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.     

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。     

视网膜缺血再灌注损伤后Caspases家族的表达及bFGF对其的影响

目的探讨大鼠视网膜缺血再灌注损伤(retina ischemia/reperfusion injury,RIRI)后半胱天冬氨酸酶(Caspases)家族的表达及碱性成纤维细胞因子(basic fibroblast growth factor,bFGF)对其表达的影响。方法采用升高眼内压的方法,制作实验性RIRI的大鼠模型。将28只Wistar大鼠随机分为正常组、缺血组和治疗组,其中后两组又分为再灌注后1、6、12、24、48、72h组。应用SP免疫组化法检测大鼠RIRI后视网膜组织中Caspase-2、Caspase-3的表达及玻璃体腔内注射外源性bFGF后对其表达的影响。结果正常组视网膜组织Caspase-2、Caspase-3不表达。缺血组6h开始出现Caspase-2的表达,24h达高峰,48h和72h下降;Caspase-3在视网膜组织的表达时段与Caspase-2的表达规律相似。bFGF治疗组各观察指标变化趋势基本同缺血组,但各时间段的表达强度明显减弱,两组比较,于再灌注6、12、24h时段差别具有统计学意义。结论Caspases家族参与了RIRI,导致神经细胞的凋亡;bFGF可以抑制Caspase-2、Caspase-3蛋白的表达,因此,bFGF在RIRI中可能通过抑制细胞凋亡而达到其保护作用。     

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。     

E-64d对大鼠视网膜缺血再灌注损伤中Calpain和Caspase-3表达的调控作用

目的 探讨E-64d（半胱氨酸蛋白酶抑制剂及钙激活中性蛋白酶抑制剂）在大鼠视网膜缺血再灌注损伤（RIRI）中对Calpain／Caspase-3表达的调节作用。方法 将80只SD大鼠随机分为正常组、视网膜缺血再灌注组、对照组和E-64d治疗组，并分为1、3、6、24、72h5个时间段。通过前房穿刺加压法制成RIRI动物模型，采用Western—blot以及RT—PCR方法测定在大鼠RIRI模型中的Calpian、Caspase-3蛋白和mRNA表达情况。结果缺血再灌注24hm—Calpain、Caspase-3蛋白的表达缺血再灌注组较正常组上升，而E-64d组表达与正常组相似，比缺血再灌注组下降。E-64d能下调视网膜缺血再灌注后m—calpain／calpastatin的mRNA比值，在24h与缺血组有显著统计学差异。结论 E-64d能降低视网膜缺血再灌注时m—Calpain、Caspase-3蛋白的表达，调控m—Calpain和Calpastatin的mRNA比值。     

替普瑞酮对大鼠视网膜缺血-再灌注损伤的保护作用

背景视网膜缺血-再灌注损伤是眼科常见的病理过程,可引起永久性的视力障碍,严重影响患者的视功能,是目前国内外研究的重点课题之一。目的探讨替普瑞酮对大鼠视网膜缺血-再灌注损伤的保护作用。方法44只SD大鼠按随机数字表法分为正常对照组4只、单纯模型组20只和替普瑞酮治疗组20只。单纯模型组及替普瑞酮治疗组造模前分别给予大鼠生理盐水和替普瑞酮灌胃,1周后采用前房加压法制作大鼠视网膜缺血-再灌注损伤模型,并分别于再灌注后6、24、48、72h制备视网膜铺片,经苏木精-伊红染色观察各组大鼠视网膜组织结构的变化,采用免疫组织化学法检测大鼠视网膜中热休克蛋白70（HSP70）和caspase-3的表达。结果视网膜缺血-再灌注后1～6h单纯模型组大鼠角膜及视网膜出现水肿,24h视网膜水肿加重,72h视网膜水肿减轻、结构较紊乱。替普瑞酮治疗组大鼠各时间点视网膜水肿较单纯模型组轻,缺血-再灌注后72h大鼠视网膜结构损害较单纯模型组减轻。正常对照组大鼠视网膜未见HSP70及caspase-3呈阳性表达。单纯模型组大鼠在再灌注后6h可见视网膜神经节细胞（RGCs）中HSP70呈阳性表达,再灌注后24h达高峰,之后逐渐下降。替普瑞酮治疗组各时间点HSP70在大鼠RGCs中的表达较单纯模型组明显增强,差异均有统计学意义（P〈0．05）。再灌注后6h可见单纯模型组大鼠RGCs中caspase-3表达,24h时caspase-3的表达量达高峰,48h后开始下降,72h仅有少量表达,而各时间点替普瑞酮治疗组视网膜中caspase-3的表达较单纯模型组大鼠明显减弱,差异均有统计学意义（P〈0．05）。结论在视网膜缺血-再灌注损伤大鼠模型中,替普瑞酮可通过上调视网膜中HSP70的表达和下调caspase-3的表达对RGCs起保护作用。     

