Yasuaki Hata

Brilliant Blue G Selectively Stains The Internal Limiting Membrane/brilliant Blue G–assisted Membrane Peeling

Purpose: To report the use of the dye brilliant blue G (BBG) for staining of the internal limiting membrane (ILM) during macular hole (MH) and epiretinal membrane (ERM) surgery. Methods: This study was designed as an interventional, noncomparative, prospective, clinical case series. Twenty eyes from 20 consecutive patients with MH or ERM underwent BBG-assisted ILM and ERM removal. In MH cases, a posterior vitreous detachment was created, followed by the injection of 0.25 mg/mL BBG solution into the vitreous cavity and immediate washout of the BBG. This technique improved visualization of the ILM, enabling peeling and surgery to be performed successfully. However, in ERM cases, staining of the ERM could not be confirmed at this concentration. Finally, the ILM including the ERM was removed in all cases. Preoperative and postoperative ophthalmic examinations were performed. Results: Postoperatively, 17 patients (85%) had visual acuity improved by at least 2 Snellen lines. No adverse effects were observed postoperatively during the observation period (mean follow-up ± SD, 7.3 ± 1.0 months). Conclusions: BBG selectively stains the ILM. This technique can facilitate the management of MH and ERM surgery without any adverse effects, as was shown in this short-term study.

Role of TGF-β in proliferative vitreoretinal diseases and ROCK as a therapeutic target

Cicatricial contraction of preretinal fibrous membrane is a cause of severe vision loss in proliferative vitreoretinal diseases such as proliferative diabetic retinopathy (PDR) and proliferative vitreoretinopathy (PVR). TGF-β is overexpressed in the vitreous of patients with proliferative vitreoretinal diseases and is also detectable in the contractile membranes. Therefore, TGF-β is presumed to contribute to the cicatricial contraction of the membranes, however, the underlying mechanisms and TGF-βs importance among various other factors remain to be elucidated. Vitreous samples from PDR or PVR patients caused significantly larger contraction of hyalocyte-containing collagen gels, compared with nonproliferative controls. The contractile effect was strongly correlated with the vitreal concentration of activated TGF-β2 (r = 0.82, P < 0.0001). PDR or PVR vitreous promoted expression of α-smooth muscle actin (α-SMA) and phosphorylation of myosin light chain (MLC), a downstream mediator of Rho-kinase (ROCK), both of which were dramatically but incompletely suppressed by TGF-β blockade. In contrast, fasudil, a potent and selective ROCK inhibitor, almost completely blocked the vitreous-induced MLC phosphorylation and collagen gel contraction. Fasudil disrupted α-SMA organization, but it did not affect its vitreal expression. In vivo, fasudil significantly inhibited the progression of experimental PVR in rabbit eyes without affecting the viability of retinal cells by electroretinographic and histological analyses. These results elucidate the critical role of TGF-β in mediating cicatricial contraction in proliferative vitreoretinal diseases. ROCK, a key downstream mediator of TGF-β and other factors might become a unique therapeutic target in the treatment of proliferative vitreoretinal diseases.

Biocompatibility of brilliant blue G in a rat model of subretinal injection.

Purpose: To evaluate the toxicity of brilliant blue G (BBG) compared with those of indocyanine green (ICG) and trypan blue (TB) in a rat model of subretinal injection. Methods: Retinal detachment was produced by subretinal injection of the dyes. The biocompatibility of BBG (0.25 mg/mL) was evaluated over 2 months and 2 weeks by ophthalmic examinations. The eyes were enucleated and analyzed by light, fluorescence, as well as transmission electron microscopy. Apoptotic cell death was detected by TdT-dUTP terminal nick-end labeling. The results were compared with those for ICG (5 mg/mL) and TB (1 mg/mL). Results: ICG caused retinal degeneration and retinal pigment epithelial (RPE) cell atrophy 2 weeks after subretinal injection. Apoptotic cell death was detected in the inner and outer nuclear layers and the RPE layer, especially the photoreceptors. TB caused less retinal degeneration, mainly in the area detached by the subretinal injection. BBG had no detectable toxic effects after 2 months and 2 weeks. Apoptotic cell death was detected in the ICG and TB groups, mainly in the photoreceptors. Conclusions: Subretinal injection of the dyes caused retinal cell degeneration at lower concentrations than those reported for intravitreous injection. However, subretinal injection of BBG at 0.25 mg/mL appeared to provide satisfactory biocompatibility.

Functional characteristics of connective tissue growth factor on vitreoretinal cells.

