Objective This investigation was to determine the relationship between changes in the expression levels of miR-134 and the E2F transcription factor 6 (E2F6) in mediating control of apoptosis in N-methyl-D-aspartate (NMDA)-induced glaucomatous mice. Methods Morphological and structural changes were quantitatively analyzed along with apoptosis in the retinal ganglion cell (RGC) layer, internal plexiform layer and RGCs. Glaucomatous RGCs were transfected, and cell viability and apoptosis were examined. The targeting relationship between miR-134 and E2F6 was analyzed, as well as their expression pattern. Results Intravitreal injection of NMDA induced a significant reduction in the number of RGCs and thinning of IPL thickness. miR-134 was highly expressed and E2F6 was lowly expressed in glaucoma mice. Suppression of miR-134 or E2F6 overexpression inhibited apoptosis in the glaucomatous RGCs and instead their proliferative activity. MiR-134 targeted inhibition of E2F6 expression. Suppressing rises in E2F6 expression reduced the interfering effect of miR-134 on glaucomatous RGC development. Conclusion Depleting miR134 expression increases, in turn, E2F6 expression levels and in turn reduces glaucomatous RGC apoptosis expression.

Excitotoxicity from the impairment of glutamate uptake constitutes an important mechanism in neurodegenerative diseases such as Alzheimer’s, multiple sclerosis, and Parkinson's disease. Within the eye, excitotoxicity is thought to play a critical role in retinal ganglion cell death in glaucoma, diabetic retinopathy, retinal ischemia, and optic nerve injury, yet how excitotoxic injury impacts different retinal layers is not well understood. Here, we investigated the longitudinal effects of N-methyl-D-aspartate (NMDA)-induced excitotoxic retinal injury in a rat model using deep learning-assisted retinal layer thickness estimation. Before and after unilateral intravitreal NMDA injection in nine adult Long Evans rats, spectral-domain optical coherence tomography (OCT) was used to acquire volumetric retinal images in both eyes over 4 weeks. Ten retinal layers were automatically segmented from the OCT data using our deep learning-based algorithm. Retinal degeneration was evaluated using layer-specific retinal thickness changes at each time point (before, and at 3, 7, and 28 days after NMDA injection). Within the inner retina, our OCT results showed that retinal thinning occurred first in the inner plexiform layer at 3 days after NMDA injection, followed by the inner nuclear layer at 7 days post-injury. In contrast, the retinal nerve fiber layer exhibited an initial thickening 3 days after NMDA injection, followed by normalization and thinning up to 4 weeks post-injury. Our results demonstrated the pathological cascades of NMDA-induced neurotoxicity across different layers of the retina. The early inner plexiform layer thinning suggests early dendritic shrinkage, whereas the initial retinal nerve fiber layer thickening before subsequent normalization and thinning indicates early inflammation before axonal loss and cell death. These findings implicate the inner plexiform layer as an early imaging biomarker of excitotoxic retinal degeneration, whereas caution is warranted when interpreting the ganglion cell complex combining retinal nerve fiber layer, ganglion cell layer, and inner plexiform layer thicknesses in conventional OCT measures. Deep learning-assisted retinal layer segmentation and longitudinal OCT monitoring can help evaluate the different phases of retinal layer damage upon excitotoxicity.

