BDNF diminishes caspase-2 but not c-Jun immunoreactivity of neurons in retinal ganglion cell layer after transient ischemia.

PURPOSE: Retinal ischemia-reperfusion injury induces apoptosis of retinal neurons. The purpose of this study was to examine the association of c-Jun, caspase-1, -2, and -3 immunoreactivities and neuronal apoptosis in the retinal ganglion cell layer (GCL) and to study the effects of intravitreal brain-derived neurotrophic factor (BDNF) on the expression of these gene products in a rat model of retinal ischemia-reperfusion injury. METHODS: After 60 minutes of ischemia, eyes were enucleated after 3, 6, 12, 24, and 168 hours of reperfusion. The numbers of c-Jun-, caspase-1-, caspase-2-, caspase-3, and TdT-dUTP terminal nick-end labeling (TUNEL)-positive cells in the GCL were counted. Recombinant human BDNF (5 microg) or vehicle was injected intravitreally immediately after reperfusion. At 6, 24, and 168 hours, the numbers of immunoreactive cells in BDNF- and vehicle-treated groups were compared. RESULTS: Expression of c-Jun and caspase-2 was found in dying cells in flat-mounted retinas. The numbers of caspase-1- and caspase-3-positive cells were fewer than c-Jun- or caspase-2-positive cells. Cell death in the retinal GCL was suppressed by an intravitreal injection of BDNF. The numbers of TUNEL- and caspase-2-positive cells were lower in the BDNF-treated group at 6 hours after reperfusion (P<0.01). The number of c-Jun-positive cells in the treated retinas was not altered by the treatment. CONCLUSIONS: Expression of c-Jun and caspase-2 is associated with neuronal cell apoptosis in the GCL. Suppression of caspase-2 expression may explain the neuroprotective effects of BDNF.     

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.     

Apoptosis and caspases after ischemia-reperfusion injury in rat retina

PURPOSE: Extensive cell loss in the retinal ganglion cell layer (RGCL) and the inner nuclear layer (INL) was noted in a rat model of retinal ischemia-reperfusion injury by transient elevated intraocular pressure (IOP). The possible involvement of apoptosis and caspases was examined in this model of neuronal loss. METHODS: Transient elevated IOP was induced in albino Lewis rats through the insertion of a needle into the anterior chamber connected to a saline column. Elevated IOP at 110 mm Hg was maintained for 60 minutes. Groups of animals were euthanatized at various times after reperfusion, and their retinas were evaluated by morphology, agarose gel electrophoresis of DNA, in situ terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling (TUNEL), immunohistochemistry of caspases II (ICH1) and III (CPP32), and morphometry. YVAD.CMK, a tetrapeptide inhibitor of caspases, was used to examine the involvement of caspases. RESULTS: A marked ladder pattern in retinal DNA gel analysis, typical of internucleosomal DNA fragmentation and characteristic of apoptosis, was present 12 and 18 hours after reperfusion. Labeling of nuclei in the RGCL and the inner nuclear layer (INL) by TUNEL was noted between 8 and 18 hours after reperfusion. Histologic and ultrastructural features typical of apoptosis were also observed in the inner retina after ischemia. YVAD.CMK administered during the ischemic period inhibited apoptotic fragmentation of retinal DNA and ameliorated the tissue damage. When administered intravitreally 0, 2, or 4 hours after reperfusion, YVAD.CMK was also effective in preserving the inner retina but had no significant effect when administered 6 or 8 hours after reperfusion. The inner retina showed transient elevated immunoreactivity of caspases II and III 4 and 8 hours after reperfusion. CONCLUSIONS: Retinal ischemia-reperfusion after transient elevated IOP induced apoptosis of cells in the retinal ganglion cell layer and the INL. Caspases may have a pivotal role in the early events of the apoptotic pathway(s). Rescue by using anti-apoptotic agents after ischemia-reperfusion is feasible.     

