Electron microscope observations on demyelination and remyelination in experimental allergic neuritis

Journal of the neurological Sciences

1

Elsevier PublishingCompany,Amsterdam- Printed in The Netherlands

Electron Microscope Observations on Demyelination and Remyelination in Experimental Allergic Neuritis Part 1. Demyelination R. H. M. BALLIN AND P. K. THOMAS Institute of Neurology, London (Great Britain)

(Received 10 May, 1968)

INTRODUCTION Extensive studies on experimental allergic encephalomyelitis (EAE) have now been published, since the original demonstration by HURST(1932) of its production by the repeated inoculation of normal homologous or heterologous brain into rabbits, and the observation that this effect could be enhanced by the use of adjuvants (FREtmD AND MCDERMOTT 1942). The production of a peripheral neuropathy by the injection of peripheral nerve tissue combined with Freund's adjuvants was first achieved by WAKSMANANDADAMS(1955), who termed the condition experimental allergic neuritis (EAN). Their histological studies revealed that the lesions consist of multiple perivascular foci of mononuclear cells in relation to which demyelination occurs. Destruction of axons also results, but this is less evident. From the examination of isolated nerve fibres, CRAGGAND THOMAS(1964) showed that the demyelination begins in the region of the nodes of Ranvier, but may extend to involve whole internodal segments, several adjacent segments sometimes becoming denuded of myelin. Species differences in the distribution of the lesions exist (WAKSMAN AND ADAMS1956). In rabbits and mice, the lesions tend to be restricted to the dorsal roots and dorsal root ganglia, whereas in guinea-pigs there are in addition more widespread lesions in the peripheral nerve trunks. EAE and EAN are at present generally considered to be initiated by sensitized mononuclear cells which enter the tissues through the vessel walls and give rise to the demyelination. Support for this view has been provided by the observations of PATERSON (1960) and /]kSTR()M AND WAKSMAN(1962), which showed that passive transfer of the disease by lymphocytes from affected animals is possible, whereas serum is ineffective. From this it appears that mononuclear cell infiltration is the primary event, the parenchymal damage being a secondary phenomenon (WAKSMANAND ADAMS1962). J. neuroL Sci. (1968) 8:1-18

2

R.H.M. BALLIN,P. K. THOMAS

Electron microscope studies on EAE by LAMPERT AND CARPENTER (1965) and LAMPERT (1965, 1967) have indicated that two separate patterns of demyelination occur. One type consists of a vesicular breakdown of the myelin lamellae that takes place extracellularly and which is frequently preceded by separation of the lamellae. The second type is produced by invasion of the myelin sheaths by mononuclear cells which can be observed to insinuate themselves between the lamellae or to strip the myelin away from the axons. The myelin is then removed by macrophages. Electron microscope observations on the process of demyelination and remyelination in EAN have not so far been published. We have investigated this condition as an example of a demyelinating neuropathy in order to obtain further knowledge as to the way in which the demyelination takes place, and if possible to correlate the morphological alterations with the changes in nerve conduction that are known to occur. This communication reports our findings on demyelination in EAN in guineapigs. The appearances associated with remyelination will be dealt with in a subsequent paper (BALLIN AND THOMAS 1969). MATERIALAND METHODS Thirty-eight adult albino guinea-pigs of both sexes weighing between 420 and 950 g were injected with a homogenate of rabbit sciatic nerve and Freund's adjuvants by the method of WAKSMANAND ADAMS (1955) as employed by CRAG~ AND THOMAS(1964). The injectant was prepared as follows. The whole length of both sciatic nerves and their branches were removed from 2 adult albino rabbits. The nerves were then cut cross-wise into small pieces, sectioned at 15 # on a freezing microtome, and recut several times at 5 # until a fine brei was formed. This was collected and weighed. Ten ml of Freund's incomplete adjuvant and 30 mg of Mycobacterium butyricum were added together and well shaken. A volume of this mixture in ml equal to 1.5 times the weight of Zhe nerve in g was added to the latter and the mixture emulsified in a fast electric blender. Sterile apparatus was employed throughout. The final emulsion contained approximately 0.4 mg sciatic nerve, 0.5 mg Bayol F, 0.1 ml Arlacel A and 2 mg bacilli per ml. Nine animals were injected intradermally in each footpad with 0.01 ml of the emulsion. The remainder were all given 0.02 ml per footpad. The incidence of clinically-detectable neurological illness varied considerably between different batches of animals in these experiments and there was no obvious correlation between the weight or sex of the animals and the production of overt illness. Twenty of the 38 animals were affected, but 5 died before biopsy was performed. The interval between the injection of the emulsion and the appearance of the first signs of weakness varied from 13 to 27 days, with a mean of 17 days. Biopsies were obtained from 12 animals between 13 and 32 days after injection, whilst showing signs of paralysis. Three affected animals were allowed to survive and were biopsied after recovery for examination of remyelination; the findings in these animals will be given in the second part of this study (BALLIN AND THOMAS 1969). Material was also obtained from 4 normal animals for control observations. The biopsies were performed under ether anaesthesia after the preliminary intraperitoneal J. neurol. Sci. 0968) 8:1-18

