The effect of trigeminal nerve and ganglion manipulation on recurrence of ocular herpes simplex in rabbits.

Latent herpes simplex virus (HSV) has been demonstrated in the trigeminal ganglia of experimentally infected rabbits between episodes of spontaneous ocular recurrence. In three experiments reported here, the normal pattern of recurrence was modified by manipulation of the trigeminal nerve and ganglion. Temporary retrobulbar disruption of trigeminal nerve function in chronically infected animals significantly decreased the number of ocular HSV isolations obtained during the 20 weeks immediately following surgery. Stereotaxic interruption of intracranial trigeminal nerve function prior to initial HSV infection dramatically reduced the incidence of peripheral recurrence of HSV. In chronically infected animals, stereotaxic stimulation of the trigeminal ganglion caused a marked increase in positive cultures within 2 days. These studies provide additional evidence for the theory that the reservoir for latent ocular HSV in rabbits is the trigeminal ganglion. Moreover, the studies suggest that the transmission of latent HSV from the trigeminal ganglion to its infectious form in the peripheral tissues involves the trigeminal nerve. We have shown that mechanical and stereotaxic stimulation of the trigeminal ganglion is a reliable and rapid means of precipitating peripheral ocular shedding of HSV on command, a finding which should prove most productive in future research.     

Reactivation of murine latent HSV infection by epinephrine iontophoresis

Iontophoresis of epinephrine into the cornea of previously infected mice was used in an attempt to induce reactivation of latent herpes simplex virus (HSV) infection of the trigeminal ganglia. BALB/c mice infected with HSV-1 strain McKrae following corneal scarification developed a latent infection of the trigeminal ganglia within 15 days. At 28 days postinfection, mice were subjected to a 3-day cycle of iontophoresis of epinephrine (0.01%) into the cornea. Ocular shedding of HSV occurred in 16/23 (70%) of stimulated mice; these animals did not shed HSV in the 3-day period prior to iontophoresis. Spontaneous shedding of HSV, however, was noted in 3/97 (3%) mice not subjected to epinephrine iontophoresis. "Infectious" virus was isolated only from the trigeminal ganglia of stimulated mice, whereas "latent" virus was isolated from the trigeminal ganglia of both stimulated and nonstimulated mice. All virus isolates were verified to be HSV by neutralization with a known HSV-1 antiserum. This ocular system thus allows for the study of the full spectrum of latent HSV infections, including latency, ganglionic reactivation, and peripheral virus shedding.     

单纯疱疹病毒Ⅰ型眼部潜伏感染的实验研究

纯系新西兰大白兔经角膜接种ＨＳＶ－ｌｓｔｏｋｅｋ株，成功建立单疱病毒性角膜炎模型。自愈后４５天，以原病变角膜为供体，健康新西兰白兔为受体，行穿通性角膜移植术（ＰＫＰ）同时，取患侧１０份及健侧１０份三叉神经节，应用多聚酶链反应（ＰｏｌｙｍｅｒａｓｅＣｈａｉｎＲｅａｃｔｉｏｎ，ＰＣＲ）检测ＨＳＶ－１ＤＮＡ。术后５０天，取术侧角膜植片１０只及术侧三叉神经节１０份作相同检测。结果表明，患侧三叉神经节１０份中８份ＨＳＶ－１呈阳性，而健侧１０份中３份阳性；角膜植片１０只有７只阳性，术侧三叉神经节未检测到ＨＳＶ－１ＤＮＡ。由此提示，单纯疱疹病毒Ｉ型（ＨＳＶ－１）既可在同侧三叉神经节形成潜伏感染，又可能在对侧三叉神经节形成潜伏，而且角膜极可能为ＨＳＶ－１的另一潜伏部位     

Effect of immunization and immunosuppression on induced ocular shedding and recovery of herpes simplex virus in infected rabbits

Immunization and immunosuppression were evaluated during latent ocular herpes simplex virus, type 1 (HSV-1) infection in the rabbit, using the following parameters: (1) ability to recover virus from preocular tearfilm cultures; (2) reactivation of latent infection by direct electrical stimulation; and (3) recovery of virus from latently infected ganglia by whole-cell co-cultivation. Immunization prior to ocular inoculation of virus significantly reduced both the titer of virus shed into the tearfilm and the duration of virus shedding during primary ocular infection. Half of the non-immunized control rabbits died secondary to virus encephalitis, whereas none of the immunized rabbits died. The immunized rabbits could not be induced to shed virus by electrically stimulating the trigeminal ganglion directly. Immunosuppression of latently infected rabbits with high-dose cyclophosphamide (300 mg kg-1) enhanced virus shedding in the tearfilm and increased mortality due to viral encephalitis. Low-dose cyclophosphamide immunosuppression (40 mg kg-1) did not increase mortality because of viral encephalitis. Tearfilm virus shedding secondary to electrical induction in high-dose and low-dose cyclophosphamide animals was higher than that of control, non-immunosuppressed animals.     

