Mouse models of retinal ischemic tolerance

PURPOSE: A brief period of noninjurious retinal ischemia, termed preconditioning, has been documented in rats to afford transient protection from retinal ischemic injury, a phenomenon known as ischemic tolerance. The present study was undertaken to develop and systematically characterize mouse models of ischemic tolerance. METHODS: Retinal ischemic injury was caused by elevating intraocular pressure for 30, 45, or 60 minutes in chloral hydrate-anesthetized ND4 Swiss-Webster mice. Random animals were preconditioned 24 hours earlier with either 5 minutes of retinal ischemia or by exposing conscious animals to hypoxia (11% oxygen) for 2 hours. Flash electroretinograms were recorded 1 day and 1 week after ischemia. At 1 or 4 weeks after ischemia, eyes were perfusion fixed for microscopic examination and quantification of layer thickness and cell counts. RESULTS: Retinal ischemia resulted in significant, duration-dependent reductions in inner retinal layer thickness and cell loss in the inner nuclear and ganglion cell layers. A duration-dependent attenuation in a- and b-wave amplitudes was concomitantly noted. The ischemic and hypoxic preconditioning treatments significantly attenuated the ischemia-induced changes in retinal morphology and function, even after 4 weeks of recovery. Tolerance was observed at 24 hours after ischemic preconditioning, but not at 72 hours. CONCLUSIONS: Two models of retinal ischemic tolerance are presented wherein ischemic or hypoxic preconditioning afforded morphologic and functional evidence of protection from retinal ischemic injury in mice. These two murine models should be useful for studies in mutant mice to elucidate endogenous genetic and molecular mechanisms of retinal protection that may then be used to design treatments for ischemic retinopathies.     

Quantification of ischemic damage in the rat retina: a comparative study using evoked potentials, electroretinography, and histology

PURPOSE: To identify objective criteria to quantify visual function in the rat for developing therapeutic strategies to protect neuronal cells after ischemia. The impact of ocular ischemia on luminance and frequency-modulated contrast vision was compared with the function of outer retinal cells and the number of intact retinal ganglion cells (RGCs). METHOD: Ischemia was induced in Brown-Norway rats by elevating the intraocular pressure to 120 mm Hg for 30, 45, 60, and 90 minutes. Visual function was evaluated by visual evoked potentials (VEPs) in awake, freely moving rats. Retinal function was analyzed with scotopic and photopic electroretinography (ERG). RGCs were quantified in retinal flatmounts after postischemic injection of tracer into the superior colliculus. RESULTS: The response to flicker stimulation in VEP recordings decreased as the ischemic episodes increased. The susceptibility to ischemic damage was more pronounced when potentials were evoked with stimuli at higher frequencies. In ERG recordings, ischemia reduced oscillatory potentials and photopic flicker responses more intensely than scotopic a- and b-waves. In counting the RGCs, the reduced cell density correlated significantly with all electrophysiological parameters. The duration of ischemia with half-maximal inhibitory effect was between 36 and 58 minutes for VEPs and between 36 and 41 minutes for ERG, and it was 51 minutes for RGCs. CONCLUSIONS: The amounts of reduction in VEPs, ERG, and RGCs differed as the duration of ischemia increased. The electrophysiological parameters presented in this study may serve as a useful addition to morphologic evaluations in future neuroprotection studies in vivo.     

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。     

盐酸氟桂嗪对家兔视网膜缺血—再灌注损伤保护作用的ERG观察

目的 动态观察盐酸氟桂嗪对家兔视网膜缺血－再灌注损伤的保护作用。方法 应用前房灌注加压法使前房内压力升高至16kPa。维持１h后再灌注,再灌注后第2、7、14天分别记录其ERG,与缺血前ERG相比,观察b波的变化。药物治疗组在造成缺血前12h,缺血－再灌注即刻及再灌注后12h静脉给予药物治疗。结果 药物治疗组ERG的b波与正常眼相比无明显差异。结论 盐酸氟桂嗪对视网膜缺血－再灌注损伤有保护作用。     

