JOURNAL OF PATHOLOGY,

MICROBIOLOGY AND IMMUNOLOGY

APMIS 126: 693–699 © 2018 APMIS. Published by John Wiley & Sons Ltd.
DOI 10.1111/apm.12864

Evaluation of the gastrointestinal tract in mdx mice: an

experimental model of Duchenne muscular dystrophy

DAVID FEDER,1 MARIANA IERARDI,1 ANA LAURA COVRE,1 GIULIANA PETRI,1 ALZIRA
ALVES DE SIQUEIRA CARVALHO,2 FERNANDO LUIZ AFFONSO FONSECA3 and BRUNO
MACHADO BERTASSOLI1
1
Pharmacology Department, Faculdade de Medicina do ABC, Santo Andr
e; 2Neurosciences Department,
Faculdade de Medicina do ABC, Santo Andr
e; and 3Clinical Analysis Department, Faculdade de Medicina
do ABC, Santo Andr
e, SP, Brazil

Feder D, Ierardi M, Covre AL, Petri G, Carvalho AAS, Fonseca FLA, Bertassoli BM. Evaluation of the
gastrointestinal tract in mdx mice: an experimental model of Duchenne muscular dystrophy. APMIS 2018; 126:
693–699.
This study describes the functional and morphological alterations in the intestines of mdx mice (n = 4) compared with
the intestinal features of C57BL/10 mice (n = 7) at 2 months of age. The whole gut transit time (carmine red) and the
upper gut transit time (activated charcoal) were measured, and light microscopy was utilized to view stained sections
(H&E and picrosirius red) for histological analysis. No significant difference in mean evacuation time for the whole gut
was observed between the two groups, but a significant delay in activated charcoal passage was observed in the mdx
mice. Visually, a higher concentration of collagen fibers in the submucosal region was apparent in the mdx mice. The
concentration of collagen fibers in the stomach and small intestine suggests a direct relationship with the decrease in
motility of the upper gastrointestinal tract in the mdx mice. Further experimental studies should be conducted to
develop therapeutic alternatives to collagen inhibition to control these manifestations.
Key words: Duchenne muscular dystrophy; gut transit; intestinal motility; mdx mice; muscular dystrophy.
David Feder, Pharmacology Department, Faculdade de Medicina do ABC, Avenida Lauro Gomes, 2000 - Vila Saca-
dura Cabral – Santo Andr
e/SP. CEP: 09060-780; Brazil. e-mail: feder2005@gmail.com

Duchenne muscular dystrophy (DMD) is the most
common hereditary myopathy in childhood, affecting
one in every 5000 males at birth (1). It occurs due to a
mutation in the gene that encodes dystrophin, which
is the second largest protein in the human body and
occurs in the sarcolemma. Dystrophin is located on
the cytoplasmic face of skeletal and cardiac muscle
membrane and constitutes approximately 5% of sar-
colemmal cytoskeletal protein (2, 3).
In all stages of the disease, the extracellular
matrix is overly increased due to the excess deposi-
tion of fibronectin, laminin, and collagen IV. In
addition, in regenerated fibers, there is an accumu-
lation of reticular fibers and collagen I and III (2).
DMD patients show decreased or no dystrophin
protein expression, which leads to reduced connec-
tions with the dystrophin-associated protein com-
plex. As a result, disorders and/or destruction of the
cytoskeleton and extracellular matrix occur, leading
Received 7 March 2018. Accepted 6 June 2018
to the rupture and necrosis of muscle fibers (4).
DMD patients have difficulty swallowing in the first
decades of life, a problem that tends to worsen with
age (5).
The exacerbation of swallowing difficulties con-
tributes to the broader dysphagia framework in
patients with DMD. One outcome is eating disor-
ders due to structural and neurological disorders
related to the complex process of swallowing and
the decreased strength of masticatory muscles (6).
DMD also compromises the function of the gas-
trointestinal tract (GIT), mainly by affecting the
smooth muscles of the wall, and it causes serious
motility disorders (7).
Patients with DMD experience progressive mus-
cle wasting and abnormal digestive tract motility,
including protracted diarrhea or constipation. Stud-
ies of colon segments from an animal model of
DMD, the muscular dystrophy (mdx) mouse, have
demonstrated the frequent generation of abnormal
colonic contraction (8).

