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Assessing retinal ganglion cell death and neuroprotective agents using real time imaging
摘要： The evaluation of retinal ganglion cell (RGC) death is a key part of retinal disease care. Previously, we used a Sytox Orange (SO)-based real-time imaging method to assess the RGCs in mice that underwent optic nerve crush. Here, we used N-methyl-D-aspartate (NMDA) injury in rats to confirm our model and assess the effect of neuroprotective agents on RGCs. The rats received NMDA injury and the intravitreal injection of SO, a cell-impermeant dyeing compound that targets nucleic acid. After ten minutes, non-invasive confocal scanning laser ophthalmoscopy visualized damaged or dying cells. Finally, the retinas were flat-mounted for histological confirmation of RGC death, with retrograde Fluorogold labeling and Alexa Fluor 488 Annexin V-conjugate (Annexin V) staining. This also revealed the time course of retinal cell death and the neuroprotective effect of SNJ-1945. Real-time imaging showed that SO-positive cells significantly increased starting 2 hours after NMDA injection and reached an approximate plateau at 3 hours. SO-positive cells were positive for Fluorogold and Annexin V in the isolated retinas. Moreover, the number of SO-positive retinal cells was significantly lower after treatment with SNJ-1945, compared to carboxymethyl cellulose. These results were confirmed in the isolated retinas. Thus, real-time imaging with SO allows the quick quantification of NMDA-induced RGC damage and death, and evaluation of neuroprotective agents. This technique may aid research into the development of new neuroprotective therapies.
关键词： retinal ganglion cell,Real-time imaging,SYTOX orange,neuroprotection
更新于2025-11-21 11:24:58
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The molecular chaperone sigma 1 receptor mediates rescue of retinal cone photoreceptor cells via modulation of NRF2
摘要： Sigma 1 receptor (Sig1R), a putative molecular chaperone, has emerged as a novel therapeutic target for retinal degenerative disease. Earlier studies showed that activation of Sig1R via the high-affinity ligand (+)-pentazocine ((+)-PTZ) induced profound rescue of cone photoreceptor cells in the rd10 mouse model of retinitis pigmentosa; however the mechanism of rescue is unknown. Improved cone function in (+)-PTZ-treated mice was accompanied by reduced oxidative stress and normalization of levels of NRF2, a transcription factor that activates antioxidant response elements (AREs) of hundreds of cytoprotective genes. Here, we tested the hypothesis that modulation of NRF2 is central to Sig1R-mediated cone rescue. Activation of Sig1R in 661W cone cells using (+)-PTZ induced dose-dependent increases in NRF2-ARE binding activity and NRF2 gene/protein expression, whereas silencing Sig1R significantly decreased NRF2 protein levels and increased oxidative stress, although (+)-PTZ did not disrupt NRF2-KEAP1 binding. In vivo studies were conducted to investigate whether, in the absence of NRF2, activation of Sig1R rescues cones. (+)-PTZ was administered systemically for several weeks to rd10/nrf2+/+ and rd10/nrf2-/- mice. Through post-natal day 42, cone function was significant in rd10/nrf2+/+, but minimal in rd10/nrf2-/- mice as indicated by electroretinographic recordings using natural noise stimuli, optical coherence tomography and retinal histological analyses. Immunodetection of cones was limited in (+)-PTZ-treated rd10/nrf2-/-, though considerable in (+)-PTZ-treated rd10/nrf2+/+mice. The data suggest that Sig1R-mediated cone rescue requires NRF2 and provide evidence for a previously-unrecognized relationship between these proteins.
