Intravitreal Injection Of N-methyl-D-aspartate Research Articles

Early inner plexiform layer thinning and retinal nerve fiber layer thickening in excitotoxic retinal injury using deep learning-assisted optical coherence tomography

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.

Idiosyncratic responses of RGCs to injury and protection

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.

Ferroptosis and glaucoma: implications in retinal ganglion cell damage and optic nerve survival.

Ferroptosis and glaucoma: implications in retinal ganglion cell damage and optic nerve survival.

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.

Magnesium acetyltaurate prevents retinal damage and visual impairment in rats through suppression of NMDA-induced upregulation of NF-κB, p53 and AP-1 (c-Jun/c-Fos).

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.

Norrin Protects Retinal Ganglion Cells from Excitotoxic Damage via the Induction of Leukemia Inhibitory Factor.

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.

Systemic Administration of an Apelin Receptor Agonist Prevents NMDA-Induced Loss of Retinal Neuronal Cells in Mice

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.

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.

Neuroprotective effect of a dietary supplement against glutamate-induced excitotoxicity in retina.

To evaluate the neuroprotective effect of a dietary supplement (ClearVision EX®; CV) against glutamate-induced excitotoxicity in retina. We evaluated the protective effects CV on glutamate-induced cell toxicity of an immortalized mouse hippocampal cell line (HT-22) in vitro and N-methyl-D-aspartate (NMDA) induced retinal injury in vivo. Once-daily oral administration of CV or vehicle (5% Arabic gum) was started the day before the NMDA injection and continued until the end of the study. Electroretinograms (ERGs) were recorded to evaluate the retinal function at 2d after NMDA injection. Furthermore, a histological evaluation, Western blot analysis, and immunohistochemistry were performed for assessing the signal transduction pathway. HT-22 cell death was induced by the addition of glutamate and co-incubation with CV protected against it. Oral administration of CV inhibited the decrease in scotopic threshold response amplitudes induced by the intravitreal injection of NMDA and those of the thickness of the inner retinal layer in the histological evaluation. The increased phosphorylated levels of extracellular signal-regulated kinase (ERK) but not cAMP response element binding protein (CREB) or Akt were observed 1h after NMDA injection in both the vehicle- and CV-treated rats; however, pERK activation was no more upregulated at 3h after NMDA injection. pERK upregulation was observed in Müller cells. CV shows a protective effect against both glutamate-induced HT-22 cell death and NMDA-induced retinal damage. pERK upregulation in the Müller cells plays a key role in the protective effect of CV against glutamate-induced retinal toxicity.

Dose-dependent effects of NMDA on retinal and optic nerve morphology in rats.

To investigate dose-dependent effects of N-methyl-D-aspartate (NMDA) on retinal and optic nerve morphology in rats. Sprague Dawley rats, 180-250 g in weight were divided into four groups. Groups 1, 2, 3 and 4 were intravitreally administered with vehicle and NMDA at the doses 80, 160 and 320 nmol respectively. Seven days after injection, rats were euthanized, and their eyes were taken for optic nerve toluidine blue and retinal hematoxylin and eosin stainings. The TUNEL assay was done for detecting apoptotic cells. All groups treated with NMDA showed significantly reduced ganglion cell layer (GCL) thickness within inner retina, as compared to control group. Group NMDA 160 nmol showed a significantly greater GCL thickness than the group NMDA 320 nmol. Administration of NMDA also resulted in a dose-dependent decrease in the number of nuclei both per 100 µm GCL length and per 100 µm2 of GCL. Intravitreal NMDA injection caused dose-dependent damage to the optic nerve. The degeneration of nerve fibres with increased clearing of cytoplasm was observed more prominently as the NMDA dose increased. In accordance with the results of retinal morphometry analysis and optic nerve grading, TUNEL staining demonstrated NMDA-induced excitotoxic retinal injury in a dose-dependent manner. Our results demonstrate dose-dependent effects of NMDA on retinal and optic nerve morphology in rats that may be attributed to differences in the severity of excitotoxicity and oxidative stress. Our results also suggest that care should be taken while making dose selections experimentally so that the choice might best uphold study objectives.