大鼠视网膜缺血-再灌注损伤后P38丝裂原活化蛋白激酶和半胱氨酸天冬氨酸蛋白酶表达的变化及尼莫地平对二者的影响

目的探讨L型钙通道阻滞剂尼莫地平对大鼠视网膜缺血一再灌注损伤的保护作用及其对细胞信号转导通路的影响。方法Wistar大白鼠95只,随机分为4组。A组正常（空白）对照组5只,B组视网膜缺血-再灌注组（实验对照组）30只,C组视网膜缺血-再灌注＋尼莫地平组（实验组）30只,D组低压灌注组（假手术组）30只,以前房灌注升高眼压的方法制备大鼠视网膜缺血-再灌注模型,分别于再灌注发生后2、6、12、24、72、168h各处死5只大鼠,石蜡包埋后切片。以原位杂交方法检测各时间点视网膜P38丝裂原活化蛋白激酶类（P38MAPK）mRNA表达情况,以免疫组化法检测视网膜半胱氨酸天冬氨酸蛋白酶3（caspase-3）表达情况。结果于Metamorph软件上处理,取平均吸光度值作统计分析。结果视网膜缺血-再灌注损伤后,P38MAPK和caspase-3表达增强。视网膜P38MAPK mRNA原位杂交信号位于节细胞层和内核层的细胞核内,正常视网膜只有少量表达。P38于B组缺血-再灌注后,6h表达即明显增加,至12h达到顶峰,持续至24h,72h后逐渐下降。C组使用尼莫地平后,P38表达趋势与B组相同,但表达水平下降。caspase-3表达情况与P38相似。2h开始见内核层细胞核散在阳性,6h后节细胞层内核层细胞核阳性数增加,至24h达到顶峰。C组caspase-3表达趋势同B组,阳性细胞数及着染深度均小于B组。对平均吸光度值行统计学分析表明：P38MAPK mRNA表达,在6、12、24、72h,B组与A、C组,C组与A、D组间差异有统计学意义。caspase-3表达,除0h、168h这两个时间点外,其他各时间点A、D组与B组,A、D组与C组,B组与C组间差异均有统计学意义。A组与D组间差异无统计学意义。结论P38 MAPK和caspase-3均参与了视网膜缺血-再灌注损伤中视网膜神经细胞信号的转导,尼莫地平通过下调P38MAPK和caspase-3的表达而实现对视网膜的保护作用。（中华眼科杂志,2006,42：435-442）     

Y27632对急性视网膜缺血再灌注大鼠视网膜组织形态学的影响

研究Rho激酶抑制剂Y27632 对视网膜缺血再灌注损伤大鼠视网膜组织形态学的影响。方法：实验研究。将60只SD大鼠随机分为4组，每组15只：正常对照组（正常组）、急性缺血再灌注损伤组（IRI组）、0.9%氯化钠溶液对照组（生理盐水组）、Y27632治疗组（Y27632组）。再灌注损伤后24 h（10只）和168 h（5只）处死各组动物，行HE染色、ADP酶染色检查，光镜下观察大鼠视网膜组织病理学变化及视网膜厚度变化。数据采用单因素方差分析。结果：正常组大鼠视网膜结构清晰，三层细胞结构排列整齐。IRI组于再灌注24 h后视网膜厚度增加，内外丛状层组织疏松，视网膜节细胞、内外核层细胞水肿明显、排列紊乱，视网膜节细胞减少。168 h后，视网膜水肿消退、厚度变薄、呈萎缩状，神经节细胞及内外核层细胞数量减少，在视网膜前和神经纤维层可见毛细血管。再灌注 24 h后，IRI组视网膜厚度较正常组增加（P=0.005），Y27632组视网膜厚度低于生理盐水组（P=0.032）。再灌注168 h后，IRI组视网膜厚度低于正常组（P<0.001），Y27632组视网膜厚度较生理盐水组增加（P=0.025）。正常组大鼠视网膜血管自视乳头发出，向四周呈放射状均匀分布，毛细血管网结构清晰。再灌注24 h后，IRI组视网膜血管管径变细，走行较僵直，分支减少，视乳头周围及中周部视网膜可见大片无灌注区，无灌注区周围可见新生血管芽渐成网状。Y27632组可见视乳头周围及中周部视网膜局部无灌注区形成，无灌注区周围可见新生血管。后极部4PD无灌注区面积明显小于IRI组及生理盐水组。结论：Y27632玻璃体腔注射可以减轻视网膜缺血再灌注早期的视网膜水肿，减少视网膜神经节细胞的凋亡，减少视网膜新生血管生成，减轻再灌注晚期的视网膜萎缩，具有视神经保护作用。     

