Expression of peroxisome proliferator-activated receptorγ in rat retina during development

AIM: To evaluate the spatiotemporal expression pattern of PPARγ in embryonic and early postnatal stages of rat retina.METHODS: Fetal rats were collected at 13-18 d of gestation(GD) from pregnant females and postnatal rats at 1d(P1) and 5d(P5) after birth were also used. We used RT-PCR to detect PPARγ m RNA and immunohistochemical to observe PPARγ protein. And at last, we chose HE staining showed the structural changes of rat retina during development.· RESULTS: RT-PCR analysis showed that PPARγm RNA was expressed as early as GD13 and gradually decreased as maturation continued. However, the PPARγgene expression significantly increased after birth,especially in P5. Immunohistochemical analysis showed PPARγ protein was expressed throughout the retinal neuroepithelium at GD13 and GD14, and then decreased during late embryogenesis but remained relatively high in the predicted ganglion cell zone. During postnatal development, PPARγ protein was remarkably increased and the positive signals were mainly located in nerve fiber layer(NFL), ganglion cell layer(GCL) and outer layers of the retina.CONCLUSION: The spatiotemporal changes of PPARγexpression demonstrated that PPARγ might play a role in regulating the differentiation and maturation of retinal cells.

Beta-amyloid precursor protein cleavage enzyme-1 expression in adult rat retinal neurons in the early period after lead exposure

Previous studies have reported that non-human primates and rodents exposed to lead during brain development may become dependent on the deposition of pre-determined β-amyloid protein （Aβ）,and exhibit upregulation of β-site amyloid precursor protein expression in old age.However,further evidence is required to elucidate the precise relationship and molecular mechanisms underlying the effects of early lead exposure on excessive Aβ production in adult mammals.The present study investigated the effects of lead exposure on expression of β-amyloid precursor protein cleavage enzyme-1 （BACE-1） in the rat retina and the production of Aβ in early development,using the retina as a window for studying Alzheimer＇s disease.Adult rats were intraocularly injected with different doses of lead acetate （10μmol/L,100μmol/L,1 mmol/L,10 mmol/L and 100 mmol/L）.The results revealed that retinal lead concentration,BACE-1 and its cleavage products β-C-terminal fragment and retina Aβ1-40 were all significantly increased in almost all of the lead exposure groups 48 hours later in a dose-dependent manner.The only exception was the 10μmol/L group.The distribution of BACE-1 in the retina did not exhibit obvious changes,and no distinctive increase in the activation of retinal microglia was apparent.Similarly,retinal synaptophysin expression did not exhibit any clear changes.These data suggest that lead exposure can result in the upregulation of retinal neuron BACE-1 expression in the early period of development and further increase the overproduction of Aβ1-40 in the retina.Our results provided novel insight into the molecular mechanisms underlying environmentally-induced Alzheimer＇s disease.

TrkB and p-trkB expression in brain-derived neurotrophic factor-pretreated rat retina following acute high intraocular pressure

