Effect of erythropoietin axotomy-induced apoptosis in rat retinal ganglion cells

PURPOSE: Erythropoietin (EPO) modulates erythropoiesis by inhibiting apoptosis in erythrocyte progenitors. Recently, EPO has been shown to be protective in experimental models of mechanical trauma, neuroinflammation, cerebral and retinal ischemia, and even in a human stroke trial. However, little is known about EPO signal transduction in vivo and the usefulness of EPO in the prevention of the chronic, purely apoptotic neuronal cell death that contributes to vision loss in glaucoma and the progression of neurodegenerative diseases. METHODS: EPO's effects and signaling in the retinal ganglion cell axotomy paradigm were studied by Western blot analysis and immunohistochemistry, receptor expression was characterized in the retina before and after lesion. EPO was injected into the vitreous body to investigate neuroprotection of axotomized rat RGCs. Moreover, EPO's effects were studied in cultures of immunopurified retinal ganglion cells. Signal-transduction pathways transmitting neuroprotective EPO effects in vivo were characterized by the use of specific kinase inhibitors, immunohistochemistry, and Western blot analysis. RESULTS: EPO receptors (EPORs) were expressed on RGC somata and dendrites in vivo. EPOR expression did not significantly change after axotomy. Application of EPO prevented death of neurotrophic-factor-deprived immunopurified rat RGCs in vitro, rescued axotomized RGCs in vivo, and prevented caspase-3 activation. EPO-induced Akt phosphorylation and survival-promoting EPO effects were completely abolished by inhibition of PI-3-kinase. EPO neuroprotection followed a bell-shaped dose-response curve in vitro and in vivo, whereas toxic EPO effects were never observed, even at high concentrations. CONCLUSIONS: These data support a potential role for EPO as a therapeutic molecule against predominantly apoptotic neuronal cell death in the context of glaucoma or neurodegenerative diseases and delineate the PI-3-K/Akt pathway as the predominant mediator of EPO neuroprotection in this in vivo paradigm of neuronal cell death.     

Retinal neuronal death induced by intraocular administration of a nitric oxide donor and its rescue by neurotrophic factors in rats

PURPOSE: To investigate the neurotoxic outcome in the rat retina exposed to nitric oxide (NO) released from an NO donor and to evaluate the effects of neurotrophic factors on the survival of NO-damaged retinal cells. METHODS: An NO releasing compound, N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino) ethanamine (NOC 12), was intravitreously injected into a rat's right eye. The influences of NOC 12 on retinal neurons and the neuroprotective effects of ciliary neurotrophic factor (CNTF) or brain-derived neurotrophic factor (BDNF) on NOC 12-mediated damage were estimated by counting cells in the ganglion cell layer (GCL) and by measuring the thickness of retinal layers. The exact count of retinal ganglion cells (RGCs) was also confirmed by means of retrograde labeling with a fluorescent tracer. RESULTS: Morphometric analyses of retinal damage in the NOC 12-exposed eyes demonstrated a significant and dose-dependent decrease in cell density in the GCL and a reduction in thickness of the inner plexiform layer and inner nuclear layer, but not of the outer nuclear layer. TdT-dUTP terminal nick-end labeling of retinal sections after intravitreous injection of NOC 12 demonstrated that NO could trigger apoptotic cell death. The counting of the RGCs labeled with a fluorescent tracer suggested that a decrease in GCL cell density induced by NOC 12 reflects a loss in RGCs. Treatment with CNTF (1 microg) or BDNF (1 microg) before the intravitreous injection of NOC 12 (400 nmol) demonstrated that these trophic factors have protective effects against NO-induced neuronal cell death in the retina. CONCLUSIONS: Exogenous NO induces retinal neurotoxicity, suggesting that NO plays a pathogenic role in degenerative retinal diseases. BDNF and CNTF protect retinal neurons from NO-mediated neurotoxicity.     

