hand, and by the clearance enzymes on the other hand, constitutes a key factor in endothelium physiology. Perturbations of this equilibrium, as seen in cardiovascular diseases, should have profound consequences, therefore misleading the cellular and paracrine signaling pathways regulating vascular homeostasis.
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References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] Ferguson JE III, Kelley RW, PattersonC. Mechanisms of endothelial differentiation in embryonic vasculogenesis. Arterioscler Thromb Vasc Biol 2005; 25: 2246-2254. Ratajska A, Czarnowska E, Kolodzinska A, Kluzek W, Lesniak W. Vasulogenesis of the embryonic heart: origin of blood island-like structures. Anat Rec A Discov Mol Cell Evol Biol 2006; 288: 223-232. Schmidt A, Brixius K, Bloch W. Endothelial precursor cell migration during vasulogenesis. Circ Res 2007; 101: 125-136. Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins: Identification by morphologic criteria. J Clin Invest 1973; 52: 2745-2756. Marin V, Kaplanski G, Gres S, Farnarier C, Bongrand P. Endothelial cell culture: protocol to obtain and cultivate human umbilical endothelial cells. J Immunol Methods 2001; 254: 183-190. Jacobi J, Kristal B, Chezar J, Shaul SM, Sela S. Exogenous superoxide mediates pro-oxidative, proinflammatory, and procoagulatory changes in primary endothelial cell cultures. Free Radic Bol Med 2005; 39: 1238-1248. Aird WC. Phenotypic heterogeneity of the endothelium: I. Structure, function, and mechanisms. Circ Res 2007; 100: 158-173. Turner RR, Beckstead JH, Warnke RA, Woods GS. Endothelial cell phenotypic diversity. In situ demonstration of immunologic and enzymatic heterogeneity that correlates with specific morphologic subtypes. Am J Clin Pathol 1987; 87: 569-575. Fina L, Molgaard HV, Robertson D, Bradley NJ, Monaghan P, Delia D, Sutherland DR, Baker MA, Greaves MF. Expression of the CD34 gene in vascular endothelial cells. Blood 1990; 75: 2417-2426. Unger RE, Krump-Konvalinkova V, Peters K, Kirkpatrick CJ. In vitro expression of the endothelial phenotype: comparative study of primary isolated cells and cell lines, including the novel cell line HPMECST1.6R. Microvascular Res 2002; 64: 384-397. Simionescu M, Gafencu A, Antohe F. Transcytosis of plasma macromolecules in endothelial cells: a cell biological survey. Microsc Res Tech 2002; 57: 269-288. Marks DS, Vita JA, Folts JD, Keaney JF, Welch GN, Loscalzo J. Inhibition of neointimal proliferation in rabbits following vascular injury by a single treatment with a protein adduct of nitric oxide. J Clin Invest 1995; 96: 2630-2638. Fltou M, Vanhoutte PM. Endothelial dysfunction: a multifaceted disorder. Am J Physiol Heart Circ Physiol 2006; 291: H985H1002. Hare JM, Stamler JS. NO/redox disequilibrium in the failing heart and cardiovascular system. J Clin Invest 2005; 115: 509-517. Linke A, Recchia F, Zhang X, Hintze TH. Acute and Chronic Endothelial Dysfunction: Implications for the Development of Heart Failure. Heart Failure Rev 2003; 8: 87-97. Bauersachs J, Bouloumie A, Fraccarollo D, Hu K, Busse R, Ertl G. Endothelial dysfunction in chronic myocardial infarction despite increased vascular endothelial nitric oxide synthase and soluble guanylate cyclase expression: Role of enhanced vascular superoxide production. Circulation 1999; 100: 292298. Seddon M, Looi YH, Shah AM. Oxidative stress and redox signalling in cardiac hypertrophy and heart failure. Heart 2007; 93: 903-907. Cai H. Hydrogen peroxide regulation of endothelial function: Origin, mechanism and consequences. Cardiovasc Res 2005; 68: 26-36. Faraci FM, Didion SP. Vascular Protection: Superoxide Dismutase Isoforms in the Vessel Wall. Arterio-
[17] [18] [19]
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scler. Thromb. Vasc. Biol. 2004; 24: 1367-1373. [20] Stralin P, Karlsson K, Johansson BO, Marklund SL. The interstitium of the human arterial wall contains very large amounts of extracellular superoxide dismutase. Arterioscler Thromb Vasc Biol 1995; 15: 2032-2036. [21] Suttorp N, Toepfer W, Roka L. Antioxidant defense mechanisms of endothelial cells: glutathione redox cycle versus catalase. Am J Physiol 1986; 251: C671-C680. [22] Kang SW, Chae HZ, Seo MS, Kim K, Baines IC, Rhee SG. Mammalian peroxiredoxin isoforms can reduce hydrogen peroxide generated in response to growth factors and tumor necrosis factor-alpha. J Biol Chem 1998; 273: 6297-6302. [23] Ramachandran A, Moellering D, Go YM, Shiva S, Levonen AL, Jo H, Patel RP, Parthasarathy S, DarleyUsmar VM. Activation of c-Jun N-terminal kinase and apoptosis in endothelial cells mediated by endogenous generation of hydrogen peroxide. Biol Chem 2002; 383: 693-701. [24] Ingram DA, Krier TR, Mead LE, McGuire C, Prater DN, Bhavsar J, Saadatzadeh MR, Bijangi-Vishehsaraei K, Li F, Yoder MC, Haneline LS. Clonogenic endothelial progenitor cells are sensitive to oxidative stress. Stem Cells 2007; 25: 297-304. [25] La Rocca G, Di Stefano A, Eleuteri E, Anzalone R, Cappello F, Magno F, Corrao S, Loria T, Farina F, Martorana A, Di Gangi C, Colombo M, Sansone F, Patan F, Rinaldi M, Giannuzzi P, Zummo G: Oxidative stress induces myeloperoxidase expression by HUVEC and Chronic Heart Failure endocardial endothelial cells. Lab Invest, 2007, submitted. [26] Rauen U, Li T, de Groot H. Inhibitory and enhancing effects of NO on H(2)O(2) toxicity: dependence on the concentrations of NO and H(2)O(2). Free Radic Res 2007; 41: 402-412. [27] Saitoh S, Zhang C, Tune JD, Potter B, Kiyooka T, Rogers PA, Knudson JD, Dick GM, Swafford A, Chilian WM. Hydrogen peroxide: A feed-forward dilator that couples myocardial metabolism to coronary blood flow. Arterioscler Thromb Vasc Biol 2006; 26: 2614-2621. [28] Li W-G, Miller FJ Jr, Zhang HJ, Spitz DR, Oberley LW, Weintraub NL. H2O2-induced O2 production by a non-phagocytic NAD(P)H oxidase causes oxidant injury. J Biol Chem 2001; 276: 29251-29256. [29] Seshiah PN, Weber DS, Rocic P, Valppu L, Taniyama Y, Griendling KK. Angiotensin II stimulation of NAD(P) H oxidase activity. Upstream mediators. Circ Res 2002; 91: 406-413. [30] Chalupsky K, Cai H. Endothelial dihydrofolate reductase: Critical for nitric oxide bioavailability and role in angiotensin II uncoupling of endothelial nitric oxide synthase. PNAS USA 2005; 102: 9056-9061. [31] Moens AL, Kass DA. Tetrahydrobiopterin and cardiovascular disease. Arterioscler Thromb Vasc Biol 2006; 26: 2439-2444.
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METODICHE MICROPERfUSIONALI E SEPSI

[Microcirculation and sepsis] Francesco Carini, Chiara Lo Piccolo, Giuseppe Alessandro Scardina*, Pietro Messina* e Vincenzo Valenza
Dipartimento di Medicina Sperimentale, Sezione di Anatomia Umana Normale - *Dipartimento di Scienze Stomatologiche, Universit degli Studi di Palermo (I)
Parole chiave: Microcircolo, Shock settico Key words: Microcircol, Septic shock Riassunto. La sepsi una complessa sindrome caratterizzata da infiammazione sistemica in risposta allinfezione. Lincidenza della sepsi aumentata negli ultimi decenni e si prospetta un ulteriore aumento. Lalta mortalit e il peso sul sistema sanitario nazionale ci convince che c urgenza necessit di migliorare la diagnosi e la gestione del paziente settico. La micro circolazione uno dei pi grandi organi del corpo e la funzione micro circolatoria il principale prerequisito per adeguare lossigenazione tissutale con la funzione dorgano. Il suo scopo trasportare lossigeno e nutrire le cellule, assicurarne adeguate funzioni immunologiche e nella patologia liberare sostanze terapeutiche. Immagini ottenute con OPS mostrano le prime osservazioni cliniche in organi umani interni e sono state messe in relazione le anomalie micro circolatorie ed i vari stadi della sepsi. Recentemente con il sistema SDF le osservazioni del micro circolo hanno offerto migliori dettagli. SDF utilizza una nuova metodica di immagine beneficiando del fatto che la uce che illumina e la luce riflessa hanno via riflessa. La micro circolazione visualizzata da una analisi del movimento cellulare la quale permette di misurare quantitativamente il flusso dei globuli rossi nei capillari; questa misura ritenuta una parametro sicuro indicativo di funzione o disfunzione cardiovascolare. Caratteristiche morfologiche della micro circolazione, densit capillare e morfologia dei micro vasi possono essere misurati usando SDF. In questa review discutiamo sul ruolo della disfunzione micro circolatoria nello sviluppo e nel trattamento dei difetti di distribuzione circolatori associati a sepsi. Abstract. Sepsis is a complex sindrome characterized by systemic inflammation in response to infection. The incidence of sepsis has increased in recent decades and is predicted to continue to rise. The high sepsis related mortalities and the burden on healthcare system means there is an urgent need to improve the diagnosis and management of sepsis patients. The microcirculation is one of the largest organs in the body and microcirculatory function is the main prerequisite for adequate tissue oxygenation and thus organ function. Its purpose is to transport oxygen and nutrients to tissue cells, ensure adequate immunological function and in disease to deliver therapeutic drugs to target cells. Orthogonal polarization spectral imaging allowed the first clinical observation in the microcirculation in human internal organ and has identified the pivotal role of microcirculatory abnormalities in defining the severity of sepsis. Recently sublingual sidestream dark field imaging (SDF) has been introducted allowing observations of the
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Francesco Carini, Chiara Lo Piccolo, Giuseppe Alessandro Scardina, Pietro Messina e Vincenzo Valenza
microcirculation in even greater detail. SDF imaging utilizes a novel method of reflectance avoidance in which the illuminated light and light travel independent pathways. The microcirculation is achieved by an analysis of the moving cells in the images, which permits the quantitative measurement of red blood cell flow in the capillaries; this measurement is belived to represent a truly sensitive measurement which s indicative of cardiovascular function or disfunction Morphological characteristics of the microcirculation such as functional capillary density and micro vessel morphology can be measured using SDF. In this review we discuss the role of microcirculatory dysfunction in the development and treatment of the circulatory distributive defect associated with sepsis.
I contributi pi recenti della letteratura suggeriscono un aumento dellincidenza della sepsi; tale aumento dovuto a vari fattori quali lavanzare dellet media della popolazione, il numero crescente di procedure invasive, limpiego di farmaci immunosoppressivi. Laumento dellincidenza della sepsi porta un danno endoteliale localizzato in vari distretti; stato questo aspetto che ci ha condotto a focalizzare i nostri studi sullendotelio e sulle indagini microperfusionali in vivo, al fine di tentare di portare un contributo scientifico in tale campo In questo lavoro viene analizzato lo stato dellarte su un tema attuale e complesso che impegna quotidianamente tutti coloro che sono coinvolti nellassistenza al paziente critico La perfusione tissutale dipende dal numero dei capillari che vengono irrorati dal sangue proveniente dalle arteriole progenitrici. In condizioni fisiologiche, lapporto ematico agli organi varia secondo le esigenze funzionali e metaboliche delle cellule. I meccanismi che consentono di aumentare il flusso ematico in un organo sono rappresentati dalle variazioni del diametro delle arteriole e dallaumento del flusso ematico attraverso i capillari. Le variazioni di diametro del vaso sono dovute allattivit del muscolo liscio vascolare che risponde a svariate stimolazioni quali: pressoria,. nervosa, ormonale, metabolica locale, endoteliale, dipendente dal flusso I batteri e le tossine possono determinare una profonda alterazione della circolazione periferica. La concentrazione di citochine infiammatorie contribuisce a determinare il danno a carico dellendotelio vascolare e degli organi bersaglio. I leucociti sono attivati e aderiscono alla parete dei vasi e la capacit di agglutinarsi causa di alterazioni della circolazione del sangue capace di determinare importanti lesioni ossidative del sistema vascolare. La pressione arteriosa si pu ridurre in modo marcato, in conseguenza di uno stato endotossico in grado di aumentare la permeabilit del sistema vascolare con conseguente edema. Nelle situazioni pi gravi pu determinarsi una condizione severa di coagulopatia da consumo. La maggior parte dei pazienti che muore a causa della sepsi presenta
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Metodiche microperfusionali e sepsi
basse concentrazioni di anticoagulanti come la proteina C e lantitrombina e la terapia di rimpiazzo di queste proteine pu risultare efficace. Si sviluppa una complessa interazione tra endotelio, infiammazione e sistema della coagulazione. Inoltre, lattivazione dei fattori umorali e cellulari, con particolare riferimento allinterazione endotelio-neutrofili, altera la barriera endoteliale e la regolazione vasale causando disfunzioni nel trasporto di ossigeno e nel suo utilizzo da parte dei tessuti. La cascata della coagulazione attivata facilmente nei diversi modelli sperimentali; in clinica, la sindrome da coagulopatia da consumo e in particolare i fenomeni di trombosi rappresentano unevenienza abbastanza frequente e in grado di peggiorare il decorso della sepsi. La presenza di trombi intravascolari diffusi nel circolo periferico pu determinare una condizione di coagulazione intravasale disseminata. Le citochine proinfiammatorie interagiscono con il sistema della coagulazione mediante lattivazione del fattore tissutale, e sono in grado di alterare in modo profondo il profilo della cascata della coagulazione. La deplezione della proteina C, i ridotti livelli di ATIII e dellinibitore C1 delle esterasi, la conseguente inibizione della fibrinolisi sono tutti fattori in grado di determinare unimportante azione procoagulante. Lattivazione del sistema della coagulazione che alimenta la reazione infiammatoria in grado di automantenersi e amplificare gli effetti deleteri sulla perfusione degli organi. I fattori che interagiscono sono molteplici, tra laltro la trombina in grado di determinare una sovraregolazione della selectina P ed E; tale condizione attiva il fattore di contatto ed in grado di stimolare la produzione di bradichinina, con conseguente peggioramento del quadro dipotensione e dipoperfusione. In studi clinici stato dimostrato che i livelli di ATIII e di proteina C, in corso di sepsi, si riducono drasticamente; la mortalit dei pazienti settici inversamente correlata con i valori di questi due elementi. Lipotensione definita da valori della pressione sistolica arteriosa < 90 mmHg e della pressione arteriosa media < 60mmHg, o da una riduzione > 40 mmHg dal valore di partenza, malgrado adeguato riempimento e in assenza di altre cause; quando il sistema circolatorio non pi in grado di assicurare una perfusione tissutale adeguate si realizza lo shock. Nello shock settico si ha usualmente un profilo caratterizzato da una iniziale gittata cardiaca elevata e da basse resistenze vascolari sistemiche con ipotensione, maldistribuzione del flusso ematico nel microcircolo e compromissione della perfusione tissutale. Sul piano fisiopatologico compendia le caratteristiche dello shock ipovolemico, ostruttivo, cardiogeno, distributivo, citotossico. I pazienti settici che richiedono un supporto emodinamico sono per definizione instabili e hanno spesso anche una compromissione dorgano preesistente di grado diverso. La maldistribuzione di una normale gittata cardiaca pu compromettere la perfusione dorgano; allinterno dellorgano, la cattiva distribuzione per la compromis81
sione delle resistenze vascolari esacerba la disfunzione. Inoltre lazione sul metabolismo cellulare da parte dei mediatori della sepsi porta ad uninadeguata utilizzazione dellossigeno e degli altri nutrienti. Lendotelio polmonare svolge un ruolo fondamentale nella dinamica fisiopatologia della sepsi; sia perch il polmone pu ospitarne la causa o perch pu esserne il bersaglio Linsulto infiammatorio allendotelio vascolare ne provoca laumento di permeabilit alle proteine e danneggia il sistema di autoregolazione del tono arteriolare. Questi eventi contribuiscono allalterazione dellequilibrio tra pressioni oncotiche e idrostatiche, favorendo la fuoriuscita dei fluidi dai vasi. Il processo naturalmente coinvolge anche il circolo polmonare ed la causa delledema interstiziale, caratteristica anatomopatologica tipica dellALI/ ARDS. Il fattore fondamentale che spiega la patogenesi delledema interstiziale si riassume nellequilibrio tra i gradienti idrostatici e oncotici tra linterstizio e i vasi in rapporto al grado di permeabilit capillare. Quando la quantit di fluidi che si raccolgono nel tessuto polmonare non riesce ad essere drenata (attraverso il sistema linfatico), si accumula il liquido extravascolare. In corso di sepsi ci possono essere diversi gradi di permeabilit capillare, quindi il gradiente di pressione idrostatica rispetto a quello oncotico varia man mano che le molecole responsabili per il mantenimento della pressione oncotica attraversano le barriere endoteliali divenute molto permeabili. Laccumulo di liquido extravascolare e lessudato di proteine plasmatiche nello spazio alveolare crea ledema interstiziale che porta allARDS. Lipertensione polmonare nellARDS ha origine multifattoriale, ma indipendente dalla causa che la sottende; i pazienti che presentano un aumento significativo della resistenza vascolare polmonare hanno indiscutibilmente prognosi peggiore. Ledema perivascolare che predomina nelle fasi iniziali dellARDS ha un ruolo importante nella patogenesi dellipertensione vascolare; alleffetto delledema si aggiungono quello della vasocostrizione causata dallipossia alveolare e da altri mediatori vasoattivi come trombossani ed endoteline, oltre allostruzione causata da trombi piastrinici. Nellevoluzione della patologia il progredire dellipertensione riflette lo svilupparsi del processo di fibrosi, anchesso responsabile dellobliterazione de letto vascolare. Bench laumento della pressione dellarteria polmonare sia un evento caratteristico in corsi di ARDS, la resistenza vascolare polmonare in genere solo poco o moderatamente elevata e ci dovuto essenzialmente alla ridotta gittata cardiaca. La causa principale dipossiemia associata a sepsi legata allo shunt intrapolmonare. NellARDS lo shunt dovuto al persistere della perfusione di alveoli atelettasici e riempiti di liquido e dallabolizione del fisiologico riflesso di vasocostrizione ipossica. Dopo il danno iniziale al polmone si sviluppa un gradiente lungo lasse gravitazionale, le zone di polmone dipendenti (e quindi maggiormente perfuse) si consolidano diventando la fonte principale di shunt. Lo shunt del sangue attraverso le parti di polmone non ventilato spiega la natura refrattaria dellipossiemia nellARDS.