693
 FEDER et al.
Some researchers have found little correlation
between the degree of skeletal muscle involvement
and the presence or severity of gastrointestinal dis-
turbance. However, other authors have reported a
positive correlation between the duration of the
skeletal muscle disease and gastrointestinal distur-
bance (9). In some patients, the impairment of gas-
trointestinal function occurs sufficiently gradually
that they adapt to it with little or no awareness of
disturbance, and only a thorough anamnesis may
elicit the recall of possible symptoms (9).
Several studies with dystrophic patients and ani-
mal models have been performed with focus on the
stomach (11–13), small intestine (4, 14) and large
intestine (15) and describe the changes in the func-
tions of these GIT segments that occur in DMD.
The number of experimental models for studying
DMD is high, exceeding 60 (1). Stem cell models
and non-mammalian models also have roles in
understanding disease pathogenesis and the effects
of treatments on disease pathophysiology. While
each model has its limitations, the following model
systems have significantly contributed to our under-
standing of DMD (16).
Among natural rodent and canine animal models
of DMD, the mdx mouse model is the most com-
monly studied (1, 17). The mdx (X chromosome-
linked muscular dystrophy) mouse originated from
spontaneous mutation of the gene (18). This model
is widely used because it is not only genetically uni-
form but also easy to handle and breed (13, 19).
Despite the histological alterations found in
this model, similar to human muscle, the disease
has a milder phenotype and is not lethal to the
model (1, 20). From day 18 after birth, muscle
necrosis can be observed along with high levels
of inflammatory infiltrates in the muscle, which
is followed by rapid muscle regeneration after
the 5th week (1).
In the later stages of the disease, the skeletal, car-
diac and smooth muscle fibers are replaced by fat
and fibrous tissue (16). Clinical descriptions of the
disorder focus principally on skeletal and cardiac
muscle degeneration; however, gastrointestinal
function in DMD has not been rigorously studied
(11). Furthermore, there are few studies of dys-
trophic mice that have examined the functioning of
smooth muscle in the GIT and the absorption of
nutrients (8, 12, 14, 15). Therefore, a detailed anal-
ysis of the intestinal tract in relation to motility
and morphological alterations is of utmost impor-
tance. Our aims were to evaluate the impacts of
increased collagen on the GIT, describe the abnor-
malities of mdx mice, and compare gastrointestinal
features between mdx and C57BL/10 mice.
694
MATERIALS AND METHODS
Animals
Two-month-old male wild-type (C57BL/10) and mdx mice
(C57BL/10 mdx) were purchased from the Animal’s
House of the Biomedical Sciences Institute of S~ ao Paulo
University. During the study, the mice were weighed,
housed in polypropylene boxes (five animals per box)
under controlled temperature (24  2 °C) and lighting
(12 h light/12 h dark) conditions and fed ration and water
ad libitum. All protocols were approved by the Research
Ethics Committee of the Faculdade de Medicina do ABC
(Santo Andr
e, SP, Brazil).
The animals were divided into two groups: a wild-type
group (n = 4) and an mdx group (n = 7).
Total gastrointestinal transit time
Carmine red, which cannot be absorbed from the lumen of
the gut, was used to study total GI transit time (32). A solu-
tion of carmine red (300 lL; 6%; Sigma-Aldrich, Germany)
suspended in 0.5% methylcellulose (Sigma-Aldrich, Ger-
many) was administered by gavage. The time at which gav-
age took place was recorded as T0. After gavage, each
animal was placed in an individual cage, and fecal pellets
were monitored at 10-minute intervals for the presence of
carmine red. Total GI transit time was considered as the
interval between T0 and the time of the first observance of
carmine red in stool.
Upper gut transit test
After 1 week, a different marker was used to test the same
group of animals: activated charcoal 10% in 5% gum ara-
bic solution, 0.5 mL/animal through gavage.
After 45 min, the animals were intraperitoneally
euthanized using an anesthetic association of ketamine
(50 mg/kg) and xylazine (2 mg/kg). Immediately there-
after, the intestine was removed, from the pylorus to the
beginning of the cecum. The total length of the intestine
and the distance traveled by the active charcoal suspension
were measured. The results were expressed in percentage
of total intestine length.
Histological analysis
Each tissue type was sampled at the same site for all of
the animals, thereby standardizing for comparison. Sam-
ples from stomach and small and large intestine were fixed
in 4% paraformaldehyde (PFA) for 48 h at room temper-
ature. After dehydration in a graded ethanol series, the
samples were diaphanized in xylol and embedded in paraf-
fin. Five-micrometer serial sections (Leica RM 2065) were
obtained at different tissue depths and stained with Harris
Hematoxylin & Eosin (H&E) and picrosirius red stains.
The latter samples were analyzed under polarized light to
classify the different types of collagen (orange type I, yel-
low type II, or green type IV) in all of the stomach and
intestine layers for later comparison between the mdx and
control groups. Slides were viewed by optic microscopy
using a BX41 Olympus microscope (Olympus, America
© 2018 APMIS. Published by John Wiley & Sons Ltd
 INTESTINAL MOTILITY IN MDX MICE