Iron-chelating agents attenuate NMDA-Induced neuronal injury via reduction of oxidative stress in the rat retina

Excitoneurotoxicity is regarded as one of the mechanisms of the death of retinal ganglion cells induced by retinal central artery occlusion and glaucoma. Oxidative stress is at least in part involved in excitoneurotoxicity. Fenton reaction, which is catalyzed by Fe2+, is known to cause formation of hydroxyl radical, one of reactive oxygen species, suggesting that chelation of iron may be protective against excitoneurotoxicity. In the present study, we histologically evaluated whether zinc-deferoxamine (Zn-DFO) and deferasirox (DFX), common iron-chelating agents, were protective against N-methyl-D-aspartate (NMDA)-induced retinal injury in the rat in vivo. Male Sprague–Dawley rats were subjected to intravitreal NMDA injection (200 nmol/eye). Zn-DFO (1, 3, 10, and 30 mg/kg), Zn (0.1, 0.2 and 0.6 mg/kg) and DFX (20 mg/kg) were intraperitoneally administered. Morphometric evaluations using paraffin-embedded retinal sections, and detection of Fe2+ using SiRhoNox-1, a fluorescent probe of labile Fe2+ in the retinal frozen sections were carried out. Intravitreal NMDA resulted in strong positive signals of SiRhoNox-1 in the ganglion cell layer 24 h after NMDA injection, suggesting that intravitreal NMDA caused Fe2+ accumulation in the retinal ganglion cells. Intravitreal NMDA induced retinal ganglion cell loss 7 days after NMDA injection. Zn-DFO (1, 3, 10, and 30 mg/kg), ZnCl2 (0.2 mg/kg, a corresponding dose of 1 mg/kg Zn-DFO) and DFX (20 mg/kg) prevented the damage of retinal ganglion cells, whereas 0.6 mg/kg ZnCl2, which is a corresponding dose of 3 mg/kg Zn-DFO, did not show any protective effects. Zn-DFO (30 mg/kg) significantly decreased the intensity of the fluorescence of SiRhoNox-1 and the transferrin immunofluorescence 24 h after NMDA injection, the number of TUNEL-positive cells 24 h after NMDA injection, that of 8-OHdG-positive cells, and that of 4-hydroxy-2-nonenal-positive cells 12 and 24 h after NMDA injection. These data suggest that iron-chelating agents protected retinal neurons against excitoneurotoxicity via reduction of iron content and oxidative stress in the rats in vivo. We proposed that treatment with iron-chelating agents would be a new strategy for the retinal diseases caused by excitoneurotoxicity.

The Relationship Between the Renin-Angiotensin-Aldosterone System and NMDA Receptor-Mediated Signal and the Prevention of Retinal Ganglion Cell Death.

Excitotoxicity, which is due to glutamate-induced toxic effects on the retinal ganglion cell (RGC), is one of several mechanisms of RGC loss. The renin-angiotensin-aldosterone system (RAAS) has also been implicated in RGC death. Therefore, it is important to determine the exact relationship between the RAAS and N-methyl-d-aspartate (NMDA) receptor-mediated signal in order to prevent RGC death. N-methyl-d-aspartate or aldosterone was injected into the vitreous body. After intravitreal injection of NMDA or aldosterone, animals were treated with spironolactone or memantine. Retinal damage was evaluated by measuring the number of RGCs at 4 weeks after local administration of aldosterone or at 2 weeks after local administration of NMDA. Vitreous humor levels of aldosterone were measured using enzyme immunoassay kits. A significantly decreased number of RGCs were observed after intravitreal injection of NMDA. Although spironolactone did not show any neuroprotective effects, memantine significantly reduced NMDA-induced degeneration in the retina. Furthermore, a significant decrease in the number of RGCs was observed after an intravitreal injection of aldosterone. While memantine did not exhibit any neuroprotective effects, spironolactone caused a significant reduction in the aldosterone-induced degeneration in the retina. There was no change in the aldosterone concentration in the vitreous humor after an NMDA injection. Our findings indirectly show that there is no relationship between the RAAS and NMDA receptor-mediated signal with regard to RGC death.

