Yuta Inokuchi

Bilberry and its main constituents have neuroprotective effects against retinal neuronal damage in vitro and in vivo.

Our aim was to determine whether a Vaccinium myrtillus (bilberry) anthocyanoside (VMA) and/or its main anthocyanidin constituents (cyanidin, delphinidin, and malvidin) can protect retinal ganglion cells (RGCs) against retinal damage in vitro and in vivo. In RGC cultures (RGC-5, a rat ganglion cell-line transformed using E1A virus) in vitro, cell damage and radical activation were induced by 3-(4-morpholinyl) sydnonimine hydrochloride (SIN-1, a peroxynitrite donor). Cell viability was measured using a water-soluble tetrazolium salt assay. Intracellular radical activation within RGC-5 cells was evaluated using 5-(and-6)-chloromethyl-2,7-dichlorodihydrofluorescein diacetate acetyl ester (CM-H(2)DCFDA). Lipid peroxidation was assessed using the supernatant fraction of mouse forebrain homogenates. In mice in vivo, we evaluated the effects of VMA on N-methyl-D-aspartic acid (NMDA)-induced retinal damage using hematoxylin-eosin and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) stainings. VMA and all three anthocyanidins (i) significantly inhibited SIN-1-induced neurotoxicity and radical activation in RGC-5, (ii) concentration-dependently inhibited lipid peroxidation in mouse forebrain homogenates. Intravitreously injected VMA significantly inhibited the NMDA-induced morphological retinal damage and increase in TUNEL-positive cells in the ganglion cell layer. Thus, VMA and its anthocyanidins have neuroprotective effects (exerted at least in part via an anti-oxidation mechanism) in these in vitro and in vivo models of retinal diseases.

Design and evaluation of a liposomal delivery system targeting the posterior segment of the eye

The purpose of this study was to evaluate the potential of submicron-sized liposomes (ssLips) as a novel system for delivering ocular drugs to the eyes posterior segment. Fluorescence emission of coumarin-6 formulated into ssLip was obvious in that segment in mice after eyedrop administration of the liposomal suspension. Such fluorescence was not observed after administration of either multilamellar vesicles or dimethyl sulfoxide (DMSO) solution containing the same amount of coumarin-6. The highest fluorescence of ssLip occurred 30 min after eyedrop administration, and all fluorescence disappeared after 180 min. The ssLip based on l-alpha-distearoyl phosphatidylcholine (DSPC ssLip) showed higher fluorescence emission in the retina than that based on egg phosphatidylcholine (EPC ssLip). These results confirmed that the magnitude of fluorescence in the retina was closely related to both liposome rigidity and particle size. Images of the entire eye showed that ssLip was delivered via the non-corneal pathway after administration. The liposomes tested in ocular cells showed little cytotoxicity. These results suggest that ssLip can be used to deliver drugs to the posterior segment of the eye.

Astaxanthin, a dietary carotenoid, protects retinal cells against oxidative stress in-vitro and in mice in-vivo.

We have investigated whether astaxanthin exerted neuroprotective effects in retinal ganglion cells in‐vitro and in‐vivo. In‐vitro, retinal damage was induced by 24‐h hydrogen peroxide (H2O2) exposure or serum deprivation, and cell viability was measured using a WST assay. In cultured retinal ganglion cells (RGC‐5, a rat ganglion cell‐line transformed using E1A virus), astaxanthin inhibited the neurotoxicity induced by H2O2 or serum deprivation, and reduced the intracellular oxidation induced by various reactive oxygen species (ROS). Furthermore, astaxanthin decreased the radical generation induced by serum deprivation in RGC‐5. In mice in‐vivo, astaxanthin (100 mg kg−1, p.o., four times) reduced the retinal damage (a decrease in retinal ganglion cells and in thickness of inner plexiform layer) induced by intravitreal N‐methyl‐d‐aspartate (NMDA) injection. Furthermore, astaxanthin reduced the expressions of 4‐hydroxy‐2‐nonenal (4‐HNE)‐modified protein (indicator of lipid peroxidation) and 8‐hydroxy‐deoxyguanosine (8‐OHdG; indicator of oxidative DNA damage). These findings indicated that astaxanthin had neuroprotective effects against retinal damage in‐vitro and in‐vivo, and that its protective effects may have been partly mediated via its antioxidant effects.

