Postkeratoplasty atopic sclerokeratitis in keratoconus patients.

PURPOSE: To estimate the incidence of and investigate possible risk factors for postkeratoplasty atopic sclerokeratitis in keratoconus patients undergoing keratoplasty. DESIGN: Retrospective, noncomparative, interventional case series. PARTICIPANTS: Two hundred forty-seven eyes with keratoconus. METHODS: We reviewed the medical records of all keratoplasty cases of keratoconus between May 2000 and December 2005 at Tokyo Dental College Ichikawa General Hospital. The incidence and clinical details of cases consistent with postkeratoplasty atopic sclerokeratitis were recorded. MAIN OUTCOME MEASURES: Cases with acute sclerokeratitis during the early postoperative period were retrospectively evaluated. RESULTS: A total of 247 keratoconus eyes were followed at our clinic after keratoplasty (mean follow-up, 18.5+/-13.0 months). Thirty-five eyes of 29 patients (14.2%) had a history of atopic dermatitis, of which 6 eyes of 5 patients (2.4%) developed postkeratoplasty atopic sclerokeratitis. Mean age of postkeratoplasty atopic sclerokeratitis patients was 29 years (range, 23-39). The mean period between keratoplasty and onset of postkeratoplasty atopic sclerokeratitis was 26 days (range, 11-41). Loosening of running sutures and wound leakage were observed in 3 eyes; persistent epithelial defects in 3 eyes; and graft melting in 2 eyes, 1 of which was perforated. Preoperative atopic blepharitis and corneal neovascularization were identified as risk factors for postkeratoplasty atopic sclerokeratitis. CONCLUSIONS: Postkeratoplasty atopic sclerokeratitis is a potentially severe complication in atopic patients undergoing keratoplasty. Systemic immunosuppression may be considered in patients with active blepharitis and corneal neovascularization.

Cataract surgery after deep anterior lamellar keratoplasty and penetrating keratoplasty in age- and disease-matched eyes.

PURPOSE: To assess the efficacy and safety of cataract surgery after deep anterior lamellar keratoplasty (DALK) and penetrating keratoplasty (PKP). SETTING: Tokyo Dental College Ichikawa General Hospital, Chiba, Japan. DESIGN: Retrospective case series. METHODS: Age-matched and disease-matched eyes that had phacoemulsification and intraocular lens insertion after DALK or PKP were studied. Graft clarity was assessed at the final follow-up. The difference in the endothelial cell density (ECD), corrected distance visual acuity (CDVA), spherical equivalence, and refractive error between the expected values and values 1, 3, 6, and 12 months after cataract surgery were compared between the 2 groups. RESULTS: Indications for keratoplasty were corneal stromal scar (22 eyes), lattice dystrophy (2 eyes), keratoconus (2 eyes), and postherpetic keratitis (4 eyes). All 30 eyes in each group had successful cataract surgery after keratoplasty. Graft clarity rates were 90.0% and 80.0% in the DALK group and PKP group, respectively (P = .47). The decrease in ECD at 12 months was significantly greater in the PKP group than in the DALK group (8.7% [SD] ± 21.7% versus 26.3% ± 27.8%) (P = .043). The CDVA was significantly improved in both groups. At 1 month, the mean refractive error was -0.5 diopter (D) ± 2.4 (SD) in the DALK and -0.4 ± 1.9 D in the PKP groups and remained stable thereafter. CONCLUSIONS: Cataract surgery was successfully performed in eyes that had DALK or PKP, providing excellent visual and refractive outcomes. In cases of combined cataract and corneal pathology, and in the absence of endothelial involvement, DALK followed by cataract surgery might cause less endothelial damage.

Amniotic membrane immobilized poly(vinyl alcohol) hybrid polymer as an artificial cornea scaffold that supports a stratified and differentiated corneal epithelium.

