Molecular and cell-based approaches for neuroprotection in glaucoma

A hallmark of glaucomatous optic nerve damage is retinal ganglion cell (RGC) death. RGCs, like other central nervous system neurons, have a limited capacity to survive or regenerate an axon after injury. Strategies that prevent or slow down RGC degeneration, in combination with intraocular pressure management, may be beneficial to preserve vision in glaucoma. Recent progress in neurobiological research has led to a better understanding of the molecular pathways that regulate the survival of injured RGCs. Here we discuss a variety of experimental strategies including intraocular delivery of neuroprotective molecules, viral-mediated gene transfer, cell implants and stem cell therapies, which share the ultimate goal of promoting RGC survival after optic nerve damage. The challenge now is to assess how this wealth of knowledge can be translated into viable therapies for the treatment of glaucoma and other optic neuropathies.     

Triamcinolone attenuates macrophage/microglia accumulation associated with NMDA-induced RGC death and facilitates survival of Müller stem cell grafts

Retinal ganglion cell (RGC) death in glaucoma models is associated with accumulation of activated microglia, a sign of neural degeneration which has been shown to constitute a barrier for transplant cell survival and migration. This study investigated the use of triamcinolone (TA) to control macrophage/microglia accumulation in a model of RGC depletion to create a permissive environment for stem cell grafting. Intravitreal NMDA alone or in combination with TA was used to induce rapid onset of RGC death in 3-4 week old Lister hooded (LH) rat eyes prior to Müller stem cell transplantation into the vitreoretinal space. The effect of NMDA on RGC death and microglial accumulation was assessed immuno-histochemically, whilst electroretinography (ERG) was used to assess RGC function. Post transplantation, survival of grafted cells and their association with microglia were also examined by immunohistochemical methods. Intravitreal injection of NMDA alone resulted in severe macrophage/microglia accumulation associated with extensive RGC death 4-7 days post-treatment. Combination of NMDA with TA significantly reduced microglial numbers in the RGC when compared to NMDA only treated eyes while still depleting the retina of RGC. At the same time, NMDA/TA treatment also caused functional RGC loss as demonstrated by reduction of the scotopic threshold response. Upon transplantation with Müller stem cells, NMDA/TA treatment caused significant reduction in the number of transplant associated macrophage/microglia compared to eyes treated with NMDA alone. On this basis it is proposed that intravitreal injection of TA may be useful as an effective anti-inflammatory agent to control macrophage/microglia accumulation induced by RGC death, thereby creating a retinal environment permissive to cell transplantation studies.     

Glaucoma 2.0: neuroprotection, neuroregeneration, neuroenhancement

Glaucoma is a progressive neurodegenerative disease of retinal ganglion cells (RGCs) associated with characteristic axon degeneration in the optic nerve. Clinically, our only method of slowing glaucomatous loss of vision is to reduce intraocular pressure (IOP), but lowering IOP is only partially effective and does not address the underlying susceptibility of RGCs to degeneration. We review the recent steps forward in our understanding of the pathophysiology of glaucoma and discuss how this understanding has given us a next generation of therapeutic targets by which to maintain RGC survival, protect or rebuild RGC connections in the retina and brain, and enhance RGC function.     

Use of an adult rat retinal explant model for screening of potential retinal ganglion cell neuroprotective therapies

