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Morphological and functional study of extensor digitorum longus muscle regeneration after iterative crush lesions in mdx mouse
Neuromusc.Disord.,Vol. 5. No. 6, pp. 489-500, 1995
Pergamon
Copyright © Elsevier Science Ltd Printed in Great Britain. All fights reserved 0960-8966/94 $9.50 + .00
096049t~tgS)00006-2 MORPHOLOGICAL A N D F U N C T I O N A L S T U D Y OF EXTENSOR DIGITORUM LONGUS MUSCLE R E G E N E R A T I O N AFTER ITERATIVE CRUSH LESIONS IN M D X MOUSE J. P LOUBOUTIN,*I" V. FICHTER-GAGNEPAIN,t C. PASTORET,* E. THAON,§ J. NOIREAUD,I" A. SI~BILLE,:~ M. FARDEAUI[ ~"Unit6 CNRS 1340. Hrpital GR La~nnec-BP 1005, 44035 Nantes Cedex 01; :~Laboratoirede Physiologic,Facult6 de M~decine Saint-Antoine, 27 rue de Chaligny, 75571 Paris Cedex, 12; ~Clinique Neuroiogique,Hrpital GR La~nnec, BP 1005, 44035 Nantes Cedex 01; IlUnit6Inserm 153-17, rue du Fer AMoulin, 75005 Pads, France (Received 16 November 1994; revised 12 February 1995; accepted 9 March 1995)
Abstract--The regenerative capacity of mdx Extensor Digitorum Longus (EDL) muscle after iterative muscle crush injuries was examined and compared with that of age-matched control C57BL/10 mice. Muscle crush injuries were performed at 8 weeks and repeated at 12 and 16 weeks. Contralateral non-crushed EDLs from mdx and C57BL/10 mice were used as internal controls for histopathology, histoenzymology, morphometry and for the study of the contractile properties. Morphological examinations were performed at 12, 16 and 20 weeks, respectively one month after a single, a second or a third crush. Contractile properties were studied at 12 and 20 weeks. By 20 weeks, no difference in the number of fibres with internal nuclei could be observed between crushed EDL from both strains, and non-crushed mdx EDL; the area and the diameter of crushed EDL from mdx mice were, respectively, 1.5- and 1.2-fold higher than the ones from crushed EDL from C57BL/10 strain. By 20 weeks, diameter distribution of crushed EDL muscles from C57BL/10 mice were shifted towards smaller fibre diameter, whereas in mdx mice, diameter distribution of crushed EDL muscles paralleled that of non-crushed EDL muscles. By 20 weeks, crushed mdx and C57BL/10 EDL muscles produced 77 and 47% of normalized tetanus tension respectively of non-crushed mdx and C57BL/10 EDL muscles. Following crush injury, both 12- and 20-week mdx and C57BL/10 EDL exhibited a slowed time to peak (TTP) and half-relaxation time (H1/2R) of twitch. There was no difference in posttetanic potentiation between the different groups. Crushed EDL of both strains showed an increased resistance to fatigue, compared to the non-crushed controls. The present study provides morphological and functional evidence for the greater recovery of mdx muscle compared to C57BL/10 muscle following iterative crush injury; however, the recovery does not completely prevent the appearance of necrosis/regeneration features. Key words: mdx mouse, muscle regeneration, mouse model, EDL, crush injury.
in m d x mice is followed by a spontaneous regeneration of clinical, morphological and physiological characteristics [2] and the adult mdx phenotype is benign compared with D M D . Moreover, the m d x mouse muscles do not show significant fibrosis [4], except in the diaphragm muscle [6, 7] and dystrophin-negative muscles from the m d x strain are not weak, as are muscles from D M D patients. Dystrophin, the high molecular weight protein product of the human D M D gene localizes to the cell periphery of normal skeletal muscle, while biopsy specimens of D M D patients are characterized by a complete
INTRODUCTION
In 1984, Bulfield et al. [1] described a mutant mouse (mdx) which arose spontaneously from the C57BL/10 ScSn strain, characterized by an X-linked skeletal myopathy. The m d x mouse myopathy and Duchenne muscular dystrophy ( D M D ) share some genetic, biochemical and histopathological features. At an early age, skeletal muscle from m d x mice exhibits degeneration and necrosis [2-5]. However, by contrast with D M D , necrosis of muscle fibres *Author to whom correspondence should be addressed. 489
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absence of dystrophin [8-14]. In mdx mice, the absence of dystrophin [8,9], is due to a point mutation in the dystrophin gene [15]. Its subcellular localization and its amino-acid sequence suggest that dystrophin is a membrane-associated cytoskeletal protein of the skeletal muscle sarcolemma [10] involved in the membrane stability. Because of the genetic and biochemical similarities with DMD, the mdx mouse has become an attractive model currently used in the development of potential therapies for the replacement of dystrophin [16, 17]. The ability of mature skeletal muscle to regenerate has been documented in many works [18, 23]. The extent and success of regeneration vary with the nature of the injury, but in all situations, the process involves revascularization, cellular infiltration, phagocytosis of necrotic damaged muscles, proliferation of muscle precursor cells and their fusion and finally, reinnervation. In the same way, the properties of regenerating mdx skeletal muscles following experimental damage have been studied in different models. Degeneration/regeneration process of the soleus (SOL) muscle after free muscle grafting are very similar in normal and mdx mice [24]. Similarly, using autoradiographic techniques to specifically investigate the timing of muscle precursor cell replication [25], little difference in the regenerative capacity was found between mdx and C57BL/10 mice after crush injury of Extensor Digitorum Longus (EDL) muscle. Following a denervation and devascularization insult, the morphological and functional recovery of the mdx EDL muscle may be greater compared to C57BL/10 muscle [26-27]. In the present work, iterative muscle crush lesions have been performed in order to determine whether recovery of some morphological and plzysiological properties occurred in the regenerated dystrophic myofibres of the mdx EDL muscle after multiple injuries and not only after a unique lesion. MATERIAL AND METHODS
Animals Establishment and maintenance of a purebred rndx stock for use in experiments was achieved by selection of animals expressing the
mdx gene as assessed by muscle biopsy. They were maintained in routine animal house conditions on a standard commercial diet. Nonmyopathic mice of the C57BL/10 ScSn strain were used as controls. Surgical procedure In all experiments, eight-week-old male mdx and C57BL/10 mice were used for the initial lesion in all experiments. Mice were anaesthetised with phenobarbital (1:10) and received an extensive muscle crush injury to the EDL muscle of the right leg. EDL muscle of the contralateral leg was left intact to be used as a control. The skin of the right leg was opened and one arm of a small pair of forceps was inserted down the side of the EDL beneath the muscle and the EDL crushed transversely. The muscle crush was about 4 mm long and extended throughout the depth of the muscle and 1.5 mm wide. Although the crush was severe, longitudinal continuity of the muscle belly was maintained. Skin wounds overlying the lesion were closed and sutured. This procedure was performed from one to three times, with an interval of four weeks between each crush. Crushed EDL muscles were examined three times after surgery: (1) four weeks after a single crush (12-week old group); (2) four weeks after a second crush; (16-week old group); (3) four weeks after a third crush; (20week old group). For each time point morphological studies were carried out in crushed (right) leg and non-crushed (left) leg EDL muscles respectively from mdx and C57BL/10 strains. Physiological investigations were performed on 12-week and 20-week-old groups of crushed and non-crushed EDL from mdx and C57BL/10 mice.