Poly(vinyl alcohol) (PVA) is a biocompatible, transparent hydrogel with physical strength that makes it promising as a material for an artificial cornea. In our previous study, type I collagen was immobilized onto PVA (PVA-COL) as a possible artificial cornea scaffold that can sustain a functional corneal epithelium. The cellular adhesiveness of PVA in vitro was improved by collagen immobilization; however, stable epithelialization was not achieved in vivo. To improve epithelialization in vivo, we created an amniotic membrane (AM)-immobilized polyvinyl alcohol hydrogel (PVA-AM) for use as an artificial cornea material. AM was attached to PVA-COL using a tissue adhesive consisting of collagen and citric acid derivative (CAD) as a crosslinker. Rabbit corneal epithelial cells were air-lift cultured with 3T3 feeder fibroblasts to form a stratified epithelial layer on PVA-AM. The rabbit corneal epithelial cells formed 3-5 layers of keratin-3-positive epithelium on PVA-AM. Occludin-positive cells were observed lining the superficial epithelium, the gap-junctional protein connexin43-positive cells was localized to the cell membrane of the basal epithelium, while both collagen IV were observed in the basement membrane. Epithelialization over implanted PVA-AM was complete within 2 weeks, with little inflammation or opacification of the hydrogel. Corneal epithelialization on PVA-AM in rabbit corneas improved over PVA-COL, suggesting the possibility of using PVA-AM as a biocompatible hybrid material for keratoprosthesis.

Penetrating Keratoplasty Performed by Residents Compared With an Experienced Cornea Transplant Surgeon.

BACKGROUND: Reports on keratoplasty by residents are rare compared to those on cataract surgery. The aim of this study was to compare the results and surgery time of penetrating keratoplasty performed by residents and an experienced cornea surgeon. METHODS: The medical records and surgery videos of the first 5 penetrating keratoplasty procedures by 4 residents were reviewed and compared with those in 20 penetrating keratoplasty procedures by an experienced cornea surgeon. Patients underwent penetrating keratoplasty at the Department of Ophthalmology, Tokyo Dental College, Chiba, Japan. The main outcome measures are graft survival, best-corrected visual acuity, postoperative astigmatism, decreased rate of graft endothelial cell density, and details of surgery time. RESULTS: Graft survival, best-corrected visual acuity, and postoperative astigmatism were not significantly different between residents group and the experienced surgeon group. The average decrease of graft endothelial cell density owing to surgery in residents and the experienced surgeon were 19.0 ± 17.5 and 18.5 ± 12.1%, respectively (p = 0.27, Mann-Whitney U test). The average surgery time taken by residents was 66.9 ± 18.1 minutes, and that by the experienced surgeon it was 28.2 ± 7.9 minutes (p = 0.0071, Mann-Whitney U test). Residents required the most time for running suture technique. CONCLUSIONS: The results of keratoplasty by residents were similar with those done by the experienced surgeon. Surgery time was the only statistically significant difference (p = 0.0071), which did not influence surgical results.

Dacryoadenitis associated with Acanthamoeba keratitis.

OBJECTIVE: To report the incidence of dacryoadenitis as a symptom associated with Acanthamoeba keratitis. METHODS: We investigated all cases of Acanthamoeba keratitis (20 patients and 21 eyes) diagnosed and treated at Tokyo Dental College, Ichikawa General Hospital, Ichikawa, Japan, between May 1, 1994, and November 30, 2005. We recorded the incidence of dacryoadenitis diagnosed using clinical signs of lacrimal gland swelling and pain on pressure, computed tomography, magnetic resonance imaging, and histopathologic analysis. RESULTS: Eight eyes (38%) of 8 patients had dacryoadenitis simultaneously with Acanthamoeba keratitis. Dacryoadenitis was diagnosed using histopathologic analysis and computed tomography in 1 patient, histopathologic analysis and magnetic resonance imaging in 1, magnetic resonance imaging in 2, and clinical signs alone in 4. Histopathologic examination in 2 patients revealed moderate infiltration of lymphocytes and plasma cells in the lacrimal gland compatible with dacryoadenitis. No Acanthamoeba organisms were found in the lacrimal gland. The standard protocol for Acanthamoeba keratitis was performed without particular treatment of dacryoadenitis in all patients. Lacrimal gland swelling improved after a mean of 10 weeks (range, 4-17 weeks) in conjunction with symptoms of keratitis; however, 1 patient (patient 1) required levator muscle surgery and blepharoplasty for residual ptosis. CONCLUSION: Dacryoadenitis is a clinical finding associated with Acanthamoeba keratitis.

