Immunohistochemical localization of somatostatin receptor subtypes sst1 and sst2 in the rat retina.

PURPOSE: To investigate the distribution of somatostatin receptor subtypes sst1 and sst2 in the rat retina by immunohistochemistry and to characterize further the neurotransmitters of the sst1- and sst2-immunoreactive cells. METHODS: Polyclonal antibodies raised against sst1 and sst2 receptors were applied to 12-microm cryostat sections of rat retinas fixed in paraformaldehyde. Further, immunofluorescence double labeling was performed for the sst1 and sst2 receptors with somatostatin, tyrosine hydroxylase (TH) and glutamate decarboxylase (GAD). RESULTS: Immunoreactivity for sst1 was present in somatostatinergic amacrine cells located in the inner nuclear layer (INL) and in displaced amacrine cells in the ganglion cell layer of the retina. Also, a small number of ganglion cells were sst1 immunoreactive. Immunoreactivity for sst2 was observed in many medium-sized amacrine cells in the middle part of the INL, with a central process projecting to the sublaminae of the inner plexiform layer. Furthermore, sst2 immunoreactivity was found in large amacrine cells of the INL. These cells also contained TH. Inner segments of cone receptors were stained with the sst2 antiserum. Immunostaining for sst2, and to a minor extent for sst1, was found in Müller cell fibers. None of the somatostatin receptors colocalized with GAD. CONCLUSIONS: These findings suggest that the sst1 receptor may function as an autoreceptor on retinal somatostatinergic cells. The presence of sst2 receptors on the TH-immunoreactive amacrine cells indicates an influence of somatostatin on the secretion of dopamine in rat retina.     

Expression of the P2Y2 receptor on the rat ocular surface during a 1-year rearing period

Purpose

P2Y₂ receptors are expressed on ocular surface tissues. Diquafosol ophthalmic solution (DIQUAS^® ophthalmic solution 3 %; Santen Pharmaceutical Co., Ltd.) acts on these receptors and promotes the secretion of water and mucin. It has been shown to be an efficient dry eye treatment. If P2Y₂ receptor expression on the ocular surface decreases with age, the effect of diquafosol may be reduced in elderly persons. In this study, we investigated the changes in P2Y₂ receptor expression on the rat ocular surface over an extended period of time.

Methods

P2Y₂ receptor expression in the conjunctiva, cornea, meibomian gland and lacrimal glands of male and female Sprague–Dawley rats was examined from 5 weeks until 53 weeks of age using immunostaining and quantitative-PCR.

Results

In the immunohistological examinations, P2Y₂ receptor expression was observed in the conjunctival epithelium containing goblet cells, corneal epithelium, meibomian gland ductal epithelium and lacrimal gland ductal epithelium. However, its expression was not significantly different between each age group or between sexes. Regarding P2Y₂ receptor mRNA expression, there was an age-related increase in the bulbar conjunctiva. In particular, a significant increase was observed in the 53-week-old age group as compared to the 5-week-old female age group. However, age-related changes in expression were not observed in the cornea or meibomian gland in males or females.

Conclusions

We observed no significant age-related decrease was observed for P2Y₂ receptor protein and mRNA expression on rat ocular surface tissues.     

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。     

Distinct P2Y receptor subtypes regulate calcium signaling in human retinal pigment epithelial cells

