大鼠视网膜神经元NOS与Parvalbumin的共存研究

用NADPH脱氢酶组化及Parvalbumin免疫组化双标记技术观察了正常大鼠视网膜一氧化氮合酶(NOS)与Parvalbumin(PV)的分布,结果显示NOS阳性神经元主要位于内核层内缘带第二列,少数位于节细胞层,胞体圆形/卵圆形,直径8～12μm,细胞一侧发出突起伸向内网层1、3、5亚层,以第3亚层最为明显,PV免疫反应(PV—Ⅰ)神经元位于内核层最内缘第一列,少数位于第二列、中间部及节细胞层,胞体卵圆形,直径6～10μm,由胞体一端发出突起伸向内网层第1、5亚层.神经纤维层可见PV～Ⅰ纤维.内核层内缘第二列可见少数双标阳性细胞,在它们的PV免疫反应胞质内散布有NOS颗粒.实验结果表明 NOS阳性神经元与PV～Ⅰ神经元均为无长突细胞,分属不同的亚型,少效PV～Ⅰ神经元属节细胞,个别双标细胞可能为另一种亚型的无长突细胞,提示NOS与PV在视觉信息传递中可能存在某些联系.     

34 移植视网膜含P物质神经元的发育及其与上丘的联系

<正>妊娠14天SD大鼠胚胎视网膜移植至新生第1天的大鼠中脑.应用免疫细胞化学和HRP逆行追踪方法,观察手术后10天至40天的移植视网膜和正常视网膜的P物质免疫反应(SP-IR)神经元的出现时间、定位分布和发育规律,以及移植视网膜与宿主上丘的联系.结果表明,移植视网膜和同龄正常视网膜有着极其相似的结构和发育规律.SP-LR神经元的定位分布在移植视网膜和正常视网膜是一致的,它们的胞体位于内核层和节细胞层,其突起进入内网层并分布全层,有时密集成亚层,在神经纤维层内亦见SP—IR纤维.出生后第3天(即术后第13天)的视网膜出现SP—LR神经元,第11～14天达高峰,约于第20天趋于正常成年水平.在宿主上丘定位注入HRP后,移植视网膜(包括宿主视网膜)内可见HRP阳性细胞和纤维,移植视网膜的阳性细胞在生后第14天比生后第4天有明显增多,亦可见HRP—SP双标记细胞.上述结果提示,移植的视网膜不但能存活,而且保持原有的发育规律,具有正常的组织结构特征,并含有神经活性物质一P物质,实验发现,含SP神经元在生后第4天已经与上丘建立形态学上的联系,SP—IR神经元是无长突细胞,移后无长突细胞和少数节细胞.这为研究移植视网膜视觉投射及其功能,神经再生及其可塑性都有重要意义.     

酪氨酸羟化酶样免疫反应神经元在幼年虎斑蝾螈视网膜的定位研究

本文应用免疫细胞化学方法对幼年虎斑蝾螈视网膜酪氨酸羟化酶(Tyrosine-Hydroxylase,TH)样免疫反应神经元进行了定位研究。90%免疫反应阳性细胞位于内核层最内排的无长突细胞,而10%的细胞位于节细胞层,暂称为异位无长突细胞。TH样免疫反应细胞的突起在内网层第一亚层分支最密,呈致密带,第3和5亚层相对地只有少量分布;在内核层不到1%的TH样免疫反应细胞,从胞体发出短突向外网层伸延,然而这些突起没有跨越过整个内核层,外网层也没有发现免疫阳性反应细胞的突起。视网膜平储片显示,TH样免疫反应无长突细胞和异位无长突细胞的分布遍及视网膜中央和周边部,TH祥免疫反应无长突细胞的密度每平方毫米为49±13个细胞(平均数±标准误)。绝大多数的细胞呈星形,发出3个或3个以上的初级突起,一些细胞在胞体相对两极发出突起,其突起一般分支很少,有时末端分支膨大呈念珠状。某些突起很长与相邻的TH样免疫反应无长突细胞的树突野之间有大量的重叠,而且常常看到在内网层多于一个平面上相互连接成网。     

