血红素氧合酶-1与视网膜

血红素氧合酶(heme oxygenase,HO)是体内唯一一种催化血红素分解代谢的限速酶,他可以氧化降解血红素,将其分解为一氧化碳、自由铁和胆绿素。其中诱导型血红素氧合酶-血红素氧合酶-1(HO-1)是机体最重要的内源性保护物质之一,广泛参与组织细胞的抗氧化应激损伤,被认为是在细胞受损维持其氧化和抗氧化动态平衡的关键因素,本文就HO-l与视网膜的关系作如下综述。     

Localization of heme oxygenase-2 and modulation of cGMP levels by carbon monoxide and/or nitric oxide in the retina

Heme oxygenase-2 (HO-2) synthesizes carbon monoxide (CO), a modulator of soluble guanylate cyclase (sGC). To examine this signal transduction pathway in the retina, we immunocytochemically localized HO-2, and investigated the effects of CO on cGMP levels. In turtle, HO-2-like immunoreactivity (-LI) was in all photoreceptors, some amacrine cells, and in numerous bipolar and ganglion cells. HO-2-LI colocalized with sGC activity in many cells. In rat, HO-LI was found only in the inner retina, in ganglion and amacrine cells. In turtle, stimulation with CO alone primarily increased cGMP-LI in bipolar cells in the visual streak. Stimulation with a combination of CO and nitric oxide (NO) dramatically increased cGMP-LI throughout the retina, in comparison to the smaller increases seen with NO or CO alone. These data suggest that CO is an endogenous modulator of the sGC/cGMP signaling pathway in many retinal neurons, and can dramatically amplify NO-stimulated increases in cGMP.     

Interleukin-1beta mediates ischemic injury in the rat retina.

Two types of experiment were performed to examine the role of interleukin-1beta in ischemia-induced damage in the rat retina. In the in vivo study, enzyme-linked immunosorbent assay was used to investigate the expression of immunoreactive interleukin-1beta in the rat retina following a hypertension-induced ischemia/reperfusion, while the effect of a recombinant human interleukin-1 receptor antagonist or an anti-interleukin-1beta neutralizing antibody on the ischemia-induced damage was examined histologically. A transient increase in the expression of immunoreactive interleukin-1beta was observed in the retina 3-12 hr after reperfusion, and morphometric evaluation at 7 days after the ischemia showed a decrease in cell numbers in the ganglion cell layer and a decreased thickness of the inner plexiform layer with no change in the other retinal layers. Intravitreal injection of interleukin-1 receptor antagonist (1 or 10 ng per eye) or anti-interleukin-1beta antibody (50 or 500 ng per eye) 5 min before the onset of the ischemia reduced the damage. In the in vitro study, interleukin-1 receptor antagonist (500 ng ml(-1)) significantly reduced glutamate-induced neurotoxicity in rat cultured retinal neurons. These results suggest that interleukin-1 plays an important role in mediating ischemic and excitotoxic damage in the retina, and that interleukin-1 inhibitors may be therapeutically useful against neuronal injury caused by optic nerve or retinal diseases such as glaucoma and central retinal artery or vein occlusion.     

血红素氧合酶-1与糖尿病性视网膜病变:氧化应激的作用(英文)

糖尿病性视网膜病变(diabetic retinopathy,DR)是糖尿病最常见且严重的微血管并发症之一,其发病机制复杂,是多因素、多阶段作用的结果。近年研究表明,DR的发生与氧化应激密切相关,抗氧化治疗有助病情改善。血红素氧合酶-1(heme oxygenase-1,HO-1)是一种广泛存在的抗氧化防御酶,可对抗氧化应激造成的损伤,具有重要的生理作用。研究表明,在高血糖环境中,视网膜内HO-1的表达被诱导增高,且通过人为调节HO-1的表达水平可以加速或延缓病情的进展,提示将HO-1应用于DR的诊治有良好的应用前景。本文从氧化应激的角度对二者加以概述。     

Quantitation of ischemic damage in the rat retina.