EM OBSERVATIONS IN EXPTL. ALLERGIC NEURITIS, PART 1 (DEMYELINATION)

3

injection of pentobarbitone sodium (Nembutal). Specimens were taken from the dorsal roots and dorsal root ganglia in the lumbo-sacral region and from the sural nerve in all instances, and also from the peroneal nerve in some animals. These were fixed for 4 h at 4°C by immersion in 1% osmium tetroxide in mammalian Ringer solution, buffered to pH 7.4 with veronal-acetate. After dehydration in graded concentrations of ethanol, some specimens were stained in block for 3 h with 1 °/o phosphotungstic acid in absolute ethanol. After embedding in Araldite, the material was sectioned with the Cambridge or Porter-Blum MT2 ultramicrotomes and collected on carbon-coated copper mesh grids. Sections from the material not treated with phosphotungstic acid were stained on the grids with saturated aqueous uranyl acetate followed by 1% lead citrate (VENABLEAND COGGESHALL1965). Because of the focal nature of the lesions, large sections of the order of 1.5 # in thickness were usually first obtained, fixed to a glass coverslip by gentle heating and either stained with toluidine blue, or a combination of toluidine blue, malachite green and basic fuchsin (GRIMLEY 1964). After staining, the sections were mounted in Canada balsam on a glass slide and examined by optical microscopy. Affected areas of nerve were identified and the blocks trimmed accordingly. The electron microscope observations were made using a Siemens Elmiskop 1. RESULTS

Cellular infiltration

The details of the leucocyte emigration from the blood vessels and the nature of the cellular infiltration were not studied in detail. The presence of collections of leucocytes in the endoneurial spaces, often in relation to blood vessels, was confirmed (Fig. 1). The endoneurial spaces tended to be expanded and oedematous. The question as to whether the leucocytes consistently appear before demyelination takes place cannot be answered from the present results, as animals were not biopsied before clinical signs of paralysis were evident. It was sometimes difficult to detect mononuclear cells in nerves showing early demyelination, but sampling difficulties in electron microscope studies of this nature are considerable. The infiltration consisted predominantly of lymphocytes and monocytes, plasma cells being encountered only rarely. Polymorphonuclear leucocytes were present in moderate numbers, particularly in severely affected animals, a feature previously commented upon in EAN in guinea-pigs by WAKSMAN AND ADAMS (1956). Cells containing myelin remains and displaying morphological similarities to monocytes were commonly observed in the endoneurial spaces (Fig. 1). Similar cells were also seen within Schwann tubes, that is, within the spaces enclosed by the Schwann cell basement membranes (THOMAS1964), in relation to dernyelinated axons. It is not possible to state whether these are derived from mononuclear leucocytes that have invaded the Schwann tubes or whether they are Schwann cells that have undergone transformation into macrophages. The same difficulty arises in the identification of the origin of the macrophages present in the endoneurial spaces. Undoubted polymorphonuclear leucocytes were occasionally seen within the Schwann tubes (Fig. 5A), but were only encountered in relation to axons that had been completely demyelinated. The alterations in the vasa nervorum have also not been examined in detail in the J. neurol. Sci. (1968) 8:1-18

4

R . H . M . BALLIN, P. K. THOMAS

Fig. 1. Focal collection of cells in endoneurium of sural nerve 22 days after injection. In the lower portion of the figure are a neutrophil granulocyte (ngc) and a mononuclear leucocyte, probably a monocyte (mc). At the upper right of the figure is a macrophage (mp) containing myelin debris (dmy) lying free in the endoneurium and at the upper left is a Schwann tube, the basement membrane of which encloses a demyelinated axon (ax) and a cell of similar appearance (mp) also containing myelin debris. Phosphotungstic acid stain.

present study. T h e changes that were observed were limited to r ed u p l i cat i o n o f the basement m e m b r a n e a n d p r o b a b l e endothelial cell proliferation. Th e c y t o p l a s m o f the endothelial cells tended to appear m o r e bulky t h a n n o r m a l and to co n t ai n m a n y p i n o cy t o t i c vesicles, suggesting active fluid transport.

