GABA_A和GABA_C受体各亚单位基因在鲫鱼视网膜内的分布──原位杂交组织化学法研究

应用原位杂交组织化学技术，利用同位素［￣（３５）Ｓ］－ｄＡＴＰ标记的寡核苷酸探针，在鲫鱼视网膜观察了含ＧＡＢＡＡ受体α１、α３、α４、α６，β１－３，γ１－２及ＧＡＢＡＣ受体ρ１亚单位ｍＲＮＡ的神经元分布。在外核层，所有测试的亚单位均无表达；而在内核层和神经节细胞层，除α４和γ２亚单位外，均有不同程度的表达。在不同区域标记神经元的数量和标记强度各不相同，α１亚单位广泛分布在内核层的远端、中部及神经节细胞层，呈强阳性；α３亚单位相对稀少，主要分布在内核层近端和神经节细胞层，呈中等阳性；α４和α６亚单位几乎无阳性表达，仅α６亚单位在神经节细胞层呈弱阳性。β１和β２亚单位在内核层及神经节细胞层呈中等阳性；β３亚单位主要分布在内核层，在神经节细胞层标记细胞较少，呈弱阳性。γ１亚单位分布在整个内核层，在神经节细胞层有零星阳性表达。ＧＡＢＡＣ受体主要分布在内核层，ρ１亚单位主要分布在内核层的远端及中间部分，呈强阳性，而在神经节细胞层表达相对较弱。这种独特的表达型式与其功能密切相关。     

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     

大鼠视神经切断后视网膜双极细胞PKC-α和recoverin的表达

为了探讨视神经切断后视网膜内部是否存在突触可塑性改变,本实验采用大鼠视神经切断模型,通过免疫组织化学方法检测视神经切断后视网膜双极细胞PKC-α和recoverin的表达变化。结果显示:正常视网膜中,PKC-α和recoverin阳性产物主要见于视网膜内核层、内网层及节细胞层,另外外核层也可见少量recoverin阳性细胞。视神经切断后3d,大鼠视网膜内网层高倍镜下可见PKC-α和recoverin免疫阳性终末的数量开始增加,14d时增至最高,21d、28d呈现逐渐减少的趋势。本研究结果提示视神经切断后视网膜双极细胞与节细胞之间的突触可能存在早期增生,后期溃变的可塑性变化。     

Enhanced protein expressions of sortilin and p75NTR in retina of rat following elevated intraocular pressure-induced retinal ischemia

Elevated introcular pressure (IOP)-induced retinal neuron ischemic death includes an early phase of necrosis and prolonged phase of apoptosis. We used this ischemic model to observe the changes of sortilin and p75(NTR) protein expressions in rat retina. The results of Western blot analysis showed the expression of p75(NTR) at the band of 75 (mature form), 60 (non-glycosylated pieces) and 50 kDa (ectodomain shedding pieces), and the expression of sortilin at the 95 and 90 kDa (the mature form). The protein expressions of p75(NTR) (60 and 50 kDa pieces) and sortilin (90 kDa) increased significantly (p < 0.05) at days 3, 5 and 7 after retinal ischemia. This effect was also confirmed by immunofluorescence staining. Sortilin was primarily present in cell membrane of the ganglion cells layer (GCL) and large ganglion cell bodies by immunofluorescence labeling. There was little expression of p75(NTR) in the normal retina, while expression increased extensively in GCL, inner plexiform layer (IPL) and inner nuclear layer (INL) after retinal ischemia. p75(NTR) was shown to co-localize with neurofilament in the axons of neuronal cells by double-labeling. These results suggested that the protein expressions of 60 and 50 kDa forms of p75(NTR), and the 90 kDa mature form of sortilin increased in ischemia-induced retinal neuron of rats.     