Objectives: The objectives of the study were to investigate the responses of different retinal ganglion cell (RGC) populations to diverse injuries [transient ischemia induced by acute ocular hypertension (AOH), excitotoxicity induced by intravitreal injection of 100 mM N‐methyl D‐aspartate (NMDA) or intra‐orbital optic nerve transection (IONT)] and protection with selective agonists of the tropomyosin related kinase B (TrkB) receptor, brain‐derived neurotrophic factor (BDNF) or 7,8‐Dihydroxyflavone (DHF) or with a voltage‐dependent calcium channel blocker (ITH12657).Methods: Adult Sprague–Dawley rats were used. In the first group, the left eye received a single intravitreal injection of 5 μL vehicle or 5 μg BDNF and was connected to a saline reservoir elevated above the eye to induce an acute ocular hypertension (AOH) and transient ischemia for 75 min. These rats were analysed at different survival intervals from 3 to 45 days.A second group were treated daily subcutaneously with vehicle or ITH5263 (10 mg/kg), or intraperitoneally with vehicle or DHF (5 mg/kg), starting 12 h prior to an intraocular injection of 100 mM N‐methyl‐D‐aspartate (NMDA) to induce retinal excitotoxicity. These rats were analysed at different survival intervals from 3 to 21 days.A third group received a left intra‐orbital optic nerve transection (IONT) to induce axotomy of the RGC population and were treated daily with an intraperitoneal injection of vehicle (0.9% NaCl containing 1% DMSO) or DHF (5 mg/kg diluted in vehicle). These rats were analysed at survival intervals from 3 to 60 days.The retinas were prepared as wholemounts and immunolabelled for Brn3a, melanopsin (m), Osteopontin (OPN) and the T‐box transcription factor T‐brain‐2 (Tbr2) to identify the following retinal ganglion cell populations: Brn3a+, melanopsin+, α‐like (OPN+), α‐ON sustained RGCs (OPN+Tbr2+), α‐ON transient RGCs (OPN+ Brn3a− Tbr2−) and α‐OFF like (OPN+Brn3a+). The labelled RGCs were quantified automatically (Brn3a) or dotted manually and quantified with a graphic software, and distribution of the different populations were represented on topographical maps.Results: Our studies document that:(i) AOH induces progressive loss of Brn3a+RGCs and a significant but not progressive loss of m+RGCs. Treatment with BDNF afforded significant long‐lasting protection for both RGC populations.(ii) Intravitreal injection of NMDA resulted at 7 days in the abrupt significant loss of Brn3a+RGCs, αRGC and αONs‐RGCs without further progression, whereas αOFF‐RGCs died massively. Both m+RGCs and αONtRGCs appear fully resistant to NMDA‐induced excitotoxicity. Both ITH12657 or DHF protect Brn3a+RGC, αRGCs and αONs‐RGCs, but not αOFF‐RGCs.(iii) IONT and vehicle treatment result in a characteristic rapid loss of Brn3a, melanopsin RGCs, αRGCs, αONs‐RGCs and αOFF‐RGCs. DHFs protect Brn3a + RGC, m + RGCs, αRGCs and αONs‐RGCs, but not αOFF‐RGCs.Conclusions: Thus, three different RGC populations (Brn3a+, m+ and α‐like) respond distinctly to different injuries and neuroprotective agents, further adding to the idea that each type of RGC has a unique response to injury and protection. Deciphering the molecular mechanisms of each RGC type will help understand neuronal responses to injury and its possible prevention.

The lateral habenula (LHb) has received special attention due to its role in modulating motivated behavior, stress response, and rewarding and aversive stimuli through monoamine transmission. In the present study, the involvement of the N-methyl-d-aspartate (NMDA) receptors of the LHb in the expression and acquisition phases of morphine-induced conditioned place preference (CPP) was studied in male rats. Bilateral injections of agonist/antagonist (MK-801) of NMDA receptor were performed during the conditioning sessions of the acquisition phase. In other separate groups, drugs were also injected into the LHb before the test session during the expression phase of CPP. A 5-day CPP bias paradigm was used to study the effect of injections of NMDA and MK-801 into the LHb on morphine reward-related behavior. Different doses of NMDA plus morphine reduced the CPP score during the acquisition phase, whereas MK-801 significantly increased conditioning scores during the acquisition phase of CPP. The injection of agonists and antagonists of NMDA receptors in LHb had no significant effect on CPP scores and locomotion during the expression phase of CPP, whereas the motor activity in the acquisition phase was affected by the drugs. The reduction effect of NMDA on the CPP scores during the acquisition phase was blocked by pretreatment with MK-801. Our findings also suggest that NMDA receptors in the LHb may be involved in the acquisition phase of morphine-induced CPP.

