Loss of cholinergic and dopaminergic amacrine cells in streptozotocin-diabetic rat and Ins2Akita-diabetic mouse retinas

PURPOSE: To identify amacrine cells that are vulnerable to degeneration during the early stages of diabetes. METHODS: Whole retinas from streptozotocin (STZ)-diabetic rats and Ins2(Akita) mice were fixed in paraformaldehyde. Apoptotic cells in the retina were quantified using terminal dUTP nick-end labeling (TUNEL) and active caspase-3 (CM-1) immunohistochemistry. Immunohistochemical markers for choline acetyltransferase (ChAT) and tyrosine hyroxylase (TH) were also used to quantify populations of amacrine cells in the Ins2Akita mouse retinas. RESULTS: The number of TUNEL-positive nuclei increased from 29+/-4 in controls to 72+/-9 in the STZ-diabetic rat retinas after only 2 weeks of diabetes. In rats, CM-1-immunoreactive (IR) cells were found primarily in the inner nuclear and ganglion cell layers after 2, 8, and 16 weeks of diabetes. At each end point, the number of CM-1-IR cells in the retina was elevated by diabetes. Approximately 2% to 6% of the CM-1-IR cells in the inner nuclear layer (INL) were double-labeled for TH immunoreactivity. After 6 months of diabetes in the Ins2Akita mouse, the morphology of the labeled ChAT-IR and TH-IR amacrine cell somas and dendrites appeared normal. A quantitative analysis revealed a 20% decrease in the number of cholinergic and a 16% decrease in dopaminergic amacrine cells in the diabetic mouse retinas, compared with the nondiabetic control. CONCLUSIONS: Dopaminergic and cholinergic amacrine cells are lost during the early stages of retinal neuropathy in diabetes. Loss of these neurons may play a critical role in the development of visual deficits in diabetes.     

Polyol pathway activity in streptozotocin-diabetic rat lens

We have examined polyol pathway kinetics in the lenses of rats made diabetic with streptozotocin. At up to 11 days after diabetes induction, the lenses were isolated and subjected to 'pulse-chase' studies: the lenses were incubated with [13C]glucose and lens metabolism followed by [13C]nuclear magnetic resonance (NMR) spectroscopy. Proton NMR spectroscopy was also performed to measure the hexose monophosphate shunt (HMPS) activity. The results showed that (1) the activity of aldose reductase increased initially and decreased after 11 days of diabetes; (2) the fructose pool increased initially but started to decline after 3 days; (3) the HMPS activity increased nearly 40% immediately after diabetes induction; and (4) the turnover rates of glucose, alpha-glycerophosphate (GP), lactate, sorbitol, and fructose were 80.8 +/- 2.6, 10.1 +/- 1.4, 47.7 +/- 3.7, 7.9 +/- 0.9 and 5.2 +/- 2.2 nmol hr-1 lens-1 (34 mg wet weight lens-1), respectively. Up to 35% of lactate appeared to derive from the polyol pathway. Further, GP was rapidly metabolized, although its fate is currently unknown. These results reveal a far more complex pattern of glucose metabolism in the diabetic lens than that in lenses incubated in high glucose.     

Nitric oxide synthase expression in ischemic rat retinas

PURPOSE: To investigate the expression of nitric oxide synthase (NOS) in the ischemic retina. METHODS: Retinal ischemia was induced in rats by bilateral common carotid artery occlusion (BCCAO) for various lengths of time. Using the retina after BCCAO, expression of neuronal NOS (nNOS) and inducible NOS (iNOS) and identification of their positive cells were studied by histological and immunohistochemical examinations. RESULTS: Histological examinations revealed significant reduction in the thickness of the inner plexiform layer and the outer plexiform layer of the retina. Expression of nNOS was detected in retinal ganglion cells, amacrine cells, and Müller cells after BCCAO. The expression of nNOS and iNOS detected in Müller cells became stronger and persisted long after BCCAO. CONCLUSIONS: In the ischemic retina, Müller cells and retinal ganglion cells expressed nNOS and iNOS. These phenomena may be involved in the ischemic damage to the retina.     

