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Immunocytochemical localization of herpes simplex virus antigen in the trigeminal ganglia of experimentally infected mice
Journal of the Neurological Sciences, 1984, 66:67-75 Elsevier
67
Immunocytochemical Localization of Herpes Simplex Virus Antigen in the Trigeminal Ganglia of Experimentally Infected Mice Yasuto Itoyama ~, Tsuyoshi Sekizawa 2, Harry Openshaw 3, Kyuya Kogure 2 and Yoshigoro Kuroiwa 1 ~Department of Neurology, Kyushu University, Fukuoka 812 (Japan); 2Department of Neurology, School of Medicine, Tohoku University,Sendai 980 (Japan); and 3Departmentof Neurology, City of Hope National Medical Center, Duarte. CA 91010 (U.S.A.) (Received 13 March, 1984) (Revised, received 29 May, 1984) (Accepted 29 May, 1984)
SUMMARY
The peroxidase anti-peroxidase (PAP) technique was used to study the distribution of herpes simplex virus (HSV) antigens in mouse ganglia during the acute infection and the transition into the latent infection. At 2 days after HSV inoculation by the corneal route, immunoperoxidase staining was present in occasional isolated neurons of the trigeminal ganglion and also in scattered satellite cells. By 4 days, more cells were stained with the infection centered in the medial portion of the ganglion. Inflammatory cells were present around PAP-labeled fragments from lysed cells. Stained satellite cells often with a hypertrophic appearance surrounded labeled or unlabeled neurons in a ring-like array. At 6 days after HSV inoculation, there was a decrease both in the number of cells stained and in the intensity of staining. By 8 days, HSV antigens could be detected by weak PAP staining only in neurons. Otherwise, these neurons appeared morphologically normal. No irnmunoperoxidase staining was present after the 8th day. These results are compatible with retrograde axoplasmic transport of HSV and cell to cell spread of virus in ganglia. Also the appearance of infected ganglion cells during the transition to latency suggests that neurons can be switched from an HSV-permissive to a non-permissive (latent) state.
Correspondence and reprint requests to: Yasuto Itoyama, Department of Neurology, Neurological Institute, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka 812, Japan, Tel. (092)641-1151
0022-510X/84/$03.00 © 1984 Elsevier Science Publishers B.V.
68 Key words: Herpes simplex virus - Herpetic infection - Immunocytochemical observation - Latent infection o f H S V - Trigeminal ganglia
INTRODUCTION
Herpes simplex virus (HSV) is present in a latent form in the trigeminal ganglion of man (Bastian etal. 1972; Baringer and Sowveland 1973; Warren etal, 1977). Herpetic recurrences follow HSV reactivation in ganglia, migration of virions down axons to the skin surface, and viral replication in epithelial cells. Details of the pathogenesis of ganglionic infection has been derived mostly from work in animal models (Stevens and Cook 1971; Walz et al. 1974; Kristensson et al. 1979; Openshaw et al. 1979; Sekizawa et al. 1980; McLennan and Darby 1980). In the experimental system, HSV is usually inoculated on the skin surface; and the course of acute, latent, and reactivated infection is studied in the corresponding sensory ganglion. A critical question in this system concerns the mechanism whereby latency is established during the transition from the acute to the latent state of the infection. This transition occurs 12-14 days after viral inoculation of an immunocompetent animal. Prior to the transition, infectious virus can be detected in cell-free ganglionic homogenates (acute stage of the infection). After 14 days (the latent stage), infectious virus can no longer be detected in homogenates but HSV can be recovered by explantation of ganglia in organ culture (in vitro reactivation). The unlabeled antibody peroxidase anti-peroxidase (PAP) method (Sternberger et al. 1970) is highly sensitive and specific for detection of various viral antigens including HSV in formalin.fixed paraffm-embedded sections ( K u m ~ s h i and Hirano 1978; Esiri 1982; Itoyama et al. 1982; Kristensson et al. 1982). In the present report, we used the PAP method in mouse ganglia to study the time course of the acute HSV infection and the transition from the acute to the latent stage. MATERIALS AND METHODS
Animals and virus inoculation
Twenty-two 6-8 week-old-female Balb/c mice were inoculated with the F strain of HSV type 1. Mice were infected by placing one drop of stock HSV containing 108 plaque forming units/ml on both corneas and scratching the epithelium with a I9-gauge needle (Openshaw et al. 1979). The inoculated mice were killed 2, 4, 6, 8, 10 and 14 days after virus inoculation. They were anesthetized with ether and peffused for 10 rain via the heart with a mixture of mercuric chloride and formalin (Sternberger et al. 1978). The trigeminal roots and ganglia were carefully removed, dissected, postfLxed overnight in the same fixative, dehydrated in a graded series of ethanol and p-dioxane, and embedded in paraffin.