Neuroprotective effects of D-allose against retinal ischemia-reperfusion injury

PURPOSE: To investigate the effect of D-allose, a rare sugar, against ischemia reperfusion injury in the rat retina. METHODS: Retinal ischemia was induced by increasing intraocular pressure to 130 mm Hg and maintaining that level for 45 minutes. Morphometric studies were performed to study the effect of D-allose on the histologic changes induced by ischemia in the rat retina. Glutamate release from the rat retina and intravitreal P(O2) profiles were monitored during and after ischemia with a microdialysis biosensor and oxygen-sensitive microelectrodes. The release of hydrogen peroxide stained with diaminobenzidine hydrochloride was monitored by an in vitro retinal ischemia model. RESULTS: Seven days after the ischemia, significant reductions in both the number of ganglion cells and the thickness of the inner plexiform layer were observed. Pretreatment with D-allose significantly inhibited the ischemic injury of the inner retina. A large release of glutamate occurred during the ischemia. After the recirculation, glutamate levels were increased again and reached a maximum in approximately 20 minutes. The increases in extracellular glutamate during and after ischemia tend to be suppressed by administration of d-allose. d-Allose attenuated the increase in intravitreal P(O2) during reperfusion. After the ischemia, production of hydrogen peroxide was detected within approximately 30 minutes. D-allose suppressed the production of hydrogen peroxide. CONCLUSIONS: These results suggest that D-allose may protect neurons by decreasing extracellular glutamate and attenuating oxidative stress in ischemic insult.     

Inhibitory effect of ischemic preconditioning on leukocyte participation in retinal ischemia-reperfusion injury

PURPOSE: Recent reports have shown that ischemic preconditioning induces strong protection against retinal damage by subsequent prolonged ischemia and that this protection is mediated by mechanisms involving the adenosine A1 receptor. This study was designed to evaluate quantitatively the effects of ischemic preconditioning on leukocyte-mediated reperfusion injury after transient retinal ischemia and to define the role of the adenosine A1 receptor in these effects. METHODS: Transient retinal ischemia was induced in male rats by temporary ligation of the optic nerve. Ischemic preconditioning (5 minutes of ischemia) was induced 24 hours before 60 minutes of ischemia. The adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) was administered intramuscularly immediately after ischemic preconditioning. Leukocyte behavior in the retina after 60 minutes of ischemia was evaluated in vivo with acridine orange digital fluorography. RESULTS: Ischemic preconditioning inhibited leukocyte rolling. The maximum number of rolling leukocytes was reduced to 3.0% at 12 hours after reperfusion (P < 0.01). Subsequent leukocyte accumulation was also decreased with ischemic preconditioning. The maximum number of accumulated leukocytes was reduced to 22.6% at 24 hours after reperfusion (P < 0.01). These inhibitory effects were suppressed by administration of DPCPX (P < 0.0001). The numbers of rolling leukocytes at 12 hours after reperfusion and accumulated leukocytes at 24 hours after reperfusion were 102.7% (NS) and 83.4% (P < 0.01), respectively, compared with the number without ischemic preconditioning. CONCLUSIONS: The present study demonstrates the inhibitory effects of ischemic preconditioning on leukocyte rolling and subsequent leukocyte accumulation during retinal ischemia-reperfusion injury. Furthermore, the adenosine A1 receptor may play an important role in these inhibitory effects.     

Protective effect of trimetazidine in a model of ischemia-reperfusion in the rat retina

Trimetazidine is an anti-ischemic agent which is frequently prescribed as a prophylactic treatment of episodes of angina pectoris and as a symptomatic treatment of vertigo and tinnitus. It has also shown beneficial effects in models of visual dysfunction, but the mechanism(s) by which this occurs is as yet undefined. The present study was intended to evaluate the influence of trimetazidine on retinal damage induced by ischemia-reperfusion in a rat model. Retinal ischemia was induced by increasing intraocular pressure to 160 mm Hg for 60 min. Trimetazidine or buffer controls were administered 3 days before the ischemia or in the postischemic period. The degree of retinal damage was assessed after 15 and 30 days of reperfusion after the ischemic insult by histopathologic study according to Hughes' quantification of ischemic damage. Retinal ischemia led to significant reductions in thickness and cell number, mainly in the inner retinal layers. The results from the study demonstrate that treatment with intraperitoneally injected trimetazidine conferred significant protection against retinal ischemic damage. Better results were obtained in the pretreatment group after 15 days of reperfusion. Trimetazidine protects the rat retina from pressure-induced ischemic injury and might be considered a potential therapeutic modality for combating retinal ischemia.     

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.     

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.     

