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Latent herpes simplex virus trigeminal ganglionic infection in mice and demyelination in the central nervous system
Journal of the Neurological Sciences, 1979, 43:253-264 © Elsevier/North-Holland Biomedical Press
253
LATENT HERPES SIMPLEX VIRUS T R I G E M I N A L G A N G L I O N I C INFECTION IN MICE AND DEMYELINATION IN THE CENTRAL NERVOUS SYSTEM
KRISTER KRISTENSSON1, BO SVENNERHOLM2, LENNART PERSSON, ANDERS VAHLNE2 and ERIK LYCKE2 2 Department of Virology, Institute of Medical Microbiology, University of Gi~teborg, GSteborg, and 1 Neuropathological Laboratory, Department of Pathology II, University t~fLinkb'ping (Sweden)
(Received 22 February, 1979) (Accepted 4 April, 1979)
SUMMARY Mice were inoculated with herpes simplex virus (HSV) type 1 by gently scraping the skin of the nose with a fine needle. About 80 ~o of the animals developed latent inapparent HSV infections in trigeminal ganglia. Virus was demonstrable for at least 6 months post inoculation (p.i.) by cocultivation of ganglionic tissue with G M K cells. Histologically, trigeminal ganglia revealed infiltrations of inflammatory cells even 6 months p.i. In addition, lesions occurred in the brainstem corresponding to the entry of trigeminal roots, trigeminal tracts and nuclei. Inflammatory cell infiltration, disruption of myelin sheaths and macrophages laden with myelin degradation products were observed 7 days p.i. Fourteen to 30 days p.i. electron microscopy demonstrated completely naked axons. In the transitional region of the trigeminal root denuded axons occurred in the central part of the region while the peripheral myelin, bordering the demyelinated central segments, was intact. Small areas of demyelination were still detectable 3 and 6 months p.i. but there were then also signs of remyelination. Possible mechanisms causing the demyelination are discussed. INTRODUCTION Herpes simplex virus (HSV) may remain as a latent infection in trigeminal ganglia of both humans and experimental animals (Stevens and Cook 1971; Baringer This study was supported by grants from the Swedish Medical Research Council, project hr. B79-12X-04480-05B and 4514. Correspondence to: Dr. Krister Kristensson, Neuropathological Laboratory, University of Link6ping, S-581 85 Link6ping, Sweden.
254 and Swoveland 1973; Forghani et al. 1977). It is not known if HSV during latency persists in the ganglion slowly replicating or in a state of arrested multiplication (cf. Klein 1976). Even 16 weeks after corneal HSV inoculation an occasional degenerated neuron containing virus particles in the trigeminal ganglia can be found (Baringer and Swoveland 1974), but it is not known if this represents virus newly formed after reactivation or a persistent infection. Nor is it known to what extent latent HSV infection is associated with pathological changes other than those in ganglia harbouring the infection. Previously, it was found that during acute HSV infection the virus may spread along the trigeminal root to the brain stem causing severe changes in the central myelin, while the peripheral myelin remained intact (Townsend and Baringer 1978a; Kristensson et al. 1978). In the present report we have examined the trigeminal root and brain stem in clinically inapparent or latent HSV infections of mice with especial regard to the appearance of the central myelin. M A T E R I A L A N D METHODS
Virus
Six HSV-1 strains were used. Strain F originated from Dr. B. Roizman, University of Chicago, the others were isolated and typed in our laboratory (Vahlne et al. 1975). The strains were propagated in GMK cells, in which pfu also were assayed. Virus isolated from trigeminal ganglia was typed by immunoelectroosmophoresis against type-specific antisera (Jeansson 1972).
Cells Green monkey kidney (GMK AH-1) cells were cultured in Eagle's minimum essential medium (MEM) supplemented with 10 ~ calf serum, 100 i.u. of penicillin and 100 #g of streptomycin per ml. For maintenance the same medium supplemented with 2 ~ serum only was used.
Inoculation technique and preparation of trigeminal ganglionic tissue Five-6-week old Swiss albino mice of both sexes of our own laboratory breed were anesthetized by ether. Virus inoculation of the eye by corneal scratching, of the side of the nose by injection or scraping with a needle, and, thirdly, inoculation of the nostrils, were the routes tested. The most satisfactory results were obtained when a drop of the virus suspension was placed on one side of the nose (left) and virus inoculated by gentle scraping with a fine needle, and this technique was used in the present study. For preparation of ganglionic tissue ether-anesthetized mice were perfused with BSS through the left cardiac ventricle, and the brains were removed for exposure of the trigeminal ganglia. Left and right side trigeminal ganglia were removed separately and placed in tubes in cell culture medium at room temperature. Before being added to cell culture bottles the ganglia were cut in pieces with scissors.
Cell culture technique A method modified from that used by Stevens and Cook (1971) was used for
255 reactivation of latent HSV of trigeminal ganglia. The ganglia were cut by scissors and the pieces cultured in 30 ml plastic bottles seeded with GMK cells (5 × 105 cells per bottle) at the time of adding the pieces of ganglia. Ganglia from one side were cocultivated with GMK cells in one bottle with Eagle's MEM. Thus, two cultures were prepared per mouse. Culture medium was replaced twice a week. Microscopic readings of cultures were performed every day for 4 weeks and virus from cultures demonstrating the cytopathogenic effect (cpe) of GMK cells was passaged on new GMK cultures before typing of the strains. Ganglia from 20 mock-inoculated animals were collected 30 days post inoculation (p.i.), when an equal number of virus-inoculated animals were examined concomitantly. From none of the mock-inoculated mice was HSV isolated, indicating that both the preparation of ganglia and the cocultivation could be performed without virus contamination.
