The ultrastructure of sciatic nerves affected by fowl paralysis (Marek's disease)

J. CAMP. PATH. 1969. VOL. 79.

THE

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P. A. L. WICJHT Agicuikml

March

Council’s Poultry ResearchCentm, King’s Buildings, West Mains Road, &&zbwgh INTRODUCTION

Fowl paralysis is a disease of the peripheral nerves of the domestic fowl (Ma,&, 1907). The present communication records an electron microscopic study of sciatic nerves from the spontaneously-occurring,

ClasSicalform of the disease.

The morphological classification of the peripheral nerve lesions used in previous studies was employed (Wight, 1962). Briefly, in type I the predominant feature of the nerves is a cellular infiltration mostly of mature lymphocytes, type I1 is character&d by oedema and collagenosis with a relatively sparse infiltration of lymphocytes and plasma cells, while in type III

there is a massive infiltration

of neoplastic lymphoblasts. MATERIALS

AND

METHODS

Brown Leghorn fowls of the Poultry Research Centre flock in which the sciatic nerves were affected by naturally-occuring, classical fowl paralysis and nineteen healthy control birds of the samebreed and age range were used. The birds were decapitated and the sciatic nerve quickly removed. Two fixatives were employed for each case as no single solution was found which gave adequate preservation. The first, 5 per cent. phosphate buffered glutaraldehyde followed by 2 per cent. osmium tetroxide, preserved the axons, the infiltrating cells and the Schwann cells except for their myelin lamellae which were often artefactually distorted. The second, 2 per cent. potassium permanganate solution (Luft, 1956), gave excellent electron contrast of the myelin lamellae and cell membranes. Tissuesfixed by both methods were dehydrated, embedded in Epoxy resin* and thin sectionswere stained with uranyl acetate followed by lead citrate. Sections 2 p thick from the Epoxy resin blocks were also stained with toluidine blue for light microscopy. The sciatic nerve on each side of the piece taken for electron microscopy was fixed in susafor examination by light microscopy. Forty

RESULTS

Control Nerves Electron microscopic examination revealed that the normal sciatic nerves con-

tained myelinated (Fig. 1) and unmyelinated fibres. The Schwann cells had a distinct external lamina (Fig. 2). The architecture of the myelin sheaths (Fig. 3) was the same as described in the classical report on those of the embryonic chick * Aralditc:

Ciba (ARL) Ltd.

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by Geren (1954). Other kinds of cells were rare, although mast cell and lymphocyte were present.

PARALYSIS

an occasional

fibroblast,

Ajfected Nerves Schwa~tn c&s. Many Schwann cells were abnormal in fowl paralysis (Fig. 4j. Cytoplasmic hyperttophy and proliferation of diffuse ribosomes gave the cell an electron-dense appearance after glutaraldehyde fixation. The cytoplasm, particularly in oedematous nerves (type II), often contained many vesicles which probably arose from dilation of the cisternae of the profuse granular and agranular endoplasmic reticulum (Fig. 5). Mitochondria were slightly more numerous than in the normal cell. Small dense bodies limited by a single membrane, perhaps lysosomes, were sometimes seen, but were not numerous. Clusters of membranelimited, electron dense bodies of a rather different appearance were also seen in a small proportion of neurites in the Schwann cell cytoplasm between the two innermost major dense lines of the myelin lamellae (Fig. 6). Cytoplasmic filaments were present. Membrane bound globules of pale, electron-translucent material resembling lipid were detected in some Schwann cells. External laminae were present (Figs. 5, 6, 8, 9 and 17), although sometimes widely separated from the Schwann cell plasma membrane. In some examples an undulating lamina surrounded the Schwann cell processes, a demyelinated axon which had been displaced to the exterior of the cell, or even more than one Schwann cell. In fowl paralysis of Type II, cytoplasmic processes were numerous and occasionally very elongated, but they were usually distinguishable from the processes of macrophages by their external laminae (Fig. 9). Sometimes they were layered around the neurite, intermixed with collagen and the processes of macrophages forming the so-called “onion bulbs” (Figs. 8 and 9). Mitotic figures were occasionally observed. Degenerated myelin was present in some Schwann cells, either as disintegrated larnellae in the form of osmiophilic membranous whorls (Fig. 10) or as irregular shapes exhibiting varying degrees of electron density (Fig. 11). Myelin. The only myelin abnormality shown by some neurites was disintegration of the inner lamellae into circular profiles adjacent to the axolemma (Fig. 13), the architecture of most of the remainder of the myelin sheath being unaffected. The intraperiod line adjacent to these changes was sometimes lost for a short distance and the region between the major dense lines was dilated and had increased the electron density (Fig. 12). Th e external lamellae were less often affected by this early change, while the mesaxons seemed relatively resistant. In other examples, particularly in type II, the myehn sheaths were thinner relative to the diameter of their axons, than in control nerves (Figs. 7 and 9), so that only a few lamellationa surrounded the axon. Sometimes these sparse lamellations were wavy, irregularly spaced and ruptured (Figs. 8, 11 and 17.) Complete disintegration of the myelin sheath and the occurrence of osmiophilic debris in the Schwann cytoplasm, as described earlier, was most often seen in types I and III. Sometimes in severe casesof type III, only a few, apparently normally-myelinated, axons were present. Except for a few clumps of o~iop~ic material, presumably myelin debris, the other neurites had apparmfly been destroyed by the massof infiltrating cells.

