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.     

Selective loss of retinal ganglion cells in albino avian glaucoma

Retinal ganglion cell loss was investigated in the retinae of albino quails before and after the development of glaucoma. The isodensity maps of ganglion cells, the total number of ganglion cells, and the histograms of the cell size in the central region of the retina were similar between albino quails without glaucoma and pigmented quails. However, ganglion cells in the intermediate and peripheral regions of the albino quails retina without glaucoma were significantly smaller than those of the pigmented quail retina. In albino quails with moderate glaucoma in 3 months of age, 11% to 55% of all the retinal ganglion cells had disappeared, with the loss of medium-sized cells (30-60 micron2 occurring earlier than that of small and large cells. In albino quails with advanced glaucoma, there was marked cupping around the optic nerve head, and only small ganglion cells remained in the ganglion cell layer.     

Specific transcellular staining of microglia in the adult rat after traumatic degeneration of carbocyanine-filled retinal ganglion cells.

The present work was undertaken to assess the fate of ganglion cell debris in the axotomized retina of adult rats and employed a new technique to label phagocytosing microglia via the internalized material. In the main experiment, transection axotomy was performed on the intraorbital segment of the optic nerve, and a fast-transported, vital fluorescent styryl dye (4Di-10ASP) was deposited at the ocular stump of the nerve in order to pre-label retrogradely the ganglion cells destined to die because of the axotomy. Optic nerve transection resulted in progressive degradation of ganglion cell axons, perikarya, and dendrites within the retina and in release of fluorescent material, which was then incorporated into cells identified as microglia. No other retinal cells stained, although astrocytes and Müller's cells also responded to neuron degeneration by accumulating glial fibrillary acidic protein. Incorporation of labelled material into microglia topo-chronologically paralleled the ganglion cell degeneration starting within the optic fibre layer (OFL) and proceeding towards the ganglion cell layer (GCL) and the inner plexiform layer (IPL) of the affected retina. Long-term labelling of microglia monitored up to 3 months after optic nerve transection indicated that labelled microglial cells persisted within the retina. Microglia displayed a strong territorial arrangement within the GCL and IPL, and staggered, bilaminated distribution in both layers. These studies directly prove that microglia in the retina can be transcellularly labelled during traumatic degeneration of ganglion cells. The findings suggest that microglial cells play an important role in axotomy-induced wound healing and removal of cell debris.     

Flavonoids protect retinal ganglion cells from ischemia in vitro

Retinal ischemia is a common cause of visual impairment and blindness. However, despite the significant advances that have been made in understanding the pathophysiology of retinal ischemia, effective treatments are still lacking. The goal of these studies was to use an in vitro model to identify molecules that could be neuroprotective for retinal ganglion cells exposed to ischemia. Ischemia was induced in the rat retinal ganglion cell line, RGC-5, using iodoacetic acid (IAA). Brief treatment with IAA resulted in RGC-5 cell death within 24 h by a non-apoptotic mechanism. Similar to ischemia in vivo, IAA treatment caused a rapid loss of ATP to approximately 50% of control levels. In contrast, changes in markers of oxidative stress occurred more slowly and included an increase in reactive oxygen species and a decrease in glutathione. Specific flavonoids were able to prevent the cell death caused by IAA treatment. Some of the flavonoids also prevented the loss of ATP as well as the changes in markers of oxidative stress. In contrast, classical antioxidants had only a very modest effect on IAA-induced cell death. These results suggest that specific flavonoids may be useful in preventing ischemia-induced retinal ganglion cell death in vivo.     