Microscopic studies, fibre typing and morphometry Animals were killed by cervical dislocation, with five individuals being studied in each group at each time point. For the morphological characterization, EDL muscles were carefully dissected and a cross-section of the muscle, taken from the midbelly region, was frozen immediately in isopentane cooled in liquid nitrogen (-165°C). Blocks were stored at -80°C prior to sectioning. Transverse cryostat sections (10 ~tm thick) were stained with haematoxylin and eosin, Gomori's trichrome,
Recovery in mdx EDL Muscle After Crush Injury
Sudan black and red. Serial sections were stained to demonstrate myofibrillar ATPase activity [28] after preincubation at pH 10.4, 4.63 and 4.35 [29], and the mitoebondrial oxydative enzymes NADH-tetrazolium reductase (NADH-TR) activity [30]. Fibre classification was made in serial sections stained with myofibrillar ATPase. Fibres exhibiting central myonuclei and type 1, 2A and 2B fibres were counted in transverse sections of muscle; the numerical ratio of each variable was calculated as a percentage of at least 200 fibres from 5 different regions within the same section. In type 1 and 2 fibres, cross-sectional area (CSA) and diameter were measured on ATPase-stained sections. At least 200 fibres from 6 different regions within a section of muscle were examined under light microscopy using a home-made image analyser (Summagraphics/Tandon PC AT). A cursor with a light-emitting diode was used to trace the outline of fibres. The image analyser was linked to the computer programmed to calculate CSA and diameters of fibres. Results were displayed in histogram form to show the size frequency distribution of the fibres.
Contractile experiments Bundles of 100 /~m diameter were isolated~ and dissected in their whole length under a binocular microscope. The preparation was then transferred to the experimental chamber and mounted. The two ends of the muscle were carefully snared by fine platinum wire loops one fixed in the experimental bath and the other to the tip of an isometric force transducer (displacement measuring system Kaman KD 2300, Colorado Springs, Co., U.S.A.). Twitches were displayed on a storage oscilloscope (Tektronix 5155 N, Beaverton, Oregon, U.S.A.) upon stimulation by current pulses (0.5 ms duration, twice the threshold value, frequency 0.07 Hz) applied between two platinum plate electrodes on each side of the channel and the maximum twitch tension was determined (Pt). Tetanic contractions were elicited with trains of stimuli at 100 Hz for the time necessary to get a steady-state tension (duration of trains: 0.7-1.5 s) and estimate the maximum tetanic tension (Po). Post-tetanic potentiation (PTP) was then measured by stimulating muscle with a single pulse (pre-twitch) followed 60 s later by a tetanus (duration: 1 s).
491
After 20 s, post-twitch was elicited. PTP was determined as the ratio between the amplitude of post-twitch and the amplitude of pre-twitch. The fatigue profile of the muscle was measured by stimulating the muscle every 30 s, 10 times with 8 s tetani. The tension of the last tetanus divided by the tension of the initial tetanus was taken as an index of fatigue (Fi). The fast-flow perfusion stream (20 ml/min) coupled to the small chamber volume allowed a total bath medium change in less than 0.2 s. Resting tension, twitches, tetanus and contractures were recorded continuously on a chart recorder (SE 120, BBC Goerz Metrawatt, Ntirnberg, Germany).
Solutions. The normal physiological solution contained (mM): Na ÷, 140; K ÷, 6; Ca 2+, 3; Mg 2+, 2; CI-, 156; HEPES, 5; pH was adjusted to 7.4 with Tris-aminomethane. The solutions were equilibrated with 100% 02. Calcium was added as a 1 M-CaC12 solution (B.D.H. Poole, U.K., volumetric standard Analar grade) to a concentration of 3 mM-Ca, unless its concentration was varied as part of the experiment. When the bathing C a 2+ concentration was reduced, an equivalent amount of MgCI 2 was added. The experiments were conducted at room temperature (19-22°C). Statistical analysis. Results were expressed as mean ±S.D. Statistical evaluation was performed with the non-parametric MannWhitney test between different groups. The test was considered to be significant for P < 0.05. RESULTS
Morphology Histopathology At the different periods of time chosen for the study, no abnormalities could be observed in the non-crushed EDL muscles from C57BL/10 mice. In C57BL/10 mice, four weeks after a single crush (Fig. l a), EDL muscles exhibited 23.3 +3.1% of fibres with internal nuclei, whereas 65.3 +3.2 and 71.3 +12% of fibres contained centrally placed nuclei, respectively four weeks after a second and a third crush (Fig. l b). Fig. 2 summarizes the percentages of fibres with central nuclei in crushed and non-crushed EDL muscles from C57BL/10 and mdx mice. In non-crushed EDL muscles from
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Fig. 1. Cryostat sections of crushed (a-b, e-f) and non-crushed (e-
mdx mice, results were similar to those previously described [2, 4, 5, 7]. By 12 weeks, EDL muscles contained 74.4 ±4.5% of fibres with central nuclei (Fig. lc) and this percentage increased with time: 77.2 ±5.6 and 79.1 ±7.3% of fibres had centrally placed nuclei by 16 and 20 weeks respectively. Continued necrosis, with necrotic muscle fibres, sometimes invaded by phagocytes and with mononuclear infiltration could be observed at the different time points.
In the same way, even at 20 weeks, there were occasional clusters of small regenerating fibres with centrally placed vesicular nuclei and basophilic cytoplasm. At 20 weeks, fibres with central nuclei have grown to reach approximately the same diameter as the surviving original muscle fibres; however, there was a great variation in fibre size (Fig. Id). No connective tissue proliferation was observed. In crushed EDL muscles from mdx mice, the percentage of
Recovery in mdx EDL Muscle After Crush Injury
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tration could be observed at the different time points (Fig. le). Abnormally large fibres were less frequently observed by 20 weeks in crushed EDL muscles from mdx mice than in the noncrushed. There was no increase of connective tissue (Fig. If).
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(n = 5).
fibres with central nuclei were respectively 72.5 +6.5, 78.5 +4.9 and 78.6 +11.9%, respectively one month after a single, a second and a third crush. However, if we consider the distance between two nuclei on longitudinal section, the percentages of fibres with internal nuclei approached probably 100% by 20 weeks. After crush injury, necrotic fibres and cellular infil-
The EDL muscle of mice consisted mostly of type 2 fibres. The proportions of different types of fibres are summarized in Table 1. Compared to contralateral muscles, an increase of type 2A fibres was observed both in crushed EDL from C57BL/10 and mdx mice after a third crush lesion (P < 0.05). In addition to type I, IIA and liB, a fourth staining was found, which exhibited moderate staining for N A D H - T R and for myosin ATPase at both acid and alkaline preincubations. These fibres are similar to what some authors refer to as type IIC fibres. However, only the small, central nucleated muscle fibres showed the type IIC pattern typical of regenerating fibres. However, four weeks after the crush (first, second or third), they represented only 1-2% of the muscle fibres, both in mdx and C57BL/10 strains. There was no significant difference in the proportion of type IIC fibres between agematched mdx and C57BL/10 crushed EDL muscles. In the same way, although type IIC fibres may be found relatively frequently in uninjured EDL muscles from four- to six-weekold mdx mice, at the time where regenerative process is at its maximum, they were quite infrequent in uninjured EDL muscles from mdx strain by 12 weeks and later. Finally, in noncrushed C57BL/10 EDL muscles, type IIC fibres were rare. No significant difference in the proportion of type IIC fibres was found between age-matched non-crushed EDL muscles from mdx and C57BL/10 strains.
