视神经夹持损伤模型大鼠视网膜和视神经小胶质细胞的活化

背景 外伤性视神经病变(TON)是继发于外力创伤下的急性视神经损伤,预后较差.小胶质细胞作为中枢神经系统中重要的免疫细胞,参与了中枢神经系统疾病与多种眼科疾病的病理生理过程.然而,小胶质细胞在TON的病理发展及损伤修复过程中的作用尚不明确. 目的 比较大鼠视神经夹持损伤后视神经与视网膜中小胶质细胞的形态变化、激活数量、分布情况及活化水平的差异. 方法 将35只SPF级健康雌性成年Sprague-Dawley(SD)大鼠按照随机数字表法分为正常对照组,造模后6h、3d、7d、14 d、30 d组和假手术组,每组5只大鼠.造模后各时间点组用夹持钳以50 g的夹持力在大鼠眼球后约2 mm处钳夹视神经10s,建立大鼠视神经夹持模型,假手术组大鼠行相同的手术操作但不夹持视神经,正常对照组不做任何处理.分别于上述时间点制备大鼠视神经和视网膜冰冻切片,采用lectin-FITC荧光标记抗体检测各组大鼠视神经和视网膜中的小胶质细胞数量和激活的小胶质细胞数量. 结果 正常对照组和假手术组大鼠视网膜中小胶质细胞主要位于内丛状层(IPL),少部分位于内核层(INL)和神经节细胞层(GCL),外核层(ONL)和外丛状层(OPL)未见小胶质细胞分布.正常对照组大鼠视网膜小胶质细胞的细胞体较小,以分支状为主,突触细长,可见二级分支.各模型组大鼠视网膜中小胶质细胞主要位于GCL和IPL,小胶质细胞在GCL的数量明显多于假手术组,小胶质细胞多为阿米巴状,部分呈半激活态,少见分支静息态.正常对照组、假手术组及造模后6h、3d、7d、14 d和30 d组大鼠视网膜中小胶质细胞数分别为6.40-±-1.52、7.20±2.05、12.00±3.54、14.00±4.06、18.00±4.36、18.40±3.13和10.80±1.92,造模后各时间点大鼠视网膜中小胶质细胞数量均明显多于正常对照组,造模后30 d小胶质细胞数量明显少于造模后7d和14d组,差异均有统计学意义(均P＜0.05);造模后3、7和14d组大鼠视网膜中激活小胶质细胞数量明显多于假手术组,差异均有统计学意义(P=0.024、0.009、0.023).正常对照组和假手术组大鼠视神经中小胶质细胞较小,呈棒状或分枝状,分布均匀且稀疏.造模后各时间点组小胶质细胞较假手术组细胞体积增大,呈阿米巴状并分布在近视神经夹持部位.造模后6h、3d、7d、14d大鼠视神经中小胶质细胞数量明显多于正常对照组,差异均有统计学意义(P=0.007、0.001、0.003、0.014).造模后30 d大鼠视神经中小胶质细胞数量明显少于造模后3d、7d和14 d组,差异均有统计学意义(均P＜0.05).造模后6h、3d和7d组大鼠视神经中活化小胶质细胞数量明显多于假手术组,差异有统计学意义(P=0.005、0.004、0.030),造模后14d、30 d大鼠视神经中活化的小胶质细胞数量较造模后3d组明显减少,差异均有统计学意义(P=0.021、0.004),造模后6h组视神经中激活态小胶质细胞增加并持续到造模后14d.结论 大鼠视神经夹持损伤后一定时间内视网膜及视神经中小胶质细胞增加并活化,视神经中小胶质细胞的活化及其衰减均早于视网膜,视神经中小胶质细胞活化程度更明显.     