Increased axonal transport of trigeminal ganglion proteins in an electrode model

This investigation uses an electrical stimulation model of ocular herpes simplex virus (HSV) reactivation to elucidate the relationship between neuronal protein synthesis, axonal transport and electrical stimulation. In this study, healthy adult albino rabbits were implanted with electrodes over the trigeminal ganglion (TG) and stimulated chronically twice a week for 28-34 days (previously shown to induce HSV reactivation in infected rabbits). The TG was visualized surgically and injected with 3H-proline to label newly synthesized protein. After various times, the TG, corneas and ophthalmic nerve were analyzed for labeled proteins. In nonimplanted and implanted rabbits (whether stimulated or not), the rate of fast anterograde axonal transport in the ophthalmic nerve was 347 mm/day and 326 mm/day, respectively. A lag period of 45 min occurred between initiation of protein labeling and onset of axonal transport. Only a small percentage of newly synthesized neuronal proteins were transported in the ophthalmic nerve (1-4% in the proximal 10 mm) or deposited in the cornea (less than 0.5%) after 24 hr. Neither the rate of protein synthesis in the TG nor the rate of axonal transport was changed by electrical stimulation. However, the amount of radioactively labeled proteins undergoing axonal transport in the stimulated group was five times that of the sham control group. Thus, the routing and loading of TG proteins for axonal transport is enhanced in the electrical stimulation model.     

Peripheral adrenergic stimulation and indomethacin in experimental ocular shedding of HSV

The application of 6-hydroxydopamine to the cornea by iontophoresis, followed by topical epinephrine, effectively induces herpes simplex virus (HSV) shedding from the external eye of latently infected rabbits. In this study the beta adrenergic blocker, Timolol, reduced virus shedding when applied immediately before the epinephrine, but continued administration resulted in increased viral shedding. While indomethacin, a prostaglandin synthesis inhibitor decreased HSV replication in cell culture, it failed to decrease virus shedding when applied topically to the eye in adrenergically stimulated animals. Timolol may act then by its effect on the peripheral cells of the eye rather than by stimulation of virus production in ganglionic neurons. These same animals were subsequently tested for latent infection of the trigeminal and superior cervical ganglia and corneas 14 months after primary infection. Only 2 of 14 animals had virus in the trigeminal ganglia, a finding which suggests that latent virus may be depleted by repeated reactivations. Virus was recovered from corneas of five rabbits by co-cultivation so it is possible that corneal latency occurs in this rabbit model as it does in humans.     

Herpes simplex virus recovery in neural tissues after ocular HSV shedding induced by epinephrine iontophoresis to the rabbit cornea

Ocular HSV-1 shedding from latently infected rabbits was induced by iontophoresis of 0.01% epinephrine into the eye. Anodal Iontophoresis of epinephrine was performed at 0.8 mAmps for 8 min once a day for 3 consecutive days. Shedding was determined by the presence of HSV-1 in the preocular tear film obtained via eye swabs. Bilateral epinephrine iontophoresis performed on selected days during 220-280 days after inoculation resulted in HSV-1 shedding in 75% of the eyes (30/40) and 100% of the rabbits (20/20). Following the induction of ocular HSV-1 shedding, rabbits were killed and selected neural tissues were homogenized. Cell-free preparations were assayed for the presence of infectious virions using primary rabbit kidney cell monolayers. When the tissues were homogenized immediately after death, virus was detected in only one neural tissue, the trigeminal ganglia. However, when the tissues were incubated in vitro for 18-24 hours prior to the homogenization, infectious HSV-1 was recovered from homogenates of the trigeminal ganglion, superior cervical ganglion, the ophthalmic branch of the trigeminal nerve, and the root entry zone of the trigeminal nerve. A relationship was noted between the time of the last ocular shedding and recovery of infectious HSV from the tissue homogenates. Furthermore, a positive correlation in 11 eyes between the recovery of HSV-1 from the perocular tear film and HSV-1 recovery from one or more corresponding neural tissues was found. These results suggested that epinephrine iontophoresis to the cornea triggered an "alteration" in the state of the virus in the neural tissues of the latently infected rabbits and that the change can be related to the induced ocular shedding.     

Peripheral neural circuits regulating IOP?

