Inhibition of cytochrome oxidase and blue-light damage in rat retina

The activity of cytochrome oxidase, outer nuclear layer thickness, and edema were quantitatively evaluated in the blue-light exposed rat retina. Dark-adapted or cyclic-light reared rats were exposed to blue light with a retinal dose of 380 kJ/m². Immediately, 1, 2, and 3 day(s) after exposure, the retinas of six rats from each adaptation group were examined. There was no difference between the dark-adapted and cyclic-light reared rats. Immediately after light exposure, cytochrome oxidase activity decreased. The activity in the inner segments remained low at day 1, while severe edema was observed in the inner and outer segments. The outer nuclear layer thickness decreased 1–3 days after exposure. The blue-light exposure inhibited cytochrome oxidase activity and caused retinal injury. Similarity of the injury process in the dark-adapted and cyclic-light reared retinas suggests that rhodopsin was not involved. The inhibition of cytochrome oxidase could be a cause of retinal damage.     

大鼠出生后发育过程中视网膜Nogo受体蛋白表达的研究

目的 观察Nogo受体（NgR）蛋白在大鼠出生后发育过程中视网膜上的表达及其意义。 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48只大鼠出生后0、3、7、14、21、35、49、63 d视网膜NgR蛋白的表达情况。每组6只大鼠。 结果 NgR蛋白在出生后整个发育过程中的大鼠视网膜上表达均为阳性，荧光染色位于细胞膜及突起。 结论 NgR蛋白的阳性表达提示髓磷脂蛋白抑制因子和NgR之间的相互作用对神经元塑形的过程可能不仅是抑制作用，而是促进和抑制双向作用，从而达到对神经元塑形过程的调控。     

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.     

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 and cellular localization of prolactin and the prolactin receptor in mammalian retina

Prolactin (PRL), originally associated with milk secretion, is known to have a wide variety of biological actions and diverse sites of production beyond the pituitary gland. Recent studies have demonstrated that PRL is synthesized in retinal tissue. To gain insights into the functional role of PRL in the mammalian retina, we mapped the distribution of the PRL protein and the expression and localization of the PRL receptor (PRLR) in the retina of adult rats and green monkeys. PRL was examined in retinal sections by double immunolabeling combining anti-PRL antibodies with antibodies specific for glutamine synthetase (labeling Müller cells), glial fibrillary acidic protein (labeling astrocytes), or neuronal nuclei protein (labeling neurons). PRL was detected throughout the rat retina: in the photoreceptor outer segments, Müller cells, interneurons, ganglion cells, and astrocytes. The PRLR was examined by RT-PCR, in situ hybridization, immunohistochemistry, and Western blot. The long isoform of the PRLR was localized in the photoreceptor nuclear layer, inner nuclear layer, and ganglion cell layer of rat retina. The monkey retina showed a similar distribution of PRL and PRLR immunoreactivities. These findings suggest that PRL functions as a local regulator of various cell types in the mammalian retina.     

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.     

