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

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.

Age-Dependent Changes in the Severity of Capillary Degeneration in Rat Retina Following N-Methyl-D-Aspartate-Induced Neurotoxicity

Purpose: Previous studies have shown that injury to the retinal vasculature, including capillary degeneration, occurs following N-methyl-d-aspartate (NMDA)-induced neuronal cell loss, but it is unclear whether there are age-dependent differences in the severity of vascular damage. The purpose of the present study was to determine age-related changes in retinal capillary degeneration in NMDA-induced retinal damage rat model.Materials and Methods: A single intravitreal injection of NMDA (200 nmol/eye) was performed in 1-, 2-, 3-, 7- and 15-week-old male Sprague–Dawley rats. Seven days after NMDA injection, damage to retinal neurons and blood vessels were assessed by measuring the number of cells in the ganglion cell layer and the length of empty basement membrane sleeves, left behind by regressing endothelial cells, respectively.Results: At all ages examined, the NMDA-induced neurotoxicity exhibited similar levels of ganglion cell death. However, the degeneration of capillaries in NMDA-treated retina was severe during the early stages of retinal vascular development, and the degree of capillary degeneration decreased with age.Conclusion: The degree of retinal capillary degeneration in NMDA-induced retinal damage rat model decreases in an age-dependent manner. Retinal capillaries may be resistant to retinal neuronal damage in young adults.

Neuroprotective effects of C3 exoenzyme in excitotoxic retinopathy

The purpose of this study is to evaluate the neuroprotective effects of C3 exoenzyme (C3) on N-methyl-d-aspartate (NMDA)-induced retinopathy in rats. C3 was expressed in Escherichia. coli and purified by affinity chromatography. Immunofluorescence was performed in NIH 3T3 cells treated with C3 to verify the cellular uptake of the protein. NMDA was injected intravitreally into rat eyes with or without C3. At various time points after injection, eyes were enucleated. Hematoxylin/eosin staining was performed on retina cross-sections for morphological analysis. Survival and apoptosis of cells in the ganglion cell layer (GCL) were assessed by cresyl violet staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) on retina flat-mounts. RhoA levels in retina cells were evaluated by Western blot to detect C3 uptake in vivo. The cellular uptake of C3 was verified by immunofluorescence. Damage including a decrease in inner plexiform layer (IPL) thickness and reduction of cell density in the GCL, corresponding to apoptosis of neurons, was induced by intravitreal injection of NMDA. Protection against this damage was observed following co-injection of C3 and NMDA. RhoA ADP-ribosylation induced by C3 was confirmed by Western blot. Our results suggest that C3 exerts neuroprotective effects against excitotoxic damage induced by NMDA.

Single-Cell Resolution Imaging of Retinal Ganglion Cell Apoptosis In Vivo Using a Cell-Penetrating Caspase-Activatable Peptide Probe

Peptide probes for imaging retinal ganglion cell (RGC) apoptosis consist of a cell-penetrating peptide targeting moiety and a fluorophore-quencher pair flanking an effector caspase consensus sequence. Using ex vivo fluorescence imaging, we previously validated the capacity of these probes to identify apoptotic RGCs in cell culture and in an in vivo rat model of N-methyl- D-aspartate (NMDA)-induced neurotoxicity. Herein, using TcapQ488, a new probe designed and synthesized for compatibility with clinically-relevant imaging instruments, and real time imaging of a live rat RGC degeneration model, we fully characterized time- and dose-dependent probe activation, signal-to-noise ratios, and probe safety profiles in vivo. Adult rats received intravitreal injections of four NMDA concentrations followed by varying TcapQ488 doses. Fluorescence fundus imaging was performed sequentially in vivo using a confocal scanning laser ophthalmoscope and individual RGCs displaying activated probe were counted and analyzed. Rats also underwent electroretinography following intravitreal injection of probe. In vivo fluorescence fundus imaging revealed distinct single-cell probe activation as an indicator of RGC apoptosis induced by intravitreal NMDA injection that corresponded to the identical cells observed in retinal flat mounts of the same eye. Peak activation of probe in vivo was detected 12 hours post probe injection. Detectable fluorescent RGCs increased with increasing NMDA concentration; sensitivity of detection generally increased with increasing TcapQ488 dose until saturating at 0.387 nmol. Electroretinography following intravitreal injections of TcapQ488 showed no significant difference compared with control injections. We optimized the signal-to-noise ratio of a caspase-activatable cell penetrating peptide probe for quantitative non-invasive detection of RGC apoptosis in vivo. Full characterization of probe performance in this setting creates an important in vivo imaging standard for functional evaluation of future probe analogues and provides a basis for extending this strategy into glaucoma-specific animal models.

