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Demyelination Associated with HSV-1-Induced Facial Paralysis
Experimental Neurology 178, 68 –79 (2002) doi:10.1006/exnr.2002.8035
Demyelination Associated with HSV-1-Induced Facial Paralysis 1 Hiroyuki Wakisaka,* Naohito Hato,† Nobumitsu Honda,† Hirotaka Takahashi,† Hisanobu Kisaki,† Shingo Murakami,‡ Kiyofumi Gyo,† Katsumi Mominoki,* Naoto Kobayashi,* and Seiji Matsuda* *Department of Anatomy and †Department of Otolaryngology, Ehime University School of Medicine, Ehime 791-0925, Japan; and ‡Department of Otolaryngology, Nagoya City University School of Medicine, Nagoya, Japan Received December 17, 2001; accepted July 18, 2002
trigeminal nerve (7, 10, 18, 19). These reports suggested that HSV-1 infection in the animal trigeminal nerve causes nerve degeneration, not only in the ganglia, but also in the central part of the trigeminal root (root entry zone) in the brain stem. In contrast, HSV-1 involvement in motor nerves and central nervous system (CNS) motor centers was neglected until Openshaw and William (13) reported on experimental HSV-1 infection in the hypoglossal nerve in 1983. They inoculated HSV-1 into the tongue muscle of mice and demonstrated HSV-1 capsids in astrocytes in the central part of the hypoglossal root and HSV-1 antigen in the hypoglossal nucleus. These findings indicated that HSV-1 can produce focal brainstem encephalitis in motor nerves, as well as HSV-1 infection in the trigeminal nerve. However, since paralysis of tongue movement did not develop in that model, they did not report a relationship between HSV-1 infection and the pathogenesis of motor nerve paralysis. In 1995, we developed the first animal model of transient homolateral facial nerve paralysis (Fig. 1) by inoculating HSV-1 into the auricle (15). In this model, facial paralysis involving loss of eye closure or loss of nose movement was observed 7 to 14 days after inoculation in about 70% of mice. This facial nerve paralysis model is well suited to understanding the role of HSV-1 infection in the pathogenesis of motor nerve dysfunction. Therefore, the present study investigated this mouse model histopathologically to clarify the mechanism of facial nerve paralysis induced by HSV-1.
In 1995, we developed an animal model of transient homolateral facial nerve paralysis by inoculating Herpes simplex virus type 1 (HSV-1) into the auricle of mice. This study examined the mechanism of facial nerve paralysis in this model histopathologically. Using the immunofluorescence technique with antiHSV-1 antibody, the time course of viral spread and the site of viral replication were investigated over the entire course of the facial nerve. Furthermore, viral replication and nerve degeneration at the site of viral replication were observed by electron microscopy. On the 7th day after inoculation, facial paralysis was observed in more than 60% of mice. Immunofluorescence study revealed HSV-1 in the geniculate ganglion, the descending root, and the facial nucleus at this stage. On the 9th day, the descending root in the sections stained with osmium looked pale, because prominent demyelination had occurred in this region; electron micrographs showed many degenerated oligodendrocytes and large naked axons. In contrast, the facial nucleus neurons showed no remarkable degeneration, despite HSV-1 particles in their cytoplasm. From these findings, we concluded that facial nerve paralysis in this model is caused mainly by facial nerve demyelination in the descending root. © 2002 Elsevier Science (USA) Key Words: Herpes simplex virus type 1; demyelination; facial nerve; motor nerve dysfunction; Bell’s palsy.
INTRODUCTION
MATERIALS AND METHODS
Herpes simplex virus type 1 (HSV-1) causes several neuronal diseases. It is commonly known to spread in sensory axons and to infect sensory neurons in the ganglia of the peripheral nervous system (4, 7, 14, 21). Therefore, in order to understand the mechanism of HSV-1 infection, many investigators have conducted animal experiments using sensory nerves, such as the
Virus and Animals It is well known that the neuropathogenicity of HSV varies among virus strains. The KOS strain is commonly used in experiments and has moderate neuropathogenicity. Blyth et al. (3) inoculated 10 5 pfu of strains SC16 and KOS into the ears of mice following Hill’s method (5). They reported that the incidence of herpetic eruptions in both strains was essentially the same, while the mortality rate with SC16 was much
1 This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, of Japan (11770996).
0014-4886/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
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higher than with KOS; half of the mice inoculated with SC16 died. The characteristics of KOS make it suitable for producing an animal model of acute, transient facial paralysis. Then, the KOS strain of HSV-1 was prepared in Vero cells and plaque titrated at 6.7 ⫻ 10 7 pfu/ml. Four-week-old, female, specific-pathogen-free BALB/cAJcl mice were purchased from Clea Japan, Inc. (Tokyo, Japan). All animals were cared for in our laboratory animal center, and studies were carried out in compliance with the Guide for Animal Experimentation of Ehime University School of Medicine. Virus Inoculation and Evaluation of Facial Nerve Paralysis Following anesthesia by intraperitoneal injection of sodium pentobarbital (50 mg/kg), virus was inoculated into the posterior aspect of the auricle on day 0. The surface of the auricle was scratched 20 times with a 27-gauge needle before HSV-1 inoculation, which was a modification of Hill’s method (5). Twenty-five microliters of virus solution (1.7 ⫻ 10 6 pfu) were placed on the right auricle. After virus inoculation, blink reflex and vibrissae movements were carefully observed daily, so as to detect and evaluate facial nerve paralysis. The blink reflex was elicited by blowing air into the eye using a 5-ml syringe with an 18-gauge needle. Immunofluorescence From the 1st to the 9th day after inoculation, 54 mice (6 mice for each day; from the 7th to the 9th day after inoculation, mice with facial paralysis were used) were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg/kg) and perfused transcardially, first with 100 ml PBS and then with 500 ml 4% formaldehyde in 0.1 M phosphate buffer (pH 7.4). The brain stem, including the descending root of the facial nerve, the facial nucleus, and the bilateral facial nerves including the geniculate ganglion, were dissected, and 40-m slices were prepared using a microslicer. These sections were stained with anti-gC (HSV-1 envelope glycoprotein C) monoclonal antibody labeled with FITC (Syva Co.) and then observed under fluorescent microscopy. Electron Microscopy On the 3rd, 4th, 5th, 6th, 9th, 14th, and 18th days after inoculation, 21 mice (3 mice for each day; on the 14 and the 18 days after inoculation, mice which had a almost complete recovery of facial paralysis were used) were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg/kg) and perfused transcardially, first with 100 ml PBS and then with 500 ml 4% formaldehyde and 4% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4).
FIG. 1. A mouse with facial nerve paralysis in the right side. The right eye cannot be closed and the nose is shifted to the left. This paralysis was observed from the 7th to 14th days after virus inoculation.
