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Central demyelination produced by diphtheria toxin: an electron microscopic study
281
Journal of the neurological Sciences Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
Central Demyelination Produced by Diphtheria Toxin" an Electron Microscopic Study B. M. H A R R I S O N , W. I. M c D O N A L D AND J. O C H O A National Hospital, Queen Square, London W.C.1 (Great Britain,) (Received 9 May, 1972)
INTRODUCTION
Demyelination of central nerve fibres occurs in a variety of human and experimental diseases, but the mechanism has been elucidated in only one - experimental allergic encephalomyelitis (EAE). In EAE there appear to be two ways in which myelin is broken down. In one, demyelination is associated with an invasion of exogenous cells and is executed by phagocytes which strip myelin lamellae from otherwise intact sheaths (Bubis and Luse 1964; Lampert 1965 ; Raine, Wi~niewski and Prineas 1969). In the other process, the myelin shows a number of changes such as interlamella splitting and vesiculation of lamellae that are not directly associated with phagocytic stripping or cellular invasion (Prineas, Raine and Wi~niewski 1969; Lampert 1965). Do these processes operate in other circumstances? Suzuki, Andrews, Waltz and Terry (1969) found no evidence of phagocytic stripping in multiple sclerosis, but as they point out, their observations were made on six biopsy specimens which may not have included an acute lesion. Phagocytic stripping was not described in the cerebrospinal fluid exchange lesion (Bunge, Bunge and Ris 1960) and demyelination was not associated with cellular invasion. Similarly, exposure of spinal cord cultures to EAE serum produced demyelination in a situation where no cellular invasion could occur (Raine and Bornstein 1970). In view of the small amount of information available about the mechanism of demyelination in vivo, and the fact that phagocytic stripping has been observed only in EAE it seemed appropriate to study a lesion which on a priori grounds we would not expect to have an allergic basis. Systemically-administered diphtheria toxin produces demyelination in the peripheral nervous system but because the blood-brain barrier is impermeable to the toxin, there is no demyelination of central nerve fibres (Waksman 1961). McDonald and Sears (1969) showed that focal central demyelination could be produced, when the blood-brain barrier was by-passed, by injecting a minute amount of diphtheria toxin directly into the spinal cord. Since peripheral demyelination by diphtheria toxin is independent of the immune system (Waksman, Adams and Mansmann 1957) J. neuroL Sci., 1972, 17:281-291
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it seemed probable that the same would be true of the central diphtheria toxin lesion. We have, therefore, made a detailed electron microscopic study of this lesion. In a recent paper we dealt with the predominantly paranodal distribution of demyelination at the edges of the lesion (Harrison, McDonald, Ochoa and Ohlrich 19721. In the present paper we describe the evolution of demyelination, and in the following paper we shall discuss the question of remyelination. A short report of some aspects of this work has been published (Harrison, McDonald, Ochoa and Sears 1970). Since completing this study a paper by Wigniewski and Raine (1971) describing the central diphtheria toxin lesion in six rabbits and two cats has appeared. Although we confirm a number of their observations, we have made additional observations, and there are important differences in interpretation deriving from the study of lesions both earlier and later than those studied by Wigniewski and Raine.
METHOD
A total of 25 cats was studied. Under pentobarbitone anaesthesia and with aseptic precautions, a laminectomy was performed in the thoracolumbar region. Injections of diphtheria toxin were made into the dorsolateral sulcus of the spinal cord using the technique described previously (McDonald and Sears 1970a). The wound was then closed and the animals were allowed to survive for varying lengths of time. Crystalline diphtheria toxin was kindly supplied by Dr. Edwards of the Wellcome Research Laboratories. The toxin was dissolved in borate buffer at pH 7.4. The volume of liquid injected into the cord was 3 /~1 in all except the 1-month animal (5 #l). Sixteen animals were injected with 0.001-0.003 fl0cculation units of diphtheria toxin. These animals were perfused after 6 hr (2), 13 hr (1), 1 day (1), 2 days (1), 3 days (1), 5 days (1), 7 days (2), 13 days (1), 14 days (1), 1 month (1), 2 months (1)~ 3 months (1), 4 months (1), and 1 year (1). Other animals were examined as controls. Five animals were normal. One animal was injected with buffer alone and examined after 7 days. In one animal two laminectomies were performed, which were separated by two cord segments. At the upper site a needle was inserted into the cord in the usual way but nothing was injected. At the lower site, the dura was simply opened and not followed by insertion of the needle. This animal was perfused after 7 days. Two cats were perfused 2 and 3 days after transection of the posterior columns to produce Wallerian degeneration. After intracardiac injection of adrenalin (1 mg/kg) (Cammermeyer 1968), the spinal cord was fixed by retrograde perfusion through the abdominal aorta at 150--200 mm of mercury. 300 ml of saline was followed by 600 ml of 4-5 ~o glutaraldehyde in M/I 5 phosphate buffer (pH 7.4), then by 400 ml of chrome-buffered osmium tetroxide (Dalton 1955). The fixatives were at 4°C. The tissue was prepared for electron microscopic examination in the conventional way. Representative 1.5 # thick sections of the whole cross-section of the spinal cord were cut at intervals of approx. 1 mm over a distance of 1 cm on either side of the injection site and counter-stained with toluidine blue for light microscopy. Ultra-thin sections from areas selected after examination of thick sections, were examined with a Siemens Elmiskop la electron microscope. J. neurol. Sci., 1972, 17:281--291
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Counts of myelin incisures Counts of incisures in central myelin were performed in normal and experimental animals. Thin transverse sections from the dorsal root entry zone were scanned at a working magnification of 8,000-9,500 diameters. 10-15 micrographs were exposed with 5-25 fibres on each plate. For convenience in counting, fields with fibres greater than 3 #m were avoided. Apart from this, electron micrographs were taken at random, care being taken not to photograph the same fibre twice. The plates were enlarged to a final magnif, cation of 20,000-24,000 diameters. At this magnification it was easy to identify the fibres that had incisures because the cytoplasm could be seen clearly between myelin lamellae. For each animal, the number of fibres with incisures was expressed as a percentage of the total number of fibres counted. The significance of the difference between the mean of the results from normal and diphtheria toxininjected animals was tested using the Z2 (chi-square) distribution and applying the Yates' correction for binomial distributions.
