Diosmin protects rat retina from ischemia/reperfusion injury

Abstract Objective: Diosmin, a natural flavone glycoside, possesses antioxidant activity and has been used to alleviate ischemia/reperfusion (I/R) injury. The aim of this study was to clarify whether the administration of diosmin has a protective effect against I/R injury induced using the high intraocular pressure (IOP) model in rat retina, and to determine the possible antioxidant mechanisms involved. Methods: Retinal I/R injury was induced in the rats by elevating the IOP to 110?mmHg for 60?min. Diosmin (100?mg/kg) or vehicle solution was administered intragastrically 30?min before the onset of ischemia and then daily after I/R injury until the animals were sacrificed. The levels of malondialdehyde (MDA) and the activities of total-superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) in the retinal tissues were determined 24?h after I/R injury. At 7 days post-I/R injury, electroretinograms (ERGs) were recorded, and the density of surviving retinal ganglion cells (RGCs) was estimated by counting retrograde tracer-labeled cells in whole-mounted retinas. Retinal histological changes were also examined and quantified using light microscopy. Results: Diosmin significantly decreased the MDA levels and increased the activities of T-SOD, GSH-Px, and CAT in the retina of rats compared with the ischemia group (P<0.05), and suppressed the I/R-induced reduction in the a- and b-wave amplitudes of the ERG (P<0.05). The thickness of the entire retina, inner nuclear layer, inner plexiform layer, and outer retinal layer and the number of cells in the ganglion cell layer were significantly less after I/R injury (P<0.05), and diosmin remarkably ameliorated these changes on retinal morphology. Diosmin also attenuated the I/R-induced loss of RGCs of the rat retina (P<0.05). Conclusion: Diosmin protected the retina from I/R injury, possibly via a mechanism involving the regulation of oxidative parameters.     

Interleukin-6 in retinal ischemia reperfusion injury in rats

PURPOSE: To study the role of interleukin (IL)-6 after retinal ischemia-reperfusion (I/R) injury in rats. METHODS: Intraocular pressure of adult male Lewis albino rats was raised to create retinal ischemia for 1 hour. Retinal reperfusion was reestablished, and the animals were killed at various time points after the injury. Their eyes were enucleated and processed for immunohistochemistry to detect IL-6 and ED-1 (a marker of microglial/phagocytic cells), enzyme-linked immunosorbent assay (ELISA) of IL-6 protein, and semiquantitative real-time RT-PCR for IL-6 mRNA. The neuroprotective effect of IL-6 was evaluated by giving intravitreal injections of 150 or 300 ng rat recombinant IL-6 to eyes immediately after I/R injury and counting cresyl violet-stained retinal ganglion cell layer cells (RGCLCs) and fluorochrome-labeled retinal ganglion cells (RGCs) on flat preparations of retinas at 7 days. RESULTS: IL-6-positive cells appeared after I/R injury in the inner plexiform layer (IPL) and the inner nuclear layer (INL). Their numbers were significantly higher 18 hours after the injury, and most of these cells were also ED-1 positive. ELISA showed noticeable increases in endogenous retinal IL-6 protein levels 8 hours after I/R injury. Semiquantitative real-time RT-PCR showed significant increases in endogenous retinal IL-6 mRNA levels between 2 and 18 hours. Exogenously added IL-6 prevented between 50% and 70% of RGC loss after I/R injury. CONCLUSIONS: IL-6 is upregulated after retinal I/R injury, and its expression by microglia/phagocytic cells may protect RGC layer neurons from I/R injury. Exogenously added IL-6 protects the inner retina after I/R injury.     

Gap Junctional Coupling Between Retinal Astrocytes Exacerbates Neuronal Damage in Ischemia-Reperfusion Injury

