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Identification of regenerated dystrophic minced muscle transplanted in normal mice
Journal o[the neurological Sciences, 1975, 24:33 38 ~ Elsevier Scientific Publishing Company, Amsterdam
33 Printed in The Netherlands
Identification of Regenerated Dystrophic Minced Muscle Transplanted in Normal Mice J. S. N E E R U N J U N
AND g . D U B O W I T Z
Department O/Paediatrics, Hammersmith Hospital, Du Cane Road, London WI2 0 H S (Great Britain) (Received 3 June, 1974)
INTRODUCTION
In the past few years there has been considerable support for a neural origin of muscular dystrophy (Dubowitz 1967, 1971 ; McComas and Mro~ek 1967; McComas and Sica 1970; McComas, Sica and Currie 1970; McComas, Sica and Campbell 1971 ; Bradley 1971 ; Harris and Wilson 1971 a,b; Salafsky 1971 ; Harris, Wallace and Wing 1972; Gallup and Dubowitz 1973; Hironaka and Miyata 1973). Two of these studies have shown that minced and whole dystrophic mouse muscles, when transplanted into normal hosts, develop normal twitch characteristics (Salafsky 1971: Hironaka and Miyata 1973). However, neither study showed whether the fully reconstituted transplanted muscle is formed from precursor cells derived from within the transplanted tissue or from those of the neighbouring muscle of the host. In the present study we have investigated the source of the resulting muscular elements by transplanting minced dystrophic and normal tibialis anterior muscles labelled with tritiated thymidine into normal unlabelled hosts. MATERIALS AND METHODS
The animals used in this study were homozygous normal ( + / + ), heterozygous (dy/+ ) and homozygous dystrophic (dy/dy) littermates of the 129 ReJ Bar Harbor strain of mice with hereditary muscular dystrophy. Labelled muscles were obtained from donor mice which came from litters born to 9 heterozygous females, each of which received intraperitoneally 50 pCi of tritiated thymidine ([-3H]thymidine, TRK 61; Amersham/Searle) on the day prior to delivery. Each newborn mouse then received 0.5 pCi of [3H]thymidine daily for 10 days, after which labelling was carried out at a dosage of 1 pCi/g body weight until both dystrophic and normal littermates were 60 days old. The total dose of [3H]thymidine administered to each dystrophic donor This work was supported by grants from the Medical Research Council and the Muscular Dystrophy Group of Great Britain.
34
J. S. NEERUNJUN, V. D U B O W I T Z
mouse ranged from 450 to 500 ItCi, whilst each normal donor mouse received 550--650 /LCi. Tibialis anterior muscles from labelled normal and dystrophic mice were removed, finely minced and transplanted into the legs of unlabelled normal mice of the same age, as described by Studitsky (see Carlson 1972). The peroneal nerve was left in the vicinity of the mince. In this series a total of 25 normal to normal and 25 dystrophic to normal transplantations were perfbrmed. After 20 days 4 normal and 4 dystrophic transplants were removed together with the host extensor digitorum longus (EDL) muscles and processed for autoradiography by the stripping film technique (Rogers 1967). One micron thick sections from 3° i, glutaraldehyde-fixed, Araldite-embedded specimens were exposed for 15 days to Kodak AR 10 stripping film. After exposure the preparations were developed and stained with 0.5" ii toluidine blue in acetone saturated with borax. The remaining transplants were removed at varying time intervals and the level of radioactivity in them was counted in a Packard Tri-Carb liquid scintillation spectrometer. The muscle samples were frozen, sliced, minced and dissolved in NCS solubiliser (Amersham/Searle) at 37 C. The counting fluid was toluene containing 7.0 g PPO (2,5-diphenyl-oxazole) and 0.5 g POPOP (l,4-bis-[2-(4-methyl-5-phenyloxazolyl)]-benzene) per litre. Radioactivity was also determined in the EDL and the contralateral tibialis anterior muscles of the host. The efficiency of 3H-counting was 30 i}ii. The rate at which radioactivity was lost from the intact labelled muscle was followed over the same time intervals in 13 normal and 13 dystrophic labelled mice. A further series of 5 dystrophic to normal and 4 normal to normal unlabelled transplants were also performed for histological examination. After 20 days the transplants were removed together with the adjacent host EDL muscles and frozen in isopentane cooled in liquid nitrogen. Cryostat sections, 10 ~m thick, were stained with Harris haematoxylin and eosin.
