Urokinase-type plasminogen activator promotes corneal epithelial migration and nerve regeneration.

Urokinase-type plasminogen activator (uPA) is a serine protease that plays a central role in the pericellular fibrinolytic system, mediates the degradation of extracellular matrix proteins and activation of growth factors, and contributes to the regulation of various cellular processes including cell migration and adhesion, chemotaxis, and angiogenesis. The corneal epithelium responds rapidly to injury by initiating a wound healing process that involves cell migration, cell proliferation, and tissue remodeling. It is innervated by sensory nerve endings that play an important role in the maintenance of corneal epithelial homeostasis and in the wound healing response. We here investigated the role of uPA in corneal nerve regeneration and epithelial resurfacing after corneal injury with the use of uPA-deficient mice. Both the structure of the corneal epithelium and the pattern of corneal innervation in uPA-/- mice appeared indistinguishable from those in uPA+/+ mice. Whereas the cornea was completely resurfaced by 36-48 h after epithelial scraping in uPA+/+ mice, however, such resurfacing required at least 72 h in uPA-/- mice. Restoration of epithelial stratification was also impaired in the mutant mice. Fibrin zymography revealed that the expression of uPA increased after corneal epithelial scraping and returned to basal levels in association with completion of re-epithelialization in wild-type animals. Staining of corneal whole-mount preparations for ßIII-tubulin also revealed that the regeneration of corneal nerves after injury was markedly delayed in uPA-/- mice compared with uPA+/+ mice. Our results thus demonstrate an important role for uPA in both corneal nerve regeneration and epithelial migration after epithelial debridement, and they may provide a basis for the development of new treatments for neurotrophic keratopathy.

Urokinase-type plasminogen activator negatively regulates α-smooth muscle actin expression via Endo180 and the uPA receptor in corneal fibroblasts.

Corneal fibroblasts are embedded within an extracellular matrix composed largely of collagen type 1, proteoglycans, and other proteins in the corneal stroma, and their morphology and function are subject to continuous regulation by collagen. During wound healing and in various pathological conditions, corneal fibroblasts differentiate into myofibroblasts characterized by the expression of α-smooth muscle actin (α-SMA). Endo180, also known as urokinase-type plasminogen activator (uPA) receptor-associated protein (uPARAP), is a collagen receptor. Here we investigated whether targeting of Endo180 and the uPA receptor (uPAR) by uPA might play a role in the regulation of α-SMA expression by culturing corneal fibroblasts derived from uPA-deficient (uPA-/-) or wild-type (uPA+/+) mice in a collagen gel or on plastic. The expression of α-SMA was upregulated, the amounts of full-length Endo180 and uPAR were increased, and the levels of both transforming growth factor-ß (TGF-ß) expression and Smad3 phosphorylation were higher in uPA-/- corneal fibroblasts compared with uPA+/+ cells under the collagen gel culture condition. Antibodies to Endo180 inhibited these effects of uPA deficiency on α-SMA and TGF-ß expression, whereas a TGF-ß signaling inhibitor blocked the effects on Smad3 phosphorylation and α-SMA expression. Our results suggest that uPA deficiency might promote the interaction between collagen and Endo180 and thereby increase α-SMA expression in a manner dependent on TGF-ß signaling. Expression of α-SMA is thus negatively regulated by uPA through targeting of Endo180 and uPAR.

Multicenter prospective observational study of fungal keratitis in Japan: analyses of culture-positive cases.