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Axons. The axons seemed relatively resistant to morphological changes. Thus in types I and II, particularly the latter, often only a few spirals of Schwann cytoplasm surrounded a large, but structurally normal axon. Sometimes remnants of the myelin sheath were in one part of the hypertrophied Schwann cell while an apparently unchanged axon lay in another part (Fig. 10) or even outside the cell. Schwann cell pseudopodia were seen partially encircling relatively large, naked axons (Fig. 15). Occasionally these axons had increased numbers of mitochondria and vesicles (Figs. 8 and 16), and circular profiles, often delicately lamellated, were present. The neurofibrils were sometimes coarsened and neurotubules with dilated lamina became numerous. The axolemma was occasionally deficient (Figs. 17 and 18). Alongside these changes small regenerating axons were seen in the periphery of the cytoplasm of Schwann cells (Fig. 19). Only in severely infiltrated cases of types I or III was there extensive destruction of axons. Very infrequently, small vesicles with dense cores were seen in the axoplasm in both diseased and normal nerves. Infiltrating cells. The kind, proportion and numbers of extraneous cells varied with the histological types of fowl paralysis. Mature lymphocytes (Fig. 20) were the predominant infiltrating cell in the nerves of types I and II. They had a dense nucleus, narrow cytoplasm containing ribosomes, a few granular endoplasmic reticula, a few small mitochondria and a Golgi apparatus. They often had several short, cytoplasmic pseudopodia. Plasma cells, identified by their profuse granular endoplasmic reticulum, were most often seen in type II. They had occasional small cytoplasmic projections. Their ultrastructure was not abnormal. Mast cells (Fig. 20) were most numerous in type II and least in type III. They often had a “fibrocyte” shape with elongated processes, but were easily identified by their specific granules. Lymphoblasts predominated in fowl paralysis of type III. This form of immature cell (Figs. 4 and 21) had a large, round or indented nucleus containing abundant heterochmmatin and moderately electron-dense nuclear matrix and euchromatin. Hetemchromatin was condensed on the nuclear membrane, sometimes forming large, dense masses. A prominent nucleolus was present, occasionally with a nucleolonema and pars amorpha. Nuclear “pockets” were common (Fig. 22). The outline of the cytoplasm was approximately circular. Mitochondria, usually in a group, were fairly numerous. There were a few small, electron dense membrane-bound bodies, probably lysosomes, an occasional multivesicular body, and both granular and agmnular endoplasmic reticula were present, but were not plentiful. Diffuse ribosomes were always present, but the number varied so that some lymphoblasts appeared light while others were relatively dark and electron-dense. Occasional degenerating lymphoblasts were seen and mitotic figures were present (Fig. 21). Macrophages were prominent in types I and II. Their irregularly shaped nucleus contained much chromatin. They had most extensive processes which, because of the thinness of the sections, were often seen as short lengths or circular profiles. From the surface of the macrophages short cytoplasmic projections were budded out, apparently constricted off at the base and left free in the interstitium as membrane bound profiles (Fig. 14) which often contained cytoplasmic con-

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stituents. These were very numerous in types I and II. Ribosomes and granular endoplasmic reticulum were very numerous suggesting that these cells elaborate protein. The endoplasmic reticulum was often widely dilated and filled with granular or flocculent material, particularly in oedematous nerves. The macrophages also frequently contained large, membrane-bound, electron-translucent globules which resembled lipid inclusions (Fig. 5). Osmiophilic debris, perhaps degenerated myelin, was less common. These cells were often closely adjacent to collagen fibrils. Other structures. In type II, the interneurite spaces were greatly enlarged and contained a fine matrix and thick arrays of collagen fibres of typical periodicity (Figs. 14 and 19). Collagen fibres were proliferated in types I and II and in the latter they often formed a dense hedge around the neurite (Figs. 7, 11 and 17). In addition, the dilated spaces between the neurites often contained circular profiles consisting of a unit membrane surrounding a pale interior in which were some vesicles, mitochondria or endoplasmic reticulum. As recorded above, these structures may have been cross-sections of macrophage or Schwann cell processes, or cytoplasmic globules budded off from the macrophages. DLSCUSSIOX