BACKGROUND： Exogenous brain-derived neurotrophic factor （BDNF） promotes retinal ganglion cell survival. However, the protective mechanisms remain unclear. OBJECTIVE： To investigate changes in retinal tyrosine kinase receptor B （trkB） expression and effects of exogenous BDNF on trkB activation in a rat model of acute high intraocular pressure （HtOP）. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Department of Anatomy and Neurobiology, Xiangya Medical School, Central South University from January 2004 to August 2006. MATERIALS： Rabbit anti-BDNF and anti-trkB.FL（full-length） polyclonal antibodies were purchased from Santa Cruz Biotechnology, USA; rabbit anti-p-trkB polyclonal antibodies were purchased from Cellsignal, USA. METHODS： A total of 48 healthy, adult, Sprague Dawiey rats were randomly assigned to acute HIOP （without BDNF pre-treatment） and BDNF pre-treated groups, with 24 animals in each group. In the BDNF pre-treated group, the left eyes were intravitreally injected with 3 pg/kg BDNF 2 days prior to HIOP. Rats in the acute HIOP group were not pre-treated with BDNE HIOP models were established by increased intraocular pressure in the left eyes until the b-wave of flash electroretinogragh disappeared and pressure was maintained for 60 minutes. The right eyes of all rats were not treated and served as the normal controls. MAIN OUTCOME MEASURES： Retinal structure and cell numbers in the ganglion cell layer （GCL） were detected by Nissl staining; expression of trkB and phosphorylated trkB in the rat retina were determined by immunohistochemistry. RESULTS： A greater number of GCL neurons were observed in the pre-treated group compared to the acute HIOP group （P 〈 0.05）. TrkB expression was significantly increased following HIOP at days 1 and 3 （P 〈 0.05）, but expression varied between retinal areas. Although trkB expression decreased at 7 days, phosphorylated trkB dramatically decreased with increasing time （P 〈 0.05）. TrkB expression in BDNF pre-treated rats was similar to the acute HIOP group at early injury time points. Nevertheless, trkB expression was significantly decreased compared to the acute HIOP group at 7 days （P 〈 0.05）, and phosphorylated trkB expression was significantly greater compared to the acute HIOP group at each time point （P〈 0.05）. CONCLUSION： TrkB expression displayed temporal and spatial changes in the rat retina following acute HIOP, and trkB up-regulation suggested that more BDNF was required for treating the injured retina. Exogenous BDNF partially ameliorated decreased expression of phosphorylated trkB and provided protection to the injured retina, to a certain degree, following HIOP.

IMMUNOHISTOCHEMICAL DISTRIBUTION OF ERYTHROPOIETIN AND ITS RECEPTOR EXPRESSION IN POSTNATAL RAT RETINA DEVELOPMENT

Objective To investigate the distribution of erythropoietin (EPO) and erythropoietin receptor (EPOR) expression in the postnatal rat retina development. Methods Forty-two male Sprague-Dawley rats were divided into 7 groups according to their various postnatal days: postnatal 1 d (D1 group), 3 d (D3 group), 1 week (W1 group), 2 weeks (W2 group), 3 weeks (W3 group), 4 weeks (W4 group) and 8 weeks (W8 group) (n=6). Single eye was randomly chosen from each rat for the study. The retinal sections were stained with hematoxylin and eosin (HE) and used for the retina development observation. Immunohistochemical staining was used to localize EPO and EPOR expressions in retinas of different stages of development, and the expression intensities were determined by an image plus 4 program. Results The retinal inner nuclear layer (INL) and outer nuclear layer (ONL) were mixed together and had not yet fully differentiated in D1 and D3 groups. The INL and ONL formed their own independent regions and the outer plexiform layer (OPL) appeared between two layers in W1 group. With the postnatal retinal development, the inner plexiform layer (IPL), rods and cones layer (RCL), and OPL were gradually widened and stabilized in W2 to W3 groups. EPO/EPOR expressions located prominently in the inner part of the postnatal rat developing retinas. The expression of EPO in GCL and INL gradually increased from D1 to W4, then the expression decreased in W8. Expression of EPOR in GCL gradually increased from D1 to W1, then decreased in W2; and it gradually increased again from W3 to W8. Expression of EPOR in INL gradually increased from D1 to W1, then decreased in W2; and it continued to decrease from W3 to W8. Expression of EPOR in the external segment of RCL gradually increased from D1 to W8. However, expression in the internal segment of RCL gradually decreased from D1 to W3, then no obvious expression was seen in the internal segment of RCL in W4 and W8. Conclusion EPO/EPOR expressions locate prominently in the inner part of the postnatal rat developing retina. And EPO/EPOR expressions in the rat retinas exist the dynamic changes during the postnatal retina development period.

