Activation of the aldosterone/mineralocorticoid receptor system and protective effects of mineralocorticoid receptor antagonism in retinal ischemia-reperfusion injury

The purpose of this project was to investigate the effects of the mineralocorticoid receptor antagonist against retinal ischemia-reperfusion injury and identify the aldosterone/mineralocorticoid receptor (MR) system in the rat retina. Retinal ischemia was induced by increasing intraocular pressure to 130?mmHg. Rats were treated with the angiotensin II type 1 receptor (AT1-R) antagonist (candesartan), MR antagonist (spironolactone), or aldosterone. Retinal damage was evaluated at 7 days after the ischemia by measuring the retinal thickness and the number of retinal ganglion cells. Pretreatment with candesartan, spironolactone, or candesartan and spironolactone significantly inhibited retinal ischemic injury. However, there was no protective effect against retinal ischemia-reperfusion injury provided by the combined aldosterone with candesartan treatment. Additionally, pretreatment with aldosterone alone also did not provide any neuroprotective effects against retinal ischemia-reperfusion injury. When rats were treated via local administration of aldosterone in the absence of ischemia, the number of retinal ganglion cells decreased while the retinal thickness remained unchanged. The present findings demonstrated the existence of a local aldosterone/MR system in the retina. Our results also demonstrated that an MR antagonist can attenuate subsequent ischemic damage in the rat retina.     

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.     

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.     

Neuroprotective effect of transcorneal electrical stimulation on ischemic damage in the rat retina

Some previous studies have showed that transcorneal electrical stimulation (TES) could protect retinal neurons in certain rodent models. However, it is not yet clear whether TES could also definitely protect retinal neurons against ischemic insults. In the present study, we hypothesized that TES had such a neuroprotective effect and further investigated its underlying mechanism. Adult female Sprague–Dawley (SD) rats received TES treatment every other day after ocular ischemia was induced by elevating the intraocular pressure to 120 mmHg for 60 min. Retinal ganglion cells (RGCs) were labeled retrogradely 7 days before ischemia and were counted 7 and 14 days later. At the same time points, retinal function was assessed by scotopic electroretinography (ERG), combined with retinal histological analysis. The glutamine synthetase (GS) immunoreactivity was compared between ischemic retinas with TES and those with sham stimulation under identical confocal laser microscope conditions. The immunohistochemical indications were confirmed by Western blot analysis. Higher mean density of RGCs was quantified in TES treated retinas compared to retinas with sham stimulation on days 7 and 14 after ischemia. Similarly, histological analysis showed that TES better preserved the mean thickness of separate retinal layers. ERG studies indicated that by undergoing TES treatment, the b-wave amplitude was also significantly preserved on day 7 after ischemia and recovered robustly on day 14. Immunohistochemical and Western blot analysis both revealed that GS levels remarkably increased after TES and lasted for at least 7 days. Our results indicate that TES can protect retinal neurons against ischemic insults, probably related to increasing levels of GS localized in Müller cells. These findings suggest a new approach for potential clinical application to ocular ischemic diseases.     

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.     

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.     

Retinal ganglion cell death induced by retinal ischemia. neuroprotective effects of two alpha-2 agonists

We have investigated in adult Sprague-Dawley rats the neuroprotective effects of two alpha-2-selective agonists [AGN 191,103 (AGN) and brimonidine tartrate (BMD)] on retinal ganglion cell (RGC) survival after transient retinal ischemia. RGCs were labelled with Fluorogold (FG) applied to both superior colliculi. Seven days later, 90 min of retinal ischemia were induced in the left eyes by ligature of the ophthalmic vessels (LOV). In one group of animals, vehicle or AGN (0.01 mg/kg) were administered systemically 1 hr before ischemia. In another group of animals, two 5 microl drops of vehicle, AGN (0.05%) or BMD (0.1%) were administered topically in the left eye 1 hr before ischemia. The animals were processed 7 or 21 days later. RGC survival was estimated by counting FG-labelled cells in 12 standard areas of each retina. In control retinas of systemically pretreated animals, mean densities of labelled RGCs were 2372 +/- 49 cells/mm(2) (mean +/- SEM; n = 6). In experimental retinas of systemically pretreated animals, mean RGC densities had decreased 7 days after ischemia to 53% (n = 6) or 81% (n = 6) of control in the groups treated with vehicle or AGN, respectively. Twenty-one days after ischemia, mean RGC densities had decreased to 38% (n = 6) or 79% (n = 6) of control in the groups treated with vehicle or AGN, respectively. In control retinas of topically pretreated animals, mean densities of labelled RGCs were 2208 +/- 29 cells/mm(2) (n = 6). In experimental retinas of topically pretreated animals, mean RGC densities had decreased 7 days after ischemia to 54% (n = 6), 95% (n = 6) or 96% (n = 6) of control in the groups treated with vehicle, AGN or BMD, respectively. These results indicate that pretreatment with a single systemic or topical dose of AGN or BMD can prevent completely the early rapid phase of RGC loss and abolish the delayed RGC loss observed after 90 min of retinal ischemia induced by ligature of the ophthalmic vessels.     

