青光眼视网膜神经节细胞凋亡的研究进展

青光眼视神经损伤的最后共同通路为视网膜神经节细胞的凋亡。但确切机制尚未阐明。为此,人们进行了大量相关体内、体外实验并取得一定成果。本文从凋亡的激发因素、信号传导及基因调控加以阐述。     

Hypothermia Protects and Prolongs the Tolerance Time of Retinal Ganglion Cells against Ischemia

Purpose

Hypothermia has been shown to be neuroprotective in the therapy of ischemic stroke in the brain. To date no studies exist on the level of the inner retina and it is unclear if hypothermia would prolong the ischemic tolerance time of retinal ganglion cells, which are decisive in many ischemic retinopathies.

Methods

Bovine eyes were enucleated and stored either at 21°C or 37°C for 100 or 340 minutes, respectively. Afterwards the globes were dissected, the retina was prepared and either the spontaneous ganglion cell responses were measured or the retina was incubated as an organotypic culture for additional 24 hours. After incubation the retina was either processed for histology (H&E and DAPI staining) or real-time PCR (Thy-1 expression) was performed.

Results

Hypothermia prolonged ganglion cell survival up to 340 minutes under ischemic conditions. In contrast to eyes kept at 37°C the eyes stored at 21°C still showed spontaneous ganglion cell spiking (56.8% versus 0%), a 5.8 fold higher Thy-1 mRNA expression (not significant, but a trend) and a preserved retinal structure after 340 minutes of ischemia.

Conclusion

Hypothermia protects retinal ganglion cells against ischemia and prolongs their ischemic tolerance time.     

Autophagy in Retinal Ganglion Cells in a Rhesus Monkey Chronic Hypertensive Glaucoma Model

Primary open angle glaucoma (POAG) is a neurodegenerative disease characterized by physiological intraocular hypertension that causes damage to the retinal ganglion cells (RGCs). In the past, RGC damage in POAG was suggested to have been attributed to RGC apoptosis. However, in the present study, we applied a model closer to human POAG through the use of a chronic hypertensive glaucoma model in rhesus monkeys to investigate whether another mode of progressive cell death, autophagy, was activated in the glaucomatous retinas. First, in the glaucomatous retinas, the levels of LC3B-II, LC3B-II/LC3B-I and Beclin 1 increased as demonstrated by Western blot analyses, whereas early or initial autophagic vacuoles (AVi) and late or degraded autophagic vacuoles (AVd) accumulated in the ganglion cell layer (GCL) and in the inner plexiform layer (IPL) as determined by transmission electron microscopy (TEM) analysis. Second, lysosome activity and autophagosome-lysosomal fusion increased in the RGCs of the glaucomatous retinas, as demonstrated by Western blotting against lysosome associated membrane protein-1 (LAMP1) and double labeling against LC3B and LAMP1. Third, apoptosis was activated in the glaucomatous eyes with increased levels of caspase-3 and cleaved caspase-3 and an increased number of TUNEL-positive RGCs. Our results suggested that autophagy was activated in RGCs in the chronic hypertensive glaucoma model of rhesus monkeys and that autophagy may have potential as a new target for intervention in glaucoma treatment.     

Synaptic Degeneration of Retinal Ganglion Cells in a Rat Ocular Hypertension Glaucoma Model

Aims Glaucoma is a common neurodegenerative disease that affects retinal ganglion cells (RGCs) and their axons. Little is known of the synaptic degeneration involved in the pathophysiology of glaucoma. Here we used an experimental ocular hypertension model in rats to investigate this issue. Methods Elevated intraocular pressure (IOP) was induced by laser coagulation of the episcleral and limbal veins. RGCs were retrogradely labeled with Fluoro-Gold (FG). The c-fos protein was used as a neuronal connectivity marker. Expression of c-fos in the retinas was investigated by immunohistochemistry at 5 days and 2 weeks after the induction of ocular hypertension. Both surviving RGCs as revealed by retrograde FG-labeled and c-fos-labeled RGCs were counted. Results The c-fos protein was mainly expressed in the nuclei and nucleoli of cells in the ganglion cell layer and inner nuclear layer in the normal retina. We also confirmed that c-fos was also expressed in the nuclei and nucleoli of RGCs retrogradely labeled with FG. There was no significant RGC loss at 5 days but about 13% RGC loss at 2 weeks after the induction of ocular hypertension. The number of RGCs expressing c-fos was significantly lower in the experimental animals at both 5 days and 2 weeks than normal. Conclusion Our study suggests that there is synaptic disconnection for RGCs after ocular hypertension and it may precede the cell death in the early stage. It may provide insight into novel therapeutic strategies to slow the progress of glaucoma. Qing-ling Fu and Xin Li contributed equally to this work.     