Acetylcholine neuroprotection against glutamate-induced excitotoxicity in adult pig retinal ganglion cells is partially mediated through alpha4 nAChRs

In the mammalian retina, excess glutamate release has been shown to be involved in retinal ganglion cell (RGC) death associated with various diseases. Recent studies have determined that activation of alpha7 nicotinic acetylcholine receptors (nAChRs) partially protect isolated RGCs from glutamate-induced excitotoxicity. In this study, we further classify the types of nAChRs involved in neuroprotection against glutamate-induced excitotoxicity using isolated adult pig RGCs. Cells were isolated with a modified two-step immunoselective panning technique designed to isolate RGCs from other retinal neurons. Once isolated, nAChR subunits were identified using a combination of pharmacological and immunocytochemical techniques. In cell culture experiments, a variety of alpha4 nAChR specific agonists were found to have a partial neuroprotective against glutamate-induced excitotoxicity. This neuroprotection was abolished in the presence of the alpha4 nAChR antagonist, dihydro-beta-erythroidine (DHbetaE). Immunocytochemical results localized several nAChR subunits on isolated adult pig RGCs; in particular alpha4, alpha7 and beta2 nAChR subunits. Large RGCs exclusively immunostained with antibodies against alpha7 nAChR subunits whereas alpha4 and beta2 subunits exclusively immunostained only small RGCs. Double label experiments provided evidence that alpha4 and beta2 subunits co-localize on small RGCs. Knowledge of the receptor subtypes responsible for neuroprotection may lead to treatments associated with glutamate-induced excitotoxicity.     

Survival and axonal regeneration of retinal ganglion cells in adult cats

Axotomized retinal ganglion cells (RGCs) in adult cats offer a good experimental model to understand mechanisms of RGC deteriorations in ophthalmic diseases such as glaucoma and optic neuritis. Alpha ganglion cells in the cat retina have higher ability to survive axotomy and regenerate their axons than beta and non-alpha or beta (NAB) ganglion cells. By contrast, beta cells suffer from rapid cell death by apoptosis between 3 and 7 days after axotomy. We introduced several methods to rescue the axotomized cat RGCs from apoptosis and regenerate their axons; transplantation of the peripheral nerve (PN), intraocular injections of neurotrophic factors, or an antiapoptotic drug. Apoptosis of beta cells can be prevented with intravitreal injections of BDNF+CNTF+forskolin or a caspase inhibitor. The injection of BDNF+CNTF+forskolin also increases the numbers of regenerated beta and NAB cells, but only slightly enhances axonal regeneration of alpha cells. Electrical stimulation to the cut end of optic nerve is effective for the survival of axotomized RGCs in cats as well as in rats. To recover function of impaired vision in cats, further studies should be directed to achieve the following goals: (1) substantial number of regenerating RGCs, (2) reconstruction of the retino-geniculo-cortical pathway, and (3) reconstruction of retinotopy in the target visual centers.     

Mechanism of retinal ganglion cells death in secondary degeneration of the optic nerve

In central nervous system injury, the secondary degeneration process is known to play a major role in determining the final extent of impairment. Here, we investigated the mechanism of retinal ganglion cell (RGC) death in secondary degeneration of the optic nerve using a unique model that allows morphological separation between primary and secondary degeneration. A partial transection model was applied unilaterally in 110 Wistar rat eyes. The rate of apoptosis was evaluated in primary and secondary degeneration over a period of 6 months using the Hoechst staining technique. The involvement of caspase 3 and members of the Bcl-2 family (Bax, Bad, Bcl-2 and Bcl-xl) was evaluated at multiple time points for 6 months after the injury by immunohistochemistry and RT-PCR. We found that in secondary degeneration of the optic nerve, RGCs died by apoptosis from day 3-6 months following the injury, peaking at 3 months (16.3% ± 2.5% apoptotic cells, p < 0.01). Both primary and secondary degeneration of the optic nerve resulted in caspase 3 activation, which was longer and more intense in the former. Similarly, both primary and secondary degeneration led to significant (p < 0.05) downregulation of the pro-survival genes Bcl-2 and Bcl-x-L and up-regulation of the pro-apoptotic genes Bax and Bad (p < 0.05), with a suggested delay in secondary degeneration. Thus, secondary degeneration of the optic nerve leads to RGC apoptosis over long periods in a similar mechanism as in primary degeneration.     

