Tomomi Metoki

Activation of mitochondrial calpain and release of apoptosis-inducing factor from mitochondria in RCS rat retinal degeneration.

The present study was performed to investigate changes of cytosolic and mitochondrial calpain activities, and effects of intravitreously injected calpain inhibitor on photoreceptor apoptosis in Royal College of Surgeons (RCS) rats. Time courses of activities for both cytosolic and mitochondrial calpains and amount of calpastatin in RCS rat retina were analyzed by subcellular fractionation, calpain assay and western blotting. Calpain assay was colorimetrically performed using Suc-LLVY-Glo as substrate. Effects of intravitreously injected calpain inhibitor (ALLN and PD150606) on RCS rat retinal degeneration were analyzed by TUNEL staining. Effects of mitochondrial calpain activity on activation and translocation of apoptosis-inducing factor (AIF) were analyzed by western blotting. Mitochondrial calpain started to be significantly activated at postnatal (p) 28 days in RCS rat retina, whereas cytosolic micro-calpain was activated at p 35 days, although specific activity of mitochondrial calpain was 13% compared to cytosolic micro-calpain. Intravitreously injected ALLN and PD150606 effectively inhibited photoreceptor apoptosis only when injected at p 25 days, but did not inhibit photoreceptor apoptosis when injected at p 32 days. Parts of AIF were truncated/activated by mitochondrial calpains and translocated to the nucleus. These results suggest that 1), calpain presents not only in the cytosolic fraction but also in the mitochondrial fraction in RCS rat retina; 2), mitochondrial calpain is activated earlier than cytosolic calpain during retinal degeneration in RCS rats; 3), photoreceptor apoptosis may be regulated by not only calpain systems but also other mechanisms; 4), mitochondrial calpain may activate AIF to induce apoptosis; and 5), calpain inhibitors may be partially effective to inhibit photoreceptor apoptosis in RCS rats. The present study provides new insights into the molecular basis for photoreceptor apoptosis in RCS rats and the future possibility of new pharmaceutical treatments for retinitis pigmentosa.

Effect of nilvadipine on central visual field in retinitis pigmentosa: a 30-month clinical trial.

Purpose: To assess the effects of nilvadipine on the progression of central visual field defect in retinitis pigmentosa (RP). Design: Prospective, randomized, nonmasked, single-center trial. Methods: Patients with RP were randomly divided into a treated group receiving oral nilvadipine at 4 mg/day for ≧30 months and a control group receiving tocopherol nicotinate at 300 mg/day, helenien at 15 mg/day or no medication for the same periods. Progression of RP was evaluated using the 10-2 SITA Fast Program of the Humphrey Visual Field Analyzer, and regression coefficients calculated from the time courses of mean deviation (MD slope) were compared between groups. Results: Nineteen patients in the treated group and 14 patients in the control group completed the follow-up for ≧30 months. The mean (±standard deviation) duration of observation was 48.8 ± 11.8 months (median 48 months, range 30–66 months) for the treated group and 49.2 ± 18.1 months (median 48 months, range 30–90 months) for the control group (p = 0.94). Mean (±standard error of the mean, SEM) regression coefficients of the averaged MD values for the initial 30 months were –0.35 ± 0.17 dB/year in the treated group and –0.75 ± 0.06 dB/year in the control group (p < 0.01). Mean (±SEM) MD slopes for total observational periods were –0.49 ± 0.17 dB/year in the treated group and –0.89 ± 0.16 dB/year in the control group (mean ± SEM, p = 0.042). Conclusion: Nilvadipine at 4 mg/day significantly retarded progression of central visual field defects in RP in this small patient series.

Study of pharmacological effects of nilvadipine on RCS rat retinal degeneration by microarray analysis.

