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Electron microscopic observations on a focal experimental demyelinating lesion in the cat spinal cord
409
Journal of the neurological Sciences
Elsevier PublishingCompany,Amsterdam- Printed in The Netherlands
Short Report
Electron Microscopic Observations on a Focal Experimental Demyelinating Lesion in the Cat Spinal Cord
INTRODUCTION
A technique has been devised recently for producing focal experimental demyelination in the central nervous system by the direct micro-injection of diphtheria toxin into the spinal cord. The light microscopic features of this lesion have been described briefly (MCDONALDAND SEARS 1969a). Here we report electron-microscopic findings to illustrate some ultrastructural features of this lesion.
MATERIAL AND METHODS
A total of 10 cats was studied. Five cats were normal and the rest were injected with diphtheria toxin and examined either 2, 3, 5 or 7 days later. The technique of injection previously described (MCDONALD AND SEARS 1969a) was used in the present experiments. Under pentobarbitone anaesthesia 0.001--0.003 flocculation units of toxin in 0.003 ml borax buffer were injected into the dorsolateral sulcus of the thoracolumbar cord of the cat. After 2-7 days the animals were again anaesthetized and fixed by retrograde perfusion through the abdominal aorta, at a pressure of 150 mm Hg. The perfusion began with 300 ml of normal saline. This was followed by fixative at 4°C: first 600 ml of 5% glutaraldehyde in M / 1 5 phosphate buffer at pH 7.4, then 400 ml of Dalton's fluid. The previous laminectomy site was re-opened. Complete transverse sections of the cord 1-2 mm thick were removed, and placed in flesh Dalton's fluid at 4°C for 2-3 hours. After dehydration and embedding in epon, 1-2 # thick sections of whole cord were cut and stained with toluidine blue. Areas of demyelination in these sections were located by light microscopy. Thin longitudinal or transverse sections were then cut from the blocks at sites corresponding with the demyelinated zones and stained with zinc uranyl acetate followed by lead citrate and examined with a Siemens Elmiskop 1 electron microscope.
RESULTS
Electron-microscopic observations of thin transverse sections have confirmed and extended the previously reported light-microscopic observations. A zone of oedema extends for several mm around the injection site and involves the ipsilateral posterior This work was supported by a grant from the Medical Research Council.
J. neurol. Sci., 1970, 10:409-413
410
SHORT REPORT
Fig. I. Electron micrograph o f a transverse section o f posterior column at T ~ . Lesion induced 7 days previously. Bar represents I /~. ~ 28,000.
column and the posterior part of the lateral column. Both Wallerian degeneration and demyelination are seen around the injection site. Demyelination predominates towards the edges of the lesion both inside and outside the oedematous zone, with Wallerian degeneration predominating in the middle. Wallerian degeneration is seen elsewhere in the cord corresponding with the distribution of distal portions of fibres interrupted at the injection site itself. Different fibres from the same lesion show varying degrees of myelin destruction. Fig. 1 is from an animal with a 7-day lesion. A fibre with damaged myelin (a) lies close to another fibre (b) which is surrounded by an apparently normal compact myelin sheath. The axoplasm of both these fibres appears normal. The damaged myelin is split in various parts of the sheath into layers made up of single or multiple lamellae. In other places the lamellar pattern is blurred and amorphous. A broad band of granular electron-dense material containing vesicles, widely separates layers of compact myelin. This appearance is quite different from that due to fixation artefact where the myelin lamellae are irregularly separated without intervening electron-dense material. Fig. 2 shows a satellite cell from a 7-day-old lesion, undergoing mitotic division. Within the dark granular cytoplasm there is much osmiophilic lamellar material presumably derived from myelin breakdown. At the lower pole, an axon of normal appearance, 5.6/~ in diameter, is surrounded by processes from the cell. This is shown J. neurol. Sci., 1970, 10:409~-13
SHORT REPORT
411
Fig. 2. Same lesion as Fig. 1. Cf. the diameter of the intact myelinated fibre at the upper pole and the naked axon at the lower pole. Bar represents 2/,. × 6250; inset x 9500.
at higher magnification in the inset. In o u r n o r m a l material, axons o f this size are always myelinated. It is i m p r o b a b l e t h a t such a relatively large axon d e v o i d o f myelin represents a swollen u n m y e l i n a t e d fibre, because o f the n o r m a l , o r slightly increased, density o f a x o p l a s m i c organelles. M o r e o v e r , the structure o f an a x o n cut t h r o u g h a n o d e as described b y PETERS (1966) a n d confirmed in o u r own n o r m a l material, is J. neuroL Sci., 1970, I0:409-413
412
SHORT REPORT
Fig. 3. Same lesion as Fig. 1. The thinly myelinated axon is surrounded by a mass of glial processes, many of which contain bundles of fibrils. Myelin debris lies close to the thinly myelinated axon. The arrows indicate a cluster of small processes referred to in the text. Bar represents 1 /z. ~," 8000.
