视网膜光化学损伤大鼠视紫红质基因mRNA表达水平的原位杂交技…

目的：研究视网膜光化学损伤状况下视紫红质基因ｍＲＮＡ表达水平的变化规律与其与形态学改变的关系，方法：应用原位杂交和电镜技术对光化学损伤大鼠视网膜视紫红质基因ｍＲＮＡ表达情况及视网膜形态学损伤性改变进行动态观察。结果；视紫红质ｍＲＮＡ原位杂交信号主要分布于视网膜光感觉细胞层，尤其是其内段和外段，随连续光照时间的延续，其表达迅速下降，且先于视网膜形态学的损伤性改变；同一视网膜中，上方及后极部杂交信号的     

光化学损伤下大鼠视网膜超氧化物歧化酶基因mRNA表达的实验研究

通过建立大鼠视网膜光化学损伤模型，应用原位杂交技术对正常及光化学损伤大鼠视风膜超氧化物歧化酶基因ｍＲＮＡ表达情况进行动态观察。结果表明，视网膜组织各层均可见超氧化物歧化酶基因ｍＲＮＡ的表达；连续光照可使其表达水平迅速下降，原位杂交为研究视网膜基因表达状况的一种良好方法。     

非诺贝特对糖尿病小鼠视网膜神经组织中miR-26a-5p/PTEN表达的影响

目的：研究非诺贝特对糖尿病小鼠视网膜神经损伤的保护作用并观察其对miR-26a-5p及其靶基因PTEN的影响。方法：构建糖尿病小鼠模型，并进行非诺贝特灌胃，H&E及透射电镜观察视网膜神经损伤情况，Real-time PCR检测视网膜组织中miR-26a-5p的表达，Western blotting检测同源性磷酸酶-张力蛋白(PTEN)在视网膜组织中的表达，并观察NF-κB及IL-1β的水平及视网膜神经上皮的结构变化。结果：与糖尿病组相比，非诺贝特治疗组糖尿病小鼠视网膜神经节细胞损伤及神经纤维层萎缩明显减轻，视网膜中miR-26a-5p表达升高，PTEN mRNA和蛋白表达下降，炎性介质NF-κB及IL-1β mRNA表达水平下降(P<0.05)。结论：非诺贝特通过上调miR-26a-5p抑制PTEN的表达，降低炎症因子水平，减轻视网膜细胞损伤，发挥糖尿病视网膜神经保护作用。     

银杏叶提取物对大鼠视神经挫伤后视网膜形态及视神经GAP-43表达的影响

目的观察银杏叶提取物（EGb761）对大鼠视神经夹挫伤后视网膜形态学及视神经GAP-43表达的影响。方法SD大鼠60只随机分为正常组12只、对照组24只、治疗组24只。后两组建立视神经不完全损伤模型,从伤后1 h开始,隔日1次,对照组给予生理盐水球后注射,治疗组给予EGb761球后注射。在伤后3 d、7 d、14 d、28 d取材,HE染色观察两组视网膜形态学改变;RT-PCR检测两组视神经中GAP-43 mRNA的表达水平。结果各时间点视网膜病理学改变治疗组轻于对照组,GAP-43 mRNA的表达治疗组较对照组高（P〈0.05）。结论EGb761可以抑制神经节细胞的损伤,使视神经组织中GAP-43 mRNA表达增加,促进视神经夹挫伤后视神经的再生。     

神经营养因子影响视神经夹伤后视网膜GAP-43mRNA表达变化

目的：观察大鼠视神经夹伤后视网膜上GAP-43基因的变化,观察玻璃体腔内注射睫状神经营养因子（ciliary neurotrophic factor,CNTF）和腺病毒介导脑源性神经营养因子（adenovirally delivered brain-derived neurotrophic factor,Ad-BDNF）对视网膜上GAP-43mRNA的影响.方法：成年SD大鼠球后2mm处作视神经夹伤模型,经巩膜玻璃体腔内注射微量神经营养因子（neurotraphic factors,NFs）,应用原位杂交法观察视网膜的GAP-43mRNA的变化.结果：正常SD大鼠视网膜上仅在节细胞层检测到少数细胞存在GAP-43mRNA杂交信号,对照组和CNTF治疗组视神经夹伤后1wk内可观察到视网膜神经节层中存在较强的GAP-43mRNA杂交信号,伤后2wk时已减弱至伤前,Ad-BDNF治疗组在视神经夹伤后4wk内在视网膜上均能观察到GAP-43mRNA的杂交信号,其中在夹伤后1～2wk时杂交信号相对较强.结论：视神经夹伤能上调视网膜上GAP-43mRNA表达,玻璃体腔内注射Ad-BDNF在伤后4wk内均能上调视网膜上GAP-43mRNA表达.     