69
lmmunoperoxidase staining A modification (Itoyama et al. 1980) of the peroxidase anti-peroxidase (PAP) method (Sternberger et al. 1970) was used with anti-HSV rabbit serum as the primary antibody. Sections 6 #m thick were cut and mounted serially on numbered glass slides to study the ganglion in three dimensions. Sections were deparaffmized by serial immersions in xylene, a xylene-ethanol mixture, absolute ethanol, and 95 ~o ethanol. For PAP staining, sections were treated sequentially with 3 ~/o normal sheep serum, rabbit anti-HSV serum diluted 1:500, sheep anti-rabbit IgG diluted 1:40, rabbit PAP diluted 1:80, and 3,3'-diaminobenzidine HC1 with hydrogen peroxide. All dilutions were made with 0.5 M Tris buffer. The final steps included counterstaining with hematoxylin, immersion in osmium tetroxide, dehydration, and the addition of cover slips. Anti-HSV antibody (500 neutralizing units) was obtained from a New Zealand white rabbit after multiple intravenous inoculations of 108 plaque-forming units of H SV (Openshaw et al. 1979). One neutralizing unit of antibody represents the reciprocal of the highest 2-fold serum dilution in 0.2 ml which produces a 50~ reduction of HSV plaques. Controls included trigeminal ganglion sections from uninfected mice incubated with the same solutions and also sections from infected mice incubated with nonimmune rabbit serum. Both of these controls were invariably negative for immunoperoxidase staining. RESULTS
The time course of the infection was studied by immunoperoxidase staining of HSV antigens in trigeminal ganglion tissue section (Fig. 1). Two days after HSV inoculation by the corneal route, there was PAP staining of only occasional neurons and satellite cells (Fig. 1A). The stained cells were morphologically normal and were confined to the medial portion of the ganglion. PAP staining was intense but positive cells occurred usually in only one of several serial sections. At this stage, there was no inflammation and no PAP staining of Schwann cells, myelinated fibers, or endothelial cells. At 4 days after HSV inoculation (Figs. 1B, 2-4), the number of PAP-positive cells was greatly increased with the infection centered in the medial portion of the ganglion (Fig. 2). Immunoperoxidase staining occurred in both large and small neurons, in satellite cells, and in occasional Schwann cells and myelinated fibers. Some heavily stained fibers were degenerated to form small stained fragments, and mononuclear inflammatory cells infiltrated around these fragments (Fig. 3). In most stained neurons, HSV reactive products Were preferentially found in the cytoplasm, and in some PAPpositive neurons the nuclei were not stained. Heavily stained and hypertrophic satellite cells surrounded some unstained neurons in a ring-like pattern (Fig. 4A). The stained satellite cells showing this pattern usually occurred in clusters (Fig. 4B) forming a link offing-like cells with contact to a heavily stained neuron at one part of the link (Fig. 1B). By 6 days after HSV inoculation, there was a decrease in both the intensity of PAP staining and in the number of cells stained (Fig. 1C). Most of PAP-positive cells
70
A