Table 1. Percentages of type 1, 2A and 2B fibres in non-crushed and crushed EDL muscles from C57BL/10 and mdx strains at the different times after surgery (n = 5) Control 1
Crushed
2A
2B
1
2A
2B
C57BL/10 12-w~k 16-w~k 20-w~k
0.5±0.1 0.5±0.1 0.6±0.1
46.1±0.8 47.1±0.7 48.8±0.6
53.4±0.8 52.3±0.6 50.6±0.5
5.5±0.5 6.2±0.4 1.5±0.5
50.7±0.6 50.6±6.7 56.1±0.6
43.7±0.6 43.2±6.8 42.4±0.5
m~ 12-w~k l~w~k 20-w~k
0.5±0.2 0.5±0.1 0.6±0.1
47.7±2.5 47.8±2.1 48.5±2.3
51.8±2.5 51.6±2.1 50.8±2.3
1.3±0.5 2.1±0.4 0.6±0.4
49.4±0.2 54.1±3.6 55.2±4.6
49.2±0.2 43.8±3.8 ~.2±4.5
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Table 2. Mean diameter and CSA of crushed and non-crushed EDL muscles from C57BL/10 and mdx strains at the different times after surgery (n = 5) Control CSA ~m-')
Crushed
Diameter (ttm)
CSA (urn 2)
Diameter (,urn)
C57BL/10 12-week 16-week 20-week
1620.6 +44.6 1680.5 _+30.2 1743.9 _+35.3
38.9 _+ 1.1 39.1 -+ 1.3 39.4 % 1.2
1507.8 +22.6 1076.6 _+75.4 937.7 _+67.8
38.7 + 1.6 32.2 _+1.6 30.1 +4.8
mdx 12-week 16-week 20-week
1609.3 -+22.3 1650.3 +42.2 1677.3 _+67.4
38,5 _+0.9 38.6 _+0.6 38.6 -+0,8
1495.5 _+99.1 1483.5 +78.3 1392.1 _+105.2
37.6 _+0.9 36.8 +0.4 36.3 _+3.2
Morphometry
Isometric' tension strength
Diameter and CSA of crushed and noncrushed EDL muscle from C57BL/10 and mdx mice are summarized in Table 2. Whatever the time after surgery, there was no difference in CSA and diameter when C57BL/10 EDL muscle was compared with that in mdx mice. In the C57BL/10 strain, when comparing crushed with control muscles, CSA and diameter were reduced by 7, 36, 47% and by 0.6, 18, 24% after one, two and three crushes respectively. In mdx mice, crush injury induced also a decrease in CSA and diameter; however, CSA and diameter were reduced by 8, 9, 17% and by 2.4, 4.7, 6% at 12, 16 and 20 weeks respectively. In mdx mice, by 20 weeks, the CSA and diameter of crushed EDL muscle were increased by a factor 1.5 (P < 0.02) and 1.2 (P < 0.04) respectively compared to the values measured in the C57BL/10 strain. By 12 and 16 weeks, diameter distribution of crushed and non-crushed EDL muscles in both C57BL/10 and mdx mice were not different. However, a slight shift of diameter distribution of crushed EDL muscles from C57BL/10 mice towards smaller fibre could be observed by 16 weeks. By 20 weeks, diameter distribution of crushed EDL muscles from C57BL/10 mice was shifted toward smaller fibre diameter, whereas in mdx mice, diameter distribution of crushed EDL muscles paralleled that of non-crushed EDL muscles (Fig. 3).
When expressed relatively to muscle weight, there was no difference in the strength of twitch and tetanic tension developed by C57BL/10 EDL muscles at 12 and 20 weeks. Such a similarity was also observed in the mdx EDL muscles. By 12 weeks, mdx EDL muscles generated less twitch and tetanus tensions than in C57BL/10 EDL (P < 0.02). In mdx EDL muscles at 20 weeks, both Po/mwt and Pt/mwt were reduced from the control (P < 0.05). Within the C57BL/10 strain at 12 weeks of age, when comparing intact unoperated muscles to those which have been crushed, all tension values were lower in the operated groups (P < 0.05). At 20 weeks, both tension parameters of the same operated group were still lower than non-crushed EDL (P < 0.01). Crush injury in mdx mice induced the same modifications as in C57BL/10 mice: a decrease in Po/mwt and Pt/mwt at both 12 and 20 weeks in crushed EDL muscles. However, by 20 weeks, crushed EDL from mdx mice produced more tension than age-matched crushed C57BL/10 EDL (P < 0.02): when comparing unoperated muscles to those which have been crushed by 20 weeks, Po/mwt and Pt/mwt were reduced respectively by 23 and 36% in the mdx strain and by 53 and 64% in the C57BL/10 one.
Contractile properties The isometric tension parameters measured in intact and crushed EDL muscles from C57BL/10 and mdx mice (12 and 20-week-old) are summarized in Table 3.
TTP and H1/2R There was no difference in TTP and H1/2R between 12-week and 20-week non-crushed EDL muscles both in the C57BL/10 and the mdx strains. Whatever the age of the animals, there was also no difference in either parameter between the non-crushed C57BL/10 EDL muscles and their age-matched non-crushed mdx.
Recovery in mdx EDL Muscle After Crush Injury
C57BL/10
495
mdx 12 weeks
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Fig. 3. Fibre size distribution in non-crushed (unfilled bars) and crushed (solid bars) EDL muscles from C57BL/10 (left side) and mdx (right side) by 12 (A, D), 16 (B, E) and 20 weeks (C, F). Note the shift of diameter distribution of crushed EDL from normal strain towards smaller fibre diameter by 20 weeks (C) (n = 5).
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Table 3. Contractile properties of E D L muscles from C57BL/10 and mdx strains (n = 5)
12-week
Strain Control Po/mwt (mN/mg)
C57BL/10
Pt/mwt (mN/mg)
C57BL/10
TTP (msec)
C57BL/10
HI/2R (msec)
C57BL/10
PTP
C57BL/10
(%)
mdx
Fi
(%)
mdx mdx rod,: mdx
C57BL/10
mdx
20-week Crushed
Control
Crushed
20.9 + 1.8 16.8 + 1.3
17.1 _+ 1.8 15.5 _+ 1.3
22.6 _+2.4 18.8 +2.6
10.6 + 1.1 14.5 _+ 1.5
2.4 _+0.1 1.6 _+0.2
2.1 _+0.2 1.5 _+0.2
2.6 _+0.1 2.1 _+0.2
0.9 _+0.2 1.3 +0.2
22.2 _+2.4 25.2 _+ 1
23.2 _+3 30.6 +4.6
24.4 +_2.2 26.2 _+2.4
32.8 _+3 34.6 _+2.4
26.2 _+3 25.8 _+ 1
27.2 _*3 33 _+6.8
26.4 _+2.4 28.2 _+ 1
33.2 _+3.2 35.2 _+4.2
101.2 _+2.3 110.8 +9.1
100.1 +_ 1.3 106.5 _+4.2
103.4 _+2.8 101.7 _+ 1.7
102.3 _+2.5 104.5 _+3.9
18.1 _+ 1.5 12.3 _+ 1.2
52.6 +4.5 44.3 _+3.8
19.1 _+1.3 17.4 _+1.5
55.2 +3.7 44.8 +4.4
Following crush injury, both 12 and 20-week mdx and C57BL/10 EDL muscles exhibited an increased TTP and H1/2R (P < 0.05). Post-tetanic potentiation In Table 3, the values obtained for PTP are also included. There was no difference in PTP between C57BL/10 and mdx EDL muscles, crushed or not, at either age. Fatigue In both strains, the crushed EDL muscles showed increased fatigue resistance compared with their respective non-crushed contralateral muscles. This increase of fatigue resistance was more marked at 20 weeks (P < 0.01) than at 12 weeks (P < 0.02) (Fig. 4). DISCUSSION
The aim of the present work was to study morphological and functional recovery of mdx compared to C57BL/10 muscle. The most striking feature of the dystrophindeficient mdx strain of mouse is the spontaneous regenerative capacity after early degeneration [2-5, 7]. The recovery to normal fibre type proportion and mean fibre CSA [2, 5, 7] is accompanied by the ability to generate normal tensions [2, 31, 32]. Recent works have established that the plasticity of mdx muscle recovery from imposed injury may be greater than that of normal muscles [24, 26, 27]. However, in these studies, only one lesion was performed, and short-term recovery solely was examined [26].