This electron microscopic investigation confirms the observations previously made by light microscopy and adds new information about the changes which occur at the ultrastructural level. The Schwann cell activation observed with the light microscope (Wight, 1962) is confirmed by the cytoplasmic hypertrophy, distention of the endoplasmic reticulum and proliferation of ribosomes and mitochondria which were seen by electron microscopy. Such changes are not specific to fowl paralysis since they are similar to those recorded in man and other animals in experimental forms of neuritis, neuropathies and Wallerian degeneration (Webster, Spiro, Waksman and Adams, 1961; Fisher and Turano, 1963 ; Barton, 1962; Nathaniel and Pease, 1963a; Weller, 1967; Lampert and Schochet, 1968). In experimental diphtheritic neuropathy in the fowl, Weller (1965) observed hypertrophied Schwann cell cytoplasm containing large mitochondria and many vesicles. He also saw dense bodies, perhaps lysosomes involved in myelin fragmentation, between the iruler myelin lamellae, and these seem identical in appearance and location to those seen in the present study. Vesicular dilation of the endoplasmic reticulum is characteristic of irritated cells. The abundant ribosomes and mitochondria indicate increased enzymic activity and protein elaboration for the internal requirements of the cell, perhaps including the elimination of myelin products and growth and repair of the cell. The proliferation of the granular endoplasmic reticulum suggests that protein is being manufactured for external uses, one of which could be collagen formation. The cause of the change in the Schwann cells was not determined, but the fact that it is prominent and occurs early in the disease suggests that this cell is either a primary target for the causative agent of fowl paralysis or is particularly susceptible to its subsequent effects. Because of the integration of Schwann cell, myelin and axon in saltatory conduction, Schwann cell changes could play a

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most important part in the induction of the clinical paralysis. Demyelination (Wight, 1962 ; Payne and Bii, 1967), is confirmed in the present study in which demyelinated axons, disorganised myelin lamellae and denatured myelin detritus were seen in the cytoplasm of the Schwann cells. There has been controversy as to whether mammalian Schwann cells or macrophages phagocytose degenerating myelin (McMinn, 1967). In the present study Schwann cells were undoubtedly the major cells involved in the myelin disintegration and initial denaturing of the sheath whereas macmphages frequently contained non-otiophilic, electron-translucent lipid. The latter probably indicated that a later molecular degradation of myelin had occurred and corresponds to the sudanophilic lipid seen with the light microscope (Wight, 1962). Myelin breakdown products in affected nerves have also been demonstrated by chemical means (Heald, Badman, Fumival and Wight, 1964). The relative importance of the Schwann cell reaction and axon degeneration is of interest. By using a counting method, Wight (1964) showed that destruction of axons may occur in the disease, but is only severe when there is a massive invasion of primitive lymphoid cells (type III). The present electron microscopic study confirms this observation. In fowl paralysis of types I and II axons persisted although they were surrounded by only a few lamellae, were entirely devoid of myelin or even lay outside the Schwann cell. In this respect type II was similar to experimental diphtheritic neuritis in guinea pigs (Webster et al., 1961) and chickens (Weller, 1965) in which it was thought that either the Schwann cell membrane system or the Schwann cell as a whole was the primary target. Demyelination in fowl paralysis followed a pattern similar to that described in neuropathies associated with Schwann cell damage (Lampert and Schochet, 1968). An indication of the resistance of the axons in types I and II is the unusual way in which relatively large, solitary axons are enveloped by Schwann cell pseudopodia. Usually small regenerating mammalian axons come to lie under the basement membrane and sink into gradually deepening gutters on the Schwann cell surface before being enfolded by spirals of Schwann cell cytoplasm (Causey, 1960; Nathaniel and Pease, 1963b). This was sometimes seen and confirms the previously recorded capacity of the a&ted nerves to regenerate (Wight, 1965). Th e f ormer observations, however, suggest that myelin breakdown, expulsion of axons and possibly degeneration of Schwann cells leaves naked but viable, adult axons in the interstitium which the Schwann cells reinvest. The ultrastructural changes typical of axonal reaction to Wallerian degeneration (Vial, 1958 ; Barton, 1962 ; Fisher and Turano, 1963) were observed in fowl paralysis of all types, but were most frequent in type III and severe type I. Such observations suggest that two processes may be taking place; if there is a massive invasion of lymphoid cells as in type III or severe type I, extensive destruction of axons takes place perhaps due to the pressure of invading cells. In less severe type I and in type II, a more subtle process occurs which primarily affects the Schwann cells and results in degeneration of their myelin lamellae. The lymphoid series is the most numerous, non-neurological cellular constituent of fowl paralysis affected nerves. Although plasma cells and lymphoblasts, and even mature lymphocytes and macrophages, often contained large numbers of ribosomes which would be pyroninophilic under the light microscope (Biggs,