PURPOSE. To validate an established adult organotypic retinal explant culture system for use as an efficient medium-throughput screening tool to investigate novel retinal ganglion cell (RGC) neuroprotective therapies. METHODS. Optimal culture conditions for detecting RGC neuroprotection in rat retinal explants were identified. Retinal explants were treated with various recognized, or purported, neuroprotective agents and cultured for either 4 or 7 days ex vivo. The number of cells surviving in the RGC layer (RGCL) was quantified using histologic and immunohistochemical techniques, and statistical analyses were applied to detect neuroprotective effects. RESULTS. The ability to replicate previously reported in vivo RGC neuroprotection in retinal explants was verified by demonstrating that caspase inhibition, brain-derived neurotrophic factor treatment, and stem cell transplantation all reduced RGCL cell loss in this model. Further screening of potential neuroprotective pharmacologic agents demonstrated that betaxolol, losartan, tafluprost, and simvastatin all alleviated RGCL cell loss in retinal explants, supporting previous reports. However, treatment with brimonidine did not protect RGCL neurons from death in retinal explant cultures. Explants cultured for 4 days ex vivo proved most sensitive for detecting neuroprotection. CONCLUSIONS. The current adult rat retinal explant culture model offers advantages over other models for screening potential neuroprotective drugs, including maintenance of neurons in situ, control of environmental conditions, and dissociation from other factors such as intraocular pressure. Verification that neuroprotection by previously identified RGC-protective therapies could be replicated in adult retinal explant cultures suggests that this model could be used for efficient medium-throughput screening of novel neuroprotective therapies for retinal neurodegenerative disease.     

Biomechanics of the sclera and effects on intraocular pressure

Accumulating evidence indicates that glaucoma is a multifactorial neurodegenerative disease characterized by the loss of retinal ganglion cells (RGC), resulting in gradual and progressive permanent loss of vision. Reducing intraocular pressure (IOP) remains the only proven method for preventing and delaying the progression of glaucomatous visual impairment. However, the specific role of IOP in optic nerve injury remains controversial, and little is known about the biomechanical mechanism by which elevated IOP leads to the loss of RGC. Published studies suggest that the biomechanical properties of the sclera and scleral lamina cribrosa determine the biomechanical changes of optic nerve head, and play an important role in the pathologic process of loss of RGC and optic nerve damage. This review focuses on the current understanding of biomechanics of sclera in glaucoma and provides an overview of the possible interactions between the sclera and IOP. Treatments and interventions aimed at the sclera are also discussed.     

优视胶囊对急性高眼压家兔眼压及神经节细胞的影响

目的：观察优视胶囊对急性高眼压兔眼压的影响及对视网膜视神经的保护作用。方法：采用自行设计的上巩膜静脉扎法建立18只兔眼急性高眼压动物模型,于造模前1周至造模后3天共10天内给予具活化血瘀、开窍明目功效的优视胶囊灌胃,18只造模眼随机分为模型组、低剂量组和高剂量组,每组6眼,与18只正常眼进行对照。实验过程中测量眼压并行视网膜神经节细胞计数。结果：造模后即可获得平均眼压高于6．83kPa并能持续3天以上的高眼压动物模型,优视胶囊高、低剂量组表现出轻微的降眼压作用。①持续性的高眼压可造成视网膜神经节细胞减少,但高、低剂量组表现出轻微的降眼压作用。②持续性的高眼压可造成视网膜神经节细胞减少,但高、低剂量组经优视胶囊治疗后高眼压模型眼神经节细胞数高于模型组,提示优视胶囊具有保护或改善急性高眼压后兔眼视网膜神经节细胞的作用。结论：优视胶囊对急性高眼压兔视网膜神经具有保护的作用。     

Neuroprotection of retinal ganglion cells with GDNF-Loaded biodegradable microspheres in experimental glaucoma

Glaucoma is the second leading cause of blindness worldwide, and also the most common optic neuropathy. The ultimate cause of vision loss in glaucoma is thought to be retinal ganglion cell (RGC) death. Neuroprotection of RGC is therefore an important goal of glaucoma therapy. Currently, glaucoma treatment relies on pharmacologic or surgical reduction of intraocular pressure (IOP). It is critical to develop treatment approaches that actively prevent the death of RGCs at risk in glaucoma. Neurotrophic factors have the ability to promote the survival and influence the growth of neurons. Neurotrophic factor deprivation has been proposed as one mechanism leading to RGC death in glaucoma. Effective neuroprotection in glaucoma likely requires the consistent availability of the active agent for prolonged periods of time. Biodegradable microspheres are especially attractive as drug delivery vehicles for a number of reasons. Sustained GDNF delivery by biodegradable microspheres offers significant neuroprotection to injured RGC in experimental glaucoma. PLGA microsphere-delivered GDNF represents an important neuroprotective strategy in the treatment of glaucomatous optic neuropathy and provides direction for further investigations of this hypothesis.     

