NMDA-induced Retinal Ganglion Cell Death Research Articles

Fasting alleviates NMDA-induced retinal ganglion cell death in mice

Fasting alleviates NMDA-induced retinal ganglion cell death in mice

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.

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

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.

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.

Triamcinolone attenuates macrophage/microglia accumulation associated with NMDA-induced RGC death and facilitates survival of Müller stem cell grafts

Retinal ganglion cell (RGC) death in glaucoma models is associated with accumulation of activated microglia, a sign of neural degeneration which has been shown to constitute a barrier for transplant cell survival and migration. This study investigated the use of triamcinolone (TA) to control macrophage/microglia accumulation in a model of RGC depletion to create a permissive environment for stem cell grafting. Intravitreal NMDA alone or in combination with TA was used to induce rapid onset of RGC death in 3–4 week old Lister hooded (LH) rat eyes prior to Müller stem cell transplantation into the vitreoretinal space. The effect of NMDA on RGC death and microglial accumulation was assessed immuno-histochemically, whilst electroretinography (ERG) was used to assess RGC function. Post transplantation, survival of grafted cells and their association with microglia were also examined by immunohistochemical methods. Intravitreal injection of NMDA alone resulted in severe macrophage/microglia accumulation associated with extensive RGC death 4–7 days post-treatment. Combination of NMDA with TA significantly reduced microglial numbers in the RGC when compared to NMDA only treated eyes while still depleting the retina of RGC. At the same time, NMDA/TA treatment also caused functional RGC loss as demonstrated by reduction of the scotopic threshold response. Upon transplantation with Müller stem cells, NMDA/TA treatment caused significant reduction in the number of transplant associated macrophage/microglia compared to eyes treated with NMDA alone. On this basis it is proposed that intravitreal injection of TA may be useful as an effective anti-inflammatory agent to control macrophage/microglia accumulation induced by RGC death, thereby creating a retinal environment permissive to cell transplantation studies.

The role of PKCζ in NMDA-induced retinal ganglion cell death: Prevention by aspirin

Intravitreal NMDA injection has been shown to induce the excitotoxic loss of retinal cells. The retinal ganglion cell apoptosis induced by NMDA is thought to play an important role in retinal ischemia injury and NMDA-injected rat has been used as a model of neuronal loss in diseases such as glaucoma. In this experimental model, we studied the early effects of NMDA leading to the degeneration of retinal ganglion cells. PKCzeta regulates the NF-kappaB pathway in cellular responses to various stresses and we have shown that aspirin inhibits purified human PKCzeta. We therefore investigated the molecular mechanism by which retinal cells limit ocular injury following NMDA treatment. We found that the NMDA-induced apoptosis of ganglion cells was mediated, at least partly, by PKCzeta. This enzyme was activated early in the cellular response to NMDA. Prolonged activation was followed by PKCzeta cleavage, and nuclear translocation of the C-terminal region of this protein-a critical event for the survival of retinal cells. We also found that pretreatment with aspirin or the coinjection of NMDA with a specific PKCzeta inhibitor counteracted the effects of NMDA. These findings provide new insight into the role played by PKCzeta in neuronal loss in glaucoma.

Activation of Matrix Metalloproteinase-9 via Neuronal Nitric Oxide Synthase Contributes to NMDA-Induced Retinal Ganglion Cell Death

Understanding the mechanism of neuronal cell death in retinal diseases like glaucoma is important for devising new treatments. One factor involves excitatory amino acid stimulation of N-methyl-D-aspartate (NMDA)-type glutamate receptors, excessive Ca2+ influx, and formation of nitric oxide (NO) via neuronal NO synthase (nNOS). Another factor is the abnormal activation of matrix metalloproteinases (MMPs), in particular MMP-9, which triggers an extracellular signaling cascade leading to apoptosis. This study was designed to investigate the mechanism of excitotoxic retinal ganglion cell (RGC) death in vivo and its relationship to MMP activation. NMDA and glycine were injected into the vitreous of the eye in rats and in nNOS-deficient mice (nNOS-/-) versus control. Gelatinolytic activity of MMP-9 and MMP-2 by zymography and cellular localization by immunohistochemistry were examined, and the effect of MMP inhibition on NMDA-induced RGC death was tested. NMDA was found to upregulate the proform of MMP-9 in the retina and to increase MMP-9 gelatinolytic activity. Retrograde labeling with aminostilbamidine to identify RGCs confirmed that MMP activity occurred only in these retinal neurons and not in glial or other retinal cell types after excitotoxic insult. Deconvolution fluorescence microscopy revealed that MMP activity colocalized with immunoreactive S-nitrosylated protein. NMDA-induced MMP activation was diminished in the retina of nNOS-/- mice, implying that S-nitrosylation of MMP had indeed occurred. In addition, the broad-spectrum MMP inhibitor GM6001 protected RGCs after intravitreal NMDA injection. These findings suggest that an extracellular proteolytic pathway in the retina contributes to RGC death via NO-activated MMP-9.