Electrophysiologic characteristics of human and rat retinas in vitro

To promote studies on the human retina, we investigated the survival of function in postmortem specimens. Visual pigment has been regenerated in normal human retinas, 5 to 58 hours postmorten, by exposure to retinal isomers in the dark. Levels from 0.1 to 0.41 nmol/ mg protein were reached. Photoresponses were obtained in 9 of 13 retinas: P III maximum amplitudes ranged from 20–398 V and thresholds, taking the criterion amplitude as 3 V, ranged from 8.8–1340 quanta/m ². In three cases, the b-wave was also seen. The P III amplitude vs. log intensity curves gave values of n between 0.6 and 1.0, and (the stimulus intensity for a half maximal response) between 132–3700 quanta/m². Recovery of sensitivity did not always correspond to that of maximum response.     

Immunohistochemical detection of blood-retinal barrier breakdown in streptozotocin-diabetic rat

Immunohistochemical staining for albumin (69 kDa) and fibrinogen (340 kDa), as markers of blood-retinal barrier (BRB) breakdown of the retinas of streptozotocin-treated diabetic rats, was performed. The number of rats with BRB breakdown was three of nine at 6 months, four of seven at 12 months, and three of three at 18 months. Extravasation of albumin from the retinal vessels was detected in the retinas of rats maintained for 6, 12, and 18 months, while extravasation of fibrinogen was detected only in the retinas of rats maintained for 18 months. These findings suggested that the duration of diabetes has an influence on BRB breakdown and that each substance in the blood starts to permeate the vascular wall individually.     

Evaluation of indocyanine green toxicity to rat retinas

PURPOSE: To investigate the toxicity of indocyanine green (ICG) on retinal cells using cultured retinal pigment epithelium (RPE) cells and the effects of intravitreous injection of ICG into rat eyes. METHODS: Cultured RPE cells were exposed to various concentrations of ICG for 2 min, a viability assay was performed 1 day after exposure. For an in vivo study, 5 microl of ICG (5 or 25 mg/ml) were injected into the vitreous cavity of rat eyes, which were examined 1, 3 and 7 days after the injection by histological and glutamine synthetase (GS) immunohistological evaluation. RESULTS: Viabilities of RPE cells were decreased dependent on the ICG dose. In the histological evaluation, we observed differences of effects of ICG between the central retinal area and the peripheral area. ICG injection caused degeneration of all retinal layers in the central retinal area. GS immunoreactivities decreased by ICG injection, which corresponded to an area of severe destruction. CONCLUSION: A high concentration of ICG may cause toxic effects on retinal cells. Mueller cell dysfunction may play some role in the retinal toxicity caused by ICG.     

Glutathione peroxidase induced in rat retinas to counteract photic injury

PURPOSE: To examine the hypothesis that glutathione peroxidase (GPX) is induced at different time points after retinal exposure to light and localizes in different retinal cells. METHODS: The rats were kept in cyclic light for 2 weeks before the experiments. The animals were maintained in 12-hour light-dark cycles, before and after exposure to intense white fluorescent light, for as long as 24 hours and then returned to cyclic light. Expression of GPX was measured by immunohistocytochemistry and Western and Northern blot analyses. Light-induced retinal damage was determined by the thickness of the outer nuclear layer (ONL) thickness in relation to total retinal thickness. RESULTS: GPX labeling did not appear in the photoreceptor inner segments, and slight labeling was observed in the photoreceptor outer segments or the retinal pigment epithelial (RPE) cells in the normal retina kept in cyclic light. In retinal specimens maintained in light for 12 and 24 hours, GPX labeling was induced in the photoreceptor outer segments and RPE cells. High expression of GPX in the RPE was sustained until day 7 after challenge. In contrast, GPX expression in the photoreceptor outer segments decreased on day 1 and disappeared on days 3 and 7 after exposure. Intense GPX labeling was seen from the internal limiting membrane to the ganglion cell layer. GPX labeling was constantly localized in both high-intensity white light and cyclic conditions, suggesting no induction of GPX in those areas. In addition, GPX labeling was apparent at the posterior retinal pole but not at the peripheral retina. We observed marked upregulation of GPX mRNA in rats kept in high-intensity white light. One, 3, and 7 days after exposure to high-intensity white light, there was a significant difference (P < 0.0001) between the control and experimental groups in the ratio of the outer nuclear layer thickness to the entire retina. CONCLUSIONS: GPX was induced at different time points after exposure to high-intensity white light and localized in different retinal cells. Changes in expression of GPX after exposure to light may be related to the difference in susceptibility of the retina to damage by light.     