In our recent study, we found that the Ca(2+) antagonist, nilvadipine caused significant preservation of photoreceptor cells in The Royal College of Surgeons (RCS) rats [Invest. Ophthalmol. Vis. Sci. 43 (2002) 919]. Here, to elucidate the mechanisms of nilvadipine-induced effects we analyzed altered gene expression of 1101 genes commonly expressed in rodent by DNA microarray analysis in the retinas of nilvadipine-treated and untreated RCS rats and SD rat. In the total number of genes, the expression of 30 genes was altered upon administration of nilvadipine to RCS rats, including several genes related to the apoptotic pathway and other mechanisms. Remarkably, neurotrophic factors, FGF-2 and Arc, known to suppress the apoptosis in the central nervous system, were up-regulated. These changes were also confirmed by real-time quantitative (Taqman) RT-PCR and Western blot analysis. Therefore, our present data suggested that administration of nilvadipine to RCS rats increases the expression of endogenous FGF-2 and Arc in retina, and potentially has a protective effect against retinal degeneration.

Inhibitory effects of trehalose on fibroblast proliferation and implications for ocular surgery

Trehalose is a disaccharide which plays an important role in preserving cells from completely dehydrated circumstances. In this study, we investigated effects of trehalose on proliferative activity of fibroblasts and epithelial cells both in vitro and in vivo. As in vitro assessment, normal human dermal fibroblasts and normal human epidermal keratinocytes were cultured in media containing various concentrations of trehalose. Growth activities of cells were evaluated with MTT assay and diff-quick™ staining. Expressions of vimentin and α smooth muscle actin (α-SMA) changed by trehalose were semiquantitatively measured by Western blot. As an in vivo study, 5% or 10% trehalose was topically instilled onto rabbit eyes after simple conjunctival incision or trabeculectomy. Condition of the surgical wound was evaluated by morphologically and immunohistochemically using isolectin B4 and antibodies specific for vimentin and α-SMA. Intraocular pressures (IOPs) after trabeculectomy were compared between eyes treated with trehalose and 0.04% mitomycin C (MMC). Results obtained by in vitro experiments showed that growth activities of cultured fibroblasts and keratinocytes were inhibited by trehalose in a dose-dependent manner. Fibroblasts were strongly inhibited by trehalose concentrations ≧ 5% of trehalose, whereas keratinocytes were less inhibited compared to fibroblasts. Expressions of vimentin and α-SMA were reduced by trehalose. With in vivo experiments, postoperative application of trehalose resulted in less firm adhesion between conjunctiva and sclera compared to controls. Immunohistochemical studies showed reduced staining of isolectin B4, vimentin and α-SMA in conjunctival wounds treated by topical trehalose. Also, after trabeculectomy, IOP remained in a low range during instillation of topical trehalose solution. We concluded that trehalose has inhibitory effects on proliferation of fibroblasts and vascular tissues, partially due to inhibition of transformation of fibroblasts into myofibroblasts in wound tissues. The present results imply that trehalose can be a potential agent for preventing postoperative fibrous scar formation after ocular surgery such as glaucoma filtration surgery.

Solid Hyaluronic Acid Film and the Prevention of Postoperative Fibrous Scar Formation in Experimental Animal Eyes

OBJECTIVE To investigate the inhibitory effect of solid hyaluronic acid-carboxymethyl cellulose film (hyaluronic acid film) on the formation of postoperative wound adhesion on rabbit eyes. METHODS We first created a conjunctival flap under which hyaluronic acid film was inserted. Then, we performed trabeculectomy on other rabbit eyes with hyaluronic acid film applied under and above the scleral flaps. Expression of proliferative cell nuclear antigen and alpha-smooth muscle actin (alpha-SMA) were histologically and immunohistochemically examined. RESULTS Hyaluronic acid film significantly prevented adhesions after both kinds of surgery. Particularly, subconjunctival scar formation was significantly inhibited when the film was simply inserted under the wound. Furthermore, the adhesion around the scleral flap of trabeculectomy was less formed in eyes treated with hyaluronic acid film than in control eyes. Immunoreactivity to proliferative cell nuclear antigen almost disappeared after 28 days postoperatively in both treated and control groups. The alpha-SMA-positive cells appeared much less around the film-treated wound than the control eye. CONCLUSION The present results indicate that hyaluronic acid film can inhibit the formation of postoperative adhesion around the conjunctiva and sclera. CLINICAL RELEVANCE The results of this study indicate that this substance has potential benefits for improving ophthalmic surgery, such as filtering surgery for glaucoma.