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Le cellule endoteliali liberano NO e PGI2 in rapporto a una serie di stimolazioni che si esercitano sulla superficie delle cellule. La PGI2 deriva dallacido arachidonico per azione dellenzima ciclossigenasi. Viene liberata dallendotelio e determina rilasciamento del muscolo liscio vascolare, aumentando i livelli intracellulari di cAMP. La PGI2 ha notevoli effetti antiaggreganti sulle piastrine, contribuendo alla perfusione dei tessuti periferici. Il NO liberato dalle cellule endoteliali induce lattivazione di una guanilatociclasi solubile, che attiva il meccanismo di formazione di cGMP nelle cellule muscolari lisce vascolari. Il risultato finale la riduzione dei flussi di calcio e il rilasciamento del muscolo liscio, che causa vasodilatazione. Laminoacido L-arginina funge da substrato per la formazione di NO, pertanto la sua somministrazione pu aumentare la formazione e la liberazione di NO. Il fattore iperpolarizzante di origine endoteliale il terzo fattore, che contribuisce alla dilatazione delle arteriole e allaumento del flusso ematico. La sua costituzione biochimica ancora da chiarire in molti distretti vascolari, ma in genere viene liberato dallendotelio e determina iperpolarizzazione delle cellule muscolari lisce, dando luogo a vasodilatazione. Tra i fattori che causano vasocostrizione vi sono le endoteline (ET1,ET2,ET3), i trombossani, che derivano dallacido arachidonico, lungo la catena di reazioni attivate da ciclossigenasi. Sono stati descritti anche fattori costrittori che sono indipendenti dalla ciclossigenasi. Dunque lendotelio pu modulare le risposte vasocostrittorie del muscolo liscio vascolare, agendo come una barriera tra il sangue e le cellule muscolari, limitando la quantit di sostanze attive sul muscolo liscio e liberando al contempo NO e fattori rilascianti, che provocano vasodilatazione Le variazioni di flusso ematico nelle arteriole sono in grado di indurre risposte endoteliali capaci di regolare il tono vascolare. Se aumenta il flusso ematico in unarteria, questa si dilata facilitando la riduzione della resistenza idraulica del sistema. Il meccanismo della vasodilatazione indotta da un aumento del flusso ematico dovuto alla liberazione di NO e PGI2 indotto dalle forze tangenziali che agiscono sulle cellule endoteliali (shear stress). Aumentando lo shear stress, si ha una dilatazione arteriolare, che contribuisce alla regolazione rapida della resistenza durante liperemia funzionale. Vi sono recettori endoteliali sensibili allo shear stress, che rispondono liberando NO e dando seguito allinterno delle cellule a messaggi trasferiti a livello nucleare. Sono, infatti, stimolati il fattore nucleare kappa B e il fattore proteico nucleare di attivazione I, che sono capaci di legarsi a elementi di risposta allo shear stress a livello di geni che rispondono alle stimolazioni meccaniche. Sepsi e disfunzioni dei sistemi di regolazione I predetti meccanismi autoregolatori e le loro funzioni microocircolatorie sono spesso
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alterati durante la sepsi e la loro disfunzione spesso da mettere in relazione a un definito fattore nella patofisiologia della sepsi. La disfunzione microcircolatoria caratterizzata da eterogenee anomalie nel flusso sanguigno con la presenza di alcuni capillari sottoperfusi, mentre altri hanno normale o alto flusso di sangue. La unit funzionali microcircolatorie divenute ipossiche, spiegano lestrazione deficitaria di ossigeno durante la sepsi. In queste condizioni, la pressione parziale di ossigeno scende sotto la pO2 venosa. La disparit stata chiamata lapertura pO2, ed una misura della severit dello shunt funzionale, avvenimento che pi severo nella sepsi che nella emorragia. Nella sepsi il microcircolo non adatto a compiere la funzione regolatoria perch disturbato dallinsieme di segnali di trasduzione e di comunicazioni elettrofisiologiche. Il sistema ossido nitrico, componente centrale nel controllo autoregolatorio microcircolatorio, severamente disturbato nella sepsi da espressione eterogenea delle iNOS in differenti aree di organo, risultando shunt patologico di flusso. Poich iNOS non espressa omogeneamente in sistemi dorgano, aree mancanti di iNOS hanno meno vasodilatazione NO-indotta e diventano ipoperfuse. Le cellule del muscolo liscio che circondano le arteriole e regolano la perfusione perdono la loro sensitivit adrenergica e il tono durante la sepsi. I globuli rossi diventano meno deformabili e si aggregano di pi. I globuli rossi giocano anche un importante ruolo nella regolazione del flusso sanguigno microcircolatorio determinato dalla loro capacit di rilasciare NO in presenza di ipossia e causare vasodilatazione. Questa propriet regolatoria dei globuli rossi pu anche essere assente durante la sepsi. Questi severi difetti, insieme con i disturbi della coagulazione durante la sepsi, impediscono ulteriormente la perfusione e la funzione microcircolatoria. In pi, lattivazione dei leucociti da infiammazione settica genera particolari reazione allossigeno che danneggia direttamente le strutture microcircolatorie, le interazioni cellulari e le funzioni coagulatorie. Questi e altri mediatori infiammatori alterano le funzioni di barriera nella microcircolazione, incluse le giunzioni tra le cellule dando edema tissutale e ulteriore deficitaria estrazione di ossigeno. La disfunzione microcircolatoria determina distress respiratorio delle cellule parenchimali risultandone fallimento dorgano. Sepsi, microcircolo, D.I.C. - Distress mitocondriale Se la causa primaria del deficit estrattivo di ossigeno nella sepsi causato da un debole shunt, lipossia microcircolatoria e lincapacit mitocondriale a processare ossigeno attualmente causa di intenso dibattito. Il mitocondrio funziona da centrale dintegrazione degli stimoli apoptoici. Questi possono essere di diversa natura (caspasi, ceramide, chinasi) e sono in grado di determinare lapertura di un complesso proteico chiamato poro di transizione mitocondriale (PTPC Permeability Transit Pore Complex) localizzato in alcuni punti di contatto tra le due membrane
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mitocondriali. Questevento fa cadere la differenza di potenziale, per uscita dei protoni, ed ingresso di molecole prima interdette allingresso. Come risultato finale, il mitocondrio si riempie di liquido e la membrana esterna scoppia liberando nel citoplasma fattori stimolanti lapoptosi come lAIF (Apoptosis Inducing Factor), che in grado di raggiungere il nucleo ed attiva una via indipendente delle caspasi in grado di degradare il DNA, ed il citocromo e che si lega alle proteine Apaf-1 (apoptotic protease activating factor) e caspasi 9 ed una molecola di ATP formando un complesso definito apoptosoma. La caspasi 9 presente diviene in grado di attivare altre caspasi che danno il via ad una cascata mitocondriale che si conclude con la degradazione del DNA ad opera di fattori nucleari. Ai processi di alterazione della permeabilit del mitocondrio prendono parte anche i membri della famiglia di Bcl-2, composta da almeno 16 proteine, le quali sono in grado di interagire con le membrane mitocondriali. Tale famiglia proteica contiene elementi sia antiapoptotici, come Bcl-2 e Bcl-XL, sia proapoptotici, come Bax, Bid, Bcl-XS. Tali membri possono unirsi formando omodimeri ed eterodimeri che hanno attivit sia proapoptotica sia antiapoptotica. Levento chiave consiste nellabbondanza dei fattori proaptotici rispetto a quelli protettivi. Se questo evento avviene potranno allora formarsi dimeri in grado di alterare la permeabilit del mitocondrio. In alcuni studi effettuati su cuore di topo con sepsi fu osservata endotossemia nellarea ipossica; tuttavia in questo modello non fu trovata disfunzione mitocondriale, come evidenziato dalla normale risposta dello stato energetico mitocondriale allipossia in situ. Probabilmente con il progredire a sepsi severa si potrebbe presentare la disfunzione mitocondriale, accompagnata o anche possibilmente causata da pi gravi disfunzioni microcircolatorie. Brealey et al. [33] mostrano che la disfunzione mitocondriale effettivamente gioca un importante ruolo nella sepsi dove i livelli di disfunzione respiratoria dei mitocondri sono correlati con loutcome del paziente. Fallimento mitocondriale associato con sepsi contribuisce al distress respiratorio, specialmente in aree ipossiche e pu alimentare il distress tissutale conducendo a disfunzione dorgano. Microcircolazione e sindrome di distress mitocondriale Leventuale recupero di un fallimento circolatorio associato con sepsi consiste nella correzione del sistema emodinamico e dalle variabili ossigeno-derivate, ma quando il distress respiratorio persiste si parla di sindrome da distress mitocondriale e microcircolatorio (MMDS). Questo concetto stato formulato per identificare il compartimento fisiologico vulnerabile mascherato dal sistema circolatorio e responsabile del trasporto di ossigeno e della respirazione cellulare che diventa da disfunzione a sepsi, e pu condurre a disfunzione dorgano. Gli elementi definiti della natura e della severit della sepsi includono la natura delliniziale colpo conducente alla sepsi, comorbidit, mappaggio
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genetico individuale, precedenti terapie e tempo di trattamento. Il tempo che la sindrome durata e la precedente terapia ricevuta hanno una responsabilit ed effetti modulatori sulla patofisiologia e definiscono le subclassi della sindrome. La natura patogenetica del tempo fu dimostrata dagli studi di Rivers dove un trattamento precoce mostrava essere associato con improvviso outcome. La MMDS quando terapia e tempo sono inclusi nella sua definizione indica che le valutazioni integrative di questi determinati fattori microcircolatori e funzionali mitocondriali sono necessario per la valutazione della severit della sindrome. Strategie di salvezza della microcircolazione La presenza di distress respiratorio, nonostante la rianimazione basata sullemodinamica e su derivati dellossigeno, suggerisce fortemente che il fallimento microcircolatorio un fattore chiave da mettere in relazione anche nellelevato livello di lattato, ai disturbi del bilancio acido base ed alti livelli di CO2 evidenziati talvolta in queste condizioni. Il fallimento microcircolatorio pu avvenire con la presenza di normali o sopranormali variabili emodinamiche e ossigeno derivate, con distress microcircolatorio essendo mascherato dalla circolazione sistemica dallinsieme di shunts. Cos grande importanza assumono le tecniche di monitoraggio per Immagine statica di videocapillaroscopia verificare le strategie da assumere e per valutare la microcircolazione.
Sidestream dark field imaging 86
Immagine ottenuta con SDF. possibile visualizzare e quantizzare il flusso del sangue ed evidenziare il movimento degli elementi corpuscolati
Monitorare la microcircolazione per ottimizzare il trattamento. Svariati metodi sono stati usati per monitorare la microcircolazione durante fallimento circolatorio in chirurgia e in terapia intensiva. Questi includono le misure di CO2 sublinguale, buccale e i livelli di CO2 sottocutanea, cos come lassorbimento, la riflettanza e la spettroscopia a infrarosso, per misurare la saturazione di emoglobina microcircolatoria. La spettroscopia ortogonale polarizzante (OPS) fu introdotta in chirurgia e mostrava la prima diretta osservazione della microcircolazione di organi interni umani. La tecnica mostra visualizzazione microscopica del microcircolo profondo e il flusso dei globuli rossi nella variabilit del microcircolo. Quando applicata sotto la lingua OPS provvede a una buona specificit per misurare la severit del difetto distributivo nella sepsi non realizzabile col monitoraggio convenzionale dellemodinamica sistemica e dellossigeno-derivati. La capnografia sublinguale combinata con lOPS immaging stata usata per investigare le relazioni tra la microcircolazione e lo stato metabolico durante la rianimazione. In cardiochirurgia, misure simultanee di spettroscopia a infrarosso sublinguale di diverse regioni misurate profondamente e spettrofotometria di regioni microcircolatorie superficiali, davano informazioni integrative circa la redistribuzione di ossigenazione microcircolatoria occorrente tra questi comparti durante cardiochirurgia. Tale combinazione, guardando ai differenti comparti funzionali della microcircolazione, pu accertare la distribuzione del trasporto di ossigeno durante sepsi, shock settico, e terapie che non sono previste dal monitoraggio convenzionale dellemodinamica sistemica e ossigeno-derivati. Studi di De Backer [7,10,2] sulla microcircolazione sublinguale usando OPS in pazienti settici hanno direttamente associato il grado di distress respiratorio con la severit della malattia e la risposta alla terapia. Questi studi con OPS mostrano che il difetto distributivo associato con sepsi caratterizzato da stasi del flusso nei piccoli capillari e normale flusso nei pi grandi vasi vicini. Questo sottolinea la necessit del monitoraggio clinico del flusso sanguigno in questi piccoli capillari.