Connective tissue growth factor (CTGF) level is elevated in eyes with proliferative vitreoretinal diseases, such as proliferative diabetic retinopathy and proliferative vitreoretinopathy (PVR), as we previously reported, but its functional characteristics on vitreoretinal cells are yet to be clarified. In this study, we demonstrated a growth-promoting effect of CTGF on cultured hyalocytes and bovine retinal pigment epithelial cells (BRPEs) with the induction of p44/p42 mitogen-activated protein kinase phosphorylation and [3H]thymidine incorporation. CTGF also stimulated the synthesis of fibronectin by hyalocytes and BRPEs without significant effect on collagen gel contraction by these cells. On the other hand, CTGF had no direct effects on the proliferation, migration, or in vitro tube formation by vascular endothelial cells. Nevertheless, CTGF promoted vascular endothelial growth factor (VEGF) gene expression by hyalocytes and BRPEs. Although the concentrations of both CTGF and VEGF in the human vitreous samples with proliferative vitreoretinal diseases were elevated, there was no significant correlation between these concentrations. These findings indicate that CTGF appears to be involved in the formation of proliferative membranes without direct regulation of their cicatricial contraction in the pathogenesis of proliferative vitreoretinal diseases. Whereas CTGF might have no direct effects or minimal effects, if any, on retinal neovascularization, it is possible that CTGF has indirect effects by modulating the expression of VEGF.

Evaluating adjunctive surgical procedures during vitrectomy for diabetic macular edema.

Purpose: To evaluate the long-term outcomes of adjunctive surgical procedures during pars plana vitrectomy (PPV) for the treatment of diabetic macular edema (DME). Methods: In this nonrandomized study, we retrospectively analyzed 57 eyes of 54 patients who had DME and had undergone PPV. We performed PPV using three different surgical procedures: conventional PPV (group PVD; 13 eyes), triamcinolone acetonide (TA)–assisted PPV (group TA; 22 eyes), and TA-assisted PPV combined with internal limiting membrane (ILM) peeling (group ILM; 22 eyes). We also evaluated the preoperative and postoperative best-corrected visual acuity (BCVA) results. Results: The overall mean preoperative BCVA was 0.86 logarithm of the minimal angle of resolution unit. In groups PVD, TA, and ILM, BCVAs were 0.99, 0.90, and 0.74 (P = 0.310), respectively. The mean postoperative BCVA for all patients improved to 0.68 (P = 0.005). The postoperative BCVA improved in 47% of the treated eyes, it remained unchanged in 37% of the treated eyes, and it deteriorated in 16% of the treated eyes. However, we observed no significant difference in the mean postoperative BCVAs between the three groups. Furthermore, we found that there was no significant difference in postoperative BCVA improvements between any of the groups (P = 0.450). Conclusion: The present study suggests that these 3 PPV approaches do not significantly affect postoperative BCVAs after 18 months of DME treatment.

Interleukin‐18 regulates pathological intraocular neovascularization

Recently, the proinflammatory cytokine IL‐18 has been shown to have a role in angiogenesis. This study aimed to elucidate its role in abnormal neovascularization (NV) in an oxygen‐induced retinopathy (OIR) mouse model of the retinopathy seen in human premature newborns. IL‐18 was constitutively expressed in the retina in C57BL/6 mice, but expression transiently dropped on Day 17 after birth in mice exposed to 75% oxygen for 5 days between Days 7 and 12. Coincident with the IL‐18 reduction in oxygen‐treated mice, vascular endothelial growth factor was expressed in the retina, and OIR developed. By Day 24, NV in the retina had regressed to normal levels. By contrast, IL‐18 knockout mice, exposed to elevated oxygen concentrations, developed more severe OIR on Day 17, and it is important that this persisted until Day 24. This suggested that IL‐18 negatively regulated retinal NV. To investigate this further, we administrated recombinant IL‐18 to C57BL/6 mice during the development of OIR but found no significant inhibition of retinopathy. However, when IL‐18‐binding protein was administered during the OIR recovery phase to neutralize endogenous IL‐18, OIR was still apparent on Day 24. We therefore concluded that IL‐18 regulates pathogenic retinal NV by promoting its regression rather than inhibiting its development. This suggests some useful, new approaches to treating retinopathy in humans.

