大鼠胚胎视网膜干细胞的分离培养

目的分离培养大鼠胚胎视网膜干细胞。方法从胎龄第16d的SD大鼠视网膜神经上皮分离视网膜细胞并进行悬浮培养,观察细胞增殖以及自发分化情况,采用免疫细胞化学方法检测Nestin、β-Tubulin、GFAP和Recoverin的表达。结果原代细胞可形成悬浮生长的神经球,传代后能形成新的细胞球。原代及传代细胞大部分表达神经干细胞标记物Nestin,分化后的细胞部分表达神经元标记物β-Tubulin或神经胶质细胞标记物GFAP,少数细胞表达光感受器细胞标记物Recoverin。结论分离培养的SD胚鼠视网膜神经干细胞可以在体外扩增并具有多分化潜能。     

人视网膜星形胶质细胞发育及起源的研究

背景 视网膜星形胶质细胞是视网膜主要的神经胶质细胞,其起源及演变过程一直是国内外研究的热点和难点. 目的 探讨人胚胎眼视网膜星形胶质细胞的起源及发育.方法 收集33例自愿终止妊娠的流产人胚胎眼标本,其中8 ～12孕周者20例,15～ 17孕周者2例,19～ 23孕周者4例,25～ 28孕周者4例,30 ～32孕周者3例.对眼球壁切片进行常规组织病理学检查以观察不同胚龄人视网膜发育的形态学变化,分别采用免疫组织化学法及免疫荧光法动态观察不同胚龄人视网膜星形胶质细胞起源位点及发育过程中胶质纤维酸性蛋白( GFAP)表达的变化.结果 人胚6～7周视杯处于视网膜分层发育阶段,9周时视杯内层原无细胞层出现分化不成熟的圆短梭形细胞；胚龄15周时视网膜主要层次可见,分化的细胞增加,但未发现GFAP阳性细胞；胚龄19周视网膜可见梭形细胞从返折部原始神经上皮迁出,并可见这些细胞中GFAP呈阳性表达；胚龄25～ 26周后极部视网膜可见GFAP表达阳性的梭形细胞,这些细胞围绕视网膜血管分布,与血管壁联系密切,邻近锯齿缘处的视网膜内层可见表达GFAP的星形或梭形细胞与睫状体非色素上皮相连,但锯齿缘稍后与赤道区之间并未见GFAP阳性细胞；胚龄28周,视网膜星形胶质细胞呈典型的星状,其突起伸达视网膜内网状层. 结论 人视网膜星形胶质细胞至少存在3个起源位点,即血管前体细胞/周皮细胞、视盘旁原始神经上皮及邻近锯齿缘的睫状体无色素上皮.     

巢蛋白和神经胶质纤维酸性蛋白在大鼠视网膜发育中的动态表达

目的 观察巢蛋白(nestin)和神经胶质纤维酸性蛋白(GFAP)在大鼠视网膜发育中的动态变化.方法 48只Wistar大鼠,其中24只大鼠分为出生后1 d,1、2、3、4、7、12、20周8组,每组3只.制作眼球矢状位冰冻切片,采用共聚焦激光显微镜观察nestin和谷氨酰胺合成酶(GS)以及GFAP和GS免疫荧光染色情况.18只大鼠分为出生后id,1、2、3、4、12周,每组3只.提取大鼠视网膜总RNA,实时定量逆转录聚合酶链反应(RT-PCR)检测nestin、GFAP和GS mRNA表达.选择6只出生后7～12 d新生鼠,取眼球体外培养Müller细胞,行GS和(或)nestin免疫荧光染色,共聚焦激光显微镜观察荧光染色情况.结果 出生后1 d,nestin免疫阳性细胞贯穿神经视网膜全层,主要定位于视网膜前体细胞放射状排列的细长纤维中,在视网膜内侧出现GFAP阳性的星形胶质细胞.出生后1周出现表达GS的Müller细胞,同时表达nestin,但不表达GFAP;GFAP阳性细胞仍然位于视网膜内侧.出生后2～12周,nestin在Mfiller细胞上的表达逐渐减少直至消失,GFAP在星形胶质细胞中的表达强度投有显著变化.体外培养的Müller细胞表达nestin,不表达GFAP.Nestin和GFAP mRNA在视网膜中的表达与免疫荧光染色结果相一致.结论 随大鼠视网膜不断发育,Müller细胞上nestin表达逐渐减少,成年大鼠视网膜Müller细胞不再表达nestin;新生鼠和成年鼠Müller细胞均不表达GFAP.     