Effect of an inducer of BiP, a molecular chaperone, on endoplasmic reticulum (ER) stress-induced retinal cell death.

PURPOSE The effect of a preferential inducer of 78 kDa glucose-regulated protein (GRP78)/immunoglobulin heavy-chain binding protein (BiP; BiP inducer X, BIX) against tunicamycin-induced cell death in RGC-5 (a rat ganglion cell line), and also against tunicamycin- or N-methyl-D-aspartate (NMDA)-induced retinal damage in mice was evaluated. METHODS In vitro, BiP mRNA was measured after BIX treatment using semi-quantitative RT-PCR or real-time PCR. The effect of BIX on tunicamycin (at 2 microg/mL)-induced damage was evaluated by measuring the cell-death rate and CHOP protein expression. In vivo, BiP protein induction was examined by immunostaining. The retinal cell damage induced by tunicamycin (1 microg) or NMDA (40 nmol) was assessed by examining ganglion cell layer (GCL) cell loss, terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining, and CHOP protein expression. RESULTS In vitro, BIX preferentially induced BiP mRNA expression both time- and concentration-dependently in RGC-5 cells. BIX (1 and 5 microM) significantly reduced tunicamycin-induced cell death, and BIX (5 microM) significantly reduced tunicamycin-induced CHOP protein expression. In vivo, intravitreal injection of BIX (5 nmol) significantly induced BiP protein expression in the mouse retina. Co-administration of BIX (5 nmol) significantly reduced both the retinal cell death and the CHOP protein expression in GCL induced by intravitreal injection of tunicamycin or NMDA. CONCLUSIONS These findings suggest that this BiP inducer may have the potential to be a therapeutic agent for endoplasmic reticulum (ER) stress-induced retinal diseases.

Coenzyme Q10 protects retinal cells against oxidative stress in vitro and in vivo

PURPOSE To investigate the neuroprotective effects of coenzyme Q10 and/or a vitamin E analogue on retinal damage both in vitro and in vivo. METHODS We employed cultured retinal ganglion cells (RGC-5, a rat ganglion cell-line transformed using E1A virus) in vitro. Cell damage was induced by 24-h hydrogen peroxide (H2O2) exposure, and cell viability was measured using tetrazolium salt (WST-8). To examine the retinal damage induced by intravitreal N-methyl-d-aspartate (NMDA) injection in mice in vivo, coenzyme Q10 at 10 mg/kg with or without alpha-tocopherol at 10 mg/kg was administered orally (p.o.) each day for 14 days, with NMDA being intravitreally injected on day 7 of this course. RESULTS In RGC-5, a combination of coenzyme Q10 and trolox, a water-soluble vitamin E analogue (a derivative of alpha-tocopherol), prevented cell damage more effectively than either agent alone. Coenzyme Q10 and alpha-tocopherol (separately or together) reduced the retinal damage, number of TUNEL-positive cells in the ganglion cell layer (GCL), and 4-hydroxyl-2-nonenal (4-HNE) expression induced by NMDA in mice in vivo. CONCLUSIONS Coenzyme Q10 and/or these vitamin E analogues exert neuroprotective effects against retinal damage both in vitro and in vivo.

Edaravone, a Free Radical Scavenger, Protects against Retinal Damage in Vitro and in Vivo

Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), a free radical scavenger, is used for the treatment of acute cerebral infarction. In this study, we investigated whether edaravone is neuroprotective against retinal damage. In vitro, we used a radical-scavenging capacity assay using reactive oxygen species-sensitive probes to investigate the effects of edaravone on H2O2, superoxide anion (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document}), and hydroxyl radical (·OH) production in a rat retinal ganglion cell line (RGC-5). The effect of edaravone on oxygen-glucose deprivation (OGD)-induced RGC-5 damage was evaluated using a 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt assay of cell viability. Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) significantly decreased radical generation and reduced the cell death induced by OGD stress. In vivo, retinal damage was induced by intravitreous injection of N-methyl-d-aspartate (NMDA; 5 nmol) and was evaluated by examining ganglion cell layer cell loss, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, and the expressions of two oxidant-stress markers [4-hydroxy-2-nonenal (4-HNE) and 8-hydroxy-2-deoxyguanosine (8-OHdG)]. In addition, activations of mitogen-activated protein kinases (MAPKs) [extracellular signal-regulated protein kinases (ERK), c-Jun NH2-terminal kinases (JNK), and p38 MAPK], as downstream signal pathways after NMDA receptor activation, were measured using immunoblotting and immunostaining. Edaravone at 5 and 50 nmol intravitreous injection or at 1 and 3 mg/kg i.v. significantly protected against NMDA-induced retinal cell death. At 50 nmol intravitreous injection, it 1) decreased the retinal expressions of TUNEL-positive cells, 4-HNE, and 8-OHdG and 2) reduced the retinal expressions of NMDA-induced phosphorylated JNK and phosphorylated p38 but not that of phosphorylated ERK. These findings suggest that oxidative stress plays a pivotal role in retinal damage and that edaravone may be a candidate for the effective treatment of retinal diseases.