白蒺藜对光损伤小鼠光感受器细胞的保护作用

目的 探讨白蒺藜对小鼠视网膜光损伤的保护作用.方法 采用10 000 lux白炽光光照30 min建立视网膜光损伤模型,将BALB/c小鼠分为正常对照组、模型组和治疗组,每组各6只.光照前0.5h治疗组腹腔注射白蒺藜水煎液100 μL(生药100mg/20 g),正常对照组和模型组腹腔注射等量生理盐水.光照后3h、7d分别通过OCT检测小鼠视网膜形态学改变;造模后7d处死小鼠,取眼球固定、包埋、切片进行HE染色,视紫红质和短波敏感视蛋白(short wave-length sensitivity opsin,M-opsin)的免疫荧光化学染色,观察视网膜病理改变情况.结果 正常对照组内外核层结构清楚、界限明显,视网膜视杆细胞内外节排列整齐;模型组视网膜结构层次模糊且外核层变薄,与正常对照组差异显著;治疗组小鼠视网膜结构与正常对照组没有显著差异.与正常对照组比较,模型组视紫红质和M-opsin表达降低,治疗组显著提高了视紫红质和M-opsin的表达.模型组较正常对照组视网膜外核层厚度显著变薄(P＜0.05);治疗组较模型组能显著性预防小鼠视网膜外核层变薄(P＜0.05).结论 小鼠视网膜光损伤模型造模成功,白蒺藜对光损伤视网膜具有显著的保护作用.     

胰岛素样生长因子1受体反义寡核苷酸对豚鼠形觉剥夺性近视眼的抑制作用

目的观察豚鼠形觉剥夺性近视眼后极部视网膜胰岛素样生长因子1受体（insulin-like growth factor 1 receptor,IGF-1R）基因表达水平的动态变化,以及IGF-1R反义寡核苷酸玻璃体腔注射对形觉剥夺性近视眼屈光度及眼轴长度的影响,探讨视网膜IGF-1R在实验性近视眼发病中的作用。方法3周龄的三色豚鼠64只,随机均分为8组（每组8只）,A组：单眼遮盖7d;B组：未遮盖7d;C组：单眼遮盖14d;D组：未遮盖14d;E组：单眼遮盖14d后去遮盖7d;F组：未遮盖21d;G组：单眼遮盖14d＋玻璃体腔注射40μg（20μl）IGF-1R正义寡核苷酸;H组：单眼遮盖14d＋玻璃体腔注射40μg（20μl）IGF-1R反义寡核苷酸。检测各组的近视屈光度、眼轴长度以及后极部视网膜IGF-1R mRNA和蛋白质的表达水平。结果遮盖14d后实验眼眼轴明显延长,形成近视,去遮盖7d后,近视屈光度减低,眼轴增长减缓;随着遮盖时间的延长,后极部视网膜IGF-1R表达水平明显上调,去遮盖后,表达水平降低（P=0.000）。形觉剥夺14d后,IGF-1R正义寡核苷酸注射眼的眼轴长度 、近视屈光度以及后极部视网膜IGF-1R表达水平与单纯遮盖组无显著差异（P=0.664,0.797,0.312,0.117）;但IGF-1R反义寡核苷酸注射眼的近视屈光度显著减低,眼轴变短,后极部视网膜IGF-1R mRNA和蛋白质表达水平均明显下调（P=0.000）。结论形觉剥夺能上调豚鼠眼后极部视网膜IGF-1R的表达水平,去遮盖后,豚鼠近视屈光度减低,眼轴增长减缓,后极部视网膜IGF-1R的表达水平下调。利用反义寡核苷酸技术抑制视网膜IGF-1R基因的表达,可以抑制近视的发展。     