巢蛋白和神经胶质纤维酸性蛋白在大鼠视网膜发育中的动态表达

目的 观察巢蛋白(nestin)和神经胶质纤维酸性蛋白(GFAP)在大鼠视网膜发育中的动态变化.方法 48只Wistar大鼠,其中24只大鼠分为出生后1 d,1、2、3、4、7、12、20周8组,每组3只.制作眼球矢状位冰冻切片,采用共聚焦激光显微镜观察nestin和谷氨酰胺合成酶(GS)以及GFAP和GS免疫荧光染色情况.18只大鼠分为出生后id,1、2、3、4、12周,每组3只.提取大鼠视网膜总RNA,实时定量逆转录聚合酶链反应(RT-PCR)检测nestin、GFAP和GS mRNA表达.选择6只出生后7～12 d新生鼠,取眼球体外培养Müller细胞,行GS和(或)nestin免疫荧光染色,共聚焦激光显微镜观察荧光染色情况.结果 出生后1 d,nestin免疫阳性细胞贯穿神经视网膜全层,主要定位于视网膜前体细胞放射状排列的细长纤维中,在视网膜内侧出现GFAP阳性的星形胶质细胞.出生后1周出现表达GS的Müller细胞,同时表达nestin,但不表达GFAP;GFAP阳性细胞仍然位于视网膜内侧.出生后2～12周,nestin在Mfiller细胞上的表达逐渐减少直至消失,GFAP在星形胶质细胞中的表达强度投有显著变化.体外培养的Müller细胞表达nestin,不表达GFAP.Nestin和GFAP mRNA在视网膜中的表达与免疫荧光染色结果相一致.结论 随大鼠视网膜不断发育,Müller细胞上nestin表达逐渐减少,成年大鼠视网膜Müller细胞不再表达nestin;新生鼠和成年鼠Müller细胞均不表达GFAP.     

巢蛋白和神经胶质纤维酸性蛋白在大鼠视网膜发育中的动态表达

目的 观察巢蛋白(nestin)和神经胶质纤维酸性蛋白(GFAP)在大鼠视网膜发育中的动态变化.方法 48只Wistar大鼠,其中24只大鼠分为出生后1 d,1、2、3、4、7、12、20周8组,每组3只.制作眼球矢状位冰冻切片,采用共聚焦激光显微镜观察nestin和谷氨酰胺合成酶(GS)以及GFAP和GS免疫荧光染色情况.18只大鼠分为出生后id,1、2、3、4、12周,每组3只.提取大鼠视网膜总RNA,实时定量逆转录聚合酶链反应(RT-PCR)检测nestin、GFAP和GS mRNA表达.选择6只出生后7～12 d新生鼠,取眼球体外培养Müller细胞,行GS和(或)nestin免疫荧光染色,共聚焦激光显微镜观察荧光染色情况.结果 出生后1 d,nestin免疫阳性细胞贯穿神经视网膜全层,主要定位于视网膜前体细胞放射状排列的细长纤维中,在视网膜内侧出现GFAP阳性的星形胶质细胞.出生后1周出现表达GS的Müller细胞,同时表达nestin,但不表达GFAP;GFAP阳性细胞仍然位于视网膜内侧.出生后2～12周,nestin在Mfiller细胞上的表达逐渐减少直至消失,GFAP在星形胶质细胞中的表达强度投有显著变化.体外培养的Müller细胞表达nestin,不表达GFAP.Nestin和GFAP mRNA在视网膜中的表达与免疫荧光染色结果相一致.结论 随大鼠视网膜不断发育,Müller细胞上nestin表达逐渐减少,成年大鼠视网膜Müller细胞不再表达nestin;新生鼠和成年鼠Müller细胞均不表达GFAP.     

