NMDA-induced Retinal Ganglion Cell Research Articles

Neuroprotective and anti-inflammatory effects of eicosane on glutamate and NMDA-induced retinal ganglion cell injury.

To investigate the protective effects, antioxidant potential, and anti-inflammatory mechanisms of eicosane on glutamate-induced cell damage and on N-methyl-D-aspartate (NMDA)-induced retinal ganglion cell (RGC) injury in a mouse model of glaucoma. The protective effects of eicosane on the rat R28 retinal precursor cell line were assessed using cell counting kit-8 assays and Hoechst-propidium iodide staining. Intracellular reactive oxygen species (ROS) production was measured using the fluorescent probe 2'-7'-dichlorofluorescin diacetate and flow cytometry. The protective role of eicosane on NMDA-induced RGC injury in a mouse glaucoma model was determined by immunostaining of frozen sections of retina. The effects of eicosane on the metabolome of the retina in mice with NMDA-induced RGC damage were evaluated by liquid chromatography-mass spectroscopy (LC-MS) and untargeted metabolomics analyses. Eicosane treatment significantly attenuated glutamate-induced damage to R28 cells in vitro. Eicosane also protected RGCs against NMDA-induced injury in a mouse glaucoma model. Untargeted metabolomics analyses showed that eicosane increased multiple metabolites, including L-arginine and L-carnitine, in the retina. Eicosane has protective effects, antioxidant potential, and anti-inflammatory properties in an in vitro model of glutamate-induced cell damage and in an in vivo model of NMDA-induced RGC injury in mouse glaucoma through modulation of L-arginine and/or L-carnitine metabolism.

Melatonin protects against NMDA-induced retinal ganglion cell injury by regulating the microglia-TNFα-RGC p38 MAPK pathway.

Glaucoma, one of the most common ocular neurodegenerative diseases worldwide, is characterized by retinal ganglion cell (RGC) loss. There is a large body of literature that describes the neuroprotective role of melatonin against neurodegenerative diseases by regulating neuroinflammation, although the exact mechanism through which melatonin acts on RGC is still uncertain. This study assessed the protective effects of melatonin using a NMDA-induced RGC injury model, and studied the possible mechanisms involved in this process. Melatonin promoted RGC survival, improved retinal function, and inhibited the apoptosis and necrosis of retinal cells. To understand the mechanism of the neuroprotective effects of melatonin on RGC, microglia and inflammation-related pathways were assessed after melatonin administration and microglia ablation. Melatonin promoted RGC survival by suppressing microglia-derived proinflammatory cytokines, in particular TNFα, which in turn inhibited the activation of p38 MAPK pathway. Inhibiting TNFα or manipulating p38 MAPK pathway protected damaged RGC. Our results suggest that melatonin protects against NMDA-induced RGC injury by inhibiting the microglial TNFα-RGC p38 MAPK pathway. It should be considered a candidate neuroprotective therapy against retinal neurodegenerative diseases.

Fasting alleviates NMDA-induced retinal ganglion cell death in mice

Fasting alleviates NMDA-induced retinal ganglion cell death in mice

Isozyme-specific histone deacetylase 1/2 inhibitor K560 attenuates oxidative stress-induced retinal cell death

Oxidative stress-induced damage is an underlying mechanism in the pathogenesis of age-related retinal diseases. Here, we examined the effects of K560, a potential candidate drug for the treatment of these diseases, on oxidative stress-induced retinal cell death. K560 is a novel isozyme-specific inhibitor of histone deacetylase 1 and 2 (HDAC1/2). Histone acetylation in retinal lysates and dissociated retinal cells was detected with a western blot analysis and cell-based enzyme-linked immunosorbent assay (ELISA), respectively. The viability of mouse retinal cells was measured with an alamarBlue assay. We used immunohistochemistry for RNA binding protein with multiple splicing (RBPMS) to visualize the retinal ganglion cells (RGCs) of mice. An ELISA analysis indicated that histone acetylation was enhanced in dissociated mouse retinal cells treated with K560. The cell viability assay indicated that K560 attenuated both exogenous hydrogen peroxide–induced and endogenous oxidative stress–induced cell death in dissociated retinal cells. Western blot analysis indicated that intravitreal K560 administration enhanced the acetylation of histones H3 and H4 in mouse retinal lysates. To examine the effect of K560 on oxidative stress–induced RGC death, we performed whole-mount immunohistochemistry for RBPMS on retinas dissected from eyes treated with K560 or vehicle on day one, and K560 or vehicle and NMDA on day two. Quantification of RBPMS-immunopositive cells indicated that K560 attenuated NMDA-induced RGC death. Taken together, our findings suggest that administration of a HDAC1/2-specific inhibitor K560 may be effective in the treatment of oxidative stress–mediated retinal degeneration and have less cytotoxicity than other known HDAC inhibitors, which are known to target a wide range of HDAC family members.