大鼠视神经夹伤模型中莫尼定的视网膜节细胞保护作用

目的 :研究莫尼定对大鼠视神经夹伤模型视网膜神经节细胞的保护作用。方法 :实验用SD大鼠 2 0只随机分为用药组 8只和对照组 12只。所有大鼠右眼用 40 g微型视神经夹紧贴球后夹持视神经 60秒 ,左眼未做夹持。用药组于夹伤前1小时及夹伤后每日腹腔注射莫尼定 1mg/kg ,阴性对照组于夹伤前 1小时及夹伤后每日腹腔注射生理盐水 5ml/kg ,实验观察2 8天。实验结束前 4天双上丘注射 3 %荧光金逆行标记视网膜神经节细胞。做视网膜铺片 ,距离视乳头中心上下左右各2mm拍摄照片 ,使用CPAS图像分析软件做节细胞定量分析 ,节细胞存活率 =右眼节细胞密度 /左眼节细胞密度× 10 0。结果 :用药组、对照组节细胞存活率分别为 61 0 1%和 53 48% ,两者之间存在显著性差异 (P =0 .0 3 5)。结论 :在大鼠视神经夹伤模型中 ,莫尼定具有明显的视网膜节细胞保护作用     

Simvastatin upregulates Bcl-2 expression and protects retinal neurons from early ischemia/reperfusion injury in the rat retina

Simvastatin has been shown to enhance the survival of retinal ganglion cells (RGCs) following ischemia-reperfusion (IR) injury by mediating the expression of stress proteins. The purpose of this study was to investigate the effect of simvastatin on retinal neurons and the expression of apoptotic proteins in a rat IR model. Wistar rats received intravitreal injection of simvastatin immediately after retinal reperfusion. Retinal ischemia was induced by increasing intraocular pressure to 150 mmHg for 60 min. The number of viable RGCs was measured after retrograde labeling with Fluoro-Gold. Ischemia-induced apoptotic cell death was studied using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). We found that simvastatin treatment enhanced RGC survival after retinal ischemia by approximately 40% and decreased retinal neuronal apoptosis. Using western blot analysis, we found that simvastatin upregulated the expression of Bcl-2 in the retina. In contrast, the level of the protein Bax was unaffected by simvastatin treatment. Our results suggest that RGC loss induced by retinal IR may be prevented by simvastatin and that the mechanism underlying this process possibly involves an alteration in the apoptotic pathway.     

