Morin Ryu

Critical role of Nrf2 in oxidative stress-induced retinal ganglion cell death

NF‐E2 related factor 2 (Nrf2) is a key transcription factor that plays a pivotal role in endogenous protection against oxidative stress. However, the role of Nrf2 in visual disorders remains unclear. It has been reported that oxidative stress is thought of as one of the causes of glaucoma. Here, we investigate whether the function of Nrf2 in oxidative stress‐induced retinal ganglion cell (RGC) death. This study used adult male Nrf2 deficient mice (Nrf2 KO) and age‐ and sex‐matched wild‐type (WT) mice. We dissociated and purified N‐4‐[4‐didecylaminostryryl]‐N‐methyl‐pyridinium iodide‐labeled RGCs with fluorescence‐activated cell sorting, and tried to detect the Nrf2 and Keap1 genes. In the absence of nerve crush (NC), the number of RGCs in Nrf2 KO mice was almost same as that in WT mice. 1‐(2‐cyano‐3‐, 12‐dioxooleana‐1, 9 (11)‐dien‐28‐oyl) imidazole (CDDO‐Im), an Nrf2 activator, prevented NC‐induced loss of RGCs in WT mice. Seven days after NC, without treatment, the number of RGCs in Nrf2 KO mice was significantly lower than in WT mice. In addition, after CDDO‐Im treatment, quantitative RT‐PCR showed increased expression of antioxidant and phase II detoxifying enzymes. These results suggest that up‐regulation of Nrf2 signaling after CDDO‐Im treatment may be a novel therapeutic strategy for the protection of RGCs, especially in glaucoma.

Waveform Analysis of Ocular Blood Flow and the Early Detection of Normal Tension Glaucoma

PURPOSE To investigate waveform changes in blood flow (BF) in the optic nerve head (ONH) and to evaluate their usefulness in identifying normal tension glaucoma (NTG). METHODS Sixty-one eyes of 61 patients with NTG and 21 eyes of age-matched healthy control subjects were included in this study. The NTG eyes were divided into the following three groups based on the progression of their visual field defects: mild (mean deviation [MD] greater than -6.0 decibels [dB]), moderate (MD between -6.0 and -12.0 dB), and severe (MD less than -12.0 dB). The ONH BF analysis was performed with laser speckle flowgraphy (LSFG) and included waveform variables such as skew, acceleration time index (ATI), and blowout time. RESULTS In the ONH, LSFG skew variables were significantly lower in the NTG eyes than in the control eyes (P < 0.001), and ATI was significantly higher (P < 0.01), despite similar systemic characteristics in the four groups. The differences were most marked in the mild NTG group. Multiple linear regression analysis showed that MD, average thickness of the circumpapillary retinal nerve fiber layer, and pulse rate were predictive factors for both skew and ATI. A receiver operating characteristic (ROC) curve analysis also revealed that skew (area under the ROC curve, 0.89) and ATI (area under the ROC curve, 0.80) had the greatest power to differentiate normal eyes from eyes with mild NTG. CONCLUSIONS These results suggest that LSFG measurements of waveform changes in ONH BF can differentiate healthy eyes from eyes with NTG, particularly those with mild NTG.

Tumor Necrosis Factor-α Mediates Photoreceptor Death in a Rodent Model of Retinal Detachment