Investigating the role of Ca2+/calmodulin-dependent protein kinase II in the survival of retinal ganglion cells.

Investigating the role of Ca2+/calmodulin-dependent protein kinase II in the survival of retinal ganglion cells.

Novel use of a chemically modified siRNA for robust and sustainable in vivo gene silencing in the retina

Despite efficient and specific in vitro knockdown, more reliable and convenient methods for in vivo knockdown of target genes remain to be developed particularly for retinal research. Using commercially available and chemically modified siRNA so-called Accell siRNA, we established a novel in vivo gene silencing approach in the rat retina. siRNA designed for knockdown of the house keeping gene Gapdh or four retinal cell type-specific genes (Nefl, Pvalb, Rho and Opn1sw) was injected into the vitreous body, and their retinal mRNA levels were quantified using real-time PCR. Intravitreal injection of siRNA for Gapdh resulted in approximately 40–70% reduction in its retinal mRNA levels, which lasted throughout a 9-day study period. Furthermore, all the selected retinal specific genes were efficiently down-regulated by 60–90% following intravitreal injection, suggesting injected siRNA penetrated into major retinal cell types. These findings were consistent with uniform distribution of a fluorescence-labeled siRNA injected into the vitreous body. Interestingly, gene silencing of Grin1, a core subunit of NMDA receptor, was accompanied by significant prevention from NMDA-induced retinal ganglion cell death. Thus, we provide single intravitreal injection of Accell siRNA as a versatile technique for robust and sustainable in vivo retinal gene silencing to characterize their biological functions under physiological and pathophysiological conditions.

Cyclin-Dependent Kinase Inhibitor 2b Mediates Excitotoxicity-Induced Death of Retinal Ganglion Cells.

Glutamate excitotoxicity seems to contribute to retinal ganglion cell (RGC) death in various eye diseases, but the underlying molecular mechanisms are not fully understood. We studied the roles of cyclin-dependent kinase inhibitors Cdkn2a and Cdkn2b, known as cellular stress-related senescence markers, in N-methyl-d-aspartate (NMDA)-induced RGC death. Gene expression was analyzed using quantitative reverse transcription (qRT)-PCR, in situ hybridization, and immunochemistry. Cdkn2a and Cdkn2b gain- and loss-of-function experiments were performed using the adeno-associated virus type 2 (AAV2)-mediated gene delivery system. AAV2-CRISPR-Cas9-mediated knockout of Cdkn2a or Cdkn2b was validated using cultured cells by T7 endonuclease I assay and Western blot analysis. The effects of altered expression of Cdkn2a and Cdkn2b on NMDA-induced RGC death were evaluated by quantification of RNA binding protein with multiple splicing (Rbpms)-immunoreactive RGCs. Intravitreal NMDA injection resulted in upregulation of Cdkn2a and Cdkn2b expression in RGCs of the mouse retina. AAV2-mediated overexpression of Cdkn2b led to increased expression of Cdkn2a in RGCs, but not vice versa. Overexpression of Cdkn2b, but not Cdkn2a, resulted in a further reduction in RGC viability in NMDA-injected retinas. However, excessive levels of Cdkn2a or Cdkn2b had no effect on RGC viability in healthy mice. AAV2-CRISPR-Cas9-mediated knockout of either Cdkn2a or Cdkn2b attenuated NMDA-induced RGC death. Cdkn2a and Cdkn2b have pivotal roles in the regulation of excitotoxic RGC degeneration under NMDA-induced pathologic conditions. Our findings imply that Cdkn2a and Cdkn2b are novel therapeutic targets for ocular diseases displaying excitotoxicity-induced neuronal degeneration.