PURPOSE: To investigate the clinical characteristics and causative fungi in patients with fungal keratitis in Japan, and to determine factors related to the prognosis. STUDY DESIGN: Multicenter prospective observational study. METHODS: Eligible patients were enrolled from November 2011 to October 2013 at the 1st stage and from April 2015 to March 2016 at the 2nd stage. The corneal foci were scraped, and the scrapings were cultured in potato dextrose agar. The isolated fungi were identified by gene analyses. Data were collected from the clinical records and statistically analyzed by Cox and logistic regression analyses. RESULTS: Ninety-four fungal strains were isolated from 93 cases, including 42 yeast-like fungi and 52 filamentous fungi. The fungi affected the deep layers of the cornea in 23 cases (24.7%) and the peripheral cornea in 29 cases (31.2%). The incidences of lid swelling/redness, ciliary injection, anterior chamber cells/flare, anterior chamber fibrin, and hyphate ulcer in cases of filamentous fungi were significantly higher than in yeast-like fungi. No history of topical steroids, absence of a main lesion in the peripheral cornea, and best-corrected visual acuity (BCVA) of more than 0.04 at the first visit were related to a shorter healing time. No history of ocular surgery, absence of lesion at one-third deep stromal layer and BCVA of more than 0.04 at the first visit were correlated with BCVA at 3 months after the initial examination. CONCLUSION: Fungal keratitis is caused by various species of fungi and can become refractory due to poor prognosis factors.

Multicenter prospective observational study of fungal keratitis in Japan: analyses of in vitro susceptibility tests for combinations of drugs.

PURPOSE: To determine the effects of a combination of two antifungal drugs against causative fungi of fungal keratitis in Japan. STUDY DESIGN: Multicenter prospective observational study. METHODS: Eighteen isolates of yeast-like fungi and 22 isolates of filamentous fungi collected by the Multicenter Prospective Observational Study of Fungal Keratitis in Japan were studied. Specially manufactured minimum inhibitory concentration (MIC) measurement plates were used to test the effectiveness of 10 combinations of two antifungal drugs against the isolates. The combinations were pimaricin (PMR) + voriconazole (VRCZ), PMR + fluconazole (FLCZ), PMR + miconazole (MCZ), PMR + micafungin (MCFG), VRCZ + FLCZ, VRCZ + MCZ, VRCZ + MCFG, VRCZ + amphotericin-B (AMPH-B), MCZ + FLCZ, and MCZ + MCFG. The checkerboard microdilution method was used, and the fractional inhibitory concentration (FIC) index was calculated based on the guidelines of The Clinical & Laboratory Standards Institute (CLSI). RESULTS: In yeast-like fungi, additive effects were observed between PMR and MCFG in 77.8% of the isolates, and they were also observed between the azoles. Synergistic effects were observed on 11.1% of the isolates for MCZ and FLCZ. On the other hand, antagonistic effects were present between PMR and azoles with 88.9% between PMR and VRCZ, 72.2% between PMR and FLCZ, and 94.4% between PMR and MCZ. In filamentous fungi, additive effects were observed between PMR and MCFG in 40.9% of the isolates, and between VRCZ and MCZ in 40.9% of the isolates. Antagonistic effects were observed for PMR and the azoles. CONCLUSIONS: The combination of drugs prescribed for fungal keratitis incurs a possibility of synergistic, additive, indifferent, or antagonistic effects, depending on drug combinations and fungal strains.

Severe Ocular Complications Associated With Wearing of Contact Lens in Japan.

OBJECTIVES: To examine the severe ocular complications associated with contact lens wearing in Japan. METHOD: A questionnaire was sent to 964 ophthalmologist training facilities inquiring for cases of contact lens-associated complications from April 2016 to March 2018. The inclusion criteria were as follows: (1) corrected distance visual acuity ≤0.1 decimal after treatment for 3 months, (2) corneal perforation observed during follow-up, and (3) requiring surgery. A secondary analysis was conducted, inquiring for further information on the type of contact lens, clinical manifestations, and course of treatment. RESULTS: Forty-two patients with infectious keratitis met the inclusion criteria. Eight patients were users of rigid gas-permeable contact lens, and 34 were users of soft contact lens. Microbiological tests were positive in 73.0%. The organisms isolated in microbiological culture were bacteria in 11 patients (Pseudomonas aeruginosa in 9 patients), fungi in 2 patients, and Acanthamoeba in 14 patients. Ten patients were treated with local antibiotics, 11 with a combination of systemic antibiotics, and 21 with a combination of surgical approaches, including 13 with corneal transplantation. CONCLUSIONS: The major cause of serious contact lens-associated ocular complications was microbial keratitis, and P. aeruginosa and Acanthamoeba were the major pathogens in Japan.