Structural and biochemical changes in vitamin A--deficient rat retinas.

The levels of rhodopsin and opsin were investigated in relation to the maintenance of retinal structure in retinas of vitamin A--deficient rats in low levels of cyclic illumination (1.5 to 2 foot-candles). Rhodopsin levels decreased in the deficient retinas to approximately 20% of control at 9 weeks, and this level was retained through 39 weeks on the deficient diet. Opsin levels decreased at a slower rate but reached about 20% of control levels at 32 weeks. Despite the decrease in rhodopsin levels, obvious deterioration of disc structure was not observed until 16 weeks of deficiency, when opsin levels had already decreased to 60% to 70% of control. The structural disruption of photoreceptor outer segments was localized initially in discs of the distal third. Rod cell degeneration preceded cone cell degeneration in vitamin A--deficient retinas. Most of the rods and cones persisted in the posterior retina at 23 weeks on the deficient diet; however, by 40 weeks, only 11% of the rod nuclei remained. In contrast, about 63% of the cone nuclei were present at 40 weeks of deficiency. The photoreceptor cells were affected by the deficiency to a greater extent in the inferior hemisphere than in the superior hemisphere of the eye.     

Evaluation of toxicity due to vital stains in isolated rat retinas

PURPOSE: We investigated whether artificial aqueous humours are adequate incubation media compared with artificial cerebrospinal fluid (aCSF), and evaluated the retinal toxicity of two vital stains--trypan blue (TB) and indocyanine green (ICG)--and triamcinolone acetonide (TA) using isolated rat retinas incubated in artificial aqueous humours. METHODS: In experiment 1, retinal segments were isolated and incubated in aCSF, BSS plus, Opeguard Neo Kit, or phosphate-buffered saline (PBS). In experiment 2, retinal tissues were exposed to one of the agents and incubated in BSS plus. Retinal damage was assessed by morphological examination and biochemical assay, which measured lactate dehydrogenase (LDH) in the medium once every hour. RESULTS: In experiment 1, BSS plus was confirmed as a suitable incubation medium. In experiment 2, there were no significant changes in the retinas exposed to TB or TA. Tissues exposed to ICG showed damage in every retinal layer and significantly higher release of LDH. CONCLUSION: Exposure to ICG caused retinal damage in isolated rat retina tissue in our experimental model (in vitro).     

Effects of moderate-intensity light on vitamin A-deficient rat retinas

The effects of moderate-intensity light (150 to 200 ft-cd) on retinal structure were compared between retinol-adequate and retinol-deficient rats after 1 to 6 days of light exposure during the 12 hr light phase of the cycle. Both damage to the outer segments and loss of photoreceptor cells were accelerated in retinol-adequate rats. Outer segments in retinas of retinol-adequate rats showed an abnormal staining pattern and disruption of disc structure in the distal portion about 2 days before those of retinol-deficient rats. After 4 days of exposure 24% of the photoreceptor cells had degenerated in the retinol-adequate retinas, but only 6% in the retinol-deficient retinas. By 6 days 65% and 41% of the photoreceptors had degenerated in the retinol-adequate and retinol-deficient retinas, respectively. Thus light exposure induced more rapid degeneration of photoreceptor cells in rats receiving adequate retinol than in those deficient in this vitamin.     