Inc., Melville, NY, USA) equipped with an Olympus
Q-Color 3 digital camera for image capture.
Statistical analysis
The experimental results were expressed as the mean 
standard deviation. Data were analyzed with Student
t-tests using a statistical software package (GraphPad
Prism statistical software; San Diego, CA, USA) and
presented as the means  SEM. (p < 0.05 was considered
statistically significant.). All of the analyses were per-
formed blindly. The researchers who treated the animals
differed from the researchers who analyzed the data.
RESULTS
The carmine marker (3 g carmine in 50 mL of
0.5% methylcellulose) did not cause diarrhea in any
of the mice. The red-stained stool samples appeared
similar to the normal samples. The mean time until
excretion (whole gut transit time) was approxi-
mately 105 min (27.5) in the mdx group and 112
(29.5) minutes in the C57BL/10 group. No signifi-
cant difference was observed between the two
groups (Fig. 1).
However, 1 week following the previous experi-
ment, the upper gut transit test using activated
charcoal revealed a significant decrease (p < 0.05)
in intestinal motility in mdx mice (15.2) relative
to that in C57BL/10 mice (10.3) (Fig. 2).
After histological processing, light microscopy
revealed that all of the structures (layers) of the
stomach and intestine were preserved in both groups.
The amounts of the three types of collagen were
evaluated in the dystrophic animals (Figs 3A–C;
4A–C; and 5A–C) and control animals (Figs 3D–F;

Fig. 1. Whole gut transit time. (A) Schematic of the time to gastric/intestinal emptying. (B) Mean time in minutes of gut
transit in wild-type (C57BL10) and dystrophic (mdx) mice. Values are the mean  SEM of n = 10 animals (p < 0.05).

Fig. 2. Percentage of distance traveled by the activate charcoal in the intestine. (A) Schematic of the distance traveled in
the intestine. (B) Mean distance traveled (as a percentage) of charcoal in the intestine of wild-type (C57BL10) and dys-
trophic (mdx) mice. Values are the mean  SEM of n = 10 animals. *p < 0.05

© 2018 APMIS. Published by John Wiley & Sons Ltd 695

 FEDER et al.

Fig. 3. Histological analysis of the stomach layers of mdx and C57BL10 mice. (A, B, and C) mdx mice; (D, E, and F)
C57BL10 mice. Arrows show the submucosal layer. Stain: picrosirius red/polarized light. Bar: 100 lm.

Fig. 4. Histological analysis of the upper intestine layers of mdx and C57BL10 mice. (A, B, and C) mdx mice; (D, E, and
F) C57BL10 mice. Arrows show the submucosal layer. Stain: picrosirius red/polarized light. Bar: 100 lm.

4D–F; and 5D–F). Visually, a thicker layer of colla- of the mdx mice, the collagen fibers were concen-
gen was apparent in the mdx mice in the stomach trated in the region of the submucosa (Figs 3B; 4B;
and the large and small intestine. In addition, in all and 5B).

