Optic Neuropathy As the Initial Presenting Sign of N-methyl-d-aspartate (NMDA) Encephalitis.

A 52-year-old woman presented with painless vision loss for 3 months. She was in custody for allegedly robbing a bank and had recently been diagnosed with paranoid schizophrenia. She had 20/100 VA OD, a 2+RAPD, and optic atrophy. Extensive diagnostic workup including MRI, Fluorescein Angiography, Infectious Disease Panel, lumbar puncture, and leptomeningeal biopsy were unrevealing. Vision in her right eye declined to NLP and her left eye declined to 20/200 VA. Anti N-methyl-D-aspartate (NMDA) Autoimmune Encephalitis was diagnosed based on CSF serology and clinical suspicion. Her clinical course improved as she was treated with corticosteroids and rituximab.

AMPA receptor desensitization is the determinant of AMPA receptor mediated excitotoxicity in purified retinal ganglion cells.

The ionotropic glutamate receptors (iGLuR) have been hypothesized to play a role in neuronal pathogenesis by mediating excitotoxic death. Previous studies on iGluR in the retina have focused on two broad classes of receptors: NMDA and non-NMDA receptors including the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptor (AMPAR) and kainate receptor. In this study, we examined the role of receptor desensitization on the specific excitotoxic effects of AMPAR activation on primary retinal ganglion cells (RGCs). Purified rat RGCs were isolated from postnatal day 4-7 Sprague-Dawley rats. Calcium imaging was used to identify the functionality of the AMPARs and selectivity of the s-AMPA agonist. Phosphorylated CREB and ERK1/2 expression were performed following s-AMPA treatment. s-AMPA excitotoxicity was determined by JC-1 mitochondrial membrane depolarization assay, caspase 3/7 luciferase activity assay, immunoblot analysis for α-fodrin, and Live (calcein AM)/Dead (ethidium homodimer-1) assay. RGC cultures of 98% purity, lacking Iba1 and GFAP expression were used for the present studies. Isolated prenatal RGCs expressed calcium permeable AMPAR and s-AMPA (100 µM) treatment of cultured RGCs significantly increased phosphorylation of CREB but not that of ERK1/2. A prolonged (6 h) AMPAR activation in purified RGCs using s-AMPA (100 µM) did not depolarize the RGC mitochondrial membrane potential. In addition, treatment of cultured RGCs with s-AMPA, both in the presence and absence of trophic factors (BDNF and CNTF), did not increase caspase 3/7 activities or the cleavage of α-fodrin (neuronal apoptosis marker), as compared to untreated controls. Lastly, a significant increase in cell survival of RGCs was observed after s-AMPA treatment as compared to control untreated RGCs. However, preventing the desensitization of AMPAR with the treatment with either kainic acid (100 µM) or the combination of s-AMPA and cyclothiazide (50 µM) significantly reduced cell survivability. Activation of the AMPAR in RGCs does not appear to activate a signaling cascade to apoptosis, suggesting that RGCs in vitro are not susceptible to AMPA excitotoxicity as previously hypothesized. Conversely, preventing AMPAR desensitization through differential agonist activation caused AMPAR mediated excitotoxicity. Activation of the AMPAR in increasing CREB phosphorylation was dependent on the presence of calcium, which may help explain this action in increasing RGC survival.

Sigma-1 receptor stimulation protects retinal ganglion cells from ischemia-like insult through the activation of extracellular-signal-regulated kinases 1/2.