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1961), plasma cells of classical ultrastructural appearance were easily distinguished. It was not possible definitely to identify the “Marek’s disease” cells observed by Payne and Biggs (1967), although these may correspond to the lymphoblasts with a heterochromatin-rich nucleus and many cytoplasmic ribosomes. In parafhn sections, the nucleus of the “Marek’s disease” cell is said to have a rather unusual appearance and, as well as mitoses, nuclear “pockets” were seen in the present study. Similar structures occur in human leukaemic cells, in Burkitt’s lymphoma and in human patients with chromosomal abnormalities. Smith and O’Hara (1967) considered them to be cytoplasmic material surrounded by elements of the nuclear envelope but, because they also occurred in normal human and certain animal leukocytes, they did not think they were specific for any disease. There are some similarities between acute Marek’s disease and the human leukaemias, but without careful quantitative studies the significance of these structures cannot be assessed. Although Biggs (19’61) showed by histochemical techniques that macrophages are much less numerous than lymphoid cells, the present study reveals that they play an important part in the phagocytosis of myelin debris and in the production of collagen. Furthermore, their abundant granular endoplasmic reticulum, ribosomes, organelles and profuse cytoplasmic budding may indicate other activities. The circular profiles of the present study may be transverse sections of projections, since ruffling of the surface cytoplasm of macrophages in association with pinocytosis and phagocytosis is well known, but in many examples they appeared to be isolated fragments. Macrophages are often involved in virus diseases and they may transfer information to immunologically competent cells (Cohn, 1965). The occurrence of mast cells, their ultrastructure and the increase in their numbers in type II and the decrease in numbers in type III have been reported previously (Wight, 1967). Their association with inflammatory oedema, chronic inflammation and with the unusual amount of mucopolysaccharides which may be present in nerves affected with fowl paralysis of type II, was discussed. Excessive collagen in nerves has been observed at the ultrastructural level by Deutsch and Siller (196 1) who considered fibrosis a characteristic change in fowl paralysis. In type II, fibrosis may be detected by light microscopy and is an important component of the “onion bulb” which may form around the neurites (Wight, 1962). Collagenosis occurs in other forms of neuritis although there is some doubt as to whether it is produced by mesodermal cells (Thomas, 1964) or by Schwann cells (Barton, 1962; Nathaniel and Pease, 1963~). Electron microscopy reveals that the “onion bulbs” are essentially similar to those in human chronic neuropathies (Webster, Schroder, Asbury and Adams, 1967), except that, as well as the collagen fibrils and circumferential Schwann cell processes which surround the neurite core, macrophages were also sometimes involved. Virus-like particles have been observed in lymphocytes of venous blood from acute Marek’s disease (Wight, Wilson, Campbell and Fraser, 1967) and there is convincing evidence from studies based on tissue culture that a herpes-type virus may be a causative agent of the disease (Churchill and Biggs, 1967 ; Nazerian, Solomon, Witter and Burrnester, 1968). Virus particles were not seen in the present examination of peripheral nerves taken directly from the naturally

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affected bird. Nakagawa (1965) also failed to find virus particles under similar circumstances. The reason for the in&ation of the haematogenous cells into the nerves remains obscure. Am these cells attracted by previously damaged neurological tissue or are the lymphoid cells first altered by the aetiological agent so that they are attracted to, and subsequently cause damage to the peripheral nerves? This important question has been examined by Payne and Biggs (1967), who, after studying the pathogenesis of the experimentally transmitted disease suggest that it is primarily a lymphoid condition with a predilection for neural tissue. Other workers (Campbell, 1956; Wight, 1962) have considered the disease to be primarily inflammatory. The present study shows that involvement of the Schwann cells was a prominent and relatively early occurrence but, as the material was from clinically affected natural cases in which some degree of lymphoid cell infiltration was invariably present, it was not possible to decide which cells were affected first. SUMMARY

