Retinal Ganglion Cell Damage Research Articles

Color vision impairment in multiple sclerosis points to retinal ganglion cell damage.

Multiple Sclerosis (MS) results in color vision impairment regardless of optic neuritis (ON). The exact location of injury remains undefined. The objective of this study is to identify the region leading to dyschromatopsia in MS patients' NON-eyes. We evaluated Spearman correlations between color vision and measures of different regions in the afferent visual pathway in 106 MS patients. Regions with significant correlations were included in logistic regression models to assess their independent role in dyschromatopsia. We evaluated color vision with Hardy-Rand-Rittler plates and retinal damage using Optical Coherence Tomography. We ran SIENAX to measure Normalized Brain Parenchymal Volume (NBPV), FIRST for thalamus volume and Freesurfer for visual cortex areas. We found moderate, significant correlations between color vision and macular retinal nerve fiber layer (rho = 0.289, p = 0.003), ganglion cell complex (GCC = GCIP) (rho = 0.353, p < 0.001), thalamus (rho = 0.361, p < 0.001), and lesion volume within the optic radiations (rho = -0.230, p = 0.030). Only GCC thickness remained significant (p = 0.023) in the logistic regression model. In the final model including lesion load and NBPV as markers of diffuse neuroaxonal damage, GCC remained associated with dyschromatopsia [OR = 0.88 95 % CI (0.80-0.97) p = 0.016]. This association remained significant when we also added sex, age, and disease duration as covariates in the regression model. Dyschromatopsia in NON-eyes is due to damage of retinal ganglion cells (RGC) in MS. Color vision can serve as a marker of RGC damage in MS.

Metalloproteinase 9 and TIMP-1 expression in retina and optic nerve in absolute angle closure glaucoma

PurposeGlaucoma is one of the most important reason causes of the blindness, associated with retinal ganglion cells (RGC) death. This process is not fully understood, however apoptosis due to hypoxia is one of the most important processes leading to RGC death. Glaucomatous optic neuropathy is characterized by remodeling of the extracellular matrix due to metalloproteinase activation, which leads to loss of RGC and axons at the optic nerve head.The aim of the study was to evaluate metalloproteinase 9 (MMP-9) and tissue metalloproteinase inhibitor-1 (TIMP-1) expression in the retinal ganglion cells and optic nerve axons in 33 eyes with absolute primary glaucoma. Material/methodsTo evaluate MMP-9 and TIMP-1 expression primary polyclonal goat antibodies against MMP-9 and TIMP-1 were used. The control group was composed of 8 cases of eyes enucleated and fixed in the first day after trauma. ResultsMMP-9 expression was observed in retinal ganglion cells and in the inner nuclear layer of the retina in all the examined cases. In 28 out of 33 glaucomatous eyes, MMP-9 expression was observed in the proliferating glial cells surrounding the optic nerve axons. TIMP-1 expression was observed in 10 out of 33 glaucomatous eyes, only in retinal ganglion cells. None of the examined injured eyes showed MMP-9 and TIMP-1 expression. ConclusionsMMP-9 activation rather than TIMP-1 may by associated with the pathomechanism of retinal ganglion cell and optic nerve damage in absolute glaucoma.

Role of Glutathione Peroxidase 4 in Glutamate-Induced Oxytosis in the Retina.

