EXPERIMENTAL

NEUROLOGY

Transplantation

TETSUJI Dcpartnmt

47,

1-15

(1975)

of Skeletal Dystrophic HIRONAKA

of

Pkarrrlacology, Dcrltal Unizwsity, Rt,cciz,cd

Muscle Mice

AND Faculty of Batrlkyo-ku, Scptcrnber

WUHEI

in Normal

MIYATA

and

1

Medicine, Tokyo Medical Tokyo 113, Japa?t

arld

20, 1974

The entire extensor digitorum longus muscles were cross-transplanted between 25-40 day-old littermates of normal and dystrophic mice (strain C57BL/6J). Between 120 and 150 days after operation, muscle weight, histological features and physiological properties of the muscles were examined. Reinnervation was well established for a normal extensor digitorum longus muscle transplanted into normal host (Nn) or dystrophic host (Nd), and a dystrophic extensor digitorum longus muscle transplanted into normal host (Dn). However, the degree of reinnervation was 70% for a dystrophic extensor digitorum longus muscle transplanted into dystrophic host (Dd). The twitch contractions of Nn or Dn extensor digitorum longus muscles were comparable to the fast twitch contractions of the nonoperated normal muscles. Those of Nd or Dd extensor digitorum longus muscles became as slo’w as the nonoperated dystrophic muscles. The maximum isometric twitch and tetanic tension per unit cross sectional area for Dn and Nd extensor digitorum longus muscles were intermediate between those for the two controls of Nn and Dd extensor digitorum longus muscles. The mean fiber diameter of Dn and Nd extensor digitorum longus muscles was intermediate between Dn extensor digithose for Nn and Dd extensor digitorum longus muscles. torum longus muscles showed an increase in lveight comparable to Nn extensor digitorum longus muscles. Conversely, Nd or Dd extensor digitorum longus muscles deteriorated. The results of the present study suggest an important role for some extramuscular, possibly neural, factor(s) in murine muscular dystrophy.

1 Dr. Hironaka’s present address is : Department of Pharmacology, Teikyo Univcrsity School of Medicine, Itabashi-ku, Tokyo 173, Japan. We thank Professor M. Otsuka for encouraging this study, reading our manuscript and offering helpful suggestions. We were also indebted to the Central Institute for Experimental Animal for providing dystrophic mice. This work was supported in part by a grant from the Ministry of Health and Welfare, Japan.

Copyright All rights

0 1975 by Academic Press, Inc. of reproduction in any form reserved.

HIRONAKA

AND

MIYATA

INTRODUCTION Michelson, Russell and Harman (28) have provided an inbred strain of laboratory mice with hereditary muscular dystrophy which has become a useful tool for research in human muscular dystrophy. Many investigations have been made to determine the etiology of murine dystrophy. It has been assumed that muscular dystrophy was within the ambit of the concept of primary myopathy (2, 21, 33, 41). However, several recent studies suggest that the dystrophic process may originate primarily in the nervous system (11, 22, 26, 27, 37) and there is strong evidence for various types of trophic interaction between nerve and muscle (16, 19, 20). In the present study, the interactions between the muscle and the host (nerve) was analyzed with regard to mechanical properties and histochemical features, using the cross-transplantation technique of entire extensor digitorum longus muscles (24) between normal and dystrophic mice. The results suggest that a neural disorder plays an important role in the dystrophic processes. Parts of this study have appeared elsewhere (23,25).

METHODS Strain C57BL/6J-dy mice of three genotypes were used: dystrophic (dy/dy ) , hetrozygous normal (Dy/dy ) and homozygous normal (Dy/Dy ) . Dystrophic mice were identified by their abnormal movements 25-40 days after birth (21)) during which the transplantation of extensor digitorum longus muscle was performed. The pairs of mice between which extensor digitorum longus muscles were cross-transplanted were of four different types : normal donor-normal recipient (Nn) ; normal donor-dystrophic recipient (Nd) ; dystrophic donor-normal recipient (Dn) ; dystrophic donor-dystrophic recipient (Dd). In all cases, the pairs were littermates of the same sex in order to avoid possible immunological reactions. No reactive cell infiltration into the transplanted muscle area was observed (see also 24). The characteristics of the transplants were examined at 120-150 days after transplanation. Two pairs of Nn and Dd mice were used for operative controls. Contralateral normal and dystrophic extensor digitorum longus muscles were used for nonoperative controls. Surgical Procedures. Under ether anesthesia,estensor digitorum longus muscleswere exposed in the right hind limbs of the paired mice and rapidly transplanted into the area vacated by removal of the corresponding muscle of the other animal. Both tendons of the muscle were tied to the neighboring tissues with a fine thread in order to maintain some muscle tension. Maintenance of tension at the ends of the muscle was found to promote viability. For example, in preliminary experiments where extensor digi-

