Yuta Ohno

Oral administration of crocetin prevents inner retinal damage induced by N-methyl-d-aspartate in mice

Crocetin, an aglycone of crocin, is found in stigmas of the saffron crocus (Crocus starus L.) and has been used in traditional medicine. We investigated the effects of oral administration of crocetin on damage induced by N-methyl-D-aspartate (NMDA) in the murine retina. Crocetin was orally administered before and after intravitreal injection of NMDA. A histological analysis was conducted by counting the cell number of ganglion cell layer (GCL). Cell apoptosis was assessed by counting cells positive for terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Retinal functions were measured in terms of a- and b-wave amplitudes using an electroretinogram (ERG). Activation of caspase-3/7 and cleaved caspase-3 expression were assayed. Calpain activity was evaluated by immunoblotting assays for proteolysis of α-spectrin. NMDA injection decreased the cell number in the GCL, and crocetin at a dose of 100 mg/kg inhibited this reduction. TUNEL-positive cells were observed in both GCL and inner nuclear layer (INL) after NMDA injection, and crocetin inhibited the increase in number of TUNEL-positive cells. ERG analysis showed that both a- and b-wave amplitudes were decreased by NMDA injection. Crocetin inhibited the reduction in the b-wave amplitude, but not in the a-wave. NMDA injection activated caspase-3/7 and increased expression of cleaved caspsase-3 in the GCL and INL, but both of these processes were inhibited by crocetin. NMDA injection also induced cleavage of α-spectrin, but crocetin did not affect this process. These findings indicate that oral administration of crocetin prevented NMDA-induced retinal damage via inhibition of the caspase pathway.

Progranulin, a Major Secreted Protein of Mouse Adipose-Derived Stem Cells, Inhibits Light-Induced Retinal Degeneration

Adipose tissue stromal vascular fraction contains mesenchymal stem cells, which show protective effects when administered to damaged tissues, mainly through secreted trophic factors. We examined the protective effects of adipose‐derived stem cells (ASCs) and ASC‐conditioned medium (ASC‐CM) against retinal damage and identified the neuroprotective factors in ASC‐CM. ASCs and mature adipocytes were isolated from mouse subcutaneous tissue. ASCs were injected intravitreally in a mouse model of light‐induced retinal damage, and ASC injection recovered retinal function as measured by electroretinogram and inhibited outer nuclear layer, thinning, without engraftment of ASCs. ASC‐CM and mature adipocyte‐conditioned medium were collected after 72 hours of culture. In vitro, H2O2‐ and light‐induced cell death was reduced in a photoreceptor cell line with ASC‐CM but not with mature adipocyte‐conditioned medium. In vivo, light‐induced photoreceptor damage was evaluated by measurement of outer nuclear layer thickness at 5 days after light exposure and by electroretinogram recording. ASC‐CM significantly inhibited photoreceptor degeneration and retinal dysfunction after light exposure. Progranulin was identified as a major secreted protein of ASCs that showed protective effects against retinal damage in vitro and in vivo. Furthermore, progranulin phosphorylated extracellular signal‐regulated kinase, cAMP response element binding protein, and hepatocyte growth factor receptor, and protein kinase C signaling pathways were involved in the protective effects of progranulin. These findings suggest that ASC‐CM and progranulin have neuroprotective effects in the light‐induced retinal‐damage model. Progranulin may be a potential target for the treatment of the degenerative diseases of the retina.