In order to determine thresholds for irreversible cellular injury in the rat retina, timed acute no-flow ischemic episodes of 30-180 min duration were produced by elevation of intraocular pressure (IOP) above systolic pressure. Quantitation of irreversible degeneration and cell loss following a 2-week post-ischemic interval was performed by computer-assisted measurements from histologic sections. Alterations of thickness of retinal layers and linear cell density were determined for ischemia of selected durations (30, 60, 80, 90, 120 and 180 min). Different thresholds were evident for inner and outer retinal damage. Neurons of the inner nuclear layers showed extensive loss with episodes at 60 min. Decrease in the thickness of the inner plexiform layer provided the best index of this inner nuclear damage. The outer retina was more resistant, with photoreceptors showing extensive damage only after 90 min in conjunction with pigment epithelial metaplasia and degeneration. Two-hour episodes produced full-thickness degeneration with loss of pigment epithelium and sparing of the peripheral retina. Greater sensitivity of the inner retina suggested problems with restoration of the retinal circulation. Horseradish peroxidase infusions did reveal central microcirculatory defects in retinal wholemounts of some specimens with episodes longer than 60 min. Refinements of the methods resulted in outcomes sufficiently reproducible for quantitative assessment of acute ischemic injury. The rat retina provides an economical basic tissue model of acute ischemic injury affecting neurons, glia, and microvasculature. Quantitation of this injury promises great utility in testing agents with potentially protective effects on acute ischemic injury.     

Heme oxygenase-1 induced in muller cells plays a protective role in retinal ischemia-reperfusion injury in rats

PURPOSE: To investigate the protective roles played by heme oxygenase (HO)-1 and -2 in the rat retina after ischemia-reperfusion injury. METHODS: Retinal ischemia was induced in rats by increasing the intraocular pressure to 110 mmHg for 60 minutes. The expression of HO-1 and -2 in the retina was determined by Western blot, real-time polymerase chain reaction (PCR), and immunohistochemistry. To inhibit the upregulation of HO-1, short interfering (si)RNA of HO-1 was injected intravitreally before ischemia and that of green fluorescent protein (GFP) was used as the control. Muller cell damage was assessed by counting the number of S-100-positive cells. The number of macrophages invading the retina was determined by counting the number of ED-1-positive cells. RESULTS: The expression of HO-1 mRNA and protein was upregulated at 6 hours after reperfusion and peaked at 12 to 24 hours, whereas that of HO-2 was not altered. HO-1 immunoreactivities were detected in Muller cells at 24 hours after reperfusion, and HO-2 immunoreactivities were detected in retinal cells. The HO-1 expression in the retina treated with siRNA of HO-1 was reduced at 12 and 24 hours after reperfusion compared with that injected with siRNA of GFP. The number of S-100-positive cells at 24 hours after reperfusion decreased significantly in retinas treated with HO-1 siRNA (P <0.01). The number of macrophages that had infiltrated the retina was increased in retinas pretreated with the siRNA of HO-1 compared with those treated with siRNA of GFP. On day 14 after reperfusion, HO-1 siRNA-treated retinas showed severe retinal injury and destruction of the retinal architecture. CONCLUSIONS: HO-1 promotes the survival of Muller cells after ischemia-reperfusion injury. Because inhibition of the upregulation of HO-1 resulted in an infiltration of inflammatory cells and destruction of the retina, the authors conclude that HO-1 induced in Muller cells plays a protective role in retinal ischemia-reperfusion.     