Sigma-1 receptor (σ-1) activation and mitogen-activated protein kinases (MAPKs) have been shown to protect retinal ganglion cells (RGCs) from cell death. The purpose of this study was to determine if σ-1 receptor stimulation with pentazocine could promote neuroprotection under conditions of an ischemia-like insult (oxygen glucose deprivation (OGD)) through the phosphorylation of extracellular signal regulated kinase (pERK)1/2. Primary RGCs were isolated from P3-P7 Sprague-Dawley rats and purified by sequential immunopanning using Thy1.1 antibodies. RGCs were cultured for 7 days before subjecting the cells to an OGD insult (0.5% oxygen in glucose-free medium) for 6 h. During the OGD, RGCs were treated with pentazocine (σ-1 receptor agonist) with or without BD 1047 (σ-1 receptor antagonist). In other experiments, primary RGCs were treated with pentazocine in the presence or absence of an MEK1/2 inhibitor, PD098059. Cell survival/death was assessed by staining with the calcein-AM/ethidium homodimer reagent. Levels of pERK1/2, total ERK1/2, and beta tubulin expression were determined by immunoblotting and immunofluorescence staining. RGCs subjected to OGD for 6 h induced 50% cell death in primary RGCs (p < 0.001) and inhibited pERK1/2 expression by 65% (p < 0.001). Cell death was attenuated when RGCs were treated with pentazocine under OGD (p < 0.001) and pERK1/2 expression was increased by 1.6 fold (p < 0.05) compared to OGD treated RGCs without pentazocine treatment. The co-treatment of PD098059 (MEK1/2 inhibitor) with pentazocine significantly abolished the protective effects of pentazocine on the RGCs during this OGD insult. Activation of the σ-1 receptor is a neuroprotective target that can protect RGCs from an ischemia-like insult. These results also established a direct relationship between σ-1 receptor stimulation and the neuroprotective effects of the ERK1/2 pathway in purified RGCs subjected to OGD. These findings suggest that activation of the σ-1 receptor may be a therapeutic target for neuroprotection particularly relevant to ocular neurodegenerative diseases that effect RGCs.

Sigma-1 receptor stimulation attenuates calcium influx through activated L-type Voltage Gated Calcium Channels in purified retinal ganglion cells.

Sigma-1 receptors (σ-1rs) exert neuroprotective effects on retinal ganglion cells (RGCs) both in vivo and in vitro. This receptor has unique properties through its actions on several voltage-gated and ligand-gated channels. The purpose of this study was to investigate the role that σ-1rs play in regulating cell calcium dynamics through activated L-type Voltage Gated Calcium Channels (L-type VGCCs) in purified RGCs. RGCs were isolated from P3-P7 Sprague-Dawley rats and purified by sequential immunopanning using a Thy1.1 antibody. Calcium imaging was used to measure changes in intracellular calcium after depolarizing the cells with potassium chloride (KCl) in the presence or absence of two σ-1r agonists [(+)-SKF10047 and (+)-Pentazocine], one σ-1r antagonist (BD1047), and one L-type VGCC antagonist (Verapamil). Finally, co-localization studies were completed to assess the proximity of σ-1r with L-type VGCCs in purified RGCs. VGCCs were activated using KCl (20 mM). Pre-treatment with a known L-type VGCC blocker demonstrated a 57% decrease of calcium ion influx through activated VGCCs. Calcium imaging results also demonstrated that σ-1r agonists, (+)-N-allylnormetazocine hydrochloride [(+)-SKF10047] and (+)-Pentazocine, inhibited calcium ion influx through activated VGCCs. Antagonist treatment using BD1047 demonstrated a potentiation of calcium ion influx through activated VGCCs and abolished all inhibitory effects of the σ-1r agonists on VGCCs, implying that these ligands were acting through the σ-1r. An L-type VGCC blocker (Verapamil) also inhibited KCl activated VGCCs and when combined with the σ-1r agonists there was not a further decline in calcium entry suggesting similar mechanisms. Lastly, co-localization studies demonstrated that σ-1rs and L-type VGCCs are co-localized in purified RGCs. Taken together, these results indicated that σ-1r agonists can inhibit KCl induced calcium ion influx through activated L-type VGCCs in purified RGCs. This is the first report of attenuation of L-type VGCC signaling through the activation of σ-1rs in purified RGCs. The ability of σ-1rs to co-localize with L-type VGCCs in purified RGCs implied that these two proteins are in close proximity to each other and that such interactions regulate L-type VGCCs.

Evaluation of Same-Day versus Next-Day Implantation of Intracanalicular Dexamethasone for the Control of Postoperative Inflammation and Pain Following Cataract Surgery.