Activation of retinal glial cells contributes to the degeneration of ganglion cells in experimental glaucoma

Elevation of intraocular pressure (IOP) is a major risk factor for neurodegeneration in glaucoma. Glial cells, which play an important role in normal functioning of retinal neurons, are well involved into retinal ganglion cell (RGC) degeneration in experimental glaucoma animal models generated by elevated IOP. In response to elevated IOP, mGluR I is first activated and Kir4.1 channels are subsequently inhibited, which leads to the activation of Müller cells. Müller cell activation is followed by a complex process, including proliferation, release of inflammatory and growth factors (gliosis). Gliosis is further regulated by several factors. Activated Müller cells contribute to RGC degeneration through generating glutamate receptor-mediated excitotoxicity, releasing cytotoxic factors and inducing microglia activation. Elevated IOP activates microglia, and following morphological and functional changes, these cells, as resident immune cells in the retina, show adaptive immune responses, including an enhanced release of pro-inflammatory factors (tumor neurosis factor-α, interleukins, etc.). These ATP and Toll-like receptor-mediated responses are further regulated by heat shock proteins, CD200R, chemokine receptors, and metabotropic purinergic receptors, may aggravate RGC loss. In the optic nerve head, astrogliosis is initiated and regulated by a complex reaction process, including purines, transmitters, chemokines, growth factors and cytokines, which contributes to RGC axon injury through releasing pro-inflammatory factors and changing extracellular matrix in glaucoma. The effects of activated glial cells on RGCs are further modified by the interplay among different types of glial cells. This review is concluded by presenting an in-depth discussion of possible research directions in this field in the future.     

Optic nerve transection in monkeys may result in secondary degeneration of retinal ganglion cells

PURPOSE: Interest in neuroprotection for optic neuropathies is, in part, based on the assumption that retinal ganglion cells (RGCs) die, not only as a result of direct (primary) injury, but also indirectly as a result of negative effects from neighboring dying RGCs (secondary degeneration). This experiment was designed to test whether secondary RGC degeneration occurs after orbital optic nerve injury in monkeys. METHODS: The superior one third of the orbital optic nerve on one side was transected in eight cynomolgus monkeys (Macaca fascicularis). Twelve weeks after the partial transection, the number of RGC bodies in the superior and inferior halves of the retina of the experimental and control eyes and the number and diameter of axons in the optic nerve were compared by detailed histomorphometry. Vitreous was obtained for amino acid analysis. A sham operation was performed in three additional monkeys. RESULTS: Transection caused loss of 55% +/- 13% of RGC bodies in the superior retina of experimental compared with fellow control eyes (mean +/- SD, t-test, P < 0.00,001, n = 7). Inferior RGCs, not directly injured by transection, decreased by 22% +/- 10% (P = 0.002). The loss of superior optic nerve axons was 83% +/- 12% (mean +/- SD, t-test, P = 0.0008, n = 5) whereas, the inferior loss was 34% +/- 20% (P = 0.02, n = 5). Intravitreal levels of glutamate and other amino acids in eyes with transected nerves were not different from levels in control eyes 12 weeks after injury. Fundus examination, fluorescein angiography, and histologic evaluation confirmed that there was no vascular compromise to retinal tissues by the transection procedure. CONCLUSIONS: This experiment suggests that primary RGC death due to optic nerve injury is associated with secondary death of surrounding RGCs that are not directly injured.     