RESULTS
By 20 days both normal and dystrophic transplants showed extensive regeneration in the normal host. Figs. I A, B, C and D illustrate the histology of a typical intact normal and dystrophic tibialis anterior muscle and that of a normal to normal and dystrophic to normal 20-day-old transplant. Both transplants were separated from the host EDL muscle by a layer of connective tissue. A number of fibres in the transplants had central nuclei and were undergoing splitting. Consequently there was some variation in fibre size. Central nuclei were also present in some of the host muscle fibres at the junction between the host and the transplanted tissue. Transplants processed for autoradiography consisted of muscle fibres with labelled nuclei (Fig. 2). On the other hand, none of the host muscle fibres had radioactive nuclei. A count of labelled nuclei in 400 fibres indicates that 60'),~, of the muscle fibres in both types of transplant had radioactive nuclei. A similar count on the labelled muscle at the time of transplantation showed that 72'!/o of the nuclei were labelled. Both central and peripheral nuclei of the regenerated fibres were labelled. The intensity of labelling of nuclei varied considerably in both normal and dystrophic trans-
IDENTIFICATION OF REGENERATED DYSTROPHIC MINCED MUSCLE
A
B
C
I)
35
Fig. 1. Histology of an intact normal (A) and dystrophic (B) tibialis anterior muscle~ a 20-day-old normal to normal (C) and dystrophic to normal (D) transplant, H = h o s t . T=transplant. HE, x 320.
plants in that the central nuclei appeared to have more silver grains over them than those situated peripherally. Radioactive mononuclear cells were also seen in the endomysial connective tissue. Counts of radioactivity confirmed the autoradiographic findings. Most of the activity was localised within the transplants whilst the EDL and the contralateral tibialis anterior host muscles had very low levels of activity (Figs. 3 A and B). Even after 30 days the transplants had more activity than the host muscles. Although both the dystrophic and normal donor muscles were adequately labelled at the time of transplantation, the amount of radioactivity fell rapidly so that by 50 days the levels of activity in the transplants were similar to those in the host muscles. A similar pattern of loss of activity was observed in the intact muscles of labelled dystrophic and normal mice, thus indicating that dilution of radioactivity in the transplants was
36
J. S. NEERUNJUN, V. DUBOWI]Z
Fig. 2. A 20-day-old dystrophic to normal transplant showing labelled nuclei only within ll~e mmsplanl. H = host, T = transplant. Toluidine blue-Borax, x 800.
2750.
~
1800! __e u 900.
~aoo] i
~
~°~
E
-..._ E 400ci u
2750..
\
E 4oo-
[3
200.
900-
u 200I
100 t
~° $__.~;~------ I
50 -~ / 0-----~ 0 _ _ Q ._--..------0 ~V.---.....v/v ~ V 0
10
20 30 40 Time (days)
50
•
t 6~
C',
10
20 30 40 Time (days!