MUSCULAR

DYSTROPHY

FIG. 1 Isometric twitch contractions of nonoperated extensor digitorum longus muscles of normal (A) and dystrophic (B) mice. The twitches were produced by nerve stimulation irt sitzc at 30°C in 27 and 30 day-old mice, respectively. The cross sectional area of the muscles was calculated as in Table 1. Histochemical features (succinic dehydrogenase) of the corresponding normal (C) and dystrophic (D) muscles.

torum longus muscles were transplanted to littermates of the same sex and body weight with fixation of the tendons, the ratio of weight of transplants to that of nonoperated extensor digitorum longus muscles of contralateral side was 69.1 k 6.3% (mean * SE) 150 days after transplantation. On the other hand, when both ends of the muscle were tied but not fixed to the neighboring tissues, the muscle weight ratio was 43.7 -+ 5.S%. This difference was statistically significant (P < 0.02). Operations were performed under a dissecting microscope and care was taken not to injure the muscles. The left hind limb was left unoperated. The muscles were transplanted in many cases enclosed by Saranwrap distally (see Fig. 1 in Hironaka and Miyata, 23). This device prevented adhesions between the transplants and surrounding tissues so that nerves and blood vessels were stimulated to reinnervate and revascularize the transplanted muscles in the proximo-distal direction. Animals were kept in aseptic conditions throughout the operation and maintained on penicillin for a few days afterwards. Physiological Procedures. Transplants were examined 120-150 days after operation.

The

animals

were

anesthetized

with

intraperitoneal

injection

of

sodium pentobarbitone (Nembutal. 75 “g/kg). The extensor digitorum longus muscles were exposed and the distal two-thirds dissected free, so the main

blood

vessels supplying

these muscles

were

kept intact.

The

an-

terior tibialis muscle was then removed and peroneal muscles separated from the transplant as much as possible to avoid their accidental excitation when the transplant was directly stimulated. All branches of the sciatic

nerve

to muscles

other

than the extensor

digitorum

longus

muscle

4

HIRONAKA

AND

MIYATA

were sectioned and the nerve was then cut centrally. The distal tendon was connected by a thin wire to a mechano-electronic transducer (5734A, Toshiba). Total mechanical compliance was less than 1 X 1O-4 cm/g. Muscles were either stimulated directly through a pair of massive platinum plate electrode or indirectly via their nerves using supramaximal square electrical pulses of 20-30 +ec duration. Muscle contractions were evoked under comparable conditions of initial tension (the initial tension was adjusted to produce maximum twitch contraction). The experiments were performed with the muscles bathed in saline at 28-30” C. Histological and Histochemical Procedures. After recording the contractile properties, transplanted muscles were excised, weighed and then frozen on dry ice in isotonic saline. Serial sections of lo-20 p thickness were prepared for histological (hematoxylin and eosin) and/or histochemical observation. Total cross sectional area of an entire muscle was measured in the largest of the serial sections. Sections used for succinic dehydrogenase reaction were dried in a small vacuum desicator and stained by the method of Nachlas, Tsou, Souza, Cheng and Seligman (30). Incubation time was 30 min at 37” C and the sections were finally mounted in glycerine jelly. Muscle fiber diameter was measured with these preparations. RESULTS Nonoperated Muscles: Dynamic Properties. At the time when the muscles were transplanted 24-40 days after birth, small differences in the dynamic properties of extensor digitorum longus muscles between normal and dystrophic mice were seen (Fig. 1) . Results are summarized in Table 1. The twitch and tetanic tensions of normal muscles at this time were closely similar to those of the fully matured muscles. However, contraction time and half relaxation time were rather slower reflecting immaturity of the muscle fibers (see also Fig. 1C and 8, 10, 17, 18). The twitch and tetanic tensions of the dystrophic muscles were significantly smaller than those of the normal muscles (P < 0.01). The twitch contraction and half relaxation times of the dystrophic muscles were also significantly slower than those of the normal muscles (P < 0.05). The mean ratio of twitch tensions produced by nerve stimulation to those produced by muscle stimulation was slightly less than unity for dystrophic mice (Table 1) . Histological Properties. In the normal extensor digitorum longus muscle at 25-40 days after birth, the average number of muscle fibers was 948 and average muscle weight was 6.5 mg (Table 2). The total number of muscle fibers in dystrophic mice at this time was 466 and the muscle weight was 4.1 mg on average (Table 2). The difference in muscle length

