Corneal Scarification Research Articles

KLEIP Deficiency in Mice Causes Progressive Corneal Neovascular Dystrophy

The BTB-kelch protein KLEIP/KLHL20 is an actin binding protein that regulates cell-cell contact formation and cell migration. The aim of our study was to characterize KLEIP's function in ocular health and disease in mice. KLEIP(-/-) mice were generated, and corneas were examined histologically and stained for keratin-1, loricrin, keratin-12, keratin-14, CD31, LYVE-1, F4/80, E-cadherin, and Ki67. Corneal abrasions were performed after eyelid opening. Corneas of KLEIP(+/+) and KLEIP(-/-) mice were indistinguishable at birth. After eyelid opening corneal epithelial hyperplasia started to manifest in KLEIP(-/-) mice, showing a progressive epithelial metaplasia leading to total corneal opacity. In KLEIP(-/-) mice the initial stratified squamous corneal epithelium was altered to an epidermal histo-architecture showing several superficial keratinized cells, cell infiltrations into the stroma, and several apoptotic cells. Skin markers keratin 1 and loricrin were positive, and surface disease was accompanied by deep stromal vascularization. Expression analysis for E-cadherin in KLEIP(-/-) corneas showed acellular areas in the squamous epithelium, indicating a progressive fragile corneal integrity. Removal of the virgin epithelium accelerated strongly development of the epithelial and stromal alterations, identifying mechanical injuries as the major trigger for corneal dystrophy formation and scarification in KLEIP(-/-) mice. The data identify KLEIP as an important molecule regulating corneal epithelial integrity.

Herpesvirus Entry Mediator and Nectin-1 Mediate Herpes Simplex Virus 1 Infection of the Murine Cornea

Herpes simplex virus 1 (HSV-1) is a ubiquitous human pathogen that enters cells by the receptor-mediated fusion of the viral envelope with a host cell membrane. The envelope glycoprotein gD of HSV must bind to one of its receptors for entry to take place. Recent studies using knockout (KO) mice demonstrated that the gD receptors herpesvirus entry mediator (HVEM) and nectin-1 are the primary entry receptors for HSV-2 in the mouse vagina and brain. Nectin-1 was most crucial for the neuronal spread of HSV-2, particularly in the brain. HVEM was dispensable for infection in these models, but when both HVEM and nectin-1 were absent, infection was completely prevented. We sought to determine the receptor requirements of HSV-1 in an ocular model of infection using knockout mice. Wild-type, HVEM KO, nectin-1 KO, and HVEM/nectin-1 double-KO mice were infected via corneal scarification and monitored for clinical signs of infection and viral replication in various tissues. We report that either HVEM or nectin-1 must be present for HSV-1 infection of the cornea. Additionally, we observed that the infection was attenuated in both HVEM KO and nectin-1 KO mice. This is in contrast to what was reported for studies of HSV-2 in vagina and brain and suggests that receptor requirements for HSV vary depending on the route of inoculation and/or serotype.

Clinical and Antiviral Efficacy of an Ophthalmic Formulation of Dexamethasone Povidone-Iodine in a Rabbit Model of Adenoviral Keratoconjunctivitis

To determine the efficacy of a new formulation of topical dexamethasone 0.1%/povidone-iodine 0.4% (FST-100) in reducing clinical symptoms and infectious viral titers in a rabbit model of adenoviral keratoconjunctivitis. Rabbit corneas were inoculated bilaterally with 2×10(6) plaque-forming-units (PFU) of adenovirus type 5 (Ad5) after corneal scarification. Animals were randomized 1:1:1:1 (five rabbits per group) to FST-100, 0.5% cidofovir, tobramycin/dexamethasone (Tobradex; Alcon Laboratories, Fort Worth, TX) ophthalmic suspension, and balanced salt solution (BSS; Alcon Laboratories). Treatment began 12 hours after viral inoculation and continued for 7 consecutive days. The eyes were clinically scored daily for scleral inflammation (injection), ocular neovascularization, eyelid inflammation (redness), friability of vasculature, inflammatory discharge (pus), and epiphora (excessive tearing). Eye swabs were collected daily before treatment for the duration of the study. Virus was eluted from the swabs and PFU determined by titration on human A549 cells, according to standard procedures. The FST-100 treatment resulted in significantly lower clinical scores (P<0.05) than did the other treatments. The 0.5% cidofovir exhibited the most ocular toxicity compared with FST-100, tobramycin/dexamethasone, and balanced salt solution treatments. FST-100 and 0.5% cidofovir significantly (P<0.05) reduced viral titers compared with tobramycin/dexamethasone or balanced salt solution. FST-100 was the most efficacious in minimizing the clinical symptoms of adenovirus infection in rabbit eyes. FST-100 and 0.5% cidofovir were both equally effective in reducing viral titers and decreasing the duration of viral shedding. By providing symptomatic relief in addition to reducing infectious virus titers, FST-100 should be a valuable addition to treatment of epidemic adenoviral keratoconjunctivitis.

