Induction of ocular herpes simplex virus shedding by iontophoresis of epinephrine into rabbit cornea

Ocular herpes simplex virus type 1 (HSV-1) shedding from the latently infected rabbit was induced by iontophoresis of 0.01% epinephrine into the eye. The iontophoresis of epinephrine was at 0.8 mAmp for 8 min once a day for 3 consecutive days. Shedding was determined by the presence of HSV-1 in the tear film obtained with eye swabs. Unilateral epinephrine iontophoresis performed 60 days after inoculation of the virus resulted in ipsilateral HSV-1 shedding in all cases (7/7). Bilateral epinephrine iontophoresis performed on selected days during 170 to 365 days after inoculation resulted in HSV-1 shedding in 75% of the eyes (21/28) and 100% of the rabbits (14/14). All shedding was initiated within 3 days after the third treatment with epinephrine iontophoresis. The shedding frequency induced by epinephrine iontophoresis was significantly higher (pb less than 0.05) than that induced by the other methods employed. HSV-1 was detected in one or both cocultivated explants of trigeminal and superior cervical ganglia for every eye in all experimental groups, indicating that all eyes had the potential to shed. In conclusion, epinephrine iontophoresis induced ocular HSV-1 shedding reliably and with a high frequency in the latently infected rabbits. Furthermore, we suggest that this easily reproducible model of viral shedding offers a system for studying the factors involved in recurrent HSV-1 ocular infections.     

Iontophoresis of epinephrine isomers to rabbit eyes induced HSV-1 ocular shedding

Iontophoresis of 0.01% levo(-) epinephrine for 8 min at 0.8 mAmp once daily for 3 consecutive days induces ocular shedding of herpes simplex virus type 1 (HSV-1) in latently infected rabbits. In the present experiment, we tested dextro(+) and levo(-) epinephrine for their comparative effects on induced HSV-1 ocular shedding. One hundred percent of the eyes shed virus after either 0.01% dextro(+) or levo(-) epinephrine iontophoresis (8 min, 0.8 mAmp). However, the shedding frequency caused by 0.005% levo(-) epinephrine was significantly higher (P less than 0.05) than that by 0.005% dextro(+) epinephrine when the iontophoresis was conducted at 0.4 mAmp for 4 min. Iontophoresis of 0.001% levo(-) epinephrine for 8 min at 0.8 mAmp once daily for 3 consecutive days and iontophoresis of 0.001% dextro(+) epinephrine for 8 min at 0.8 mAmp once daily for 3 consecutive days did not induce HSV-1 ocular shedding in latently infected rabbits. The data suggest that the mechanism of induction of HSV-1 ocular shedding by epinephrine is correlated to the receptor potency of levo(-) epinephrine.     

Herpes simplex virus mediated gene transfer to primate ocular tissues.

We evaluated the feasibility of delivering a gene into monkey eyes using a replication-competent herpes simplex virus (HSV) type 1 ribonucleotide reductase mutant (hrR3) expressing the Escherichia coli lacZ gene. To determine the efficiency of in vitro HSV-mediated gene transfer, cultured human trabecular meshwork (HTM) and human ciliary muscle (HCM) cells were infected with hrR3 and beta-galactosidase activity was measured histochemically. Six cynomolgus monkey eyes received viral injections into the anterior chamber (2 x 10(7) pfu) and/or the vitreous (5 x 10(7) pfu), and the distribution of cells expressing lacZ was evaluated. In vitro, both cultured HTM and HCM cells displayed multiplicity-dependent beta-galactosidase activity. In vivo, intracameral and/or intravitreal injection resulted in transgene expression in TM cells and in non-pigmented ciliary epithelial cells (NPE), but not in CM cells. Transgene expression was also detected in retinal pigmented epithelial (RPE) cells and sporadic retinal ganglion cells (RGC) in eyes receiving virus intracamerally and intravitreally respectively. We observed significant inflammation in the anterior chamber, TM and CM in virus-injected eyes, along with mild vitritis and retinitis. This study demonstrates successful gene transfer using hrR3 as a vector in human ocular cells and in ocular tissues in living monkeys. Further investigation of the etiology of the inflammatory response, possible cytotoxicity, and limited duration of transgene expression is necessary in order to make this technique clinically applicable.     