Bimatoprost protects retinal neuronal damage via Akt pathway

Worldwide, prostaglandin analogs, such as bimatoprost, have become the major therapeutic class for medical treatment of glaucoma because of their efficacy and generally well tolerated systemic safety profile. However, the detailed mechanism of the direct action of bimatoprost on retinal ganglion cells (RGC) has rarely been understood. Thus, in this study, we elucidated the mechanism of the protective effects of bimatoprost on RGC against oxidative stress. To examine the protective effects of bimatoprost, cultured RGC with various concentrations of bimatoprost (in both free acid and amide form) were exposed to l-buthionin-(S,R)-sulfoximine (BSO) plus glutamate or serum depletion in vitro and intravitreal injection of N-methyl-D-aspartate (NMDA) was used to induce retinal damage in vivo. To elucidate the protective mechanism of bimatoprost, we used western blot analysis to investigate the phosphorylation of Akt and extracellular signal-regulated kinase (ERK). Bimatoprost significantly reduced BSO plus glutamate- and serum deprivation-induced death in concentration-dependent manners. Bimatoprost induced activation of Akt and ERK, and a phosphatidylinositol 3-kinase inhibitor, LY294002, attenuated the protective effect of bimatoprost. On the other hand, a mitogen-activated protein kinase kinase inhibitor, U0126, exhibited protective effect unexpectedly. Moreover, ERK was more phosphorylated by attenuation of Akt activity in cultured RGC. In an in vivo study, bimatoprost reduced NMDA-induced RGC death. Taken together, these findings indicate that bimatoprost has protective effects on in vitro and in vivo retinal damage, suggesting that the mechanism underlying may be via the Akt pathway, which may modulate the ERK pathway.

Protective Effects of Antiplacental Growth Factor Antibody Against Light-Induced Retinal Damage in Mice

PURPOSE Placental growth factor (PlGF) is part of the VEGF family and is known to be involved in angiogenesis, vasopermeability, and neuroprotection. Recently, PlGF has been reported as a novel therapeutic target for wet AMD. However, there are few reports about the effect of PlGF against dry AMD. Previously, we reported that PlGF has protective effects against retinal neuronal cell damage in vitro. Therefore, we investigated the effects of PlGF against photoreceptor degeneration. METHODS In this study, mice were exposed to white light at 8000 lx for 3 hours to induce retinal damage, which was evaluated by recording the electroretinogram amplitude and measuring the outer nuclear layer (ONL) thickness. The mice were injected intravitreally with PlGF before light exposure, PlGF after light exposure, or anti-PlGF antibody before light exposure. RPE-choroid-sclera flat mounts were immunostained with anti-ZO-1 antibody to evaluate the disruption of retinal pigmented epithelium (RPE) cell-cell junctional integrity after light exposure. Furthermore, the expression of VEGF receptor in the retina and RPE-choroid complex after light exposure was measured using Western blot analysis. RESULTS Contrary to the expected outcome, PlGF treatment exacerbated the light-induced retinal functional damage and ONL thinning. In contrast, anti-PlGF treatment significantly improved the light-induced retinal degeneration. The disruption of RPE cell-cell junctional integrity after light exposure was suppressed by anti-PlGF treatment. Moreover, the VEGF receptor, which is involved in blood-retinal barrier breakdown, was up-regulated after light exposure. CONCLUSIONS These findings suggest that anti-PlGF antibody has protective effects against light-induced retinal degeneration in the murine retina through inhibition of RPE breakdown after light exposure. Thus, anti-PlGF antibody may be useful therapeutic agents in dry AMD.

Metallothionein-III deficiency exacerbates light-induced retinal degeneration.

PURPOSE Retinal photoreceptor damage is a common feature of ophthalmic disorders, such as age-related macular degeneration and retinitis pigmentosa. Oxidative stress has a key role in these diseases. Metallothioneins (MTs) are a family of cysteine-rich proteins, and various physiologic functions have been reported, including protection against metal toxicity and antioxidative potency. We investigated the functional role of MT-III in light-induced retinal damage. METHODS The expression of retinal MT-I, -II, and -III mRNA was evaluated by real-time reverse-transcription PCR in retina exposed to light. Retinal damage in MT-deficient mice was induced by exposure to white light at 16,000 lux for 3 hours after dark adaptation. Photoreceptor damage was evaluated histologically by measuring the thickness of the outer nuclear layer (ONL) 5 days after light exposure and by electroretinogram recording. In an in vitro experiment, the MT-III siRNAs were tested for their effects on light-induced mouse photoreceptor cell (661W) damage. RESULTS The mRNAs of the MTs were increased significantly in murine retina after light exposure. The ONL in the MT-III-deficient mice was remarkably thinner compared to light-exposed wild-type (WT) mice, and a- and b-wave amplitudes were decreased; the damage induced in MT-I/-II-deficient mice was comparable to that observed in WT mice. MT-III knockdown by siRNA in 661W exacerbated the cell damage and increased the production of reactive oxygen species in response to light exposure. CONCLUSIONS These findings suggested that MT-III can help protect against light-induced retinal damage compared to MT-I/II. Some of these effects may be exerted by its antioxidative potency.

