Number Of Retinal Ganglion Cells Research Articles

Algae Oil Treatment Protects Retinal Ganglion Cells (RGCs) via ERK Signaling Pathway in Experimental Optic Nerve Ischemia.

Background: We investigated the therapeutic effects and related mechanisms of algae oil (ALG) to protect retinal ganglion cells (RGCs) in a rat model of anterior ischemic optic neuropathy (rAION). Methods: Rats were daily gavaged with ALG after rAION induction for seven days. The therapeutic effects of ALG on rAION were evaluated using flash visual evoked potentials (FVEPs), retrograde labeling of RGCs, TUNEL assay of the retina, and ED1 staining of optic nerves (ONs). The levels of inducible nitric oxide synthase (iNOS), IL-1β, TNF-α, Cl-caspase-3, ciliary neurotrophic factor (CNTF), and p-ERK were analyzed by using western blots. Results: Protection of visual function in FVEPs amplitude was noted, with a better preservation of the P1–N2 amplitude in the ALG-treated group (p = 0.032) than in the rAION group. The density of RGCs was 2.4-fold higher in the ALG-treated group compared to that in the rAION group (p < 0.0001). The number of ED1-positive cells in ONs was significantly reduced 4.1-fold in the ALG-treated group compared to those in the rAION group (p = 0.029). The number of apoptotic RGCs was 3.2-fold lower in number in the ALG-treated group (p = 0.001) than that in the rAION group. The ALG treatment inhibited ERK activation to reduce the levels of iNOS, IL-1β, TNF-α, and Cl-caspase-3 and to increase the level of CNTF in the rAION model. Conclusion: The treatment with ALG after rAION induction inhibits ERK activation to provide both anti-inflammatory and antiapoptotic effects in rAION.

Expression and role of autophagy related protein p62 and LC3 in the retina in a rat model of acute ocular hypertension.

To investigate the expression and possible role of the autophagy related protein p62 and LC3 in the retina based on a rat model of acute ocular hypertension. Fifty rats were randomized into five groups: control group A, B, C, and D. Groups A to D all received normal saline perfusion into the anterior chamber with pressure of 80 mm Hg for one hour, and retina tissue was obtained at 6, 12, 24 and 48h after perfusion respectively, to investigate the activation of autophagy following ischemia-reperfusion. The distribution and semi-quantification of autophagy related protein p62 and LC3 in the retina were detected using immunohistochemistry technique. The expression level of these two proteins was evaluated using Western blot. The number of retinal ganglion cells (RGCs) decreased with increasing reperfusion time, and significant reduction in the retinal thickness was observed 48h after perfusion. In normal adult rats, LC3 protein was mainly expressed in the ganglion cell layer (GCL), and p62 protein was expressed in the nerve fiber layer (NFL), GCL, inner plexiform layer (IPL), inner nuclear layer (INL) and outer plexiform layer (OPL). In comparison to the control group, the expression level of LC3- II was higher in all the experimental groups (P<0.05), with the peak expression at 12h after reperfusion. Additionally, the expression level of p62 was higher in all the experimental groups than the control (P<0.05, except for group A), with the peak level occurred 24h after reperfusion. Both p62 and LC3 show low level and uneven expression in the retina of normal adult rats. Acute ocular hypertension can lead to upregulation of LC3- II and p62 expression in the retina. Autophagy flux is damaged 12h after reperfusion, potentially resulting in further loss of RGCs.

Carvedilol Promotes Retinal Ganglion Cell Survival Following Optic Nerve Injury via ASK1-p38 MAPK Pathway.

Carvedilol, which is considered as a nonselective β-adrenoreceptor blocker, has many pleiotropic activities. It also causes great impact on neuroprotection because of its antioxidant ability, which suggested that carvedilol may be effective in protecting RGCs from increased oxidative stress. To examine the effects of carvedilol on preventing Retinal Ganglion Cell (RGC) death in a mouse model of Optic Nerve Injury (ONI). C57BL/6J mice were subjected to Optic Nerve Injury (ONI) model and treated with carvedilol or placebo. Histological and morphometric studies were performed; the RGC number, the amount of neurons in the ganglion cell layer and the thickness of the Inner Retinal Layer (IRL) was quantified. The average thickness of Ganglion Cell Complex (GCC) was determined by the Spectral- Domain OCT (SD-OCT) assay. Immunohistochemistry, western blot and quantitative real-time PCR analysis were also applied. Daily treatment of carvedilol reduced RGC death following ONI, and in vivo retinal imaging revealed that carvedilol can effectively prevent retinal degeneration. The expression of chemokines important for micorglia recruitment was deceased with carvedilol ingestion and the accumulation of retinal microglia is reduced consequently. In addition, the ONI-induced expression of inducible nitric oxide synthase in the retina was inhibited with carvedilol treatment in the retina. We also discovered that carvedilol suppressed ONI-induced activation of Apoptosis Signal-regulating Kinase-1 (ASK1) and p38 Mitogen-Activated Protein Kinase (MAPK) pathway. The results of this study indicate that carvedilol can stimulate neuroprotection and neuroregeneration, and may be useful for treatment of various neurodegenerative diseases.