神經營養因子對混合培養鼠視網膜神經節細胞的影響

目的觀察兩種神經營養因子腦源性神經營養因子BDNF和碱性成纤維生長因子bFGF對混合培養鼠視網膜神經節細胞(retinal ganglion cells,RGCs)的影響,并節選出其有效濃度.方法采用胰酶消化法將12衹生後2～3天的Sprague-Dawley(SD)乳鼠視網膜制成細胞悬液,接種于鼠尾原包被的96孔培養板(5×104個細胞/孔).分别加入各種濃度梯度的BDNF和bFGF,在37℃、5%CO2恒温培養箱中培養,于1、3、5天,用MTT微量自動比色法測量存活細胞的吸光度A值.用Thy-l抗體、NSE抗體和GFAP抗體進行細胞免疫化學檢查以鍳定RGCs.結果培養在鼠尾原上的細胞生長良好,大部分伸出突起.培養1天、3天,各種濃度的BDNF和bFGF實驗組吸光度A值舆對照組比較均具有顯著性差异(p<0.01,p<0.05);培養5天,BDNF(40ng/ml、50ng/ml)、bFGF(15ng/ml)和BDNF+bFGF組吸光度A值高于對照組,具有顯著性差异(P＜0.01,P＜0.05).結論各種濃度的BDNF和bFGF均能促進鼠RGCs在體外的存活,BDNF具有濃度依賴性,BDNF的作用優于bFGF;同時應用BDNF和bFGF無叠加作用.     

依托咪酯对成年大鼠视神经切断后视网膜神经节细胞的保护作用

目的:观察依托咪酯(ET)对成年大鼠视神经切断后视网膜神经节细胞(RGC)存活的作用.方法:成年雌性SD大鼠42只,眶内距视神经根部1mm处切断左侧视神经,残端留置浸有荧光金(50g/L)的明胶海绵逆行标记RGC.术后大鼠随机分为ET(4mg/kg,ip,1次/d)治疗组、1,2-丙二醇(PG)溶剂对照组、生理盐水对照组和正常对照组.再根据术后不同存活时间将前3组动物分为7d和14d两个亚组,正常对照组动物则存活2d.于相应存活时间点处死动物,取出各组大鼠左侧视网膜平铺后计数存活RGC并得出RGC的平均密度.结果:术后7dET治疗组存活RGC平均密度为1 307±55/mm2,显著高于PG对照组(1 128±75/mm2)和生理盐水对照组(1 068±75/mm2,P＜0.001).然而,未能在术后14d观察到ET的这种保护作用,因为ET治疗组存活RGC平均密度(210±36/mm2)与PG对照组(215±20/mm2)和生理盐水对照(208±19/mm2)间无显著差异(P＞0.05).结论:ET在视神经切断后一定时期内对RGC具有神经保护作用.     

Neuroprotective effect of inosine on axotomized retinal ganglion cells in adult rats

PURPOSE: To explore the potential survival-promoting effect of inosine on axotomized retinal ganglion cells (RGCs) of adult rats in vivo. METHODS: The left optic nerves (ON) in the subject rats were transected at 1.5 mm from the optic disc. Repeated intraperitoneal injections or single intraocular injection of inosine were administered. The RGCs were retrogradely labeled with a gold fluorescent dye and the density of surviving RGCs in number per square millimeter of retina was calculated in wholemounted retinas. The functional integrity of the blood-retinal barrier (BRB) after ON transection was evaluated with an intravenous injection of Evans blue. RESULTS: In control animals, the mean density of surviving RGCs (number per square millimeter) of the whole retina was 2007 +/- 68 at 2 days (taken as the normal value), 927 +/- 156 at 7 days, and 384 +/- 33 at 14 days after surgery. Repeated intraperitoneal injections (75 mg/kg for each injection) of inosine significantly enhanced RGC survival at 14 days after ON transection (500 +/- 38), whereas no significant difference in the densities was detected at 7 days (974 +/- 101), even when the dosage of inosine was doubled (1039 +/- 61). At this time point, however, a single intraocular injection of inosine significantly increased the density of surviving RGCs (1184 +/- 156). Moreover, more RGCs around the optic disc were rescued when inosine, administered either intraperitoneally or intraocularly, showed a beneficial effect on RGC survival. No breakdown of the BRB after ON transection was detected with the method used in the study. CONCLUSIONS: These findings demonstrate that inosine could protect axotomized RGCs in vivo after ON transection.     