RESULTS
Normal spinal cord The thoracolumbar region of the spinal cord contains both myelinated and unmyelinated fibres. Two normal features require comment because of their relevance to the interpretation of the pathological material. To distinguish in experimental animals between normally unmyelinated fibres and demyelinated fibres, it was
Fig. 1. Normal cat. Region near dorsal root entry zone. A small axon, 0.3/~m in diameter, is surrounded by myelin which is typical of the central nervous system. Bar, 0.2 #m. J. neurol. Sci., 1972, 17:281-291
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necessary to determine the critical axonal size for myelination. Axons as small its 0.3 /~m had myelin sheaths (Fig. 1) and no axons greater than 1 /~m were found without myelin. Throughout this work, axons were not accepted as demyelinated unless their diameter was greater than 1 #m. Although dendrites may sometimes be confused with axons in the border zones between grey and white matter, this was not a problem in the regions studied in the present experiments. lncisures resembling those of Schmidt and Lantermann in the peripheral nervous system have been described in detail in the central nerve fibres of the rat (Blakemore 1969; Hirano, Zimmerman and Levine 1969), and were easily recognised in the present material. In transverse sections they appear as separations of the lamellae at the major dense line with electron-dense, granular cytoplasm (derived from the myelin-forming oligodendrocyte) filling the space between the separated lamellae. Incisures occurred in fibres of all sizes, and counting showed that at any level they were present in 2.3 ~o of fibres less than 3 #m in diameter. In longitudinal section they appeared as a series of loops filled with cytoplasm arranged in a step-wise fashion in consecutive lamellae.
Diphtheria toxin lesion A survey of all the lesions showed that there was an approximate correlation between the amount of toxin injected and the size of the lesion. The lesion itself was clearly orientated to the needle track. There were no perivascular accumulations of mononuclear cells. At every stage there was a central zone of degeneration where
7day diphtheria toxin injection 1 cm
Fig. 2. Diagram to show the distribution of changes within the spinal cord 7 days after the injection of diphtheria toxin. Solid black: Many fibres show WaUerian-type degeneration. Hatched: The majority of the axons appear normal but many myelin sheaths are abnormal. The extracellular space in these two zones is expanded. Elsewhere in the cord, around the injection site, the extraeellular space is normal. Most axons are normal but some are surrounded by abnormal myelin sheaths. The approximate distribution of these fibres with abnormal myelin is shown by dots in the diagram.
many fibres showed disintegration of both axons and myelin (Fig. 2). The size of this zone varied, being scarcely wider than the needle in the control preparation in which a needle alone was inserted into the cord, and many times larger than this after the injection of toxin. For several mitlimetres around the central zone, the extraceUular space was expanded, presumably by oedema. There were many abnormal fibres here too, but the vast majority of these fibres had abnormal myelin sheaths but normal axons. Outside this was a region in which there were still abnormal fibres, but the extracellular space appeared to be of normal size. This zone gradually faded over about 5 mm into normal spinal cord. J. neurol. Sci.. 1972. 17:281-291
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In the following description of the diphtheria toxin lesions we shall describe only those changes which occurred outside the central zone, that is away from the region of Wallerian-type degeneration. Demyelination had already commenced at 6 hr. Myelin debris was present in the expanded extracellular space around the needle track and consisted of irregular clumps of electron-dense material. Usually the lamellar pattern was lost, leaving an amorphous mass, but in some fragments it was preserved. A few scattered, completely demyelinated axons were present and a few large diameter axons had inappropriately thin myelin sheaths. Between 24 and 72 hr there was a marked increase in the amount of myelin debris and in the numbers of demyelinated axons. At the centre of the lesion the majority of the axons showed changes typical of Wallerian-type degeneration, but more peripherally the axoplasm of demyelinated fibres appeared normal. At 3 days demyelinated axons occurred in small groups. The size of the groups increased until they contained up to 30 large diameter demyelinated axons at 13 days (Fig. 3). At 5 days the axons within a group were in contact with each other (Fig. 4), but from 7 days on the fibres were increasingly separated by invading cellular processes. Some of these cellular processes contained myelin debris. Others contained fibrils suggesting that they were derived from astrocytes. Fragments of basement lamina were seen in relation to some of the processes.