MK-801对大鼠慢性高眼压视网膜神经节细胞的保护作用

目的：探讨MK-801对大鼠慢性高眼压视网膜神经节细胞(retinal ganglial cells, RGC)的保护作用。 方法：制备SD大鼠慢性高眼压模型,36只SD大鼠随机分为空白对照组、慢性高眼压＋生理盐水对照组、慢性高眼压＋MK-801处理组,各组12 只。观察并比较各组大鼠在不同时间点（处理后1, 4, 7和10d）谷氨酸含量、视网膜总厚度和内层厚度、RGC数目及RGC凋亡情况。 结果：在各时间点,慢性高眼压大鼠（MK-801处理组和生理盐水对照组）视网膜谷氨酸含量均较空白对照组高（P<0.05）; MK-801处理组视网膜谷氨酸含量比同期生理盐水对照组明显降低,差异有统计学意义（P<0.05）。HE染色表明MK-801处理组和空白对照组视网膜总厚度和内层厚度均大于生理盐水对照组（P＜0.05）,RGC数目高于生理盐水对照组（P＜0.05）。MK-801处理组RGC 层凋亡阳性细胞的表达少于生理盐水对照组。 结论：MK-801可通过阻断谷氨酸介导的神经毒性作用,对慢性高眼压RGC起到保护作用。     

Simvastatin upregulates Bcl-2 expression and protects retinal neurons from early ischemia/reperfusion injury in the rat retina

Simvastatin has been shown to enhance the survival of retinal ganglion cells (RGCs) following ischemia-reperfusion (IR) injury by mediating the expression of stress proteins. The purpose of this study was to investigate the effect of simvastatin on retinal neurons and the expression of apoptotic proteins in a rat IR model. Wistar rats received intravitreal injection of simvastatin immediately after retinal reperfusion. Retinal ischemia was induced by increasing intraocular pressure to 150 mmHg for 60 min. The number of viable RGCs was measured after retrograde labeling with Fluoro-Gold. Ischemia-induced apoptotic cell death was studied using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). We found that simvastatin treatment enhanced RGC survival after retinal ischemia by approximately 40% and decreased retinal neuronal apoptosis. Using western blot analysis, we found that simvastatin upregulated the expression of Bcl-2 in the retina. In contrast, the level of the protein Bax was unaffected by simvastatin treatment. Our results suggest that RGC loss induced by retinal IR may be prevented by simvastatin and that the mechanism underlying this process possibly involves an alteration in the apoptotic pathway.     

Ganglion cell death in rat retina by persistent intraocular pressure elevation

Glaucoma is characterized by loss of retinal ganglion cells (RGCs) and their axons. Retrograde axoplasmic transport blockade and excitotoxicity were proposed to be a major cause of RGC apoptosis. We conducted this study to characterize the episcleral vessel cauterization glaucoma model in the rat with respect to decreased retrograde axoplasmic flow and subsequent apoptotic RGC death. After episcleral vessels were cauterized in Sprague-Dawley rats, Fluorogold was injected into their superior colliculi by stereotactic method. Retrograde axoplasmic flow and TUNEL-stained apoptotic dead cells were observed microscopically. Elevated intraocular pressure was maintained for up to 6 weeks during follow-up. Retrograde axoplasmic flow to the rat retina was significantly decreased. Apoptotic RGC was selectively TUNEL-stained in the retina, especially at the ganglion cell layers. We concluded that elevated intraocular pressure caused apoptotic RGC death through retrograde axoplasmic flow blockage. Further studies will elucidate the neuroprotection strategies in glaucoma patients.     