Rapamycin prevents N‐methyl‐D‐aspartate‐induced retinal damage through an ERK‐dependent mechanism in rats

Recent studies have demonstrated that inhibition of the mammalian target of rapamycin (mTOR) protects against neuronal injury, but the mechanisms underlying this protection are not fully understood. The present study investigates whether rapamycin, an inhibitor of the mTOR pathway, protects against N-methyl-D-aspartate (NMDA)-induced retinal neurotoxicity and whether the extracellular signal-regulated kinase (ERK) pathway contributes to this protective effect in rats. Significant cell loss in the ganglion cell layer and a reduction in thickness of the inner plexiform layer were observed 7 days after a single intravitreal injection of NMDA (200 nmol/eye). These NMDA-induced morphological changes were significantly reduced by rapamycin (20 nmol/eye). The number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive apoptotic cells had increased 6 hr after NMDA injection, an effect that was significantly attenuated by rapamycin. The ERK inhibitor U0126 (1 nmol/eye) almost completely abolished rapamycin's inhibition of NMDA-induced apoptosis. Immunohistochemical studies showed that NMDA caused a time-dependent increase in levels of the phosphorylated form of the ribosomal protein S6 (pS6), a downstream indicator of mTOR activity. The increased pS6 levels were markedly decreased by rapamycin. Both NMDA and rapamycin increased the level of phosphorylated ERK (pERK) in Müller cells, and coinjection of both agents further increased pERK levels. These results suggest that rapamycin has a neuroprotective effect against NMDA-induced retinal neurotoxicity and that this effect could be patially mediated by activation of the ERK pathway in retinal Müller cells.

Hydrogen sulfide attenuates NMDA-induced neuronal injury via its anti-oxidative activity in the rat retina

Although hydrogen sulfide (H2S) is generally thought to be a toxic gas, it has been reported to protect various tissues against ischemia-reperfusion injury. In the present study, we histologically investigated whether H2S, using sodium hydrosulfide (NaHS) as its donor, had a protective effect on N-methyl-d-aspartate (NMDA)-induced retinal injury in the rat in vivo. Under ketamine/xylazine anesthesia, male Sprague–Dawley rats were subjected to intravitreal NMDA injection. NaHS (0.163–120 μmol/kg) was intraperitoneally administered 15 min before NMDA injection. Morphometric evaluation at 7 days after NMDA injection showed that intravitreal NMDA injection resulted in ganglion cell loss. NaHS dose-dependently prevented this damage. NaHS (120 μmol/kg) significantly decreased the numbers of TUNEL-positive, 4-hydroxy-2-nonenal-positive, and 8-OHdG-positive cells 12 h after NMDA injection. In another experimental series, we demonstrated that NaHS (120 μmol/kg) significantly reduced the retinal injury induced by intravitreal NOC12 (400 nmol/eye), which was a nitric oxide donor and reported to induce oxidative stress, in the retina, 7 days after intravitreal injection. These results suggested that H2S protects retinal neurons against the injury induced by intravitreal NMDA in rats in vivo. Anti-oxidative activity of H2S are possibly involved in underlying protective mechanisms.

ISO-1, a macrophage migration inhibitory factor antagonist, prevents N-methyl-d-aspartate-induced retinal damage

Macrophage migration inhibitory factor (MIF) has been shown to play an important role in a variety of inflammatory and immune-mediated diseases. The inflammatory responses contribute to retinal neuronal degeneration. However, the role of MIF in the progression of retinal degeneration has not yet been elucidated. In this study, we determined whether pharmacological inhibition of MIF protects against the retinal damage induced by N-methyl-d-aspartate (NMDA) in rats. Intravitreal injection of NMDA (200nmol) resulted in (1) cell loss in the ganglion cell layer and reduction in the thickness of the inner plexiform layer, (2) an increase in apoptotic cells, (3) a decrease in parvalbumin-positive amacrine cells, (4) accumulation of leukocytes, and (5) microglia activation. Injection of (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1, 100nmol), a MIF antagonist, significantly attenuated these NMDA-induced responses. These findings suggest that ISO-1 exerts protective effects against retinal injuries and that MIF may be a target for neuroprotective intervention in retinal diseases associated with glutamate-induced excitotoxicity.