Involvement of calpain isoforms in ischemia-reperfusion injury in rat retina

PURPOSE: Much evidence has accumulated suggesting that activation of calpain causes neuronal cell death in ischemic brain. However, little is known about the involvement of calpain in retinal cell death in ischemic injury. Thus, the purpose of present study was to investigate the involvement of calpain isoforms (m- and mu-calpain) in ischemia-reperfusion injury in retina from rat. METHODS: Retinal ischemia was produced by occlusion of the central retinal artery for one hour, and this was followed by reperfusion for seven days. Calpain mRNAs, calpain activities, total calcium content and proteolysis of alpha-spectrin were determined in retina. Effect of a calpain inhibitor SJA6017 was histologically tested in retinal injury after ischemia-reperfusion. RESULTS: Following retinal ischemia, most of cells in the ganglion cell layer were sloughed off by day 1 after reperfusion, followed by loss of cells in the inner plexiform layer on day 3 and loss of cells in the inner nuclear layer by day 5. These morphologic changes were accompanied by several presumptive biochemical indicators of calpain activation: increased calcium, proteolysis of alpha-spectrin (a sensitive substrate for calpains), decreased caseinolytic activity for both calpains (suggesting calpain activation followed by autolytic degradation), increased mRNA levels for mu-calpain and calpastatin - the endogenous inhibitor of calpains - and decreased mRNA levels for mu-calpain. Moreover, the calpain inhibitor SJA6017 protected the reduction of cell density in the ganglion cell layer after ischemia-reperfusion. CONCLUSION: These results suggest that calpain isoforms may play an important role in neuronal cell death induced by retinal ischemia-reperfusion injury in rat.     

Administration of 17beta-estradiol attenuates retinal ischemia-reperfusion injury in rats

PURPOSE: Accumulating evidence has suggested that 17beta-estradiol exerts protective effects against ischemic damage in various organs. In addition, leukocytes that accumulate in postischemic tissues are thought to play a central role in ischemia-reperfusion injury. This study was designed to evaluate quantitatively the inhibitory effects of 17beta-estradiol on leukocyte accumulation during ischemia-reperfusion injury and on subsequent retinal damage after transient retinal ischemia. METHODS: Transient (60 minutes) retinal ischemia was induced in male rats by temporary ligation of the optic nerve. Thirty minutes before induction of ischemia, 17beta-estradiol (0.1 mg/kg) was administered intraperitoneally. At 6, 12, 24, and 48 hours after reperfusion, leukocyte accumulation in the retina was evaluated in vivo by means of acridine orange digital fluorography. Histologic and electroretinographic (ERG) studies were carried out to evaluate retinal damage. RESULTS: Treatment with 17beta-estradiol significantly inhibited postischemic leukocyte accumulation; the maximum number of accumulating leukocytes was reduced by 35.7% at 24 hours after reperfusion (P = 0.01). Histologic examination showed that administration of 17beta-estradiol significantly reduced retinal damage, which was most obvious in the inner retina, 168 hours after reperfusion (P = 0.0001). ERG studies at 12 and 168 hours after reperfusion showed that recovery of the b-wave amplitude was significantly improved with treatment of 17beta-estradiol (P = 0.023). CONCLUSIONS: The present study demonstrated the inhibitory effects of 17beta-estradiol on leukocyte accumulation and subsequent tissue injury during retinal ischemia-reperfusion injury.     

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.     

Induction of a 55-kDa PKN cleavage product by ischemia/reperfusion model in the rat retina

PURPOSE: To investigate the physiological role of a protein kinase, PKN, and its relation to apoptosis in vivo. METHODS: An ischemia/reperfusion model of the rat retina was created by elevating the intraocular pressure. Retinal samples were obtained after ischemic insult (15-45 minutes) followed by reperfusion (1-7 days). The effect of ischemia on the fragmentation of PKN was examined by immunoblotting and immunocytochemical procedures using the antibody against PKN. N-methyl-D-aspartate (NMDA) or a caspase-3 inhibitor (DEVD-CHO) was administered intravitreally to investigate its effect on the induction of PKN fragmentation. The retinal cell loss in each sample was evaluated by toluidine blue staining. RESULTS: Ischemia induced a 55-kDa PKN cleavage fragment corresponding to the molecular size of the constitutively active fragment of PKN. The appearance of the cleavage fragment depended on the duration of reperfusion and correlated with the occurrence of retinal cell loss. Immunocytochemical analysis revealed that ischemia increased PKN immunoreactivity in the inner layers of the retina. DEVD-CHO significantly inhibited the appearance of the 55-kDa fragment and protected against retinal cell loss. The administration of NMDA also induced cleavage of PKN. CONCLUSIONS: PKN is specifically cleaved by caspase-3 or a related protease during apoptosis in vivo, and PKN cleavage is at least partially initiated by activation of the NMDA receptor.     