成人睫状体-视网膜区域神经巢蛋白阳性细胞的分布特征

目的探讨成人眼睫状体-视网膜区域神经巢蛋白nestin阳性细胞的分布特征。方法由广东省眼库提供8只新鲜瘁死的健康成人眼球和3只婴儿眼球,分别采用免疫组织化学、逆转录聚合酶链反应（RT-PCR）等方法检测神经干细胞特异性抗原神经巢蛋白nestin在睫状体、视网膜中的表达,通过透射电镜观察其超微结构和分布特点。结果睫状体、视网膜中均有少量nestin阳性细胞表达,其散在分布于睫状体色素上皮层和视网膜内核层靠近外核层的一侧;RT-PCR检测到睫状体、视网膜中均有nestin基因mRNA的表达,睫状体中的表达量高于视网膜区域;透射电镜观察可见睫状体和视网膜组织中均存在一些核大而圆、核仁明显、细胞器较幼稚的具有神经干细胞特征的细胞,位于睫状体色素上皮靠近基底膜的区域及视网膜内核层与外核层之间。结论成人睫状体-视网膜中存在一些具有神经干细胞特征的神经巢蛋白nestin阳性细胞,主要分布于睫状体色素上皮层和视网膜内核层。     

急性眼压升高所致视网膜、视神经及视交叉胶质细胞的早期反应北大核心CSCD

背景中枢神经系统以及视网膜中的胶质细胞与神经元关系十分紧密,胶质细胞在神经元损伤和修复过程中发挥着重要作用。急性眼压升高引起的视网膜、视神经及视交叉各部位胶质细胞的早期反应特点以及其与视神经损伤的关系目前尚不清楚。目的探讨大鼠视网膜、视神经及视交叉的胶质细胞对急性高眼压的早期反应,同时观察神经前体细胞标志物巢蛋白（nestin）在反应性胶质细胞中的表达。方法成年雌性Wistar大鼠9只,分为正常对照组3只和急性高眼压组6只,急性高眼压组大鼠采用右眼前房灌注生理盐水的方法升高大鼠眼压至110mmHg,持续60min。于术后第3天和第7天用过量麻醉法处死各组动物各3只,摘出眼球分离视神经和大脑标本,并制作冰冻切片。利用Nissl染色的方法测量高眼压眼视网膜内层厚度,观察视网膜和视交叉的大体形态。用BIU-tubu｜in免疫荧光染色法标记视神经内的视网膜神经节细胞（RGCs）轴突,用胶质纤维酸性蛋白（GFAP）和nestin双重标记显示视网膜、视神经及视交叉的胶质细胞反应,并对两组结果进行比较。结果正常大鼠的视网膜、视神经以及视交叉内均可见到一定量的GFAP阳性胶质细胞,但nestin的表达量很低。急性眼压升高后的第3天,视网膜内丛状层厚度明显变薄,RGCs数目较损伤前减少约46％。视网膜内胶质细胞GFAP的表达显著增加,细胞突起由神经纤维层伸展至整个视网膜,增生的胶质细胞内可见nestin的明显表达。视神经内RGCs轴突发生变性样改变,GFAP阳性胶质细胞内nestin的表达较眼压升高前明显增加。同损伤眼相对应的一侧视交叉的横断面积减小,出现大量星状GFAP和nestin共表达的胶质细胞。以上改变在眼压升高后第7天更趋明显。结论急性眼压升高早期即可引起RGCs的丢失及轴突的变性,视觉神经元改变的同时伴随胶质细胞的反应,增生的胶质细胞表达神经前体细胞的标志物。视网膜与视神经和视交叉的改变在时间上具有一定的同步性。     

