skeletal muscle regeneration after crush injury in dystrophic mdx mice: an autoradiographic study

The X-linked dystrophic animal model, the mdx mouse, shows an extraor- dinary capacity for sustained muscle regeneration compared with X-linked dystrophic golden retriever dogs and humans with Duchenne muscular dystrophy. To test the hypothesis that muscles of mdx mice might have inherently superior muscle regeneration to that in nondystrophic animals, muscle regeneration in response to a crush injury was examined in mdx mice and in the nondystrophic, parental strain C57B1/1 OSn. Autoradiographic tech- niques were used specifically to investigate the timing of muscle precursor replication after crush injury. Little difference in the regenerative capacity, either histologically or with respect to the timing of muscle precursor replication, was found between mdx mice and C57BI/lOSn or other nondystrophic strains of mice. Key words: dystrophy mdx mouse muscle regeneration autoradiogra- PhY

MUSCLE & NERVE 15:580-586 1992 ~~ ~

SKELETAL MUSCLE REGENERATION AFTER CRUSH INJURY IN DYSTROPHIC MDX MICE: AN AUTORADIOGRAPHIC STUDY

MIRANDA D. GROUNDS, BSc, PhD, and JOHN K. McGEACHIE, BDSc, PhD, FDSRCS

T h e mdx mouse' is of increasing interest as an animal model for X-linked Duchenne muscular dystrophy (DMD), particularly for investigating potential myoblast replacement therapy for this and other myopathies.20721 The myopathy in humans with DMD, in the mdx and mdx-like mice,6 and also in the X-linked dystrophic golden retriever and cat,4 are due to mutations22 in a very large gene on the X-chromosome which re- sult in the absence of a subsarcolemmal protein called dy~trophin, '~ the function of which is un- known.

From the Departments of Pathology (Dr. Grounds) and Anatomy and Hu- man Biology (Dr. McGeachie), The University of Western Australia, Ned- lands, Western Australia.

Acknowledgments: We thank Dr. L. Austin of the Department of Bio- chemistry, Monash University, Melbourne for the generous gift of the rndx and C5781/10Sn mice. We are grateful to A. Minten, K. Cole, and M. Seats of the Department of Pathology and M. Holmes, M Lee, and M. Thompson of the Department of Anatomy and Human Biology for techni- cal assistance This work was supported by the National Health and Medical Reserach Council (Dr Grounds) and the Nicholas and Eliza Mc- Cluskey Memorial Bequest (Dr. McGeachie).

Address reprint requests to Dr. M. Grounds, University Department of Pathlogy, Queen Elizabeth I1 Medical Centre, Nedlands, Western Austra- lia, 6009.

Accepted for publication May 9, 1991

CCC 01 48-639X/92/050580-07 $04.00 0 1992 John Wiley & Sons, Inc.

In the X-linked myopathies in humans and dogs, the dystrophic muscle fibers persistently un- dergo cycles of necrosis, muscle regeneration be- comes impaired, and the muscle is progressively replaced by fat and connective tissues, resulting in clinical wasting. In mdx mice, muscle regeneration is effective, and there is no long-term muscle weakness, although an increase in collagen con- tent of the muscles and a change in fiber type dis- tribution has been r e p ~ r t e d . ' ~ There is conflicting evidence regarding the extent of muscle wasting in aging mdx mice. Early reports indicate that mdx muscle necrosis and regeneration, which is conspicuous around 3 or 4 weeks of age, is essentially transient and is greatly reduced in older ani- m a l ~ . ~ ' ' ~ However, other studies suggest that the pattern of muscle necrosis and regeneration is relatively constant or only sli htl reduced throughout the life of mdx mice." !?.? * ' 23

In this study we test the possibility that mdx mice might have inherently more effective muscle regeneration than normal animals. Muscle regeneration after crush injury was examined in mdx mice and in the parental (nondystrophic) control strain (C57BVlOsn), using autoradiographic tech- niques to investigate specifically the timing of muscle precursor cell replication. Similar autoradiographic studies have been carried out previ-

580 Regeneration in mdx Mice MUSCLE & NERVE May 1992

ously in our laboratories using BALB/ci8 and SJL/J mice.”