Brazilian Green Propolis Protects against Retinal Damage In Vitro and In Vivo

Propolis, a honeybee product, has gained popularity as a food and alternative medicine. Its constituents have been shown to exert pharmacological (anticancer, antimicrobial and anti-inflammatory) effects. We investigated whether Brazilian green propolis exerts neuroprotective effects in the retina in vitro and/or in vivo. In vitro, retinal damage was induced by 24 h hydrogen peroxide (H2O2) exposure, and cell viability was measured by Hoechst 33342 and YO-PRO-1 staining or by a resazurin–reduction assay. Propolis inhibited the neurotoxicity and apoptosis induced in cultured retinal ganglion cells (RGC-5, a rat ganglion cell line transformed using E1A virus) by 24 h H2O2 exposure. Propolis also inhibited the neurotoxicity induced in RGC-5 cultures by staurosporine. Regarding the possible underlying mechanism, in pig retina homogenates propolis protected against oxidative stress (lipid peroxidation), as also did trolox (water-soluble vitamin E). In mice in vivo, propolis (100 mg kg−1; intraperitoneally administered four times) reduced the retinal damage (decrease in retinal ganglion cells and in thickness of inner plexiform layer) induced by intravitreal in vivo N-methyl-d-aspartate injection. These findings indicate that Brazilian green propolis has neuroprotective effects against retinal damage both in vitro and in vivo, and that a propolis-induced inhibition of oxidative stress may be partly responsible for these neuroprotective effects.

Systemic administration of a free radical scavenger, edaravone, protects against light-induced photoreceptor degeneration in the mouse retina

Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), a free radical scavenger, is used for the clinical treatment of acute cerebral infarction. In this study, we investigated the protective effects of edaravone against light-induced retinal damage in the mouse. Retinal damage in the mouse was induced by exposure to white light at 8000lx for 3h after dark adaptation. Photoreceptor damage was evaluated by measuring the outer nuclear layer thickness at 5days after the light exposure and recording the electroretinogram (ERG). Retinal cell damage was also detected by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and the expression of 8-hydroxy-2-deoxyguanosine (8-OHdG) and the phosphorylation of mitogen-activated protein kinases (MAPKs) such as extracellular signal regulated protein kinases (ERK), c-Jun N-terminal kinases (JNK), and p38 were analyzed in the retinal samples by immunohistochemistry and immunoblotting. According to evaluation of outer nuclear layer thickness, 3mg/kg, i.p. of edaravone and 1mg/kg. i.v. of edaravone significantly protected against light-induced photoreceptor degeneration at 5days after exposure to light. In ERG measurement, 3mg/kg, i.p. of edaravone inhibited retinal dysfunction at 5days after exposure to light. In addition, 3mg/kg, i.p. of edaravone decreased the numbers of TUNEL-positive cells, 8-OHdG, phosphorylated JNK, and phosphorylated p38, but not that of phosphorylated ERK, in the whole retina at 6h after light exposure. These findings suggest that oxidative stress plays a pivotal role in light-induced retinal damage and that systemic administration of edaravone may slow the progression of photoreceptor degeneration.

Reduced retinal function in amyloid precursor protein-over-expressing transgenic mice via attenuating glutamate-N-methyl-d-aspartate receptor signaling.