MUSCULAR

DYSTROPHY

TABLE DYNAMIC

PROPERTIES

OF NONOPERATED

MUSCLES

OF NORMAL

extensor

digitorum

EXTENSOR

AND

longus

DICITORUM

DYSTROPHIC

4RT (mscc)

CT (msec)

PIl/Plll

Normal

1 LONGUS

MICE~

Twitch tension (kg/cm”)

Tetanic tension (!ig/cm”)

muscle

A. 25-40 days n = 11

1.04 f

0.11

24.1 zt 0.1

22.2 rt 1.6

0.23 zk 0.02

1.57 f

B. 145-190 days n = 21

1.08 f

0.06

22.1 f

18.1 & 1.8

0.32 f 0.02 n = 17

1.88 f 0.12 n = 17

29.2 f

2.4

0.10 f

0.02

0.50 f. 0.10

43.0

4.7

0.09 f

0.01

0.56 f

Dystrophic

extensor

digitorum

longus

1.4

muscle

A. 25-40 days n = 11

0.90

zk 0.05

27.2

B.

0.70

4~ 0.06

37.0 h 3.4

145-190 n=7

days

0.19

zk 1.1

f

0.10

a Mean values f SE of measurements on a number (n) of normal dystrophic extensor (B) days after birth. The digitorum longus muscles from mice 25-40 (A) and 145-190 abbreviations above the columns are for maximum isometric twitch contraction produced by nerve stimulation (Pn), that produced by muscle stimulation (Pm), and isometric twitch contraction time (CT) and half relaxation time (4RT). The wet weight of the muscle divided by the length of superficial muscle fiber is used as a measure of muscle cross-sectional area, assuming the density of the muscle to be unity. The tetanic tension was induced by repetitive stimulation for 200 msec at a rate of 100 count/set and the maximum tension was measured.

that may be assumed proportional to differences in tibia length was very slight between normal and dystrophic mice (Table 2), and mean diameter of muscle fibers was almost the same between the two groups of animals (Table 2), so that the lesser muscle weight of the whole dystrophic muscle at this stage should be entirely due to a decrease in number of muscle fibers. The difference in the dynamic and histological properties between normal and dystrophic mice became more pronounced with the lapse of time (Figs. 1, 2; Table 1). Histochemical Properties. With succinic dehydrogenase staining techniques, the staining properties are related to the diameter of the muscle fibers (32, 39). The three types of muscle fibers seen in this study are called A, B and C fibers after Stein and Padykula (39). The percentages of each fiber type in normal extensor digitorum longus muscle, which were calculated from the measurements in nine fully matured mice 135-190 days after birth,

HIRONAKA

AND

TABLE

MIYATA

2

PROPERTIES OF NONOPERATED EXTENSOR DIGITOHUM LONGUS MUSCLES AND THE TIHA OF NORMAL AND DYSTRO~HIC MICE’ Muscle weight bw) Normal

extensor

A. 25-40

A. 25-40

longus

days

B. 145-190

Dystrophic

digitorum

days

extensor

12.2 f 0.4 n = 43

days

B. 145-190

a The values show mean f sured for fiber size. n : number

Fiber size (m)

Tibia length (mm)

948 f 50 n = 11

50.4 f 1.8 n = 11

14.7 f 0.1 n = 12

859 f ?2=8

62.5 f n=7

17 8 f 0.1 n = 12

muscle

6.5 i 0.3 n = 16

digitorum

Number of muscle fibers

longus

44

3.3

muscie

4.1 f 0.2 ?a = 14

466 f 23 n = 11

49.4 f 1.2 n = 11

13.4 i ?&=a

4 5 f 0.2 n = 20

423 f 43 n = 10

55.9 f n=7

16.6 f 0.1 n = 10

SE. The largest diameter at transverse of muscle or bone measured.