Histopathological changes in retinas and F-ERG features of streptozotocin-induced diabetic rats treated with ozone

AIM： To study the histopathological changes in the retina and flash electroretinogram （F-ERG） features of ozone-treated streptozotocin （STZ）-induced diabetic rats. METHODS： Seventy male Sprague Dawley rats were grouped as follows： blank group （GB, n=10）, model control group （GM, n=18）, ozone group （GOs, n=19）, and oxygen group （GO2, n=18）. The model was induced by single intraperitoneal injection of STZ. Ozone or oxygen enteroclysm was given twice per week for 4wk. F-ERG and histopathological examinations were performed one month after treatment. RESULTS： Under dark adaption, as compared to GB, the other groups each had differential decreases in the a-wave amplitudes （P〈0.05）; the latencies were delayed in GM, GO2, and GO3 rats （P〈0.05）. Similar results were observed under light adaption, with the exception that the a-wave of the amplitudes （F=0.28, P〉0.05）. There were significant differences in the apoptosis index among the groups （P〈0.05）. Under ozone treatment, apoptosis was decreased in GO3 as compared to GM and GO2 . CONCLUSION： Ozone administration alleviates nerve damage and reduces pathology and apoptosis in the retinas of diabetic rats.

Evans blue staining to detect deep blood vessels in peripheral retina for observing retinal pathology in early-stage diabetic rats

AIM: To observe and compare the statistical significance of superficial and deep vascular leakage in the pathological changes of the diabetic rats retina after the Evans blue(EB) perfusion, and utilize the modified whole-retina spreading method to make the slides while protecting the periphery of the retina. METHODS: The Sprague-Dawley(SD) rats were randomly divided into 6 groups. Each group named as the normal groups for 4, 8, and 12 wk and the diabetic groups for 4, 8, and 12 wk. The EB was injected into the cardiovascular system of the rats at the different time points. The retina of each group was obtained for observation.RESULTS: The superficial vascular leakage was found in all 6 groups. The size of leakage area of superficial retinal blood vessels was(0.54±0.23)%,(0.65±0.11)%,and(0.58±0.10)% in normal group. No notable leakage was found in the deep blood vessels [(0.03±0.04)%,(0.03±0.05)%, and(0.03±0.05)%]. The deep retinal vascular leakage was found in the peripheral retina of diabetic rats. The size of leakage area of superficial retinal blood vessels in diabetic group were(0.53±0.22)%,(0.69±0.16)%, and(0.52±0.11)%. The leakage areas of deep blood vessels were(0.54±0.50)%,(1.42±0.16)%, and(1.80±0.07)% at 4, 8, and 12 wk, respectively. There was a statistically difference of the leakage area between the 8 th week and the 4 th week of diabetes group(P=0.003). The statistically significant difference between the diabetes and the control groups was noted at 4 wk and 8 wk(P<0.001).CONCLUSION: The main retinal pathological changes of early-stage diabetic rats are the vascular leakage of the periphery of deep retina. Diabetic rats modeled after 8 wk have semi-quantitative statistical difference compared with the normal rats, thus early intervention treatment research can start at this time point.

Differences in expression of retinal proteins between diabetic and normal rats

瞄准：为了在在正常、糖尿病的老鼠之间的网膜的蛋白质的表示比较并且识别差别，并且分析网膜的疾病的分子的病原的机制，由糖尿病引起了。方法：在从糖尿病、正常的老鼠的网膜的纸巾的蛋白质表示的变化用 2-dimensional polyacrylamide 胶化电气泳动(2-DE ) 被观察。展出统计上重要的变化的一些蛋白质点(P 【 0.05 ) 随机被选择并且由双人脚踏车团分光术识别了并且由生物信息学分析了。结果：2-DE 证明表示在 5 网膜的蛋白质是起来调整的，在 23 网膜的蛋白质下面调整，并且消失在 8 网膜的蛋白质。八个点被双人脚踏车团分光术(MS/MS ) 从 36 个点识别并且由生物信息学分析了。Guanylate 激酶 1， triosephosphate 异构酶 1， 2 玩了的 ATP synthase 子单元 d，白朊和 dimethylarginine dimethylaminohydrolase 在信号转导变异的一个重要角色。Triosephosphate 异构酶 1，晶状体蛋白高山哈 B， ATP synthase 子单元 d 和 peroxiredoxin 6 涉及网膜的纸巾的能代谢。1 玩了的 Guanylate 激酶在网膜的杆光敏电阻器房间的相片刺激的一个重要角色。晶状体蛋白贝它 A1 是否在糖尿病的视网膜起一个作用，到目前为止是未知的。结论：在在糖尿病、正常的老鼠之间的网膜的蛋白质的表示有差别。这些蛋白质可以涉及糖尿病引起的网膜的疾病的机制和预后。