Study of effects of antiglaucoma eye drops on N-methyl-D-aspartate-induced retinal damage.

PurposeTo study the effects of antiglaucoma eye drops on N-methyl-d-aspartate (NMDA)-induced retinal damage.MethodsSeveral antiglaucoma eye drops, β-blockers, α/β-blockers, an α1-blocker, an α2-agonist, and a prostaglandin derivative, were topically administrated to NMDA-treated rat eyes daily for 2 weeks, and the retinal thickness, the number of retrograde-labeled retinal ganglion cells (RGCs), and the results of a cDNA microarray analysis were studied.ResultsIntravitreal administration of NMDA caused a significant decrease in the thickness of the retinal layers and induced upregulation of glial fibrillary acidic protein (GFAP). Topical administration of β-blockers (timolol, betaxolol, and carteolol) and a prostaglandin derivative (latanoprost) showed almost no significant effects on retinal thickness, the number of RGCs, or expression of GFAP. In contrast, the α/β-blockers (nipradilol and levobunolol), the α1-blocker (bunazosin HCl), and the α2-agonist (brimonidine) showed preservation effects on retinal thickness and the number of RGCs, and marked suppression of NMDA-induced upregulation of GFAP. Among 1101 genes related to cellular regulatory mechanisms, the expression of two genes, both for insulin-like growth factors, (IGF-1) and ErbB3, was altered upon administration of the α/β-blockers, the α1-blocker, and the α2-agonist.ConclusionOur present study suggests that modulations of the α-adrenergic receptor, α1-blocking and α2-stimulation, by antiglaucoma eye drops may cause beneficial effects on NMDA-induced retinal damage in the rat. Jpn J Ophthalmol 2005;49:453–461

Misregulation of rhodopsin phosphorylation and dephosphorylation found in P23H rat retinal degeneration

To examine rhodopsin (Rho) functions in P23H rat, kinetics of Rho regeneration and dephosphorylation were investigated by spectrophotometric analysis and immunofluorescence labeling method using specific antibodies toward phosphorylated 334Ser or 338Ser site. Rho dephosphorylation at both sites was extremely delayed in P23H retina as compared to normal ones. Kinetics of Rho regeneration was not altered between normal and P23H rats under dark adaptation. Next, to study the effects of several Ca2+channel blockers on this model, retinal function and morphology were evaluated. Among them, nilvadipine showed a significant protective effect against P23H retinal degeneration. Neurotrophic factor, fibroblast growth factor-2 and Arc, known to suppress the apoptosis in the central nervous system, were significantly upregulated upon administration of nilvadipine. The present study indicates that misregulation of Rho phosphorylation may be involved as an important step in retinal degeneration of P23H and administration of nilvadipine may be a potential therapeutic agent for the retinal degenerations.

Effects of intravitreal injection of bevacizumab on inflammatory cytokines in the vitreous with proliferative diabetic retinopathy.