HSV antibodies Serum samples were collected from 7 virus-inoculated and 6 uninoculated mice and tested for presence of HSV antibodies 7 days p.i. using the K-test (Wildy 1972).
Histological technique For histological examination groups of 5-10 mice were killed 7, 14, 30, 60, 90 and 180 days p.i. with undiluted strain F, see below. The mice were anesthetized with ether, perfused through the heart with 5 ~ glutaraldehyde in phosphate buffer and the trigeminal ganglia, the trigeminal roots and the brain stems including the areas of the trigeminal nuclei and tracts were dissected. The specimens were postfixed in osmic acid, dehydrated in alcohols and embedded in Epon 812. One-/~m thick sections were cut and stained with toluidine blue for light microscopy. Ultrathin sections were cut from selected areas and contrasted with uranyl acetate and lead citrate for electron microscopy. RESULTS
Latent HSV-1 infection in trigeminal ganglia In pilot experiments the strain of HSV-1, the concentration of virus and the route of inoculation were selected for establishment of latent HSV infection of trigeminal ganglia in a high and reproducible percentage of inoculated mice. Of 6 HSV-1 strains observed the F strain yielded latent trigeminal infection in 75-85 ~ of the animals. As this strain caused no overt signs of disease and, thus, no deaths of inoculated animals, it could be used undiluted in a concentration of 8 log pfu/ml. In general, strains highly pathogenic for mice causing fulminant infections of the CNS were less satisfactory for establishment of latent infections and by diluting the strains to avoid loss of animals the percentage of latently infected animals decreased. HSV plaque-inhibiting antibodies on GMK monolayer cultures were revealed in all virusinoculated mice but in none of the uninoculated animals. The time elapsing between the onset of cultivation of ganglionic tissue and appearance of cpe was 7-16 days with
256 TABLE 1 Days post inoculation
No. of mice
7 14 30 60 180
22 20 60 21 20
~o virus-positive animals left ganglion
right ganglion
70.6 62.5 77.5 76.1 70.0
11.2 22.5 5.8 4.7 5.0
Total
81.8 85.0 83.3 80.8 75.0
a mean of 11 days. The mean incubation time for cultures of mice killed 7 or 14 days p.i. did not differ statistically from that of cultures of mice observed at 180 days p.i. Table 1 shows that latent infection of trigeminal ganglia was demonstrable 7 days after inoculation of the animals and persisted for at least 180 days. Infections of homolateral ganglia were observed in 62,5-77.5 ~ of the mice and in 4.7-22.5 ~ the contra lateral ganglia were harbouring virus. HSV was, thus, isolated from on average 80 ~ of the latently infected animals.
Morphological observations Trigeminal ganglia From each mouse 2-4, or in some instances up to 12, sections from the ganglia were examined light-microscopically. Scattered small infiltrations of mononuclear
Fig. 1. Infiltration of mononuclear inflammatory cells in trigeminal ganglia 180 days p.i. × 944.
257
Fig. 2. Inflammatory cells and myelin degeneration in central part of trigeminal nerve transitional region 30 days p.i. Intact peripheral myelin in lower part of the picture, x 694. Fig. 3. Vesicular disruption of CNS myelin sheaths close to macrophage with myelin debris. Brain stem 7 days p.i. x 10,220. Fig. 4. Process of macrophage extending into degenerated myelin sheath. Brain stem 7 days p.i. x 5426. l~ig. 5. Necrotic cell in brain stem 7 days p.i. with virus nucleocapsids (arrows). x 19,760.
258
Fig. 6. Area of demyelination in the brain stem 14 days p.i. x 2975. Fig. 7. Naked axons in area of demyelination. Brain stem 14 days p,i. × 14,356.