The brainstem and bilateral facial nerves were dissected and 70-m slices were cut with a microslicer. These specimens were postfixed with 2% osmium tetroxide, dehydrated, and embedded in epoxy resin. Ultrathin sections were prepared from specimens of the facial nerve, including the geniculate ganglion, the descending root, and the facial nucleus in the brain stem. These sections were stained with uranyl acetate and lead citrate and then observed under electron microscopy (Hitachi H-800, Japan). RESULTS
One hundred twelve mice were inoculated with HSV-1, and then 6 mice for each day were used for immunofluorescence. On the second day after inoculation, erosion or ulceration of the skin was observed in all animals at the site of inoculation. On the third day, 7 mice became much less vigorous; their bodies were curled up and their fur stood erect. They also showed palsy of the extremities and trembling of the body, suggesting the presence of encephalitis. Since they died on the 4th day without developing facial paralysis, these mice were excluded from the study. On the 7th day, facial paralysis (Fig. 1) was observed in 37 of 57 mice (65%). Ten mice with facial paralysis were ob-
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FIG. 2. Immunofluorescence over the entire course of the facial nerve with anti-gC monoclonal antibody labeled with FITC. a: Scheme showing the geniculate ganglion, the descending root, and the facial nucleus in the course of the facial nerve. b: The geniculate ganglion on the 5th day after inoculation. Many immunopositive cells are observed. c: The descending root of the facial nerve in the brain stem. Many immunopositive cells (arrows) were observed near the root entry zone on the 5th day after inoculation. d: The facial nucleus contained many immunopositive cells on the 5th day after inoculation. e: The time course of immunopositive cell appearance. Immunopositive cells appeared in the geniculate ganglion by the 3rd day, in the descending root by the 4th day, and in the facial nucleus by the 5th day after inoculation.
served to evaluate the natural course of the palsy. All of them showed almost complete recovery by the 14th day. Immunofluorescence The entire course of the facial nerve was examined under fluorescent microscopy, from the periphery to the facial nucleus. HSV-1 immunopositive cells were observed only in the geniculate ganglion (Fig. 2a), the descending root (Fig. 2b), and the facial nucleus (Fig.
2c). Figure 2 shows representative immunofluorescence in these areas and the data obtained from all the animals. Immunoreactivity was observed only on the inoculated side. Immunopositive cells were first observed in the geniculate ganglion 3 days after inoculation, in the descending root after 4 days, and in the facial nucleus after 5 days. The number of the immunopositive cells increased after the 5th day, reached a peak on the 7th day, and thereafter decreased abruptly. There were
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few immunopositive cells on the 9th day, and their immunoreactivity was very weak. Electron Microscopy of the Geniculate Ganglion Area On the 3rd or 4th day after inoculation, HSV-1 particles were observed in some neurons, which had a well-preserved, fine structure. Replicated HSV-1 particles were frequently observed, beginning on the 4th day (Fig. 3a). These replicated virus particles were observed in the cytoplasm of neurons (Fig. 3b) and, rarely, in Schwann cells. Nerve degeneration, such as demyelination or destruction of axon (axonotmesis), induced by viral infection was not observed in either the sensory or the motor nerve area in the first 4 days. However, demyelinated axons were observed from the 5th day (Fig. 3c), when viral particles were observed in neurons more frequently. Vacuolar changes and dilatation of the rough endoplasmic reticulum were also observed in these infected neurons. On the 9th day, HSV-1-infected neurons continued to produce viral particles, which infected satellite and Schwann cells surrounding the neurons (Figs. 3d and 3e). Demyelination was observed in the facial motor nerve in the geniculate ganglion area (Fig. 3f), where some axonotmesis was also observed. Some neurons underwent lysis, releasing cellular debris. In contrast, the contralateral geniculate ganglion neurons and facial motor nerve had a well-preserved, fine structure on the 9th day (Fig. 3g). Electron Microscopy of the Descending Root of the Facial Nerve in the Brain Stem On the 3rd day after inoculation, no HSV-1 particles were observed in astrocytes, and oligodendrocytes had a well-preserved, fine structure. On the 5th day, the astrocytes forming the descending root were heavily infected, while infection was not observed in other glial cells (Fig. 4a), and numerous replicated virus particles were found in astrocyte nuclei (Fig. 4b). However, these infected astrocytes vanished from the descending root on the 6th day and much cell debris was observed instead. In contrast, HSV-1 replicated in many oligodendrocytes, and demyelination was observed with these infected oligodendrocytes (Fig. 4c). On the 9th day, nerve degeneration became prominent on the inoculated side and the descending root could no longer be observed (Figs. 4d and 4e). An inflammatory infiltrate consisting of lymphocytes and mononuclear cells accompanied the appearance of cell debris and myelin breakdown in the descending root. The process of demyelination was remarkable with many degenerated oligodendrocytes, and there were many demyelinated large axons (Fig. 4f). Some of these axons showed axonotmesis. As expected from the immunofluorescence, no viruses were observed at this stage. On the 14th day, facial movement had essentially recovered to nor-
mal in all the paralyzed mice, and the descending root could be observed once again (Fig. 4g). On the same day, although some degeneration was still seen in the cytoplasm of oligodendrocytes (Fig. 4h), many of the large axons were remyelinated, although myelin on these axons appeared thinner than normal (Fig. 4i). A few axons were naked in places. On the 18th day, the descending root could be observed clearly (Fig. 4j). Electron Microscopy of the Facial Nucleus Until the 5th day after inoculation, no neurons with HSV-1 particles were observed. From the 6th to the 9th days after inoculation, the facial nucleus was well preserved (Figs. 5a and 5b). Although HSV-1 particles were observed in some neurons, these infected neurons had a well-preserved, fine structure (Figs. 5c and 5d). Even on the 9th day, HSV-1 infection was restricted to neurons; infected glial cells or degenerated nerves were not seen in the facial nucleus. None of these infected neurons underwent lysis. DISCUSSION
In our model, using polymerase chain reaction (PCR) analysis of HSV-1 DNA, we traced the migration route of the virus at the intratemporal facial nerve including geniculate ganglion and the homolateral brain stem on the 3rd and 10th days after inoculation (11). We detected HSV-1 DNA in the intratemporal facial nerve on the 3rd day and in both the intratemporal facial nerve and the brain stem on the 10th day. However, in that paper we were unable to demonstrate the detailed time course of viral spread throughout the facial nerve or the site of viral replication, because of the limitations of the PCR technique, so the role of HSV-1 infection in the pathogenesis of facial paralysis remained unclear. Therefore, we conducted the present histopathological study, which is the first to demonstrate the relationship between viral replication and nerve degeneration in the mechanism of facial paralysis. The facial nerve is composed of the facial motor nerve and the sensory nerve with the geniculate ganglion. These two fibers run together from the periphery to the end of the descending root. The geniculate ganglion neurons are located in the temporal bone, and the facial nucleus motor neurons are located in the brain stem (Fig. 2a). In this study, HSV-1 began to replicate in geniculate ganglion neurons on the 3rd or 4th day after inoculation. How does HSV-1 reach the geniculate ganglion from the auricle? Many studies have reported that the main transport of virus from the periphery is intraaxonal transport (1, 2, 8, 10, 13). Therefore, we tried to find HSV-1 particles in the axons of sensory and motor nerves around the geniculate ganglion on the 1st or 2nd day. However, we failed to find HSV-1 particles in
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FIG. 3. Electronmicrographs of the geniculate ganglion. a: Neuron with numerous virus capsids in the rough endoplasmic reticulum. This neuron has a well-preserved, fine structure. Arrows indicate the area in which virus particles were located (4th day after inoculation, scale bar: 10 m). b: HSV-1 particles in the rough endoplasmic reticulum (high magnification of the square area in a, 4th day after inoculation, scale bar: 1 m). c: Naked axons (arrows) are observed in some nerve fibers. Some Schwann cells have vacuolar changes in the cytoplasm (arrowheads) (5th day after inoculation, scale bar: 5 m). d: Electronmicrograph showing HSV-1 infected neuron, satellite, and Schwann cells. Virus particles in the neuron are observed in the rough endoplasmic reticulum. Vacuolar changes (arrowheads), destruction of mitochondria, and dilatation of the rough endoplasmic reticulum are observed in the cytoplasm of another neuron. Arrows indicate HSV1 particles (scale bar: 5 m). e: These virus particles were uniform and approximately 150 nm in diameter (high magnification of the square area in d, 9th day after inoculation, scale bar: 1 m). f: Facial motor nerve area on the 9th day after inoculation. Many Schwann cells have intracytoplasmic vacuolar changes, and many axons show demyelination (A). Despite more severe demyelination, the fine structures of the axons themselves are relatively well preserved. Some axonotmesis (B) was also observed (scale bar: 1 m). g: The facial motor nerve area on the contralateral side on the 9th day after inoculation. The fine structures and myelin of Schwann cells are preserved (scale bar: 1 m).