50
6~
Fig. 3. Radioactivity counts/min at varying time intervals in (A) intact labelled normal tibialis anterior (z% A), normal to normal transplant ([] []), host EDL muscle adjacent to the transplant ( 0 0), contralateral tibialis anterior muscle of the host (V - V ), and (B) intact labelled dystrophic tibialis anterior ( • - - • ) , dystrophic to normal transplant (g---ml), host EDL muscle adjacent to the transplant (O • ) , contralateral thibialis anterior muscle of the host ( • - &).
due to turnover of nucleic acid and not due to infiltration of host muscle precursor cells into the transplants. DISCUSSION
The results of our investigation show that: (a) precursor muscle cells involved in the reconstitution o f the transplanted muscle originate from within the donor tissue: and
IDENTIFICATION OF REGENERATED DYSTROPHIC MINCED MUSCLE
37
(b) murine dystrophic muscle regenerates successfully in the environment of a normal host. Morphologically both normal and dystrophic transplants show similar features of extensive cellular infiltration, muscle fibres with central nuclei and variation in fibre size. Although the reconstituted muscles appear to be formed from muscle elements which originated from the transplants themselves, it is not known whether the myoblasts are derived entirely by budding from pre-existing multinucleated muscle fragments or from satellite cells. According to Waldeyer (1865), Volkmann (1893). Le Gros Clark (1946), Godman (1957)and Reznik (1969, 1970), damaged muscle repairs itself by a continuous process which involves budding of myoblasts from existing muscle fibres. On the other hand, the discovery of satellite cells (Mauro 1961 ) has led several investigators (Church, Noronha and Allbrook 1966; Shafiq, Gorycki and Milhorat 1967: Ontell 1974) to suggest that these cells are precursor myoblasts which undergo rapid multiplication during physical and chemical injury in order to repair the damaged muscle. As both transplants show similar histological features, it appears that the dystrophic transplants fare just as well as the normal transplants in the environment of the normal hosts. Since physiological studies on dystrophic muscles transplanted for longer periods show normal physiological twitch characteristics (Salafsky 1971 ; Hironaka and Miyata 1973), and normal and dystrophic transplants appear similar in morphology, it is quite likely that the dystrophic muscle becomes normal. Furthermore, as minced muscles undergo abortive regeneration in denervated rat limbs (Zhenevskaya 1958; Studitsky, Zhenevskaya and Rumyantseva 1963), it seems likely that the normal motor innervation determines the state of the regenerated transplanted dystrophic muscle. This is substantiated by the fact that dystrophic mice do not support the regeneration of either normal or dystrophic transplants (Laird and Timmer 1966; Neerunjun and Dubowitz 1974). ACKNOWI.EDGEMENTS
We thank Miss C. Heinzman for technical assistance and Mrs. C. Maunder for valuable help and advice on autoradiography. The operations were carried out under Home Office Licence No. 33867. SUMMARY
The origin of the reconstituted normal and dystrophic transplants in normal mice of the Bar Harbor 129 ReJ strain was investigated by transplanting [3H]thymidinelabelled minced tibialis anterior muscles into the legs of unlabelled hosts. After 20 days the transplants were processed for autoradiography and histology. At varying time intervals between 0 and 50 days radioactivity counts were made on the transplants and compared with those from the adjacent EDL and contralateral tibialis anterior muscles of the hosts. Both autoradiography and radioactivity counts showed that the transplanted muscles were formed from muscle cells derived from within the donor tissue. Moreover, normal and dystrophic transplants from normal hosts were histologically similar.