PurposeThe purpose of the present study was to investigate the role of glutathione peroxidase 4 (GPx4) in glutamate-induced oxytosis in the retina.MethodsFor in vitro studies, an immortalized rat retinal precursor cell line R28 was used. Cells were transfected with siRNA specifically silencing GPx4 or with scrambled control siRNA. Lipid peroxidation was evaluated by 4-hydroxy-2-nonenal (4-HNE) immunostaining. Cytotoxicity and cell death were evaluated using an LDH activity assay and annexin V staining, respectively. Cells transfected with GPx4 siRNA or control siRNA were treated with glutamate (1 or 2 mM), and the cytotoxicity was evaluated using the LDH activity assay. For in vivo studies, retinal ganglion cell damage was induced by intravitreal injection of 25-mM N-methyl-D-aspartate (NMDA, 2 μL/eye) in GPx4+/+ and GPx4+/− mice. The evaluation of lipid peroxidation (4-HNE immunostaining), apoptosis (TUNEL staining), and cell density in the ganglion cell layer (GCL) were performed at 12 h, 1 day, and 7 days after the NMDA injection.ResultsGPx4 knockdown significantly increased LDH activity by 13.9-fold (P < 0.01) and increased peroxidized lipid levels by 3.2-fold in R28 cells (P < 0.01). In cells transfected with scrambled control siRNA, treatment with glutamate at 1 or 2 mM did not increase LDH activity; whereas, in cells transfected with GPx4 siRNA, glutamate treatment significantly increased LDH activity (1.52-fold, P < 0.01). GPx4+/− mice exhibited higher levels of lipid peroxidation in retinas treated with NMDA than GPx4+/+ mice (1.26-fold, P < 0.05). GPx4+/− mice had more TUNEL-positive cells induced by NMDA in GCL (1.45-fold, P < 0.05). In addition, the cell density in GCL of GPx4+/− mice was 19% lower than that in GPx4+/+ mice after treatment with NMDA (P < 0.05).ConclusionThese results suggest that defective GPx4 expression is associated with enhanced cytotoxicity by glutamate-induced oxytosis in the retina.

Neuroprotective effect of edaravone in experimental glaucoma model in rats: a immunofluorescence and biochemical analysis.

To evaluate the neuroprotective activity of systemically administered edaravone in early and late stage of experimental glaucoma in rats. In this study, 60 Wistar albino rats were used. Experimental glaucoma model was created by injecting hyaluronic acid to the anterior chamber once a week for 6wk in 46 of 60 subjects. Fourteen subjects without any medication were included as control group. Edaravone administered intraperitoneally 3 mg/kg/d to the 15 of 30 subjects starting at the onset of glaucoma induction and also administered intraperitoneally 3 mg/kg/d to the other 15 subjects starting at three weeks after the onset of glaucoma induction. The other 16 subjects who underwent glaucoma induction was administered any therapy. Retinal ganglion cells (RGCs) have been marked with dextran tetramethylrhodamine (DTMR) retrograde at the end of the sixth week and after 48h, subjects were sacrificed by the method of cardiac perfusion. Alive RGC density was assessed in the whole-mount retina. Whole-mount retinal tissues homogenized and nitric oxide (NO), malondialdehyde (MDA) and total antioxidant capacity (TAC) values were measured biochemically. RGCs counted with Image-Pro Plus program, in the treatment group were found to be statistically significantly protected, compared to the glaucoma group (Bonferroni, P<0.05). The neuroprotective activity of edaravone was found to be more influential by administration at the start of the glaucoma process. Statistically significant lower NO levels were determined in the glaucoma group comparing treatment groups (Bonferroni, P<0.05). MDA levels were found to be highest in untreated glaucoma group, TAC levels were found to be lower in the glaucoma induction groups than the control group (Bonferroni, P<0.05). Systemic administration of Edaravone in experimental glaucoma showed potent neuroprotective activity. The role of oxidative stress causing RGC damage in glaucoma was supported by this study results.

T-helper1/T-helper2 cytokine imbalance in the iris of patients with glaucoma.

The mechanistic study of glaucoma pathogenesis has shifted to seeking to understand the effects of immune responses on retinal ganglion cell damage and protection. Cytokines mediate the biological effects of the immune system, and our previous study revealed an imbalance of T-helper (Th) 1-derived and Th2-derived cytokines in the serum of patients with glaucoma. In this study, we collected irises from normal individuals and patients with primary open-angle closure (POAG) or chronic angle-closure glaucoma (CACG). We used real-time polymerase chain reaction (PCR) to measure the expression of Th1 (interleukin (IL)-2, interferon-gamma (IFN-γ)), Th2 (IL-4, IL-6, IL-10), and Th3 (transforming growth factor-beta (TGF-β)) cytokines. We then performed immunohistochemical staining to characterize the localization of the upregulated cytokines in iris cryosections. We observed an upward trend in the expression of IL-2 and IFN-γ and a downward trend in IL-6 expression in the iris of POAG and CACG patients. Expression of TGF-β also increased. Immunohistochemistry revealed that IL-2 expression in POAG and CACG patients was localized in the anterior surface of the blood vessel wall in the stroma of the iris, in the cytoplasm of some cells, in the anterior epithelium, and in the posterior pigment epithelium. These findings indicate that immune status differed between the iris tissues of POAG and CACG patients and those of normal individuals. A T-helper cytokine imbalance may modulate the immune microenvironment in glaucomatous eyes and thus influence optic neuropathy.