~neurol. Sci. (1968) 8:1-18

EM OBSERVATIONS IN EXPTL. ALLERGIC NEURITIS, PART

1 (DEMYELINATION)

5

Changes at the nodes of Ranvier The earliest detectable changes at the nodes of Ranvier consisted of separation and retraction of the terminal loops of the myelin lamellae and of the Schwann cell nodal processes from the axon. The appearances were examined both in transverse and longitudinal sections of the nerve, the latter in general being more informative. The loss of attachment of the terminal myelin loops to the axolemma was sometimes restricted to the inner (Fig. 3A) or outer lamellae (Fig. 2A, B), or involved the full width of the terminal myelin. If the outer lamellae were selectively affected, the terminal loops tended to withdraw from the node and to lie surrounded by the paranodal Schwann cell cytoplasm, often directed away from the surface of the axon (Fig. 2A, B). The Schwann cell nodal processes likewise became separated from the axolemma and later became lost, the Schwann cell cytoplasm often extending down into the nodal gap formerly occupied by the nodal processes (Fig. 8B). The detached terminal myelin loops underwent a vesicular breakdown (Figs. 3A and 6A). The details of this have not yet been established with certainty, because of the problems of preparative artefact. Artefactual separation of the myelin lamellae tends to occur especially in the myelin at the nodes of the larger nerve fibres and is probably responsible for much of the separation evident in Figs. 2A and B, and 3A. Perfusion fixation with glutaraldehyde fails to eliminate this. It is tentativelyconsidered that the vesicles probably form as spaces resulting from splitting of the major dense lines of the myelin after prior separation at the intraperiod line, as was found by LAMPERT(1967) in EAE, by MASUROVSKVet al. (1967) following X-irradiation of myelinated nerve fibres in tissue culture and by THOMAS(1968) during the extracellular breakdown of myelin in Wallerian degeneration of peripheral nerve. This is likely to be the result of hydration of the myelin. More severely-affected nodes displayed a widening of the distance between the myelin of the adjacent Schwann cells, the nodal axon either being surrounded by a cuff of Schwann cell cytoplasm (Fig. 6A) or, less commonly, devoid of any covering except the Schwann cell basement membrane. The Schwann cell cytoplasm around the nodal axon and adjacent to the terminal myelin lamellae, at this stage but not earlier, frequently contained dense bodies and vacuoles within which there was granular or lamellar material. Their morphological features suggested that they are probably lysosomal in nature, but histochemical confirmation is required. Schwann cell nodal processes were no longer evident. Mononuclear cells were never seen in direct relationship to the myelin at this stage. Segmental demyelination The early changes during breakdown of the internodal myelin proved difficult to evaluate, nerve fibres tending to show either intact internodal myelin or extensive breakdown. It is therefore possible that once breakdown begins, the process advances rapidly so that the early changes are infrequently observed. An example of extensive breakdown of the internodal myelin is shown in Fig. 4B, the axon being surrounded by multiple small ovoids or spheres of disintegrated myelin. Such ovoids occasionally lay apparently extracellularly within clefts in the axon (Fig. 7C). "Ribbon" forms were also observed, the terminations of the myelin lamellae tending to become separated and to undergo vesicular disintegration (Fig. 7B). Sometimes only the inner myelin J. neurol. Sci. (1968) 8 : 1-18

6

R . H . M . BALLIN~ P. K. THOMAS

A

Fig. 2A. Longitudinal section through abnormal node from dorsal root 22 days after injection. In the lower portion of the figure the outer terminal loops (tl) of the myelin (my) have retracted from the nodal region on both sides. The nodal axoplasm (ax) contains two dense lamellar structures (db). Phosphotungstic acid stain. Fig. 2B. Detail from A, showing the retraction of the outer terminal myelin loops (tl) from the node apparently into the Schwann cell cytoplasm (Sc) and the presence of a vesicular bleb (v) between the myelin lamellae.

J. neurol. Sci. (1968) 8 : 1 - 1 8

EM OBSERVATIONS IN EXPTL. ALLERGIC NEURITIS~ PART 1 (DEMYELINATION)

7

A

B

Fig. 3A. Longitudinal section through an abnormal node from sural nerve 22 days after injection. The inner myelin lamellae at the lower left of the figure have undergone vesicular breakdown (v). Phosphotungstic acid stain. Fig. 3B. Longitudinal section through completely demyelinated axon (ax) from sural nerve 22 days after injection. The axon is surrounded by the collapsed basement membrane (brn) of the original Schwann tube. Phosphotungstic acid stain.

J. neurol. Sci. (1968) 8:1-18

8

R . H . M . BALLIN, P. K. THOMAS

A

Fig. 4A. Longitudinal section through axon (ax) from dorsal root 22 days after injection. Internal to a narrow layer of myelin (my), the Schwann cell cytoplasm contains multiple vacuoles (v) and dense bodies (db). Phosphotungstic acid stain. Fig. 4B. Longitudinal section through demyelinated axon (ax) surrounded by multiple small ovoids of degenerating myelin (dmy). Sural nerve 15 days after injection. Phosphotungstic acid stain.

