Masahiro Omoto

Deep lamellar keratoplasty (DLKP) in keratoconus patients using viscoadaptive viscoelastics.

Purpose: To demonstrate the effectiveness and safety of using viscoadaptive viscoelastics in deep lamellar keratoplasty (DLKP) for keratoconus. Methods: A nonrandomized, comparative, interventional case series was performed on 12 eyes of 10 consecutive keratoconus patients without a history of acute hydrops. Patients were treated with DLKP using ophthalmic viscosurgical devices (OVDs) to dissociate Descemet membrane (DM) from the stroma before suturing of the donor cornea. Eight eyes were operated using Viscoat® (dispersive), and 4 eyes were operated using Healon V® (viscoadaptive). Results: Six out of 8 eyes in the Viscoat group were successfully treated, and 2 eyes were converted to PKP because of tears in the DM. Another 3 eyes had double chambers as a result of perforations or tears in DM, which were treated by injecting air into the anterior chamber. All 4 eyes in the Healon V group were successfully operated, with 1 case with microperforation of DM during suturing. Healon V offered better control of the area to be dissociated and served as a pseudostroma that protects the exposed DM while the overlying stroma is maneuvered. Conclusion: Healon V is a valuable tool for performing DLKP in difficult cases such as keratoconus.

The use of human mesenchymal stem cell-derived feeder cells for the cultivation of transplantable epithelial sheets.

PURPOSE To report the efficacy of human bone marrow-derived mesenchymal stem cells as a source of feeder cells for the cultivation of transplantable corneal epithelial cell sheets. METHODS Human mesenchymal stem cells (marrow adherent stem cells; MASCs) were cultured in alpha-modified Eagles medium with 10% serum and were treated with mitomycin C. Expression of cytokines in MASCs was confirmed by reverse transcription-polymerase chain reaction. Human limbal epithelial cells were cocultured with MASCs or 3T3 feeder cells to compare colony-forming efficiency (CFE). Limbal epithelial cells were cultured on MASCs or 3T3 feeder cells at the air-liquid interface to allow stratification, and stratified epithelial sheets were analyzed by immunohistochemistry against cytokeratin 3 (K3), K15, p63alpha, and ABCG2. Rabbit limbal epithelial cell sheets were cultivated with MASC feeder cells and transplanted to the ocular surface of the limbal-deficient rabbits. Epithelial grafts were observed by slit lamp microscopy for 4 weeks and then evaluated by histology and immunohistochemistry against K3 and K4. RESULTS MASC feeder cells expressed keratinocyte growth factor, hepatocyte growth factor, and N-cadherin. The CFE of human limbal epithelial cells was similar in MASC and 3T3 feeder groups. Stratified cell sheets were successfully cultivated with MASC feeder cells expressing K3, K15, p63alpha, and ABCG2. Transplanted epithelial sheets regenerated the corneal phenotype in limbal-deficient rabbits. CONCLUSIONS MASC-derived feeder cells are suitable for the engineering of epithelial sheets, avoiding the use of potentially hazardous xenologic feeder cells.

Early visual results with the 1CU accommodating intraocular lens.

Purpose: To prospectively assess the clinical outcome after implantation of the 1CU accommodating intraocular lens (IOL) and a foldable acrylic IOL (AcrySof, Alcon). Setting: Department of Ophthalmology, Tokyo Dental College, Ichikawa Hospital, Ichikawa, and Minami Aoyama Eye Clinics, Tokyo, Yokohama, Japan. Methods: Twenty‐two eyes of 16 patients with cataract had phacoemulsification implantation of 1CU accommodating IOL. Twenty eyes of 10 age‐matched and sex‐matched patients with cataract had the same surgery but with a foldable acrylic IOL. All patients had assessments of the amplitude of accommodation, refraction, uncorrected and best corrected distance and near visual acuity, and distance corrected near visual acuity before surgery up to 12 months after surgery. Contrast visual acuities were measured 1 year after surgery. Anterior segment photography, intraocular pressure measurements, specular microscopy, and computerized topography were also performed. Results: The final best corrected distance visual acuity was above 20/25 in all eyes with the 1CU and the AcrySof IOLs. The mean distance corrected near visual acuity was significantly higher in the 1CU IOL group than in the acrylic IOL group after 3 months. None of the eyes with the AcrySof IOL implants displayed an accommodative response at any examination. The peak mean amplitude of accommodation with the 1CU IOLs was observed at 3 months and was 0.5 diopters ± 0.44 (SD). Accommodation amplitude declined after 6 months. Conclusion: The 1CU IOL provided additional near acuity postoperatively, but the benefit disappeared at 12 months with a concomitant decrease in accommodation amplitude owing to an increase in anterior and posterior capsular opacities.

