Yoshimi Nakajima

Fasudil, a Rho kinase (ROCK) inhibitor, protects against ischemic neuronal damage in vitro and in vivo by acting directly on neurons

BACKGROUND AND PURPOSE Recently, fasudil, a Rho kinase (ROCK) inhibitor, was reported to prevent cerebral ischemia in vivo by increasing cerebral blood flow and inhibiting inflammatory responses. However, it is uncertain whether a ROCK inhibitor can directly protect neurons against ischemic damage. Our purpose was to evaluate both the involvement of ROCK activity in ischemic neuronal damage and any direct neuroprotective effect of fasudil against cerebral infarction. METHODS In vivo, focal cerebral ischemia was induced by permanent middle cerebral artery occlusion in mice, and the resulting infarction was evaluated 24 h later. ROCK expression and activity were assessed using Western blotting and immunohistochemistry. In vitro, the effects of fasudil and hydroxyfasudil (a main metabolite of fasudil) were examined on oxygen-glucose deprivation (OGD)-induced PC12 cell death and on glutamate-induced neurotoxicity in primary cerebral neuronal culture. RESULTS ROCK expression and activity increased in the striatum, especially in axons, in the early phase of ischemia. Fasudil reduced this ROCK activity and protected against cerebral infarction in vivo. Hydroxyfasudil inhibited OGD-induced PC12 cell death, and fasudil and hydroxyfasudil each attenuated glutamate-induced neurotoxicity in vitro. CONCLUSIONS These findings indicate that ROCK plays a pivotal role in the mechanism underlying ischemic neuronal damage and that a direct effect of fasudil on neurons may be partly responsible for its protective effects against such damage.

Docosahexaenoic acid (DHA) has neuroprotective effects against oxidative stress in retinal ganglion cells.

We examined the radical-scavenging activity of docosahexaenoic acid (DHA) and its effects on the neuronal cell death induced by oxidative or hypoxic stress in cultured retinal ganglion cells (RGC-5, a rat ganglion cell-line transformed using E1A virus). The radical-scavenging activity [hydrogen peroxide (H(2)O(2)), superoxide anion (O(2)*(-)), and hydroxyl radical (*OH)] of DHA in RGC-5 cells was measured using the ROS-sensitive probes CM-H(2)DCFDA and APF. DHA concentration-dependently scavenged the intracellular radical productions induced by H(2)O(2) radical, O(2)*(-), and *OH (minimum effective DHA concentrations 0.1, 10 and 100 microM, respectively). Cell damage was induced by H(2)O(2), oxygen-glucose deprivation (OGD), or tunicamycin (an endoplasmic reticulum-stress inducer), and cell viability was assessed by Hoechst 33342 nuclear staining or by the tetrazolium salt (WST-8) cell-viability assay. H(2)O(2) (0.3 mM for 24 h), 4-h OGD exposure followed by 18-h reoxygenation, or tunicamycin at 2 microg/ml for 24 h induced apoptotic cell death accompanied by nuclear condensation and/or fragmentation, and each maneuver decreased cell viability. Treatment with DHA at 0.1 and 1 microM significantly inhibited the decrease in cell viability induced by H(2)O(2). Treatment with DHA at 0.1, 1, or 10 microM significantly inhibited the decrease in cell viability induced by OGD/reoxygenation exposure. However, DHA (0.1 to 10 microM) had no effect on the decrease in cell viability induced by tunicamycin. These results indicate that DHA may be protective against oxidative or hypoxic stress-induced cell damage in retinal ganglion cells.

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.

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.

Neuroprotective effects of Brazilian green propolis and its main constituents against oxygen-glucose deprivation stress, with a gene-expression analysis.

Our purpose was to investigate the neuroprotective effects (and the underlying mechanism) exerted by water extract of Brazilian green propolis (WEP) and its main constituents against the neuronal damage induced by oxygen‐glucose deprivation (OGD)/reoxygenation in retinal ganglion cells (RGC‐5, a rat ganglion cell‐line transformed using E1A virus). Cell damage was induced by OGD 4 h plus reoxygenation 18 h exposure. In RGC‐5, and also in PC12 (rat pheochromocytoma, neuronal cells), WEP and some of its main constituents attenuated the cell damage. At the end of the period of OGD/reoxygenation, RNA was extracted and DNA microarray analysis was performed to examine the gene‐expression profile in RGC‐5. Expression of casein kinase 2 (CK2) was down‐regulated and that of Bcl‐2‐related ovarian killer protein (Bok) was up‐regulated following OGD stress, results that were confirmed by quantitative reverse transcriptase‐PCR (qRT‐PCR). These effects were normalized by WEP. Our findings indicate that WEP has neuroprotective effects against OGD/reoxygenation‐induced cell damage and that certain constituents of WEP (caffeoylquinic acid derivatives, artepillin C, and p‐coumaric acid) may be partly responsible for its neuroprotective effects. Furthermore, the protective mechanism may involve normalization of the expressions of antioxidant‐ and apoptosis‐related genes (such as CK2 and Bok, respectively). Copyright

Zeaxanthin, a retinal carotenoid, protects retinal cells against oxidative stress.

Purpose: To investigate whether zeaxanthin, the predominant carotenoid pigment of the macular pigments in human retina, provides neuroprotection against retinal cell damage. Methods: We used in vitro cultured retinal ganglion cells (RGCs), specifically RGC-5, an E1A virus-transformed rat cell line. Cell damage was induced either by a 24-hr exposure to hydrogen peroxide (H2O2) or by serum deprivation. Cell viability was measured using the tetrazolium salt, WST-8. The scavenging capacity of zeaxanthin for H2O2, superoxide anion radical (O2·−), and hydroxyl radical (HO ·) was measured using a radical scavenging capacity assay with CM-H2DCFDA, a reactive oxygen species (ROS)-sensitive probe. Results: When added to RGC-5 cell cultures, 0.1, 10, and 1 μM zeaxanthin scavenged the free radicals induced by H2O2, O2·−, and HO ·, respectively. In addition, pretreatment with 1 μ M zeaxanthin permitted scavenging of staurosporine-induced intracellular radicals. Zeaxanthin also inhibited the neurotoxicity induced by H2O2 or serum deprivation and scavenged the intracellular radicals induced by H2O2 or serum deprivation. Conclusions: Our results suggest that zeaxanthin provides effective protection against oxidative stress-induced retinal cell damage.