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FIG. 3—Continued
these axons. On the other hand, we first detected HSV-1 particles in geniculate ganglion neurons, not in nonneuronal cells. IF HSV-1 particles were transported from one Schwann cell to another or via extracellular fluid, then with viral infection viral particles or cell damage would first be observed in the nonneuronal cells. Therefore, we conclude that HSV-1 transport in
this model is intra-axonal, although no virus was detected in the axons. Furthermore, we think that this intra-axonal transport occurs not only in the sensory nerve, but also in the motor nerve. Since HSV-1 particles were first detected in facial nucleus motor neurons, not in nonneuronal cells, as observed in the geniculate ganglion, we conclude that HSV-1 inoculated in
FIG. 4. Electron and light micrographs of the descending root. a: Heavily infected astrocyte with numerous virus capsids in the nucleus (5th day after inoculation, scale bar: 5 m). b: HSV-1 particles in the astrocyte (square area in a). These viruses had capsids and tegments. They were approximately 150 nm in diameter (scale bar: 0.5 m). c: Infected oligodendrocyte in the descending root on the 6th day after inoculation. It contains numerous virus capsids in its nucleus. Two axons show demyelination (arrows) (scale bar: 5 m). d: Light micrograph of the brain stem stained with osmium on the 9th day after inoculation. A few fibers in the descending root of the facial nerve on the inoculated side can be observed. e: High magnification of the square area in d. Some of the descending root can be observed (9th day after inoculation). f: Large demyelinated axons and oligodendrocytes (Ol) with some myelin-like bodies are observed. A mononuclear cell (*) infiltrates near the damaged oligodendrocyte. One axon shows demyelination (arrows) (9th day after inoculation, scale bar: 5 m). g: Light micrograph of the brainstem stained with osmium 14 days after inoculation. The descending root of the facial nerve can be observed on the inoculated sides. h: Although a few myelin-like bodies (arrows) are observed in oligodendrocytes (Ol), the axons beside the oligodendrocytes are myelinated (14 days after inoculation, scale bar: 5 m). i: There are many large myelinated axons; their myelin sheaths appear thin and there are bare sections on some axons (arrows). j: Light micrograph of the brain stem stained with osmium 18 days after inoculation. The descending roots of the facial nerve appear similar on both sides. 74
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FIG. 4—Continued
the auricle ascends from the auricle in both sensory and motor axons. However, the possibility that the virus released from the dead sensory neuron was taken by motor axons at the geniculate ganglion cannot be excluded. In this study, both demyelination and axonotmesis were observed in facial motor nerves and in sensory nerves in the geniculate ganglion area. How does this nerve degeneration occur in facial motor fibers? The demyelination in the geniculate ganglion can be simply explained. Under electron microscopy, remarkable replication of HSV-1 was seen in the geniculate ganglion neurons. Furthermore, some neurons were lysed and released cellular debris and HSV-1 to the extracellular fluid space. Some of these HSV-1 spread directly to neighboring satellite and Schwann cells (Fig. 3d). Fi-
nally, these infected Schwann cells showed vacuolar changes and demyelination (Fig. 3f). On the other hand, axonotmesis, which means severe axonal damage or neuronal lysis, cannot be explained only by the phenomenon that occurred in the geniculate ganglion. While sensory nerve axonotmesis can be explained by the geniculate ganglion neuronal lysis, in considering the motor nerve axonotmesis we must consider the phenomena seen in the descending root and facial nucleus. In the facial nucleus, no degenerated neurons were observed, despite HSV-1 infection (Figs. 5b and 5c). Furthermore, no infected glial cells or degenerated nerves were seen in the facial nucleus. Therefore, the main reason for the motor nerve axonotmesis is the severe damage to the descending root, not the degeneration of the facial nucleus neurons (Figs. 4d– 4g).
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FIG. 4—Continued
Under electron microscopy, marked demyelination and some destroyed axons were observed in the descending root, and this is likely the cause of the motor nerve axonotmesis in the geniculate ganglion area. There are two possible reasons why the nerve degen-
eration in this area is so severe compared with that in other peripheral areas, including the geniculate ganglion. The first is the structural difference between the myelin-forming cells in the peripheral nervous system (PNS) and those in the CNS. Schwann cells make my-
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FIG. 5. Electron and light micrographs of the facial nucleus on the 9th day after inoculation. a: Light micrograph of the facial nucleus stained with osmium; there are no abnormalities. b: Light micrograph of the facial nucleus stained with toluidine blue; there are no abnormalities (scale bar: 100 m). c: Electron micrograph of the facial nucleus on the 9th day after inoculation; HSV-1 particles have replicated in the rough endoplasmic reticulum. However, the neuron has a well-preserved, fine structure (scale bar: 5 m) d: High magnification of the square area in c; HSV-1 particles in the rough endoplasmic reticulum were approximately 150 nm in diameter (scale bar: 1 m).