Differential temporal and spatial expression of immediate early genes in retinal neurons after ischemia-reperfusion injury

PURPOSE: To investigate genes that are up- and downregulated in rat retinal ischemia-reperfusion injury systematically by using an oligonucleotide microarray system and to determine temporal and spatial expression changes of some genes that showed upregulation in the analysis. METHODS: Retinal ischemia was induced in rats by increasing intraocular pressure to 110 mm Hg for 1 hour. Gene expression at 12 hours after reperfusion was compared with that in the control retina by using oligonucleotide microarrays that display a total of 8800 genes and expressed sequence tags (ESTs). Temporal and spatial expression changes of immediate early genes and cell-cycle-related genes were studied by using real-time polymerase chain reaction (PCR) and immunohistochemical methods. RESULTS: At 12 hours after reperfusion, 135 genes and ESTs were found to be up- or downregulated. The upregulated genes were classified into seven groups: (1) immediate early genes; (2) cell-cycle-related genes; (3) stress-responsive protein genes; (4) cell-signaling protein genes; (5) cell-adhesion and cell surface protein genes; (6) genes for translation and protein turnover; and (7) genes for metabolism. Real-time PCR analyses showed peaks of Fra-1 expression at 6 hours after reperfusion, whereas those for c-Jun, Jun B, and cyclin D1 were at 24 hours. Fra-1 and Jun B immunoreactivities were found in Müller cells, whereas c-Jun and cyclin D1 immunoreactivities were found in apoptotic retinal neurons. CONCLUSIONS: Gene expression changes after a retinal ischemia-reperfusion injury were profiled by using an oligonucleotide microarray system. Seven groups of genes were found to be upregulated by the injury. Among the immediate early genes, Fra-1 and Jun B immunoreactivities were found in Müller cells whereas c-Jun and cyclin D1 immunoreactivities were found in apoptotic retinal neurons.     

电刺激大鼠小脑顶核对视网膜缺血再灌注损伤的保护作用

目的　探讨电刺激大鼠小脑顶核对视网膜缺血再灌注损伤的保护作用。方法　大鼠随机分为缺血再灌注组、电刺激组和假手术组。观察视网膜形态学改变 ;用NADPH黄递酶组织化学染色法 (NADPH NDP)观察视网膜内诱导型一氧化氮合酶 (iNOS)的表达 ;采用TUNEL法检测视网膜细胞凋亡情况。结果　 (1)缺血再灌注组的内视网膜层 (包括内核层、内丛状层、节细胞 )、神经纤维层和内界膜厚度增加 ,尤其是内丛状层厚度明显高于假手术组 (t=3 6 80 ,P <0 0 1) ;电刺激组的内视网膜厚度与假手术组相比 ,差异无显著意义 (t=1 0 6 4 ,P >0 0 5 ) ;(2 )光镜观察可见缺血再灌注组有明显的细胞核染色质致密浓缩、核碎裂等改变 ,电刺激组仅见少量核浓缩及碎裂 ;(3)电刺激组的iNOS阳性的神经节细胞数明显低于缺血再灌注组 ,其差异有显著意义 (t=3 32 6 ,P <0 0 1) ;(4)电刺激组大鼠发生凋亡的视网膜细胞数明显低于缺血再灌注组 ,其差异有显著意义 (t=4 0 38,P <0 0 1)。结论　电刺激大鼠小脑顶核对缺血再灌注所导致的视网膜组织损伤具有保护作用。     

Post-treatment at 12 or 18 hours with 3-aminobenzamide ameliorates retinal ischemia-reperfusion damage