The In Vitro and In Vivo Evaluation of ddC as a Topical Antiviral for Ocular Adenovirus Infections

To evaluate the antiviral activity of 2', 3'-dideoxycytidine (ddC) in vitro against a panel of ocular adenovirus serotypes and in vivo in the ocular Ad5/NZW rabbit replication model. In vitro, the 50% inhibitory concentrations (IC(50)) of ddC and cidofovir were determined using standard plaque-reduction assays. In vivo, 40 rabbits were topically inoculated in both eyes with Ad5 after corneal scarification. On day 1, the rabbits were equally divided into four topical treatment groups: 3% ddC; 2% ddC; 0.5% cidofovir; and saline. ddC and saline eyes were treated four times daily for 7 days, and cidofovir-treated eyes were treated twice daily for 7 days. Eyes were cultured for virus a multiple times over 2 weeks. The in vitro IC(50) for ddC ranged from 0.18 to 1.85 microg/mL, whereas those for cidofovir ranged from 0.018 to 5.47 microg/mL. ddC was more potent than cidofovir for seven of nine serotypes. In vivo, 3% ddC, 2% ddC, and 0.5% cidofovir significantly reduced the number of Ad5-positive cultures per total (days 1-14), mean Ad5 ocular titer (days 1-5), and duration of shedding (among other outcome measures) compared with the saline control. The 3% and 2% ddC treatments were significantly more efficacious than the 0.5% cidofovir treatment in the parameters listed above. ddC demonstrated potent antiadenovirus activity in vitro and in vivo. Systemic safety studies after topical ocular administration are needed to evaluate ddC as a topical antiviral treatment for adenoviral ocular infections in the target population.

Expression of Matrix Metalloproteinases 2 and 9 in Experimental Corneal Injury and Fungal Keratitis

Levels of matrix metalloproteinases (MMPs) can be modulated during corneal infection, but little is known about MMP profiles during fungal keratitis. The purpose of this study was to determine the effect of corneal trauma and immunosuppressive treatment on the expression kinetics of MMP-2 and MMP-9 during experimental keratomycosis. Corneas of immunocompetent and cyclophosphamide-treated adult BALB/c mice were topically inoculated with 1 x 10 culturable units of Fusarium solani or mock-inoculated with or without superficial corneal scarification. Eyes were scored daily for disease severity and processed for zymography after 1.5 hours, 6 hours, 1 day, 4 days, or 8 days. Gelatinase activity was densitometrically quantitated and normalized to MMP-2 and MMP-9 controls. MMP-9 levels in nontraumatized eyes transiently increased at 6 hours after fungal exposure, but this increase was inhibited by cyclophosphamide treatment. Corneal injury significantly induced early MMP-9 expression that returned to baseline levels within 4 days. Cyclophosphamide pretreatment reduced and delayed MMP-9 after scarification. Fusarium exposure dampened the MMP-9 response to corneal trauma in immunocompetent and cyclophosphamide-treated animals. Ocular levels of MMP-2 were not affected by scarification, fungal exposure, or immunosuppressive treatment. Ocular MMP-9 levels, but not MMP-2 levels, increased soon after corneal injury. A similar, although muted, MMP-9 response occurs during early filamentous fungal keratitis, with a kinetic profile similar to corneal disease progression. The early stage of ulcerative keratitis may involve selective regulation of corneal matrix metalloproteinases, suggesting an initial opportunity for therapeutic intervention.