Alteration of Neurotrophic Factors After Transplantation of Bone Marrow Derived Lin-ve Stem Cell in NMDA-Induced Mouse Model of Retinal Degeneration.

Retinal ganglion cell layer (RGCs) is one of the important layers of retina, depleted in Glaucoma. Loss of RGC neurons is a major cellular mechanism involved in its pathogenesis resulting in severe vision loss. Stem cell therapy has emerged as a potential strategy to arrest the apoptotic loss of RGCs and also replace the degenerative cells in damaged retina. Here, we have investigated the incorporation and survival of mouse bone marrow derived Lin-ve stem cells in N-methyl-d-aspartate (NMDA)-induced mouse model of retinal degeneration. Two days after intravitreal injection of NMDA (100 mM) showed significant decrease in ganglion cell number and increase in TUNEL positive apoptotic cells in retinal layers. The injury was further characterized by immunohistochemical expression of Brn3b, GFAP, Bcl2, pCREB, CNTF, GDNF, and BDNF in retinal layers. Lin-ve cells (100,000 dose) were intravitreally transplanted after 2 days of injury and evaluated after 7, 14, and 21 days of transplantation. Transplanted cells were found to have migrated from intravitreal space and incorporated into injured retina at 7, 14, and 21 days post-transplantation. At 21 days Brn3b, CNTF, and BDNF expression was found to be upregulated whereas GDNF was downregulated when compared to respective injury time points. Molecular data showed decrease in the expression of Brn3b, BDNF, CNTF, and GDNF post transplantation when compared with injury groups. This study reveals that Lin-ve stem cells may exert neuroprotective effect in damaged retina mediated by participation of neurotrophic factors induced by stem cell transplantation at the site of injury. J. Cell. Biochem. 118: 1699-1711, 2017. © 2016 Wiley Periodicals, Inc.

Apelin protects against NMDA-induced retinal neuronal death via an APJ receptor by activating Akt and ERK1/2, and suppressing TNF-α expression in mice

Glutamate excitotoxicity mediated by N-methyl-d-aspartate (NMDA) receptors is an important cause of retinal ganglion cell death in glaucoma. To elucidate whether apelin protects against retinal neuronal cell death, we examined protective effects of exogenous and endogenous apelin on neuronal cell death induced by intravitreal injection of NMDA in the retinas of mice. An intravitreal injection of NMDA induced neuronal cell death in both the retinal ganglion cell layer and inner nuclear layer, and reduced the amplitudes of scotopic threshold response (STR) in electroretinography studies. Both cell death and STR amplitudes decrease induced by NMDA were prevented by a co-injection of [Pyr1]-apelin-13, and were facilitated by apelin deficiency. The neuroprotective effects of [Pyr1]-apelin-13 were blocked by an apelin receptor APJ antagonist, and by inhibitors of Akt and extracellular signal-regulated kinase 1/2 signaling pathways. Additionally, an intravitreal injection of tumor necrosis factor-α (TNF-α) neutralizing antibody prevented NMDA-induced retinal neuronal cell death, and exogenous and endogenous apelin suppressed NMDA-induced upregulation of TNF-α in the retina. These results suggest that apelin protects neuronal cells against NMDA-induced death via an APJ receptor in the retina, and that apelin may have beneficial effects in the treatment of glaucoma.