PURPOSE: The window of protection afforded by 3-aminobenzamide (3-ABA), a poly-(ADP-ribose) polymerase (PARP) inhibitor, against apoptotic loss of inner retinal elements after ischemia-reperfusion insult in rats was examined. METHODS: Ischemia-reperfusion injury to the retinas in albino Lewis rats was induced by elevated intraocular pressure (IOP) through cannulation of the anterior chamber with a needle connected to a saline column delivering a pressure of 110 mm Hg. The ischemic period was held at 60 minutes, and reperfusion was established immediately afterward. 3-Aminobenzamide (3-ABA) was administered intravitreally at 0, 4, 8, 12, 18, or 24 hours after reperfusion and its effect evaluated by morphology and morphometry of the inner retinas at 7 days after reperfusion. Immunohistochemistry of poly-(ADP-ribose), a product of PARP activity, and Western blot analysis for PARP were performed on retinas at 0, 4, 8, 12, 18, and 24 hours after reperfusion. RESULTS: Morphology and morphometry showed significantly better preserved inner retinas in animals receiving 3-ABA between 12 and 18 hours after reperfusion. Immunohistochemical study of poly-(ADP-ribose) showed elevated levels at the retinal ganglion cell layer and the inner nuclear layer at 12 and 18 hours after reperfusion. Western blot analysis of PARP showed a notable increase in the 116-kDa band (PARP) from 4 to 18 hours after reperfusion. CONCLUSIONS: Administration of 3-ABA at 12 or 18 hours after ischemia, when there was accumulation of poly-(ADP-ribose) in the inner retina, significantly ameliorated retinal ischemia-reperfusion injury. These findings, together with earlier reports from our laboratory, are consistent with a late and pivotal role of PARP in apoptotic loss of inner retinal elements after ischemia-reperfusion insult to the retina.     

大鼠视网膜缺血再灌注后HIF-1α的表达与视网膜细胞凋亡的研究

目的:探讨大鼠视网膜细胞缺血再灌注后低氧诱导因子(HIF-1α)的表达、视网膜细胞凋亡的发生以及二者之间的关系.方法:采用前房灌注生理盐水升高大鼠眼压到110mmHg(1kPa=7.5mmHg)持续1 h的方法制作实验性视网膜缺血再灌注损伤模型,分别于再灌注不同时间点取材,行免疫组织化学法染色观察HIF-1α在视网膜细胞的阳性表达;采用DNA原位末端标记(terminal dUTP nick end labelling,TUNEL)法定位凋亡的视网膜细胞来检测视网膜凋亡细胞.结果:缺血再灌注2h组视网膜神经节细胞层及内核层细胞出现HIF-1α弱阳性表达,12h组阳性表达至高峰,24h组HIF-1α表达渐减少.视网膜组织细胞凋亡见于缺血再灌注12,24及48h组,凋亡细胞主要位于内核层,且24h组凋亡阳性表达最强.结论:缺血再灌注后大鼠视网膜HIF-1α的表达增加.参与视网膜缺血再灌注损伤;视网膜细胞的损伤部分以凋亡的形式发生,HIF-1α的表达可能与视网膜细胞凋亡有密切的关系.     

腺病毒载体介导的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:观察重组腺病毒介导的色素上皮衍生因子(Ad-PEDF)对大鼠视网膜缺血再灌注损伤的保护作用及机制。方法:选用健康大鼠96只,随机分为正常组、缺血再灌注组、缺血再灌注+Ad-CMV组,缺血再灌注+Ad-PEDF组,以前房加压的方法制备大鼠视网膜缺血再灌注模型,缺血再灌注+Ad-CMV组,缺血再灌注+Ad-PEDF组分别玻璃体腔注射Ad-CMV或Ad-PEDF1μL(滴度3.8×109/PFU),每组按照时间点12,24,72,168h,为4亚组,光学显微镜观察视网膜组织切片情况,并测量视网膜内层厚度及神经节细胞层神经节细胞数量。以TUNEL方法观察大鼠视网膜神经节细胞凋亡情况。结果:Ad-PEDF组视网膜内层厚度均超过缺血组及缺血+Ad-CMV组,Ad-PEDF组神经节细胞数目多于缺血组及Ad-CMV组,Ad-PEDF组视网膜神经节细胞凋亡细胞少于缺血组及Ad-CMV组,凋亡程度减轻,上述差异均具有显著性(P<0.05)。结论:腺病毒介导的色素上皮衍生因子玻璃体腔注射能够恢复大鼠视网膜缺血再灌注损伤所致的视网膜内层厚度降低,神经节细胞密度减少,具有保护作用。     