视神经切断激活小胶质细胞后血-视网膜屏障的功能状态

目的观察视神经切断激活视网膜小胶质细胞后血-视网膜屏障(BRB)的功能状态。方法自眶内切断18只成年大鼠左侧视神经，12只近侧断端留置浸有50g／L荧光金的明胶海绵，分别于术后7d或14d(每时间点各6只)处死动物，取左眼视网膜后固定，荧光显微镜下观察小胶质细胞的反应。另外6只存活7d或14d(每时间点各3只)后，股静脉注射30g/L伊文思蓝。2h后，以温生理盐水灌注动物，立即摘除双侧眼球，行冰冻切片，荧光显微镜下观察；右眼球为内对照。结果视神经切断后视网膜节细胞层活化的小胶质细胞随时间逐渐增多，部分小胶质细胞位于视神经层。术后7d和14d均未发现伊文思蓝渗漏至手术侧视网膜。结论视神经切断后视网膜内增多的活化小胶质细胞尚不足以破坏BRB，BRB的功能性完整得以维持。     

大鼠视神经损伤视网膜内P38丝裂原活化蛋白激酶活性的表达

目的:动态观察视神经损伤后视网膜中P38丝裂原活化蛋白激酶(MAPK)活性的表达变化和早期细胞凋亡情况。方法:制作大鼠视神经钳夹伤模型后设立对照组、假手术组和视神经夹伤组,应用免疫组化方法及流式细胞仪分别检测视神经损伤后1,6,12,24h;15,30d共6个时间点3组大鼠视网膜中磷酸化(活化)P38MAPK的表达和早期细胞凋亡率,同时对视网膜形态学改变进行观察。结果:视神经损伤诱导视网膜神经节细胞(RGC)严重丧失,损伤后1~15dRGC快速减少,15d后缓慢减少。在正常对照组、假手术组磷酸化P38MAPK表达阴性,视神经损伤后P38MAPK活性的表达于6h检测到表达,逐渐增加至24h阳性表达达高峰,15d表达下降,30d消失,具有统计学意义(P<0.01)。视神经损伤后早期细胞凋亡率逐渐上升,24h达最高8.9%,随后下降。结论:视神经不完全损伤刺激了大鼠视网膜中P38MAPK的活性表达,与早期细胞凋亡率变化相似。P38MAPK通路与视神经损伤诱导的大鼠视网膜RGC凋亡密切相关。     

大鼠视神经再生的组织病理学机制

目的探讨大鼠视神经再生的组织病理学机制。方法 40只Wistar大鼠按随机数字表法随机分为正常组（n=8）、单纯视神经损伤组（n=16）和视神经损伤联合晶状体损伤组（n=16）3组。单纯视神经损伤组：单纯的视神经完全性横断性损伤并保存中央血管完好;视神经损伤联合晶状体损伤组：视神经的完全性横断性损伤并保存中央血管完好,同时损伤晶状体形成白内障。4周后处死动物,处死前3d,用毛细玻璃管注入大鼠玻璃体内4～5μL质量分数2.5%的罗丹明B异硫氰酸盐（RITC）。大鼠视神经和视网膜行常规组织病理学检查并计数视网膜神经节细胞（RGCs）的数量。结果正常视神经可以观察到神经纤维及神经胶质细胞。单纯视神经损伤组视神经断端可见呈团块状的胶质瘢痕,细胞较密集,排列紊乱。视神经损伤联合晶状体损伤组视神经断端无胶质瘢痕,且细胞排列较疏松,并有纵向排列的趋势,伴有大量巨噬细胞浸润。视神经损伤联合晶状体损伤组周边部RGCs的数量为4.06±1.45,单纯视神经损伤组为4.06±1.45,2组比较差异有统计学意义（P〈0.01）。结论视神经再生的关键是克服视神经断端作为物理性屏障的胶质瘢痕和提高RGCs的存活数量。     