Protective effect of magnesium acetyltaurate against NMDA-induced retinal damage involves restoration of minerals and trace elements homeostasis

Glutamate-mediated excitotoxicity involving N-methyl-d-aspartate (NMDA) receptors has been recognized as a final common outcome in pathological conditions involving death of retinal ganglion cells (RGCs). Overstimulation of NMDA receptors results in influx of calcium (Ca) and sodium (Na) ions and efflux of potassium (K). NMDA receptors are blocked by magnesium (Mg). Such changes due to NMDA overstimulation are also associated with not only the altered levels of minerals but also that of trace elements and redox status. Both the decreased and elevated levels of trace elements such as iron (Fe), zinc (Zn), copper (Cu) affect NMDA receptor excitability and redox status. Manganese (Mn), and selenium (Se) are also part of antioxidant defense mechanisms in retina. Additionally endogenous substances such as taurine also affect NMDA receptor activity and retinal redox status. Therefore, the aim of this study was to evaluate the effect of Mg acetyltaurate (MgAT) on the retinal mineral and trace element concentration, oxidative stress, retinal morphology and retinal cell apoptosis in rats after-NMDA exposure. One group of Sprague Dawley rats received intravitreal injection of vehicle while 4 other groups similarly received NMDA (160nmolL−1). Among the NMDA injected groups, 3 groups also received MgAT (320nmolL−1) as pre-treatment, co-treatment or post-treatment. Seven days after intravitreal injection, rats were sacrificed, eyes were enucleated and retinae were isolated for estimation of mineral (Ca, Na, K, Mg) and trace element (Mn, Cu, Fe, Se, Zn) concentration using Inductively Coupled Plasma (DRC ICP-MS) techniques (NexION 300D), retinal oxidative stress using Elisa, retinal morphology using H&E staining and retinal cell apoptosis using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Intravitreal NMDA injection resulted in increased concentration of Ca (4.6 times, p<0.0001), Mg (1.5 times, p<0.01), Na (3 times, p<0.0001) and K (2.3 times, p<0.0001) compared to vehicle injected group. This was accompanied with significant increase of Ca/Mg and Na/K ratios, 3 and 1.27 times respectively, compared to control group. The trace elements such as Cu, Fe and Zn also showed a significant increase amounting to 3.3 (p<0.001), 2.3 (p<0.0001) and 3 (p<0.0001) times respectively compared to control group. Se was increased by 60% (p<0.005). Pre-treatment with MgAT abolished effect of NMDA on minerals and trace elements more effectively than co- and post-treatment. Similar observations were made for retinal oxidative stress, retinal morphology and retinal cell apoptosis. In conclusion, current study demonstrated the protective effect of MgAT against NMDA-induced oxidative stress and retinal cell apoptosis. This effect of MgAT was associated with restoration of retinal concentrations of minerals and trace elements. Further studies are warranted to explore the precise molecular targets of MgAT. Nevertheless, MgAT seems a potential candidate in the management of diseases involving NMDA-induced excitotoxicity.

Neuroprotective Effect of Tauroursodeoxycholic Acid on N-Methyl-D-Aspartate-Induced Retinal Ganglion Cell Degeneration.

Retinal ganglion cell degeneration underlies the pathophysiology of diseases affecting the retina and optic nerve. Several studies have previously evidenced the anti-apoptotic properties of the bile constituent, tauroursodeoxycholic acid, in diverse models of photoreceptor degeneration. The aim of this study was to investigate the effects of systemic administration of tauroursodeoxycholic acid on N-methyl-D-aspartate (NMDA)-induced damage in the rat retina using a functional and morphological approach. Tauroursodeoxycholic acid was administered intraperitoneally before and after intravitreal injection of NMDA. Three days after insult, full-field electroretinograms showed reductions in the amplitudes of the positive and negative-scotopic threshold responses, scotopic a- and b-waves and oscillatory potentials. Quantitative morphological evaluation of whole-mount retinas demonstrated a reduction in the density of retinal ganglion cells. Systemic administration of tauroursodeoxycholic acid attenuated the functional impairment induced by NMDA, which correlated with a higher retinal ganglion cell density. Our findings sustain the efficacy of tauroursodeoxycholic acid administration in vivo, suggesting it would be a good candidate for the pharmacological treatment of degenerative diseases coursing with retinal ganglion cell loss.

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.