Nerve growth factor (NGF) reduces and NGF antibody exacerbates retinal damage induced in rabbit by experimental ocular hypertension

• Background: It has been shown that intravitreal injection of NGF inhibits ganglion cell degeneration after optic nerve transection and ischemic injury. The aim of our study was to investigate the presence of NGF in aqueous humor and its involvement in retinal damage during ocular hypertension. • Methods: We used an experimental model of ocular hypertension in rabbit. Before treatment and 4, 10 and 15 days after induction of hypertension, we evaluated histological retinal damage and NGF levels in aqueous humor using an immunoenzymatic assay. Polyclonal anti-NGF antibodies were injected intravitreally into one eye of each rabbit (n = 6), and the animals were killed after 4 days of hypertension. Another group of rabbits (n =12) was injected retro-ocularly with NGF and killed after 10 or 15 days of treatment for histologic evaluation of the retina. • Results: Our data show that experimental ocular hypertension in adult rabbits induces retinal damage and enhances local NGF levels. The highest NGF value was found after 4 days of intraocular hypertension; high levels persisted, though to a lesser extent, for up to 15 days. Histological examination revealed that the number of retinal ganglion cells (RGC) remained unchanged during the first 4 days but decreased at 10 days. These studies also showed that retro-ocular administration of NGF reduced RGC loss, whereas intraocular injection of NGF antibodies, which inhibited the endogenous NGF, exacerbated the retinal insult. • Conclusion: These findings demonstrate a protective effect of NGF on RGC damaged by ocular hypertension and prompt further investigations to evaluate a possible therapeutic use of NGF to retard RGC death in humans.     

Light-driven retinal ganglion cell responses in blind rd mice after neural retinal transplantation

PURPOSE: Light-elicited retinal ganglion cell (RGC) responses after fetal neural retinal transplantation have not been demonstrated in animal or human subjects blind from outer retinal degeneration, despite apparent morphologic success. This study was designed to test the hypothesis that the functional success of retinal transplantation may be enhanced by using a young host retina (13 days old). METHODS:At postnatal day (P)13 C3H/HeJ (rd/rd) retinal degenerate mice received a subretinal transplant, in one eye only, of neural retinal tissue isolated from newborn normal C57/BL6J mice. Between 33 and 35 days after transplantation, local electroretinograms (ERGs) and ganglion cell responses were recorded directly from the retinal surface using a differential bipolar surface electrode. Measurements were performed both with and without light stimulation. Similar recordings were also performed in age-matched eyes subjected to sham transplantation, in control eyes that were not subjected to surgery, and in animals eyes that underwent transplantation at 8 weeks of age. After the recordings, the eyes were processed for light and transmission electron microscopy. RESULTS:Three of 10 mice showed bursts of ganglion cell action potentials (ON response only) as well as recordable intraocular ERGs over the transplant in response to 1-second and 200-msec light stimuli. Light-driven ganglion cell responses could not be recorded in areas outside the transplant in all transplant-recipient eyes, age-matched control eyes, and sham-transplantation eyes. Light responses also could not be recorded in animal eyes that received transplants at an older age (8 weeks). Electron microscopic examination confirmed the presence of photoreceptor outer segments in the areas affected by transplantation. CONCLUSIONS: This study demonstrates the presence of light-driven ganglion cell responses after subretinal transplantation in a retinal degenerate model. This finding may reflect functional integration of the transplant with the host, but a rescue effect on remaining host photoreceptors cannot be ruled out. The findings suggest, however, that modification of host parameters, such as host age, may be important approaches for improving the functional success of retinal transplantation.     

Neurotrophin roles in retinal ganglion cell survival: Lessons from rat glaucoma models

The neurotrophin (NT) hypothesis proposes that the obstruction of retrograde transport at the optic nerve head results in the deprivation of neurotrophic support to retinal ganglion cells (RGC) leading to apoptotic cell death in glaucoma. An important corollary to this concept is the implication that appropriate enhancement of neurotrophic support will prolong the survival of injured RGC indefinitely. This hypothesis is, perhaps, the most widely recognized theory to explain RGC loss resulting from exposure of the eye to elevated intraocular pressure (IOP). Recent studies of NT signaling using rat glaucoma models, have examined the endogenous responses of the retina to pressure exposure as well as studies designed to augment NT signaling in order to rescue RGC from apoptosis following pressure-induced injury. The examination of these studies in this review reveals a number of consistent observations and provides direction for further investigations of this hypothesis.     