胶质纤维酸性蛋白在视神经横断后大鼠视网膜神经胶质细胞中的表达

目的探讨胶质纤维酸性蛋白（GFAP）存视神经横断后大鼠视网膜神经胶质细胞中的表达情况及可能的意义。方法制作大鼠视神经横断模型,分别于术后1、3、7d取材,制作视网膜矢状伉冰冻切片,用谷氨酰胺合成酶（Gs）标记视网膜Muller细胞,行GS／GFAP免疫荧光双标。提取视网膜总RNA行GFAPReal—TimePCR半定量分析。结果正常大鼠视网膜中GFAP阳性染色仅位于视网膜内层的星形胶质细胞上。术后1d,在Muller细胞上出现GFAP的诱导表达,术后3d,GFAP在Muller细胞上的表达进一步增强,术后1周,GFAP在Muller细胞上的表达保持在较高水平,Real-TimePCR半定量分析与以上结果吻合。结论视神经横断后GFAP在Muller细胞上的诱导表达是Muller细胞对视网膜损伤产生的一种胶质化反应,GFAP的表达量与病程进展相一致。GFAP在Muller细胞上的诱导表达尤其在足板处的强烈表达可能对视网膜神经节细胞具有保护作用。     

急性眼压升高所致视网膜、视神经及视交叉胶质细胞的早期反应

背景 中枢神经系统以及视网膜中的胶质细胞与神经元关系十分紧密,胶质细胞在神经元损伤和修复过程中发挥着重要作用.急性眼压升高引起的视网膜、视神经及视交叉各部位胶质细胞的早期反应特点以及其与视神经损伤的关系目前尚不清楚. 目的 探讨大鼠视网膜、视神经及视交叉的胶质细胞对急性高眼压的早期反应,同时观察神经前体细胞标志物巢蛋白( nestin)在反应性胶质细胞中的表达. 方法 成年雌性Wistar大鼠9只,分为正常对照组3只和急性高眼压组6只,急性高眼压组大鼠采用右眼前房灌注生理盐水的方法升高大鼠眼压至110 mmHg,持续60 min.于术后第3天和第7天用过量麻醉法处死各组动物各3只,摘出眼球分离视神经和大脑标本,并制作冰冻切片.利用Nissl染色的方法测量高眼压眼视网膜内层厚度,观察视网膜和视交叉的大体形态.用βⅢ-tubulin免疫荧光染色法标记视神经内的视网膜神经节细胞(RGCs)轴突,用胶质纤维酸性蛋白(GFAP)和nestin双重标记显示视网膜、视神经及视交叉的胶质细胞反应,并对两组结果进行比较.结果 正常大鼠的视网膜、视神经以及视交叉内均可见到一定量的GFAP阳性胶质细胞,但nestin的表达量很低.急性眼压升高后的第3天,视网膜内丛状层厚度明显变薄,RGCs数目较损伤前减少约46％.视网膜内胶质细胞GFAP的表达显著增加,细胞突起由神经纤维层伸展至整个视网膜,增生的胶质细胞内可见nestin的明显表达.视神经内RGCs轴突发生变性样改变,GFAP阳性胶质细胞内nestin的表达较眼压升高前明显增加.同损伤眼相对应的一侧视交叉的横断面积减小,出现大量星状GFAP和nestin共表达的胶质细胞.以上改变在眼压升高后第7天更趋明显.结论 急性眼压升高早期即可引起RGCs的丢失及轴突的变性,视觉神经元改变的同时伴随胶质细胞的反应,增生的胶质细胞表达神经前体细胞的标志物.视网膜与视神经和视交叉的改变在时间上具有一定的同步性.     