Fig. 1. Time course of the acute HSV infection in trigeminal ganglia. Paraffin sections were immunostained with antisera to H SV, counterstained with hematoxylin, and photographed at a final magnification of × 240. A: At 2 days after HSV inoculation by the corneal route, an isolated neuron stained intensely with PAP. B: At 4 days, more cells are labeled. Satellite cells containing HSV antigens form a ring-like array around neurons and make contact with a heavily stained neuron (arrow). C: At 6 days, the PAP staining is less intense and most of the labeled cells are neurons. D: At 8 days, only neurons are labeled (arrow). They appear morphologically normal with faint peroxidase staining. at this stage were neurons. There was a further d e c r e a s e in the intensity a n d the n u m b e r o f stained cells at 8 d a y s after H S V inoculation (Fig. 1D). By this time, P A P - p o s i t i v e cells were exclusively neurons. W e a k l y positive granular staining was present in the nuclei or c y t o p l a s m b u t otherwise the n e u r o n s a p p e a r e d to b e morphologically normal. I m m u n o p e r o x i d a s e staining was negative at d a y 10 a n d 14 after H S V inoculation. M o n o n u c l e a r inflammatory ceils persisted in the medial p o r t i o n o f the ganglion as a residual o f the acute infection (Fig. 5). DISCUSSION In c o m p a r i s o n to immunofluorescence, the P A P technique shows higher sensitivity a n d specificity, gives a relative p e r m a n e n t r e c o r d o f the results, a n d provides superior histological detail.
71
i ¸ ¸¸¸I¸¸¸¸!¸~¸
Fig. 2. Trigeminal ganglion section 4 days after HSV inoculation by the corneal route (PAP, hematoxylin counterstain, x 60). A number of stained cells were confined to the medial portion of the ganglion (left side of the ganglion in this picture corresponds to the medial part of the ganglion).
With this technique, we could detect HSV antigens in trigeminal ganglia up to 8 days after viral inoculation of mice. A comparable study with indirect immunofluorescence reported a 6-day limit of detection in mouse ganglia (Cook and Stevens 1973). In contrast, Green et al. (1981) using a rabbit model demonstrated HSV antigens into the latent stage of the infection. In their study, indirect immunofluorescence was used in ganglion sections with the primary antisera directed against the immediate early HSV polypeptide VP 175. In the present study, cells bearing HSV antigens were clustered in the medial aspect of the ganglia. This finding is compatible with retrograde axoplasmic transport of HSV (Cook and Stevens 1973; K_ristensson et al. 1971; Tullo et al. 1982) since the medial portion of the ganglion contains those neurons that correspond to nerve terminals on the cornea (Arvidsson 1975; Kristensson et al. 1978). By day 4 after virus inoculation, a productive H SV infection was present with lysis of some neurons and recruitment of inflammatory cells. Satellite cells beating HSV antigens appeared in a ring-like array around infected and apparently unifected neurons. These arrays usually occurred in clusters forming a link of ring-like cells with contact
72
Fig. 3. Trigeminal ganglion section 4 days after HSV inoculation by the corneal route (PAP, hematoxylin counterstain, x 480). Immunostaining with anti-HSV serum is more prominent in the cytoplasm than in the nucleus of neurons. Mononuclear inflammatory cells occur in the vicinity of infected, degenerating neurons (arrows).
Fig. 4. Trigeminal ganglion sections 4 days after HSV inoculation by the corneal route (PAP, hematoxylin counterstain). A: Hypertrophic immunostained satellite cells surrounding an unstained neuron ( x 24ff). B: Cluster of hypertrophic infected satellite cells forming ring-like arrays ( × 550),
73
Fig. 5. Trigeminal ganglion section 10 days after HSV inoculation by the corneal route (PAP, hematoxylin counterstain, x 480). There are mononuclear inflammatory cells in the medial portion of the ganglion, however immunostaining was negative in all cells.