Dorzolamide and timolol saves retinal ganglion cells in glaucomatous adult rats.

PURPOSE: This study was designed to evaluate the effects of a dorzolamide-timolol combination or dorzolamide on retinal ganglion cell (RGC) density and intraocular pressure (IOP) in glaucomatous eyes of adult rats. METHODS: Glaucoma was induced in the right eye of adult Wistar rats by episcleral venous occlusion. One experimental group was administered dorzolamide 2%-timolol 0.5% combination eye drops, while the other experimental group was administered dorzolamide 2% eye drops. Control groups had surgery without drug administration. Drug application was initiated either 2 weeks before surgery (Group A), from the day of surgery (Group B), 2 weeks after surgery (Group C), or 4 weeks after surgery (Group D). RGCs were labeled by intratectal Fluorogold injections and counted from flat-mount preparations, and IOP was measured using Tonopen. RESULTS: Both dorzolamide-timolol combination and dorzolamide, when applied topically, significantly reduced IOP and improved RGC densities in experimental eyes when compared to control eyes. Earlier initiation, as well as longer duration of drug application, resulted in higher RGC densities. CONCLUSIONS: Topical application of a dorzolamide-timolol combination or dorzolamide saved RGCs to a significant extent and reduced IOP in glaucomatous rat eyes.     

重组人促红细胞生成素对离体培养视网膜神经节细胞的影响

目的研究重组人促红细胞生成素（recombinant humanerythropoiain，rhEPO）对培养的视网膜神经节细胞（retinal ganglion cells，RGCs）存活、突起生长及生长相关蛋白43（growth associmed protein43，GAP-43）表达的影响，探讨rhEPO对培养RGCs可能的作用机制。方法分别用DMEM及含有rhEPO的DMEM对RGCs进行离体培养，观察RGCs在体外存活的时间，测量2d、4d、6dRGCs的最长突起长度；免疫细胞化学检测GAP-43蛋白表达，并测定平均灰度值。结果DMEM组离体培养RGCs于6～8d死亡，而rhEPO组RGCs能存活10～12d，存活时间较DMEM组显著延长（P〈0．01）。培养2d、4d、6d时，DMEM组最长突起长度依次为（42．90±4．71）μm、（79．74±8．49）μm、（110．02±10．79）μm，rhEPO组依次为（55．47±7．07）μm、（100．16±7．78）μm、（118．63±11．50）μm，培养2d、4d时rhEPO组与DMEM组相比差异非常显著（P〈0．01），6d时差异显著（P〈0．05）。2组细胞在培养2d时GAP-43蛋白的表达水平较高，4d时GAP-43蛋白表达到高峰。6d时GAP-43蛋白的表达水平明显降低。各时间点rhEPO组RGCsGAP-43蛋白的表达均较DMEM组有明显增高，与DMEM组相比均有非常显著差异（P〈0．01）。结论rhEPO可延长离体培养RGCs的存活时间和促进其突起的生长。上调离体培养RGCs GAP-43蛋白的表达。rhEPO促进RGCs突起生长的作用可能通过上调RGCs GAP-43蛋白的表达来实现。[眼科新进展2007；27（3）：138-192]     

新生SD大鼠视网膜神经节细胞体外原代培养

目的:建立简便易行的新生SD大鼠视网膜神经节细胞(RGCs)体外原代培养方法。方法:取出生后24h的SD大鼠视网膜,胰蛋白酶消化制成单细胞悬液,接种于多聚赖氨酸包被的预先置入盖玻片的24孔培养板中培养,倒置相差显微镜观察细胞生长规律,使用Thy-1mAb免疫细胞化学法鉴定RGCs。结果:该方法培养的RGCs体外存活时间可达5d,免疫细胞化学法显示RGCs的纯度约为80%。结论:使用不添加附加成分的普通培养液SD大鼠RGCs体外培养成功,且RGCs较纯化,是一种较理想的细胞培养实验模型。     

