Tohru Sakimoto

Laser eye surgery for refractive errors

Several laser and non-laser refractive surgical procedures have been used to modify the shape of the cornea and correct myopia, hyperopia, astigmatism, and presbyopia. Introduction of the excimer laser to reshape the cornea has resulted in remarkable developments in the correction of these refractive errors. Combined with other advanced ophthalmological instruments, laser refractive eye surgery has resulted in a substantial rise in the safety, efficacy, and predictability of surgical outcomes. Despite these advances, certain limitations and complications persist. In this review, we describe the history, preoperative assessment, surgical techniques, outcomes, and complications of laser refractive surgery.

Increased Tear Fluid Production as a Compensatory Response to Meibomian Gland Loss: A Multicenter Cross-sectional Study

PURPOSE To compare tear film parameters as well as meibomian gland morphologic features and function among patients with meibomian gland dysfunction (MGD), those with non-Sjögren syndrome aqueous-deficient dry eye (non-SS ADDE), those with non-SS ADDE and MGD, and normal subjects. DESIGN Multicenter, cross-sectional, observational case series. PARTICIPANTS Forty-one eyes of 41 patients (all women; mean age ± standard deviation, 62.1±9.9 years) with non-SS ADDE, 70 eyes of 70 patients (all women; 66.0±8.7 years) with MGD, 17 eyes of 17 patients (all women; 72.4±7.8 years) with non-SS ADDE and MGD, and 70 eyes of 70 normal control subjects (all women; 65.0±7.1 years). METHODS Ocular symptoms were scored from 0 to 14 and lid margin abnormalities from 0 to 4 according to their respective number. Meibomian gland changes were scored from 0 to 6 (meiboscore) on the basis of noncontact meibography findings, and meibum was graded from 0 to 3 depending on its volume and quality. Conjunctival and corneal epithelial damage were scored from 0 to 9 (fluorescein score). Tear film break-up time (TBUT) was measured as an index of tear film stability, and tear fluid production was evaluated with Schirmers test. MAIN OUTCOME MEASURES Ocular symptom score, lid margin abnormality score, meiboscore, meibum grade, fluorescein score, TBUT, and Schirmers test value. RESULTS The ocular symptom score did not differ significantly between the MGD and non-SS ADDE groups (P = 0.762). The lid margin abnormality score, meiboscore, and meibum grade were significantly higher in the MGD group than in the non-SS ADDE group (P = 0.0012, P < 0.0001, and P < 0.0001, respectively). The fluorescein score, TBUT, and Schirmers test value were significantly worse in the non-SS ADDE group than in the MGD group (P < 0.0001, P = 0.0061, and P < 0.0001, respectively). The meiboscore correlated significantly with Schirmers test value only in the MGD group (ρ = 0.508, P = 8.3×10(-6)). CONCLUSIONS An increase in tear fluid production likely compensates for loss of meibomian glands in individuals with MGD.

Pretranscriptional Regulation of Tgf-β1 by PI Polyamide Prevents Scarring and Accelerates Wound Healing of the Cornea After Exposure to Alkali

Corneal alkali burns are a serious clinical problem that often leads to permanent visual impairment. In this process, transforming growth factor (Tgf)-β1 is upregulated and involved in the response to corneal injury and the process of corneal stromal scarring. To develop an efficient compound to inhibit Tgf-β1 in the cornea, we designed GB1201, a pyrrole-imidazole (PI) polyamide targeting rat Tgf-β1 gene promoter to the activator protein-1 (AP-1) binding site. GB1201 showed a high binding affinity to the target DNA sequence in the gel mobility shift and Biacore assays. GB1201 significantly inhibited the rat Tgf-β1 gene promoter activity in HEK (human embryonic kidney) 293 cells in a concentration-dependent manner. Topically administrated GB1201 was distributed immediately to the nuclei of all cell layers of the cornea and remained for 24 hours. A corneal alkali burn model in rats was used to evaluate the therapeutic efficacy of GB1201. GB1201 suppressed the upregulation of Tgf-β1 in the burned cornea, both in the mRNA and protein levels. Moreover, daily treatment with GB1201 for a week significantly improved the corneal tissue wound healing, reduced corneal stromal scarring, and prevented corneal haze formation. Our data suggest that PI polyamide may open new opportunities for therapeutic intervention in the treatment of chemically burned corneas.Corneal alkali burns are a serious clinical problem that often leads to permanent visual impairment. In this process, transforming growth factor (Tgf)-beta1 is upregulated and involved in the response to corneal injury and the process of corneal stromal scarring. To develop an efficient compound to inhibit Tgf-beta1 in the cornea, we designed GB1201, a pyrrole-imidazole (PI) polyamide targeting rat Tgf-beta1 gene promoter to the activator protein-1 (AP-1) binding site. GB1201 showed a high binding affinity to the target DNA sequence in the gel mobility shift and Biacore assays. GB1201 significantly inhibited the rat Tgf-beta1 gene promoter activity in HEK (human embryonic kidney) 293 cells in a concentration-dependent manner. Topically administrated GB1201 was distributed immediately to the nuclei of all cell layers of the cornea and remained for 24 hours. A corneal alkali burn model in rats was used to evaluate the therapeutic efficacy of GB1201. GB1201 suppressed the upregulation of Tgf-beta1 in the burned cornea, both in the mRNA and protein levels. Moreover, daily treatment with GB1201 for a week significantly improved the corneal tissue wound healing, reduced corneal stromal scarring, and prevented corneal haze formation. Our data suggest that PI polyamide may open new opportunities for therapeutic intervention in the treatment of chemically burned corneas.