In the present study, the significant difference in fibre diameter and CSA of crushed EDL muscles between 20-week-old mdx and C57BL/10 mice suggests that the mdx EDL muscle has a greater potential to recover from iterative crush injuries than C57BL/10 EDL muscle. A similar finding has been described two weeks after denervation and devascularization of the EDL muscles from mdx and C57BL/10 strains [26]. However, crush injury and denervation/devascularization lesions differ by some features. After crush injury, the vascular and nerve supply are severely interrupted [33]. Furthermore, the basal lamina, which is used as an important scaffold for aligning regenerating myofibres [34, 35], and the sarcolemma [36, 37] are disrupted in association with a segmental degeneration of the sarcomeres. Denervation and devascularization treatment is similar to muscle autografting [34] or free grafting [21], but without disruption of tendon attachments and with preservation of the longitudinal disposition of external lamina sheaths. After crush injury, necrotic fibres and cellular infiltration have been shown at each of the different time points, suggesting that the structural recovery of mdx EDL muscle after crush lesion does not completely prevent the appearance of necrosis/degeneration features. Similar results were observed in mdx EDL muscle after bupivacaine injection (unpublished results), and after denervation/devascularization after a short [26] or a long period (unpublished results) of recovery. The present work confirms that twitch and tetanic tensions developed by mdx EDL muscle, when expressed relatively to muscle
Recovery in mdx EDL Muscle After Crush Injury A
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Fatigue profiles showing the decline of the relative tetanus tension with time. A: 12-week-old EDL muscles; B: 20-week-old EDL muscles (n = 5).
weight, were lower than in age-matched C57BL/10 strain [2, 31, 32]. The most striking aspect of the results obtained during the contractile experiments was the extent to which crushed mdx EDL muscles recovered tensionproducing capabilities at 20 weeks, as compared with the non-crushed C57BL/10 EDL. By 12 weeks, crushed C57BL/10 EDL muscle produced 81 and 85% of normalized tetanus and twitch tensions compared to agematched controls. By 20 weeks, after a third crush injury, only 47 and 43% of normal values could be obtained. As in the C57BL/10 mice, at 12 weeks, the mdx crushed EDL muscle developed twitch and tetanic tensions similar to the
ones measured in contralateral muscles (83 and 88% respectively). However, by 20 weeks, the mdx crushed EDL already produced 77% of normalized tetanus tension and 64% of normalized twitch tension of the non-crushed agematched mdx controls. This seems to be a clear demonstration that mdx EDL muscle was able to recover more fully tension-producing capabilities following iterative injuries than the C57BL/10 one. Moreover, the tensions of the mdx EDL muscles measured 12 weeks after the 3rd crush lesion were not significatively different from the ones measured four weeks after the third lesion (unpublished personal results). In addition, the necrosis did not increase 12
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weeks after the third injury and the mdx EDL muscle did not finally become weaker 12 weeks after the third crush lesion (personal results). Similar results were reported after denervation/devascularization in mdx muscle by Mechalchuk and Bressler [27]. On the other hand, Moens et al. [24] have demonstrated that the regeneration occurring after transplantation of the SOL muscle induced a similar decrease of maximal isometric force in the mdx and C57BL/10 strains. It has been postulated that overabundance of matrix fibroblast growth factor (FGF) in mdx hindlimb muscles may be related to an increase in both satellite cells and regenerative capacity [38]. FGF level has not been determined in crushed muscles from the mdx strain. It would be of interest to measure it in crushed muscles from both mdx and C57BL/10 strains, in order to compare the levels of this growth factor in muscles from C57BL/10 and mdx strains after such a trauma. However, the role of other putative growth factors in muscle regeneration in the mdx strain remains to be determined. The overabundance of such growth factors in mdx muscles may explain why they recover as well as they do after each crush lesion and do not become weaker and weaker, despite the presence of dystrophic features. There was no difference in the kinetics of the contraction between any of the non-crushed EDL muscles. However, by 20 weeks, crushed EDL muscles of both strains exhibit larger TTP and H1/2R than non-crushed. These findings are in agreement with previous work performed after denervation/devascularization of EDL muscle from normal and mdx strains [27]. Similar slowing of the contractile responses has been also reported in regenerating rat muscle [18]. A normal characteristic of fast-twitch EDL muscle is a lack of resistance to fatigue. As already shown in previous work [2, 27, 32], in the present study, the C57BL/10 and mdx noncrushed EDL muscles, aged 12 and 20 weeks, became fatigued to the same relative tension in both age groups. Similarly, mdx slow-twitch SOL muscle shows no difference in the resistance to fatigue compared to normal muscle. However, resistance to fatigue was found to be higher in mdx diaphragm muscle compared to control muscle [39, and unpublished personal results]. A resistance to fatigue appears after a single crush in both strains and by 20 weeks,
resistance to fatigue is slightly higher in crushed EDL muscles from C57BL/10 strain compared with muscles from mdx strain. This finding may be in relation with the increase in oxidative fibre proportion observed after crush injury in the two strains. After denervation/devascularization, the operated mdx EDL muscle, which initially showed an increased resistance to fatigue, became almost as fatigueable as C57BL/10 non-operated EDL by 16 weeks after the surgical intervention [27]. In the cat EDL after transplantation, Faulkner et al. [40] noted that fatigue profile never returns to normal levels. As already reported [2, 27, 32], no significant differences were noted for PTP. PTP is a characteristic of fast-twitch skeletal muscle. We measured PTP according to the protocol described by Anderson et al. [2] and Mechalchuk and Bressler [27] in order to compare the fatigue of the uninjured or injured EDL muscles from mdx mice. Thus, it is suggested that following crush injury, as after denervation/devascularization [27], EDL muscle from mdx and C57BL/10 strains, does not lose some of its fast-twitch characteristics. We have shown that EDL muscles from C57BL/10 and mdx strains were mainly composed of type IIA and type IIB fibres. However, it is probable that IIX fibres were also present [41]. In this way, since IIA fibres are indistinguishable by ATPase staining from the 25-30% IIX fibres, it is not surprising that the numbers of type IIA fibres are higher than is typical for mouse EDL. Any intermediate fibres (I/IIA, IIA/IIX, IIX/IIB) are also likely to be included in the IIA category by ATPase staining. In this way, immunohistochemistry seems an important way to study the precise distribution of fibres in muscles. Thus, the present study provides morphological and functional evidence for a greater recovery of mdx compared to C57BL/10 muscle, but also for the recurrence of dystrophic features in mdx muscle regenerating from iterative crush injuries. Acknowledgements--The authors are most grateful to Dr Y. Ch6rel and Prof. M. Wyers for use of the image analysis system. This work was supported by a grant from the Association Franqaise contre les Myopathies (AFM). We thank Dr. S. Baudet for his critical evaluation of the English.
Recovery in mdx EDL Muscle After Crush Injury REFERENCES
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2. Anderson J E, Bressler B H, Ovalle W K. Functional regeneration in the hind limb skeletal muscle of the mdx mouse. J Muscle Res Cell Motil 1988; 9: 449-515. 3. Bridges L R. The association of cardiac muscle necrosis and inflammation with the degenerative and persistent myopathy of mdx mice. J Neurol Sci 1986; 72: 147-157. 4. Tanabe Y, Esaki K, Nomura T. Skeletal muscle pathology in X-crhomosome-linked muscular dystrophy (mdx) mouse. Aeta Neuropathol 1986; 69: 91-95. 5. Torres F B and Duchen L W. The mutant mdx: inherited myopathy in the mouse. Brain 1987; 110: 269-299. 6. Stedman H H, Sweeney H L, Shrager J B e t al. The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy. Nature 1991; 352: 536-539. 7. Louboutin J P, Fichter-Gagnepain V, Thaon E, Fardeau M. Morphometric analysis of mdx diaphragm muscle fibres. Comparison with hindlimb muscles. Neuromusc Disord 1993; 3: 463-469. 8. Hoffman E P, Brown R H, Kunkel L M. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 1987; 51: 919-928. 9. Bonilla E, Samitt C E, Miranda A F et al. Duchenne muscular dystrophy: deficiency of dystrophin at the muscle cell surface. Cell 1988; 54: 447-452. 10. Koenig M, Monaco A P, Kunkel L M. The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein. Cell 1988; 53: 219-228. 11. Koenig M and Kunkel L M. Detailed analysis of the repeat domain of dystrophin reveals four potential hinge segments that may confer flexibility. J Biol Chem 1990; 265: 4560-4566. 12. Arahata K, Ishiura S, Ishiguro T et al. Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne dystrophy peptide. Nature 1988; 333: 861-866. 13. Hoffman E P, Fischbeck K H, Brown R H et al. Characterization of dystrophin in muscle-biopsy specimens from patients with Duchenne's or Becker's muscular dystrophy. N Eng J Med 1988; 318: 1363-1368. 14. Zubrzycka-Gaarn E E, Bulman D E, Karpati G e t al. The Duchenne muscular dystrophy gene product is localized in sarcolemma of human skeletal muscle. Nature 1988; 333: 466--469. 15. Sicinski P, Geng Y, Ryder-Cook A S, Barnard E A, Darlison M G, Bernard P J. The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science 1989; 244: 1578-1580. 16. Partridge T A, Morgan J E, Coulton J R, Hoffman E P, Kunkel L M. Conversion of mdx myofibres from dystrophin-negative to -positive by injection of normal myoblasts. Nature 1989; 337: 176-179. 17. Karpati G, Pouliot Y, Zubrzycka-Gaarn E E et al. Dystrophin is expressed in mdx skeletal muscle fibres after normal myoblast implantation. Am J Pathol 1989; 135: 27-32. 18. Carlson B M and Gutman E. Development of contractile properties of minced muscle regenerates in the rat. Exp Neural 1972; 36: 239-249. 19. Carlson B M and Gutman E. Regeneration in free grafts of normal and denervated muscles in the rat: morphology and histochemistry. Anat Rec 1975; 183: 47~i2. 20. Carlson B M and Gutman E. Contractile and histo-
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chemical properties of sliced muscle grafts regenerating in normal and denervated rat limbs. Exp Neurol 1976; 50: 319-329. 21. Carlson B M, Hnik P, Tucek S, Bader D M, Faulkner J A. Comparison between grafts with intact nerves and standard free grafts of the rat extensor digitorum longus muscle. Physiol Bohemoslov 1981; 30: 505-513.