Neuroprotective therapy for glaucoma

Glaucoma is the second leading cause of blindness worldwide.It is mainly caused by glaucomatous optic neuropathy characterized by retinal ganglion cells (RGCs) loss, which leads to visual field loss and blindness.There are many risk factors other than intraocular pressure (IOP) elevation are thought to be responsible for RGCs damage induced by glaucoma, such as neurotrophic factors deprivation, excitotoxicity, oxidative stress and enhanced microglia activity, and these factors are essential for glaucomatous optic neuropathy, especially in normal tension glaucoma (NTG). Up to date, the major attempt of glaucoma therapy is to protect optic nerve function by lowering IOP through surgery and drugs.However, the therapies can not arrest RGCs damage although effectively lowing IOP in a number of patients.Novel study is turning to find and develop some new approaches to solve neuroprotection problem targeting to the pathogenic factors of glaucomatous optic neuropathy out of IOP.This review paper mainly focused on the neuroprotective therapies that are developed in the past few years. Key words: Glaucoma/therapy; Optic neuropathy; Retinal ganglion cells; Neuroprotection

S100 alone has the same destructive effect on retinal ganglion cells as in combination with HSP 27 in an autoimmune glaucoma model.

As previously shown, immunization with ocular antigens, like heat shock protein 27 (HSP 27), leads to retinal ganglion cell loss in an autoimmune glaucoma model. Aim of this study was to assess how immunization with S100 alone and in combination with HSP 27 affects retinal ganglion and macroglia cells. Rats were immunized with S100 or S100 plus HSP 27 (COMB). Neuronal cell density was evaluated on Nissl-stained flatmounts. Immunized groups showed a significant neuronal cell loss (S100, p = 0.005; COMB, p = 0.0005). A significant loss of retinal ganglion cells was also observed in both immunized groups on Brn-3a stained retinal cross-sections (S100, p = 0.003; COMB, p = 0.001). An increase in GFAP(+) area was noted in both groups (S100, p = 0.01; COMB, p = 0.001). In contrary, vimentin staining was not altered (S100/COMB, p > 0.05). In summary, immunization with solely S100 leads to retinal ganglion cell damage and reactive gliosis. While the combination of S100 plus HSP 27 also caused retinal ganglion cell loss and a glia response, the combination of the two antigens did not cause additional damage or more severe cell loss. We assume that both antigens might interact, possibly having inhibitory effects on each other and thus preventing additional damage to the retina.

Acute optic neuritis: a clinical paradigm for evaluation of neuroprotective and restorative strategies?

What makes the acute optic neuritis model unique in an era of trials for neuroprotective and myelin repair agents? Acute optic neuritis (AON) is a common, and often the earliest manifestation of central nervous system (CNS) inflammatory demyelinating disorders like multiple sclerosis (MS) and neuromyelitis optica (NMO). It affects at least half the patients with MS and is the presenting feature in 15–20% of patients. Vision loss in AON secondary to NMO tends to be more severe with less potential for recovery and greater axonal degeneration, as measured by optical coherence tomography (OCT) (Bennett et al., 2014). Several unique features of the afferent visual pathway (AVP) make the AON model an ideal system to study disease pathogenesis and evaluate potential neuroprotective and myelin repair strategies.