The peripheral nervous system is classically separated into a somatic division containing both afferent and efferent pathways and an autonomic division composed of efferents only. The somatic afferent division is divided in A- and B-neurons. The B-neurons are supposed to be autonomic afferents as part of a reflex system involved in homeostasis. Recent data obtained by neuronal tracing and immunohistochemical experiments concerning the eye related peripheral nervous system endorse the existence of these peripheral reflex systems. Somatic afferents of trigeminal origin synaptically innervate parasympathetic neurons in the pterygopalatine ganglion. This probably represents a pathway mediating autonomically regulated ocular activity in response to sensory stimulation. In addition, it has been hypothesized that trigeminal sensory nerve fibres have an efferent function in response to noxious stimuli e.g. the ocular injury response. Sympathetic nerve fibres originating in the superior cervical ganglion course through the trigeminal and pterygopalatine ganglion without forming direct synaptic contacts. These fibres, however, contain clusters of vesicles suggesting some kind of interneural communication. Parasympathetic nerve fibres of pterygopalatine origin course through the ciliary ganglion. These nerve fibre terminals also contain clusters of vesicles without direct synaptic contacts. Experimental data concerning the distribution of neuropeptides revealed a more detailed knowledge of the anterior eye segment innervation. These experimental data are subject to some debate. The pros and cons of different techniques are discussed. Neural circuits regulating IOP have long been postulated. The possible role of peripheral reflex systems in the regulation of IOP is discussed.     

The biology of herpes simplex virus infection in humans

Herpes simplex virus is a frequent cause of recurrent ocular, oral, genital or cutaneous eruptions in man. Lesions are highly localized and tend to recur at the same site. Among the most consistent factors provoking recurrence is root section of the trigeminal nerve. Clinical and experimental data suggest that herpes simplex virus is commonly resident within the trigeminal ganglia of man, where it may be responsible for recurrent oral or lip lesions, and is less frequently a resident of the second or third sacral ganglia where it might be responsible for genital eruptions. Generally, the trigeminal virus is type 1 and the sacral virus is type 2; the virus is only rarely recoverable from other sensory ganglia. Factors provoking the reactivation from the virus' latent site and the mechanism for reactivation remain largely unknown. Further study is needed to understand the behavior of HSV and other viruses in nervous system tissue.     

Studies on chronic herpetic keratitis — Estimation of virus-harboring ganglion cells

The reservoiring site of herpes simplex virus (HSV) during the chronic phase of herpetic keratitis was confined to the trigeminal ganglion. Other ocular tissues, the cerebrum and the cerebellum were also examined and not found positive for the isolation of HSV by cocultivation with green monkey kidney cells 28–224 days after infection. The percentage of virus-harboring trigeminal ganglionic cells was estimated by determining the number of dispersed ganglionic cells needed to produce cytopathology of HSV on 50% of Vero cell monolayers. The percentages of infected ganglionic cells were calculated to range from 0.05% to 0.3% during this chronic period.     

Neuronal propagation of HSV1 from the oral mucosa to the eye

PURPOSE: To identify possible neuronal pathways leading to herpetic ocular disease after primary oral infection in mice. METHODS: The SC16 strain of herpes simplex virus (HSV)-1 (10(6) plaque-forming units) was injected into the mucocutaneous border of the left upper lip. Animals were killed 2 to 10 days postinoculation (DPI). Spread of the virus in neural structures was studied by immunochemistry. RESULTS: HSV1 first replicated at the site of inoculation and then at the superior cervical ganglion (at 2 DPI). The trigeminal ganglion and the facial nerve fibers were infected by 4 DPI. Infection of the ciliary body and iris occurred at 6 DPI, together with several brain stem nuclei belonging to the autonomic or sensory pathways. Between 8 and 10 DPI, the neural infection gradually cleared up, except for the ipsilateral sympathetic ganglion, and ipsilateral keratitis appeared in some animals. CONCLUSIONS: The pattern of viral dissemination in this mouse model suggests that infection of iris and ciliary body results from transfer of virus in the superior cervical ganglion from sympathetic neurons innervating the lip to neighboring neurons innervating the anterior uvea. Later, zosteriform spread of virus from the trigeminal system may have contributed to the clinical and histologic findings.     

A quantitative assay of retrograde transported HSV in the trigeminal ganglion.