696 © 2018 APMIS. Published by John Wiley & Sons Ltd

 INTESTINAL MOTILITY IN MDX MICE
Fig. 5. Histological analysis of the lower intestine layers of mdx and C57BL10 mice. (A, B, and C) mdx mice; (D, E, and
F) C57BL10 mice. Arrows show the submucosal layer. Stain: picrosirius red/polarized light. Bar: 100 lm.
DISCUSSION
This study provides evidence that mdx mice show
alterations in gastrointestinal motility, with a signif-
icant delay in small intestinal transit relative to that
in control mice. The results suggest that the loss of
dystrophin and the increased collagen in the sub-
mucosal tissue have important in vivo effects on
intestinal motility.
Different studies have shown that activated char-
coal (23) and carmine dye (24) are effective markers
for gut motility analysis (25).
In this study, the first method used to observe
gut activity used carmine red as a marker to mea-
sure whole gut transit time. With this approach,
activity (time) is determined by the oral administra-
tion of a non-absorbable marker such as carmine
red and subsequent monitoring for the first appear-
ance of the marker in stool. This method is widely
used to evaluate gut activity (21, 22, 24). The sec-
ond method used active charcoal as marker to
observe intestinal transit time. With this approach,
activity is determined by the distance traveled by
the marker in the small intestine of a treatment
group relative to the distance traveled in one or
more other groups. This method is also widely used
to evaluate gut activity (23, 25). Here, both meth-
ods (carmine red and active charcoal) were applied
to the same animals. Therefore, we used a smaller
© 2018 APMIS. Published by John Wiley & Sons Ltd
number of animals and obtained more accurate
data in the current study.
The results showed no difference in whole gut tran-
sit (carmine red method) between the two groups;
however, progression in the upper intestinal transit
(activated charcoal method) was delayed in the mdx
mice when compared with that in the WT animals
(C57BL10). These results indicate that DMD affects
smooth muscle. Some clinical trials have shown that
gastrointestinal manifestations, such as distension,
satiety sensation and gastric emptying, are delayed in
DMD (10, 11). Different clinical manifestations,
including gastric dilatation and intestinal pseudo-
obstruction, have been reported in DMD patients,
indicating a direct or indirect association with the
lack of dystrophin in smooth muscle (11, 26).
In a study of the stomach of mdx mice, Bertassoli
et al. (13) described a high concentration of collagen
fibers in the submucosal region of the stomach (fun-
dic portion) that appeared as an enlarged interglan-
dular space in the glandular region. In our study,
these collagen fibers were observed in both groups
(C57BL10 and mdx mice); however, in the mdx mice,
the concentration of these fibers was higher, consis-
tent with Bertassoli et al. (13). The observed fibrosis
might be the cause of the observed decreased motility
of the upper GIT.
The presence of fibrosis in our samples suggests
that fibrosis might explain the motility problems
697
 FEDER et al.
observed in DMD patients, as we observed such
problems in our animals. It is known that mdx mice
have a mild DMD phenotype. Life expectancy is
reduced by 25% in mdx mice, whereas it is reduced
by approximately 75% in human DMD patients (1,
20). Therefore, in addition to the immobility and
weakness of the abdominal wall muscles, impair-
ment of the smooth muscle of the colon might help
explain the high frequency of constipation reported
in DMD patients (12, 15).
Severe and even fatal cases of acute gastric dilata-
tion and pseudo-intestinal obstruction have been
associated with histological evidence of smooth mus-
cle fibrosis in the whole GIT (9, 10, 27), as we found
in our samples. Some DMD patients suffer from
constipation; however, such patients have not been