前列地尔对体外培养类缺血再灌注损伤模型视网膜神经节细胞的影响

目的 探讨前列地尔对体外培养类缺血再灌注损伤模型视网膜神经节细胞(retinal ganglion cells,RGC)生长的影响及其作用机制.方法 以原代培养SD大鼠RGC为研究对象,将培养的细胞随机分为正常组、模型组、模型+前列地尔组.建立类缺血再灌注损伤模型,模型+前列地尔组在缺血期与再灌注期均加入浓度分别为15 μg·L-1、45 μg·L-1、135 μg·L-1前列地尔.类缺血模型建立后24 h、48 h、72 h应用MTT比色法分别测定各组细胞的吸光度值(A值),确定前列地尔的最佳有效作用浓度与时间;采用荧光定量PCR方法分别测定各组细胞中Bax、Bcl-2的相对表达量.结果 RGC培养48 h后,可见RGC伸出突起,随着培养时间的延长,细胞突起变长,部分细胞突起相互连接.使用抗大鼠Thy-1单克隆抗体对RGC进行细胞免疫组织化学检测,可见RGC胞浆及轴突染成棕黄色.模型建立后24 h、48 h、72 h时,与正常组、模型+前列地尔组比较,模型组A值均明显降低,差异均有统计学意义(均为P ＜0.05);模型建立后24 h时,模型+45 μg·L-1前列地尔组A值与正常组差异无统计学意义(P＞0.05),而正常组、模型+45 μg·L-1前列地尔组与模型+15 μg· L-1前列地尔组、135 μg· L-1前列地尔组比较,差异均有统计学意义(均为P ＜0.05),模型+15 μg· L-1前列地尔组与模型+ 135μg·L-1前列地尔组组间差异无统计学意义(P＞0.05);模型建立后48 h、72 h时,正常组、模型+前列地尔组之间A值差异均无统计学意义(均为P＞0.05).正常组Bcl-2的相对表达量为0.999±0.035,模型组为0.657±0.012,模型+45 μg·L-1前列地尔组为1.715±0.016(P ＜0.05);正常组Bax相对表达量为1.001±0.048,模型组为1.712±0.089,模型+45 μg· L-1前列地尔组为1.508±0.061(P ＜0.05).结论 前列地尔对体外培养类缺血再灌注损伤模型RGC具有保护作用;前列地尔可能通过上调细胞内Bcl-2的表达,同时下调Bax的表达,进而发挥细胞保护作用.     

Rescue of axotomized retinal ganglion cells by BDNF gene electroporation in adult rats

PURPOSE: To determine whether the brain-derived neurotrophic factor (BDNF) gene can be transfected into retinal ganglion cells (RGCs) by electroporation and whether axotomized RGCs can be rescued after transfection by BDNF in adult rats. METHODS: Mouse BDNF cDNA was injected intravitreally followed by in vivo electroporation in adult rats. The expression of BDNF in RGCs was confirmed by Western immunoblot analysis and immunohistochemistry. After introduction of BDNF cDNA, the survival of axotomized RGCs was estimated by the TdT-dUTP terminal nick-end labeling (TUNEL) method and measured by counting the number of RGCs that were labeled retrogradely by 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyamine percholorate (diI) applied to the superior colliculus (SC). RESULTS: Eyes with injection of the BDNF gene followed by in vivo electroporation showed a significantly higher level of expression of BDNF in the RGC layer, a higher rescue ratio, and a lower number of TUNEL-positive cells than the control samples. CONCLUSIONS: These findings demonstrate that electroporation is an effective method for the direct delivery of genes into RGCs, and that the BDNF gene transferred into RGCs by in vivo electroporation can protect axotomized RGCs against apoptosis.