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Limmagine OPS limitata, le immagine dei capillari sono talvolta macchiate e non possono essere sempre determinate. A quanto detto gli studi di Ince sviluppano una nuova modalit di immagini per osservare la microcircolazione chiamato sidestream dark field imaging (SDF). SDF consiste di una leggera guida circondata da 530 nm diodi a emissione di luce (LEDs), una lunghezza donda di luce che assorbita dallemoglobina dei globuli rossi, permettendo la loro osservazione come cellule scure che scorrono nella microcircolazione. I LEDs alla punta della guida sono otticamente isolati dallinterno dellimmagine conducente e danno luce profonda nei tessuti illuminando la microcircolazione. Questa illuminazione in campo oscuro applicata evita riflessi di superficie, dando chiare immagini delle strutture microcircolatorie e del flusso del sangue. Ci si aspetta che le immagini con SDF miglioreranno le immagini della microcircolazione specialmente per i capillari. Scelte terapeutiche Numerose scelte terapeutiche sono disponibili per migliorare la microcircolazione nel paziente settico. Volume rianimatorio. Se i meccanismi di autoregolazione sono intatti questi assicurano la rianimazione dallo shock ipovolemico attraverso volume, che effettivamente recupera letto microcircolatorio. Il volume fornito ripristina anche la funzione di barriera microcircolatoria e promuove il trasporto di ossigeno microcircolatorio [23,50,52]. Tuttavia induce emodiluizione, effetto che pu causare una redistribuzione della distribuzione di ossigeno. Il significato di una redistribuzione di approvvigionamento di ossigeno e il suo ruolo nella patofisiologia della sepsi e nella rianimazione, tuttavia, ancora da essere stabilito. Il sangue il miglior trasportatore di ossigeno, migliore dei cristalloidi e dei colloidi. Let della riserva dei globuli rossi tuttavia, pu modificare le propriet del sangue e ci deve essere preso in considerazione [53]. Il trasporto di ossigeno con lemoglobina, anche se valido non pu essere adoperato come routine clinica. Inibitori di iNOS e steroidi. Lossido di azoto, impropriamente chiamato ossido nitrico, uno dei pi potenti mediatori biochimici che gli organismi viventi producono; sicuramente degno di nota il fatto che a questa sostanza sia legato il premio Nobel 1998 per la Medicina/Fisiologia, attribuito al ricercatore americano Louis Ignaro per le sue scoperte riguardanti lossido nitrico come molecola segnale nel sistema cardiovascolare. Sei anni prima la rivista Science aveva eletto lNO come molecola dellanno. LNO una sostanza prodotta a partire dallamminoacido L-arginina in una reazione multi-step catalizzata dallenzima ossido nitrico sintetasi. Questultimo esiste in numerose isoforme, alcune costitutive (cellule endoteliali, piastrine, sistema nervoso) ed altre inducibili (macrofagi,
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leucociti, cellule muscolari lisce, epatociti). LNO agisce come importante messaggero intra ed intercellulare regolando numerose funzioni. Le cellule endoteliali producono NO che diffonde nel circolo, riducendo laggregabilit delle piastrine e ladesivit dei leucociti alle pareti dei vasi sanguigni e, in parte raggiunge la sottostante muscolatura liscia vascolare inducendone il rilasciamento. I conseguenti effetti antiaggreganti, antinfiammatori ed antiipertensivi sono di grande importanza. Oltre alleffetto sullendotelio il NO ha altre funzioni: a livello cerebrale (controllo dellapprendimento e della memoria), gastrointestinali (modulazione dele secrezioni e della motilit), respiratorio (modulazione del tono della muscolatura liscia bronchiale). Sono in corso studi sulle sue possibili azioni nei confronti delle infezioni batteriche e nel controllo della crescita dei tumori. Spesso nella sepsi, i meccanismi di autoregolazione sono danneggiati [55]. Semplici fluidi, possono correggere lemodinamica sistemica e recuperare deboli aree della microcircolazione ipossica [11,12]. Questa distribuzione di flusso in relazione, fra altri meccanismi, alla eterogenea espressione delle iNOS in parti differenti di letti dorgano risultando shunt patologici di flusso [15,16]. Di nota che deficienze di iNOS nel topo non espongono le disfunzioni circolatorie associate con le endotossine che sopravvengono in topi di tipo selvatico, sottolineando limportanza del controllo di iNOS nella sepsi [57]. In studi recenti di maiali con sepsi, liquidi combinati con inibitori di iNOS, reclutavano letto circolatorio nellintestino [50, 58]. Linibizione di iNOS protegge anche le funzioni di barriera della microcircolazione e pu essere guardata come una misura per il reclutamento microcircolatorio [59]. Agenti antinfiammatori, cos come gli steroidi, sono estremamente validi allinibizione di iNOS e possono prevenire lipotensione indotta dalle endotossine. Somministrandoli in ritardo, tuttavia, non danno inbibizione di iNOS dovuto dagli inibitori di NO sepsi evocata dei recettori dei glucocorticoidi [60]. Tali studi mostrano la razionalit di una terapia precoce. Gli steroidi migliorano anche la funzione autoregolatoria come osservato in studi su ratto delle propriet autoregolatorie di un cuore isolato in sepsi [55]. La maggior parte degli studi sperimentali tuttavia usa quantit elevate di steroidi e lorientamento clinico mette al corrente contro luso di alti livelli di steroidi nel trattamento della sepsi [61]. Ci nonostante questi studi indicano che riducendo lespressione di iNOS si pu avere un controllo importante nella distribuzione emodinamica deficitaria nella sepsi. Vasodilatatori e vasopressori Reclutare la perfusione microcircolatoria sotto normovolemia pu essere realizzato da terapia vasodilatatoria perch ci aumenta la pressione giuda del flusso sanguigno allingresso della microcircolazione [62]. In un modello di maiale con sepsi, NO somministrato in combinazione con liquidi migliora lossigenazione microcircolatoria intestinale e corregge
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la pressione parziale gastrica di CO2 mentre ci non avviene somministrando solo fluidi [63]. In uno studio clinico di pazienti in shock settico, dove la microcircolazione sublinguale fu osservata con OPS, la pressione-guida risultava in conseguenza del flusso nei larghi vasi ma non nei capillari, dove rimaneva lenta o stagnante. Questo scenario visualizza direttamente lazione dello shunt e identifica la microcircolazione come il luogo dei difetti di distribuzione durante sepsi. Terapia vasodilatatoria da nitroglicerina con adeguati supporti di volume, tuttavia, fu capace di reclutare questo flusso stazionario di capillari e ripristinare la microcircolazione sublinguale [10]. De Backer et al. riportarono simili anomalie microcircolatorie in pazienti settici [7]. Essi mostrarono ulteriormente che la risposta vasodilatatoria endoteliale fu intatta dimostrando che lapplicazione topica di acetilcolina era effettiva nel reclutamento dellapertura/chiusura dei capillari. Studi di immagini di OPS sublinguale in pazienti con sepsi trovarono che mentre la pressione guida rianimatoria era effettivamente in ripristino, la pressione sanguigna sistemica non ha un effetto correttivo sulla perfusione microcircolatoria [2]. Da una prospettiva microcircolatoria, i vasopressori dovrebbero essere applicati con attenzione e sotto condizioni di monitoraggio microcircolatorio. Uno studio di Dubois et al. [84] dimostra che la pressione sistemica sanguigna fu ripristinata da vasopressina in pazienti con shock. Qui dirette osservazioni della circolazione sublinguale con immagini OPS non mostravano effetti dannosi sulla perfusione microcircolatoria. Tuttavia, in altri casi di pazienti studiati con shock settico, la vasopressina anche se aumentava la pressione del sangue e le urine eliminate, causava una completa cessazione del flusso microcircolatorio sublinguale, comprimendo la circolazione regionale e portando a morte. Esperimenti su animali hanno anche mostrato risultati conflittuali: alcuni studi hanno mostrato che la vasopressina ha effetti benefici sulla microcircolazione renale [65], mentre altri hanno mostrato che la vasopressina causa sospensione dellattivit microcircolatoria intestinale [66]. Terapia con multiple azioni I fluidi in combinazione con vasoattivi e supporto inotropo reclutano effettivamente la microcircolazione, anche se i loro effetti sulla microcircolazione non si possono dedurre solo con le variazioni sistemiche [2, 38]. I pazienti dove la microcircolazione non era responsabile di rianimazione, tuttavia, hanno una cattiva prognosi [2]. Il reclutamento della microcircolazione pu essere portato a termine con differenti modi e combinazioni di terapie. In questo modo, un agente donatore di NO pu aprire la microcircolazione e la terapia perfusionale lentamente recuperare le unit microperfusionali, mentre un agente antinfiammatorio o specifici inibitori di iNOS possono ridurre lo shunt patologico e reinstradare il flusso sanguigno a reclutare lentamente le unit microcircolatorie. Questo pu apparire paradossale da una certa posizione, ma entrambi le terapie reclutano effettivamente la
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microcircolazione e potrebbero essere teoricamente combinate. chiaro che quando applicate come combinazione di terapia la loro efficacia sul reclutamento microcircolatorio necessita di essere verificato per differenti sistemi dorgano. Prendendo in considerazione che la rianimazione dalle forme di sepsi multifattoriale, medicine con multipli siti di azione possono provvedere a uneffettiva strategia di trattamento per reclutare le funzioni microcircolatorie durante sepsi. Attivata la proteina C (APC) [67] provvede solo cos a un approccio integrato agendo su diversi meccanismi coinvolti in distress respiratorio. stato mostrato per esempio, che APC inibisce lespressione di iNOS e protegge contro lipotensione endotossina indotta [68]. Inoltre, attraverso la sua azione sul fattore nucleare kB, APC riduce anche il livello del fattore di necrosi tumorale, un effetto non visto quando gli inibitori di iNOS sono somministrati da soli [69]. In pi APC riduce lattivazione leucocitaria e la liberazione di specie reattive di ossigeno, cos come intervenendo sulla via coagulatoria [70]. Ulteriori studi hanno mostrato che queste azioni multifattoriali migliorano la microcircolazione in animali con sepsi [71,72]. La proteina C scatena un numero di effetti che possono essere visti come una liberazione strategica per la disfunzione microcircolatoria nella sepsi. Tuttavia, parecchie questioni rimangono nelle modalit di azione della proteina C. In questa review si discusso sul ruolo delle disfunzioni microcircolatorie nello sviluppo e sul monitoraggio e trattamento della distribuzione circolatoria deficitaria associata con sepsi. Lemodinamica sistemica tradizionale e le variabili derivate dovrebbero scoprire le disfunzioni microcircolatorie e le risposte alla terapia. Disfunzioni microcircolatorie possono dare distress cellulare e portare a disfunzione dorgano. Da queste prospettive la microcircolazione si pu guardare come motore della sepsi. Ulteriori monitoraggi delle funzioni microcircolatorie contribuiranno alla diagnosi e al trattamento della sepsi.
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Bibliografia [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] Spronk PE, Zandstra DF, Ince C: Bench to bedside: Sepsi is a disease of the microcirculation. Crit Care 2004, 8:462-468. Sakr Y, Dubois MJ, De Backer D, Creteur J, Vincent JL : Persistent microcircolatry alterations are associated with organ failure and death in patients with septic shock. Crit Care Med 2004, 32:1825-1831. Meakins JL, Marshall JC: The gastrointestinal tract: the motor of MOF. Arch Surg 1986, 121:197-204. Vallet B: Endothelial cell dysfunction and abnormal tissue perfusion. Crit Care Med 2002, 30(supll. 5):S229-S234. Lidington D, Tyml K, Quallette Y: Lipopolysaccheride-induced reduction in cellular coupling correlate with tyrosine phos-phorystion of connexin. J Cell Physiol 2002, 193:373-379. Bateman RM, Sharpe MD, Ellis CG: Bench-to bedside review:microvascular dysfunction in sepsis-hemodynamics, oxygen transport,and nitric oxide. Crit Care 2003, 7:359-373. De Backer D, Creteur J, Presier JC, Dubois MJ, Vincent JL: Microvascular blood flow in altered in patients with sepsis. Am J Respir Crit Care Med 2002, 166:98-104. Lam C, Tyml K, Martin C, Sibbald W: Microvascular perfusion in impaired in a rat model of normotensive sepsis. J Clin Invest 1994, 94:2077-2083. Nakijma Y, Baudry N, Durante J, Vicaut E: Microcirculation in intestinal villi : a comparison between hemorrhagic and endotoxin shock. Am J Respir Crit Care Med 2001, 164:1526-1530. Spronk PE, Ince C, Gardien MJ, Mathura KR: Nytroglicerin promotes microvascular recruitment in septic shock after intravascular volume resuscitation. Lancet 2002,360:1395-1396. Goldmann D, Baterman RM, Ellis CG: Effect of sepsis on skeletal muscle oxygen consumption and tissue oxygenation. Interpreting capillary oxygen transport data using a mathematical model. Am J Physiol Heart Circ Physiol 2004, 287:H2535-H2544. Ince C, Sinaappel M: Microcircolatory oxygenation and shunting in sepsis and shock. Crit Care Med 1999, 27:1369-1377. Sinaappel M, Ince C: Microvascular oxygen pressure measurements in the intestine during hemorrhagic shock and resuscitation. J Phisiol 1999, 514:245-253. Schwarte LA, Fournell A, von Bommel J, Ince c: Redistribution of intestinal microcircolatory oxygenation during acute hemodilution in pigs. J Appl Physiol 2005, 98:1070-1075. Morin MJ, Unno N, Hodin RA, Fink MP: Differential expression of indicibile nitric oxide synthase messanger RNA along the longitudinal and crypt-villus axes of the intestine in endotoxemic rats. Crit Care Med 1998, 26:1258-1264. Revelly J.P, Ayuse T, Brienza N, Fessier HE, Robotham Jl : Endotoxic shock alters distribution of blood flow within the intestinal wall. Crit Care Med 1996, 24:1345-1351. Baker CH, Wilmoth FR: Microvascular responses E.coli endotoxin with altered adrenergic activity. Circ Shock 1984, 12: 165-176. Price SA, Spain DA, Wilson MA, Harris PD, Garrison RN: Subacute sepsis impairs vascular smooth muscle contractile machinery and alters vasoconstrictor and dilator mechanisms. J Surg Res 1999, 83:75-80. Baskurt OK, Temiz A, Meiselman HJ : Red blood cell aggregation in experimental sepsis. J Lab Clin Med 1997, 130:183-190. Piagnerelli M, Boudjeltia KZ, Vanhaeverbeek M, Vincent JL: Red blood cell rheology in sepsis. Intensive Care Med 2003, 29: 1052-1061. Siegemund M, Hardeman MR, van Bommel J, Stegenga ME, Lind A, Ince D: Red blood cell deformability
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in two different doses of LPS in a porcine model of endotoxemia. Intensive Care Med 1999, 25:S21. [22] Cosby K, Partovi KS, Crawford JH, Patel Rp Reiter CD Martyr S, Yang BZ, waclawin MA, Zalos G, Xu X, et al: Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation. Nat Med 2003, 9:1498-1505. [23] Singel DJ, Starnier JS: Chemical physiology of blood flow regulation by red blood cells: the role of nitric oxide and S-nitroso-hemoglobin. Ann Rev Physiol 2005, 67:99-145. [24] Cervinka Wh, Coopr D, Krieglstein CF, Ross CR, McCord JM, Granger DN : Superoxide mediates endotoxin-induced platelet-endothelial cell adhesion in intestinal venules. Am J Physiol Heart Circ Physiol 2003, 284:H535-H541. [25] Martins PS, Kalias EG, Neto MC, Dalboni MA, Blecher S, Salomao R: Upregulation of reactive oxygen species generation and phagocytosis and increased apoptosis in human neutrophilis during severe sepsis and septic shock. Shock 2003, 20:208-212. [26] Victor VM, Rocha M, De la Fuente M: Immune cells: free radicals and antioxidants in sepsis. Int Immunopharmacol 2004, 4:327-347. [27] Fink MP: Intestinal epithelial hyperpermeability: update on the pathogenesis of gut mucosal barrier dysfunction in critical illness. Curr Opin Crit Care 2003, 9:143-151. [28] van den Berg BM, Vink H. Spaan JA: The endothelialglycocalyx protects against myocardial edema. Circ Res 2003, 92:592-594. [29] Fink MP: Cytopathic hypoxis in sepsis. Acta Anaesthesiol Scand (Suppl) 1997, 110:87-95. [30] Ince C: Microcirculatory weak units: an alternative explanation. Crit Care Med 2000, 28:3128-3129. [31] Avontuur JAM, Bruining HA, Ince C. Inhibition of nitric oxide synthesis causes myocardial ischemia in endotoxemic rats. Circ Res 1995, 76:418-425. [32] Eerbeek O, Milstein DMJ, Inca C: Microcirculatory dysfunction in Langendorff endotoxemic rat in hearts. Shock 2004 21:81. [33] Brealey D, Brand M, Hargreaves I, Heales S, Land J, Singer M. Associations between mitochonrial dysfunction and severity and outcome of septic shock. Lancet 2002, 360:219-223. [34] Dubois MJ, de Backer D, Creteur J, Anana S, Vincent JL. Effect of vasopressin on sublingual microcirculation in a patient with distributive shock. Intensive Care Med 2003, 29:1020-1023. [35] Spronk PE, Kanoore-Edul VS, Ince C. Microcircolatory and mitochondrial distress syndrome (MMDS): a new look at sepsis. In Functional Hemodynamic Monitoring. Edited by Pinsky MR, Payen D. Berlin: Springer Verlag 2004. Update in Intensive Care Emergency Medicine 2004, 42: 47-69. [36] Rivera E, Nguyen B, Havstad S, Ressler J, Muzzin A, Knoblich B, Peterson E, Tomlanovich M, Early GoalDirected Therapy Collaborative Group: Early goal directed therapy in the treatment of severe sepsis and septic shock. N Engl J Med 2001, 345:1368-1377. [37] Siegemund M, van Bommel J, Ince C: Assessment of regional tissue oxygenation. Intensive Care Med 1999, 25: 1044-1060. [38] Creteur J, Backer D, Sakr Y, Koch M, Vincent JL: Determinant of sublingual pCO2 in patients with septic shock. Crit Care Med (Suppl) 2004, 31:419. [39] Guzman JA, Dikin MS, Kruse JA: Lingual splanchnic and systemic hemodynamic and carbon dioxide tension changes during endotoxic shock and resuscitation. J Appl Physiol 2005, 98:108-113. [40] Venkatesh B, Morgan TJ, Hall J, Willgoss EZ: Subcutaneous gas tensions closely track ileal mucosal gas tensions in a model of endotoxemia without anaerobism. Intensive Care Med 2005, 31:447-454. [41] Weil MH, Nakagawa Y, Tang W, Sato Y, Ercoli F, Finegan R, Grayman G, Bisera J: Sublingual capnometry: a new noninvasive measurement for diagnosis and quantitation of severity of circulatory shock. Crit Care Med 1999, 27:1225-1229.