关键词： retinitis pigmentosa,NRF2-KEAP1,retinal neuroprotection,retina,rd10 mouse,NRF2-Neh luciferase assay,oxidative stress
更新于2025-11-21 11:08:12
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Retinal Neuroprotection From Optic Nerve Trauma by Deletion of Arginase 2
摘要： Our previous studies have implicated expression of the mitochondrial isoform of the arginase enzyme arginase 2 (A2) in neurovascular injury during ischemic retinopathies. The aim of this study was to characterize the specific involvement of A2 in retinal injury following optic nerve crush (ONC). To accomplish this, wild-type (WT) or A2 knockout (A2?/?) mice were subjected to ONC injury. The contralateral eye served as sham control. Quantitative RT-PCR and western blot were used to evaluate mRNA and protein expression. Retinal ganglion cell (RGC) survival was assessed in retinal whole mounts. Axonal sprouting was determined by anterograde transport of Cholera Toxin B (CTB). These analyses showed increased A2 expression following ONC. Numbers of NeuN-positive neurons as well as Brn3a- and RBPMS-positive RGC were decreased in the WT retinas at 14 days after ONC as compared to the sham controls. This ONC-induced neuronal loss was diminished in the A2?/? retinas. Similarly, axonal degeneration was ameliorated by A2 deletion whereas axon sprouting was enhanced. Significant retinal thinning was also seen in WT retinas at 21 days after ONC, and this was blocked in A2?/? mice. Cell death studies showed an increase in TUNEL positive cells in the RGC layer at 5 days after ONC in the WT retinas, and this was attenuated by A2 deletion. ONC increased glial cell activation in WT retinas, and this was significantly reduced by A2 deletion. Western blotting showed a marked increase in the neurotrophin, brain derived neurotrophic factor (BDNF) and its downstream signaling in A2?/? retinas vs. WT after ONC. This was associated with increases in the axonal regeneration marker GAP-43 in A2?/? retinas. Furthermore, A2?/? retinas showed decreased NLRP3 inflammasome activation and lower interleukin (IL-) 1β/IL-18 levels as compared to WT retinas subjected to ONC. Collectively, our results show that deletion of A2 limits ONC-induced neurodegeneration and glial activation, and enhances axonal sprouting by a mechanism involving increases in BDNF and decreases in retinal inflammation. These data demonstrate that A2 plays an important role in ONC-induced retinal damage. Blockade of A2 activity may offer a therapeutic strategy for preventing vision loss induced by traumatic retinal injury.
关键词： optic nerve crush,retinal ganglion cells,retina,brain derived neurotrophic factor,neuroprotection,arginase 2
更新于2025-09-23 15:23:52
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Pre-conditioning with Remote Photobiomodulation Modulates the Brain Transcriptome and Protects Against MPTP Insult in Mice
摘要： Transcranial photobiomodulation (PBM), which involves the application of low-intensity red to near-infrared light (600-1100nm) to the head, provides neuroprotection in animal models of various neurodegenerative diseases. However, the absorption of light energy by the human scalp and skull may limit the utility of transcranial PBM in clinical contexts. We have previously shown that targeting light at peripheral tissues (i.e. “remote PBM”) also provides protection of the brain in an MPTP mouse model of Parkinson’s disease, suggesting remote PBM might be a viable alternative strategy for overcoming penetration issues associated with transcranial PBM. This present study aimed to determine an effective pre-conditioning regimen of remote PBM for inducing neuroprotection and elucidate the molecular mechanisms by which remote PBM enhances the resilience of brain tissue. Balb/c mice were irradiated with 670nm light (4J/cm2 per day) targeting dorsum and hindlimbs for 2, 5 or 10 days, followed by injection of the parkinsonian neurotoxin MPTP (50mg/kg) over two consecutive days. Despite no direct irradiation of the head, 10 days of pre-conditioning with remote PBM significantly attenuated MPTP-induced loss of midbrain tyrosine hydroxylase-positive dopaminergic cells and mitigated the increase in FOS-positive neurons in the caudate-putamen complex. Interrogation of the midbrain transcriptome by RNA microarray and pathway enrichment analysis suggested upregulation of cell signaling and migration (including CXCR4+ stem cell and adipocytokine signaling), oxidative stress response pathways and modulation of the blood-brain barrier following remote PBM. These findings establish remote PBM preconditioning as a viable neuroprotective intervention and provide insights into the mechanisms underlying this phenomenon.
关键词： MPTP,microarray,Parkinson’s disease,mouse model,neuroprotection,photobiomodulation
更新于2025-09-23 15:23:52
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Pharmacological interference of adrenergic receptor signaling preserve photoreceptors after retinal detachment through inhibition of oxidative stress and inflammation
摘要： Background and Purpose: The current strategy is not sufficient to halt progression of photoreceptor death and subsequent visual impairment related to retinal detachment (RD) which is observed in various retinal disorders. This study investigated the neuroprotective effects of adrenergic receptor (AR)-targeting pharmaceuticals, the α1-AR antagonist doxazosin or the α2-AR agonist guanabenz, against photoreceptor cell death in RD. Experimental Approach: Brown-Norway rats were created with experimental RD by subretinal injection of sodium hyaluronate. Oxidative stress biomarkers and cytokine production were quantified with ELISA. Protein expression levels and immunofluorescent labelling were determined in rats with RD and controls for mechanistic elucidation. The effects of systemic administration of doxazosin, guanabenz on photoreceptor apoptosis, retinal histology and electroretinography were evaluated in rats with RD compared to vehicle controls. Key Results: Photoreceptors were the major source of RD-induced ROS overproduction in the rat retina through the regulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Systemic administration of doxazosin or guanabenz significantly alleviated the RD-induced production of ROS and proinflammatory cytokines, including IL-1β and MCP-1, and suppressed retinal gliosis, resulting in attenuation of photoreceptor death and preservation of retinal structures and functions in RD. Conclusions and Implications: Our findings point to adrenergic receptors as novel therapeutic targets for photoreceptor protection and suggest that both doxazosin and guanabenz, two FDA-approved drugs, could be further explored to treat retinal diseases.