Dynamic Expression of Chemokines and the Infiltration of Inflammatory Cells in the HSV-Infected Cornea and its Associated Tissues

Background: The chemotactic signals regulating cell trafficking in the herpes simplex virus type 1 (HSV-1) infected cornea are well documented, however, those in the cornea-associated tissues, such as the trigeminal ganglion (TG) and draining lymph nodes (LNs), are largely unknown. Objectives: To examine chemokine expression and subsequent cell infiltration in the HSV-1 infected cornea and its associated tissues. Study design: Eight-week-old female BALB/c mice were infected with 10 μ l HSV-1 (CHR3 strain: 5 × 106 PFU/ml) by corneal scarification. Total RNAs were extracted from the corneas, TGs, and LNs at pre-inoculation, 3 days post-inoculation (P.I.) and 7 days P.I. The mRNA for 28 different chemokines in the extracts was amplified by RT-PCR. Infiltrating cells were identified by immunohistochemistry. Result: After the HSV-1 infection, the corneal stroma became edematous by infiltrated cells under the eroded epithelium. The TG and LNs were markedly swollen. The cornea was infiltrated with granulocytes and CD11b+ cells at 3 days P.I., followed by CD4+ and CD8+ T cells at 12 days P.I. In the TG, CD11b+ cells, but no granulocytes, infiltrated throughout the observation period. T cells migrated into the TG earlier than into the cornea. Gene expressions of neutrophil-attracting chemokines (CXCL1, 2, 3, and 5) increased in the cornea, but they did not enhance in the TG or LNs. On the other hand, gene expressions of chemokines which attract CD11b+ cells such as CCL2, 8, 7, 12, CCL3, 4, and CCL5, increased in the cornea and TG with its peak at 3 days P.I. Gene expressions of chemokines those work on T cells and B cells, such as CCL19, CCL21, CXCL9, CXCL13, CXCL10, XCL1, and CXCL16, were up-regulated and peaked at 3 days P.I. in the cornea and in the TG. Thus, pattern of chemokine gene expression was similar in the cornea and in the TG. On the contrary, gene expressions of chemokines in the draining LNs affecting CD11b+ cells and T cells were temporarily down-regulated. Conclusion: Upon HSV-1 infection, dynamic gene expression of chemokines was observed not only in the inoculated cornea but also in its associated tissues.

Apolipoprotein E Modulates Establishment of HSV-1 Latency and Survival in a Mouse Ocular Model

Purpose: To evaluate and compare the neuroinvasiveness and neurovirulence after ocular HSV-1 infection in ApoE knockout (ApoE−/−) and control C57BL/6 (ApoE+/+) mice. Methods: Age-matched (14 weeks of age) C57BL/6J (ApoE+/+) female mice and female ApoE knockout (ApoE−/−) mice were inoculated by corneal scarification with HSV-1 strain 17Syn+. Analysis of HSV-1 replication in the mouse cornea was assessed through infectious virus assays of ocular (tear film) swabs at 1 to 5 days postinoculation (PI), slit-lamp examination (SLE) of corneas at PI days 1 to 7, and survival of infected mice. The contribution of apoE to the efficient establishment of latency was measured by real-time PCR quantitation of the latent viral genome in the trigeminal ganglia (TG) of infected mice. Results: These studies showed that HSV-1 strain 17Syn+ replicates efficiently in the eyes, regardless of the host ApoE genotype. Neither the scoring of corneal pathology via SLE nor the infectious virus assay of the tear film resulted in any statistical differences between ApoE knockout (−/−) mice or the C57BL/6 (ApoE+/+) mice. In mice latently infected with HSV-1, our real-time PCR data showed significantly lower viral copy numbers of HSV-1 DNA in ApoE knockout (ApoE−/−) mice compared with C57BL/6 (ApoE+/+) mice. C57BL/6 (ApoE+/+) mice are more susceptible to the neurovirulence of HSV-1 strain 17Syn+ than female ApoE knockout (−/−) mice, as demonstrated by the fact that 50% (7/14) of the female C57BL/6 (ApoE+/+) mice inoculated with 17Syn+ died, as opposed to none (0/14) of the age- and sex-matched ApoE knockout mice. Conclusions: These data indicate that age (14 weeks) and sex-matched (female) wild mice with an ApoE null background (ApoE−/−) are more resistant and less efficient in the establishment of latency compared with ApoE+/+ mice in the C57BL/6 background.