人胎视网膜超氧化物歧化酶和波形蛋白免疫阳性细胞的发育

目的：观察人胎视网膜超氧化物歧化酶（SOD）和波形蛋白（VIM）免疫阳性细胞的分布发育。方法：不同孕龄的人胎16例,ABC免疫细胞化学方法显示视网膜SOD和VIM免疫阳性细胞,结果：（1）SOD免疫阳性细胞：E15w节细胞层开始出现SOD免疫阳性细胞;D20W和E28W SOD免疫阳性细胞排列较整齐,分布于视网膜的外核层,内核层,节细胞层,其数量增多,其中内核层SOD免疫阳性细胞增多明显。（2）VIM免疫阳性细胞的发育：E15w内界膜开始出现Muller细胞的VIM免疫阳性终足,并见VIM免疫阳性突起伸向外界膜;E20 人VIM免疫阳性物质集中于内界膜,并见VIM免疫阳性突起伸向外界膜;E28wVIM免疫阳性物质的数量较E20w以前各孕龄明显增多,除色素上皮和视杆视锥层外均有VIM免疫阳性物质出现,除伸向外界膜的VIM免疫阳性突起外,还内网层,内核层,节细胞层和神经纤维层还可见水平走行的细胞突起,结论：（1）视网膜发育基本成熟后,视网膜SOD可能主要来源于内核层的SOD免疫阳性细胞。（3）视网膜神经纤维髓鞘是从内向外逐渐形成的。     

兔胎视网膜内P物质免疫反应神经元的发生

本文用免疫细胞化学ＡＢＣ法，研究了新西兰白兔１８、２２、２５、２６、２８和３０ｄ胎龄视网膜内Ｐ物质免疫反应（ＳＰＩＲ）神经元的发生。在胎龄１８和２２ｄ兔视网膜未见ＳＰＩＲ细胞体和纤维。在胎龄２５ｄ视网膜的节细胞层最先出现ＳＰＩＲ神经元，胞体浅染呈卵圆形，突起不明显，在神经纤维层偶见串珠状ＳＰＩＲ纤维，其平均细胞密度为１０４．６个细胞／ｍｍ￣２。到胎龄２６和２８ｄ时，在节细胞层的ＳＰＩＲ神经元的胞体渐深染，可见个别ＳＰＩＲ神经元发出粗而短的突起伸向内网层，平均细胞密度分别为３８７和７７９．５个细胞／ｍｍ２。到胎龄３０ｄ时ＳＰＩＲ神经元开始出现于内核层的内排细胞，但数量很少，胞体呈卵圆形，发出细突起伸入内同层，在节细胞层的ＳＰＩＲ神经元的突起分支增加。此时ＳＰＩＲ神经元平均细胞密度为３５７．４个细胞／ｍｍ￣２。     

脑啡肽与生长抑素在鸡视网膜无长突细胞共存的免疫组织化学研究

本文介绍用免疫组织化学的单标和双标技术研究脑啡肽(ENK)和生长抑素(SOM)在鸡视网膜无长突细胞的定位和共存。单标的实验结果表明,一些SOM免疫反应阳性无长突细胞的形态、胞体在内核层的位置及其突起在内网层的分支式样与某些ENK免疫反应阳性无长突细胞相似,虽然其突起在内网层的第3、4亚层形成的丛网不象ENK免疫反应阳性突起那样丛密,在内网层的第5亚层也未见SOM免疫阳性突起。双标的实验结果表明,一些无长突细胞显示ENK和SOM两种免疫阳性反应,而另一些无长突细胞分别只显示ENK或SOM阳性免疫反应。文中还对视网膜神经多肽间或与经典神经递质的共存进行了讨论。     

GABAc受体ρ1亚单位mRNA在大鼠视网膜和移植视网膜的定位

<正> 应用原位杂交组织化学技术及放射自显影术,通过[~35S]-dATP标记的寡聚核苷酸探针,研究GABA受体ρ1亚单位mRNA在正常SD大鼠视网膜和移植视网膜的定位.本实验分正常组和移植组.将孕14天SD大鼠胚胎视网膜移植至新生第1天SD大白鼠中脑背面偏左侧,于此同时摘除宿主右眼.SD大鼠从受精到出生的时间平均约22天,因此术后第9天移植视网膜的发育时间与正常视网膜生后第1天相当.实验用的探针是合成的反义寡聚核苷酸探针.共40个碱基.探针序列为:(1301-1342)5-CCTCTGTGGGGAGCTCCTCTCTGAGGCCAGGGTCAGCTGCAC-3用(35)~S来标记ρ1探针,探针与组织细胞内ρ1亚单位mRNA形成的杂交体可通过放射自显影来显示其位置.mRNA存在于细胞核和细胞质中,所以标记银粒主要位于胞体上.阳性标准为细胞银粒密度是本底5倍以上.杂交反应阳性细胞意味着细胞内含有ρ1亚单位MRNA,细胞膜上存在GABAc受体.实验结果发现:ρ1亚单位mRNA在大鼠视网膜和移植视网膜的分布相似.它们都分布在内核层中部和外侧部(即近外网层侧),胞体呈椭圆形或卵园形.正常大鼠视网膜ρ1亚单位mRNA杂交阳性细胞最先出现于生后第12天;移植视网膜出现于术后第18天(即相当于正常视网膜生后第10天).根据杂交阳性细胞细胞的     