PurposeRetinal astrocytes abundantly express connexin 43 (Cx43), a transmembrane protein that forms gap junction (GJ) channels and unopposed hemichannels. While it is well established that Cx43 is upregulated in retinal injuries, it is unclear whether astrocytic Cx43 plays a role in retinal ganglion cell (RGC) loss associated with injury. Here, we investigated the effect of astrocyte-specific deletion of Cx43 (Cx43KO) and channel inhibitors on RGC loss in retinal ischemia/reperfusion (I/R) injury and assessed changes in expression and GJ channel and hemichannel function that occur in I/R injury. The effect of Cx43 deletion on neural function in the uninjured retina was also assessed.MethodsCx43 expression, astrocyte density and morphology, and RGC death in wild-type and Cx43KO mice after I/R injury were determined using immunohistochemistry and Western blotting. Visual function was assessed using ERG recordings. GJ coupling and hemichannel activity were evaluated using tracer coupling and uptake studies, respectively.ResultsLoss of RGCs in I/R injury was accompanied by an increase of Cx43 expression in astrocytes. Functional studies indicated that I/R injury augmented astrocytic GJ coupling but not Cx43 hemichannel activity. Importantly, deletion of astrocytic Cx43 improved neuronal survival in acute ischemia but did not affect RGC function in the absence of injury. In support, pharmacologic inhibition of GJ coupling provided neuroprotection in I/R injury.ConclusionsThe increase in Cx43 expression and GJ coupling during acute I/R injury exacerbates RGC loss. Inhibition of astrocytic Cx43 channels might represent a useful strategy to promote RGC survival in pathologic conditions.     

Retinal ischemia and reperfusion causes capillary degeneration: similarities to diabetes

PURPOSE: Retinal neurons and vasculature interact with each other under normal conditions, and occlusion of the retinal vasculature can result in damage to retinal neurons. Whether damage to the neural retina will damage the vasculature, however, is less clear. This study was conducted to explore the relationship between vascular and nonvascular cells of the retina. The response of the retinal vasculature to an injury (ischemia and reperfusion; I/R) that is known to cause neuronal degeneration was studied. METHODS: I/R injury to the retinas was induced in Lewis rats and C57BL/6J mice by elevating intraocular pressure (IOP), and reperfusion was established immediately afterward. Some rats were pretreated with aminoguanidine (AMG, 50 mg/Kg BW in drinking water) before the procedure. Poly(ADP-ribose) polymerase (PARP) activity and expression of inducible nitric oxide synthase (iNOS), and cycloxygenase-2 (COX-2) were measured by Western blot analysis, and levels of TNF-alpha and intercellular adhesion molecule (ICAM)-1 mRNA were measured by qPCR at 2 and 7 days after the procedure. Also at 2 and 7 days after the I/R injury, apoptosis of retinal neural cells (demonstrated by TUNEL assay), density of cells in the ganglion cell layer, and thickness of retinas were quantitated, and the number of TUNEL-positive capillary cells and degenerated capillaries were assessed. Retinal neurodegeneration and capillary degeneration were also examined in C57BL/6J mice 2, 5, 8, and 14 days after I/R injury. RESULTS: As expected, loss of cells in the retinal ganglion cell layer was apparent 2 days after I/R injury in the rat and mouse models. In contrast, the retinal vasculature had essentially no pathology at this time in either model. Surprisingly, the number of degenerated capillaries increased greatly by 7 to 8 days after the injury. Administration of aminoguanidine significantly inhibited the I/R-induced capillary degeneration as well as neurodegeneration in the rat model. Retinal I/R caused increased PARP activity (detected by poly(ADP-ribosy)lated proteins), as well as upregulation of iNOS, COX-2, TNF-alpha, and ICAM-1 levels in rats, consistent with an inflammatory process. CONCLUSIONS: Capillary degeneration is an unrecognized component of acutely elevated IOP and develops only after neurodegeneration is severe. Thus, this finding raises the possibility that damage to the neural retina contributes to capillary degeneration. Aminoguanidine, a nonspecific inhibitor of iNOS, inhibited I/R-induced degeneration of both neuronal and vascular cells of the retina. The model of retinal ischemia and reperfusion will be a useful tool for investigating the relationship between neuronal damage and vascular damage in glaucoma and other diseases such as diabetic retinopathy.     