Ketogenic Diet Attenuates NMDA-Induced Damage to Rat's Retinal Ganglion Cells in an Age-Dependent Manner

Objective: This study was conducted to investigate neuroprotective effects of a high fat/low carbohydrate and protein diet (ketogenic diet, KD) in a model of N-methyl <smlcap>D</smlcap>-aspartate (NMDA)-induced retinal ganglion cell (RGC) damage in juvenile and young adult rats. Methods: Juvenile (30-35 days old) and young adult (56-70 days old) female Brown Norway rats were fed the KD for 21 days; rats exposed to a standard rodent diet (SRD) served as controls. The main constituents of the KD used in the present study were approximately 80% fats, 8% proteins, and less than 1% carbohydrates. On day 14 of exposure to the KD (or the SRD in the control group), each rat received a single intravitreal injection of NMDA; RGCs were then retrogradely labelled by hydroxystilbamidine on day 19 and collected on day 21 to assess the degree of damage induced by NMDA. Blood biomarkers to confirm the expected metabolic response to the KD (i.e. ketosis and hypoglycaemia) were also assessed. Results: Although both the juvenile and young adult rats developed comparable ketosis and hypoglycaemia when fed the KD, NMDA-induced loss in RGCs was significantly attenuated only in juvenile rats exposed to the KD in comparison with those fed the SRD; exposure to the KD had no protective effect in young adult rats. In summary, exposure to the KD had a neuroprotective effect in NMDA-induced RGC damage in juvenile rats, but not in young adult rats. Conclusion: These results support further exploration of metabolic interventions to treat optic neuropathies associated with neurodegeneration.

Tumor necrosis factor alpha has an early protective effect on retinal ganglion cells after optic nerve crush.

BackgroundGlaucoma is an optic neuropathy that is characterized by the loss of retinal ganglion cells (RGCs) initiated by damage to axons in the optic nerve. The degeneration and death of RGCs has been thought to occur in two waves. The first is axogenic, caused by direct insult to the axon. The second is somatic, and is thought to be caused by the production of inflammatory cytokines from the activated retinal innate immune cells. One of the cytokines consistently linked to glaucoma and RGC damage has been TNFα. Despite strong evidence implicating this protein in neurodegeneration, a direct injection of TNFα does not mimic the rapid loss of RGCs observed after acute optic nerve trauma or exposure to excitotoxins. This suggests that our understanding of TNFα signaling is incomplete.MethodsRGC death was induced by optic nerve crush in mice. The role of TNFα in this process was examined by quantitative PCR of Tnfα gene expression, and quantification of cell loss in Tnfα−/− mice or in wild-type animals receiving an intraocular injection of exongenous TNFα either before or after crush. Signaling pathways downstream of TNFα were examined by immunolabeling for JUN protein accumulation or activation of EGFP expression in NFκB reporter mice.ResultsOptic nerve crush caused a modest increase in Tnfα gene expression, with kinetics similar to the activation of both macroglia and microglia. A pre-injection of TNFα attenuated ganglion cell loss after crush, while ganglion cell loss was more severe in Tnfα−/− mice. Conversely, over the long term, a single exposure to TNFα induced extrinsic apoptosis in RGCs. Müller cells responded to exogenous TNFα by accumulating JUN and activating NFκB.ConclusionEarly after optic nerve crush, TNFα appears to have a protective role for RGCs, which may be mediated through Müller cells.

Determination of morphological, biometric and biochemical susceptibilities in healthy Eurasier dogs with suspected inherited glaucoma.

In both humans and dogs, the primary risk factor for glaucoma is high intraocular pressure (IOP), which may be caused by iridocorneal angle (ICA) abnormalities. Oxidative stress has also been implicated in retinal ganglion cell damage associated with glaucoma. A suspected inherited form of glaucoma was recently identified in Eurasier dogs (EDs), a breed for which pedigrees are readily available. Because of difficulties in assessing ICA morphology in dogs with advanced glaucoma, we selected a cohort of apparently healthy dogsfor the investigation of ICA morphological status, IOP and plasma concentrations of oxidative stress biomarkers. We aimed to establish correlations between these factors, to identify predictive markers of glaucoma in this dog breed. A cohort of 28 subjects, volunteered for inclusion by their owners, was selected by veterinary surgeons. These dogs were assigned to four groups: young males, young females (1–3 years old), adult males and adult females (4–8 years old). Ocular examination included ophthalmoscopy, tonometry, gonioscopy, biometry and ultrasound biomicroscopy (UBM), and the evaluation of oxidative stress biomarkers consisting of measurements of plasma glutathione peroxidase (GP) activity and taurine and metabolic precursor (methionine and cysteine) concentrations in plasma. The prevalence of pectinate ligament abnormalities was significantly higher in adult EDs than in young dogs. Moreover, in adult females, high IOP was significantly correlated with a short axial globe length, and a particularly large distance between Schwalbe's line and the anterior lens capsule. GP activity levels were significantly lower in EDs than in a randomized control group of dogs, and plasma taurine concentrations were higher. Hence, ICA abnormalities were associated with weaker antioxidant defenses in EDs, potentially counteracted by higher plasma taurine concentrations. This study suggests that EDs may constitute an appropriate canine model for the development of glaucoma. This cohort will be used as a sentinel for longitudinal monitoring.