Fig. 3. 13-day lesion. Posterior columns. A large number of demyelinated axons are closely packed. Some are completely surrounded by dark cytoplasm (not myelin). Debris-laden cells are scattered throughout the region. Bar, 20 #m. J. neurol. Sci., 1972, 17:281-291
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Fig. 4. 5-day lesion. Six demyelinatcd axons lie within an expanded extracellular space. Fheir axolemmas are m close contact. Bar. 2 Ira1
Fig. 5. l-month lesion. A naked axon (3/~m) is embedded in cell cytoplasm. An arrow points to a mesaxonlike structure. The cell itself is surrounded by a basement lamina (BL). N. nucleus: C. collagen fibres. Bar, 1 Itm.
.I. neuroL Sci., 1972, 17: 2Zl 291
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Fig. 6. 6-hr lesion. An axon is surrounded by a ring of elliptical profiles that are derived from the myelin sheath. There is an intra-period line between the membrane of each profile and the remaining compact myelin (arrow). Bar, 0.2/~m.
From 13 days onwards a few individual axons were surrounded by cells with a complete investment of basement lamina (Fig. 5). Collagen was frequently present in the vicinity of these cells which appear to be Schwann cells. Further evidence for this conclusion will be presented in the following paper. The astrocyte reaction increased with time, and by 2 months the expanded extracellular space was filled with densely-packed astrocytic processes. At 2 months the number of fibres in the lesion was reduced, and surviving fibres were widely separated. Many fibres remained completely demyelinated and as in all earlier lesions some axons had inappropriately thin myelin. Myelin debris still persisted up to 4 months after induction of the lesion. After 1 year, debris was no longer present, but apart from an increase in the collagen the lesion resembled that at 2 months.
Early changes in myelin The appearance of damaged myelin around a normal axon took several forms. In some fibres the intra-period line was split leaving a clear space between adjacent J. neurol. Sci., 1972, 17:281-291
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major dense lines. No cellular process intervened between the split lamellae. Although separation of lamellae at the intra-period line is a common preparation artefact, wc think that it is an important part of the pathological process since in the earliest lesions it is most prominent at the nodes while the nearby internodal myelin appears well-fixed and intact. A second change was vesiculation of myelin lamellae both on the outer and inner surfaces of the sheath (Fig. 6). This change was seen in the 6-hr. and in some, but not all, older lesions. A third feature was an increase in the number of incisures in the myelin of the toxin-injected animals. In four normal animals a mean of 2.3 ~/o of fibres up to 3 /~m in diameter (see Method) showed incisures. By contrast, there were incisures in a mean of 6.1 ~o of fibres in lesions from 6-hr- to
Fig. 7. 2-day lesion. Lateral columns. Transverse section through Schmidt-Lantermann-like incisure. Some myelin lamellae are separated along the major dense line (arrows) by electron-dense granular cytoplasm. GP, glial processes on the outer surface of the sheath. Bar. 0.25 pm.
4-month-old (10)lesions. The difference between the mean of the normal and diphtheria toxin-injected animals is highly significant (P=0.002). Structurally the incisures in the lesion (Fig. 7) resembled normal incisures but the granular material often formed a bulge to one side of the sheath and here there were fewer myelin lamellae than on the other side of the sheath. All these changes in the myelin were visible before the first appearance of phagocytes, which was at 24 hr. The phagocytic invasion increased markedly in the first 3 days. Intracellular debris was first seen at 24 hr, and was very prominent from 3 days to 13 days. In contrast to the findings reported in EAE, however, phagocytes were never seen to strip apparently normal myelin lamellae from otherwise intact sheaths. J. neurol. Sci., 1972, 17:281 291
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Control animals
Dural opening alone produced no lesion after 7 days. Injection of 3 #1 of buffer produced after 7 days a lesion only slightly larger than that produced by simple insertion of a needle. The fibres in the needle track were undergoing Wallerian degeneration. Around the needle track were a few completely-demyelinated and a few thinly-myelinated axons. There was an increase in numbers of fibres showing incisure-like clefts. DISCUSSION
The present experiments have shown that in the central diphtheria toxin lesion the breakdown of the regular lamellar pattern of myelin to irregular clumps of debris occurs prior to ingestion of myelin by phagocytes. At no stage do phagocytes strip apparently normal lamellae from an otherwise intact myelin sheath. The evolution of demyelination in this lesion is thus fundamentally different from that in EAE in vivo where a major mechanism for myelin destruction involves initial phagocytosis of intact lamellae (Lampert 1965). It is possible that the very early myelin changes are contributed to by trauma from the injection needle. However, by 48 hr the lesion is much larger than that produced by the injection of buffer. We, therefore, attribute the changes at this stage to the action of the toxin. The means by which diphtheria toxin produces demyelination is unknown. Although it has been shown to inhibit protein synthesis in cell-free systems (Collier 1967) the relevance of this to its in vivo action is uncertain. We are not in a position to decide whether diphtheria toxin attacks the myelin sheath directly or whether it damages the myelin-forming cell (oligodendrocyte) first. The importance of vesiculation of myelin sheaths in the demyelinating process in the central diphtheria toxin lesion has recently been stressed by Wi~niewski and Raine (1971). However, because in our experiments vesiculation was not seen at all in some