3.7

sections

0.1

was mea-

are as follows : A fiber: 45.4 + 2.09’76 (large pale) I3 fiber : 268 + 1.29% (intermediate) C fiber: 24.8 f 1.41% (small dense) where the values show the mean * SE, respectively (see also Fig. 3). In normal mice, the three types of muscle fiber were distinguishable 25-40 days after birth (Fig. 1C). As the occupancy of A fiber in terms of cross-sectional area was more than SO%, estimated from above data and those of Stein and Padykula (39), the contractile properties of the extensor digitorum longus muscles analyzed in the present study predominantly represent the characteristics of A fiber, i.e., fast twitch fibers. In the dystrophic mice, muscle fibers of varying diameters were present, but the enzymatic reaction of small fibers was weaker than that of normal mice and haphazard degeneration was observed (Fig. 1D). The contractile properties of dystrophic extensor digitorum longus muscles with the characteristics similar to those of slow skeletal muscles may not be due to a preferential involvetnent of ,4 fibers in the dystrophic processesbut due to the abortive “maturation” of all types of muscle fibers (cf. 8, 10). Transplanted Mzrscles: Suroiznl atld Nistorhenziral Features. The four types of transplanted muscles (Ivn, Dn, Nd and Dd) could all survive the

MUSCULAR

FIG. 2 transplanted extensor extensor extensor hlethods

DYSTROPHY

Histochemical features (succinic dehydrogenase activity) of nonoperated extensor digitorum longus muscles 120-150 days after operation. A, digitorum longus muscle; 13, D extensor digitorum longus muscle; C, digitorum longus muscle; E, digitorum longus muscle : D, Dd extensor digitorum longus muscle ; F, Nd extensor digitorum longus muscle. for designation of the types of muscles.

7

and N Nn Dn See

transplantation well some 120-150 days after the operation. The color of the transplants looked more reddish than the surrounding muscles, suggesting a good blood supply for the muscles. When normal or dystrophic extensor digitorum longus muscles were transplanted into normal host the normal mosaic pattern of the muscle fiber types (Fig. 2-4) was replaced by a pattern characterized by islands or clusters of a similar type (Fib.m 2C, E) as ~vas observed after self-reinnervation (36, 42). Histochemical features of the Dn extensor digitorum longus

8

HIRONAKA

AND

MIYATA

FIG. 3 Isometric twitch contractions of transplanted extensor digitorum longus muscles and nonoperated extensor digitorum longus muscles of contralateral side. All records of the contractions were obtained from the muscles 120-150 days after the operation in situ at 28-30°C. The largest cross sectional area in serial sections of the muscle was used in calculation. A, N extensor digitorum longus muscle; B, D extensor digitorum longus muscle; C, Nn extensor digitorum longus muscle;D, Dd extensor digitorum longus muscle;E, Dn extensordigitorum longusmuscle; F, Nd extensor digitorum longus muscle, For designation of the types of muscles, see the

Methods. muscle (Fig. 2E) closely resembles that of the Nn extensor digitorum longus muscle (Fig. 2C), both of which contain the three types of muscle fibers similar to those of the N extensor digitorum longus muscle (Fig. 2A). Degeneration of muscle fibers was scarcely observed in these two cases. When normal or dystrophic muscles were transplanted into dystrophic host, on the other hand, there was haphazard degeneration of muscle fibers (Fig. 2D, F) which was more prominent in the latter case (Dd, Fig. 2D). Lowered succinic dehydrogenase activity in smaller fibers and accelerated activity in hypertrophied fibers (see 2 for hypertrophied fibers) were observed (Fig. 2D, F). In Nd extensor digitorum longus muscle (Fig. 2F) as well as in D extensor digitorum longus muscle (Fig. 2B) target fibers were observed (cf. 15). Tension Development and Extent of Reinnervation. All types of transplants were capable of developing tension when the nerve was stimulated (Fig. 3). The mean ratio of twitch tensions produced by nerve stimulation to those by muscle stimulation was approximately unity for N, Nn, Dn and Nd extensor digitorum longus muscles (Tables 1, 3)) suggesting full reinnervation of the muscles. The ratio for Dd and D extensor digitorum longus muscleswas 0.7, suggesting that “functional denervation” occur when both muscle and host are dystrophic. Contraction Time and Half Relaxation Time. Figure 3 and Table 3 show that the contraction and half relasation times of Nn and N estensor digitorum longus muscles were approximately equal. Similarly, Dd and