Herpes simplex virus latency in the cornea

We tried to identify herpes simplex virus type 1 (HSV-1) latency in the cornea obtained at the time of penetrating keratoplasty from patients with herpetic stromal keratitis in the non-active (subsided) stage. The subject consisted of 8 patients (3 males and 5 females; average age 42.3 years) who were diagnosed as having herpetic stromal keratitis and underwent penetrating keratoplasty during a period without active lesions (subsided stage) between August, 1984 and July, 1988. No infective virus was detected in the centrifugation supernatant following each corneal homogenization. Latent virus was detected from the culture supernatant of sections of the corneas in 4 of the 8 patients. Although the ganglion trigger theory of Hill et al. has been conventionally supported as the mechanism(s) of herpetic keratitis recurrence, our results suggest that HSV-1 proliferation from latency in the cornea (peripheral tissue) might stimulate the ganglion (ganglion and skin trigger theory). The present study is the first to demonstrate HSV-1 latency in the cornea.     

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.     

The effects of Xalatan on the recovery of ocular herpes simplex virus type 1 (HSV-1) in the induced reactivation and spontaneous shedding rabbit models.

PURPOSE: Xalatan treatment has been reported both clinically and experimentally to promote recurrences of herpetic keratitis. Our goal was to determine the effects of topical Xalatan and its components on the recovery of ocular herpes simplex virus type 1 (HSV-1) in the Induced Reactivation (IR) and Spontaneous Shedding (SS) HSV-1/NZW rabbit latency models using virological outcome measures. METHODS: HSV-1 latently-infected rabbits in both the IR and SS studies were divided into different topical treatment groups to evaluate commercial Xalatan, its preservatives, and vehicle against appropriate negative and positive controls. In the IR Studies, 91 rabbits received intra-stromal injections of water in both eyes to promote ocular shedding of latent HSV-1. All eyes were then treated and cultured for 10 days. In the SS Studies, 65 rabbits were treated and cultured in both eyes for 30 days. RESULTS: Dexamethasone, a positive control, promoted extensive ocular shedding of HSV-1 in both the IR and SS Models. In general, neither Xalatan nor its components demonstrated any adverse effects, but some experimental variation was noted. All groups demonstrated comparable recovery of latent HSV-1 from respective trigeminal ganglia. CONCLUSIONS: Our experimental studies support the world wide clinical epidemiological experience that commercial Xalatan does not appear to promote HSV-1 ocular shedding.     

Corneal nerves contain intra-axonal HSV-1 after virus reactivation by epinephrine iontophoresis

Experimental ocular models of herpes simplex virus type 1 (HSV-1) reactivation have been used to monitor viral shedding in the tear film and the appearance of corneal epithelial lesions, but the temporal correlation between reactivation and the presence of viral particles in the corneal nerves has not been made. Two New Zealand white rabbits were inoculated with 20 microliters of HSV-1 McKrae strain (5.0 x 10(6) PFU/ml) in each eye. Beginning on postinfection day 82, ocular iontophoresis (0.8 mAmps for 8 min) of 0.01% epinephrine was done once a day for 3 consecutive days to induce reactivation. Ten limbal nerves from four corneas processed for transmission electron microscopy contained 883 unmyelinated and 40 myelinated axons. Seven nerves were positive for virus. Viral particles were found only in unmyelinated axons, and in low frequency (24/883). Virus was not found in Schwann cells, perineurium, or adjacent stroma nor were virus particles seen exiting axons. No enveloped virions were found. Axons from six nerves of four control corneas from rabbits with latent, but not reactivated, HSV-1 did not contain virus particles. Induction by corneal iontophoresis of epinephrine suggests that HSV-1 is translocated from the ganglion to the cornea through axonal transport mechanisms. For the first time, evidence of anterograde, intra-axonal transport of HSV-1 particles in response to epinephrine reactivation is demonstrated.     

The distribution of gentamicin in the rabbit cornea following iontophoresis to the central cornea.

The purpose of this study was to evaluate the penetration of gentamicin into the central, midperipheral and peripheral cornea of rabbits following iontophoresis to the central 3 mm of the cornea. Four groups (groups 1-4) of five rabbits (one eye per rabbit) underwent corneal iontophoresis using gentamicin dissolved in agar. Low (1 mg/ml) and high (10 mg/ml) concentrations of gentamicin in agar were used for one or ten minutes. Two control groups (groups 5 and 6) of five eyes each underwent mock iontophoresis with low and high concentrations of agar-gentamicin mixture. Following sacrifice of the rabbits, the central, midperipheral and peripheral parts of each cornea were excised. Gentamicin concentration was determined in each part of every cornea. High concentrations of gentamicin (951.6 +/- 369.4 microg/ml to 26.6 +/- 41.34 microg/ml) were obtained in the central parts of all the iontophoresis-treated corneas. In each group, except group 6, central corneas had higher concentrations of gentamicin compared to midperipheral corneas (p = 0.038 to p = 0.021), and midperipheral corneas had higher levels than peripheral corneas (p = 0.038 to p = 0.021). Following iontophoresis, gentamicin is found in all portions of the corneas; however, the highest concentration of the drug remains in the central cornea.     