lesions with extensive demyelination and was present in only a few fibres in those lesions in which it was seen, we do not believe that it represents a necessary stage in myelin breakdown in this lesion. One of the early features of the diphtheria toxin lesion was the increase in the number of fibres in cross-section that had incisures. This has not previously been reported in the central nervous system. Morphologically similar appearances have been reported in cases of Alzheimer's presenile dementia (Terry, Gonatas and Weiss 1964) and in Jakob-Creutzfeldt disease (Gonatas, Terry and Weiss 1965). It has been reported that the number of incisures that occur in the peripheral nervous system increases during Wallerian degeneration (Webster 1965). Recent evidence suggests that this increase is due to the expansion of pre-existing incisures (Williams and Hall 1971), rather than the formation of new ones. In either case, the change represents a reaction by the myelin-forming cell. In the diphtheria toxin lesion, an increase in incisures occurred in the outer parts of the lesion well away from the central zone of axonal degeneration, and was present long after degeneration was completed. It seems likely that an increase in the number of incisures is an early phase of demyelination which-does not necessarily go on to complete myelin breakdown. This lesion is a useful model for the study of central demyelination. Although J. neurol. Sci., 1972, 17:281-291
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diphtheria toxin may produce extensive axonal degeneration in the central part ot the lesion, outside this is a zone of demyelination virtually free from degenerating fibres. This contrasts with the situation in EAE where fibres undergoing Walleriantype degeneration are commonly intermingled with demyelinated fibres (Lampert 1967). Wigniewski and Raine (1971) took a different view because of the extent of Wallerian-type degeneration and scarring in their experiments. However, their method differs from ours in that they used under-neutralised toxin-antitoxin mixture instead of pure toxin, and infused a hundred times the volume we used. The relative purity of the outer zone of demyelination in the lesion produced by our method and the fact that the lesion can be placed at a predetermined site in the central nervous system have allowed the physiological consequences of demyelination of central nerve fibres to be established (McDonald and Sears 1970b). The lesion has proved useful in studying the pattern of demyelination in single fibres (Harrison et al. 1972), and should be helpful in both morphological and biochemical studies of demyelination and remyelination in the central nervous system.
ACKNOWLEDGEMENTS
We would like to thank Dr. D. N. Landon and Dr. J. F. Hallpike for their helpful discussion of this paper and Mr. K. Yogendran for technical assistance. Dr. Ochoa is a Wellcome Senior Research Fellow. We are grateful to Mr. H. Long for the preparation of the figures for this paper. The work was supported in part by a grant from the Medical Research Council.
SUMMARY
The direct micro-injection of diphtheria toxin into the spinal cord produces a lesion with a central zone of Wallerian-type degeneration and an outer zone of demyelination. Demyelination commences within 6 hr of induction of a lesion, well before the first appearance of phagocytic cells at 24 hr. It is extensive at 3 days but at no stage are cells seen to strip myelin lamellae from intact sheaths. There are no perivascular accumulations of mononuclear cells. The evolution of demyelination in this lesion is thus fundamentally different from that produced in vivo by experimental allergic encephalomyelitis. In the diphtheria toxin lesion the myelin sheaths undergo a number of changes: The lamellar pattern is disrupted and there is an increase in the number of Schmidt-Lantermann-like incisures. A feature of the diphtheria toxin lesions at all stages is that large axons are frequently surrounded by inappropriately thin myelin sheaths. Within the first fortnight a number of naked axons are engulfed by Schwann cells. Demyelination is still widespread 1 year after toxin injection. The diphtheria toxin lesion is a useful model for the study of central demyelination.
REFERENCES BLAKEMORE, W. F. (1969) Schmidt-Lantermann incisures in the central nervous system, J. Ultrastruct. Res., 29: 496-498.
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Bums, J. J. AND S. A. LUSE (1964) An electron-microscope study of experimental allergic encephalomyelitis in the rat, Amer. J. Path., 44: 299-318. BUNGE, R. P., M. B. BUNGE AND H. RIS (1960) Electron-microscopic study of demyelination in an experimentally induced lesion in adult cat spinal cord, J. biophys, biochem. Cytol., 7: 685~596. CAMMERMEYER,J. (1968) Peripheral vasoconstriction with epinephrine for selective fixation of the central nervous system by perfusion, Aeta neuropath., 11 : 368 371. COLLIER, R. J. (1967) Effect of diphtheria toxin on protein synthesis: Inactivation of one of the transfer factors, J. mol. Biol., 25: 83-98. DALTON, A. J. (1955) A chrome-osmium fixative for electron microscopy, Anat. Rec., 121 : 281. GONATAS, N. K., R. D. TERRY AND M. WEISS (1965) An electron-microscope study in two cases of JakobCreutzfeldt disease, J. Neuropath. exp. Neurol., 24: 575-598. HARRISON, B. M., W. I. MCDONALD, J. OCHOA AND G. D. OHLRICH (1972) Paranodal demyelination in the central nervous system, J. neurol. Sei., 16: 489-494. HARRISON, B. M., W. I. McDONALD, J. OCHOAAND T. A. SEARS(1970) Electron-microscope observations on a focal experimental demyelinating lesion in the cat spinal cord, J. neurol. Sci., 10: 409-413. HIRANO, A., H. M. ZIMMERMANAND S. LEVINE (1969) Electron microscope observations of peripheral myelin in a central nervous system lesion, Acta neuropath., 12: 348-365. LAMPERT, P. W. (1965) Demyelination and remyelination in experimental allergic encephalomyelitis, J. Neuropath. exp. Neurol., 24: 371-385. LAMPERT, P. W. (1967) Electron