Protective effects of autophagy inhibitor 3-methyladenine on ischemia-reperfusion-induced retinal injury.

To investigate the protective effects of autophagy inhibitor 3-methyladenine (3-MA) in a rat model of ischemic-reperfusion injury (IRI). Forty Sprague-Dawley male rats (weight 220-250g) were randomly divided into four groups: a control group (NC, n = 10), a Sham surgery group (n = 10), an IRI group (n = 10), and a 3-MA-treated IRI group [10 μL 3-MA (10mmol/L) was injected in vitreous after the injury, n = 10]. The retinal IRI was induced by elevating the intraocular pressure to 110mmHg for 60min. Hematoxylin and eosin (HE) staining was used to calculate the number of retinal ganglion cells (RGCs). The level of microtubule-associated protein 1A/1B light chain 3 (LC3), Beclin-1, and Caspase-3 in the retina was detected using the immunofluorescence staining method. The LC3, Beclin-1, B-cell lymphoma/leukemia-2 (Bcl-2), and Caspase-3 protein levels were examined by Western blotting. The number of RGCs in IRI group was significantly lower than that in NC group (P < 0.05), demonstrated by HE staining. Western blotting results indicated that the protein expression of LC3 and Beclin-1 in the IRI group was significantly elevated compared with those in the NC group (P < 0.05). However, with 3-MA treatment, the number of RCGs in 3-MA-treated IRI group was elevated and protein levels of LC3, Beclin-1 were down-regulated, compared with those in the IRI group (P < 0.05). Further immunohistochemistry staining and Western blot showed that 3-MA-treated IRI group presented down-regulated Caspase-3 and up-regulated Bcl-2 protein expression with comparison of IRI group (P < 0.05). Retina IRI-caused RGCs loss involved activated autophagy pathway and apoptosis, which could be prevented by autophagy inhibitor 3-MA. Autophagy inhibitor 3-MA may act as a potent therapeutic tool in attenuating retina IRI.

Intravitreal application of AAV-BDNF or mutant AAV-CRMP2 protects retinal ganglion cells and stabilizes axons and myelin after partial optic nerve injury

Secondary degeneration following an initial injury to the central nervous system (CNS) results in increased tissue loss and is associated with increasing functional impairment. Unilateral partial dorsal transection of the adult rat optic nerve (ON) has proved to be a useful experimental model in which to study factors that contribute to secondary degenerative events. Using this injury model, we here quantified the protective effects of intravitreally administered bi-cistronic adeno-associated viral (AAV2) vectors encoding either brain derived neurotrophic factor (BDNF) or a mutant, phospho-resistant, version of collapsin response mediator protein 2 (CRMP2T555A) on retinal ganglion cells (RGCs), their axons, and associated myelin. To test for potential synergistic interactions, some animals received combined injections of both vectors. Three months post-injury, all treatments maintained RGC numbers in central retina, but only AAV2-BDNF significantly protected ventrally located RGCs exclusively vulnerable to secondary degeneration. Behaviourally, treatments that involved AAV2-BDNF significantly restored the number of smooth-pursuit phases of optokinetic nystagmus. While all therapeutic regimens preserved axonal density and proportions of typical complexes, including heminodes and single nodes, BDNF treatments were generally more effective in maintaining the length of the node of Ranvier in myelin surrounding ventral ON axons after injury. Both AAV2-BDNF and AAV2-CRMP2T555A prevented injury-induced changes in G-ratio and overall myelin thickness, but only AAV2-BDNF administration protected against large-scale myelin decompaction in ventral ON. In summary, in a model of secondary CNS degeneration, both BDNF and CRMP2T555A vectors were neuroprotective, however different efficacies were observed for these overexpressed proteins in the retina and ON, suggesting disparate cellular and molecular targets driving responses for neural repair. The potential use of these vectors to treat other CNS injuries and pathologies is discussed.