Interleukin-6 in retinal ischemia reperfusion injury in rats

PURPOSE: To study the role of interleukin (IL)-6 after retinal ischemia-reperfusion (I/R) injury in rats. METHODS: Intraocular pressure of adult male Lewis albino rats was raised to create retinal ischemia for 1 hour. Retinal reperfusion was reestablished, and the animals were killed at various time points after the injury. Their eyes were enucleated and processed for immunohistochemistry to detect IL-6 and ED-1 (a marker of microglial/phagocytic cells), enzyme-linked immunosorbent assay (ELISA) of IL-6 protein, and semiquantitative real-time RT-PCR for IL-6 mRNA. The neuroprotective effect of IL-6 was evaluated by giving intravitreal injections of 150 or 300 ng rat recombinant IL-6 to eyes immediately after I/R injury and counting cresyl violet-stained retinal ganglion cell layer cells (RGCLCs) and fluorochrome-labeled retinal ganglion cells (RGCs) on flat preparations of retinas at 7 days. RESULTS: IL-6-positive cells appeared after I/R injury in the inner plexiform layer (IPL) and the inner nuclear layer (INL). Their numbers were significantly higher 18 hours after the injury, and most of these cells were also ED-1 positive. ELISA showed noticeable increases in endogenous retinal IL-6 protein levels 8 hours after I/R injury. Semiquantitative real-time RT-PCR showed significant increases in endogenous retinal IL-6 mRNA levels between 2 and 18 hours. Exogenously added IL-6 prevented between 50% and 70% of RGC loss after I/R injury. CONCLUSIONS: IL-6 is upregulated after retinal I/R injury, and its expression by microglia/phagocytic cells may protect RGC layer neurons from I/R injury. Exogenously added IL-6 protects the inner retina after I/R injury.     

Retinal neuroprotection against ischemic injury mediated by intraocular gene transfer of pigment epithelium-derived factor

PURPOSE. To determine whether intraocular gene transfer of pigment epithelium-derived factor (PEDF) protects the retina from ischemia-reperfusion injury. METHODS. Four days before induction of pressure-induced ischemia, Lewis rats received intravitreous injection of 3 x 10(9) particles of an adenovirus vector expressing PEDF (AdPEDF.11) in one eye and 3 x 10(9) particles of an empty adenovirus vector (AdNull.11) in the contralateral eye. Seven days after reperfusion, eyes were enucleated and processed for morphometric analysis. Apoptotic cells stained by TdT-dUTP terminal nick-end labeling (TUNEL) in the retina were counted 12 hours after initiation of reperfusion. Retina levels of PEDF were measured by enzyme-linked immunosorbent assay. RESULTS. PEDF levels in retinal homogenates from eyes receiving AdPEDF.11 injection were well above the background levels in the untreated baseline and control eyes (P = 0.04). Retinal thickness was preserved in AdPEDF.11-treated eyes. Retinal cell density was significantly greater in the ganglion cell layer (GCL; P = 0.014), inner nuclear layer (INL; P = 0.008), and outer nuclear layer (ONL; P = 0.008) of AdPEDF.11-treated eyes compared with the corresponding layers in AdNull.11-treated eyes. AdNull.11-treated eyes also had significantly more TUNEL-positive cells in these layers than AdPEDF.11-treated eyes (P < 0.05). CONCLUSIONS. Adenoviral vector-mediated intraocular expression of PEDF significantly increases cell survival after ischemia-reperfusion injury of the retina. The protective effect may result from inhibition of ischemia-induced apoptosis. This study provides proof of concept for a gene transfer approach directed at interrupting programmed cell death induced by retinal ischemic insult.     