玻璃体内细胞移植或细胞衍生物注入在视神经损伤中的作用

视网膜神经节细胞绝大部分位于神经节细胞层,可通过视神经通路将光感受器接收的视觉信息传递至高级视觉中枢,产生视觉。视网膜神经节细胞损伤和视神经轴突的中断,往往会导致视力下降甚至失明。近年来研究证实,玻璃体内细胞移植对损伤的视神经具有保护作用,为视神经损伤的修复治疗提供了新的方向。本文就玻璃体内细胞移植或细胞衍生物注入在视神经损伤中的作用展开综述。     

Transplantation of neuroblastic progenitor cells as a sheet preserves and restores retinal function

Diseases affecting the outer retina are incurable once photoreceptors are lost, and these diseases usually cause retinal pigment epithelium (RPE) dysfunction. However, the inner retina can remain functional for some time, even though retinal remodeling occurs as compensation for photoreceptor loss. If the damaged part can be replaced with neuroblastic progenitor and RPE cells as sheets with a beneficial effect on function, vision loss may be prevented and vision may be restored. This review presents an overview of the research of transplanting sheets of neural retina, with or without its RPE, to the subretinal space. In different animal models of retinal degeneration, retinal transplants can morphologically reconstruct a damaged retina, and restore visual sensitivity. Good morphological integration of transplants with the host retina can occur, whereas other transplants exhibit a glial barrier. Synaptic connections between transplant and host have been indicated by transsynaptic tracing. Retinal transplants can restore and preserve visual responses in a small area of the superior colliculus corresponding to the placement of the transplant in the retina. The beneficial effect of retinal transplantation likely involves two mechanisms: trophic effects, e.g., rescue of host cones; and synaptic connectivity between transplant and host retina.     

Stem cell based therapies for age-related macular degeneration: The promises and the challenges

Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly in developed countries. AMD is classified as either neovascular (NV-AMD) or non-neovascular (NNV-AMD). Cumulative damage to the retinal pigment epithelium, Bruch's membrane, and choriocapillaris leads to dysfunction and loss of RPE cells. This causes degeneration of the overlying photoreceptors and consequential vision loss in advanced NNV-AMD (Geographic Atrophy). In NV-AMD, abnormal growth of capillaries under the retina and RPE, which leads to hemorrhage and fluid leakage, is the main cause of photoreceptor damage. Although a number of drugs (e.g., anti-VEGF) are in use for NV-AMD, there is currently no treatment for advanced NNV-AMD. However, replacing dead or dysfunctional RPE with healthy RPE has been shown to rescue dying photoreceptors and improve vision in animal models of retinal degeneration and possibly in AMD patients. Differentiation of RPE from human embryonic stem cells (hESC-RPE) and from induced pluripotent stem cells (iPSC-RPE) has created a potentially unlimited source for replacing dead or dying RPE. Such cells have been shown to incorporate into the degenerating retina and result in anatomic and functional improvement. However, major ethical, regulatory, safety, and technical challenges have yet to be overcome before stem cell-based therapies can be used in standard treatments. This review outlines the current knowledge surrounding the application of hESC-RPE and iPSC-RPE in AMD. Following an introduction on the pathogenesis and available treatments of AMD, methods to generate stem cell-derived RPE, immune reaction against such cells, and approaches to deliver desired cells into the eye will be explored along with broader issues of efficacy and safety. Lastly, strategies to improve these stem cell-based treatments will be discussed.     