Neuroprotection by trans-resveratrol in rats with N-methyl-D-aspartate (NMDA)–induced retinal injury: Insights into the role of adenosine A1 receptors

The β-cell dedifferentiation is one of the critical mechanisms in diabetic β-cell loss. Long-term activation of N-methyl-D-aspartate (NMDA) receptors plays an essential role in the development of diabetes, but the underlying mechanisms have not been fully elucidated. This study aims to investigate the effect of prolonged activation of NMDA receptors on islet β-cell dedifferentiation. Male C57BL/6 mice were randomly divided into a normal control group (control group) and an NMDA group. The mice in the NMDA group were intraperitoneally injected with NMDA (8 mg/kg body weight) and those in the control group were injected with the same volume of saline every day for 6 months. At the end of the 6 th month, glucose tolerance and enzyme linked immunosorbent assay (ELISA) were used to detect the function of islets, and pancreatic tissues were taken for immunofluorescence staining to detect the expressions of insulin, glucagon, and proliferating cell nuclear antigen (PCNA). Real-time PCR was used to detect the mRNA expression of pancreatic β cells, α cells, and islet progenitor cell markers.The primary islets were treated with NMDA to observe the effect of NMDA on the dedifferentiation of β cells. The nuclear factor kappa-B (NF-κB) inhibitor BAY 11-7082 was used at the cellular level via detecting insulin secretion and the expression of endocrine cell markers. Compared with the control group, the mice in the NMDA group had higher blood glucose levels at each time point after glucose injection, and the area under the glucose tolerance curve was significantly increased ( P <0.05). The serum insulin content and insulin stimulatory index of the mice in the NMDA group were significantly lower than those in the control group at 30 min after glucose injection (both P <0.05). The double immunofluorescence staining for insulin and glucagon showed that the number of insulin-positive β cells in the pancreatic tissues of mice was significantly decreased after intraperitoneal injection of NMDA in mice for 6 months, while the number of glucagon-positive α cells was significantly increased. Real-time PCR results showed that β-cell markers ( Insulin , Pdx1 , Neurod1 , and Mafa ) were significantly down-regulated in mouse pancreatic tissues after intraperitoneal injection of NMDA for 6 months, while pancreatic progenitor cell markers ( Neurog3 , Gata6 , Hnf4a , Notch1, and Hes1 ) were significantly down-regulated; α-cell markers ( Glucagon , Arx , Irx2 , Mafb , Pou6f2 , Fev , Kcnj3, and Sv2b ) were significantly up-regulated. NMDA treatment of mouse primary islets for 48 h cause significant down-regulation of β-cell marker gene expression ( P <0.05 or P <0.01), accompanied by significant up-regulation of pancreatic progenitor cell markers and α-cell markers ( P <0.05, P <0.01 or P <0.001). The NF-κB inhibitor BAY 11-7082 significantly blocked the down-regulation of β-cell marker expression (all P <0.05) and the up-regulation of α-cell and pancreatic progenitor cell marker after NMDA treatment of islets for 48 h ( P <0.05 or P <0.01). Prolonged activation of NMDA receptors induces islet β-cell dedifferentiation via regulating the NF-κB pathway.

Early detection of such retinal diseases as glaucoma and age-related macular degeneration (AMD) is important to prevent blindness. There have been reports of changes in some components in the tears of glaucoma and AMD patients, suggesting tears' potential usefulness in screening for retinal diseases. We hypothesized that retinal damage might alter gene expression in the lacrimal gland, leading to those changes in tear components. We caused retinal damage in mice by intravitreal injection of N-methyl-d-aspartate (NMDA) or excessive light exposure. Hematoxylin and eosin staining showed no histological changes in the lacrimal glands of animals whose retinas had been damaged. However, RNA sequencing of lacrimal glands on the 3rd day after NMDA injection or light exposure revealed changes in the expression of 491 genes (268 up-regulated; 223 down-regulated) in the NMDA group and 531 genes (311 up-regulated; 220 down-regulated) in the light group. Further gene-set enrichment analysis indicated that both types of retinal damage activated the immune system in the lacrimal glands. This is the first demonstration that retinal damage can alter gene expression in the lacrimal glands, and it might lead to a novel non-invasive screening method for early detection of retinal diseases.

