Yukiko Morita

Protection of Human Corneal Epithelial Cells from TNF-α-Induced Disruption of Barrier Function by Rebamipide.

PURPOSE TNF-α disrupts the barrier function of cultured human corneal epithelial (HCE) cells. We investigated the effects of the cytoprotective drug rebamipide on this barrier disruption by TNF-α as well as on corneal epithelial damage in a rat model of dry eye. METHODS The barrier function of HCE cells was evaluated by measurement of transepithelial electrical resistance. The distribution of tight-junction (ZO-1, occludin) and adherens-junction (E-cadherin, β-catenin) proteins, and the p65 subunit of nuclear factor-κB (NF-κB) was determined by immunofluorescence microscopy. Expression of junctional proteins as well as phosphorylation of the NF-κB inhibitor IκB-α and myosin light chain (MLC) were examined by immunoblot analysis. A rat model of dry eye was developed by surgical removal of exorbital lacrimal glands. RESULTS Rebamipide inhibited the disruption of barrier function as well as the downregulation of ZO-1 expression, and the disappearance of ZO-1 from the interfaces of neighboring HCE cells induced by TNF-α. It also inhibited the phosphorylation and downregulation of IκB-α, the translocation of p65 to the nucleus, the formation of actin stress fibers, and the phosphorylation of MLC induced by TNF-α in HCE cells. Treatment with rebamipide eyedrops promoted the healing of corneal epithelial defects as well as attenuated the loss of ZO-1 from the surface of corneal epithelial cells in rats. CONCLUSIONS Rebamipide protects corneal epithelial cells from the TNF-α-induced disruption of barrier function by maintaining the distribution and expression of ZO-1 as well as the organization of the actin cytoskeleton. Rebamipide is, thus, a potential drug for preventing or ameliorating the loss of corneal epithelial barrier function associated with ocular inflammation.

Quantitative analysis of collagen lamellae in the normal and keratoconic human cornea by second harmonic generation imaging microscopy.

PURPOSE To characterize the structural properties of collagen lamellae in the normal and keratoconic human corneal stroma, we measured their width and angle relative to Bowmans layer (BL). METHODS Thirteen normal and four keratoconic corneas were examined. Collagen lamellae in tissue blocks from the central cornea were visualized by second harmonic generation imaging microscopy. Images obtained in 1-μm steps from BL to Descemets membrane (DM) were subjected to three-dimensional reconstruction. The reconstructed data sets were divided into 10 layers of equal depth (L1-L10) for analysis. The width of lamellae adherent to BL (L0) was also determined. RESULTS For the normal cornea, the width (mean ± SD) of collagen lamellae was 6.5 ± 1.7 μm at L0, decreased to 4.3 ± 1.3 μm at L1, and then increased gradually with progression toward DM to 122.2 ± 34.5 μm at L10, whereas the angle of lamellae was 20.9° ± 5.4° at L1 and decreased initially to 10.6° ± 3.2° at L2 before declining gradually to 2.7° ± 2.2° at L10. The width and angle of collagen lamellae in the keratoconic cornea were significantly larger and smaller, respectively, relative to those in the normal cornea. CONCLUSIONS In the normal human cornea, collagen lamellae adjacent to BL are narrow and form a steep angle with BL, whereas they increase in width and their angle relative to BL flattens with progression toward DM. These properties of collagen lamellae are altered in keratoconus and are likely related to abnormalities of corneal shape.

Effect of preoperative duration of stromal edema in bullous keratopathy on early visual acuity after endothelial keratoplasty.