Collagen-immobilized poly(vinyl alcohol) as an artificial cornea scaffold that supports a stratified corneal epithelium.

The cornea is a transparent tissue of the eye, which is responsible for the refraction of incoming light. Both biological corneal equivalents and synthetic keratoprostheses have been developed to replace donor tissue as a means to restore vision. However, both designs have drawbacks in terms of stability and biocompatibility. Clinically available synthetic devices do not support an intact epithelium, which poses a risk of microbial infection or protrusion of the prosthesis. In the present study, type I collagen was immobilized onto poly(vinyl alcohol) (PVA-COL) as a possible artificial cornea scaffold that can sustain a functional corneal epithelium. Human and rabbit corneal epithelial cells were air-lift cultured with 3T3 feeder fibroblasts to form a stratified epithelial layer on PVA-COL. The epithelial sheet expressed keratin 3/12 differentiation markers, the tight junction protein occludin, and had characteristic microvilli structures on transmission electron microscopy. Functionally, the stratified epithelium contained normal glycogen levels, and an apical tight-junction network was observed to exclude the diffusion of horseradish peroxidase. Furthermore, the epithelium-PVA-COL composite was suturable in the rabbit cornea, suggesting the possibility of using PVA-COL as a biocompatible material for keratoprosthesis.

Role of the endothelial pump in flap adhesion after laser in situ keratomileusis.

PURPOSE: To investigate the role of the endothelial pump in the attachment of the corneal flap during the early period after laser in situ keratomileusis (LASIK). SETTING: Department of Ophthalmology, Tokyo Dental College, Tokyo, Japan. METHODS: The study comprised 30 porcine eyes. In all eyes, a corneal flap was made with a microkeratome. Five minutes after the flap was placed over the bed, the adhesion strength was measured with a load-testing machine. To inhibit the endothelial pump, ouabain was injected into the anterior chamber (10 eyes) or the endothelium was mechanically scraped (10 eyes). The control group included 10 eyes. RESULTS: The load needed to lift the flap was significantly lower in the ouabain group (0.21 gram force [gf]; P<.01) and the endothelial scrape group (0.22 gf; P<.01) than in the control group (0.61 gf). CONCLUSION: The endothelial pump plays an important role in flap attachment in the early postoperative period after LASIK.

Detection of neutrophils and possible involvement of interleukin-8 in diffuse lamellar keratitis after laser in situ keratomileusis.

PURPOSE: To investigate the cell populations in diffuse lamellar keratitis (DLK) infiltration after laser in situ keratomileusis and the possible mechanism underlying the infiltration. SETTING: Department of Ophthalmology, Tokyo Dental College, Chiba, Japan. METHODS: To develop DLK in rabbit eyes, 25 microL of lipopolysaccharide (LPS) solution at a concentration of 50 microg/mL was applied to the stromal bed beneath corneal flaps. For control rabbits, phosphate-buffered saline was applied. Postoperative examination by slitlamp microscopy was performed for 3 days after surgery. Rabbit eyes were excised and examined for histopathology with hematoxylin and eosin staining. Immunohistochemical analysis for interleukin (IL)-8 was performed. RESULTS: Diffuse lamellar keratitis-like inflammation composed mainly of neutrophils was reproduced by LPS instillation in rabbit eyes. In eyes with severe inflammation, IL-8 immunoreactivity was found in the stromal keratocytes and infiltrating neutrophils. CONCLUSIONS: The major cell type in the DLK infiltration induced by LPS instillation in rabbit eyes was the neutrophil. Interleukin-8, a prototype of CXC chemokine produced by keratocytes and neutrophils, may contribute to the development of DLK.

Collagen-poly(N-isopropylacrylamide)-based membranes for corneal stroma scaffolds.