金雀异黄酮对大鼠视网膜缺血-再灌注损伤的保护作用

目的　探讨金雀异黄酮（GEN）对大鼠视网膜缺血-再灌注（I/R）损伤的保护作用。方法　选取30只健康SPF级大鼠，按照随机数字表法将其分为假手术组、I/R组和I/R+GEN组，每组各10只。I/R组和I/R+GEN组大鼠采用前房灌注生理盐水升高眼压法制备视网膜I/R损伤大鼠模型，I/R组大鼠术后每天给予注射生理盐水（1 mL·kg^-1·d^-1）；I/R+GEN组大鼠术后每天给予注射GEN （40 mL·kg^-1·d^-1）；假手术组大鼠行相应假手术后每天予以注射生理盐水（1 mL·kg^-1·d^-1），连续7 d后，颈椎脱臼法处死各组大鼠，取眼部组织。HE染色观察各组大鼠视网膜的形态以及内丛状层（IPL）、内核层（INL）和神经节细胞层（GCL）厚度；免疫荧光分析观察各组大鼠视网膜神经节细胞（RGC）的存活率；TUNEL染色检测各组大鼠视网膜细胞凋亡水平；检测各组大鼠视网膜组织中过氧化氢酶（CAT）、丙二醛(MDA)及超氧化物歧化酶(SOD)水平以反映视网膜氧化应激状态；Western blot分析三组大鼠视网膜中NLRP3、ASC、Caspase-1的蛋白表达。结果　I/R组、I/R+GEN组和假手术组大鼠视网膜总厚度分别为(114.37±7.32）μm、（155.31±6.83）μm和（178.98±13.65）μm（t=14.284，P<0.01），I/R组低于假手术组和I/R+GEN组，差异均有统计学意义（均为P<0.01）。I/R组大鼠视网膜IPL、INL和GCL厚度分别为（18.95±5.06）μm、（17.62±4.69）μm和（19.03±4.74）μm，I/R+GEN组大鼠视网膜IPL、INL和GCL厚度分别为（20.69±8.13）μm、（25.74±6.78）μm和（26.71±7.85）μm，假手术组大鼠视网膜IPL、INL和GCL厚度分别为（11.73±3.15）μm、（11.97±3.56）μm和（12.59±3.24）μm（均为P<0.05），I/R组与假手术组和I/R+GEN组三个指标比较差异均有统计学意义（均为P<0.01）。I/R组、I/R+GEN组和假手术组大鼠视网膜中RGC相对存活率分别为（26.87±3.12）%、（73.46±7.80）% 和（100.00±5.64）%（t=16.825，P<0.01），I/R组RGC相对存活率低于假手术组和I/R+GEN组，差异均有统计学意义（均为P<0.01）。I/R组、I/R+GEN组和假手术组大鼠视网膜中CAT、MDA、SOD含量总体比较差异均有统计学意义（均为P<0.01），I/R组与I/R+GEN组和假手术组三个指标比较差异均有统计学意义（均为P<0.05）。Western blot检测结果显示，三组大鼠视网膜中NLRP3、ASC、Caspase-1的蛋白表达总体比较差异均有统计学意义（均为P<0.01），组间两两比较差异均有统计学意义（均为P<0.05）。结论　GEN可通过提高视网膜细胞抗炎和抗氧化能力抑制视网膜细胞凋亡，从而起到对大鼠视网膜I/R损伤的保护作用。     

Neuroprotective Effects of Granulocyte Colony-Stimulating Factor on Ischemia-Reperfusion Injury of the Retina

Purpose: It has been reported that granulocyte colony-stimulating factor (G-CSF) provides neuroprotection in models in which neuronal cell death is induced. This research was designed to investigate the effects of G-CSF on neurodegeneration of the inner retinal layer in a rat model of ischemic reperfusion (I/R) injury. Materials and Methods: Retinal ischemia was induced by increasing the intraocular pressure to 110 mm Hg for 45 min in the left eyes of the rats. A sham operation was carried out on the right eyes. G-CSF (100 μg/kg/day in 0.3 ml saline) or the same volume of saline was intraperitoneally injected just before the operation and continued for 4 consecutive days (a total of 5 consecutive days). Morphological examinations, including the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, were performed 7 days after I/R induction. The expression of phosphorylated AKT in the retina was examined by Western blot analysis and immunohistochemistry. Results: Cell loss in the ganglion cell layer was more significantly reduced in the I/R-induced eyes of the G-CSF-injected rats than in the I/R-induced eyes of the saline-injected rats (20.3 vs. 6.6%). The inner retinal thickness ratios, such as the inner plexiform layer to the inner limiting membrane/outer nuclear layer and the inner nuclear layer/outer nuclear layer, were significantly better preserved in the I/R-induced eyes of the G-CSF-injected rats than in the I/R-induced eyes of the saline-injected rats. TUNEL assays showed fewer apoptotic cells in the retinal sections of the I/R-induced eyes of the G-CSF-injected rats. The phosphorylation of AKT (p-AKT/AKT) was upregulated in the retinas of the I/R-induced eyes of the G-CSF-injected rats. Conclusion: Our results demonstrated that systemic injection of G-CSF can protect retinal ganglion cells and inner retinal layers from I/R injury. The effects could be associated with the activation of AKT.     