259 inflammatory cells were present in about a 4th of the ganglia even at 180 days p.i. (Fig. 1). The cells were clustered among nerve cell bodies in the ophthalmo-maxillary part of the ganglion. No degenerating nerve cells were detected and ultrastructurally no virus particles were identified. There were no myelin changes in the ganglia. Brain stems In several of the virus-inoculated mice lesions occurred in the brainstem corresponding to the entry of trigeminal roots, trigeminal tracts and nuclei. Seven days p.i. changes were observed in 5 of 10 mice, consisting of foci of inflammatory cell infiltration, disruption of myelin sheaths and macrophages laden with myelin degradation products. Ultrastructurally, the myelin sheaths showed prominent intramyelinic vacuoles and disruption of myelin lamellae. Often there was a honey-comb shaped vesiculation of the myelin (Fig. 3). Occasionally cytoplasmic tongues of macrophages appeared to remove the altered myelin (Fig. 4). Also completely denuded axons were observed. In remnants of a few necrotic cells HSV nucleocapsids could be identified (Fig. 5). In about half the number of mice killed at 14 and 30 days p.i., respectively, areas of demyelination with completely naked axons were seen (Figs. 6 and 7). In these areas macrophages with myelin debris were scattered and inflammatory cells also occurred. At 14 days p.i. some axons surrounded by only a few myelin lamellae were observed electron-microscopically (Fig. 8), and at 30 days p.i. several axons with an abnormally thin myelin sheath could be seen, even at the light-microscopical level. Ninety and 180 days p.i. several mice showed areas within the trigeminal pathways where nerve fibres had abnormally thin myelin sheaths (Fig. 9) and some were still completely denuded. In these areas there was a scanty and mainly perivascular infiltration of inflammatory cells. Transitional region of the trigeminal root Central nervous tissue extends into the trigeminal root and borders on peripheral nervous tissue in a transitional region, which contains numerous astrocytic processes; the transition in a myelinated nerve fibre between peripheral and central myelin generally occurs at a node of Ranvier (cf. Berthold and Carlstedt 1977). From each mouse one or two sections of the trigeminal root including the transitional region were examined. Seven and 14 days p.i. small collections of inflammatory ceils occurred in a few mice (Fig. 2). Thirty days p.i. denuded axons or axons with a thin myelin sheath occurred in the central part of the region in half the number of mice. Within the same fibre intact peripheral myelin next to completely demyelinated central segments could be seen. In these demyelinated areas inflammatory cells and macrophages containing myelin debris were present. Even 90 and 180 days p.i. small areas of demyelination in the central part of the transitional region occurred in a few mice (Fig. 10).
260
Fig. 8. Axons surrounded by a few myelin lamellae indicating remyelination. Brain stem 30 days p.i. x 17,580. Fig. 9. Area in the brain stem within the left trigerninal tract showing several axons with an abnormally thin myelin sheath 180 days p.i. × 944.
261
Fig. 10. Demyelinatedsegmentsof axons in the central part of the transitionalregionof the trigeminal root 180 days p.i. × 944.
DISCUSSION The F strain of HSV-1, inoculated into the skin of the nose of 5-6-week-old mice, caused latent trigeminal ganglionic infections in a high and reproducible percentage of the animals. The strain, demonstrating low pathogenicity also for suckling mice (Kristensson et al. 1978), produced no overt signs of disease in 5-6week-old animals and proved to be superior to a number of more pathogenic HSV strains for the establishment of latent infections in trigeminal ganglia, suggesting that low virulence of the virus might be a property positively correlated to the development of latency. Once established the latent infection persisted in a reactivable state for a long period, at least 6 months. This mouse model was used by us for observations on the histo-pathological changes of latently HSV-infected animals. The most interesting findings were the changes within the trigeminal pathways in the CNS with areas of demyelination leaving naked, intact axons. The demyelination was most pronounced after 14 and 30 days p.i., but 180 days p.i. nerve fibres still showed segmental demyelination in the central part of the transitional region of the trigeminal root. As in acute HSV infection the peripheral myelin remained intact (Townsend and Baringer 1978a; Kristensson et al. 1978), and in the transitional region the difference in reaction between peripheral and central myelin could be well delineated. There are different possible ways by which demyelination may be induced in a virus disease including direct cytopathic effects of the virus on myelin-forming
262 oligodendroglial cells and myelin changes secondary to an immune response (cf. Wisniewski 1977; Kristensson and Wisniewski 1978; Lampert 1978). In previous studies (Kristensson et al. 1978; Townsend and Baringer 1978a) a few oligodendroglial cells have been found containing virus particles and it is possible that some of the necrotic cells found 7 days p.i. represented oligodendroglia. Thus, the mechanism with direct virus effect on the oligodendroglia may have prevailed. However, when myelin changes were observed there was also infiltration of inflammatory cells in the tissue. Recently, Townsend and Baringer (1978b) reported that immunosuppression prior to corneal HSV inoculation in mice markedly reduced the extent of mononuclear cell infiltration and myelin destruction, while there was no significant decrease in virus titers in the central part of the trigeminal root. The demyelination observed by us may therefore, alternatively, be a result of immune reactions initiated by the HSV infection in the CNS. Such an immune response may be due to cross-reactivity between viral and myelin antigens, to autosensitization to removed damaged myelin or to immune reactions directed against virus infected oligodendroglial cells, or, as a "bystander" effect, against other infected cells in the vicinity of the myelin. HSV absorbs better to astrocytes than to nerve cell perikarya (Vahlne et al. 1978) and in acute CNS infection of mice astrocytes, which are particularly abundant in the transitional region of the trigeminal root, are frequently infected (Kristensson et al. 1978; Townsend and Baringer 1978a). Immune reactions directed against infected astrocytes might possibly lead to myelin changes as a "bystander" effect (Wisniewski and Bloom 1975). Recently it has been shown that neutral proteases secreted by stimulated macrophages can degrade basic protein in myelin, which may provide a possible mechanism for induction of such a "bystander" demyelination (Cammer et al. 1978). The differences between PNS and CNS myelin might then reflect a different susceptibility to noxious factors released by the inflammation similar to their different modes of reaction to toxic agents (Scheinberg et al. 1966; Persson et al. 1978). Fourteen and 30 days p.i. axons surrounded by only a few myelin lamellae were found. This may indicate onset of remyelination since later p.i. axons with abnormally thin myelin sheaths appeared to be numerous. Remyelination by oligodendroglia in the CNS following demyelination caused by toxic or infectious agents has been well documented (Blakemore et al. 1977; Raine and Schaumberg 1977). We have not yet evaluated the possibility of an invasion of Schwann cells into the CNS compartment of the root. It is evident that the latent HSV infection of trigeminal ganglia in mice involves the trigeminal root and the brain stem with areas of demyelination. Whether this is so also in humans is not known, but cases of herpetic brain stem encephalitis have been reported (Dayan et al. 1972). In this context, one further interesting case should be mentioned with latent HSV infection of the trigeminal ganglia in combination with multiple sclerosis and extensive demyelination in the trigeminal entry zones in the brain stem (Warren et al. 1977). To further elucidate the demyelinating capacity of HSV infections it would be of interest to study the effects on myelin of experimentally repeated reactivation of latent ganglionic infections.
