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Phenotype of dystrophinopathy in old MDX mice.

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Phenotype of Dystrophinopathy in Old MDX Mice
Laboratoire de Physiologie (J.P.L., A.S.), Facult6 de Mkdecine Saint-Antoine, Paris,
France; and Department of Histopathology (C.P.), Charing Cross and Westminster Medical
School, London, United Kingdom
Background: Mdx mutant mice, like patients with Duchenne Muscular Dystrophy (DMD), lack dystrophin, a subsarcolemmal
protein, that results in myofiber necrosis. However young mdx mice, in
contrast to DMD children, exhibit a successful muscle regeneration and not
an extensive fibrosis.
Methods: Old mdx mice were monitored clinically up to their spontaneous
death, and most of their organs were studied histologically to look for differences with those of the wild C57BL/10 mice strain.
Results: In old mdx mice (at least 20 months of age), we report clinical and
pathological features of muscular dystrophy, i.e., progressive motor weakness and loss of myofibers replaced by extensive connective tissue, similar
to the phenotype of dystrophinopathy observed in DMD patients. Various
degrees of dystrophic involvement were observed in cardiac, respiratory,
postural, and hindlimb skeletal mdx muscles and also in smooth muscles of
the digestive and urinary tracts. No gross histological abnormalities were
found in other tissue than muscular tissue.
Conclusions: Late in life, mdx mice develop a muscular dystrophy close to
DMD dystrophinopathy. We suggest that the study of the effects of ageing
in mdx mice would give clues to better understand the pathophysiology of
DMD. o 1995 Wiley-Liss, Inc.
Key words: mdx mice, Duchenne muscular dystrophy, Dystrophinopathy,
Smooth muscle, Fibrosis, Ageing
The rndx mutant was identified from a colony of
C57BL/lOScSn inbred mice a s a murine X-linked muscular dystrophy (Bulfield et al., 1984). Further genetic
studies showed that the affected gene in rndx mice was
homologous with that of patients suffering from Duchenne Muscular Dystrophy (DMD) (Sicinski et al.,
1989). Like DMD patients, m d x mice lack dystrophin, a
membrane-associated cytoskeletal protein of the skeletal muscle sarcolemma (Hoffmann et al., 1987). The
absence of dystrophin is likely to reduce the stability of
muscle plasma membrane (Menke and Jockush, 1991)
which may result in a n increase of intracellular free
calcium levels (Turner et al., 1991) and segmental necrosis of the muscle fibers (Weller et al., 1990). However, there is a discrepancy between DMD patients and
rndx mice. On one hand, DMD children exhibit muscular dystrophy (dystrophinopathy), defined as a clinical
motor deficit resulting from a pattern of myofiber degeneration and fibrosis at muscle biopsy (Bell and
Conen, 1968). On the other hand, young and adult rndx
mice do not show any skeletal muscle weakness and
present histological aspects of successful muscle regeneration (Tanabe et al., 1986; Coulton e t al., 1988; Cooper, 1989). Thus, different authors regarded rndx mice
as a model of spontaneous muscle regeneration (Dangain and Vrbova, 1984; Anderson et al., 1987, 1988)
rather than a model of DMD, considering that myofiber
loss, fibrosis and clinical motor deficit do not occur in
these mutants (Weller et al., 1991).
Stedman et al. (1991) discovered dystrophic features
in the diaphragm of 16-month-old m d x mice, highlighting the effects of ageing in rndx muscle. Later, myopathic changes not only in old but also in adult m d x
diaphragm were confirmed (Dupont-Versteegden and
McCarter, 1992; Louboutin et al., 1993). Data are more
controversial concerning the dystrophic involvement of
m d x cardiac muscle. Some authors found extensive
muscle necrosis or fibrosis in m d x myocardium
(Bridges, 1986; Sicinski and Barnard, 1990), but others
did not (Tanabe et al., 1986; Torres and Duchen, 1987).
Extensive fibrosis in skeletal muscle was first observable in a figure of proximal hindlimb muscle cross section in a 18-month-old m d x mice, published by Hoffman and Kunkel(19891, who did not report any clinical
weakness. The aim of this study is to describe in relation to clinical signs the histopathological features observed in most tissues of older mdx mice up to their
spontaneous death.