Astragalus membranaceus Injection Protects Retinal Ganglion Cells by Regulating the Nerve Growth Factor Signaling Pathway in Experimental Rat Traumatic Optic Neuropathy.

Activation of the nerve growth factor (NGF) signaling pathway is a potential method of treatment for retinal ganglion cell (RGC) loss due to traumatic optic neuropathy (TON). The present study aimed to explore the biological effects of injecting Astragalus membranaceus (A. mem) on RGCs in an experimental TON model. Adult male Wistar rats were randomly divided into three groups: sham-operated (SL), model (ML), and A. mem injection (AL). The left eyes of the rats were considered the experimental eyes, and the right eyes served as the controls. AL rats received daily intraperitoneal injections of A. mem (3 mL/kg), whereas ML and SL rats were administered the same volume of normal saline. The TON rat model was induced by optic nerve (ON) transverse quantitative traction. After two-week administration, the number of RGCs was determined using retrograde labeling with Fluoro-Gold. The protein levels of NGF, tyrosine kinase receptor A (TrkA), c-Jun N-terminal protein kinase (JNK), JNK phosphorylation (p-JNK), and nuclear factor kappa-B (NF-κB) were assessed using western blotting. The levels of p75 neurotrophin receptor (p75NTR) and NF-κB DNA binding were examined using real-time PCR and an electrophoretic mobility shift assay. In addition, the concentrations of JNK and p-JNK were assessed using an enzyme-linked immunosorbent assay. Results. The number of RGCs in ML was found to be significantly decreased (P < 0.01) relative to both AL and SL, together with the downregulation of NGF (P < 0.01), TrkA (P < 0.05), and NF-κB (P < 0.01); upregulation of p75NTR mRNA (P < 0.01); and increased protein levels of JNK (P < 0.05) and p-JNK (P < 0.05). Treatment using A. mem injection significantly preserved the density of RGCs in rats with experimental TON and markedly upregulated the proteins of NGF (P < 0.01), TrkA (P < 0.05), and NF-κB (P < 0.01) and downregulated the mRNA level of p75NTR(P < 0.01), as well as the proteins of JNK (P < 0.05) and p-JNK (P < 0.01). Thus, A. mem injection could reduce RGC death in TON induced by ON transverse quantitative traction by stimulating the NGF signaling pathway.

Fortified S-Allyl L-Cysteine: Animal Safety, Effect on Retinal Ischemia, and Role of Wnt in the Underlying Therapeutic Mechanism.

Purpose Retinal ischemia is a medical condition associated with numerous retinal vascular disorders, such as age-related macular degeneration, glaucoma, and diabetic retinopathy. This in vitro cell and in vivo animal study investigated not only the protective effect of S-allyl L-cysteine (SAC, an active component of garlic) against retinal ischemia but also its associated protective mechanisms. Methods Retinal ischemia was mimicked by raising the intraocular pressure to 120 mmHg for 1 hour in one eye. The effects of pre-/postischemic administration of vehicle vs. SAC 0.18 mg vs. SAC 0.018 mg vs. SAC 0.0018 mg treatments on retina cells were evaluated through cellular viability (MTT assay), flash electroretinograms (ERGs), and fluorogold retrograde labelling (retinal ganglion cell (RGC) counting). Also, protein immunoblot was utilized to assess the role of Wnt, hypoxia inducible factor (HIF)-1α, and vascular endothelium factor (VEGF) in the proposed anti-ischemic mechanism. Lastly, the safety of drug consumption was investigated for changes in the animal's body weight, ERG waves, and blood biochemical parameters (e.g., glucose levels). Results The characteristic ischemic changes including significant reduction in ERG b-wave ratio and RGC number were significantly counteracted by pre- and postischemic low dose of SAC. Additionally, ischemia-induced overexpression of Wnt/HIF-1α/VEGF protein was ameliorated significantly by preischemic low dose of SAC. In terms of the animal safety, no significant body weight and electrophysiological differences were observed among defined different concentrations of SAC without following ischemia. In low SAC dosage and vehicle groups, various blood biochemical parameters were normal; however, high and medium concentrations of SAC significantly lowered the levels of uric acid, Hb, and MCHC. Conclusion This study shows that preischemic administration of low SAC dosage has been proved to be safe and most effective against rat retinal ischemia electrophysiologically and/or histopathologically. Moreover, counteracting the ischemia-induced overexpression of Wnt/HIF-1α/VEGF might presently explain SAC's anti-ischemic mechanism.