Brain-derived neurotrophic factor and repair of retinal ganglion cells after injury

Blocking of retrograde transport after the optic nerve injury results in deprivation of neurotrophic support to retinal ganglion cells (RGCs), and leads to apoptotic cell death in glaucoma. As an important member of neurotrophins, the brain-derived neurotrophic factor (BDNF) plays a substantial role in the repair of retinal ganglion cells injury, but its signaling pathway in the pathogenesis of glaucoma remains unclear. This review focuses on the structure, distribution and receptors of BDNF and its effects on RGC survival, axon regeneration and relevant signaling pathway, to provide theoretical foundation for neuroprotective treatment of glaucoma.     

无血清神经基质培养基培养纯化的视网膜神经节细胞

目的 建立无血清培养基培养纯化原代视网膜神经节细胞（RGCs）的方法，为研究青光眼等引起的RGCs损伤及防护提供理想的细胞模型。 方法 出生后1～3 d的SD大鼠视网膜细胞悬液，分别经抗大鼠信号调节蛋白（CD172G）单克隆抗体筛除巨噬细胞、抗大鼠Thy-1单克隆抗体筛选RGCs的两步筛选法，得到纯化的RGCs。用加入B27、睫状神经营养因子等的无血清神经元专用神经基质（neurobasal）培养基进行培养，通过细胞形态学观察、Thy-1免疫荧光细胞化学染色、钙黄绿素-乙酰乙酸酯（AM）染色等方法观察原代培养的视网膜神经细胞及纯化培养的RGCs生长并进行鉴定。 结果 原代培养的视网膜细胞中约91%为神经细胞。纯化培养的RGCs接种后24 h有突起长出;第4～8天可见细胞形态均一，细胞体较大，细胞突起较长;至第14天，仍有超过60%的细胞有活性。Thy-1的免疫荧光细胞染色结果显示RGCs纯度约为90%。钙黄绿素-AM染色存活的RGCs，结果显示RGCs细胞体较大，多数细胞有长度大于细胞体直径2倍的突起。 结论 该方法培养的RGCs大小均一、形态理想、纯度高，适宜研究各种因素引起的RGCs损伤及保护剂效果评价。 (中华眼底病杂志, 2006, 22: 200-203)     

Activation of matrix metalloproteinase-9 via neuronal nitric oxide synthase contributes to NMDA-induced retinal ganglion cell death

PURPOSE: Understanding the mechanism of neuronal cell death in retinal diseases like glaucoma is important for devising new treatments. One factor involves excitatory amino acid stimulation of N-methyl-D-aspartate (NMDA)-type glutamate receptors, excessive Ca2+ influx, and formation of nitric oxide (NO) via neuronal NO synthase (nNOS). Another factor is the abnormal activation of matrix metalloproteinases (MMPs), in particular MMP-9, which triggers an extracellular signaling cascade leading to apoptosis. This study was designed to investigate the mechanism of excitotoxic retinal ganglion cell (RGC) death in vivo and its relationship to MMP activation. METHODS: NMDA and glycine were injected into the vitreous of the eye in rats and in nNOS-deficient mice (nNOS-/-) versus control. Gelatinolytic activity of MMP-9 and MMP-2 by zymography and cellular localization by immunohistochemistry were examined, and the effect of MMP inhibition on NMDA-induced RGC death was tested. RESULTS: NMDA was found to upregulate the proform of MMP-9 in the retina and to increase MMP-9 gelatinolytic activity. Retrograde labeling with aminostilbamidine to identify RGCs confirmed that MMP activity occurred only in these retinal neurons and not in glial or other retinal cell types after excitotoxic insult. Deconvolution fluorescence microscopy revealed that MMP activity colocalized with immunoreactive S-nitrosylated protein. NMDA-induced MMP activation was diminished in the retina of nNOS-/- mice, implying that S-nitrosylation of MMP had indeed occurred. In addition, the broad-spectrum MMP inhibitor GM6001 protected RGCs after intravitreal NMDA injection. CONCLUSIONS: These findings suggest that an extracellular proteolytic pathway in the retina contributes to RGC death via NO-activated MMP-9.     