ROLE OF CASPASE-3 IN ACUTE LIGHT DAMAGE TO RETINA OF RATS

Objective To investigate the role of Caspase-3 in retinal damage caused by light exposure in rats. Methods Light injury to retina was induced by persistent exposure to illumination (intensity:30000±50lux) of operating microscope for 30 minutes in the right eyes of Sprague-Dawley rats. The pathological changes of retina were observed under optical and electron microscopies at different time points, which were 6 hours, 1, 3, 7, and 15 days after the light exposure. Apoptosis of retinal cells was analyzed by flow cytometry. The activity of Caspase-3 was evaluated by using the Caspase-3 assay kit. At the same time, the expression of Caspase-3 protease was determined with Western blot analysis. Results The examination results of optical and transmission electron microscopes showed that edema of inner and outer segments of the retina, especially the chondriosome inside the inner segment, became obvious 6 hours after the light exposure. The change was deteriorated along with the increasing time. The structures of the discoidal valve dissociated in the outer segment simultaneously. Disorderly arranged nuclei, karyopycnosis, and thinning in the outer nuclear layer were observed. The retinal pigment epithelium almost disappeared during the later stage. The staining results of Annexin-V combined with PI demonstrated that the proportion of apoptotic cells increased with time. The proportion between 7th day (82.7%) and 15th day (80.4%), however, showed no significant difference. Caspase-3 became remarkably active with the lapse of time, which increased from 0.02 at 6th hour to the peak of 9.8 at 7th day before it started to descend. The Western blot detected a expression of the active form of Caspase-3 at 7th day and 15th day.Conclusion Apoptosis of photoreceptor cells is markedly involved in the light damage and Caspase-3 protease may play an important role in the apoptotic process of the retina after light exposure in rats.

Hazardous effects of fried potato chips on the development of retina in albino rats

Objective:To evaluate the hazardous effects of fried potato chips upon the retina of two developmental stages of the albino rats aged 7 and 14 days from parturition.Methods:Pregnant rats were arranged into two groups:control pregnant rats and consequently their delivered newborns until reaching 7 and 14 days old from parturition and fried potato chips group in which pregnant rats at the 6th day of gestation maintained on diet formed of fried potato chips supplied from the market mixed with standard diet at a concentration of 50%per each till 7 and 14 postpartum.Three fold integrated approaches were adopted,namely,histological,ultrastructural and proteomic analysis.Results:Histological examination of the retina of the experimental offsprings revealed many histopathological changes,including massive degeneration,vacuolization and cell loss in the ganglion cell layer,as well as general reduction in retinal size.At the ultrastructural level,the retina of experimental offsprings exhibited number of deformities,including ill differentiated and degenerated nuclear layer,malformed and vacuolated pigment epithelium with vesiculated and fragmented rough endoplasmic reticulum,degenerated outer segment of photoreceptors,as well as swollen choriocapillaris and loss of neuronal cells.Proteomic analysis of retina of the two experimental developmental stages showed variations in the expressed proteins as a result of intoxication which illustrated the adverse toxic effects of fried potato chips upon the retina.Conclusions:It can be concluded that the effect of fried potato chips on the development of retina in rats may be due to the presence of acrylamide or its metabolite.