Protective Efficacy of a Dietary Supplement Based on Forskolin, Homotaurine, Spearmint Extract, and Group B Vitamins in a Mouse Model of Optic Nerve Injury.

Glaucoma is a multifactorial blinding disease with a major inflammatory component ultimately leading to apoptotic retinal ganglion cell (RGC) death. Pharmacological treatments lowering intraocular pressure can help slow or prevent vision loss although the damage caused by glaucoma cannot be reversed. Recently, nutritional approaches have been evaluated for their efficacy in preventing degenerative events in the retina although mechanisms underlying their effectiveness remain to be elucidated. Here, we evaluated the efficacy of a diet supplement consisting of forskolin, homotaurine, spearmint extract, and vitamins of the B group in counteracting retinal dysfunction in a mouse model of optic nerve crush (ONC) used as an in vivo model of glaucoma. After demonstrating that ONC did not affect retinal vasculature by fluorescein angiography, we determined the effect of the diet supplement on the photopic negative response (PhNR) whose amplitude is strictly related to RGC integrity and is therefore drastically reduced in concomitance with RGC death. We found that the diet supplementation prevents the reduction of PhNR amplitude (p < 0.001) and concomitantly counteracts RGC death, as in supplemented mice, RGC number assessed immunohistochemically is significantly higher than that in non-supplemented animals (p < 0.01). Major determinants of the protective efficacy of the compound are due to a reduction of ONC-associated cytokine secretion leading to decreased levels of apoptotic markers that in supplemented mice are significantly lower than in non-supplemented animals (p < 0.001), ultimately causing RGC survival and ameliorated visual dysfunction. Overall, our data suggest that the above association of compounds plays a neuroprotective role in this mouse model of glaucoma thus offering a new perspective in inflammation-associated neurodegenerative diseases of the inner retina.

Intravitreal Triamcinolone Acetonide Injection in a Rodent Model of Anterior Ischemic Optic Neuropathy: Response.

We would like to thank Dr. Harvey for his interest in our study titled “Intravitreal triamcinolone acetonide injection in a rodent model of anterior ischemic optic neuropathy” (1). He points out the limitations of using a rodent model to investigate anterior ischemic optic neuropathy (AION). We are in agreement that animal models will always fall short of studying human disease. However, they are often the most appropriate first step. There is no question that rodents have different responses to tissue injury than do primates. We also agree that, unlike AION in humans, the current animal models of AION, both rodent and nonhuman primate, are potentially different from human AION. With all animal models, we know exactly when damage occurred; however, with humans, we do not know when damage began—only when symptoms were first detected. Positive studies are of most value. If drug studies in rodents and other lower animals (e.g., rodents, rabbits, and hamsters) work, then we have a basis on which to test drugs in nonhuman primates or, in some cases, directly in humans. However, if they do not work, we still do not know with certainty that they would not work in humans. Such negative studies do have value in that they serve as essential building blocks, providing direction for future studies and at times justification for funding. Dr. Harvey voices several specific concerns. He asserts that laser-induced optic AION should be viewed as “a homolog” to end-stage AION. This presumption may or may not be accurate. But, even if accurate, our findings would still suggest that steroids lack benefit at this stage of disease, which may be at or shortly after appreciation of visual symptoms. He further argues that edema may play a role in the pathophysiology of AION, at different phases of the disease, and in some cases, preceding infarction. This likely is correct, and it is possible that steroids could have benefit in a subset of patients, specifically those with progressive axonal ischemia related to secondary compartmental compression. As we stated in our discussion section, we agree that our findings do not negate the possibility that steroids may have benefit in all or possibly a subset of human patients. In closing, we agree with Dr. Harvey that findings in a rodent model of AION far from perfectly parallel human disease. However, it would be unwise to assume animal studies such as this are entirely without value. They are, in many cases, the most appropriate initial step. We maintain that our data are sufficient to exclude a significant impact of intravitreal triamcinolone, with a final intravitreal concentration of 1 mg/mL, on retinal ganglion cells number in rodent eyes with nonarteritic ischemic optic neuropathy (NAION). Our findings do not exclude the possibility that anti-inflammatory therapy may be beneficial in all or in subsets of human patients with NAION. A detailed account of our study's limitations and implications comprises the bulk of the discussion section of the article (1). We are hopeful that these data will benefit future the design of more focused studies.

Enriched environment provides neuroprotection against experimental glaucoma.