视紫红质基因启动子的分离及视网膜特异表达载体的构建

目的 分离视紫红质基因启动子，构建以绿色荧光蛋白(GFP)为报告基因的视网膜特异表达载体，为今后视网膜细胞特异的靶向基因转移，特别是为视网膜疾病的基因治疗提供T具。方法 根据小鼠视紫红质基因5’端DNA顺序合成引物，通过PCR技术从小鼠基因组DNA中扩增524bp DNA片段，然后插入质粒pEGFP-1GFP编码基因上游的多克隆位点处，构建表达载体pmRho-EGFP。采用脂质体包裹pmRho-EGFP，转染体外培养的人视网膜色素上皮(RPE)细胞及其他来源的细胞；注射到SD大鼠视网膜下或玻璃体腔内；或通过电穿孔直接转移到RCS大鼠腓肠肌内。GFP表达采用荧光显微镜观察。结果 pmRho-EGFP在体外培养的人RPE细胞中的表达水平明显高于其他组织来源的细胞；体内转染实验证明pmRho-EGFP可在大鼠视网膜神经细胞中表达，但在大鼠腓肠肌中不表达。结论 小鼠视紫红质基因5’端524hp片段具有基本的启动子活性，能够调控基因在视网膜神经细胞及色素上皮细胞中表达，并具有一定的组织和细胞特异性。     

微量分光光度法检测光损伤状况下大鼠视网膜视紫红质含量的变化

目的：探索视紫红质在鼠视网膜光化学损伤中的变化规律。方法：用微量可见紫外分光光度仪分别测定接受连续光照１、２、３、４、天后以及光照１和３天，置暗环境恢复２、７、１４天后大鼠视网膜紫红含量的变化。结果：连续光照致视紫红质含量在光照开始的１～２天内急骤下降，以后趋于水平缓。在一定范围内延长光照时间可使视紫红 一水平更为低下。光照后暗环境中其含量的恢复并非直线上升，在１周左右表现为一个回落。微量分光光度     

兔眼慢性高眼压模型视网膜损害相关基因的克隆及筛选

目的　构建慢性高眼压条件下兔眼视网膜组织差异表达基因的消减cDNA文库并鉴定、克隆及筛选与慢性高眼压视网膜损害相关的基因表达片段。方法　于兔眼前房注射1%甲基纤维素每周1次,连续5周,制作慢性高眼压模型。提取实验组和对照组兔眼视网膜组织总RNA及纯化mRNA。采用原位杂交技术,进行视网膜组织差异表达基因抑制性消减文库的构建及初步的基因筛选。对一个上调的cDNA片段用地高辛标记,采用原位杂交的方法在实验组和对照组的视网膜中进行相关基因片段的细胞定位。结果　成功构建慢性高眼压条件下兔眼视网膜损害相关基因的抑制性消减cDNA文库,经基因片段测序及同源性检索,得到有效基因序列16个,经NCBI BLAST信息途径分析,发现高度同源序列(同源性>98% )2个。其中一个编号F9基因片段与Ras家族基因相似,原位杂交结果确定其大量表达于实验组视网膜神经节细胞及内核层细胞中,而对照组视网膜组织未表达。结论　慢性高眼压条件下视网膜组织基因表达失调,F9片段在慢性高眼压视网膜损害过程中表达明显增高。建立慢性高眼压条件下视网膜损害相关基因的抑制性消减cDNA文库,可为今后青光眼视神经损害机制及视神经保护的研究奠定基础。     

Light treatment enhances photoreceptor survival in dystrophic retinas of Royal College of Surgeons rats.

PURPOSE: To determine whether treatment with bright light elicits a protective response that enhances photoreceptor survival in Royal College of Surgeons (RCS) rats with inherited retinal degeneration. METHODS: RCS rats were illuminated for 10 to 12 hours with 130 foot-candles (fc) of white or green light. Untreated littermates that were kept under low cyclic light levels were used as control subjects. Photoreceptor survival was determined by quantitative analysis of photoreceptor nuclei and ultrastructural assessment of cellular organization. Basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF) gene expression were determined at the mRNA and protein levels. RESULTS: Treatments of RCS rats with a single dose of bright light on postnatal day 23 (P23) greatly enhanced photoreceptor survival. Ultrasturctural analysis revealed intact inner segments in light-treated retinas, whereas in untreated retinas only remnants of inner segments were observed. By P42, numerous viable nuclei were counted in the posterior retina of light-treated rats, whereas most of the remaining nuclei in untreated RCS rat retinas were highly pyknotic. At 2.5 days after treatment with a single dose of bright light, bFGF gene expression was significantly higher than in untreated RCS rat retinas. By P42, bFGF protein levels were still significantly higher in the treated retinas. CONCLUSIONS: Exogenous bFGF has been shown to promote photoreceptor survival in the RCS rat retina. Thus, the increased bFGF expression that was measured in the light-treated RCS rat retinas may be a protective response to light stress, which supports the observed rescue of photoreceptors in light-treated RCS rat retinas.     