PURPOSE: To clarify the relationship between visual acuity after Descemet‐stripping automated endothelial keratoplasty (DSAEK) and the preoperative duration of stromal edema. SETTING: Yamaguchi University Hospital, Yamaguchi, Japan. DESIGN: Comparative case series. METHODS: Patients who had DSAEK were divided into 2 groups based on whether the preoperative duration of stromal edema was less than 12 months (Group A) or more than 12 months (Group B). No patient had postoperative conditions that might have affected postoperative visual outcomes. Postoperatively, the corrected distance visual acuity (CDVA) was measured and the morphology of the anterior cornea evaluated by in vivo laser confocal microscopy. RESULTS: The postoperative CDVA in Group A ranged from 20/50 to 20/16 at 3 months and from 20/66 to 20/16 at 6 months; the maximum CDVA was 20/40 to 20/13. The postoperative CDVA in Group B ranged from 20/66 to 20/40 at 3 months and 6 months; the maximum CDVA was 20/66 to 20/33. The structure of the anterior cornea was normal in patients in Group A but was abnormal with fibroblastic cells in the anterior stroma in patients in Group B. CONCLUSIONS: Patients with a preoperative duration of stromal edema of more than 12 months had pathologic changes in the corneal stroma that may have adversely affected visual acuity after DSAEK. Given that stromal edema, including that associated with bullous keratopathy, has been proposed to be a progressive condition, DSAEK may be most effective when performed early after the onset of edema, before the occurrence of pathologic changes in the stroma. Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned.

Expression of semaphorin 3A in the rat corneal epithelium during wound healing.

The neural guidance protein semaphorin 3A (Sema3A) is expressed in corneal epithelial cells of the adult rat. We have now further investigated the localization of Sema3A in the normal rat corneal epithelium as well as changes in its expression pattern during wound healing after central corneal epithelial debridement. The expression pattern of Sema3A was compared with that of the tight-junction protein zonula occludens-1 (ZO-1), the gap-junction protein connexin43 (Cx43), or the cell proliferation marker Ki67. Immunofluorescence analysis revealed that Sema3A was present predominantly in the membrane of basal and wing cells of the intact corneal epithelium. The expression of Sema3A at the basal side of basal cells was increased in the peripheral epithelium compared with that in the central region. Sema3A was detected in all layers at the leading edge of the migrating corneal epithelium at 6h after central epithelial debridement. The expression of Sema3A was markedly up-regulated in the basal and lateral membranes of columnar basal cells apparent in the thickened, newly healed epithelium at 1 day after debridement, but it had largely returned to the normal pattern at 3 days after debridement. The expression of ZO-1 was restricted to superficial epithelial cells and remained mostly unchanged during the wound healing process. The expression of Cx43 in basal cells was down-regulated at the leading edge of the migrating epithelium but was stable in the remaining portion of the epithelium. Ki67 was not detected in basal cells of the central epithelium at 1 day after epithelial debridement, when Sema3A was prominently expressed. Immunoblot analysis showed that the abundance of Sema3A in the central cornea was increased 1 day after epithelial debridement, whereas that of ZO-1 or Cx43 remained largely unchanged. This increase in Sema3A expression was accompanied by up-regulation of the Sema3A coreceptor neuropilin-1. Our observations have thus shown that the expression of Sema3A is increased markedly in basal cells of the newly healed corneal epithelium, and that this up-regulation of Sema3A is not associated with cell proliferation. They further suggest that Sema3A might play a role in the regulation of corneal epithelial wound healing.

Spontaneous Healing of Corneal Perforation after Temporary Discontinuation of Erlotinib Treatment

Purpose: To report a case of corneal perforation associated with oral administration of erlotinib and its spontaneous healing after temporary discontinuation of drug treatment. Case Report: A 65-year-old man with metastatic lung cancer was treated with erlotinib (150 mg/day), a specific tyrosine kinase inhibitor of the epidermal growth factor receptor. He was referred to our corneal service for the treatment of bilateral corneal disorders, diagnosed with mild aqueous-deficient dry eye, and treated by insertion of punctal plugs. His corneal epithelial disorders initially improved, but subsequently worsened, as manifested by the development of bilateral corneal ulceration with corneal perforation in the right eye. The oral administration of erlotinib was interrupted in preparation for tectonic keratoplasty, but 2 days later the corneal perforation of the right eye and the bilateral epithelial defects had healed spontaneously. Treatment with erlotinib was resumed at half the initial dose, and the cornea of both eyes has remained apparently healthy. Discussion: Erlotinib may be secreted into tear fluid and thereby adversely affect the corneal epithelium. The development of corneal epithelial disorders in patients receiving this drug may be reversed by reducing its dose.