Neuronal activity regulates viral replication of herpes simplex virus type 1 in the nervous system

Herpes simplex virus types 1 and 2 (HSV-1, -2) infect and also establish latency in neurons. In the present study, the authors investigated the influence of neuronal activity on the replication of HSV-1. The results showed that the sodium channel blocker tetrodotoxin (TTX) and the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) could significantly increase viral replication in primary neuronal cultures, by two- to fourfold. In contrast, KCl reduced viral production by at least 80% in the same cultures. Inhibitors of GABA(A) receptors completely abolished the effects of GABA. Intravitreously injected TTX in a mouse corneal scarification model enhanced the viral titers > 10-fold in both the trigeminal ganglia and the brain. At 2 h post infection, both TTX and GABA significantly up-regulated the levels of transcription for the viral immediate early (IE) genes ICP0, ICP4, and ICP27, as revealed by real time PCR. These results indicate that the neuronal excitation status may dictate the efficiency of HSV-1 viral replication, probably by regulating the levels of viral IE gene expression. These are the first findings connecting neuronal activity to the molecular mechanisms of HSV replication in the nervous system, which may significantly influence our view of herpesvirus infection and latency.

Immunosuppression affects the severity of experimental Fusarium solani keratitis.

We have established a mouse model of corneal fusariosis that permits the evaluation of fungal infection and pathogenesis. Corneas of immunocompetent and cyclophosphamide-treated adult BALB/c mice were topically inoculated with Fusarium solani after corneal scarification. Eyes were scored for corneal involvement daily for 8 days and at 2 weeks after infection. Eyes were enucleated at various time points for quantitative fungal recovery and histopathological examination. An inoculum-dose response was observed in cyclophosphamide-treated mice, and fungi were recovered from the infected eyes by quantitative microbial culturing. Treatment with cyclophosphamide increased disease severity and delayed fungal clearance. Fungal hyphae, inflammatory cells, and stromal edema were histologically evident within corneal tissue and correlated with disease severity. Although the mouse cornea resists fungal infections, F. solani keratitis could be induced in immunosuppressed mice after surface scarification, which resulted in infection and clinical disease that could be evaluated both in vivo and in vitro.

Glycoprotein C of herpes simplex virus type 1 is required to cause keratitis at low infectious doses in intact rabbit corneas

Purpose. To determine whether the herpes simplex virus type 1 (HSV-1) viral glycoprotein C (gC) plays a role in induction of keratitis in unscarified and scarified rabbit eyes.Materials and methods. A gC deletion mutant (ΔgC) was constructed and then rescued back to wild type (wt) for use as a control. Following ocular infection with each virus in rabbit eyes, with or without prior corneal scarification, keratitis was compared.Results. At low infection doses of 2 × 103 and 2 × 104 plaque-forming units (PFU)/eye, in unscarified cornea, ΔgC produced significantly less keratitis than did wt virus (p = 0.007 and 0.03, respectively). In contrast, the keratitis induced by ΔgC was similar to that induced by the wt virus (p > 0.60) in scarified cornea. At high infection dose (2 × 105 PFU/eye), keratitis induced by ΔgC was similar in scarified and unscarified cornea, and the severity of disease was similar to that seen in scarified eyes at the low-dose ΔgC infections. Interestingly, although ΔgC induced keratitis with or without corneal scarification at high infection doses, the severity of disease was significantly less than that induced by wt infection. At all infection doses, keratitis induced by wt infection was similar in scarified and unscarified eyes.Conclusions. These results suggest that (1) at low infection doses, in unscarified corneas, gC is required for HSV-1 induced keratitis; (2) corneal scarification prior to infection can circumvent the need for gC at low doses, but (3) at higher doses, gC is required for wild-type levels of keratitis even in scarified cornea.