家兔视网膜缺血-再灌注损伤后ERG的动态变化

目的 动脉观察家兔视网膜缺血－再灌注损伤后ＥＲＧ的ｂ波的变化。方法 应用前房灌注加压法使前房内压力高至１６ＫＰＡ,维持１小时,再灌注后的第２、７、１４天分别记录真ＥＲＧ,与缺血前ＥＲＧ相比较,观察Ｂ波的变化幅度。结果 缺血－再灌注后第２天,ＥＲＧ的Ｂ波已经开始下降,随时间延开,Ｂ波振幅呈性、进行性下降。结论 视网膜缺血－再灌注损伤在再灌注呈持续性、进行性改变。     

Local hypothermia protects the retina from ischemia. A quantitative study in the rat

We developed a quantitative histologic method for assessing injury in the rat retina due to transient ischemia. We used this technique to test the effectiveness of local hypothermia and allopurinol, an inhibitor of oxygen-free radical formation, in reducing ischemia/reperfusion injury in the rat retina. Retinal ischemia and reperfusion was produced by transient ligation of the optic nerve. Histologic evaluation by a masked observer was based on the average count of nonpyknotic nuclei in the inner nuclear layer of the retina from eight high power fields (X100) in one 5 microns thick sagital section at or near the optic nerve. A sharp increase in tissue damage occurs between 90 and 120 min of ischemia. Ischemia for periods of 60 and 90 min produced mild damage while periods of 120 and 240 min produced severe damage. Hypothermia protected the retina significantly from 120 min of ischemic injury (P less than 0.001 student t-test, compared to 120 min control), while allopurinol had no protective effect.     

视网膜缺血再灌注损伤中WTp53蛋白、CyclinD1表达的研究及bFGF对其的影响

目的探讨碱性成纤维细胞生长因子(bFGF)在大鼠视网膜缺血再灌注损伤(RIRI)中的治疗作用及机制。方法将28只Wistar大鼠随机分为正常组、缺血组和bFGF治疗组,其中后两组又按再灌注时间不同分为再灌注后1、6、12、24、48和72h6个时间段。通过前房穿刺加压法制作成RIRI动物模型,并在以bFGF(治疗组)或平衡盐溶液(缺血组)玻璃体腔注射后按不同时段处死动物,通过免疫组织化学链酶卵白素-生物素复合体(SABC)法检测各时段视网膜组织中野生型p53蛋白(WTp53)和细胞周期蛋白D1(CyclinD1)的表达变化。结果缺血组视网膜在再灌注后6h可发现有WTp53蛋白及CyclinD1的表达,24h达到高峰,48h仍持续强表达,72h表达已明显下降;bFGF治疗组各观察指标变化规律基本与缺血组相似,但表达量相对明显减弱,两组比较,在再灌注6-48h时段差别P值均<0.05,具有统计学意义。结论bFGF可抑制RIRI时WTp53蛋白和CyclinD1在视网膜表达的增高。     