Neuroprotective role of sphingolipid rheostat in excitotoxic retinal ganglion cell death

The intrathecal (i.t.) injection of substance P (SP) and N-methyl-D-aspartate (NMDA) induce transient nociceptive response by activating neurokinin (NK) 1 and NMDA receptors, respectively. We have recently reported that angiotensin (Ang) (1-7), an N-terminal fragment of Ang II, could alleviate several types of pain including neuropathic and inflammatory pain by activating spinal MAS1. Here, we investigated whether Ang (1-7) can inhibit the SP- and NMDA-induced nociceptive response. The nociceptive response induced by an i.t. injection of SP or NMDA was assessed by measuring the duration of hindlimb scratching directed toward the flank, biting and/or licking of the hindpaw or the tail for 5 min. Localization of MAS1 and either NK1 or NMDA receptors in the lumbar superficial dorsal horn was determined by immunohistochemical observation. The nociceptive response induced by SP and NMDA was attenuated by the i.t. co-administration of Ang (1-7) (0.03-3 pmol) in a dose-dependent manner. The inhibitory effects of Ang (1-7) (3 pmol) were attenuated by A779 (100 pmol), a MAS1 antagonist. Moreover, immunohistochemical analysis showed that spinal MAS1 co-localized with NK1 receptors and NMDA receptors on cells in the dorsal horn. Taken together, the i.t. injection of Ang (1-7) attenuated the nociceptive response induced by SP and NMDA via spinal MAS1, which co-localized with NK1 and NMDA receptors. Thus, the spinal Ang (1-7)/MAS1 pathway could represent a therapeutic target to effectively attenuate spinal pain transmission caused by the activation of NK1 or NMDA receptors.

Magnesium acetyltaurate (MgAT) has been shown to have a protective effect against N-methyl-D-aspartate (NMDA)-induced retinal cell apoptosis. The current study investigated the involvement of nuclear factor kappa-B (NF-κB), p53 and AP-1 family members (c-Jun/c-Fos) in neuroprotection by MgAT against NMDA-induced retinal damage. In this study, Sprague-Dawley rats were randomized to undergo intravitreal injection of vehicle, NMDA or MgAT as pre-treatment to NMDA. Seven days after injections, retinal ganglion cells survival was detected using retrograde labelling with fluorogold and BRN3A immunostaining. Functional outcome of retinal damage was assessed using electroretinography, and the mechanisms underlying antiapoptotic effect of MgAT were investigated through assessment of retinal gene expression of NF-κB, p53 and AP-1 family members (c-Jun/c-Fos) using reverse transcription-polymerase chain reaction. Retinal phospho-NF-κB, phospho-p53 and AP-1 levels were evaluated using western blot assay. Rat visual functions were evaluated using visual object recognition tests. Both retrograde labelling and BRN3A immunostaining revealed a significant increase in the number of retinal ganglion cells in rats receiving intravitreal injection of MgAT compared with the rats receiving intravitreal injection of NMDA. Electroretinography indicated that pre-treatment with MgAT partially preserved the functional activity of NMDA-exposed retinas. MgAT abolished NMDA-induced increase of retinal phospho-NF-κB, phospho-p53 and AP-1 expression and suppressed NMDA-induced transcriptional activity of NF-κB, p53 and AP-1 family members (c-Jun/c-Fos). Visual object recognition tests showed that MgAT reduced difficulties in recognizing the visual cues (i.e. objects with different shapes) after NMDA exposure, suggesting that visual functions of rats were relatively preserved by pre-treatment with MgAT. In conclusion, pre-treatment with MgAT prevents NMDA induced retinal injury by inhibiting NMDA-induced neuronal apoptosis via downregulation of transcriptional activity of NF-κB, p53 and AP-1-mediated c-Jun/c-Fos. The experiments were approved by the Animal Ethics Committee of Universiti Teknologi MARA (UiTM), Malaysia, UiTM CARE No 118/2015 on December 4, 2015 and UiTM CARE No 220/7/2017 on December 8, 2017 and Ethics Committee of Belgorod State National Research University, Russia, No 02/20 on January 10, 2020.