Transient ischemic injury in the rat retina caused by thrombotic occlusion-thrombolytic reperfusion

PURPOSE: To establish a clinically relevant model of transient retinal ischemia by thrombotic occlusion-thrombolytic reperfusion of the central retinal artery of the rat. METHODS: Thrombus was photochemically induced in the central retinal artery by the combination of intravenous injection of photo-sensitive dye, rose bengal, and green laser irradiation focused on the artery. Transient retinal ischemia for 60 minutes was achieved by a subsequent systemic administration of tissue-type plasminogen activator to reperfuse the occluded vessel. Samples of retinas were excised from the animals killed 3, 9, 12, 24, 48, and 78 hours after the reperfusion. The experimental data were processed using the TdT-dUTP terminal nick-end labeling (TUNEL) method to detect apoptotic cells. RESULTS: The transient retinal ischemia caused time-sequential apoptotic changes in the retinal cells as evaluated by counting the number of TUNEL-positive cells. The most remarkable changes occurred in the central area of retina, and further on the sections taken 24 hours after reperfusion. The peripheral area was less affected, and the outer nuclear cell layer was almost unaffected throughout the observation period. CONCLUSIONS: The proposed method to cause retinal transient ischemia is highly reproducible, and it is easy to simulate the progress and topographical distribution of retinal changes observed in the clinical cases of central retinal arterial occlusion and its subsequent thrombolytic reperfusion. This may provide a useful tool for constructing the effective thrombolytic strategies against the central retinal arterial occlusion and for evaluating the effects of neuroprotective agents.     

Signal transduction mechanisms involved in ischemic preconditioning in the rat retina in vivo

Ischemic preconditioning (IPC) protects the rat retina against the injury that ordinarily follows severe ischemia. We showed previously that release of adenosine and de novo protein synthesis were required for IPC protection. The mechanisms of IPC were studied in the rat retina by examining the signal transduction mediators responsible, in particular, those theorized to be downstream of adenosine receptors. In addition, we examined the hypothesis that nitric oxide and hydroxyl radicals were involved in the IPC protective phenomenon.Retinal ischemia was produced for 60 min in ketamine/xylazine-anesthetized Sprague-Dawley rats, and recovery was measured using electroretinography. We tested the effects on the protective effect of IPC resulting from antagonism of protein kinase C, potassium ATP channels, nitric oxide synthase, or hydroxyl radicals. The effects of the inhibition of de novo protein synthesis or of protein kinase C, and blockade of potassium ATP channels on the mimicking of IPC by adenosine receptor agonists was examined.IPC protection was strongly attenuated by inhibition of protein kinase C and by blockade of potassium ATP channels, but unaffected by the inhibition of hydroxyl radicals. Blockade of nitric oxide synthase produced a trend toward enhancement of IPC protection. Mimicking of IPC protection by adenosine receptor agonists was inhibited by blockade of protein synthesis or of protein kinase C, as well as by potassium ATP channel antagonism.These results demonstrate that protein kinase C and potassium ATP channels are mediators of the protective effect produced by IPC. In addition, the results show that stimulation of adenosine receptor subtypes A1 and A2a is responsible for IPC protection via downstream stimulation of protein kinase C, the opening of potassium ATP channels, and de novo protein synthesis.     

Expression of osteopontin in the rat retina: effects of excitotoxic and ischemic injuries

PURPOSE: The cytokine osteopontin (OPN) has been localized to the retinal ganglion cell layer in the normal rodent retina, prompting the suggestion that it could serve as a useful marker for identifying and quantifying such neurons in models of retinal and optic nerve neurodegeneration. In the present study, we characterized the time course and cellular localization of OPN expression in the rat retina after excitotoxic and ischemic injuries. METHODS: Excitotoxicity and ischemia-reperfusion experiments were performed by using standard techniques. Rats were killed at various time points, and the retinas were removed either for mRNA analysis or to be processed for immunohistochemistry. RESULTS: In the normal retina, double-labeling immunofluorescence indicated that OPN is expressed by the majority of, if not all, RGCs, since OPN was associated with more cells than Brn-3, but was colocalized with Thy1.1. NMDA, kainic acid, and ischemia-reperfusion all caused decreases in the total retinal levels of Thy1 and Brn-3 mRNAs, reflecting injury to RGCs, but a dramatic, short-lived upregulation in OPN mRNA. The source of the increased OPN signal after excitotoxic-ischemic insults is unlikely to be injured RGCs, as no alteration in the intensity of OPN immunostaining in RGCs was apparent. Instead, additional cells, mostly contained within the IPL, were identified as positive for OPN. Double-labeling immunofluorescence showed that ED1 always colocalized with OPN in these cells, indicating their status as activated microglia. CONCLUSIONS: OPN is exclusively expressed by RGCs in the physiological retina, but in response to retinal neurodegeneration is synthesized de novo by endogenous, activated microglia.     

