Journal of the Neurological Sciences, 1978, 38:77-82 © Elsevier/North-Holland Biomedical Press

77

F U R T H E R OBSERVATIONS ON M Y E L I N A T E D A X O N N U M B E R S I N N O R M A L A N D D Y S T R O P H I C MICE

A. MONTGOMERY and L. SWENARCHUK Department of Zoology, University of Toronto, Toronto, Ont. MSS 1A1 (Canada)

(Recieved 7 December, 1977) (Accepted 30 March, 1978)

SUMMARY Counts of the number of myelinated axons in the nerves to slow-twitch soleus and fast-twitch plantaris muscles of 129 ReJ dy/dy dystrophic mice at 4, 15 and 24 weeks of age have shown statistically significant reductions when compared to normal values. In addition muscle fibre losses had occurred in both muscles at the earliest age point studied. There was no suggestion of a progressive loss of myelinated axons. There was a greater percentage reduction in the soleus than in the plantaris nerves. The number of myelinated axons in the nerves to the plantaris and soleus muscles of the 129 ReJ - k / + normal animals was approximately the same, in contrast to the C57BL/6 J strain where the soleus nerve always contained a greater number. Statistically significant reductions in the number ofmyelinated axons in the nerves to the medial gastrocnemius muscles of C57BL/6J dy2J/dy 2J dystrophic mice have been observed at both 15 and 72 weeks of age. Muscle fibre losses have also been recorded in 72 weeks dystrophic medial gastrocnemius muscles.

INTRODUCTION Recent studies (Montgomery, 1976; Montgomery and Swenarchuk, 1977) have shown that between 1 week and 15 weeks of age a progressive reduction in the number of muscle fibres of both fast and slow-twitch muscles of the C57BL/6Jdy2Z/dy2J dystrophic

This work was supported by funds provided by the Muscular Dystrophy Association of Canada. Present address: Muscular Dystrophy Research Laboratories, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne NE4 6BE, Great Britain.

78 strain of mice takes place. At I week there was already a demonstrable reduction in the number of myelinated axons in the soleus nerve. By 4 weeks similar reductions were apparent in the nerve innervating the plantaris muscle. In view of these findings, it was felt that a study of the soleus and plantaris nerves and muscles of young and old 129 ReJ dystrophic mice was necessary to determine if similar losses could also be identified in this strain. It has been shown (Bray and Aguayo 1975) that the nerve to the medial gastrocnemius muscle of 129 B6F1 dy/dy dystrophic mice had fewer myelinated axons present than the nerves of normal animals. However, only a small number of animals were used and it was felt that a statistical analysis should be carried out if possible. This we have obtained for 15- and 72-week-old C57BL/6J dy2J/dyzJ dystrophic and normal mice and for 24-week-old 129 ReJ dy/dy dystrophic and normal animals. In addition we examined the medial gastrocnemius muscles of the oldest animals to try to determine if muscle fibre losses had occurred. MATERIALSAND METHODS Procedure for nerves

The techniques used in preparing transverse sections were the same as those previously described (Montgomery and Swenarchuk 1977). Myelinated axon counts were made from nerves innervating the soleus and plantaris muscles of 129 ReJ dy/dy dystrophic and 129 ReJ -k-/-t- normal animals. In each case an easily accessible unifascicular portion of the nerve was available from which the total number of myelinated axons could be counted. At least 7 values were obtained for each of 3 age points, 4, 15 and 24 weeks; this latter age is about the average lifespan of the 129 ReJ dy/dy dystrophic animals. When breeding from animals heterozygous for dystrophy 4 weeks is a stage in development at which dystrophic mice can be clearly distinguished from their (homozygous and heterozygous) clinically normal littermates. The nerve to the medial gastrocnemius muscle also presented a readily accessible unifascicular portion. This nerve was examined in 24-week 129 ReJ normal and dystrophic animals and also in both 15- and 72-week C57BL/6J dy2J/dy2J dystrophic and C57BL/6J + / + normal animals. Total muscle fibre numbers were obtained from transverse sections of 129 ReJ +/q- normal and 129 ReJ dy/dy dystrophic soleus muscles at the 3 age points of 4, 15 and 24 weeks. The plantaris and medial gastrocnemius muscles have a complex architecture, thus it was not possible to obtain the total number of muscle fibres simply by counting those seen in one transverse section taken through the belly of the muscle. However, it was felt that a reasonable estimate of any muscle fibre losses could be made by obtaining a transverse section halfway along the muscle length, counting the total number of muscle fibres seen and then comparing normal and dystrophic values. This procedure was adopted for 4-, 15- and 24-week-old normal and dystrophic 129 ReJ plantaris muscles, for 24-week normal and dystrophic 129 ReJ medial gastrocnemius and 72-week C57BL/6J normal and dystrophic medial gastrocnemius muscles.