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     

大鼠视网膜不完全性缺血引起细胞凋亡及bcl-2表达的研究

目的 :通过结扎双侧颈总动脉造成大鼠视网膜不完全性缺血 ,用 TUNEL及免疫组化方法观察视网膜细胞凋亡和 bcl- 2的表达。结果 :缺血 1天即见节细胞层和内核层出现凋亡细胞 ,7天在外核层也出现 ;高峰时间是缺血 14天 ,术后 6 0天仍可见凋亡细胞。缺血 1天 Mul　¨ le's细胞内侧部 bcl- 2表达明显 ,7天着色区域扩大至Mul　¨ le's细胞外侧部 ,14天阳性反应局限于 Mul　¨ le's细胞内侧部 ,并且着色明显 ,持续至缺血 30天。缺血 6 0天bcl- 2表达明显减弱。结论 :细胞凋亡参与视网膜缺血损伤 ,缺血后 bcl- 2表达增强是细胞防御功能增强的体现     

N-methyl-D-aspartate-induced excitotoxicity in adult rat retina is antagonized by single systemic injection of MK-801

Intravitreal injection of N-methyl-D-aspartate (NMDA) produced a substantial damage to the adult rat retina that was largely restricted to inner retinal layers, including the ganglion cell layer (GCL), inner nuclear layer (INL), inner, and outer plexiform layers. This retinal damage was significantly reduced by a systemic injection of a low dose of MK-801 (0.5 mg/kg), a potent NMDA-receptor antagonist. This neuroprotection was dose dependent and was most effective when the antagonist was given 1 h before NMDA insult. An intraperitoneal injection of 0.5 mg/kg MK-801 provided a virtually complete protection to the retina to the NMDA-induced toxicity, as indicated quantitatively by the number of DiI-filled retinal ganglion cells, the number of cells in the GCL and INL that undergo DNA fragmentation, and the edematous changes in retinal thickness. A post-lesion administration of MK-801 was still able to provide an effective neuroprotective effect to the retina, but this protection was lost when MK-801 was given 4 h after NMDA exposure. The current results indicate a therapeutic potential of systemic application of MK-801 in protecting the adult rat retina from neurologic disorders related to excessive activation of NMDA receptors.     

Spatial and temporal patterns of apoptosis during differentiation of the retina in the turtle

We investigated patterns of cell death in the turtle retina that could potentially be associated with the innervation of the optic tectum, and looked for mechanisms of retinal development that might be common to reptilian and homeotherm vertebrates. We used retinas of turtle embryos between the 23rd day of incubation (E23) (before the first optic fibres reach the optic tectum) and hatching (when all the optic fibres have established synaptic connections). Dying retinal neurons were identified in paraffin sections by the TUNEL technique, which specifically labels fragmented DNA. Apoptotic cells were found in the ganglion cell layer (GCL), the inner nuclear layer (INL), and the outer nuclear layer (ONL). Cell death in the GCL was intense between E29 and E47, and had disappeared by the day of hatching. In the INL, dead and dying cells were most abundant between E31 and E34, and progressively disappeared. The temporal pattern in the ONL was similar to the INL although the density was very low. In all the nuclear layers cell death spread from the dorso-temporal area of the central retina to the periphery. Additional dorsal to ventral and temporal to nasal gradients were distinguishable in a quantitative TUNEL analysis. The patterns of cell death observed in the developing turtle retina were thus similar to those found in birds and mammals. This process could be under the control of differentiation gradients in all the vertebrate classes.     