大鼠视神经损伤后Toll样受体4在视网膜中的表达

背景Toll样受体4（TLR4）是一种重要的免疫相关受体,在多种疾病的发生中起致炎作用。研究发现,视神经损伤后继发的炎症反应可进一步引起视网膜损伤,因此视神经损伤后TLR4的表达及其效应值得研究。目的研究大鼠视神经损伤后视网膜TLR4的表达情况。方法选取成年健康SPF级SD大鼠24只,按随机数字表法随机分为视神经损伤3d组和视神经损伤7d组。取大鼠右眼用视神经钳夹法制备视神经损伤模型,左眼不予处理为对照组。分别于视神经损伤后3d和7d用过量麻醉法处死大鼠并分离视网膜,采用免疫荧光法检测各组大鼠视网膜中TLR4的表达;分别采用逆转录PCR法（RT—PCR）和Westernblot法检测大鼠视网膜中TLR4mRNA及其蛋白的表达;采用TUNEL染色法观察各组大鼠视网膜神经节细胞（RGCs）的凋亡情况。结果视网膜免疫荧光法检测结果显示,TLR4在大鼠视网膜中呈绿色荧光,视神经损伤3d组和视神经损伤7d组造模眼视网膜中的荧光强度较对照组左眼均明显增强,绿色荧光主要分布在视网膜内层。RT—PCR法检测表明,模型眼视网膜损伤后3d和7d视网膜中TLR4mRNA相对表达量分别为2．92±0．06和3．92±0．12,对照眼TLR4mRNA的相对表达量分别为2．87±0．12和3．44±0．17,大鼠模型眼TLR4mRNA表达的灰度值较对照眼明显增加,差异均有统计学意义（t3d=-12．888,P〈0．001;t7d=-4．669,P=0．010）。Westernblot法检测显示,大鼠模型眼视网膜损伤3d和7d视网膜中TLR4蛋白的相对表达量分别为1．14±0．05和1．49±0．03,对照眼TLR4蛋白的相对表达量分别为0．99±0．09和1．38±0．07,模型眼视网膜中TLR4蛋白表达量明显高于对照眼,差异均有统计学意义（t3d=-11．324,P〈0．001;t7d=-5．638,P=0．005）。TUNEL染色显示,模型眼RGCs凋亡数较对照眼增多。结论TLR4在视神经损伤大鼠视网膜内层的表达明显上调,提示TLR4通路可能参与RGCs的损伤。     

蛇毒神经生长因子对大鼠视神经夹伤保护的电镜观察

目的研究蛇毒神经生长因子在视神经损伤后对视网膜神经节细胞的保护作用。方法将Wistar大鼠40只随机分为实验对照组和实验治疗组。制作实验性视神经夹伤模型,用头部宽1mm的微型血管夹夹伤大鼠右眼视神经后,实验治疗组向伤眼玻璃体腔内注入蛇毒神经生长因子100BU(0.025mL)。实验对照组向伤眼玻璃体腔内注入0.025mL平衡盐液。于损伤后第3d、7d、14d、30d、60d取材,用透射电镜观察不同时间段各组视网膜形态学变化。结果电镜下大鼠视网膜改变:实验治疗组和对照组电镜下均可见坏死和凋亡。伤后14d,实验治疗组视网膜微管数目比实验对照组较多,排列比较整齐。结论在视神经损伤早期,蛇毒神经生长因子能减轻视神经夹伤后微管的损坏,提高视网膜神经节细胞的存活数量,对视网膜神经节细胞有明显的保护作用。     

小鼠视神经损伤后视神经小胶质细胞数目和形态变化

目的：研究小鼠视神经损伤后不同时间点视神经小胶质细胞的数目与形态变化。方法：实验研究。选取32只成年雄性健康CX3CR1—/GFP转基因杂交小鼠,按照随机数字表法将小鼠随机分为正常对照组、视神经损伤1、7、14 d组,每组8只。视神经损伤组均在左眼建立视神经夹伤模型,右眼不处理,正常对照组不做任何处理。分别于上述时间点制备小鼠视神经冰冻切片,每根视神经取3张切片（30 μm）,采用共聚焦显微镜在距离眼球端500 μm处拍摄图像,比较小胶质细胞的数量和形态变化。4组小胶质细胞数目比较采用单因素方差分析。结果：正常对照组、视神经损伤1、7和14 d组的视神经小胶质细胞数目分别为（438±16）个/mm2、（323±15）个/mm2、（1 252±107）个/mm2、（1 474±113）个/mm2。视神经损伤7、14 d组小胶质细胞数量明显多于正常对照组和视神经损伤1 d组,差异均有统计学意义（均 P<0.001）。正常对照组的视神经小胶质细胞均匀分布,细胞核较小,分枝细长并向四周伸展。视神经损伤1 d组,小胶质细胞分枝数量减少,细胞核形态变化不明显。损伤7 d组的视神经小胶质细胞大量激活,排列较紊乱,存在少量细胞聚集现象,分枝短而粗,且越靠近细胞核分枝越粗,细胞核体积明显增大。视神经损伤14 d组,视神经小胶质细胞形态与损伤7 d组类似。结论：小鼠视神经夹持损伤后,视神经小胶质细胞初期数目减少,随着损伤时间延长,小胶质细胞数目大量增加,且形 态由分枝状变为阿米巴样。     