经瞳孔温热疗法对急性高眼压大鼠视网膜神经节细胞的保护作用

目的研究低阈值经瞳孔温热疗法（TTT）对急性高眼压大鼠视网膜神经节细胞（RGC）是否具有保护作用。设计实验研究。研究对象BN大鼠。方法采用810nm二极管激光机对10只大鼠视网膜进行热刺激，照射光斑1．2mm，能量50mW，照射时间20s，干预后3d光镜下观察视网膜形态结构的改变，免疫组化方法检测HSP70、HSP27在视网膜组织表达。采用上述激光参数，照射视网膜后3d，制作急性高眼压模型（TTT＋I/R组，n=10），采用TUNEL法检测RGC层细胞凋亡数量，及计数高倍镜下RGC层细胞数，与未干预的急性高眼压模型组（I/R组，n=10）、单纯TTT干预组（TTT组）及正常对照组（n=6）进行比较。主要指标免疫组化染色RGC细胞数及RGC层细胞凋亡数。结果采用低阈值TTT可诱导BN大鼠视网膜神经节细胞HSP70及HSP27表达，且光镜下未出现明显视网膜脉络膜形态的改变。TTT＋I／R组RGC层细胞凋亡数量明显少于I／R组（P=0．048），且前者RGC层细胞数量明显多于后者（辟0．016）；TTT组与正常对照组比较RGC层细胞凋亡数量无显著性差异（P=0．882），但RGC层细胞数明显少于正常对照组（P=0．001）。结论低阈值TTT可诱导BN大鼠视网膜HSP70、HSP27表达，并在急性高眼压损伤下对大鼠RGC凋亡具有抑制作用。（眼科，2007,16：48—51）     

Quantitative confocal imaging of the retinal microvasculature in the human retina

Purpose. We investigated quantitatively the distribution of blood vessels in different neural layers of the human retina. Methods. A total of 16 human donor eyes was perfusion-fixed and labeled for endothelial f-actin. Retinal eccentricity located 3 mm superior to the optic disk was studied using confocal scanning laser microscopy. Immunohistochemical methods applied to whole-mount and transverse sections were used to colocalize capillary networks with neuronal elements. Capillary morphometry, diameter, and density measurements were compared among networks. Results. Four different capillary networks were identified and quantified in the following regions: Nerve fiber layer (NFL), retinal ganglion cell (RGC) layer, border of the inner plexiform layer (IPL) and superficial boundary of the inner nuclear layer (INL), and boundary of the deep INL and outer plexiform layer. The innermost and outermost capillary networks demonstrated a laminar configuration, while IPL and deep INL networks displayed a complex three-dimensional configuration. Capillary diameter in RGC and IPL networks were significantly less than in other networks. Capillary density was greatest in the RGC network (26.74%), and was significantly greater than in the NFL (13.69%), IPL (11.28%), and deep INL (16.12%) networks. Conclusions. The unique metabolic demands of neuronal sub-compartments may influence the morphometric features of regional capillary networks. Differences in capillary diameter and density between networks may have important correlations with neuronal function in the human retina. These findings may be important for understanding pathogenic mechanisms in retinal vascular disease.     

The effect of thalidomide on vascular endothelial growth factor and tumor necrosis factor-α levels in retinal ischemia/reperfusion injury

Background To evaluate the effects of thalidomide treatment on the temporal course of TNF-α, VEGF production and the histopathological changes in ischemia/reperfusion (I/R) injured guinea pigs retina. Methods Control, ischemia, and thalidomide/ischemia groups including seven animals each were formed. Retinal ischemia was induced in male guinea pigs by cannulating anterior chambers and lifting the bottle to a height of 205 cm for 90 min in the ischemia and thalidomide/ischemia groups. The thalidomide/ischemia group received thalidomide (300 mg/kg/day) via nasogastric tube 24 h before ischemia and during 7 days of reperfusion. Guinea pigs were sacrificed for histopathological examination to evaluate the mean thickness of the inner plexiform layer (IPL), polymorphonuclear leukocyte (PMNL) infiltration, and biochemical analysis of retinal VEGF and TNF-α levels by ELISA. Results The mean retinal VEGF and TNF-α levels of the control, ischemia, and thalidomide/ischemia groups were 10.22 ± 2.58 and 270.41 ± 69.77 pg/ml; 35.80 ± 5.97 and 629.93 ± 146.41 pg/ml; 19.01 ± 3.01 and 340.93 ± 158.26 pg/ml, respectively. The retinal VEGF levels were significantly higher in I/R injured groups. The thalidomide/ischemia group retinal VEGF level was significantly lower versus the ischemia group. The retinal TNF-α levels were significantly elevated in the ischemia group, but no difference was observed between the thalidomide/ischemia and control groups. Also, the retinal TNF-α level was significantly lower in the thalidomide/ischemia group versus the ischemia group. The mean thickness of IPL and PMNL infiltration showed no difference between the control and thalidomide/ischemia groups. However, there was a significant difference between the control and ischemia groups. Conclusion Thalidomide treatment decreases PMNL infiltration, retinal edema, VEGF, and TNF-α synthesis following I/R injury to the guinea pig retina. This study supported by Firot University Research Fund.     