New antioxidant SkQ1 is an effective protector of rat neural retina under conditions of long-term organotypic cultivation

During life human eye is constantly exposed to sunlight and artificial light, the sources of reactive oxygen species (ROS)—the main cause of age-related eye pathology. A novel mitochondria-targeted antioxidant SkQ1 has recently been invented to reduce mitochondrial ROS by cleaning the mitochondria matrix, “the dirtiest place in the cell” in respect of ROS production and accumulation. Earlier we studied SkQ1 effects upon retinal pigment epithelium and choroid in the rat eye posterior cups exposed to long-term 3D organotypic culturing. It was found that under in vitro conditions 20 nM SkQ1 effectively reduced cell death in retinal pigment epithelium and choroid and protected the tissues from disintegration and cell withdrawal. In the present study we used same ex vivo conditions to examine the effect of SkQ1 upon the rat neural retina kept in the content of the posterior eye cup. Eye cups were isolated and cultured in vitro during 7, 14, and 30 days under rotation in the presence and absence of 20 nM SkQ1 in the culture medium. Serial sections of cultivated eye cups were subjected to histology, computer morphometry and immunohistochemistry. Obtained results show that SkQ1 operates as a strong protective agent, preventing neuronal cell death and other degenerative processes in the neural retina. Cell rescue by SkQ1 was more vivid in the central part of the retina than at the periphery. That, in turn, suggests SkQ1 effectiveness in treatment of some age-related eye diseases when central part of the retina, including macula, is most susceptible to degeneration.

硫辛酸烟酸二联体对蓝光致大鼠视网膜损伤的防治作用

目的:探讨硫辛酸烟酸二联体(N2L)对蓝光致SD大鼠视网膜损伤的防治作用及最佳药物剂量,探寻其可能存在的保护机制。方法:选取150-200 g的SPF级雄性SD大鼠36只,随机分为正常对照组、蓝光损伤组、N2L低剂量组(1.0 mg/kg)、N2L中剂量组(2.5 mg/kg)、N2L高剂量组(5.0 mg/kg)及生理盐水组,每组各6只。正常对照组12 h明暗循环饲养,其余组每日接受9 h日常光照,3 h波长455 nm、强度3000±50 lx蓝光照射及12 h黑夜来建立损伤模型,持续14 d。同时每日腹腔注射1 mL对应剂量的药物。14 d后,所有组常规12 h明暗循环再饲养5 d,采用视网膜电图检查。过量麻醉法处死大鼠制备标本,HE染色,在光学显微镜下观察外核层厚度;CheKineTM超氧化物歧化酶(SOD)活性检测试剂盒检测SOD活性;Western Blot检测大鼠视网膜Caspase-3、醌氧化还原酶1(NQO1)、谷胱甘肽巯基转移酶(GST)、Bcl-2和Bax蛋白表达量。结果:蓝光损伤组暗视ERG 3.0、10.0(cd·s)/m^(2)刺激光下b波、明视ERG 3.0(cd·s)/m^(2)刺激光下b波振幅及震荡电位第2个波峰振幅显著低于正常对照组(均P<0.01),N2L中剂量组较蓝光损伤组振幅显著提高(均P<0.05),且与正常对照组无显著差异;蓝光损伤组较正常对照组视网膜ONL厚度下降(P<0.001),N2L中剂量组厚于蓝光损伤组(P<0.001),与正常对照组无显著差异;N2L中剂量组超氧化物歧化酶活性显著高于其余5组(P<0.05);蓝光损伤组Caspase-3、Bax及NQO1表达量较正常对照组更高(均P<0.01),N2L中剂量组Bax、Caspase-3表达量较蓝光损伤组显著降低(均P<0.001),而GST、NQO1及Bcl-2显著增加(均P<0.01)。结论:2.5 mg/kg N2L能有效拮抗蓝光对SD大鼠视网膜的损伤作用,有望成为其防治药物。

Electroretinogram and Histopathologic Changes of the Retina after Methanol Intoxication