Short-term Interim Results of Clinical Outcomes and Complications After Implantation of Boston Keratoprosthesis in Japanese Patients.

Keratoprosthesis is used for corneal transplantation in high-risk patients who require penetrating keratoplasty (PKP). Boston keratoprosthesis (BKpro) is a representative type of keratoprosthesis used worldwide. In Japan, the first BKpro was implanted in an eye after multiple corneal graft failures in 2008, but its use remains limited. A recent patient survey revealed that among the Japanese patients who had previously undergone multiple PKPs, the retention rate of BKpro was significantly higher than that of PKP at 5 years postoperatively (100% vs. 26%; P < 0.01). Patients with implanted BKpro also had better best corrected visual acuity of 20/200 or higher than those with PKP at 5 years postoperatively (80.0% vs. 17.6%; P = 0.03). Regarding the postoperative complications, retroprosthetic membrane formation was observed in 88.9%, infectious keratitis in 33.3%, and glaucoma progression in 11.1% of cases. Another retrospective analysis showed that fungal keratitis occurred in 0.09 patients per year and severely affected visual acuity. Furthermore, because it is difficult to accurately examine intraocular pressure after BKpro implantation, the intraocular pressure of patients with implanted BKpro was prospectively estimated using a transpalpebral tonometer (Diaton). In conclusion, BKpro implantation is effective and safe for Japanese patients, given the reported improvements in visual acuity and low rates of complications.

Corneal Perforation during Combination Chemotherapy including Cetuximab in a Patient with a History of Herpetic Keratitis.

PURPOSE: To report a case of corneal perforation, in a patient with a history of herpetic keratitis, during combination chemotherapy including cetuximab. CASE: We report the case of a 71-year-old man who was diagnosed with a hypopharyngeal carcinoma and received radiation therapy combined with cetuximab, the epidermal growth factor receptor (EGFR) inhibitor monoclonal antibody. He was referred to us because of ocular hyperemia and corneal perforation in his left eye. In spite of conservative therapy, his corneal perforation was exacerbated, with iris incarceration into the wound site and exposure to the surface of the cornea. He therefore discontinued treatment with the combination chemotherapy and underwent lamellar keratoplasty using a preserved donor cornea. After treatment with cetuximab resumed, there was no recurrence of the corneal perforation. CONCLUSION: We have presented the first case of cetuximab-related corneal perforation in a patient who had a history of recurrent herpetic keratitis. EGFR inhibitors, such as cetuximab, can induce corneal perforation in cases with a history of herpetic stromal keratitis.

Microsporidial keratitis retrospectively diagnosed by ultrastructural study of formalin-fixed paraffin-embedded corneal tissue: a case report.

BACKGROUND: The utility of formalin-fixed paraffin-embedded (FFPE) corneal tissue specimens for retrospective diagnosis of microsporidial keratitis was evaluated by transmission electron microscopy (TEM) analysis and the possible second case of microsporidial keratitis after Descemet stripping automated endothelial keratoplasty (DSAEK) was described. CASE PRESENTATION: A 68-year-old man presented with multiple crystalline opacities in the corneal stroma that progressed extremely slowly after DSAEK. Fungiflora Y staining of corneal scrapings from the affected regions revealed an oval microorganism. Topical voriconazole administration was ineffective and penetrating keratoplasty was performed. Histological and molecular analyses were carried out on the excised cornea. Ziehl-Neelsen staining revealed an acid-fast, oval organism that was visible by ultraviolet illumination after Fungiflora Y and Uvitex 2B staining, whereas periodic acid-Schiff and Grocott's staining did not yield any significant findings. Microsporidium was detected by TEM of FFPE tissue. Nosema or Vittaforma sp. was suspected as the causative microorganism by PCR of FFPE tissue and by the fact that those species are known to cause eye infection. The corneal graft has maintained transparency at 1 year and half postoperatively. CONCLUSIONS: This is the first known case of microsporidial keratitis diagnosed retrospectively by molecular and ultrastructural study of FFPE tissue, and the possible second case of microsporidial keratitis after DSAEK. Microsporidial keratitis should be considered when corneal opacity refractory to conventionally known therapy would occur after DSAEK. Our findings suggest that more microsporidial keratitis cases than have been reported to date can be identified by TEM or PCR examination of FFPE corneal specimens.