Recovery of the Corneal Stroma Associated With Rapid Reepithelialization Induced by the Fibronectin-Derived Peptide PHSRN in 2 Cases of Corneal Perforation Due to a Persistent Epithelial Defect.

Purpose: To report 2 cases of corneal perforation associated with a persistent epithelial defect (PED), which were treated with eye drops containing the fibronectin-derived peptide PHSRN (Pro-His-Ser-Arg-Asn). Methods: A 67-year-old man and a 58-year-old man presented with corneal perforation associated with a PED caused by lagophthalmos. PHSRN eye drops were administered 4 times a day to both patients. Results: Both patients experienced healing of the epithelial defect and closure of corneal perforation within 3 or 4 days after the onset of PHSRN treatment. Anterior segment optical coherence tomography also revealed recovery of corneal stromal thickness at the lesion site. Conclusions: PHSRN eye drops were effective for the treatment of corneal perforation due to the PED, with rapid reepithelialization being followed by full restoration of stromal thickness.

Peters' anomaly imaged with an infrared anterior segment camera

Peters’ anomaly is a congenital ocular condition characterized by the absence of the posterior cornea or adherence of the iris to Descemet’s membrane. The corneal stroma of affected individuals is partially oedematous as a result of the lack of endothelium, making it difficult to observe the condition of the iris or the spatial relation between the cornea and the iris. The lattice theory proposed by Maurice states that light of visible wavelengths cannot pass through the oedematous corneal stroma because of the associated irregular arrangement of collagen fibres, with the result that the light rays are scattered at the stroma. Light of longer wavelengths, such as infrared radiation, can penetrate the irregular organization of collagen fibres induced by stromal oedema, however. Indeed, infrared imaging was found to be effective for observation of equine corneas affected by stromal oedema, superficial neovascularization or infectious keratitis. We have therefore now applied an infrared anterior segment camera, Meibopen (JFC, Tokyo, Japan), which was developed for observation of Meibomian glands, to observe the iris and its relation to the cornea in three individuals with Peters’ anomaly. Institutional Review Board approval was obtained for this study, and the patients provided written informed consent. The right eyes of a 75-year-old woman (Fig. 1a) and a 10-year-old boy (Fig. 1c) with Peters’ anomaly were examined. The cornea of each eye was oedematous, and it was thus difficult to observe the condition of the iris with light of visible wavelengths. When we changed the wavelength of the light source for the camera to infrared, however, the iris was clearly visualized and was found to adhere to the posterior cornea in both individuals (Fig. 1b,d). We also examined the right eye of a 1-year-old girl with corneal opacity of unknown aetiology. Light of visible wavelengths was scattered by the corneal lesion (Fig. 1e), but infrared light revealed the iris to be adherent to the posterior cornea (Fig. 1f), allowing a diagnosis of Peters’ anomaly.

Persistence of Structural Changes at the Anterior Cornea in Bullous Keratopathy Patients after Endothelial Keratoplasty