MATERIALS AND METHODS

Fifteen inbred male mdx mice, aged 42 to 50 days, and 15 male C57B1110Sn mice (35 to 49 days old) received a single crush injury to the tibialis ante- rior (TA) muscle of the right leg.’’ Animal proce- dures were approved by the Animal Welfare Committee of the University of W.A. and the Na- tional Health and Medical Research Council of Australia.

To label replicating muscle precursor cells, tritiated thymidine (3H-Tdr: specific activity 5 Ci/ mol: Amersham, Australia) was injected intraperi- toneally (IP) into all mice at 1 p.Ci/g body weight, at times ranging from 24 to 120 hours after injury (Table 1). Each mouse received one injection of 3H-Tdr. Later, mice were anesthetised and killed by cervical dislocation. Regenerated TA muscles and uninjured TA muscles from the opposite leg were sampled and processed for autoradiography.” Muscle from 1 mdx and 1 C57BVlOSn mouse were removed 1 hour after 3H-Tdr injection at 72 hours, to quantitate labeling of premitotic nuclei.18 The progeny of muscle precursor cells labelled with 3H-Tdr were identified later as labeled muscle nuclei in multinucleated young muscle cells (myotubes) in samples taken at 10 days after injury (Table 1). The proportion of la-

beled myotube nuclei and grain counts per nucleus were analyzed in regenerated mdx and control muscles. In each sample, 200 myotube nuclei were counted. In uninjured muscles of control nondystrophic C57Bl/lOSn mice, where no myotubes were present, 200 myonuclei were analyzed.

RESULTS

Histology. Histologically, crush lesions sampled 10 days after muscle injury were similar in mdx mice and control C57Bl/lOSn mice. Large central areas of fibrous and cellular connective tissue with scattered myotubes were surrounded by a zone of well-formed myotubes (Fig. 1A and B). In mdx muscles, the precise limits of the lesion were diffi- cult to define, as many of the associated “uninjured” dystrophic myofibers had central nuclei due to the progressive regeneration, however, there were always sufficient numbers of myotubes clearly within the lesion. In the uninjured muscles of mdx mice, many myofibers had central nuclei and scattered small foci of newly formed myotubes were present. Uninjured muscles of control C57BV10Sn mice consisted of healthy myofibers devoid of central nuclei.

Autoradiography mdx Mice. In transverse sections of crush le-

sions from mdx mice injected with 3H-Tdr at 24

~~ ~~ ~~

Table 1. Injured and uninjured muscle from rndx mice, and Injured control C579111OSn muscle percentages of labeled myotube nuclei in regenerated muscles

Uninjured mdx Injured C57Bl/lOSn Injured mdx muscle muscle muscle

Time of 3H-Tdr injection % myotube nuclei % myotube nuclei % rnyotube nuclei Age of rndx mice at time of after injury (hours) labeled (*) labeled (*) labeled (*) 3H-Tdr injection (days)

24

30

36

48

72

96

120

1 (5) 1 (5)

10 (9) 7 (10)

13 (18) 5 (8)

26 (27) 19 (19) 20 (1 3) 16 (20) 12 (22) 10 (26) 10 (17)

10 (14)

43 43 43 43 43 51 52 52 53 45 46 46 47 47

Animals were injected with a single dose (1 pCiIg) of 3H-Tdr at 24 to 120 hours after injury, and sampled 10 days affer injury. *Maximum grain counts over myotube nuclei.

Regeneration in mdx Mice MUSCLE & NERVE May 1992 581

FIGURE 1. Low-power views of regenerated crush lesions 10 days after injury in (A) mdx and (B) C576111OSn mice. Areas of fibrous and cellular connective tissue with scattered myotubes are adjacent to relatively intact muscle tissue.