Glaucoma is one of the most frequent causes of visual impairment worldwide, and involves selective damage to retinal ganglion cells (RGCs) and their axons. We analyzed the effect of enriched environment (EE) housing on the optic nerve, and retinal alterations in an induced model of ocular hypertension. For this purpose, male Wistar rats were weekly injected with vehicle or chondroitin sulfate (CS) into the eye anterior chamber for 10weeks and housed in standard environment or EE. EE housing prevented the effect of experimental glaucoma on visual evoked potentials, retinal anterograde transport, phosphorylated neurofilament-immunoreactivity, axon number, microglial/macrophage reactivity (ionized calcium binding adaptor molecule 1-immunoreactivity), and astrocytosis (glial fibrillary acidic protein-immunostaining), as well as oligodendrocytes alterations (luxol fast blue staining, and myelin basic protein-immunoreactivity) in the proximal portion of the optic nerve. Moreover EE prevented the increase in ionized calcium binding adaptor molecule-1 levels, and RGC loss (Brn3a-immunoreactivity) in the retina from hypertensive eyes. EE increased retinal brain-derived neurotrophic factor levels. When EE housing started after 6weeks of ocular hypertension, a preservation of visual evoked potentials amplitude, axon, and Brn3a(+) RGC number was observed. Taken together, these results suggest that EE preserved visual functions, reduced optic nerve axoglial alterations, and protected RGCs against glaucomatous damage.

JIP1 Deficiency Protects Retinal Ganglion Cells From Apoptosis in a Rotenone-Induced Injury Model.

Retinal ganglion cells (RGCs) undergo apoptosis after injury. c-Jun N-terminal kinase (JNK)-interacting protein 1 (JIP1) is a scaffold protein that is relevant to JNK activation and a key molecule known to regulate neuronal apoptosis. However, the specific role of JIP1 in the apoptosis of RGCs is currently undefined. Here, we used JIP1 gene knockout (KO) mice to investigate the importance of JIP1-JNK signaling in the apoptosis of RGCs in a rotenone-induced injury model. In adult JIP1 KO mice, the number and electrophysiological functions of RGCs were not different from those of wild-type (WT) mice. Ablation of JIP1 attenuated the activation of JNK and the cleavage of caspase-3 in the retina after rotenone injury and contributed to a lower number of TUNEL-positive RGCs, a greater percentage of surviving RGCs, and a significant reduction in the electrophysiological functional loss of RGCs when compared to those in WT controls. We also found that JIP1 was located in the neurites of primary RGCs, but accumulated in soma in response to rotenone treatment. Moreover, the number of TUNEL-positive RGCs, the level of activation of JNK and the rate of cleavage of caspase-3 were reduced in primary JIP1-deficient RGCs after rotenone injury than in WT controls. Together, our results demonstrate that the JIP1-mediated activation of JNK contributes to the apoptosis of RGCs in a rotenone-induced injury model in vitro and in vivo, suggesting that JIP1 may be a potential therapeutic target for RGC degeneration.

HIF inhibitor topotecan has a neuroprotective effect in a murine retinal ischemia-reperfusion model.

PurposeThe therapeutic approach for retinal ganglion cell (RGC) degeneration has not been fully established. Recently, it has been reported that hypoxia-inducible factor (HIF) may be involved with retinal neurodegeneration. In this study, we investigated neuroprotective effects of a HIF inhibitor against RGC degeneration induced in a murine model of retinal ischemia-reperfusion (I/R).MethodsEight-weeks-old male C57/BL6J mice were treated with intraperitoneal injection of a HIF inhibitor topotecan (1.25 mg/kg) for 14 days followed by a retinal I/R procedure. Seven days after the I/R injury, the therapeutic effect was evaluated histologically and electrophysiologically.ResultsThe increase of HIF-1α expression and the decrease of retinal thickness and RGC number in I/R were significantly suppressed by administration of topotecan. Impaired visual function in I/R was improved by topotecan evaluated with electroretinogram and visual evoked potentials.ConclusionsTopotecan administration suppressed HIF-1a expression and improved RGC survival resulting in a functional protection against retinal I/R. These data indicated that the HIF inhibitor topotecan may have therapeutic potentials for RGC degeneration induced with retinal ischemia or high intraocular pressure.