38
.1. S. NEERUNJUN. V. I)UBOWITZ REFERENCES
BRADLEY, W. G. (1971) Nerve, muscle and muscular dystrophy, Devehq~. Med. Child Neurcd. t ~ : 528 531 CARLSON, B. M. (1972) The Regeneration (~[Mineed Muscles, Karger, Basel. CHURCH, J. C. T., R. F. X. NORONHAAND D. B. AELnRCX~K(1966) Satellite cells and skeletal muscle regeneration, Brit. J. Surg., 53: 638-642. CUARK, W. E. LE GROS (1946) An experimental study of the regeneration of m a m m a l i a n striped muscle, J. Anat.( Lond.), 80: 24-36. D/'Bowlrz, V. (1967) Pathology of experimentally re-innervated skeletal muscle. J. Ncu;o/ Neurosur~l. Psychiat., 30:99 110. DUBOWITZ, V. (1971 ) Muscular dystrophy where is the lesion ? Develop. Med. Child Neuro/.. t 3 : 238--24(I. GALLUP, B. AND V. DUBOWITZ (1973} Failure of 'dystrophic" neurones to support functional regeneration of normal or dystrophic muscle in culture, Nature (Lond.), 243:287-289. GODMAN, G. C. (1957)On the regeneration and redifferentiation of m a m m a l i a n striated muscle, J Mot. phol.. 100 : 27- 82. HARRIS, J. B. AND P. WILSON (197ta) Denervation in murine dystrophy, Nature ~Lond. 2, 22~): t~l-62. HARRIS, J. B. AND P. WILSON (1971b) Mechanical properties of dystrophic muscle, J. Neuro/ Neurosur¢l. Ps.l'chiat., 34 : 512-520. HARRIS, J. B., C. WALLACE AND I. WING (1972) Myelinated nerve fibre counts in nerves of normal and dystrophic mouse muscle, J. Neurol. Sci., 15: 245-249. HIRONAKA. T. AND Y. MIYATA (1973) Muscle transplantation in the aetiological elucidation of murine muscular dystrophy. Nature New Biology, 244: 221-223. LAIRD. J. L. AND R. F. TIMMER (1966} Transplantation of skeletal muscle into a host with muscular dystrophy, Texas Rep. Biol. Med., 24:169 179. MCCOMAS, A. J. AND K. MROZEK (1967) Denervated muscle fibres in hereditary mouse dystrophy, J. Neurol. Neurosurg. Psychiat., 30: 526-530. McCOMAS. A. J. AND R. E. P. SICA (1970) Muscular dystrophy: myopathy or neuropathy'.' La,cet, i: 1119. M(-COMAS, A. J.. R. E. P. SI('A AND S. CURRIE (1970) Muscular dystrophy: evidence for a neural factor, Nature ( Lond.), 226:1263 1264. McComAs, A. J., R. E. P. SICA AND M. J. CAMP1~ELL(1971) ~Sick" motor neurones. A unifying concept of muscle disease, Lancet, i: 321 325. MAURO, A. (1961) Satellite cell of skeletal muscle fibres, J. biophys, biochem. ('ytol., 9:493-495. NEERUNJUN, J. S. AND V. DuBowrrz (1974) In preparation. ONTEt, L, M. (1974) Muscle satellite cells: A validated technique l\3r light microscopic identification and ~ quantitative study of changes in their population following denervation, Anat. Rec.. 178 : 211 228. REZNIK, M. (1969) Origin of myoblasts during skeletal muscle regeneration. Electron microscopic observations. Lab. hn~est., 20: 353-363. REZN1K, M. (1970) Satellite cells, myoblasts, and skeletal~nuscle regeneration. In : A. Maurr~, S A. SHM I~) AND A. T. MIL~IORA'r {Eds.), Regeneration (~['Striatcd Muscle, and Myogenesis (Proceedings of an International Conference held under the auspices of the Muscular Dystrophy Associations of America, New York. N. Y., 1969) (International Congress Series, No. 218) Excerpta Medica, Amsterdam, p p 133-156. ROGERS. A. W. (1967) Techniques o[Autoradiography, Elsevier, Amsterdam, London, New York, SM At-SKY, B. (1971) Functional studies of regenerated muscles from normal and dystrophic mice, Nature (Loud.), 229 : 270 273. SHMqQ, S. A,, M. A. GORYCKI AND A. T. MILHORAT (1967) An electron microscopic study oF regeneration and satellite cells in h u m a n muscle, Neurology (Minneap.), 17: 567-574. SrIIDITSKY, A. N., R. P. ZHENEVSKAYAAND O. RUMYANTSEVA(1963) The role of neurotrophic influences upon the restitution of structure and function of regenerating muscles. In : E. GurmANN ~nD P. HniK (Eds.), The El~[Pct (?[' Use and Disuse in Neuromuscular Functions, Publishing House Czechoslovak Acad. Sci., Prague, pp. 71-81. VOEKr*IaNN, R. (1893) Ueber die Regeneration des quergestreiften Muskelgewebes beim Menschen und Saugetier, Beitr. path. Anat., 12 : 233-332. WALDEYER, W. (1865) Ueber die Verfinderungen der quergestreiften Muskel bei der Entzfindung und dem Typhus-prozess, sowie fiber die Regeneration derselben nach Substanz-defecten, Virchows Arch. path. Anat. Physiol., 34: 473. ZHENEVSKAYA, R. P. (1958) The role of nervous connections on the early stages of muscle regeneration, Dokl. Akad. Nauk. SSSR, 121: 182-185.