J. neurol. Sci. (1968) 8: I 18

A

B

Fig. 5A. Transverse section through Schwann tube from sural nerve 22 days after injection. The basement membrane (bin) encloses a demyelinated axon (ax) partially surrounded by a neutrophil granulocyte (ngc), external to which is a crescentic process that is probably a portion of a Schwann cell (Sc). Phosphotungstic acid stain. Fig. 5B. Transverse section through Schwann tube, the basement membrane (bin) of which encloses a completely demyelinated axon (ax). The axon contains mitochondria (m) and a lamellar body (lb). Sural nerve 22 days after injection. Phosphotungstic acid stain.

I0

R . H . M . BALLIN, P. K. THOMAS

lamellae a n d S c h w a n n cell cytoplasm appeared to be affected, single-walled vesicles lying between the a x o l e m m a a n d the i n n e r m o s t surviving myelin lamella. These appearances resembled those f o u n d in X-irradiated tissue cultures of dorsal root ganglia by MASUROVSKY et al. (1967). At other times, the S c h w a n n cell cytoplasm internal to

A

B

Fig. 6A. Longitudinal section through axon (ax) from sural nerve 15 days after injection showing paranodal demyelination. The terminal regions of the myelin (my) show vesicular breakdown (v). The demyelinated axon is surrounded by a cuff of Schwann cell cytoplasm; on the right, this contains a dense body (db). Phosphotungstic acid stain. Fig. 6B. Longitudinal section through axon (ax) at the junction between a myelinated and a demyelinated region. To the right of the figure, the axon is surrounded by myelin (my) that terminates in a heminode of normal appearance, except that the Schwann cell cytoplasm (Sc) is prolonged around the demyelinated axon to the left. Dorsal root 22 days after injection. Phosphotungstic acid stain.

J. neurol. Sci. (1968) 8:1-18

A

B

C

Fig. 7A. Longitudinal section through Schwann tube from sural nerve 22 days after injection. The tube encloses a demyelinated axon (ax) and a cell containing myelin debris (dmy). This cell extends through the basement membrane (bm) of the Schwann tube in the lower part of the figure. Phosphotungstic acid stain. Fig. 7B. Demyelination from dorsal root 22 days after injection. The myelin (my) is undergoing breakdown into multiple vesicular profiles (v). Uranyl acetate and lead citrate stain. Fig. 7C. Demyelination from dorsal root 22 days after injection, with small multilamellar ovoids of myelin (dmy) lying in a cleft in the axon (ax). Uranyl acetate and lead citrate stain.

J. neurol. Sei. (1968) 8:1-18

A

B Fig. 8A. Schwann tube, the basement membrane (bin) of which encloses a demyelinated axon (ax) surrounded by a Schwann cell (Sc) and a number of smaller cell processes (Scp) probably also derived from Schwann cells. The latter are surrounded by new basement membrane. Dorsal root 32 days after injection. Phosphotungstic acid stain. Fig. 8B. Longitudinal section through portion of node of Ranvier from sural nerve 22 days after injection. To the left of the figure, the axon (ax) protrudes between the terminal myelin lamellae (my). The axonal protrusion contains a mitochondrion (in), two dense bodies (db) and vesicular structures. Phosphotungstic acid stain.

EM OBSERVATIONS IN EXPTL. ALLERGIC NEURITIS, PART 1 (DEYMELINATION)

13

the myelin contained multiple vacuoles and dense bodies (Fig. 4A). Frequently, the degree of disorganization observed suggested a generalized cytolysis of the Schwann cell, with disintegration of both myelin and cytoplasm. Necrosis of a Schwann cell nucleus, however, was only detected in one instance. An appearance of different type encountered quite commonly was for an intact axon to be displaced to one side of a Schwann tube and in direct contact with the Schwann cell basement membrane, the remainder of the tube containing a crescentic mass of myelin in which the lamellar pattern was preserved. As in the region of the nodes, leucocytes were again not seen within the Schwann tubes in direct relationship to the disintegrating myelin. Completely demyelinated axons were often observed as isolated structures within the Schwann tubes, surrounded by the persisting Schwann cell basement membrane (Figs. 3B and 5B). At other times, the demyelinated axons shared the Schwann tubes with leucocytes (Fig. 5A) or with cells that contained myelin breakdown products and which were sometimes observed extending through gaps in the basement membrane (Fig. 7A). The nature of such cells, as noted earlier, is diffficult to ascertain on morphological grounds, it being uncertain whether they are Schwann cells or histiocytes that have entered the tubes. At the junction between a demyelinated axon and a preserved internodal segment, processes usually extended out from the intact Schwann cell and surrounded the demyelinated axon for a distance of up to 10/~ (Fig. 6B). These processes were sometimes multiple and layered one upon the other. In the biopsies taken at the later stages after injection, the Schwann tubes enclosing demyelinated axons also contained multiple cell processes that were considered most probably to be derived from Schwann cells (Fig. 8A). The outer processes tended to become separated from those in direct relation to the demyelinated axon and to become surrounded by new basement membrane. Axonal changes