睫状神经生长因子与眼病的相关研究

睫状神经营养因子(ciliary neurotrophic factor,CNTF)是神经保护因子中被研究的最多的因子之一,细胞包囊技术(encapsulated cell technology,ECT)为CNTF的临床应用提供了安全有效的给药途径.大量的研究证据表明,CNTF对视网膜色素变性、青光眼、年龄相关性黄斑变性、视神经损伤等眼部疾病具有神经保护作用.本文就CNTF与眼病的相关研究进展进行综述.     

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

AIM: To evaluate the neuroprotective activity of systemically administered edaravone in early and late stage of experimental glaucoma in rats. METHODS: 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. RESULTS: 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). CONCLUSION: 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.     

促红细胞生成素对视神经视网膜的保护作用

促红细胞生成素(Erythropoietin,EPO)主要作用于骨髓巨核前体细胞,刺激红系造血祖组织及早幼红细胞形成成熟的红细胞集落。近年来,大量的研究显示EPO和EPO受体在神经系统上有功能表达,在体外培养及动物试验中都显示了显著的神经保护功能。在眼科,青光眼、视网膜脱离、视网膜色素变性等疾病的共同病理机制是视网膜神经节细胞、光感受器细胞等神经元的凋亡,最终导致视力的丧失。因此,EPO的神经保护作用对治疗这些疾病有一定的实际意义。现总结了EPO的神经保护性质分子机制及信号传导途径,探讨EPO用于青光眼视神经保护治疗的基础和可能性。     

青光眼与Th1、Th2相关细胞因子研究进展

青光眼是导致不可逆盲的首要原因,包括视野缺损和视神经的慢性退行性病变,如视网膜神经节细胞(RGCs)的凋亡和视神经轴突的逐步缺失.目前普遍认为高眼压是青光艰的主要危险因素,降低眼压是减缓青光眼发生和发展的首选治疗方法.近年来发现免疫因素是青光眼视神经损害的非压力依赖性危险因素之一.大部分免疫,甚至非免疫性生物效应都通过细胞因子来调控,而CD4+辅助性T细胞是细胞因子产生和调节的主要来源,其中Th1和Th2相关细胞因子在青光眼的发病机制中起着不可或缺的作用,并关系着RGCs的存活和凋亡.本文就近年Th1和Th2主要的相关细胞因子及Th1/Th2平衡与青光眼潜在关系的研究进展进行综述.     

Detection of early neuron degeneration and accompanying microglial responses in the retina of a rat model of glaucoma

PURPOSE: To characterize the early reaction of retinal ganglion cells (RGCs) in a rat model of glaucoma using in vivo imaging and to examine the involvement of retinal microglia in glaucomatous neuropathy. METHODS: Glaucoma was induced in adult female Sprague-Dawley rats by cauterizing two episcleral veins, which resulted in a 1.6-fold increase in intraocular pressure (IOP). Retinal ganglion cells were retrogradely labeled with the fluorescent dye, 4-[didecylaminostyryl]-N-methyl-pyridinium-iodide (4-Di-10ASP) and monitored in vivo after elevation of IOP using fluorescence microscopy imaging. The number of RGCs was quantified on retinal flatmounts. Dying RGCs were surrounded by activated microglia that became visible after taking up the fluorescent debris. Immunocytochemistry was conducted to characterize further the ganglion cells and microglia. RESULTS: Cauterizing two of the four episcleral veins resulted in a consistent increase of IOP to 25.3 +/- 2.0 mm Hg, as measured with a handheld tonometer. IOP remained high for at least 3 months in glaucomatous eyes. The earliest sign of RGC death was detected in anesthetized animals 20 hours after induction of glaucoma. RGCs continued to decrease in number over time, with 40% of RGCs having degenerated after 2.5 months. Fundoscopic examination of the optic nerve head revealed cupping 2 months after induction of glaucoma. In addition, microglia were detected on retinal flatmounts as early as 72 hours after induction. Activated microglia and RGCs were also identified immunocytochemically, with an antibody against ionized calcium-binding adaptor molecule (Iba)-1 and an antibody specific to the 200-kDa subunit of the neurofilament protein, respectively. CONCLUSIONS: Occlusion of episcleral veins is a reproducible method that mimics human glaucoma, with chronically elevated IOP-induced RGC loss. This study shows that in vivo imaging permits the detection of ganglion cells in the living animal in the early stages of the disease and highlights the importance of in vivo imaging in understanding ophthalmic disorders such as glaucoma. Secondly, activation of intraretinal microglia coincides with degeneration of RGCs in glaucoma.     