microscopic studies on ordinary and hyperacute experimental allergic encephalomyelitis, Aeta neuropath., 9: 99-126. McDONALD, W. I. AND T. A. SEARS (1969) Effect of demyelination on conduction in the central nervous system, Nature (Lond.), 221: 182-183. McDONALD, W. I. AND T. A. SEARS (1970a) Focal experimental demyelination in the central nervous system, Brain, 93:575 582. McDONALD, W. I. AND T. A. SEARS (1970b) Effects of experimental demyelination on conduction in the central nervous system, Brain, 93: 583-598. PRINEAS, J., C. S. RAINEAND H. WI~NIEWSKI(1969) An ultra-structural study of experimental demyelination and remyelination, III. Chronic experimental allergic encephalomyelitis in the central nervous system, Lab. Invest., 21 : 472-483. RAINE, C. S. AND M. B. BORNSTEIN (1970) Experimental allergic encephalomyelitis: An ultra-structural study of experimental demyelination in vitro, J. Neuropath. exp. Neurol., 29: 177-191. RAINE,C. S., H. WIgNIEWSlClANDJ. PRINEAS(1969)An ultra-structural study of experimental demyelination and remyelination, II. Chronic experimental allergic encephalomyelitis in the peripheral nervous system, Lab. Invest., 21 : 316-327. SUZUKI, K., J. M. ANDREWS, J. M. WALTZ AND R. D. TERRY (1969) Ultra-structural studies of multiple sclerosis, Lab. Invest., 20:444 454. TERRY, R. D., N. K. GONATASAND M. WEISS (1964) Ultra-structural studies in Alzheimer's pre-senile dementia, Amer. J. Path., 44: 269-298, WAKSMAN,B. H. (1961) Experimental study of diphtheritic polyneuritis in the rabbit and guinea pig, III. Blood-nerve barrier in the rabbit, J. Neuropath. exp. Neurol., 20: 35-77. WAKSMAN, B. H., R. D. ADAMS AND H. C. MANSMANN (1957) An experimental study of diphtheritic polyneuritis in the rabbit and guinea pig, I. Immunologic and histopathologic observations, J. exp. Med., 105: 591~14. WEBSTER, H. DE F. (1965) The relationship between Schmidt-Lantermann incisures and myelin segmentation during Wallerian degeneration, Ann. N.Y. Acad. Sci., 122: 29-38. WILLIAMS,P. L. AND S. M. HALL (1971) Prolonged in vivo observations of normal peripheral nerve fibres and their acute reactions to crush and deliberate trauma, J. Anat., 108: 397-408. WlgNIEWSKI,H. AND C. S. RAINE (1971) An ultra-structural study of experimental demyelination and remyelination, V. Central and peripheral nervous system lesions caused by diphtheria toxin, Lab. Invest., 25: 73-80.
J. neurol. Sci., 1972, 17:281-291
Journal of the neurological Sciences Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
Central Demyelination Produced by Diphtheria Toxin" an Electron Microscopic Study B. M. H A R R I S O N , W. I. M c D O N A L D AND J. O C H O A National Hospital, Queen Square, London W.C.1 (Great Britain,) (Received 9 May, 1972)
INTRODUCTION
Demyelination of central nerve fibres occurs in a variety of human and experimental diseases, but the mechanism has been elucidated in only one - experimental allergic encephalomyelitis (EAE). In EAE there appear to be two ways in which myelin is broken down. In one, demyelination is associated with an invasion of exogenous cells and is executed by phagocytes which strip myelin lamellae from otherwise intact sheaths (Bubis and Luse 1964; Lampert 1965 ; Raine, Wi~niewski and Prineas 1969). In the other process, the myelin shows a number of changes such as interlamella splitting and vesiculation of lamellae that are not directly associated with phagocytic stripping or cellular invasion (Prineas, Raine and Wi~niewski 1969; Lampert 1965). Do these processes operate in other circumstances? Suzuki, Andrews, Waltz and Terry (1969) found no evidence of phagocytic stripping in multiple sclerosis, but as they point out, their observations were made on six biopsy specimens which may not have included an acute lesion. Phagocytic stripping was not described in the cerebrospinal fluid exchange lesion (Bunge, Bunge and Ris 1960) and demyelination was not associated with cellular invasion. Similarly, exposure of spinal cord cultures to EAE serum produced demyelination in a situation where no cellular invasion could occur (Raine and Bornstein 1970). In view of the small amount of information available about the mechanism of demyelination in vivo, and the fact that phagocytic stripping has been observed only in EAE it seemed appropriate to study a lesion which on a priori grounds we would not expect to have an allergic basis. Systemically-administered diphtheria toxin produces demyelination in the peripheral nervous system but because the blood-brain barrier is impermeable to the toxin, there is no demyelination of central nerve fibres (Waksman 1961). McDonald and Sears (1969) showed that focal central demyelination could be produced, when the blood-brain barrier was by-passed, by injecting a minute amount of diphtheria toxin directly into the spinal cord. Since peripheral demyelination by diphtheria toxin is independent of the immune system (Waksman, Adams and Mansmann 1957) J. neuroL Sci., 1972, 17:281-291
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it seemed probable that the same would be true of the central diphtheria toxin lesion. We have, therefore, made a detailed electron microscopic study of this lesion. In a recent paper we dealt with the predominantly paranodal distribution of demyelination at the edges of the lesion (Harrison, McDonald, Ochoa and Ohlrich 19721. In the present paper we describe the evolution of demyelination, and in the following paper we shall discuss the question of remyelination. A short report of some aspects of this work has been published (Harrison, McDonald, Ochoa and Sears 1970). Since completing this study a paper by Wigniewski and Raine (1971) describing the central diphtheria toxin lesion in six rabbits and two cats has appeared. Although we confirm a number of their observations, we have made additional observations, and there are important differences in interpretation deriving from the study of lesions both earlier and later than those studied by Wigniewski and Raine.