Received October 13, 1994; accepted January 3, 1995.
Address reprint requests to Jean-Pascal Lefaucheur, Laboratoire de
Physiologie, Faculte de MBdecine Saint-Antoine, 27 rue Chaligny,
7551 Paris Cedex 12, France.
--u- mdx mice
C57BL/lO mice
Fig. 1. Percentages of surviving mdx mice ( n = 14) and age-matched C57BW10 mice from 18 to 24
months of age.
Breeding colonies of rndx and wild C57BL/10 mice
were maintained since 1988 in our laboratory, with
commercial diet, tap water ad libitum, and natural
light cycle. The life span of rndx mice (n = 14) and agematched C57BL/10 mice (n = 13) was monitored from
18 months of age up t o their spontaneous death. In
other groups of 23-month-old rndx mice (n = 4) and agematched C57BL/10 mice (n = 4), the following muscles
were removed under anesthesia (chloral hydrate 3.5%,
1.5 m1/100 g body weight, injected intraperitoneally):
quadriceps, tibialis anterior, extensor digitorum longus, gastrocnemius, soleus, plantaris, diaphragm, sternomastoid, and masseter. The samples were mounted
in a cork piece with tragacanth gum, frozen in isopentane cooled in liquid nitrogen, and stored at -80°C.
After sacrifice, the whole neck and a piece of chest
wall were removed, decalcified, and paraffin-embedded. The following organs were also removed and
either frozen or paraffin-embedded: eye, brain, tongue,
lung, heart, aorta, liver, spleen, pancreas, esophagus,
stomach, duodenum, kidney, suprarenal glands, ovary,
vagina, uterus, and bladder. In all the cases, 10 pm
thick sections were cut, stained with standard procedures, and observed with conventional light microscope.
To quantify rndx smooth muscfe abnormalities, sections were cut every 100 pm all along the stomach and
the bladder in the four animals of each group. Sections
were matched between rndx and C57BL/10 mice on the
basis of the level of the section and the size of the lumen area. One slide was selected at five levels distributed along each organ for each animal. On magnified
projection of each selected slide, the thickness of the
smooth muscle layers was measured at four cardinal
points, and these four data were summed. The respective sum values of rndx and C57BL/10 groups (five levels, four animals) were compared by a Mann-Whitney
test for each organ.
Clinical Features
The lifetime of mdx mice was greatly reduced (Fig.
1). In our colony, all rndx mice died before 24 months of
age, while about half of the C57BW10 mice were still
alive and active. Skeletal muscle weakness was not
observed in mdx mice up to 18 months of age. Thereafter, rndx mice reduced by steps their peregrination
and exhibited progressively an extensive motor deficit.
Figure 2 shows symptomatic posture with dropped
head, lumbar kyphosis, and hindlimb flexion. These
mice were unable to straighten up their head, hindering them from feeding, unless cube diet and water were
at the level of the sawdust. Despite gross thoracic deformity (X-ray examination on Fig. 2), older mdx mice
did not seem to exhibit severe respiratory failure, up t o
the few days preceding their death. The body weight of
23-month-old rndx mice (mean 2s.d.: 13.3 g k1.9) was
59%lighter compared to that of age-matched C57BL/10
mice (mean 2s.d.: 32.3 g k3.5). As shown in Table 1,a
loss of weight was also found in hindlimb muscles (52%
to 57% in tibialis anterior, extensor digitorum longus,
soleus, and plantaris), with more pronounced reduction
in larger muscles (66% to 70% in quadriceps and gastrocnemius).
Histopathological Features
Eye, brain, lung, liver, spleen, pancreas, kidney, suprarenal glands, and ovary in old m& did not show
macroscopic o r microscopic differences compared to the
organs of old C57BL/10 mice, in contrast to smooth or
striated muscle tissue and myocardium.
In old mdx mice, both the circular and the longitudinal smooth muscle layers in the urinary tract and the
circular smooth muscle layer in the digestive tract appeared thinned, and extensive connective tissue was
observed between the muscularis mucosae and the circular smooth muscle layer (Fig. 3). Smooth muscle
thickness was significantly reduced in rndx bladder
Fig. 2.23-month-old mdz mice photograph (left) and body X-rays (right).