葡萄糖调节蛋白在大鼠视网膜缺血再灌注损伤中的时相性表达及意义

目的 检测葡萄糖调节蛋白(glucose-regulated protein 78,grp78)在大鼠视网膜缺血再灌注后不同时期的表达情况,探讨内质网应激在视网膜缺血再灌注损伤中的作用及机制.方法 36只Wistar大鼠随机分为6组:缺血再灌注6 h、12 h、24 h、48 h和72 h组,以及正常对照组,每组6只大鼠.缺血组大鼠均行单眼生理盐水前房高压灌注(110 mmHg×60 min,1 kPa=7.5 mmHg)的方法 建立视网膜缺血再灌注模型,用免疫组织化学和半定量RT-PCR方法 检测grp78 在视网膜中的定位及时相性表达,取各组平均光密度值进行统计分析.结果 grp78在正常大鼠视网膜中少量表达,主要分布于视网膜内核层和神经节细胞层,免疫组织化学和RT-PCR检测均发现在视网膜缺血再灌注后6 h grp78的表达量开始升高(A值为0.778±0.004,与正常对照组0.756±0.007相比P＜0.05);再灌注后24 h其表达量达到峰值(A值为0.851±0.040),与正常对照组比较P＜0.01,与12 h组(A值为0.799±0.010)相比P＜0.01;再灌注48 h grp78表达开始下降(A值为0.825±0.007,与24 h相比P＜0.05),但仍高于正常对照组(P＜0.01);72 h组grp78的表达与正常组相比,差异无统计学意义.结论 grp78参与了大鼠视网膜缺血再灌注损伤机制,若能在损伤早期内质网应激环节上加以干预,可为临床治疗和干预提供一定的理论依据.     

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.     

A histological analysis of the protective effect of ischemic preconditioning in the rat retina

PURPOSE: Ischemic preconditioning (IP) protects the retina from the damaging effect of subsequent ischemia in vivo. We aimed to investigate the histological alterations induced by the protective effect of IP to the retina. METHODS: The eyes of the rats were rendered ischemic by intra-ocular pressure (IOP) elevation. IP procedure consisted of producing ischemia for 5 minutes. Sham operation was similar to IP procedure except the pressure elevation. The operational eyes of sham and IP group underwent 60 minutes of ischemia 24 hours after the first procedure. The eyes contralateral to the experimental eyes made up the control group. The eyes were histologically analysed one week after the ischemia. RESULTS: The total retinal thickness of the sham group was significantly less than total retinal thickness of the control group (p < 0.001). There was not a significant difference between control and IP group regarding the total retinal thickness (p > 0.05). The thickness of the inner retinal layers of the sham group were significantly less than corresponding retinal layers of the control group (p < 0.001). The inner plexiform layer (IPL) and inner nuclear layer (INL) thickness values of the sham group were significantly less than same layers of the IP group (p < 0.001). Ganglion cell layer (GCL) thickness of the IP group was significantly less than GCL thickness of the control group (p < 0.001). IPL thickness of the IP group was significantly less than that of control group's (p < 0.05). The GCL and total retinal thickness of the IP group were significantly more than thickness of the corresponding layers of the sham group (p < 0.05). CONCLUSION: IP considerably protects inner retinal layers from subsequent ischemic damage in a high IOP ischemic model. This endogenous process could further be utilized to tailor specific neuroprotective strategies for retinal cells.     