The Relationship between Visual Field Index and Estimated Number of Retinal Ganglion Cells in Glaucoma

Purpose

To evaluate the relationship between visual field index (VFI) and the estimated number of retinal ganglion cells (RGCs) in glaucoma.

Methods

A multicenter study of 1,245 healthy, glaucomatous and suspected glaucomatous eyes of 1,245 subjects recruited from the Diagnostic Innovations in Glaucoma Study (DIGS) and African Descent and Glaucoma Evaluation Study (ADAGES). All eyes underwent standard automated perimetry (SAP) and time-domain optical coherence tomography (TD-OCT). Estimates of RGC count and percentage of RGCs remaining, compared to age-matched healthy eyes, were calculated from TD-OCT using a previously described formula. Smoothing spline curves were fitted to examine the relationship between VFI and the percent remaining RGCs. The first derivative (i.e., slopes) of these curves was used to explore the relationship between changes in these measures.

Results

The relationships between the VFI and both estimated RGC counts and the percent remaining RGCs were nonlinear. A unit number of VFI loss corresponded to substantially greater loss of estimated RGCs and estimated percentage of RGCs remaining in early compared to late disease.

Conclusions

The relationship between VFI and estimated RGC counts is nonlinear and the index substantially underestimates the amount of neural loss early in the disease. Disease severity should be taken into account when interpreting rates of VFI change over time.     

青光眼视网膜神经节细胞凋亡机制研究进展

青光眼是由视网膜神经节细胞(Retinal ganglion cells,RGCs)死亡引起的一种疾病,最终能导致失明。近年来,关于高眼压(elevated intraocular pressure,IOP)引发的视网膜的特定分子途径等方面的信息逐渐增多。青光眼中视网膜神经节细胞的状态取决于视网膜神经节细胞促存活和促死亡途径之间的平衡,而有关这些反应的具体机制有较多的研究,但仍只能解释部分现象。本文综述了关于视网膜神经节细胞的凋亡、凋亡通路途径及可能引发损伤条件的最新研究进展。     

Pharmacological Inhibition of Caspase-2 Protects Axotomised Retinal Ganglion Cells from Apoptosis in Adult Rats

Severing the axons of retinal ganglion cells (RGC) by crushing the optic nerve (ONC) causes the majority of RGC to degenerate and die, primarily by apoptosis. We showed recently that after ONC in adult rats, caspase-2 activation occurred specifically in RGC while no localisation of caspase-3 was observed in ganglion cells but in cells of the inner nuclear layer. We further showed that inhibition of caspase-2 using a single injection of stably modified siRNA to caspase-2 protected almost all RGC from death at 7 days, offering significant protection for up to 1 month after ONC. In the present study, we confirmed that cleaved caspase-2 was localised and activated in RGC (and occasional neurons in the inner nuclear layer), while TUNEL⁺ RGC were also observed after ONC. We then investigated if suppression of caspase-2 using serial intravitreal injections of the pharmacological inhibitor z-VDVAD-fmk (z-VDVAD) protected RGC from death for 15 days after ONC. Treatment of eyes with z-VDVAD suppressed cleaved caspase-2 activation by >85% at 3–4 days after ONC. Increasing concentrations of z-VDVAD protected greater numbers of RGC from death at 15 days after ONC, up to a maximum of 60% using 4000 ng/ml of z-VDVAD, compared to PBS treated controls. The 15-day treatment with 4000 ng/ml of z-VDVAD after ONC suppressed levels of cleaved caspase-2 but no significant changes in levels of cleaved caspase-3, -6, -7 or -8 were detected. Although suppression of caspase-2 protected 60% of RGC from death, RGC axon regeneration was not promoted. These results suggest that caspase-2 specifically mediates death of RGC after ONC and that suppression of caspase-2 may be a useful therapeutic strategy to enhance RGC survival not only after axotomy but also in diseases where RGC death occurs such as glaucoma and optic neuritis.     