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096049t~tgS)00006-2 MORPHOLOGICAL A N D F U N C T I O N A L S T U D Y OF EXTENSOR DIGITORUM LONGUS MUSCLE R E G E N E R A T I O N AFTER ITERATIVE CRUSH LESIONS IN M D X MOUSE J. P LOUBOUTIN,*I" V. FICHTER-GAGNEPAIN,t C. PASTORET,* E. THAON,§ J. NOIREAUD,I" A. SI~BILLE,:~ M. FARDEAUI[ ~"Unit6 CNRS 1340. Hrpital GR La~nnec-BP 1005, 44035 Nantes Cedex 01; :~Laboratoirede Physiologic,Facult6 de M~decine Saint-Antoine, 27 rue de Chaligny, 75571 Paris Cedex, 12; ~Clinique Neuroiogique,Hrpital GR La~nnec, BP 1005, 44035 Nantes Cedex 01; IlUnit6Inserm 153-17, rue du Fer AMoulin, 75005 Pads, France (Received 16 November 1994; revised 12 February 1995; accepted 9 March 1995)
Abstract--The regenerative capacity of mdx Extensor Digitorum Longus (EDL) muscle after iterative muscle crush injuries was examined and compared with that of age-matched control C57BL/10 mice. Muscle crush injuries were performed at 8 weeks and repeated at 12 and 16 weeks. Contralateral non-crushed EDLs from mdx and C57BL/10 mice were used as internal controls for histopathology, histoenzymology, morphometry and for the study of the contractile properties. Morphological examinations were performed at 12, 16 and 20 weeks, respectively one month after a single, a second or a third crush. Contractile properties were studied at 12 and 20 weeks. By 20 weeks, no difference in the number of fibres with internal nuclei could be observed between crushed EDL from both strains, and non-crushed mdx EDL; the area and the diameter of crushed EDL from mdx mice were, respectively, 1.5- and 1.2-fold higher than the ones from crushed EDL from C57BL/10 strain. By 20 weeks, diameter distribution of crushed EDL muscles from C57BL/10 mice were shifted towards smaller fibre diameter, whereas in mdx mice, diameter distribution of crushed EDL muscles paralleled that of non-crushed EDL muscles. By 20 weeks, crushed mdx and C57BL/10 EDL muscles produced 77 and 47% of normalized tetanus tension respectively of non-crushed mdx and C57BL/10 EDL muscles. Following crush injury, both 12- and 20-week mdx and C57BL/10 EDL exhibited a slowed time to peak (TTP) and half-relaxation time (H1/2R) of twitch. There was no difference in posttetanic potentiation between the different groups. Crushed EDL of both strains showed an increased resistance to fatigue, compared to the non-crushed controls. The present study provides morphological and functional evidence for the greater recovery of mdx muscle compared to C57BL/10 muscle following iterative crush injury; however, the recovery does not completely prevent the appearance of necrosis/regeneration features. Key words: mdx mouse, muscle regeneration, mouse model, EDL, crush injury.
in m d x mice is followed by a spontaneous regeneration of clinical, morphological and physiological characteristics [2] and the adult mdx phenotype is benign compared with D M D . Moreover, the m d x mouse muscles do not show significant fibrosis [4], except in the diaphragm muscle [6, 7] and dystrophin-negative muscles from the m d x strain are not weak, as are muscles from D M D patients. Dystrophin, the high molecular weight protein product of the human D M D gene localizes to the cell periphery of normal skeletal muscle, while biopsy specimens of D M D patients are characterized by a complete
INTRODUCTION
In 1984, Bulfield et al. [1] described a mutant mouse (mdx) which arose spontaneously from the C57BL/10 ScSn strain, characterized by an X-linked skeletal myopathy. The m d x mouse myopathy and Duchenne muscular dystrophy ( D M D ) share some genetic, biochemical and histopathological features. At an early age, skeletal muscle from m d x mice exhibits degeneration and necrosis [2-5]. However, by contrast with D M D , necrosis of muscle fibres *Author to whom correspondence should be addressed. 489
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absence of dystrophin [8-14]. In mdx mice, the absence of dystrophin [8,9], is due to a point mutation in the dystrophin gene [15]. Its subcellular localization and its amino-acid sequence suggest that dystrophin is a membrane-associated cytoskeletal protein of the skeletal muscle sarcolemma [10] involved in the membrane stability. Because of the genetic and biochemical similarities with DMD, the mdx mouse has become an attractive model currently used in the development of potential therapies for the replacement of dystrophin [16, 17]. The ability of mature skeletal muscle to regenerate has been documented in many works [18, 23]. The extent and success of regeneration vary with the nature of the injury, but in all situations, the process involves revascularization, cellular infiltration, phagocytosis of necrotic damaged muscles, proliferation of muscle precursor cells and their fusion and finally, reinnervation. In the same way, the properties of regenerating mdx skeletal muscles following experimental damage have been studied in different models. Degeneration/regeneration process of the soleus (SOL) muscle after free muscle grafting are very similar in normal and mdx mice [24]. Similarly, using autoradiographic techniques to specifically investigate the timing of muscle precursor cell replication [25], little difference in the regenerative capacity was found between mdx and C57BL/10 mice after crush injury of Extensor Digitorum Longus (EDL) muscle. Following a denervation and devascularization insult, the morphological and functional recovery of the mdx EDL muscle may be greater compared to C57BL/10 muscle [26-27]. In the present work, iterative muscle crush lesions have been performed in order to determine whether recovery of some morphological and plzysiological properties occurred in the regenerated dystrophic myofibres of the mdx EDL muscle after multiple injuries and not only after a unique lesion. MATERIAL AND METHODS
Animals Establishment and maintenance of a purebred rndx stock for use in experiments was achieved by selection of animals expressing the
mdx gene as assessed by muscle biopsy. They were maintained in routine animal house conditions on a standard commercial diet. Nonmyopathic mice of the C57BL/10 ScSn strain were used as controls. Surgical procedure In all experiments, eight-week-old male mdx and C57BL/10 mice were used for the initial lesion in all experiments. Mice were anaesthetised with phenobarbital (1:10) and received an extensive muscle crush injury to the EDL muscle of the right leg. EDL muscle of the contralateral leg was left intact to be used as a control. The skin of the right leg was opened and one arm of a small pair of forceps was inserted down the side of the EDL beneath the muscle and the EDL crushed transversely. The muscle crush was about 4 mm long and extended throughout the depth of the muscle and 1.5 mm wide. Although the crush was severe, longitudinal continuity of the muscle belly was maintained. Skin wounds overlying the lesion were closed and sutured. This procedure was performed from one to three times, with an interval of four weeks between each crush. Crushed EDL muscles were examined three times after surgery: (1) four weeks after a single crush (12-week old group); (2) four weeks after a second crush; (16-week old group); (3) four weeks after a third crush; (20week old group). For each time point morphological studies were carried out in crushed (right) leg and non-crushed (left) leg EDL muscles respectively from mdx and C57BL/10 strains. Physiological investigations were performed on 12-week and 20-week-old groups of crushed and non-crushed EDL from mdx and C57BL/10 mice.