关键词： G protein-coupled receptors,adrenergic receptors,retinal detachment,photoreceptor neuroprotection,NADPH oxidase
更新于2025-09-23 15:23:52
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Protective role of melatonin on retinal ganglionar cell: In vitro an in vivo evidences
摘要： Oxidative stress triggers ocular neurodegenerative diseases, such as glaucoma or macular degeneration. The increase of reactive oxygen and nitrogen species in retinal ganglion cells (RGCs) causes damage to the structure and function of the axons that make up the optic nerve, leading to cell death arising from apoptosis, necrosis or autophagy in the RCGs. The use of antioxidants to prevent visual neurodegenerative pathologies is a novel and possibly valuable therapeutic strategy. To investigate in vitro and in vivo neuroprotective efficacy of melatonin (MEL) in RGCs, we used a model of oxidative glutamate (GLUT) toxicity in combination with L-butionin-S, R-sulfoximine (BSO), which induces cell death by apoptosis through cytotoxicity and oxidative stress mechanisms. Histological sectioning and immunohistochemical assays using the TUNEL technique were performed to determine the damage generated in affected cells and to observe the death process of RGCs. Whit BSO-GLUT the results revealed a progressive RGCs death without any significant evidence of a decreased retinal function after 9 days of treatment. In this way, we were able to develop a retinal degeneration model in vivo to carry out treatment with MEL and observed an increase in the survival percentage of RGCs, showing that BSO-GLUT could not exert an oxidant effect on cells to counteract the effect of MEL. These findings reveal that MEL has a neuroprotective and antiapoptotic effect as evidenced by the reduction of oxidative stress damage. MEL demonstrated in this model makes it a promising neuroprotective agent for the treatment of ocular neurodegenerative diseases when administered locally.
关键词： Ganglion retinal cells (RGCs),Oxidative stress,Neuroprotection,Melatonin
更新于2025-09-23 15:23:52
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Intravitreal Injection of Proinsulin-Loaded Microspheres Delays Photoreceptor Cell Death and Vision Loss in the <i>rd10</i> Mouse Model of Retinitis Pigmentosa
摘要： PURPOSE. The induction of proinsulin expression by transgenesis or intramuscular gene therapy has been shown previously to retard retinal degeneration in mouse and rat models of retinitis pigmentosa (RP), a group of inherited conditions that result in visual impairment. We investigated whether intraocular treatment with biodegradable poly (lactic-co-glycolic) acid microspheres (PLGA-MS) loaded with proinsulin has cellular and functional neuroprotective effects in the retina. METHODS. Experiments were performed using the Pde6brd10 mouse model of RP. Methionylated human recombinant proinsulin (hPI) was formulated in PLGA-MS, which were administered by intravitreal injection on postnatal days (P) 14 to 15. Retinal neuroprotection was assessed at P25 by electroretinography, and by evaluating outer nuclear layer (ONL) cellular preservation. The attenuation of photoreceptor cell death by hPI was determined by TUNEL assay in cultured P22 retinas, as well as Akt phosphorylation by immunoblotting. RESULTS. We successfully formulated hPI PLGA-MS to deliver the active molecule for several weeks in vitro. The amplitude of b-cone and mixed b-waves in electroretinographic recording was significantly higher in eyes injected with hPI-PLGA-MS compared to control eyes. Treatment with hPI-PLGA-MS attenuated photoreceptor cell loss, as revealed by comparing ONL thickness and the number of cell rows in this layer in treated versus untreated retinas. Finally, hPI prevented photoreceptor cell death and increased AktThr308 phosphorylation in organotypic cultured retinas. CONCLUSIONS. Retinal degeneration in the rd10 mouse was slowed by a single intravitreal injection of hPI-PLGA-MS. Human recombinant proinsulin elicited a rapid and effective neuroprotective effect when administered in biodegradable microspheres, which may constitute a future potentially feasible delivery method for proinsulin-based treatment of RP.