MUSCULAR

DYSTROPHY

TABLE DYNAMIC Type

Nn

Dn

Nd

PKOPERTIES of muscle

OF TRANSPLANTED

3 EXTENSOR

DIGITOKCM

LONGUS

Pn/Pm

CT (msec)

+RT (msec)

Twitch tension (kg/cm~)

MUSCLES~ Tetanic tension (kg/my

extensor digitorum

longus

1.05 hO.11 ?2=8

20.1 * 1.4 n = 11

17.2 f 1.2 n = 11

0.10 &0.02 n=6

0.55 f n=6

extensor digitorum

longus

1.13 f 0.06 ?I = 12

22.1 f 1.8 n = 12

17.7 f 0.9 n = 12

0.06 f 0.01 n=7

0.40 -I 0.06 n = 7

0.96 f 0.16

31.2 zt 4.1

29.4 f 4.4

0.06 f 0.01

0.40 Ik 0.07

0.70 & 0.05

36.9 f 4.7

32.9 zk 4.4

0.01 f

0.06 AZ 0.02

extensor digitorum n=6

longus

Dd extensor digitorum n=6

longus

0.003

0.10

a The measurements were made with the muscles 120-150 days after transplantation. The abbreviations used are the same as those in Table 1 (see also Methods). For condition measuring tetanic tension, see Table 1. The cross sectional area of the muscles was measured through histological procedures in this case as the largest of the serial sections of each case. The tetanic tension per cross sectional area revaluated by this histological procedures for N extensor digitorum longus muscles in Table 1 was 1.13 kg/cm2 instead of 1.88 kg/cm’.

D extensor digitorum longus muscles had comparable speed of contraction. These results suggest that the operative procedure alone did not significantly alter the time course in twitch contraction of transplanted muscles. After dystrophic and normal muscles were cross transplanted into normal and dystrophic hosts respectively, the contractile properties of Dn and Nd extensor digitorum longus muscles were reversed (Fig. 3 E, F) and significantly different from one another (P < 0.01, Table 3). It has been shown in cross union experiments that the kinetic properties of myosin ATPase, calcium uptake of endoplasmic reticulum and so on, which are correlated with the time course of tlvitch contraction (3, 14), are regulated by the innervation (4, 7, 29, 35). Thus, it seems likely that the altered contractile properties of dystrophic muscles may be due to an abnormality (e.g., altered trophic function) of the nervous system in dystrophic animals. Isometric Twitch ad Tetanic Tcmion. The peak twitch tension was about the same for both the Dn and Nd extensor digitorum longus muscles, but it was rather larger in the Nn extensor cligitorum longus

10

HIRONAKA

AND

TABLE

MIYATA

4

PROPERTIES OF TRANSPLANTED EXTENSOR DIGITORUM LONGUS MUSCLES Type

Nn

of muscle

Muscle weight (4

Number of muscle fibers

Fiber size

Number of muscle fibers / 1 X 104fim2

t.4

extensor digitorum

8.7 f 0.6 ?I = 19

725 f n=6

68

61.1 f n=7

2.5

4.0 f n=7

0.3

longus

Dn extensor digitorum

4.6 f 0.5 n = 26

410 f n=8

42

55.5 f n=7

3.9

4.3 f n=7

0.6

longus

extensor digitorum

5.5 f 0.7 n = 12

497 f n=7

9

55.1 zk 1.6 n=7

3.6 f n=7

0.3

longus

extensor digitorum

1.9 f n=8

19.5 f n=7

37

48.4 f 72=5

2.8 f n=5

0.6

longus

Nd

Dd

0.4

2.1

0 The measurements were made with the muscles 120-150 days after transplantation. The values show mean f SE. For designation of type of muscle, see Methods. Fiber size was measured as the largest diameter at transverse sections through middle of muscles.

muscle, though the difference between the Dn and Nn extensor digitorum longus muscles was not so highly significant (P < 0.1). The Dd muscle still showed much smaller tension output than the others in this evaluation. In tetanic tension, the Dn and Nd extensor digitorum longus muscles attained rather lower tension output than the Nn extensor digitorum longus muscles (Table 3), although the tension output for these three groups of muscles showed no statistically significant differences from each other. Since the cross sectional area measured contained the degenerating fibers and connective tissues, if one should revaluate the area using the mean diameter of the muscle fibers and the number of fibers occupying a constant area (Table 4)) one could obtain a roughly comparable amount of tension output for the Nd extensor digitorum longus muscle to that for the Nn extensor digitorum longus muscle in tetanus as well as in twitch. In contrast, the Dd extensor digitorum longus muscle attained substantially smaller tension output in tetanus than the others. In experiments for evaluating the effect of operation, the isometric tetanic tension for the Nn extensor digitorum longus muscle was greatly reduced to 49% of that of the N extensor digitorum longus muscle 1201.50 days postoperatively. This operative effect gives rather equivocal analysis for tension output.