METHOD
A total of 25 cats was studied. Under pentobarbitone anaesthesia and with aseptic precautions, a laminectomy was performed in the thoracolumbar region. Injections of diphtheria toxin were made into the dorsolateral sulcus of the spinal cord using the technique described previously (McDonald and Sears 1970a). The wound was then closed and the animals were allowed to survive for varying lengths of time. Crystalline diphtheria toxin was kindly supplied by Dr. Edwards of the Wellcome Research Laboratories. The toxin was dissolved in borate buffer at pH 7.4. The volume of liquid injected into the cord was 3 /~1 in all except the 1-month animal (5 #l). Sixteen animals were injected with 0.001-0.003 fl0cculation units of diphtheria toxin. These animals were perfused after 6 hr (2), 13 hr (1), 1 day (1), 2 days (1), 3 days (1), 5 days (1), 7 days (2), 13 days (1), 14 days (1), 1 month (1), 2 months (1)~ 3 months (1), 4 months (1), and 1 year (1). Other animals were examined as controls. Five animals were normal. One animal was injected with buffer alone and examined after 7 days. In one animal two laminectomies were performed, which were separated by two cord segments. At the upper site a needle was inserted into the cord in the usual way but nothing was injected. At the lower site, the dura was simply opened and not followed by insertion of the needle. This animal was perfused after 7 days. Two cats were perfused 2 and 3 days after transection of the posterior columns to produce Wallerian degeneration. After intracardiac injection of adrenalin (1 mg/kg) (Cammermeyer 1968), the spinal cord was fixed by retrograde perfusion through the abdominal aorta at 150--200 mm of mercury. 300 ml of saline was followed by 600 ml of 4-5 ~o glutaraldehyde in M/I 5 phosphate buffer (pH 7.4), then by 400 ml of chrome-buffered osmium tetroxide (Dalton 1955). The fixatives were at 4°C. The tissue was prepared for electron microscopic examination in the conventional way. Representative 1.5 # thick sections of the whole cross-section of the spinal cord were cut at intervals of approx. 1 mm over a distance of 1 cm on either side of the injection site and counter-stained with toluidine blue for light microscopy. Ultra-thin sections from areas selected after examination of thick sections, were examined with a Siemens Elmiskop la electron microscope. J. neurol. Sci., 1972, 17:281--291
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Counts of myelin incisures Counts of incisures in central myelin were performed in normal and experimental animals. Thin transverse sections from the dorsal root entry zone were scanned at a working magnification of 8,000-9,500 diameters. 10-15 micrographs were exposed with 5-25 fibres on each plate. For convenience in counting, fields with fibres greater than 3 #m were avoided. Apart from this, electron micrographs were taken at random, care being taken not to photograph the same fibre twice. The plates were enlarged to a final magnif, cation of 20,000-24,000 diameters. At this magnification it was easy to identify the fibres that had incisures because the cytoplasm could be seen clearly between myelin lamellae. For each animal, the number of fibres with incisures was expressed as a percentage of the total number of fibres counted. The significance of the difference between the mean of the results from normal and diphtheria toxininjected animals was tested using the Z2 (chi-square) distribution and applying the Yates' correction for binomial distributions.
RESULTS
Normal spinal cord The thoracolumbar region of the spinal cord contains both myelinated and unmyelinated fibres. Two normal features require comment because of their relevance to the interpretation of the pathological material. To distinguish in experimental animals between normally unmyelinated fibres and demyelinated fibres, it was
Fig. 1. Normal cat. Region near dorsal root entry zone. A small axon, 0.3/~m in diameter, is surrounded by myelin which is typical of the central nervous system. Bar, 0.2 #m. J. neurol. Sci., 1972, 17:281-291
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necessary to determine the critical axonal size for myelination. Axons as small its 0.3 /~m had myelin sheaths (Fig. 1) and no axons greater than 1 /~m were found without myelin. Throughout this work, axons were not accepted as demyelinated unless their diameter was greater than 1 #m. Although dendrites may sometimes be confused with axons in the border zones between grey and white matter, this was not a problem in the regions studied in the present experiments. lncisures resembling those of Schmidt and Lantermann in the peripheral nervous system have been described in detail in the central nerve fibres of the rat (Blakemore 1969; Hirano, Zimmerman and Levine 1969), and were easily recognised in the present material. In transverse sections they appear as separations of the lamellae at the major dense line with electron-dense, granular cytoplasm (derived from the myelin-forming oligodendrocyte) filling the space between the separated lamellae. Incisures occurred in fibres of all sizes, and counting showed that at any level they were present in 2.3 ~o of fibres less than 3 #m in diameter. In longitudinal section they appeared as a series of loops filled with cytoplasm arranged in a step-wise fashion in consecutive lamellae.