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[42] Buise MP, Ince C, Tilanus HW, van Bommel J: The effect of nitroglycerin perfusion and oxy-generation during gastric tube reconstruction. Anesth Analg 2005, 100:1107-1111. [43] Atasever B, van der Veen A, Goedhart P, Ince C: Sublingual NIRS and reflectance spectrophotometry: new methods to monitor sublingual oxygen availability. Crit Care 2005, 8 (suppl. 1):P73. [44] Mathura KR, Vollebrecht KC, Boer K, de Graaf JC, Ince C: Comparison of OPS imaging to intravital capillaroscopy of nail fold microcirculation. J Appl Physiol 2001, 91:74-78. [45] Groner W, Winkelman JW, Harris AG, Ince C, Bouma GJ, Messmer K, Nadeau R: Orthogonal polarization spectral imaging: a new method for study of the microcirculation. Nat Med 1999, 5:1209-1212. [46] Mathura KR, Alic R, Ince C: Initial clinical experience with OPS imaging. In Yearbook of Intensive Care and Emergency Medicine. Edited by Vincent JL. Berlin: Springer-Verlag 2001:233-244. [47] Mathura KR, Bouma GJ, Ince C: Abnormal microcirculation in brain tumours during surgery. Lancet 2001, 358:1698-1699. [48] Pennings F, Bouma GJ, Ince C: Direct observation of the human cerebral microcirculation during aneurysm surgery reveals increased arteriolar contractility. Stroke 2004, 35:1284-1288. [49] Ince C: Sidestream dark field (SDF) imaging: an improved technique to observe sublingual microcirculation. Crit Care 2005, B(suppl 1): P72. [50] Siegmund M, van Bommel J, Schwarte LA, Emons M, Rademacher P, Ince C: Selective blockade of iNOS by 1400W restores the gut oxygenation in a pig model of low-dose endotoxaemia. Intensive Care Med 2005, 31:985-992. [51] Van Iterson M, Siegemund K, Burhop K, Ince C: Heart and gut microvascular oxygenation in pigs after resuscitation from hemorrhage by different doses of a hemoglobin based oxygen carrier. J Trauma 2003, 55:1111-1124. [52] Anning PB, Finney SJ, Singh S, Winlove CP, Evans TW: Fluids reverse the early lipopolysacchardeinduced albumin leakage in rodent mesenteric venules. Intensive Care Med 2004, 30:1944-1949. [53] Raat NU, Verhoeven AJ, Mik EG, Gouwerok CW, Verhaar R, Goedhart PT, Ince C: The effect of storage time of human red cells on intestinal microcircolatory oxygenation in a rat isovolemic exchange model. Crit Care Med 2005, 33:39-45. [54] Van Iterson M, Ince C: Resuscitation of the microcirculation with haemglobin based oxygen carriers following hemorrhage. In Yearbook of Intensive Care and Emergency Medicine. Edited by Vincent JL Berlin: Springer-Verlag; 2004, 762-779. [55] Avontuur JA, Bruining HA, Ince C: Nitric Oxide causes dyfunction of coronary autoregulation in endotoxemic rats. Cardiovasc Res 1997, 35:368-376. [56] Groneveld AB, van Lambalgen AA, van den Bos GC, Bronsveld W, Nauta J, Thijs G: Maldistribution of heterogeneous coronary blood flow during canine endotoxin shock. Cardiovasc Res 1991, 25:80-88. [57] Hollemberg SM, Broussard M, Osman J, PariloJE: Increased microvascular reactivity and improved mortality in septic mice lacking inducible nitric oxide synthase. Circ Res 2000, 86:774-779. [58] Pitner A, Nalos M, Asfar P, Yang Y, Ince C, Georgeff M, Bruckner UB, Radermacher P, Froba G: Mechnisms of inducible nitric oxide synthase (iNOS) inhibition-related improvement of gut mucosal acidosis during hyperdynamic porcine endotoxemia. Intensive Care Med 2003, 29:312-316. [59] Wang F, Patel M, Razavi Hm, Weicher S, Joseph MG, McCormack DG, Mehta S: Role of inducible nitric oxide synthas in pulmonary microvascular protein leak in murine sepsis. Am J Respir Crit Care Med 2002, 165:1634-1639. [60] Duma D, Silva-Santos JE, assreuy J: Inhibition of glucocorticoid receptor binding by nitric oxide in endotoxemic rats. Crit Care Med 2004, 32.2304-2310. [61] Keh D, Sprung CL: Use of corticosteroid therapy in patients with sepsis and septic shock: an evidence-
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based review: Crit Care Med 2004, 32:S527-S533. [62] Buwaida M, Ince C: Opening the microcirculation: can vasodilatators be useful in sepsis? Intensive Care Med 2002, 28: 1208-1217. [63] Siegemund M, van Bommel J, Vollebrecht K, Dries J, Ince C: Influence of NO donor SIN-1 on the gut oxygenation in a normodynamic, porcine model of low-dose endotoxemia. Intensive Care Med 2000, 26:S362. [64] Boema EC, van der Voort, Ince C: Sublingual mcrocircolatory flow is impaired by the vasopressin-analogue terlipressin in a patient with catecholamine-resistant septic shock. Acta Anaesth Scand 2005, 1011. [65] Albert M, Losser MR, Hayon D, Faivre V, Payen D: Systemic and renal macro and microcircolatory responses to arginine vasopressin in endotoxic rabbits. Crit Care Med 2004, 32:1891-1898. [66] Westphal M, Freise H, Kehe BE, Bone HG, van Aken H: Argenine vasopressin compromises gut mucosal microcirculation in septic rats. Crit Care Med 2004, 32:194-200. [67] Bernard GR, Vincent JL, Laterre P, Larosa SP, Dhainaut JF: Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001, 344:699-709. [68] Isobe H, Okajiama K, Mizutani A, Harada N, Nagasaki A, Okabe K: Activated protein C prevents endotoxin-induced hypotension in rats by inhibiting excessve production of nitric oxide. Circulation 2001, 104: 1171-1175. [69] Brueckmann M, Hoffmann U, Lang S, Kaden J, Haase KK: drotrecogin alpha inhibits NF-Kappa B activation and MIP-1 alpha release from isolated mononuclear cells of patients with severe sepsis. Inflamm Res 2004, 53:528-533. [70] Yamaji K, Wang Y Liu Y, Uchimura T, Iwamoto H, Maruyama I: Activated protein C, a nature anticoagulant protein, has antioxidant prperties and inhibits lipid peroxidation and advanced glycation and products formation. Thromb Res 2005, 115:319-325. [71] Hoffmann JN, Volimar B, Laschke MW, Inthom D, Ferttmann J, Schildberg FW, Menger MD: Microhemodynamic and cellular mechanism of activated protein action during endotoxemia. Crit Care Med 2004, 32: 1011-1017. [72] Iba T, Kidokoro M, Nagasaki K, Shirahama A, Ida Y: Activated protein Cimproves the visceral microcirculation by attenuating the leukocyte andothelial interaction in a rat lipopolysaccharide model. Crit Care Med 2005, 33:368-372. [73] Ince C: The microcirculation in distress: a new resuscitation end-point? Crit Care Med 2004, 32: 1963-1964.
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IMMUNOHISTOCHEMICAL EXPRESSION Of INOS AND MATRIX METALLOPROTEINASES MMP-2 AND MMP-9 IN AORTIC ANEURYSMS
[Espressione immunoistochimica della iNOS e delle metalloproteinasi MMP-2 e MMP-9 negli aneurismi aortici] Giovanni Francesco Spatola
DI.ME.S., Sezione di Istologia ed Embriologia, Facolt di Medicina, Universit degli Studi di Palermo (I)
Key words: Aortic aneurysm, iNOS, Metalloproteinases, MMP-2, MMP-9 Parole chiave: Aneurisma aortico, iNOS, Metalloproteinasi, MMP-2, MMP-9 Abstract. Aortic aneurysms (AA) is a degenerative vascular disease characterized by localized dilatation of the aortic wall as a result of altered matrix composition (elastin and collagen degradation). Howewer the pathogenesis of the changes is elusive and unclear. Some experimental evidences suggest that iNOS (who synthesize a large amount of NO in inflammatory processes) and the metalloproteinases (MMP) are implicated in the pathogenesis of AA but the relationship between NO and MMP to aneurismal disease is currently unknown. The aim of this study is to investigate the immunohistochemical expression of iNOS and MMP-2 and MMP-9 in human aneurysmal tissues. Riassunto. Gli aneurismi aortici (AA) sono una patologia vascolare degenerativa caratterizzata da una locale dilatazione della parete aortica derivante da una alterazione della matrice extracellulare (in particolare deriva da una degradazione delle fibre collagene ed elastiche). Tuttavia, a tuttoggi non si sono del tutto chiariti i meccanismi patogenetici. Diverse evidenze sperimentali suggeriscono che liNOS (che sintetizza grandi quantit di NO durnate i processi infiammatori) e le metalloproteinasi (MMP) sono implicate nella patogenesi degli AA anche se la relazione tra NO e MMP nella patologia aneurismatica attualmente poco chiara. Lo scopo di questo studio quello di investigare lespressione immunoistochimica della iNOS e delle MMP-2 e MMP-9 in tessuti aneurismatici umani.
Introduction The cellular components of blood vessels wall are supported and organized by a complex structure of collagens, elastins, laminins, fibronectins, and proteoglycans known as the extracellular matrix (ECM) [1]. Researchers in nearly every medical discipline are examining the ECM in their quest to arrest disease, with many of the most promising advances taking place in cardiovascular research. Much of this cardiovascular research is focused on the family of ECM-remodeling enzymes collectively termed the matrix metalloproteinases (MMPs). Twenty-three MMPs have been described in humans, although they share a high degree of homology in their structure. Most MMPs are dismissed freely into the extracellular space immediately after synthesis as proenzymes, but some are stored
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Giovanni Francesco Spatola
within cells (eg, MMP-9 in neutrophil granules), and others are bound to cell surface membranes (eg, MT1-MMP) [2,3]. Rigorous regulation of MMP production and activity is a crucial part of ECM homeostasis. MMPs are formed as inactive proenzymes and are activated by proteolysis in the extracellular fluid, a process that is tightly regulated by other proteases and by endogenous MMP inhibitors. Plasma proteins and tissue inhibitors of metalloproteinases (TIMPs) are the primary endogenous inhibitors of MMPs, although they also serve other physiologic functions [2,3]. For example, TIMP-2 inhibits MMP-2, but is also required for MT1-MMPmediated activation of proMMP-2 [4]. The vascular microenvironment provides several specific modes of MMP regulation. For example, the cyclic strain on endothelial cells created by arterial pulsation has been shown to increase expression and activity of MMP-2 and its activator, MT1-MMP [5,6,7]. Other recent studies clarified a leading role of MMP2 in the angiogenesis and in the hypoxia. The endothelial cells overexpression MMP2 not induced by MT1-MMP reduced during hypoxia on the membrane cell, caused a migration of endothelial cells in accordance with the proangiogenetic role ascribed to MMP2. The involvement of this protease in the hypoxia-related death of endothelial cells support an additional apoptotic role of this protease [8,9,10,11,12]. Furthermore, emerging evidence suggests that nitric oxide inhibits gene expression of MMP-2 from endothelial cells, and increase a production of MMP-9 from endothelial cells and vascular smooth muscle cells. MMPs contribute to many normal and necessary physiologic processes through their modification of the ECM. Inflammation accompanied by increased MMP activity also contributes to many disease processes several are sequestered in inflammatory cells. In fact, increased inflammation and loss of MMP regulation is the hallmark of many pathologic states, including many of the disease processes treated by vascular surgeons [13,14]. The permanent and irreversible expansion of a tract of artery is defined aneurysm. Related of the seat distinguish, thoracic (TAA), abdominal thoracic (TAAA) and abdominal (AAA) aneurysms. The TAA can be localized to level of the ascending aorta, with eventual interest of the aortic valve, arc or the descending aorta. The TAAA and the AAA imply the involvement, with variable extension, of the descendant thoracic aorta and the abdominal aorta [15,16]. The pathogenesis of several types of aneurysms often differ based of their localization: in particular the TAA are in kind to degenerate, dilatative character or dissecting [17,18] while the AAA recognize a multifactorial genesis and an inflammatory origin (infective processes, aortities type Takayasu etc.) [19,20]. According the anatomo-pathologic point of view, the aneurysms have a degenerative
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Espressione Immunoistochimica della inos e delle metalloproteinasi MMP-2 e MMP-9 negli aneurismi aortici
vascular basis that are caused by altering of the cellular components of ECM with degradation of elastic and collagen fibers. Recent experimental studies have clarified that MMP are responsible of these alteration particularly the MMP-2 and the MMP-9. The MMP-2 is physiologic formed by tiny quantity in the muscle cells and in the fibroblast [21,22] and only a little quantity is formed by macrophages. Furthermore, the MMP-2 as we have seen before, it is perceptible in the hipoxia and it makes up one case of the apoptotic endothelial processes because it is produced also by these cells in particulary pathologic situation [8,9,10,11,12]. This production, localized, in the first time, in the aneurysm wall it will be exalted because it is the primum movens of degradative processes of the collagens fibres and causes the display of elastic fibres. The MMP-9 are formed by a huge quantity by macrophages [21,22] and only in one little side by the fibroblasts, act, on the contrary, degrading mostly elastic fibres. In addition, the inflammatory processes, that are involved in the first step of the pathological aneurysm process, provoke in the vessel wall forms an infiltrate consisting of, in the main way, macrophages that are producing a huge quantity of MMP-9. MMP-9, working at the same time with MMP-2, reduces in a full way the extracellular matrix by causing the dilatation by deterioration of the vessel [22]. The aim of our study is to investigate and compare the immunohistochemical expression of MMP-2 and MMP-9 and iNOS in human probable inflammatory abdominal aneurysmal tissues and in the dilatative and dissecting thoracic aneurysm. Materials and Methods Fragments of human 10 AAA, 10 dilatative TAA and 10 dissecting TAA were obtained during surgical procedure. However, during surgical aorto-coronaric bypass were obtained 10 punches of normal aorta in the side of joint of bypass, to use like normal control (the sample came from GENURTO O.U. of Cardiac surgery and O.U. of Vascular surgery, University of Palermo). All the specimens were fixed in Bouins mixture and embedded in paraffin; obtained sections were processed with anti MMP-9 (Chemicon International), monoclonal anti MMP-2 (Chemicon International), anti iNOS (Transduction laboratories) by EnVision+System HRP (AEC) (Dako Cytomation). In the same time, the negative controls have been realized on sections that are near by and processed on the base of the same protocol but without the passage of the primary antibody. Other sections near by those processed have been submitted at the histological stain using the method of Mallory Azan, and this to look forward to seeing the fibrous component and the miocytes. All the samples have been studied with microscope Leica DM1000 and Nikon OPTIPHOT 2.