In order to study the functional and structural alterations of the retina in SD rat model after methanol intoxication,35 rats were divided randomly into five groups administrated with saline,3-day high dose,7-day high dose,3-day low dose and 7-day low dose methanol separately.The retinal function of each group was assessed by flash electroretinogram(F-ERG) 3 and 7 days after methanol poisoning.The microstructure and ultrastructure of the retina were observed at the same time.The high-dose methanol intoxication induced irreversible retinal functional and structural damages 3 days after poisoning,which included prolonged latency and reduced amplitude of the Max-reaction of F-ERG.These injuries were aggravated 7 days after poisoning.Meanwhile,the latency and amplitude of the Cone-reaction of F-ERG were also affected 3 days after poisoning,but there were no further worsening tendency 7 days after poisoning.The retinal histological analysis showed cellular edema,heteromorphy and disarrangement,tissular loosen of the inner nuclear layer and photoreceptors layer.The mitochondrial damage began at the photoreceptors layer and developed further into the inner nuclear layer.The low-dose methanol intoxication only caused transient damage of the retina.Our results showed that the function and structure of the photoreceptor and inner nuclear layer were the primary target of methanol intoxication and that the rod cells were more sensitive to methanol intoxication than the cone cells.The mitochondrial damage developed from outer layer to inner layer of the retina.

褪黑素减轻高糖诱导的大鼠视网膜米勒细胞氧化应激

目的:研究褪黑素对高糖(HG)诱导的大鼠视网膜米勒细胞的保护作用。方法:从新生大鼠视网膜分离培养米勒细胞,用CCK⁃8实验检测含不同浓度葡萄糖的培养液对米勒细胞活性的影响。用褪黑素和高糖培养液分别处理米勒细胞,乳酸脱氢酶(LDH)检测试剂盒检测培养上清中LDH含量,超氧化物歧化酶(SOD)活性检测试剂盒检测米勒细胞中SOD的活性,用反应性活性氧(ROS)检测试剂盒检测米勒细胞中ROS的水平。结果:高糖培养液可导致米勒细胞培LDH释放增多(P<0.05),细胞内SOD活性降低(P<0.05),ROS含量升高(P<0.05);褪黑素可减少高糖培养液导致的LDH释放(P<0.05),并增加米勒细胞中SOD活性及降低ROS的含量(P<0.05)。结论:褪黑素能减轻高糖培养液诱导的大鼠米勒细胞氧化应激损伤。

Long Evans大鼠视网膜缺血再灌注损伤模型的特征及其造模前后谷氨酸的含量

目的观察Long Evans大鼠视网膜缺血再灌注(retinal ischemia reperfusion,RIR)损伤后视网膜的功能、结构及谷氨酸含量的变化,为视网膜的损伤及可能保护机制研究提供参考。方法随机选取30只成年SPF级Long Evans大鼠,对其左眼前房持续60 min灌注高压生理盐水(132 mmHg),构建RIR损伤模型,右侧眼不处理作为自身对照。在造模后1、3、7和14 d,利用闪光全视网膜电图(flash electroretinogram,f-ERG)检测视网膜电生理功能的变化情况;在造模前及造模后3、7、14 d,利用光学相干断层技术(optical coherence tomography,OCT)测量视网膜厚度,眼底成像观察眼底血管的变化情况;于造模后14 d处死大鼠,进行石蜡包埋、苏木精伊红(hematoxylin eosin,HE)染色和缺口末端标记法(TdT-mediated dUTP nick end labeling,TUNEL)荧光染色观察视网膜形态结构、细胞凋亡及分布情况,ELISA检测视网膜谷氨酸的含量。结果与对照眼相比,造模眼从第1天开始视网膜电图b波振幅极显著下降(P<0.01),潜伏期极显著延迟(P<0.01);OCT显示从第3天开始视网膜神经节细胞复合体(retinal ganglion cell complex,GCC)厚度极显著变薄(P<0.01),从第7天开始全层视网膜厚度极显著变薄(P<0.01),且都随着时间延长越来越薄(P<0.05);眼底照片显示视网膜从第3天开始出现明显的缺血,一直到第14天都没有恢复到正常水平;第14天HE染色切片结果显示视网膜萎缩,内层明显变薄,视网膜神经节细胞(ganglion cells,RGCs)减少;TUNEL荧光染色结果显示视网膜各层出现明显的细胞凋亡;ELISA结果显示造模后视网膜谷氨酸含量增加(P<0.05)。结论Long Evans大鼠RIR损伤引起视觉电生理功能严重损伤,视网膜萎缩,尤其GCC厚度减少最明显,且随着时间延长损伤加剧,不可逆转,RGCs凋亡,眼底血管缺血,视网膜谷氨酸含量增加,为视网膜损伤类疾病的研究提供良好的动物模型。