Protective effect of halothane anesthesia on retinal light damage: inhibition of metabolic rhodopsin regeneration

PURPOSE: To determine whether the volatile anesthetic halothane protects against light-induced photoreceptor degeneration in the rodent retina. METHODS: Albino mice and rats were anesthetized with halothane and exposed to high levels of white or blue light. Nonanesthetized animals served as controls. Retinal morphology was assessed by light microscopy, and apoptosis of photoreceptor cells was verified by detection of fragmented genomic DNA and in situ staining of apoptotic nuclei (TUNEL assay). Rhodopsin regeneration after bleaching was determined by measuring rhodopsin levels in retinas of mice or rats at different time points in darkness. RESULTS: Halothane anesthesia reversibly inhibited metabolic rhodopsin regeneration and thus prevented rhodopsin from absorbing high numbers of photons during light exposure. Consequently, photoreceptors of mice and rats anesthetized with halothane were completely protected against degeneration induced by white light. In remarkable contrast, however, halothane anesthesia did not protect against blue-light-induced photoreceptor cell death. CONCLUSIONS: After the initial bleach, halothane impeded photon absorption by rhodopsin by inhibiting metabolic rhodopsin regeneration. Apparently, the rhodopsin-mediated uptake of the critical number of photons to initiate white light-induced retinal degeneration was prevented. In contrast, halothane did not protect the retina against blue light. Blue light can efficiently restore functional rhodopsin from bleaching intermediates through a process termed photoreversal of bleaching. This process does not depend on the visual cycle via the pigment epithelium but nevertheless enables rhodopsin molecules to absorb the critical number of photons required to induce retinal degeneration.     

Prosaposin gene expression in normal and dystrophic RCS rat retina

PURPOSE: To identify proteins secreted by the retinal pigment epithelium (RPE) and to analyze their cellular distribution in normal and pathologic rat retinas at various stages of eye development. METHODS: A cDNA library was constructed with RNA isolated from porcine RPE sheets and screened by using the yeast signal sequence trap system. In situ hybridization, immunohistochemistry, and semiquantitative RT-PCR analysis were performed on rat retinas. RESULTS: The cDNA encoding prosaposin was isolated. This is the first time this gene has been shown to be expressed in the retina. Prosaposin mRNA was detected in the rat RPE cell monolayer and in ganglion cells 14, 21, and 45 days after birth. The amount of prosaposin mRNA increased between days 14 and 45 after birth in normal retinas (rdy+), but not in the pathologic retinas (rdy-) of RCS rats. CONCLUSIONS: Several techniques were used to determine the localization of prosaposin in rat retinas. The increase in the amount of prosaposin mRNA in normal retinas coincided with the maturation of photoreceptor cells and the beginning of the phagocytosis process. In addition, the RCS rdy- RPE cells, characterized by the abrogation of the ingestion phase of the photoreceptor outer segments, are deficient in prosaposin expression.     

Dexamethasone ameliorates retinal photic injury in albino rats.