Active form of gelatinases in tear fluidin patients with corneal ulcer or ocular burn

PURPOSE To investigate the expression of the active form of gelatinases (gelatinase A: matrix metalloproteinase-2 [MMP-2] and gelatinase B: matrix metalloproteinase-9 [MMP-9]) in ocular surface disorders, we determined the presence of the active form of these gelatinases in the tear fluid of patients with corneal ulcer or ocular burn. METHODS The subjects consisted of the ulcer group comprising 13 eyes, the burn group comprising 6 eyes, and the normal group comprising 10 eyes. Tear samples were taken by the method of the Schirmer test I. The tears were eluted using extraction buffer containing 0.01 M phosphate buffer solution (pH 7.2), 1% Tween 20 and 1 M NaCl, and analyzed by gelatin zymography. RESULTS Only the proforms of MMP-2, and MMP-9 were detected in the normal group and in uncomplicated herpetic keratitis or herpetic keratitis cases complicated by mixed infection in the ulcer group. Active MMP-9 was detected in mild corneal ulcer cases and mild ocular burn cases. Both active MMP-2 and active MMP-9 were detected in endogenous corneal ulcer cases and severe burn cases, including perforation cases. CONCLUSIONS Both active gelatinases were detected in tears of severe corneal ulcer or severe ocular burn cases. The active form of gelatinase expression may be related to the severity of ulceration.

Metalloproteinases in corneal diseases: degradation and processing.

Abstract: Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases with the potential to degrade all types of extracellular matrix. The ADAM (a disintegrin and metalloproteinase) family of peptidases was recently identified as cleaving the extracellular domain of transmembrane proteins. This was termed ectodomain shedding. We investigated the MMP expression in patients with corneal diseases and the potential role of ADAMs in corneal pathophysiology. We detected upregulation of the active form of MMP-2 and MMP-9 in the tear fluid from patients with corneal melting or recurrent corneal erosion. Using human corneal epithelial cells, we observed ADAM17-dependent ectodomain shedding of soluble tumor necrosis factor receptor 1 and soluble interleukin-6 (IL-6) receptor (sIL-6R). The production of sIL-6R was also induced by messenger RNA splicing in the human corneal epithelial cells. IL-6/sIL-6R-induced signal transducer and activator of transcription 3 phosphorylation was observed in cultured human corneal fibroblasts, suggesting that IL-6 trans-signaling induced inflammatory cellular signaling in the human corneal fibroblasts. We demonstrated that MMPs are significantly upregulated in collagen-destructive disorders of the cornea. Additionally, we observed that ectodomain shedding by ADAMs in corneal epithelial cells mediated the production of soluble cytokine receptors. Trans-signaling of IL-6 can induce an inflammatory response in corneal stroma, indicating the significance of IL-6 trans-signaling in ocular surface inflammation. Thus, MMPs and ADAMs play an important role in the pathophysiology of corneal diseases.

