Yuichi Ohashi

Fibronectin appears at the site of corneal stromal wound in rabbits

Fibronectin (FN) is present both in plasma and in extracellular matrix, and is postulated to be involved in wound healing. The appearance of FN was investigated in the wounded rabbit cornea after corneal stroma was injured with a sharp knife or after lamellar keratoplasty was performed. An indirect immunofluorescence technique was employed to detect the presence of FN. Normal, unwounded rabbit cornea showed an intensive fluorescence at Descemets membrane. Strong specific fluorescence was observed at the edge of a stromal wound and beneath the sliding epithelial cells after non-perforating incisions. These could be detected as early as 3 hours after injury. The fluorescence became the strongest at 6 hours, and and then gradually decreased in its intensity, disappearing by about 7 days when epithelial cells entirely filled the stromal wound. In case of lamellar keratoplasty, FN was detected at the interface of the graft and the recipient cornea. These results suggest that FN plays an important role in the stromal wound healing.

Reversal of Abnormal Corneal Epithelial Cell Morphologic Characteristics and Reduced Corneal Sensitivity in Diabetic Patients by Aldose Reductase Inhibitor, CT-112

PURPOSE A randomized clinical study was undertaken to determine whether a topically applied aldose reductase inhibitor, CT-112, was capable of reversing the abnormal morphologic characteristics of corneal epithelial cells, as well as the reduced corneal sensitivity, in diabetic patients. METHODS Thirty-nine diabetic patients were randomly divided into two groups: one group was treated with topical aldose reductase inhibitor (CT-112) in an ophthalmic preparation, and a control group was treated with the same preparation without the inhibitor. Specular microscopy was performed to analyze the morphologic characteristics of corneal epithelial cells before and after the treatment. Corneal sensitivity was measured by means of the Cochet-Bonnet esthesiometer. RESULTS The anterior surface area of superficial cells in the group treated with CT-112 was significantly decreased from a mean value of 881 to 728 microns2 (P < .0001), whereas the control group showed no significant changes. Corneal sensitivity remained decreased in the control group, whereas that in the group treated with CT-112 significantly improved, from 5.36 to 1.37 g/mm2 (P < .0001). CONCLUSIONS These results indicate that treatment with topical CT-112 is capable of reversing abnormal morphologic characteristics of corneal epithelial cells and reduced corneal sensitivity in diabetic patients.

Demonstration of Herpes Simplex Virus DNA in Idiopathic Corneal Endotheliopathy

A 56-year-old man developed idiopathic corneal endotheliopathy. The lesion consisted of severe stromal edema at the lower half of the cornea along with a number of associated keratic precipitates and steadily progressed to the upper half of the cornea. By polymerase chain reaction, herpes simplex virus DNA was demonstrated in the aqueous humor of this patient. Corneal stromal edema was resolved in response to treatment with topically applied and systemic acyclovir. Herpes simplex virus DNA was repeatedly demonstrated in the aqueous humor when the endothelial lesion recurred later. This evidence strongly indicates that this unique endothelial disorder is of viral origin.

Hanganutziu-Deicher Heterophile Antigen in Human Retinoblastoma Cells

We investigated the possible existence of Hanganutziu-Deicher antigen (HD antigen) on retinoblastoma cells by means of indirect membrane immunofluorescence and a complement-dependent antibody-mediated cytotoxicity test. We prepared antiserum in the chicken by immunization with purified equine hematoside highly specific to HD antigen. Three kinds of retinoblastoma cell lines, Y-79, WERI-Rb 1, and TOTL-1, showed positive membrane staining. Y-79 and TOTL-1 cells were also confirmed by the cytotoxicity test to possess HD antigen. Retinoblastoma cells freshly isolated from two patients had a positive membrane immunofluorescence. These results indicated that HD antigen was expressed in human retinoblastoma cells in vitro as well as in vivo.

Aldose reductase inhibitor (CT-112) eyedrops for diabetic corneal epitheliopathy.

We treated two diabetic patients with corneal epithelial disorder that resisted conventional medical therapy with topical CT-112 (5-[3-ethoxy-4-pentyloxyphenyl]-2,4-thiazolidinedione), a newly synthesized aldose reductase inhibitor. One patient had developed recurrent corneal erosion after vitrectomy and the other had spontaneously developed superficial punctate keratopathy. The corneal lesion in each patient responded to topical CT-112 in two to four weeks and was almost cleared within two months. A similar corneal lesion recurred in both patients soon after CT-112 was discontinued, but it disappeared again when the drug was resumed.

Distribution of epidermal growth factor in rat ocular and periocular tissues.

Using a sensitive radioimmunoassay (RIA) specific for rat epidermal growth factor (rEGF), we investigated the presence of rEGF in a variety of rat ocular and periocular tissues. Immunoreactive rEGF (IR-rEGF) was present in tear fluids (25.5 ± 5.8 ng/ml), exorbital lacrimal gland (6.73 ng/g wet weight), intraorbital lacrimal gland (2.80 ng/g wet weight), Harderian gland (1.90 ng/g wet weight), and conjunctiva (0.16 ng/g wet weight). EGF was not detectable in aqueous humor, cornea, iris and ciliary body, lens, or the posterior part of the globe (retina, choroid, and vitreous body). Gel exclusion chromatography and reverse-phase high-performance liquid chromatography revealed that IR-rEGF in the above ocular fluids and tissues was indistinguishable from standard rEGF. Using enzyme-linked immunohistochemistry, rEGF was demonstrated to be localized in the duct epithelial cells of lacrimal glands. These findings reveal that EGF is preferentially localized in the ocular surface and lacrimal apparatus.