PURPOSE: Nucleotide signaling plays a role in retinal pigment epithelial (RPE) function, and receptors for nucleotides are potential therapeutic targets for various ocular diseases. The purpose of this study was to investigate the expression of P2Y receptor subtypes in native and cultured human RPE cells. METHODS: Intracellular Ca(2+) levels were monitored using real-time fluorescence imaging in cultured human RPE cells loaded with Fura-2. Expression of P2Y receptors in native and cultured RPE cells was determined by quantitative RT-PCR and Western blot analysis. RESULTS: Adenosine triphosphate (ATP), uridine triphosphate (UTP), adenosine diphosphate (ADP), 2-methylthio ATP (2MeSATP), and uridine diphosphate (UDP) produced concentration-related increases in [Ca(2+)](i) in cultured RPE cells. However, differences between the magnitude and shape of agonist responses were observed. ATP and UTP showed similar response characteristics, including a distinct Ca(2+) influx component. ATP and UTP were equipotent (EC(50), 6 muM) and maximum responses were equivalent, suggesting activation of a P2Y(2) receptor. Maximal responses to ADP and 2MeSATP were equivalent with EC(50)s of 1 muM and 0.3 muM. The P2Y(1) antagonist MRS 2179 (10 muM) inhibited these responses, confirming functional expression of P2Y(1) receptors. The presence of a response to UDP suggested P2Y(6) expression. There was no influx component to P2Y(1)- and P2Y(6)-mediated responses. mRNA for P2Y(1), P2Y(2,) P2Y(4), and P2Y(6) receptor subtypes was found in cultured RPE cells, and for P2Y(1), P2Y(2,) P2Y(4,) P2Y(6), and P2Y(12) it was found in native RPE cells. Expression of P2Y(1), P2Y(2), and P2Y(6) protein was found in native and cultured RPE cells. CONCLUSIONS: These data define the expression profile of P2Y receptors in human RPE and show that different P2Y subtypes control distinct calcium responses in these cells.     

Identification of P2Y receptor subtypes in human muller glial cells by physiology, single cell RT-PCR, and immunohistochemistry

PURPOSE: Retinal Müller glial cells are known to express metabotropic P2Y receptors. The present study was conducted to identify certain subtypes of P2Y receptors in human Müller cells. METHODS: The patch-clamp technique was used to measure increases of Ca(2+)-dependent K+ currents mediated by the activation of P2Y receptors in freshly isolated human Müller cells. Several P2 agonists were used. Subsequently, the cells were harvested into the patch pipette and a single cell RT-PCR was performed. Moreover, retinal tissue from organ donors was used for immunohistochemistry. RESULTS: The electrophysiological data were consistent with the expression of P2Y1, P2Y2, P2Y4, and P2Y6 receptor subtypes. RT-PCR revealed that mRNA for all these subtypes was present in Müller cells. However, the incidence of P2Y2 receptor mRNA was significantly lower than that of the other subtypes. Immunoreactivity for all four subtypes was found in retinal tissue, partly colocalized with immunoreactivity for vimentin. CONCLUSIONS: The presented data obtained by different techniques revealed that human Müller cells express P2Y1, P2Y2, P2Y4, and P2Y6 receptors. The specific roles of these receptor subtypes in retinal physiology and/or pathophysiology remain to be investigated in future studies.     

Angiotensin II and its receptor subtypes in the human retina

PURPOSE: To quantify and evaluate the distribution of angiotensin II (Ang II) and its receptors in the human retina. METHODS: Donor eyes were obtained within 12 hours postmortem and classified as hypertensive or normotensive and diabetic or nondiabetic, based on the donors' medical histories. Ang II in retina and vitreous was quantified by RIA. Ang II receptors were characterized and quantified by competitive membrane-binding assays. Ang II, its heptapeptide metabolite Ang-(1-7), and AT1 and AT2 receptors were localized by immunohistochemistry and confocal imaging. RESULTS: Levels of Ang II in the retina were significantly higher than in vitreous (P < 0.05). Ang II in the diabetic retina had a higher median compared with that in the nondiabetic retina. Ang II and Ang-(1-7) colocalized in retinal Müller cells. The retina had the highest levels of Ang II receptors that were significantly higher than the optic nerve, retinal pigment epithelium-choroid complex, and ciliary body-iris complex (P < 0.05). AT1 receptors were more abundant than AT2 receptors in the retina. Immunoreactivity for AT1 was detected in Müller cells and on blood vessels. AT2 receptors were localized throughout the Müller cells and nuclei of ganglion cells and neurons in the inner nuclear layer. CONCLUSIONS: In the human retina, identification of Ang II and its bioactive metabolite Ang-(1-7) in Müller cells suggests that these glial cells are able to produce and process Ang II. Ang receptors were localized in the blood vessels and neural cells. Local Ang II signaling may thus allow for autoregulation of neurovascular activity. Such an autonomous system could modulate the onset and severity of retinovascular disease.     