Differential effects of LY294002 and wortmannin on neurons and vascular endothelial cells in the rat retina

BackgroundNeuronal damage leads to capillary degeneration in an N-methyl-D-aspartate (NMDA)-induced retinal degeneration model; however, the mechanisms underlying this phenomenon are not fully understood. The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway has been recognized as an intracellular pro-survival signaling system. Therefore, we used the PI3K inhibitors LY294002 and wortmannin to investigate the role of this pathway in neuronal and blood vessel injury in the rat retina treated with NMDA. MethodsMale Sprague-Dawley rats weighing 220–240g were used in this study. NMDA combined with LY294002, wortmannin, or vehicle was administered intravitreally, and histological evaluation was performed at 2 and 7 days after injection. The effects of LY294002 or wortmannin alone were also evaluated. ResultsThe number of cells in the ganglion cell layer (GCL) was significantly reduced at 2 and 7 days after intravitreal injection of NMDA, whereas enhanced capillary degeneration was observed at 7 days. Simultaneous injection of LY294002 with NMDA significantly attenuatedNMDA-induced retinal cell loss and capillary degeneration at 7 days. However, simultaneous injection of wortmannin with NMDA did not affect cell loss, but enhanced capillary degeneration. Treatment with LY294002 alone showed no effect on neuronal or vascular cells, whereas wortmannin induced capillary degeneration without significantly affecting the cell number in the GCL. ConclusionsAlthough both LY294002 and wortmannin are known as PI3K inhibitors, they exhibit differential effects on neurons and vascular endothelial cells in the rat retina. Therefore, the results obtained using these inhibitors should be carefully interpreted. However, our finding that LY294002 was protective against NMDA-induced retinal damage suggests that this compound may be an effective candidate for preventing the development of retinal diseases associated with glutamate-induced excitotoxicity.

Thickness mapping of the inner retina by spectral-domain optical coherence tomography in an N-methyl-d-aspartate-induced retinal damage model

Spectral-domain optical coherence tomography (SD-OCT) is an interferometric optical tomography technique and provides high resolution and noninvasive visualization of retinal morphology. The purpose of this study was to assess the utility of thickness maps and quantitative thickness measurements of the ganglion cell complex (GCC: retinal nerve fiber layer, ganglion cell layer, and inner plexiform layer) obtained by SD-OCT of a mouse model of N-methyl-d-aspartate (NMDA)-induced retinal damage. SD-OCT imaging was performed in ddY mice at 1, 3, and 7 days and 1 month after intravitreal injection of NMDA. GCC thickness maps and circle cross-sectional OCT images were made from volumetric OCT images. The GCC thickness was measured on a cross-sectional OCT image on a circle with a radius 300 μm from the center of the optic nerve disc. Histological analysis was conducted by measuring the GCC thickness at the same time intervals. The thickness maps and the quantitative thickness values of GCC showed thickness changes at each time point in the NMDA-treated mice when compared with normal and vehicle-treated mice. Both the OCT sectional images and the histological images revealed increases in GCC thickness at 1 day, followed by decreases from 3 days to 1 month after NMDA injection. The GCC thickness measured using OCT sectional images correlated with the thickness measured using histological images. In conclusion, GCC thickness mapping is a useful method for evaluating NMDA-induced retinal degeneration in mice.

Neuroprotective effect of epigallocatechin‐3‐gallate against N‐methyl‐D‐aspartate‐induced excitotoxicity in the adult rat retina

Epigallocatechin-3-gallate (EGCG), the major polyphenol of green tea, has been suggested to reduce glutamate excitotoxicity. We therefore investigated the potentially protective effects of EGCG against N-methyl-d-aspartate (NMDA)-induced excitotoxicity in the retina. Female Wistar rats (n = 171) were divided into a normal control group (n = 9); saline control group with intravitreal saline injections (n = 54); NMDA control group with an intravitreal NMDA injection and intraperitoneal saline injections (n = 54); and NMDA study group (n = 54) receiving an intravitreal NMDA injection plus intraperitoneal EGCG (25 mg/kg) injections. Starting at 2 days prior to the intravitreal NMDA injection, the intraperitoneal injections were performed daily for the whole study period. At 12 hr, 1, 2, 3 days, 1 and 2 weeks after the intravitreal NMDA injection, the animals were killed. We counted the neurons in the retinal ganglion cell layer (GCL) on histological sections, measured the thickness of Thy-1 immunoreactivity and assessed the expression of Thy-1 mRNA by real-time polymerase chain reaction. At all time-points, GCL cell density, thickness of Thy-1 immunoreactivity and expression of Thy-1 mRNA were significantly (all p < 0.05) lower in the NMDA control group than in the NMDA study group, in which the parameters were significantly (all p < 0.05) lower than in the saline control group and the normal control group. In both groups with an intravitreal NMDA injection, GCL cell density, thickness of Thy-1 immunoreactivity and expression of Thy-1 mRNA decreased significantly with increasing follow-up time. Intraperitoneal application of EGCG resulted in a significantly less marked NMDA-associated loss of retinal ganglion cells.