The ultrastructure of the peripheral nerves and infiltrating cells in naturallyoccurring, classical fowl paralysis has been described. There was activation of the Schwann cells including hypertrophy and cytoplasmic process formation, dilation of the endoplasmic reticulum, proliferation of ribosomes and other organelles and phagocytosis of myelin. Affected sheaths were thin, fragmented and degenerated but, unless there was extensive cellular infiltration, the axons were preserved. Collagenosis and “onion bulb” formation were recorded. Lymphocytes, plasma cells and mast cells were present, but their morphology was not specifically abnormal. Lymphoblasts were rich in ribosomes and heterochromatin, and nuclear “pockets” were often present. Macrophagcs were active and contained lipid which was probably derived from denatured myelin. Their structure indicated that they may have been involved in protein elaboration, perhaps including collagenosis. Virus-like particles were not seen. It was not possible to decide whether fowl paralysis was primarily lymphoidal or neurological, but Schwann cell changes were prominent and occurred relatively early in the disease. REFERENCES

Barton, A. A. (1962). Brain, 85, 799. Biggs, P. M. (1961). C&on Papers, 13, 83. Campbell, J. G. (1956). Vet. Rec., 68, 527. Causey, G. (1960). The Cell of Schwann, E. & S. Livingstone Ltd.; Edinburgh. Churchill, A. E., and Biggs, P. M. (1967). Nature, Lond., 215, 528. Cohn, 2. A. (1965). In The In@mzatory PTOC~JS, Ed. by Zweifach, B. W., Grant, I,., and McCluskey, R. T., Ch. 8, p. 347, Academic Press; New York and London. Deutsch, K., and Siller, W. G. (1961). Res. vet. Sci., 2, 19. Fisher, E. R., and Turano, A. (1963). Archs. Path., 75, 517. Geren, B. B. (1954). &p. cell Res., 7, 558. Heald, P. J., Badman, H. G., Furnival, B. F., and Wight, P. A. L. (1964). Poultry sci., 43, 701.

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Lampert, P. W., and Schochet, S. S. (1968). J. Neuropath. exp. Neurol., 27, 527. Luft, J. H. (1956). j. biophys. biochem. Cytol., 2, 799. Marek, J. (1907). Dtsch. tieriirztl. Wschr., 15, 417. McMinn, R. M. H. (1967). Znt. Rev. Cytol., 22, 64. Nakagawa, M. (1965). /up. 1. vet. Res., 13, 55. Nathaniel, E. J. H., and Pease,D. C. (1963a). 1. ultrastruct. Res., 9, 511; (1963b). Ibid., 533; (1963c). Ibid., 550. Nazerian, K., Solomon, J. J., Witter, R. L., and Burmester, B. R. (1968). PYOC. Sot. exp. Biol.

Med.,

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Payne, L. N., and Biggs, P. M. (1967). /. nutn. Cancer Inst., 39, 281. Smith, G. F., and O’Hara, P. T. (1967). J. ultrastruct. Res., 21, 415. Thomas, P. K. (1964). /. cell Biol., 23, 375. Vial, J. D., (1958). J. biophys. biochem. Cytol., 4, 551. Webster, H. D., Spiro, D., Waksman, B., and Adams, R. D. (1961). /. Neuropath. exp. Neural.,

20, 5.

Webster, H. D., Schroder, J. M., Asbury, A. K., and Adams, R. D. (1967). Ibid., 26, 276.

Well% ?.I?.

(1965). /. Path. Bact., Ss, 591; (1967). J. Neural.

Neurosurg.

Psychiat.,

Wight,‘P. A: L. (1962). J. camp. Path., 72, 40; (1964). Res. vet. Sci., 5, 46; (1965). Brit. vet. J., 121, 278; (1967). Experientiu, 23, 836. Wight, P. A. L., Wilson, J. E., Campbell, J. G., and Fraser, E. (1967). Nature, Lond., 216, 804. [Received

Key to abbreviations Ax AXOIl AxL Axolemma B Mast cell Collagen Dense bodies &3 DE Dilated endoolasmic External mekxon % Granular endoplasmic H Macrophage im Internal mesaxon Lymphocyte JL Lamina externa

for

publication,

February

LP LY M Mt my reticulum reticulum

Es OM E S-L

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19691

Lipid Lysosome Myelin sheath Mitochondrion Multivesicular body Nucleolonema Schwann cell nucleus Osmiophilic myelin debris Schwann cell pseudopodia Schwann cell cytoplasm Schmidt-Lanterman cleft