小鼠视网膜缺血-再灌注后核因子-κB的激活

目的 研究小鼠视网膜缺血-再灌注所致视网膜神经细胞凋亡中,核因子-κB的表达。方法 通过升高小鼠眼内压造成视网膜缺血,用计算机图像分析方法测量视网膜再灌注后神经细胞凋亡的比例和视网膜厚度的改变。免疫组化标记核因子-κB p65亚单位,并与原位缺口末端标记（TUNEL）做双重荧光标记,分析核因子-κB的表达与细胞凋亡之间的时相关系。结果 视网膜缺血-再灌注后最初24h,视网膜内层厚度增加,至168h,厚度显著减少。再灌注后6h,神经节细胞和内核细胞层中p65的免疫表达增强,至24h达到高峰,这一过程与TUNEL标记的时相一致。结论 视网膜缺血-再灌注损伤后,核因子-κB的激活对于视网膜神经细胞的凋亡有重要作用,对于其起促进凋亡还是抑制凋亡的作用,则有待于进一步研究。     

Lipid peroxidation and peroxynitrite in retinal ischemia-reperfusion injury

PURPOSE: To investigate whether lipid peroxides play a role in retinal cell death due to ischemia-reperfusion injury, whether recombinant human thioredoxin (rhTRX) treatment reduces production of lipid peroxides of the retina, and whether such treatment reduces the number of cells expressing c-Jun and cyclin D1. METHODS: Retinal ischemia was induced in rats by increasing the intraocular pressure to 110 mm Hg for 60 minutes. After reperfusion, immunohistochemical staining for lipid peroxide, peroxynitrite, c-Jun, and cyclin D1 and propidium iodide (PI) staining were performed on retinal sections from animals treated intravenously with and without rhTRX, a free radical scavenger. Quantitative analyses of PI-, c-Jun-, and cyclin D1-positive cells were performed after the ischemic insult. Concentration of lipid peroxides in the retina was determined by the thiobarbituric acid assay. RESULTS: Specific immunostaining for lipid peroxides was seen in the ganglion cell layer at 6 hours after reperfusion, in the inner nuclear layer at 12 hours, and in the outer nuclear layer at 48 hours. Time course studies for PI-positive cells in the three nuclear layers coincided with those of specific immunostaining for lipid peroxides. The specific immunostaining was weakened by pre- and posttreatment with 0.5 mg of rhTRX. The number of PI-, c-Jun-, and cyclin D1-positive cells and the concentration of lipid peroxides were significantly decreased by treatment with rhTRX compared with those of vehicle-treated control rats (P: < 0. 01). CONCLUSIONS: Lipid peroxides formed by free radicals may play a role in neuronal cell death in retinal ischemia-reperfusion injury.     

缺血预处理对大鼠视网膜缺血再灌注损伤保护作用

目的：探讨缺血预处理是否对视网膜缺血再灌注损伤有保护作用及其机理,方法：利用前房灌注生理盐水形成高眼压的视网膜缺血再灌注损伤的动物模型,视网膜缺血时间为1h分别于缺血前30min、24h或72h对大鼠一只眼5min短暂缺血即预处理,24h或72h后行视网膜电图（ERG）、电镜、光镜、丙二醛（MDA）及热休克蛋白70（HSP70）检测,或者一侧眼行5min假处理,24h后行1h缺血,24h或72h再行上述检测,所有对侧眼不作处理作对照,结果：与假处理相相比,缺血前24、72h进行预处理后的大鼠视网膜光镜、电镜表现损害明显减轻,ERGb波明显恢复（P＜0．01）,MDA含量降低（P＜0．01）,缺血前30min预处理的视网膜表现严重的损害,ERGb波几安全消失,结论：缺血预处理对视网膜缺血再灌注损伤有保护作用,且有一定时限性。     

Retinal ischemia-reperfusion injury attenuated by blocking of adhesion molecules of vascular endothelium