elin sheaths in the PNS, while myelin sheaths in the CNS are made by oligodendrocytes. The same degree of damage in these two types of myelin-forming cells results in much more severe demyelination in the CNS than in the PNS. The second reason is the difference in the “tightness” of Schwann cells and astrocytes. This difference was reported to be the reason that HSV-1 first infects astrocytes, in several studies of HSV-1
infection of the trigeminal nerve (4, 6, 10, 18, 19, 21). In the PNS, the basal lamina of Schwann cells acts as a barrier to axonal virions. In the PNS–CNS junction, however, the foot processes of astrocytes abut directly on axons. Itoyama et al. (6) speculated that an intraaxonal virus may gain entry from these foot processes of astrocytes in the PNS–CNS junction area. We noted that HSV-1 infection occurred in the descending root
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and that this infection led to severe degeneration in this area, which is consistent with their speculation. The demyelination in the descending root can be classified into three stages. In the first stage, lysis of the infected astrocytes is prominent. HSV-1 first infects astrocytes in the descending root on the 5th day (Figs. 4a and 4b). This infection of astrocytes is similar to the HSV-induced entry zone lesions that have been reported in the trigeminal root (4, 6, 10, 18, 19, 21). Some authors (10, 18, 19) have reported that these infected astrocytes produce numerous viral particles and undergo lysis. In our model, almost all of the astrocytes in the descending root were infected with HSV-1 on the 5th day, yet no astrocytes were observed in this region on the 6th day. This is consistent with the report of Itoyama et al. (6), who used immunohistochemistry with glial cell markers. Most of these infected astrocytes likely undergo lysis, releasing cellular debris and numerous viral particles that may trigger the infiltration of mononuclear cells (9, 17, 22). We observed that an inflammatory infiltrate consisting of lymphocytes and mononuclear cells accompanied the appearance of cellular debris on the 6th day. In the second stage, the released viruses infect oligodendrocytes and start to replicate in their nuclei on the 6th day (Fig. 4c). These infected oligodendrocytes begin to show demyelination. At 9 days, there are many large demyelinated axons with many degenerated oligodendrocytes (Fig. 4g). In the third stage, severe degeneration is induced by a secondary immune response (10, 19, 20). Townsend and Baringer (20) concluded that early infection of oligodendrocytes might lead to partial demyelination followed by a macrophage response that causes further demyelination. Furthermore, the infiltrating cells destroy some axons in the descending root. This nerve destruction seems to lead to Wallerian degeneration and cause axonotmesis. In conclusion, the demyelination observed on the 6th day may occur as a direct cytopathic effect of the virus, and the demyelination and axonotmesis noted after the 9th day may result from a secondary immune response. Facial paralysis and degeneration in the descending root begin at almost the same time after inoculation. Moreover, remyelination of the descending root was observed accompanied with facial nerve recovery. These findings indicate that degeneration of the descending root, especially demyelination, is the main cause of facial paralysis in our model. In this study, 65% of the mice developed transient facial paralysis, while 6% of the mice died very quickly in the early stage of infection without developing facial paralysis. The symptoms of the dying mice suggested encephalitis. However, it is still unclear why HSV inoculation into the ear killed only some of the mice, and did so very quickly, while other mice at the same stage did not show pathological degeneration in the facial
nerve or brain stem. To solve this puzzle, it is very important to carry out a pathological study of the dying mice at different stages of their illness. However, collecting dying mice at different stages would be very difficult, because their illness progresses so rapidly. Such a study, while important, is beyond the scope of this paper, as the dead mice never developed facial paralysis. We plan to examine this in a future study. In 1996, we detected HSV-1 genomes in clinical samples of endoneurial fluid of facial nerve that were obtained from Bell’s palsy patients (12). This report strongly suggested that HSV-1 can be the cause of Bell’s palsy and we demonstrated the mechanism of facial nerve paralysis resulting from primary HSV-1 infection in the present study. However, since Bell’s palsy is thought to arise from latent HSV-1 infection in the geniculate ganglion, the mechanism of facial palsy in the present study using primary infection model may not be the same as that in the reactivation process of Bell’s palsy. Recently, we succeeded in reactivating latently infected virus and producing facial nerve paralysis in another animal model (16). Using this model, further investigations are currently underway in our laboratory to clarify whether the mechanism of this palsy is more similar to that of Bell’s palsy. ACKNOWLEDGMENT We thank Professor Naoaki Yanagihara for helpful discussion.
REFERENCES 1.
2.
3.
4.
5.
6.
7.
8.
Bak, J. J., Ch. Markham, M. L. Cook, and J. G. Steavens. 1977. Intra-axonal transport of herpes simplex virus in the rat central nervous system. Brain Res. 136: 415– 429. Bak, J. J., Ch. Markham, M. L. Cook, and J. G. Steavens. 1978. Ultrastructural and immunoperoxidase study of striatonigral neurons by means of retrograde axonal transport of herpes simplex virus. Brain Res. 136: 361–368. Blyth, W. A., D. A. Harbour, and T. J. Hill. 1984. Pathogenesis of Zosteriform spread of herpes simplex virus in the mouse. J. Gen. Virol. 65: 1477–1486. Cook, M. L., and J. G. Stevens. 1973. Pathogenesis of herpetic neuritis and ganglionitis in mice: Evidence for intra-axonal transport of infection. Infect. Immunol. 7: 272–288. Hill, T. J., H. J. Field, and W. A. Blyth. 1975. Acute and recurrent infection with herpes simplex virus in the mouse: A model for studying latency and reccurrent disease. J. Gen. Virol. 28: 341–353. Itoyama, Y., T. Sekizawa, H. Openshaw, K. Kogure, and I. Goto. 1991. Early loss of astrocytes in herpes simplex virus-induced central nervous system demyelination. Ann. Neurol. 29: 285– 292. Itoyama, Y., T. Sekizawa, H. Openshaw, K. Kogure, and Y. Kuroiwa. 1984. Immunocytochemical localization of herpes simplex virus antigen in the trigeminal ganglia of experimentally infected mice. J. Neurol. Sci. 66: 67–75. Kristensson, K., I. Nennesmo, L. Persson, and E. Lycke. 1982. Neuron to neuron transmission of herpes simplex virus. Trans-
FACIAL PARALYSIS INDUCED BY HSV-1
9.
10.
11.
12.
13.
14. 15.
port of virus from skin to brainstem nuclei. J. Neurol. Sci. 54: 149 –156. Kristensson, K., H. Thormar, and H. M. Wisniewski. 1979. Myelin lesions in the rabbit eye model as a by-stander effect of herpes simplex and Visna virus sensitization. Acta Neuropathol. (Berl.) 48: 215–217. Kristensson, K., A. Vahlne, L. A. Persson, and E. Lycke. 1978. Neural spread of herpes simplex virus type 1 and 2 in mice after corneal or subcutaneous (footpad) inoculation. J. Neurol. Sci. 35: 331–340. Murakami, S., N. Hato, M. Mizobuchi, T. Doi, and N. Yanagihara. 1996a. Role of herpes simplex virus infection in the pathogenesis of facial paralysis in mice. Ann. Otol. Rhinol. Laryngol. 105: 49 –53. Murakami, S., M. Mizobuchi, Y. Nakashiro, T. Doi, N. Hato, and N. Yanagihara. 1996b. Bell’s palsy and herpes simplex virus: Identification of viral DNA in endoneural fluid and muscle. Ann. Intern. Med. 124: 27–30. Openshaw, H., and G. E. William. 1983. Herpes simplex virus infection of motor neurons: Hypoglossal model. Infect. Immunol. 42: 409 – 413. Stevens, J. G., and M. L. Cook. 1971. Latent herpes simplex virus in spinal ganglia of mice. Science 173: 843– 845. Sugita, T., S. Murakami, N. Yanagihara, Y. Fujiwara, and Y. Hirata. 1995. Facial nerve paralysis induced by herpes simplex
16.
17.
18.
19.
20.
21.
22.
79
virus in mice, an animal model of acute and transient facial paralysis. Ann. Otol. Rhinol. Laryngol. 104: 574 –581. Takahashi, H., Y. Hitsumoto, N. Honda, N. Hato, M. Mizobuchi, S. Murakami, H. Kisaki, H. Wakisaka, and K. Gyo. 2001. Mouse model of Bell’s palsy induced by reactivation of herpes simplex virus type 1. J. Neuropathol. Exp. Neurol. 60: 621– 627. Townsend, J. J. 1981. The relationship of astrocytes and macrophages to CNS demyelination after experimental herpes simplex virus infection. J. Neuropathol. Exp. Neurol. 40: 369 –379. Townsend, J. J., and J. R. Baringer. 1976. Comparative vulnerability of peripheral and central nervous tissue to herpes simplex virus. J. Neuropathol. Exp. Neurol. 35: 100. Townsend, J. J., and J. R. Baringer. 1978. Central nervous system susceptibility to herpes simplex infection. J. Neuropathol. Exp. Neurol. 37: 255–262. Townsend, J. J., and J. R. Baringer. 1979. Morphology of central nervous system disease in immunosuppressed mice after peripheral herpes simplex virus inoculation. Lab. Invest. 40(2): 178 –182. Townsend, J. J., and P. K. Collins. 1986. Peripheral nervous system demyelination with herpes simplex virus. J. Neuropathol. Exp. Neurol. 45: 419 – 425. Wisniewski, H. M., and B. R. Bloom. 1975. Primary demyelination as a nonspecific consequence of a cell-mediated immune reaction. J. Neuropathol. Exp. Neurol. 141: 346 –359.