Assessing retinal ganglion cell death and neuroprotective agents using real time imaging

The evaluation of retinal ganglion cell (RGC) death is a key part of retinal disease care. Previously, we used a Sytox Orange (SO)-based real-time imaging method to assess the RGCs in mice that underwent optic nerve crush. Here, we used N-methyl-D-aspartate (NMDA) injury in rats to confirm our model and assess the effect of neuroprotective agents on RGCs.The rats received NMDA injury and the intravitreal injection of SO, a cell-impermeant dyeing compound that targets nucleic acid. After ten minutes, non-invasive confocal scanning laser ophthalmoscopy visualized damaged or dying cells. Finally, the retinas were flat-mounted for histological confirmation of RGC death, with retrograde Fluorogold labeling and Alexa Fluor 488 Annexin V-conjugate (Annexin V) staining. This also revealed the time course of retinal cell death and the neuroprotective effect of SNJ-1945.Real-time imaging showed that SO-positive cells significantly increased starting 2 h after NMDA injection and reached an approximate plateau at 3 h. SO-positive cells were positive for Fluorogold and Annexin V in the isolated retinas. Moreover, the number of SO-positive retinal cells was significantly lower after treatment with SNJ-1945, compared to carboxymethyl cellulose. These results were confirmed in the isolated retinas.Thus, real-time imaging with SO allows the quick quantification of NMDA-induced RGC damage and death, and evaluation of neuroprotective agents. This technique may aid research into the development of new neuroprotective therapies.

Systemic administration of an apelin receptor agonist protects against NMDA-induced retinal ganglion cell death

Systemic administration of an apelin receptor agonist protects against NMDA-induced retinal ganglion cell death

Puerarin Attenuates N-Methyl-D-aspartic Acid-induced Apoptosis and Retinal Ganglion Cell Damage Through the JNK/p38 MAPK Pathway.

To explore the protective effect of puerarin on N-methyl-D-aspartic acid (NMDA)-induced retinal ganglion cells (RGCs) injury and its underlying mechanism. Primary RGCs were isolated from P3-P7 Sprague-Dawley rats and purified by sequential immunopanning using Thy1.1 antibodies. NMDA was used to mimic the glutamate activation, cell apoptosis, reactive oxygen species (ROS), malondialdehyde levels, SOD and NO production, nNOS and iNOS expression, as well as caspase-3 activity, Bcl-2, and Bax expression in the RGCs were analyzed by ELISA, RT-PCR, and Western blotting. A rat model of retinal injury was used to detect the protective effect of puerarin. Puerarin protected against NMDA-induced RGCs injury in a dose-dependent manner. Compared with the NMDA-treated group, puerarin pretreatment significantly reduced ROS and malondialdehyde levels, promoted SOD and NO production, and downregulated nNOS and iNOS expression in the RGCs. Mechanism analysis showed that pretreatment with puerarin could effectively offset the increase of Bax expression and caspase-3 activity brought by NMDA, and promote Bcl-2 expression in the RGCs. Puerarin pretreatment also effectively inhibited NMDA-induced JNK and p38 phosphorylation in the RGCs, whereas pretreatment with either JNK agonist anisomycin or p38 agonist P79350 could significantly compensate the effects caused by puerarin. Furthermore, puerarin prevented RGCs loss in the retinal injury induced by intravitreal NMDA in a rat model. The present results of this study demonstrated that puerarin protected against NMDA-induced apoptosis and RGCs damage through the JNK/p38 MAPK pathway.

Apelin-36 is protective against N-methyl-D-aspartic-acid-induced retinal ganglion cell death in the mice

Retinal ganglion cell death in glaucoma is caused at least in part by a large Ca2+ influx through N-methyl-D-aspartic acid (NMDA) receptors. Apelin is a peptide originally found in the tissue extracts of bovine stomach. Recent studies have been shown that apelin protects against the ischemic-reperfused injury in the brain. We examined whether apelin had protective effects on the NMDA-induced retinal ganglion cell (RGC) death using B6.Cg-TgN(Thy1-CFP)23Jrs/J transgenic mice, which express the enhanced cyan fluorescent protein in RGCs in the retina, in vivo. The mice were anesthetized by ketamine and xylazine, and NMDA (40 nmol/eye) was intravitreally injected. We evaluated the effects of apelin-13, [Glp1]-apelin-13, a potent agonist of apelin receptor, and apelin-36 on the NMDA-induced retinal ganglion cell death. NMDA-induced retinal ganglion cell loss was clearly seen 7 days after NMDA injection. Intravitreal apelin-36 (0.33 nmol/eye), but not apelin-13 (1 nmol/eye) nor [Glp1]-apelin-13 (1 nmol/eye), simultaneously injected with NMDA significantly reduced the cell loss. The protective effect of apelin-36 was not reduced by ML221 (0.1 nmol/eye; 5-[(4-Nitrobenzoyl)oxy]-2-[(2-pyrimidinylthio)methyl]-4H-pyran-4-one), an apelin receptor antagonist, GF109203X (0.03 nmol/eye), a protein kinase C inhibitor, U0126 (0.2 nmol/eye), a MAPK/ERK kinase inhibitor, LY294002 (0.1 nmol/eye), a phosphoinositide 3-kinase inhibitor, Akti 1/2 (0.05 nmol/eye), an Akt inhibitor, or 4,5,6,7-tetrabromobenzotriazole (0.2 nmol/eye), a casein kinase-2 inhibitor. In addition, human apelin-36 did not affect the kainic-acid (20 nmol/eye)-induced ganglion cell death. The present study suggests that apelin-36 protects against the NMDA-induced ganglion cell death independently of the activation of apelin receptor in the murine retina in vivo.