Chronic bilateral common carotid artery occlusion: a model for ocular ischemic syndrome in the rat

Background Ocular ischemic syndrome is a devastating eye disease caused by severe carotid artery stenosis. The reduction of blood flow produced by bilateral common carotid artery occlusion (BCCAO) of rats for 7 days induces events related to gliosis with no evident histological damage. However, retinal degeneration and cellular death occur after 90 days of BCCAO. Our purpose has been to investigate the effects of BCCAO for 30 days in the retina of adult rats. Methods Adult Wistar rats were submitted to BCCAO or sham surgery. Both direct and consensual pupillary light reflexes were investigated before and after surgery, everyday for the first week and weekly for 30 days. After 1 month, eyes were enucleated and embedded in paraffin. The retinal ganglion cell (RGC) density and thickness of the internal plexiform (IPL), internal nuclear, outer plexiform, and outer nuclear layers were estimated. Results Four rats of the BCCAO group (50%) lost the direct pupillary reflex in both eyes, three rats (37%) lost this reflex in one eye, and only one (13%) maintained it in both eyes. RGC density (cells/mm) was diminished in the BCCAO group, and a significant decrease was found in the total retina and IPL thickness; however, no changes were evident in the other layers. BCCAO pupillary-reflex-negative rats presented with a significant decrease in total retinal thickness and retinal ganglion cell density compared with the sham group. Both BCCAO pupillary-reflex-positive) and -negative rats showed a decrease in IPL compared with the sham group. Conclusion This study demonstrates that BCCAO for 30 days induces functional and morphological damage to the retina with loss of the pupillary reflex and a decrease in IPL thickness and RGC number. We suggest that this protocol might be used as a model for ocular ischemic syndrome in the rat.     

Resveratrol prevents hypoxia-induced retinal ganglion cell death related with ErbB2

AIM: To confirm the changes in proteins related with hypoxia-induced retinal cell death and to assess the effects of resveratrol (Res). METHODS: The therapeutic effect of Res was verified using an ischemic/reperfusion (I/R) model in vivo and a hypoxia modelin retinal ganglion cells (RGCs) in vitro. Death of RGCs were confirmed by TUNEL assay. Protein expression was confirmed by Western blotting and immunohistochemistry. In addition, flow cytometric analysis was used to confirm the response in the cell unit to obtain more accurate data. RESULTS: ErbB2 expression and apoptosis in the ganglion cell layer (GCL) increased after I/R injury. Treatment of Res rescued I/R-induced ganglion cell death, downregulated apoptosis and ErbB2 protein expression in the retina. In subsequent in vitro models, Res affects apoptosis by regulating the phosphorylation and expression of mouse double minute 2 homolog (MDM2), along with those of ErbB2. These results suggest that Res reverses GCL-specific apoptosis via downregulation of ErbB2 in ischemic injury. CONCLUSION: In light of Res favorable properties, it should be evaluated in the treatment of RGC death and related retinal disease characterized by ErbB2 and MDM2 expression. Therefore, Res is appropriate therapeutic agent for treating ischemic injury-related eye diseases by targeting the expression of ErbB2 and MDM2.     

circRNA＿0051079通过靶向miR-26a-5p/PTEN轴诱导视网膜神经变性的分子机制

目的 探讨circ＿0051079对缺血/再灌注(I/R)损伤诱导的视网膜神经变性的影响及机制。方法 从C57BL/6J乳鼠眼球中分离视网膜神经节细胞(RGC),随机分为对照组、siRNA组(转染阴性siRNA)、si＿circ＿0051079组(转染si-circ-0051079干扰RNA)、模拟物对照组(转染Scr mimic)、miR-26a-5p组(转染miR-26a-5p mimic)、miR-26a-5p+vector组(转染pcDNA 3.1)、miR-26a-5p+PTEN组(转染pcDNA 3.1-PTEN),分别在正常、缺氧(体积分数1%O₂暴露)或氧化应激(50μmol·L^-1H₂O₂暴露)条件下培养24 h,进行RT-qPCR、CCK-8、TUNEL、RNA免疫沉淀(RIP)等检测。于15只C57BL/6小鼠左眼中建立I/R损伤模型,对侧眼保持正常眼压作为对照,I/R损伤后0 d、3 d和7 d各取5只小鼠收集视网膜检测circ＿0051079表达。另取20只C57BL/6小...     

