Journal of the Neurological Sciences, 1977, 33:243-249

243

© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

A B N O R M A L L Y C O M B I N E D M Y E L I N A T E D AND U N M Y E L I N A T E D NERVES IN D Y S T R O P H I C MICE

E. OKADA, V. MIZUHIRA and H. NAKAMURA

Department of Cell Biology ( EO, VM), Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-Ku, Tokyo, and Division of Neuropathology (HN), Institute of Neurological Sciences, Tottori University School of Medicine, 86 Nishi-Cho, Yonago 683 (Japan)

(Received 31 January, 1977)

SUMMARY Abnormally combined M- and U-nerves were observed in the peripheral nerves of C57BL dystrophic mice. They were counted in the sciatic nerves of the dystrophic mice 3 times more frequently than in control mice. The myelin disappeared beyond the nodes of Ranvier just as in the dysmyelination of dystrophic mice. Some of the U-axons inside the myelin were enveloped by Schwann cell cytoplasm while others were not. The myelinated U-axons contained cored vesicles, dilated endoplasmic reticulum and degenerate mitochondria. These abnormalities might cause disordered function and be related to the pathophysiology of murine muscular dystrophy.

INTRODUCTION In cutaneous nerves of the human fetus from 10-22 weeks, bundles of unmyelinated (U-) axons were not enveloped by cytoplasm or basement membranes of Schwann cells. These were called "very immature nerves" by Gamble (1966). Demyelination in peripheral neuropathy has been described in many types of study on humans and experimental animals (Greenfield, Blackwood, McMenemey, Meyer and Norman 1958; Davison and Peters 1970; Bradley 1974; Dyck, Thomas and Lambert 1975). Demyelination with an increase in the number of Schwann nuclei is observed in hypertrophic interstitial neuritis, leprosy, peroneal muscular atrophy and in many other conditions (Cammermeyer 1956; Greenfield et al. 1958; Simpson and Fowler 1966). In the central nervous system, demyelination is observed in chronic experimental allergic encephalomyelitis and multiple sclerosis (Raine, Snyder, Valsamis and Stone This work was supported in part by a research grant for specificdisease from the Ministry of Health and Welfare.

244

1974). Dysmyelination during the developing stage has been shown in dystrophic mice of both the Bar Harbor 129 strain (Bradley and Jenkison 1973, 1975: Biscoe. Caddy, Pallot, Pehrson and Stirling 1974; Stifling 1975) and the C57BL/6J strain. We have also published accounts of neural lesions in the C57 strain (Okada, Mizuhira aqd Nakamura 1975, 1976). The objective of our work has been to determine how widespread the neural lesion is in the dystrophic mouse mutant and this paper gives an account of abnormally combined myelinated (M-) and U-nerves in the C57 strain. M A T E R I A L A N D METHODS

Three control mice (C57BL/6J-++ and C57BL/6J-dy+) and 4 dystrophic mice (C57BL/6Jodydy) ranging in age from 3-16 weeks were used. The control mice weighed from 6-15 g and the dystrophic mice from 5-9 g. They were kept in an airconditioned room and given laboratory chow ad libitum. Under ether anesthesia, the mice were fixed by cardiac perfusion for 10 min with 2.5 % glutaraldehyde in 0.1 M phosphate buffer of pH 7.2, at room temperature. The flow rate was 2 ml/min. The

Brachlal plex us e ~ _ _ ~

nerve

~j]~sciatinerve c •~

Sclalicr~lch

D Fig. 1. Graphic illustration of the parts of the peripheral nerve which were dissected out: area of the brachial plexus (PI), the thoracic nerves (Th), area of the sciatic roots (R) used for calculation, the portion just proximal to the sciatic notch (P) used for calculation, and the portion distal t o the sciatic notch (D) used for calculation.

245

Fig. 2. Electron micrograph of a cross-section of abnormally combined M- and U-nerves observed m the thoracic nerve of a 4-week-old dystrophic mouse. The cluster of U-fibres is partially enveloped by the cytoplasm of Schwann cells (Sc and arrows). Double-stained with uranyl acetate and lead tartrate. The bar is 1/~m. Fig. 3. A longitudinal section from the sciatic distal part of a nerve from an 8-week-old dystrophic mouse showing thin myelin which disappears at the node of Ranvier before reaching the neighbouring segment. Double staining. The bar is 1/~m. In the inset, cored vesicles are seen in the U-axons (arrow). Double staining. The bar is 1 t~m.