Demyelination Associated with HSV-1-Induced Facial Paralysis 1 Hiroyuki Wakisaka,* Naohito Hato,† Nobumitsu Honda,† Hirotaka Takahashi,† Hisanobu Kisaki,† Shingo Murakami,‡ Kiyofumi Gyo,† Katsumi Mominoki,* Naoto Kobayashi,* and Seiji Matsuda* *Department of Anatomy and †Department of Otolaryngology, Ehime University School of Medicine, Ehime 791-0925, Japan; and ‡Department of Otolaryngology, Nagoya City University School of Medicine, Nagoya, Japan Received December 17, 2001; accepted July 18, 2002
trigeminal nerve (7, 10, 18, 19). These reports suggested that HSV-1 infection in the animal trigeminal nerve causes nerve degeneration, not only in the ganglia, but also in the central part of the trigeminal root (root entry zone) in the brain stem. In contrast, HSV-1 involvement in motor nerves and central nervous system (CNS) motor centers was neglected until Openshaw and William (13) reported on experimental HSV-1 infection in the hypoglossal nerve in 1983. They inoculated HSV-1 into the tongue muscle of mice and demonstrated HSV-1 capsids in astrocytes in the central part of the hypoglossal root and HSV-1 antigen in the hypoglossal nucleus. These findings indicated that HSV-1 can produce focal brainstem encephalitis in motor nerves, as well as HSV-1 infection in the trigeminal nerve. However, since paralysis of tongue movement did not develop in that model, they did not report a relationship between HSV-1 infection and the pathogenesis of motor nerve paralysis. In 1995, we developed the first animal model of transient homolateral facial nerve paralysis (Fig. 1) by inoculating HSV-1 into the auricle (15). In this model, facial paralysis involving loss of eye closure or loss of nose movement was observed 7 to 14 days after inoculation in about 70% of mice. This facial nerve paralysis model is well suited to understanding the role of HSV-1 infection in the pathogenesis of motor nerve dysfunction. Therefore, the present study investigated this mouse model histopathologically to clarify the mechanism of facial nerve paralysis induced by HSV-1.
In 1995, we developed an animal model of transient homolateral facial nerve paralysis by inoculating Herpes simplex virus type 1 (HSV-1) into the auricle of mice. This study examined the mechanism of facial nerve paralysis in this model histopathologically. Using the immunofluorescence technique with antiHSV-1 antibody, the time course of viral spread and the site of viral replication were investigated over the entire course of the facial nerve. Furthermore, viral replication and nerve degeneration at the site of viral replication were observed by electron microscopy. On the 7th day after inoculation, facial paralysis was observed in more than 60% of mice. Immunofluorescence study revealed HSV-1 in the geniculate ganglion, the descending root, and the facial nucleus at this stage. On the 9th day, the descending root in the sections stained with osmium looked pale, because prominent demyelination had occurred in this region; electron micrographs showed many degenerated oligodendrocytes and large naked axons. In contrast, the facial nucleus neurons showed no remarkable degeneration, despite HSV-1 particles in their cytoplasm. From these findings, we concluded that facial nerve paralysis in this model is caused mainly by facial nerve demyelination in the descending root. © 2002 Elsevier Science (USA) Key Words: Herpes simplex virus type 1; demyelination; facial nerve; motor nerve dysfunction; Bell’s palsy.
INTRODUCTION
MATERIALS AND METHODS
Herpes simplex virus type 1 (HSV-1) causes several neuronal diseases. It is commonly known to spread in sensory axons and to infect sensory neurons in the ganglia of the peripheral nervous system (4, 7, 14, 21). Therefore, in order to understand the mechanism of HSV-1 infection, many investigators have conducted animal experiments using sensory nerves, such as the
Virus and Animals It is well known that the neuropathogenicity of HSV varies among virus strains. The KOS strain is commonly used in experiments and has moderate neuropathogenicity. Blyth et al. (3) inoculated 10 5 pfu of strains SC16 and KOS into the ears of mice following Hill’s method (5). They reported that the incidence of herpetic eruptions in both strains was essentially the same, while the mortality rate with SC16 was much
1 This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, of Japan (11770996).
0014-4886/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
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higher than with KOS; half of the mice inoculated with SC16 died. The characteristics of KOS make it suitable for producing an animal model of acute, transient facial paralysis. Then, the KOS strain of HSV-1 was prepared in Vero cells and plaque titrated at 6.7 ⫻ 10 7 pfu/ml. Four-week-old, female, specific-pathogen-free BALB/cAJcl mice were purchased from Clea Japan, Inc. (Tokyo, Japan). All animals were cared for in our laboratory animal center, and studies were carried out in compliance with the Guide for Animal Experimentation of Ehime University School of Medicine. Virus Inoculation and Evaluation of Facial Nerve Paralysis Following anesthesia by intraperitoneal injection of sodium pentobarbital (50 mg/kg), virus was inoculated into the posterior aspect of the auricle on day 0. The surface of the auricle was scratched 20 times with a 27-gauge needle before HSV-1 inoculation, which was a modification of Hill’s method (5). Twenty-five microliters of virus solution (1.7 ⫻ 10 6 pfu) were placed on the right auricle. After virus inoculation, blink reflex and vibrissae movements were carefully observed daily, so as to detect and evaluate facial nerve paralysis. The blink reflex was elicited by blowing air into the eye using a 5-ml syringe with an 18-gauge needle. Immunofluorescence From the 1st to the 9th day after inoculation, 54 mice (6 mice for each day; from the 7th to the 9th day after inoculation, mice with facial paralysis were used) were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg/kg) and perfused transcardially, first with 100 ml PBS and then with 500 ml 4% formaldehyde in 0.1 M phosphate buffer (pH 7.4). The brain stem, including the descending root of the facial nerve, the facial nucleus, and the bilateral facial nerves including the geniculate ganglion, were dissected, and 40-m slices were prepared using a microslicer. These sections were stained with anti-gC (HSV-1 envelope glycoprotein C) monoclonal antibody labeled with FITC (Syva Co.) and then observed under fluorescent microscopy. Electron Microscopy On the 3rd, 4th, 5th, 6th, 9th, 14th, and 18th days after inoculation, 21 mice (3 mice for each day; on the 14 and the 18 days after inoculation, mice which had a almost complete recovery of facial paralysis were used) were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg/kg) and perfused transcardially, first with 100 ml PBS and then with 500 ml 4% formaldehyde and 4% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4).
FIG. 1. A mouse with facial nerve paralysis in the right side. The right eye cannot be closed and the nose is shifted to the left. This paralysis was observed from the 7th to 14th days after virus inoculation.