The relationship between the dose of Herpes simplex virus type 1 (HSV) inoculated in the cornea and the amount of actively replicating virus recovered from mouse trigeminal ganglion cells 5 d after corneal scratch and inoculation was investigated with a tissue culture plaque assay. A dose response curve of productive viral replication was obtained. The estimated dose of HSV that produces half-maximal recovery of virus within the ganglion was 9.15 x 10(3) plaque forming units per eye, and the maximal amount of HSV recovered was 1.34 x 10(4) pfu per ganglion. This definition of infectivity as a function of dose will be useful for studying the effects of potential inhibitors of the binding, uptake, and transport of HSV by productively or latently infected trigeminal neurons.     

Protective immunity against herpetic ocular disease in an outbred mouse model

Immunization of outbred mice by subdermal (footpad) inoculation with the F strain of herpes simplex virus type 1 (HSV-1) induces an immune response which protects the animals against herpetic ocular disease and encephalitis, and reduces the incidence of latent trigeminal ganglion infections following corneal challenge with the RE strain of HSV type 1. The protective effects are proportional to the dose of virus used for immunization. Heat-killed virus preparations also protected the mice against encephalitis and stromal keratitis, but failed to prevent epithelial keratitis and establishment of latency.     

Intentional reactivation of latent ocular herpes infection during BVDU therapy

Sixteen adult New Zealand white rabbits with previously confirmed herpes simplex virus type 1 (HSV-1) infections were stimulated by iontophoresis of 6-hydroxydopamine into the cornea and were followed-up by topical epinephrine treatment to confirm latency. A total of 224 ocular cultures were obtained, of which 73 were positive for HSV during the seven day cycle. Twenty-seven of the 32 eyes (84%) had at least one positive culture. Animals were randomly divided into two treatment groups. Upon repeat stimulation (Cycle 2), concurrent with oral and topical bromovinyl-deoxyuridine (BVDU) therapy, only 3/104 ocular cultures were HSV positive, while 24/112 ocular cultures from placebo-treated animals were positive. Anti-HSV serum titers were comparable before and after BVDU therapy and the HSV isolates from BVDU treated animals did not develop drug resistance (i.e., ED-50 values were approximately 0.1-0.2 ug/ml both before and after therapy). It was concluded that BVDU had a demonstrable therapeutic effect on the recovery of HSV-1 from ocular cultures during intentional reactivation, but the latent ganglionic infection was not eliminated.     

HSV-1 quantitation from rabbit neural tissues after epinephrine-induced reactivation

Epinephrine iontophoresis into the eye can induce ocular herpes simplex virus type-1 (HSV-1) shedding with a high frequency from latently infected rabbits. The present study was designed to qualify and quantify infectious HSV-1 from neural tissues of latently infected rabbits after ocular epinephrine iontophoresis. Epinephrine iontophoresis was performed daily for 3 consecutive days on selected days during 220-227 days postinoculation. The induced ocular shedding was detected in the tear film with a frequency of 83% (10/12) within 72 hr after the initial iontophoresis. The rabbits were killed 24 hr after the last iontophoretic treatment, and the corneas and neural tissues were homogenized immediately. The cell-free supernatants were inoculated on primary rabbit kidney cell monolayers for qualitative assays of infectious virus and later titrated on CV-1 monolayers. The frequencies of the recovery of infectious HSV-1 from the cell-free homogenates were 0% of the corneas (0/12), 83% (10/12) from the superior cervical ganglion (SCG), 100% (12/12) from the trigeminal ganglion (TG), 42% (5/12) from the ophthalmic branch of the trigeminal nerve (TN), 8% (1/12) from the root entry zone of the trigeminal nerve into the brain-stem (REZ), and 0% (0/12) from the cerebellum. The authors conclude that epinephrine iontophoresis can reactivate the latent HSV-1 in neural tissues and infectious virus can be quantified from the cell-free homogenates. To the best of our knowledge, this is the first report to quantify HSV-1 with a high frequency from neural tissues following induced reactivation.     

A rat model of radiofrequency ablation of trigeminal innervation via a ventral approach with stereotaxic surgery

Neurotrophic keratopathy (NK), a consequence of sensory denervation of the cornea, must be better understood in order to develop new approaches to therapy. The purpose of this study was to create a rat model for neurotrophic keratopathy by denervating the trigeminal nerve through a ventral approach with stereotaxic surgery. Stereotaxic coordinates were measured in 46 male Sprague Dawley rat cadavers for localization of V1. After further refining the coordinates in nine live animals, radiofrequency ablation was chosen as an effective method of disrupting the innervation to the cornea. Fifty-two live rats were treated with radiofrequency ablation to define the anatomical localization of the lesion by utilizing gross and histopathological studies. A gross lesion of the trigeminal nerve and/or ganglion was observed in 47 (90%) of the 52 animals. Histopathological studies revealed that all 52 animals had anatomical damage of the trigeminal innervation to the eye. Low mortality and little morbidity were observed in these animals. We have developed a rat model for neurotrophic keratopathy that is simple to produce, accurate in creating a lesion by utilizing stereotaxic techniques combined with radiofrequency ablation, and successful in decreasing morbidity and mortality.     