Numbers of Retinal Ganglion Cells in Mice with Genetic Defects in Different A1 Adrenoreceptor Subtypes

Objective. To perform a comparative study of the numbers of retinal ganglion cells (RGC) in mice with genetic defects affecting different subtypes of α1 adrenoreceptors. Materials and methods. Studies were run on 36 male laboratory mice aged over 18 months with genetic defects affecting one α1 adrenoreceptor subtype (α1a, α1b, α1d). The control group consisted of intact laboratory mice (C57Bl/6NTac) of the same age and sex distribution. An immunofluorescent method was used for differential visualization of cells in retinal wholemount preparations using the Brn3a marker allowing these cells to be counted. Results. Mice with genetic defects of α1b and α1d adrenoreceptors had more and mice with defective α1a adrenoreceptors had fewer RGC than intact mice of the same age and sex. The total numbers of retinal cells in mice of all the study strains were essentially identical. Conclusions. The results obtained here provide evidence that each α1 adrenoreceptor subtype (α1a, α1b, α1d) makes a specific contribution during the process of forming retinal topography.

Soriano award lecture: Light, circadian rhythms and sleep: Mechanisms to new therapeutics

Until recently, it seemed inconceivable to most vision researchers and ophthalmologists that there could be an unrecognised class of light sensor (photoreceptor) within the eye. However, by studying how circadian rhythms and sleep are regulated by the dawn/dusk cycle we demonstrated that there exists a “3rd class” of photoreceptor within the eye based upon a small number of photosensitive retinal ganglion cells (pRGCs) that utilise the blue light sensitive photopigment melanopsin (OPN4). The first part of the presentation will consider the discovery and clinical importance of the “third” photoreceptor system of the eye. The second part of the talk will address how these photoreceptors signal light to the molecular clockwork and by what means this pathway is modified by sleep history. There has been remarkable progress in understanding the complex intracellular mechanisms that generate circadian rhythms, the molecular pathways whereby the pRGCs entrain circadian biology and sleep has remained poorly understood. The suprachiasmatic nuclei (SCN) are the site of the primary circadian pacemakers within the mammalian brain. Until recently, the model for entrainment involved a simple linear pathway whereby glutamate release from the pRGCs resulted in Ca2+ influx and raised intracellular cAMP in SCN neurones, which in turn resulted in CREB phosphorylation leading to increased transcription of two key clock genes, Per1 and Per2. This signal then advanced or delayed the molecular clockwork. However, an important feature of entrainment is that circadian responses to light are limited – as typified by jet-lag. Full recovery from jet-lag requires a day for every time-zone crossed. We addressed this issue and have identified and characterized a key role for Salt Inducible Kinase 1 (SIK1) and the CREB-regulated transcription co-activator 1 (CRTC1) in clock re-setting. However, our most recent findings have shown that light entrainment also involves the parallel activation of a Ca2+-ERK1/2-AP-1 signalling pathway. Thus both CRE and AP-1 regulatory elements drive light-induced clock gene expression. In addition, whilst light activation of the Ca2+-ERK1/2-AP-1 signalling pathway increases Per1 and Per2 expression, sleep/wake behaviour alters the effects of light on the clock. Our proposed mechanism suggests that adenosine acts as a signalling molecule that encodes wake duration. Adenosine acts via inhibitory A1 receptors on the SCN to inhibit the Ca2+-ERK1/2-AP-1 signalling pathway, which in turn, reduces the expression of Per1 and Per2. Thus sleep/wake history, encoded by adenosine, reduces the phase shifting effects of light upon the circadian system, altering sleep/wake timing. Finally, the talk will explore how such signalling pathways provide a new target for the regulation of circadian rhythms and the “pharmacological” replacement of light for sleep/wake re-setting in individuals lacking eyes or other individuals with severe circadian rhythm disruption.

β-alanine supplementation induces taurine depletion and causes alterations of the retinal nerve fiber layer and axonal transport by retinal ganglion cells

To study the effect of taurine depletion induced by β-alanine supplementation in the retinal nerve fiber layer (RNFL), and retinal ganglion cell (RGC) survival and axonal transport. Albino Sprague-Dawley rats were divided into two groups: one group received β-alanine supplementation (3%) in the drinking water during 2 months to induce taurine depletion, and the other group received regular water. After one month, half of the rats from each group were exposed to light. Retinas were analyzed in-vivo using Spectral-Domain Optical Coherence Tomography (SD-OCT). Prior to processing, RGCs were retrogradely traced with fluorogold (FG) applied to both superior colliculi, to assess the state of their retrograde axonal transport. Retinas were dissected as wholemounts, surviving RGCs were immunoidentified with Brn3a, and the RNFL with phosphorylated high-molecular-weight subunit of the neurofilament triplet (pNFH) antibodies. β-alanine supplementation decreases significantly taurine plasma levels and causes a significant reduction of the RNFL thickness that is increased after light exposure. An abnormal pNFH immunoreactivity in some RGC bodies, their proximal dendrites and axons, and a further diminution of the mean number of FG-traced RGCs compared with Brn3a+RGCs, indicate that their retrograde axonal transport is affected. In conclusion, taurine depletion causes RGC loss and axonal transport impairment. Finally, our results suggest that care should be taken when ingesting β-alanine supplements due to the limited understanding of their potential adverse effects.