大鼠视网膜干细胞与神经干细胞体外增殖、分化特点比较

目的 通过对大鼠视网膜干细胞和神经干细胞体外增殖、分化特点进行比较,进一步明确视网膜于细胞自我更新及向神经细胞方向分化的能力.方法 实验研究.分离出生10 d大鼠睫状体区组织及新生大鼠脑组织,分别应用酶消化法将两种组织制成单细胞悬液,放入含有20 μg/L碱性成纤维细胞生长因子、20μg/L表皮生长因子及1×B27添加剂的无血清DMEM/F12培养液中培养,观察并比较视网膜干细胞及神经干细胞体外增殖的特点.分别取第2代细胞,应用免疫细胞化学染色法检测干细胞特异性抗原神经巢蛋白（nestin）及细胞分裂增殖标志物5-溴脱氧尿核苷（BrdU）的表达.同时,应用含50 ml/L的胎牛血清及1×N2添加剂的培养基促进其分化,观察两种细胞向神经细胞方向分化的特点：分别应用免疫细胞化学染色法对分化后的细胞nestin、神经无标志物神经元特异性烯醇酶（NSE）、神经胶质细胞标志物神经胶质酸性蛋白（GFAP）进行检测,并应用卡方检验对两种干细胞分化后的NSE及GFAP阳性细胞数比例进行比较.结果 两种细胞原代培养48 h后均可见小的球状细胞团悬浮生长,折光性强.原代培养约7 d后传代,传代后细胞能重新形成球状细胞团,应用免疫细胞化学染色方法均可检测到细胞内nestin及BrdU的阳性表达,视网膜干细胞体外增殖的能力较神经干细胞弱.两种细胞均可在血清诱导条件下转变为贴壁生长,并分化出具有神经细胞形态的细胞.诱导第7天进行免疫细胞化学染色,检测出两种干细胞表达nestin阳性细胞数比例分别为（9.5±3.5）%及（9.1±0.7）%.视网膜干细胞分化后NSE及GFAP的阳性细胞数比例分别为（11.2±2.8）%及（18.9＋2.1）%,低于神经干细胞分化后两种标志物阳性细胞数比例[（34.1±63）%及（41.9±3.3）%],NSE、GFAP在两组表达的差异均有统计学意义（x2=103.23,P＜0.05;x2=74.36,P＜0.05）.结论 来源于睫状体区的大鼠视网膜干细胞形态、体外增殖及可分化特性与神经干细胞相似,但其增殖及分化为神经细胞的能力较神经干细胞弱.     

Distribution of Müller stem cells within the neural retina: Evidence for the existence of a ciliary margin-like zone in the adult human eye

Much interest has been generated by the identification of neural stem cells in the human neural retina and ciliary body. However, it is not clear whether stem cells identified in these ocular compartments are of the same origin or whether they ontogenically derive from different cell populations. This study examined the in situ anatomical distribution of these cells within the neural retina and ciliary body, as well as their ability to proliferate in response to EGF. Human retinae and ciliary body were examined for co-expression of Nestin, cellular retinaldehyde binding (CRALBP) or Vimentin, and the stem cell markers SOX2, CHX10, NOTCH1 and SHH. Retinal explants were cultured with epidermal growth factor (EGF) to assess retinal cell proliferation. Intense Nestin and CRALBP staining was observed in the neural retinal margin, where cells formed bundles of spindle cells (resembling glial cells) that lacked lamination and co-stained for SOX2, CHX10 and SHH. This staining differentiated the neural retina from the ciliary epithelium, which expressed SOX2, CHX10 and NOTCH1 but not Nestin or CRALBP. Nestin and CRALBP expression decreased towards the posterior retina, where it anatomically identified a population of Müller glia. All Vimentin positive Müller glia co-stained for SOX2, but only few Vimentin positive cells expressed Nestin and SOX2. Cells of the retinal margin and the inner nuclear layer (INL), where the soma of Müller glia predominate, re-entered the cell cycle upon retinal explant culture with EGF. Lack of lamination and abundance of Müller glia expressing stem cell markers in the marginal region of the adult human retina resemble the ciliary marginal zone (CMZ) of fish and amphibians. The findings that cells in this CM-like zone, as well in the inner nuclear layer proliferate in response to EGF suggest that the adult human retina has regenerative potential. Identification of factors that may promote retinal regeneration in the adult human eye would provide efficient treatments for retinal degenerative conditions for which treatments are not yet available.     