模拟长期失重对白化与非白化大鼠眼部影响的比较

目的 比较白化鼠与非白化鼠在长期模拟失重后眼部结构与功能的改变,完善和补充长期微重力暴露所致眼部损伤疾病动物模型构建方法。方法 将正常雄性Sprague-Dawley(SD)大鼠(白化鼠)和Brown-Norway(BN)大鼠(非白化鼠)各12只随机分为SD正常对照组(N_(S))、BN正常对照组(N_(B))、SD模型组(M_(S))和BN模型组(M_(B)),每组6只,模型组采取30°头低位尾悬吊8周模拟长期失重,在造模结束后利用视网膜电流图(ERG)和视觉诱发电位(VEP)检测大鼠的视觉功能,石蜡切片HE染色观察视网膜形态,同时通过小动物眼底成像系统观察大鼠眼底血管改变情况。结果 在8周尾吊模拟失重后,M_(S)组与N_(S)组相比,暗适应3.0 ERG反应振幅明显下降(P<0.01),VEP的P2波峰时值明显延长(P<0.01),HE染色显示外核层变薄(P<0.01),眼底可见脉络膜血管血流增多,而M_(B)组与N_(B)组相比,视网膜结构与功能均无明显变化。结论 与非白化大鼠相比,白化大鼠在长期模拟失重情况下更容易出现眼部损伤,视网膜色素上皮细胞中黑色素合成相关蛋白可能是研究对抗模拟长期失重导致视网膜退行性变的重要分子。

黑米花青素对大鼠视网膜光化学损伤感光细胞凋亡和Caspase-1表达的影响

目的探讨黑米花青素(black rice anthocyanins,BRACs)对视网膜光化学损伤(retinal photochemical damage,RPD)感光细胞凋亡及Caspase-1表达的影响。方法 60只Sprague-Dawley大鼠随机分为对照组(n=30)和BRACs组(n=30),均饲以基础饲料,12h明/12h暗循环光照。BRACs组按100mg/kg.bw剂量给予BRACs,每天灌胃1次;对照组给予等量生理盐水灌胃1次。15d后,两组动物同时给予(3 000±200)lux强度的白色荧光持续光照0～24h,原位末端标记(TUNEL)法检测视网膜细胞凋亡,逆转录聚合酶链反应(RT-PCR)法和免疫组织化学法检测视网膜内Caspase-1mRNA和蛋白表达水平。结果光照能诱导大鼠视网膜感光细胞凋亡(P<0.05),灌胃给予BRACs能降低光照诱导的感光细胞凋亡(P<0.05);光照后Caspase-1mRNA和蛋白表达增加,BRACs干预后Caspase-1表达显著降低(P<0.05);光照后外核层和内核层出现大量的Caspase-1阳性细胞,而BRACs组阳性细胞数量明显少于对照组。结论 BRACs能抑制RPD中感光细胞的凋亡,防护视网膜光化学损伤,该作用可能与其下调Caspase-1表达相关。