to a heavily stained neuron at one part of the link. One possible explanation for this pattern is that replicated virus in neurons may further spread to neighboring satellite cells. Extracellular spread of virus in ganglion also occurs since some PAP-stained cells appear at distant foci in ganglia. Extracellular spread is also suggested by the observation that ganglion cells capable of reactivating HSV in vitro are not confined to one portion of the trigeminal ganglia but present throughout (Seldzawa, unpublished). Evidence from many laboratories indicate that neurons and not satellite cells harbor the latent HSV genome (McLennan and Darby 1980; Cook et al. 1974; Galloway et al. 1979; Tenser et al. 1982). In the present study, at 8 clays after virus inoculation, HSV antigens were still present in some neurons but no longer present in satellite cells. The PAP staining at this stage was much less intense and the labeled neurons were morphologically normal. The results suggest that an infection productive of HSV antigens in a neuron can be modulated without cell lysis of the neuron, i.e. that neurons can be switched from an HSV-permissive to a non-permissive state (Openshaw et al. 1981). It is probable that these surviving neurons are the ones that harbor HSV DNA during latency.
74 ACKNOWLEDGEMENT
We thank Dr. A. L. Notkins for his generous gift of antiserum against HSV, Prof. Y. Iwasaki and Mrs. M. Ohara for their critical review of the manuscript, and Miss N. Itoh for editorial assistance.
REFERENCES Arvidsson, J. (1975) Location of cat trigeminal ganglion cells innervating dental pulp of upper and lower canines studied by retrograde transport of horseradish peroxidase, Brain Res., 99: 135-139. Baringer, J. R. and P. Sowveland (1973) Recovery of herpes-simplex virus from human trigeminal ganglions, N. Engl. J. Med., 288: 648-650. Bastian, F. O., A. S. Rabson, C. L. Yee and T. S. Tralka (1972) Herpes virus hominis - - Isolation from human trigeminal ganglion, Science, 178: 306-307. Cook, M. L. and J. G. Stevens (1973) Pathogenesis of herpetic neuritis and ganglionitis in mice - - Evidence for intra-axonal transport of infection, Infect. lmmun., 7: 272-288. Cook, M.L., V.B. Bastone and J.G. Stevens (1974) Evidence that neurons harbor latent herpes simplex virus, Infect. Immun., 9: 946-951. Esiri, M. M. (1982) Herpes simplex encephalitis - - An immunohistological study of the distribution of viral antigen within the brain, J. Neurol. Sci., 54: 209-226. Galloway, D. A., C. Fenoglio, M. Shevchuk and J.K. MeDougall (1979) Detection of herpes simplex RNA in human sensory ganglia, Virology, 95: 265-268. Green, M.T., R.J. Courteney and E.C. Dunkel (1981) Detection of an immediate early herpes simplex virus type 1 polypeptide in trigeminal ganglia from latentty infected animals, Infect. Immun., 34: 987-992. Itoyama, Y., N.H. Sternberger, M.W. Kies, S.R. Cohen, E.P. Richardson, Jr. and H. deF. Webster (1980) Immunocytochemical method to identify myelin basic protein in oligodendroglia and myelin sheaths of the human nervous system, Ann. NeuroL, 7: 157-166. Itoyama, Y., H. deF. Webster, N. H. Sternberger, E.P. Richardson, Jr., D. L. Walker, R. H. Quarles and B. L. Padgett (1982) Distribution of papovavirus, myelin-associated glyeoprotein, and myelin basic protein in progressive