Therapeutic Retrobulbar Inhibition of STAT3 Protects Ischemic Retina Ganglion Cells.

Astrocytes play an important role in the pathogenesis of glaucoma. Abnormal activation and/or proliferation of astrocytes, termed astrogliosis, have been observed during optic nerve degeneration. Our previous study identified signal transducer and activator of transcription 3 (STAT3) signaling as an important regulator of astrogliosis in the optic nerve in a rat transient ischemia/reperfusion model. In this study, we used pharmacological inhibition of STAT3 activation in the same model to assess whether it could attenuate reactive astrogliosis and to observe its influence on optic nerve damage and retinal ganglion cell (RGC) damage. Our findings show that retrobulbar inhibition of STAT3 in optic nerve head astrocytes leads to (a) increased nerve fiber bundle survival in the optic nerve, (b) increased nerve fiber bundle and RGC survival in the retina, (c) decreased astrocyte reactivation in the optic nerve (d) decreased remodeling of astrocytes in the optic nerve, and (e) no influence of astrocyte reactivation inside the retina. Taken together, the Janus kinase/STAT3 pathway contributes to astrocyte reactivation in the optic nerve, which plays a pivotal role in neurodegeneration after transient ischemia/reperfusion in vivo. Inhibition of this pathway provides a potential therapeutic strategy for the treatment of glaucomatous neuropathy, and could extend to other neurodegenerative diseases.

Experimental scleral cross-linking increases glaucoma damage in a mouse model

The purpose of this study was to assess the effect of a scleral cross-linking agent on susceptibility to glaucoma damage in a mouse model.CD1 mice underwent 3 subconjunctival injections of 0.5 M glyceraldehyde (GA) in 1 week, then had elevated intraocular pressure (IOP) induced by bead injection. Degree of cross-linking was measured by enzyme-linked immunosorbent assay (ELISA), scleral permeability was measured by fluorescence recovery after photobleaching (FRAP), and the mechanical effects of GA exposure were measured by inflation testing. Control mice had buffer injection or no injection in 2 separate glaucoma experiments. IOP was monitored by Tonolab and retinal ganglion cell (RGC) loss was measured by histological axon counting. To rule out undesirable effects of GA, we performed electroretinography and detailed histology of the retina. GA exposure had no detectable effects on RGC number, retinal structure or function either histologically or electrophysiologically. GA increased cross-linking of sclera by 37% in an ELISA assay, decreased scleral permeability (FRAP, p = 0.001), and produced a steeper pressure–strain behavior by in vitro inflation testing. In two experimental glaucoma experiments, GA-treated eyes had greater RGC axon loss from elevated IOP than either buffer-injected or control eyes, controlling for level of IOP exposure over time (p = 0.01, and 0.049, multivariable regression analyses). This is the first report that experimental alteration of the sclera, by cross-linking, increases susceptibility to RGC damage in mice.

Estrogen prevents high-glucose-induced damage of retinal ganglion cells via mitochondrial pathway.

Diabetic retinopathy (DR) is a leading cause of acquired blindness in adults. Previous research has shown that the apoptosis of retinal ganglion cells(RGCs) plays an important role in the initiation and development of diabetic retinopathy. The positive effect of estrogen on the nervous system is currently attracting increasing attention. In this study, we investigated whether17β-estradiolum (E2) has protective effects on RGCs in a high-glucose environment. The cell survival rates were measured by Cell Counting Kit-8, the apoptosis was detected by flow cytometry, the intracellular reactive oxygen species (ROS) levels were examined by immunofluorescence method, and the intracellular mitochondrial membrane potential was examined by confocal microscopy. The expression levels of cytochrome C, Bcl-2, and Bax were analyzed by Western blot method. The effect of estrogen receptor blocker tamoxifen on the RGCs was also evaluated. It was found that E2 stabilizes the mitochondrial membrane potential, reduces intracellular ROS levels, up-regulates Bcl-2 expression, inhibits Bax expression, decreases the generation of cytochrome C, and finally reduces the apoptosis of RGC-5 cells in a high-glucose environment. These protective functions could be attributed to E2 receptors, which make E2 a prospective patent application candidate in the treatment of DR. Furthermore, when cells were pre-cultured with tamoxifen, we found that tamoxifen inhibited the shielding effects of E2. E2 has a broad application prospect in the treatment of DR.