三种方法对大鼠视网膜固定效果的比较研究

目的研究三种不同方法对大鼠视网膜石蜡切片的固定效果。方法根据离体眼球的不同固定方法,将9只SD大鼠随机分为A组(改良固定液固定,n=3)、B组(4%多聚甲醛固定,n=3)和C组(4%多聚甲醛心脏灌注法固定,n=3)。24 h后制作标本进行苏木精-伊红染色,分析比较固定效果。结果 A组固定的眼杯不变形,B组固定的有少许变形,C组固定的基本不变形。显微镜观察显示A组眼球视网膜各层组织结构完整,B组和C组视网膜均有断层、分离和脱片等现象。结论改良固定液固定眼球的方法优于单纯4%多聚甲醛和4%多聚甲醛心脏灌注固定的方法,适用于视网膜石蜡切片的后期研究。

神经生长因子对糖尿病大鼠神经视网膜超微结构的影响

目的:通过观测实验性糖尿病动物的神经视网膜的超微结构变化,从视网膜的神经功能角度来探讨糖尿病视网膜病变的发生机制以及相关药物干预后的影响作用。方法:用链脲佐菌素制作糖尿病大鼠动物模型,分为正常对照组(CON组)、糖尿病对照组(DM组)、糖尿病神经生长因子治疗组(D+N组)。分别于病程3,6,9,12mo取大鼠眼球制备石蜡切片,将视网膜组织行HE染色,并将上述标本制备超薄切片,用透射电镜观察并分析。结果:从病程3mo开始,DM组视网膜血管内皮细胞及周细胞的核变形、线粒体肿胀变性、基底膜增厚。视神经节细胞水肿,胞器减少,线粒体变性。光感受器细胞(视锥、视杆)膜盘间隙扩大,线粒体及核也有病理改变。上述病变随病程延长而逐渐加重。经过NGF治疗后,上述DM改变有所减轻,主要表现在感光细胞外节膜盘间隙较DM组缩小,平行度好转;神经细胞突起水肿减轻,胞器水肿好转。各组DM大鼠的视网膜血管、神经网膜及视神经的糖尿病性病变之间未见明显的先后因果关系。结论:神经生长因子对DM大鼠视网膜形态学上所见的视网膜血管、感光细胞和神经节细胞的超微结构改变有着明确的改善作用。在DR的发病过程中,视网膜血管的病变与视网膜神经组织的病变可能是同时存在、互相促进,共同造成糖尿病视网膜病变和视功能的损害。

早期糖尿病大鼠视网膜蛋白质表达变化的初步研究

目的观察2个月病程大鼠视网膜组织蛋白质变化情况;建立并优化视网膜蛋白质组分析所需要的双向凝胶电泳(2-DE)技术,提高其分辨率及重复性。方法提取视网膜蛋白质进行2-DE,对影响2-DE结果的各种因素进行调整、优化。用PDQUest7.3.2软件分析凝胶图谱以获得差异表达的蛋白点。结果成功获得了视网膜组织的2-DE图谱。比较后得到36个表达差异有统计学意义(P<0.05)的蛋白质点,包括上调5个,下调23个,消失8个。结论2个月病程糖尿病大鼠视网膜已经有蛋白质表达的差异。已建立的视网膜蛋白质2-DE技术将为进一步开展视网膜疾病的蛋白质组学研究奠定基础。

大鼠视网膜细胞间隙连接蛋白Cx43的表达

目的:探讨细胞间隙连接蛋白connexin43(Cx43)在BN大鼠视网膜中的分布特点。方法:以雄性挪威棕色大鼠(Brown Norway)为研究对象,应用免疫组化方法观察Cx43在BN大鼠视网膜中的分布特点。结果:正常视网膜内界膜,视神经纤维层以及神经节细胞层明显表达,神经节细胞表达的主要位置是细胞质和细胞膜,色素上皮细胞全层表达,内外颗粒层及内外层状层无表达。结论:正常视网膜的Cx43主要分布在内界膜,神经纤维层,视网膜节细胞层和视网膜色素上皮层。