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.     

Retinal ischemic injury rescued by sodium 4-phenylbutyrate in a rat model

Retinal ischemia is a common cause of visual impairment for humans and animals. Herein, the neuroprotective effects of phenylbutyrate (PBA) upon retinal ischemic injury were investigated using a rat model. Retinal ganglion cells (RGCs) were retrograde labeled with the fluorescent tracer fluorogold (FG) applied to the superior collicoli of test Sprague-Dawley rats. High intraocular pressure and retinal ischemia were induced seven days subsequent to such FG labeling. A dose of either 100 or 400 mg/kg PBA was administered intraperitoneally to test rats at two time points, namely 30 min prior to the induction of retinal ischemia and 1 h subsequent to the cessation of the procedure inducing retinal ischemia. The test-rat retinas were collected seven days subsequent to the induction of retinal ischemia, and densities of surviving RGCs were estimated by counting FG-labeled RGCs within the retina. Histological analysis revealed that ischemic injury caused the loss of retinal RGCs and a net decrease in retinal thickness. For PBA-treated groups, almost 100% of the RGCs were preserved by a pre-ischemia treatment with PBA (at a dose of either 100 or 400 mg/kg), while post-ischemia treatment of RGCs with PBA did not lead to the preservation of RGCs from ischemic injury by PBA as determined by the counting of whole-mount retinas. Pre-ischemia treatment of RGCs with PBA (at a dose of either 100 or 400 mg/kg) significantly reduced the level of ischemia-associated loss of thickness of the total retina, especially the inner retina, and the inner plexiform layer of retina. Besides, PBA treatment significantly reduced the ischemia-induced loss of cells in the ganglion-cell layer of the retina. Taken together, these results suggest that PBA demonstrates a marked neuroprotective effect upon high intraocular pressure-induced retinal ischemia when the PBA is administered prior to ischemia induction.     

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...     

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.     

Protective effects of catalase on retinal ischemia/reperfusion injury in rats

Retinal ischemia/reperfusion (I/R) injury causes profound tissue damage, especially retinal ganglion cell (RGC) death. The aims of the study were to investigate whether catalase (CAT) has a neuroprotective effect on RGC after I/R injury in rats, and to determine the possible antioxidant mechanism. Wistar female rats were randonmized into four groups: normal control group (Control group), retinal I/R with vehicle group (I/R with vehicle group), retinal I/R with AAV-CAT group (I/R with AAV-CAT group), and normal retina with AAV-CAT group (normal with AAV-CAT group). One eye of each rat was pretreated with recombinant adeno-associated virus containing catalase gene (I/R with AAV-CAT group or normal with AAV-CAT group) and recombinant adeno-associated virus containing GFP gene (I/R with vehicle group) by intravitreal injection 21 days before initiation of I/R injury. Retinal I/R injury was induced by elevating intraocular pressure to 100 mmHg for 1 h. The number of RGC and inner plexiform layer (IPL) thickness were measured by fluorogold retrograde labeling and hematoxylin and eosin staining at 6 h, 24 h, 72 h and 5d after injury. Hydrogen peroxide (H₂O₂), the number of RGC, IPL thickness, malondialdehyde(MDA), 8-hydroxy-2-deoxyguanosine (8-OHdG), CAT activity and nitrotyrosine were measured by fluorescence staining, immunohistochemistry and enzyme-linked immunosorbent assay analysis at 5 days after injury. Electroretinographic (ERG) evaluation was also used. Pretreatment of AAV-CAT significantly decreased the levels of H₂O₂, MDA, 8-OHdG and nitrotyrosine, increased the catalase activity, and prevented the reduction of a- and b- waves in the I/R with AAV-CAT group compare with the I/R with vehicle group (p < 0.01). Catalase attenuated the I/R-induced damage of RGC and IPL and retinal function. Therefore, catalase can protect the rat retina from I/R-induced injury by enhancing the antioxidative ability and reducing oxidative stress, which suggests that catalase may be relevant for the neuroprotection of inner retina from I/R-related diseases.     

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.     