PURPOSE Photoreceptor degeneration is a major cause of visual loss in various retinal diseases, including retinal detachment (RD) and neovascular AMD, but the underlying mechanisms remain elusive. In this study, the role of TNFα in RD-induced photoreceptor degeneration was investigated. METHODS RD was induced by subretinal injection of hyaluronic acid. Photoreceptor degeneration was assessed by counting the number of apoptotic cells with TdT-dUTP terminal nick-end labeling (TUNEL) 3 days after RD and measurement of the outer nuclear layer (ONL) thickness 7 days after RD. As the target of anti-inflammatory treatment, the expression of TNFα, with or without dexamethasone (DEX) was examined in rats by real-time PCR. To understand the role of TNFα in photoreceptor degeneration, RD was induced in mice deficient in TNFα or its receptors (TNFR1, TNFR2, and TNFR1 and -2), or in wild-type (WT) mice by using a functionally blocking antibody to TNFα. CD11b(+) cells in the outer plexiform layer (OPL) and subretinal space were counted by immunohistochemistry (IHC). RESULTS Treatment with DEX (P = 0.001) significantly suppressed RD-induced photoreceptor degeneration and the expression of TNFα. RD-induced photoreceptor degeneration was significantly suppressed with specific blockade of TNFα (P = 0.032), in mice deficient for TNFα (P < 0.001), TNFR2 (P = 0.001), or TNFR1 and -2 (P < 0.001). However, lack of TNFR1 did not protect against RD-induced photoreceptor degeneration (P = 0.060). Müller cell activation was unchanged in WT and TNFα(-/-) mice. Recruitment of CD11b(+) monocytes was significantly lower in the TNFα(-/-) mice compared to WT mice (P = 0.002). CONCLUSIONS TNFα plays a critical role in RD-induced photoreceptor degeneration. This pathway may become an important target in the prevention of RD-induced photoreceptor degeneration.

Critical role of calpain in axonal damage-induced retinal ganglion cell death

Calpain, an intracellular cysteine protease, has been widely reported to be involved in neuronal cell death. The purpose of this study is to investigate the role of calpain activation in axonal damage‐induced retinal ganglion cell (RGC) death. Twelve‐week‐old male calpstatin (an endogenous calpain inhibitor) knockout mice (CAST KO) and wild‐type (WT) mice were used in this study. Axonal damage was induced by optic nerve crush (NC) or tubulin destruction induced by leaving a gelatin sponge soaked with vinblastine (VB), a microtubule disassembly chemical, around the optic nerve. Calpain activation was assessed by immunoblot analysis, which indirectly quantified the cleaved α‐fodrin, a substrate of calpain. RGCs were retrogradely labeled by injecting a fluorescent tracer, Fluoro‐Gold (FG), and the retinas were harvested and flat‐mounted retinas prepared. The densities of FG‐labeled RGCs harvested from the WT and CAST KO groups were assessed and compared. Additionally, a calpain inhibitor (SNJ‐1945, 100 mg/kg/day) was administered orally, and the density of surviving RGCs was compared with that of the vehicle control group. The mean density of surviving RGCs in the CAST KO group was significantly lower than that observed in the WT group, both in NC and in VB. The mean density of surviving RGCs in the SNJ‐1945‐treated group was significantly higher than that of the control group. The calpain inhibitor SNJ‐1945 has a neuroprotective effect against axonal damage‐induced RGC death. This pathway may be an important therapeutic target for preventing this axonal damage‐induced RGC death, including glaucoma and diabetic optic neuropathy and other CNS diseases that share a common etiology.

ERK1 plays a critical protective role against N-methyl-D-aspartate-induced retinal injury

Excitotoxicity has been implicated in several ischemic diseases of the retina, including retinal vessel occlusion and diabetic retinopathy. Glutamate signaling mediated through the N‐methyl‐D‐aspartate (NMDA) receptor contributes to ischemic cell death. The NMDA receptor antagonists MK‐801 and memantine have substantial neuroprotective effects in experimental retinal disease models, but the mechanisms by which NMDA receptor activity leads to cell death is not clear. Here we describe a previously unknown role for retinal glial cells in NMDA‐induced retinal injury that involves the activation of ERK1/2. Within 1 hr after injecting NMDA intravitreally, activation of ERK1/2 and c‐Fos induction were observed in retinal Müller cells. The roles of activated ERK1/2 in neuronal damage were examined using ERK1 gene deficient mice (homozygous ERK1−/− mice). NMDA‐induced ERK1/2 activation in retina was significantly suppressed in ERK1−/− mice, and these mice had significantly higher numbers of TUNEL‐positive retinal cells than wild‐type mice 24 hr after NMDA injection. These data suggest that, during NMDA injury, Müller cells are activated and play a protective role against NMDA‐induced retinal cell death. ERK1 appears to play a major role in this process. These new findings on retinal glial cell response during NMDA injury offer an important new therapeutic target for preventing many retinal disorders associated with excitotoxicity.