Mitochondria and Autophagy Dysfunction in Glucocorticoid-Induced Ocular Hypertension/Glaucoma Mice Model

ABSTRACTPurpose: This study is aimed to investigate the effects of periocular steroids induction on intraocular pressure (IOP), retinal ganglion cells (RGCs) and trabecular meshwork (TM) ultrastructure in glucocorticoid-induced ocular hypertension mice model.Materials and Methods: Dexamethasone-21-acetate (Dex-Ace) was administered through periocular conjunctival fornix injection every 3 days in C57BL/6J mice. Intraocular pressure was measured weekly by rebound tonometry. RGCs were examined with immunofluorescent staining of BRN3a at week 1, 4, and 8. TM morphology was visualized with electron microscopy. Autophagy was evaluated with immunoblotting in TM tissues.Results: Dex-Ace rapidly and significantly induced IOP, which peaked at week 4. The absolute increase in IOP in the Dex-Ace-treated mice was 8.1 ± 1.4 mmHg, a 60% induction (p < .0001) compared with that in the vehicle-treated mice. The IOP sustained a higher level in the Dex-Ace group from week 4 to week 8. Dex-Ace treatment decreased the number of RGCs in a time-dependent manner, suggesting that high IOP resulted in optic neuropathy. In addition, Dex-Ace thickened trabecular beams and decreased intertrabecular spaces, with marked accumulation of fibrillar and amorphous granular extracellular material. Moreover, Dex-Ace induced swollen and elongated mitochondria in TM cells. The average mitochondria area was 0.090 ± 0.044 µm2 in the vehicle-treated mice, and increased to 0.161 ± 0.094 µm2 (p < .0001), 0.121 ± 0.029 µm2 (p = .0223) and 0.171 ± 0.076 µm2 (p < .0001) in the Dex-Ace-treated mice at weeks 1, 4 and 8, respectively. Autophagy was also increased by Dex-Ace treatment, indicating by the upregulation of LC3-I, LC3-II and beclin-1, and downregulation of p62.Conclusion: Dex-Ace administration decreased RGCs and changed TM ultrastructure, mimicking hallmarks of human glucocorticoid-induced glaucoma (GIG). In addition, mitochondria and autophagy dysfunction suggested abnormal energy metabolism in TM cells, which warranted further study to fully elucidate the pathogenesis of GIG.

Effects of siRNA-Mediated Knockdown of GSK3β on Retinal Ganglion Cell Survival and Neurite/Axon Growth.

There are contradictory reports on the role of the serine/threonine kinase isoform glycogen synthase kinase-3β (GSK3β) after injury to the central nervous system (CNS). Some report that GSK3 activity promotes axonal growth or myelin disinhibition, whilst others report that GSK3 activity prevents axon regeneration. In this study, we sought to clarify if suppression of GSK3β alone and in combination with the cellular-stress-induced factor RTP801 (also known as REDD1: regulated in development and DNA damage response protein), using translationally relevant siRNAs, promotes retinal ganglion cell (RGC) survival and neurite outgrowth/axon regeneration. Adult mixed retinal cell cultures, prepared from rats at five days after optic nerve crush (ONC) to activate retinal glia, were treated with siRNA to GSK3β (siGSK3β) alone or in combination with siRTP801 and RGC survival and neurite outgrowth were quantified in the presence and absence of Rapamycin or inhibitory Nogo-A peptides. In in vivo experiments, either siGSK3β alone or in combination with siRTP801 were intravitreally injected every eight days after ONC and RGC survival and axon regeneration was assessed at 24 days. Optimal doses of siGSK3β alone promoted significant RGC survival, increasing the number of RGC with neurites without affecting neurite length, an effect that was sensitive to Rapamycin. In addition, knockdown of GSK3β overcame Nogo-A-mediated neurite growth inhibition. Knockdown of GSK3β after ONC in vivo enhanced RGC survival but not axon number or length, without potentiating glial activation. Knockdown of RTP801 increased both RGC survival and axon regeneration, whilst the combined knockdown of GSK3β and RTP801 significantly increased RGC survival, neurite outgrowth, and axon regeneration over and above that observed for siGSK3β or siRTP801 alone. These results suggest that GSK3β suppression promotes RGC survival and axon initiation whilst, when in combination with RTP801, it also enhanced disinhibited axon elongation.