Genetically induced retinal degeneration leads to changes in metabotropic glutamate receptor expression

In the retina, neurotransmission from photoreceptors to ON-cone and rod bipolar cells is sign reversing and mediated by the metabotropic glutamate receptor mGluR6, which converts the light-evoked hyperpolarization of the photoreceptors into depolarization of ON bipolar cells. The Royal College of Surgeons (RCS) rat retina undergoes progressive photoreceptor loss due to a genetic defect in the pigment epithelium cells. The consequences of photoreceptor loss and the concomitant loss of glutamatergic input to second-order retinal neurons on the expression of the metabotropic glutamate receptor was investigated in the RCS rat retina from early stages of photoreceptor degeneration (P17) up to several months after complete rod and cone degeneration (P120). The expression of the gene encoding mGluR6 was studied by in situ hybridization in the retina, using an [(35)S]dATP-labeled oligonucleotide probe. In congenic control and RCS retina, we found mRNA expression of mGluR6 receptor only in the outer half of the inner nuclear layer (INL) on emulsion-coated retinal sections. Quantitative analysis of the hybridization signal obtained from the autoradiographic films revealed decreased expression levels of the mGluR6 mRNA at early stages of photoreceptor degeneration (P17). On the contrary, increased expression levels were observed at late stages of degeneration (P60 and P120) in RCS compared to congenic control retina. In conclusion, our data demonstrate that the metabotropic glutamate receptor-6 mRNA levels are altered in the young and adult RCS rat retina and suggest that the genetically induced degeneration of photoreceptors affects the expression of this receptor by the INL retinal neurons.     

泛素蛋白酶系统在四氧嘧啶诱导的糖尿病大鼠视网膜中的表达

目的:建立泛素蛋白酶系统(UPS)在正常和8周糖尿病大鼠视网膜的表达图谱,探讨其在糖尿病视网膜病变(DR)发生发展中可能的分子机制。方法:在限制片段差异显示PCR(RFDD-PCR)技术建立正常和8周糖尿病大鼠视网膜基因表达谱的基础上,对差异片段进行生物信息学分析,筛选UPS DR相关基因,并以免疫组织化学、半定量RT-PCR技术进行验证。结果:RFDD-PCR结果显示,泛素蛋白酶系统DR相关基因UBS3A、PSMD8和PSMD11在糖尿病组表达上调。RT-PCR结果显示,糖尿病组UBS3A表达比正常组明显增高,PSMD8和PSMD11则仅在糖尿病组中表达。免疫组织化学结果显示,正常组UBE3A阳性免疫反应物见于内丛状层,外丛状层和锥、杆体细胞层,PSMD8和PSMD11未见阳性细胞;糖尿病组UBE3A、PSMD8和PSMD11阳性细胞明显增多,见于节细胞层、内核层和外核层。结论:UPS与DR发生发展有关。     

Expression of the 40 kDa catecholamine regulated protein in the normal and injured rat retina

Catecholamine regulated protein 40 (CRP40) has been shown to be expressed in the central nervous system (CNS) of several mammalian species where it may function in a similar manner to members of the heat shock protein (HSP) family. Immunohistochemical and immunoblotting techniques were utilized to investigate whether CRP40 is expressed in normal rat retinas. In addition, changes in CRP40 expression were studied following optic nerve transection. The immunohistochemical results showed that CRP40 is expressed in the normal rat retina. The protein was found to be highly expressed in the ganglion cell layer (GCL), the inner nuclear layer (INL) and the outer plexiform layer (OPL). In addition, a low level of CRP40 was found in the inner plexiform layer (IPL), and in the inner segment layer (ISL). No expression was found in the outer nuclear layer (ONL) of normal rat retina. The immunoblotting results show that CRP40 expression decreased in a time-dependent fashion after the optic nerve transection. This decrease indicates that the expression of CRP40 is dependent on the neuron's normal physiological state and that it plays an important function in physiological and pathological conditions in the retina.     