Ganglion cell and inner plexiform layer thickness determined by spectral domain optical coherence tomography in patients with brain lesions

PurposeTo evaluate the thickness of the macular ganglion cell and inner plexiform layer (GCIPL) using spectral domain optical coherence tomography (SD OCT) in patients with brain lesions.MethodsThis case-control study included...

Hemifield pattern electroretinogram in ocular hypertension: comparison with frequency doubling technology and optical coherence tomography to detect early optic neuropathy.

BackgroundTo assess the sensitivity and specificity of hemifield pattern electroretinogram (HF-PERG) for detecting early retinal ganglion cell (RGC) damage in ocular hypertensive (OH) patients.MethodsFifty-two OH patients (mean age 56±9.6 years) with an intraocular pressure (IOP) .21 mmHg were assessed. All subjects underwent HF-PERG, optical coherence tomography (OCT), and frequency doubling technology (FDT) visual field.ResultsOH patients showed a significant increase of peak-time of the N95 (P=0.027) compared to controls. The amplitude of the N95 of the lower and upper HF-PERG showed significant differences (P=0.037 and P=0.023, respectively) between the two groups. A significant intraocular (P=0.006) and interocular (P=0.018) asymmetry of N95 amplitude was found. Receiver operating characteristic (ROC) curve analysis revealed a sensitivity of 93% for the N95 of the lower HF-PERG, whereas full-field pattern electroretinogram (PERG) N95 peak-time had a sensitivity of 88%. In OH patients, we found a thinning of OCT - retinal nerve fiber layer (RNFL), especially in the superior and inferior quadrant, although not statistically significant, and a significantly higher FDT pattern standard deviation (FDT-PSD; P=0.001). In the OCT-RNFL inferior quadrant, a sensitivity of 82% was recorded. Finally, the sensitivity of the FDT-PSD was 92%.ConclusionOur study shows that HF-PERG is a very sensitive test for detecting early damage of the RGC.

Hemifield patter electroretinogram and macular thickness asymmetry parameters in ocular hypertension patients

AbstractPurpose To assess which is the most sensitive and specific exam for detecting an early retinal ganglion cells (RGC) damage between hemifield test pattern electroretinogram (PERG HF) and the posterior pole asymmetry analysis with optical coherence tomography (OCT‐PPAA) in ocular hypertensive patients (OH).Methods Forty‐two OH patients (mean age of 56.0±5.6 yrs.) with an intraocular pressure (IOP)&gt;21mmHg and forty healthy controls (mean age of 55.2±6.4 yrs.) with IOP&lt;21mmHg were assessed. All patients had normal visual acuity, normal optic disc appearance and normal standard achromatic perimetric (SAP) indices. All subjects underwent an OCT‐PPAA with the OCT‐Spectralis and PERG HF examination. Data were analyzed with Wilcoxon test and Receiver Operating Characteristic (ROC) curve analysis taking p &lt; 0.05 as significant.Results We found that OH patients compared to healthy controls with full field PERG had a not significant decrease of the N95 wave amplitude (p&lt;0.097) with a significant increase of its peak‐time (103.88 ± 11.11 vs 92.78 ± 4.30 msec; p&lt;0.027). With PERG HF we found a significant intraeye asymmetry of the N95 wave (‐1.99±1.58 vs ‐0.67± 0.35 µVolt; p&lt;0.023). With OCT‐PPAA in OH patients we found a decrease in macular thickness but not a statistical significant intraeye hemispheric asymmetry (3.94± 2.89 vs 5.98±4.74 µ; p&lt;0.116). ROC curve analysis revealed a sensitivity of 80% and specificity of 87.6% for PERG HF whereas by using OCT‐PPAA we found a sensitivity of 70% and a specificity of 74%.Conclusion Our study shows that at the moment the PERG HF is the most sensitive and specific technique than OCT‐PPAA for detecting early RGC damages in eyes with ocular hypertension.