Metabolic stress response implicated in diabetic retinopathy: The role of calpain, and the therapeutic impact of calpain inhibitor

To describe how a high fat diet (HFD) and hyperglycemia initiate a sequence of calpain activation and oxidative stress associated with neuro-degenerative changes in diabetic retinopathy (DR), hyperglycemia was induced with streptozotocin in mice lacking the gene for calpastatin (CAST KO), and in mice lacking the gene for the transcription factor NF-E2 related factor 2 (Nrf2 KO). All animals were fed a HFD. Retinal ganglion cell (RGC) density was estimated by labeling with fluorogold and immunohistochemistry. A potent calpain inhibitor, SNJ-1945, was administered daily until the animals were sacrificed. In vitro, oxidative stress-induced RGC loss was evaluated in a high glucose culture medium with and without SNJ-1945. Retinal mRNA of calpain-1 and calpain-2 was measured by quantitative RT-PCR. Pre-apoptotic substrates of cleaved α-fodrin and synaptophysin protein were quantified by immunoblot analysis. Axonal damage was examined in transverse sections of the optic nerve. A HFD and hyperglycemia significantly increased RGC and axonal degeneration 3 weeks into the experiment. Levels of cleaved α-fodrin were increased. In the CAST KO mice, the neurotoxicity was augmented significantly. Gene manipulation of CAST and orally administered SNJ-1945 successfully modified calpain levels in the retina and prevented RGC death. In vitro, a high-glucose culture of retinal cells without antioxidants showed more RGC death than that with antioxidant treatment. The expression of synaptophysin was significantly suppressed by SNJ-1945 treatment. These results suggest that calpain plays a crucial role in metabolic-induced RGC degeneration caused by hyperglycemia and oxidative stress. Antioxidant and calpain inhibition offers important opportunities for future neuroprotective treatment against RGC death in various metabolic stress-induced diseases including DR.

The Influence of Posture Change on Ocular Blood Flow in Normal Subjects, Measured by Laser Speckle Flowgraphy

Abstract Purpose: To investigate, using laser speckle flowgraphy (LSFG), the autoregulation of ocular blood flow (BF) in response to posture change. Methods: This study comprised 20 healthy volunteers (mean age 30.0 ± 8.5). The mean blur rate (MBR) of the ocular circulation in the subjects was assessed in both a sitting and a supine position every 2 min over the course of 10 min. Baseline measurements of the MBR at the optic nerve head (ONH) and the choroid were taken in a sitting position. Increases in the MBR ratio in a supine position were calculated with reference to this baseline. Intraocular pressure (IOP), systemic blood pressure and heart rate in the brachial artery were also recorded. Results: In the ONH, the MBR ratio increased significantly over the baseline after 2 min (104.8 ± 5.0%, p = 0.001) and 4 min (104.4 ± 5.6%, p = 0.005), in a supine position, but decreased to the initial level after only 6 min. In the choroid, on the other hand, while the MBR ratio also increased significantly after 2 min in a supine position (113.7 ± 8.1%, p < 0.001), it kept this significant increase over the time course of 10 min. After 10 min in a supine position, IOP increased significantly (p < 0.001), systolic blood pressure decreased significantly (p < 0.001), but diastolic blood pressure did not change significantly compared to the baseline. (p = 0.07) Conclusions: ONH and choroidal circulation have significantly different hemodynamics in response to posture change in healthy volunteers. This finding suggests that LSFG enables us to assess the autoregulation of BF in the ONH.