MUSCULAR

DYSTROPHY

11

Histological Analysis. Change in number of muscle fibers: The number of muscle fibers exhibited a postoperative decrease in all cases (Tables 2, 4). It can be said, however, that the viability of dystrophic muscle fibers was well retained in normal host, while that of normal and dystrophic muscle fibers was greatly reduced in dystrophic host (Tables 2, 4). In this case the normal muscles bvere rather more resistant to degenerating change than the dsytrophic muscle when transplanted into the dystrophic hosts. Change in fiber size: The largest diameter of each muscle fibers was used as the estimate of fiber size. This parameter manifested as a difference between normal (Nn) and dystrophic (Dd) mice after the operative treatment (P < 0.01, Tables 2, 4). When extensor digitorum longus muscles were cross transplanted betiveen dystrophic and normal mice, these two muscles attained intermediate size between the two controls of Nn and Dd estensor digitorum longus muscles (Table 4). Change in number of muscle fibers per a cross sectional area: The degree of degeneration is numerically represented in Table 4, with the number of muscle fibers occupying a cross-sectional area, 1 X lo4 p2. As will also be seen in Fig. 2C-2F degeneration of muscle fibers was more prominent in dystrophic host than in normal ones, and in dystrophic hosts the dystrophic muscle (Dd) was prominent in degeneration than normal muscle (Nd). Change in muscle weight: The muscle growth was variably retarded in each type of muscle after transplantation (Tables 2, 4). In spite of such substantial operative effect on muscle weight, it is evident that the dystrophic changes in weight of the transplanted muscles strongly depend on the difference in types of hosts. DISCUSSION When entire estensor digitorum longus muscles of mice were transplanted, the transplants regenerated well to complete replicae (24). In the present study, cross transplantation of the extensor digitorum longus muscles was performed between normal and dystrophic mice. The dystrophic muscle reinnervated by normal nerve and the normal muscle reinnervated by dystrophic nerve may provide a suitable model for analyzing interactions between muscle and nerve. The transplants (Dn and Nd) were well reinnervated by each host nerve fibers as suggested by ratios of Pn/Pm in Table 3. But the dystrophic extensor digitorum longus muscles transplanted into the dystrophic host (Dd) showed “functional denervation” as suggested by the ratio of 0.7 (Table 3). These results indicate that both muscle and host failures were concomitantly required for developing the “functional denervation.”

12

HIRONAKA

AND

MIYATA

The muscle failure necessary for the “functional denervation” should primarily originate in the muscle tissue itself, because if it were brought about secondarily through a host side failure, Dd extensor digitorum longus muscles should have attained as much the reinnervation as Nd extensor digitorum longus muscles, considering the fact that the transplanted muscles mostly regenerated (24). The dystrophic muscles (Dn) were fully reinnervated by the nerve fibers of normal mice and well preserved for a long period of time (Table 3). This experimental fact suggests that the host side failure necessary was not brought about secondarily for the “functional denervation” through the muscle failure. An Exclusive Decrease in Tension in Transplanted Muscles. Maximum tension per unit cross-sectional area of Nn extensor digitorum longus muscle was markedly reduced, by about SO%, compared with that of Nextensor digitorum longus muscle (Tables 1, 3). Since the contraction time, half relaxation time and twitch/tetanus tension ratio of Nn extensor digitorum longus muscles were almost the same with those of N extensor digitorum longus muscles (Tables 1, 3), it is probable that the processes that would bring the contractile elements to the active or inactive state were well retained after transplantation (3, 13). This remarkable reduction of tension output, therefore, might be attributable to a disproportionate decrease of contractile proteins per cross sectional area and/or to some structural change in the proteins as would result in relatively smaller tension output for a given activation of the contractile system. There is a possibility that about half the fibers might have lost the ability to contract. Histochemical and histological observations revealed that Nn extensor digitorum longus muscles contained enzymatically well matured fibers and no target fiber (Fig. 2C), and attained no substantial decrease in the fiber size as in the case of denervation atrophy (Table 4 ; also see 20). These experimental facts suggest that the individual fibers of the Nn extensor cligitorum longus muscle received all their own innervation. This possibility, therefore, may not be the case. Etiology of Murine Dystrophy. In the interpretation of the results in the present experiments, the origin of the regenerated muscle is a critical point (23). Accumulating evidence suggests that it is of the donor muscle (12,35). We have obtained the results that reflect differences in characteristics of hosts, e.g., a. contraction and half relaxation times, b. number of muscle fibers per a muscle and a cross-sectional area, and c. muscle weight, which led us to assume that some regulatory disorder may exist in the nervous system of dystrophic mice, as already discussed in results. Although this experimental model used in the present study is able to re-