Diphtheria toxin lesion A survey of all the lesions showed that there was an approximate correlation between the amount of toxin injected and the size of the lesion. The lesion itself was clearly orientated to the needle track. There were no perivascular accumulations of mononuclear cells. At every stage there was a central zone of degeneration where
7day diphtheria toxin injection 1 cm
Fig. 2. Diagram to show the distribution of changes within the spinal cord 7 days after the injection of diphtheria toxin. Solid black: Many fibres show WaUerian-type degeneration. Hatched: The majority of the axons appear normal but many myelin sheaths are abnormal. The extracellular space in these two zones is expanded. Elsewhere in the cord, around the injection site, the extraeellular space is normal. Most axons are normal but some are surrounded by abnormal myelin sheaths. The approximate distribution of these fibres with abnormal myelin is shown by dots in the diagram.
many fibres showed disintegration of both axons and myelin (Fig. 2). The size of this zone varied, being scarcely wider than the needle in the control preparation in which a needle alone was inserted into the cord, and many times larger than this after the injection of toxin. For several mitlimetres around the central zone, the extraceUular space was expanded, presumably by oedema. There were many abnormal fibres here too, but the vast majority of these fibres had abnormal myelin sheaths but normal axons. Outside this was a region in which there were still abnormal fibres, but the extracellular space appeared to be of normal size. This zone gradually faded over about 5 mm into normal spinal cord. J. neurol. Sci.. 1972. 17:281-291
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In the following description of the diphtheria toxin lesions we shall describe only those changes which occurred outside the central zone, that is away from the region of Wallerian-type degeneration. Demyelination had already commenced at 6 hr. Myelin debris was present in the expanded extracellular space around the needle track and consisted of irregular clumps of electron-dense material. Usually the lamellar pattern was lost, leaving an amorphous mass, but in some fragments it was preserved. A few scattered, completely demyelinated axons were present and a few large diameter axons had inappropriately thin myelin sheaths. Between 24 and 72 hr there was a marked increase in the amount of myelin debris and in the numbers of demyelinated axons. At the centre of the lesion the majority of the axons showed changes typical of Wallerian-type degeneration, but more peripherally the axoplasm of demyelinated fibres appeared normal. At 3 days demyelinated axons occurred in small groups. The size of the groups increased until they contained up to 30 large diameter demyelinated axons at 13 days (Fig. 3). At 5 days the axons within a group were in contact with each other (Fig. 4), but from 7 days on the fibres were increasingly separated by invading cellular processes. Some of these cellular processes contained myelin debris. Others contained fibrils suggesting that they were derived from astrocytes. Fragments of basement lamina were seen in relation to some of the processes.
Fig. 3. 13-day lesion. Posterior columns. A large number of demyelinated axons are closely packed. Some are completely surrounded by dark cytoplasm (not myelin). Debris-laden cells are scattered throughout the region. Bar, 20 #m. J. neurol. Sci., 1972, 17:281-291
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Fig. 4. 5-day lesion. Six demyelinatcd axons lie within an expanded extracellular space. Fheir axolemmas are m close contact. Bar. 2 Ira1
Fig. 5. l-month lesion. A naked axon (3/~m) is embedded in cell cytoplasm. An arrow points to a mesaxonlike structure. The cell itself is surrounded by a basement lamina (BL). N. nucleus: C. collagen fibres. Bar, 1 Itm.
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Fig. 6. 6-hr lesion. An axon is surrounded by a ring of elliptical profiles that are derived from the myelin sheath. There is an intra-period line between the membrane of each profile and the remaining compact myelin (arrow). Bar, 0.2/~m.
From 13 days onwards a few individual axons were surrounded by cells with a complete investment of basement lamina (Fig. 5). Collagen was frequently present in the vicinity of these cells which appear to be Schwann cells. Further evidence for this conclusion will be presented in the following paper. The astrocyte reaction increased with time, and by 2 months the expanded extracellular space was filled with densely-packed astrocytic processes. At 2 months the number of fibres in the lesion was reduced, and surviving fibres were widely separated. Many fibres remained completely demyelinated and as in all earlier lesions some axons had inappropriately thin myelin. Myelin debris still persisted up to 4 months after induction of the lesion. After 1 year, debris was no longer present, but apart from an increase in the collagen the lesion resembled that at 2 months.
Early changes in myelin The appearance of damaged myelin around a normal axon took several forms. In some fibres the intra-period line was split leaving a clear space between adjacent J. neurol. Sci., 1972, 17:281-291
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major dense lines. No cellular process intervened between the split lamellae. Although separation of lamellae at the intra-period line is a common preparation artefact, wc think that it is an important part of the pathological process since in the earliest lesions it is most prominent at the nodes while the nearby internodal myelin appears well-fixed and intact. A second change was vesiculation of myelin lamellae both on the outer and inner surfaces of the sheath (Fig. 6). This change was seen in the 6-hr. and in some, but not all, older lesions. A third feature was an increase in the number of incisures in the myelin of the toxin-injected animals. In four normal animals a mean of 2.3 ~/o of fibres up to 3 /~m in diameter (see Method) showed incisures. By contrast, there were incisures in a mean of 6.1 ~o of fibres in lesions from 6-hr- to
Fig. 7. 2-day lesion. Lateral columns. Transverse section through Schmidt-Lantermann-like incisure. Some myelin lamellae are separated along the major dense line (arrows) by electron-dense granular cytoplasm. GP, glial processes on the outer surface of the sheath. Bar. 0.25 pm.