Protective effects of the β3-adrenoceptor agonist CL316243 against N-methyl-D-aspartate-induced retinal neurotoxicity

We have previously reported that β(3)-adrenoceptor agonists dilate retinal blood vessels, but their effects on retinal neurons have been unclear. In this study, we examined the action of the β(3)-adrenoceptor agonist CL316243 against retinal damage induced by intravitreal injection of N-methyl-D-aspartate (NMDA) in rats. CL316243 was injected into the vitreous cavity before, with, or after intravitreal NMDA injection. Seven days after NMDA injection, cell loss in the ganglion cell layer (GCL) and thinning of the inner plexiform layer were observed. The reduction in the number of cells in the GCL was diminished by injection of CL316243 at 15, 30, 60, or 120min after NMDA injection, whereas no significant protective effect was observed when CL316243 was administered 240min after NMDA injection. Neither preinjection of CL316243 30min before NMDA nor simultaneous injection of CL316243 with NMDA exerted any protective effect. The β(3)-adrenoceptor antagonist L748337 almost completely abolished the protection conferred by CL316243 injection 120min after NMDA injection. The number of parvalbumin-positive amacrine cells was decreased in eyes examined 1day after NMDA treatment, but this was prevented by CL316243 injection at 120min after NMDA injection. These results suggest that CL316243 exerts protective effects against NMDA-induced damage by stimulation of β(3)-adrenoceptors. β(3)-adrenoceptor agonists may be effective candidates for the treatment of retinal diseases associated with glutamate-induced excitotoxicity, including glaucoma and diabetic retinopathy.

Compound Isolated from the Leaves of Phyllostachys nigra Protects NMDA-Induced Retinal Degeneration

The luteolin 6-C-(6″-O-trans-caffeoylglucoside) (PN6) isolated from Phyllostachys nigra is effective against both the negative influence of N-methyl-D-aspartate (NMDA) to the rat retina and the oxidative stress induced transformed retinal ganglion cells (RGC-5) death. The PN6 concentration-dependently inhibited sodium nitroprusside-induced lipid peroxidation. Treatment of the RGC-5 with PN6 decreased the apoptotic proteins of poly (adenosine diphosphate-ribose) polymerase (PARP) and cleaved caspase-3, and increased the antioxidant proteins of superoxide dismutase (SOD)-2, catalase and glutathione peroxidase (GPx-1) expressions by Western blot analysis. The PN6 reduced the thickness of the inner plexiform layer using hematoxylin and eosin staining and decreased the number of terminal deoxynucleotidyl transferase 2′-deoxyuridine 5′-triphosphate nick-end labeling (TUNEL)-positive cells using TUNEL kit assay. Moreover, PN6 attenuated upregulation of apoptotic proteins (PARP and cleaved caspase-3) and downregulation of antioxidant proteins (SOD-2, catalase and GPx-1) caused by NMDA in the rat retina. Practical Applications The aim of this study was to determine whether the luteolin 6-C-(6″-O-trans-caffeoylglucoside) (PN6) previously isolated from Phyllostachys nigra is effective at blunting the negative influence of N-methyl-D-aspartate (NMDA)-induced excitotoxicity in situ. The treatment of the PN6 was evaluated by lipid peroxidation studies, which showed that PN6 significantly inhibited lipid peroxidation with an half maximal inhibitory concentration (IC50) of 1.05 μM. PN6 decreased the upregulation of poly (adenosine diphosphate-ribose) polymerase (PARP) and cleaved caspase-3 and the downregulation of superoxide dismutase (SOD-2), catalase and glutathione peroxidase (GPx -1) in retinal ganglion cells. Intravitreal injection of NMDA to the rat retina reduced the thickness of the inner plexiform layer and increased the number of terminal deoxynucleotidyl transferase 2′-deoxyuridine 5′-triphosphate nick-end labeling-positive cells, which was partially, but significantly blunted by the presence of PN6. Moreover, PN6 blunted the changes in PARP, cleaved caspase-3, SOD-2, catalase and GPx-1 proteins in the rat retina. Evidence is therefore provided to show that P. nigra can be considered as a candidate neuroprotectant for the treatment of various neurodegenerative diseases.