quite different from the structure o f this axon. We therefore conclude that such an axon is demyelinated. In animals studied 5 days or longer after an injection of diphtheria toxin, some fibres were surrounded by a few myelin iamellae. In the middle o f Fig. 3 is an axon with a myelin sheath m u c h thinner than would be expected for an axon o f this size (cJ~ the surrounding smaller axons in the same section). At one pole there is a cluster o f small processes (see arrows in the Figure). DISCUSSION O u r evidence thus suggests that demyelination followed by remyelination has occurred. Similar appearances have been reported in experimental allergic encephalomyelitis (LAMPERT AND CARPENTER 1965) and barbotage (BUNGE et al. 1960) lesions. In the present lesion changes are seen within 2 days o f diphtheria toxin injection. This illustrates the speed with which demyelination can occur after direct introduction of the toxin into the central nervous system and in this respect resembles the rapid demyelination seen in tissue culture (BORNSTEINAND APPEL 1961). The process may be contrasted with Wallerian degeneration in which the axon disintegrates while the lamellar pattern o f the myelin sheath is still intact (LAMPERT 1967). J. neurol. Sci., 1970, 10:409-413
SHORT REPORT
413
The varying degrees o f myelin d a m a g e shown by different fibres in lesions o f the same age is interesting in the light o f the g r a d e d physiological effects o f these lesions on c o n d u c t i o n (MCDONALD AND SEARS 1969a, b; 1970). C o n d u c t i o n in some fibres is n o r m a l ; in others, the c o n d u c t i o n is c o m p l e t e l y b l o c k e d at the level o f the lesion. I n yet o t h e r fibres c o n d u c t i o n continues t h r o u g h the lesion, but at a r e d u c e d velocity. However, it is n o t yet possible to link p a r t i c u l a r m o r p h o l o g i c a l changes with specific physiological abnormalities.
ACKNOWLEDGEMENTS W e are grateful to Dr. W. G . P. M a i r a n d the M u s c u l a r D y s t r o p h y G r o u p for the use o f the electron m i c r o s c o p e a n d to Professor J. B. C a v a n a g h for allowing us access to facilities in the M . R . C . R e s e a r c h G r o u p in A p p l i e d N e u r o b i o l o g y . W e wish to t h a n k Miss C a r o l A l d r i d g e , Miss F r a n c i s D r a i n a n d Mr. Brian Y o u n g for their technical assistance. W e w o u l d also like to t h a n k Professor R . W . Gilliatt for helpful criticism a n d advice. Institute of Neurology, Queen Square London, W.C.1 (Great Britain.)
B. M. HARRISON W. I. McDONALD J. OCHOA T. A. SEARS
(Received 26 J a n u a r y , 1970) REFERENCES
BORNSTEIN,M. B. ANDS. H. APPEL(1961) The application of tissue culture to the study of experimental "allergic" encephalomyelitis, Part 1 (Patterns of demyelination), J. Neuropath. exp. Neurol., 20: 141-157. BUNGE, R. P., MARYB. BUNGEAND H. RIS (1960) Electron microscopic study of demyelination in an experimentally induced lesion in adult cat spinal cord, J. biophys, biochern. Cytol., 7: 685-696. LAMPERT,P. W. (1967) A comparative electron microscopic study of reactive, degenerating, regenerating and dystrophic axons, J. Neuropath. exp. Neurol., 26: 345-368. LAMPERT, P. AND S. CARPENTER(1965) Electron microscopic studies on the vascular permeability and the mechanism of demyelination in experimental allergic encephalomyelitis, J. Neuropath. exp. Neurol., 24: 11-24. MCDONALD, W. I. AND T. A. SEARS(1969a) Effect of demyelination on conduction in the central nervous sytem, Nature (Lond.), 221 : 182-183. MCDONALD, W. I. ANDT. A. SEARS(1969b) The effects of demyelination on conduction in the central nervous system, Trans. Amer. neurol. Ass., 94.' In press. MCDONALD,W. I. ANDT. A. SEARS(1970) Effect of a demyelinating lesion on conduction in the central nervous system studied in single nerve fibres, J. Physiol. (Lond.), P: In press. PETERS, A. (1966) The node of Ranvier in the central nervous system. Quart. J. exp. Physiol., 51 : 229-236.