582 Regeneration in rndx Mice MUSCLE & NERVE May 1992

FIGURE 2. Examples of myotube nuclei in a crush lesion removed 10 days after injury from a mdx mouse. This animal had been injected with 3H-Tdr at 48 hours after injury. Autoradiographic grains in the photographic emulsion overlying the nuclei are in focus and, therefore, the underlying nuclei are slightly out of focus.

hours after injury, and sampled at 10 days, there was no significant labeling of myotube nuclei (Ta- ble l ) , indicating that muscle precursors had not been synthesizing DNA at 24 hours. The small percentage of myotube nuclei labeled (1%) and the low grain counts (5 5) can probably be attrib- uted to reutilization of 3H-Tdr. 10218 A substantial proportion of myotube nuclei were labeled (5% to 26%) and grain counts were much higher in crush lesions of mdx mice injected with 3H-Tdr from 30 to 120 hours after injury (Fig. 2). However, the proportion of myotube labeling, which was due to precursor cell replication resulting directly from the crush injury, was complicated by the ongoing regeneration in uninjured mdx muscles. The extent of this “background” labeling in uninjured mdx muscles was extremely variable (Table 1). These mdx mice were aged between 43 and 53 days at the time of 3H-Tdr injection (Table 1). the highest proportion of labeled myotube nuclei was 6%, although labeled myotube nuclei were not seen in 3 uninjured mdx muscles. Labeled myotube nuclei of uninjured mdx muscles were often in discrete foci of about 4 in a microscopic field (Fig. 3).

In some samples of both injured and uninjured mdx muscle, postmitotic labeling was noted in peripheral nuclei lying in the satellite cell position: these labeled myotube nuclei were not counted because they were not in the typical central position within the myotube.

In 1 mdx mouse (aged 45 days) injected with ’H-Tdr at 72 hours after injury, samples were removed after 1 hour to provide data on (premitotic) labeled myogenic cells, before they had time to divide. In uninjured mdx muscles, 0.63% of nuclei lying within the contour of the muscle fiber were labeled. These represent myonuclei lying within the sarcoplasm, either in a central or subsarcolemmal position, plus satellite cells located outside the plasmalemma but within the external lamina. Of the total population of muscle nuclei lying within the mdx muscle fibers, 53% (1000 of 1900) were located centrally, and none of these were labeled. Labeled premitotic nuclei were seen only in the peripheral, juxtasarcolemmal position, and represented 1.34% of the peripheral nuclear population.

In these regenerated muscles sampled at 10 days after crush in-

Control C57BlIlOSn Mice.

Regeneration in rndx Mice MUSCLE & NERVE May 1992 583

FIGURE 3. A group of 3 labeled nuclei (arrows) in adjacent myotubes in uninjured (control) dystrophic muscle of a mdx mouse. The muscle sample was removed 8 days after 3H-Tdr injection.

jury, few (1% and 2%) myotube nuclei were labeled, and grain counts were low (5 4) in mice which had been injected with 3H-Tdr at 24 hours after injury (Table 1). In mice injected at 30 and 36 hours, grain counts were slightly higher (up to 8 graidmyotube nucleus) although only 3.5% of myotube nuclei were labeled. Substantial labeling of myotube nuclei (1 1%) was seen at 48 hours, and the highest proportion of labeled myotube nuclei (33%) was at 72 hours after injury; labeling of myotube nuclei was also present in mice which had been injected at 96 hours, but was reduced in mice which had been injected at 120 hours after injury (Fig. 4).

There was essentially no labeling of muscle nuclei in uninjured muscles of the control (nondystrophic) C57BVlOSn mice, either in the 15 postmitotic samples or in the single premitotic sample.

DISCUSSION

The pattern of muscle precursor replication in crush injured mdx muscle is complicated by the persistent background regeneration in uninjured mdx muscle, so this will be discussed first.

Uninjured mdx Muscle. The labeling of myotube nuclei in uninjured, dystrophic muscles clearly shows that detectable numbers of muscle precursors are replicating in muscles of mdx mice aged 6 to 8 weeks. The autoradiographic labeling identi- fies only those muscle precursors which had been replicating within 1 hour of ‘H-Tdr injection. If these data are extrapolated throughout the TA muscle, then it appears that regeneration is very active. Furthermore, the small foci of labeled myotube nuclei, often in adjacent myotubes, suggest that relatively few muscle precursor stem cells contribute to the regeneration of discrete areas of focal necrosis.