缺血后适应对视网膜缺血-再灌注损伤的保护作用

背景研究证明,缺血后适应（IPC）对多种组织器官的缺血缺氧损伤均有一定的抵抗作用,但其对视网膜缺血缺氧的作用仍受到关注。目的探讨IPC对大鼠视网膜缺血-再灌注损伤（RIRI）后视网膜结构和功能的保护作用。方法将36只健康雄性Wistar大鼠以随机数字表法分为正常对照组、伪手术组、缺血-再灌注组、IPC组。利用前房灌注生理盐水升高眼压至100mmHg（1mmHg=0．133kPa）维持60min的方法制备RIRI大鼠模型,实施IPC处理鼠亚分为再灌注后即刻、1min、10min组（即IPCⅠ组、IPCⅡ组、IPCⅢ组）,分别于实验后1d、7d行大鼠视网膜电图（ERG）检测,然后用过量麻醉法处死大鼠并制备视网膜切片,行苏木精-伊红染色,对各组大鼠视网膜厚度的变化和视网膜形态进行观察。采用SPSS13．0统计学软件的单因素方差分析对各组大鼠ERG各波振幅恢复率和视网膜厚度值的差异进行比较。结果实验后1d,与正常对照组大鼠比较,伪手术组大鼠视网膜结构接近正常,而缺血-再灌注组及IPCⅠ组、IPCⅡ组、IPCⅢ组大鼠视网膜均出现水肿,可见空泡变性,主要在内丛状层（IPL）及内核层（INL）。缺血-再灌注组及IPCⅠ组、IPCⅡ组、IPCⅢ组大鼠视网膜全层、INL、IPL及视网膜外层厚度值均明显高于正常对照组,差异均有统计学意义（均P〈0．05）。再灌注后7d,缺血-再灌注组大鼠视网膜全层厚度值明显低于正常对照组,差异均有统计学意义（均P〈0．05）,尤以INL、IPL显著。IPCⅠ组、IPCⅡ组、IPCⅢ组大鼠视网膜全层、INL、IPL及视网膜外层厚度值均明显高于缺血-再灌注组,差异均有统计学意义（均P〈0．05）。再灌注后7d,缺血-再灌注组、IPC各组大鼠ERG a波、b波和OPs振幅恢复率明显低于伪手术组和正常对照组大鼠,差异均有统计学意义（均P〈0．05）;而IPCⅠ组、IPCⅡ组、IPCⅢ组大鼠ERG a波、b波和OPs振幅恢复率明显高于缺血-再灌注组,差异均有统计学意义（均P〈0,05）。结论IPC对RIRI具有保护作用,在大鼠模型中,这种保护作用在再灌注后即刻至1min时最强。     

Heme oxygenase-1-related carbon monoxide and flavonoids in ischemic/reperfused rat retina

PURPOSE: There is increasing evidence to show cytoprotective effects of various flavonoid-rich extracts and the tissue-protective capacity of flavonoid-rich extract of sour cherry is due to flavonoid components of seeds. Sour cherry seed flavonoids were evaluated for their contribution to postischemic recovery related to endogenous carbon monoxide (CO) production in rat retinas subjected to ischemia/reperfusion. METHODS: Rats were orally treated with selected doses of flavonoid-rich extract of sour cherry seeds for 2 weeks. Animals were anesthetized, and a suture was placed behind the globe including the central retinal artery. Next, retinas were subjected to 90 minutes of ischemia followed by 24 hours of reperfusion. After this procedure, heme oxygenase-1 (HO-1)-related protein expression and enzyme activity, HO-1-related endogenous CO production, and ionic imbalance including tissue Na(+), K(+), and Ca(2+) in untreated and treated ischemic/reperfused retinas were measured. RESULTS: Retinal ischemia/reperfusion resulted in a significant reduction (to 10%) in HO-1 protein expression, enzyme activity, and HO-1-related endogenous CO production in the retina. These changes were accompanied by increases in retinal Na(+) and Ca(2+) gains and loss of K(+). In rats treated with 10 and 30 mg/kg of sour cherry flavonoid-rich extract, after 24 hours of reperfusion, tissue Na(+) and Ca(2+) accumulation and K(+) loss were prevented in comparison with the drug-free control. CONCLUSIONS: Sour cherry seed flavonoid-rich extract showed a protective effect against reperfusion-induced injury through its ability to reduce the changes in concentrations of retinal ions through HO-1-related endogenous CO production in the ischemic/reperfused retina.     