263 REFERENCES Baringer, J. R. and P. Swoveland (1973) Recovery of herpes-simplex virus from human trigeminal ganglions, New Engl. J. Med., 288 : 648-650. Baringer, J. R. and P. Swoveland (1974) Persistent herpes-simplex virus infection in rabbit trigeminal ganglia, Lab. Invest., 30: 230-240. Berthold, C.-H. and T. Carlstedt (1977) General organization of the transitional regions on $1 dorsal rootlets, Acta physiol, stand., Suppl. 446: 23-42. Blakemore, W. F., R. A. Eames, K. J. Smith and W. I. McDonald (1977) Remyelination in the spinal cord of the cat following intraspinal injections oflysolecithin, J. neurol. Sci., 33 : 31-43. Cammer, W., B. R. Bloom, W. T. Norton and S. Gordon (1978) Degradation of basic protein in myelin by neutral proteases secreted by stimulated macrophages - - A possible mechanism of inflammatory demyelination, Proc. nat. Acad. Sci., 75: 1554-1558. Dayan, A. D., W. Gooddy, M. J. G. Harrison and P. Rudge (1972) Brain stem encephalitis caused by herpesvirus hominis, Brit. med. J., 4: 405-406. Forghani, B., T. Klassen and J. R. Baringer (1977) Radioimmunoassay of herpes simplex virus antibody - - Correlation with ganglionic infection, J. gen. Virol., 36: 371-375. Jeansson, S. (1972) Differentiation between herpes simplex virus tyre I and type 2 strains by immunoelectroosmophoresis, Appl. Microbiol., 24: 96-100. Klein, R. J. (1976) Pathogenetic mechanisms of recurrent herpes simplex virus infections, Arch. Virol., 51 : 1-13. Kristensson, K. and H. M. Wisniewski (1978) Arrest of myelination and demyelinatien in rabbit retina induced by herpes simplex virus infection, Neuropath. appl. Neurobiol., 4: 25-36. Kristensson, K., A. Vahlne, L. A. Persson and E. Lycke (1978) Neural spread of herlzes simplex virus type 1 and 2 in mice after corneal or subcutaneous (footpad) inoculation, J. neurol. Sci., 35:331340. Lampert, P. (1978) Autoimmune and virus-induced demyelinating diseases, Amer. J. Path., 91: 176-208. Persson, L. A., B. Norlander and K. Kristensson (1978) Studies on hexachlorophene-induced myelin lesions in the trigeminal root transitional region in developing and adult mice, Acta neuropath. (Berl.), 42:115-120. Raine, C. S. and H. H. Schaumberg (1977) The neuropathology of myelin diseases. In: P. Morell (Ed.), Myelin, Plenum Press, New York, pp. 271-323. Scheinberg, I_,. C., J. M. Taylor, I. Herzog and S. Mandell (1966) Optic and peripheral nerve response to triethyltin intoxication in the rabbit --- Biochemical and ultrastructural studies, J. Neuropath. exp. Neurol., 25 : 202-213. Stevens, J. C. and M. L. Cook (1971) Latent herpes simplex virus in spinal ganglia of mice, Science, 173 : 343-345. Townsend, J. J. and J. R. Baringer (1978a) Central nervous system susceptibility to herpes simplex infection, J. Neuropath. exp. Neurol., 37: 255-262. Townsend, J. J. and J. R. Baringer (1978b) Alteration of herpes simplex induced CNS lesions by immunosuppression, J. Neuropath. exp. Neurol., 37: 701. Vahlne, A., J. Blomberg, S. Olofsson and E. Lycke (1975) Subtyping of herpes simplex virus, Acta path. microbiol, scand., 83: 506-512. Vahlne, A., B. Nystr6m, M. Sandberg, A. Hamberger and E. Lycke (1978) Attachment of herpes simplex virus to neurons and glial cells, J. gen. Virol., 40: 359-371. Warren, K. G., M. Devlin, D. H. Gilden, Z. Wroblewska, S. M. Brown, J. Subak-Sharpe and H. Koprowski (1977) Isolation of herpes simplex virus from human trigeminal ganglia, including ganglia from one patient with multiple sclerosis, Lancet, 2 (8039) : 637-639. Wildy, P. (1972) Herpes simplex virus and antigens. In: P. M. Biggs, G. de Th6 and L. N. Payne (Eds.), Oncogenesis and Herpes viruses, Int. Agency Res. Cancer, Lyon, pp. 409-419. Wisniewski, H. M. (1977) Immunopathology of demyelination in autoimmune diseases and virus infections, Brit. med. Bull., 33 (1): 54-59. Wisniewski, H. M. and B. R. Bloom (1975) Primary demyelination as a nonspecific consequence of a cell-mediated immune reaction, J. exp. Med., 141 : 346-359.