水通道蛋白在大鼠眼组织视网膜中的分布

目的：研究水通道蛋白1（AQP1）在正常大鼠眼组织视网膜中的分布。方法：对正常Wistar鼠眼组织切片进行免疫酶组织化学方法染色，光镜观察。正常Wistar鼠肾脏的冰冻切片为阳性对照。结果：AQP1在正常Wistar鼠视网膜的内核层有明显表达，首次发现在神经节细胞层也有明显表达，阴性对照未见。结论：AQP1在视网膜的内核层及神经节细胞层的分布，可能AQP1或AQP1和AQP4协同在高眼压和视网膜缺血状态下对维持视网膜神经细胞正常功能及细胞内外水电平衡起一定的调节作用。     

视网膜缺血再灌注损伤中Ref-1的表达及bFGF对其的影响

目的研究缺血再灌注损伤视网膜组织中无嘌呤／无嘧啶核酸内切酶／氧化还原因子-1（APE／Ref—1，Ref—1）表达的变化及玻璃体腔内注射碱性成纤维细胞生长因子（bFGF）对其的影响。方法建立大鼠视网膜缺血再灌注损伤动物模型，于再灌注开始即刻玻璃体腔内注射bFGF2μg，利用SABC免疫组织化学法检测不同时段Ref—1蛋白在视网膜组织表达的变化，分析Ref—1蛋白与细胞凋亡的关系以及bFGF对其表达的影响。结果Ref—1蛋白在视网膜组织中的表达随着缺血再灌注时间的延长明显减少。bFGF治疗组Ref-1蛋白表达规律与缺血组相同，但其阳性表达率较同一时段缺血再灌注组有所增加。结论大鼠玻璃腔注射bFGF，可以上调Ref—1的表达，对视网膜缺血再灌注损伤起到修复的作用。     

Inducible nitric oxide synthase inhibitors abolished histological protection by late ischemic preconditioning in rat retina

Brief ischemia was reported to protect retinal cells against injury induced by subsequent ischemia-reperfusion with de novo protein synthesis, and this phenomenon is known as late ischemic preconditioning. The aims of the present study were to determine whether nitric oxide synthase (NOS) was involved in the mechanism of late ischemic preconditioning in rat retina using pharmacological tools. Under anesthesia with pentobarbital sodium, male Sprague-Dawley rats were subjected to 60 min of retinal ischemia by raising intraocular pressure to 130 mm Hg. Ischemic preconditioning was achieved by applying 5 min of ischemia 24 hrs before 60 min of ischemia. Retinal sections sliced into 5 microm thick were examined 7 days after ischemia. Additional groups of rats received NG-nitro-L-arginine and NG-monomethyl-L-arginin, non-selective NO synthase inhibitors, 7-nitroindazole, a neuronal NOS inhibitor, and aminoguanidine and L-N6-(1-iminoethyl) lysine, inducible NO synthase (iNOS) inhibitors before preconditioning, and were subjected to 60 min of ischemia. In the non-preconditioned group, cell loss in the ganglion cell layer and thinning of the inner plexiform and inner nuclear layer were observed 7 days after 60 min of ischemia. Ischemic preconditioning for 5 min completely protected against the histological damage induced by 60 min of ischemia applied 24 hrs thereafter. Treatment of rats with aminoguanidine and L-N6-(1-iminoethyl) lysine, but not NG-nitro-L-arginine, NG-monomethyl-L-arginine or 7-nitroindazole, wiped off the protective effect of ischemic preconditioning. The inhibitory effect of aminoguanidine was abolished by L-arginine, but not D-arginine. The results in the present study suggest that NO synthesized by iNOS is involved in the histological protection by late ischemic preconditioning in rat retina.