246

Fig. 4. A longitudinal section from the proximal part of Fig. 3. The bar is I /~m. peripheral nerves were carefully dissected from the roots down to the distal portions. After removal, the nerves were further fixed at 4 °C for 2 hr with the same fixative as above, and washed for 10 min in 0.1 M phosphate buffer containing 8 I~ sucrose. They were then fixed in 2 ~ osmium tetroxide for 2 hr in 0.1 M phosphate buffer o f p H 7.2 with 8 ~ sucrose, and dehydrated by immersion in increasing concentrations of ethanol and then in propylene oxide. The tissue was then embedded in Epon. Tissue blocks were prepared from various portions as shown in Fig. 1. The blocks were trimmed and cross-sections of the nerve trunks were cut on an MT-1 or MT-2 Porter-Blum microtome using glass knives. Ultrathin sections were doubly stained with uranyl acetate and lead tartrate, and examined with Hitachi HS-7S (Fig. 2), HU-1 IDS (for the counts of Table 1) and HS-9 (Figs. 3 and 4) electron microscopes. For the calculations, ultrathin cross-sections of the nerve trunks were cut with the same microtomes and were mounted on one-hole-grid meshes of 0.8 m m in diameter. Sections were doubly stained. Electron micrographs with an original magnification of 900 times were prepared to reconstruct cross-sections of the sciatic nerve trunks, paying special attention under high magnification to make sure that the neurofilaments and neurotubules were being viewed in a perfect cross-section. The images of each trunk were reconstructed with about 100 pictures enlarged to 3 times the original magnification (Okada et al. 1975). The total number of M- and U-fibres in the nerve trunks was counted in the reconstructed pictures. Abnormally combined M- and U-nerves (Fig. 2) were then tentatively calculated in the montaged pictures of the cross-sections in each part of the sciatic nerves. RESULTS Abnormalities of the peripheral nerves were observed in the C57BL/6J strain. In the cross-section, many U-axons, from 2-20, were surrounded by thinner layers of

247 TABLE 1 CALCULATION OF CHANGES IN COMBINED NERVES (A) Total number of M- and U-axons

(B) Number of combined axons

C16 R P D

14,445 14,539 7,763

1 4 0

C 4R

12,609

0

p

- -

- -

D

13,684

0

C3R P D

-12,679 7,179

-0 0

Total axons (B)/(A) x 105

82,898

5 6.O3

D16

7,774 12,854 9,442

0 1 2

D8

R P D P D

15,836 9,442

-2 5

D 4-1 R P D

5,494 14,185 2,354

0 7 1

D 4-2R P D

-15,095 6,292

-2 0

Total axons (B)/(A) x 105

98,748

R

--

20 20.25 a

a Dystrophic mice showed a ratio 3 times higher than in control mice (0.05 < P < 0.1). C = control mice, D = dystrophic mice (Nos. : age in weeks), R = sciatic radix, P = proximal portion near the sciatic notch, D = distal portion near the sciatic notch. myelin sheaths t h a n in the c o n t r o l s (Fig. 2). W i t h i n the myelin sheath o f these adults, each U - a x o n was p a r t i a l l y e n v e l o p e d in the c y t o p l a s m o f S c h w a n n cells. Occasionally, there were spaces between U - a x o n s n o t occupied by S c h w a n n cell c y t o p l a s m . In l o n g i t u d i n a l section, the myelin sheaths d i s a p p e a r e d b e y o n d a n o d e o f R a n v i e r (Figs. 3 a n d 4). Several g r o u p s o f U - a x o n s were n o t e n v e l o p e d in the b a s e m e n t m e m b r a n e a n d c y t o p l a s m o f S c h w a n n cells. I n these groups, there were c o r e d vesicles, dilated e n d o p l a s m i c r e t i c u l u m a n d d e g e n e r a t e d m i t o c h o n d r i a in the U - a x o n s (Figs. 3 a n d 4). T h e y were o b s e r v e d in every b r a c h i a l plexus, t h o r a c i c nerve a n d sciatic nerve o f the d y s t r o p h i c mice. T h e frequency o f a p p e a r a n c e o f c o m b i n e d nerves was c o u n t e d in the sciatic nerves a n d is s h o w n in T a b l e 1. A b n o r m a l i t i e s o f axons were o b s e r v e d in b o t h c o n t r o l a n d d y s t r o p h i c mice. In the c o n t r o l mice, the r a t i o o f a p p e a r a n c e was 6.0 p e r 100,000 a n d in d y s t r o p h i c mice, 20.3 per 100,000 (0.05 < P < 0.1). It is higher in the