Oral administration of crocetin prevents inner retinal damage induced by N-methyl-d-aspartate in mice

Crocetin, an aglycone of crocin, is found in stigmas of the saffron crocus (Crocus starus L.) and has been used in traditional medicine. We investigated the effects of oral administration of crocetin on damage induced by N-methyl-d-aspartate (NMDA) in the murine retina. Crocetin was orally administered before and after intravitreal injection of NMDA. A histological analysis was conducted by counting the cell number of ganglion cell layer (GCL). Cell apoptosis was assessed by counting cells positive for terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Retinal functions were measured in terms of a- and b-wave amplitudes using an electroretinogram (ERG). Activation of caspase-3/7 and cleaved caspase-3 expression were assayed. Calpain activity was evaluated by immunoblotting assays for proteolysis of α-spectrin. NMDA injection decreased the cell number in the GCL, and crocetin at a dose of 100mg/kg inhibited this reduction. TUNEL-positive cells were observed in both GCL and inner nuclear layer (INL) after NMDA injection, and crocetin inhibited the increase in number of TUNEL-positive cells. ERG analysis showed that both a- and b-wave amplitudes were decreased by NMDA injection. Crocetin inhibited the reduction in the b-wave amplitude, but not in the a-wave. NMDA injection activated caspase-3/7 and increased expression of cleaved caspsase-3 in the GCL and INL, but both of these processes were inhibited by crocetin. NMDA injection also induced cleavage of α-spectrin, but crocetin did not affect this process. These findings indicate that oral administration of crocetin prevented NMDA-induced retinal damage via inhibition of the caspase pathway.

Effect of Nafamostat on &lt;i&gt;N&lt;/i&gt;-Methyl-&lt;small&gt;D&lt;/small&gt;-aspartate-Induced Retinal Neuronal and Capillary Degeneration in Rats

We examined the effects of the serine protease inhibitor nafamostat mesilate on neuronal and vascular injury in rat retinas treated with N-methyl-D-aspartate (NMDA). The degree of neuronal degeneration was assessed by measuring the number of cells in the ganglion cell layer and the thickness of the inner plexiform layer. The degree of capillary degeneration was assessed by measuring the number of empty basement membrane sleeves that were left as remnants of the vessels. Significant neuronal and capillary degeneration was observed 7 d after a single intravitreal injection of NMDA into the eye. Both forms of degeneration were significantly prevented by simultaneous injection of nafamostat mesilate with NMDA. These results indicate that nafamostat mesilate affords protection against the neuro/vascular injury seen in NMDA-treated retinas. Nafamostat mesilate may be considered as a candidate for neuro/vascular protective interventions in retinal diseases associated with glutamate-induced excitotoxicity, such as glaucoma and diabetic retinopathy.

Functional and morphological effects of β-estradiol in eyes with N-methyl- d-Aspartate-induced retinal neurotoxicity in rats

Glutamate-mediated excitotoxicity, mainly induced by N-methyl- d-aspartate (NMDA) receptors, is known to cause retinal ganglion cell death in retinal ischemia, glaucoma, and several other retinal diseases. We evaluated the effects of β-estradiol (E2) against a single intravitreal injection of NMDA using a functional and morphological approach. Male rats were randomly divided into 3 treatment groups: (1) Control; (2) NMDA (intravitreal injection of 5 mM NMDA); and (3) NMDA + E2 (intravitreal injection of 5 mM NMDA and pretreatment with subcutaneous E2 implantation). Seven days after NMDA injection, full-field electroretinograms (ERGs) and quantitative morphological analyses using transverse sections of the retina were conducted. In the NMDA group, full-field ERGs showed reductions in the amplitudes of the negative-scotopic threshold response, rod response b-wave, oscillatory potentials, flicker response second b-wave and cone response b-wave. Morphological evaluations of transverse sections of the retina demonstrated a reduction in the thickness of the inner plexiform layer, increases in the thickness of the outer plexiform and outer nuclear layers, and a loss of cells in the ganglion cell layer. In the NMDA + E2 group, pretreatment with E2 prevented the aggravations in the amplitudes of the ERGs except for oscillatory potential 2 (OP2); however, no morphological differences between the NMDA and NMDA + E2 groups were seen. These findings indicate that E2 can protect retinal function against NMDA-induced neurotoxicity. In addition, these indications suggested that the effect of E2 may have therapeutic benefits in NMDA related diseases, such as retinal ischemia and glaucoma.