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。     

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。     

负透镜诱导型近视豚鼠视网膜中谷氨酸及其受体的表达

目的　观察谷氨酸及其离子型受体Ｎ-甲基-Ｄ-天冬氨酸受体（Ｎ-ｍｅｔｈｙｌ-Ｄ-ａｓｐａｒｔａｔｅｒｅｃｅｐｔｏｒ,ＮＭＤＡＲ）功能亚单位ＮＲ２Ａ在近视豚鼠视网膜上的动态表达,探讨其在近视发病过程中的作用。方法　选取３周龄健康三色短毛豚鼠１２０只,采用随机数字表法分为０周正常组、２周正常组、２周近视组、４周正常组、４周近视组,每组２４只;正常组不作任何干预,近视组右眼均戴－１０Ｄ透镜,左眼不戴镜作为对照。入组前与处死前均进行屈光度及眼轴长度检测,腹腔注射过量水合氯醛处死,并分离视网膜,采用高效液相色谱分析检测视网膜中谷氨酸的含量变化,实时荧光定量ＰＣＲ以及ＥＬＩＳＡ分别检测豚鼠视网膜中ＮＲ２ＡｍＲＮＡ及其蛋白水平的表达变化。结果　造模前各组屈光度及眼轴长度差异均无统计学意义（均为Ｐ＞０．０５）。造模后各组与造模前比较,眼轴长度及屈光度差异均有统计学意义（２周正常组Ｐ＜０．０５,２周近视组Ｐ＜０．００５,４周正常组Ｐ＜０．０１,４周近视组Ｐ＜０．００１）。造模后,正常组间眼轴长度和屈光度比较差异均有统计学意义（Ｆ＝２０．３２,Ｐ＜０．０１;Ｆ＝１９９．６５,Ｐ＜０．０１）;２周近视组与４周近视组比较,右眼眼轴长度和屈光度差异均有统计学意义（Ｆ＝７８．９６,Ｐ＜０．００１;Ｆ＝２５２．１０,Ｐ＜０．００１）,左眼眼轴长度和屈光度差异均无统计学意义（Ｆ＝１３．６６,Ｐ＞０．０５;Ｆ＝２１．２０,Ｐ＞０．０５）;２周近视组与４周近视组右眼分别与相应的左眼比较,眼轴长度差异均有统计学意义（２周近视组:ｔ＝９．５１５,Ｐ＜０．００５;４周近视组:ｔ＝９．４４９,Ｐ＜０．００１）,屈光度差异同样均有统计学意义（２周近视组:ｔ＝８．８９７,Ｐ＜０．００１;４周近视组:ｔ＝１７．２３５,Ｐ＜０．００１）。视网膜中谷氨酸含量正常组之间差异均无统计学意义（均为Ｐ＞０．０５）;造模后２周近视组与４周近视组比较,右眼视网膜中谷氨酸含量逐渐增加,差异有统计学意义（ｔ＝３６．２３０,Ｐ＜０．０１）。造模后２周近视组较２周正常组右眼谷氨酸含量增加,差异有统计学意义（ｔ＝－２３．２４０,Ｐ＜０．０５）;同样４周近视组较４周正常组右眼谷氨酸含量亦增加,差异亦有统计学意义（ｔ＝－５３．６９０,Ｐ＜０．０１）。视网膜中ＮＲ２ＡｍＲＮＡ的表达量,正常组之间差异均无统计学意义（均为Ｐ＞０．０５）。造模后４周近视组与２周近视组比较,右眼视网膜ＮＲ２ＡｍＲＮＡ表达量明显上调,差异有统计学意义（ｔ＝５５．６６０,Ｐ＜０．００１）;２周近视组与２周正常组右眼比较表达量增加,差异有统计学意义（ｔ＝－１５．０８６,Ｐ＜０．００５）,同样４周近视组与４周正常组右眼比较差异亦有统计学意义（ｔ＝－４３．２７６,Ｐ＜０．００１）。正常组之间视网膜中ＮＲ２Ａ蛋白表达量,差异无统计学意义（均为Ｐ＞０．０５）。４周近视组与２周近视组比较,右眼视网膜ＮＲ２Ａ蛋白表达量明显上调,差异有统计学意义（ｔ＝４３．２１０,Ｐ＜０．００５）;２周近视组与２周正常组右眼比较ＮＲ２Ａ蛋白表达量增加,差异有统计学意义（ｔ＝－２．３６５,Ｐ＜０．０５）,同样４周近视组与４周正常组右眼比较差异亦有统计学意义（ｔ＝－５．５１８,Ｐ＜０．０１）。结论负透镜诱导型近视豚鼠视网膜中谷氨酸及其ＮＭＤＡＲ受体亚单位ＮＲ２Ａ在透镜诱导眼中表达上调,并随透镜诱导时间的延长和近视程度的加深而增加。     

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。     

人视网膜发育分化过程中基质金属蛋白酶9表达的变化

目的 观察人视网膜发育和分化过程中基质金属蛋白酶9(MMP-9)的表达水平与细胞分布特点,探讨其在人视网膜发育和分化过程中的可能作用.方法 胚胎发育早期、中晚期及成人视网膜石蜡切片,抗MMP-9免疫组织化学染色,观察MMP-9表达水平和分布的变化.结果 胚胎发育早期的视网膜仅在外成神经细胞层的外侧可见弱阳性细胞;胚胎发育中期外成神经细胞层的外侧,即将来要发育成盘膜处出现明显的特异性染色,而其他细胞染色仍然较弱;成人视网膜的外核层出现显著的特异性染色细胞,胞质明显着色,胞体轮廓清晰,并可观察到锥体的结构以及突触的特异染色,判断这些细胞主要是光感受器细胞中的视锥细胞.结论 MMP-9在视网膜中的表达与视网膜的成熟和细胞分化关系密切,在光感受器中的视锥细胞高水平表达,提示MMP-9可能参与视觉信号的处理.     

视神经损伤时视网膜睫状神经营养因子受体的表达

目的研究睫状神经营养因子受体于视神经损伤后不同时间在视网膜中的表达情况,探讨外源性的睫状神经营养因子在神经损伤疾病中的应用价值。方法采用钳夹视神经的方法建立神经损伤的模型,在损伤后1、7、14、28d获取视网膜,提取总RNA及总蛋白,用半定量逆转录聚合酶链反应(RT-PCR)测定视网膜中睫状神经营养因子受体(CNTFR)αmRNA的表达,用WesternBlot方法了解损伤后不同时间CNTFRα蛋白水平的表达。结果在正常大鼠视网膜内CNTFRαmRNA无表达,在损伤后的1、7、14、28d均有一定水平的CNTFRαmRNA的表达,与正常对照比较差别具有显著性意义(P<0.01);损伤后备时间均有CNTFRα蛋白的表达,但CNTFRα蛋白的表达较弱。结论神经损伤后CNTF的表达先短暂升高以后持续下调,而CNTFRα-mRNA在损伤后4周内均有一定量的表达,提示补充外源性CNTF可能改善神经再生的微环境而发挥保护效应。