The World Health Organization and the French Health Safety Agency (ANSES) recognize that the expressed pain and suffering of electromagnetic field hypersensitivity syndrome (EHS) people are a lived reality requiring daily life adaptations to cope. Mechanisms involving glutamatergic N-methyl d-aspartate (NMDA) receptors were not explored yet, despite their possible role in hypersensitivity to chemicals. Here, we hypothesized that radiofrequency electromagnetic field (RF-EMF) exposures may affect pain perception under a modulatory role played by the NMDA receptor. The rats were exposed to RF-EMF for four weeks (five times a week, at 0 (sham), 1.5 or 6 W/kg in restraint) or were cage controls (CC). Once a week, they received an NMDA or saline injection before being scored for their preference between two plates in the two-temperatures choice test: 50 °C (thermal nociception) versus 28 °C. Results in the CC and the sham rats indicated that latency to escape from heat was significantly reduced by −45% after NMDA, compared to saline treatment. Heat avoidance was significantly increased by +40% in the 6 W/kg, compared to the sham exposed groups. RF-EMF effect was abolished after NMDA treatment. In conclusion, heat avoidance was higher after high brain-averaged specific absorption rate, affording further support for possible effect of RF-EMF on pain perception. Further studies need to be performed to confirm these data.

PurposeGlaucoma is a group of chronic optic neuropathies characterized by the degeneration of retinal ganglion cells (RGCs) and their axons, and they ultimately cause blindness. Because neuroprotection using neurotrophic factors against RGC loss has been proven a beneficial strategy, extensive attempts have been made to perform gene transfer of neurotrophic proteins. This study used the inner retinal injury mouse model to evaluate the neuroprotective effect of tyrosine triple mutated and self-complementary adeno-associated virus (AAV) encoding brain-derived neurotrophic factor (BDNF; tm-scAAV2-BDNF).MethodsC57BL/6J mice were intravitreally injected with 1 μl of tm-scAAV2-BDNF and its control AAV at a titer of 6.6 E+13 genome copies/ml. Three weeks later, 1 μl of 2 mM N–methyl-D-aspartate (NMDA) was administered in the same way as the viral injection. Six days after the NMDA injection, we assessed the dark-adapted electroretinography (ERG). Mice were sacrificed at one week after the NMDA injection, followed by RNA quantification, protein detection, and histopathological analysis.ResultsThe RNA expression of BDNF in retinas treated with tm-scAAV2-BDNF was about 300-fold higher than that of its control AAV. Meanwhile, the expression of recombinant BDNF protein increased in retinas treated with tm-scAAV2-BDNF. In addition, histological analysis revealed that tm-scAAV2-BDNF prevented thinning of the inner retina. Furthermore, b-wave amplitudes of the tm-scAAV2-BDNF group were significantly higher than those of the control vector group. Histopathological and electrophysiological evaluations showed that tm-scAAV2-BDNF treatment offered significant protection against NMDA toxicity.ConclusionsResults showed that tm-scAAV2-BDNF-treated retinas were resistant to NMDA injury, while retinas treated with the control AAV exhibited histopathological and functional changes after the administration of NMDA. These results suggest that tm-scAAV2-BDNF is potentially effective against inner retinal injury, including normal tension glaucoma.