253
LATENT HERPES SIMPLEX VIRUS T R I G E M I N A L G A N G L I O N I C INFECTION IN MICE AND DEMYELINATION IN THE CENTRAL NERVOUS SYSTEM
KRISTER KRISTENSSON1, BO SVENNERHOLM2, LENNART PERSSON, ANDERS VAHLNE2 and ERIK LYCKE2 2 Department of Virology, Institute of Medical Microbiology, University of Gi~teborg, GSteborg, and 1 Neuropathological Laboratory, Department of Pathology II, University t~fLinkb'ping (Sweden)
(Received 22 February, 1979) (Accepted 4 April, 1979)
SUMMARY Mice were inoculated with herpes simplex virus (HSV) type 1 by gently scraping the skin of the nose with a fine needle. About 80 ~o of the animals developed latent inapparent HSV infections in trigeminal ganglia. Virus was demonstrable for at least 6 months post inoculation (p.i.) by cocultivation of ganglionic tissue with G M K cells. Histologically, trigeminal ganglia revealed infiltrations of inflammatory cells even 6 months p.i. In addition, lesions occurred in the brainstem corresponding to the entry of trigeminal roots, trigeminal tracts and nuclei. Inflammatory cell infiltration, disruption of myelin sheaths and macrophages laden with myelin degradation products were observed 7 days p.i. Fourteen to 30 days p.i. electron microscopy demonstrated completely naked axons. In the transitional region of the trigeminal root denuded axons occurred in the central part of the region while the peripheral myelin, bordering the demyelinated central segments, was intact. Small areas of demyelination were still detectable 3 and 6 months p.i. but there were then also signs of remyelination. Possible mechanisms causing the demyelination are discussed. INTRODUCTION Herpes simplex virus (HSV) may remain as a latent infection in trigeminal ganglia of both humans and experimental animals (Stevens and Cook 1971; Baringer This study was supported by grants from the Swedish Medical Research Council, project hr. B79-12X-04480-05B and 4514. Correspondence to: Dr. Krister Kristensson, Neuropathological Laboratory, University of Link6ping, S-581 85 Link6ping, Sweden.
254 and Swoveland 1973; Forghani et al. 1977). It is not known if HSV during latency persists in the ganglion slowly replicating or in a state of arrested multiplication (cf. Klein 1976). Even 16 weeks after corneal HSV inoculation an occasional degenerated neuron containing virus particles in the trigeminal ganglia can be found (Baringer and Swoveland 1974), but it is not known if this represents virus newly formed after reactivation or a persistent infection. Nor is it known to what extent latent HSV infection is associated with pathological changes other than those in ganglia harbouring the infection. Previously, it was found that during acute HSV infection the virus may spread along the trigeminal root to the brain stem causing severe changes in the central myelin, while the peripheral myelin remained intact (Townsend and Baringer 1978a; Kristensson et al. 1978). In the present report we have examined the trigeminal root and brain stem in clinically inapparent or latent HSV infections of mice with especial regard to the appearance of the central myelin. M A T E R I A L A N D METHODS
Virus
Six HSV-1 strains were used. Strain F originated from Dr. B. Roizman, University of Chicago, the others were isolated and typed in our laboratory (Vahlne et al. 1975). The strains were propagated in GMK cells, in which pfu also were assayed. Virus isolated from trigeminal ganglia was typed by immunoelectroosmophoresis against type-specific antisera (Jeansson 1972).
Cells Green monkey kidney (GMK AH-1) cells were cultured in Eagle's minimum essential medium (MEM) supplemented with 10 ~ calf serum, 100 i.u. of penicillin and 100 #g of streptomycin per ml. For maintenance the same medium supplemented with 2 ~ serum only was used.
Inoculation technique and preparation of trigeminal ganglionic tissue Five-6-week old Swiss albino mice of both sexes of our own laboratory breed were anesthetized by ether. Virus inoculation of the eye by corneal scratching, of the side of the nose by injection or scraping with a needle, and, thirdly, inoculation of the nostrils, were the routes tested. The most satisfactory results were obtained when a drop of the virus suspension was placed on one side of the nose (left) and virus inoculated by gentle scraping with a fine needle, and this technique was used in the present study. For preparation of ganglionic tissue ether-anesthetized mice were perfused with BSS through the left cardiac ventricle, and the brains were removed for exposure of the trigeminal ganglia. Left and right side trigeminal ganglia were removed separately and placed in tubes in cell culture medium at room temperature. Before being added to cell culture bottles the ganglia were cut in pieces with scissors.
Cell culture technique A method modified from that used by Stevens and Cook (1971) was used for
255 reactivation of latent HSV of trigeminal ganglia. The ganglia were cut by scissors and the pieces cultured in 30 ml plastic bottles seeded with GMK cells (5 × 105 cells per bottle) at the time of adding the pieces of ganglia. Ganglia from one side were cocultivated with GMK cells in one bottle with Eagle's MEM. Thus, two cultures were prepared per mouse. Culture medium was replaced twice a week. Microscopic readings of cultures were performed every day for 4 weeks and virus from cultures demonstrating the cytopathogenic effect (cpe) of GMK cells was passaged on new GMK cultures before typing of the strains. Ganglia from 20 mock-inoculated animals were collected 30 days post inoculation (p.i.), when an equal number of virus-inoculated animals were examined concomitantly. From none of the mock-inoculated mice was HSV isolated, indicating that both the preparation of ganglia and the cocultivation could be performed without virus contamination.