MUSCULAR

DYSTROPHY

13

fleet various types of host side effects, some other extramuscular factors than neural ones, e.g., hormonal, nutritional and a reduced metabolic rate, seems unlikely to be of significance for dystrophic processes (1, 5, 6, 9, 31, 34,40). The data analyzed in the present study showed a multiplicity of factors. Different from the results of Salafsky (34) w‘1IO used the minced tibialis anterior muscles, we could obtain the normal and dystrophic fm~ctional regenerates (Nd and Dd) in the dystrophic host limbs (Tables 3, 4 ; Fig. 2 D, F). Results obtained in this manner reflected dystrophic failure of not only host but also transplanted muscle. The parameters are: a. the fraction of muscle reinnervation (Pn/Pm), b. twitch and tentanic tension, and c. mean fiber size (Fig. 3 ; Tables 3, 4). The 120-150 days of period after which the transplants were examined would be long enough for Nd and Dn extensor digitorum longus muscles to attain their interactions between muscle tissue and host (24). A simple interpretation of these results is that dystrophic origin on these parameters may lie in both muscular and estramuscular factors. We did not obtain the results reflected by muscle characteristics alone. This fact suggests that muscular dystrophy of mice is not a primary myopathy, because if it were, the dystrophic muscle would have retained its dystrophic condition even in the normal host. The most likely problem in dystrophic failure of mice may be in the nervous system. REFERENCES 1. BAKER, N., M. TUBIS, and W. H. BLAHD. 1958. Metabolic and nutritional studies in mice with a hereditary myopathy (Dystrophia muscularis). ilrjzcr. J. Physiol. 193 : 525-529. 2. BANKER, B. Q., and D. DENNY-BROWN. 1959. A study of denervated muscle in normal and dystrophic mice. J. Nruropafh. hp. Ncnrol. 18: 517-530. 3. BARANY, M. 1967. ATPase activity of myosin correlated with speed of muscle shortening. J. Gor. Plrysiol. 50 : [Supp., Part 21 197-218. 4. BARANY, M., and R. I. CLOSE. 1971. The transformation of myosin in crossinnervated rat muscles. J. PI~~‘siol. 213 : 455474. 5. BERG, L., F. G. EBAVGH JR., G. M. SHY, B. HORVATH, and D. J. CCMMINGS. 1955. Blood content of dystrophic muscles. J. .4ppl. Physiol. 8: 31-32. 6. BOND, C. F., and S. L. LEONARD. 1959. Total blood, erythrocyte and plasma volumes in muscular dystrophic mice. Poor. Sot. Exp. Biol. Md. 100: 189191. 7. BULLER, A. J., W. F. H. M. MOJIMAERTS, and Ii. SERAYDARIAN. 1969. Enzymic properties of myosin in fast and slow twitch muscles of the cat following crossinnervation. J. Pl~.vsiol. 205 : 581-597. 8. BULLER, A. J., J. C. ECCLES, and R. M. ECCLES. 1960. Differentiation of fast and slow muscles in the cat hind limb. J. Physiol. 150: 399-416. 9. CANAL, N., and FRATTOLA, L. 1962. Studies on the pentose phosphate pathway in hereditary muscular dystrophy in mice. Med. Exp. 7: 27-31.

14

fIIRONAI

Transplantation of skeletal muscle in normal and dystrophic mice.

EXPERIMENTAL NEUROLOGY Transplantation TETSUJI Dcpartnmt 47, 1-15 (1975) of Skeletal Dystrophic HIRONAKA of Pkarrrlacology, Dcrltal Unizwsity...
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