Disruption of the complement cascade delays retinal ganglion cell death following retinal ischemia-reperfusion

Recent reports have indicated that components of the complement cascade are synthesized during the degeneration of retinal ganglion cells (RGC) in glaucoma. While complement deposition in the retina may simply serve to aid phagocytosis of damaged RGC, activation of the complement cascade can also contribute to neuronal loss in neurodegenerative diseases. This study was designed to determine if disruption of the complement cascade affects RGC survival in a murine model of retinal ischemia-reperfusion (I/R) injury. We induced retinal ischemia in the eyes of normal mice and mice with a targeted disruption of the complement component 3 (C3) gene. Tissue was harvested 7 and 21 days after induction of I/R and retinal complement synthesis was determined by quantitative PCR and immunohistochemical methods. RGC death and associated axon loss was evaluated through histological examination of the optic nerve and retina. Our data show that retinal I/R induces the expression and deposition of complement components. C3 deficient mice clearly exhibited reduced optic nerve damage and substantial preservation of RGC 1 week after I/R when compared to normal animals (p = 0.005). Three weeks after the ischemic event C3 deficient mice retained more RGC cell bodies although the degree of optic nerve damage was similar between both groups. These findings demonstrate that inhibition of the complement cascade delays optic nerve axonal and RGC degeneration in retinal I/R. It appears that injured RGC are targeted and actively destroyed through complement mediated processes. These results may have implications for the pathophysiology and clinical management of ischemic retinal conditions.     

Expression of kynurenine aminotransferases in the rat retina during development

The study investigates the cellular expression of kynurenine aminotransferases (KAT I and II) in the rat retina during development. At P1 (the day of birth) and P7 (the 7th day after birth), KAT I expression was observed in the inner plexiform layer (IPL), the fiber layer (FL), and in vertically running processes in the ganglion cell layer (GCL) (but not in the cell bodies). At P14 (the 14th day after birth) a strong KAT I immunoreactivity was observed in Müller cell endfeet. KAT II was expressed in the IPL, the FL, and in cells in the GCL at P1 and P7. From P14 on, KAT II expression in the IPL decreased. Double labeling revealed that KAT I was expressed in Müller cell endfeet, whilst KAT II both on retinal ganglion cells (RGC) and Müller cell endfeet. In conclusion, KAT I and II are present in the rat retina during development. The heterogeneity of the KAT developmental profiles possibly reflects a neuromodulatory role in the retinal differentiation.     

Protective effects of antithrombin III on retinal ischemia/reperfusion injury in rats: a histopathologic study

PURPOSE: To investigate the effect of antithrombin III (AT III) on retinal ischemia/reperfusion (I/R) injury in rats. METHODS: The study was carried out on 10 Wistar albino rats (20 eyes) and four-vessel occlusion method was employed to induce retinal ischemia in this study. Rats were divided into two groups: Group I (control group, 10 eyes) and Group II (AT III, 10 eyes). In both groups, vertebral arteries were occluded bilaterally an electric needle coagulator under an operating microscope. A total of 48 hours after the initial procedure, the rats were re-anesthetized and both common carotid arteries were clamped to interrupt blood flow. In Group II, rats were injected intravenously with 250 U/kg of AT III 5 minutes before the induction of ischemia. Duration of ischemia was 30 minutes. At the end of this period, clamp was removed for the reperfusion of the eye for 4 hours. Following the reperfusion period, the animals were killed by decapitation. Retinal sections were evaluated under light and electron microscope. The signs of I/R injury at the microscopic level, i.e., cellular degeneration, vacuolization between retinal layers, increase in the retinal thickness due to edema, mononuclear cell infiltration, and apoptotic cells, were recorded for each group. RESULTS: Retinal sections obtained from the rats in the AT III group revealed a well preserved retinal structure. When average thickness values of the two groups were compared to each other, the difference was significant with respect to inner nuclear and inner plexiform layers indicating increased retinal thickness values in Group I due to tissue edema resulting from I/R injury. Similarly, mononuclear cell infiltration and apoptotic cell counts were found to be significantly higher in control group compared to AT III group showing the inhibitory effect of AT III on leukocyte infiltration and apoptotic cell death in rat retina. CONCLUSIONS: Antithrombin III attenuated I/R injury in rat retina.     