Local hypothermia protects the retina from ischaemic injury in vitrectomy

AIMS—Hypothermic irrigating solutions were used during vitrectomy in pressure induced ischaemic eyes so that their effects on retinal function and histological changes could be investigated.
METHODS—After anaesthetised albino rabbits underwent closed vitrectomy, their vitreous cavities were continuously irrigated for 30 minutes at a perfusion pressure of 140 mm Hg. The rabbits were divided into three groups according to their intraocular perfusion temperatures—8°C, 22°C, and 38°C. Electroretinograms were taken before and after irrigation. Glutamate levels in the vitreous were examined after irrigation. Eyes were enucleated on the seventh postoperative day and examined histologically.
RESULTS—On the seventh postoperative day, the recovery rate of a-wave amplitudes was significantly lower in the 38°C group than in the 8°C group, and that of b-wave amplitudes was significantly lower in the 38°C group than in either the 8°C or 22°C group. Retinal damage in the 38°C group revealed more severe histological impairment than in either the 8°C or 22°C group. Oedema of the inner retinal layer was significant in both the 22°C and 38°C groups. Glutamates reached peak values 30 minutes after the end of ischaemia in the 38°C group. However, no significant glutamate increases were detected 15 to 60 minutes after ischaemia in either the 8°C or 22°C group.
CONCLUSION—Local hypothermia during vitrectomy in acute ischaemic eyes appears to decrease retinal damage.

     

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.     

视网膜缺血再灌注损伤后HSP70、NF-κB的表达和细胞凋亡的关系

目的探讨大鼠视网膜缺血再灌注损伤后热休克蛋白70（HSP70）、核蛋白因子-κB（NF-κB）表达和损伤神经细胞凋亡的关系。方法前房加压法制作大鼠视网膜缺血再灌注损伤模型，28只大鼠随机分为正常组和缺血再灌注组，后者又按照再灌注后不同时间段分为1、6、12、24、48、72h组。应用末端脱氧核酸转移酶介导的脱氧三磷酸尿苷缺口末端标记（TUNEL）法检测视网膜神经细胞凋亡指数（AI），过氧化物酶标记的链酶卵白素（SP）免疫组织化学法检测视网膜组织中HSP70、NF-κB的表达。结果视网膜神经细胞的凋亡出现于再灌注后6h，并逐渐递增，24h达到高峰．48h开始下降，72h不明显。HSP70在再灌注后1h即有表达，随时间段延长表达逐渐增加，至再灌注后24h达到高峰．之后渐下降。NF-κB的表达变化与凋亡细胞变化的规律基本一致。结论视网膜缺血再灌注损伤导致神经节细胞的凋亡：HSP70和NF-κB在视网膜缺血再灌注损伤中表达升高，对神经细胞的凋亡起着重要的调节作用。     

Heterogeneous populations of microglia/macrophages in the retina and their activation after retinal ischemia and reperfusion injury