Therapeutic Effects of Puerarin Against Anterior Ischemic Optic Neuropathy Through Antiapoptotic and Anti-Inflammatory Actions.

This study investigated the therapeutic effects of puerarin (PR) on a rat model of anterior ischemic optic neuropathy (rAION). The neuroprotective effects of PR on rAION were evaluated using flash visual-evoked potentials (FVEP), retrograde labeling of retinal ganglion cells (RGCs), TUNEL assay of the retina, optical coherence tomography (OCT) images of optic nerve width, and ED1 staining of the optic nerve (ON). The inflammatory response of ON and Akt signaling pathways were analyzed through Western blot. M2 polarization was determined by immunostaining and immunoblotting in ONs. In FVEP analysis, the amplitude of P1-N2 and the RGC density in the PR-treated group were 2.3- and 1.6-fold higher than those in the PBS-treated group, respectively (P < 0.05). The number of apoptotic RGC in the PR-treated group was 2.8-fold lower than that in the PBS-treated group. OCT images demonstrated that PR treatment-reduced ON edema in the acute phase compared to PBS treatment (P < 0.05). Macrophage infiltration was reduced by 5.2-fold by PR treatment compared with the PBS treatment (P < 0.05). PR treatment inhibited the levels of iNOS, IL-1β, and TNF-α, induced the levels of IL-10, Arg1, and Fizz1 in the rAION model. The levels of p-Akt1 and C/EBPβ in the PR-treated group increased by 3.4-fold and 5.89-fold compared with those in the PBS-treated group (P < 0.05). Inhibition of Akt activation reduced the number of M2 macrophage in the PR-treated group (P < 0.05). PR treatment provided the neuroprotective effects in the rAION model, which may lead to new clinical applications.

Age-Related Decline of Retinal Oxygen Extraction in Healthy Subjects.

To investigate the age-dependence of total retinal blood flow and total retinal oxygen extraction in healthy subjects and determine their possible correlations with structural optical coherence tomography (OCT) parameters. This observational cross-sectional study consisted of 68 healthy subjects (mean ± SD age, 45.6 ± 16.3 years; 47% female). Total retinal oxygen extraction was calculated based on measurement of total retinal blood flow using bi-directional Doppler OCT and measurement of oxygen saturation using spectroscopic reflectometry. Retinal nerve fiber layer thickness was measured using OCT, and the total number of retinal ganglion cells was estimated based on a previous published model. Correlation of these parameters with age was studied and the association between structural OCT parameters and hemodynamic vascular parameters was calculated. Both structural and vascular parameters showed a significant decline with increasing age. The correlation coefficients were between r = -0.25 and r = -0.41. Furthermore, structural and vascular parameters were significantly correlated with each other. The strongest association was found between the level of total retinal oxygen extraction and the number of retinal ganglion cells (r = 0.75, P < 0.001). We showed that there was an age-related decline of retinal oxygen extraction. Levels of retinal oxygen extraction are correlated to retinal nerve fiber layer thickness and number of retinal ganglion cells. Our data partially explain the wide inter-individual variability in retinal blood flow values in healthy subjects. Longitudinal studies are required to study the time course of vascular and neuronal loss in humans.

Detailed Evaluation of Possible Ganglion Cell Loss in the Retina of Zucker Diabetic Fatty (ZDF) Rats

A thinning of the inner retina is one of the earliest potential markers of neuroretinal damage in diabetic subjects. The histological background is uncertain; retinal ganglion cell (RGC) loss and changes in the structure or thickness of the inner plexiform layer (IPL) have been suspected. Studies conducted on animal models on RGC pathology gave contradictory results. Hereby we present RGC numbers, distribution patterns and IPL thickness from Zucker Diabetic Fatty (ZDF) rats. After labelling RGCs on retinal whole mounts, isodensity maps were constructed, RGC numbers and distribution patterns analysed using a custom-built algorithm, enabling point-by-point comparison. There was no change in staining characteristics of the antibodies and no significant difference in average RGC densities was found compared to controls. The distribution patterns were also comparable and no significant difference was found in IPL thickness and stratification or in the number of apoptotic cells in the ganglion cell layer (GCL). Our results provide a detailed evaluation of the inner retina and exclude major RGC loss in ZDF rats and suggest that other factors could serve as a potential explanation for inner retinal thinning in clinical studies. Our custom-built method could be adopted for the assessment of other animal or human retinas.