RNA Sequence Reveals Mouse Retinal Transcriptome Changes Early after Axonal Injury

Glaucoma is an ocular disease characterized by progressive retinal ganglion cell (RGC) death caused by axonal injury. However, the underlying mechanisms involved in RGC death remain unclear. In this study, we investigated changes in the transcriptome profile following axonal injury in mice (C57BL/6) with RNA sequencing (RNA-seq) technology. The experiment group underwent an optic nerve crush (ONC) procedure to induce axonal injury in the right eye, and the control group underwent a sham procedure. Two days later, we extracted the retinas and performed RNA-seq and a pathway analysis. We identified 177 differentially expressed genes with RNA-seq, notably the endoplasmic reticulum (ER) stress-related genes Atf3, Atf4, Atf5, Chac1, Chop, Egr1 and Trb3, which were significantly upregulated. The pathway analysis revealed that ATF4 was the most significant upstream regulator. The antioxidative response-related genes Hmox1 and Srxn1, as well as the immune response-related genes C1qa, C1qb and C1qc, were also significantly upregulated. To our knowledge, this is the first reported RNA-seq investigation of the retinal transcriptome and molecular pathways in the early stages after axonal injury. Our results indicated that ER stress plays a key role under these conditions. Furthermore, the antioxidative defense and immune responses occurred concurrently in the early stages after axonal injury. We believe that our study will lead to a better understanding of and insight into the molecular mechanisms underlying RGC death after axonal injury.

Progression of Visual Field Defects in Eyes With Different Optic Disc Appearances in Patients With Normal Tension Glaucoma

Purpose:To investigate the relationship between the optic disc appearance and the progression of visual field defects in eyes with normal tension glaucoma (NTG). Methods:Two hundred nine patients with NTG, who were being treated with topical antiglaucoma drugs and had been followed for at least 3 years, were studied. The baseline optic disc appearance was classified into 4 types: focal ischemic (FI), myopic glaucomatous (MY), senile sclerotic (SS), and generalized cup enlargement (GE). The progression of the NTG was assessed by the slope of the mean deviations (MDs) obtained from the visual field results collected during the follow-up examinations. The baseline and mean intraocular pressures (IOPs) were also followed. Results:Twenty-seven patients were placed in the FI group, 63 into the MY group, 24 into the SS group, and 43 into the GE group. Fifty-two patients (24.9%) could not be classified. There were no significant differences in the percentage reduction of the IOP among the 4 groups. The MD slope in the GE group (−0.51±0.74 dB/y) was significantly steeper than that in the other groups. Regression analyses showed that the factors most associated with the MD slope were the age in the FI (r, −0.495) and the GE (r=0.496) groups, and the relative reduction of the IOP (r=0.413) in the SS group. None of the factors in the MY group was significantly associated with the MD slope. Conclusions:The rate of progression of the field defects, the MD slope, in patients with NTG is possibly dependent on the baseline optic disc appearance. Thus, the optic disc appearance may be useful for the management of patients with NTG.

Suppression of phagocytic cells in retinal disorders using amphiphilic poly(γ-glutamic acid) nanoparticles containing dexamethasone

To investigate the potential of nanoparticles (NPs) composed of poly(γ-glutamic acid) conjugated with l-phenylalanine (γ-PGA-Phe NPs) for the treatment of retinal diseases, γ-PGA-Phe NPs (200nm) were tested with macrophages and microglia in vitro or by intravitreal administration into normal or pathological rat eyes. The anti-inflammatory effects of the NPs containing dexamethasone (DEX-NPs) were examined using qRT-PCR in vitro by counting activated microglia and Fluorogold-labeled retinal ganglion cells in the retinas under excitotoxicity or by counting TUNEL (+) photoreceptors in the detached retinas. The NPs were taken up efficiently by cultured macrophages or microglia. At day 7, 60-80% of the diffuse signal remained in the cytoplasm of these cells. In normal rat eyes, the NPs did not accumulate in the retinas and no inflammatory cells were recruited. Conversely, under pathological conditions, the NPs were localized in activated CD11b-positive cells in the retina. DEX-NPs suppressed the expression of TNFα and MCP-1 in cultured macrophages or microglia, the activation of microglia, the loss of retinal ganglion cells (RGCs) in excitotoxic retinas, and the number of TUNEL (+) photoreceptors in detached retinas. These data suggest that γ-PGA-Phe NPs can be a powerful tool for suppressing inflammatory cells in pathological conditions in the retina.