视网膜中的自主感光神经节细胞

视网膜中少数神经节细胞能够合成感光蛋白--黑视素(melanopsin),因此具备了自主感光的能力,被称为自主感光神经节细胞(intrinsically photosensitive retinal ganglion cells,ipRGCs).ipRGCs可根据树突形态和分层位置的差异分为五个不同的亚型,其轴突主要投...     

Edible Seaweed, Eisenia bicyclis, Protects Retinal Ganglion Cells Death Caused by Oxidative Stress

The purpose of the present study was to determine whether edible seaweed, Eisenia bicyclis, is effective in blunting the negative influence of N-methyl-D-aspartate (NMDA) on rat retinas and of oxidative stress-induced transformed retinal ganglion cell (RGC-5 cell line) death. The ethanol extract of E. bicyclis (EEEB) significantly attenuated the negative insult of L: -buthionine-(S,R)-sulfoximine plus glutamate on RGC-5 cells. Treatment of the RGC-5 cells with EEEB reduced the reactive oxygen species and recovered the reduced glutathione level caused by various radical species such as H(2)O(2), OH·, or O(2)·(-). Moreover, EEEB inhibited lipid peroxidation on rat brain homogenates caused by sodium nitroprusside. Applying NMDA to the retina affected the thickness of the inner plexiform layer (IPL) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) produced a positive effect on ganglion cells. Importantly, EEEB protected the thinning of IPL and increased TUNEL positive cells in the ganglion cell layer (GCL). Five phlorotannin derivatives were isolated using chromatographic methods and liquid chromatography-mass spectroscopy analysis which has been known as an antioxidant. In conclusion, EEEB has a neuroprotective effect in vitro and in vivo. Furthermore, the major constituents of this extract, phlorotannins, could possibly be active compounds due to their antioxidative potency.     

Lactate-Mediated Protection of Retinal Ganglion Cells

Loss of retinal ganglion cells (RGCs) is a leading cause of blinding conditions. The purpose of this study was to evaluate the effect of extracellular l-lactate on RGC survival facilitated through lactate metabolism and ATP production. We identified lactate as a preferred energy substrate over glucose in murine RGCs and showed that lactate metabolism and consequently increased ATP production are crucial components in promoting RGC survival during energetic crisis. Lactate was released to the extracellular environment in the presence of glucose and detained intracellularly during glucose deprivation. Lactate uptake and metabolism was unaltered in the presence and absence of glucose. However, the ATP production declined significantly for 24 h of glucose deprivation and increased significantly in the presence of lactate. Finally, lactate exposure for 2 and 24 h resulted in increased RGC survival during glucose deprivation. In conclusion, the metabolic pathway of lactate in RGCs may be of great future interest to unravel potential pharmaceutical targets, ultimately leading to novel therapies in the prevention of blinding neurodegenerative diseases, for example, glaucoma.     

Retinal Ganglion Cell Dendritic Atrophy in DBA/2J Glaucoma

Glaucoma is a complex disease affecting an estimated 70 million people worldwide, characterised by the progressive degeneration of retinal ganglion cells and accompanying visual field loss. The common site of damage to retinal ganglion cells is thought to be at the optic nerve head, however evidence from other optic neuropathies and neurodegenerative disorders suggests that dendritic structures undergo a prolonged period of atrophy that may accompany or even precede soma loss and neuronal cell death. Using the DBA/2J mouse model of glaucoma this investigation aims to elucidate the impact of increasing intraocular pressure on retinal ganglion cell dendrites using DBA/2J mice that express YFP throughout the retinal ganglion cells driven by Thy1 (DBA/2J.Thy1(YFP)) and DiOlistically labelled retinal ganglion cells in DBA/2J mice. Here we show retinal ganglion cell dendritic degeneration in DiOlistically labelled DBA/2J retinal ganglion cells but not in the DBA/2J.Thy1(YFP) retinal ganglion cells suggesting that a potential downregulation of Thy1 allows only ‘healthy’ retinal ganglion cells to express YFP. These data may highlight alternative pathways to retinal ganglion cell loss in DBA/2J glaucoma.     