Impaired retinal vasodilator response to acetylcholine in a rat model of NMDA-induced retinal degeneration

Previous studies have shown that degeneration of retinal capillaries occurs following N-methyl-d-aspartate (NMDA)-induced retinal neurotoxicity, but it is unclear whether vasodilatory mechanisms are altered in retinal blood vessels. The purpose of the present study was to determine whether retinal vasodilator responses are affected in a rat model of NMDA-induced retinal damage. At 14 days after a single intravitreal injection of NMDA (200 nmol), retinal vasodilator responses were assessed by measuring the diameter of retinal arterioles in fundus images. Acetylcholine-induced vasodilation of retinal arterioles was significantly reduced in NMDA-treated retinas, whereas retinal vasodilatory effects of the nitric oxide (NO) donor NOR3, the β2-adrenoceptor agonist salbutamol, and the β3-adrenoceptor agonist CL316243 were unaltered. The vasodilator response to acetylcholine observed under the combined blockade of NO synthase and cyclooxygenase with NG-nitro-l-arginine methyl ester (30 mg/kg, i.v.) plus indomethacin (5 mg/kg, i.v.), possibly an endothelium-derived hyperpolarizing factor-mediated response, was also reduced. These results suggest that endothelium-dependent vasodilatory mechanisms in retinal blood vessels are impaired in the rat model of NMDA-induced retinal degeneration. Glutamate-induced neurotoxicity is implicated in several retinal diseases; therefore, abnormal retinal circulation would contribute to the progression of the diseases.

Preventive effects of rapamycin on inflammation and capillary degeneration in a rat model of NMDA-induced retinal injury.

Inhibitors of the mammalian target of rapamycin (mTOR) have been shown to protect against neuronal injury, but the mechanisms underlying this effect are not fully understood. The present study aimed to examine the effects of rapamycin, an inhibitor of the mTOR pathway, on inflammation and capillary degeneration in a rat model of N-methyl-D-aspartate (NMDA)-induced retinal neurotoxicity. Inflammation and capillary degeneration were evaluated by counting the numbers of CD45-positive leukocytes and Iba1-positive microglia, and by measuring the length of empty basement membrane sleeves, respectively. Marked increases in the numbers of leukocytes and microglia were observed 1 d after intravitreal injection of NMDA (200 nmol), and significant capillary degeneration was observed after 7 d. These NMDA-induced changes were significantly reduced by the simultaneous injection of rapamycin (20 nmol) with NMDA. These results suggest that rapamycin has preventive effects on inflammation and capillary degeneration during retinal injury.

Regression of retinal capillaries following N‐methyl‐D‐aspartate‐induced neurotoxicity in the neonatal rat retina

Degeneration of retinal capillaries occurs following N-methyl-D-aspartate (NMDA)-induced retinal neurotoxicity, and the degree of capillary degeneration decreases in an age-dependent manner. To determine the role of vascular endothelial growth factor (VEGF) in the high susceptibility of capillaries to neuronal damage during the early postnatal stage, this study compares the vascular regression patterns between NMDA-treated retinas and retinas treated with N-[2-chloro-4-{(6,7-dimethoxy-4-quinazolinyl)oxy}phenyl]-N'-propylurea (KRN633), a VEGF receptor tyrosine kinase inhibitor, in neonatal rats. Two days after a single intravitreal injection of NMDA (200 nmol/eye) on postnatal day (P) 7, substantial retinal neuron loss and delayed expansion of the retinal vascular bed were observed. The reduction in the capillary density in the central retina reached statistical significance 4 days after NMDA treatment. In retinas of rats injected subcutaneously with KRN633 (10 mg/kg) on P7 and P8, simplified vasculature attributable to capillary regression and prevention of endothelial cell growth were seen on P9, whereas no visible changes in the morphology of the retinal layers were observed. The degree of capillary degeneration in NMDA-treated retinas was less than that in KRN633-treated retinas. No apparent changes in immunoreactivities for VEGF were found 2 days after NMDA treatment. These results indicate that neuronal cell loss in the retina precedes retinal capillary degeneration following NMDA treatment, and VEGF-dependent immature capillaries might be more susceptible to NMDA-induced neuronal damage.