Bis(7)-tacrine protects retinal ganglion cells against excitotoxicity via NMDA receptor inhibition.

To investigate whether bis(7)-tacrine, a multifunctional drug, inhibits N-methyl-D-aspartate (NMDA) -activated current in retinal ganglion cells(RGC) and provides neuroprotection against retinal cell damage. Purified RGC cultures were obtained from retinas of 1-3 days old Sprague-Dawley(SD) rats, following a two-step immunopanning procedure. After 7 days of cultivation, the inhibition of NMDA-activated current by bis(7)-tacrine was measured by using patch-clamp recording techniques. In animal experiments, RGCs were damaged after intravitreal injection of NMDA (5µL, 40nmol) in adult rats. Bis(7)-tacrine(0.05, 0.1, 0.2mg/kg) or memantine(20mg/kg) was intraperitoneal administered to the rats fifteen minutes before intravitreally injection of NMDA. RGC damage was analyzed by histologic techniques, TUNEL and retrograde labeling techniques. Whole-cell patch-clamp recordings demonstrated that NMDA (30µmol/L) resulted in approximately -50 pA inward currents that were blocked by bis(7)-tacrine(1µmol/L). Histological examination and retrograde labeling analysis revealed that bis(7)-tacrine induced a significant neuroprotective effect against NMDA-induced cell damage 7 days after NMDA injection. TUNEL staining showed that pretreatment with bis(7)-tacrine was effective in ameliorating NMDA-induced apoptotic cell loss in the retinal ganglion cell layer 18 hours after injection. Bis(7)-tacrine possesses remarkable neuroprotective activities against retinal excitotoxicity through inhibition of NMDA receptors.

Kinesin-1 and degenerative changes in optic nerve axons in NMDA-induced neurotoxicity

We examined the histologic findings of optic nerve axons and changes in kinesin-1, which is involved in axonal flow, in N-methyl- d-aspartate (NMDA)-induced neurotoxicity in rats. Substantial degenerative changes visualized as black profiles and pale large axons were observed 72 h after NMDA injection, but those degenerative changes were not apparent in axons 12 and 24 h after injection. Morphometric analysis showed a significant, approximately 40% reduction in the number of axons 72 h after NMDA injection. Immunohistochemical study showed that there was a recognizable loss of neurofilament-immunopositive dots, but myelin basic protein immunostaining was unchanged 72 h after NMDA injection. Western blot analysis showed early elevation of kinesin-1 (KIF5B) protein levels in the retina 24 and 72 h after NMDA injection. Conversely, significant decreases in KIF5B protein levels in the optic nerve were seen during the same time course. Immunohistochemical study also showed that there was a reduction in KIF5B immunoreactivity in axons, but neurofilament immunostaining was unchanged 24 h after NMDA injection. These findings suggest that the intravitreal injection of NMDA causes neurofilament loss without myelin alteration in the early stage. The depletion of kinesin-1 precedes axonal degeneration of the optic nerve in NMDA-induced neurotoxicity.

Neuroprotective Effects of C-Type Natriuretic Peptide on Rat Retinal Ganglion Cells