79 TABLE1 MEAN MYELINATED AXON NUMBERS FROM THE NERVES TO THE SOLEUS AND PLANTARIS MUSCLES OF 129 ReJ dy/dy DYSTROPHIC AND 129 ReJ ÷ / ÷ NORMAL MICE AT 4, 15 AND 24 WEEKS OF AGE Soleus nerve

Plantaris nerve

Age (weeks)

Age (weeks)

4 Myelinated axon numbers from normal 129 ReJ ÷ / + mice mean 82.3 SE 2.40 N 8 Myelinated axon number from dystrophic 129 ReJ dy/dy mice mean 67.7 SE 3.03 N 7 t-value 9.08 Degrees of freedom 13 P < 0.001

15

24

4

15

24

86.5 1.39 7

85.2 1.45 15

85.0 2.14 8

87.4 2.36 7

85.7 2.32 12

67.0 2.85 7 15.07 12 < 0.001

65.6 1.80 16 36.0 29 < 0.001

77.7 2.15 7 6.13 13 < 0.001

78.4 2.91 7 5.85 12 < 0.001

78.5 1.76 18 2.95 28 < 0.001

RESULTS

129 ReJ soleus and plantaris nerves The mean myelinated axon numbers from the nerves to the plantaris and soleus muscles of 129 ReJ q-/q- normal and 129 ReJ dy/dy dystrophic animals are presented in Table 1. At each age point there was a reduction in the mean number of myelinated axons present in the nerves of dystrophic animals when compared to normal. The differences between normal and dystrophic mean values were all statistically significant, using Student's t-test (P = 0.05). The percentage reductions of the dystrophic mean values were always larger in the soleus nerves, about 20 %, than in the plantaris nerves, about 10 ~o. There was no evidence of a progressive loss of myelinated axons in either nerve. It should be noted that in this strain the mean numbers of myelinated axons in the soleus and plantaris nerves of normal animals were about the same.

129 ReJ soleus and plantaris muscles The muscle fibre numbers of normal and dystrophic soleus muscles at each of the age points were similar to previously reported values (Rowe and Goldspink 1969a and b). The counting technique employed with the plantaris muscle showed mean percentage reductions of 47.5 ~o; 53.9 % and 52.4 ~o at 4, 15 and 24 weeks respectively.

C57BL/6J and 129 ReJ medial gastrocnemius nerves Table 2 shows the mean myelinated axon numbers from the medial gastrocnemius

80 nerves of 15- and 72-week-old C57BL/6J +/-+ normal and C57BL/6J dyZa/dy~,j dystrophic mice. Also shown are the myelinated axon numbers from the nerve to the medial gastrocnemius muscles of 24-week normal and dystrophic 129 ReJ animals. In each case there was a statistically significant difference (Student's t-test, P =- 0.05) between the respective 15-, 24- and 72-week normal and dystrophic mean values. Statistically significant differences were also apparent between the 15- and 72-week normal C57BL/6J mean values and between the 15- and 72-week dystrophic C57BL/6J mean values, the older animals having the lower mean value in each case. It is also of interest to note the higher absolute values obtained from the nerves of both normal and dystrophic 129 ReJ animals.

C57BL/6J and 129 ReJ medial gastrocnemius muscles The mean percentage reduction in the muscle fibre numbers of 129 dy/dy dystrophic medial gastrocnemius muscles was similar to those reported by Rowe and Goldspink (1969b) and Johnson and Montgomery (1975) for tibialis anterior muscles, that is about 48 ~ . For the C57BL/6J dyZJ/dyzJ dystrophic medial gastrocnemius muscles the figure was 50.3 ~ . DISCUSSION The 20 ~ reduction of the mean myelinated axon number in the soleus nerve of 129 ReJ dy/dy dystrophic mice reported here confirms that finding of Law and Atwood (1974). The difference between the absolute mean values obtained from these two observations probably reflects the use of the light and electron microscope techniques used for counting purposes.

TABLE2 MEAN MYELINATED AXON NU MBERS FROM THE MEDIAL GASTROCNEMIUS NERVE oF 15- AND 72-WEEK C57BL/6J dy2~/dy~a DYSTROPHIC AND C57BL/6J + / + NORMAL AN~IMALS. ALSO SHOWN ARE THE NUMBERS FROM 24-WEEK 129 ReJ dy/dy DYSTROPHIC AND 129 ReJ + / + NORMAL MICE Myelinated axon numbers of Mean medial gastrocnemius nerve 15 weeks C57BL/6J + / + normal 15 weeks C57BL/6J dy2a/dy 2J dystrophic 72 weeks C57BL/6J + / + normal 72 weeks C57BL/6J dyga/dy2a dystrophic 24 weeks 129 ReJ + / + normal 24 weeks 129 ReJ dy/dy dystrophic