PURPOSE: To investigate the feasibility of using the biocompatibility of collagen-based blended biomaterials as cell-delivery systems in ocular surface reconstruction in vivo. METHODS: Collagen-based composites that were blended with synthetic acrylamide-based polymers [poly(N-isopropylacrylamide), pNIPAAm] were transplanted into corneal pockets of white rabbits, with a 3-mm epithelial window. Epithelial cells were allowed to migrate onto the polymer. Transplanted eyes were examined daily for up to 30 days, after which animals were killed for histologic examination. Immunohistochemistry was performed for vimentin, alpha-smooth muscle actin (alpha-SMA), CD4, and CD8. Gold-chloride staining was performed to observe neuronal regrowth. Human amniotic membranes (AMs) and sham-operated corneas served as controls. All animals received topical antibiotics (levofloxacin) without the use of steroids or other immunosuppressive agents. RESULTS: The pNIPAAm polymer allowed smooth epithelialization of the cornea, which was similar to the epithelialization observed in sham controls and AM-transplanted eyes. Histology revealed that epithelium overlying the polymer was bundled into several layers, without the orientation observed with AM and sham controls. The polymer gradually thinned and was gradually replaced by host tissue. Vimentin- and alpha-SMA-positive cells were found in stromal pockets up to 1 month following polymer transplantation. These cells were responsible for slight subepithelial haze near the wound edge. CD4- and CD8-positive lymphocytes were also observed in the vicinity of the polymer. Gold-chloride staining showed nerve regrowth in the wound edge after 1 month and subepithelial branches after 3 months. CONCLUSION: Collagen-pNIPAAm blended polymers may be effective as biomaterials to be used in the early stages of lamellar stromal replacement

Validation of Na,K-ATPase pump function of corneal endothelial cells for corneal regenerative medicine.

Tissue-engineering approaches to cultivate corneal endothelial cells (CECs) or induce CECs from stem cells are under investigation for the treatment of endothelial dysfunction. Before clinical application, a validation method to determine the quality of these cells is required. In this study, we quantified the endothelial pump function required for maintaining the corneal thickness using rabbit CECs (RCECs) and a human CEC line (B4G12). The potential difference of RCECs cultured on a permeable polyester membrane (Snapwell), B4G12 cells on Snapwell, or B4G12 cells on a collagen membrane (CM6) was measured by an Ussing chamber system, and the effect of different concentrations of ouabain (Na,K-ATPase specific inhibitor) was obtained. A mathematical equation derived from the concentration curve revealed that 2 mM ouabain decreases pump function of RCECs to 1.0 mV, and 0.6 mM ouabain decreases pump function of B4G12 on CM6 to 1.0 mV. Ouabain injection into the anterior chamber of rabbit eyes at a concentration of <2 mM maintained the corneal thickness, while those over 3 mM significantly increased the corneal thickness. B4G12 cell sheets transplanted into rabbit eyes treated with 0.6 mM ouabain maintained the corneal thickness, while 3.5 mM ouabain significantly increased the corneal thickness. Taken together, pump function >1.0 mV is required to maintain the corneal thickness. These results can be used for standardization of CEC pump function and validation of tissue-engineered CEC sheets for clinical use.

Functional lacrimal gland regeneration by transplantation of a bioengineered organ germ.

The lacrimal gland has a multifaceted role in maintaining a homeostatic microenvironment for a healthy ocular surface via tear secretion. Dry-eye disease, which is caused by lacrimal gland dysfunction, is one of the most prevalent eye diseases that cause corneal epithelial damage and results in significant loss of vision and a reduction in the quality of life. Here we demonstrate orthotopic transplantation of bioengineered lacrimal gland germs into adult mice with an extra-orbital lacrimal gland defect, a mouse model that mimics the corneal epithelial damage caused by lacrimal gland dysfunction. The bioengineered lacrimal gland germs and harderian gland germs both develop in vivo and achieve sufficient physiological functionality, including tear production in response to nervous stimulation and ocular surface protection. This study demonstrates the potential for bioengineered organ replacement to functionally restore the lacrimal gland.

Prospective, randomized study of the efficacy of systemic cyclosporine in high-risk corneal transplantation.