VEGF TrapR1R2 suppresses experimental corneal angiogenesis

Purpose To determine the effect of vascular endothelial growth factor (VEGF) TrapR1R2 on bFGF-induced experimental corneal neovascularization (NV). Methods Control pellets or pellets containing 80 ng bFGF were surgically implanted into wild-type C57BL/6 and VEGF-LacZ mouse corneas. The corneas were photographed, harvested, and the percentage of corneal NV was calculated. The harvested corneas were evaluated for VEGF expression. VEGF-LacZ mice received tail vein injections of an endothelial-specific lectin after pellet implantation to determine the temporal and spatial relationship between VEGF expression and corneal NV. Intraperitoneal injections of VEGF TrapR1R2 or a human IgG Fc domain control protein were administered, and bFGF pellet-induced corneal NV was evaluated. Results NV of the corneal stroma began on day 4 and was sustained through day 21 following bFGF pellet implantation. Progression of vascular endothelial cells correlated with increased VEGF-LacZ expression. Western blot analysis showed increased VEGF expression in the corneal NV zone. Following bFGF pellet implantation, the area of corneal NV in untreated controls was 1.05±0.12 mm2 and 1.53±0.27 mm2 at days 4 and 7, respectively. This was significantly greater than that of mice treated with VEGF Trap (0.24±0.11 mm2 and 0.35±0.16 mm2 at days 4 and 7, respectively; p<0.05). Conclusions Corneal keratocytes express VEGF after bFGF stimulation and bFGF-induced corneal NV is blocked by intraperitoneal VEGF TrapR1R2 administration. Systemic administration of VEGF TrapR1R2 may have potential therapeutic applications in the management of corneal NV.

Comparison of topical dexamethasone and topical FK506 treatment for the experimental allergic conjunctivitis model in BALB/c mice.

PurposeTo evaluate the efficacy of topical dexamethasone and topical FK506 treatment on allergic inflammation in conjunctiva, we performed a comparative study using a mouse experimental allergic conjunctivitis model.MethodsThe experimental allergic conjunctivitis model was developed by ovalbumin (OVA) instillation after OVA sensitization by intraperitoneal application of a mixed solution of OVA and aluminum hydroxide. The Balb/c mice of the experimental allergic conjunctivitis model were divided into three groups, depending on whether or not they received topical treatment and which type of treatment they received. The allergy group comprised six mice without topical treatment; the dexamethasone (Dx) group comprised six mice receiving topical 0.1% dexamethasone ointment treatment; and the FK506 (FK) group comprised six mice receiving topical 0.1% FK506 ointment treatment. The negative control group comprised six mice with neither sensitization nor topical treatment. For the histological examination, frozen sections of the eyelids, eyeballs, and lacrimal glands were stained with acid Giemsa stain or, in the immunohistochemical method, with alkaline phosphatase–antialkaline phosphatase (APAAP), for CD4. Densities of eosinophils and CD4-positive lymphocytes in the conjunctival tissue were counted. Productive C ε gene expression in the conjunctival tissue, including the lacrimal gland, was also evaluated by reverse transcriptase polymerase chain reaction (RT-PCR).ResultsIn the histological study, the cell density of eosinophils infiltrating into subconjunctival tissues was 66.3 ± 34.7 cells/mm2 (mean ± SD) for the allergy group, 2.5 ± 4.2 cells/mm2 for the Dx group, 4.2 ± 8.6 cells/mm2 for the FK group, and 3.7 ± 7.6 cells/mm2 for the negative control group. The cell density of CD4-positive cells infiltrating into subconjuctival tissues was 145.9 ± 66.4 cells/mm2 for the allergy group, 10.0 ± 12.9 cells/mm2 for the Dx group, 10.1 ± 15.5 cells/mm2 for the FK group, and 21.7 ± 17.8 cells/mm2 for the negative control group. The allergy group showed significant differences in the density of eosinophils and CD4-positive cells compared with those of the Dx (P < 0.0001) and FK (P < 0.0001) groups. In the RT-PCR study, the expression of productive C ε gene was positive in the Dx group, but negative in the allergy group, the FK group, and the negative control group.ConclusionsTopical Dx and FK506 treatments were both efficacious in suppressing eosinophil and lymphocyte infiltration into subconjunctival tissue in the mouse experimental allergic conjunctivitis model. However, the influence of their IgE productive mechanisms seemed to differ, causing different efficacy of productive C ε gene expression in the conjunctival tissue. Jpn J Ophthalmol 2005;49:205–210© Japanese Ophthalmological Society 2005

