Determination of conventional protein kinase C isoforms involved in high intraocular pressure-induced retinal ischemic preconditioning of rats

Evidence indicates that conventional protein kinase C (cPKC) plays a pivotal role in the development of retinal ischemic preconditioning (IPC). In this study, the effect of high intraocular pressure (IOP)-induced retinal IPC on cPKC isoform-specific membrane translocation and protein expression were observed. We found that cPKCγ membrane translocation increased significantly at the early stage (20 min-1 h), while the protein expression levels of cPKCα and γ were markedly elevated in the delayed retinal IPC (12-168 h) of rats. The increased protein expressions of cPKCα at 72 h and cPKCγ at 24 h after IPC were further confirmed by immunofluorescence staining. In addition, we found that cPKCγ co-localized with retinal ganglion cell (RGC)-specific marker, neurofilaments heavy chain (NF-H) by using double immunofluorescence labeling. These results suggest that increased cPKCγ membrane translocation and up-regulated protein expressions of cPKCα and γ are involved in the development of high IOP-induced rat retinal IPC.     

色素上皮衍生因子对大鼠视网膜缺血 再灌注损伤的保护作用

目的已证实色素上皮衍生因子(pigment epithelium-deriv ed factor,PEDF)对中枢神经细胞有抗凋亡作用。本实验评价其对压力诱发的视网膜缺血再灌注的影响。方法经前房灌注维持眼压110 mm Hg (1 mm Hg = 0.133kpa),45 min,建立大鼠视网膜缺血再灌注模型。 随后立即向玻璃体注射10　μ1(0.1μg/μl)PEDF,分别于2d和7d摘除眼球,测量塑 料包埋切片的平均视网膜内层厚度(mean thickness of inner retinal layer,MTIRL) 和视网膜节细胞(retinal ganglion cells,RGC)计数。结果PEDF玻璃体注射7d后治疗组的MTIRL和RGC明显高于对照组[(118.1±5．0) μm对(949±3.0)μm, P<0.05;(6.0±1．0)个/100 μm对(4.5±0.5)个/100 μm, P<0.05]。结论玻璃体内注射PEDF有助于防止视网膜缺血再灌注后神经变性和细胞死亡。(中华眼底病杂志,2001,17:138-140)     

High intraocular pressure-induced ischemia and reperfusion injury in the optic nerve and retina in rats

• Purpose: The purpose of this paper is to describe the damage caused to the retina and the axons of the optic nerve by acute ischemia-reperfusion injury and the extent to which optic nerve damage correlates with the duration if ischemia due to high intraocular pressure (IOP). • Methods: Acute ischemia in the retina and optic disc was induced in albino rats by increasing the IOP to 110 mmHg for a period of 45–120 min. Thereafter, the eyes were reperfused at normal IOP after 7 days. The retina and optic nerve were examined by light and electron microscopy, and morphometrical counts of the optic nerve axons were performed. • Results: After 45 min of ischemia, electron microscopic examination revealed swelling of mitochondria and degeneration of neurotubules on axons in cross sections of the optic nerve. The axonal counts in eyes subjected to 45 min of ischemia were 29% lower than in control eyes. After 60 min of ischemia, there were distinct disruptions of mitochondria and degeneration of the axons. After 90 min of ischemia, numerous axons showed degeneration with disordered myelin sheaths. Neuronal cell death was seen in the retina, mainly in the ganglion cell layer. • Conclusion: Damage to the retinal ganglion cell layer and the optic nerve was evident after only 45 min of ischemia in normal eyes. This experiment suggests that seriously injured eyes must be protected from high IOP; if IOP elevation is required during vitrectomy, it is essential to reduce the duration of interruption of blood flow to a minimum.     

Involvement of oxygen free radicals in experimental retinal ischemia and the selective vulnerability of retinal damage.