PURPOSE. To evaluate the potential neuroprotective effects of C-type natriuretic peptide (CNP) on rat retinal ganglion cells (RGCs). METHODS. Cultured adult rat retinal cells were treated with vehicle, CNP, or atrial natriuretic peptide (ANP), followed by cytotoxic insults (glutamate, TNFalpha, or withdrawal of trophic factor). RGC survival was analyzed by counting Thy-1-positive cells in each well. For in vivo evaluation, N-methyl-d-aspartate (NMDA) with or without CNP was injected intravitreally into rat eyes. At various time points after injection, retinal cross-sections were analyzed for thickness changes in the retinal layers, and retinal flat mounts were assessed by counting cresyl violet-labeled or TUNEL-positive cells. Expressions of natriuretic peptide receptor-B (NPRB) and apoptosis-related genes in retina, including Bcl-xL, BAX, and micro-calpain, were analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS. At 50 and 500 nM, CNP, but not ANP, significantly (P < 0.05) protected against glutamate-insult and trophic factor withdrawal-induced RGC death in vitro. Neither peptide significantly affected TNFalpha-induced cytotoxicity. Intravitreal injection of NMDA (20 nanomoles) significantly (P < 0.05) decreased the thickness of the inner plexiform layer (IPL), induced cell loss, increased the number of TUNEL-positive cells in the RGC layer, and upregulated the expression of Bcl-xL, BAX, and micro-calpain. All these effects were significantly (P < 0.05) alleviated by concomitant injection of CNP (4.5 nmol, 10 microg). The neuroprotective effects of CNP were maintained up to 14 days after CNP injection. CONCLUSIONS. CNP protects rat RGCs against the apoptotic damage induced by insults such as excitatory amino acid, both in vitro and in vivo.

Toxicity Assessment of Intravitreal Triamcinolone and Bevacizumab in a Retinal Explant Mouse Model Using Two-Photon Microscopy

Intravitreal drug administration leads to high intraocular concentrations with potentially toxic effects on ocular tissues. This study was an assessment of the toxicity of triamcinolone and bevacizumab in living retinal explants using two-photon (2P) microscopy. Wild-type mice received intravitreal injections of triamcinolone, bevacizumab, or vehicle. Ten and 45 days after injection, wholemounted retinal explants were incubated with the fluorescent dye sulforhodamine 101 (SR101) to analyze morphology and tissue damage with 2P microscopy ex vivo. Retinas that received the same treatment were stained for apoptosis (TUNEL) and glial activation (GFAP). An intravitreal injection of NMDA (N-methyl-d-aspartate) was used as a positive control to ensure the fidelity of detection of retinal damage with ex vivo 2P microscopy. Overall retinal morphology was undisturbed after all procedures and time points. NMDA injection resulted in a strong increase in the number of SR101-labeled cells and increased apoptosis and glial activation when compared with sham-injected eyes. This result was in contrast to exposure to bevacizumab, which caused no appreciable damage. After triamcinolone treatment, marked damage in the inner retina was observed. However, damaged cells were restricted to sharply demarcated areas, and only mild changes in TUNEL-positive cells and GFAP activation was observed when compared to sham-injected eyes. 2P microscopy in combination with SR101 staining allows fast morphologic assessment of living retinal explants and can be used to evaluate adverse effects on retinal viability of test substances. Bevacizumab treatment did not cause any detectable retinal damage, whereas triamcinolone was associated with substantial, although spatially restricted, damage.

Effect of an Inducer of BiP, a Molecular Chaperone, on Endoplasmic Reticulum (ER) Stress-Induced Retinal Cell Death

The effect of a preferential inducer of 78 kDa glucose-regulated protein (GRP78)/immunoglobulin heavy-chain binding protein (BiP; BiP inducer X, BIX) against tunicamycin-induced cell death in RGC-5 (a rat ganglion cell line), and also against tunicamycin- or N-methyl-D-aspartate (NMDA)-induced retinal damage in mice was evaluated. In vitro, BiP mRNA was measured after BIX treatment using semi-quantitative RT-PCR or real-time PCR. The effect of BIX on tunicamycin (at 2 microg/mL)-induced damage was evaluated by measuring the cell-death rate and CHOP protein expression. In vivo, BiP protein induction was examined by immunostaining. The retinal cell damage induced by tunicamycin (1 microg) or NMDA (40 nmol) was assessed by examining ganglion cell layer (GCL) cell loss, terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining, and CHOP protein expression. In vitro, BIX preferentially induced BiP mRNA expression both time- and concentration-dependently in RGC-5 cells. BIX (1 and 5 microM) significantly reduced tunicamycin-induced cell death, and BIX (5 microM) significantly reduced tunicamycin-induced CHOP protein expression. In vivo, intravitreal injection of BIX (5 nmol) significantly induced BiP protein expression in the mouse retina. Co-administration of BIX (5 nmol) significantly reduced both the retinal cell death and the CHOP protein expression in GCL induced by intravitreal injection of tunicamycin or NMDA. These findings suggest that this BiP inducer may have the potential to be a therapeutic agent for endoplasmic reticulum (ER) stress-induced retinal diseases.