Retinal Ganglion Cells are Resistant to Photoreceptor Loss in Retinal Degeneration

The rapid and massive degeneration of photoreceptors in retinal degeneration might have a dramatic negative effect on retinal circuits downstream of photoreceptors. However, the impact of photoreceptor loss on the morphology and function of retinal ganglion cells (RGCs) is not fully understood, precluding the rational design of therapeutic interventions that can reverse the progressive loss of retinal function. The present study investigated the morphological changes in several identified RGCs in the retinal degeneration rd1 mouse model of retinitis pigmentosa (RP), using a combination of viral transfection, microinjection of neurobiotin and confocal microscopy. Individual RGCs were visualized with a high degree of detail using an adeno-associated virus (AAV) vector carrying the gene for enhanced green fluorescent protein (EGFP), allowed for large-scale surveys of the morphology of RGCs over a wide age range. Interestingly, we found that the RGCs of nine different types we encountered were especially resistant to photoreceptor degeneration, and retained their fine dendritic geometry well beyond the complete death of photoreceptors. In addition, the RGC-specific markers revealed a remarkable degree of stability in both morphology and numbers of two identified types of RGCs for up to 18 months of age. Collectively, our data suggest that ganglion cells, the only output cells of the retina, are well preserved morphologically, indicating the ganglion cell population might be an attractive target for treating vision loss.     

Directional Summation in Non-direction Selective Retinal Ganglion Cells

Retinal ganglion cells receive inputs from multiple bipolar cells which must be integrated before a decision to fire is made. Theoretical studies have provided clues about how this integration is accomplished but have not directly determined the rules regulating summation of closely timed inputs along single or multiple dendrites. Here we have examined dendritic summation of multiple inputs along On ganglion cell dendrites in whole mount rat retina. We activated inputs at targeted locations by uncaging glutamate sequentially to generate apparent motion along On ganglion cell dendrites in whole mount retina. Summation was directional and dependent13 on input sequence. Input moving away from the soma (centrifugal) resulted in supralinear summation, while activation sequences moving toward the soma (centripetal) were linear. Enhanced summation for centrifugal activation was robust as it was also observed in cultured retinal ganglion cells. This directional summation was dependent on hyperpolarization activated cyclic nucleotide-gated (HCN) channels as blockade with ZD7288 eliminated directionality. A computational model confirms that activation of HCN channels can override a preference for centripetal summation expected from cell anatomy. This type of direction selectivity could play a role in coding movement similar to the axial selectivity seen in locust ganglion cells which detect looming stimuli. More generally, these results suggest that non-directional retinal ganglion cells can discriminate between input sequences independent of the retina network.     

The Wnt Signaling Pathway Protects Retinal Ganglion Cell 5 (RGC-5) Cells from Elevated Pressure

The Wnt pathway is an essential signaling cascade that regulates survival and differentiation in the retina. We recently demonstrated that retinal ganglion cells (RGCs) have constitutively active Wnt signaling in vivo. However, the role of Wnt in RGC viability or function is unknown. In this study, we investigated whether Wnt protects the retinal ganglion cell line RGC-5 from elevated pressure, oxidative stress, and hypoxia injuries. Expression of RGC marker genes in the RGC-5 cultures was confirmed by immunocytochemistry and PCR. We demonstrated that the Wnt3a ligand significantly reduced pressure-induced caspase activity in RGC-5 cells (n = 5, P = 0.03) and decreased the number of TUNEL-positive cells (n = 5, P = 0.0014). Notably, Wnt3a-dependent protection was reversed by the Wnt signaling inhibitor Dkk1. In contrast, Wnt3a did not protect RGC-5 cells from oxidative stress or hypoxia. Furthermore, Wnt3a significantly increased growth factor expression in the presence of elevated pressure but not in the presence of oxidative stress and hypoxia. These results indicate that Wnt3a induces injury-specific survival pathways in RGC-5 cells, potentially by upregulating neuroprotective growth factors. Therefore, activation of the Wnt pathway by Wnt3a could be investigated further as a tool to develop novel molecular therapeutic strategies for the prevention of RGC death in retinal disease.     