FRO 2014: MMP‐2 as a potential benefactor in zebrafish optic nerve regeneration

AbstractThe zebrafish retinotectal system proves to be a valuable research model to investigate regenerative strategies of damaged retinal ganglion cells (RGCs) and thus holds great promise towards identifying new regenerative molecules. Recent research performed in our lab shows that retinal broad‐spectrum MMP inhibition seriously reduces retinotectal regeneration after optic nerve crush (ONC) in the adult zebrafish. Based on preliminary results, we now intend to determine the exact role of MMP‐2 in zebrafish RGC axonal regrowth and identify underlying molecules, which might ultimately serve as new therapeutic targets for the injured mammalian CNS. Hereto, we will pursue the following goals: (1) Determination of the regenerative potential of RGCs after retinal MMP‐2 inhibition/knockdown in zebrafish subjected to ONC. (2) Proteomic identification of MMP‐2 regulated pathways underlying a successful regeneration in the zebrafish visual system.

The role of calpain in an in vivo model of oxidative stress-induced retinal ganglion cell damage

PurposeIn this study, we set out to establish an in vivo animal model of oxidative stress in the retinal ganglion cells (RGCs) and determine whether there is a link between oxidative stress in the RGCs and the activation of calpain, a major part of the apoptotic pathway. Materials and methodsOxidative stress was induced in the RGCs of C57BL/6 mice by the intravitreal administration of 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH, 30mM, 2μl). Control eyes were injected with 2μl of vehicle. Surviving Fluorogold (FG)-labeled RGCs were then counted in retinal flat mounts. Double staining with CellROX and Annexin V was performed to investigate the co-localization of free radical generation and apoptosis. An immunoblot assay was used both to indirectly evaluate calpain activation in the AAPH-treated eyes by confirming α-fodrin cleavage, and also to evaluate the effect of SNJ-1945 (a specific calpain inhibitor: 4% w/v, 100mg/kg, intraperitoneal administration) in these eyes. ResultsIntravitreal administration of AAPH led to a significant decrease in FG-labeled RGCs 7days after treatment (control: 3806.7±575.2RGCs/mm2, AAPH: 3156.1±371.2RGCs/mm2, P<0.01). CellROX and Annexin V signals were co-localized in the FG-labeled RGCs 24h after AAPH injection. An immunoblot assay revealed a cleaved α-fodrin band that increased significantly 24h after AAPH administration. Intraperitoneally administered SNJ-1945 prevented the cleavage of α-fodrin and had a neuroprotective effect against AAPH-induced RGC death (AAPH: 3354.0±226.9RGCs/mm2, AAPH+SNJ-1945: 3717.1±614.6RGCs/mm2, P<0.01). ConclusionAAPH administration was an effective model of oxidative stress in the RGCs, showing that oxidative stress directly activated the calpain pathway and induced RGC death. Furthermore, inhibition of the calpain pathway protected the RGCs after AAPH administration.

A rat experimental model of glaucoma incorporating rapid-onset elevation of intraocular pressure.

Glaucoma is a chronic disease that causes structural and functional damage to retinal ganglion cells (RGC). The currently employed therapeutic options are not sufficient to prevent vision loss in patients with glaucoma; therefore, there is a need to develop novel therapies, which requires the creation of functional, repeatable and easy-to-utilize animal models for use in pre-clinical studies. The currently available models ensure only low to moderate damage in optic nerves, with high variation in the outcomes and poor repeatability. We have developed an effective and reproducible rat glaucoma model based on a previous idea for a “Bead Model” in mice, which could be useful in future glaucoma research. Additionally, in an attempt to achieve rapid elevation of Intraocular Pressure (IOP), we included an initial “high-pressure injury” as part of this method, which serves as the equivalent of a severe glaucoma attack. These modifications made it possible to achieve longer lasting IOP elevation with chronic damage of retinal ganglion cells.