adequately studied in relation to colonic motility.
This study showed a decrease in upper intestinal
motility in a mouse model of DMD.
However, some experimental studies have related
the impairment in intestinal motility in mdx mice to
alterations in the production/release of nitric oxide
(NO) (28). In addition to being produced by smooth
muscle cells in the stomach of mammals (29), NO is
considered the main inhibitory neurotransmitter
released from nonadrenergic noncholinergic
(NANC) nerve fibers of the smooth muscle, and it is
a mediator of gastrointestinal relaxation (30).
In the current study, no significant difference in
mean evacuation time was observed between dys-
trophic mice and control mice. However, there was
a higher concentration of collagen fibers in the sub-
mucosal region of the stomach and small intestine
in the mdx group than in the control group, sug-
gesting that the accumulation of collagen fibers
might be responsible for the decreased motility
levels of the whole gut tract in mdx mice.
Therefore, these results corroborate with Alves
et al. (4), evidencing that the protein dystrophin
plays an important role in the preservation of both
the microstructure and ultrastructure of mice intes-
tine, while exerting a minor but important role con-
cerning the intestinal contractile responsiveness and
calcium handling.
The findings here reported increase our under-
standing of the mechanism of intestinal dysfunction
in DMD. Due to the relevance of intestinal abnor-
malities in DMD patients, further experimental
studies should be conducted to identify therapeutic
alternatives for collagen inhibition to control these
manifestations.
CONFLICT OF INTEREST
We declare that there is no conflict of interest.
698
REFERENCES
1. Mcgreevy JW, Hakim CH, Mcintosh MA, Duan D.
Animal models of Duchenne muscular dystrophy:
from basic mechanisms to gene therapy. Dis Model
Mech 2015;8:195–213.
2. Lessa TB, Carvalho RC, Franciolli ALR, Oliveira LJ,
Barreto RS, Feder D, et al. Muscle reorganisation
through local injection of stem cells in the diaphragm
of mdx mice. Acta Vet Scand 2012;54:1–7.
3. Lessa TB, Abreu DK, Bertassoli BM, Ambrosio CE.
Arquitetura comparativa dos pulm~ oes de camundon-
gos normais e afetados pela Distrofia Muscular de
Duchenne. Pesq Vet Bras 2015;35:56–60.
4. Alves GA, Silva LR, Rosa EF, Aboulafia J, Freymuller-
Haapalainen E, Souccar C, et al. Intestine of dystrophic
mice presents enhanced contractile resistance to stretch-
ing despite morphological impairment. Am J Physiol
Gastrointest Liver Physiol 2014;306:191–9.
5. Pane M, Vasta I, Messina S, Sorleti D, Aloysius A,
Sciarra F, et al. Feeding problems and weight gain in
Duchenne muscular dystrophy. Eur J Paediatr Neurol
2006;10:231–6.
6. Lisboa MJS, De Oliveira Lima MF, Stabille SR,
Zanoni JN, Gagliardo KM, Souto MS, et al. Supple-
mentation action with ascorbic acid in the morphology
of the muscular layer and reactive acetylcholinesterase
neurons of ileum of mdx mice. Auton Neurosci
2017;205:57–66.
7. Abdel-Salam E, Abdel-Meguid I, Korraa SS. Markers
of degeneration and regeneration in Duchenne muscu-
lar dystrophy. Acta Myol 2009;28:94–100.
8. Tameyasu T, Ogura S, Ogihara K. The effect of e-, i-,
and n-nitric oxide synthase inhibition on colonic
motility in normal and muscular dystrophy (mdx)
mice. Jpn J Physiol 2004;6:555–66.
9. Bellini M, Biagi S, Stasi C, Costa F, Mumolo MG,
Ricchiuti A, et al. Gastrointestinal manifestations in
myotonic muscular dystrophy. World J Gastroenterol
2006;12:1821–8.
10. Bellini M, Alduini P, Costa F, Tosetti C, Pasquali L,
Pucciani F, et al. Gastric emptying in myotonic dys-
trophic patients. Dig Liver Dis 2002;34:484–8.
11. Borrelli O, Salvia G, Mancini V, Santoro L, Tagliente
F. Evolution of gastric electrical features and gastric
emptying in children with Duchenne and Becker mus-