Restoring Visual Function to Blind Mice with a Photoswitch that Exploits Electrophysiological Remodeling of Retinal Ganglion Cells

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Expression of Aquaporin-6 in Rat Retinal Ganglion Cells

Several aquaporins (AQPs) have been identified to be present in the eyes, and it has been suggested that they are involved in the movement of water and small solutes. AQP6, which has low water permeability and transports mainly anions, was recently discovered in the eyes. In the present study, we investigate the localization of AQP6 in the rat retina and show that AQP6 is selectively localized to the ganglion cell layer and the outer plexiform layer. Along with the gradual decrease in retinal ganglion cells after a crushing injury of optic nerve, immunofluorescence signals of AQP6 gradually decreased. Confocal microscope images confirmed AQP6 expression in retinal ganglion cells and Müller cells in vitro. Therefore, AQP6 might participate in water and anion transport in these cells.     

Targeted Expression of Channelrhodopsin-2 to the Axon Initial Segment Alters the Temporal Firing Properties of Retinal Ganglion Cells

The axon initial segment (AIS) is essential for initiating action potentials and maintaining neuronal excitability in axon-bearing neurons in the CNS. There is increasing interest in the targeting of optogenetic tools to subcellular compartments, including the AIS, to gain precise control of neuronal activity for basic research and clinical applications. In particular, targeted expression of optogenetic tools in retinal ganglion cells (RGCs) has been explored as an approach for restoring vision after photoreceptor degeneration. Thus, understanding the effects of such targeting on spiking abilities and/or patterns is important. Here, we examined the effects of recombinant adeno-associated virus (rAAV)-mediated targeted expression of channelrhodopsin-2 (ChR2)-GFP with a Na_V channel motif in mouse RGCs. We found that this targeted expression disrupted Na_V channel clustering at the AIS and converted the spike firing patterns of RGCs from sustained to transient. Our results suggest that the clustering of membrane channels, including Na_V channels, at the AIS is important for the ability of RGCs to generate sustained spike firing. Additionally, the targeting of optogenetic tools to the AIS with the Na_V channel motif may offer a way to create transient light responses in RGCs for vision restoration.     

Treatment In vitro of Retinal Cells with IL-4 Increases the Survival of Retinal Ganglion Cells: The Involvement of BDNF

Interleukin 4 (IL-4) is a pleiotropic cytokine involved in many functions during the development as well as in adult life. Previous work from our group demonstrated, in vitro, that this interleukin is able to prevent rat retinal ganglion cells death after axotomy. The aim of the present study was to investigate the signaling pathways involved in this trophic effect, particularly the cAMP pathway and also to demonstrate the expression of IL-4 in retinas at different stages of post natal development. Our results show that the trophic effect of IL-4 on rat retinal ganglion cells is dependent on the activation of Janus Kinase 3, Protein Kinase A, c-Jun N-terminal Kinase and Tropomyosin related Kinase receptors, on the increase in intracellular calcium levels, on polypeptide release and on the endogenous Brain Derived Neurotrophic Factor (BDNF). We also observed that treatment with IL-4 enhances c-AMP response element binding and Mitogen Activated Protein Kinase phosphorylation and increases the expression of BDNF. Concerning the IL-4 expression our data show an increase in IL-4 levels during post natal development. Taken together our results demonstrate that the trophic effect of IL-4 on retinal ganglion cells of newborn rats is mediated by cAMP pathway and BDNF release.     

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase^1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina^5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation⁶. In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.Download video file.^{(32M, mov)}