晶状体损伤对视神经损伤后RGCs的保护作用及其机制

背景大鼠Miiller细胞提取液能够促进离体培养的视网膜神经节细胞（RGCs）存活及轴突的再生,伴晶状体损伤的视神经外伤眼RGCs存活率提高,但Milller细胞和晶状体损伤在促进RGCs存活方面的关系鲜见报道。目的探讨伴晶状体损伤的视神经外伤眼Mtiller细胞对RGCs存活的促进作用及其机制。方法清洁级成年Wistar大鼠48只按随机数字表法随机分为伪手术组、视神经损伤组、晶状体联合视神经损伤组。伪手术组大鼠手术中暴露但不损伤视神经,视神经损伤组大鼠行视神经横断伤,晶状体联合视神经损伤组行视神经横断伤联合晶状体针刺伤,并导致晶状体混浊。术后7d及14d各组分别取8只大鼠处死后制备视网膜标本。采用苏木精一伊红染色观察各组大鼠视网膜和RGCs的形态学改变,采用免疫组织化学法检测各组大鼠视网膜内核层胶质纤维酸性蛋白（GFAP）标记的Muller细胞,光学显微镜下计数各组大鼠RGCs数量及GFAP阳性标记的Muller细胞数量。结果术后7d及14d,伪手术组大鼠RGCs的数量分别为（52．98±1．90）个／高倍视野和（51．81±3．09）个／高倍视野,差异无统计学意义（t=0．910,P=0．378）;术后14d视神经损伤组大鼠RGCs数量为（22．67±1．94）个／高倍视野,明显少于术后7d的（36．61±1．69）个／高倍视野,差异有统计学意义（t=15．312,P=0．000）;术后14d晶状体联合视神经损伤组RGCs数量为（35．69±1．80）个／高倍视野,明显少于术后7d的（50．76±2．77）个／高倍视野,差异有统计学意义（t=12．920,P=0．000）。术后7d及14d,晶状体联合视神经损伤组存活的RGCs数量均多于视神经损伤组,差异均有统计学意义（7d：t=102．840,P=0．000;14d：t=164．020,P=0．000）;术后14d晶状体联合视神经损伤组存活的RGCs：牧量少于伪手术组,差异有统计学意义（t=187．04,P=0．034）。术后7d及14d,伪手术组大鼠视网膜内核层均未见GFAP阳性标记的Muller细胞;视神经损伤组大鼠内核层GFAP阳性标记Muller细胞数量分别为（29．38＋2．04）个／高倍视野和（19．07±2．14）个／高倍视野,差异有统计学意义（t=-9．868,P=0．000）。晶状体联合视神经损伤组大鼠内核层GFAP阳性标记的Muller细胞数量分别为（48．96±2．80）个／高倍视野和（46．73±1．50）个／高倍视野,差异无统计学意义（t=1．987,P=0．067）。术后7d及14d,晶状体联合视神经损伤组大鼠内核层GFAP阳性Muller细胞数量均较视神经损伤组增多,差异均有统计学意义（7d：t=-15．997,P=0．000;14d：t=-29．938,P=0．000）。结论在视神经损伤合并晶状体刺伤时,晶状体损伤可诱导Muller细胞活化,进而促进视神经损伤后RGCs的存活。     

视网膜下移植睫状神经营养因子编码基因修饰的细胞保护SD大鼠视神经横断伤后视网膜节细胞变性

目的探讨移植表达睫状神经营养因子(CNTF)的细胞对SD大鼠视神经横断伤后视网膜节细胞的保护作用。方法通过脂质体将CNTF表达质粒转移至人胚肺成纤维细胞,建立稳定、高水平表达CNTF的细胞株。采用双侧背外侧膝状体及上丘核团注射3%荧光金逆行标记视网膜节细胞。将标记后的大鼠分为两组,于标记后7d手术切断眶内段视神经其中一组左眼不做手术作为正常对照组,右眼切断视神经作为手术对照组;另一组双眼均手术切断视神经,左眼注射PBS作为治疗对照组,右眼视网膜下移植表达CNTF的细胞作为实验组。术后5、14、17、21及28d取出眼球,铺片后荧光显微镜观察并计数视网膜内存活的节细胞。结果手术切断眶内段视神经后2周,视网膜内节细胞数减少6744%,视网膜下移植表达CNTF的细胞后第5、17、21d视网膜内存活的节细胞数明显多于治疗对照组(P<005)。结论视网膜下移植高水平表达CNTF的细胞对视网膜节细胞有保护作用。     