The ketone bodies (KB), β-hydroxybutyrate (BHB) and acetoacetate, have been proposed for the treatment of acute and chronic neurological disorders, however, the molecular mechanisms involved in KB protection are not well understood. KB can substitute for glucose and support mitochondrial metabolism increasing cell survival. We have reported that the D-isomer of BHB (D-BHB) stimulates autophagic degradation during glucose deprivation in cultured neurons increasing cell viability. Autophagy is a lysosomal degradation process of damaged proteins and organelles activated during nutrient deprivation to obtain building blocks and energy. However, impaired or excessive autophagy can contribute to neuronal death. The aim of the present study was to test whether D-BHB can preserve autophagic function in an in vivo model of excitotoxic damage induced by the administration of the glutamate receptor agonist, N-methyl-Daspartate (NMDA), in the rat striatum. D-BHB was administered through an intravenous injection followed by either an intraperitoneal injection (i.v+i.p) or a continuous epidural infusion (i.v+pump), or through a continuous infusion of D-BHB alone. Changes in the autophagy proteins ATG7, ATG5, BECLIN 1 (BECN1), LC3, Sequestrosome1/p62 (SQSTM1/ p62) and the lysosomal membrane protein LAMP2, were evaluated by immunoblot. The lesion volume was measured in cresyl violet-stained brain sections. Autophagy is activated early after NMDA injection but autophagic degradation is impaired due to the cleavage of LAMP2. Twenty-four h after NMDA intrastriatal injection, the autophagic flux is re-established, but LAMP2 cleavage is still observed. The administration of D-BHB through the i.v+pump protocol reduced the content of autophagic proteins and the cleavage of LAMP2, suggesting decreased autophagosome formation and lysosomal membrane preservation, improving autophagic degradation. D-BHB also reduced brain injury. The i.v+i.p administration protocol and the infusion of D-BHB alone showed no effect on autophagy activation or degradation.

Objective To investigate the protective effects of different concentrations of chloroquine on RGC in n-methyl-d-aspartate (NMDA) injured mice and its possible mechanisms. Methods Fifty-four healthy male C57/BL6 mice were randomly divided into three groups, 18 in each group. The mice in low-dose chloroquine group were intraperitoneally injected with chloroquine solution at a dose of 10 mg/kg daily. Mice in high-dose chloroquine group were intraperitoneally injected with chloroquine solution at a dose of 100 mg/kg, and the mice in control group were intraperitoneally injected with the same volume of PBS. NMDA intravitreal injection was performed 2 days after intraperitoneal injection, 5 nmoles NMDA was injected into the left eye, and the same volume of PBS was injected into the right eye as a control. The RGC staining of retinal plaques were performed 7 days after NMDA injection, and the number of alive RGC was calculated. The visual acuity and electroretinogram were used to evaluate the electrophysiological functions of RGC at 9 and 10 days after modeling. Real-time quantitative PCR and retinal frozen sections and glial fibrillary acidic protein (GFAP) immunofluorescence staining were performed 11 days after NMDA injection to evaluate the glial activation of the retina. The density, visual acuity, and the amplitude of PhNR-wave of RGC between groups were compared by one-way analysis of variance. Results At 7 days after NMDA injection, the density of RGC in retinal patch of low-dose chloroquine group was significantly higher than that of intraperitoneal injection of PBS control group (F=54.41, P 0.05). The visual acuity was higher than control group, and the difference was statistically significant (F=9.10, P<0.05). The amplitude of PhNR-wave was significantly higher in low-dose chloroquine group than that of the control group (F=17.60, P<0.01). The mRNA level of inflammatory factor and GFAP positive signal was also significantly lower than that of the control group (F=23.66, P<0.05). The amplitude of PhNR-wave, the expression of GFAP (F=110.20, P<0.01) and the mRNA level of inflammatory factors (F=167.60, 17.78; P<0.01) in the high-dose chloroquine group were higher than the other two groups, and the differences were statistically significant. Conclusions In NMDA injury retinal model, low-dose chloroquine significantly increased the survival and physiological function of RGC, and the mechanism may be related to the inhibition of glial activation and inflammatory response. High-dose of chloroquine would aggravate the apoptosis of RGC. Key words: Chloroquine; Retinal ganglion cells; N-methylaspartate; Toxic actions; Animal experimentation