Fibres undergoing complete axonal degeneration were observed, but this was much less common than demyelination with survival of the axon. The demyelinated axons displayed comparatively few abnormalities, but occasionally contained concentric lamellar structures (Fig. 5B), similar to those present during Wallerian degeneration (WEBSTER 1962) and shown to be lysosomal in nature by HOLTZMAN AND NOVIKOFF (1965). Protrusions of the paranodal axon between the terminal myelin loops were sometimes noted (Fig. 8B) and mitochondria, dense bodies and lameUar structures tended to be sequestered in these axonal out-pouchings. They could be observed at nodes that otherwise showed only minor abnormalities, such as the loss of the Schwann cell nodal processes, as in Fig. 8B. DISCUSSION

In the present study, a clear relationship between the presence of mononuclear cells in the tissues and evidence of demyelination was not always detectable. However, the demonstration by light microscopy that mononuclear cell invasion is the primary event (WAt:SMAN AND ADAMS 1962) seems more likely to be correct in view of the sampling difficulties inherent in the use of ultrathin sections. On the other hand, it was evident J. neurol. ScL (1968) 8:1-18

14

R.H.M. BALLIN, P. K. THOMAS

that the initial stages of demyelination at the nodes of Ranvier did not involve the direct contact of leucocytes with the nodal structures. This therefore suggests that the demyelination is initiated by a chemical process, possibly by the local production of antibodies by leucocytes. For the more extensive myelin breakdown involving longer lengths of the internode, it again appeared that this could take place without the direct proximity of leucocytes, although here, because of the difficulty in the identification of cells within the Schwann tubes, the presence of intratubal mononuclear leucocytes could not be excluded with the same degree of certainty. Myelin breakdown unrelated to direct contact with mononuclear cells was observed in EAE by LA~PERT (1967). The second pattern of demyelination in EAE observed by LAMPERT, that is, the stripping of the myelin from the axon by mononuclear cells or the insinuation of their processes between the myelin lamellae, was not seen. The early changes at the nodes of Ranvier consist of the separation of the terminal myelin loops from the axon and the loss of the Schwann cell nodal processes. Observations on the control nerves (BALLIN 1967) have confirmed that the terminal myelin loops in peripheral nerve, as in the central nervous system (LAATSCH AND COWAN 1966), are closely related to the axon, lying in grooves in the axolemma. The outer lamina of the trilaminar unit membrane of the adjacent axolemma bears a regular annular or spiral series of thickenings. LAATSCH AND COWAN suggested that this arrangement might either serve as a mechanical attachment for the myelin, or to restrict the ionic exchanges to the nodal region. In EAN, the terminal myelin loops pull away from the nodes. This indicates that retraction of the myelin from the nodes is part of the process that leads to widening of the nodal gap, the tendency possibly being resisted by the attachments to the axon under normal circumstances. Perhaps this could be the consequence of the action of surface tension forces, which YOCNG (1944) believed were operative in myelin breakdown during Wallerian degeneration. The retracted myelin loops then undergo vesicular disintegration. The breakdown of the internodal myelin often appeared to include breakdown of the Schwann cell cytoplasm, so that a large portion of the cell, or perhaps the whole cell, underwent cytolysis. The presence of polymorphonuclear leucocytes may be related to the cellular necrosis that takes place. Further observations on the details of myelin breakdown in EAN are necessary, but the present results have indicated that the disrupting myelin tends to form small ovoids with lamellar walls or vesicles with walls composed of a single membrane. The smaller size of the ovoids as compared with those that develop during Wallerian degeneration is presumably related to the limited space available within the Schwann tubes because of the continued presence of the axon. The myelin breakdown products may indent the axon, giving rise to deep clefts, as was noted on light microscopy by HALL (1967a). Ribbon-shaped lamellar forms are also observed, the ends of which show separation at the intraperiod lines and then dissolution into single-walled vesicles. A further pattern is for a Schwann tube to contain an intact axon displaced to one side and in direct contact with the basement membrane, the tube also containing a large crescentic mass of myelin in which the lamellar pattern may be comparatively well preserved. Similar appearances have been reported in experimental diphtheritic neuritis by WELLER0965). J. neurol. Sci.