黄芪多糖对急性高眼压诱导大鼠视网膜神经节细胞的保护作用

背景青光眼患者视神经保护的问题日益引起关注。黄芪多糖（APS）是黄芪的主要活性成分,可增加再生神经蛋白的表达并促进损伤的周围神经修复,但其对视网膜神经节细胞（RGCs）再生作用的研究少见报道。目的探讨APS对急性高眼压状态下RGCs的保护作用。方法采用抽签法将40只SPF级sD大鼠随机分为正常对照组、模型对照组、低剂量APS组和高剂量APS组,每组各10只。低剂量APS组和高剂量APS组大鼠自实验开始每日分别给予APS500mg／kg、2000mg／kg（均溶于2．5ml生理盐水）灌胃,模型对照组仅给予2．5ml生理盐水灌胃,正常对照组不做任何处理。用药2周后,除正常对照组外,其余3个组均抽取0．2ml房水继而单眼前房注射等体积甲基纤维素使眼压升高至22mmHg（1mmHg=0．133kPa）以上制作急性高眼压模型。造模后5d,过量麻醉法处死动物,摘除该眼球制作视网膜石蜡切片,常规组织病理学观察视网膜的形态结构变化,采用免疫组织化学法检测caspase-3蛋白在大鼠视网膜中的表达,采用TUNEL染色法观察和计算各组大鼠RGCs的凋亡率。ImageProPlus5．1软件测量各组大鼠视网膜厚度和神经纤维层厚度。结果大鼠成模后5d,模型对照组、低剂量APS组和高剂量APS组大鼠的眼压均明显高于正常对照组,差异均有统计学意义（t=-8．900、-10．700、-11．300,P〈0．01）。正常对照组大鼠视网膜形态正常;模型对照组大鼠视网膜水肿,细胞排列紊乱;低剂量APS组视网膜可见空泡样变性,但视网膜细胞排列较模型对照组整齐,视网膜水肿减轻;高剂量APS组视网膜水肿较明显。低剂量APS组视网膜厚度、外颗粒层及视神经纤维层厚度值均明显低于模型对照组,差异均有统计学意义（t=-23．700、-14．770、-11．640,P〈0．01）,但高剂量APS组外颗粒层及视神经纤维层厚度与模型对照组比较差异均无统计学意义（t=-0．780、-0．460,P〉0．05）。低剂量APS组大鼠caspase-3蛋白阳性RGCs百分比及RGCs凋亡百分率均明显低于模型对照组（caspase-3蛋白：F=87．710,P=0．001;RGCs凋亡：F=272．840,P〈0．01）,差异均有统计学意义（t=-11．700、-8．600,P〈0．01）,高剂量APS组与模型对照组比较caspase-3蛋白阳性RGCs百分比及RGCs凋亡百分率的差异均有统计学意义（t=-7．900、-6．400,P〈0．05）。结论500mg／kgAPS可有效抑制急性高眼压模型大鼠的视网膜水肿及RGCs的凋亡率,对急性高眼压大鼠的RGCs有保护作用。     

白皮杉醇对青光眼大鼠视网膜神经节细胞的保护作用

目的 观察白皮杉醇对青光眼大鼠视网膜神经节细胞(retinal ganglion cells,RGCs)的保护作用并探讨其可能的机制.方法 将40只大鼠随机分为对照组、模型组、白皮杉醇低剂量组和白皮杉醇高剂量组.采用光凝法建立青光眼大鼠模型,白皮杉醇低、高剂量组分别给予100 mg·kg-和200 mg·kg-1白皮杉醇灌胃.测量各组大鼠眼压;荧光金逆行标记各组大鼠RGCs并计数;HE染色观察各组大鼠视网膜组织病理形态;免疫印迹法检测各组大鼠视网膜组织中磷酸化原癌基因蛋白Jun(proto oncogene protein Jun,c-Jun)氨基末端激酶(phosphorylation c-Jun N-terminal kinase,p-JNK)、磷酸化c-Jun(phosphorylation c-jun,p-c-Jun)、磷酸化细胞外调节蛋白激酶(phosphorylation extracellular regulated protein kinases,p-ERK)、磷酸化丝裂原活化蛋白激酶p38 (phosphorylation mitogen activated protein kinases p38,p-p38 MAPK)和肿瘤坏死因子-α(tumor necrosis factor-alpha,TNF-α)蛋白表达.结果 模型组眼压、p-JNK、p-c-Jun、p-ERK、p-p38 MAPK和TNF-α蛋白表达均显著高于对照组(均为P＜0.05),RGCs低于对照组(P＜0.05),视网膜组织结构可见空泡和水肿样改变.与模型组相比,白皮杉醇低剂量组和白皮杉醇高剂量组大鼠RGCs显著增多(P =0.003、P=0.002),视网膜组织水肿和空泡样状况改善,p-JNK、p-c-Jun、p-ERK、p-p38 MAPK和TNF-α蛋白表达明显减少(均为P＜0.05),且高剂量组的改善作用更为显著.结论 白皮杉醇具有保护青光眼大鼠RGCs的作用,其作用机制可能与抑制MARK信号通路有关.     

