P.1.c.
Basic and clinical neuroscience − Neuropharmacology
and mechanical stimuli were used as outcome measures of neu-
ropathic pain in sham and nerve-injured rats (n = 8 per group) by
using the plantar, acetone and von Frey test, respectively. These
tests were performed during the day portion of the circadian
cycle (09:00−16:00 h). A period of 20−30 min was allowed for
behavioral accommodation. After cage exploration and major
grooming activities ceased, we made measurements to determine
baseline values, and then carried out the pain tests 3, 5, 7, 10 and
14 day after the surgery.
The data were analyzed by repeated measure ANOVA test with
SPSS software. Lithium decreased thermal haperalgesia score with
dose of 5, 10 and 15 mg/kg and allodynia score with dose of
10 and 15 mg/kg significantly (P < 0.001). Naloxone (1 mg/kg)
significantly reverses anti-hyperalgesic and anti-allodynic effects
of lithium (10 mg/kg) in radiant heat plantar and von Frey fila-
ment stimulation test (P?0.05) but cannot block the anti-allodynic
effects of lithium in acetone test. Our study demonstrates that
intraperitoneal injection of lithium reduces hyperalgesia and al-
lodynia in sciatic nerve ligation model of neuropathic pain with
involvement of opioid system.
References
[1] Chuang DM, Chen RW, Chalecka-Franaszek E, Ren M, Hashimoto R,
Senatorov V, Kanai H, Hough C, Hiroi T, Leeds P. Neuroprotective
effects of lithium in cultured cells and animal models of diseases.
Bipolar Disord. 2002 Apr;4(2):129−36.
[2] Alborzi A, Mehr SE, Rezania F, Badakhshan S, Mombeini T, Shafa-
roodi H, Moezi L, Ravan MN, Sharifian M, Dehpour AR The effect of
lithium chloride on morphine-induced tolerance and dependence in iso-
lated guinea pig ileum. Eur J Pharmacol. 2006 Sep 18;545(2−3):123−8.
[3] Seltzer Z, Dubner R, Shir Y. A novel behavioral model of neuropathic
pain disorders produced in rats by partial sciatic nerve injury. Pain.
1990 Nov;43(2):205−18.
P.1.c.011 Influence of the structural analogue of
AVP(6−9) on training of rats followed with
positive and negative reinforcement
A. Belyakova1 ° , O.G. Voskresenskaya1 . 1 Moscow State
University, Human and Animal Physiology Department Faculty
of Biology, Moscow, Russia
The positive influence of both vasopressin and its analogues
on learning processes and memory is now of no doubts. The
C-terminal fragments of arginine-vasopressin (AVP) are known to
be crucial for the development of behavioral effects. In our preced-
ing researches we have shown that sintetic analogue C-terminal
fragment of AVP − Ac-D-MPRG (wherein Cys6 was changed
to D-Met) improves learning process, increase the orienting and
exploratory, decreases the anxiety level in white rats and may be
used as antidepressant drug. On the basis of theoretical analysis
and conformational designing of original tetrapeptide structurally
related to the C-terminal fragment of AVP(6−9) − N-Ac-D-Ser-
Pro-Arg-Gly-NH2 (Ac-D-SPRG) − was synthesized.
The given researches was aimed at investigation of influence
of the original structural analogue of AVP(6−9) Ac-D-SPRG on
production of a conditioned active avoidance of a painful stimulus
and production of conditioned food-procuring reaction on site.
The work was carried out on mature white male rats with weight
of 220–250 g. The preparation was given via intranasal injection
in doses of 0.01, 0.1, 1.0, and 10.0 mg/kg in volume of 1 ml per
10 g of body weight in 5 min before testing. The control animals
were treated with equivalent volume of distilled water.
S241
Ac-D-SPRG exhibited no significant influence on production
of conditioned food-procuring site reaction in case of intranasal
injection.
When producing passive avoidance reaction, Ac-D-SPRG had
positive influence on animals’ training capacities. They manifested
in all test parameters in case of dose of 10 mg/kg. As for dose of
1 mg/kg, only increase of duration of animal standing in light part
of maze. Injection of low doses of Ac-D-SPRG seemed to have
no significant effect on parameters.
Ac-D-SPRG positively influenced the production of reaction of
active avoidance. The significant increase of number of performed
reactions was registered in experimental group of rats by the fourth
training day for all concentrations.
Conclusively, the investigated tetrapeptide either improves train-
ing process or has no influence on it. Ac-D-SPRG appeared to
be more effective in training with negative reinforcement. The
stimulating action of peptide on training processes is probably
dependent on character of produced habit and signal initiating
production of conditioned reaction. The “complex maze” is the
least stressful among used tests, the reactions of passive and active
avoidance follow according to increase of stress effect. One cannot
exclude, that stressful conditions of experiment lead to activation
of biogenic amins system which consequently potentiates the
vasopressinergic system. The injection of exogenous peptides may
in its turn alter the condition of both systems thus leading to
corresponding effects.