(1968) 8:1-18

EM OBSERVATIONS IN EXPTL. ALLERGIC NEURITIS, PART

1 (DEMYELINATION)

15

Following demyelination, the myelin remnants are removed by macrophages. How far these are the result of "transformation" of Schwann cells, as has been stated to occur in tissue culture (WEISS AND WANG 1945) and during Wallerian degeneration (HoLTZMAN AND NOVIKOFF 1965), or how far they are derived from tissue histiocytes that invade the Schwann tubes, cannot at present be decided. The solution of this question will probably require the employment of cell-labelling techniques. Schwann cells associated with myelinated axons are known to be more sensitive to ischaemia than the axons themselves. Thus ischaemia from occlusive vascular disease in man has been shown to produce both restricted paranodal demyelination and demyelination of whole internodal segments (LAMES AND LANGE 1967; CHOPRA AND HLrRWITZ 1967). Similar changes have also been noted for the paralysis produced by compression with a tourniquet in the cat (DENNY-BROWN AND BRENNER 1944) and which is probably the result of ischaemia. The question thus arises as to whether any of the demyelination in EAN could be the consequence of abnormalities in the blood vessels. This seems improbable, as structural alterations in the vasa nervorum are not conspicuous and their nature does not suggest that they are likely to give rise to ischaemia. It is not yet clear whether the pattern of demyelination that we have observed is specific for EAN, or whether it may occur in other demyelinating neuropathies such as diphtheritic or ischaemic neuropathy. Electron microscope observations on diphtheritic neuropathy have already been published by WEBSTER e t al. (1961) and by WELLER (1965), but details as to the early nodal changes were not given. WELLERwas impressed by the presence of lysosomes in the paranodal Schwann cell cytoplasm before evidence of myelin breakdown was apparent. He suggested that the diphtheria toxin damaged the metabolic activity of the Schwann cell so that it was no longer able to maintain the myelin, which was accordingly broken down by lysosomal activity. We have not so far studied lysosomal activity in EAN, and it is therefore not possible to state whether the situation differs in this condition, but there was no morphological evidence to indicate the presence of lysosomes at the nodes prior to myelin breakdown. WEBSTER and his associates, on the other hand, suggested that the diphtheria toxin directly damaged the myelin which broke down and was removed by macrophages. The axonal changes shown by demyelinated axons in EAN consist of the presence of lamellar structures in the axoplasm and "aneurysmal" protrusions between the terminal myelin lamellae at nodes that otherwise may show only minor alterations. These protrusions may contain such lamellar structures, together with dense bodies and mitochondria. Neither of these abnormalities is commonly observed, the large majority of demyelinated axons showing no detectable abnormality. Somewhat similar "reactive" changes in the axons in EAE have been recorded by LAMPERT(1967) in the form of accumulations of mitochondria, membranous dense bodies, vesicular elements and neurofilaments. Electrophysiological observations in EAN (KAESER AND LAMBERT 1962; KAESER 1962; CRAGG AND THOMAS 1964; HALL 1967b) have demonstrated that in the acute stage, affected myelinated nerve fibres may either exhibit a conduction block, or continue to conduct but at a reduced rate. Although an abnormality of the axon membrane cannot be excluded, the comparatively slight nature of the ultrastructural changes J. neurol. Sci. (1968) 8:1-18

16

R . H . M . BALLIN, P. K. THOMAS

in the axons leads to the possibility that the reduction in conduction velocity and the conduction block may be related to the loss of some factor dependent upon the presence of an intact Schwann cell-axon relationship. On the basis of the widening of the nodal gap seen by light microscopy, it has been postulated (KAESERAND LAMBERT1962) that the reduction in conduction velocity might be attributable to an increase in the area of the exposed axon membrane. It was suggested that this might be due to an increase in nodal activation time because of a decrease in the density of current flow across the axon membrane. However, the present studies have revealed that the axon at the widened nodes is not necessarily exposed and is often surrounded by a collar of Schwann cell cytoplasm. Moreover, as pointed out by CRAGG AND THOMAS(1964), widening of the gap between the adjacent myelin segments is observed during Wallerian degeneration (CAUSEY AND PALMER 1952) but is associated with only a slight reduction in conduction velocity (GUTMANN AND HOLUBA~ 1950; KAESERAND LAMBERT1962). Similarly, MORGAN-HUGHES(1968) has recently found that nodal widening in diphtheritic neuropathy, which precedes the development of detectable functional disability, is not associated with any marked reduction of conduction velocity. The present observations have shown that, apart from retraction and disintegration of the terminal myelin loops, loss of the Schwann cell nodal processes takes place. WILLIAMS AND LANDON (1963) have suggested that the paranodal Schwann cell cytoplasm, which contains large numbers of mitochondria, may be involved in providing metabolic support for the axon in connection with impulse propagation. Energy-rich substances might be synthesized in the Schwann cell and transferred to the axon through the nodal processes which come into intimate contact with the axolemma. However, this suggestion is purely speculative, and it will not be possible to predict the consequences of alterations in nodal structure until more is understood about the function of these various morphological specializations. CRAGG AND THOMAS(1964), on the basis of the electrophysiological properties of nerves affected by EAN, raised the possibility that the severe reduction in conduction velocity that may be encountered in this condition might be explained in terms of the continuous propagation of the nerve impulse across demyelinated regions as in non-myelinated nerve fibres. Some support for this view has recently been provided by MORGAN-HUGHES (1968), who found that in diphtheritic neuropathy, the gross reduction of conduction velocity that occurs is accompanied by myelin destruction involving complete internodal segments, and before evidence of remyelination was detectable. From the present findings, this hypothesis could be extended by postulating that the conduction block is related to the complete denudation of the axon of Schwann cell cytoplasm and myelin. Return of conduction at a reduced velocity by continuous conduction might occur when the axon becomes ensheathed by proliferating Schwann cells. The morphological situation at this latter stage would then be comparable to that of non-myelinated axons. Saltatory conduction at a faster velocity would be expected to recommence once remyelination begins. From observations on normal developing chick nerve, CARPENTER AND BERGLAND (1957) and BERGLAND (1960) demonstrated that conduction begins before myelination is evident and that the onset ofmyelination is associated with a sharp increase in conduction velocity. HowJ. neurol. Sci.