Background: To investigate the effects of preoperative intravitreal injection of bevacizumab (IVB) on the levels of 27 inflammatory cytokines, including interleukins (ILs) and vascular endothelial growth factor. Methods: From among 200 patients who had proliferative diabetic retinopathy and underwent vitrectomy in our department from September 2009 to October 2010, 8 study subjects met the enrollment criteria in which both eyes at nearly equivalent stages underwent vitrectomy. The first vitrectomy for each patient was performed without IVB (control group), whereas the second vitrectomy on the contralateral eye was performed with IVB treatment (1.25 mg/0.05 mL) 3 days before surgery (IVB group). Undiluted vitreous fluid was collected at the start of each vitrectomy. A multiplex assay was used to simultaneously determine the levels of 27 inflammatory cytokines and growth factors. Results: Mean vascular endothelial growth factor levels were significantly lower in the IVB group (519.69 pg/mL) than in the control group (11,807.44 pg/mL) (P = 0.012, Wilcoxon signed rank test). Moreover, the mean levels (IVB/control, pg/mL) of IL-1RA (38.50/62.31, P = 0.036), IL-5 (27.75/34.00, P = 0.018), IL-10 (433.63/1,995.94, P = 0.012), IL-12 (246.69/1,033.69, P = 0.012), IL-13 (707.50/1,450.38, P = 0.012), and interferon &ggr; (71.13/84.69, P = 0.036) were significantly lower in the IVB group. No other significant differences were observed in the levels of the other 20 cytokines and growth factors between the 2 groups. Conclusion: Preoperative IVB reduced not only the intravitreal vascular endothelial growth factor level but also the intravitreal levels of other inflammatory cytokines, including IL-1RA, IL-5, IL-10, IL-12, IL-13, and interferon &ggr;. These results indicate the interaction of some cytokines in the vitreous fluid of proliferative diabetic retinopathy patients and suggest the possibility that preoperative IVB may not only reduce vascular proliferation by its direct antivascular endothelial growth factor effect but also modulate the inflammatory response through putative cytokine networks. None of the other cytokines examined were elevated after IVB.

Effects of Solid Hyaluronic Acid Film on Postoperative Fibrous Scar Formation After Strabismus Surgery in Animals

PURPOSE The purpose of this study was to investigate the inhibitory effect of solid hyaluronic acid-carboxymethylcellulose film on the formation of wound adhesion after strabismus surgery on rabbit eyes. METHODS The authors performed strabismus surgery on rabbit eyes with hyaluronic acid film applied under and above the muscles. Histological examination was performed 90 days postoperatively using Masson trichrome staining. The length of adhesion tissues in the operated area was quantitatively compared between film-treated and control eyes. RESULTS Hyaluronic acid film significantly prevented the formation of adhesions between muscle, conjunctiva, and sclera after strabismus surgery (t test, P < .05). CONCLUSION The current results indicate that hyaluronic acid film can inhibit the formation of postoperative adhesion around conjunctiva, muscle, and sclera to some extent. The authors conclude that this substance offers potential benefits for ophthalmic surgery, not only with strabismus surgery but also procedures such as scleral buckle.

Level of Vascular Endothelial Growth Factor in the Vitreous Fluid of Proliferative Diabetic Retinopathy Patients and Prognosis after Vitrectomy

Purpose: The aim of this study was to evaluate the relationship between the intravitreal vascular endothelial growth factor (VEGF) level and prognosis of proliferative diabetic retinopathy (PDR). Methods: The study involved 136 eyes of 114 PDR patients who underwent an initial vitrectomy between 2006 and 2008. Intravitreal VEGF levels were determined using Bio-Plex® (Bio-Rad), with levels of 5,000 pg/mL or more classified as high-VEGF (45 eyes) and levels lower than 5,000 pg/mL as low-VEGF (91 eyes). Diabetic control, PDR severity, and frequency of postoperative complications were compared between the groups. Results: There was no significant difference in preoperative status between the groups. In the low-VEGF group, a reoperation was required due to postoperative complications in 2 eyes (2.2%); 1 with vitreous hemorrhage (VH) and 1 with retinal detachment (RD). In contrast, a reoperation was required in 8 eyes (17.8%) in the high-VEGF group; 3 with VH, 2 with RD, and 3 with neovascular glaucoma. The difference between the groups was significant. There was a statistically lower postoperative corrected visual acuity logMAR (6 months after surgery) in the high-VEGF than in the low-VEGF group (p = 0.02, unpaired t test). Conclusion: Current findings indicate that careful observation is needed in patients with elevated VEGF levels.