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Results Morphological results The microscopic observation of the normal and pathologic fragments that are coloured with the Mallory-Azans method, who has pointed out the total subversion of the structure of arterial media and intima layers in aneurysmatic fragments compared to the controls. Furthermore, in the specimens of AAA aneurysm, was emphasized a plentyfull inflammation infiltrate in the media and adventitial tonaca. This infiltrate shows to be less evident in the dilatative aneurysm TAA and totally absent in those dissecant. However, the structure of the layers shows to be adulteraded in the aneurysm inflammatory, in those dilatative and in some points it becomes really difficult to distinguish it (Fig. 1,2,3,4). However, in the dissecant aneurysm, we emphasize the presence of the lesion at the level of the muscular layer that seem to be fractured in different points (Fig.5). Immunohistochemical results iNOS: In the fragments of the normal aorta hasnt been emphasized any activity of iNOS (fig. 6). In the fragments of the AAA has been emphasized a widespread immunoreactivity more intense at the level of the inflammatory cells that are in the context of the vascular wall (fig. 7). There is a weak reactivity in the dilatative TAA aneurysm endothelial cells (Fig.8) and not have reactivity in the specimens of the aortic dissection (Fig.9). MMP-2: There is a discrete reactivity of MMP-2 in the normal aortic wall localized in the tonaca media, where there are positive some miocytes, as in the tonaca adventitia, where some fibroblasts are immunopositive (Fig.10). In the aneurysm samples of inflammatory nature (AAA), we emphasize an intense MMP-2 immunoreactivity, that is located both in musculary cells and in fibroblasts. (Fig. 11). In the TAA of dilatative nature, we emphasize a strong granular reactivity in the cytoplasm of the endothelial cells (Fig.12,13). However, this reactivity shows to be absent in the dissecant TAA, while we emphasize a reactivity in the capillars and in a few fibroblasts (Fig.14). MMP-9: The fragments of the normal aorta show a weak immunoreactivity located in the tonaca media (Fig. 15). In the AAA there is an intense reactivity widespread in all components of the wall, more evident in the areas where there is much more presence of the inflammatory infiltrate (Fig. 16). In the dilatative TAA there is the presence of an intense immunoreactivity at the level of the endothelial cells (Fig.17) and moreover there is a modest immunoreactivity in some cell members of probable fibroblastic nature. This distribution is the same also in the dissecant aneurysm.
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Conclusions Our results document that the iNOS is present only in the aneurysm wall of AAA, in fact according the recent literature, they are considered as a inflammatory genesis, while it proves to be absent or present in small amount, in the TAA dilatative or dissecant and totaly absent in the normal vessels used as control. The reactivity of MMP are significantly increased in the pathologic specimens compared to those of the normal aorta. MMP-2, in AAA is more present at the level of the muscle cells and in the fibroblast, while in TAA of dilatative nature, that are present at in the endothelial cells according with the recent letterature that invest those cytotipes of angiogenetic role (the intima to be absolutely disarranged with an obvious and followed tissutal hypoxia), and, in second time, of apoptotic functions. MMP-9 in the AAA is produced in a big quantity in the inflammatory cells, while in the TAA it presents a reactivity like to MMP-2. This research gives the immunohistochemical basis to support the role of the NO and the metalloproteinases in the AAAs pathogenesis and suggests the hypothesis that the iNOS is produced in huge quantity below the inflammatory cytochines induction and it stimulates the activity of the MMP during the formation of the aneurysm. Moreover it confirm other biochemical data that give to the MMP-2 a fundamental role in the angiogenetic and apoptotic processes in the TAA.
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[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
Figures 1-8: [1] Normal Aorta Mallory-Azan 10x aortic media and adventitia with the normal parallel distribution of myocites and the presence of collagen and elastic fibers, fibroblast and vasa vasorum - [2] AAA Mallory-Azan 10x inflammatory infiltrate in the disarranged media and adventitia - [3] Dilatative TAA Mallory-Azan 20X endothelial cells and the totally disrupted intima layer - [4] Dilatative TAA MalloryAzan 20X alteration of aortic wall - [5] Dissecant TAA Mallory Azan 20X the muscular layer of aorta that seem to be fractured - [6] Aorta iNOS 10x iNOS reactivity is totally absent - [7] AAA iNOS 40x immunoreactivity iNOS more intense in the inflammatory cells - [8] Dilatative TAA iNOS 63X iNOS immunopositive endothelial cells.
[9]
[10]
[11]
[12]
[15]
[13]
[14]
[16]
[17]
Figures 9-17: [9] Dissecant TAA iNOS 20X poor and aspecific immunoreactivity - [10] Aorta MMP-2 40x presence of MMP-2 reactivity in the myocites and in adventitia layer - [11] AAA MMP-2 20x strong immunoreactivity in the muscular layer - [12] Dilatative TAA MMP-2 20x positive endothelial cells - [13] Dilatative TAA MMP-2 100x oil positive endothelial cells - [14] Dissecant TAA MMP-2 40x reactivity in the capillars and in a few fibroblasts - [15] Aorta MMP-9 20x immunoreactivity located in the tonaca media - [16] AAA MMP-9 20x strongly immunoreactivity in the media and the adventitia (macrophages positive) - [17] Dilatative TAA MMP-9 20x positive endothelial cells.
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] Kelleher CM, Mc Lean SE, Mecham RP. Vascular extracellular matrix and aortic development. Curr Top Dev Biol 2004; 62:153-88. Vise R, Nagase H. Matrix metaloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res 2003; 92:827-39. Nagase H. Woessner JF. Matrix Metalloproteinases. J Biol Chem 1999; 274:21491-4. Wang Z, Juttermann R, Soloway PD. TIMP-2 is required for efficient activation of proMMP-2 in vivo. J Biol Chem 2000; 275:26411-5. Von Offenberg Sweeney N, Cummins PM, Birney YA, Cullen JP, Redmond EM, Cahill PA. Cyclic strainmediated regulation of endothelial matrix metalloproteinase-2 expression and activity. Cardiovasc Res 2004; 63:625-34. Wang BW, Chang H, Lin S, Kuan P, Shyu KG. Induction of matrix metalloproteinases-14 and 2 by cyclical mechanical stretch is mediated by tumor necrosis factor-alpha in cultured human umbilical vein endothelial cells. Cardiovasc Res 2003;59:460-9. Hobeika MJ, Thompson MD, Muhs BE, Brooks PC, Gagne PJ. Matrix metalloproteinases in pheripheral vascular disease. Journ Vasc Surg 2007; 45: 849-57. Boyd PJ, Doyle J, Gee E, Pallan S, Haas TL. MAPK signalling regulates endothelial cell assembly into network and expression of MT1-MMP and MMP2. Am Journ Physiol 2005; 57:3 C659-C668. Puyraimond A, Fridman R, Lemesle M, Arbeille B, Menashi S. MMP2 Colocalizes with Caveolae on the surface of endothelial cell. Exp Cell Res 2001; 262: 28-36. Ben-Yosef Y, Miller A, Shapiro S, Lahat N. Hypoxia of endothelial cells leads to MMP2-dependent survival and death. Am Journ Physiol Cell Physiol 2005; 289:1321-1331. Wang L, Zhang ZG, Zhang RL, Gregg SR, Hozeska-Solgot A, LeTourneau Y, Wang Y, Chopp M. Matrix metalloproteinase 2 (MMP-2) and MMP-9 secreted by erythropoietin-activated endothelial cells promote neural progenitor cell migration. Journ Nurosci 2006; 26(22): 5996-6003. Finetti F, Solito R, Morbidelli L, Giachetti A, Ziche M, Donnini S. Prostaglandin E2 regulates angiogenesis via activation of fibroblast growth factor receptor-1. Journ Biol Chem November 2007 (paper in press). Thompson RW et al. Pathophysiology of abdominal aortic aneurysma: insights from a the elastase-induced model in mice different genetic backgrounds. Ann NY Acad Sci 2006 Nov.;1085:59-73. Sun MH et al. Expression of inducible nitric oxide synthase and matrix metalloproteinase-9 and their effects on angiogenesis and progression of hepatocellular carcinoma. World J Gastroenterol 2005 Oct 14; 11(38): 5931-7. Svensson LG, Crawford ES, Hess KR, et al. Composite valve graft replacement of the proximal aorta: comparison of techniques in 348 patients. Ann Thorac Surg 1992;54:427-39. Crawford ES, Crawford JL, Safi HJ et al. Thoracoabdominal aortic aneurysms: preoperative and intraoperative factors determining immediate and long-term results of operations in 605 patients. J Vasc Surg 1986;3:389-404. De Bakey ME, Henley WS, Cooley DA et al. Surgical management of dissecting aneurysm of the aorta. J Thorac Cardiovasc Surg 1965; 49: 130-149. Daily PO, Trueblood HW, Stinson EB, Werflein RD, Shumway NE. Management of acute aortic dissections. Ann Thorac Surg 1970; 10: 237-247. Maffei S, Di Renzo M, Bova G, Auteri A, Pasqui AL. Takayasus arteritis: a review of the literature. Intern Emerg Med 2006;1(2):105-12.
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[20] Pearce WH, Shively VP. Abdominal aortic aneurysm as a complex multifactorial disease: interactions of polymorphisms of inflammatory genes, features of autoimmunity, and current status of MMPs. Ann N Y Acad Sci 2006 Nov;1085:117-32. [21] Davis VPR et al. Matrix metalloproteinase-2 production abd its binding to the matrix are increased in abdominal aortic aneurysm. Arter Thromb Vasc Biol 18: 1625-1633, 1998. [22] Longo GM et al. Matrix metalloproteinases 2 and 9 work in concert to produce aortic aneurysms. Journ Clin Invest 110, 5, 2002.
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Cellular stress
CHAPERONOLOGY: A NOvEL RESEARCH fIELD fOR EXPERIMENTAL MEDICINE IN THE XXI CENTURY
[Chaperonologia: un moderno campo di ricerca per la Medicina Sperimentale del XXI secolo] Francesco Cappello, Fabio Bucchieri, Sabrina David, Claudia Campanella, Anna Ribbene, Antonella Marino-Gammazza, Nella Ardizzone, Anna Merendino, Vito Marcian, Giovanni Peri, Everly Conway de Macario*, Alberto J.L. Macario* and Giovanni Zummo
Dipartimento di Medicina Sperimentale, Sezione di Anatomia Umana, Universit degli Studi di Palermo, via del Vespro 129, 90127 Palermo, Italy. - * University of Maryland Biotechnology Institute (UMBI), Columbus Center, 701 E. Pratt Street, Baltimore, MD 21202, USA
Key words: Chaperones, Chaperonins, Hsp60, Hsp10, Chaperonopathies, Chaperonotherapy Parole chiave: Chaperoni, Chaperonine, Hsp60. Hsp10, Chaperonopatie, Chaperonoterapia Abstract. We have been studying for some years two mitochondrial heat shock proteins (Hsps), the chaperonins Hsp60 and Hsp10, that are necessary for folding of mitochondrial proteins. In recent times, the interest in these Hsps has been growing since it has been shown that they can also be present in the cytoplasm and secreted outside cells. We still do not know all their functions, but we are aware that they could represent important biomarkers for some tumours and inflammatory diseases. Riassunto. Da alcuni anni ci occupiamo di studiare due proteine da shock termico mitocondriali, la chaperonina Hsp60 e la sua co-chaperonina Hsp10, necessarie per il folding delle proteine mitocondriali. Linteresse nei loro confronti negli ultimi tempi aumentato perch si scoperto che queste molecole possono anche essere presenti nel citoplasma ed essere secrete allesterno della cellula. Non sono ancora note tutte le loro funzioni, che possono anche essere degli importanti biomarcatori in alcuni tumori e in alcune patologie infiammatorie.
Introduction For some years, our group has been studying morphology and function of mitochondria both in normal cells and pathological models, in order to achieve a better understanding of the mitochondrial involvement in the pathogenesis of some disease, like cancer. More recently, we have focused our attention on two mitochondrial proteins, namely Hsp60 and Hsp10, that are important for the survival of such organelles and thus of the whole cell. The Hsps constitute a heterogeneous group of molecules highly preserved during evolution as they are involved in many crucial cellular functions [1-3]. One of the most relevant is the chaperoning role, which is responsible not only for the acquisition of functional conformation of other proteins, but also of their preservation after stress caused by a variety of stressors affecting diverse tissues (Table 1). Chaperones are also involved in the
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Francesco Cappello, Fabio Bucchieri, Sabrina David, Claudia Campanella, Anna Ribbene, Antonella Marino-Gammazza, Nella Ardizzone, Anna Merendino, Vito Marcian, Giovanni Peri, Everly Conway de Macario, Alberto J.L. Macario and Giovanni Zummo
degradation of damaged proteins [4-6]. So, chaperones are proteins whose function is to promote correct folding of nascent polypeptides, refolding of partially denatured proteins, and degradation of irreversibly damaged molecules. Not all Hsps are chaperones, since other molecules, different from Hsps, have such a function, too. Table 1: Chaperone inducers (Cell stressors)a Type Description
Physical Heat, irradiation, UV light, etc Chemical Oxygen derived free radicals, hypoxia-anoxia-reperfusion Biological Infection, Inflammation Psychological Emotions, hormonal imbalance Mechanical Compression, shearing stretching Others: ethanol, methanol, tetracycline, teratogens, mutagens, carcinogens
a
Reproduced with permission from reference 9.
Chaperonin and co-chaperonins in normal and pathologic tissues Hsp60 and Hsp10 are mitochondrial chaperones, commonly named chaperonin (Hsp60) and co-chaperonin (Hsp10). It is known from studies in prokaryotes that these two chaperonins participate in the folding of nascent polypeptides (Fig.1) [7-8]. Since the mitochondrial Hsp60 and Hsp10 are phylogenetically close to those from bacteria it is generally assumed that the mitochondrial molecules have similar functions than those of the bacterial counterparts and act through similar mechanisms. A number of studies carried out by different groups have shown that Hsp60 and Hsp10, as well as other chaperones, are associated with several diseases, now referred to as
Fig. 1: Schematic representation of Hsp60-Hsp10 performing their protein-folding function as envisioned from data from the prokaryotic system. 110
Chaperonology: a novel research field for Experimental Medicine in the XXI century
chaperonopathies [9-11]. A first classification included two main classes of chaperonopathies, genetic and acquired. Some of the genetic chaperonopathies are, CharcotMarie Tooth disease, distal hereditary motor neuropathy, childhood cataracts, distal motor neuropathy, hereditary spastic paraplegia, X-linked retinitis pigmentosa, and Leber congenital amaurosis. Examples of acquired chaperonopathies are Alzheimer disease, amyotrophic lateral sclerosis, Huntingtons disease, and other pathologic conditions affecting the vascular, respiratory tract, intestinal tract, and hematopoietic tissues. More recently, a third class has been proposed, the chaperonopathies by collaborationism (or by mistake) [11], consisting of a number of tumors in which Hsps have been found overexpressed or downregulated and in which Hsps seem to play a role in tumor growth. Our recent studies have contributed to the understanding of the involvement of both Hsp60 and Hsp10 in the pathogenesis of some solid tumors. In particular, we found an overexpression of such molecules during carcinogenesis of large bowel (Fig. 2), uterine exocervix, and prostate [12-18]. By contrast, we found downregulation during bronchial carcinogenesis and vesical cancer progression [19-20]. Our data have been confirmed by other studies [21-24]. Moreover, our work has highlighted that Hsp60 and Hsp10 may also have a cytoplasmic localisation, even if not always together. We have postulated that these proteins, when present in the cytoplasm, may perform functions that are different from their canonical role in protein folding, For example, cytosolic Hsp60 and Hsp10 could participate in the mechanisms of cell proliferation and differentiation. In addition, we have shown that these molecules occur also in the peritumoral stroma [12-15]; their presence there due, perhaps, to a secretory mechanism.
Fig. 2: Hsp60 is overexpressed in colon adenocarcinoma (a), compared to normal colonic mucosa (b). 111
Perspectives Our attention is now directed to elucidating the molecular mechanisms by which increased or reduced expression of Hsp60 and Hsp10 determine the development of certain neoplasms [25-27]. Preliminary data suggest that Hsp60 plays both pro- and antiapoptotic roles, depending on the expression of other molecules, like Hsp70, and p53; therefore, Hsp60 is probably connected to other pro- and anti-proliferative molecules in a complex network with a fine regulation. Our studies are also focused on the mechanisms underlying the presence of Hsp60 and Hsp10 outside cells, since we assume that such molecules, when released in the interstitium, constitute a powerful pro-inflammatory stimulus [28]. In parallel, we are determining the levels of Hsp60 and Hsp10 in sera of patients with certain types of cancer in the hope that they could become biomarkers useful in clinical oncology. Conclusions In summary, our research pertains to the field of Chaperonology, encompassing Chaperonomics and Chaperonotherapy. Chaperonology is the study of intracellular and extracellular chaperones in all their aspects (structure, function, genetics, evolution, pathology) aiming to expand our knowledge on disease pathogenesis, and to use chaperones as diagnostic markers and prognostic indicators. Chaperonomics refers to the analysis of chaperone genes in genomes and at their products, including identification and classification of genes and pseudogenes, transcripts, polymorphisms, mutations, and inheritance. These studies should provide a solid basis for further bioinformatics analyses and experimental studies on the role of chaperone genes-proteins in ageing and associated diseases. Chaperonotherapy defines the use of chaperones in prevention and treatment of pathological conditions in which malfunctioning or absence of chaperones play a pathogenetic role and that may, therefore, benefit from the use of chaperones as therapeutic agents. Chaperonotherapy also includes the development and use of anti-chaperone agents to control conditions in which chaperones play a pro-disease role, like those types of malignant tumors mentioned above that need chaperones to grow and metastasize. We hope that research and clinico-pathological activities in these fields will be useful and will contribute to improve human health in the XXI century.