In another autoradiographic study of dystrophic muscle,2 mdx mice 4 or 32 weeks of age were injected with 3H-Tdr, samples were removed 1 hour later, and the labelling of premitotic “sublaminal” nuclei (which include both satellite cells and myocnuclei) was reported. At 2 hours after 3H-Tdr injection, 2.90 ? 0.46% and 1.50 k 0.13% of sublaminal nuclei were labeled in 4- and 32- week-old mdx mice, respectively.2 The lowe labeling in older mice corresponds with our observa- tions in a mdx mouse aged 45 days, where 1.34%


A

35

30

'5 2 5 - 0 a z -

4 2 0 - c 0 >

U 0 al

1 5 - - - n 2 10- - 0

5 -

r

-

Regenerated mdx muscle H

Regenerated C57Bl /10Sn muscle A-A Uninjured mdx muscle 0 0

0

Onset / ,,,---.\

I t ,' \

- - - _ _ It 0

I 0' OI'O 0

2 4 4 8 7 2 9 6 120 144 168 192

yoo A 0 1 9 9 1

Time of 3H-Tdr Injection after Injury (hours)

FIGURE 4. A graphic representation of the labeling of myotube nuclei in regenerated muscles removed 10 days after crush injury in mdx and control C5781/1OSn mice: the labeling in uninjured dystrophic muscles of mdx mice is also shown. Lines drawn between mid-data points are simply for ease of comparative reference.

of juxtasarcolemmal premitotic nuclei were labeled. Since 3H-Tdr is available for about 1 hour after injection, only cells synthesizing DNA at this precise time are labeled. Of the muscle nuclei, only satellite cells are capable of replication, and indeed labelling was not seen in the centrally located nuclei, which represented about half of all muscle nuclei in the TA muscle of the 45-day-old mdx mouse. Satellite cells normally represent only 1% to 5% of all muscle nuclei, and are essentially quiescent in adult host muscle fibers.' The proportion of labeled (premitotic) satellite cells in mdx muscle (0.63% of total muscle nuclei) repre- sents a 50-fold increase over the minimal satellite cell turnover (0.014%) of uninjured muscles of adult BALB/c mice,lg and is slightly higher (0.5%) than in denervated BALB/c m u ~ c l e . ' ~

Regenerated Muscles of Dystrophic and Other Strains of Mice. The histological appearance of regenerated lesions in both mdx and control C57BVlOSn strains were similar, and showed a large central area of fibrous and cellular connective tissue surrounded by a peripheral zone of myotubes, corresponding with the appearance of crush lesions at 10 days in BALB/c mice.'' This

contrasts with the more effective muscle regeneration and limited fibrosis after a similar injury in muscles of SJL/J The similarity of regeneration in mdx and control C57Bl/lOSn mice indicates that dystrophic muscles of mdx mice do not have any exceptional capacity for muscle regeneration.

In mdx mice, muscle precursor replication was not apparent at 24 hours and started around 30 hours after injury. This corresponds with the ear- liest onset of DNA synthesis in muscle precursor cells after cut and crush injury in BALB/c mice.18 However, replication of muscle precursor cells was consistently seen earlier, at 24 hours after injury, in crush lesions of SJL/J mice, although not prior to this time.'' In contrast with the data for mdx, BALB/c, and SJL/J mice, few muscle precursor cells were replicating in control C57BVlOSn mice at 30 or 36 hours after injury. The reason(s) for this are not clear. It is possible that very few muscle precursor cells were available initially in C57BVlOSn muscle, as this strain appears to have low numbers of satellite cells compared with some other strains. l 3 Pronounced labeling of myotube nuclei (reflecting substantial muscle precursor cell replication) was first noted in 1 control C57B1/

Regeneration in mdx Mice MUSCLE & NERVE May 1992 585

10% mouse at 48 hours. In regenerating mdx muscle, the peak of muscle precursor cell replication around 48 to 72 hours, and the decrease in replication by 120 hours after crush injury, corre- sponded closely with previous data from male BALB/c and male and female SJL/J mice." Data from control C57BVlOSn mice also fit this pat-

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skeletal muscle regeneration after crush injury in dystrophic mdx mice: an autoradiographic study

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