大鼠视神经损伤的病理学和组织化学研究

目的:拟通过对于视神经损伤后轴突的病理变化和GFAP及星形胶质细胞分泌的ECM阻抑性大分子CSPG的表达分布情况,观察视神经损伤后的修复反应,为进一步研究中枢神经纤维的再生提供实验资料。方法:首先建立大鼠视神经不完全损伤的挤压伤模型,于损伤后不同时间段取材进行HE染色和GFAP及CSPG的免疫组织化学研究。结果:HE染色示大鼠视神经损伤后3～7d小胶质细胞增殖吞噬功能活跃,伤后14d,损伤处可见多处新生血管。免疫组织化学结果示大鼠视神经损伤后7d,GFAP在损伤区缺乏表达,而CSPG在损伤区中心呈强阳性表达。结论:CNS轴突再生失败可能与损伤局部瘢痕形成的机械性阻碍和CSPG沉积的化学性阻碍有关。     

Distribution and expression of RhoA in rat retina after optic nerve injury

BACKGROUND: RhoA is a small guanosine triphosphatase which participates in signaling pathways of axonal repellents or inhibitors. However, the distribution and expression of RhoA in the rat retina after optic nerve injury has not been elucidated yet. OBJECTIVES: To study the distribution and expression of RhoA in the rat retina after optic nerve injury. METHODS: Immunohistochemistry was used to determine the distribution of RhoA in rat retina after optic nerve injury. The expression of RhoA was analyzed by Western blot. RESULTS: In normal retina and the retina 1 day after optic nerve injury, RhoA was distributed in the retinal ganglion cell (RGC) layer. Three days after optic nerve injury, it existed in RGCs and the inner plexiform layer. However, 7 days after surgery its immunoreactivity was abundant not only in the RGC and inner plexiform layers but also in the inner nuclear and outer plexiform layers. Western blot analysis showed that the expression of RhoA increased significantly in the retina after optic nerve injury in comparison with normal retina. CONCLUSION: These results indicate that the distribution and expression of RhoA were extended and enhanced after optic nerve injury, and that RhoA plays an important role in optic nerve regeneration.     

c-Jun expression in surviving and regenerating retinal ganglion cells: effects of intravitreal neurotrophic supply

PURPOSE: To investigate c-jun expression in surviving and axon-regenerating retinal ganglion cells (RGCs) and the effect of intravitreal neurotrophic supply on c-jun expression. METHODS: All animals underwent optic nerve transection (ONT) 0.5 mm behind the eyeball. Some animals underwent a replacement of the optic nerve with an autologous sciatic nerve graft (SNG) to allow axonal regrowth. To provide a neurotrophic supply, a peripheral nerve (PN) segment or brain-derived neurotrophic factor (BDNF)/ciliary neurotrophic factor (CNTF) was applied intravitreally. The time course of c-jun expression was first examined in both surviving and regenerating RGCs. Then, c-jun expression was examined in surviving and regenerating RGCs 3 weeks after intravitreal BDNF/CNTF treatment. Animals with vehicle eye injection were used as the control. Fluorescent dye was used for retrograde labeling of surviving (applied behind the eyeball) and regenerating (applied at the distal end of the SNG) RGCs. All retinas were immunohistochemically stained for c-jun. RESULTS: c-Jun was not detected in normal RGCs, but weak expression was seen in surviving RGCs after ON injury. The proportion of c-jun-positive (+) RGCs among surviving cell population was 52.6% to 86.5% 2 to 6 weeks after ONT. Among regenerating RGCs, more than 80% expressed c-jun in all treatment groups, a proportion that was significantly higher after CNTF treatment (90.7%). In addition, c-jun expression was much stronger in intensity and the c-jun(+) nuclei were much larger in regenerating than in surviving RGCs. CONCLUSIONS: c-Jun expression in RGCs was upregulated after injury. Most regenerating RGCs were c-jun(+), and the intensity of c-jun expression was higher in regenerating than in surviving RGCs. CNTF also upregulated c-jun expression in RGCs.     