Neuroprotective Effect of Lutein on NMDA-Induced Retinal Ganglion Cell Injury in Rat Retina.

Lutein injection is a possible therapeutic approach for retinal diseases, but the molecular mechanism of its neuroprotective effect remains to be elucidated. The aim of this study was to investigate its protective effects in retinal ganglion cells (RGCs) against N-methyl-D-aspartate (NMDA)-induced retinal damage in vivo. Retinal damage was induced by intravitreal NMDA injection in rats. Each animal was given five daily intraperitoneal injections of Lutein or vehicle along with intravitreal NMDA injections. Electroretinograms were recorded. The number of viable RGCs was quantified using the retinal whole-mount method by immunofluorescence. Proteins were measured by Western blot assays. Lutein reduced the retinal damage and improved the response to light, as shown by an animal behavior assay (the black-and-white box method) in rats. Furthermore, Lutein treatment prevented the NMDA-induced reduction in phNR wave amplitude. Lutein increased RGC number after NMDA-induced retina damage. Most importantly, Bax, cytochrome c, p-p38 MAPK, and p-c-Jun were all upregulated in rats injected with NMDA, but these expression patterns were reversed by continuous Lutein uptake. Bcl-2, p-GSK-3β, and p-Akt in the Lutein-treated eyes were increased compared with the NMDA group. Lutein has neuroprotective effects against retinal damage, its protective effects may be partly mediated by its anti-excitability neurotoxicity, through MAPKs and PI3K/Akt signaling, suggesting a potential approach for suppressing retinal neural damage.

Ketogenic Diet Attenuates NMDA-Induced Damage to Rat's Retinal Ganglion Cells in an Age-Dependent Manner

Objective: This study was conducted to investigate neuroprotective effects of a high fat/low carbohydrate and protein diet (ketogenic diet, KD) in a model of N-methyl <smlcap>D</smlcap>-aspartate (NMDA)-induced retinal ganglion cell (RGC) damage in juvenile and young adult rats. Methods: Juvenile (30-35 days old) and young adult (56-70 days old) female Brown Norway rats were fed the KD for 21 days; rats exposed to a standard rodent diet (SRD) served as controls. The main constituents of the KD used in the present study were approximately 80% fats, 8% proteins, and less than 1% carbohydrates. On day 14 of exposure to the KD (or the SRD in the control group), each rat received a single intravitreal injection of NMDA; RGCs were then retrogradely labelled by hydroxystilbamidine on day 19 and collected on day 21 to assess the degree of damage induced by NMDA. Blood biomarkers to confirm the expected metabolic response to the KD (i.e. ketosis and hypoglycaemia) were also assessed. Results: Although both the juvenile and young adult rats developed comparable ketosis and hypoglycaemia when fed the KD, NMDA-induced loss in RGCs was significantly attenuated only in juvenile rats exposed to the KD in comparison with those fed the SRD; exposure to the KD had no protective effect in young adult rats. In summary, exposure to the KD had a neuroprotective effect in NMDA-induced RGC damage in juvenile rats, but not in young adult rats. Conclusion: These results support further exploration of metabolic interventions to treat optic neuropathies associated with neurodegeneration.