Protective effects of CV-3611, a free radical scavenger, on retinal ischemic injury in the rat and on glutamate-induced cytotoxicity in a cell line were evaluated. Transient retinal ischemia was induced by raising intraocular pressure of rats to 110 mm Hg for 45 min, and the electroretinogram (ERG) was measured to evaluate retinal function. No ERG could be recorded immediately after reperfusion, and thereafter the ERG gradually recovered. Recovery of the a-wave latency and the amplitudes of the a and b waves in the CV-3611-treated (10 mg/kg, p.o.) group were significantly better than those in the control group up to 24 h after reperfusion. In both the control and CV-3611 group, the b wave showed better recovery than the a wave up to 6 h after reperfusion, while the relationship was reversed after 24-hour reperfusion. Glutamate (10 mM)-induced cytotoxicity in the N18-RE-105 cell, a neural retina-neuroblastoma hybridoma, was quantified by measuring lactate dehydrogenase. Three and 10 microM of CV-3611 significantly attenuated the glutamate-induced cytotoxicity in N18-RE-105 cells. Thus, the radical scavenger (CV-3611) promoted the recovery of retinal function after ischemia-reperfusion injury and ameliorated glutamate-induced cytotoxicity. These results suggest that oxygen free radicals play an important role in the early phase of retinal ischemic injury. Moreover, differential recovery processes of the a and b waves after ischemia suggest that the selective vulnerability of the retina to ischemia could change functionally during the period of reperfusion.     

慢性高眼压状态下大鼠视网膜内神经营养因子表达的变化

目的检测高眼压状态下大鼠视网膜神经营养因子的表达变化。方法通过烙闭大鼠巩膜上静脉制作慢性高眼压模型,并对模型进行评估。应用免疫组化技术和RT-PCR检测正常大鼠和高眼压下大鼠视网膜上神经营养因子及其受体的表达水平。结果大鼠造模后1d、3d、5d、1周、2周、1个月和2个月的早晨800眼压分别为(14.387±3.527)mmHg(1kPa=7.5mmHg)、(13.833±3.473)mmHg、(13.833±3.473)mmHg、(13.388±3.473)mmHg、(14.350±2.727)mmHg、(15.310±3.495)mmHg、(15.053±3.467)mmHg,升高幅度达到基础眼压的50%以上。高眼压状态下,神经生长因子出现微弱表达,其mRNA水平及受体TrkA表达水平低下;脑源性神经营养因子在造模后2周出现较强阳性表达,并持续到造模后2个月,其mRNA水平短期增高,受体TrkB的mRNA水平降低;神经营养素-3在内核层出现阳性表达增强,mRNA水平逐渐降低,神经营养素-3受体TrkC表达水平随时间延长表达强度逐渐增加,mRNA水平一过性增高;神经营养素-4蛋白和mRNA水平在造模后都出现一过性表达增强;p75的mRNA水平低于正常大鼠。结论神经营养因子及其受体的蛋白和mRNA水平随着高眼压时间变化而相应改变,蛋白与mRNA变化趋势一致,但是神经营养因子及其受体变化的水平趋势不完全一致。     

Comparison of intraocular treatment of DMTU and SOD following retinal ischemia in rats.

The effect of intravitreal injections of DMTU (dimethylthiourea) and SOD (superoxide dismutase), two free radical scavengers, was evaluated in a rat model of retinal ischemia induced by elevated intraocular pressure. The drugs were administered just before or just after a 60 min ischemia. At days 2 and 7 after reperfusion, retinal recovery was evaluated by electroretinography. At day 7, layer thicknesses and cell rows were measured from histologic sections of paraffin-embedded retinas. In the vehicle-treated control group, we observed a decrease in the inner retinal layers and b-wave amplitude impairment. SOD injection (6 units/eye) protected the retina from ischemia/reperfusion injury. At day 2 after reperfusion, electroretinographic recovery was more efficient when SOD was administered just after ischemia (99%) than after pretreatment with SOD (81%) (p<0.03). In the DMTU-treated group (75 microg/eye), only the pretreatment induced significant electrophysiologic (40%) (p<0.001) and morphologic recovery.     

Ischemic preconditioning attenuates hypoperfusion after retinal ischemia in rats.