Prediction and verification of miRNA expression in human and rat retinas

PURPOSE: MicroRNAs (miRNAs) play a global role in regulating gene expression and have important tissue-specific functions. Little is known about their role in the retina. The purpose of this study was to establish the retinal expression of those miRNAs predicted to target genes involved in vision. METHODS: miRNAs potentially targeting important "retinal" genes, as defined by expression pattern and implication in disease, were predicted using a published algorithm (TargetScan; Envisioneering Medical Technologies, St. Louis, MO). The presence of candidate miRNAs in human and rat retinal RNA was assessed by RT-PCR. cDNA levels for each miRNA were determined by quantitative PCR. The ability to discriminate between miRNAs varying by a single nucleotide was assessed. The activity of miR-124 and miR-29 against predicted target sites in Rdh10 and Impdh1 was tested by cotransfection of miRNA mimics and luciferase reporter plasmids. RESULTS: Sixty-seven miRNAs were predicted to target one or more of the 320 retinal genes listed herein. All 11 candidate miRNAs tested were expressed in the retina, including miR-7, miR-124, miR135a, and miR135b. Relative levels of individual miRNAs were similar between rats and humans. The Rdh10 3'UTR, which contains a predicted miR-124 target site, mediated the inhibition of luciferase activity by miR-124 mimics in cell culture. CONCLUSIONS: Many miRNAs likely to regulate genes important for retinal function are present in the retina. Conservation of miRNA retinal expression patterns from rats to humans supports evidence from other tissues that disruption of miRNAs is a likely cause of a range of visual abnormalities.     

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。     

血小板反应蛋白-1在糖尿病大鼠早期视网膜中的表达及意义

目的 检测血小板反应蛋白-1(thrombospondin-1,TSP-1)在正常及糖尿病大鼠视网膜组织中的表达并探讨其在早期糖尿病视网膜病变中的作用.方法 36只雄性Wistar大鼠随机分为正常对照组6只,实验组30只.实验组大鼠采用链脲佐菌素诱导大鼠糖尿病模型,其中3只于造模后1个月血糖恢复正常作为血糖恢复组,余27只随机分成3组:糖尿病1月组(D1组)、糖尿病2月组(D2组)和糖尿病3月组(D3组),每组各9只大鼠,HE染色观察各组视网膜病理变化,免疫组织化学方法检测TSP-1蛋白在各组大鼠视网膜中的表达.结果HE染色可见血糖恢复组、D1组和D2组视网膜各层排列与正常对照组无明显差异,而D3组表现为视网膜内血管不均匀并膨胀,视网膜神经节细胞肿胀,排列紊乱,内界膜增厚,内丛状层有肿胀.各观察时间点血糖恢复组大鼠视网膜TSP-1蛋白阳性着色的积分光密度值与正常对照组差异均无统计学意义(均为P＞0.05);DI组、D2组和D3组大鼠视网膜TSP-1蛋白阳性着色的积分光密度值分别为21.50 3.02、19.58±2.70和15.93±2.41,均较正常对照组1个月、2个月和3个月时的26.34±3.40、25.38±3.20和25.91 ±3.31低,差异均有统计学意义(均为P＜0.05).结论 TSP-1蛋白在实验性糖尿病大鼠视网膜中的表达明显降低并与病程有关,提示TSP-1表达不足可能是糖尿病视网膜病变的原因之一.     