The brainstem and bilateral facial nerves were dissected and 70-m slices were cut with a microslicer. These specimens were postfixed with 2% osmium tetroxide, dehydrated, and embedded in epoxy resin. Ultrathin sections were prepared from specimens of the facial nerve, including the geniculate ganglion, the descending root, and the facial nucleus in the brain stem. These sections were stained with uranyl acetate and lead citrate and then observed under electron microscopy (Hitachi H-800, Japan). RESULTS
One hundred twelve mice were inoculated with HSV-1, and then 6 mice for each day were used for immunofluorescence. On the second day after inoculation, erosion or ulceration of the skin was observed in all animals at the site of inoculation. On the third day, 7 mice became much less vigorous; their bodies were curled up and their fur stood erect. They also showed palsy of the extremities and trembling of the body, suggesting the presence of encephalitis. Since they died on the 4th day without developing facial paralysis, these mice were excluded from the study. On the 7th day, facial paralysis (Fig. 1) was observed in 37 of 57 mice (65%). Ten mice with facial paralysis were ob-
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FIG. 2. Immunofluorescence over the entire course of the facial nerve with anti-gC monoclonal antibody labeled with FITC. a: Scheme showing the geniculate ganglion, the descending root, and the facial nucleus in the course of the facial nerve. b: The geniculate ganglion on the 5th day after inoculation. Many immunopositive cells are observed. c: The descending root of the facial nerve in the brain stem. Many immunopositive cells (arrows) were observed near the root entry zone on the 5th day after inoculation. d: The facial nucleus contained many immunopositive cells on the 5th day after inoculation. e: The time course of immunopositive cell appearance. Immunopositive cells appeared in the geniculate ganglion by the 3rd day, in the descending root by the 4th day, and in the facial nucleus by the 5th day after inoculation.
served to evaluate the natural course of the palsy. All of them showed almost complete recovery by the 14th day. Immunofluorescence The entire course of the facial nerve was examined under fluorescent microscopy, from the periphery to the facial nucleus. HSV-1 immunopositive cells were observed only in the geniculate ganglion (Fig. 2a), the descending root (Fig. 2b), and the facial nucleus (Fig.
2c). Figure 2 shows representative immunofluorescence in these areas and the data obtained from all the animals. Immunoreactivity was observed only on the inoculated side. Immunopositive cells were first observed in the geniculate ganglion 3 days after inoculation, in the descending root after 4 days, and in the facial nucleus after 5 days. The number of the immunopositive cells increased after the 5th day, reached a peak on the 7th day, and thereafter decreased abruptly. There were
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few immunopositive cells on the 9th day, and their immunoreactivity was very weak. Electron Microscopy of the Geniculate Ganglion Area On the 3rd or 4th day after inoculation, HSV-1 particles were observed in some neurons, which had a well-preserved, fine structure. Replicated HSV-1 particles were frequently observed, beginning on the 4th day (Fig. 3a). These replicated virus particles were observed in the cytoplasm of neurons (Fig. 3b) and, rarely, in Schwann cells. Nerve degeneration, such as demyelination or destruction of axon (axonotmesis), induced by viral infection was not observed in either the sensory or the motor nerve area in the first 4 days. However, demyelinated axons were observed from the 5th day (Fig. 3c), when viral particles were observed in neurons more frequently. Vacuolar changes and dilatation of the rough endoplasmic reticulum were also observed in these infected neurons. On the 9th day, HSV-1-infected neurons continued to produce viral particles, which infected satellite and Schwann cells surrounding the neurons (Figs. 3d and 3e). Demyelination was observed in the facial motor nerve in the geniculate ganglion area (Fig. 3f), where some axonotmesis was also observed. Some neurons underwent lysis, releasing cellular debris. In contrast, the contralateral geniculate ganglion neurons and facial motor nerve had a well-preserved, fine structure on the 9th day (Fig. 3g). Electron Microscopy of the Descending Root of the Facial Nerve in the Brain Stem On the 3rd day after inoculation, no HSV-1 particles were observed in astrocytes, and oligodendrocytes had a well-preserved, fine structure. On the 5th day, the astrocytes forming the descending root were heavily infected, while infection was not observed in other glial cells (Fig. 4a), and numerous replicated virus particles were found in astrocyte nuclei (Fig. 4b). However, these infected astrocytes vanished from the descending root on the 6th day and much cell debris was observed instead. In contrast, HSV-1 replicated in many oligodendrocytes, and demyelination was observed with these infected oligodendrocytes (Fig. 4c). On the 9th day, nerve degeneration became prominent on the inoculated side and the descending root could no longer be observed (Figs. 4d and 4e). An inflammatory infiltrate consisting of lymphocytes and mononuclear cells accompanied the appearance of cell debris and myelin breakdown in the descending root. The process of demyelination was remarkable with many degenerated oligodendrocytes, and there were many demyelinated large axons (Fig. 4f). Some of these axons showed axonotmesis. As expected from the immunofluorescence, no viruses were observed at this stage. On the 14th day, facial movement had essentially recovered to nor-
mal in all the paralyzed mice, and the descending root could be observed once again (Fig. 4g). On the same day, although some degeneration was still seen in the cytoplasm of oligodendrocytes (Fig. 4h), many of the large axons were remyelinated, although myelin on these axons appeared thinner than normal (Fig. 4i). A few axons were naked in places. On the 18th day, the descending root could be observed clearly (Fig. 4j). Electron Microscopy of the Facial Nucleus Until the 5th day after inoculation, no neurons with HSV-1 particles were observed. From the 6th to the 9th days after inoculation, the facial nucleus was well preserved (Figs. 5a and 5b). Although HSV-1 particles were observed in some neurons, these infected neurons had a well-preserved, fine structure (Figs. 5c and 5d). Even on the 9th day, HSV-1 infection was restricted to neurons; infected glial cells or degenerated nerves were not seen in the facial nucleus. None of these infected neurons underwent lysis. DISCUSSION
In our model, using polymerase chain reaction (PCR) analysis of HSV-1 DNA, we traced the migration route of the virus at the intratemporal facial nerve including geniculate ganglion and the homolateral brain stem on the 3rd and 10th days after inoculation (11). We detected HSV-1 DNA in the intratemporal facial nerve on the 3rd day and in both the intratemporal facial nerve and the brain stem on the 10th day. However, in that paper we were unable to demonstrate the detailed time course of viral spread throughout the facial nerve or the site of viral replication, because of the limitations of the PCR technique, so the role of HSV-1 infection in the pathogenesis of facial paralysis remained unclear. Therefore, we conducted the present histopathological study, which is the first to demonstrate the relationship between viral replication and nerve degeneration in the mechanism of facial paralysis. The facial nerve is composed of the facial motor nerve and the sensory nerve with the geniculate ganglion. These two fibers run together from the periphery to the end of the descending root. The geniculate ganglion neurons are located in the temporal bone, and the facial nucleus motor neurons are located in the brain stem (Fig. 2a). In this study, HSV-1 began to replicate in geniculate ganglion neurons on the 3rd or 4th day after inoculation. How does HSV-1 reach the geniculate ganglion from the auricle? Many studies have reported that the main transport of virus from the periphery is intraaxonal transport (1, 2, 8, 10, 13). Therefore, we tried to find HSV-1 particles in the axons of sensory and motor nerves around the geniculate ganglion on the 1st or 2nd day. However, we failed to find HSV-1 particles in
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FIG. 3. Electronmicrographs of the geniculate ganglion. a: Neuron with numerous virus capsids in the rough endoplasmic reticulum. This neuron has a well-preserved, fine structure. Arrows indicate the area in which virus particles were located (4th day after inoculation, scale bar: 10 m). b: HSV-1 particles in the rough endoplasmic reticulum (high magnification of the square area in a, 4th day after inoculation, scale bar: 1 m). c: Naked axons (arrows) are observed in some nerve fibers. Some Schwann cells have vacuolar changes in the cytoplasm (arrowheads) (5th day after inoculation, scale bar: 5 m). d: Electronmicrograph showing HSV-1 infected neuron, satellite, and Schwann cells. Virus particles in the neuron are observed in the rough endoplasmic reticulum. Vacuolar changes (arrowheads), destruction of mitochondria, and dilatation of the rough endoplasmic reticulum are observed in the cytoplasm of another neuron. Arrows indicate HSV1 particles (scale bar: 5 m). e: These virus particles were uniform and approximately 150 nm in diameter (high magnification of the square area in d, 9th day after inoculation, scale bar: 1 m). f: Facial motor nerve area on the 9th day after inoculation. Many Schwann cells have intracytoplasmic vacuolar changes, and many axons show demyelination (A). Despite more severe demyelination, the fine structures of the axons themselves are relatively well preserved. Some axonotmesis (B) was also observed (scale bar: 1 m). g: The facial motor nerve area on the contralateral side on the 9th day after inoculation. The fine structures and myelin of Schwann cells are preserved (scale bar: 1 m).