PURPOSE: Retinal blood flow (RBF) was measured in rats to test the hypotheses that hypoperfusion follows severe ischemia in the retina and that ischemic preconditioning (IPC) attenuates this change in blood flow. METHODS: Male Sprague-Dawley rats were anesthetized with halothane and mechanically ventilated by tracheostomy to maintain normocarbia and normoxia. Retinal ischemia was induced for 0, 5, 30, 60, 75, or 90 minutes. RBF was measured 60 and 150 minutes after the end of ischemia using the radioactive microsphere blood flow method, and electroretinography was performed during the first 120 minutes after ischemia to quantitate the extent of functional recovery. Additional groups received IPC (5 minutes of ischemia) 24 hours before 30, 60, or 75 minutes of ischemia. RESULTS: Control (0 minutes' ischemia) RBF was 22+/-3 ml/100 g per minute (mean +/- SE). At 60 minutes after 5, 30, 60, 75, or 90 minutes of ischemia, RBF was 15+/-2 (NS), 11+/-1, 8+/-2, 8+/-1, and 10+/-1 ml/100 g per minute, respectively (significance, P<0.05 versus control). At 150 minutes after 5, 30, 60, 75, or 90 minutes of ischemia, RBF was 18+/-3 (NS), 13+/-1, 12+/-3, 12+/-2, and 11+/-1 ml/100 g per minute respectively (significance, p<0.05 versus control). With prior IPC, RBF after 30 and 60 minutes of ischemia was 21+/-1 and 19+/-3 ml/100 g per minute (both NS compared with control; P<0.05 compared with 30 or 60 minutes of ischemia without IPC). When ischemia was 75 minutes in duration, IPC did not prevent postischemic hypoperfusion. The extent of recovery of the electroretinogram b wave was inversely related to the length of ischemia. CONCLUSIONS: Postischemic hypoperfusion is present in the rat retina 60 minutes after ischemia, does not resolve by 150 minutes after ischemia, and is attenuated by IPC when ischemia is 60 minutes or less in duration. Maintenance of postischemic perfusion in the retina may be one of the mechanisms involved in the neuroprotection afforded by IPC.     

Vascular endothelial growth factor-165b protects the blood-retinal barrier from damage after acute high intraocular pressure in rats

AIM: To elucidate the role of vascular endothelial growth factor-165b (VEGF-165b) in blood-retinal barrier (BRB) injury in the rat acute glaucoma model. METHODS: In this study, the rat acute high intraocular pressure (HIOP) model was established before and after intravitreous injection of anti-VEGF-165b antibody. The expression of VEGF-165b and zonula occludens-1 (ZO-1) in rat retina was detected by double immunofluorescence staining and Western blotting, and the breakdown of BRB was detected by Evans blue (EB) dye. RESULTS: The intact retina of rats expressed VEGF-165b and ZO-1 protein, which were mainly located in the retinal ganglion cell layer and the inner nuclear layer and were both co-expressed with vascular endothelial cell markers CD31. After acute HIOP, the expression of VEGF-165b was up-regulated; the expression of ZO-1 was down-regulated at 12h and then recovered at 3d; EB leakage increased, peaking at 12h. After intravitreous injection of anti-VEGF-165b antibody, the expression of VEGF-165b protein was no significantly changed; and the down-regulation of the expression of ZO-1 was more obvious; EB leakage became more serious, peaking at 3d. EB analysis also showed that EB leakage in the peripheral retina was greater than that in the central retina. CONCLUSION: The endogenous VEGF-165b protein may protect the BRB from acute HIOP by regulating the expression of ZO-1. The differential destruction of BRB after acute HIOP may be related to the selective loss of retinal ganglion cells.     

蛇毒神经生长因子对大鼠视网膜缺血再灌注损伤超微结构的影响

目的 探讨玻璃体内注射蛇毒神经生长因子(venom nerve growth factor,vNGF)对实验性大鼠视网膜缺血再灌注(retinal ischemia reperfusion,RIR)损伤的视网膜超微结构是否具有保护作用.方法 采用升高眼压的方法制作实验性RIR损伤大鼠模型.将Spregue-Dawley(SD)大鼠随机分为正常组、缺血组、实验组及对照组.实验组及对照组于再灌注开始时分别注入vNGF和平衡盐溶液各20 μL.通过透射电镜观察各组大鼠视网膜超微结构变化.结果 缺血组主要是视网膜神经节细胞(retinal ganglion cells,RGCs)和光感受器细胞等结构的水肿.对照组再灌注后1 d出现膜盘排列紊乱、变形,RGCs核染色质浓缩、边集、变性,胞浆内细胞器肿胀、空泡化且大量减少.神经纤维内大量的线粒体肿胀、空泡化.至再灌注后7 d膜盘溶解,RGCs出现变性坏死及凋亡;而实验组于再灌注后1 d主要为光感受器细胞排列紊乱、肿胀及RGCs肿胀、少许变性为主,至再灌注后7 d膜盘排列尚整齐,细胞肿胀减轻,胞浆内细胞器较丰富.实验组大鼠视网膜超微结构的改变较对照组明显轻.结论 大鼠玻璃体内注射vNGF对实验性RIR损伤大鼠的视网膜超微结构具有保护作用.     