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Chaperonology: a novel research field for Experimental Medicine in the XXI century
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] Ohtsuka K, Kawashima D, Gu Y, Saito K. Inducers and co-inducers of molecular chaperones. Int J Hyperthermia 2005 Dec;21(8):703-11. Walsh D, Grantham J, Zhu XO, Wei Lin J, van Oosterum M, Taylor R, Edwards M. The role of heat shock proteins in mammalian differentiation and development. Environ Med 1999 Dec;43(2):79-87. Bensaude O, Bellier S, Dubois MF, Giannoni F, Nguyen VT. Heat-shock induced protein modifications and modulation of enzyme activities. EXS 1996;77:199-219. Latchman DS. Heat shock proteins and cardiac protection. Cardiovasc Res. 2001 Sep;51(4):637-46. Latchman DS. Protective effect of heat shock proteins in the nervous system. Curr Neurovasc Res 2004 Jan;1(1):21-7. Alsbury S, Papageorgiou K, Latchman DS. Heat shock proteins can protect aged human and rodent cells from different stressful stimuli. Mech Ageing Dev 2004 Mar;125(3):201-9. Richardson A, Schwager F, Landry SJ, Georgopoulos C. The importance of a mobile loop in regulating chaperonin/ co-chaperonin interaction humans versus Escherichia coli. J Biol Chem 2001 Feb 16;276(7):4981-7. Dubaquie Y, Looser R, Rospert S. Significance of chaperonin 10-mediated inhibition of ATP hydrolysis by chaperonin 60. Proc Natl Acad Sci (USA) 1997 Aug 19;94(17):9011-6. Macario AJL, Conway de Macario E. Sick chaperones, cellular stress, and disease. N Engl J Med 2005 Oct 6;353(14):1489-501. Macario AJL, Conway de Macario E. Chaperonopathies and chaperonotherapy. FEBS Lett 2007 Jul 31;581(19):3681-8. Macario AJL, Conway de Macario E. Chaperonopathies by defect, excess, or mistake. Ann N Y Acad Sci 2007 May 4; [Epub ahead of print] Cappello F, Bellafiore M, Palma A, Marciano V, Martorana G, Belfiore P, Martorana A, Farina F, Zummo G, Bucchieri F. Expression of 60-kD heat shock protein increases during carcinogenesis in the uterine exocervix. Pathobiology 2002-2003;70(2):83-8. Cappello F, Bellafiore M, Palma A, David S, Marciano V, Bartolotta T, Sciume C, Modica G, Farina F, Zummo G, Bucchieri F. 60KDa chaperonin (HSP60) is over-expressed during colorectal carcinogenesis. Eur J Histochem 2003;47(2):105-10. Cappello F, Rappa F, David S, Anzalone R, Zummo G. Immunohistochemical evaluation of PCNA, p53, HSP60, HSP10 and MUC-2 presence and expression in prostate carcinogenesis. Anticancer Res 2003 Mar-Apr;23(2B):1325-31. Cappello F, Bellafiore M, David S, Anzalone R, Zummo G. Ten kilodalton heat shock protein (HSP10) is overexpressed during carcinogenesis of large bowel and uterine exocervix. Cancer Lett 2003 Jun 30;196(1):35-41. Cappello F. HSP60 and HSP10 as diagnostic and prognostic tools in the management of exocervical carcinoma. Gynecol Oncol 2003 Dec;91(3):661. Cappello F, David S, Rappa F, Bucchieri F, Marasa L, Bartolotta TE, Farina F, Zummo G. The expression of HSP60 and HSP10 in large bowel carcinomas with lymph node metastase. BMC Cancer 2005 Oct 28;5:139. Cappello F, Di Stefano A, DAnna SE, Donner CF, Zummo G. Immunopositivity of heat shock protein 60 as a biomarker of bronchial carcinogenesis. Lancet Oncol 2005 Oct;6(10):816. Czarnecka AM, Campanella C, Zummo G, Cappello F. Mitochondrial chaperones in cancer. From molecular biology to clinical diagnostics. Cancer Biol Ther 2006 Jul;5(7):714-20.
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[20] Cappello F, Di Stefano A, David S, Rappa F, Anzalone R, La Rocca G, DAnna SE, Magno F, Donner CF, Balbi B, Zummo G. Hsp60 and Hsp10 down-regulation predicts bronchial epithelial carcinogenesis in smokers with chronic obstructive pulmonary disease. Cancer 2006 Nov 15;107(10):2417-24. [21] Urushibara M, Kageyama Y, Akashi T, Otsuka Y, Takizawa T, Koike M, Kihara K. HSP60 may Predict Good Pathological Response to Neoadjuvant Chemoradiotherapy in Bladder Cancer. Jpn J Clin Oncol 2007 Jan;37(1):56-61. [22] Johansson B, Pourian MR, Chuan YC, Byman I, Bergh A, Pang ST, Norstedt G, Bergman T, Pousette A. Proteomic comparison of prostate cancer cell lines LNCaP-FGC and LNCaP-r reveals heatshock protein 60 as a marker for prostate malignancy. Prostate 2006 Sep 1;66(12):1235-44. [23] Mori D, Nakafusa Y, Miyazaki K, Tokunaga O. Differential expression of Janus kinase 3 (JAK3), matrix metalloproteinase 13 (MMP13), heat shock protein 60 (HSP60), and mouse double minute 2 (MDM2) in human colorectal cancer progression using human cancer cDNA microarrays. Pathol Res Pract 2005;201(12):777-89. [24] Castle PE, Ashfaq R, Ansari F, Muller CY. Immunohistochemical evaluation of heat shock proteins in normal and preinvasive lesions of the cervix. Cancer Lett 2005 Nov 18;229(2):245-52. [25] Cappello F, Zummo G. HSP60 expression during carcinogenesis. A molecular proteus of carcinogenesis? Cell Stress Chaperones 2005 Winter;10(4):263-4. [26] Cappello F, Zummo G. HSP60 expression during carcinogenesis. Where is the pilot? Pathol Res Pract 2006;202(5):401-2. [27] Czarnecka AM, Campanella C, Zummo G, Cappello F. Heat shock protein 10 and signal transduction. a capsula eburnea of carcinogenesis? Cell Stress Chaperones 2006 Winter;11(4):287-94. [28] Cappello F, Czarnecka AM, Rocca GL, Stefano AD, Zummo G, Macario AJ. Hsp60 and Hsp10 as Antitumor Molecular Agents. Cancer Biol Ther 2007 Apr 1;6(4).
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Brain, behavior
THE SUBSTANTIA NIGRA DOPAMINERGIC NEURONS

[I Neuroni Dopaminergici della Substantia Nigra] Arcangelo Benigno, Attilio Licciardi and Giuseppe Di Giovanni
Dipartimento di Medicina Sperimentale, Sezione di Fisiologia Umana, G. Pagano, Universit degli Studi di Palermo (I)
Key words: Parkinsons Disease, Neurogenesis, Apoptosis, Neuroprotection Parole chiave: Malattia di Parkinson, Neurogenesi, Apoptosi, Neuroprotrezione Abstract. Since the 1950s, when dopamine (DA) was discovered in the mammalian central nervous system (CNS), an enormous amount of experimental evidence has revealed the pivotal role of this biogenic amine in a number of cognitive and behavioural functions. Moreover, DAergic neurons, although their numbers are few, are of clinical importance because it is implicated in several psychiatric disorders. The lost of DAergic neurons of the substantia nigra compacta (SNc) is associated with one of the most prominent human neurological disorders, Parkinsons disease (PD). The mechanisms whereby nigral dopaminergic neurons may degenerate still remain controversial. Hitherto, several data have shown that the earlier cellular disturbances occurring in dopaminergic neurons include oxidative stress, excitotoxicity, inflammation, mitochondrial dysfunction, and altered proteolysis. These alterations, rather than killing neurons, trigger subsequent deathrelated molecular pathways, including elements of apoptosis. In this review, the characteristics of the SNc dopaminergic neurons and their life cycle from birth to death are discussed. Furthermore, the new evidence of a possible de novo neurogenesis in the SNc of adult animals and in PD patients will also be examined. Riassunto. Dal 1950, quando la dopamina (DA) stata scoperta nel sistema nervoso centrale (SNC) dei mammiferi, una enorme quantita` di evidenze sperimentali ha rilevato il ruolo fondamentale di questa amina biologica in numerose funzioni cognitive e comportamentali. Inoltre, i neuroni dopaminergici, sebbene pochi di numero, sono di importanza clinica perch implicati in numerose malattie psichiatriche. La perdita dei neuroni dopaminergici della substantia nigra pars compacta (SNc) associata con una delle principali malattie neurologiche, il morbo di Parkinson (PD). Il meccanismo con cui i neuroni dopaminergici degenerano rimane ancora controverso. Fino ad ora numerosi dati sperimentali hanno mostrato che anomalie cellulari avvengono in stadi precoci come stress ossidativo, citossicita`, infiammazione e disfunzione dei mitocondri e alterata proteolisi. Queste alterazioni, piuttosto che uccidere i neuroni, scatenano un meccanismo molecolare di morte cellulare, probabilmente lapoptosi. In questa review verranno trattate le caratteristiche dei neuroni dopaminergici e il loro ciclo vitale dalla nascita alla morte. Inoltre, verranno esaminate le nuove evidenze di una possible neurogenesi nella SNc in animali adulti e nei parkisoniani.
Parkinsons disease (PD) is the second most common neurodegenerative disorder in the elderly population for which, unfortunately, there is no cure as yet [1-3]. The cardinal motor symptoms are the result of the loss of dopaminergic (DAergic) neurons in the substantia
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Arcangelo Benigno, Attilio Licciardi and Giuseppe Di Giovanni
nigra pars compacta (SNc), which causes a consequent reduction of dopamine (DA) levels in the striatum [4,5]. Since the underlying mechanisms of neuronal loss in patients are not known, current therapies are mainly symptomatic and do not halt the progression of the disease [1,3,6]. It appears clear that understanding the etiopathogenesis of PD, the modalities whereby the neurodegenerative process begins and progresses, is fundamental for the development of drugs to slow or prevent the progression of PD. There have certainly been major advances in these areas over the past few years, but, the modalities whereby the neurodegenerative process begins and progresses remain unclear. The situation is complicated further by the large number of factors that seem to be involved in the onset of this disease, such as aging, genetic vulnerability, exogenous or endogenous toxins, hydroxyl radicals (.OH) production, neuronal metabolic disturbances and inflammation (Fig. 1) [1,3,7-9]. Thus, the cumulative neuronal insults attributable to these metabolic stress factors may promote premature SNc DAergic degeneration through the activation of apoptotic programs [10] (Table). The SNc DAergic neurons Dopamine is one of the most intensively studied neurotransmitters in the brain due to its involvement in several mental and neurological disorders, such as schizophrenia, depression and PD. The most prominent DAergic cell group resides in the ventral part of mesencephalon, which contains approximately 90% of the total number of brain DAergic cells. Essentially, they are restricted to two nuclei, the ventral tegmental area of Tsai (VTA; A10) and the lateral SNc (A9). Nevertheless, cells expressing tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of catecholamines, have also been described in the striatum of rodents, monkeys and even humans [11-13]. The DAergic neurons localised in the SNc preferably project to the caudate nucleus and the putamen, i.e. the dorsal part of the striatum, and therefore this pathway is often called nigrostriatal DAergic system. The substantia nigra (SN) has been cytoarchitecturally divided into three different parts: the SNc, a horizontal sheet of densely packed medium and large cells that occupies its dorsal one-third; the SN pars reticulata (SNr), a more diffuse and cell-poor division, containing small and medium neurons lying between the SNc and the cerebral peduncles, and the SN pars lateralis (SNL), a small cluster of medium cells that extends rostrocaudally along the lateral border of SNc and SNr [14,15]. According to their neurotransmitter, nigral neurons were classified into DAergic and -aminobutyric acid (GABA)-ergic neurons [16]. Most DAergic neurons are localized in the SNc, some of them in the SNr, and to a lesser extent, in the SNL. The majority (>90%) of cells in the SNc are medium sized aspiny DAergic neurons with sparsely branching dendritic trees. Most pars compacta DAergic neurons also send one or two very long dendrites ventrally into pars reticulata that may be over 1
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The Substantia Nigra Dopaminergic Neurons
mm in length. The axon is thin and unmyelinated, and does not give off local collaterals. Electrophysiologically, DAergic neurons in the SNc display the typical firing properties of DAergic neurons i.e. broad action potential (mean biphasic = 2.17 ms), slow firing rate (mean = 4.15 Hz) and regular firing pattern [17] (Fig. 2). They are composed of subsets of neurochemically different neurons, and these chemical differences may be involved in their physiological properties and vulnerability to aggression. DA-nigral cells are far from being a homogeneous group, considering the distribution of imureactivity for calbindin-D28k (CB) the SNc is compartmentally organized along the lines of a nigrosome-matrix [18]. According to Damier and colleagues [19,20], 60% of DA neurons in the human SNc are sparsely distribuited whithin the large region of intense CB staining, which they named nigral matrix; the other 40% of DA-containing neurons are included within 5 different nigrosomes, numered from 1 to 5. The birth of SNc DAergic neurons The number of nigral neurons declines during normal aging, and it is possible that many normal elderly subjects, as well as would-be PD cases, do not develop signs of the condition because DAergic cell loss has not reached the threshold level. Hence, it is theoretically feasible that individuals born with smaller numbers of nigral neurons might be more susceptible to reaching the critical level of neuronal loss, for example by exposure to environmental toxins or even through aging. The quality of the contact and/or the degree of trophic support in early life might be important in determining the number, health and length of survival of cells such as DAergic neurons. Moreover, the transcription factors involved are expressed throughout life in the basal ganglia, suggesting that they have a role in maintaining the health of specific neurons. Recent advances in molecular biology and mouse genetics have helped to unravel the mechanisms involved in the development of mesodiencephalic DAergic (mdDA) neurons, including their specification, migration and differentiation, as well as the processes that govern axonal pathfinding and their specific patterns of connectivity and maintenance [21]. The precise time point of origin of the first postmitotic mdDA neurons is still a matter of debate. The problem has been complicated by the fact that the mdDA system is not a homogenous group of neurons. The development of mdDA neurons follows a number of stages marked by distinct events. The first mDA neurons are born around embryonic day (E)12 in Sprague-Dawley rats, and develop from a single embryological cell group that originates at the mesencephalic-diencephalic junction, known as the isthmus. Developmental studies of the pathways involved, have led to the identification of several factors that influence the final formation of midbrain DA-neurons in the adult animal. The specification of the permissive region for DA neuron generation occurs through the secretion by the isthmus of two secreted signalling proteins;
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the sonic hedgehog (SHH) and the fibroblast growth factor 8 (FGF8). This permissive region is also defined by a specific pattern of gene expression in the mesodiencephalon ventricular zone; i.e. orthodenticle homologue 2 (OTX2), gastrulation brain homeobox 2 (GBX2) and transforming growth factor-b (TGF b). Studies analyzing the functions of these factors have not only increased the understanding of how DAergic neurons are generated in vivo, but also allowed for the development of new strategies in stem cells for engineering DAergic neurons in vitro. These results may be significant in terms of the development of future therapies for PD patients [21]. The death of the SNc DAergic neurons Considerable differences exist in the numbers of midbrain DAergic cell bodies in various mammals ranging from about 45,000 in the rat, 165,000 in the macaca monkey, to 590,000 in human beings [1]. This latter number applies to humans in their fourth decade of life but drops to an average of about 350,000 during the sixth decade of life [1]. Such an age-dependent decrease in the numbers of SNc DA-cells has also been reported for nonhuman primates. It is intriguing to note that parkinsonian neurodegeneretion it is not simply an accelerated form of cell loss seen during the normal aging, even though they share some pathological characteristics. For example, the pattern of loss is opposite to ageing, with greatest in the ventral part of SNc [22]. DAergic neurons are peculiarly prone to oxidative stress due to their high rate of oxygen metabolism, low levels of antioxidants, and high contents of iron and neuromelanin pigment (NM). Moreover, DA is thought to be capable of generating toxic reactive oxygen species (ROS) via both its enzymatic and non-enzymatic catabolism [2,7]. Furthermore, the midbrain region that encompasses the SN is particularly rich in microglia [2,7], therefore, activation of nigral microglia and the release of these pro-inflammatory neurotoxic factors may be a crucial component of the degenerative process of DAergic neurons in PD. We are far from seeing the whole picture; the mechanisms responsible for nigral DA cell death are only beginning to be understood. It is well known that DAergic neurons degenerate in PD in a disease-duration pattern [19]. Human DA nigral neurons in the calbindin-CB-poor nigrosomes in contrast to those in the CB-rich matrix are more likely to degenerate in PD. Within the nigrosomes, cell loss follows a strict order, depletion being maximal, with a maximum cell loss of 98%, in nigrosome 1 located in the caudal and mediolateral part of the SNc and then to other nigrosomes and finally to the matrix. The reason for such differential vulnerability of DAergic neurons is not known. Yet, the different nigral compartments may differ in terms of their content of growth factor, receptors, compounds related to exocitoxicity, agents involved in oxidative metabolism, and activity of potentially predisposing genes such as those for a-synuclein and parkin. Much attention has been
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given to the role of neuromelanin (NM), which has long been recognized as a marker of increased oxidative stress. Interestingly, the distribution pattern of NM within the SNc is inverse to the density of CB-immunostaining intensity and clearly overlaps the degeneration disease-duration pattern showed by Damier et al. [19,20]. In so far as ventral SNc DAergic neurons of nigrosome 1, the first to degenerate in PD, contain a high degree of pigmentation. NM pigment could be toxic to aminergic neurons as it physically interferes with intracellular communication, causing a macromolecular crowding effect, thereby interfering with the synthesis and degradation of cellular proteins. How the DAergic neurons die Two major mechanisms of neuronal demise have been discussed in neurodegeneration: apoptosis and necrosis. These cell death types are different, frequently divergent, but sometimes overlapping cascades of cellular breakdown. Apoptosis, a specific form of gene-directed programmed cell death (pcd) brings about the removal of unnecessary, aged or damaged cells and is distinguished by distinct morphological and biochemical features. It is performed by an intrinsic suicidal machinery of the cell and can be set off by environmental stimuli including irradiation leading to DNA damage, oxidative stress, toxins, viruses, withdrawal of neurotrophic support, etc. It is well known that adult mature neurons die at a low rate during the normal aging process but at an accelerated pace in cases of neurodegenerative disorders, like PD. Some groups have reported that dying neurons displaying the morphological features of apoptosis are present in the post mortem human brain of patients with neurodegenerative disorders. These features include cell shrinkage, chromatic condensation, DNA fragmentation, and increased expression of both proapototic, and antiapoptotic proteins, or DNA repair enzymes [23]. However, other groups have observed little or no evidence of apoptotic neuronal cell death associated with neurodegenerative diseases. The current view about the apoptotic mechanism underlying nigrostriatal DA neuron degeneration in PD is quite mixed. Recent evidence in experimental models of PD, point to the fact that neuroapoptosis might quite possibly be an early pathological event, and may or may not be present at the end of disease stage, when postmortem samples are collected and analyzed. The SNc DAergic neurons resurrection Recently, another dogma of science has been disproved; the adult mammalian brain does have the potential to generate new neurons and to integrate them into existing circuits. Neurogenesis has been shown at least in two rather discrete areas of the brain, the dentate gyrus of the hippocampal formation and the subventricular zone (SVZ) [24,25].