Here, we examined whether amyloid‐β (Aβ) protein participates in cell death and retinal function using three types of transgenic (Tg) mice in vivo [human mutant amyloid precursor protein (APP) Tg (Tg 2576) mice, mutant presenilin‐1 (PS‐1) knock‐in mice, and APP/PS‐1 double Tg mice]. ELISA revealed that the insoluble form of Aβ1‐40 was markedly accumulated in the retinas of APP and APP/PS‐1, but not PS‐1 Tg, mice (vs. wild‐type mice). In APP Tg and APP/PS‐1 Tg mice, immunostaining revealed accumulations of intracellular Aβ1–42 in retinal ganglion cells and in the inner and outer nuclear layers. APP Tg and APP/PS‐1 Tg, but not PS‐1 Tg, mice had less NMDA‐induced retinal damage than wild‐type mice, and the reduced damage in APP/PS‐1 Tg mice was diminished by the pre‐treatment of N‐[N‐(3,5‐difluorophenacetyl)‐l‐alanyl]‐S‐phenylglycine t‐butyl ester, a γ‐secretase inhibitor. Furthermore, the number of TUNEL‐positive cells was significantly less in ganglion cell layer of APP/PS‐1 Tg mice than PS‐1 Tg mice 24 h after NMDA injection. The phosphorylated form of calcium/calmodulin‐dependent protein kinase IIα (CaMKIIα), but not total CaMKIIα or total NMDA receptor 1 (NR1) subunit, in total retinal extracts was decreased in non‐treated retinas of APP/PS‐1 Tg mice (vs. wild‐type mice). CaMKIIα and NR2B proteins, but not NR1, in retinal membrane fraction were significantly decreased in APP/PS‐1 Tg mice as compared with wild‐type mice. The NMDA‐induced increase in p‐CaMKIIα in the retina was also lower in APP/PS‐1 Tg mice than in wild‐type mice. In electroretinogram and visual‐evoked potential recordings, the implicit time to each peak from a light stimulus was prolonged in APP/PS‐1 mice versus wild‐type mice. Hence, Aβ may impair retinal function by reducing activation of NMDA‐receptor signaling pathways.

A Novel Calpain Inhibitor, ((1S)-1-((((1S)-1-Benzyl-3-cyclopropylamino-2,3-di-oxopropyl)amino)carbonyl)-3-methylbutyl)carbamic Acid 5-Methoxy-3-oxapentyl Ester (SNJ-1945), Reduces Murine Retinal Cell Death In Vitro and In Vivo

We examined whether ((1S)-1-((((1S)-1-benzyl-3-cyclopropylamino-2,3-di-oxopropyl)amino)carbonyl)-3-methylbutyl)carbamic acid 5-methoxy-3-oxapentyl ester (SNJ-1945), a new orally available calpain inhibitor, might reduce retinal cell death in vivo and/or in vitro. Retinal cell damage was induced in vivo in mice by intravitreal injection of N-methyl-d-aspartate (NMDA), and SNJ-1945 was intraperitoneally or orally administered twice. NMDA-induced calpain activity (measured as the cleaved products of α-spectrin) and its substrate, p35 (a neuron-specific activator for cyclin-dependent kinase 5), in the retina were examined by immunoblotting. In RGC-5 (a rat retinal ganglion cell line) cell culture, cell damage was induced by a 4-h oxygen-glucose deprivation (OGD) treatment followed by an 18-h reoxygenation period. In mouse retinas, SNJ-1945 (30 or 100 mg/kg i.p., 100 or 200 mg/kg p.o.) significantly inhibited the cell loss in the ganglion cell layer (GCL) and the thinning of the inner plexiform layer induced by NMDA. Furthermore, the number of positive cells for terminal deoxynucleotidyl transferase dUTP nick-end labeling was significantly reduced in the GCL and the inner nuclear layer of retinas treated with SNJ-1945 compared with vehicle-treated retinas 24 h after NMDA injection. Levels of cleaved α-spectrin products increased and p35 decreased 6 h after NMDA injection or later, and their effects were attenuated by SNJ-1945. In vitro, SNJ-1945 (10 and 100 μM) inhibited the OGD stress-induced reduction in cell viability. In conclusion, SNJ-1945 may afford valuable neuroprotection against retinal diseases, because it was effective against retinal damage both in vitro and in vivo. Our results also indicate that calpain activation and subsequent p35 degradation may be involved in the mechanisms underlying retinal cell death.