Incidence of Prominent Corneal Nerves in Multiple Endocrine Neoplasia Type 2A

We studied the increased visibility of corneal nerves inside an 8-mm diameter central corneal area in 14 patients with multiple endocrine neoplasia type 2A, one patient with multiple endocrine neoplasia type 2B, five patients with nonhereditary medullary thyroid carcinoma, ten patients with anterior keratoconus, and ten normal subjects. We used a grading system (grade 0 through grade 4) for nerve visibility based on slit-lamp biomicroscopic examination and photographic documentation. All 20 normal eyes showed either grade 0 or grade 1, which indicated no pathologic thickening of corneal nerves. Sixteen of the 28 eyes (57%) with multiple endocrine neoplasia type 2A, however, were evaluated as grade 2 or higher, which indicated thickened corneal nerves. The incidence of high nerve visibility in eyes with multiple endocrine neoplasia type 2A was significantly greater compared to normal eyes (P less than .0001), anterior keratoconus (P less than .0001), and nonhereditary medullary thyroid carcinoma (P = .0012). Furthermore, eight of the 28 eyes (29%) with multiple endocrine neoplasia type 2A showed markedly prominent corneal nerves (grade 3 and 4), a prominence similar to those seen in eyes with multiple endocrine neoplasia type 2B. There was no definite relationship among prominent nerve, age of the patient, and occurrence of pheochromocytoma. These findings suggest that over half of all patients with multiple endocrine neoplasia type 2A show corneal nerves pathologically thickened to different degrees.

The effects of aldose reductase inhibitor on the corneal endothelial morphology in diabetic rats

Diabetic rats were produced by intravenous injection of streptozotocin. Of these, eleven rats were treated with topical instillation of 0.5% aldose reductase inhibitor (ARI), while ten received vehicle alone. The corneal endothelium of these diabetic rats was examined by specular microscopy and compared to age-matched nondiabetic rats (ten rats). Computerized morphometric analysis of individual cells demonstrated that the endothelium of the untreated diabetic rats had marked polymegathism (increased coefficient of variation in cell area) and pleomorphism (decreased percentage of hexagonal cells), as previously observed in diabetic patients. Similar endothelial changes were also noted in the ARI-treated diabetic rats, but to a significantly lesser extent. These results suggest that topically applied ARI can be effective in reducing morphologic changes of the diabetic endothelium, and that activation of the sorbitol pathway may be implicated in the etiology of such endothelial changes.

Protective effects of anti-glycoprotein D monoclonal antibodies in murine herpetic keratitis.

The protective effects of passive immunization with two kinds of anti-glycoprotein D (anti-gD) monoclonal antibodies, having different antiviral activities, were investigated in murine herpetic keratitis. One monoclonal antibody, designated M1, had high virus-neutralizing antibody titers, along with undetectable levels of complement-dependent cytolysis (CDC) and antibody-dependent cellular cytotoxicity (ADCC); the other, designated M12, exhibited extremely low titers of virus-neutralization with high level of CDC and ADCC. When systemically administered 24 hours prior to virus inoculation to the cornea, both M1 and M12 almost completely prevented the development of stromal keratitis. The protective efficacy of both was observed to be dose-dependent. Pepsin-treated M1 retained its efficacy in suppressing stromal keratitis, whereas pepsin-treated M12 did not. When the administration of M1 and M12 were delayed, both provided significant (but less complete) protection, up to 24 hours after virus inoculation. These results suggest that both virus neutralization and CDC/ADCC play an important role in preventing virus growth in the corneal stroma during the early stage of corneal infection.

Herpetic keratitis therapy to reduce recurrence

Since the herpes simplex virus locates latently in the ganglia and is reactivated by certain triggers, the important problem in treatment is to prevent or reduce recurrence. The present study investigates the role of mechanical debridement in lessening the recurrence of dendritic keratitis in patients with herpetic epithelial keratitis. Group A was treated with mechanical debridement plus IDU, Group B with IDU alone. Also studied was the effect of steroid subconjunctival injection on herpetic recurrence in patients with herpetic stromal keratitis: Group C was treated with intravenous infusion of pepsin-treated gamma-globulin (PTGG) and steroid subconjunctival injection, Group D with steroid subconjunctival injection alone and Group E with intravenous infusion of PTGG alone. The recurrence rates of dendritic keratitis were 19% in Group A and 40% in Group B (average follow-up periods: 24 and 25 months). The recurrence rates of stromal keratitis were 61% in Group C, 64% in Group D, and 36% in Group E (average follow-up periods: 20, 19 and 15 months). The difference between Groups D and E was statistically significant (chi 2 test, P less than 0.05). The recurrence rates of dendritic keratitis were 18% in Group C, 21% in Group D and 7% in Group E. In conclusion, mechanical debridement tended to reduce the recurrence of herpetic epithelial keratitis and steroid subconjunctival injection to increase the recurrence of epithelial or stromal keratitis.