Kinetics and Function of Mesenchymal Stem Cells in Corneal Injury

PURPOSE Bone marrow-derived mesenchymal stem cells (MSCs) hold great promise for wound healing and tissue regeneration. In the present study, we investigated the impact of corneal injury on the homeostasis of endogenous MSCs, and the potential of MSCs to home to injured tissue and promote corneal repair. METHODS Corneal injury in mice was induced by thermal cauterization. Circulating MSCs were quantified by flow cytometric analysis. Ex vivo expanded red Q-dot-labeled or GFP+ bone marrow-derived MSCs were intravenously injected after injury and detected using epifluorescence microscopy. Corneal fluorescein staining was performed to evaluate epithelial regeneration. RESULTS Following the induction of corneal injury in mice, a 2-fold increase in the frequency of circulating endogenous MSCs was observed within 48 hours of injury, which was accompanied by increased levels of the stem cell chemoattractants, substance P and SDF-1, in both the injured cornea and blood. Systemically administered MSCs homed to the injured cornea, but not to the normal cornea, and showed long-term survival. In addition, in the setting of corneal injury, MSC administration showed significant and rapid corneal epithelial regeneration. CONCLUSIONS These findings provide novel evidence that corneal injury causes significant mobilization of endogenous MSCs into blood, and that MSCs home specifically to the injured cornea and promote regeneration, highlighting the therapeutic implications of MSC-mediated tissue repair in corneal injury.

The CCR6/CCL20 Axis Mediates Th17 Cell Migration to the Ocular Surface in Dry Eye Disease

PURPOSE Th17 cells are believed to be the primary effector cells in the pathogenesis of dry eye disease (DED). However, the mechanisms by which Th17 cells migrate from the lymphoid tissues to the ocular surface are unknown. The purpose of this study was to investigate the role of the C-C chemokine receptor 6/C-C chemokine ligand 20 (CCR6/CCL20) chemokine axis in mediating Th17 cell migration in DED. METHODS DED was induced by housing C57BL/6 mice in a low-humidity environment supplemented with scopolamine treatment. Th17 cell expression of CCR6 was evaluated using flow cytometry and ocular surface expression of CCL20 was measured using PCR and ELISA assays. CCL20 neutralizing antibody was administered subconjunctivally to DED mice and disease severity, including the frequency of conjunctival Th17 cells, was evaluated. RESULTS CCR6 is preferentially expressed by Th17 cells in both normal and DED mice and DED significantly upregulates ocular surface expression of CCL20. Disruption of CCR6/CCL20 binding with CCL20 neutralizing antibody decreases T-cell migration in vitro and reduces Th17 cell infiltration of the conjunctiva when administered in vivo, significantly improving clinical signs of DED. These changes were accompanied by a decrease in ocular surface inflammatory cytokine levels and corneal CD11b+ cell frequencies. Treatment also significantly reduced the generation of Th17 cells. CONCLUSIONS Local neutralization of CCL20 decreases Th17 cell infiltration of the ocular surface in DED, leading to improvement in clinical signs of disease. This suggests that CCR6/CCL20 interactions direct Th17 cell migration in DED and that disruption of this axis may be a novel therapeutic approach to this condition.

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.

VEGF-trap aflibercept significantly improves long-term graft survival in high-risk corneal transplantation.

Background Graft failure because of immune rejection remains a significant problem in organ transplantation, and lymphatic and blood vessels are important components of the afferent and efferent arms of the host alloimmune response, respectively. We compare the effect of antihemangiogenic and antilymphangiogenic therapies on alloimmunity and graft survival in a murine model of high-risk corneal transplantation. Methods Orthotopic corneal transplantation was performed in hemevascularized and lymph-vascularized high-risk host beds, and graft recipients received subconjunctival vascular endothelial growth factor (VEGF)-trap, anti-VEGF-C, sVEGFR-3, or no treatment, beginning at the time of surgery. Fourteen days after transplantation, graft hemeangiogenesis and lymphangiogenesis were evaluated by immunohistochemistry. The frequencies of Th1 cells in regional lymphoid tissue and graft-infiltrating immune cells were evaluated by flow cytometry. Long-term allograft survival was compared using Kaplan-Meier curves. Results VEGF-trap significantly decreased graft hemangiogenesis as compared to the control group and was most effective in reducing the frequency of graft-infiltrating immune cells. Anti-VEGF-C and sVEGFR3 significantly decreased graft lymphangiogenesis and lymphoid Th1 cell frequencies as compared to control. VEGF-trap (72%), anti-VEGF-C (25%), and sVEGFR-3 (11%) all significantly improved in the 8-week graft survival compared to control (0%), although VEGF-trap was significantly more effective than both anti-VEGF-C (P < 0.05) and sVEGFR-3 (P < 0.05). Conclusion In a clinically relevant model of high-risk corneal transplantation in which blood and lymphatic vessels are present and treatment begins at the time of transplantation, VEGF-trap is significantly more effective in improving long-term graft survival as compared to anti-VEGF-C and sVEGFR-3, but all approaches improve survival when compared to untreated control.

Mesenchymal stem cells home to inflamed ocular surface and suppress allosensitization in corneal transplantation.