multifocal leukoencephalopathy lesions, Ann. Neurol., 11: 396-407. Kristensson, K., E. Lycke and J. Sj6strand (1971 ) Spread of herpes simplex virus in peripheral nerves, A cta Neuropath. (Berl.), 17: 44-53. Kristensson, K., A. Vahlne, L. A. Persson and E. Lycke (1978) Neural spread of herpes simplex virus type 1 and 2 in mice al~er corneal or subcutaneous (footpad) inoculation, J. Neurol. Sci., 35: 331-340. Kristensson, K., B. Svennerholm, L. Persson, A. Vahlne and E. Lycke (1979) Latent herpes simplex virus trigeminal ganglionic infection in mice and demyelination in the central nervous system, J. Neurol. Sci., 43: 253-264. Kristensson, K., I. Nennesmo, L. Persson and E. Lycke (1982) Neuron to neuron transmission of herpes simplex virus --Transport of virus from skin to brainstem nuclei, J. Neurol. Sci., 54: 149-156. Kumanishi, R. and A. Hirano (1978) An immunoperoxidase study of herpes simplex virus encephalitis. J. Neuropath. Exp. Neurol., 37: 790-795. McLennan, J. L. and G. Darby (1980) Herpes simplex latency - - The cellular location of virus in dorsal root ganglia and the fate of the infected cells following virus activation, J. Gen. Virol., 51 : 233-243. Openshaw, H., L.V.S. Asher, C. Wohlenberg, T. Sekizawa and A.L. Notkins (1979) Acute and latent infection of sensory ganglia with herpes simplex virus - - Immune control and virus reactivation, J. Gen. Virol., 44: 205-215. Openshaw, H., T. Sekizawa, C. Wohlenberg and A. L. Notkins (1981) Latency and reactivation of HSV - Role of immunity. In: A.J. Nahmias, W. R. Dowdle and R. F. Schinazi (Eds.), The Human Herpesviruses An Interdisciplinary Perspective, Elsevier/North-HoUand, New York, pp. 289-296. Sekizawa, T., H. Openshaw, C. Wohle-aberg and A.L. Notkins (1980) Latency of herpes simplex virus in absence of neutralizing antibody - - Model for reactivation, Science, 210: 1026-1028. Sternberger, L.A., P.H. Hardy, Jr., J.J. Cuculis and H.G. Mayer (1970) The unlabeled antibody enzyme method ofimmunohistochemistry - - Preparation and properties of soluble aatigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirocheters. J. Histochern. Cytochem., 18:315-333.
75 Sternberger, N. H., Y. Itoyama, M.W. Kies and H. deF. Webster (1978) Immunocytochemical method to identify basic protein in myelin-forming oligodendrocytes of newborn rat CNS, J. Neurocytol., 7: 251-263. Stevens, J.G. and M.L. Cook (1971) Latent herpes simplex virus in spinal ganglia of mice, Science, 173:843-845. Tenser, R. B., M. Dawson, S.J. Ressel and M. E. Dunstan (1982) Detection of herpes simplex virus mRNA in latently infected trigeminal ganglion neurons by in situ hybridization, Ann. Neurol., 11: 285-291. Tullo, A.B., C. Shimeld, W.A. Blyth, T.J. Hill and D.L. Easty (1982) Spread of virus and distribution of latent infection following ocular herpes simplex in the non-immune and immune mouse, J. Gen. Virol., 63: 95-101. Walz, M.A., R.W. Price and A.L. Notkins (1974) Latent ganglionic infection with herpes simplex virus type 1 and 2 - - Viral reactivation in vivo aRer neurectomy, Science, 184 : 1185-1187. Warren, K. G., D. H. Gilden, S.M. Brown, M. Devlin, Z. Wroblewska, J. Subak-Sharpe and H. Koprowski (1977) Isolation of herpes simplex virus from human tfigeminal ganglia, including ganglia from one patient with multiple sclerosis, Lancet, ii: 637-639.