关键词： photoreceptors,neuroprotection,retinitis pigmentosa
更新于2025-09-23 15:22:29
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Enhancement of corneal epithelium cell survival, proliferation and migration by red light: Relevance to corneal wound healing
摘要： The aim of the present study was to analyse how short wave blue and long wave red light differentially affect corneal epithelial (HCE-2) cells in culture. The corneal epithelium in situ is exposed to more blue light than in the past because of Light Emitting Diodes (LEDs) used for indoor lighting and computer, television and phone screens. Compared with cultures maintained in the dark, low intensity blue light, such as that emitted from computer screens, reduced the proliferation rate of HCE-2 cells and caused cell death at greater intensities in a dose-dependent manner. In contrast, red light at high intensity slightly enhanced the proliferation rates of HCE-2 cells and importantly blunted the negative influence of blue light on cell survival when delivered after the insult. The toxic influence of blue light on HCE-2 cells involves mitochondrial dysfunction and the activation of AIF, p38-MAPK and HO-1. Importantly, red light blocks the effects caused by blue light and enhances mitochondrial function when delivered independently. The mechanism of action of red light is to directly stimulate mitochondrial function, suggested by staining with JC-1, which results in the activation of multiple biochemical mechanisms and the ability to blunt a variety of death pathways. As a consequence, even sodium azide-induced toxicity to HCE-2 cells in culture is blunted by red light. We interpret our studies on HCE-2 cell cultures to suggest that red light can be used prophylactically to protect the corneal epithelial in situ and is also able to counteract a variety of potential environmental insults to the tissue that includes blue light. This might be of particular significance when the cornea is already affected as, for example, in dry eye.
关键词： corneal epithelial cells – mitochondria – red light – neuroprotection
更新于2025-09-23 15:22:29
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Preconditioning with carbon monoxide inhalation promotes retinal ganglion cell survival against optic nerve crush via inhibition of the apoptotic pathway
摘要： Optic neurodegeneration, in addition to central nervous trauma, initiates impairments to neurons resulting in retinal ganglion cell (RGC) damage. Carbon monoxide (CO) has been observed to elicit neuroprotection in various experimental models. The present study investigated the potential retinal neuroprotection of preconditioning with CO inhalation in a rat model of optic nerve crush (ONC). Adult male Sprague-Dawley rats were preconditioned with inhaled CO (250 ppm) or air for 1 h prior to ONC. Animals were euthanized at 1 or 2 weeks following surgery. RGC densities were quantified by hematoxylin and eosin (H&E) staining and FluoroGold labeling. Visual function was measured via flash visual evoked potentials (FVEP). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and caspase-9 and caspase-3 activity in the retinas, were assessed at 2 weeks post-ONC. The RGC density of CO + crush rats was significantly increased compared with that of the corresponding crush-only rats at 2 weeks (survival rate, 66.2 vs. 48.2% as demonstrated by H&E staining, P<0.01; and 67.6 vs. 37.6% as demonstrated by FluoroGold labeling, P<0.05). FVEP measures indicated a significantly better-preserved latency and amplitude of the P1 wave in the CO + crush rats compared with the crush-only rats. The TUNEL assays demonstrated fewer apoptotic cells in the CO + crush group compared with the crush-only group, accompanied by the suppression of caspase-9 and caspase-3 activity. The results of the present study suggested that inhaled CO preconditioning may be neuroprotective against ONC insult via inhibition of neuronal apoptosis.
关键词： neuroprotection,optic nerve crush,carbon monoxide,preconditioning,retinal ganglion cell
更新于2025-09-23 15:22:29
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Age-dependent neuroprotection of retinal ganglion cells by tempol-C8 acyl ester in a rat NMDA toxicity model
摘要： Background: The efficacy of tempol and its acyl derivative tempol-C8 as retinoprotective agents was compared in a rat model of NMDA-induced retinal ganglion cell (RGC) damage. Material and methods: Tempol or tempol-C8 in different doses was administered intraperitoneally to 6 weeks old (pre-adolescent) and 9-10 weeks old (young adult) rats before and after an intravitreous NMDA injection. Retinal ganglion cell were retrogradely labeled with the fluorescent tracer hydroxystilbamidine and RGC counting was performed on retinal flatmounts. Results: Intravitreal NMDA reduced RGC counts by about 90%, independently of age (p < 0.001). In pre-adolescent animals tempol-C8, but not tempol unmodified, showed a significant, dose-dependent RGC rescue effect, with peak activity at 5.8 μmol/kg (p < 0.001). In young adult animals, however, no neuroprotective effect was found for either tempol or tempol-C8. Conclusions: In contrast to tempol itself, tempol-C8 acyl ester was neuroprotective in pre-adolescent rats in the NMDA-induced RGC damage model. Therefore, neuroprotection by tempol acyl esters seems to be superior to that of tempol under certain conditions.
关键词： tempol acyl ester,ganglion cell,NMDA,neuroprotection,tempol
更新于2025-09-23 15:22:29