Subepithelial fibrosis (SEF) and the transdifferentiation of keratocytes into fibroblasts or myofibroblasts (Fbs/MFbs) have been detected in the cornea of individuals with bullous keratopathy. We examined the anterior cornea of bullous keratopathy patients for such changes after Descemet’s stripping automated endothelial keratoplasty (DSAEK). Twenty-two individuals who underwent unilateral DSAEK at Yamaguchi University Hospital were enrolled in the study. The subjects were divided into groups A (n = 10) and B (n = 12) with a preoperative duration of stromal edema of less than or at least 12 months, respectively. The structure of the anterior stroma was examined by in vivo laser confocal microscopy at various times after surgery. SEF was detected in 1 (10.0%) and 11 (91.7%) cases in groups A and B, respectively, before surgery as well as in 0 (0%) and 7 (58.3%) cases, respectively, at 6 months after DSAEK. Fb/MFb transdifferentiation was detected in 0 (0%) and 8 (66.7%) cases in groups A and B, respectively, before surgery as well as in 0 and 1 (8.3%) case, respectively, at 6 months postsurgery. Anterior stromal scattering (ASS) was detected in 10 (100%) and 12 (100%) cases in groups A and B, respectively, before surgery as well as in 0 (0%) and 6 (50.0%) cases, respectively, at 6 months after DSAEK. Changes in anterior stromal structure apparent before surgery were thus also detected in bullous keratopathy patients after DSAEK. SEF and ASS persisted for more than 6 months in a substantial proportion of individuals with a preoperative duration of stromal edema of at least 12 months.

Differential Changes in Intraocular Pressure and Corneal Manifestations in Individuals with Viral Endotheliitis after Keratoplasty

Purpose: To investigate the clinical characteristics of viral endotheliitis after keratoplasty, we evaluated clinical parameters in individuals with anterior chamber inflammation after keratoplasty classified according to the absence or presence of DNA from various viruses in aqueous humor. Methods: A total of 29 eyes of 27 subjects with anterior chamber inflammation after keratoplasty were enrolled in the study. The subjects were classified into the herpes simplex virus (HSV) group (7 patients, 8 eyes), cytomegalovirus (CMV) group (6 patients, 6 eyes), varicella zoster virus group (2 patients, 2 eyes), and nonviral group (12 patients, 13 eyes) on the basis of detection of viral DNA in aqueous humor. The interval between keratoplasty and the onset of inflammation, changes in intraocular pressure (IOP), and corneal manifestations of inflammation were retrospectively reviewed. Results: Sixteen of the 29 eyes developed anterior chamber inflammation within 2 years after keratoplasty. IOP during active inflammation was significantly increased in the HSV and CMV groups compared with preinflammation values, and the increased IOP was significantly ameliorated in association with resolution of inflammation. Linear-type keratoprecipitates were frequently detected in the HSV (5 of 8 eyes), CMV (3 of 6 eyes), and nonviral (6 of 13 eyes) groups. Corneal edema was also commonly observed in the HSV (7 of 8 eyes) and nonviral (13 of 13 eyes) groups. Conclusions: Changes in IOP and corneal manifestations may provide a basis for differentiation of viral endotheliitis from allograft rejection and among viral pathogens in individuals with anterior chamber inflammation after keratoplasty, and they may thus allow initiation of appropriate treatment before viral DNA is identified.

Remodeling of the Corneal Epithelial Scaffold for Treatment of Persistent Epithelial Defects in Diabetic Keratopathy

Background: To develop a strategy based on surgical removal of a degenerated corneal epithelial scaffold for treatment of persistent epithelial defects (PEDs) in diabetic keratopathy. Case Presentation: Three diabetic patients with PEDs were initially treated with eyedrops containing the fibronectin-based peptide PHSRN (Pro-His-Ser-Arg-Asn) or both the substance P-derived peptide FGLM-NH2 and the insulin-like growth factor-1-derived peptide SSSR. A degenerated Bowman’s layer or calcified lesion thought to be responsible for incomplete healing was surgically removed after confirmation of reactivity to the peptide eyedrops. All three patients achieved complete epithelial wound closure after surgery. Two cases treated by phototherapeutic keratectomy or lamellar keratoplasty did not show PED recurrence during 6 or 36 months of follow-up, respectively. One case treated by mechanical removal of a degenerated Bowman’s layer manifested recurrence after 1 month, but resurfacing of the defect was again achieved after repeat surgery. Conclusion: We propose a new strategy for treatment of diabetic PEDs based on surgical remodeling of the corneal epithelial scaffold for patients who respond to peptide eyedrops but fail to achieve wound closure.