促红细胞生成素与促红细胞生成素受体在大鼠脱离视网膜中的表达

目的 观察促红细胞生成素（EPO）和促红细胞生成素受体（EPOR）在大鼠脱离视网膜中的表达情况。方法48只雄性SD大鼠,随机分为正常对照组、视网膜脱离（RD）后1、3、6、12、24、48、72 h组,每组6只大鼠12只眼。视网膜下腔单次注射1.4%透明质酸钠致上半侧视网膜隆起建立RD模型,采用逆转录聚合酶链式反应（RT-PCR）和蛋白免疫印迹（Western blot）测定不同时间点EPO和EPOR的mRNA和蛋白水平的表达情况,同时采用免疫组织化学方法观察EPO和EPOR在视网膜中定位表达的情况。结果 EPO和EPOR的mRNA表达水 平在RD后均上调,均于RD后48 h达到高峰,分别于RD后6、12 h显著高于正常对照组,差异具有统计学意义（P＜0.05）;同样,EPO和EPOR的蛋白表达水平也在RD后均增高并在RD后48 h达到高峰,均于RD后3 h显著高于正常对照组,差异具有统计学意义（P＜0.05）。免疫组织化学结果显示正常视网膜自神经节细胞层至光感受器细胞的内外节均有EPO弱表达,RD后48 h视网膜相应部位呈强阳性表达;正常视网膜自神经节细胞层至光感受器细胞的内节均有EPOR表达,RD后48 h视网膜相应部位呈强阳性着染。结论 RD后大鼠视网膜EPO和EPOR表达均逐渐增强,48 h达到高峰;大鼠神经视网膜大部分层次能表达EPO和EPOR。     

Expression of thromboxane synthase and the thromboxane-prostanoid receptor in the mouse and rat retina

Experimental models of the diabetic retina have suggested a pathological role for thromboxane. To date however, little information is available as to the cellular locations of retinal thromboxane synthase (TxS), or its receptor, even in non-diabetic controls. In this study, C57BL/6 mice and Wistar rats were injected with streptozotocin to induce diabetes, or with buffer for non-diabetic controls. Four weeks following the injection, eyes were enucleated and labeled for TxS and the thromboxane-prostanoid (TP) receptor. Immunofluorescent intensity was quantified in the ganglion cell plus inner plexiform layers, inner nuclear layer, outer plexiform layer, outer nuclear layer, and photoreceptor inner segment. Even in control mice and rats, all layers of the retina showed immunoreactivity for TxS and the TP receptor: however, the pattern of expression demonstrated an inverse relationship, with the highest TxS staining in the inner retina, and the highest TP receptor staining in the outer retina (more specifically, in the photoreceptor inner segment). Four weeks of hyperglycemia did not increase the retinal levels of TxS or TP receptor; however, TP receptor intensities in the outer retina of diabetic rats were highly variable (mostly high but some low), with no values from the photoreceptor inner segment in the same range as obtained from controls.     