大鼠视网膜缺血-再灌注损伤超微结构观察及机制探讨

目的研究大鼠视网膜缺血-再灌注损伤中的超微结构改变以及凋亡相关基因表达的变化,探讨其损伤机制。方法将28只大鼠随机分为正常组和手术组,手术组按照再灌注后不同时间段分为1h、6h、12h、24h、48h、72h组。前房加压法制作大鼠视网膜缺血-再灌注损伤模型,透射电镜检测视网膜超微结构改变,免疫组织化学法检测视网膜组织中bcl-2、bax、Fas的表达。结果(1)正常组视网膜神经纤维中微管及线粒体清晰可见;视网膜神经节细胞(retinal ganglion cells,RGCs)核大,电子密度低,核仁明显,细胞器丰富;再灌注损伤后RGCs核膜肿胀,线粒体嵴模糊不清,可见凋亡小体,神经纤维中微管模糊、减少甚至消失,以再灌注后24h为甚;(2)再灌注后6h,bax表达逐渐递增,24h达到高峰,48h开始下降,72h不明显;(3)bcl-2在视网膜神经节细胞层、纤维层及内核层有微弱表达,各个时间段变化不明显;(4)Fas表达改变与bax基本一致。结论视网膜缺血-再灌注损伤中,细胞凋亡是引起视网膜内核层和神经节细胞层细胞死亡的主要方式,其损伤机制与凋亡相关基因bcl-2、bax、Fas的表达变化有关。     

N-乙酰-5-羟色胺对视网膜缺血-再灌注损伤大鼠视网膜 Fas、FasL 蛋白表达的影响

目的　探讨N-乙酰-5-羟色胺（N-acetylserotonin，NAS）对视网膜缺血-再灌注损伤（retina ischemia-reperfusion injury，RIRI）大鼠视网膜Fas、FasL蛋白表达的影响。方法　取健康成年Sprague Dawley大鼠54只，将大鼠随机分为正常组（6只）、RIRI组（24只）与NAS组（24只）；采用高眼压法建立大鼠RIRI模型，依据造模后不同时间点将RIRI组与NAS组大鼠又分为6 h、12 h、24 h及72 h四个亚组。NAS组于造模前30 min腹腔注射NAS（5 mg·kg^-1），RIRI组腹腔注射等剂量的生理盐水。通过HE染色在光学显微镜下观察各组大鼠视网膜形态学变化，并记录各组大鼠视网膜厚度及视网膜神经节细胞数，采用免疫组织化学染色法检测NAS对RIRI大鼠视网膜Fas、FasL蛋白表达的影响。结果　HE染色显示，正常组大鼠视网膜各层细胞分界清晰，形态正常，神经细胞排列整齐；RIRI组大鼠再灌注后6 h视网膜各层出现水肿，以神经节细胞层及内核层较显著，神经节细胞数较正常组减少；随后视网膜水肿进一步加重，神经节细胞继续减少；NAS组大鼠在再灌注后6 h、12 h、24 h 视网膜水肿程度较 RIRI组轻，NAS组在再灌注后72 h视网膜厚度较 RIRI组厚，NAS组各时间点神经节细胞数均较 RIRI组多，差异均有统计学意义（均为P＜0.05）。免疫组织化学染色显示，正常组几乎未见 Fas⁺细胞。再灌注后6 h，RIRI组视网膜神经节细胞及内核层开始出现少量 Fas⁺细胞；再灌注后12 h，RIRI组视网膜 Fas⁺细胞表达逐渐增多；再灌注后24 h视网膜Fas⁺细胞数达到高峰，棕色阳性染色细胞分布在视网膜神经节细胞层、内丛状层、内核层及神经纤维层；再灌注后 72 h 视网膜 Fas⁺细胞较再灌注后 24 h 减少。NAS组在再灌注后6 h、12 h、24 h、72 h 视网膜 Fas⁺细胞数均较 RIRI组各时间点减少，再灌注后24 h，Fas⁺细胞数达较高水平，随后下降，差异均有统计学意义（均为P＜0.05）。正常组视网膜可见 FasL 全层低表达。RIRI组再灌注后 6 h，视网膜神经节细胞层和神经纤维层存在少量 FasL⁺细胞；再灌注后12 h FasL蛋白表达逐渐增多；再灌注后24 h FasL⁺细胞数达高峰，可见深棕色的细胞膜及细胞质染色细胞分布在视网膜神经节细胞层、内丛状层、内核层及神经纤维层；再灌注后72 h FasL蛋白的阳性表达逐渐减少。NAS组再灌注后6 h、12 h、24 h、72 h 视网膜FasL⁺细胞数均少于 RIRI组各时间点阳性细胞数，差异均有统计学意义（均为P＜0.05）。结论　NAS可通过抑制RIRI大鼠视网膜细胞Fas、FasL蛋白的表达，减轻缺血再灌注对大鼠视网膜细胞造成的损伤。     