TABLE 1. Hindlimb muscle weights (mg, mean
C57BU10 mice
m.dx mice
Tibialis anterior
45.7 2 5.3
20.1 0.7
s.dJ of 23-month-old C57BL/lO (n= 4) and m&
digitorum longus
10.5 1.1
4.5 0.3
Fig. 3. Histopathological aspects of the stomach and bladder walls
respectively in 23-month-old C57BLi10 (A, C? and mdx (B, D) mice.
Note the thinning of the circular smooth muscle layer (star) in mdx
stomach (B) and of both the circular (star? and longitudinal (double
11.9 2 0.5
5.7 k 0.7
13.1 2 0.8
6.1 2 0.5
227.1 ? 18.0
68.2 2 6.3
mice (n = 4)
158.1 11.9
53.8 9.7
star) smooth muscle layers in rndx bladder (D). An extensive connective tissue (arrow) can be observed between the muscularis mucosa
and the circular smooth muscle layer in both mdx organs (B, D?.
Haematoxylin and eosin stained cross-cryosections (A-D, x 250).
Fig. 4. Histopathological aspects of the muscular tissue in the heart, the tongue, and the esophagus
respectively in 23-month-old C57BL/10(A, C , E) and mdx (B, D, F)mice. Muscle fibrosis is advanced in
mdx myocardium and tongue (B, D), but less pronounced in mdz esophagus (F).Haematoxylin and eosin
stained cryosections (A-D, x 100; E,F, x 250).
(P=0.028), but not in rndx stomach wall (P=0.474). phragm (Fig. 51, and sternomastoid muscles, but less
Such differences were not observed in vessel walls, pronounced in masseter (data not shown). Hindlimb
skeletal muscles were also dystrophic, with histological
uterus, and vagina.
Foci of inflammation and fibrosis were found in mdx features similar to those observed in DMD patients
muscular tissue of the heart and the tongue (Fig. 4). (Dubowitz, 1985): pronounced fibrosis in perimysium
Striated muscle layers of rndx esophagus were disorga- and endomysium with moderate inflammatory cell innized, showing a wide range of myofiber size, and dis- filtrates, increased variability in the size of the muscle
crete fibrosis developed (Fig. 4). Signs of dystrophy, i.e., fibers due to the presence of atrophic, splitted, or hymost atrophic muscle fibers surrounded by extensive pertrophied myofibers (Fig. 51, and even changes in
fibrosis, were severe in intercostal, paraspinal, dia- myofiber architecture as giant conglomerates of
Fig. 5. Histopathological aspects of intercostal (A), paraspinal (B),
diaphragm (C), tibialis anterior (D), extensor digitorum longus (El,
soleus (F), gastrocnemius (GI, and quadriceps (H) muscles in 23month-old mclx mice. Note the extensive fibrosis (arrow) in respira-
tory or postural muscles (A, B, C, F) and the wide range of myofiber
sizes in larger muscles (G, HI. Haematoxylin and eosin stained cross
sections of paraffin-embedded (A, B) or frozen (C-H) tissues (A, B
X 100;C, D X 250; E-H, x 400).
whorled fibers. In contrast with DMD muscle, adipose
tissue scarcely developed in m d x muscle. Foci of myofiber necrosis or phagocytosis and foci of small-diameter regenerating centronucleated fibers were constantly observed in m d x mice up to 20 months of age,
but became very rare in older animals. The signs of
dystrophy in hindlimb muscles were more pronounced
in the soleus and the larger muscles (i.e., quadriceps
and gastrocnemius muscles), and less pronounced in
the extensor digitorum longus and the plantaris.