Rabbit retinal ganglion cell survival after optic nerve section and its effect on the inner plexiform layer

Structural modifications of the inner retina were studied after optic nerve section (ONS) in the rabbit. Retinal ganglion cells (RGC) were labelled by injection of Fast Blue into the optic nerve, and counted under fluorescent light in control retina and retina 7, 14, 21 and 26 days post-axotomy. Studies on retinal cross-sections were also performed. For this purpose, retinal sections were stained with haematoxylin-eosin and immunohystochemistry for alpha1 and beta2/beta3 sub-units of the GABA(A) receptors.One week after axotomy, there was no significant loss in the number of ganglion cells with respect to control counts (1086+/-173cellsmm(-2) in the visual streak and 119+/-46cellsmm(-2) in the periphery, mean+/-SD, n=5). At 14 days post-axotomy, 271+/-46cellsmm(-2) remained in the visual streak and 33+/-6cellsmm(-2) in the periphery, corresponding to a mean survival of 27%. The number of ganglion cells decreased further on the following days, reaching 7.54% 1 month after ONS. A significant reduction in the thickness of the inner plexiform and ganglion cell layers was also observed in retinal cross-sections. Immunocytochemical studies show a remarkable disorganization of the layer stratification in the inner plexiform layer (IPL). We conclude that after ONS, RGC death occurs mainly between 7 and 14 days post-axotomy and a progressive death up to 26 days, causing a decrease in the thickness of the IPL and subsequent disorganization of its layers.     

The protective effects of melatonin,vitamin E and octreotide on retinal edema during ischemia-reperfusion in the guinea pig retina

PURPOSE: The purpose of this study is to provide evidence that free radical damage is a component of retinal ischemia-reperfusion (I/R) injury, and to determine whether melatonin, vitamin E and octreotide can protect retina from this injury. METHODS: The right eyes of 50 male guinea pigs weighing 500-600 g were used. The animals were randomly assigned to group 1 (control), group 2 (I/R), group 3 (melatonin + I/R), group 4 (vitamin E + I/R) and group 5 (octreotide + I/R). Groups 3, 4 and 5 received four subcutaneous injections at six-hour intervals for total dosage of 10 mg/kg melatonin, 150 mg/kg vitamin E and 22 microg/kg octreotide respectively. The first dose of each substance was administered 5 minutes before retinal ischemia. Retinal ischemia was induced for 1.5 hours, then followed by reperfusion for 24 hours. Infections of all three substances were repeated at 6, 12 and 18 hours during reperfusion. The animals were killed at 24 hours of reperfusion. Sagittal sections of 4 microm were cut and stained with hematoxylin and eosin for light microscopic evaluation. The average thickness (edema) of the inner plexiform layer for each eye was measured in sagittal sections near the optic nerve and expressed in microns. RESULTS: The efficacy of each compound had the following relationships: melatonin>vitamin E>octreotide in preventing retinal damage by ischemia-reperfusion. The mean thickness of the inner plexiform layer was 13.3 +/- 0.8 microm, 25.9 +/- 2. 0 microm, 20.0 +/- 0. 7 microm, 21.6 +/- 0.7 microm, 23.9 +/- 0.8 microm respectively in the control, I/R, I/R plus melatonin, I/R plus vitamin E and I/R plus octreotide groups. The thickness of the inner plexiform layer in group 1 (control) was significantly less than the other groups (p<0.001). The inner plexiform layer was thicker in the I/R group than with I/R plus melatonin, I/R plus vitamin E and I/R plus octreotide (all p < 0.01). The inner plexiform layer was thicker in the I/R plus octreotide group than the I/R plus vitamin E and I.R plus melatonin groups both (p < 0.05). Compared to the I/R plus melatonin group, the inner plexiform layer was significantly thicker in the I/R plus vitamin E group (p < 0. 05). CONCLUSIONS: This study demonstrates a protective effect of melatonin, vitamin E and octreotide on the retina during retinal ischemia-reperfusion injury.     