Neuroprotective effect of small interfering RNA targeted to caspase-3 on rat retinal ganglion cell loss induced by ischemia and reperfusion injury

Purpose: To investigate neuroprotective effects of siRNA targeted to caspase-3 against ischemia and reperfusion (I/R) injury in rat eyes. Methods: Retinal ischemia was induced in Wistar rats by increasing the intraocular pressure (IOP) to 110 mmHg for 120 min. To examine the effect of siRNA on rat caspase-3, siRNA was injected into the vitreous cavity 24 h prior to induction of retinal ischemia. Eyes were removed at 2, 7 or 14 days later, and then analyzed for the number of retinal ganglion cells (RGCs), the retinal thickness and the amount of apoptosis of the retinal neural cells (as demonstrated by the TUNEL assay). The amount of caspase-3 mRNA was analyzed by rt-PCR. Differences between groups were evaluated by an unpaired t test. Results: The numbers of RGCs in the saline and non-silencing siRNA controls were reduced significantly at 2 and 7 days after the I/R injury. RGCs were significantly retained in eyes pretreated with siRNA targeted to caspase-3 as compared to the control eyes at 2 days after the I/R injury. Inner retinal thickness in the control eyes was significantly thinner as compared to the treated eyes at 2 and 7 days after the I/R injury. After siRNA treatment, the amount of caspase-3 mRNA was significantly lower when compared to the saline control group. Conclusions: The injection of siRNA targeted to caspase-3 into the vitreous cavity of rat eyes may block caspase-3, and may thus be able to prevent retinal cell death associated with ischemic injury. As inhibition of the apoptosis pathway may provide a neuroprotective effect, examination of new strategies for treating these disorders needs to be undertaken.     

巩膜上静脉烙闭法建立大鼠高眼压模型对视网膜微观结构的影响

目的　观察巩膜上静脉烙闭法建立的大鼠高眼压模型对视网膜微观结构的影响，为青光眼视神经损伤及保护机制提供研究基础。方法　选取不同体质量SD大鼠40只，分为A组（150~200 g）、B组（>200~250 g）、C组（>250~300 g）、D组（>300~350 g），每组各10只，分别测量3 d白天及夜间基线眼压。随机选取SD大鼠（雌雄随机，250~300 g）40只，术前测量3 d基线眼压，以右眼为实验眼，烙闭实验眼3~4支巩膜上静脉，以左眼为对照眼，于术后即刻、1~7 d、14 d、21 d、28 d分别测量实验眼和对照眼眼压。于术前及术后28 d分别用光学相干断层扫描(optical coherence tomography，OCT)仪测量视网膜厚度。于术后28 d处死大鼠，行石蜡包埋、苏木精伊红（hematoxylin eosin，HE）染色，计数视网膜神经节细胞（retinal ganglion cells，RGCs）并测量视网膜厚度。结果　不同体质量大鼠昼夜眼压比较差异均有统计学意义（均为P<0.001）。A组大鼠眼压较其他3组眼压低（均为P<0.05）。巩膜上静脉烙闭术后即刻实验眼眼压达到峰值，较对照眼高122%（P<0.001），之后缓慢下降，到术后14 d、21 d实验眼眼压较对照眼分别升高约41%、20%（均为P<0.001），到术后28 d实验眼与对照眼眼压差异无统计学意义（P>0.05）。OCT提示内层视网膜变薄（均为P<0.001）。HE染色切片结果提示RGCs数量减少，内、中、外层视网膜均变薄（均为P<0.05），且以内层变化最明显（P<0.001）。结论　大鼠基线眼压昼夜存在差异，白天眼压较夜间眼压低，低体质量（150~200 g）大鼠眼压偏低。烙闭大鼠巩膜上静脉能维持3周的眼压升高，且能使视网膜变薄、RGCs数量减少，故巩膜上静脉烙闭法是建立大鼠高眼压模型的有效方法。     

JAK/STAT pathway mediates retinal ganglion cell survival after acute ocular hypertension but not under normal conditions

Intraocular pressure (IOP) elevation is an important cause of glaucoma. Animal models of ocular hypertension have been widely used to mimic glaucoma to investigate the mechanisms underlying retinal ganglion cell (RGC) death and search for possible cure. The aim of the present study was to examine the role of JAK/STAT pathway in RGC viability in normal condition or after acute IOP elevation. Retinal explants obtained from intact or IOP-elevated eyes were firstly used to examine the effect of the JAK/STAT pathway inhibitors, AG490 and Jak Inhibitor I, on RGC viability in vitro. The role of this signal pathway was further investigated and confirmed in vivo. AG490 and Jak Inhibitor I were applied into the left eye on days 3, 9, and 15 post 2-h IOP elevation at 110mmHg. Fluorescence dye Fluorogold was used to retrogradely label surviving RGCs. Because macrophage recruitment was seen in the IOP-elevated eyes after inhibition of this pathway, clodronate liposomes were used to remove phagocytic cells in the eye and examine the role of JAK/STAT pathway in RGC survival independent of macrophages. Activities and location of JAK/STAT pathway in the retina were examined using Western blotting and immunohistochemistry. We found that inhibition of JAK/STAT pathway did not affect RGC survival in the retinal explants derived from intact eye but caused RGC death in the retinal explants that were derived from IOP-elevated eye. Importantly, the detrimental effect of JAK/STAT pathway inhibition on RGC survival was also observed in vivo following acute IOP elevation, but not in intact eye. In addition, both in vitro and in vivo experiments confirmed a detrimental action of phagocytic cells following acute IOP elevation and the pathway inhibition. Compatible with what were observed in vivo, Western blotting and immunohistochemistry showed that JAK/STAT activities were not present in intact retina, but acute IOP elevation activated JAK/STAT pathway in the retina, in the regions of inner nuclear layer and ganglion cell layer, including RGCs. The IOP elevation-induced JAK/STAT activities were effectively abolished by intravitreal application of AG490. This study thus shows that (1) acute IOP elevation activates JAK/STAT pathway in RGCs, and (2) JAK/STAT pathway mediates RGC survival following IOP elevation but not under normal condition.     