NADPH-diaphorase colocalization with somatostatin receptor subtypes sst2A and sst2B in the retina

PURPOSE: To investigate the differential localization of somatotropin release-inhibitory factor (SRIF) receptor subtypes (sst2A and sst2B) and their possible colocalization with reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase in the rat and rabbit retina. METHODS: Polyclonal antibodies raised against sst2A and sst2B receptors were applied to 10- to 14-microm cryostat sections of rat and rabbit retinas fixed in paraformaldehyde. NADPH-diaphorase reactivity was assessed histochemically. Double labeling was performed for sst2A or sst2B receptors with NADPH-diaphorase, and with markers for the cell types present in the retina (protein kinase C [PKC], tyrosine hydroxylase; [TH], calbindin, and recoverin). RESULTS: sst2A immunoreactivity was detected in rod bipolar cells and colocalized with NADPH-diaphorase in the rabbit, but not the rat, retina. sst2B was present only in photoreceptor cells of the rat and colocalized with NADPH-diaphorase. CONCLUSIONS: These results suggest that SRIF, acting through sst2A receptors in bipolar cells and sst2B receptors in photoreceptor cells, may affect nitric oxide function in the rabbit and rat retina.     

Molecular and functional mapping of regional differences in P2Y receptor expression in the rat lens

Extracellular ATP has been shown to mobilize intracellular Ca²⁺ in cultured ovine lens epithelial cells and in human lens epithelium, suggesting a role for purines in the modulation of lens transparency. In this study, we characterized the expression profiles of P2Y receptor isoforms throughout the rat lens at both the molecular and the functional levels. RT-PCR indicated that P2Y₁, P2Y₂, P2Y₄ and P2Y₆ are expressed in the lens, while P2Y₁₂, P2Y₁₃ and P2Y₁₄ are not. Immunohistochemistry, using isoform specific antibodies, indicated that the epithelium does not express P2Y₁ and P2Y₂, but that the underlying fiber cells, which differentiate from the epithelial cells, exhibit strong membranous labeling. Although co-expressed in fiber cells, differences in P2Y₁ and P2Y₂ expression were apparent. P2Y₁ expression extended deeper into the lens than P2Y₂, and its expression co-localized with Cx50 gap junction plaques, while P2Y₂ did not. Labeling for P2Y₄ and P2Y₆ receptors were observed in both epithelial cells and fiber cells, but the labeling was predominantly cytoplasmic in nature. While purine agonist (ATP, ADP, UTP and UDP) application to the lens induced mobilization of intracellular Ca²⁺ in cortical fiber cells, little to no effect was observed in the anterior and equatorial epithelium. Thus the inability of UTP and UDP to mobilize intracellular Ca²⁺ in the epithelium and the predominately cytoplasmic location of P2Y₄ and P2Y₆ suggests that these receptors may represent an inactive pool of receptors that may be activated under non-physiological conditions. In contrast, our results indicated that P2Y₁ and P2Y₂ are functionally active in fiber cells and their differential subcellular expression patterns suggest they may regulate distinct processes in the lens under steady state conditions.     

NgR蛋白在新生SD大鼠视网膜中的表达

目的观察NgR（Nogo-66 receptor）蛋白在出生24hsD大鼠视网膜中的表达。方法使用免疫荧光组织化学技术及激光共聚焦显微镜观察6只出生24h的正常SD大鼠视网膜中NgR蛋白的表达。结果在6只出生24h的正常SD大鼠视网膜神经节细胞及其突起上可以观察到NgR蛋白的表达，且该蛋白主要分布在视网膜神经节细胞核的周围。结论出生24h的SD大鼠视网膜中就已经存在NgR蛋白，为进一步研究哺乳动物中枢神经系统损伤后轴突再生修复奠定了基础。[眼科新进展2007；27（2）：81-83]     

Expression of GABA transporter subtypes (GAT1, GAT3) in the adult rabbit retina.

PURPOSE: GABA transporters (GATs) are of importance for GABA signal systems. They have previously not been examined in rabbit retina, nor has their correlation with neurotransmitter GABA and GABA receptors been examined in the retina of any species. METHODS: The distribution of GATs, GABA and GABA receptors was examined with immunohistochemical methods. RESULTS: Both GAT1 and GAT3 immunoreactivities were found in the inner plexiform layer and in amacrine cells. GAT3 was also present in Müller cells. GAT1 appeared in amacrine cells that also had a high GABA concentration, but not in cells with moderate to low GABA concentration. GAT1 was also present in amacrine cells that did not show GABA immunoreactivity, possibly indicating a postsynaptic GABA uptake system. CONCLUSION: GAT3 is probably involved in both neuronal and glial GABA uptake whereas GAT1 is involved in predominantly neuronal uptake, and possibly also into non-GABA-ergic amacrine cells. Further, there may be at least two populations of GABA containing neurons.     