J. neurol. Sci., 1970, 10:409~,13
Journal of the neurological Sciences
Elsevier PublishingCompany,Amsterdam- Printed in The Netherlands
Short Report
Electron Microscopic Observations on a Focal Experimental Demyelinating Lesion in the Cat Spinal Cord
INTRODUCTION
A technique has been devised recently for producing focal experimental demyelination in the central nervous system by the direct micro-injection of diphtheria toxin into the spinal cord. The light microscopic features of this lesion have been described briefly (MCDONALDAND SEARS 1969a). Here we report electron-microscopic findings to illustrate some ultrastructural features of this lesion.
MATERIAL AND METHODS
A total of 10 cats was studied. Five cats were normal and the rest were injected with diphtheria toxin and examined either 2, 3, 5 or 7 days later. The technique of injection previously described (MCDONALD AND SEARS 1969a) was used in the present experiments. Under pentobarbitone anaesthesia 0.001--0.003 flocculation units of toxin in 0.003 ml borax buffer were injected into the dorsolateral sulcus of the thoracolumbar cord of the cat. After 2-7 days the animals were again anaesthetized and fixed by retrograde perfusion through the abdominal aorta, at a pressure of 150 mm Hg. The perfusion began with 300 ml of normal saline. This was followed by fixative at 4°C: first 600 ml of 5% glutaraldehyde in M / 1 5 phosphate buffer at pH 7.4, then 400 ml of Dalton's fluid. The previous laminectomy site was re-opened. Complete transverse sections of the cord 1-2 mm thick were removed, and placed in flesh Dalton's fluid at 4°C for 2-3 hours. After dehydration and embedding in epon, 1-2 # thick sections of whole cord were cut and stained with toluidine blue. Areas of demyelination in these sections were located by light microscopy. Thin longitudinal or transverse sections were then cut from the blocks at sites corresponding with the demyelinated zones and stained with zinc uranyl acetate followed by lead citrate and examined with a Siemens Elmiskop 1 electron microscope.
RESULTS
Electron-microscopic observations of thin transverse sections have confirmed and extended the previously reported light-microscopic observations. A zone of oedema extends for several mm around the injection site and involves the ipsilateral posterior This work was supported by a grant from the Medical Research Council.
J. neurol. Sci., 1970, 10:409-413
410
SHORT REPORT
Fig. I. Electron micrograph o f a transverse section o f posterior column at T ~ . Lesion induced 7 days previously. Bar represents I /~. ~ 28,000.
column and the posterior part of the lateral column. Both Wallerian degeneration and demyelination are seen around the injection site. Demyelination predominates towards the edges of the lesion both inside and outside the oedematous zone, with Wallerian degeneration predominating in the middle. Wallerian degeneration is seen elsewhere in the cord corresponding with the distribution of distal portions of fibres interrupted at the injection site itself. Different fibres from the same lesion show varying degrees of myelin destruction. Fig. 1 is from an animal with a 7-day lesion. A fibre with damaged myelin (a) lies close to another fibre (b) which is surrounded by an apparently normal compact myelin sheath. The axoplasm of both these fibres appears normal. The damaged myelin is split in various parts of the sheath into layers made up of single or multiple lamellae. In other places the lamellar pattern is blurred and amorphous. A broad band of granular electron-dense material containing vesicles, widely separates layers of compact myelin. This appearance is quite different from that due to fixation artefact where the myelin lamellae are irregularly separated without intervening electron-dense material. Fig. 2 shows a satellite cell from a 7-day-old lesion, undergoing mitotic division. Within the dark granular cytoplasm there is much osmiophilic lamellar material presumably derived from myelin breakdown. At the lower pole, an axon of normal appearance, 5.6/~ in diameter, is surrounded by processes from the cell. This is shown J. neurol. Sci., 1970, 10:409~-13
SHORT REPORT
411
Fig. 2. Same lesion as Fig. 1. Cf. the diameter of the intact myelinated fibre at the upper pole and the naked axon at the lower pole. Bar represents 2/,. × 6250; inset x 9500.
at higher magnification in the inset. In o u r n o r m a l material, axons o f this size are always myelinated. It is i m p r o b a b l e t h a t such a relatively large axon d e v o i d o f myelin represents a swollen u n m y e l i n a t e d fibre, because o f the n o r m a l , o r slightly increased, density o f a x o p l a s m i c organelles. M o r e o v e r , the structure o f an a x o n cut t h r o u g h a n o d e as described b y PETERS (1966) a n d confirmed in o u r own n o r m a l material, is J. neuroL Sci., 1970, I0:409-413
412
SHORT REPORT
Fig. 3. Same lesion as Fig. 1. The thinly myelinated axon is surrounded by a mass of glial processes, many of which contain bundles of fibrils. Myelin debris lies close to the thinly myelinated axon. The arrows indicate a cluster of small processes referred to in the text. Bar represents 1 /z. ~," 8000.