Aqueous and vitreous penetration of levofloxacin after topical and/or oral administration

Purpose. To investigate the aqueous and vitreous penetration of levofloxacin, the drug was administered topically and/or orally to patients undergoing vitrectomy. Methods. Thirty-six patients undergoing initial vitrectomy with phacoemulsification and aspiration (PEA) were enrolled, and were divided randomly into three groups. Group 1 was treated with topical application of levofloxacin (three times on the day before surgery and seven times on the day of surgery), Group 2 received oral administration of levofloxacin (200 mg twice on the day before surgery and 200 mg at 3 hours before surgery), and Group 3 received both topical and oral levofloxacin according to the above schedules. The concentration of levofloxacin was measured in aqueous humor and vitreous fluid samples obtained during surgery. Results. In Groups 1, 2, and 3, the mean levofloxacin concentration in aqueous humor was 0.765±0.624 μg/mL, 1.279±0.440 μg/mL, and 1.823±0.490 μg/mL, respectively, while the mean levofloxacin concentration in vitreous fluid was <0.02 μg/mL, 1.455±0.445 μg/mL, and 1.369±0.530 μg/mL, respectively. Conclusions. Oral administration of levofloxacin at a dose of 400 mg/day was sufficient for the prophylaxis of ocular infections, because the drug concentrations in both aqueous humor and vitreous fluid were higher than the MIC90 values for major ocular pathogens. Topical application of levofloxacin achieved adequate drug levels in aqueous humor, but not in vitreous fluid, while combined topical and oral administration had an additive effect on the drug concentration in aqueous humor.

Triamcinolone acetonide-assisted pars plana vitrectomy improves residual posterior vitreous hyaloid removal: ultrastructural analysis of the inner limiting membrane.

Objective: To determine whether triamcinolone acetonide (TA) can facilitate residual posterior vitreous hyaloid removal in pars plana vitrectomy (PPV), we examined the ultrastructure of inner limiting membrane (ILM) removed in TA-assisted PPV for diabetic macular edema (DME). Patients and Methods: In this retrospective series of 38 eyes of 37 patients who underwent PPV and ILM removal for diffuse DME with posterior hyaloid attachment, 24 eyes underwent standard PPV without TA (control group), and 14 eyes underwent TA-assisted PPV (TA group). Excised ILMs during PPV were examined by transmission electron microscopy (control group, n = 20; TA group, n = 10) or scanning electron microscopy (control group, n = 4; TA group, n = 4). Results: Transmission electron microscopy clearly demonstrated that the ratio of the posterior vitreous hyaloid remaining on ILM was significantly lower (P = 0.0187) in the TA group than in the control group and also that TA-assisted PPV successfully removed posterior hyaloid in five of seven eyes with TA granules remaining on the retinal surface even after surgical separation of the posterior vitreous. Scanning electron microscopy enabled spatial analysis of the residual posterior hyaloid on ILM, which appeared in a patchy fashion in the control group. Conclusions: TA-assisted PPV clearly demonstrated the residual posterior hyaloid on ILM and allowed more efficient removal of the posterior hyaloid than standard PPV.

The internal limiting membrane peeling with brilliant blue G staining for retinal detachment due to macular hole in high myopia

Internal limiting membrane (ILM) peeling without dye is technically difficult in retinal detachment due to macular hole in high myopia (MHRD) with chorioretinal atrophy because of poor visualisation of ILM. Accordingly, a dye such as indocyanine green (ICG) or trypan blue (TB) is essential for facilitating ILM peeling. However, recent reports have emerged about retinal damage caused by ICG and TB both in experimental and clinical use.1–4 There is a need to develop new dyes for effective and safe staining in order to facilitate ILM peeling. In the present study, we investigated the efficacy of a new dye: brilliant blue G (BBG) assisted ILM peeling for MHRDs.nnThis study was designed as a …

Preclinical investigation of fluorometholone acetate as a potential new adjuvant during vitreous surgery

ObjectiveTo examine the effects of intravitreal fluorometholone acetate (FMT) on the morphology and function of the retina and to investigate its possible use for vitreous surgery.MethodsBrown Norway rat eyes (nu2009=u20096, 12 groups) were injected with 0.05xa0ml of SF6 gas for vitrectomization. Four weeks later, FMT solution was injected into the vitreous cavity/subretinal space of the vitrectomized eyes at doses of 10, 20, and 40xa0mg/ml (0.05xa0ml/eye, nu2009=u200912 for each group). The retinal function was evaluated by electroretinography (ERG) at 4 and 8xa0weeks after FMT injection. Retinal toxicity was also assessed histologically by a light microscopy. Sham-operated eyes (0.05xa0ml of irrigating solution, nu2009=u200912) were used as control animals. FMT-assisted pars plana vitrectomy with internal limiting membrane (ILM) peeling was performed in primate eyes (nu2009=u20092). Retinal toxicity was assessed by ophthalmoscope, fluorescein angiography and electron microscopy three months after the vitreous surgery.ResultsThere was no remarkable reduction in any ERG waves at either time interval at 4 and 8xa0weeks after the intravitreal/subretinal injection of FMT. No obvious histological change was observed in any of the rat eyes either. Using ophthalmoscope, fluorescein angiography and electron microscopy, the appearance of the primate retinas remained to be in a non-pathological condition.ConclusionFMT appears to be a potentially useful tool in assisting vitreous surgery including safe ILM peeling.