Acetylcholine protection of adult pig retinal ganglion cells from glutamate-induced excitotoxicity

PURPOSE: To determine which glutamate receptor (GluR) subtypes are responsible for glutamate-induced excitotoxicity in cultured adult pig retinal ganglion cells (RGCs) and to characterize the neuroprotective effect of acetylcholine (ACh) on pig RGCs. METHODS: Adult pig RGCs were isolated from other retinal tissue by a modified panning technique using Thy 1.1 antibody. Isolated RGCs were cultured in control media and media containing: glutamate, NMDA, or KA; glutamate and CNQX, MK-801, or AP-7; ACh, nicotine or muscarine; ACh and alpha-bungarotoxin (Bgt) or methyllycaconitine (MLA); and glutamate and choline or glutamate, choline, and MLA. To determine cell viability, cells were loaded with calcein and counted. RESULTS: Ninety-eight percent of isolated cells were immunolabeled with Thy 1.1 antibody. Chronic exposure to 500 microM glutamate decreased the number of surviving large and small RGCs, compared to control conditions. This glutamate-induced excitotoxicity was mediated through both NMDA and non-NMDA GluRs. In neuroprotective studies, ACh, nicotine, and choline significantly reduced glutamate-induced excitotoxicity in adult pig RGCs through alpha-Bgt-sensitive nicotinic ACh receptors (nAChRs). DISCUSSION: This was the first report of a modified panning technique to isolate adult pig RGCs. Cell viability was relatively high using this method, and both large and small RGCs grew extensive neurites in culture. The finding that both NMDA and non-NMDA GluRs were involved in glutamate-induced excitotoxicity suggests that isolated pig RGCs provide a good model for glaucoma. In addition, activation of AChRs may be useful in protecting RGC from excitotoxic insults occurring in neurodegenerative diseases such as glaucoma.     

Cataractogenic lens injury prevents traumatic ganglion cell death and promotes axonal regeneration both in vivo and in culture

PURPOSE: To examine and quantify neuroprotective and neurite-promoting activity on retinal ganglion cells (RGCs) after injury of the lens. METHODS: In adult albino rats, penetrating lens injury was performed by intraocular injection. To test for injury-induced neuroprotective effects in vivo, fluorescence-prelabeled RGCs were axotomized by subsequent crush of the optic nerve (ON) with concomitant lens injury to cause cataract. The numbers of surviving RGCs were determined in retinal wholemounts and compared between the different experimental and control groups. To examine axonal regeneration in vivo, the ON was cut and replaced with an autologous piece of sciatic nerve (SN). Retinal ganglion cells with axons that had regenerated within the SN under lens injury or control conditions were retrogradely labeled with a fluorescent dye and counted on retinal wholemounts. Neurite regeneration was also studied in adult retinal explants obtained either after lens injury or without injury. The numbers of axons were determined after 1 and 2 days in culture. Putative neurotrophins (NTs) were studied within immunohistochemistry and Western blot analysis. RESULTS: Cataractogenic lens injury performed at the same time as ON crush resulted in highly significant rescue of 746 +/- 126 RGCs/mm(2) (mean +/- SD; approximately 39% of total RGCs) 14 days after injury compared with controls without injury or with injection of buffer into the vitreous body (30 +/- 18 RGCs/mm(2)). When lens injury was performed with a delay of 3 days after ON crush, 49% of RGCs survived, whereas delay of 5 days still rescued 45% of all RGCs. In the grafting paradigm virtually all surviving RGCs after lens injury appeared to have regenerated an axon within the SN graft (763 +/- 114 RGCs/mm(2) versus 79 +/- 17 RGCs/mm(2) in controls). This rate of regeneration corresponds to approximately 40% of all RGCs. In the regeneration paradigm in vitro preceding lens injury and ON crush 5 days previous resulted in a maximum of regeneration of 273 +/- 39 fibers/explant after 1 day and 574 +/- 38 fibers/explant after 2 days in vitro. In comparison, in control retinal pieces without lens injury 28 +/- 13 fibers/explant grew out at 1 day, and 97 +/- 37 fibers/explant grew out at 2 days in culture. Immunohistochemical and Western blot analysis of potential NTs in the injured lens revealed no expression of ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), NT-4, nerve growth factor (NGF), and basic fibroblast growth factor (bFGF). CONCLUSIONS: The findings indicate that the lens contains high neuroprotective and neuritogenic activity, which is not caused by NT. Compared with the data available in the literature, this neuroprotection is quantitatively among the highest ever reported within the adult rat visual system.     