Increased susceptibility to herpes simplex virus type I (HSV-I) ocular infection in rats fed with low-protein diets

In order to study the susceptibility to Herpes Simplex Virus type 1 (HSV-1) infection in malnourished rats, groups of ten animals were fed with diets containing 8% or 20% of protein for 15 days before infection. Then, animals were infected in the eye by corneal scarification with 10 5 plaque forming units of HSV-1, strain F. Significant increases ( p < 0.05) in morbility and mortality as well as higher viral titers in ocular washings were observed in the malnourished group. This group showed a diminution in lymphocyte antigen dependent proliferation compared with control, and a significantly lower delayed type hypersensitivity reaction against inactivated virus ( p < 0.05). Neutralizing antibodies titers in serum were lower in the malnourished group ( p < 0.05). When malnourished animals were shifted to a diet containing 20% protein for 15 days, all parameters showed no significant differences compared with control. Thus, we conclude that malnutrition diminished the capability of the animals to overcome HSV-1 infection.

Experimental keratomycosis in a mouse model.

To establish a murine model of corneal candidiasis that permits molecular evaluation of fungal adherence and invasion. Corneas of immunocompetent, methylprednisolone-treated, and cyclophosphamide-treated adult NIH Swiss and BALB/c mice were topically mock inoculated or inoculated with 10-fold increasing amounts between 100 and 100 million colony-forming units (CFU) of Candida albicans after unilateral corneal scarification. Mock-inoculated eyes served as the control. Eyes were scored daily on a 12-point scale to categorize corneal inflammation and were enucleated for quantitative fungal cultures, analysis by polymerase chain reaction (PCR), and histopathologic examination. At least 100 CFU of C. albicans initiated measurable corneal infection, but 1 million or more colony-forming units were needed to induce consistent keratitis. Treatment with methylprednisolone increased disease severity in infected BALB/c mice and fungal persistence in both BALB/c and NIH Swiss mice. Treatment with cyclophosphamide increased disease severity and fungal persistence in both strains of mice. Infectious organisms were recovered by quantitative culture, and candidal DNA was detectable by PCR. C. albicans, inflammatory cells, and stromal necrosis were histologically evident within ocular tissue. Although mice are innately resistant to Candida infection after corneal inoculation, moderate to severe keratomycosis can be established in immunocompromised mice by the route of corneal scarification. Although differences between mouse strains and among immunosuppressive regimens remain to be explored, this murine model provides the basis for understanding the pathogenesis of fungal infections of the cornea.

Mapping of genes involved in murine herpes simplex virus keratitis: identification of genes and their modifiers.

Herpes simplex keratitis (HSK) is an inflammatory response to viral infection and self antigens in the cornea and is a major cause of blindness. Using two strains of mice which are susceptible (129/SVEV) and resistant (C57BL/6) to herpes simplex virus (HSV) strain KOS, (129/SVEV x C57BL/6)F(2) mice were generated and examined for their disease susceptibility in terms of clinical symptoms, ocular disease, and antibody production following corneal scarification with HSV (KOS). A genome-wide screen was carried out using microsatellite markers to determine the genetic loci involved in this response. Loci on chromosomes 4, 5, 12, 13, and 14 were shown to be involved in general susceptibility to clinical disease, whereas loci on chromosomes 10 and 17 were shown to be unique to ocular disease.

Internal and external contamination of donor corneas before in situ excision: bacterial risk factors in 93 donors.

Most studies of corneal donor contamination concentrate on postenucleation contamination of the eyeball. The purpose of the present study was to evaluate the relative contamination of in situ excised corneal tissue and relevance to final success or rejection by recipients of the corneal grafts. Ninety-three donors underwent anterior chamber puncture (ACP) and corneal epithelium scarification (CS) before and after disinfection with 5% povidone-iodine. Following in situ excision, corneas were preserved in culture medium at +35 degrees C. Morphological and bacteriological assessment was carried out after culture, and recipients were followed up over a 2-year period. Samples taken by ACP, CS before disinfection, CS after disinfection and a culture medium sample were contaminated by bacteria in, respectively, 8 (8.6%), 23 (24.7%) 4 (4.3%) and 5 (5.4%) donors. Contamination of aqueous humor was significantly associated with age, death-to-sample time and premortem systemic infection. Contamination of epithelium significantly increased culture medium contamination. External bacteria on donor cornea are mainly skin bacteria (especially Staphylococcus) and can be partially eliminated by a povidone-iodine wash. Internal bacteria are mainly gut bacteria and may be due to perimortem bacteriemia. However, bacterial infection at the time of death appears to have no effect on the incidence of endophthalmitis in recipients and should no longer prevent use of such corneal tissue in grafts.