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.     

Expression and role of autophagy related protein p62 and LC3 in the retina in a rat model of acute ocular hypertension

AIM: To investigate the expression and possible role of the autophagy related protein p62 and LC3 in the retina based on a rat model of acute ocular hypertension.METHODS: Fifty rats were randomized into five groups: control group A, B, C, and D. Groups A to D all received normal saline perfusion into the anterior chamber with pressure of 80 mm Hg for one hour, and retina tissue was obtained at 6, 12, 24 and 48 h after perfusion respectively, to investigate the activation of autophagy following ischemiareperfusion. The distribution and semi-quantification of autophagy related protein p62 and LC3 in the retina were detected using immunohistochemistry technique. The expression level of these two proteins was evaluated using Western blot.RESULTS: The number of retinal ganglion cells(RGCs) decreased with increasing reperfusion time, and significant reduction in the retinal thickness was observed 48 h after perfusion. In normal adult rats, LC3 protein was mainly expressed in the ganglion cell layer(GCL), and p62 protein was expressed in the nerve fiber layer(NFL), GCL, inner plexiform layer(IPL), inner nuclear layer(INL) and outer plexiform layer(OPL). In comparison to the control group, the expression level of LC3-II was higher in all the experimental groups(P<0.05), with the peak expression at 12 h after reperfusion. Additionally, the expression level of p62 was higher in all the experimental groups than the control(P<0.05, except for group A), with the peak level occurred 24 h after reperfusion. CONCLUSION: Both p62 and LC3 show low level and uneven expression in the retina of normal adult rats. Acute ocular hypertension can lead to upregulation of LC3-II and p62 expression in the retina. Autophagy flux is damaged 12 h after reperfusion, potentially resulting in further loss of RGCs.     

Distribution of p27(KIP1), cyclin D1, and proliferating cell nuclear antigen after retinal detachment

Purpose To examine the expression of the p27(KIP1), cyclin D1, and proliferating cell nuclear antigen (PCNA) in the retina and retinal pigment epithelium (RPE) after retinal detachment.Methods Normal eyes and eyes at 2 or 4 days after retinal detachment with the C57B16 mouse were analyzed by immunocytochemistry using anti-p27(KIP1), anti-cyclin D1, and anti-proliferating cell nuclear antigen (PCNA) antibodies as well as anti-glutamate synthetase (GS) antibody.Results The p27(KIP1) positive nuclei were distributed in the inner nuclear layer (INL) and the RPE of the normal mice eye. In the INL, p27(KIP1) was detected in the middle sublayer, where the nuclei of glutamate synthetase positive Müller cells were situated. In contrast, cyclin D1 was not detected either in the retina or in the RPE. At 2 and 4 days after the retinal detachment, RPE cells under the detached retina were negative for p27(KIP1) and positive for cyclin D1 and PCNA. In the INL of the detached retina, p27(KIP1) was detected after 2 days, but was not detected after 4 days. In contrast, PCNA was not detected in the INL after 2 days, but was detected after 4 days. Cyclin D1 was detected in the middle sublayer of the INL at both 2 and 4 days after the retinal detachment.Conclusion These results suggested that degradation of p27(KIP1) and expression of cyclin D1 was involved in the proliferation of the Müller cells as well as RPE cells after retinal detachment.