Activation of Microglia/macrophages has been observed in ischemia-reperfusion injury of the brain. This study was undertaken to investigate the different subpopulations of microglia/macrophages in the normal rat retina and their activation after retinal ischemia. Retinal ischemia was induced by elevation of intraocular pressure to 120 mmHg for 60 min. Microglia/macrophages were identified on frozen retinal sections by four antibodies, namely OX42, 5D4, OX6 and ED1. In the normal retina, there were heterogeneous populations of resident microglia/macrophages as characterized by their differences in morphology, antigen expression and distribution. OX42+ cells had delicate processes and were located in the inner layers of the retina, while 5D4+ cells were highly ramified and mostly scattered in the inner plexiform layer (IPL) and the outer plexiform layer. Few amoeboid ED1+ cells were also seen in the ganglion cell layer and IPL. OX6+ (MHC-II antigen presenting) cells were not detected in the normal retinas. Double labeling with OX42 and 5D4 antibodies on normal retinal sections showed few microglia exhibited positive labeling with both OX42 and 5D4, while the majority of the microglia were labeled with either OX42 or 5D4 antibodies. After retinal ischemia single labeling with these antibodies showed increased number of these antigen-expressing cells, disappearance of normal cellular processes, and rounding or amoeboid like appearance of the cell bodies. At 1 day after ischemia, there was a significant infiltration of round OX42+, ED1+ and OX6+ cells with loss of the cellular processes in the inner retina. From 3 to 14 days, all subpopulations of microglia/macrophages differentiated cellular processes and became dendritic again. Double labeling on retinas after 1 day of recovery showed OX42+ cells were co-labeled with ED1+ or OX6+ cells, but not with 5D4+ cells. Scattered amoeboid OX42+, 5D4+, and ED1+ cells were noted in the subretinal space 3-14 days after ischemia. In summary, there were heterogeneous populations of resident microglia/macrophages in the normal inner retina and they were activated early after ischemia-reperfusion injury and exhibited different antigenic expression which were further altered in the recovery phase.     

Heterogeneous populations of microglia/macrophages in the retina and their activation after retinal ischemia and reperfusion injury

Activation of Microglia/macrophages has been observed in ischemia-reperfusion injury of the brain. This study was undertaken to investigate the different subpopulations of microglia/macrophages in the normal rat retina and their activation after retinal ischemia. Retinal ischemia was induced by elevation of intraocular pressure to 120 mmHg for 60 min. Microglia/macrophages were identified on frozen retinal sections by four antibodies, namely OX42, 5D4, OX6 and ED1. In the normal retina, there were heterogeneous populations of resident microglia/macrophages as characterized by their differences in morphology, antigen expression and distribution. OX42+ cells had delicate processes and were located in the inner layers of the retina, while 5D4+ cells were highly ramified and mostly scattered in the inner plexiform layer (IPL) and the outer plexiform layer. Few amoeboid ED1+ cells were also seen in the ganglion cell layer and IPL. OX6+ (MHC-II antigen presenting) cells were not detected in the normal retinas. Double labeling with OX42 and 5D4 antibodies on normal retinal sections showed few microglia exhibited positive labeling with both OX42 and 5D4, while the majority of the microglia were labeled with either OX42 or 5D4 antibodies. After retinal ischemia single labeling with these antibodies showed increased number of these antigen-expressing cells, disappearance of normal cellular processes, and rounding or amoeboid like appearance of the cell bodies. At 1 day after ischemia, there was a significant infiltration of round OX42+, ED1+ and OX6+ cells with loss of the cellular processes in the inner retina. From 3 to 14 days, all subpopulations of microglia/macrophages differentiated cellular processes and became dendritic again. Double labeling on retinas after 1 day of recovery showed OX42+ cells were co-labeled with ED1+ or OX6+ cells, but not with 5D4+ cells. Scattered amoeboid OX42+, 5D4+, and ED1+ cells were noted in the subretinal space 3–14 days after ischemia. In summary, there were heterogeneous populations of resident microglia/macrophages in the normal inner retina and they were activated early after ischemia-reperfusion injury and exhibited different antigenic expression which were further altered in the recovery phase.     

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.     

大鼠视网膜缺血再灌注后IL一1β的免疫组化研究

观察大鼠视网膜缺血再灌注(retinal ischemiareperfusion,RIR)后,白细胞介素1β(IL-1β)多肽在视网膜表达变化.方法采用前房灌注生理盐水,形成1 30mmHg(17.3kPa)高眼压,诱导大鼠视网膜缺血 60min,解除高眼压,建立RIR模型.缺血 60min,再灌注1 2h、48h作视网膜冰冻切片,IL-1β免疫组化观察.结果正常对照组未见IL β表达,RIR后12h、48h,视网膜神经节细层可见IL-1β表达.结论结果提示IL-1β多肽在蛋白质水平参与RIR损伤发生.     

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.