Retinal ganglion cell death and optic nerve degeneration by genetic ablation in adult mice

Despite the magnitude of the problem, no effective treatments exist to prevent retinal ganglion cell (RGC) death and optic nerve degeneration from occurring in diseases affecting the human eye. Animal models currently available for developing treatment strategies suffer from cumbersome procedures required to induce RGC death or rely on mutations that induce defects in developing retinas rather than in mature retinas of adults. Our objective was to develop a robust genetically engineered adult mouse model for RGC loss and optic nerve degeneration based on genetic ablation. To achieve this, we took advantage of Pou4f2 (Brn3b), a gene activated immediately as RGCs begin to differentiate and expressed throughout life. We generated adult mice whose genomes harbored a conditional Pou4f2 allele containing a floxed-lacZ-stop-diphtheria toxin A cassette and a CAGG-Cre-ER™ transgene. In this bigenic model, Cre recombinase is fused to a modified estrogen nuclear receptor in which the estrogen-binding domain binds preferentially to the estrogen agonist tamoxifen rather than to endogenous estradiol. Upon binding to the estrogen-binding domain, tamoxifen derepresses Cre recombinase, leading to the efficient genomic deletion of the floxed-lacZ-stop DNA sequence and expression of diphtheria toxin A. Tamoxifen administered to adult mice at different ages by intraperitoneal injection led to rapid RGC loss, reactive gliosis, progressive degradation of the optic nerve over a period of several months, and visual impairment. Perhaps more reflective of human disease, partial loss of RGCs was achieved by modulating the tamoxifen treatment. Especially relevant for RGC death and optic nerve degeneration in human retinal pathologies, RGC-ablated retinas maintained their structural integrity, and other retinal neurons and their connections in the inner and outer plexiform layers appeared unaffected by RGC ablation. These events are hallmarks of progressive optic nerve degeneration observed in human retinal pathologies and demonstrate the validity of this model for use in developing stem cell therapies for replacing dead RGCs with healthy ones.     

In vivo toxicity study of rhodamine 6G in the rat retina

PURPOSE: To investigate the intraocular effect of rhodamine 6G (R6G) on retinal structures and function in an in vivo rat model and to develop an in vivo method for accurate evaluation of new dyes for intraocular surgery. METHODS: R6G in physiologic saline solution (PSS) was injected into the vitreous of adult Brown Norway rats at concentrations of 0.0002%, 0.002%, 0.02%, 0.2%, and 0.5%. Control animals received only PSS. Retinal toxicity was assessed by retinal ganglion cell (RGC) counts, light microscopy 7 days later, photopic electroretinography (ERG), and measurement of scotopic sensitivity and recovery of dark adaptation 48 hours and 7 days after intravitreous injection. RESULTS: R6G at concentrations of 0.2% and 0.5% led to a dose-dependent loss of RGC. The most significant loss occurred at 0.5%. Lower concentrations (0.0002%, 0.002%, and 0.02%) produced no statistically significant retinal ganglion cell loss. Analysis of the eyes by light microscopy showed no structural changes in the central retina, although injections of 0.5% R6G were followed by impressive degenerative changes adjacent to the injection sites. ERGs showed no effects of the highest R6G concentration on rods, kinetics of rhodopsin recovery after bleaching, or cone-driven responses. CONCLUSIONS: R6G can be safely injected in doses of up to 0.02% in rats, but has a toxic effect on retinal ganglion cells at higher concentrations. Accumulation of R6G may be a problem at higher concentrations, particularly at the injection site.     

青光眼视神经节细胞凋亡的作用机制研究进展

青光眼是一组以视神经退行性病变、视野缺损和视力下降为特征的不可逆性致盲性眼病。青光眼的发生主要涉及视网膜神经节细胞（retinal ganglion cells，RGC）的变性、凋亡、进行性丧失。目前，青光眼RGC凋亡的具体机制尚未明确，其中涉及多种凋亡途径和凋亡相关因子，包括死亡受体途径、线粒体途径、内质网途径等凋亡机制及caspases等凋亡因子参与。本文就RGC几种常见凋亡途径及凋亡相关因素做一简要综述，对青光眼的靶向治疗提供一定的理论依据，进而为临床治疗青光眼提供可靠的分子依据。