248 dystrophic mice than in the control group. The abnormalities were observed in only 1 of the control mice but were present in each of the dystrophic mice. DISCUSSION Nerves combining M- with U-nerves, which have not been reported previously, were observed in the C57BL strain. Such an abnormality might have occurred during the developing phase of the peripheral nerves of this strain. I f this is true, this neural abnormality may be identical to the dysmyelination which we reported previously, because the myelin is lost beyond the node of Ranvier (Okada et al. 1975, 1976). M-axon formations surrounding groups of U-axons were naked distally to the Ranvier node without basement membrane and cytoplasm of Schwann cells. These abnormalities have never been reported in developing peripheral nerves (Gamble 1966), or in peripheral neuropathy in humans and experimental animals (Greenfield et al. 1958; Bradley 1974; Dyck et al. 1975; Blackwood and Corsellis 1976). They appeared in the dystrophic mice more frequently than in the controls. This abnormality was seen in only one 16-week-old mouse of the control groups examined. It is possible that this C-16 control mouse might have had a C57BL/6J-dyq- heterogene, but it is difficult to discern the dy allele when we use healthy control mice. These abnormalities in dystrophic mice might interfere with normal nerve function in these animals by transmitting incorrect information, e.g., by combining a motor and a sensory impulse. Thus the phenomenon might have some relationship to the pathophysiology o f m u r i n e muscular dystrophy. ACKNOWLEDGEMENT We thank Dr. K. Ezaki in the Central Institute for Experimental Animals for providing dystrophic mice.

REFERENCES Biscoe, T. J., K. W. T. Caddy, D. J. Pallot, U. M. M. Pehrson and C. A. Stirling (1974) The neurological lesion in the dystrophic mouse, Brain Res., 76: 534-536. Blackwood, W. and J. A. N. Corsellis (I976) Greenfield's Neuropathology, 3rd edition, T. A. Constable Ltd, Edinburgh. Bradley, W. G. (1974) Disorders of Peripheral Nerves, Blackwell Scientific Publications, London. Bradley, W. G. and M. Jenkison (1973) Abnormalities of peripheral nerves in murine muscular dystrophy, J. neuroL ScL, 18: 227-247. Bradley, W. G. and M. Jenkison (1975) Neural abnormalities in the dystrophic mouse, J. neurol. Sci., 25 : 249-255. Cammermeyer, J. (1956) Neuropathological changes in hereditary neuropathies - - Manifestation of the syndrome heredopathia ataetica polyneuritiformis in the presence of interstitial hypertrophic potyneuropathy, J. Neuropath. exp. NeuroL, 15: 340--361. Davison, A. N. and A. Peters (1970) Myelination, Charles C. Thomas, Springfield, I11. pp. 3-79. Dyck, P. J., P. K. Thomas and E. H. Lambert (1975) Peripheral Neuropathy, W. B, Saunders Company, Philadelphia, London, Toronto. Gamble, H. J. (1966) Further electron microscope studies of human foetal peripheral nerves, J. Anat. (Lond.), 100: 487-502.

249 Greenfield, J. G., W. Blackwood, W. H. McMenemey, A. Meyer and R. M. Norman (1958) Neuropathology, Arnold, London, pp. 643-646. Okada, E., V. Mizuhira and H. Nakamura (1975) Abnormalities of the sciatic nerves of dystrophic mice, with reference to nerve counts and mean area of axons, Bull. Tokyo med. dent. Univ., 22: 25-43. Okada, E., V. Mizuhira and H. Nakamura (1976) Dysmyelination in the sciatic nerves of dystrophic mice, J. neurol. ScL, 28: 505-520. Raine, C. S., D. H. Snyder, M. P. Valsamis and S. H. Stone (1974) Chronic experimental allergic encephalomyelitis in inbred guinea pigs - - An ultrastructural study, Lab. Invest., 31: 369-379. Simpson, D. A. and M. Fowler (1966) Two cases of localized hypertrophic neurofibrosis, J. Neurol. Neurosurg. Psychiat., 29: 80-84. Stirling, C. A. (1975) Abnormalities in Schwann cell sheath in spinal nerve roots of dystrophic mice, J. Anat. (Lond.), 119: 169-180.

Abnormally combined myelinated and unmyelinated nerves in dystrophic mice.

Journal of the Neurological Sciences, 1977, 33:243-249 243 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands A B N O...
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