Neuronostatin, a newly identified anorexigenic peptide, is present in the central nervous system. We tested the hypothesis that neuronostatin neurons are activated by feeding as a peripheral factor and that the glutamatergic system has regulatory influences on neuronostatin neurons. The first set of experiments analyzed the activation of neuronostatin neurons by refeeding as a physiological stimulus and the effectiveness of the glutamatergic system on this physiological stimulation. The subjects were randomly divided into three groups: the fasting group, refeeding group, and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)+refeeding group. We found that refeeding increased the phosphorylated signal transducers and transcription activator-5 (pSTAT5) expression in neuronostatin-positive neurons and that the CNQX injection significantly suppressed the number of pSTAT5-expressing neuronostatin neurons. The second set of experiments analyzed the activation pathways of neuronostatin neurons and the regulating effects of the glutamatergic system on neuronostatin neurons. The animals received intraperitoneal injections of glutamate receptor agonists (kainic acid, α-amino-3-hydroxy-5methyl-4-isoazepropionic acid (AMPA), and N-methyl-D-aspartate (NMDA)) or 0.9% NaCl. The number of c-Fos-expressing neuronostatin neurons significantly increased following the AMPA and NMDA injections. In conclusion, we found that the neuronostatin neurons were activated by peripheral or central signals, including food intake and/or glutamatergic innervation, and that the glutamate receptors played an important role in this activation.

Combined administration of N-Methyl-D-Aspartate (NMDA) and kainic acid (KA) on the inner retina was studied as a model of excitotoxicity. The right eye of C57BL6J mice was injected with 1 µL of PBS containing NMDA 30 mM and KA 10 mM. Only PBS was injected in the left eye. One week after intraocular injection, electroretinogram recordings and immunohistochemistry were performed on both eyes. Retinal ganglion cell (RGC) projections were studied by fluorescent-cholerotoxin anterograde labeling. A clear decrease of the retinal “b” wave amplitude, both in scotopic and photopic conditions, was observed in the eyes injected with NMDA/KA. No significant effect on the “a” wave amplitude was observed, indicating the preservation of photoreceptors. Immunocytochemical labeling showed no effects on the outer nuclear layer, but a significant thinning on the inner retinal layers, thus indicating that NMDA and KA induce a deleterious effect on bipolar, amacrine and ganglion cells. Anterograde tracing of the visual pathway after NMDA and KA injection showed the absence of RGC projections to the contralateral superior colliculus and lateral geniculate nucleus. We conclude that glutamate receptor agonists, NMDA and KA, induce a deleterious effect of the inner retina when injected together into the vitreous chamber.

Purpose: To investigate whether and how leukemia inhibitory factor (Lif) is involved in mediating the neuroprotective effects of Norrin on retinal ganglion cells (RGC) following excitotoxic damage. Norrin is a secreted protein that protects RGC from N-methyl-d-aspartate (NMDA)-mediated excitotoxic damage, which is accompanied by increased expression of protective factors such as Lif, Edn2 and Fgf2. Methods: Lif-deficient mice were injected with NMDA in one eye and NMDA plus Norrin into the other eye. RGC damage was investigated and quantified by TUNEL labeling 24 h after injection. Retinal mRNA expression was analyzed by quantitative real-time polymerase chain reaction following retinal treatment. Results: After intravitreal injection of NMDA and Norrin in wild-type mice approximately 50% less TUNEL positive cells were observed in the RGC layer when compared to NMDA-treated littermates, an effect which was lost in Lif-deficient mice. The mRNA expression for Gfap, a marker for Müller cell gliosis, as well as Edn2 and Fgf2 was induced in wild-type mice following NMDA/Norrin treatment but substantially blocked in Lif-deficient mice. Conclusions: Norrin mediates its protective properties on RGC via Lif, which is required to enhance Müller cell gliosis and to induce protective factors such as Edn2 or Fgf2.

This study was aimed to investigate morphological alteration of the retina with N-methyl-D-aspartate (NMDA)-induced injury in rabbits by optical coherence tomography (OCT). The right and left eyes of a total of 12 rabbits received single-intravitreal injection of vehicle and NMDA, respectively. Four out of the 12 animals underwent OCT and quantification of plasma microRNA repeatedly (4, 48, and 168 hr after dosing), followed by ocular histopathology at the end of the study. Ocular histopathology was also conducted in the eyes collected 4 or 48 hr after dosing from 4 animals at each time period. OCT revealed hyper-reflective ganglion cell complex and thickened inner retina in NMDA-treated eyes 4 hr after dosing; the inner retina shifted to thinning at later time points. The eyes given NMDA also exhibited greater thickness of the outer retina, which contains photoreceptors, after treatment, and thickened and obscured ellipsoid zone 168 hr after dosing. The plasma levels of miR-182 and miR-183, which are known to be highly expressed in photoreceptors, were higher 4 hr after dosing than pre-dosing values. Histopathologically, NMDA-induced inner retinal damage was confirmed: single-cell necrosis was observed in the ganglion cell layer and the inner nuclear layer 4 hr after dosing, the incidence of which decreased thereafter. At 168 hr after dosing, reduced number of ganglion cells was noted. No change was histopathologically observed in the outer retina. In conclusion, our results suggest involvement of photoreceptors in NMDA-induced inner retinal injury. Additionally, OCT revealed acute inner retinal findings suggestive of temporary edema.