67
Immunocytochemical Localization of Herpes Simplex Virus Antigen in the Trigeminal Ganglia of Experimentally Infected Mice Yasuto Itoyama ~, Tsuyoshi Sekizawa 2, Harry Openshaw 3, Kyuya Kogure 2 and Yoshigoro Kuroiwa 1 ~Department of Neurology, Kyushu University, Fukuoka 812 (Japan); 2Department of Neurology, School of Medicine, Tohoku University,Sendai 980 (Japan); and 3Departmentof Neurology, City of Hope National Medical Center, Duarte. CA 91010 (U.S.A.) (Received 13 March, 1984) (Revised, received 29 May, 1984) (Accepted 29 May, 1984)
SUMMARY
The peroxidase anti-peroxidase (PAP) technique was used to study the distribution of herpes simplex virus (HSV) antigens in mouse ganglia during the acute infection and the transition into the latent infection. At 2 days after HSV inoculation by the corneal route, immunoperoxidase staining was present in occasional isolated neurons of the trigeminal ganglion and also in scattered satellite cells. By 4 days, more cells were stained with the infection centered in the medial portion of the ganglion. Inflammatory cells were present around PAP-labeled fragments from lysed cells. Stained satellite cells often with a hypertrophic appearance surrounded labeled or unlabeled neurons in a ring-like array. At 6 days after HSV inoculation, there was a decrease both in the number of cells stained and in the intensity of staining. By 8 days, HSV antigens could be detected by weak PAP staining only in neurons. Otherwise, these neurons appeared morphologically normal. No irnmunoperoxidase staining was present after the 8th day. These results are compatible with retrograde axoplasmic transport of HSV and cell to cell spread of virus in ganglia. Also the appearance of infected ganglion cells during the transition to latency suggests that neurons can be switched from an HSV-permissive to a non-permissive (latent) state.
Correspondence and reprint requests to: Yasuto Itoyama, Department of Neurology, Neurological Institute, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka 812, Japan, Tel. (092)641-1151
0022-510X/84/$03.00 © 1984 Elsevier Science Publishers B.V.
68 Key words: Herpes simplex virus - Herpetic infection - Immunocytochemical observation - Latent infection o f H S V - Trigeminal ganglia
INTRODUCTION
Herpes simplex virus (HSV) is present in a latent form in the trigeminal ganglion of man (Bastian etal. 1972; Baringer and Sowveland 1973; Warren etal, 1977). Herpetic recurrences follow HSV reactivation in ganglia, migration of virions down axons to the skin surface, and viral replication in epithelial cells. Details of the pathogenesis of ganglionic infection has been derived mostly from work in animal models (Stevens and Cook 1971; Walz et al. 1974; Kristensson et al. 1979; Openshaw et al. 1979; Sekizawa et al. 1980; McLennan and Darby 1980). In the experimental system, HSV is usually inoculated on the skin surface; and the course of acute, latent, and reactivated infection is studied in the corresponding sensory ganglion. A critical question in this system concerns the mechanism whereby latency is established during the transition from the acute to the latent state of the infection. This transition occurs 12-14 days after viral inoculation of an immunocompetent animal. Prior to the transition, infectious virus can be detected in cell-free ganglionic homogenates (acute stage of the infection). After 14 days (the latent stage), infectious virus can no longer be detected in homogenates but HSV can be recovered by explantation of ganglia in organ culture (in vitro reactivation). The unlabeled antibody peroxidase anti-peroxidase (PAP) method (Sternberger et al. 1970) is highly sensitive and specific for detection of various viral antigens including HSV in formalin.fixed paraffm-embedded sections ( K u m ~ s h i and Hirano 1978; Esiri 1982; Itoyama et al. 1982; Kristensson et al. 1982). In the present report, we used the PAP method in mouse ganglia to study the time course of the acute HSV infection and the transition from the acute to the latent stage. MATERIALS AND METHODS
Animals and virus inoculation
Twenty-two 6-8 week-old-female Balb/c mice were inoculated with the F strain of HSV type 1. Mice were infected by placing one drop of stock HSV containing 108 plaque forming units/ml on both corneas and scratching the epithelium with a I9-gauge needle (Openshaw et al. 1979). The inoculated mice were killed 2, 4, 6, 8, 10 and 14 days after virus inoculation. They were anesthetized with ether and peffused for 10 rain via the heart with a mixture of mercuric chloride and formalin (Sternberger et al. 1978). The trigeminal roots and ganglia were carefully removed, dissected, postfLxed overnight in the same fixative, dehydrated in a graded series of ethanol and p-dioxane, and embedded in paraffin.