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FIG. 3—Continued
these axons. On the other hand, we first detected HSV-1 particles in geniculate ganglion neurons, not in nonneuronal cells. IF HSV-1 particles were transported from one Schwann cell to another or via extracellular fluid, then with viral infection viral particles or cell damage would first be observed in the nonneuronal cells. Therefore, we conclude that HSV-1 transport in
this model is intra-axonal, although no virus was detected in the axons. Furthermore, we think that this intra-axonal transport occurs not only in the sensory nerve, but also in the motor nerve. Since HSV-1 particles were first detected in facial nucleus motor neurons, not in nonneuronal cells, as observed in the geniculate ganglion, we conclude that HSV-1 inoculated in
FIG. 4. Electron and light micrographs of the descending root. a: Heavily infected astrocyte with numerous virus capsids in the nucleus (5th day after inoculation, scale bar: 5 m). b: HSV-1 particles in the astrocyte (square area in a). These viruses had capsids and tegments. They were approximately 150 nm in diameter (scale bar: 0.5 m). c: Infected oligodendrocyte in the descending root on the 6th day after inoculation. It contains numerous virus capsids in its nucleus. Two axons show demyelination (arrows) (scale bar: 5 m). d: Light micrograph of the brain stem stained with osmium on the 9th day after inoculation. A few fibers in the descending root of the facial nerve on the inoculated side can be observed. e: High magnification of the square area in d. Some of the descending root can be observed (9th day after inoculation). f: Large demyelinated axons and oligodendrocytes (Ol) with some myelin-like bodies are observed. A mononuclear cell (*) infiltrates near the damaged oligodendrocyte. One axon shows demyelination (arrows) (9th day after inoculation, scale bar: 5 m). g: Light micrograph of the brainstem stained with osmium 14 days after inoculation. The descending root of the facial nerve can be observed on the inoculated sides. h: Although a few myelin-like bodies (arrows) are observed in oligodendrocytes (Ol), the axons beside the oligodendrocytes are myelinated (14 days after inoculation, scale bar: 5 m). i: There are many large myelinated axons; their myelin sheaths appear thin and there are bare sections on some axons (arrows). j: Light micrograph of the brain stem stained with osmium 18 days after inoculation. The descending roots of the facial nerve appear similar on both sides. 74
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FIG. 4—Continued
the auricle ascends from the auricle in both sensory and motor axons. However, the possibility that the virus released from the dead sensory neuron was taken by motor axons at the geniculate ganglion cannot be excluded. In this study, both demyelination and axonotmesis were observed in facial motor nerves and in sensory nerves in the geniculate ganglion area. How does this nerve degeneration occur in facial motor fibers? The demyelination in the geniculate ganglion can be simply explained. Under electron microscopy, remarkable replication of HSV-1 was seen in the geniculate ganglion neurons. Furthermore, some neurons were lysed and released cellular debris and HSV-1 to the extracellular fluid space. Some of these HSV-1 spread directly to neighboring satellite and Schwann cells (Fig. 3d). Fi-
nally, these infected Schwann cells showed vacuolar changes and demyelination (Fig. 3f). On the other hand, axonotmesis, which means severe axonal damage or neuronal lysis, cannot be explained only by the phenomenon that occurred in the geniculate ganglion. While sensory nerve axonotmesis can be explained by the geniculate ganglion neuronal lysis, in considering the motor nerve axonotmesis we must consider the phenomena seen in the descending root and facial nucleus. In the facial nucleus, no degenerated neurons were observed, despite HSV-1 infection (Figs. 5b and 5c). Furthermore, no infected glial cells or degenerated nerves were seen in the facial nucleus. Therefore, the main reason for the motor nerve axonotmesis is the severe damage to the descending root, not the degeneration of the facial nucleus neurons (Figs. 4d– 4g).
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FIG. 4—Continued
Under electron microscopy, marked demyelination and some destroyed axons were observed in the descending root, and this is likely the cause of the motor nerve axonotmesis in the geniculate ganglion area. There are two possible reasons why the nerve degen-
eration in this area is so severe compared with that in other peripheral areas, including the geniculate ganglion. The first is the structural difference between the myelin-forming cells in the peripheral nervous system (PNS) and those in the CNS. Schwann cells make my-
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FIG. 5. Electron and light micrographs of the facial nucleus on the 9th day after inoculation. a: Light micrograph of the facial nucleus stained with osmium; there are no abnormalities. b: Light micrograph of the facial nucleus stained with toluidine blue; there are no abnormalities (scale bar: 100 m). c: Electron micrograph of the facial nucleus on the 9th day after inoculation; HSV-1 particles have replicated in the rough endoplasmic reticulum. However, the neuron has a well-preserved, fine structure (scale bar: 5 m) d: High magnification of the square area in c; HSV-1 particles in the rough endoplasmic reticulum were approximately 150 nm in diameter (scale bar: 1 m).