Histological Study of Conjunctiva-associated Lymphoid Tissue in Mouse

PURPOSE To investigate conjunctiva-associated lymphoid tissue (CALT) in the mouse conjunctiva by histological methods. METHODS The presumed follicular tissue in the conjunctiva of normal mice, age ranging from 4 to 6 weeks, was histologically investigated by the hematoxylin-eosin staining method. Next, we treated the mice with topical instillation of a combined solution of ovalbumin and cholera toxin B to investigate the morphological changes of conjunctival follicles to antigen challenge. The treated mice underwent sequential clinical examinations, and the conjunctival follicular tissue was examined by an immunohistochemical method using anti-CD4 antibody, anti-CD8 antibody, and anti-S-100 antibody. RESULTS Follicular tissue was present on the mouse nictitating membrane. Both size and number of follicular tissue areas increased with topical ovalbumin treatment. Immunohistochemical study revealed CD4, CD8, and S-100 positive cells in the follicular tissue. The epithelial layer, corresponding to follicular tissue, demonstrated intra-epithelial pocket and the presence of CD4-positive cells in the intra-epithelial pocket. CONCLUSION Follicular tissue at the nictitating membrane is CALT in the mouse.

Regulation of soluble interleukin-6 (IL-6) receptor release from corneal epithelial cells and its role in the ocular surface

PurposeInterleukin (IL)-6 signaling through its soluble receptor (sIL-6R) (IL-6 trans-signaling) plays an important role in various inflammatory states. We investigated production of sIL-6R in the corneal epithelium and examined the role of IL-6 trans-signaling in the cornea.MethodsIn-vitro experiments were performed using SV40-transformed human corneal epithelial cells (HCEC) and primary human corneal fibroblasts (HCF, keratocytes). Ectodomain shedding in HCEC was stimulated by adding phorbol myristate acetate (PMA, 3 μM) both with and without ectodomain shedding inhibition using TNF-α processing inhibitor-1 (TAPI-1, 250 ng/mL), and the concentration of sIL-6R in the culture medium was determined by enzyme-linked immunosorbent assay (ELISA). Expression of differential sIL-6R mRNA splicing (DS-sIL-6R) in HCEC was examined by using reverse transcription (RT)-PCR. The recombinant IL-6 or combination of recombinant IL-6/sIL-6R was added to HCF culture medium and phosphorylation of STAT3 was analyzed by Luminex assay. Tear fluid from patients with Sjögren syndrome was collected and analyzed by ELISA for sIL-6R concentration.ResultsIn HCEC culture medium, sIL-6R release was increased significantly (P < 0.01) by adding PMA and this increased release of sIL-6R was inhibited significantly by adding TAPI-1, indicating the participation of ectodomain shedding in sIL-6R production. In RT-PCR, DS-sIL-6R expression was noted in HCEC. IL-6/sIL-6R-induced STAT3 phosphorylation was recognized in cultured HCF, suggesting IL-6 trans-signaling induced inflammatory cellular signaling in HCF. In the tear fluid of the patients with Sjögren syndrome, sIL-6R expression was up-regulated (Sjögren syndrome; 2.38 ± 0.98 ng/mL, normal control; 0.16 ± 0.34 ng/mL).ConclusionsProduction of sIL-6R was induced by both ectodomain shedding and mRNA splicing in the corneal epithelium. IL-6 trans-signaling can induce an inflammatory response in corneal fibroblasts. The up-regulation of sIL-6R in inflamed ocular surfaces suggests a pivotal role of sIL-6R at the ocular surface.

Anti-inflammatory effect of IL-6 receptor blockade in corneal alkali burn

We investigated the effect of soluble IL-6R (sIL-6R) blockade on corneal inflammation. Topical instillation of either anti-IL-6R antibody (MR16-1) or phosphate buffered saline (PBS) was applied after wounding BALB/c mice corneas with alkali burn. The vascularized area was significantly reduced in the MR16-1 group. The immunoreactivity of phosphorylated STAT3, Gr-1, and F4/80 diminished significantly in the MR16-1 group. Laser capture microdissection resulted in a significant down-regulation of the mRNA expressions of ICAM-1, MCP-1, and VEGF-A in the corneal stroma of the MR16-1 group. Adding a combination of recombinant IL-6 and sIL-6R resulted in a significant increase in the release of VEGF from human corneal fibroblasts. As the infiltration of inflammatory cells, the expression of phosphorylated STAT3, and the expressions of inflammatory-related molecules in the experimental model of corneal inflammation were significantly inhibited by topical instillation of MR16-1, we deduce that IL-6 trans-signaling plays a significant role in ocular surface inflammation and that the blockade of IL-6R contributes to the reduction in corneal inflammation.