孕酮对急性高眼压大鼠视网膜神经损伤保护作用的研究

目的：通过建立大鼠急性高眼压模型,应用孕酮干预,观察孕酮是否对视网膜神经细胞具有保护作用。

方法：Wistar大鼠140只,随机分为正常对照组(A组)20只、高眼压对照组(B组)60只、高眼压孕酮干预组(C组)60只。应用生理盐水前房灌注的方法建立大鼠急性高眼压模型,C组在高眼压后即刻予腹腔注射孕酮4.0mg/kg,以后于6,24h以及隔日行皮下注射,B组于相同时间注射等量的生理盐水。于高眼压后1,3,7d分别获取各组大鼠的视网膜组织。HE染色观察视网膜病理学的变化,用免疫组化染色、Western blot检测视网膜细胞凋亡基因Caspase-3蛋白的表达情况,TUNEL法检测细胞的凋亡程度,并对各组数据进行统计学分析。

结果：B组和C组的大鼠视网膜均发生水肿, C组水肿程度明显低于B组。免疫组化染色及Western blot相对定量检测,C组Caspase-3表达程度低于B组。Caspase-3蛋白主要表达于视网膜神经节细胞层和内核层,于高眼压后第1d强度最大,以后随时间的推移逐渐减少。TUNEL检测细胞凋亡,B组和C组均出现阳性细胞,主要位于视网膜的节细胞层和内核层,C组细胞凋亡数量明显低于B组。

结论：孕酮对大鼠视网膜急性高眼压损伤有保护作用。     

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。     

青光康注射液对急性实验性高眼压大鼠视网膜节细胞代谢的作用

采用改良细胞色素氧化酶（ｃｙｔｏｃｈｒｏｍｅｏｘｉｄａｓｅ，ＣＯ）组织化学法研究青光康注射液对大鼠实验性急性高眼压视网膜代谢机能的影响。结果表明：以平均动脉压的７５％作为大鼠的前房灌注压可导致视网膜ＣＯ活性节细胞减少、灰度降低；青光康注射液具有保护和／或改善急性高眼压后大鼠视网膜ＣＯ活性节细胞的作用，表现为青光康组经青光康治疗后高眼压模型眼ＣＯ活性节细胞密度及灰度与对照组高眼压模型眼比较，差异有显著性（Ｐ＜０．０５）。     

Photically-induced retinal damage in diabetic rats

The present study examines the interaction of light damage to the retina and streptozotocin (SZ)-induced diabetes in male and female rats during the early development of the disease, when changes occur in the blood-retinal barrier and in pigment cell membranes. Exposure of rats to low illuminance was used to determine the relationship between photically-induced cell death and diabetes. Other groups of animals were exposed to a greater illuminance for shorter time periods (24 hours) in attempts to identify a specific post-treatment day for the effect of diabetes. Blood glucose levels were monitored to indicate the severity of the diabetes. Morphometric analyses and histopathologic observations demonstrated that the outer nuclear layer (ONL, photoreceptor nuclei) was reduced significantly in thickness in female rats exposed to light during a 9 day period after SZ injection, but was unchanged from the control groups when exposed beginning at 12 days after SZ treatment. Removal of the pituitary gland prior to SZ treatment and light exposure resulted in the survival of more photoreceptor cells and prevented the differential in ONL thickness observed between control and diabetic intact animals. Attempts to establish a period of greatest susceptibility of the diabetic retina to photic damage were unsuccessful, but results indicate that prior light history and/or shipment stress might be related to retinal damage from light exposure.     