Microscopic studies, fibre typing and morphometry Animals were killed by cervical dislocation, with five individuals being studied in each group at each time point. For the morphological characterization, EDL muscles were carefully dissected and a cross-section of the muscle, taken from the midbelly region, was frozen immediately in isopentane cooled in liquid nitrogen (-165°C). Blocks were stored at -80°C prior to sectioning. Transverse cryostat sections (10 ~tm thick) were stained with haematoxylin and eosin, Gomori's trichrome,
Recovery in mdx EDL Muscle After Crush Injury
Sudan black and red. Serial sections were stained to demonstrate myofibrillar ATPase activity [28] after preincubation at pH 10.4, 4.63 and 4.35 [29], and the mitoebondrial oxydative enzymes NADH-tetrazolium reductase (NADH-TR) activity [30]. Fibre classification was made in serial sections stained with myofibrillar ATPase. Fibres exhibiting central myonuclei and type 1, 2A and 2B fibres were counted in transverse sections of muscle; the numerical ratio of each variable was calculated as a percentage of at least 200 fibres from 5 different regions within the same section. In type 1 and 2 fibres, cross-sectional area (CSA) and diameter were measured on ATPase-stained sections. At least 200 fibres from 6 different regions within a section of muscle were examined under light microscopy using a home-made image analyser (Summagraphics/Tandon PC AT). A cursor with a light-emitting diode was used to trace the outline of fibres. The image analyser was linked to the computer programmed to calculate CSA and diameters of fibres. Results were displayed in histogram form to show the size frequency distribution of the fibres.
Contractile experiments Bundles of 100 /~m diameter were isolated~ and dissected in their whole length under a binocular microscope. The preparation was then transferred to the experimental chamber and mounted. The two ends of the muscle were carefully snared by fine platinum wire loops one fixed in the experimental bath and the other to the tip of an isometric force transducer (displacement measuring system Kaman KD 2300, Colorado Springs, Co., U.S.A.). Twitches were displayed on a storage oscilloscope (Tektronix 5155 N, Beaverton, Oregon, U.S.A.) upon stimulation by current pulses (0.5 ms duration, twice the threshold value, frequency 0.07 Hz) applied between two platinum plate electrodes on each side of the channel and the maximum twitch tension was determined (Pt). Tetanic contractions were elicited with trains of stimuli at 100 Hz for the time necessary to get a steady-state tension (duration of trains: 0.7-1.5 s) and estimate the maximum tetanic tension (Po). Post-tetanic potentiation (PTP) was then measured by stimulating muscle with a single pulse (pre-twitch) followed 60 s later by a tetanus (duration: 1 s).
491
After 20 s, post-twitch was elicited. PTP was determined as the ratio between the amplitude of post-twitch and the amplitude of pre-twitch. The fatigue profile of the muscle was measured by stimulating the muscle every 30 s, 10 times with 8 s tetani. The tension of the last tetanus divided by the tension of the initial tetanus was taken as an index of fatigue (Fi). The fast-flow perfusion stream (20 ml/min) coupled to the small chamber volume allowed a total bath medium change in less than 0.2 s. Resting tension, twitches, tetanus and contractures were recorded continuously on a chart recorder (SE 120, BBC Goerz Metrawatt, Ntirnberg, Germany).
Solutions. The normal physiological solution contained (mM): Na ÷, 140; K ÷, 6; Ca 2+, 3; Mg 2+, 2; CI-, 156; HEPES, 5; pH was adjusted to 7.4 with Tris-aminomethane. The solutions were equilibrated with 100% 02. Calcium was added as a 1 M-CaC12 solution (B.D.H. Poole, U.K., volumetric standard Analar grade) to a concentration of 3 mM-Ca, unless its concentration was varied as part of the experiment. When the bathing C a 2+ concentration was reduced, an equivalent amount of MgCI 2 was added. The experiments were conducted at room temperature (19-22°C). Statistical analysis. Results were expressed as mean ±S.D. Statistical evaluation was performed with the non-parametric MannWhitney test between different groups. The test was considered to be significant for P < 0.05. RESULTS
Morphology Histopathology At the different periods of time chosen for the study, no abnormalities could be observed in the non-crushed EDL muscles from C57BL/10 mice. In C57BL/10 mice, four weeks after a single crush (Fig. l a), EDL muscles exhibited 23.3 +3.1% of fibres with internal nuclei, whereas 65.3 +3.2 and 71.3 +12% of fibres contained centrally placed nuclei, respectively four weeks after a second and a third crush (Fig. l b). Fig. 2 summarizes the percentages of fibres with central nuclei in crushed and non-crushed EDL muscles from C57BL/10 and mdx mice. In non-crushed EDL muscles from
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Fig. 1. Cryostat sections of crushed (a-b, e-f) and non-crushed (e-
mdx mice, results were similar to those previously described [2, 4, 5, 7]. By 12 weeks, EDL muscles contained 74.4 ±4.5% of fibres with central nuclei (Fig. lc) and this percentage increased with time: 77.2 ±5.6 and 79.1 ±7.3% of fibres had centrally placed nuclei by 16 and 20 weeks respectively. Continued necrosis, with necrotic muscle fibres, sometimes invaded by phagocytes and with mononuclear infiltration could be observed at the different time points.
In the same way, even at 20 weeks, there were occasional clusters of small regenerating fibres with centrally placed vesicular nuclei and basophilic cytoplasm. At 20 weeks, fibres with central nuclei have grown to reach approximately the same diameter as the surviving original muscle fibres; however, there was a great variation in fibre size (Fig. Id). No connective tissue proliferation was observed. In crushed EDL muscles from mdx mice, the percentage of
Recovery in mdx EDL Muscle After Crush Injury
493
tration could be observed at the different time points (Fig. le). Abnormally large fibres were less frequently observed by 20 weeks in crushed EDL muscles from mdx mice than in the noncrushed. There was no increase of connective tissue (Fig. If).
A .Q
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Histoenzymology
°t
"6 2O
12
16
20 Age (weeks)
12
16
20 Age (weeks)
B 80 J~
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o
40
o
"6 20 0
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Fig. 2. Histograms showing mean frequency of occurrence of fibres with internal nuclei in crushed (solid bars) a n d n o n - c r u s h e d (unfilled b a r s ) E D L m u s c l e s f r o m C57BL/10 (A) and mdx (B) strains at different time points
(n = 5).
fibres with central nuclei were respectively 72.5 +6.5, 78.5 +4.9 and 78.6 +11.9%, respectively one month after a single, a second and a third crush. However, if we consider the distance between two nuclei on longitudinal section, the percentages of fibres with internal nuclei approached probably 100% by 20 weeks. After crush injury, necrotic fibres and cellular infil-
The EDL muscle of mice consisted mostly of type 2 fibres. The proportions of different types of fibres are summarized in Table 1. Compared to contralateral muscles, an increase of type 2A fibres was observed both in crushed EDL from C57BL/10 and mdx mice after a third crush lesion (P < 0.05). In addition to type I, IIA and liB, a fourth staining was found, which exhibited moderate staining for N A D H - T R and for myosin ATPase at both acid and alkaline preincubations. These fibres are similar to what some authors refer to as type IIC fibres. However, only the small, central nucleated muscle fibres showed the type IIC pattern typical of regenerating fibres. However, four weeks after the crush (first, second or third), they represented only 1-2% of the muscle fibres, both in mdx and C57BL/10 strains. There was no significant difference in the proportion of type IIC fibres between agematched mdx and C57BL/10 crushed EDL muscles. In the same way, although type IIC fibres may be found relatively frequently in uninjured EDL muscles from four- to six-weekold mdx mice, at the time where regenerative process is at its maximum, they were quite infrequent in uninjured EDL muscles from mdx strain by 12 weeks and later. Finally, in noncrushed C57BL/10 EDL muscles, type IIC fibres were rare. No significant difference in the proportion of type IIC fibres was found between age-matched non-crushed EDL muscles from mdx and C57BL/10 strains.