HSV antibodies Serum samples were collected from 7 virus-inoculated and 6 uninoculated mice and tested for presence of HSV antibodies 7 days p.i. using the K-test (Wildy 1972).
Histological technique For histological examination groups of 5-10 mice were killed 7, 14, 30, 60, 90 and 180 days p.i. with undiluted strain F, see below. The mice were anesthetized with ether, perfused through the heart with 5 ~ glutaraldehyde in phosphate buffer and the trigeminal ganglia, the trigeminal roots and the brain stems including the areas of the trigeminal nuclei and tracts were dissected. The specimens were postfixed in osmic acid, dehydrated in alcohols and embedded in Epon 812. One-/~m thick sections were cut and stained with toluidine blue for light microscopy. Ultrathin sections were cut from selected areas and contrasted with uranyl acetate and lead citrate for electron microscopy. RESULTS
Latent HSV-1 infection in trigeminal ganglia In pilot experiments the strain of HSV-1, the concentration of virus and the route of inoculation were selected for establishment of latent HSV infection of trigeminal ganglia in a high and reproducible percentage of inoculated mice. Of 6 HSV-1 strains observed the F strain yielded latent trigeminal infection in 75-85 ~ of the animals. As this strain caused no overt signs of disease and, thus, no deaths of inoculated animals, it could be used undiluted in a concentration of 8 log pfu/ml. In general, strains highly pathogenic for mice causing fulminant infections of the CNS were less satisfactory for establishment of latent infections and by diluting the strains to avoid loss of animals the percentage of latently infected animals decreased. HSV plaque-inhibiting antibodies on GMK monolayer cultures were revealed in all virusinoculated mice but in none of the uninoculated animals. The time elapsing between the onset of cultivation of ganglionic tissue and appearance of cpe was 7-16 days with
256 TABLE 1 Days post inoculation
No. of mice
7 14 30 60 180
22 20 60 21 20
~o virus-positive animals left ganglion
right ganglion
70.6 62.5 77.5 76.1 70.0
11.2 22.5 5.8 4.7 5.0
Total
81.8 85.0 83.3 80.8 75.0
a mean of 11 days. The mean incubation time for cultures of mice killed 7 or 14 days p.i. did not differ statistically from that of cultures of mice observed at 180 days p.i. Table 1 shows that latent infection of trigeminal ganglia was demonstrable 7 days after inoculation of the animals and persisted for at least 180 days. Infections of homolateral ganglia were observed in 62,5-77.5 ~ of the mice and in 4.7-22.5 ~ the contra lateral ganglia were harbouring virus. HSV was, thus, isolated from on average 80 ~ of the latently infected animals.
Morphological observations Trigeminal ganglia From each mouse 2-4, or in some instances up to 12, sections from the ganglia were examined light-microscopically. Scattered small infiltrations of mononuclear
Fig. 1. Infiltration of mononuclear inflammatory cells in trigeminal ganglia 180 days p.i. × 944.
257
Fig. 2. Inflammatory cells and myelin degeneration in central part of trigeminal nerve transitional region 30 days p.i. Intact peripheral myelin in lower part of the picture, x 694. Fig. 3. Vesicular disruption of CNS myelin sheaths close to macrophage with myelin debris. Brain stem 7 days p.i. x 10,220. Fig. 4. Process of macrophage extending into degenerated myelin sheath. Brain stem 7 days p.i. x 5426. l~ig. 5. Necrotic cell in brain stem 7 days p.i. with virus nucleocapsids (arrows). x 19,760.
258
Fig. 6. Area of demyelination in the brain stem 14 days p.i. x 2975. Fig. 7. Naked axons in area of demyelination. Brain stem 14 days p,i. × 14,356.
259 inflammatory cells were present in about a 4th of the ganglia even at 180 days p.i. (Fig. 1). The cells were clustered among nerve cell bodies in the ophthalmo-maxillary part of the ganglion. No degenerating nerve cells were detected and ultrastructurally no virus particles were identified. There were no myelin changes in the ganglia. Brain stems In several of the virus-inoculated mice lesions occurred in the brainstem corresponding to the entry of trigeminal roots, trigeminal tracts and nuclei. Seven days p.i. changes were observed in 5 of 10 mice, consisting of foci of inflammatory cell infiltration, disruption of myelin sheaths and macrophages laden with myelin degradation products. Ultrastructurally, the myelin sheaths showed prominent intramyelinic vacuoles and disruption of myelin lamellae. Often there was a honey-comb shaped vesiculation of the myelin (Fig. 3). Occasionally cytoplasmic tongues of macrophages appeared to remove the altered myelin (Fig. 4). Also completely denuded axons were observed. In remnants of a few necrotic cells HSV nucleocapsids could be identified (Fig. 5). In about half the number of mice killed at 14 and 30 days p.i., respectively, areas of demyelination with completely naked axons were seen (Figs. 6 and 7). In these areas macrophages with myelin debris were scattered and inflammatory cells also occurred. At 14 days p.i. some axons surrounded by only a few myelin lamellae were observed electron-microscopically (Fig. 8), and at 30 days p.i. several axons with an abnormally thin myelin sheath could be seen, even at the light-microscopical level. Ninety and 180 days p.i. several mice showed areas within the trigeminal pathways where nerve fibres had abnormally thin myelin sheaths (Fig. 9) and some were still completely denuded. In these areas there was a scanty and mainly perivascular infiltration of inflammatory cells. Transitional region of the trigeminal root Central nervous tissue extends into the trigeminal root and borders on peripheral nervous tissue in a transitional region, which contains numerous astrocytic processes; the transition in a myelinated nerve fibre between peripheral and central myelin generally occurs at a node of Ranvier (cf. Berthold and Carlstedt 1977). From each mouse one or two sections of the trigeminal root including the transitional region were examined. Seven and 14 days p.i. small collections of inflammatory ceils occurred in a few mice (Fig. 2). Thirty days p.i. denuded axons or axons with a thin myelin sheath occurred in the central part of the region in half the number of mice. Within the same fibre intact peripheral myelin next to completely demyelinated central segments could be seen. In these demyelinated areas inflammatory cells and macrophages containing myelin debris were present. Even 90 and 180 days p.i. small areas of demyelination in the central part of the transitional region occurred in a few mice (Fig. 10).