PURPOSE: To evaluate the safety and efficacy of a sustained-release intracanalicular dexamethasone insert for postoperative inflammation and pain implanted in a clinical setting preoperatively or on postoperative day 1. METHODS: Single-site, retrospective, contralateral eye study of patients undergoing cataract surgery. Included were subjects with a dexamethasone intracanalicular insert implanted in the clinic immediately prior to surgery in one eye (same-day) and on postoperative day 1 (POD1) in the contralateral eye. The primary outcome measure was the resolution of anterior chamber inflammation at 1 week postoperative. Secondary outcome measures included proportion of eyes requiring additional therapy for pain and inflammation through 1 month as well as the number of eyes with IOP spikes above baseline. Safety measures included adverse events through 1 month postoperative. RESULTS: Sixty-two eyes of 31 subjects were included in the case series. At 1 week postoperative, 52% of the eyes (n = 16) achieved complete resolution of inflammation in the same-day group and 58% (n = 18) met this endpoint at 1 week in the POD1 group. One subject in the same-day group required additional therapy for rebound inflammation and no eyes required additional therapy in the POD1 group. There were no reports of pain at 1 week or 1 month in either group. There were no implant-related adverse events in either group. CONCLUSION: The favorable results of this study indicate that the sustained-release dexamethasone insert can be safely implanted in the clinic either preoperatively on the day of surgery or on postoperative day 1 for the control of pain and inflammation following cataract surgery.

Neuroprotective effects of inhibitors of Acid-Sensing ion channels (ASICs) in optic nerve crush model in rodents.

PURPOSE: The purpose of the current study was to assess the potential involvement of acid-sensing ion channel 1 (ASIC1) in retinal ganglion cell (RGC) death and investigate the neuroprotective effects of inhibitors of ASICs in promoting RGC survival following optic nerve crush (ONC). RESULTS: ASIC1 protein was significantly increased in optic nerve extracts at day 7 following ONC in rats. Activated calpain-1 increased at 2 and 7 days following ONC as evidenced by increased degradation of α-fodrin, known substrate of calpain. Glial fibrillary acidic protein levels increased significantly at 2 and 7 days post-injury. By contrast, glutamine synthetase increased at 2 days while decreased at 7 days. The inhibition of ASICs with amiloride and psalmotoxin-1 significantly increased RGC survival in rats following ONC (p < 0.05, one-way ANOVA). The mean number of surviving RGCs in rats (n = 6) treated with amiloride (100 µM) following ONC was 1477 ± 98 cells/mm2 compared with ONC (1126 ± 101 cells/mm2), where psalmotoxin-1 (1 µM) treated rats (n = 6) and subjected to ONC had 1441 ± 63 RGCs/mm2 compared with ONC (1065 ± 76 RGCs/mm2). Average number of RGCs in control rats (n = 12) was 2092 ± 46 cells/mm2. Blocking of ASICs also significantly increased RGC survival from ischemic-like insult from 473 ± 80 to 842 ± 49 RGCs/mm2 (for psalmotoxin-1) and from 628 ± 53 RGCs/mm2 to 890 ± 55 RGCs/mm2 (for amiloride) with p ≤ 0.05, using one-way ANOVA. Acidification (a known activator of ASIC1) increased intracellular Ca2+ ([Ca2+]i) in rat primary RGCs, which was statistically blocked by pretreatment with 100 nM psalmotoxin-1. CONCLUSIONS: ASIC1 up-regulation-induced influx of extracellular calcium may be responsible for activation of calcium-sensitive calpain-1 in the retina. Calpain-1 induced degradation of α-fodrin and leads to morphological changes and eventually neuronal death. Therefore, blockers of ASIC1 can be used as potential therapeutics in the treatment of optic nerve degeneration. ABBREVIATIONS: 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF); acid-sensing ion channels (ASICs); analysis of variance (ANOVA); bicinchoninic acid (BCA); brain-derived neurotrophic factor (BDNF); central nervous system (CNS); ciliary neurotrophic factor (CNTF); dimethyl sulfoxide (DMSO); endoplasmic reticulum (ER); ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA); ethylenediaminetetraacetic acid (EDTA); Food and Drug Administration (FDA); glial fibrillary acidic protein (GFAP); glutamine synthetase (GS); intraocular pressure (IOP); kilodalton (kDa); Krebs-Ringer Buffer (KRB); optic nerve crush (ONC); phosphate-buffered saline (PBS); plasma membrane (PM); polymerase chain reaction (PCR); retinal ganglion cell (RGC); RNA Binding Protein With Multiple Splicing (RBPMS); room temperature (RT); standard error of the mean (SEM).