The results of our experiments obtained demonstrate that Ac-D-
SPRG is neuromodulator of functions of central nervous system,
having direct relation to processes of the perception, the consoli-
dation and the reproduction of memory traces.
P.1.c.012 Design, structural characterisation and
biocompatibility evaluation of tramadol
loaded nanoparticulate systems
L. Tartau1 ° , A. Garlea2 , V. Melnig2 . 1 University of Medicine
and Pharmacy “Gr T Popa”, Department of Pharmacology and
Algesiology Faculty of Medicine, Iasi, Romania; 2 “Al. I. Cuza”
University, Department of Physics − COMB Laboratory Faculty
of Physics, Iasi, Romania
Nanoparticle drug carriers consists of solid biodegradable par-
ticles in size ranging from 10 to 1000 nm in which the active
principle is dissolved, entrapped or encapsulated, and to which
the active principle is absorbed or attached [1]. From all nanopar-
ticulate systems, vesicles presented some advantages represented
by the low number of excipients used in their formulations,
simple procedures for preparation, high physical stability, and the
possibility of sustained drug release [2]. In the literature there
are limited studies reported about the design of tramadol carrier
systems, one of them referring to a formulation containing the
substance in sponges, prepared by freeze drying chitosan solution,
using glutaraldehyde as a cross linking agent [3].
Objective: The objective of this study consists in the obtain-
ing of lipid vesicles coated in polysaccharide chitosan which
entrapped the opioid tramadol inside, and their biocompatibility
determination in mice.
Method: Design of nanoparticulate formulations: The soft
matter vesicles obtained after tramadol immobilization inside lipid
vesicles, were realized by dissolving the lipid in chloroform and
removing the solvent by evaporation which leaded to a dry lipid
film. The film was then hydrated, by adding distilled water with
S242 P.1.c. Basic and clinical neuroscience − Neuropharmacology
tramadol. In the end, the vesicles were stabilized with a 0.5%
biodegradable polymer chitosan solution. The tramadol vesicles
were characterized for size and zeta-potential by Malvern Ze-
tasizer Nano ZS ZEN-3500 model and by Shimadzu UV-1700
PharmaSpec spectrophotometer. Using a Nikon Ti Eclipse optical
microscope the vesicles were directly viewed.
In vivo experiment was carried out on Swiss mice (20−25
g) treated orally with the same volume of solution (0.3 ml):
Group I (control): distilled water; Group II (chitosan vesi-
cles), Group III (TMD-ves): 10 mg/kbw. At different time
points (1 and 6 hours), blood samples were taken from retro-
orbitary plexus to assess blood count, phagocytic capacity of
peripheral neutrophils and serum complement activity, important
elements for biocompatibility evaluation. The data were presented
as +/− SD and significance was analyzed using SPSS for Windows
version 17.0 and ANOVA method. Experimental protocol was
implemented according to recommendations of the Gr.T. Popa
University Committee for Research and Ethical Issues.
Results: We developed new carrier formulations that entrapped
tramadol in lipid vesicles with a moderate stability. The polymer
chitosan binds to vesicles, causing the bilayer to rigidify and in
turn driving a decrease in the size of unilamellar vesicles. The
tramadol vesicles investigated, were found to have a mean zeta
potential of +16.6mV and a mean size of 699nm. Laboratory anal-
ysis showed no significant differences of blood count, phagocytic
capacity of peripheral neutrophils and serum complement activity
values, between chitosan lipid vesicles, tramadol entrapped in
vesicles and control group.
Conclusion: It was demonstrated that these vesicles are capable
to entrap tramadol water solution with a large efficiency. Both
chitosan and tramadol vesicles demonstrated similar immune
responses with control group, after oral administration in mice,
indicative of good in vivo biocompatibility.
Disclosure statement: Acknowledgements: This work was fi-
nancial supported by the Gr.T. Popa University of Medicine and
Pharmacy, Iasi, Romania, Internal Research Grant Nr. 16407/2009
and by PN II Capacities 195CPI/2008.
References
[1] Morrow, K.J., Bawa, R., Wei, C., 2007, Recent advances in basic and
clinical nanomedicine, Med. Clin. North Am., 91 (5), 805–843.
[2] Barauskas, J., Johnsson, M., Tiberg, F., 2005, Self-Assembled Lipid
Superstructures: Beyond Vesicles and Liposomes, Nano Lett., 5 (8),
1615–1619.