The neuropeptide NAP provides neuroprotection against retinal ganglion cell damage after retinal ischemia and optic nerve crush

Background  NAP, an 8-amino acid peptide (NAPVSIPQ=Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln) derived from activity-dependent neuroprotective protein (ADNP), plays an important role in neuronal differentiation and the survival of neurons in different pathological situations. We already discovered that NAP increases the survival of retinal ganglion cells (RGC) in vitro, and supports neurite outgrowth in retinal explants at femtomolar concentrations. The aim of this study was to investigate the effects of NAP on RGC survival after transient retinal ischemia and optic nerve crush. Methods  RGC of male Wistar rats were labelled retrogradely with 6 l FluoroGold injected stereotactically into both superior colliculi. Seven days later, retinal ischemia was induced by elevating the intraocular pressure to 120 mm Hg for 60 minutes or by crushing one optic nerve for 10 s after a partial orbitotomy. NAP was either injected intraperitoneally in the concentration of 100 mg/kg 1 day before, directly after, and on the first and the second days after damage, or intravitreally (0.05 or 0.5 μg/eye) directly after the optic nerve crush. Controls received the same concentrations of a control peptide. Densities of surviving RGC and activated microglial cells (AMC) were quantified in a masked fashion 10 days after damage by counting FluoroGold-labelled cells. Results  After retinal ischemia, intraperitoneal injections of NAP increased the number of surviving RGC by 40% (p < 0.005) compared to the control group. After optic nerve crush, NAP raised the number of surviving RGC by 31% (p = 0.07) when injected intraperitoneally and by 54% (p < 0.05) when administered intravitreally. Conclusions  NAP acts neuroprotectively in vivo after retinal ischemia and optic nerve crush, and may have potential in treating optic nerve diseases. Supported by the Ernst und Berta Grimmke Stiftung, Germany. IG is the incumbent of the Lily and Avraham Gildor Chair for the Investigation of Growth Factors and the Director of the Adams Super Center for Brain Research at Tel Aviv University and is the Chief Scientific Officer of Allon Therapeutics Inc., Vancouver, Canada. An erratum to this article can be found at     

Degeneration of retinal cells of the rat in pressure-induced ischemia-reperfusion damage: an electron microscopy study]

An ischaemia-reperfusion insult to the retina was produced in one eye of 29 rats by transitory elevation of the intraocular pressure. The ultrastructural morphology of the degenerating retinal cells was studied at several time points after the insult. Three morphologically distinct types of cell death could be individualized. Type I exhibited progressive karyolysis and cytolysis and was consistent with necrosis. Type II was characterized by progressive shrinkage and condensation of the nucleus and cytoplasm followed by heterophagic elimination, thus resembling apoptosis. The main features of type III cell death were homogenization of the nucleoplasm and cytoplasm and dilatation of the perinuclear cisternae; thus, they were evocative of "non-lysosomal vesiculate" cell death. Transitional stages were observed as well.     

正常大鼠和急性高眼压诱导的视网膜缺血再灌注损伤大鼠模型的视网膜电图

目的：探索闪光视网膜电图（F－ERG）对大鼠视网膜功能评价的客观性和可靠性;并探讨急性高眼压诱导的视网膜缺血再灌注损伤大鼠在视网膜神经细胞保护研究方面的可行性。方法：采用Nicolet Spirit电生理诊断仪,检测34只Sprague-Dawley（SD）大鼠的F－ERG的最大反应和Ops波;用提高眼压的方法造成视网膜缺血60分钟,然后恢复眼压形成血流再灌注造成大鼠动物模型,随后检测灌注后24h,72h以及168h时的F－ERG的最大反应和Ops波。结果：每只正常大鼠均可引出典型的ERG的a波,b波以及Ops波,但个体之间存在一定差异;模型大鼠的各波比灌注前均显著降低,而且灌注后各时间点也有一定差异。结论：本模型操作简单,可控性好以及F－ERG的最大反应和Ops波能客观反映大鼠视网膜功能,因此可以用来进行视网膜神经细胞保护方面研究。     

Effect of pressure-induced ischemia on oxygen supply in the retinal capillary area. Results of a mathematical model

This paper attempts to obtain a better understanding of the oxygen supply to tissue cells of the optic nerve head by describing all phases of the release, diffusion and consumption process by means of a mathematical model. As a result the oxygen partial pressure reduction induced by intraocular pressure elevation--as it occurs in glaucoma--seems to be mainly influenced by a pressure-induced lowering of the average blood velocity in the retinal capillaries. A reduction of this velocity to a fourth of its normal value even makes the oxygen partial pressure at the venous capillary wall drop below the critical level necessary for maintaining cell activity. Furthermore our calculations allow us to conclude that an involvement of the retinal capillary network supplied by the central retinal artery should not be neglected in glaucoma.