Neurotrophic Keratopathy after Trigeminal Nerve Block for Treatment of Postherpetic Neuralgia.

PURPOSE: To report a case of persistent corneal epithelial defect that had occurred after a trigeminal nerve block. CASE PRESENTATION: A 75-year-old female had suffered from postherpetic neuralgia for 8 years. She underwent Gasserian ganglion block surgery and noticed declining visual acuity in the right eye on the following day. She presented with severe hyperemia and corneal epithelial defects in the right eye and experienced remarkable reduction of sensitivity in the right cornea. She was diagnosed with neurotrophic keratopathy. Ofloxacin eye ointment and rebamipide ophthalmic suspension ameliorated the corneal epithelial defects but superficial punctate keratopathy, corneal superficial neovascularization, and Descemet's fold persisted. Although the epithelial defects occasionally recurred, the corneal sensation and epithelial defects, Descemet's fold, and corneal superficial neovascularization all improved around 5 months after trigeminal nerve block. The HRT II Rostock Cornea Module (RCM) could not detect any corneal subbasal nerve fibers at postoperative 4 months; however, it could detect them at postoperative 6 months. CONCLUSIONS: As the nerve block effect wore off, the corneal subbasal nerve fibers slowly regenerated. As the corneal sensation improved, the corneal epithelial defects and superficial neovascularization also improved. The HRT II RCM appeared useful for observing loss and regeneration of the corneal subbasal nerve fibers.

A Case of Neurotrophic Keratopathy Associated with Nasopharyngeal Carcinoma.

PURPOSE: To report a case of neurotrophic keratopathy associated with nasopharyngeal carcinoma. CASE REPORT: A 59-year-old man who had been diagnosed with a nasopharyngeal carcinoma was referred to the authors because of visual disturbance and pain in his right eye. Slit-lamp examination revealed a corneal epithelial defect and corneal stromal edema surrounding the epithelial defect area in his right eye. Magnetic resonance imaging showed a mass in his cavernous sinus, which was identified as nasopharyngeal carcinoma (NPC). We diagnosed neurotrophic keratopathy associated with NPC and initiated treatment with preservative-free artificial tears, antibiotic eye drops, fibronectin, a therapeutic contact lens, and amniotic membrane transplantation. However, the persistent corneal epithelial defect was unresponsive to these treatments. CONCLUSION: Neurotrophic keratopathy secondary to NPC is thought to be rare. We presented a case of neurotrophic keratopathy associated with cavernous sinus metastasis of an NPC. The development of new and more effective treatments for this refractory disease is anticipated.

Spontaneous reattachment of dislocated endothelial graft after non-Descemet stripping automated endothelial keratoplasty: a case report.

BACKGROUND: Graft detachment is a complication of non-Descemet stripping automated endothelial keratoplasty (nDSAEK). We report a case of spontaneous reattachment of an extensively dislocated graft after nDSAEK. CASE PRESENTATION: A 54-year-old male underwent penetrating keratoplasty (PKP) for keratoconus in his left eye in 2001. Following graft opacity due to rejection, a second PKP was implemented in May 2014. The graft was kept in good condition after the reoperation and yet, visual acuity (VA) declined due to cataract. PEA+IOL was then performed in May 2015. Because edema appeared in the graft 6 months after the PEA+IOL, nDSAEK was carried out in May 2016. Although the donor graft well attached immediately after the nDSAEK, the graft was almost completely dislocated 3 h later except a temporal part. Air was reinjected into the anterior chamber on the following day and the detachment was resolved. Despite of the treatment, about 1/5 of the graft remained detached and the detachment deteriorated to 3/4 of the graft 9 days later. Because the patient could not decide whether to undergo another operation immediately, we decided to follow him up first and found that the partially detached graft reattached spontaneously 1 month later during the follow-up. Although the cornea had a mild edema remaining in the superior temporal area, his BCVA improved to 1.0. Three months later, the graft remained in position and the cornea kept its transparency. CONCLUSIONS: Spontaneous reattachment was observed during the follow-up in a case that had shown a comparatively extensive graft dislocation after nDSAEK.