Protective Effects of Vitamin E Forms (Alpha-tocopherol, Gamma-tocopherol and d-alpha-tocopherol Polyethylene Glycol 1000 Succinate) on Retinal Edema During Ischemia–reperfusion Injury in the Guinea Pig Retina

Purpose: The purpose of this study is to provide evidence that free radical damage is a component of retinal ischemia–reperfusion (I/R) injury, and to determine whether alpha-tocopherol, gamma-tocopherol and d-alpha-tocopherol polyethylene glycol 1000 succinate (TPGS) can protect the retina from this injury. Methods: The right eyes of 40 male guinea pigs weighing 500–600 g were used. The animals were randomly assigned to group 1 (control), group 2 (I/R), group 3 (I/R plus alpha-tocopherol), group 4 (I/R plus gamma-tocopherol) and group 5 (I/R plus TPGS). Groups 3, 4 and 5 received four subcutaneous injections at six-hour intervals for total dosage of 800 IU/kg alpha-tocopherol, 1000 IU/kg gamma-tocopherol and 750 IU/kg TPGS, respectively. The first dose of each substance was administered 5 minutes before retinal ischemia. Retinal ischemia was induced for 90 minutes, then followed by reperfusion for 24 hours. Injections of three substances were repeated at 6, 12 and 18 hours during reperfusion. The animals were killed at 24 hours of reperfusion. Sagittal sections of 4 μm were cut and stained with hematoxylin and eosin for light microscopic evaluation. The average thickness (edema) of the inner plexiform layer for each eye was measured in sagittal sections near the optic nerve and expressed in microns. Results: All the three substances showed statistically significant protection against the formation of retinal edema during ischemia–reperfusion injury. The mean thickness of the inner plexiform layer were 15.0, 25.44, 19.81, 21.38 and 20.88 μm in control, I/R, I/R plus alpha-tocopherol, I/R plus gamma-tocopherol and I/R plus TPGS groups, respectively. The results showed that the thickness of the inner plexiform layer in group 1 (control) was significantly lower than the other groups (p<0.001). The inner plexiform layer was thicker in the I/R group than with I/R plus alpha-tocopherol (p<0.001), I/R plus gamma-tocopherol (p<0.001) and I/R plus TPGS (p<0.01). The inner plexiform layer was not thicker in the I/R plus TPGS group than in the I/R plus alpha-tocopherol and I/R plus gamma-tocopherol groups. Compared to the I/R plus alpha-tocopherol group, the inner plexiform layer was significantly thicker in the I/R plus gamma-tocopherol group (p<0.01). Conclusions: The results from these experiments indicate that vitamin E forms have protective effects on the retina during retinal ischemia–reperfusion injury, but, the effects of alpha-tocopherol and TPGS appear to be much greater than that of gamma-tocopherol.     

Retinoic acid metabolic change in retina and choroid of the guinea pig with lens-induced myopia

AIM: To investigate the role of retinoic acid (RA) and retinaldehyde dehydrogenase-2 (RALDH₂) of retina and choroid in the guinea pig lens-induced myopic eyes. METHODS: Totally 45 guinea pigs, at age of three weeks, were randomly assigned into three groups: the normal control, the lens-induced group and the recovering group. Out of focus was induced by the -6.00D concave lens on the left eye, and lasted for 15 days. All animals underwent biometric measurement (corneal radius of curvature, refraction and axial length). Subsequently, RA content in the retina and RPE/choriod complex was detected by reversed-phase high-performance liquid chromatography. RALDH₂ protein in the retina and RPE/choriod complex was evaluated by the immunohistochemical staining and Western blotting. RESULTS: After wearing -6.00D lens for 15 days, axial length of the lens-induced eye extends and myopia was formed, with RA contents increasing in both the neural retina and RPE/choroid complex. Comparing with the lens-induced group, myopic degree significantly relieved, and its RA contents in both the neural retina and RPE/choroid complex decreased in the recovering group. In the normal control, RALDH2 protein was expressed positively in the retinal nerve fiber layer (RNFL), inner plexiform layer (IPL) and lateral border of outer nuclear layer (ONL). Retinal RALDH₂ protein increased in the lens-induced group, and was also positive in the outer plexiform layer (OPL). In the recovering group, retinal RALDH₂ protein attenuated the expression in the OPL turns to negative. RALDH₂ protein was not expressed in the choroid of any group. CONCLUSION: RA of retina and chorid participates in the regulation of the lens-induced myopia in guinea pigs, which may be related with retinal RALDH2 protein.