Rapid detection of herpes simplex virus (HSV) antigen in human ocular infections

The new HERPCHEK (Dupont, No. Billerica, MA) enzyme immunosorbent assay (EIA) was used in a double-blind clinical study for rapid and specific detection of ocular herpes simplex virus (HSV) infection. This 4-hour assay can be used to demonstrate conclusively the presence of HSV antigen without culture and thereby rapidly differentiate between HSV and other clinically similar ocular infectious diseases. Ocular samples were collected from 180 individuals including 30 patients with acute HSV, 90 with latent HSV (ie, currently asymptomatic but with a positive history), 11 with acute or latent varicella zoster virus, 30 with nonherpetic infections (due to adenovirus, Acanthamoeba or bacteria), and 19 normal controls. A clinical diagnosis was made by one of us (DPL) and duplicate tear-film samples obtained by swabbing the conjunctival cul-de-sac and cornea. Coded samples were tested by routine viral culture on Vero cell monolayers and also were run independently in the HERPCHEK test. During active HSV infection, the HERPCHEK correlated 100% with clinical diagnosis, and virus culture correlated 90% with clinical diagnosis. In all latent HSV ocular infections, other nonherpetic ocular infections and normal samples, both the HERPCHEK and culture assays were negative.     

Herpes simplex virus keratitis after laser in situ keratomileusis

PURPOSE: To report two cases of herpes simplex virus (HSV) keratitis after laser in situ keratomileusis (LASIK). METHODS: Interventional small case series. Two patients underwent uneventful LASIK. History of herpes labialis in one patient and herpetic eye disease > 10 years prior to intervention in the other patient was reported. Both patients developed stromal herpetic keratitis 6 weeks and 2 years after the procedure, respectively. RESULTS: Treatment consisting of topical steroid drops and topical and systemic antiviral therapy was administered. Recurrences of the herpetic keratitis were seen after tapering of the topical steroids; four and three recurrences were observed, respectively. Final visual acuity was > 6/9 in both cases. CONCLUSIONS: Herpetic keratitis after LASIK is an uncommon, possibly under-reported, entity. Even patients without history of herpetic eye disease can present with this complication. Oral antiviral prophylaxis may be appropriate when performing LASIK on patients with a history of ocular or systemic HSV infection.     

Anti-herpes simplex virus (HSV) effect of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) in rabbit cornea

The anti-herpes simplex virus (HSV) effect and cytotoxicity of a new nucleoside analogue, 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) in rabbit cornea were studied. In tests of the anti-HSV effect of DHPG, even 0.03% ointment, given 5 times per day for 2 days, prevented lesion formation. The preventive effect of DHPG was much stronger than that of acyclovir (ACV) or 5-iodo-2'-deoxyuridine (IDU). In tests on the therapeutic effect of DHPG against dendritic ulcers, 0.3% ointment, given 5 times per day for 4 days, had a dramatic therapeutic effect. The effect was stronger than that of 3% ACV or 0.5% IDU ointment. Application of 0.3%, 1% or 3% DHPG ointment to normal rabbit corneas, 5 times per day for 2 weeks resulted in no histopathological abnormalities. The above results show that DHPG is superior to ACV or IDU for treatment of HSV infections.     