PURPOSE: To evaluate quantitatively the effects of blocking of adhesion molecules (P-selectin or intercellular adhesion molecule-1 [ICAM-1]) on leukocyte dynamics in the retinal microcirculation in vivo during ischemia-reperfusion injury and the therapeutic efficacy of the blocking of adhesion molecules on retinal ischemia-reperfusion injury. METHODS: Retinal ischemia was induced for 60 minutes in anesthetized pigmented rats by temporary ligation of the optic nerve. P-selectin or ICAM-1 monoclonal antibody (mAb) was administered at 5 minutes before reperfusion. At 4, 12, and 24 hours after onset of reperfusion, leukocyte behavior in the retinal microcirculation was evaluated in vivo with acridine orange digital fluorography. After 7 or 14 days of reperfusion, retinal damage was evaluated histologically. RESULTS: P-selectin mAb significantly inhibited leukocyte rolling along the major retinal veins after reperfusion. Subsequently, the number of accumulated leukocytes decreased in the P-selectin-inhibited rats. ICAM-1 mAb also inhibited leukocyte accumulation during the reperfusion period in a more substantial manner than P-selectin mAb. Histologic examination demonstrated the protective effect of the blocking of P-selectin or ICAM-1. In accordance with a reduction in leukocyte accumulation, the protective effect of mAb on retinal ischemia-reperfusion injury was more substantial in ICAM-1-inhibited rats. CONCLUSIONS: The present study demonstrates the inhibitory effect of P-selectin and ICAM-1 mAb on leukocyte accumulation and subsequent tissue injury during retinal ischemia-reperfusion injury.     

Protective effect of ischemic preconditioning on retinal ischemia-reperfusion injury in rats

BACKGROUND: A short period of ischemia can induce remarkable tissue resistance to the deleterious effects of subsequent ischemia and reperfusion. We performed a study to investigate the effect of ischemic preconditioning on retinal ischemia-reperfusion injury in rats. METHODS: Ten Wistar albino rats were divided into two groups of five animals (10 eyes): one group underwent 5 minutes of ischemic preconditioning (achieved by clamping the common carotid arteries at the time of vertebral artery cauterization), and the other did not (control group). In both groups, the vertebral arteries were occluded bilaterally with an electric needle coagulator under an operating microscope. Forty-eight hours later the rats were reanesthesized, and both common carotid arteries were clamped to interrupt blood flow.The duration of ischemia was 30 minutes. The clamp was then removed to enable reperfusion for 4 hours. The animals were killed by decapitation, and retinal sections were evaluated under light and electron microscopy.The signs of ischemia-reperfusion injury (cellular degeneration, vacuolization between retinal layers, increase in retinal thickness due to edema, mononuclear cell infiltration and apoptotic cell count) were recorded. RESULTS: Light microscopy of retinal sections from rats in the ischemic preconditioning group showed a well-preserved retinal structure. The mean thickness values (and standard deviation [SD]) for the inner nuclear layer (104.0 microm [2.54 microm] vs. 49.0 microm [ 10.83 microm]) and inner plexiform layer (134.8 microm [10.13 microm] vs. 88.5 microm [17.46 microm]) were significantly higher in the control group than in the preconditioning group (p = 0.009), indicating increased retinal thickness in the former group due to tissue edema resulting from ischemia-reperfusion injury.The mean mononuclear cell count (6.67 [SD 1.97] vs. 2.5 [SD 1.0]) and apoptotic cell count (18.2 [SD 5.7] vs. 5.3 [SD 1.0]) were significantly higher in the control group than in the preconditioning group (p = 0.002), indicating an inhibitory effect of ischemic preconditioning on leukocyte infiltration and apoptotic cell death. INTERPRETATION: Ischemic preconditioning attenuated ischemia-reperfusion injury in the rat retina.     

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.     

大鼠视网膜缺血再灌注后细胞间粘附分子-1的表达

目的　探讨大鼠视网膜缺血再灌注后不同时间视网膜细胞间粘附分子 1(inter cellularadhesionmolecule 1,ICAM 1)表达和白细胞浸润的变化。方法　选择健康成年Wistar大鼠 70只 ,随机分成 7组 :正常组和再灌注 0、2、6、12、2 4、4 8h组 ,每组 10只。用眼内灌注法建立视网膜缺血再灌注动物模型。制作大鼠视网膜冰冻及石蜡切片 ,分别作免疫组化SP染色和常规HE染色 ,并对SP染色结果作计算机图像分析。结果　ICAM 1在正常视网膜血管内皮细胞胞膜微量表达 ,缺血 6 0min后 ,ICAM 1表达上调 ,至再灌注2 4h达高峰 ,在再灌注 4 8h仍维持较高水平。再灌注 6h ,视网膜开始有白细胞浸润 ,随再灌注时间延长而增多 ,再灌注 2 4、4 8h组 ,视网膜中发现较多浸润的白细胞。结论　视网膜缺血再灌注早期 ,视网膜血管内皮细胞ICAM 1表达上调 ,继而 ,视网膜中白细胞浸润增多 ,这一过程是视网膜缺血再灌注损伤的重要机制之一。