CNTF and retina

Ciliary neurotrophic factor (CNTF) is one of the most studied neurotrophic factors for neuroprotection of the retina. A large body of evidence demonstrates that CNTF promotes rod photoreceptor survival in almost all animal models. Recent studies indicate that CNTF also promotes cone photoreceptor survival and cone outer segment regeneration in the degenerating retina and improves cone function in dogs with congenital achromotopsia. In addition, CNTF is a neuroprotective factor and an axogenesis factor for retinal ganglion cells (RGCs). This review focuses on the effects of exogenous CNTF on photoreceptors and RGCs in the mammalian retina and the potential clinical application of CNTF for retinal degenerative diseases.     

去乙酰化酶Sirt1在视神经视网膜疾病中的神经保护作用

作为一类广泛参与细胞能量代谢、周期控制和免疫应答等生命过程的重要蛋白,去乙酰化酶Sirt1还具有明显的神经元保护作用。同时,Sirt1在许多视网膜疾病动物模型的干预中展示了不可忽视的作用。本文对这一领域的研究进展进行了综述,主要包括：在糖尿病视网膜病变中,Sirt1能够抑制视网膜的慢性炎症反应、减少视网膜新生血管生成并清除视网膜细胞的高血糖记忆;在视神经炎和青光眼模型中,Sirt1能保护视网膜神经节细胞,减少其凋亡;而在年龄相关性黄斑变性模型中,Sirt1能保护视网膜色素上皮细胞的活力和功能,并降低补体因子引起的视网膜自身免疫反应。在脉络膜新生血管生成过程中,Sirt1可能具有促血管增生作用,但尚需进一步研究。由于Sirt1活性对于减轻多种视网膜疾病的组织损伤均具有重要作用,因此Sirt1是治疗视网膜病变很有前景的靶点。     

Activation of MAPK and CREB by GM1 induces survival of RGCs in the retina with axotomized nerve

PURPOSE: Neuronal cells undergo apoptosis when the supply of neurotrophic factor is limited by injury, trauma, or neurodegenerative disease. Ganglioside has both neuritogenic and neurotropic functions. Exogenously administered monosialoganglioside (GM1) has been shown to have a stimulatory effect on neurite outgrowth and to prevent degeneration of neuronal cells in the central nervous system. Even though GM1 has been shown to mimic, or have synergy with, neurotrophic factors, the neuroprotective mechanism of GM1 has not been well understood. In this study, optic nerve transection, or axotomy, was used as an in vivo model system for injury, to examine the protective mechanism of GM1 in injured retinal ganglion cells. METHODS: GM1 was injected into the vitreous body before axotomy, and the protective effect of GM1 observed with regard to activation of mitogen-activated protein kinase (MAPK) and phosphorylation of cAMP-responsive element-binding (CREB) protein. Activation of MAPK and CREB were examined by Western blot analysis and immunohistochemistry, and the surviving retinal ganglion cells were counted after retrograde fluorescence labeling. RESULTS: GM1 inhibited the degeneration of axotomized retinal ganglion cells. In addition, GM1 enhanced the activation of MAPK and CREB with the treatment of GM1 in the retina with axotomized nerve. Treatment of MAPK inhibitor PD98059 with GM1 reduced the protective action of GM1 and prevented GM1-induced phosphorylation of CREB. CONCLUSIONS: GM1 protected the axotomized retinal ganglion cells (RGCs) from cell death after axotomy through the activation of MAPK and CREB.     