Diagnostic Approach to Ocular Infections Using Various Techniques From Conventional Culture to Next-Generation Sequencing Analysis.

Ocular infection is caused by both endogenous (resident) and exogenous (environmental) microbes. As the ocular surface interacts with both outer environment and its own resident microbiota, clinical ocular samples are predicted to contain a diverse set of microorganisms. Microscopy of sample smears is an important step in the diagnostic process of infectious diseases to interpret the culture results. Traditional culture techniques have several limitations in the detection and/or identification of uncharacterized bacteria of environmental origin. Molecular biological techniques, such as polymerase chain reaction of pathogen-specific virulence genes, 16S rRNA gene clone library analysis, and next-generation sequencing of 16S rDNA amplicons, compensate for diagnostic culture techniques in diagnosing infectious diseases. These techniques are expected to provide novel insights into the ocular microbiota and pathology of ocular infections. In this article, we describe various ocular infections, including contact lens-related keratitis, silicone buckle infection, and dacryocystitis, which were analyzed using molecular biological techniques. The advantages and disadvantages of these highly sensitive and inclusive microbiological detection systems for ocular infections are discussed.

Cosmetic Cleansing Oil Absorption by Soft Contact Lenses in Dry and Wet Conditions.

OBJECTIVES: Previous reports showed that cosmetic cleansing oil for removing makeup, which contains mineral oil and surfactant, can deform some silicone hydrogel contact lenses (SHCLs) when applied directly to the lenses, although plasma-coated SHCLs (lotrafilcon A and B) were not affected. In the present study, we investigated hydrogel lenses and SHCLs in both wet and dry conditions. METHODS: Several brands of hydrogel and SHCLs were immersed in a cleansing oil solution containing Sudan Black B for 5 min under wet and dry conditions. The lenses under the wet condition were simply picked up from the saline, whereas those under the dry condition were blotted with paper wipes. After immersing, the excess solution remaining on the lenses was removed by finger rubbing with a multipurpose solution. The lenses were then examined using a stereomicroscope, and their mean brightness was measured and compared. RESULTS: The cosmetic cleansing oil was not absorbed by the hydrogel lenses under wet or dry conditions. However, four of seven brands of SHCLs absorbed the cosmetic cleansing oil under both conditions (dry and wet), whereas asmofilcon A absorbed it only under the dry condition. Lotrafilcon B and delefilcon A did not absorb cleansing oil even under the dry condition. CONCLUSIONS: Hydrogel lenses resist cosmetic cleansing oil. However, SHCLs have different degrees of resistance depending on the lens material. Some SHCLs absorbed cosmetic cleansing oil more under dry conditions than under wet conditions.

Intraoperative optical coherence tomography (RESCAN® 700) for detecting iris incarceration and iridocorneal adhesion during keratoplasty.

There is no report focusing on the visualization of the iris incarceration or the iridocorneal adhesion during keratoplasty by use of microscope-integrated intraoperative optical coherence tomography (MIOCT). The purpose of this study is to report the usefulness of MIOCT for detecting iris incarceration and iridocorneal adhesions during penetrating keratoplasty (PK) and deep anterior lamellar keratoplasty (DALK). MIOCT system was applied both in a patient who underwent PK for corneal leukoma and in a patient who underwent DALK for keratoconus. During the surgeries, we obtained cross-sectional images around the host-graft interface by operating the foot switch of microscope without discontinuing the surgical procedure. Intraoperative MIOCT findings and postoperative outcomes were examined. An iris incarceration at the host-graft interface was visualized during surgery after corneal suture in PK, which allowed surgeons to return the iris to its original position instantly. In DALK, misdirected air into the posterior chamber could also be seen at the end of the DALK. This iridocorneal adhesion was resolved by fluid injection through paracentesis. Secondary glaucoma and graft rejection have not occurred postoperatively in both cases. The MIOCT system provides advantages such as prevention of secondary glaucoma and rejection following PK and DALK.