ENK与CaBPs在PPE-GFP转基因小鼠视网膜细胞中的共存

目的:以PPE-GFP转基因小鼠为研究工具,观察绿色荧光蛋白(GFP)阳性的脑啡肽(ENK)能神经元与钙结合蛋白D28K(CB)、钙视网膜蛋白(CR)和小白蛋白(PV)等钙结合蛋白(CaBPs)成员在视网膜的分布及共存情况。方法:利用免疫组织化学和免疫荧光双标染色的方法。结果:GFP阳性的ENK能细胞主要分布在视网膜内核层内缘,少量分布在节细胞层。所有的GFP阳性细胞均与神经元标志物NSE共存,但不与星形胶质细胞标志物GFAP共存。GFP与CB、CR和PV均有部分共存,其中GFP/CB共存神经元占GFP阳性细胞的8.65%,占CB阳性细胞的5.84%;GFP/CR共存神经元占GFP阳性细胞的18.18%,占CR阳性细胞的14.28%,且共存细胞仅见于内核层;GFP/PV共存细胞占GFP阳性细胞的68.75%,占PV阳性细胞的91.67%,共存细胞主要位于内核层,少量见于节细胞层。结论:ENK能神经元在视网膜内具有板层特异性的分布特点和与钙结合蛋白成员有不同的共存模式,上述结果为深入研究小鼠视网膜ENK能神经元的功能意义提供了形态学依据。     

Indoleamine-accumulating cell death and endogenous glial cell reaction induced by 5,7-dihydroxytryptamine in the cat retina.

We investigated the patterns of degenerative changes of indoleamine-accumulating cells (IACs) induced by 5,7-dihydroxytryptamine (5,7-DHT, 100 microg), and the glial reaction to the neurodegenerative changes of IACs in the cat retina by using light-and electron-microscopy. The neurons accumulating 5,7-DHT in the cat retina were a few ganglion cells and displaced amacrine cells located in the ganglion cell layer (GCL), and some amacrine cells in the inner nuclear layer (INL). The cell density (per unit area, 1 mm2) of the 5,7-DHT accumulating cells in the GCL and INL was 910 and 134 cells, respectively. Most 5,7-DHT accumulating cells showed dark degeneration characterized by widening of the cellular organelles at early stage, and by darkening of the cytoplasm at a late stage. In addition, amacrine cells, showing a typical filamentous degeneration, were observed in a few cases. The degenerated neurons were phagocytosed by microglial cells and astrocytes. The immunoreactivity for glial fibrillary acidic protein (GFAP) in Muller cells was increased at early stage, but thereafter abruptly decreased. In a few cases, severe degenerative changes were observed in Miller cells. These results indicate that 5,7-DHT induces severe dark degeneration of IACs, and most degenerated cells could be eliminated by microglial cells and astrocytes in the cat retina.     

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.     

Müller细胞反应性胶质化在急性高眼压致大鼠视网膜损伤中作用

 目的 观察Müller细胞反应性胶质化在急性高眼压(AOH)大鼠视网膜中变化及其抑制对视网膜损伤的影响。方法 建立大鼠AOH青光眼模型,分为正常对照(Ctrl)、AOH和AOH+玻璃体内注射胶质毒素α-氨基己二酸(AAA)后再灌注1、3和5d组,以及单纯AAA和AOH+PBS对照组。TUNEL染色检测细胞凋亡,GFAP免疫荧光染色反应Müller细胞反应性胶质化程度,Thy-1染色标记视网膜神经节细胞(RGCs)。结果 AOH可致大鼠视网膜内丛状层和内核层明显变薄、神经节细胞层内细胞排列紊乱和数量减少,并诱发Müller细胞反应性胶质化(GFAP表达增加)。同时,AAA抑制Müller细胞反应性胶质化可明显缓解AOH所致RGCs丢失和凋亡发生。结论 Müller细胞反应性胶质化参与AOH所致视网膜损伤,抑制其反应性胶质化可能是改善高眼压性青光眼视网膜病变的一种有效治疗方法。     