Role of Heparin-Binding Epidermal Growth Factor-Like Growth Factor in Light-Induced Photoreceptor Degeneration in Mouse Retina

PURPOSE Although heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been reported to have protective effects against various neuronal cell damage, its role in the retina has not been elucidated. Here, we investigated its role in light-induced photoreceptor degeneration using retinas and ventral forebrain-specific Hb-egf knockout (KO) mice. METHODS Disruption of Hb-egf was confirmed by β-galactosidase (LacZ) staining and RT-PCR. Time-dependent changes in retinal HB-EGF were measured using quantitative RT-PCR and Western blotting. Retinal damage was induced by exposure to light. Recombinant human HB-EGF was injected intravitreally. Electroretinogram (ERG) and histological analyses were performed. To evaluate the effect of HB-EGF against light irradiation-induced cell death, 661W cells, a transformed mouse cone cell line, were used. RESULTS LacZ-positive cells were observed and Hb-egf deletion was confirmed in the retinas of Hb-egf KO mice. Hb-egf and pro-HB-EGF levels were increased after light exposure in wild-type (WT) mice. Exposure to light reduced the a- and b-wave amplitudes of the dark-adapted ERG, and also outer nuclear layer (ONL) thickness, in Hb-egf KO mice versus WT mice. Treatment with HB-EGF improved both the a- and b-wave amplitudes and the thickness of the ONL. The 661W cell death induced by light irradiation was exacerbated by Hb-egf knockdown. HB-EGF also protected against light-induced cell death and reduced reactive oxygen species (ROS) production in 661W cells. HB-EGF treatment improved the a-wave amplitudes and the thickness of the ONL in Hb-egf KO mice. CONCLUSIONS These data suggest that HB-EGF plays a pivotal role in light-induced photoreceptor degeneration. It therefore warrants investigation as a potential therapeutic target for such light-induced retinal diseases as age-related macular degeneration.

Progranulin promotes the retinal precursor cell proliferation and the photoreceptor differentiation in the mouse retina

Progranulin (PGRN) is a secreted growth factor associated with embryo development, tissue repair, and inflammation. In a previous study, we showed that adipose-derived stem cell-conditioned medium (ASC-CM) is rich in PGRN. In the present study, we investigated whether PGRN is associated with retinal regeneration in the mammalian retina. We evaluated the effect of ASC-CM using the N-methyl-N-nitrosourea-induced retinal damage model in mice. ASC-CM promoted the differentiation of photoreceptor cells following retinal damage. PGRN increased the number of BrdU+ cells in the outer nuclear layer following retinal damage some of which were Rx (retinal precursor cell marker) positive. PGRN also increased the number of rhodopsin+ photoreceptor cells in primary retinal cell cultures. SU11274, a hepatocyte growth factor (HGF) receptor inhibitor, attenuated the increase. These findings suggest that PGRN may affect the differentiation of retinal precursor cells to photoreceptor cells through the HGF receptor signaling pathway.

Thickness mapping of the inner retina by spectral-domain optical coherence tomography in an N-methyl-d-aspartate-induced retinal damage model

Spectral-domain optical coherence tomography (SD-OCT) is an interferometric optical tomography technique and provides high resolution and noninvasive visualization of retinal morphology. The purpose of this study was to assess the utility of thickness maps and quantitative thickness measurements of the ganglion cell complex (GCC: retinal nerve fiber layer, ganglion cell layer, and inner plexiform layer) obtained by SD-OCT of a mouse model of N-methyl-d-aspartate (NMDA)-induced retinal damage. SD-OCT imaging was performed in ddY mice at 1, 3, and 7 days and 1 month after intravitreal injection of NMDA. GCC thickness maps and circle cross-sectional OCT images were made from volumetric OCT images. The GCC thickness was measured on a cross-sectional OCT image on a circle with a radius 300 μm from the center of the optic nerve disc. Histological analysis was conducted by measuring the GCC thickness at the same time intervals. The thickness maps and the quantitative thickness values of GCC showed thickness changes at each time point in the NMDA-treated mice when compared with normal and vehicle-treated mice. Both the OCT sectional images and the histological images revealed increases in GCC thickness at 1 day, followed by decreases from 3 days to 1 month after NMDA injection. The GCC thickness measured using OCT sectional images correlated with the thickness measured using histological images. In conclusion, GCC thickness mapping is a useful method for evaluating NMDA-induced retinal degeneration in mice.