The neurogenic competence of progenitors from the postnatal rat retina in vitro

The mammalian retina develops from stem or progenitor cells that are of neuroectodermal origin and derive from bilateral invaginations of the neuroepithelium, the optic vesicles. Shortly after birth, around 12 days postnatal in rats, the retina is fully developed in its cellular parts. Even though different cell types in the adult might be potential sources for retinal stem cells or progenitor cells, the retina is a non-neurogenic region and the diseased retina is devoid of any spontaneous regeneration. In an attempt to link late developmental processes to the adult situation, we analyzed the presence and the neurogenic potential of retinal progenitors during the postnatal period and compared it to adult ciliary body (CB) derived retinal progenitors and subventricular zone (SVZ) derived neural stem cells. Retinal progenitor properties were identified by the capacity to proliferate and by the expression of the progenitor markers Nestin, Flk-1, Chx10, Pax6 and the radial glia marker BLBP. The neurogenic potential was assayed by the expression of the neuronal markers doublecortin, betaIII Tubulin, Map2 and NSE, the glial makers A2B5, NG2, GalC and GFAP, and by incorporation of BrdU. The number of Flk-1 positive cells and concomitantly the number of newly born betaIII Tubulin-positive cells decreased within the first postnatal week in retinal progenitor cultures and no newly generated betaIII Tubulin, but GFAP positive cells were detected thereafter. In contrast to neural stem cells derived from the adult SVZ, postnatal and adult CB derived progenitors had a lower and a restricted proliferation potential and did not generate oligodendrocytes. The work demonstrates, however, that the existence of retinal progenitor cells is not restricted to embryonic development. In the sensory retina the differentiation potential of late retinal progenitors becomes restricted to the glial lineage, whereas neurogenic progenitor cells are still present in the CB. In addition, major differences in growth and differentiation potential of adult neural stem cells and postnatal and adult retinal progenitors are presented.     

Nestin positive cells in adult human retina and in epiretinal membranes

BACKGROUND/AIM: Nestin is an intermediate filament marker for neural progenitor cells. The authors aimed to identify nestin positive cells in adult human retina and within surgically removed epiretinal membranes. METHODS: Adult human retina and epiretinal membranes were studied. Tissue was fixed and processed for semithin sections or whole mount preparations for immunohistochemical detection of nestin and glial fibrillary acidic protein (GFAP) expression. RESULTS: Nestin positive cells are most prominent at the ora serrata, possess fibrillary processes, small amounts of perinuclear cytoplasm, and are arranged radially within or superficially on the retina. In the posterior retina, speckled cytoplasmic nestin staining is seen around the nuclei of neurons. In the peripapillary retina most of the cells in the retinal ganglion cell layer are nestin positive. These cells appear to represent nestin positive neurons. Speckled cells are also seen in the myelinated portion of the optic nerve. In epiretinal membranes patches of elongated nestin positive cells were found. These cells were also positive for GFAP. CONCLUSIONS: Some neurons and glia in the adult human retina are nestin positive. Their pattern in anterior retina suggests an analogy with the ciliary marginal zone found in many other species. The role of these cells in pathological responses to retinal disease is suggested by the presence of large numbers of ectopic nestin positive cells in epiretinal membranes. The authors hypothesise that nestin positive cells represent a population of progenitor cells from normal adult human retina that differentiate to make up retinal scar tissue.     