Valproic Acid Prevents NMDA-Induced Retinal Ganglion Cell Death via Stimulation of Neuronal TrkB Receptor Signaling

Valproic acid (VPA) is widely prescribed for treatment of epilepsy, mood disorders, migraines, and neuropathic pain. It exerts its therapeutic benefits through multiple mechanisms, including enhancement of GABAergic activity, activation of prosurvival protein kinases, and inhibition of histone deacetylase. Increasing evidence suggests that VPA possesses neuroprotective properties. We examined neuroprotective effects of VPA in an N-methyl-d-aspartate (NMDA) excitotoxicity model, which mimics some of the pathological features of glaucoma. Invivo retinal imaging using optical coherence tomography revealed that NMDA-induced retinal degeneration was suppressed in the VPA-treated retina, and histological analyses confirmed that VPA reduced retinal ganglion cell death. Invivo electrophysiological analyses demonstrated that visual impairment was prevented in the VPA-treated retina, clearly establishing both histological and functional effects of VPA. Brain-derived neurotrophic factor (BDNF) expression was up-regulated in Müller glial cells, and neuroprotective effects of VPA on retinal ganglion cells were significantly reduced in a conditional knockout mouse strain with deletion of tropomyosin receptor kinase B (TrkB), a receptor for BDNF from retinal ganglion cells. The results show that VPA stimulates BDNF up-regulation in Müller glial cells and provides direct evidence that neuronal TrkB is important in VPA-mediated neuroprotection. Also, VPA suppresses oxidative stress induced by NMDA in the retina. Our findings raise intriguing possibilities that the widely prescribed drug VPA may be useful for treatment of glaucoma.

Age-dependent neuroprotection of retinal ganglion cells by tempol-C8 acyl ester in a rat NMDA toxicity model

The efficacy of tempol and its acyl derivative tempol-C8 as retinoprotective agents was compared in a rat model of NMDA-induced retinal ganglion cell (RGC) damage. Tempol or tempol-C8 in different doses was administered intraperitoneally to 6 weeks old (pre-adolescent) and 9-10 weeks old (young adult) rats before and after an intravitreous NMDA injection. Retinal ganglion cell were retrogradely labeled with the fluorescent tracer hydroxystilbamidine and RGC counting was performed on retinal flatmounts. Intravitreal NMDA reduced RGC counts by about 90%, independently of age < 0.001). In pre-adolescent animals tempol-C8, but not tempol unmodified, showed a significant, dose-dependent RGC rescue effect, with peak activity at 5.8 µmol/kg (p < 0.001). In young adult animals, however, no neuroprotective effect was found for either tempol or tempol-C8. In contrast to tempol itself, tempol-C8 acyl ester was neuroprotective in pre-adolescent rats in the NMDA- induced RGC damage model. Therefore, neuroprotection by tempol acyl esters seems to be superior to that of tempol under certain conditions.

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.

NMDA receptor subunits have different roles in NMDA-induced neurotoxicity in the retina

BackgroundLoss of retinal ganglion cells (RGCs) is a hallmark of various retinal diseases including glaucoma, retinal ischemia, and diabetic retinopathy. N-methyl-D-aspartate (NMDA)-type glutamate receptor (NMDAR)-mediated excitotoxicity is thought to be an important contributor to RGC death in these diseases. Native NMDARs are heterotetramers that consist of GluN1 and GluN2 subunits, and GluN2 subunits (GluN2A–D) are major determinants of the pharmacological and biophysical properties of NMDARs. All NMDAR subunits are expressed in RGCs in the retina. However, the relative contribution of the different GluN2 subunits to RGC death by excitotoxicity remains unclear.ResultsGluN2B- and GluN2D-deficiency protected RGCs from NMDA-induced excitotoxic retinal cell death. Pharmacological inhibition of the GluN2B subunit attenuated RGC loss in glutamate aspartate transporter deficient mice.ConclusionsOur data suggest that GluN2B- and GluN2D-containing NMDARs play a critical role in NMDA-induced excitotoxic retinal cell death and RGC degeneration in glutamate aspartate transporter deficient mice. Inhibition of GluN2B and GluN2D activity is a potential therapeutic strategy for the treatment of several retinal diseases.