elin sheaths in the PNS, while myelin sheaths in the CNS are made by oligodendrocytes. The same degree of damage in these two types of myelin-forming cells results in much more severe demyelination in the CNS than in the PNS. The second reason is the difference in the “tightness” of Schwann cells and astrocytes. This difference was reported to be the reason that HSV-1 first infects astrocytes, in several studies of HSV-1
infection of the trigeminal nerve (4, 6, 10, 18, 19, 21). In the PNS, the basal lamina of Schwann cells acts as a barrier to axonal virions. In the PNS–CNS junction, however, the foot processes of astrocytes abut directly on axons. Itoyama et al. (6) speculated that an intraaxonal virus may gain entry from these foot processes of astrocytes in the PNS–CNS junction area. We noted that HSV-1 infection occurred in the descending root
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and that this infection led to severe degeneration in this area, which is consistent with their speculation. The demyelination in the descending root can be classified into three stages. In the first stage, lysis of the infected astrocytes is prominent. HSV-1 first infects astrocytes in the descending root on the 5th day (Figs. 4a and 4b). This infection of astrocytes is similar to the HSV-induced entry zone lesions that have been reported in the trigeminal root (4, 6, 10, 18, 19, 21). Some authors (10, 18, 19) have reported that these infected astrocytes produce numerous viral particles and undergo lysis. In our model, almost all of the astrocytes in the descending root were infected with HSV-1 on the 5th day, yet no astrocytes were observed in this region on the 6th day. This is consistent with the report of Itoyama et al. (6), who used immunohistochemistry with glial cell markers. Most of these infected astrocytes likely undergo lysis, releasing cellular debris and numerous viral particles that may trigger the infiltration of mononuclear cells (9, 17, 22). We observed that an inflammatory infiltrate consisting of lymphocytes and mononuclear cells accompanied the appearance of cellular debris on the 6th day. In the second stage, the released viruses infect oligodendrocytes and start to replicate in their nuclei on the 6th day (Fig. 4c). These infected oligodendrocytes begin to show demyelination. At 9 days, there are many large demyelinated axons with many degenerated oligodendrocytes (Fig. 4g). In the third stage, severe degeneration is induced by a secondary immune response (10, 19, 20). Townsend and Baringer (20) concluded that early infection of oligodendrocytes might lead to partial demyelination followed by a macrophage response that causes further demyelination. Furthermore, the infiltrating cells destroy some axons in the descending root. This nerve destruction seems to lead to Wallerian degeneration and cause axonotmesis. In conclusion, the demyelination observed on the 6th day may occur as a direct cytopathic effect of the virus, and the demyelination and axonotmesis noted after the 9th day may result from a secondary immune response. Facial paralysis and degeneration in the descending root begin at almost the same time after inoculation. Moreover, remyelination of the descending root was observed accompanied with facial nerve recovery. These findings indicate that degeneration of the descending root, especially demyelination, is the main cause of facial paralysis in our model. In this study, 65% of the mice developed transient facial paralysis, while 6% of the mice died very quickly in the early stage of infection without developing facial paralysis. The symptoms of the dying mice suggested encephalitis. However, it is still unclear why HSV inoculation into the ear killed only some of the mice, and did so very quickly, while other mice at the same stage did not show pathological degeneration in the facial
nerve or brain stem. To solve this puzzle, it is very important to carry out a pathological study of the dying mice at different stages of their illness. However, collecting dying mice at different stages would be very difficult, because their illness progresses so rapidly. Such a study, while important, is beyond the scope of this paper, as the dead mice never developed facial paralysis. We plan to examine this in a future study. In 1996, we detected HSV-1 genomes in clinical samples of endoneurial fluid of facial nerve that were obtained from Bell’s palsy patients (12). This report strongly suggested that HSV-1 can be the cause of Bell’s palsy and we demonstrated the mechanism of facial nerve paralysis resulting from primary HSV-1 infection in the present study. However, since Bell’s palsy is thought to arise from latent HSV-1 infection in the geniculate ganglion, the mechanism of facial palsy in the present study using primary infection model may not be the same as that in the reactivation process of Bell’s palsy. Recently, we succeeded in reactivating latently infected virus and producing facial nerve paralysis in another animal model (16). Using this model, further investigations are currently underway in our laboratory to clarify whether the mechanism of this palsy is more similar to that of Bell’s palsy. ACKNOWLEDGMENT We thank Professor Naoaki Yanagihara for helpful discussion.
REFERENCES 1.
2.
3.
4.
5.
6.
7.
8.
Bak, J. J., Ch. Markham, M. L. Cook, and J. G. Steavens. 1977. Intra-axonal transport of herpes simplex virus in the rat central nervous system. Brain Res. 136: 415– 429. Bak, J. J., Ch. Markham, M. L. Cook, and J. G. Steavens. 1978. Ultrastructural and immunoperoxidase study of striatonigral neurons by means of retrograde axonal transport of herpes simplex virus. Brain Res. 136: 361–368. Blyth, W. A., D. A. Harbour, and T. J. Hill. 1984. Pathogenesis of Zosteriform spread of herpes simplex virus in the mouse. J. Gen. Virol. 65: 1477–1486. Cook, M. L., and J. G. Stevens. 1973. Pathogenesis of herpetic neuritis and ganglionitis in mice: Evidence for intra-axonal transport of infection. Infect. Immunol. 7: 272–288. Hill, T. J., H. J. Field, and W. A. Blyth. 1975. Acute and recurrent infection with herpes simplex virus in the mouse: A model for studying latency and reccurrent disease. J. Gen. Virol. 28: 341–353. Itoyama, Y., T. Sekizawa, H. Openshaw, K. Kogure, and I. Goto. 1991. Early loss of astrocytes in herpes simplex virus-induced central nervous system demyelination. Ann. Neurol. 29: 285– 292. Itoyama, Y., T. Sekizawa, H. Openshaw, K. Kogure, and Y. Kuroiwa. 1984. Immunocytochemical localization of herpes simplex virus antigen in the trigeminal ganglia of experimentally infected mice. J. Neurol. Sci. 66: 67–75. Kristensson, K., I. Nennesmo, L. Persson, and E. Lycke. 1982. Neuron to neuron transmission of herpes simplex virus. Trans-
FACIAL PARALYSIS INDUCED BY HSV-1
9.
10.
11.
12.
13.
14. 15.
port of virus from skin to brainstem nuclei. J. Neurol. Sci. 54: 149 –156. Kristensson, K., H. Thormar, and H. M. Wisniewski. 1979. Myelin lesions in the rabbit eye model as a by-stander effect of herpes simplex and Visna virus sensitization. Acta Neuropathol. (Berl.) 48: 215–217. Kristensson, K., A. Vahlne, L. A. Persson, and E. Lycke. 1978. Neural spread of herpes simplex virus type 1 and 2 in mice after corneal or subcutaneous (footpad) inoculation. J. Neurol. Sci. 35: 331–340. Murakami, S., N. Hato, M. Mizobuchi, T. Doi, and N. Yanagihara. 1996a. Role of herpes simplex virus infection in the pathogenesis of facial paralysis in mice. Ann. Otol. Rhinol. Laryngol. 105: 49 –53. Murakami, S., M. Mizobuchi, Y. Nakashiro, T. Doi, N. Hato, and N. Yanagihara. 1996b. Bell’s palsy and herpes simplex virus: Identification of viral DNA in endoneural fluid and muscle. Ann. Intern. Med. 124: 27–30. Openshaw, H., and G. E. William. 1983. Herpes simplex virus infection of motor neurons: Hypoglossal model. Infect. Immunol. 42: 409 – 413. Stevens, J. G., and M. L. Cook. 1971. Latent herpes simplex virus in spinal ganglia of mice. Science 173: 843– 845. Sugita, T., S. Murakami, N. Yanagihara, Y. Fujiwara, and Y. Hirata. 1995. Facial nerve paralysis induced by herpes simplex
16.
17.
18.
19.
20.
21.
22.
79
virus in mice, an animal model of acute and transient facial paralysis. Ann. Otol. Rhinol. Laryngol. 104: 574 –581. Takahashi, H., Y. Hitsumoto, N. Honda, N. Hato, M. Mizobuchi, S. Murakami, H. Kisaki, H. Wakisaka, and K. Gyo. 2001. Mouse model of Bell’s palsy induced by reactivation of herpes simplex virus type 1. J. Neuropathol. Exp. Neurol. 60: 621– 627. Townsend, J. J. 1981. The relationship of astrocytes and macrophages to CNS demyelination after experimental herpes simplex virus infection. J. Neuropathol. Exp. Neurol. 40: 369 –379. Townsend, J. J., and J. R. Baringer. 1976. Comparative vulnerability of peripheral and central nervous tissue to herpes simplex virus. J. Neuropathol. Exp. Neurol. 35: 100. Townsend, J. J., and J. R. Baringer. 1978. Central nervous system susceptibility to herpes simplex infection. J. Neuropathol. Exp. Neurol. 37: 255–262. Townsend, J. J., and J. R. Baringer. 1979. Morphology of central nervous system disease in immunosuppressed mice after peripheral herpes simplex virus inoculation. Lab. Invest. 40(2): 178 –182. Townsend, J. J., and P. K. Collins. 1986. Peripheral nervous system demyelination with herpes simplex virus. J. Neuropathol. Exp. Neurol. 45: 419 – 425. Wisniewski, H. M., and B. R. Bloom. 1975. Primary demyelination as a nonspecific consequence of a cell-mediated immune reaction. J. Neuropathol. Exp. Neurol. 141: 346 –359.