260
Fig. 8. Axons surrounded by a few myelin lamellae indicating remyelination. Brain stem 30 days p.i. x 17,580. Fig. 9. Area in the brain stem within the left trigerninal tract showing several axons with an abnormally thin myelin sheath 180 days p.i. × 944.
261
Fig. 10. Demyelinatedsegmentsof axons in the central part of the transitionalregionof the trigeminal root 180 days p.i. × 944.
DISCUSSION The F strain of HSV-1, inoculated into the skin of the nose of 5-6-week-old mice, caused latent trigeminal ganglionic infections in a high and reproducible percentage of the animals. The strain, demonstrating low pathogenicity also for suckling mice (Kristensson et al. 1978), produced no overt signs of disease in 5-6week-old animals and proved to be superior to a number of more pathogenic HSV strains for the establishment of latent infections in trigeminal ganglia, suggesting that low virulence of the virus might be a property positively correlated to the development of latency. Once established the latent infection persisted in a reactivable state for a long period, at least 6 months. This mouse model was used by us for observations on the histo-pathological changes of latently HSV-infected animals. The most interesting findings were the changes within the trigeminal pathways in the CNS with areas of demyelination leaving naked, intact axons. The demyelination was most pronounced after 14 and 30 days p.i., but 180 days p.i. nerve fibres still showed segmental demyelination in the central part of the transitional region of the trigeminal root. As in acute HSV infection the peripheral myelin remained intact (Townsend and Baringer 1978a; Kristensson et al. 1978), and in the transitional region the difference in reaction between peripheral and central myelin could be well delineated. There are different possible ways by which demyelination may be induced in a virus disease including direct cytopathic effects of the virus on myelin-forming
262 oligodendroglial cells and myelin changes secondary to an immune response (cf. Wisniewski 1977; Kristensson and Wisniewski 1978; Lampert 1978). In previous studies (Kristensson et al. 1978; Townsend and Baringer 1978a) a few oligodendroglial cells have been found containing virus particles and it is possible that some of the necrotic cells found 7 days p.i. represented oligodendroglia. Thus, the mechanism with direct virus effect on the oligodendroglia may have prevailed. However, when myelin changes were observed there was also infiltration of inflammatory cells in the tissue. Recently, Townsend and Baringer (1978b) reported that immunosuppression prior to corneal HSV inoculation in mice markedly reduced the extent of mononuclear cell infiltration and myelin destruction, while there was no significant decrease in virus titers in the central part of the trigeminal root. The demyelination observed by us may therefore, alternatively, be a result of immune reactions initiated by the HSV infection in the CNS. Such an immune response may be due to cross-reactivity between viral and myelin antigens, to autosensitization to removed damaged myelin or to immune reactions directed against virus infected oligodendroglial cells, or, as a "bystander" effect, against other infected cells in the vicinity of the myelin. HSV absorbs better to astrocytes than to nerve cell perikarya (Vahlne et al. 1978) and in acute CNS infection of mice astrocytes, which are particularly abundant in the transitional region of the trigeminal root, are frequently infected (Kristensson et al. 1978; Townsend and Baringer 1978a). Immune reactions directed against infected astrocytes might possibly lead to myelin changes as a "bystander" effect (Wisniewski and Bloom 1975). Recently it has been shown that neutral proteases secreted by stimulated macrophages can degrade basic protein in myelin, which may provide a possible mechanism for induction of such a "bystander" demyelination (Cammer et al. 1978). The differences between PNS and CNS myelin might then reflect a different susceptibility to noxious factors released by the inflammation similar to their different modes of reaction to toxic agents (Scheinberg et al. 1966; Persson et al. 1978). Fourteen and 30 days p.i. axons surrounded by only a few myelin lamellae were found. This may indicate onset of remyelination since later p.i. axons with abnormally thin myelin sheaths appeared to be numerous. Remyelination by oligodendroglia in the CNS following demyelination caused by toxic or infectious agents has been well documented (Blakemore et al. 1977; Raine and Schaumberg 1977). We have not yet evaluated the possibility of an invasion of Schwann cells into the CNS compartment of the root. It is evident that the latent HSV infection of trigeminal ganglia in mice involves the trigeminal root and the brain stem with areas of demyelination. Whether this is so also in humans is not known, but cases of herpetic brain stem encephalitis have been reported (Dayan et al. 1972). In this context, one further interesting case should be mentioned with latent HSV infection of the trigeminal ganglia in combination with multiple sclerosis and extensive demyelination in the trigeminal entry zones in the brain stem (Warren et al. 1977). To further elucidate the demyelinating capacity of HSV infections it would be of interest to study the effects on myelin of experimentally repeated reactivation of latent ganglionic infections.