quite different from the structure o f this axon. We therefore conclude that such an axon is demyelinated. In animals studied 5 days or longer after an injection of diphtheria toxin, some fibres were surrounded by a few myelin iamellae. In the middle o f Fig. 3 is an axon with a myelin sheath m u c h thinner than would be expected for an axon o f this size (cJ~ the surrounding smaller axons in the same section). At one pole there is a cluster o f small processes (see arrows in the Figure). DISCUSSION O u r evidence thus suggests that demyelination followed by remyelination has occurred. Similar appearances have been reported in experimental allergic encephalomyelitis (LAMPERT AND CARPENTER 1965) and barbotage (BUNGE et al. 1960) lesions. In the present lesion changes are seen within 2 days o f diphtheria toxin injection. This illustrates the speed with which demyelination can occur after direct introduction of the toxin into the central nervous system and in this respect resembles the rapid demyelination seen in tissue culture (BORNSTEINAND APPEL 1961). The process may be contrasted with Wallerian degeneration in which the axon disintegrates while the lamellar pattern o f the myelin sheath is still intact (LAMPERT 1967). J. neurol. Sci., 1970, 10:409-413
SHORT REPORT
413
The varying degrees o f myelin d a m a g e shown by different fibres in lesions o f the same age is interesting in the light o f the g r a d e d physiological effects o f these lesions on c o n d u c t i o n (MCDONALD AND SEARS 1969a, b; 1970). C o n d u c t i o n in some fibres is n o r m a l ; in others, the c o n d u c t i o n is c o m p l e t e l y b l o c k e d at the level o f the lesion. I n yet o t h e r fibres c o n d u c t i o n continues t h r o u g h the lesion, but at a r e d u c e d velocity. However, it is n o t yet possible to link p a r t i c u l a r m o r p h o l o g i c a l changes with specific physiological abnormalities.
ACKNOWLEDGEMENTS W e are grateful to Dr. W. G . P. M a i r a n d the M u s c u l a r D y s t r o p h y G r o u p for the use o f the electron m i c r o s c o p e a n d to Professor J. B. C a v a n a g h for allowing us access to facilities in the M . R . C . R e s e a r c h G r o u p in A p p l i e d N e u r o b i o l o g y . W e wish to t h a n k Miss C a r o l A l d r i d g e , Miss F r a n c i s D r a i n a n d Mr. Brian Y o u n g for their technical assistance. W e w o u l d also like to t h a n k Professor R . W . Gilliatt for helpful criticism a n d advice. Institute of Neurology, Queen Square London, W.C.1 (Great Britain.)
B. M. HARRISON W. I. McDONALD J. OCHOA T. A. SEARS
(Received 26 J a n u a r y , 1970) REFERENCES
BORNSTEIN,M. B. ANDS. H. APPEL(1961) The application of tissue culture to the study of experimental "allergic" encephalomyelitis, Part 1 (Patterns of demyelination), J. Neuropath. exp. Neurol., 20: 141-157. BUNGE, R. P., MARYB. BUNGEAND H. RIS (1960) Electron microscopic study of demyelination in an experimentally induced lesion in adult cat spinal cord, J. biophys, biochern. Cytol., 7: 685-696. LAMPERT,P. W. (1967) A comparative electron microscopic study of reactive, degenerating, regenerating and dystrophic axons, J. Neuropath. exp. Neurol., 26: 345-368. LAMPERT, P. AND S. CARPENTER(1965) Electron microscopic studies on the vascular permeability and the mechanism of demyelination in experimental allergic encephalomyelitis, J. Neuropath. exp. Neurol., 24: 11-24. MCDONALD, W. I. AND T. A. SEARS(1969a) Effect of demyelination on conduction in the central nervous sytem, Nature (Lond.), 221 : 182-183. MCDONALD, W. I. ANDT. A. SEARS(1969b) The effects of demyelination on conduction in the central nervous system, Trans. Amer. neurol. Ass., 94.' In press. MCDONALD,W. I. ANDT. A. SEARS(1970) Effect of a demyelinating lesion on conduction in the central nervous system studied in single nerve fibres, J. Physiol. (Lond.), P: In press. PETERS, A. (1966) The node of Ranvier in the central nervous system. Quart. J. exp. Physiol., 51 : 229-236.
J. neurol. Sci., 1970, 10:409~,13