Glutamate excitotoxicity via N-methyl-D-aspartate (NMDA) receptors is thought to be a factor involved in the loss of retinal neuronal cells, including retinal ganglion cells, in retinal diseases such as diabetic retinopathy and acute angle closure glaucoma. Herein we report the protective effect of systemic administration of ML233, an apelin receptor agonist, against retinal neuronal cell death induced by the intravitreal injection of NMDA into mice. Intraperitoneal administration of ML233 prevented the NMDA-induced reduction in the amplitude of scotopic threshold responses (STR), which mainly reflect the activity of the retinal ganglion cells. Immunohistochemical staining showed that ML233 inhibited the NMDA-induced loss of retinal ganglion cells and amacrine cells. In addition, ML233 suppressed the breakdown of spectrin αII, a neuronal cytoskeleton protein cleaved by calpain activation, in the retina after intravitreal injection of NMDA. Intraperitoneal administration of ML233 increased the phosphorylation of Akt, a potent anti-apoptotic protein in neurons, in the retina. Furthermore, oral administration of ML233 protected against the decrease in the STR amplitudes and the loss of retinal ganglion cells caused by NMDA. These results suggest that systemic administration of ML233 protected retinal neurons from NMDA receptor-mediated excitotoxicity and that drugs activating the apelin receptor may be a new candidate for preventing the progression of these retinal diseases.

Abstract PurposeTo study short and long term responses of the population of RGCs expressing Brn3a (Brn3a+RGCs) and the population of intrinsically photosensitive RGCs expressing melanopsin (m+RGCs) to excitotoxicity induced by intravitreal injection of N‐methyl‐D‐Aspartate (NMDA).MethodsIn adult albino Sprague Dawley rats, the left eye received an intraocular injection of 100nM NMDA and the retinas were analyzed 3 (n=9), 7 (n=6), 14 (n=10) days (d) or 15 (n=23) months (m) later. Spectral Domain Optical Coherence Tomography (SD‐OCT; Spectralis, Heidelberg) was used to image and analyze retinal thickness longitudinally in vivo at short (3 m; n=18) and long (15 m; n=18) survival intervals. Ex vivo, retinal whole mounts were immunodetected with antibodies against Brn3a and melanopsin and the numbers of surviving Brn3a+RGCs and of m+RGCs were automatically counted and represented using isodensity or neighbour maps.ResultsIntraocular injection of 100nM NMDA causes a massive loss of Brn3a+RGCs; at 3 and 14 days Brn3a+RGCs numbers diminished to 50% and 25%respectively, but there was no further loss up to 15 months. However, m+RGCs showed 3 d after injection a transient downregulation of melanopsin that recovered shortly and by 14 d and at the end of the study (15 m) the numbers of m+RGCs were comparable to their contralateral fellow eyes. SD‐OCT examination showed important reductions of the total and inner retinal thicknesses at 3 m that progressed further up to 15 m.ConclusionThe population of Brn3a+RGCs is quite sensitive to NMDA‐induced excitotoxicity that causes rapidly the loss of approximately 75% of these neurons. In contrast, m+RGCs appear fully resistant to NMDA‐induced excitotoxicity and show only a downregulation of melanopsin expression during the first two weeks. SD‐OCT can be used to assess retinal toxicity revealed by important reductions in total and inner retinal thickness at 3 m progressing up to 15 m.