Involvement of cannabinoid receptor type 2 in light-induced degeneration of cells from mouse retinal cell line in vitro and mouse photoreceptors in vivo

Abstract Earlier studies showed that the expressions of the agonists of the cannabinoid receptors are reduced in the vitreous humor of patients with age‐related macular degeneration (AMD), and the cannabinoid type 2 receptor is present in the retinas of rats and monkeys. The purpose of this study was to determine whether the cannabinoid type 2 receptor is involved in the light‐induced death of cultured 661W cells, an immortalized murine retinal cell line, and in the light‐induced retinal degeneration in mice. Time‐dependent changes in the expression and location of retinal cannabinoid type 2 receptor were determined by Western blot and immunostaining. The cannabinoid type 2 receptor was down‐regulated in murine retinae and cone cells. In the in vitro studies, HU‐308, a cannabinoid type 2 receptor agonist, had a protective effect on the light‐induced death of 661W cells, and this effect was attenuated by SR144528, a cannabinoid type 2 receptor antagonist. Because the cannabinoid type 2 receptor is a G‐protein coupled receptor and is coupled with Gi/o protein, we investigated the effects of the cAMP‐dependent protein kinase (PKA). HU‐308 and H89, a PKA inhibitor, deactivated PKA in retinal cone cells, and H89 also suppressed light‐induced cell death. For the in vivo studies, a cannabinoid type 2 receptor agonist, HU‐308, or an antagonist, SR144528, was injected intravitreally into mouse eyes before the light exposure. Electroretinography was used to determine the physiological status of the retinas. Injection of HU‐308 improved the a‐ and b‐waves of the ERGs and also the thickness of the outer nuclear layer of the murine retina after light exposure. These findings indicate that the cannabinoid type 2 receptor is involved in the light‐induced retinal damage through PKA signaling. Thus, activation of cannabinoid type 2 receptor may be a therapeutic approach for light‐associated retinal diseases. HighlightsCannabinoid receptor type 2 is decreased by light irradiation in photoreceptor cells.HU‐308, an agonist of Cannabinoid receptor type 2, reduced photoreceptor cell death induced by light irradiation.HU‐308 attenuated photoreceptor degeneration induced by light irradiation.

Rimonabant, a selective cannabinoid1 receptor antagonist, protects against light-induced retinal degeneration in vitro and in vivo

Abstract The endocannabinoid system is involved in some neurodegenerative diseases such as Alzheimers disease. An endogenous constellation of proteins related to cannabinoid1 receptor signaling, including free fatty acids, diacylglycerol lipase, and N‐acylethanolamine‐hydrolyzing acid amidase, are localized in the murine retina. Moreover, the expression levels of endogenous agonists of cannabinoid receptors are changed in the vitreous fluid. However, the role of the endocannabinoid system in the retina, particularly in the light‐induced photoreceptor degeneration, remains unknown. Therefore, we investigated involvement of the cannabinoid1 receptor in light‐induced retinal degeneration using in vitro and in vivo models. To evaluate the effect of cannabinoid1 receptors in light irradiation‐induced cell death, the mouse retinal cone‐cell line (661 W) was treated with a cannabinoid1 receptor antagonist, rimonabant. Time‐dependent changes of expression and localization of retinal cannabinoid1 receptors were measured using Western blot and immunostaining. Retinal damage was induced in mice by exposure to light, followed by intravitreal injection of rimonabant. Electroretinograms and histologic analyses were performed. Rimonabant suppressed light‐induced photoreceptor cell death. Cannabinoid1 receptor expression was upregulated by light exposure. Treatment with rimonabant improved both a‐ and b‐wave amplitudes and the thickness of the outer nuclear layer. These results suggest that the cannabinoid1 receptor is involved in light‐induced retinal degeneration and it may represent a therapeutic target in the light‐induced photoreceptor degeneration related diseases.