酒精暴露对视网膜致畸作用和细胞凋亡的影响

目的 探讨产前酒精暴露（PAE）对视网膜致畸作用和细胞凋亡的影响。方法 利用C57BL/6J小鼠建立孕期酒精暴露模型，用HE染色和
免疫荧光染色技术观察酒精对生后0d （P0）、P7、P14和P30 4个年龄点共72只子鼠的视网膜致畸性和细胞凋亡的影响。结果 高剂量酒精组中
视网膜的畸形率增加；在各剂量组Caspase-3、Caspase-8、Caspase-9 阳性细胞于内颗粒层中的表达与在节细胞层中的表达具有一致性，且具
有剂量依赖性(P<0.05)； TUNEL染色结果发现，与对照组相比，酒精组各年龄点的内颗粒层和节细胞层中细胞凋亡量均增加（P<0.05），提示
孕期酒精暴露诱导视网膜细胞发生凋亡时具有长时程效应。结论 视网膜畸形及细胞凋亡可能是导致PAE相关眼病的病理学基础。     

Immunohistochemical localization of sortilin and p75 in normal and ischemic rat retina

In our previous study, we found the increased expression of p75^NTR and sortilin by Western blotting in ischemic retina induced by elevated intraocular pressure (IOP). Cell specific expression of sortilin and p75 neurotrophin receptor (p75^NTR) was now characterized in normal and ischemic rat retina induced by elevated IOP by double-labeling immunochemistry. Two patterns of sortilin staining in normal retina were identified: punctate and consistent. The former was seen in the retinal ganglion cell (RGC) bodies, probably in Golgi apparatus. The latter was found in astrocytes and Müller glial cells (MGCs). The expression pattern of sortilin did not change in ischemic state induced by elevated IOP. p75^NTR was not found in RGCs, but in MGCs of most of the retinal layers, especially the inner plexiform layer (IPL), and outer plexiform layer (OPL) of normal retina. Taken together, the enhanced expression of sortilin in MGCs might be involved in the neuro–glial interactions in ischemic retina, but may not directly contribute to RGCs death through proNGF binding to complex receptor composed of sortilin and p75^NTR in ischemic retina.     

Dopamine- and adenosine-3':5'-monophosphate (cAMP)-regulated phosphoprotein of Mr 32,000 (DARPP-32) in the retina of cat, monkey and human.

The cellular localization of a dopamine- and cAMP-regulated phosphoprotein of Mr 32,000 (DARPP-32) was investigated in cat, monkey and human retina by immunohistochemistry. In cat, DARPP-32-immunoreactive cell bodies identified as Müller cells were demonstrated in the inner nuclear layer (INL) with processes closely surrounding the cell soma of photoreceptors in the outer nuclear layer. Some DARPP-32-IR cells were also seen in the nerve fiber layer (NFL) sending processes to the inner plexiform layer. In monkey and human retina, DARPP-32-IR cell bodies were also demonstrated in the INL, with few cells located in the NFL.     

Glutamate receptor expression in the rat retina.

The expression of five genes (GluR A; B; C; D; GluR 5) encoding functional subunits of glutamate receptors was investigated in the rat retina using in situ hybridization with oligonucleotide probes. All five genes are expressed in the retina. All probes label cell bodies in the ganglion cell layer as well as somata in the inner third of the inner nuclear layer (INL), where the amacrine cells are located. In addition GluR 5, B and D, and to a lesser extent also GluR A are found in the middle and outer part of the INL, where bipolar and horizontal cells reside. Different subsets of retinal neurons may thus use glutamate receptors of different subunit composition.     

游离锌离子在小鼠视网膜的定位研究

目的研究游离锌离子在小鼠视网膜的定位分布。方法应用ZnSe金属自显影技术(AMG)检测硒酸钠注射40 m in后小鼠视网膜内的锌离子。结果注射硒酸钠40 m in后发现游离锌离子主要分布于小鼠视网膜的色素上皮细胞层、光感受器的内节、外核层、外网层、内核层、内网层和神经节细胞层。在色素上皮细胞层、光感受器的内节和内核层与内网层交界处AMG阳性反应最为明显,在光感受器外节和神经纤维层几乎没有AMG阳性反应产物。结论小鼠视网膜内锌离子,在视网膜神经元视觉信息的传导和形成过程中可能起着重要作用。