SE

N

128.3

3.09

7

118.4

4.44

10

117.5

2.74

13

97.1

4.63

14

137.7

3.87

15

123.2

4.21

17

t-value

Degreesof freedom

P

4.35

15

< 0.001

13.26

25

< 0.00l

9.77

30

< 0.001

81 In contrast to the C57BL/6J dyZJ/dyzJ dystrophic animals the 129 ReJ dy/dy dystrophic mice showed no progressive losses of myelinated axons between 4 and 24 weeks. The respective 20 7oand 10 7olosses that were observed in the soleus and plantaris nerves of 129 ReJ dystrophic mice were already present at 4 weeks. Also at the 4-, 15and 24-week age points the percentage reductions of the dystrophic soleus and plantaris mean myelinated axon numbers were always less in the 129 ReJ than in the C57BL/6J dystrophic mice (Montgomery 1976; Montgomery and Swenarchuk 1977). In both strains, however, the soleus nerve at each age point always showed a greater deficit than the plantaris nerve. The mean numbers ofmyelinated axons in the plantaris and soleus nerves of both normal and dystrophic 129 ReJ animals were always greater than in the same nerves from respectively age-matched C57BL/6J normal and dystrophic animals. This is probably also the case in the medial gastrocnemius muscle nerves of 129 ReJ normal and dystrophic animals (see Table 2) but no age-matched normal and dystrophic C57BL/6J animals were available for direct comparison. These differences in myelinated axon number probably reflect a real strain difference. Another aspect of such a strain difference was that in C57BL/6J q-/q- normal animals at each age point, the soleus nerve always had a higher mean myelinated axon number than the plantaris nerves, whereas 129 ReJ -k/q- normal animals showed little difference between soleus and plantaris at each age point. The observations on the nerves to the medial gastrocnemius muscles showed that the lower mean numbers of myelinated axons present in the nerves of both dystrophic strains at each age point were statistically significantly different from normal. Thus the observation of Bray and Aguayo (1975) have been extended and confirmed. Although the 72-week dystrophic mean value was reduced when compared to the 15-week dystrophic value and suggested a progressive loss of myelinated axons, a similar age-related change was apparent between 15- and 72-week normal animals. Thus it appears that the medial gastrocnemius nerve probably does not show the progressive loss of myelinated axons known to occur in the soleus and plantaris nerves (Montgomery and Swenarchuk 1977). However, it may well be that it is necessary to examine these nerves at earlier ages than 15 weeks before it can be more definitely stated that this nerve does not show progressive myelinated axon losses. In each of the dystrophic muscles examined in this study from both allelic strains of dystrophic mouse, fibre losses have been recorded. In addition, myelinated axon deficits have been recorded in the nerves that innervated these several muscles. Whilst there were differences between the two strains with regard to the percentage reduction in the dystrophic mean myelinated axon number at each age point, it would, however, seem that a major difference between the two strains is one of chronology. Thus the bulk of the muscle fibre losses and possibly myelinated fibre losses in the 129 ReJ dy/dy dystrophic mice, occurred at an earlier stage of development than in the C57BL/6J dyZJ/dye~ dystrophic strain. It would be of considerable value to know the extent of muscle fibre and myelinated axon losses in neo-natal 129 ReJ dystrophic mice.

82 ACKNOWLEDGEMENTS I a m m o s t grateful to Professor H a r o l d A t w o o d for his e n c o u r a g e m e n t a n d advice a n d also to Mrs. Irene K w a n for her valuable technical assistance.

REFERENCES Bray, G. M. and A. J. Aguayo (1975) Quantitative ultrastructural studies of the axon Schwann cell abnormality in spinal nerve roots from dystrophic mice, J. Neuropath. exp. Neurol., 34: 517-530. Johnson, M. A. and A. Montgomery (1975) Parabiotic reinnervation in normal and dystrophic mice, Part 2 (Morphological studies), J. neurol. Sci., 26: 425--441. Law, P. and H. L. Atwood (1974) Does axonal sprouting occur in dystrophic mouse muscles? Experientia (Basel), 30: 155-156. Montgomery, A. (1976) Myelinated axon numbers in the peripheral nerves of normal and dystrophic mice, Int. Res. Comm. Syst. reed. Sci., 4: 561. Montgomery, A. and L. Swenarchuk (1977) Dystrophic mice show age related muscle fibre and myelinated axon losses, Nature (Lond,), 267: 167-169. Rowe, R, W. D. and G. Goldspink (1969a) Muscle fibre growth in five different muscles in both sexes of mice, Part 1 (Normal mice), J. Anat. (Lond.), 104: 519-130. Rowe, R. W. D. and G. Goidspink (1969b) Muscle fibre growth in five different muscles in both sexes of mice, Part 2 (Dystrophic mice), J. Anat. (Lond.), 104: 531-538.

Further observations on myelinated axon numbers in normal and dystrophic mice.

Journal of the Neurological Sciences, 1978, 38:77-82 © Elsevier/North-Holland Biomedical Press 77 F U R T H E R OBSERVATIONS ON M Y E L I N A T E D...
317KB Sizes 0 Downloads 0 Views