细胞因子信号转导抑制因子3(SOCS3)对视神经损伤大鼠视网膜神经节细胞存活的影响

目的 研究细胞因子信号转导抑制因子3(suppressor of cytokine signaling 3,SOCS3)对视神经损伤大鼠视网膜神经节细胞(retinal ganglion cells,RGCs)存活的影响,并探讨其潜在的分子机制.方法 采用视神经横断手术构建大鼠视神经损伤模型,术后分离RGCs.实验分为视神经损伤组(optic nerve transection,ONT组)和假手术组.采用Western blot和RT-PCR检测SOCS3在两组细胞中的表达.随后将SOCS3 siRNA分别转染假手术组和ONT组RGCs,实验进一步分为空白对照组、阴性对照组和SOCS3沉默组.CCK8和MTT法检测细胞存活,Hoechst 33342荧光染色法和流式细胞技术检测细胞凋亡.进一步将mTOR siRNA和SOCS3 siRNA共转染RGCs,检测细胞存活和细胞凋亡.结果 视神经损伤3d后,ONT组SOCS3表达水平显著高于假手术组(P =0.049),且随损伤时间延长而升高.与空白对照组相比,SOCS3沉默可显著提高视神经损伤后RGCs的存活率[空白对照组与SOCS3沉默组分别为(49.47±7.35)％和(73.24±8.70)％],降低细胞凋亡率[2组分别为(27.25±0.75)％和(10.96±1.07)％]和细胞生长抑制率[2组分别为(23.06±1.43)％和(10.65±1.77)％].Hoechst染色也表明SOCS3沉默可改善视神经损伤诱导的细胞凋亡.同时,SOCS3沉默可显著提高视神经损伤后2周mTOR活性标志蛋白pS6的表达;且与单独沉默SOCS3相比,共同沉默mTOR和SOCS3可降低细胞存活率,提高细胞生长抑制率和细胞凋亡率.结论 SOCS3沉默可以通过上调损伤后期mTOR的活性而促进损伤RGCs的存活并抑制其凋亡.     

Blocking LINGO-1 function promotes retinal ganglion cell survival following ocular hypertension and optic nerve transection

PURPOSE: LINGO-1 is a functional member of the Nogo66 receptor (NgR1)/p75 and NgR1/TROY signaling complexes that prevent axonal regeneration through RhoA in the central nervous system. LINGO-1 also promotes cell death after neuronal injury and spinal cord injury. The authors sought to examine whether blocking LINGO-1 function with LINGO-1 antagonists promotes retinal ganglion cell (RGC) survival after ocular hypertension and optic nerve transection. METHODS: An experimental ocular hypertension model was induced in adult rats using an argon laser to photocoagulate the episcleral and limbal veins. LINGO-1 expression in the retinas was investigated using immunohistochemistry and Western blotting. Soluble LINGO-1 protein (LINGO-1-Fc) and anti-LINGO-1 mAb 1A7 were injected into the vitreous body to examine their effects on RGC survival after ocular hypertension and optic nerve transection. Signal transduction pathways mediating neuroprotective LINGO-1-Fc effects were characterized using Western blotting and specific kinase inhibitors. RESULTS: LINGO-1 was expressed in RGCs and up-regulated after intraocular pressure elevation. Blocking LINGO-1 function with LINGO-1 antagonists, LINGO-1-Fc and 1A7 significantly reduced RGC loss 2 and 4 weeks after ocular hypertension and also promoted RGC survival after optic nerve transection. LINGO-1-Fc treatment blocked the RhoA, JNK pathway and promoted Akt activation. LINGO-1-Fc induced Akt phosphorylation, and the survival effect of LINGO-1 antagonists was abolished by Akt phosphorylation inhibitor. CONCLUSIONS: The authors demonstrated that blocking LINGO-1 function with LINGO-1 antagonists rescues RGCs from cell death after ocular hypertension and optic nerve transection. They also delineated the RhoA and PI-3K/Akt pathways as the predominant mediator of LINGO-1-Fc neuroprotection in this paradigm of RGC death.     