[3] Foda, N.H., Ellaithy, H.M., Tadros, M.I., 2004, Optimization of
Biodegradable sponges as controlled release drug matrices. I. Effect
of moisture level on chitosan sponge mechanical properties, Drug
Development and Industrial Pharmacy, 30, (4), 369–379.
P.1.c.013 A7 nicotinic receptor agonist enhances
neurogenesis and reduces brain inflammation
after lipopolysaccharide (LPS) or radiation
treatment
W. Zhao1 , S.M. Toler2 , P.M. Lippiello3 ° , M. Bencherif3 .
1 Wake Forest University, Radiation Oncology, Winston-Salem,
USA; 2 Targacept Inc., Translational Medicine, Winston-Salem,
USA; 3 Targacept Inc., Preclinical Research, Winston-Salem, USA
Purpose: Stem cell therapies may represent the first true disease-
modifying treatments for diverse neurological diseases and in-
juries such as Alzheimer’s disease, Parkinson’s disease, multiple
sclerosis, Huntington’s disease, ALS, spinal cord injury and stroke.
However, many hurdles remain, including not only safety but a
variety of host responses that could hinder efficacy. Adjunct ther-
apies aimed at enhancing progenitor stem cell growth and survival
and suppressing counter-productive inflammatory responses may
provide a useful strategy to optimize the stem cell approach. In this
regard, alpha7 neuronal nicotinic receptors (NNRs) may represent
a unique opportunity. A neuroprotective role for alpha7 NNRs is
supported by numerous in vitro and in vivo studies. For example,
NNR agonists decrease LDH release in hippocampal slices evoked
by oxygen and glucose deprivation in wild-type mice, but fail to
exert neuroprotection in alpha7 knockout mice. Alpha7 agonists
also protect against ethanol-induced cytotoxicity in hippocampal
neurons, possibly by reducing the formation of reactive oxygen
species (ROS) and preventing mitochondrial dysfunction. Thus
progenitor stem cell survival and differentiation in brain following
stem cell therapy may benefit from early intervention with alpha7
agonists to promote pathways implicated in cell survival. In the
present studies a highly selective alpha7 agonist TC-7020 [1]
was used to evaluate the potential benefit of targeting alpha7
pharmacology in models that mimic some aspects of stem cell
graft survival and differentiation.
Methods: Inflammatory Pathways: F344 rats were treated either
with vehicle, 10Gy of whole body irradiation (WBI), 0.3 mg/kg
TC-7020 or 10Gy plus TC-7020. Rats were euthanized and
brains were excised. Cell lysates were collected from brain cortex
and Cox-2, IL-6 and ICAM-1 levels were determined using
Western blotting. Cell Proliferation: F344 rats were treated with
vehicle, TC-7020 (0.3 mg/kg), WBI (10Gy), or 10Gy plus TC-
7020. Rats were euthanized and brains excised at 2 months post-
irradiation. Brain slices were stained with Ki-67 antibody and
Ki-67 positive cells counted in the SGZ of the dentate gyrus.
Microglial Activation: F344 rats were treated with vehicle, TC-
7020 (0.3 mg/kg), WBI (10Gy) or 10Gy plus TC-7020. Rats were
euthanized and brains were excised at 2 months post-irradiation.
Sections of Hippocampus (DG) were stained with CD-68 antibody
and CD-68 positive cells counted. Progenitor Cells: Animals were
labeled with 10% 2 H2 O during drug treatment and hippocampal
progenitor cells were isolated by Percoll fractionation. DNA was
isolated from progenitor cells, hydrolyzed and derivatized for
GC/MS analysis.
Results: TC-7020 prevented radiation-induced reduction in
hippocampal cell proliferation and associated changes in mito-
chondrial respiratory chain proteins. The mechanism appears to
be mediated in part through inhibition of radiation-induced up-
regulation of inflammatory factors, including ICAM-1, COX-2
and IL-6. In addition TC-7020 inhibited radiation-induced gener-
ation of ROS and up-regulation of P47, a protein involved in the
NADPH oxidase pathway. In separate in vivo studies TC-7020
potently enhanced neurogenesis in hippocampus and inhibited
LPS-induced microglial activation.
Conclusions: From these results we conclude that alpha7-
selective ligands such as TC-7020 have beneficial effects on pro-
genitor stem cells and inflammatory pathways that could support
the optimization of stem cell therapies.
Disclosure statement: Drs. Lippiello, Toler and Bencherif are
employees of Targacept, Inc.
References
[1] Marrero, M.B., Lucas, R., Salet, C., Hauser, T.A., Mazurov, A., Lip-
piello, P.M., Bencherif, M., 2010 An alpha7 nicotinic acetylcholine
receptor-selective agonist reduces weight gain and metabolic changes
in a mouse model of diabetes. J Pharmacol Exp Ther 332, 173–180.