Regulatory Mechanism of Collagen Degradation by Keratocytes and Corneal Inflammation: The Role of Urokinase-Type Plasminogen Activator.

Keratocytes, corneal resident cells in the corneal stroma, exist between collagen lamellae and maintain the corneal stromal structure. When the corneal stroma is damaged, keratocytes are transformed to myofibroblasts to aid corneal wound healing by phagocytizing debris. Keratocytes and extracellular collagen influence each other because keratocytes cultured in a 3D collagen gel undergo morphological changes and keratocytes produce metalloproteases that degrade extracellular collagen. IL-1 and plasminogen are critical mediators for collagen degradation. The plasminogen system contributes to tissue repair by activating matrix metalloproteinases (MMPs), releasing growth factors from the extracellular matrix and extracellular matrix degradation. Urokinase-type plasminogen activator (uPA) is thought to be involved in corneal disorders and regulates corneal wound healing. uPA is a serine protease synthesized by various cells such as corneal epithelial cells, corneal fibroblasts, vascular endothelial cells, smooth muscle cells, monocytes, macrophages, and malignant tumor cells of different origins. Here, we review the role of uPA in corneal stromal wound healing. uPA is expressed in leukocytes and corneal fibroblasts in the corneas of patients with corneal ulcerations suggesting it is a key regulator of corneal stromal wound healing. uPA is directly involved in plasmin-mediated collagen degradation induced by IL-1. Moreover, uPA is critically involved in promoting leukocyte infiltration in corneal inflammation by activating MMP-9. This activation is presumably directly and indirectly mediated by the plasminogen/plasmin cascade. Moreover, uPA mediates the release of inflammatory cytokines from corneal fibroblasts to promote leukocyte infiltration.

[Multicenter Prospective Observational Study of Fungal Keratitis--Identification and Susceptibility Test of Fungi].

PURPOSE: To investigate the causative fungi of fungal keratitis in Japan and their drug susceptibility. METHODS: Identification and antifungal susceptibility test for 8 drugs (micafungin, amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, miconazole and pimaricin) were performed using isolated fungi from patients with fungal keratitis treated at 27 facilities in Japan between November 1, 2011 and October 31, 2013. RESULTS: Fungal strains were detected in 72 (50.7%) out of 142 samples. The major isolates were Fusarium spp. (18), Candida parapsilosis (12), C. albicans (11) and Alternaria spp. (6), in all, fungi of 31 species were identified by gene analysis. In the yeast-like fungi, susceptibility rates were evident for more than 80% in voriconazole, pimaricin, flucytosine, micafungin, amphotericin B and fluconazole. In filamentous fungi, the susceptibility rate was less than 50% except for PMR (90%). Fusarium spp., which were susceptible to amphotericin B and pimaricin, showed lower susceptibility rates compared with other genera. CONCLUSIONS: Although various genera and species of fungi cause fungal keratitis, the obtained drug susceptibility data in this study demonstrates the different susceptibility patterns among the major isolates (Fusarium spp., C. parapsilosis, C. albicans and other groups). This is important evidence useful for fungal keratitis treatment.

[Multicenter Prospective Observational Study of Fungal Keratitis--Current Status of Patients' Background, Clinical Findings, Treatment and Prognosis].

