THE ANATOMICAL RECORD 24270-76 (1995) Phenotype of Dystrophinopathy in Old MDX Mice JEAN PASCAL LEFAUCHEUR, CHRISTIAN PASTORET, AND ALAIN SEBILLE 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 ABSTRACT 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 0 1995 WJLEY-LISS, INC 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. MUSCULAR DYSTROPHY I N OLD MDX MICE --u- mdx mice 71 - C57BL/lO mice Fig. 1. Percentages of surviving mdx mice ( n = 14) and age-matched C57BW10 mice from 18 to 24 months of age. MATERIALS AND METHODS 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. RESULTS 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 72 J. P. LEFAUCHEUR ET AL. 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 * f s.dJ of 23-month-old C57BL/lO (n= 4) and m& Extensor 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 Soleus 11.9 2 0.5 5.7 k 0.7 Plantaris 13.1 2 0.8 6.1 2 0.5 Quadriceps 227.1 ? 18.0 68.2 2 6.3 mice (n = 4) Gastrocnemius 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). MUSCULAR DYSTROPHY IN OLD M D X MICE 73 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 74 J. P. LEFAUCHEUK ET AL. 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). MUSCULAR DYSTROPHY IN OLD M D X MICE 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. DISCUSSION 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 mice. 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 75 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. ACKNOWLEDGMENTS 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 Program. LITERATURE CITED Anderson, J.E., B.H. Bressler, and W.K. Ovalle 1988 Functional regeneration in the hindlimb skeletal muscle of the mdx mouse. J . Muscle Res. Cell Motil., 9:499-515. Anderson, J.E., W.K. Ovalle, and B.H. 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