69
lmmunoperoxidase staining A modification (Itoyama et al. 1980) of the peroxidase anti-peroxidase (PAP) method (Sternberger et al. 1970) was used with anti-HSV rabbit serum as the primary antibody. Sections 6 #m thick were cut and mounted serially on numbered glass slides to study the ganglion in three dimensions. Sections were deparaffmized by serial immersions in xylene, a xylene-ethanol mixture, absolute ethanol, and 95 ~o ethanol. For PAP staining, sections were treated sequentially with 3 ~/o normal sheep serum, rabbit anti-HSV serum diluted 1:500, sheep anti-rabbit IgG diluted 1:40, rabbit PAP diluted 1:80, and 3,3'-diaminobenzidine HC1 with hydrogen peroxide. All dilutions were made with 0.5 M Tris buffer. The final steps included counterstaining with hematoxylin, immersion in osmium tetroxide, dehydration, and the addition of cover slips. Anti-HSV antibody (500 neutralizing units) was obtained from a New Zealand white rabbit after multiple intravenous inoculations of 108 plaque-forming units of H SV (Openshaw et al. 1979). One neutralizing unit of antibody represents the reciprocal of the highest 2-fold serum dilution in 0.2 ml which produces a 50~ reduction of HSV plaques. Controls included trigeminal ganglion sections from uninfected mice incubated with the same solutions and also sections from infected mice incubated with nonimmune rabbit serum. Both of these controls were invariably negative for immunoperoxidase staining. RESULTS
The time course of the infection was studied by immunoperoxidase staining of HSV antigens in trigeminal ganglion tissue section (Fig. 1). Two days after HSV inoculation by the corneal route, there was PAP staining of only occasional neurons and satellite cells (Fig. 1A). The stained cells were morphologically normal and were confined to the medial portion of the ganglion. PAP staining was intense but positive cells occurred usually in only one of several serial sections. At this stage, there was no inflammation and no PAP staining of Schwann cells, myelinated fibers, or endothelial cells. At 4 days after HSV inoculation (Figs. 1B, 2-4), the number of PAP-positive cells was greatly increased with the infection centered in the medial portion of the ganglion (Fig. 2). Immunoperoxidase staining occurred in both large and small neurons, in satellite cells, and in occasional Schwann cells and myelinated fibers. Some heavily stained fibers were degenerated to form small stained fragments, and mononuclear inflammatory cells infiltrated around these fragments (Fig. 3). In most stained neurons, HSV reactive products Were preferentially found in the cytoplasm, and in some PAPpositive neurons the nuclei were not stained. Heavily stained and hypertrophic satellite cells surrounded some unstained neurons in a ring-like pattern (Fig. 4A). The stained satellite cells showing this pattern usually occurred in clusters (Fig. 4B) forming a link offing-like cells with contact to a heavily stained neuron at one part of the link (Fig. 1B). By 6 days after HSV inoculation, there was a decrease in both the intensity of PAP staining and in the number of cells stained (Fig. 1C). Most of PAP-positive cells
70
A
Fig. 1. Time course of the acute HSV infection in trigeminal ganglia. Paraffin sections were immunostained with antisera to H SV, counterstained with hematoxylin, and photographed at a final magnification of × 240. A: At 2 days after HSV inoculation by the corneal route, an isolated neuron stained intensely with PAP. B: At 4 days, more cells are labeled. Satellite cells containing HSV antigens form a ring-like array around neurons and make contact with a heavily stained neuron (arrow). C: At 6 days, the PAP staining is less intense and most of the labeled cells are neurons. D: At 8 days, only neurons are labeled (arrow). They appear morphologically normal with faint peroxidase staining. at this stage were neurons. There was a further d e c r e a s e in the intensity a n d the n u m b e r o f stained cells at 8 d a y s after H S V inoculation (Fig. 1D). By this time, P A P - p o s i t i v e cells were exclusively neurons. W e a k l y positive granular staining was present in the nuclei or c y t o p l a s m b u t otherwise the n e u r o n s a p p e a r e d to b e morphologically normal. I m m u n o p e r o x i d a s e staining was negative at d a y 10 a n d 14 after H S V inoculation. M o n o n u c l e a r inflammatory ceils persisted in the medial p o r t i o n o f the ganglion as a residual o f the acute infection (Fig. 5). DISCUSSION In c o m p a r i s o n to immunofluorescence, the P A P technique shows higher sensitivity a n d specificity, gives a relative p e r m a n e n t r e c o r d o f the results, a n d provides superior histological detail.