(1968) 8:1-18

EM OBSERVATIONSIN EXPTL. ALLERGIC NEURITIS, PART 1 (DEMYELINATION)

17

ever, the precise explanation of the alterations in nerve conduction that take place in demyelinated nerve fibres is only likely to be achieved by combined electrophysiological and electron microscope studies on single nerve fibres, an investigation which will involve considerable technical difficulties. ACKNOWLEDGEMENTS

This investigation was supported by a grant from the Medical Research Council. Some of the results reported here were incorporated in a Ph.D. thesis presented to the University of London by R.H.M.B. We wish to thank Miss Ann Armstrong and Miss Theresa Tilley for technical assistance, Professor W. H. McMenemey for laboratory facilities at Maida Vale Hospital, and Dr. D. N. Landon for helpful discussion. SUMMARY

Electron microscope observations have been made on experimental allergic neuritis in guinea-pigs, produced by the intradermal inoculation of an emulsion of rabbit sciatic nerve combined with Freund's adjuvants. The leucocyte infiltration of the endoneurium consisted predominantly of lymphocytes and monocytes with a smaller proportion of polymorphonuclear leucocytes. The early changes seen at the nodes of Ranvier were separation of the terminal myelin loops from the axon, the myelin lamellae then undergoing vesicular disintegration. The Schwann cell nodal processes were lost and at times the Schwann cell cytoplasm extended into the region formerly occupied by the nodal processes. On more severely-affected fibres disruption of the internodal myelin occurred, the myelin breaking down into small ovoids with multilamellar walls or into single-walled vesicles. The myelin was then removed by macrophages, the origin of which was uncertain. Leucocytes were not identified within the Schwann tubes at the time myelin breakdown commenced, but were noted within the tubes in which the demyelination was further advanced. Occasional complete degeneration of axons was observed, but changes in demyelinated axons were not conspicuous. REFERENCES /~STROM,g. E. ANDB. H. WAKSMAN(1962) The passive transfer of experimental allergic encephalomyelitis and neuritis with living lymph node cells, J. Path. Bact., 83 : 89-106. BALLIN,R. H. M. (1967) Electron Microscope Observations on Experimental Allergic Neuritis, Ph.D.

Thesis, University of London. BALLIN,R. H. M. AND P. g . THOMAS(1969) Electron microscope observations on demyelination and

remyelination in experimental allergic neuritis, Part 2 (Remyelination), to be published. BERGLAND,R. M. (1960) Newer concepts of myelin formation correlated to functional changes, Arch. Neurol. (Chic.), 2: 260-265. CARPENTER,F. G. ANDR. M. BERGLAND(1957) Excitation and conduction in immature nerve fibers of the developingchick, Amer. J. Physiol., 190: 371-376. CAUSEY,G. ANDE. PALMER(1952) Early changes in degenerating mammalian nerves, Proc. roy. Soc.

B., 139: 597-609. CHOPRA,J. S. ANDL. J. HURWITZ(1967) Internodal length of sural nerve fibres in chronic occlusive vascular disease, J. Neurol. Neurosurg. Psychiat., 30: 207-214. CRAGG,]3. G. ANDP. K. THOMAS(1964) Changes in nerve conduction in experimental allergic neuritis, J. Neurol. Neurosurg. Psychiat., 27: 106-115. DENNY-BROWN,D. AND C. BRENNER(1944) Paralysis of nerve induced by direct pressure and by tourniquet, Arch. Neurol. (Chic.), 51 : 1-26. J. neurol. Sci. (1968) 8:1-18