PURPOSE: Immunologic rejection remains a major cause of graft failure in high-risk corneal transplantation. This study was conducted to elucidate the efficacy and safety of systemic cyclosporine (CsA) in high-risk corneal transplantation. DESIGN: Prospective, randomized, open-labeled clinical trial with a parallel-group study. METHODS: Patients underwent high-risk corneal transplantation at the Department of Ophthalmology, Tokyo Dental College, Chiba, Japan. High-risk was defined as corneal neovascularization in more than 1 quadrant or a history of corneal grafting. Patients were assigned to either a systemic CsA group or a control group. Administration of CsA was continued for at least 6 months with blood CsA concentration 2 hours after administration of approximately 800 ng/mL, unless undesirable side effects developed. The main outcome measures were graft clarity, endothelial rejection, and local and systemic complications. RESULTS: Forty patients were enrolled and 39 (18 men, 21 women; mean age, 67.4 ± 11.9 years) were analyzed. In the CsA group, CsA was discontinued within 6 months in 7 patients because of side effects. With a mean follow-up of 42.7 months, endothelial rejection developed in 6 and 2 eyes in the CsA and control groups, respectively. No differences were observed in the rates of graft clarity loss between the 2 groups (P = .16, Kaplan-Meier analysis). CONCLUSIONS: No positive effect of systemic CsA administration for suppressing rejection in high-risk corneal transplantation was observed. With a relatively high incidence of systemic side effects, the results suggest that this protocol should not be recommended for corneal transplant recipients, especially those of advanced age.

Porous silk fibroin film as a transparent carrier for cultivated corneal epithelial sheets.

Biological carriers, such as the amniotic membrane and serum-derived fibrin, are currently used to deliver cultivated corneal epithelial sheets to the ocular surface. Such carriers require being transparent and allowing the diffusion of metabolites in order to maintain a healthy ocular surface. However, safety issues concerning biological agents encouraged the development of safer, biocompatible materials as cell carriers. We examined the application of porous silk fibroin films with high molecular permeability prepared by mixing silk fibroin and poly(ethylene glycol) (PEG), and then removal of PEG from the silk-PEG films. Molecular permeability of porous silk fibroin film is higher than untreated silk fibroin film. Epithelial cells were isolated from rabbit limbal epithelium, and seeded onto silk fibroin coated wells and co-cultured with mitomycin C-treated 3T3 fibroblasts. Stratified epithelial sheets successfully engineered on porous silk fibroin film expressed the cornea-specific cytokeratins K3 and K12, as well as the corneal epithelial marker pax6. Basement membrane components such as type-IV collagen and integrin ß1 were expressed in the stratified epithelial sheets. Further more, colony-forming efficiency of dissociated cells was similar to primary corneal epithelial cells showing that progenitor cells were preserved. The biocompatibility of fibroin films was confirmed in rabbit corneas for up to 6 months. Porous silk fibroin film is a highly transparent, biocompatible material that may be useful as a carrier of cultivated epithelial sheets in the regeneration of corneal epithelium.

Transplantation of human limbal epithelium cultivated on amniotic membrane for the treatment of severe ocular surface disorders.

PURPOSE: To study the short-term clinical results of transplanting of cultivated corneal/limbal epithelial cells on human amniotic membrane (AM) for limbal deficiency. DESIGN: Noncomparative, retrospective interventional case series. PARTICIPANTS: Thirteen eyes of 13 patients with severe limbal deficiency (Stevens-Johnson syndrome in eight eyes, ocular cicatricial pemphigoid in three eyes, and chemical burns in two eyes) were treated at the department of Ophthalmology, Tokyo Dental College, Japan. INTERVENTION: Cultivated allo-limbal epithelium was transplanted onto the ocular surface of patients with severe limbal deficiency. MAIN OUTCOME MEASURES: Ocular surface reconstruction with corneal epithelialization, changes in visual acuity, and postoperative complications were studied. Histologic examinations were also performed on cultivated epithelium. RESULTS: Cultivated corneal epithelium on AM formed two to three layers with the formation of basement membrane-like structures. After the surgery, the epithelium regenerated and covered the ocular surface in eight eyes (61.5%). However, three of the eight eyes developed partial conjunctival invasion, and two eyes later developed epithelial defects. At last examination, corneal epithelialization was achieved in six eyes (46.2%). Five eyes had conjunctivalization, one eye had dermal epithelialization, and one eye was not epithelialized. Complications were corneal perforation in four eyes and infectious keratitis in two eyes. CONCLUSIONS: This study demonstrates that the success rate for transplanting cultivated allo-limbal epithelium on the AM is not different from the conventional limbal and AM transplantation for the treatment of severe limbal stem cell dysfunction.