Expression of GABA transporter subtypes (GAT1, GAT3) in the developing rabbit retina.

PURPOSE: Little is known about the expression of GABA transporters (GATs) in the developing retina. We have therefore examined the expression of GABA transporters (GAT1 and GAT3) in the developing rabbit retina. METHODS: The distribution of GATs was examined with immunohistochemical methods. RESULTS: GAT3 immunoreactivity appeared at PN0, whereas GAT1 immunoreactivity appeared first at PN3, both in the inner plexiform layer. From PN5 and onwards, both GAT1 and GAT3 immunoreactivity gradually appeared in numerous amacrine cell somas. At PN10, the immunostaining patterns and the distribution of both GAT1 and GAT3 were similar to that found in the adult rabbit retina. Full staining intensity was reached at PN20. CONCLUSION: GABA neurotransmission starts to develop already the first one to three days after birth, reaches the mature neuron pattern at about PN9 to PN10 but is not fully developed until about PN20. Neither GAT1 nor GAT3 appears to be involved in trophic actions by GABA in the prenatal retina.     

Localization of GABAA receptor subtypes in the tiger salamander retina.

Dry autoradiography was used to determine the distribution of GABAA binding sites in tiger salamander retina. High-affinity binding of [3H]-flunitrazepam [( 3H]-FNZ) was used to localize benzodiazepine receptors (BZR) and [3H]-muscimol was used to localize the GABAA recognition site. Specific [3H]-FNZ binding was present only in the inner retina, primarily in the inner plexiform layer (IPL). Co-incubation with GABA enhanced [3H]-FNZ binding by 20-50%. [3H]-muscimol binding was found throughout the IPL and in the outer plexiform layer (OPL). Mouse monoclonal antibodies 62-3G1 and BD-17, that recognize the GABAA beta 2, beta 3 polypeptides, and BD-24, that recognizes the GABAA alpha 1 polypeptide, did not label either the OPL or IPL, despite numerous variations in the fixation and immunoprocessing methods. GABAA receptor location, as revealed by [3H]-muscimol binding, matches the distribution of presumed GABAergic terminals in the OPL and IPL. We suggest that there are at least two subtypes of GABAA receptor in the tiger salamander retina: one type is present only in the inner retina, primarily in the IPL and is functionally coupled to BZRs; the other type is located in the OPL and is not coupled to the BZRs. Furthermore, GABAA receptors in the tiger salamander retina appear to be of a different epitope than GABAA receptors in numerous other preparations that are recognized by mAbs 62-3G1, BD-17, and BD-24.     

基质细胞衍生因子-1在Wistar大鼠视网膜上的生理性表达

目的研究正常成体大鼠视网膜基质细胞衍生因子（SDF-1）的生理性表达情况。方法取正常Wistar成年大鼠视网膜进行抗SDF-1和抗PCK免疫组织化学染色,镜下观察结合半定量图像分析;实时定量RT-PCR测定视网膜神经上皮层SDF-1mRNA含量,与内参基因βactin比较。结果正常大鼠视网膜神经上皮可见SDF-1表达,其阳性结果多位于视网膜内层,视神经无明显染色。PCK在视网膜神经上皮上无明显表达。实时定量RT-PCR证实在健康成体大鼠视网膜神经上皮存在一定量的SDF-1mRNA。结论正常成年大鼠视网膜神经上皮层存在SDF-1的生理性表达,其生理作用及调控机制有待进一步研究。     