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Low levels of neurogenesis also occur in various other regions of the adult CNS including the SNc [26,27], a finding, which may have profound implications for the treatment of PD. Whether DAergic neurogenesis occurs in the adult SNc in normal brain or in PD animal models is still a matter of debate [28]. The existence of endogenous neurogenesis in the striatum and the subventricular zone in PD would open possibilities for a new cell-based approach to the treatment of neurodegeneration in PD patients, bypassing the need for transplantation. However, there is some evidence supporting neurogenesis or a more general degree of plasticity in the brains of PD patients and of PD animal models [29]. Conclusions From the literature here reviewed it appears evident that the progress in understanding the neuropathological process that induces DAergic cell demise in PD has been impressive. However, despite these advances, the processes that initiate cell death remain unclear. Whether they involve energy metabolism deficiencies, inadequate control of the redox state, low amounts of neurotrophic support and/or the action of environmental and endogenous toxins, remains to be elucidated. Clearly, a better understanding of the DAergic cell biology, the mode of cell death in PD, and the molecular mechanisms that controls it, is required. Indeed, DAergic neurons are sui generis cells, involved in a large number of physiological and pathological conditions and are also very delicate. SNc DAergic cells for some unknown reason are prone to degenerate and they are very sensitive to oxidative stress and inflammation. A better comprehension of the difference in resistance among DAergic cells of the mesencenphalic region will bring further insight into their peculiar characteristics.
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Table SNc dopaminergic neurons characteristic The first DAergic neurones are born around day E12 of rat development from a single embryological cell group that originates at the the isthmus. 590,000 DAergic cells in human beings, that drops to an average of about 350,000 during the sixth decade of life. The majority of brain DAergic cells are restricted in the VTA and SNc, from which originate the mesocorticolimbic and nigrostriatal DAergic system, respectively. SNc DAergic cells are medium sized aspiny neurones, coexpresing also CCK, CR, and CB. According to CB staining the SNc is compartmentally organized along the lines of nigrosome-matrix. SNc DAergic neurones degenerate in PD in a disease-duration pattern: from nigrosome 1 to nigrosoma 5 and then to the matrix. SNc DAergic neurons are likely to die for apoptosis in PD degeneration. A de novo neurogenesis is possible that occurs in the SNc of adult brain and in the SNc and striatum of PD patients, suggesting a new interventional approach.
Fig 1. A simplified schematic of the vicious cycle that lead to the final demise of nigral DA cells. Activated microglia (subsequent to immune activation or neuronal lesion caused by exposure to toxins such as MPTP or 6-OHDA) can contribute to the degeneration of DA neurones by releasing neurotoxic factors. Cytokines can then activate receptor-mediated proapoptotic pathways within the DA neuron as well as further stimulation of the microglia in the form of iNOS and COX2 induction. The former will lead to greatly increased NO generation and resulting elevation of ROS, which damage the cell as a result of DNA damage, protein disruption and lipid peroxidation. The latter causes increased PGE2 production leading to direct toxicity to the DA neuron. Superoxide further stimulates microglial cytokine production as well as increases the quantities of ROS in the dopaminergic neuron. As signals from damaged DA cells further recruit and also stimulate microglia, the process can readily spiral out of control into full-blown neurodegeneration.
Fig. 2. Anatomical and electrophysiological characteristics of a SNc DAergic neurone. Left panel: Reconstruction of an electrophysiologically identified nigrostriatal DAergic neurone intracellularly labeled with HRP following in vivo intracellular recording. The inset at lower right shows the position of the neurone with respect to the boundaries of substantia nigra. Right panel: Identification of SNc neurones in extracellular (A) and whole-cell patch-clamp (B) recordings. (A) Example of extracellular recording from a DAergic neurone. A(1): spontaneous firing. A(2): interval histogram demonstrating regularity of spontaneous firing. A(3): shape of individual action potentials (average of 16 consecutive potentials). Note inflection on the rising phase of the action potential (arrow). A(4): inhibition of firing after bath application of dopamine (30 M). (B) Example of whole-cell patch-clamp recording. B(1): DAergic neuron. From top: Ih current induced by hyperpolarizing voltage commands (voltage-clamp), and voltage responses to step current pulses recorded in current-clamp. Note depolarizing sag of the membrane potential (arrow) evoked by hyperpolarizing current pulses. B(2): non-DAergic neurone. From above: currents induced by voltage steps (voltage-clamp), and responses to step current pulses (current-clamp).
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] Esposito E, Di Matteo V, Di Giovanni G. Death in the substantia nigra: a motor tragedy. Expert Rev Neurother 2007;7(6):677-97. Sian J, Gerlach M, Youdim MB, Riederer P. Parkinsons disease: a major hypokinetic basal ganglia disorder. J Neural Transm 1999;106(5-6):443-76. Esposito E, Di Matteo V, Benigno A, Pierucci M, Crescimanno G, Di Giovanni G. Non-steroidal anti-inflammatory drugs in Parkinsons disease. Exp Neurol 2007;205(2):295-312. Bernheimer H, Birkmayer W, Hornykiewicz O, Jellinger K, Seitelberger F. Brain dopamine and the syndromes of Parkinson and Huntington. Clinical, morphological and neurochemical correlations. J Neurol Sci 1973;20(4):415-55. Hornykiewics O. Neurochemical pathology and etiology of Parkinsons disease: basic facts and hypothetical possibilities Mt Sinai J Med 1988;55(10):11-20. Schapira AH. Present and future drug treatment for Parkinsons disease. J Neurol Neurosurg Psychiatry 2005;76(11):1472-78. Jenner P, Olanow CW. The pathogenesis of cell death in Parkinsons disease. Neurology 2006;66(10 Suppl 4):S24-36. Di Matteo V, Pierucci M, Di Giovanni G, Di Santo A, Poggi A, Benigno A, Esposito E. Aspirin protects striatal dopaminergic neurons from neurotoxin-induced degeneration: an in vivo microdialysis study. Brain Res 2006;1095(1):167-77. Di Matteo V, Benigno A, Pierucci M, Giuliano DA, Crescimanno G, Esposito E, Di Giovanni G. 7-nitroindazole protects striatal dopaminergic neurons against MPP+-induced degeneration: an in vivo microdialysis study. Ann NY Acad Sci 2006;1089:462-71. Novikova L, Garris BL, Garris DR, Lau YS. Early signs of neuronal apoptosis in the substantia nigra pars compacta of the progressive neurodegenerative mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/ probenecid model of Parkinsons disease. Neuroscience 2006;140(1):67-76. Cossette M, Lecomte F, Parent A. Morphology and distribution of dopaminergic neurons intrinsic to the human striatum. J Chem Neuroanat 2005;29(1): 1-11. Baker H, Kobayashi K, Okano H, Saino-Saito S. Cortical and striatal expression of tyrosine hydroxylase mRNA in neonatal and adult mice. Cell Mol Neurobiol 2003;23(4-5):507-18. Andn NE, Carlsson A, Dahlstroem A, Fuxe K, Hillarp NA, Larsson K. Demonstration and mapping out of nigro-neostriatal dopamine neurons. Life Sci 1964;3: 523-30. McRitchie DA, Halliday GM. Calbindin D28k-containing neurons are restricted to the medial substantia nigra in humans. Neuroscience 1995;65(1):87-91. Dahlstrom A, Fuxe K. Localization of monoamines in the lower brain stem. Experientia 1964; 20(7): 398-99. Oertel WH, Mugnaini E. Immunocytochemical studies of GABAergic neurons in rat basal ganglia and their relations to other neuronal systems. Neurosci Lett 1984; 47(3): 233-38. Di Giovanni G, De Deurwaerdere P, Di Mascio M, Di Matteo V, Esposito E, Spampinato U. Selective blockade of serotonin-2C/2B receptors enhances mesolimbic and mesostriatal dopaminergic function: a combined in vivo electrophysiological and microdialysis study. Neuroscience 1999;91(2):587-97. McRitchie DA, Hardman CD, Halliday GM. Cytoarchitectural distribution of calcium binding proteins in midbrain dopaminergic regions of rats and humans. J Comp Neurol 1996;364(1):121-50. Damier P, Hirsch EC, Agid Y, Graybiel AM. The substantia nigra of the human brain. II. Patterns of loss of dopamine-containing neurons in Parkinsons disease. Brain 1999;122(Pt 8):1437-48.
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[20] Damier P, Hirsch EC, Agid Y, Graybiel AM. The substantia nigra of the human brain. I. Nigrosomes and the nigral matrix, a compartmental organization based on calbindin D (28K) immunohistochemistry. Brain 1999;122(Pt 8):1421-36. [21] Smidt MP, Burbach JP. How to make a mesodiencephalic dopaminergic neuron. Nat Rev Neurosci 2007;8(1):21-32. [22] Fearnley JM, Lees AJ. Ageing and Parkinsons disease: substantia nigra regional selectivity. Brain 1991;114(Pt 5):2283-301. [23] Graeber MB, Grasbon-Frodl E, Abell-Aleff P, Ksel S. Nigral neurons are likely to die of a mechanism other than classical apoptosis in Parkinsons disease. Parkinsonism. Relat Disord 1999;5(4):187-92. [24] Santarelli L, Saxe M, Gross C, Surget A, Battaglia F, Dulawa S, Weisstaub N, Lee J, Duman R, Arancio O, Belzung C, Hen R. Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants. Science 2003;301(5634):805-9. [25] Kempermann G, Jessberger S, Steiner B, Kronenberg G. Milestones of neuronal development in the adult hippocampus. Trends Neurosci 2004;27(8):447-52. [26] Lie DC, Dziewczapolski G, Willhoite AR, Kaspar BK, Shults CW, Gage FH. The adult substantia nigra contains progenitor cells with neurogenic potential. J Neurosci 2002;22(15):6639-49. [27] Zhao M, Momma S, Delfani K, Carlen M, Cassidy RM, Johansson CB, Brismar H, Shupliakov O, Frisen J, Janson AM. Evidence for neurogenesis in the adult mammalian substantia nigra. Proc Natl Acad Sci (USA) 2003;100(13):7925-30. [28] Frielingsdorf H, Schwarz K, Brundin P, Mohapel P. No evidence for new dopaminergic neurons in the adult mammalian substantia nigra. Proc Natl Acad Sci (USA). 2004;101(27):10177-82. [29] Borta A. Hoglinger, G.U. Dopamine and adult neurogenesis. J Neurochem 2007;100(3):587-95.
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MULTIvARIATE ANALYSIS AS TOOL fOR THE STUDY Of BEHAvIOR

[Analisi multivariata come strumento di studio del comportamento] Maurizio Casarrubea
University of Palermo, School of Medicine, Dept. of Experimental Medicine, Human Physiology Section Palermo (I)
Key words: Multivariate analysis, Stochastic analysis, Cluster analysis, T-Pattern analysis Parole chiave: Analisi multivariata, Analisi stocastica, Cluster-analisi, T-Pattern analisi Abstract. Crucial feature of emergent phenomena is that, even if basic processes are fully elucidated and/or described, they may be impossible to predict. This idea perfectly fits with the evidence that, concerning behavioral studies, reductionistic descriptions (durations, per cent distributions and so on) of isolate events are far to be representative of the whole behavior. This last can be exhaustively understood only if inter-relations among events are taken into account. It is exactly here that traditional descriptive approaches to behavioral study give way to multivariate analysis. In fact, an important characteristic of multivariate techniques dwells in the possibility to analyze relationships between the elements of a behavioral sequence. Riassunto. Laspetto principale di un qualsiasi fenomeno emergente risiede nella sua assoluta imprevedibilit, anche se i processi di base che lo determinano sono noti e/o descritti. Questa idea si adatta perfettamente con levidenza che, per quanto concerne gli studi comportamentali, descrizioni puramente riduzionistiche (durate, frequenze, distribuzioni percentuali ecc.) di isolati eventi non sono affatto rappresentative dellintero comportamento che si prende in esame. Questultimo pu essere esaustivamente compreso solo se le inter-relazioni tra i diversi elementi che lo compongono sono prese in considerazione. esattamente qui che gli approcci puramente descrittivi allo studio del comportamento cedono il passo allanalisi multivariata. Infatti, una delle caratteristiche pi importanti delle tecniche di analisi multivariata risiede proprio nella possibilit di analizzare le relazioni esistenti tra gli elementi di una data sequenza comportamentale.
1. Introduction Multivariate analysis (MVA) encompasses techniques dedicated to the study of data sets comprising more than one variable. Along the last two decades, MVA has been considered as an useful tool to study the structure of a behavior in different experimental approaches. For instance, previous papers have illustrated different characteristics of rats social behavior [1], rodents responses in the hot plate [2-4], in the elevated plus maze test [5, 6] and in the swimming test [7]. MVA has also been used to describe effects of isolation on behavior [8], relationships between rat behavioral structure and EEG activity [9] and rat responses in specific conditions of fear or aggressiveness [10-12]. Moreover, in a recent paper, through a MVA approach, we have demonstrated specific behavioral modifications
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following administration of dopaminergic drugs, in rats tested on hot plate [13]. Aim of the present review is to present an outline of different multivariate techniques useful for the behavioral analysis. 2. Transition matrix The first step is the construction of an ethogram that is, in its simplest form, a formal list containing descriptions of animal behaviors. After that, behavioral transitions from a behavioral element to another can be noted down in a square transition matrix. Basically, a transition matrix is a table representing shifts among the behavioral elements, according to the selected ethogram. Fig. 1 summarizes the pivotal structure of a transition matrix:
Fig. 1 - Transition matrix. See text for details.
A = behavioral element A B = behavioral element B a = transitions from A to B b = transitions from B to A a = Total transitions of A b = Total transitions of B The transition matrix (fig. 1) represents the starting point of all MVA, except T-Pattern analysis (see below).