4-month-old (10)lesions. The difference between the mean of the normal and diphtheria toxin-injected animals is highly significant (P=0.002). Structurally the incisures in the lesion (Fig. 7) resembled normal incisures but the granular material often formed a bulge to one side of the sheath and here there were fewer myelin lamellae than on the other side of the sheath. All these changes in the myelin were visible before the first appearance of phagocytes, which was at 24 hr. The phagocytic invasion increased markedly in the first 3 days. Intracellular debris was first seen at 24 hr, and was very prominent from 3 days to 13 days. In contrast to the findings reported in EAE, however, phagocytes were never seen to strip apparently normal myelin lamellae from otherwise intact sheaths. J. neurol. Sci., 1972, 17:281 291
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Control animals
Dural opening alone produced no lesion after 7 days. Injection of 3 #1 of buffer produced after 7 days a lesion only slightly larger than that produced by simple insertion of a needle. The fibres in the needle track were undergoing Wallerian degeneration. Around the needle track were a few completely-demyelinated and a few thinly-myelinated axons. There was an increase in numbers of fibres showing incisure-like clefts. DISCUSSION
The present experiments have shown that in the central diphtheria toxin lesion the breakdown of the regular lamellar pattern of myelin to irregular clumps of debris occurs prior to ingestion of myelin by phagocytes. At no stage do phagocytes strip apparently normal lamellae from an otherwise intact myelin sheath. The evolution of demyelination in this lesion is thus fundamentally different from that in EAE in vivo where a major mechanism for myelin destruction involves initial phagocytosis of intact lamellae (Lampert 1965). It is possible that the very early myelin changes are contributed to by trauma from the injection needle. However, by 48 hr the lesion is much larger than that produced by the injection of buffer. We, therefore, attribute the changes at this stage to the action of the toxin. The means by which diphtheria toxin produces demyelination is unknown. Although it has been shown to inhibit protein synthesis in cell-free systems (Collier 1967) the relevance of this to its in vivo action is uncertain. We are not in a position to decide whether diphtheria toxin attacks the myelin sheath directly or whether it damages the myelin-forming cell (oligodendrocyte) first. The importance of vesiculation of myelin sheaths in the demyelinating process in the central diphtheria toxin lesion has recently been stressed by Wi~niewski and Raine (1971). However, because in our experiments vesiculation was not seen at all in some lesions with extensive demyelination and was present in only a few fibres in those lesions in which it was seen, we do not believe that it represents a necessary stage in myelin breakdown in this lesion. One of the early features of the diphtheria toxin lesion was the increase in the number of fibres in cross-section that had incisures. This has not previously been reported in the central nervous system. Morphologically similar appearances have been reported in cases of Alzheimer's presenile dementia (Terry, Gonatas and Weiss 1964) and in Jakob-Creutzfeldt disease (Gonatas, Terry and Weiss 1965). It has been reported that the number of incisures that occur in the peripheral nervous system increases during Wallerian degeneration (Webster 1965). Recent evidence suggests that this increase is due to the expansion of pre-existing incisures (Williams and Hall 1971), rather than the formation of new ones. In either case, the change represents a reaction by the myelin-forming cell. In the diphtheria toxin lesion, an increase in incisures occurred in the outer parts of the lesion well away from the central zone of axonal degeneration, and was present long after degeneration was completed. It seems likely that an increase in the number of incisures is an early phase of demyelination which-does not necessarily go on to complete myelin breakdown. This lesion is a useful model for the study of central demyelination. Although J. neurol. Sci., 1972, 17:281-291
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diphtheria toxin may produce extensive axonal degeneration in the central part ot the lesion, outside this is a zone of demyelination virtually free from degenerating fibres. This contrasts with the situation in EAE where fibres undergoing Walleriantype degeneration are commonly intermingled with demyelinated fibres (Lampert 1967). Wigniewski and Raine (1971) took a different view because of the extent of Wallerian-type degeneration and scarring in their experiments. However, their method differs from ours in that they used under-neutralised toxin-antitoxin mixture instead of pure toxin, and infused a hundred times the volume we used. The relative purity of the outer zone of demyelination in the lesion produced by our method and the fact that the lesion can be placed at a predetermined site in the central nervous system have allowed the physiological consequences of demyelination of central nerve fibres to be established (McDonald and Sears 1970b). The lesion has proved useful in studying the pattern of demyelination in single fibres (Harrison et al. 1972), and should be helpful in both morphological and biochemical studies of demyelination and remyelination in the central nervous system.
ACKNOWLEDGEMENTS
We would like to thank Dr. D. N. Landon and Dr. J. F. Hallpike for their helpful discussion of this paper and Mr. K. Yogendran for technical assistance. Dr. Ochoa is a Wellcome Senior Research Fellow. We are grateful to Mr. H. Long for the preparation of the figures for this paper. The work was supported in part by a grant from the Medical Research Council.
SUMMARY
The direct micro-injection of diphtheria toxin into the spinal cord produces a lesion with a central zone of Wallerian-type degeneration and an outer zone of demyelination. Demyelination commences within 6 hr of induction of a lesion, well before the first appearance of phagocytic cells at 24 hr. It is extensive at 3 days but at no stage are cells seen to strip myelin lamellae from intact sheaths. There are no perivascular accumulations of mononuclear cells. The evolution of demyelination in this lesion is thus fundamentally different from that produced in vivo by experimental allergic encephalomyelitis. In the diphtheria toxin lesion the myelin sheaths undergo a number of changes: The lamellar pattern is disrupted and there is an increase in the number of Schmidt-Lantermann-like incisures. A feature of the diphtheria toxin lesions at all stages is that large axons are frequently surrounded by inappropriately thin myelin sheaths. Within the first fortnight a number of naked axons are engulfed by Schwann cells. Demyelination is still widespread 1 year after toxin injection. The diphtheria toxin lesion is a useful model for the study of central demyelination.
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