Herpes simplex virus ophthalmic disease induced using two different methods of mice inoculation

Two different procedures for inoculation of HSV on corneas of BALB/c mice were evaluated. The first was by the use of HSV suspensions directly on the corneas and the other was after corneal scarification. Animals by this later method presented greater morbidity and mortality than those of first group, suggesting that inoculation of HSV without scarification of the cornea should be the method of choice for the study of HSV ophthalmic infection. This model showed also be an efficient experimental system to testing antiviral drugs.

Increased severity of herpes simplex virus type 1-induced keratitis in Hox A5 transgenic mice

Purpose. Herpes simplex virus type 1 is a major cause of stromal keratitis and blindness in humans. Understanding of the role of host genes in the pathogenesis of herpes stromal keratitis is limited. We used a transgenic mouse model to examine the effect of a host gene, Hox A5 (which binds to the TAATGARAT sequence in the promoter regions of HSV-1 immediate early genes and increases HSV-1 replication), on the pathogenesis of HSV-1 induced stromal keratitis. Methods. Corneas of wildtype and Hox A5 transgenic mice were infected with HSV-1 strain F following corneal scarification. Clinical severity of keratitis was evaluated using slit-lamp biomicroscopy. Histologic severity of keratitis was determined by light microscopic evaluation and by computerized morphometry. Ocular viral replication was measured via plaque assay. Results. Clinical lesions of stromal keratitis were more severe at 17 and 23 days post infection in Hox A5 transgenic mice than in wildtype mice. Histological evaluation and morphometric analysis confirmed that keratitis lesions were more severe in the transgenic mice. HSV-1 replication was approximately 100-fold greater in the corneas of transgenic mice than in wildtype mice. Conclusions. Our results demonstrate that a host gene (Hox A5) can increase ocular replication of HSV-1 and alter the pathogenesis of herpetic stromal keratitis.

Corneal Insult Affects the Production and Distribution of FGF-2 within the Lacrimal Gland

The purpose of this study was to determine the distribution of FGF-2 within rabbit lacrimal glands and to determine whether corneal insult affects that distribution. The scarified corneas of experimental animals were inoculated either with adenovirus type 5 or buffer. Control animals were either untreated, or animals whose corneas were scarified. Twenty-one days later all animals were killed and the lacrimal glands were studied by immunocytochemistry and Western blotting to detect FGF-2. In untreated control animals, FGF-2 was immunolocalized predominantly within a population of elongated cells in the basal epithelium of ducts, and to a lesser degree in the basal epithelium of the acini. The elongated immunopositive cells appear to be myoepithelial cells known to be present at these sites. Interstitial cells around ducts and acini, and the basement membranes of the ducts and acini, were also immunopositive for FGF-2. Twenty-one days after adenovirus inoculation and scarification of the cornea, immunopositivity for FGF-2 was dramatically decreased in basement membranes, but increased within myoepithelial cells of the duct epithelium. These myoepithelial cells were frequently enlarged, bulging toward the duct lumen. In animals whose corneas were inoculated with buffer and scarified, or animals whose corneas were simply scarified, the changes in the lacrimal gland were similar, but somewhat less pronounced, to those of adenovirus-inoculated animals. Western blots confirmed the presence of FGF-2 immunoreactivity in all groups. The major band in untreated controls was at 24kD, whereas all animals with corneal scarification had major bands at 38kD. Densitometry of Western blots demonstrated that the amount of 24kD FGF-2 present within the lacrimal gland after corneal scarification was at least 50% less than in untreated controls, whereas 38kD FGF-2 was at least ten-fold greater. Our findings indicate that corneal scarification results in an altered distribution of FGF-2 within the lacrimal gland, which involves a decrease in low molecular weight FGF-2 and a dramatic increase in a higher molecular weight isoform of FGF-2. FGF-2 may be released from myoepithelial cells apically (exocrine) into the tear fluid and basally (autocrine/paracrine) into the connective tissue, as well as from extracellular complexes within basal laminae.