Effect of immunization on herpes simplex virus type 1 latent infection in the trigeminal ganglion

PURPOSE: To quantify and characterize immune protection from herpes simplex virus (HSV) latent infection in mice following corneal challenge. METHODS: Mice immunized or mock-immunized and boosted in the flank with an HSV replication-deficient mutant were challenged by corneal inoculation with wild type (wt) or thymidine kinase-negative (TK(-)) HSV. At specified times post challenge, trigeminal ganglia were assayed for in vitro reactivation, latent and acute viral load (using quantitative PCR), acute infection, and cellular infiltration (hematoxylin and eosin stained sections). RESULTS: With wt HSV challenge infection, immunization led to reduced reactivation, significantly less latent and acute viral DNA, and no acute viral replication in ganglia, and rapid infiltration of inflammatory cells. Immunization had little effect on viral load following challenge with replication-conditional TK(-) mutant virus. CONCLUSION: These results indicate that immune protection from latent HSV infection in mouse trigeminal ganglia following ocular infection can act under these experimental conditions to block acute viral replication in ganglia and is directed to antigenic targets within the ganglia.     

Corneal nerve disruption reactivates virus in rabbits latently infected with HSV-1

Trauma, inflammation, and neuronal stimulation or damage can reactive latent herpes simplex virus type 1 (HSV-1). The innervation density of the corneal epithelium is 300-600 times that of skin and, therefore, corneal nerve disruption could provide a strong stimulus for HSV-1 reactivation. This study has documented HSV-1 ocular reactivation following three methods of corneal nerve disruption in rabbits. Twenty HSV-1 latently infected rabbits (26 eyes) were divided into three groups: 7 rabbits received uniocular cryogenic injury, 7 rabbits underwent uniocular anterior superficial keratectomy, and 6 rabbits had binocular transection of the corneal nerves at the corneoscleral limbus which, in contrast to the other treatments, produced minimal epithelial change. Opposite eyes in the first two groups of rabbits were left undisturbed to serve as HSV-1 infected controls. Three additional rabbits, not infected with HSV-1, underwent gold chloride impregnation of the corneal nerves for light microscopic documentation of corneal nerve damage induced by each procedure. On all HSV-1 infected eyes, daily HSV-1 ocular cultures were obtained for 7 consecutive days. All three procedures resulted in marked corneal nerve destruction and degeneration. HSV-1 shedding occurred in 5/7 (71%) of the eyes that underwent cryogenic lesioning; in 5/7 (71%) of the eyes that underwent anterior keratectomy; and in 8/12 (67%) of the eyes that had the corneal nerves transected at the corneoscleral limbus. Only 4 (29%) of the 14 control eyes had positive HSV-1 ocular cultures. This investigation provides strong evidence that corneal nerve disruption is correlated with ocular HSV-1 reactivation.     

The isolation of herpes simplex virus from rabbit corneas during latency

Herpes simplex virus type 1 (HSV-1) latency, as operationally defined, is a state in which cell-free infectious virus cannot be demonstrated in tissue at the time of sacrifice, but infectious virus can be isolated from the same tissue after prolonged cultivation. Latent HSV has been routinely detected in sensory ganglia of the infected dermatome. We have isolated HSV-1 (RE) from the corneas of 11% of infected rabbits which harbored virus in a latent state in trigeminal ganglia. HSV-1 (RE) was isolated from 10 of 88 cultures of corneal cells established following collagenase digestion of individual corneas taken from asymptomatic animals 118 days after infection. Virus was recovered only after prolonged primary culture and in some cases serial passage of corneal cells (range 5 to 26 days to initial cytopathic effect, n = 10). Virus was isolated from 68 of 68 trigeminal ganglia from the same rabbits by cocultivation of ganglion pieces with Vero cells (range 9 to 20 days to initial cytopathic effect, n = 68) while no cell-free virus was isolated from ganglia at the time of sacrifice. Virus isolation from corneas during the latent period occurred in a manner independent of prior antiviral or antiviral plus immunosuppressive therapy. Clinical evaluation of the corneas throughout the course of acute disease, stromal disease, and at the time of sacrifice provided no evidence that could be used to predict which corneas would yield virus. These data suggest that HSV-1 can remain in a nonreplicative state characteristic of latency in cells of rabbit corneas for long periods after infection and therapy of herpetic eye disease.