33 Printed in The Netherlands
Identification of Regenerated Dystrophic Minced Muscle Transplanted in Normal Mice J. S. N E E R U N J U N
AND g . D U B O W I T Z
Department O/Paediatrics, Hammersmith Hospital, Du Cane Road, London WI2 0 H S (Great Britain) (Received 3 June, 1974)
INTRODUCTION
In the past few years there has been considerable support for a neural origin of muscular dystrophy (Dubowitz 1967, 1971 ; McComas and Mro~ek 1967; McComas and Sica 1970; McComas, Sica and Currie 1970; McComas, Sica and Campbell 1971 ; Bradley 1971 ; Harris and Wilson 1971 a,b; Salafsky 1971 ; Harris, Wallace and Wing 1972; Gallup and Dubowitz 1973; Hironaka and Miyata 1973). Two of these studies have shown that minced and whole dystrophic mouse muscles, when transplanted into normal hosts, develop normal twitch characteristics (Salafsky 1971: Hironaka and Miyata 1973). However, neither study showed whether the fully reconstituted transplanted muscle is formed from precursor cells derived from within the transplanted tissue or from those of the neighbouring muscle of the host. In the present study we have investigated the source of the resulting muscular elements by transplanting minced dystrophic and normal tibialis anterior muscles labelled with tritiated thymidine into normal unlabelled hosts. MATERIALS AND METHODS
The animals used in this study were homozygous normal ( + / + ), heterozygous (dy/+ ) and homozygous dystrophic (dy/dy) littermates of the 129 ReJ Bar Harbor strain of mice with hereditary muscular dystrophy. Labelled muscles were obtained from donor mice which came from litters born to 9 heterozygous females, each of which received intraperitoneally 50 pCi of tritiated thymidine ([-3H]thymidine, TRK 61; Amersham/Searle) on the day prior to delivery. Each newborn mouse then received 0.5 pCi of [3H]thymidine daily for 10 days, after which labelling was carried out at a dosage of 1 pCi/g body weight until both dystrophic and normal littermates were 60 days old. The total dose of [3H]thymidine administered to each dystrophic donor This work was supported by grants from the Medical Research Council and the Muscular Dystrophy Group of Great Britain.