Therapeutic efficacy of DNA encoding IFN-a1 against corneal HSV-1 infection

Purpose. Type I interferons (IFN-a and -ß) are an innate immune component that plays a critical role in controlling herpes simplex virus type 1 (HSV-1) infection. We have previously shown that topical administration of a plasmid DNA encoding IFN-a1 onto mouse corneas prior to ocular HSV-1 infection provided prophylactic efficacy against HSV-1-induced encephalitis. As a result, the present study was undertaken to investigate the kinetics of the efficacy mediated by the IFN-a1 transgene following ocular challenge with HSV-1. Methods. Mice were ocularly infected with a lethal dose of HSV-1 (450 plaque forming units/eye, McKrae strain) following corneal scarification and topically administered the pCMV-IFN-a1 transgene or pCMV-ß (plasmid vector) starting at 12, 24, or 48 hr post infection. Cumulative survival of infected mice was recorded. In addition, the effect of the transgene on viral replication and viral gene expression was determined from tissues 3 and 6 days post infection by plaque assay and RT-PCR respectively. Results. Mice treated with the pCMV-IFNa1 transgene survived to a greater degree compared to mice topically administered the plasmid vector alone in a time-dependent manner. The protective effect correlated with a decrease in the viral load and expression of HSV-1 immediate early and early gene transcripts, infected cell protein–27 and thymidine kinase respectively in the trigeminal ganglion 6 days post infection. Conclusion. These results suggest that the application of plasmid DNA encoding IFN-a1 transgene is beneficial as a therapeutic approach when applied early after HSV-1 infection of the corneas.

The heterogeneity in the immune response and efficiency of viral dissemination in brain infected with herpes simplex virus type 1 through peripheral or central route.

Using immunohistochemistry on adjacent brain sections, we studied the correlation between the dissemination of the virus, the inflammatory responses and the expression of major histocompatibility complex (MHC) proteins in rat brain infected with herpes simplex virus (HSV-1) F strain by either corneal scarification or intracerebral injection. Our results showed that the mortality of the corneally infected rats was much higher than that of the intracerebrally infected rats, due to a more extensive dissemination of the virus in the brain, particularly in the brain stem. The inflammatory responses were similar in brains infected through either route, as demonstrated by the expression of MHC I/II antigens on infiltrating lymphocytes, leukocytes and macrophage/microglia cells. While there was strong immunoreactivity for HSV-1 antigens in the cerebral cortex, the infiltrates were only located in subcortical areas, especially the hippocampus. Therefore, the distribution of these immune cells did not always overlap with the regions of viral infection. These results suggest that HSV-1 disseminate more efficiently from the peripheral to the central nervous system (CNS) than from CNS to CNS, which is independent of the immune responses, and that the cerebral cortex may immunologically respond to HSV-1 infection differently from other brain regions.

Morphine reduces mortality in mice following ocular infection with HSV-1.

To determine the effect of chronic opioid treatment on mice infected with herpes simplex virus (HSV-1), female ICR mice were administered saline or morphine (25 mg/kg) subcutaneously (s.c.) 3 X /day and infected five days later via corneal scarification and inoculation with 150-210 plaque forming units of HSV-1. Mice deprived of morphine succumbed following infection faster than morphine- or saline-maintained mice, and morphine-maintained mice had a higher cumulative survival than either saline-maintained or morphine-deprived mice. There was no significant difference in cytokine mRNA or protein levels by RT-PCR and ELISA, respectively, in the eyes, trigeminal ganglia, cerebellum, or brain stem, comparing morphine-maintained to saline-treated mice. Similarly, there were no differences in the viral load in the eyes and trigeminal ganglia during the acute infection comparing these two groups assayed three and six days post-infection. While there were no differences in the expression of viral transcripts in the eyes and trigeminal ganglia during the acute infection, HSV-1 infected cell polypeptide 27 expression was reduced in the brain stem of morphine-maintained mice. Collectively, the results suggest that mice maintained on morphine antagonize the spread of HSV-1 in the central nervous system and thus, reduce the incidence of viral-induced encephalitis.