Expression of osteopontin in the rat retina: effects of excitotoxic and ischemic injuries

PURPOSE: The cytokine osteopontin (OPN) has been localized to the retinal ganglion cell layer in the normal rodent retina, prompting the suggestion that it could serve as a useful marker for identifying and quantifying such neurons in models of retinal and optic nerve neurodegeneration. In the present study, we characterized the time course and cellular localization of OPN expression in the rat retina after excitotoxic and ischemic injuries. METHODS: Excitotoxicity and ischemia-reperfusion experiments were performed by using standard techniques. Rats were killed at various time points, and the retinas were removed either for mRNA analysis or to be processed for immunohistochemistry. RESULTS: In the normal retina, double-labeling immunofluorescence indicated that OPN is expressed by the majority of, if not all, RGCs, since OPN was associated with more cells than Brn-3, but was colocalized with Thy1.1. NMDA, kainic acid, and ischemia-reperfusion all caused decreases in the total retinal levels of Thy1 and Brn-3 mRNAs, reflecting injury to RGCs, but a dramatic, short-lived upregulation in OPN mRNA. The source of the increased OPN signal after excitotoxic-ischemic insults is unlikely to be injured RGCs, as no alteration in the intensity of OPN immunostaining in RGCs was apparent. Instead, additional cells, mostly contained within the IPL, were identified as positive for OPN. Double-labeling immunofluorescence showed that ED1 always colocalized with OPN in these cells, indicating their status as activated microglia. CONCLUSIONS: OPN is exclusively expressed by RGCs in the physiological retina, but in response to retinal neurodegeneration is synthesized de novo by endogenous, activated microglia.     

Effect of glatiramer acetate on primary and secondary degeneration of retinal ganglion cells in the rat

PURPOSE: After crush injury to the optic nerve, elevated intraocular pressure, and glutamate toxicity, the immune modulator glatiramer acetate (GA, Cop-1; Copaxone; Teva Pharmaceutical Industries, Pitach Tikva, Israel) has been shown to reduce the delayed cell death of retinal ganglion cells (RGCs). This study was undertaken to confirm the protective effect of GA on secondary degeneration of RGCs in the rat, by using a spatial, rather than temporal, model. METHODS: A total of 131 Wistar rats divided into 10 groups underwent bilateral stereotactic injection of fluorescent tracer (Fluorogold; Fluorochrome, Denver, CO) into the superior colliculus to label RGCs. They received a concurrent subcutaneously injection of (1) GA mixed with complete Freund's adjuvant (CFA), (2) CFA alone, or (3) saline. One week later, the superior one third of the left optic nerve was transected in animals in the six partial transection groups. Optic nerves in four additional groups underwent full transection. Rats were killed and retinas harvested from both eyes 1 or 4 weeks after partial transection and 1 or 2 weeks after full transection. RGC densities were calculated from retinal wholemounts, and differences between right (control) and left (transected) eyes were compared across treatment groups. RESULTS: Among the partial transection groups, differences in the mean percentage of RGC loss in the inferior retinas were not significant at 1 or 4 weeks (ANOVA; P = 0.20, P = 0.12, respectively). After full transection, there was significantly more RGC loss in the GA group than in the CFA group when comparing whole retinas at 1 week, but not at 2 weeks (two-tailed t-test; P = 0.04, P = 0.36, respectively). CONCLUSIONS: There is no evidence that GA has a neuroprotective effect after optic nerve transection, either for primarily injured or secondarily involved RGC.     

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.     

Expression of insulin‐like growth factor 1 receptor in rat retina following optic nerve injury

Purpose: To investigate the apoptosis in retinal ganglion cells (RGCs) and insulin‐like growth factor 1 receptor (IGF‐1R) in the retina following optic nerve crush. Methods: Healthy Wistar rats (N = 70) were divided into two groups: a normal control group and an optic nerve injury group. Immunohistochemistry and flow cytometry were performed to detect the expression of IGF‐1R and to measure the apoptosis of RGCs, respectively. Results: Immunohistochemistry revealed that at 1 hr after optic nerve injury, IGF‐1R immunoreactivity began to increase and reached a maximal level at 24 hr (p < 0.05), where it remained elevated up to 14 days after injury. RGC apoptosis in the normal control group was 0.53%, while the apoptosis rate in the optic nerve injury group increased over time. The apoptosis rate in the optic nerve injury group was 1.4% at 1 hr, 4.4% at 6 hr, 5.2% at 12 hr and reached a maximal level (8.5%) at 24 hr. Subsequently, the rate declined to 1.9% 7 days after injury and 0.9% 2 weeks after injury. Conclusion: The IGF‐1R immunereactivity in the retina increased after optic nerve injury. IGF‐1R may regulate the apoptosis and regeneration of RGCs at different stages after optic nerve injury.