The effect of dexamethasone in two regimens on retinal photic injury was studied in Lewis albino rats that were exposed to 24 hr of continuous green fluorescent light. Under regimen 1, dexamethasone was given at a daily dosage of 1 mg kg-1 for 8 days, starting 6 days before light exposure. Under regimen 2, dexamethasone was given at the same daily dosage for 3 days, started 1 day before light exposure. Pathologic study of the light-exposed retina, morphometric evaluation of the photoreceptor cell loss, cell counts of the macrophages in the subretinal space, and measurements of rhodopsin levels were undertaken in the dexamethasone-treated and control retinas at various times. The administration of dexamethasone in both regimens did not produce pathologic changes in the retina before light exposure, but rhodopsin levels were significantly lowered in both treated groups when compared to corresponding vehicle treated control animals. Under regimen 1, at 6 hr after light exposure, both the treated and the control groups showed comparable loss of photoreceptor cells, degeneration of the photoreceptor elements and retinal pigment epithelium, but a significantly lowered level of rhodopsin in the treated group was noted. At 6 days after exposure, the outer nuclear layer thickness, and the outer and inner segments showed significant preservation in the treated group. Also in the treated group, the number of macrophages was significantly reduced and the retinal pigment epithelial (RPE) vacuolation was markedly less. However, there was no difference in rhodopsin levels. At 14 days after exposure, the outer nuclear layer thickness and rhodopsin levels of the treated rats had significantly higher values than the controls. Under regimen 2, however, at 6 days after exposure, an ameliorative effect in the RPE was observed but there were no differences of rhodopsin levels, the outer nuclear thickness and number of macrophages between the treated and control groups. Regimen 1 was associated with a significantly higher retinal level of dexamethasone when compared with regimen 2. The ameliorative effect of dexamethasone on rat retinal photic injury may be through inhibition of lipid peroxidation, in which a high retinal level of the steroid is required.     

Abnormal centrifugal axons in streptozotocin-diabetic rat retinas

PURPOSE: To characterize the effects of diabetes on the expression of histidine decarboxylase mRNA and on the morphology of the histaminergic centrifugal axons in the rat retina. METHODS: Rats were made diabetic by streptozotocin. After 3 months, retinal histidine decarboxylase expression was analyzed by in situ hybridization in radial sections. Flatmount retinas from a second group of rats were labeled with an antiserum to histamine or an antibody to phosphorylated neurofilament protein. RESULTS: Histidine decarboxylase mRNA was expressed in cells in the inner and outer nuclear layers of diabetic retinas, but not in normal retinas. However, immunoreactive (IR) histamine was not localized to perikarya in either the normal or the diabetic retinas. Instead, a population of centrifugal axons was labeled. These axons emerged from the optic disc and had varicose terminal branches in the inner plexiform layer (IPL) of the peripheral retina. Some branches ended on large retinal blood vessels and others in dense clusters in the IPL. In rats with streptozotocin-induced diabetes, the centrifugal axon terminals developed many large swellings that contained neurofilament immunoreactivity; these swellings were rare in normal retinas. CONCLUSIONS: Diabetes perturbs the retinal histaminergic system, causing increases in histidine decarboxylase mRNA expression in neurons or glia and abnormal focal swellings on the centrifugal axons.     

The use of adenovirus-mediated gene transfer to develop a rat model for photoreceptor degeneration

PURPOSE: To investigate the effects of recombinant adenovirus-mediated downregulation of cathepsin S (CatS) on the retinal pigment epithelium and/or neural retina in vivo. METHODS: The expression of green fluorescent protein (gfp) after subretinal injection of a recombinant adenovirus, Ad.gfp, into rat eyes was first established by in vivo fundus fluorescence photography and fluorescence microscopy. The autofluorescent debris accumulation in Ad.CatSAS (recombinant adenovirus carrying the antisense CatS gene)injected rat eyes was monitored by fluorescence microscopy, and the antisense CatS RNA expression was demonstrated by in situ hybridization. Changes in the retinal morphology were assessed by light microscopy. ResuLTS. The gfp expression was present in 30% to 90% of the injection area at 3 days and was absent 9 days after Ad.gfp injection. In Ad.CatSAS-injected eyes, the expression of antisense CatS RNA was demonstrated by in situ hybridization. Autofluorescent debris accumulation was significantly higher in Ad.CatSAS-injected eyes than in control eyes. The shortening of photoreceptor outer segments in Ad.CatSAS-injected eyes coincided with intense autofluorescent debris accumulation. The number of layers of photoreceptor cells decreased with time and were 11, 9, and 8 at 7, 14, and 28 days after Ad.CatSAS injection, respectively. In control eyes, the number of layers of photoreceptor cells (14) remained unchanged. CONCLUSIONS: These results demonstrate that recombinant adenovirus-mediated transient modulation of gene expression in retinal pigment epithelial (RPE) cells could induce changes in the retina, and, in spite of the low expression of endogenous CatS in RPE cells, this enzyme plays an important role in maintenance of normal retinal function.