cular dystrophy. Am J Gastroenterol 2005;100:695–
702.
12. Mule F, Zizzo MG, Amato A, Feo S, Serio R. Evi-
dence for a role of inducible nitric oxide synthase in
gastric relaxation of mdx mice. Neurogastroenterol
Motil 2006;18:446–54.
13. Bertassoli BM, Lessa TB, Santos AC, Oliveira DM,
Feder D, Assis Neto AC. Morphological analysis of
the stomach of dystrophic mice ‘mdx’. R Elect Vet
2013;14:1–9.
14. Zizzo MG, Mule F, Serio R. Duodenal contractile
activity in dystrophic (mdx) mice: reduction of nitric
oxide influence. Neurogastroenterol Motil 2003;15:
559–65.
15. Mule F, Amato A, Serio R. Gastric emptying, small
intestinal transit and fecal output in dystrophic (mdx)
mice. J Physiol Sci 2010;60:75–9.
© 2018 APMIS. Published by John Wiley & Sons Ltd
 INTESTINAL MOTILITY IN MDX MICE
16. Wilson K, Faelan C, Patterson-Kane JC, Rudmann
DG, Moore SA, Frank D, et al. Duchenne and
Becker muscular dystrophies: a review of animal mod-
els, clinical end points, and biomarker quantification.
Toxicol Pathol 2017;45:1–16.
17. Sch€afer BT, Silveira MP, Palombit K, Mendes CE,
Watanabe IS, Miglino MA, et al. Morphological char-
acterization of the myenteric plexus of the ileum and
distal colon of dogs affected by muscular dystrophy.
Anat Rec (Hoboken) 2017;301:673–85.
18. Feder D, Rugollini M, Santomauro A Jr, Oliveira LP,
Lioi VP, Dos Santos R, et al. Erythropoietin reduces
the expression of myostatin in mdx dystrophic mice.
Braz J Med Biol Res 2014;47:966–71.
19. Oliveira DM, Santos AC, Bertassoli BM, Viana DC,
Prado AAF, Assis Neto AC. Comparative study of
the kidneys from dystrophic mice. J Morphol Sci
2013;30:186–90.
20. Chamberlain JS, Metzger J, Reyes M, Townsend D,
Faulkner JA. Dystrophin-deficient mdx mice display
a reduced life span and are susceptible to sponta-
neous rhabdomyosarcoma. FASEB J 2007;21:
2195–204.
21. Vannucchi MG, Garella R, Cipriani G, Bcari MC.
Relaxin counteracts the altered gastric motility of dys-
trophic (mdx) mice: functional and immunohistochem-
ical evidence for the involvemen of nitric oxide. Am J
Physiol Endoncrinol Metab 2011;300:380–91.
22. Kimball ES, Palmer JM, D’Andrea MR, Hornby PJ,
Wade PR. Acute colitis induction by oil of mustard
results in later development of an IBS-like accelerated
upper GI transit in mice. Am J Physiol Gastrointest
Liver Physiol 2005;288:1266–73.
© 2018 APMIS. Published by John Wiley & Sons Ltd
23. Paganotte DM, Sannomiya M, Rinaldo D, Vilegas W,
Salgado RN. Operculina macrocarpa: chemical and
intestinal motility effect in mice. Rev Bras Farmocog
2016;26:427–32.
24. Li Z, Chalazonitis A, Huang Y, Mann JJ, Margolis
KG, Yang QM, et al. Essential roles of enteric neu-
ronal serotonin in gastrointestinal motility and the
development/survival of enteric dopaminergic neurons.
J Neurosci 2011;31:8998–9009.
25. Figueiredo EM, Michelin DC, Sannomiya M, Silva
MA, Santos LC, Almeida LFR, et al. Avaliacß~ ao
qu
ımica e da atividade antidiarr
eica das folhas de Byr-
sonima cinera DC (Malpighiaceae). Braz J Pharmacol
Sci 2005;41:80–3.
26. Byers T, Kunkel L, Watkins S. The subcellular distri-
bution of dystrophin in mouse skeletal, cardiac, and
smooth muscle. J Cell Biol 1991;115:411–21.
27. Nowak TV, Ionasescu V, Anuras S. Gastrointestinal
manifestations of the muscular dystrophies. Gastroen-
terology 1982;82:800–10.
28. Baccari MC, Nistri S, Vannucchi MG, Calamai F,
Bani D. Reversal by relaxin of altered ileal sponta-
neous contractions in dystrophic (mdx) mice through
a nitric oxide-mediated mechanism. Am J Physiol
Regul Integr Comp Physiol 2007;293:662–8.
29. Grider JR, Murthy KS, Jin JG, Makhlouf GM. Stim-
ulation of nitric oxide from smooth muscle cells by
VIP: prejunctional enhancement of VIP release. Am J
Physiol 1992;262:774–8.
30. Rand MJ. Nitrergic transmission: nitric oxide as mediator
of non-adrenergic, non-cholinergic neuro-effector trans-
mission. Clin Exp Pharmacol Physiol 1992;19:147–69.
699