辛伐他汀对糖尿病大鼠视网膜血管内皮钙黏蛋白表达的影响

目的观察链脲佐菌素（STZ）诱导的糖尿病视网膜血管内皮钙黏蛋白（VE-cadherin）的表达及辛伐他汀对VE-cadherin表达的影响,探讨他汀类药物可能的非调脂性治疗作用。方法尾静脉注射STZ法建立糖尿病大鼠模型,造模成功48只。成模次日起,24只大鼠每日给予辛伐他汀20 mg/kg灌胃为辛伐他汀组,24只等量生理盐水灌胃为糖尿病组;另选24只大鼠作为正常对照组。分别在造模后2、5、8周各组取8只大鼠采用伊凡思蓝（EB）法检测视网膜的渗透性,免疫组织化学和Western blot法检测各组大鼠视网膜VE-cadherin的表达情况。结果与正常对照组比较,辛伐他汀组和糖尿病组大鼠体重明显下降,差异均有统计学意义（P〈0.01）,血糖显著上升,差异均有统计学意义（P〈0.01）。与糖尿病组大鼠比较,各时间点辛伐他汀组大鼠体重均明显上升,而血糖均显著下降（P〈0.05）。免疫组织化学分析证实,VE-cadherin在糖尿病组大鼠视网膜血管呈黄褐色阳性表达。Western blot分析表明随着糖尿病视网膜病变（DR）的发生发展,糖尿病组视网膜血管表达VE-cadherin的量显著减少,与正常对照组和辛伐他汀组比较,差异均有统计学意义（P〈0.05）。糖尿病组和辛伐他汀组EB渗透量明显高于正常对照组（P〈0.01）,辛伐他汀组低于糖尿病组（P〈0.05）。结论STZ诱导的糖尿病大鼠视网膜血管中VE-cadherin表达量减少,辛伐他汀可改善这种改变,提示辛伐他汀对DR有预防和治疗作用。     

Long-term glial reactivity in rat retinas ipsilateral and contralateral to experimental glaucoma

Although glaucoma is known to alter glial reactivity, the long-term effect of elevated intraocular pressure (IOP) on glial change has not been fully elucidated. This study aimed to examine how chronically elevated IOP induced by episcleral vein cauterization (EVC) in unilateral eyes affect reactivities of astrocytes and Müller cells of rats in the treated as well as contralateral eyes over time. EVC in unilateral eyes of Sprague-Dawley rats were performed to produce chronically elevated IOP. Flat mounted retina preparations were made at several points until 6 months, which were subjected to immunostaining for glial fibrillary acidic protein (GFAP). Retinal homogenates were one- or two-dimensionally electrophoresed, followed by GFAP immunoblotting. EVC significantly increased IOPs up to 27.8 from 13.1 mmHg, which gradually decreased over time. In flat mounted retinas, astrocytes lost but Müller cells gained GFAP immunoreactivity at 3 days after cauterization. The glial changes were partially reversed over time but last even after IOP normalization. In the contralateral eyes, similar glial changes gradually appeared at 1 month after EVC and thereafter. Immunoblotting demonstrated not only molecular size shifts but also alteration of isoelectric focusing of GFAP both in treated and contralateral retina as compared with age-matched control retina. EVC led to opposite reactions in astrocytes and Müller cells in terms of GFAP immunoreactivity. Late-onset glial reactivity also occurred in the contralateral retina.     

Taurine fluxes in isolated cat and rat retinas: Effects of illumination

Taurine is taken up by isolated cat retinas incubated in an oxygenated medium in the light or in the dark. Onset and cessation of illumination are associated with a prompt transient release followed by reuptake of taurine; in contrast, glycine is gradually released only with onset of illumination. The uptake of taurine and the light evoked release of taurine followed by reuptake are inhibited by reduction in temperature, iodoacetate, ouabain, and absence of glucose. Similar light-evoked taurine fluxes are observed in the isolated retinas from normal rats and 30-day-old RCS rats but cannot be demonstrated in the photoreceptorless retinas from 180-day-old RCS rats. These findings support the idea that the effects of illumination on taurine fluxes depend on the viability of photoreceptor cells.     