Table 1. Percentages of type 1, 2A and 2B fibres in non-crushed and crushed EDL muscles from C57BL/10 and mdx strains at the different times after surgery (n = 5) Control 1
Crushed
2A
2B
1
2A
2B
C57BL/10 12-w~k 16-w~k 20-w~k
0.5±0.1 0.5±0.1 0.6±0.1
46.1±0.8 47.1±0.7 48.8±0.6
53.4±0.8 52.3±0.6 50.6±0.5
5.5±0.5 6.2±0.4 1.5±0.5
50.7±0.6 50.6±6.7 56.1±0.6
43.7±0.6 43.2±6.8 42.4±0.5
m~ 12-w~k l~w~k 20-w~k
0.5±0.2 0.5±0.1 0.6±0.1
47.7±2.5 47.8±2.1 48.5±2.3
51.8±2.5 51.6±2.1 50.8±2.3
1.3±0.5 2.1±0.4 0.6±0.4
49.4±0.2 54.1±3.6 55.2±4.6
49.2±0.2 43.8±3.8 ~.2±4.5
J. P. L o u b o u t i n et al.
494
Table 2. Mean diameter and CSA of crushed and non-crushed EDL muscles from C57BL/10 and mdx strains at the different times after surgery (n = 5) Control CSA ~m-')
Crushed
Diameter (ttm)
CSA (urn 2)
Diameter (,urn)
C57BL/10 12-week 16-week 20-week
1620.6 +44.6 1680.5 _+30.2 1743.9 _+35.3
38.9 _+ 1.1 39.1 -+ 1.3 39.4 % 1.2
1507.8 +22.6 1076.6 _+75.4 937.7 _+67.8
38.7 + 1.6 32.2 _+1.6 30.1 +4.8
mdx 12-week 16-week 20-week
1609.3 -+22.3 1650.3 +42.2 1677.3 _+67.4
38,5 _+0.9 38.6 _+0.6 38.6 -+0,8
1495.5 _+99.1 1483.5 +78.3 1392.1 _+105.2
37.6 _+0.9 36.8 +0.4 36.3 _+3.2
Morphometry
Isometric' tension strength
Diameter and CSA of crushed and noncrushed EDL muscle from C57BL/10 and mdx mice are summarized in Table 2. Whatever the time after surgery, there was no difference in CSA and diameter when C57BL/10 EDL muscle was compared with that in mdx mice. In the C57BL/10 strain, when comparing crushed with control muscles, CSA and diameter were reduced by 7, 36, 47% and by 0.6, 18, 24% after one, two and three crushes respectively. In mdx mice, crush injury induced also a decrease in CSA and diameter; however, CSA and diameter were reduced by 8, 9, 17% and by 2.4, 4.7, 6% at 12, 16 and 20 weeks respectively. In mdx mice, by 20 weeks, the CSA and diameter of crushed EDL muscle were increased by a factor 1.5 (P < 0.02) and 1.2 (P < 0.04) respectively compared to the values measured in the C57BL/10 strain. By 12 and 16 weeks, diameter distribution of crushed and non-crushed EDL muscles in both C57BL/10 and mdx mice were not different. However, a slight shift of diameter distribution of crushed EDL muscles from C57BL/10 mice towards smaller fibre could be observed by 16 weeks. By 20 weeks, diameter distribution of crushed EDL muscles from C57BL/10 mice was shifted toward smaller fibre diameter, whereas in mdx mice, diameter distribution of crushed EDL muscles paralleled that of non-crushed EDL muscles (Fig. 3).
When expressed relatively to muscle weight, there was no difference in the strength of twitch and tetanic tension developed by C57BL/10 EDL muscles at 12 and 20 weeks. Such a similarity was also observed in the mdx EDL muscles. By 12 weeks, mdx EDL muscles generated less twitch and tetanus tensions than in C57BL/10 EDL (P < 0.02). In mdx EDL muscles at 20 weeks, both Po/mwt and Pt/mwt were reduced from the control (P < 0.05). Within the C57BL/10 strain at 12 weeks of age, when comparing intact unoperated muscles to those which have been crushed, all tension values were lower in the operated groups (P < 0.05). At 20 weeks, both tension parameters of the same operated group were still lower than non-crushed EDL (P < 0.01). Crush injury in mdx mice induced the same modifications as in C57BL/10 mice: a decrease in Po/mwt and Pt/mwt at both 12 and 20 weeks in crushed EDL muscles. However, by 20 weeks, crushed EDL from mdx mice produced more tension than age-matched crushed C57BL/10 EDL (P < 0.02): when comparing unoperated muscles to those which have been crushed by 20 weeks, Po/mwt and Pt/mwt were reduced respectively by 23 and 36% in the mdx strain and by 53 and 64% in the C57BL/10 one.
Contractile properties The isometric tension parameters measured in intact and crushed EDL muscles from C57BL/10 and mdx mice (12 and 20-week-old) are summarized in Table 3.
TTP and H1/2R There was no difference in TTP and H1/2R between 12-week and 20-week non-crushed EDL muscles both in the C57BL/10 and the mdx strains. Whatever the age of the animals, there was also no difference in either parameter between the non-crushed C57BL/10 EDL muscles and their age-matched non-crushed mdx.
Recovery in mdx EDL Muscle After Crush Injury
C57BL/10
495
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A
D A v J~
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Fig. 3. Fibre size distribution in non-crushed (unfilled bars) and crushed (solid bars) EDL muscles from C57BL/10 (left side) and mdx (right side) by 12 (A, D), 16 (B, E) and 20 weeks (C, F). Note the shift of diameter distribution of crushed EDL from normal strain towards smaller fibre diameter by 20 weeks (C) (n = 5).
J. P. L o u b o u t i n et al.
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Table 3. Contractile properties of E D L muscles from C57BL/10 and mdx strains (n = 5)
12-week
Strain Control Po/mwt (mN/mg)
C57BL/10
Pt/mwt (mN/mg)
C57BL/10
TTP (msec)
C57BL/10
HI/2R (msec)
C57BL/10
PTP
C57BL/10
(%)
mdx
Fi
(%)
mdx mdx rod,: mdx
C57BL/10
mdx
20-week Crushed
Control
Crushed
20.9 + 1.8 16.8 + 1.3
17.1 _+ 1.8 15.5 _+ 1.3
22.6 _+2.4 18.8 +2.6
10.6 + 1.1 14.5 _+ 1.5
2.4 _+0.1 1.6 _+0.2
2.1 _+0.2 1.5 _+0.2
2.6 _+0.1 2.1 _+0.2
0.9 _+0.2 1.3 +0.2
22.2 _+2.4 25.2 _+ 1
23.2 _+3 30.6 +4.6
24.4 +_2.2 26.2 _+2.4
32.8 _+3 34.6 _+2.4
26.2 _+3 25.8 _+ 1
27.2 _*3 33 _+6.8
26.4 _+2.4 28.2 _+ 1
33.2 _+3.2 35.2 _+4.2
101.2 _+2.3 110.8 +9.1
100.1 +_ 1.3 106.5 _+4.2
103.4 _+2.8 101.7 _+ 1.7
102.3 _+2.5 104.5 _+3.9
18.1 _+ 1.5 12.3 _+ 1.2
52.6 +4.5 44.3 _+3.8
19.1 _+1.3 17.4 _+1.5
55.2 +3.7 44.8 +4.4
Following crush injury, both 12 and 20-week mdx and C57BL/10 EDL muscles exhibited an increased TTP and H1/2R (P < 0.05). Post-tetanic potentiation In Table 3, the values obtained for PTP are also included. There was no difference in PTP between C57BL/10 and mdx EDL muscles, crushed or not, at either age. Fatigue In both strains, the crushed EDL muscles showed increased fatigue resistance compared with their respective non-crushed contralateral muscles. This increase of fatigue resistance was more marked at 20 weeks (P < 0.01) than at 12 weeks (P < 0.02) (Fig. 4). DISCUSSION
The aim of the present work was to study morphological and functional recovery of mdx compared to C57BL/10 muscle. The most striking feature of the dystrophindeficient mdx strain of mouse is the spontaneous regenerative capacity after early degeneration [2-5, 7]. The recovery to normal fibre type proportion and mean fibre CSA [2, 5, 7] is accompanied by the ability to generate normal tensions [2, 31, 32]. Recent works have established that the plasticity of mdx muscle recovery from imposed injury may be greater than that of normal muscles [24, 26, 27]. However, in these studies, only one lesion was performed, and short-term recovery solely was examined [26].