263 REFERENCES Baringer, J. R. and P. Swoveland (1973) Recovery of herpes-simplex virus from human trigeminal ganglions, New Engl. J. Med., 288 : 648-650. Baringer, J. R. and P. Swoveland (1974) Persistent herpes-simplex virus infection in rabbit trigeminal ganglia, Lab. Invest., 30: 230-240. Berthold, C.-H. and T. Carlstedt (1977) General organization of the transitional regions on $1 dorsal rootlets, Acta physiol, stand., Suppl. 446: 23-42. Blakemore, W. F., R. A. Eames, K. J. Smith and W. I. McDonald (1977) Remyelination in the spinal cord of the cat following intraspinal injections oflysolecithin, J. neurol. Sci., 33 : 31-43. Cammer, W., B. R. Bloom, W. T. Norton and S. Gordon (1978) Degradation of basic protein in myelin by neutral proteases secreted by stimulated macrophages - - A possible mechanism of inflammatory demyelination, Proc. nat. Acad. Sci., 75: 1554-1558. Dayan, A. D., W. Gooddy, M. J. G. Harrison and P. Rudge (1972) Brain stem encephalitis caused by herpesvirus hominis, Brit. med. J., 4: 405-406. Forghani, B., T. Klassen and J. R. Baringer (1977) Radioimmunoassay of herpes simplex virus antibody - - Correlation with ganglionic infection, J. gen. Virol., 36: 371-375. Jeansson, S. (1972) Differentiation between herpes simplex virus tyre I and type 2 strains by immunoelectroosmophoresis, Appl. Microbiol., 24: 96-100. Klein, R. J. (1976) Pathogenetic mechanisms of recurrent herpes simplex virus infections, Arch. Virol., 51 : 1-13. Kristensson, K. and H. M. Wisniewski (1978) Arrest of myelination and demyelinatien in rabbit retina induced by herpes simplex virus infection, Neuropath. appl. Neurobiol., 4: 25-36. Kristensson, K., A. Vahlne, L. A. Persson and E. Lycke (1978) Neural spread of herlzes simplex virus type 1 and 2 in mice after corneal or subcutaneous (footpad) inoculation, J. neurol. Sci., 35:331340. Lampert, P. (1978) Autoimmune and virus-induced demyelinating diseases, Amer. J. Path., 91: 176-208. Persson, L. A., B. Norlander and K. Kristensson (1978) Studies on hexachlorophene-induced myelin lesions in the trigeminal root transitional region in developing and adult mice, Acta neuropath. (Berl.), 42:115-120. Raine, C. S. and H. H. Schaumberg (1977) The neuropathology of myelin diseases. In: P. Morell (Ed.), Myelin, Plenum Press, New York, pp. 271-323. Scheinberg, I_,. C., J. M. Taylor, I. Herzog and S. Mandell (1966) Optic and peripheral nerve response to triethyltin intoxication in the rabbit --- Biochemical and ultrastructural studies, J. Neuropath. exp. Neurol., 25 : 202-213. Stevens, J. C. and M. L. Cook (1971) Latent herpes simplex virus in spinal ganglia of mice, Science, 173 : 343-345. Townsend, J. J. and J. R. Baringer (1978a) Central nervous system susceptibility to herpes simplex infection, J. Neuropath. exp. Neurol., 37: 255-262. Townsend, J. J. and J. R. Baringer (1978b) Alteration of herpes simplex induced CNS lesions by immunosuppression, J. Neuropath. exp. Neurol., 37: 701. Vahlne, A., J. Blomberg, S. Olofsson and E. Lycke (1975) Subtyping of herpes simplex virus, Acta path. microbiol, scand., 83: 506-512. Vahlne, A., B. Nystr6m, M. Sandberg, A. Hamberger and E. Lycke (1978) Attachment of herpes simplex virus to neurons and glial cells, J. gen. Virol., 40: 359-371. Warren, K. G., M. Devlin, D. H. Gilden, Z. Wroblewska, S. M. Brown, J. Subak-Sharpe and H. Koprowski (1977) Isolation of herpes simplex virus from human trigeminal ganglia, including ganglia from one patient with multiple sclerosis, Lancet, 2 (8039) : 637-639. Wildy, P. (1972) Herpes simplex virus and antigens. In: P. M. Biggs, G. de Th6 and L. N. Payne (Eds.), Oncogenesis and Herpes viruses, Int. Agency Res. Cancer, Lyon, pp. 409-419. Wisniewski, H. M. (1977) Immunopathology of demyelination in autoimmune diseases and virus infections, Brit. med. Bull., 33 (1): 54-59. Wisniewski, H. M. and B. R. Bloom (1975) Primary demyelination as a nonspecific consequence of a cell-mediated immune reaction, J. exp. Med., 141 : 346-359.