大鼠视网膜神经元NOS与Parvalbumin的共存研究

用ＮＡＤＰＨ脱氢酶组化及Ｐａｒｖａｌｂｕｍｉｎ免疫组化双标记技术观察了正常大鼠视网膜一氧化氮合酶与Ｐａｒｖａｌｂｕｍｉｎ（ＰＶ）的分布，结果显示ＮＯＳ阳性神经元主要位于内核层内缘带第二列，少数位于节细胞层，胞体圆形／卵圆形，直径８￣１２μｍ，细胞一侧发现突出伸向内网层１、３、５亚层，以第３亚层最为明显，ＰＶ免疫反应（ＰＶ－Ｉ）神经元位于内核层最内缘第一例，少数位于第二列、中间部及节细胞层、胞体卵圆     

大鼠,金黄地鼠和家兔视网膜内一氧化氮合酶分布的比较

用ＮＡＤＰＨ黄递酶组织化学染色法观察了正常成年大鼠、金黄地鼠和家兔视网膜内一氧化氮合酶的分布，并比较了３种不同动物的区别。结果显示，在视网膜内ＮＯＳ阳性神经元主要为分布于内核层的无长突细胞、节细胞层的移位无长突细胞和少数节细胞，不同种类动物的视网膜内，ＮＯＳ阳性细胞的配布、密度和细胞形态均有差异。大鼠视网膜内ＮＯＳ阳性细胞多尾于内核层无长突细胞和节细胞层移位无长细胞，偶见于视网膜节细胞。金黄地鼠视     

神经肽Y样免疫反应神经元在蟾蜍视网膜的定位与分布

用免疫组织化学ABC方法研究神经肽Y(NPY)样免疫反应神经元在蟾蜍视网膜的定位、形态与分布。结果表明:NPY样免疫反应仅存在于无长突细胞中。在102个免疫反应阳性无长突细胞中,92%为Ⅰ型,8%为Ⅱ型。NPY样免疫反应阳性纤维分布于内网层并密集呈三条带,分别位于第1亚层,第2、3亚层交界处和第4、5亚层之间。在视网膜平铺片上,NPY样免疫反应阳性胞体均匀分布于视网膜中央区和周围区,其密度为27±8个/mm~2(均数±标准差)。它们的树突野呈对称和非对称两型,前者的树突野大小为300—500μm×100—300μm,后者的长突约为200—400μm。     

Distribution of GABA immunoreactivity in kainic acid-treated rabbit retina

Ischaemic retinal cell degeneration seems to involve both NMDA and non-NMDA receptor over stimulation. However, different retinal cell types differ largely in their susceptibility to excitatory amino acid induced neurotoxicity. We have investigated the vulnerability of GABAergic cells in the rabbit retina to the non-NMDA receptor agonist kainic acid (KA). The distribution of GABA immunoreactivity (GABA-IR) was examined in the central inferior retina at different survival times (5 h–6 days) following an intra-ocular injection of 140 nmol KA and compared to that of control and untreated retinas. In the normal retina, the majority of GABA-positive cells (79%) were located in the inner nuclear layer (INL), in one to four cell rows next to the inner plexiform layer (IPL), and in one cell row next to the outer plexiform layer (OPL). The remainder (21%) were found in the ganglion cell layer (GCL). Dense immunoreactivity was seen throughout the IPL. In the OPL, stained dots and occasional immunoreactive large processes could be seen. KA-exposed retinas processed for GABA immunocytochemistry 5 and 24 h after the injection showed an 85% reduction in the number of GABA immunoreactive cells. About the same degree of depletion was seen among GABA-IR cells located at different retinal levels. However, at these survival times, immunostaining was observed in three distinct bands in the IPL, indicating that the vulnerability to KA is not uniformly distributed among all GABAergic cells. At 48 h, an additional decrease in the number of labelled cells was noted, but immunoreactive cells were still found both in the INL and GCL. Even 6 days after KA treatment, a few stained cell bodies were seen in the INL next to the IPL, as well as a few processes in the IPL. The study shows that KA receptor overstimulation induces a marked depletion of the endogenous cellular GABA pools of the central rabbit retina, most likely as a result of GABAergic cell loss. However, a small population of GABAergic cells located in the INL appears to be less vulnerable to the toxic effects of 140 nmol KA.     