34
J. S. NEERUNJUN, V. D U B O W I T Z
mouse ranged from 450 to 500 ItCi, whilst each normal donor mouse received 550--650 /LCi. Tibialis anterior muscles from labelled normal and dystrophic mice were removed, finely minced and transplanted into the legs of unlabelled normal mice of the same age, as described by Studitsky (see Carlson 1972). The peroneal nerve was left in the vicinity of the mince. In this series a total of 25 normal to normal and 25 dystrophic to normal transplantations were perfbrmed. After 20 days 4 normal and 4 dystrophic transplants were removed together with the host extensor digitorum longus (EDL) muscles and processed for autoradiography by the stripping film technique (Rogers 1967). One micron thick sections from 3° i, glutaraldehyde-fixed, Araldite-embedded specimens were exposed for 15 days to Kodak AR 10 stripping film. After exposure the preparations were developed and stained with 0.5" ii toluidine blue in acetone saturated with borax. The remaining transplants were removed at varying time intervals and the level of radioactivity in them was counted in a Packard Tri-Carb liquid scintillation spectrometer. The muscle samples were frozen, sliced, minced and dissolved in NCS solubiliser (Amersham/Searle) at 37 C. The counting fluid was toluene containing 7.0 g PPO (2,5-diphenyl-oxazole) and 0.5 g POPOP (l,4-bis-[2-(4-methyl-5-phenyloxazolyl)]-benzene) per litre. Radioactivity was also determined in the EDL and the contralateral tibialis anterior muscles of the host. The efficiency of 3H-counting was 30 i}ii. The rate at which radioactivity was lost from the intact labelled muscle was followed over the same time intervals in 13 normal and 13 dystrophic labelled mice. A further series of 5 dystrophic to normal and 4 normal to normal unlabelled transplants were also performed for histological examination. After 20 days the transplants were removed together with the adjacent host EDL muscles and frozen in isopentane cooled in liquid nitrogen. Cryostat sections, 10 ~m thick, were stained with Harris haematoxylin and eosin.
RESULTS
By 20 days both normal and dystrophic transplants showed extensive regeneration in the normal host. Figs. I A, B, C and D illustrate the histology of a typical intact normal and dystrophic tibialis anterior muscle and that of a normal to normal and dystrophic to normal 20-day-old transplant. Both transplants were separated from the host EDL muscle by a layer of connective tissue. A number of fibres in the transplants had central nuclei and were undergoing splitting. Consequently there was some variation in fibre size. Central nuclei were also present in some of the host muscle fibres at the junction between the host and the transplanted tissue. Transplants processed for autoradiography consisted of muscle fibres with labelled nuclei (Fig. 2). On the other hand, none of the host muscle fibres had radioactive nuclei. A count of labelled nuclei in 400 fibres indicates that 60'),~, of the muscle fibres in both types of transplant had radioactive nuclei. A similar count on the labelled muscle at the time of transplantation showed that 72'!/o of the nuclei were labelled. Both central and peripheral nuclei of the regenerated fibres were labelled. The intensity of labelling of nuclei varied considerably in both normal and dystrophic trans-
IDENTIFICATION OF REGENERATED DYSTROPHIC MINCED MUSCLE
A
B
C
I)
35
Fig. 1. Histology of an intact normal (A) and dystrophic (B) tibialis anterior muscle~ a 20-day-old normal to normal (C) and dystrophic to normal (D) transplant, H = h o s t . T=transplant. HE, x 320.
plants in that the central nuclei appeared to have more silver grains over them than those situated peripherally. Radioactive mononuclear cells were also seen in the endomysial connective tissue. Counts of radioactivity confirmed the autoradiographic findings. Most of the activity was localised within the transplants whilst the EDL and the contralateral tibialis anterior host muscles had very low levels of activity (Figs. 3 A and B). Even after 30 days the transplants had more activity than the host muscles. Although both the dystrophic and normal donor muscles were adequately labelled at the time of transplantation, the amount of radioactivity fell rapidly so that by 50 days the levels of activity in the transplants were similar to those in the host muscles. A similar pattern of loss of activity was observed in the intact muscles of labelled dystrophic and normal mice, thus indicating that dilution of radioactivity in the transplants was
36
J. S. NEERUNJUN, V. DUBOWI]Z
Fig. 2. A 20-day-old dystrophic to normal transplant showing labelled nuclei only within ll~e mmsplanl. H = host, T = transplant. Toluidine blue-Borax, x 800.
2750.
~
1800! __e u 900.
~aoo] i
~
~°~
E
-..._ E 400ci u
2750..
\
E 4oo-
[3
200.