PURPOSE To investigate whether systemically injected syngeneic mesenchymal stem cells (MSCs) can home to the transplanted cornea, suppress induction of alloimmunity, and promote allograft survival. METHODS Mesenchymal stem cells were generated from bone marrow of wild-type BALB/c or GFP (green fluorescent protein)+ C57BL/6 mice, and 1×10(6) cells were intravenously injected to allografted recipients 3 hours after surgery. Mesenchymal stem cells homing to the cornea were examined at day 3 post transplantation by immunohistochemistry. MHC (major histocompatibility complex) II+CD11c+ cells were detected in the cornea and lymph nodes (LNs) 14 days post transplantation using flow cytometry. Cytokine expression of bone marrow-derived dendritic cells (BMDCs) was determined using real-time PCR. ELISPOT assay was used to assess indirect and direct host T cell allosensitization, and graft survival was evaluated by slit-lamp biomicroscopy weekly up to 8 weeks. RESULTS Intravenously injected GFP+ MSCs were found in abundance in the transplanted cornea, conjunctiva, and LNs, but not in the ungrafted (contralateral) tissue. The frequencies of mature MHC II+CD11c+ antigen-presenting cells (APCs) were substantially decreased in the corneas and draining LNs of MSC-injected allograft recipients compared to control recipients. Maturation and function of in vitro cultured BMDCs were decreased when cocultured with MSCs. Draining LNs of MSC-injected allograft recipients showed lower frequencies of IFNγ-secreting Th1 cells compared to the control group. Allograft survival rate was significantly higher in MSC-injected recipients compared to non-MSC-injected recipients. CONCLUSIONS Our data demonstrate that systemically administered MSCs specifically home to the inflamed ocular surface and promote allograft survival by inhibiting APC maturation and induction of alloreactive T cells.

The Semaphorin 3A Inhibitor SM-345431 Accelerates Peripheral Nerve Regeneration and Sensitivity in a Murine Corneal Transplantation Model

Background Peripheral nerve damage of the cornea is a complication following surgery or infection which may lead to decreased visual function. We examined the efficacy of the semaphorin 3A inhibitor, SM-345431, in promoting regeneration of peripheral nerves in a mouse corneal transplantation model. Methodology/Principal Findings P0-Cre/Floxed-EGFP mice which express EGFP in peripheral nerves cells were used as recipients of corneal transplantation with syngeneic wild-type mouse cornea donors. SM-345431 was administered subconjunctivally every 2 days while control mice received vehicle only. Mice were followed for 3 weeks and the length of regenerating nerves was measured by EGFP fluorescence and immunohistochemistry against βIII tubulin. Cornea sensitivity was also measured by the Cochet-Bonnet esthesiometer. CD31 staining was used to determine corneal neovascularization as a possible side effect of SM-345431. Regeneration of βIII tubulin positive peripheral nerves was significantly higher in SM-345431 treated mice compared to control. Furthermore, corneal sensitivity significantly improved in the SM-345431 group by 3 weeks after transplantation. Neovascularization was limited to the peripheral cornea with no difference between SM-345431 group and control. Conclusions/Significance Subconjunctival injections of SM-345431 promoted a robust network of regenerating nerves as well as functional recovery of corneal sensation in a mouse keratoplasty model, suggesting a novel therapeutic strategy for treating neurotrophic corneal disease.

In Vivo Expansion of Regulatory T Cells by Low-Dose Interleukin-2 Treatment Increases Allograft Survival in Corneal Transplantation.

Background Corneal allograft survival dramatically decreases in hosts with inflamed or vascularized recipient beds. We have previously shown that in rejected corneal allografts regulatory T cells (Treg) demonstrate diminished Foxp3 expression and immunoregulatory function. Treatment with low doses of IL-2 selectively expands Treg and has been proposed for the treatment of autoimmune diseases. In this study, we investigated the effect of low-dose IL-2 administration on Treg function and corneal allograft survival. Methods Allogeneic corneal transplantation was performed on inflamed host beds. Low-dose systemic IL-2 was administered starting 3 days before grafting until 6 weeks after transplantation. Frequencies of Treg and their immunosuppressive function and antigen specificity were assessed using flow cytometry, in vitro proliferation assays, and adoptive transfer experiments. Frequencies of effector T cells (Teff) and graft infiltrating immune cells were measured at 2 weeks posttransplantation. Long-term allograft survival was evaluated for up to 9 weeks using Kaplan-Meier survival analysis. Results Treatment with low-dose IL-2 significantly increased frequencies of CD4+CD25+Foxp3+ Treg and their immunosuppressive function. It also suppressed alloimmune response as shown by the decreased CD4+ IFN&ggr;+ T cell frequencies and graft infiltration of CD45+ and CD4+ cells. Clinical evaluation of the grafts showed significant improvement in long-term corneal allograft survival in the IL-2 treated group compared with controls. Conclusions Our study is the first to report that treatment with low-dose IL-2 increases survival of corneal allografts. We propose that IL-2-mediated Treg expansion can be an effective tool to prevent alloimmunity and to improve long-term allograft survival in transplantation.