HSV-1上调白细胞介素-18 mRNA在小鼠角膜组织中表达的研究

目的研究单纯疱疹病毒Ⅰ型(HSV-1)感染小鼠眼球后,白细胞介素-18(IL-18)在角膜组织中的表达及IL-18的表达与单纯疱疹性角膜基质炎之间的关系。方法用106PFU的HSV-1感染BALB/c鼠的角膜后,在裂隙灯显微镜下观察角膜的临床变化,组织学检查角膜的病理改变;用RT-PCR和ELISA法检测IL-18在角膜组织中的表达水平。结果HSV-1引起的角膜基质炎在病毒感染后的第10d明显可见,在病毒感染后的第14~21d达到高峰。RT-PCR检测的数据显示:HSV-1感染的早期即可诱导IL-18mRNA在角膜组织中的表达,并且表达持续存在;IL-18mRNA的表达高峰时间(3~21d)位于临床疾病出现之前和临床疾病发展的过程中。ELISA检测角膜提取物中的IL-18蛋白显示了相似的结果。结论HSV-1上调IL-18在小鼠角膜组织中的表达;IL-18的表达与角膜基质炎的发生和发展密切相关;IL-18诱导Th1细胞介导的对单纯疱疹病毒特异性的免疫反应,导致单纯疱疹性角膜基质炎。     

Valacyclovir inhibition of recovery of ocular herpes simplex virus type 1 after experimental reactivation by laser in situ keratomileusis

PURPOSE: To determine whether the systemic administration of valacyclovir (Valtrex) reduces ocular shedding of herpes simplex virus type 1 (HSV-1) after laser in situ keratomileusis (LASIK) in the New Zealand White (NZW) rabbit latency model. SETTING: Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA. METHODS: New Zealand White rabbits latently infected with HSV-1 W strain were divided into 3 groups. The first received 100 mg/kg/day of valacyclovir; the second, 200 mg/kg/day of valacyclovir; and the third (control), saline. One half the total dose of valacyclovir was delivered via intraperitoneal injections twice daily for 7 days beginning with 1 dose before LASIK. The HSV-1 ocular shedding was determined from eye cultures for 7 days after LASIK. RESULTS: The administration of both 100 mg/kg/day and 200 mg/kg/day of valacyclovir significantly reduced the number of eyes (1/16 in both groups) and the total number of HSV-1 shedding days (1/122 and 2/122, respectively) from which HSV-1 was recovered compared to the control group (7/16 [P =.0396] and 14/129 [P <.007], respectively). CONCLUSIONS: Systemic administration of valacyclovir significantly reduced HSV-1 ocular shedding after LASIK in the NZW rabbit latency model. The clinical implications of this study suggest that patients with a history of recurrent ocular herpes may be able to safely have LASIK with less risk of a recurrent herpetic episode while on valacyclovir antiviral prophylaxis.     

Pharmacokinetics of terbinafine in the rabbit ocular tissues after topical administration

OBJECTIVE: To determine the distribution of terbinafine in the cornea and aqueous humor after topical administration. METHODS: A corn oil ointment of terbinafine 0.2% (resolved in sterile corn oil) was applied to the conjunctival sac of albino rabbits twice (with a 5-min interval). The concentration of terbinafine was determined with high-performance liquid chromatography (HPLC) 5, 15, 30, 60, 120 and 240 min after administration of terbinafine. RESULTS: After topical administration, the concentration of terbinafine increased gradually, reached a peak (1.39 microg/ml at 30 min in the cornea and 82.9 ng/ml at 30 min in aqueous humor, respectively) and then decreased. The concentration was 0.18 microg/g at 240 min in the cornea, but terbinafine could not be tested at 120 min in aqueous humor. CONCLUSIONS: Topical ophthalmic terbinafine 0.2% could penetrate into the cornea and aqueous humor at concentrations adequate for inhibition of fungus.     

Dendritic cells in the cornea during Herpes simplex viral infection and inflammation

Herpes simplex keratitis is commonly caused by Herpes simplex virus type 1, which primarily infects eyelids, corneas, or conjunctiva. Herpes simplex virus type 1—through sophisticated interactions with dendritic cells (DCs), a type of antigen-presenting cell)—initiates proinflammatory responses in the cornea. Corneas were once thought to be an immune-privileged region; however, with the recent discovery of DCs that reside in the cornea, this long-held conjecture has been overturned. Therefore, evaluating the clinical, preclinical, and cell-based studies that investigate the roles of DCs in corneas infected with Herpes simplex virus is critical. With in vivo confocal microscopy, animal models, and cell culture experiments, we may further the understanding of the sophisticated interactions of Herpes simplex virus with DCs in the cornea and the molecular mechanism associated with it. It has been shown that specific differentiation of DCs using immunohistochemistry, flow cytometry, and polymerase chain reaction analysis in both human and mice tissues and viral tissue infections are integral to increasing understanding. As for in vivo confocal microscopy, it holds promise as it is the least invasive and a real-time investigation. These tools will facilitate the discovery of various targets to develop new treatments.