The involvement of matrix metalloproteinases 2 and 9 in rat retinal ischemia

Background The involvement of matrix metalloproteinases (MMPs) in ischemic tissue damage and remodeling has been reported by many investigators. Our study was designed to investigate the involvement of MMPs and of tissue inhibitors of metalloproteinases (TIMPs) in rat retinal ischemic injury, the effect of nitric oxide synthase (NOS) inhibitors on MMPs’ activity in this model and whether minocycline (an MMP inhibitor) is protective in retinal ischemia. Methods Ninety-four rats were used in the study. Ischemia was induced by 90 min elevation of intraocular pressure. MMPs’ activities and the effect of NOS inhibitors [aminoguanidine (AG) or N-nitro-L-arginine (NNA)] and minocycline on MMPs’ activities were assessed by zymography and TIMPs expression by Western analysis. Morphological damage was quantified by morphometry of hematoxylin and eosin-stained retinal sections. Results Retinal extracts exhibited activities of proMMP-9 and proMMP-2. The activity of proMMP-9 increased immediately post ischemia (PI) and peaked to 4.6 times that of normal untreated controls in ischemic retinas and to 2.6 times that of controls in retinas of fellow sham-treated eyes at 24 h PI. The relative amount of TIMP-1 increased to 1.9-fold following ischemia and 2.5-fold in fellow sham-treated eyes at 24 h PI. ProMMP-2 activity increased more than two-fold immediately, at 24 h and at 48 h PI in ischemic retinas, and insignificantly in fellow sham-treated eyes. Treatment with 25 mg/kg AG or NNA caused a non-significant increase in proMMP-9 activity at 24 h PI (3.7- and 2.9-fold, respectively, p>0.6). There was no effect of AG or NNA on the activity of proMMP-2. Minocycline significantly attenuated the retinal ischemic damage, primarily by partially preserving ganglion cells and the inner plexiform layer. Minocyline (0.5 mg/ml or 5 mg/ml) inhibited MMPs’ activities in ischemic retinal extracts in vitro. Conclusions MMPs participated in morphological ischemic damage to rat retina. Treatment with minocycline dramatically attenuated damage to the retina.     

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.     

Neuroprotective effects of brimonidine against transient ischemia-induced retinal ganglion cell death: a dose response in vivo study

The purpose of this study was to investigate the dose-response effects of topically administered brimonidine (BMD) on retinal ganglion cell (RGC) survival, short and long periods of time after transient retinal ischemia. In adult Sprague-Dawley rats, RGCs were retrogradely labeled with the fluorescent tracer fluorogold (FG) applied to both superior colliculi. Seven days later, the left ophthalmic vessels were ligated for 90 min. One hr prior to retinal ischemia, two 5 microl drops of saline alone or saline containing 0.0001, 0.001, 0.01 or 0.1% BMD were instilled on the left eye. Rats were processed 7, 14 or 21 days later and densities of surviving RGCs were estimated by counting FG-labeled RGCs in 12 standard regions of each retina. The following have been found. (1) Seven days after 90 min of transient ischemia there is loss of approximately 46% of the RGC population. (2) topical pre-treatment with BMD prevents ischemia-induced RGC death in a dose-dependent manner. Administration of 0.0001% BMD resulted in the loss of approximately 37% of the RGC population and had no significant neuroprotective effects. Administration of higher concentrations of BMD (0.001 or 0.01%) resulted in the survival of 76 or 90%, respectively, of the RGC population, and 0.1% BMD fully prevented RGC death in the first 7 days after ischemia. (3) Between 7 and 21 days after ischemia there was an additional slow cell loss of approximately 25% of the RGC population. Pre-treatment with 0.1% BMD also reduced significantly this slow cell death. These results indicate that the neuroprotective effects of BMD, when administered topically, are dose-dependent and that the 0.1% concentration achieves optimal neuroprotective effects against the early loss of RGCs. Furthermore, this concentration is also effective to diminish the protracted loss of RGCs that occurs with time after transient ischemia.     

Ocular hypertension impairs optic nerve axonal transport leading to progressive retinal ganglion cell degeneration