18

R . H . M . BALLIN~ P. K. THOMAS

LAMES, R. A. AND L. S. LANGE (1967) Clinical and pathological study of ischaemic neuropathy, J. Neurol. Neurosurg. Psychiat., 30: 215-226. FREUND, J. AND K. MCDERMOTT (1942) Sensitization to horse serum by means of adjuvants, Proc. Soc. exp. Biol. Med., 49: 548-553. GRIMLEY, P. M. (1964) A tribasic stain for thin sections of plastic-embedded, OsO4 fixed tissues, Stain Technol., 39: 220-233. GUTMANN, E. AND J. HOLUBA~ (1950) Degeneration of peripheral nerve fibres, J. Neurol. Neurosurg. Psychiat., 13: 89-105. HALL, J. I. (1967a) Studies on demyelinated peripheral nerves in guinea-pigs with experimental allergic neuritis, Part 1 (Symptomatology and histological observations), Brain, 90:297-312. HALL, J. I. (1967b)Studies on demyelinated peripheral nerves in guinea-pigs with experimental allergic neuritis, Part 2 (Electrophysiological observations), Brain, 90: 313-332. HOLTZMAN, E. AND A. B. NOVIKOFF 0965) Lysosomes in the rat sciatic nerve following crush, J. Cell Biol., 27 : 651-669. HURST, E. W. (1932) The effects of the injection of normal brain emulsion into rabbits with special reference to the aetiology of the paralytic accidents of antirabies treatment, J. Hyg. (Lond.), 32: 33-43. KAESER, H. E. (1962) Funktionspriifungen peripherer Nerven bei experimentellen Polyneuritiden und bei der Wallerschen Degeneration, Dtsch. Z. Nervenheilk., 183 : 268-304. KAESER, H. E. AND E. H. LAMBERT (1962) Nerve function studies in experimental polyneuritis, Electroenceph. clin. Neurophysiol., Suppl., 22: 29-35. LAATSCH, R. H. AND W. M. COWAN (1966) A structural specialization at nodes of Ranvier in the central nervous system, Nature (Lond.), 210: 757-758. LAMPERT, P. (1965) Demyelination and remyelination in experimental allergic encephalomyelitis. Further electron microscope observations, J. Neuropath. exp. Neurol., 24: 371-384. LAMPERT, P. (1967) Electron microscope studies on ordinary and hyperacute experimental allergic encephalomyelitis, Acta Neuropath., 9 : 99-126. LAMPERT,P. AND S. CARPENTER (1965) Electron microscope studies on the vascular permeability and the mechanism of demyelination in experimental allergic encephalomyelitis, J. Neuropath. exp. Neurol., 24: 11-24. MASUROVSKY, E. B., M. B. BUNGE AND R. P. BUNGE (1967) Cytologic studies of organotypic cultures of rat dorsal root ganglia following X-irradiation in vitro, J. Cell Biol., 32: 497-518. MORGAN-HUGHES, J. A. (1968) Experimental diphtheritic neuropathy: pathological and electrophysiological study, J. neurol. Sci., 7:157-175. PATERSON, P. Y. (1960) Transfer of allergic encephalomyelitis in rats by means of lymph node cells, J. exp. Med., l l l : 119-136. THOMAS, P. K. (1964) Changes in the endoneurial sheaths of peripheral myelinated nerve fibres during Wallerian degeneration, J. Anat., 98 : 175-182. THOMAS, P. K. 0968) Unpublished observations. VENABLE,J. H. AND R. COGGESHALE(1965) A simplified lead citrate stain for use in electron microscopy, J. Cell Biol., 25 : 407-408. WAKSMAN,B. H. AND R. D. ADAMS(1955) An experimental disease of rabbits induced by the injection of peripheral nerve tissue and adjuvants, J. exp. Med., 102: 213-236. WAKSMAN, B. H. AND R. D. ADAMS (1956) A comparative study of experimental allergic neuritis in rabbit, guinea pig and mouse, J. Neuropath. exp. Neurol., 15: 293-332. WA~:SMAN,B. H. AND R. D. ADAMS(1962) A histologic study of the early lesion in experimental allergic encephalomyelitis in the guinea pig and rabbit, Amer. J. Path., 41 : 135-162. WEBSTER, H. DE F. (1962) Transient focal accumulation of axonal mitochondria during the early stages of Wallerian degeneration, J. Cell Biol., 12: 361-384. WEBSTER, H. DE F., D. SPIRO, B. H. WAKSMANAND R. D. ADAMS(1961) Phase and electron microscopic studies of experimental demyelination, Part 2 (Schwann cell changes in guinea pig sciatic nerves during experimental diphtheritic neuritis), J. Neuropath. exp. Neurol., 20: 5-12. WEISS, P. AND H. WANG (1945) Transformation of adult Schwann cells into macrophages, Proc. Soc. exp. Biol. Med., 58: 273-275. WELLER, R. O. (1965) Diphtheritic neuropathy in the chicken: an electron microscope study, J. Path. Bact., 89: 591-598. WILLIAMS, P. L. AND D. N. LANDON (1963) Paranodal apparatus of peripheral myelinated nerve fibres of mammals, Nature (Lond.), 198: 670-673. YOUNG, J. Z. (1944) Surface tension and the degeneration of nerve fibres, Nature (Lond.), 154: 521522.

J. neurol. Sci. (1968) 8 : 1 - 1 8