900-
u 200I
100 t
~° $__.~;~------ I
50 -~ / 0-----~ 0 _ _ Q ._--..------0 ~V.---.....v/v ~ V 0
10
20 30 40 Time (days)
50
•
t 6~
C',
10
20 30 40 Time (days!
50
6~
Fig. 3. Radioactivity counts/min at varying time intervals in (A) intact labelled normal tibialis anterior (z% A), normal to normal transplant ([] []), host EDL muscle adjacent to the transplant ( 0 0), contralateral tibialis anterior muscle of the host (V - V ), and (B) intact labelled dystrophic tibialis anterior ( • - - • ) , dystrophic to normal transplant (g---ml), host EDL muscle adjacent to the transplant (O • ) , contralateral thibialis anterior muscle of the host ( • - &).
due to turnover of nucleic acid and not due to infiltration of host muscle precursor cells into the transplants. DISCUSSION
The results of our investigation show that: (a) precursor muscle cells involved in the reconstitution o f the transplanted muscle originate from within the donor tissue: and
IDENTIFICATION OF REGENERATED DYSTROPHIC MINCED MUSCLE
37
(b) murine dystrophic muscle regenerates successfully in the environment of a normal host. Morphologically both normal and dystrophic transplants show similar features of extensive cellular infiltration, muscle fibres with central nuclei and variation in fibre size. Although the reconstituted muscles appear to be formed from muscle elements which originated from the transplants themselves, it is not known whether the myoblasts are derived entirely by budding from pre-existing multinucleated muscle fragments or from satellite cells. According to Waldeyer (1865), Volkmann (1893). Le Gros Clark (1946), Godman (1957)and Reznik (1969, 1970), damaged muscle repairs itself by a continuous process which involves budding of myoblasts from existing muscle fibres. On the other hand, the discovery of satellite cells (Mauro 1961 ) has led several investigators (Church, Noronha and Allbrook 1966; Shafiq, Gorycki and Milhorat 1967: Ontell 1974) to suggest that these cells are precursor myoblasts which undergo rapid multiplication during physical and chemical injury in order to repair the damaged muscle. As both transplants show similar histological features, it appears that the dystrophic transplants fare just as well as the normal transplants in the environment of the normal hosts. Since physiological studies on dystrophic muscles transplanted for longer periods show normal physiological twitch characteristics (Salafsky 1971 ; Hironaka and Miyata 1973), and normal and dystrophic transplants appear similar in morphology, it is quite likely that the dystrophic muscle becomes normal. Furthermore, as minced muscles undergo abortive regeneration in denervated rat limbs (Zhenevskaya 1958; Studitsky, Zhenevskaya and Rumyantseva 1963), it seems likely that the normal motor innervation determines the state of the regenerated transplanted dystrophic muscle. This is substantiated by the fact that dystrophic mice do not support the regeneration of either normal or dystrophic transplants (Laird and Timmer 1966; Neerunjun and Dubowitz 1974). ACKNOWI.EDGEMENTS
We thank Miss C. Heinzman for technical assistance and Mrs. C. Maunder for valuable help and advice on autoradiography. The operations were carried out under Home Office Licence No. 33867. SUMMARY
The origin of the reconstituted normal and dystrophic transplants in normal mice of the Bar Harbor 129 ReJ strain was investigated by transplanting [3H]thymidinelabelled minced tibialis anterior muscles into the legs of unlabelled hosts. After 20 days the transplants were processed for autoradiography and histology. At varying time intervals between 0 and 50 days radioactivity counts were made on the transplants and compared with those from the adjacent EDL and contralateral tibialis anterior muscles of the hosts. Both autoradiography and radioactivity counts showed that the transplanted muscles were formed from muscle cells derived from within the donor tissue. Moreover, normal and dystrophic transplants from normal hosts were histologically similar.
38
.1. S. NEERUNJUN. V. I)UBOWITZ REFERENCES
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