Ocular hypertension (OHT) is the main risk factor of glaucoma, a neuropathy leading to blindness. Here we have investigated the effects of laser photocoagulation (LP)-induced OHT, on the survival and retrograde axonal transport (RAT) of adult rat retinal ganglion cells (RGC) from 1 to 12 wks. Active RAT was examined with fluorogold (FG) applied to both superior colliculi (SCi) 1 wk before processing and passive axonal diffusion with dextran tetramethylrhodamine (DTMR) applied to the optic nerve (ON) 2 d prior to sacrifice. Surviving RGCs were identified with FG applied 1 wk pre-LP or by Brn3a immunodetection. The ON and retinal nerve fiber layer were examined by RT97-neurofibrillar staining. RGCs were counted automatically and color-coded density maps were generated. OHT retinas showed absence of FG⁺ or DTMR⁺RGCs in focal, pie-shaped and diffuse regions of the retina which, by two weeks, amounted to, approximately, an 80% of RGC loss without further increase. At this time, there was a discrepancy between the total number of surviving FG-prelabelled RGCs and of DMTR⁺RGCs, suggesting that a large proportion of RGCs had their RAT impaired. This was further confirmed identifying surviving RGCs by their Brn3a expression. From 3 weeks onwards, there was a close correspondence of DTMR⁺RGCs and FG⁺RGCs in the same retinal regions, suggesting axonal constriction at the ON head. Neurofibrillar staining revealed, in ONs, focal degeneration of axonal bundles and, in the retinal areas lacking backlabeled RGCs, aberrant staining of RT97 characteristic of axotomy. LP-induced OHT results in a crush-like injury to ON axons leading to the anterograde and protracted retrograde degeneration of the intraocular axons and RGCs.     

Y-27632, a Rho-associated protein kinase inhibitor,attenuates neuronal cell death after transient retinal ischemia

Purpose Transient retinal ischemia induces the death of retinal neuronal cells. Postischemic damage is associated with the infiltration of leukocytes into the neural tissue through vascular endothelia. The current study aimed to investigate whether this damage was attenuated by the inhibition of Rho/ROCK (Rho kinases) signaling, recently shown to play a critical role in the transendothelial migration of leukocytes. Methods Y-27632, a selective inhibitor of ROCK, was injected intravitreally into rat eyes with transient retinal ischemia. Cell loss of the ganglion cell layer (GCL) and thinning of the inner plexiform layer (IPL) with and without the administration of Y-27632 were evaluated by histological anaysis, TUNEL assay and retrograde labeling of retinal ganglion cells (RGCs). To examine the attenuation of leukocyte infiltration in postischemic retinas with the administration of Y-27632, silver nitrate staining and immunohistochemistry using an anti-LCA antibody were performed. Results Cell loss of the GCL and thinning of the IPL were significantly attenuated when 100 nmol Y-27632 was administered within three hours of the induction of ischemia. TUNEL assay and retrograde labeling of RGCs showed a decreased number of apoptotic cells and an increased number of RGCs in Y-27632-injected retinas. Moreover, silver nitrate staining and immunohistochemical analysis using an anti-LCA antibody showed that Y-27632 injection dramatically inhibited leukocyte infiltration and endothelial disarrangement. Conclusions Our data suggest that inhibition of Rho/ROCK signaling offers neuroprotective therapy against postischemic neural damage, by regulating leukocyte infiltration in the neural tissue.     

Neuroprotective effects of alpha(2)-selective adrenergic agonists against ischemia-induced retinal ganglion cell death

PURPOSE: To investigate in adult rats the effects of two alpha(2)-selective adrenergic agonists (alpha(2)-SAs; AGN 191103 and AGN 190342) on retinal ganglion cell (RGC) survival after transient retinal ischemia. METHODS: RGCs were labeled with a Fluorogold (FG) tracer applied to both superior colliculi. Seven days later, the left ophthalmic vessels were ligated for 60 or 90 minutes. In one group, a single dose of saline or one alpha(2)-SA was administered intraperitoneally (IP) or topically 1 hour before ischemia. In another group, a single dose of AGN 190342 was administered IP, 1, 2, 4, 24, or 72 hours after ischemia. Rats were processed 7, 14, or 21 days later. Densities of surviving RGCs were estimated by counting FG-labeled cells in 12 standard retinal areas. RESULTS: Seven days after 60 or 90 minutes of retinal ischemia, death had occurred in 36% or 47%, respectively, of the RGC population, and by 21 days the loss of RGCs amounted to 42% or 62%, respectively. Systemic pretreatment with an alpha(2)-SA resulted in enhanced survival of ischemic-injured RGCs. Topical pretreatment with an alpha(2)-SA prevented up to 100% of the ischemia-induced RGC loss. Pretreatment with an alpha(2)-SA abolished the secondary slow RGC loss that occurred between days 7 and 21 after ischemia. When administered shortly after ischemia (up to 2 hours) AGN 190342 rescued substantial proportions of RGCs destined to die and diminished slow RGC death. CONCLUSIONS: Pretreatment and early posttreatment with an alpha(2)-SA induces marked long-lasting neuroprotective in vivo protection against ischemia-induced cell death in RGCs.