Brain-derived neurotrophic factor prevents axotomized retinal ganglion cell death through MAPK and PI3K signaling pathways

PURPOSE: Brain-derived neurotrophic factor (BDNF) has a potential neuroprotective effect on axotomized retinal ganglion cells (RGCs); however, the mechanism, in regard to intracellular signaling, of BDNF-induced neuroprotection of RGCs is largely unknown. Intracellular signaling was investigated, by using axotomized RGCs and the relative contribution of the two major downstream signaling routes of TrkB determined--that is, mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase (PI3K)-Akt routes, mediated by BDNF. METHODS: Neuroprotective effects of BDNF were determined by quantifying the surviving RGCs after axotomy, by retrograde labeling. The MAPK and Akt levels were determined by Western blot analysis and activity assays. Quantification of the relative contribution of the two signaling pathways was performed by use of specific inhibitors for MAPK and PI3K (i.e., U0126 and LY294002, respectively). RESULTS: Intravitreous administration of BDNF had the most profound neuroprotective effects on axotomized RGCs among the neurotrophins. Burst phosphorylation of MAPK and Akt was induced by BDNF within 1 hour and was sustained over 2 weeks in the whole retina. Immunohistochemistry revealed that phosphorylated MAPK was detected in the RGCs and retinal Müller cells, and Akt was in the RGCs. BDNF-induced phosphorylation of MAPK and Akt was suppressed by their specific inhibitors. Moreover, administration of U0126 and LY294002 decreased significantly, but only partially, the neuroprotective effect of BDNF on the axotomized RGCs. CONCLUSIONS: BDNF-mediated signaling involves activation of both MAPK and Akt on the axotomized adult rat retina, and the collaboration of both MAPK and PI3K-Akt pathways seems to be necessary in neuroprotective signaling in axotomized RGCs.     

Interleukin-6 protects retinal ganglion cells from pressure-induced death

PURPOSE: The response of retinal ganglion cells (RGCs) to ocular pressure in glaucoma likely involves signals from astrocytes and microglia. How glia-derived factors influence RGC survival at ambient and elevated pressure and whether the inflammatory cytokine interleukin-6 (IL-6) is a contributing factor were investigated. METHODS: Primary cultures of retinal astrocytes, microglia, and RGCs were prepared using immunomagnetic separation. Comparisons were made of RGC survival at ambient and elevated pressure (+70 mm Hg) and with pressure-conditioned medium from glia with, and depleted of, IL-6. RESULTS: Pressure elevated for 24 to 48 hours reduced RGC density, increased TUNEL labeling, and upregulated several apoptotic genes, including the early immediate genes c-jun and jun-B. Pressure-conditioned medium from astrocytes reduced RGC survival another 38%, while microglia medium returned RGC survival to ambient levels. These effects were unrelated to IL-6 in microglia medium. Neither astrocyte- nor microglia-conditioned medium affected ambient RGC survival unless depleted of IL-6, which induced a 63% and a 18% decrease in RGCs, respectively. Recombinant IL-6 equivalent to levels in glia-conditioned medium doubled RGC survival at elevated pressure. CONCLUSIONS: For RGCs at ambient pressure, IL-6 secreted from astrocytes and microglia under pressure is adequate to abate other proapoptotic signals from these glia. For RGCs challenged by elevated pressure, decreased IL-6 in astrocyte medium is insufficient to counteract these signals. Increased IL-6 in microglia medium counters not only proapoptotic signals from these cells but also the pressure-induced apoptotic cascade intrinsic to RGCs.     