71
i ¸ ¸¸¸I¸¸¸¸!¸~¸
Fig. 2. Trigeminal ganglion section 4 days after HSV inoculation by the corneal route (PAP, hematoxylin counterstain, x 60). A number of stained cells were confined to the medial portion of the ganglion (left side of the ganglion in this picture corresponds to the medial part of the ganglion).
With this technique, we could detect HSV antigens in trigeminal ganglia up to 8 days after viral inoculation of mice. A comparable study with indirect immunofluorescence reported a 6-day limit of detection in mouse ganglia (Cook and Stevens 1973). In contrast, Green et al. (1981) using a rabbit model demonstrated HSV antigens into the latent stage of the infection. In their study, indirect immunofluorescence was used in ganglion sections with the primary antisera directed against the immediate early HSV polypeptide VP 175. In the present study, cells bearing HSV antigens were clustered in the medial aspect of the ganglia. This finding is compatible with retrograde axoplasmic transport of HSV (Cook and Stevens 1973; K_ristensson et al. 1971; Tullo et al. 1982) since the medial portion of the ganglion contains those neurons that correspond to nerve terminals on the cornea (Arvidsson 1975; Kristensson et al. 1978). By day 4 after virus inoculation, a productive H SV infection was present with lysis of some neurons and recruitment of inflammatory cells. Satellite cells beating HSV antigens appeared in a ring-like array around infected and apparently unifected neurons. These arrays usually occurred in clusters forming a link of ring-like cells with contact
72
Fig. 3. Trigeminal ganglion section 4 days after HSV inoculation by the corneal route (PAP, hematoxylin counterstain, x 480). Immunostaining with anti-HSV serum is more prominent in the cytoplasm than in the nucleus of neurons. Mononuclear inflammatory cells occur in the vicinity of infected, degenerating neurons (arrows).
Fig. 4. Trigeminal ganglion sections 4 days after HSV inoculation by the corneal route (PAP, hematoxylin counterstain). A: Hypertrophic immunostained satellite cells surrounding an unstained neuron ( x 24ff). B: Cluster of hypertrophic infected satellite cells forming ring-like arrays ( × 550),
73
Fig. 5. Trigeminal ganglion section 10 days after HSV inoculation by the corneal route (PAP, hematoxylin counterstain, x 480). There are mononuclear inflammatory cells in the medial portion of the ganglion, however immunostaining was negative in all cells.
to a heavily stained neuron at one part of the link. One possible explanation for this pattern is that replicated virus in neurons may further spread to neighboring satellite cells. Extracellular spread of virus in ganglion also occurs since some PAP-stained cells appear at distant foci in ganglia. Extracellular spread is also suggested by the observation that ganglion cells capable of reactivating HSV in vitro are not confined to one portion of the trigeminal ganglia but present throughout (Seldzawa, unpublished). Evidence from many laboratories indicate that neurons and not satellite cells harbor the latent HSV genome (McLennan and Darby 1980; Cook et al. 1974; Galloway et al. 1979; Tenser et al. 1982). In the present study, at 8 clays after virus inoculation, HSV antigens were still present in some neurons but no longer present in satellite cells. The PAP staining at this stage was much less intense and the labeled neurons were morphologically normal. The results suggest that an infection productive of HSV antigens in a neuron can be modulated without cell lysis of the neuron, i.e. that neurons can be switched from an HSV-permissive to a non-permissive state (Openshaw et al. 1981). It is probable that these surviving neurons are the ones that harbor HSV DNA during latency.
74 ACKNOWLEDGEMENT
We thank Dr. A. L. Notkins for his generous gift of antiserum against HSV, Prof. Y. Iwasaki and Mrs. M. Ohara for their critical review of the manuscript, and Miss N. Itoh for editorial assistance.
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