Toll样受体4及相关炎性细胞因子在糖尿病大鼠视网膜中的表达

目的 观察糖尿病大鼠视网膜中Toll受体4（TLR4）、炎性细胞因子的表达水平以及视网膜中白细胞聚集与视网膜通透性改变。方法 Brown Norway大鼠120只,剔除自然死亡14只,随机分为实验组和对照组,每组均为53只大鼠。实验组大鼠腹腔注射链脲佐菌素建立糖尿病模型;对照组大鼠腹腔注射等体积柠檬酸-柠檬酸钠缓冲液。建模后4周行定量聚合酶链反应、蛋白质免疫印迹法检测,观察大鼠视网膜中TLR4的基因及蛋白表达,酶联免疫吸附试验测定大鼠视网膜匀浆上清液中肿瘤坏死因子-α（TNF-α）、白细胞介素-1β(IL-1β)、单核细胞趋化蛋白-1（MCP-1）等炎性细胞水平;吖啶橙眼底血管造影观察视网膜中白细胞密度;伊凡思蓝（EB）检测视网膜通透性。结果 对照组、实验组大鼠视网膜TLR4 mRNA、蛋白表达量比较,差异均有统计学意义（F=1.606、0.789,P＜0.05）;视网膜匀浆上清液中MCP-1、IL-1β、TNF-α表达量比较,差异均有统计学意义（F=24.622、5.758、4.829,P＜0.05）。实验组、对照组大鼠视网膜中白细胞密度分别为（6.2±0.5）×10^-5、(2.2 ±0.3）×10^-5个细胞 /像素2,实验组大鼠视网膜中白细胞密度较对照组显著增加,差异有统计学意义（F=2.025,P＜0.05）。实验组、对照组大鼠视网膜EB渗漏量分别为（23.41±4.47）、（13.22±3.59） ng/mg,实验组大鼠视网膜EB渗漏量较对照组增加,差异有统计学意义（F=21.08,P＜0.05）。结论 糖尿病大鼠视网膜中TLR4及炎性细胞因子表达均显著提高;视网膜中白细胞密度和视网膜渗漏量增加。     

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。     

TLR-4在大鼠慢性高眼压模型视网膜中的表达

】背景TLR-4是一种天然免疫受体,在免疫性中枢神经系统损伤的修复中发挥重要作用,但在青光眼视神经损伤中的具体作用机制不详。目的从基因和蛋白质水平研究正常大鼠及慢性高眼压模型大鼠视网膜组织中TLR4的表达情况,探讨高眼压视网膜神经节细胞（RGCs）损伤的免疫机制及TLR-4在RGCs修复中的可能作用。方法选择d～5周龄的雄性清洁级纯种SD大鼠150只,用烧烙3条巩膜静脉联合术中应用丝裂霉素的方法建立大鼠慢性高眼压模型。Tono—penⅡ型笔式眼压计测量造模前、造模后2h,1、3、7、14、28、56d大鼠眼压。上述各时间点分别取5只高眼压组及空白对照组大鼠视网膜行免疫组织化学染色,观察视网膜TLR4蛋白的表达情况。应用逆转录聚合酶链反应（RT．PCR）法及Westernblot法检测各组视网膜组织中TLR-4mRNA及蛋白质的表达。结果实验组手术后眼压明显升高,与空白对照组比较差异有统计学意义（P〈0．01）。免疫组织化学检测结果显示造模后早期TLR--4在视网膜组织中的表达开始增加,表达量均高于空白对照组（P〈O．05～0．01）;RT—PCR检测表明,大鼠慢性高眼压模型眼造模后各时间点TLR-4mRNA在视网膜的表达量明显高于空白对照组（P〈0．05～0．01）;WesternNot检测显示TLR4在造模后早期视网膜组织中即有表达,结果显示组间差异有统计学意义（P〈0．05～0．01）。结论TLR-4在大鼠慢性高眼压模型眼视网膜中的表达明显上调,提示其在大鼠慢性高眼压视神经退行性病变中发挥免疫调节作用。