PURPOSE: To investigate the current status of fungal keratitis in Japan. METHODS: The patients with fungal keratitis were examined at 27 facilities in Japan from November 1st 2011 to October 31st 2013, concerning isolates, patient background, clinical findings, treatment and prognosis. RESULTS: Out of 139 cases, 133 were diagnosed as fungal keratitis, of which fungi were isolated from 72 samples of 71 cases (yeast-like fungi 32 strains and filamentous fungi 40 strains). The corrected visual acuity at the first visit of 88 cases (66.2%) was less than 20/200 and 42 cases (31.6%) were involved with deep stromal lesions, indicating high proportion of severe cases in this study. Three months later, 56 cases (42.1%) were still under treatment, and corrected visual acuity of 57 cases (42.9%) was less than 20/200. In cases with yeast-like fungi, there were significantly more cases with past history of corneal diseases, ocular surgery including keratoplasty, and eye drops' use such as steroids than those with filamentous fungi. On the other hand, there were significantly more cases of filamentous fungi, with trauma on the onset and with intervention of previously attending doctors than those with yeast-like fungi. Logistic regression analyses revealed that contact lens wearing was a significant factor of good prognosis, and yeast-like fungi as one of poor outcome compared with no fungal isolation. CONCLUSION: Although the choice of antifungal drugs has been increasing, fungal keratitis is still severe, refractory and vision-threatening disease.

Effects of antiviral medications on herpetic epithelial keratitis in mice.

PURPOSE: Aciclovir (ACV), valaciclovir (VACV) and famciclovir (FCV) are used for systemic infections caused by herpes virus. In Japan, only topical ACV is permitted for use against herpetic keratitis. We investigated the effectiveness of topical ACV, oral VACV and oral FCV on mouse epithelial herpetic keratitis. METHODS: C57/BL76 mice were inoculated with HSV-1 McKrae strain in the cornea. Once infection was confirmed 4 days after inoculation, topical ACV, oral VACV and FCV were started and administered for 5 days. Control groups were given either topical or oral saline. On days 2, 4, 6 and 10 after medication started, tears, eyeballs, and trigeminal ganglia were examined using viral culture and real-time PCR. RESULTS: Viral culture of tears detected no HSV in the topical ACV group on day 4 after administration start; with similar results for the oral VACV group on day 4; and the oral FCV group on day 6. Real-time PCR of the eyeballs showed significant decrease of HSV DNA copy number in the topical ACV group on days 4 and 6 compared to the topical saline group. Real-time PCR of the trigeminal ganglia showed significant decrease of HSV DNA copy number in the oral VACV group on days 4 and 6, and in the oral FCV group on day 6 compared to the oral saline group. CONCLUSION: We suggest that 5-day administration of topical ACV, oral VACV and oral FCV are effective for mouse epithelial herpetic keratitis and sufficiently decrease HSV amounts in the ocular surface and eyeballs.

Virological and molecular biological evidence supporting herpes simplex virus type 1 corneal latency.

PURPOSE: Trigeminal and other ganglia are known as sites of latent infection by herpes simplex virus type 1 (HSV-1). In ophthalmology, HSV-1 remains latent in the trigeminal ganglia, and becomes reactivated by several factors, including stress, thermal stimulation, or immunosuppression, and may lead to herpetic keratitis. The purpose of this study was to demonstrate HSV corneal latent infection using molecular biology and virology techniques. METHODS: Six corneas obtained at penetrating keratoplasty were snap-frozen; three of them were with past history of herpetic keratitis. TaqMan Real-time PCR was used to show positive HSV DNA in the corneas. We proved negative homogenate and positive explant virologically. Using real-time RT-PCR, we showed that only latency-associated transcript (LAT) was detected and no transcriptional products of other virus genes (α, ß, γ) were detected. RESULTS: All three corneas with past history of herpetic keratitis had HSV DNA and showed negative homogenate and positive explant. LAT was detected in all three corneas. However, α, ß, or γ genes were not expressed. All the results of these corneas were consistent with the conditions of corneal latency. The other three corneas without history of herpetic keratitis showed negative homogenate and negative explant. None of them had LAT. CONCLUSION: We have shown a possibility that HSV can latently infect the cornea aside from the ganglion.