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.     

Loss of retinal ganglion cells following retinal ischemia: the role of inducible nitric oxide synthase

Following experimental, transient, retinal ischemia in the rat, there is loss of retinal neurons, which occurs over several weeks. Retinal ganglion cells (RGCs) are particularly susceptible and there is early, massive degeneration of these neurons after ischemia. We have determined the early mechanisms by which RGCs are killed following ischemia. Retinal ischemia/reperfusion was produced in rats by transient unilateral elevation of intraocular pressure above systolic blood pressure. Retinas were studied by immunohistochemistry for the presence of inducible nitric oxide synthase (NOS-2) at several time points post-ischemia and specific cell types were identified. Rats were also treated orally with L -N(6) -(1-iminoethyl)lysine 5-tetrazole amide (SC-51), a prodrug of an inhibitor of NOS-2 or with aminoguanidine (AG) for a period of 14 days. Retrograde labelling with Fluoro-Gold quantitated the loss of RGCs. NOS-2 was not present in the normal retina and was not present in the eyes that were contralateral to the ischemic eyes. Within 24hr after ischemia, polymorphonuclear leukocytes containing NOS-2 had entered the ganglion cell layer and surrounded RGCs. Within 5 days after ischemia, NOS-2 was present in many inner retina cells and in invading monocytes in the vitreous. Between 7 and 14 days post-ischemia, there were few hematogenous cells in the retina but NOS-2 was sparsely detectable in microglia and other cells of the inner retina. Two weeks after ischemia, rat eyes lost approximately 50% of the RGCs. Treatment with AG for 14 days following ischemia was partially neuroprotective; approximately 28% of the RGCs were lost. Treatment with SC-51 for 14 days following ischemia almost completely prevented the loss of RGCs. Thus, within 24hr following ischemia, polymorphonuclear leukocytes containing NOS-2 attack and kill neurons in the ganglion cell layer. For 2 weeks after ischemia, NOS-2 appears transiently in the retina in several different cell types at different times. Continuous pharmacological treatment with inhibitors of NOS-2 activity during the 2 weeks post-ischemia period provides significant neuroprotection against the loss of RGCs.     

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.     

Effect of MCI-9042, a 5-HT2 receptor antagonist,on retinal ganglion cell death and retinal ischemia

The neuroprotective effect of MCI-9042 (Mitsubishi Pharma Corporation) was investigated on glutamate-induced retinal ganglion cell (RGC) death in vitro and on rat retinal ischemia in vivo. RGCs were purified from retinal cells isolated from 6-day-old Wistar rats and cultured in serum-free media. After application of 25 microM glutamate, the viability of RGCs treated with or without several serotonin 2 (5-HT(2)) receptor antagonists: MCI-9042, M-1 (a major metabolite of MCI-9042), ketanserin, and LY-53857; was evaluated by calcein-acetoxymethyl ester staining. Retinal ischemia was induced by intraocular pressure (IOP) elevation (130 mmHg, 50 min). Rats were intraperitoneally injected with MCI-9042 at a dose of 3, 30 mg/kg or base at 30 min before and just after ischemia-reperfusion. Retinal damages were evaluated by histology, morphometric analysis and electroretinograms (ERGs) recordings at 7 days after ischemia-reperfusion. 25 microM glutamate decreased the number of viable RGCs to about 60 to 65% of untreated RGCs. MCI-9042, M-1, ketanserin, and LY-53857 significantly reduced glutamate-induced RGC death at concentrations of more than 100 nM, 1 nM, 1 microM and 100 nM, respectively. Ischemia-reperfusion caused thinning of the thickness between the inner plexiform layer and the outer plexiform layer and attenuation of a-and b-waves in ERG recordings. The intraperitoneal injection of MCI-9042 significantly reduced morphological and functional damages in retinal ischemia. Our data demonstrate that 5-HT(2) receptor antagonists including MCI-9042 and M-1 have the neuroprotective effects in cultured RGCs and that MCI-9042 protects against ischemic retinal diseases.     