The absence of clinical or histological signs of muscular dystrophy (i.e., motor weakness, muscle atrophy
with extensive fibrosis) in young rndx mice, although
they lack dystrophin like DMD patients, was initially a
source of questioning. However, evidence for extensive
fibrosis in adult rndx diaphragm suggested that ageing
or work could play a role in the development of dystrophic features (Stedman et al., 1991; Dupont-Versteegden and McCarter, 1992; Louboutin et al., 1993). In old
mdx mice, we appraised a similar involvement in
smooth and skeletal muscles, leading to clinical muscle
weakness and premature death. These mice exhibited a
symptomatic posture probably related to the weakness
of respiratory, paraspinal, and hindlimb muscles. Their
death may result from either respiratory failure, heart
attack, or nutritional deficiency. We keep in mind that
our rndx colony, which is now probably inbred, could
present any particularity enhancing the development
of muscular dystrophy. For instance, a de novo gene
mutation may have occurred in the germ line of our
Dystrophin was localized in smooth muscle fiber
membrane in various normal murine organs (Miyatake
et al., 1989), particularly in mouse stomach (Hoffman
et al., 1987), and was absent in mdx smooth muscles
(Miyatake et al., 1989). This probably explains the
marked differences in smooth muscles of the urinary
and digestive tracts between old rndx and C57BL/10
mice. Interestingly, the rndx stomach wall exhibited
thinned muscle layers with extensive connective tissue
as it was previously described in DMD (Nowak et al.,
1982; Barohn et al., 1988).Smooth muscle observations
are poorly documented in other DMD organs, but the
significant abnormalities in rndx bladder lead us to
propose that a pathology of dystrophin-deficient
smooth muscles exists and remains to be delineated.
In each striated rndx muscles, the severity of dystrophic changes was unequally advanced, although dystrophin deficiency must affect all muscles (Hoffman et
al., 1988; Hoffman and Kunkel, 1989). Necrosis in dystrophin-deficient muscle was suggested to result from
focal breakings in sarcolemma induced by contractile
activity (Weller et al., 1990; Stedman et al., 1991; Dick
and Vrbova, 1993; Moens et al., 1993; Petrof et al.,
1993). This hypothesis may explain that dystrophic
features were predominant in muscles with postural
activity (e.g., soleus, sternomastoid, or paraspinal muscles), or with an obligatory ceaseless work (e.g., diaphragm or intercostal muscles). Moreover differences
in the pathological timetable between DMD and rndx
may relate to the fact that mouse muscle fibers are
short and poorly branched, whereas human muscle fibers are long and split. In the latter case the tension of
the sarcolemma should be more important in the transmission of contractile force and damage may occur
early during muscle activity. However, heart and
esophagus seemed to be less involved, but the organization of muscular tissue is different in these organs
and in skeletal muscles. In addition, small muscles
with almost exclusive fast twitch myofibers (e.g., extensor digitorum longus and plantaris muscles) seemed
to be less involved than larger muscles with mixed
types of myofibers (e.g., quadriceps and gastrocnemius
muscles), as in DMD dystrophinopathy (Hoffman and
Kunkel, 1989). Interestingly, fibrosis occurs later in
the life of rndx mice than DMD patients. Since the
mechanisms of muscle fibrosis development remain hypothetical in DMD dystrophinopathy and could result
either from an active fibrotic process, or from a decrease in the regenerating capacity or in the available
number of muscle precursor cells, rndx mice will be
useful to discover the methods by which fibrosis onset
can be delayed.
The rndx mutant mouse appears to be a closer model
of the DMD dystrophinopathy than it was previously
appreciated. Better knowledge of old rndx mice would
offer new perspectives to approach some questions
about the pathophysiology of DMD which remain unsolved.
We are grateful to Mrs. Claude Guernier and Dr.
Nicole Maurin (Laboratoire d'Histologie, Faculte de
Medecine Saint-Antoine, Paris) for their help in paraffin methods, to Dr. Stephane Blot (Ecole Nationale
Veterinaire, Maisons-Alfort) for the mice X-rays, to
Ms. Jacqueline Chandellier for her photographic work,
to Ms. Nathalie Ouvrard and Mr. Pierre Casanovas for
animal care, and to Dr. Terence Partridge for his critical support. This work was granted by the Association
Francaise contre les Myopathies (AFM). J.P. Lefaucheur is recipient from the Ministere de la Recherche et de la Technologie (MRT). C. Pastoret is recipient from the Human Capital and Mobility EEC
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