In the present study, the significant difference in fibre diameter and CSA of crushed EDL muscles between 20-week-old mdx and C57BL/10 mice suggests that the mdx EDL muscle has a greater potential to recover from iterative crush injuries than C57BL/10 EDL muscle. A similar finding has been described two weeks after denervation and devascularization of the EDL muscles from mdx and C57BL/10 strains [26]. However, crush injury and denervation/devascularization lesions differ by some features. After crush injury, the vascular and nerve supply are severely interrupted [33]. Furthermore, the basal lamina, which is used as an important scaffold for aligning regenerating myofibres [34, 35], and the sarcolemma [36, 37] are disrupted in association with a segmental degeneration of the sarcomeres. Denervation and devascularization treatment is similar to muscle autografting [34] or free grafting [21], but without disruption of tendon attachments and with preservation of the longitudinal disposition of external lamina sheaths. After crush injury, necrotic fibres and cellular infiltration have been shown at each of the different time points, suggesting that the structural recovery of mdx EDL muscle after crush lesion does not completely prevent the appearance of necrosis/degeneration features. Similar results were observed in mdx EDL muscle after bupivacaine injection (unpublished results), and after denervation/devascularization after a short [26] or a long period (unpublished results) of recovery. The present work confirms that twitch and tetanic tensions developed by mdx EDL muscle, when expressed relatively to muscle
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i
i
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400 Time (sec)
Fatigue profiles showing the decline of the relative tetanus tension with time. A: 12-week-old EDL muscles; B: 20-week-old EDL muscles (n = 5).
weight, were lower than in age-matched C57BL/10 strain [2, 31, 32]. The most striking aspect of the results obtained during the contractile experiments was the extent to which crushed mdx EDL muscles recovered tensionproducing capabilities at 20 weeks, as compared with the non-crushed C57BL/10 EDL. By 12 weeks, crushed C57BL/10 EDL muscle produced 81 and 85% of normalized tetanus and twitch tensions compared to agematched controls. By 20 weeks, after a third crush injury, only 47 and 43% of normal values could be obtained. As in the C57BL/10 mice, at 12 weeks, the mdx crushed EDL muscle developed twitch and tetanic tensions similar to the
ones measured in contralateral muscles (83 and 88% respectively). However, by 20 weeks, the mdx crushed EDL already produced 77% of normalized tetanus tension and 64% of normalized twitch tension of the non-crushed agematched mdx controls. This seems to be a clear demonstration that mdx EDL muscle was able to recover more fully tension-producing capabilities following iterative injuries than the C57BL/10 one. Moreover, the tensions of the mdx EDL muscles measured 12 weeks after the 3rd crush lesion were not significatively different from the ones measured four weeks after the third lesion (unpublished personal results). In addition, the necrosis did not increase 12
498
J.P. Louboutin et al.
weeks after the third injury and the mdx EDL muscle did not finally become weaker 12 weeks after the third crush lesion (personal results). Similar results were reported after denervation/devascularization in mdx muscle by Mechalchuk and Bressler [27]. On the other hand, Moens et al. [24] have demonstrated that the regeneration occurring after transplantation of the SOL muscle induced a similar decrease of maximal isometric force in the mdx and C57BL/10 strains. It has been postulated that overabundance of matrix fibroblast growth factor (FGF) in mdx hindlimb muscles may be related to an increase in both satellite cells and regenerative capacity [38]. FGF level has not been determined in crushed muscles from the mdx strain. It would be of interest to measure it in crushed muscles from both mdx and C57BL/10 strains, in order to compare the levels of this growth factor in muscles from C57BL/10 and mdx strains after such a trauma. However, the role of other putative growth factors in muscle regeneration in the mdx strain remains to be determined. The overabundance of such growth factors in mdx muscles may explain why they recover as well as they do after each crush lesion and do not become weaker and weaker, despite the presence of dystrophic features. There was no difference in the kinetics of the contraction between any of the non-crushed EDL muscles. However, by 20 weeks, crushed EDL muscles of both strains exhibit larger TTP and H1/2R than non-crushed. These findings are in agreement with previous work performed after denervation/devascularization of EDL muscle from normal and mdx strains [27]. Similar slowing of the contractile responses has been also reported in regenerating rat muscle [18]. A normal characteristic of fast-twitch EDL muscle is a lack of resistance to fatigue. As already shown in previous work [2, 27, 32], in the present study, the C57BL/10 and mdx noncrushed EDL muscles, aged 12 and 20 weeks, became fatigued to the same relative tension in both age groups. Similarly, mdx slow-twitch SOL muscle shows no difference in the resistance to fatigue compared to normal muscle. However, resistance to fatigue was found to be higher in mdx diaphragm muscle compared to control muscle [39, and unpublished personal results]. A resistance to fatigue appears after a single crush in both strains and by 20 weeks,
resistance to fatigue is slightly higher in crushed EDL muscles from C57BL/10 strain compared with muscles from mdx strain. This finding may be in relation with the increase in oxidative fibre proportion observed after crush injury in the two strains. After denervation/devascularization, the operated mdx EDL muscle, which initially showed an increased resistance to fatigue, became almost as fatigueable as C57BL/10 non-operated EDL by 16 weeks after the surgical intervention [27]. In the cat EDL after transplantation, Faulkner et al. [40] noted that fatigue profile never returns to normal levels. As already reported [2, 27, 32], no significant differences were noted for PTP. PTP is a characteristic of fast-twitch skeletal muscle. We measured PTP according to the protocol described by Anderson et al. [2] and Mechalchuk and Bressler [27] in order to compare the fatigue of the uninjured or injured EDL muscles from mdx mice. Thus, it is suggested that following crush injury, as after denervation/devascularization [27], EDL muscle from mdx and C57BL/10 strains, does not lose some of its fast-twitch characteristics. We have shown that EDL muscles from C57BL/10 and mdx strains were mainly composed of type IIA and type IIB fibres. However, it is probable that IIX fibres were also present [41]. In this way, since IIA fibres are indistinguishable by ATPase staining from the 25-30% IIX fibres, it is not surprising that the numbers of type IIA fibres are higher than is typical for mouse EDL. Any intermediate fibres (I/IIA, IIA/IIX, IIX/IIB) are also likely to be included in the IIA category by ATPase staining. In this way, immunohistochemistry seems an important way to study the precise distribution of fibres in muscles. Thus, the present study provides morphological and functional evidence for a greater recovery of mdx compared to C57BL/10 muscle, but also for the recurrence of dystrophic features in mdx muscle regenerating from iterative crush injuries. Acknowledgements--The authors are most grateful to Dr Y. Ch6rel and Prof. M. Wyers for use of the image analysis system. This work was supported by a grant from the Association Franqaise contre les Myopathies (AFM). We thank Dr. S. Baudet for his critical evaluation of the English.
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