Selective excitotoxic degeneration of adult pig retinal ganglion cells in vitro

PURPOSE: Excitotoxicity is proposed to play a prominent role in retinal ganglion cell (RGC) death ensuing from diseases such as glaucoma and ischemia, but cell culture studies have used tissue from newborn rodents, yielding conflicting data that implicate either N-methyl D-aspartate (NMDA) or non-NMDA glutamate (Glu) receptor-mediated pathways. Excitotoxic RGC death was examined in vitro in this study, using adult pigs, a large-animal model for human retina. METHODS: Adult pig retina (and for comparative purposes young and adult rat retina) were dissociated and maintained in monolayer culture. Medium was supplemented with Glu or pharmacologic agonists or antagonists, and surviving RGCs and other retinal neurons were quantified using specific immunolabeling methods. Electrophysiological responses to externally applied Glu of RGCs in culture were recorded using whole-cell patch-clamp techniques. RESULTS: Application of Glu led to selective, dose-dependent losses in large RGCs (maximal 37% decrease at 1 mM; median effective dose [ED50], approximately 80 microM) and neurite damage in surviving RGCs. Application of Glu agonists and Glu receptor subclass antagonists showed that large RGC death was mediated through both NMDA and non-NMDA receptor pathways. Small RGCs, amacrine cells, and all other retinal neurons were resistant to Glu-induced death. By comparison, rat retinal cultures displayed heightened RGC vulnerability to Glu, mediated exclusively by non-NMDA receptor-mediated pathways. Amacrine cells were unaffected by NMDA but were very sensitive to kainate application (>90% loss). Other retinal neurons were unaffected by any treatment. CONCLUSIONS: The molecular pathways underlying excitotoxic RGC death in vitro (non-NMDA or NMDA-preferring Glu receptors) vary among species and developmental stages. The selective elimination of adult pig large RGCs by NMDA receptor-mediated pathways more closely resembles human and animal glaucoma in vivo than other published culture models, providing a simplified experimental system for investigating the pharmacologic and toxicologic bases of glaucoma-like neuronal death.     

Retinal concentration and protective effect against retinal ischemia of nilvadipine in rats

PURPOSE: Several calcium entry blockers have neuroprotective effects on cellular damage in the brain induced by ischemia. The purpose of this study was to determine whether nilvadipine (NID) crosses the blood-retinal barrier, and if so, whether it can then protect the photoreceptors against retinal ischemia-reperfusion injury. METHODS: Rats received an intramuscular injection of 1 mg/kg of NID and nifedipine (NIF), and the retinal and serum concentrations were measured. Ischemia was induced by raising the intraocular pressure for 45 minutes. Twenty-four hours after the reperfusion, the number of TUNEL positive cells and retinal ganglion cells (RGCs) were counted, and the thickness of the retina was measured. RESULTS: After 60 minutes, the concentration of NID, but not NIF, was higher in retina than in the serum. The number of TUNEL-positive cells was fewer and the reduction in the number of RGCs and the thickness of retina was less in the eyes that had received NID than controls. CONCLUSIONS: The findings show that NID has high permeability to retina compared with NIF, which has less fat solubility than NID, and neuroprotective effect to retinal cells. NID might be useful for the treatment of glaucoma or other retinal diseases that have some relation to apoptosis.     

Chondroitin sulfate-derived disaccharide protects retinal cells from elevated intraocular pressure in aged and immunocompromised rats

PURPOSE: A disaccharide (DS) derived from the naturally occurring compound chondroitin sulfate proteoglycan (CSPG) was recently shown to have neuroprotective activity. The authors examined the ability of this compound (CSPG-DS) to protect retinal ganglion cells (RGCs) from death caused by elevated intraocular pressure (IOP). METHODS: With the use of chronic and acute models of elevated IOP, the authors examined the effects of CSPG-DS on RGC survival in adult ( approximately 2 months old), aged (10-12 months old), and immunocompromised Lewis rats. Systemic, topical, and oral routes of administration were examined. RESULTS: CSPG-DS protected RGCs from IOP-induced death. Treatment was effective in all three examined rat populations (normal adult, aged, and immunocompromised rats) and with all routes of administration, possibly in part through its control of microglial activity. CONCLUSIONS: Results point to the therapeutic potential of CSPG-DS for glaucoma, particularly in elderly populations for whom disease prevalence is high.