The light microscopic localization of substance P- and somatostatin-like immunoreactive cells in the larval tiger salamander retina

Summary Light microscopic immunocytochemistry was utilized to localize the populations of substance P (SP)- and somatostatin (SOM)-like immunoreactive cells in the larval tiger salamander retina. Of 104 SP-immunostained cells observed, 82% were Type 1 amacrine cells. Another 8% of the SP-cells were classified as Type 2 amacrine cells, while 10% of the SP-cells had their cell bodies located in the ganglion cell layer and were designated as displaced amacrine cells. Each type of SP-like immunoreactive cell was observed in the central and peripheral retina. SP-immunopositive processes were observed in the inner plexiform layer as a sparse plexus in sublamina 1 and as a denser network of fibers in sublamina 5. Seventy-eight percent of the 110 somatostatin-immunopositive cells observed were designated as Type 1 amacrine cells. Another 12% of SOM-cells were classified as displaced amacrine cells, while only two SOM-immunopositive Type 2 amacrine cells were observed. Nine percent of the SOM-cells were designated as interplexiform cells, based on their giving rise to processes distributing in the outer plexiform layer as well as processes ramifying in the inner plexiform layer. Each type of SOM-immunoreactive cell was observed in the central and peripheral retina, with the exception of the Type 2 amacrine cells, whose somas were only found in the central retina. Lastly, SOM-immunopositive processes in the inner plexiform layer appeared as a fine plexus in sublamina 1 and as a somewhat denser network of fibers in sublamina 5.     

碱性成纤维细胞生长因子对谷氨酸毒性损伤豚鼠视网膜内生长抑素表达的影响

为了探讨过量谷氨酸毒性损伤豚鼠视网膜内生长抑素(SOM)的表达及碱性成纤维细胞生长因子(bFGF)对其表达的影响,本实验将豚鼠随机分成三组。损伤组:腹膜腔内给予谷氨酸钠3g/kg,隔天给药,连续7d;对照组:采用相同方法注射等量生理盐水;治疗组(bFGF+谷氨酸钠):在注射谷氨酸钠之前1~2h,给予bFGF800U/kg,隔天给药,连续7d。各组动物分别在存活10d后取材。采用免疫组织化学和图像分析技术,对各组豚鼠视网膜内SOM样免疫反应产物的表达进行检测。结果显示:(1)正常豚鼠视网膜内可观察到许多SOM样免疫阳性神经元,这些神经元主要分布于视网膜节细胞层(GCL)和内核层(INL);(2)损伤组相应区域内可观察到SOM样阳性细胞数较对照组明显减少(P<0.05或P<0.01),其染色强度亦明显减弱;(3)治疗组经预先给予bFGF后,在GCL和INL内可检测到SOM样阳性细胞数较损伤组明显增加(P<0.05或P<0.01),其染色强度有所增强。以上结果提示:过量谷氨酸钠可导致视网膜内SOM的表达显著降低,而bFGF则可以上调由于过量谷氨酸毒性损伤所引起的SOM的表达。     

Increased expression of ciliary neurotrophic factor receptor alpha mRNA in the ischemic rat retina

Using in situ hybridization, we investigated the expression of ciliary neurotrophic factor receptor ((CNTFRalpha) mRNA in the rat retina rendered ischemic by elevation of the intraocular pressure (IOP). The IOP was increased to 120 mmHg and maintained for 60 min. The rats were sacrificed on the day of reperfusion (DRP) 1, 3, 7, 14, and 28. In the normal retina, the signal for CNTFRalpha mRNA was present in retinal cells in the inner nuclear layer (INL) and in the ganglion cell layer (GCL). On DRP 1, numerous cells in the INL and GCL showed a CNTFRalpha mRNA signal. From DRP 3 onwards, CNTFRalpha mRNA appeared in photoreceptor cells located in the outer part of the outer nuclear layer. The signal in these cells increased up to DRP 14 and then decreased at DRP 28. Our findings suggest that cells expressing CNTFRalpha mRNA may resist the degenerative processes induced by ischemic insult in the rat retina.     