Immunocytochemical localization of interphotoreceptor retinoid-binding protein in developing normal and RCS rat retinas

Interphotoreceptor retinoid-binding protein (IRBP) was localized immunocytochemically in developing normal and RCS rat retinas. IRBP was present in normal and RCS neural retinas on the day after birth (postnatal day 2, P2) to P8 in the space between the neuroblastic layer and the retinal pigment epithelium (RPE). The presence of IRBP prior to the development of outer segments (OS) suggests that OS formation is not linked temporally with IRBP secretion. On P10, staining was confined to the interphotoreceptor space with an intense band of label adjacent to the RPE. This staining pattern persisted in normal rats throughout development and until P18 in RCS rats. On P18, anti-IRBP staining in the RCS was spread evenly throughout the OS layer with no intense band of label adjacent to the RPE and after P18, there was decreased staining with anti-IRBP. On P45 and later, no staining of the RCS retina was found with anti-IRBP. Immunoblots of normal and RCS retinas corroborated the results from immunocytochemical staining. These findings suggest that IRBP may be synthesized in the photoreceptors, but is not abnormal in amount or distribution prior to onset of retinal degeneration in the RCS rat.     

Glucose uptake by normal and dystrophic rat retinas and ciliary bodies

The accumulation of [¹⁴C]d-glucose, [¹⁴C]3-O-methyl-d-glucose, [³H]thio-d-glucose, and [³H]2-deoxy-d-glucose was investigated in retinas and ciliary bodies isolated from normal Long-Evans rats and rats afflicted with an inherited retinal dystrophy. Rats employed in this study were taken from four age groups, ranging from 15 to 60 days.The accumulation of 2-deoxy-d-glucose and d-glucose in the normal retina decreased between 15 and 60 days. In the dystrophic retina, 3-O-methyl-d-glucose, 2-deoxy-d-glucose, and d-glucose levels decreased significantly during this time period. The decrease in accumulation of d-glucose and 2-deoxy-d-glucose in the dystrophic retina occurred at a much more rapid rate than in the normal retina, the greatest decrease occurring between 15 and 27 days.Acetazolamide, phloretin, and phloridzin significantly increased the accumulation of 3-O-methyl-d-glucose in the retina. Acetazolamide and phloretin significantly decreased the accumulation of thio-d-glucose, while acetazolamide and phloridzin increased the accumulation of d-glucose and 2-deoxy-d-glucose.The accumulation of 2-deoxy-d-glucose and d-glucose decreased significantly in normal ciliary bodies, between 15 and 60 days of age. In the dystrophic ciliary bodies, 3-O-methyl-d-glucose, d-glucose, and 2-deoxy-d-glucose intracellular levels decreased during this time period. The decline in 2-deoxy-d-glucose was much more precipitous between 15 and 27 days in the dystrophic ciliary body than in the normal ciliary body. The percent of d-glucose which was phosphorylated decreased significantly in both normal and dystrophic ciliary bodies, the decrease in the dystrophic tissue occurring earlier than in the normal tissue.Phloridzin and acetazolamide significantly increased the accumulation of thio-d-glucose and d-glucose, while phloretin increased the accumulation of 3-O-methyl-d-glucose and d-glucose in the 60-day normal ciliary body.     

Taurine levels, uptake and synthesizing enzyme activities in degenerated rat retinas.

Taurine levels, cysteine oxidase and cysteine sulfinate decarboxylase activities, [³⁵S]-taurine uptake in vitro and [³⁵S]taurine accumulation in vivo were studied in the retinas of rats in which the inner or the outer cell layers were degenerated by treatment with monosodium glutamate or with iodoacetate-malate respectively.Monosodium glutamate treatment significantly reduced the cysteine oxidase activity to 12% of control and the cysteine sulfinate decarboxylase activity to 34% of control. Taurine levels were unaffected. Taurine accumulation was slightly decreased and taurine uptake in vitro was unchanged by MSG treatment.Iodoacetate-malate treatment decreased taurine levels to 75% of control and the activities of cysteine oxidase and cysteine sulfinate decarboxylase to 73 and 60% of control, respectively. Iodoacetate-malate treatment decreased taurine accumulation in vivo to only 12% of control; taurine uptake in vitro was also reduced to approximately 50% of control.