视网膜神经节细胞无血清上清培养液诱导大鼠视网膜干细胞的分化

目的　探讨视网膜神经节细胞无血清上清培养液对视网膜干细胞分化的影响。方法　分离大鼠视网膜干细胞和视网膜神经节细胞;采用免疫荧光法鉴定体外培养的大鼠视网膜干细胞与视网膜神经节细胞,视网膜干细胞以Nestin抗体进行鉴定,视网膜神经节细胞以Thy-1抗体进行鉴定;以视网膜神经节细胞无血清上清培养液培养视网膜干细胞,以不加入条件培养液培养的视网膜干细胞为对照组,收集分化细胞,采用qPCR法检测Nestin、Pax6、Thy-1及Brn-3的基因表达。结果　培养的视网膜干细胞Nestin抗体染色阳性,视网膜神经节细胞Thy-1抗体染色阳性。培养视网膜干细胞72 h后,与对照组相比,无血清上清培养液组细胞Nestin和PAX6基因相对表达量降低,差异均有统计学意义（均为P<0.000 1）;Thy-1和Brn-3基因相对表达量升高,差异均有统计学意义（均为P<0.05）。结论　视网膜神经节细胞无血清上清培养液能够诱导视网膜干细胞分化为视网膜神经节样细胞。     

Localization of retinoid-binding proteins in developing rat retina

Antibodies to cellular retinol-binding protein (CRBP) and cellular retinal-binding protein (CRALBP) were obtained from rabbits immunized with antigens purified from bovine retina. Antigens were localized on frozen sections of rat retina using indirect FITC immunofluorescence. In the retinal pigment epithelium of rats from postnatal day 1 (the day of birth) to postnatal day 32, specific staining with anti-CRBP was restricted to the cytoplasm; the nuclei were unstained. In the neurosensory retina, Muller cell endfeet were stained with anti-CRBP at all ages examined. No CRBP reactivity was found in the pigment epithelium of the ciliary body at any age examined. On postnatal days 14 and 32, in addition to Muller cell endfeet and radial processes, two fine laminae in the inner plexiform layer were faintly positive for CRBP. Anti-CRALBP stained the cytoplasm of the RPE and Muller cells in the adult rat. In developing rat retina, in addition to Muller cells and the retinal pigment epithelium, the ciliary body pigment epithelium and the outer epithelium of the iris were stained with anti-CRALBP in the first postnatal week. The intensity of staining of the ciliary body pigment epithelium decreased gradually from postnatal day 8 until postnatal day 14, at which point a clear line of demarcation was found between the positively stained retinal pigment epithelium and the adjacent, lightly stained, pigment epithelial cells of the ciliary body.     

Engraftment of adult neural progenitor cells transplanted to rat retina injured by transient ischemia

PURPOSE: To optimize delivery parameters for achieving engraftment, migration, and differentiation of adult neural progenitor cells transplanted to the retinas of rats after transient retinal ischemia. METHODS: Retinal ischemia was induced by transiently raising the intraocular pressure. Some animals then received transplantation of green fluorescent protein (GFP)-expressing cells derived from the adult rat hippocampus and were allowed to recover for 6 hours to 9 weeks. Retinal cryosections were prepared for TUNEL analysis to determine the time course of ischemia-induced cell death, and some sections were prepared for immunohistochemistry for retinal neuronal antigens. RESULTS: TUNEL analysis revealed that ischemia-induced cell death peaked at 24 hours. By 96 hours, the inner nuclear (INL) and ganglion cell (GCL) layers were largely obliterated in the central retina, sparing peripheral regions. By 2 weeks after transplantation, numerous GFP-expressing cells had engrafted into the host retina, migrated to the inner retina, and extended processes. At 4 weeks, many GFP-labeled cells were present throughout the INL and displayed horizontal-, bipolar-, and amacrine cell-like morphologies. GFP-expressing cells were also present in the GCL with fibers extending into the nerve fiber layer. At 5 weeks, many GFP-expressing cells were present at the optic nerve head, and some GFP-labeled fibers were present in the optic nerve, occasionally passing through the full extent of the lamina cribrosa. Only rarely were GFP-expressing cells found that coexpressed retinal phenotypic markers at any time point examined. CONCLUSIONS: Adult hippocampus-derived neural progenitor cells transplanted to the subretinal space readily engraft into a host retina that has undergone ischemic injury. Many cells migrate to specific retinal cellular layers and undergo limited morphologic differentiation reminiscent of retinal neurons, including extension of processes into the optic nerve. Concurrent control studies demonstrate that optimal engraftment is achieved by subretinal delivery within a specific temporal window. These results imply that certain inductive cues may be regulated after injury, and they demonstrate the potential for adult neural progenitor cell transplantation for the treatment of retinal neurodegenerative diseases.     