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Multivariate analysis as tool for the study of behavior
2.1. Stochastic analysis A relatively simple approach to the analysis of transition matrices is represented by the stochastic analysis. The stochastic analysis requires transition matrices to be transformed into probability matrices which represent relative frequencies of transition from a behavioral element to another one. Thus, within a probability matrix, the following qualifications need to be taken into account: (a) switching probability from an element to all others must be 1; (b) all elements must be between 0 and 1; (c) each row must sum 1. Matrices containing relative frequencies can be graphically expressed through corresponding stochastic pathway diagrams. Fig. 2 illustrates a pathway diagram representing probabilistic relations among five different behavioral elements in a group of 42 rats in open field. Three probability ranges were selected: between 0.10 and 0.24 (thin dotted arrows), between 0.25 and 0.49 (medium arrows), and between 0.50 and 1 (large arrows). All behavioral elements show convergences on immobile-sniffing. Also, high probabilities of transitions are shared between walking and climbing and between immobility and immobile sniffing. 2.2. Cluster analysis In the cluster analysis transition matrices are transformed into similarity matrices through an aggregative procedure which allows to identify clusters on the basis of the overall reciprocal number of transitions. It is important to mention that the similarity table (containing only absolute values concerning the affinity between two behavioral elements) is not a square from-to matrix but an half matrix where each cell indicates the vicinity between two given elements. Cluster analysis is to some extent less intuitive than stochastic analysis probably because of the underlying aggregative algorithm that converts transitions into similarity values (i.e. not expressing directions of transitions). Also, different aggregative procedures can be used to obtain an half similarity matrix. For a detailed description of specific aggregative procedures see E.F. Espejo et al. [2] or M. Casarrubea et al. [13]. The main outcome of the cluster analysis is a dendrogram, that is a tree diagram. Dendrogram in fig. 3 represents vicinity relations among the same five behavioral elements presented in fig. 2. Four elements group in two different behavioral clusters including respectively immobility - immobile sniffing (S = 100) and walking - climbing (S = 110). 2.3. Transition matrix: elaborations Statistics between groups (for instance a control and an administered group) is both an important and neuralgic part of an experiment. Concerning MVA, it goes without saying that a comparison between groups could involve matrices. However, procedures concerning
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Fig. 2 - Pathway diagram representing probabilistic relations among five different behavioral elements in a group of 42 rats in open field. Three different probabilities of transitions are indicated on the right side. Im = immobility; Cl = climbing; Wa; walking; Re = rearing; IS = immobile sniffing.
Fig. 3 - Dendrogram representing similarities between five different behavioral elements in a group of 42 rats in open field. Similarity values (y-axis) mould two different clusters (x-axis): Im-IS and Wa-Cl. See fig. 2 for abbreviations.
comparison of matrices represent a challenging issue. For example, it has been pointed out that the classical methods such as chi-square test should not be applied to transitional tables because behavioral transitions are dependent each other. So, for instance, Tavar and Colleagues proposed the so called gamma correction factor to multiply by the chi-square [14]. However, an elegant, albeit not simple, method to assess significance of individual cells within and between transition matrices is probably the one successfully used by B.S. Everitt in 1977 [15] and B.M. Spruijt in 1984 [1] and, after that, in different recent papers. In this method a transition matrix is transformed into a matrix containing adjusted residuals. In brief, for each given transition, the adjusted residual represents the difference between the observed cell value and an expected one, calculated by a specific software engine on the basis of a random distribution of transitions. Positive residuals indicate transitions occurring more often than expected, negative residuals represent transitions occurring less often than expected. The great advantage of adjusted residuals is that they can be expressed according to a Z-distribution so that P-values can be found in a Z-table.
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2.4. T-Pattern analysis Stochastic and cluster analyses have both benefits and negative aspects. Noticeable advantages provided by dendrograms and stochastic pathway diagrams is that they represent patterning among behavioral elements in a very original, simple and intuitive way. On the other hand it should be taken into account that stochastic and/or cluster analyses: need heavy additional elaborations (see par. 2.3.); represent a whole observational period, similar to snapshots of everything; are extremely sensitive to noise (i.e. disturbing behavioral elements need to be removed before running the analysis). Fig. 4 represents an observational period (T0 Tx) with various behavioral elements and few uncommon ones (for instance: Y, T, Z): they are the noise that must be removed before running a cluster or a stochastic analysis. These drawbacks are not present in a T-Pattern analysis in fact, not only the a-priori noise reduction is not necessary but, importantly, T-Patterns represent events along the session time. T-Pattern analysis is carried out through a specific software known as Theme. This program repeats a recursively test, checking the distribution of every combination of events along a specific time window [16]. If the time lag of a sequence of events is not randomly distributed a T-Pattern is defined (elements l and f in fig. 4). In a second step, detected patterns are processed again and if there is a temporal relation with other events, they are combined (l-f-b, see fig. 4) into higher order patterns, and so on, over and over again, following a bottom-up process. Since the graphical representation of T-patterns and the results of cluster analysis are both visualized through tree diagrams (fig. 4 and fig. 5), it is important to remember that cluster analysis is based on the similarities between events (fig. 3). On the other hand, the tree structure provided by Theme does not represent such similarities, but the existence of significant relationships along time. Fig. 5 shows a T-Pattern encompassing Immobility, Immobile-Sniffing and Walking in ten subjects randomly taken from the main group of animals represented in fig. 2 and fig. 3. The T-pattern structure is shown in the upper left panel. All occurrences of the responses are shown in the upper right panel. The bottom panel shows nine occurrences of one significant (p < 0.0001) T-pattern.
Fig. 4 - Observational period (T0 Tx) with various behavioral elements (letters). T-Pattern analysis is carried out through a specific software that repeats a test over and over again, checking the distribution of every combination of events along the specified time window. If a sequence of events is found and their time lag is not randomly distributed, then a simple T-Pattern is defined (l-f). In a second step, detected patterns are processed again and if there is a temporal relation with other events, they are combined (l-f-b) into higher order patterns and so on, recursively, following a bottom-up process. The final result is a tree representation. 133
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Fig. 5 - T-pattern structure is shown in the upper left panel. All occurrences of the responses are shown in the upper right panel. The bottom panel shows nine occurrences of one significant (p < 0.0001) T-pattern between immobility, immobile-sniffing and walking. Data concerning ten subjects randomly taken from the main group of 42 rats in open field. b = beginning point of the event; e = ending point. See fig. 2 for abbreviations.
3. Discussion An even swift comparison among fig. 2, fig. 3 and fig. 5 makes clear how three rather different MV approaches strengthen each other in representing animals behavior. Moreover, each representation perfectly fits with the remaining ones. In other terms, these MV techniques can be successfully used together to depict behavioral patterning from different point of views. Nonetheless, the pivotal question is why do we need to represent behavior through these unconventional approaches? Behavior is much more than simple durations or frequencies of isolate behavioral elements: the meaning of behavior largely resides in its sequential organization. It is my contention that even hundreds of purely descriptive parameters make available only a short-sighted, reductionistic, view of the behavior and, whats more, the possible illusion of deepness and exhaustivity concerning presented data. To tell it like it is, descriptive analyses reduce behavior to simple numbers. This is a reductionistic approach, extremely similar to the so called Cartesian Reductionism. The Cartesian reductionism can be easily summarized through the so called machine metaphor. The meaning of the
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machine metaphor lies in the intuitive idea of machine that we have: a machine is built up from separate pieces, nonetheless it can be reduced to those pieces without losing its machine-like qualities. This approach does not work for behavioral analysis because behavior is complex, characterized by emergent phenomena arising from the inter-relations among events. Thus, durations, per cent distributions and/or frequencies of disjointed behavioral elements cant offer answers to the crucial question in all behavioral studies: what about the relationships between the observed elements? It is exactly here that traditional descriptive approaches to behavioral study give way to MVA: the pivotal aspect of MVA techniques dwells in the possibility to analyze the existing relationships between the elements of a behavioral sequence. On the basis of these inter-relations it becomes possible to explore behavior from very different points of view, greatly beyond what the human eye can intuitively interpret.
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References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] Spruijt BM, Gispen WH. Behavioral sequences as an easily quantifiable parameter in experimental studies. Physiol Behav 1984; 32: 707-10. Espejo EF, Mir D. Structure of the rats behaviour in the hot plate test. Behav Brain Res 1993; 56: 171-6. Espejo EF, Mir D. Differential effects of weekly and daily exposure to the hot plate on the rats behavior. Physiol Behav 1994; 55: 1157-62. Espejo EF, Stinus L, Cador M, Mir D. Effects of morphine and naloxone on behaviour in the hot plate test: an ethopharmacological study in the rat. Psychopharmacology 1994; 113: 500-10. Cruz AP, Frei F, Graeff FG. Ethopharmacological analysis of rat behavior on the elevated plus-maze. Pharmacol Biochem Behav 1994; 49: 171-6 Espejo EF. Structure of the mouse behaviour on the elevated plus-maze test of anxiety. Behav Brain Res 1997; 86: 105-12. Lino-de-Oliveira C, De Lima TCM, Carobrez AP. Structure of the rat behaviour in the forced swimming test. Behav Brain Res 2005; 158: 243-50. Van Den Berg CL, Van Ree JM, Spruijt BM. Sequential analysis of juvenile isolation-induced decreased social behavior in the adult rat. Physiol Behav 1999; 67: 483-8. Van Lier H, Drinkenburg WH, Coenen AM. Strain differences in hippocampal EEG are related to strain differences in behaviour in rats. Physiol Behav 2003; 78: 91-7. Mos J, Olivier B, Van Der Poel M. Modulatory actions of benzodiazepine receptor ligands on agonistic behaviour. Physiol Behav 1987; 41: 265-78. Aguilar R, Gil L, Gray JA, Driscoll P, Flint J, Dawson GR, Gimenez-Llort L, Escorihuela RM, FernandezTeruel A, Tobena A. Fearfulness and sex in F2 Roman rats: males display more fear though both sexes share the same fearfulness traits. Physiol Behav 2003; 78: 723-32. Aguilar R, Gil L, Flint J, Gray JA, Dawson GR, Driscoll P, Gimenez-Llort L, Escorihuela RM, FernandezTeruel A, Tobena A. Learned fear, emotional reactivity and fear of heights: a factor analytic map from a large F(2) intercross of Roman rat strains. Brain Res Bull 2002; 57: 17-26. Casarrubea M, Sorbera F, Crescimanno G. Effects of 7-OH-DPAT and U 99194 on the behavioral response to hot plate test, in rats. Physiol Behav 2006; 89: 552-62. Tavar S, Altham PME. Serial dependence of observations leading to contingency tables, with corrections to c2 statistics. Biometrika 1983, 70: 139-144. Everitt BS. The analysis of contingency tables. New York: John Wiley and Sons, 1977. Magnusson MS. Repeated Patterns in Behavior and Other Biological Phenomena. Eds: Oller DK, Griebel U. Evolution of Communication Systems: A Comparative Approach, Cambridge: The MIT Press, 2004: 111-128.
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MODIfICAZIONI INDOTTE DALLALOPERIDOLO SUL COMPORTAMENTO ATTENTIvO DEL RATTO

[Modifications induced by haloperidol on rat attentive behavior] Giuseppe Crescimanno, Maurizio Casarrubea e Filippina Sorbera
Dipartimento di Medicina Sperimentale, Sezione di Fisiologia Umana, Universit di Palermo (I)
Parole chiave: Comportamento attentivo, Dopamina, Aloperidolo, Ratto Key words: Attentive behavior, Dopamine, Haloperidol, Rat Riassunto. Scopo della presente ricerca stata la individuazione di possibili effetti dellaloperidolo, antagonista della famiglia recettoriale dopaminergica D2, su alcune manifestazioni comportamentali caratterizzanti lattenzione focalizzata nel ratto. Sono stati adoperati 5 gruppi di ratti Wistar maschi analizzati nel corso di unattivit esploratoria durante la quale stimoli acustici erano erogati secondo sequenze casuali per mantenere uno stato di continua vigilanza. Un gruppo riceveva solo stimolazione acustica, uno stimolazione acustica e salina IP, e tre gruppi erano stimolati dopo la somministrazione di aloperidolo a differenti dosi (0.05, 0.1, 0.3 mg/kg). Due parametri sono stati scelti per analizzare gli episodi di attenzione focalizzata: il numero di episodi e la loro durata. Laloperidolo induceva una diminuzione del numero di episodi e un aumento della loro durata, eccetto che alla dose minore che, agendo a livello autorecettoriale, provocava effetti opposti. I risultati suggeriscono che laloperidolo potrebbe influenzare specifici meccanismi neurali coinvolti nella regolazione del comportamento attentivo, probabilmente influenzando i processi di gating sensoriale dopamino-dipendenti. Abstract. Aim of the present research was to analyze effects of haloperidol (HAL), a dopamine D2 receptor antagonist, on focused attention, in the rat. Five groups of male Wistar rats, observed during exploration of a novel environment and subjected to randomly delivered acoustic stimuli to induce a condition of attentional focusing, were used. One group received acoustic stimulation, one was stimulated and received saline i.p., and three groups were stimulated and administered different doses of HAL (0.05, 0.1, 0.3 mg/kg. i.p.). Episodes of attentional focusing were characterized by immobility, auricle erection and orientation, eyes open and head oriented toward the stimulus source. With the aid of a videoanalysis apparatus, number and duration of focusing episodes were recorded during experimental sessions lasting 20 min. HAL provoked a decrease of the episodes of focusing and an increase of their duration except for the lower dose which likely affected presynaptic receptors. Results suggest that HAL might influence specific neural mechanisms involved in the regulation of attentive behavior likely by affecting sensory-gating dopamine-dependent processes.
Introduzione stato ipotizzato che alterazioni della trasmissione dopaminergica, spesso presenti in alcune forme di psicosi [1, 6, 13], possano causare delle modificazioni dei processi di sensory gating conducendo a disordini della sfera attentiva. Laloperidolo, antagonista
137
Giuseppe Crescimanno, Maurizio Casarrubea, Filippina Sorbera
dei recettori dopaminergici appartenenti alla famiglia D2, stato dimostrato possedere unefficace attivit antipsicotica assieme a quella di controllo sui sintomi ipercinetici di alcune sindromi neurologiche [8]. stato inoltre dimostrato che laloperidolo provoca rapidi cambiamenti nei profili di attivit dei neuroni dopaminergici della pars compacta della substantia nigra (neuroni A9) e dellarea ventrale tegmentale (neuroni A10) [7, 11]. Daltra parte, lesioni di queste popolazioni neuronali provocano nel ratto deficits sensorimotori e disordini attentivi caratterizzati dalla impossibilit di orientarsi verso nuovi stimoli provenienti dallambiente [3, 10]. stato quindi possibile ipotizzare una relazione tra neuroni dopaminergici troncoencefalici e condizione attentiva ponendo in particolare i neuroni A9 in relazione ai fenomeni di shift attentivo e i neuroni A10 ai fenomeni di focalizzazione dellattenzione. La presente ricerca si prefigge di studiare se esiste una relazione tra i rapidi cambiamenti osservati nel firing dei neuroni dopaminergici A9 e A10 in seguito alla somministrazione di aloperidolo e modificazioni di alcuni parametri comportamentali riconducibili allattenzione focalizzata. Tale condizione riconoscibile per la presenza di immobilit, occhi aperti, padiglioni auricolari eretti e orientati assieme al capo verso la sorgente dello stimolo. Per mezzo di un sistema di videoregistrazione sono stati analizzati in differenti gruppi di ratti, gli effetti della somministrazione per via generale di aloperidolo sul numero e la durata degli episodi di attenzione focalizzata (focusing attentivo). Tali episodi erano riscontrabili in ratti esposti a brevi stimoli acustici erogati secondo sequenze casuali per intervallo e localizzazione. Materiali e metodi La ricerca stata condotta su ratti wistar maschi del peso di 250-300 gr stabulati in gabbie singole, in un ambiente a temperatura costante (21 + 1 C) e con libero accesso al cibo ed allacqua. Gli esperimenti si svolgevano tra le 07.00 e le 19.00 in una stanza insonorizzata e mantenuta a temperatura e illuminazione costante. Per le osservazioni comportamentali, gli animali erano posti in box di perspex (30 x 30

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Edited by Aldo Gerbino Giovanni Zummo Giuseppe Crescimanno

Dipartimento di Medicina Sperimentale

Experimental Medicine Reviews Morphophysiological Remarks

D.i.Me.S - Anatomia Umana e Istologia ed Embriologia, Palermo

D.i.Me.S - Fisiologia Umana, Palermo

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[17] [18] [19]

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