Spatial and temporal distribution patterns of enkephalinergic neurons in adult and developing retinas of the preproenkephalin-green fluorescent protein transgenic mouse

Enkephalin (ENK) peptides are present in the retina of several vertebrate species and play a crucial role in establishing specific circuits during retinal development. However, there is no information available concerning the development of ENKergic neurons in the mouse retina. To address this question, we used preproenkephalin-enhanced green fluorescent protein (GFP) transgenic mice, in which ENKergic neurons are revealed by GFP. Our results showed that most GFP-positive cells were located in the proximal part of the inner nuclear layer with a scattering of GFP-immunoreactive cells in the ganglion cell layer (GCL) in the adult retina. Double immunostaining with syntaxin indicates that GFP expression was restricted to a population of amacrine cells. The proportions of glycine transporter-1 and γ-aminobutyric acid-positive cells among ENKergic neurons were 57.3 ± 2.4% and 10.1 ± 1.8%, respectively. We then injected retrograde tracer into the superior colliculus and observed that none of the ENKergic neurons in the GCL were retrogradely labeled with the tracer. GFP-positive cells were first observed at embryonic day (E) 15 in the inner neuroblastic layer at only very low levels, which gradually increased until E18. After birth, there was a steep rise in GFP expression levels, reaching maximal activity by postnatal day (P) 7. The distribution and intensity of GFP-positive cells at P15 were similar to those of adult retina. It was found that immunoreactive processes in the inner plexiform layer formed strongly stained patches. The present results provide detailed morphological evidence of the cell type and spatial and temporal distribution of ENKergic neurons in the retina.     

大鼠视网膜缺血后Parvalbumin免疫反应神经元的变化

本文观察了大鼠视网膜缺血后Parvalbumin（PV）免疫反应神经元的变化。动物分为缺血10min组、15min组、30min组及60min组等4组．动物右眼为缺血眼，左眼做自身对照眼．结果表明PV免疫反应神经元主要位于内核层及节细胞层，其突起伸向内网层第1、5亚层，神经纤维层也可见PV免疫反应纤维。缺血10min后PV免疫反应神经元未出现变化，缺血15min后数量开始减少，内网层第5亚层PV免疫反应纤维消失、缺血30min、60min后PV免疫反应神经元比缺血15min后减少明显．表明缺血15min后即出现PV免疫反应神经元的变化，但各缺血时间点上其减少率低于其它类型的神经元，提示它对缺血有一定的耐受性。     

GABAc受体ρ1亚单位mRNA在大鼠视网膜和移植视网膜的定位

应用原位杂交组织化学技术及放射自显影技术 ,通过同位素 [35 S] -d ATP标记寡聚核苷酸探针 ,研究了 GABAc受体ρ1亚单位 m RNA在大鼠视网膜和移植视网膜的定位。实验结果发现 :ρ1亚单位 m RNA在大鼠视网膜和移植视网膜的分布相似 ,它们都分布在内核层中部和外侧部 (即近外网层侧 ) ,胞体呈椭圆形或卵圆形。正常大鼠视网膜 ρ1亚单位 m RNA杂交阳性细胞最先出现于生后第 12 d;移植视网膜出现于术后第 18d(即相当于正常视网膜生后第 10 d)。杂交阳性细胞的形态和位置提示 :表达 GABAc受体ρ1亚单位 m RNA的细胞可能是视网膜双极细胞。这为揭示视网膜信息调控提供了重要依据     

Synapsin I expression in the rat retina during postnatal development

Summary The expression of the synapsin I gene was studied during postnatal development of the rat retina at the mRNA and protein levels. In situ hybridization histochemistry showed that synapsin I mRNA was expressed already in nerve cells in the ganglion cell layer of the neonatal retina, while it appeared in neurons of the inner nuclear layer from postnatal day 4 onward. Maximal expression of synapsin I mRNA was observed at P12 in ganglion cells and in neurons of the inner nuclear layer followed by moderate expression in the adult. At the protein level a shift of synapsin I appearance was observed from cytoplasmic to terminal localization during retinal development by immunohistochemistry. In early stages (P4 and P8), synapsin I was seen in neurons of the ganglion cell layer and in neurons of the developing inner nuclear layer as well as in the developing inner plexiform layer. In the developing outer plexiform layer synapsin I was localized only in horizontal cells and in their processes. Its early appearance at P4 indicated the early maturation of this cell type. A shift and strong increase of labelling to the plexiform layers at P12 indicated the localization of synapsin I in synaptic terminals. The inner plexiform layer exhibited a characteristic stratified pattern. Photoreceptor cells never exhibited synapsin I mRNA or synapsin I protein throughout development.Abbreviations GCL ganglion cell layer - INB inner neuroblast layer - INL inner nuclear layer - IPL inner plexiform layer - ONB outer neuroblast layer - ONL outer nuclear layer - OPL outer plexiform layer