Morphological and immunohistochemical studies on degenerative changes of the retina and the optic nerve in neonatal rats injected with monosodium-L-glutamate

Degenerative changes in the retina and the optic nerve were investigated morphologically and immunohistochemically following administration of monosodium-L-glutamate (MSG) in rats. MSG (5mg/g b.w.) was injected subcutaneously every other day for 5 times after birth. The retina and the optic disc was observed ophthalmoscopically at 1, 3 and 6 months after birth. At the same stage, morphological and immunohistochemical changes were also investigated under light microscopy. The neuron of the retinal ganglion cell was identified immunohistochemically with antiserum to neurofilament 200 kD (NF). Glial cells were stained with antisera to glial fibrillary acidic protein (GFAP) and S-100 beta protein, and myelin of oligodendrocytes was also stained with antiserum to myelin basic protein (MBP). Histologically the inner layer of the retina was selectively destroyed, and the optic nerve also showed degeneration, changing into a thin strand. The retinal vessels were narrow and coarse, however, they extended to the peripheral region. Although immunohistochemical staining with NF antiserum was scarcely detected both in the retina and in the optic nerve, glial stainings with GFAP and S-100 beta protein antisera were widely observed in the perivascular space of the retina and in the glial column of the optic nerve. These findings indicated that the ganglion cells and their neurons are significantly affected by MSG, but the retinal vessels and glial cells are rarely affected. Ophthalmoscopically, the optic discs of rats treated with MSG were small and deeply excavated. The vitreous vessels persisted in most cases even at 6th months after birth.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences between the neurogenic and proliferative abilities of Müller glia with stem cell characteristics and the ciliary epithelium from the adult human eye

Much controversy has arisen on the nature and sources of stem cells in the adult human retina. Whilst ciliary epithelium has been thought to constitute a source of neural stem cells, a population of Müller glia in the neural retina has also been shown to exhibit neurogenic characteristics. This study aimed to compare the neurogenic and proliferative abilities between these two major cell populations. It also examined whether differences exist between the pigmented and non-pigmented ciliary epithelium (CE) from the adult human eye. On this basis, Müller glia with stem cell characteristics and pigmented and non-pigmented CE were isolated from human neural retina and ciliary epithelium respectively. Expression of glial, epithelial and neural progenitor markers was examined in these cells following culture under adherent and non-adherent conditions and treatments to induce neural differentiation. Unlike pigmented CE which did not proliferate, non-pigmented CE cells exhibited limited proliferation in vitro, unless epidermal growth factor (EGF) was present in the culture medium to prolong their survival. In contrast, Müller glial stem cells (MSC) cultured as adherent monolayers reached confluence within a few weeks and continued to proliferative indefinitely in the absence of EGF. Both MSC and non-pigmented CE expressed markers of neural progenitors, including SOX2, PAX6, CHX10 and NOTCH. Nestin, a neural stem cell marker, was only expressed by MSC. Non-pigmented CE displayed epithelial morphology, limited photoreceptor gene expression and stained strongly for pigmented epithelial markers upon culture with neural differentiation factors. In contrast, MSC adopted neural morphology and expressed markers of retinal ganglion cells and photoreceptors when cultured under similar conditions.This study provides the first demonstration that pigmented CE possess different proliferative abilities from non-pigmented CE. It also showed that although non-pigmented CE express genes of retinal progenitors, they do not differentiate into neurons in vitro, as that seen with Müller glia that proliferate indefinitely in vitro and that acquire markers of retinal neurons in culture under neural differentiation protocols. From these observations it is possible to suggest that Müller glia that express markers of neural progenitors and become spontaneously immortalized in vitro constitute a potential source of retinal neurons for transplantation studies and fulfil the characteristics of true stem cells due to their proliferative and neurogenic ability.