Taurine Provides Neuroprotection against Retinal Ganglion Cell Degeneration

Retinal ganglion cell (RGC) degeneration occurs in numerous retinal diseases leading to blindness, either as a primary process like in glaucoma, or secondary to photoreceptor loss. However, no commercial drug is yet directly targeting RGCs for their neuroprotection. In the 70s, taurine, a small sulfonic acid provided by nutrition, was found to be essential for the survival of photoreceptors, but this dependence was not related to any retinal disease. More recently, taurine deprivation was incriminated in the retinal toxicity of an antiepileptic drug. We demonstrate here that taurine can improve RGC survival in culture or in different animal models of RGC degeneration. Taurine effect on RGC survival was assessed in vitro on primary pure RCG cultures under serum-deprivation conditions, and on NMDA-treated retinal explants from adult rats. In vivo, taurine was administered through the drinking water in two glaucomatous animal models (DBA/2J mice and rats with vein occlusion) and in a model of Retinitis pigmentosa with secondary RGC degeneration (P23H rats). After a 6-day incubation, 1 mM taurine significantly enhanced RGCs survival (+68%), whereas control RGCs were cultured in a taurine-free medium, containing all natural amino-acids. This effect was found to rely on taurine-uptake by RGCs. Furthermore taurine (1 mM) partly prevented NMDA-induced RGC excitotoxicity. Finally, taurine supplementation increased RGC densities both in DBA/2J mice, in rats with vein occlusion and in P23H rats by contrast to controls drinking taurine-free water. This study indicates that enriched taurine nutrition can directly promote RGC survival through RGC intracellular pathways. It provides evidence that taurine can positively interfere with retinal degenerative diseases.

Norrin: Molecular and functional properties of an angiogenic and neuroprotective growth factor

Norrin is a secreted signaling molecule with structural and functional characteristics of an autocrine and/or paracrine acting growth factor. In the eye, Norrin is constitutively expressed in Müller cells. Norrin specifically binds to Frizzled-4 receptors and activates the canonical Wnt/β-catenin signaling pathway that is profoundly enhanced when Tspan12 is present at the Norrin/Frizzled-4 receptor complex. In the absence of Norrin or Frizzled-4, intraretinal capillaries are not formed during developmental angiogenesis. As a result there is considerable evidence that Norrin and Frizzled-4 are part of an essential signaling system that controls the formation of the retinal vasculature during eye development. Intriguingly, Norrin promotes vessel regrowth and induces the formation of intraretinal capillaries following oxygen-induced retinopathy in mice, an animal model of retinopathy of prematurity. Moreover, Norrin has pronounced neuroprotective properties on retinal ganglion cells (RGC) with the distinct potential to decrease the damaging effects of excitotoxic NMDA-induced RGC injury. The neuroprotective effects of Norrin similarly involve an activation of Wnt/β-catenin signaling and the subsequent induction of neuroprotective growth factor synthesis in Müller cells, such as that of fibroblast growth factor-2 (FGF2) or ciliary neurotrophic factor (CNTF). Overall, Norrin and the molecules involved in its signaling pathway appear to be promising targets to develop strategies that induce intraretinal vessel formation in patients suffering from ischemic retinopathies, or that increase RGC survival in glaucoma.

Longitudinal and Simultaneous Imaging of Retinal Ganglion Cells and Inner Retinal Layers in a Mouse Model of Glaucoma Induced byN-Methyl-d-Aspartate

To investigate the longitudinal profile of N-methyl-D-aspartate (NMDA) injection-induced damage in retinal ganglion cells (RGCs) by imaging retinal Thy 1-cyan fluorescent protein (CFP) expression and inner retinal layers using a custom-made imaging device containing short-wavelength confocal scanning laser ophthalmoscope (scSLO) and speckle noise-reduced spectral-domain optical coherence tomography (SD-OCT). Simultaneous scSLO and SD-OCT examinations were performed in Thy 1-CFP mice injected with NMDA (1-20 nanomoles). CFP-expressing RGCs were counted using scSLO images. Ganglion cell complex (GCC: retinal nerve fiber layer, ganglion cell layer, and inner plexiform layer) thickness around the optic disc was measured in SD-OCT images. The RGCs rapidly decreased 1 day after NMDA injection in a dose-dependent manner (65.3%, 71.7%, 49.5%, and 27.1% of the preinjection level, 2, 5, 10, and 20 nanomoles, respectively) and continued to decrease slightly (to 53.7%, 44.1%, 28.3%, and 20.2% of the preinjection level on days 14, 2, 5, 10, and 20 nanomoles, respectively). In contrast, dose-dependent reduction of GCC thickness was first detected 4 days after injection. The thickness further decreased to 84.6%, 75.7%, 76.5%, and 71.4% of the preinjection level on day 14 (2, 5, 10, and 20 nanomoles, respectively). NMDA-induced RGC damage is characterized by rapid RGCs loss followed by gradual reduction in GCC thickness. Simultaneous imaging of CFP expression in the RGCs and inner retinal layers provides a sensitive, reliable, and new method for longitudinal evaluation of progressive RGC damage in experimental models of glaucoma.