Neuroprotective effects of transcription factor Brn3b in an ocular hypertension rat model of glaucoma.

PURPOSE: Glaucoma is an optic neuropathy commonly associated with elevated intraocular pressure (IOP), leading to optic nerve head (ONH) cupping, axon loss, and apoptosis of retinal ganglion cells (RGCs), which could ultimately result in blindness. Brn3b is a class-4 POU domain transcription factor that plays a key role in RGC development, axon outgrowth, and pathfinding. Previous studies suggest that a decrease in Brn3b levels occurs in animal models of glaucoma. The goal of this study was to determine if adeno-associated virus (AAV)-directed overexpression of the Brn3b protein could have neuroprotective effects following elevated IOP-mediated neurodegeneration. METHODS: Intraocular pressure was elevated in one eye of Brown Norway rats (Rattus norvegicus), following which the IOP-elevated eyes were intravitreally injected with AAV constructs encoding either the GFP (rAAV-CMV-GFP and rAAV-hsyn-GFP) or Brn3b (rAAV-CMV-Brn3b and rAAV-hsyn-Brn3b). Retina sections through the ONH were stained for synaptic plasticity markers and neuroprotection was assessed by RGC counts and visual acuity tests. RESULTS: Adeno-associated virus-mediated expression of the Brn3b protein in IOP-elevated rat eyes promoted an upregulation of growth associated protein-43 (GAP-43), actin binding LIM protein (abLIM) and acetylated α-tubulin (ac-Tuba) both posterior to the ONH and in RGCs. The RGC survival as well as axon integrity score were significantly improved in IOP-elevated rAAV-hsyn-Brn3b-injected rats compared with those of the IOP-elevated rAAV-hsyn-GFP- injected rats. Additionally, intravitreal rAAV-hsyn-Brn3b administration significantly restored the visual optomotor response in IOP-elevated rat eyes. CONCLUSIONS: Adeno-associated virus-mediated Brn3b protein expression may be a suitable approach for promoting neuroprotection in animal models of glaucoma.

Endothelin B receptors contribute to retinal ganglion cell loss in a rat model of glaucoma.

Glaucoma is an optic neuropathy, commonly associated with elevated intraocular pressure (IOP) characterized by optic nerve degeneration, cupping of the optic disc, and loss of retinal ganglion cells which could lead to loss of vision. Endothelin-1 (ET-1) is a 21-amino acid vasoactive peptide that plays a key role in the pathogenesis of glaucoma; however, the receptors mediating these effects have not been defined. In the current study, endothelin B (ET(B)) receptor expression was assessed in vivo, in the Morrison's ocular hypertension model of glaucoma in rats. Elevation of IOP in Brown Norway rats produced increased expression of ET(B) receptors in the retina, mainly in retinal ganglion cells (RGCs), nerve fiber layer (NFL), and also in the inner plexiform layer (IPL) and inner nuclear layer (INL). To determine the role of ET(B) receptors in neurodegeneration, Wistar-Kyoto wild type (WT) and ET(B) receptor-deficient (KO) rats were subjected to retrograde labeling with Fluoro-Gold (FG), following which IOP was elevated in one eye while the contralateral eye served as control. IOP elevation for 4 weeks in WT rats caused an appreciable loss of RGCs, which was significantly attenuated in KO rats. In addition, degenerative changes in the optic nerve were greatly reduced in KO rats compared to those in WT rats. Taken together, elevated intraocular pressure mediated increase in ET(B) receptor expression and its activation may contribute to a decrease in RGC survival as seen in glaucoma. These findings raise the possibility of using endothelin receptor antagonists as neuroprotective agents for the treatment of glaucoma.