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.     

Time Course of Age-dependent Changes in Intraocular Pressure and Retinal Ganglion Cell Death in DBA/2J Mouse

Glaucoma is a major cause of irreversible blindness and is characterized by the death of retinal ganglion cells (RGCs) [1]. This death of RGCs is frequently associated with an elevation in intraocular pressure (IOP) [2].However, the understanding of how elevated IOP leads to cell death is hampered by the lack of an animal model that emulates the clinical time course for decades. Mouse studies have proven helpful for investigating human complex diseases. The DBA/2J mouse, which is inheri…     

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.     

Betaxolol,a beta1-adrenoceptor antagonist,protects a transient ischemic injury of the retina

In the present study, we investigated the protective effects of the topical beta-adrenoceptor antagonist Betoptic((R)) (0.25% betaxolol) in the rat retina following the ischemic injury induced by a transient increase of intraocular pressure (IOP). Like other areas of the central nervous system, the retina is highly vulnerable to ischemic-induced injury. Ischemia was induced in the rat retina by raising the IOP above the systolic blood pressure for 60min. After an ischemia/reperfusion, the thickness of the retinal layers and the immunoreactivities of choline acetyltransferase (ChAT), gamma-amino butyric acid (GABA) and tyrosine hydroxylase (TH) were examined. After a reperfusion period of 7 days, the thickness of both the inner plexiform layer and inner nuclear layer was much decreased. After a reperfusion period of 14-28 days, the thickness of the outer nuclear layer decreased markedly. Moreover, the ChAT and TH immunoreactivity had almost completely disappeared in the retinas after 7 days, while GABA immunoreactivity remained for 28 days. These results suggest that the inner retinal layers are more susceptible to ischemic-induced injury than the outer retinal layer.Histological examination demonstrated protective effects of betaxolol on ischemic-induced retinal damage, which was more substantial in the inner retinal layer. When two drops of betaxolol, once before ischemic injury and twice daily for 28 days after ischemia, were continuously administered, the reductions in the retinal ChAT, GABA and TH immunoreactivities were significantly attenuated. The present study suggests that topically applied betaxolol is an efficient neuroprotective agent and prevents the retinal cell damage induced by ischemic injury in rats.     

Protective effect of docosahexaenoic acid against retinal ischemic injury: an electroretinographic study.

PURPOSE: To determine whether docosahexaenoic acid (DHA) can reduce the retinal damage induced by transient retinal ischemia. METHODS: Retinal ischemia was induced by increasing intraocular pressure (IOP). Retinal circulation was restored by lowering IOP. An intraperitoneal injection of 1,000 mg/kg of DHA-ester (DHA-E) was given 5 h before the ischemia. Electroretinograms were recorded just before the ischemia and at 60-min intervals up to 4 h after circulation was restored. RESULTS: The ratio of the amplitudes after the ischemia to that just before ischemia was significantly higher in eyes administered DHA-E than in controls at each time point (p < 0.01). CONCLUSION: DHA-E is effective in protecting the retina against transient retinal ischemia.     

高良姜素对青光眼大鼠视网膜神经节细胞的保护作用

目的 研究高良姜素对青光眼大鼠视网膜神经节细胞(RGC)的保护作用。方法 取48只SD大鼠随机分为对照组、对照+高良姜素组、高眼压组和高眼压+高良姜素组,每组12只(12眼)。对照+高良姜素组和高眼压+高良姜素组大鼠接受高良姜素滴眼液治疗,对照组和高眼压组大鼠则接受相同剂量二甲基亚砜溶剂滴眼。于造模后第0～28天监测大鼠眼压,并于造模后第28天处死大鼠,收集各组大鼠右眼球,并采用HE染色观察大鼠视网膜形态变化,采用免疫组织化学染色分析各组大鼠视网膜中GFAP、Toll样受体4(TLR4)和NOD样受体3(NLRP3)表达。从4只4 d龄大鼠视网膜中分离RGC,分为空白组、光气组和CoCl₂+高良姜素组。光气组和光气+高良姜素组RGC与200μmol·L^-1 CoCl₂一起孵育48 h,光气+高良姜素组RGC中加入20μmol·L^-1高良姜素干预48 h。收集各组细胞,采用流式细胞术和Western blot检测RGC的凋亡率及相关蛋白的相对表达情况,并采用ELISA检测空白组、光气组和光气+高良姜素...