川芎嗪对大鼠缺血再灌注视网膜SOD,MD和NO水平及细胞凋亡的影响

目的 :观察川芎嗪对大鼠视网膜缺血再灌注后视网膜超氧化物歧化酶 (superoxidedismutase ,SOD)、丙二醛 (malondialdehyde ,MDA)、一氧化氮 (nitricoxide ,NO)水平及视网膜细胞凋亡的影响。方法 :采用大鼠视网膜压力缺血再灌注模型 ,分光光度法测定SOD、MDA和NO ,琼脂糖凝胶电泳分析DNA断裂。结果 :视网膜缺血 6 0min再灌注后SOD水平下降 ,而MDA和NO水平则升高 ;视网膜缺血 6 0min再灌注 12h ,提取DNA进行琼脂糖凝胶电泳可见凋亡样DNA断裂 (ApoptoticDNAfragmentation)。川芎嗪能显著对抗视网膜缺血再灌注时视网膜SOD水平的下降、MDA和NO水平的升高 ;同时能阻断大鼠视网膜缺血再灌注 12h后视网膜细胞DNA凋亡样断裂。结论 :川芎嗪可能通过抑制自由基的产生和提高抗氧化能力来对抗大鼠视网膜缺血再灌注诱导的细胞凋亡     

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.     

川芎嗪对大鼠缺血再灌注视网膜SOD，MDA和NO水平及细胞凋亡的影响

目的 观察川芎嗪对大鼠视网膜缺血再灌注后视网超氧化物歧化酶（SOD）,丙二醛（MDA）,一氧化氮（NO）水平及细胞凋亡的影响。方法 采用大鼠视网膜压力缺血再灌注模型,分光光度法测定SOD,MDA和NO,琼脂糖凝胶电泳分析DNA断裂。结果 川芎嗪能显著对抗视膜膜缺血再灌注后视网膜SOD水平的下降及MDA和NO水平的升高;同时能阻断缺血再灌注12h后细胞DNSA凋亡样断裂。结论 川芎嗪可能通过抑制自由基的产生和提高抗氧化能力来对抗大鼠视网缺血再灌注诱导的细胞凋亡。     

大鼠视网膜缺血再灌注损伤后脑源性神经生长因子对细胞凋亡及caspase-2和caspase-3表达的影响

目的:探讨caspase-2和caspase-3在大鼠视网膜缺血再灌注损伤中的表达与细胞凋亡的关系及脑源性神经生长因子对其的影响及对视网膜的保护作用。方法:实验于2007-02/2007-07在青岛大学医学院附属医院中心实验室完成。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手术组,其中手术组大鼠左眼为缺血再灌注组,右眼为治疗组(BDNF玻璃体腔注射),手术组又按照再灌注后不同时间段分为1,6,12,24,48,72h组。光学显微镜观察并计数视网膜神经节细胞的数量。应用末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸缺口末端标记法(TUNEL)检测视网膜神经节细胞凋亡、免疫组织化学法(SABC)和酶联免疫吸附实验(ELISA)检测视网膜组织中caspase-2和caspase-3的表达情况。结果:正常视网膜未见凋亡细胞表达,缺血后6～24h可见大量凋亡细胞表达,48h开始下降。凋亡细胞在缺血后24h达到高峰,caspase-2缺血6h后逐渐增加,24h达高峰,然后在48至72h下降。caspase-3表达改变与caspase-2改变基本一致。BDNF治疗组各观察指标表达变化规律与缺血组基本一致,但能明显抑制凋亡细胞的表达,同时使caspase-2和caspase-3的表达降低。结论:视网膜缺血再灌注损伤诱导了神经节细胞的凋亡;BDNF可抑制caspase-2和caspase-3的表达,减少神经节细胞凋亡,对视网膜缺血再灌注损伤有治疗作用。     

Light-induced Apoptosis: Differential Timing in the Retina and Pigment Epithelium

Apoptosis is a genetically regulated form of cell death. Individual cells show condensed nuclear chromatin and cytoplasm, and biochemical analysis reveals fragmentation of the DNA. Ensuing cellular components, apoptotic bodies, are removed by macrophages or neighboring cells. Genes involved in the regulation of apoptosis as well as stimuli and signal transduction systems, are only beginning to be understood in the retina. Therefore, we developed a new in vivo model system for the investigation of events leading to apoptosis in the retina and the pigment epithelium. We induced apoptosis in retinal photoreceptors and the pigment epithelium of albino rats by exposure to 3000 lux of diffuse, cool white fluorescent light for short time periods of up to 120 minutes. Animals were killed at different time intervals during and after light exposure. The eyes were enucleated and the lower central retina was processed for light- and electron microscopy. DNA fragmentation was analysed in situ by TdT-mediated dUTP nick-end labeling (TUNEL) or by gel electrophoresis of total retinal DNA. We observed that the timing of apoptosis in the photoreceptors and pigment epithelium was remarkably different, the pigment epithelium showing a distinct delay of several hours before the onset of apoptosis. In photoreceptors, apoptosis was induced within 90 minutes of light exposure, with the morphological appearance of apoptosis preceding the fragmentation of DNA. In the pigment epithelium, the morphological appearance of apoptosis and DNA fragmentation were coincident. Different regulative mechanisms may lead to apoptotic cell death in the retinal photoreceptors and pigment epithelium. This in vivo model system will allow measurement of dose-responses, a potential spectral dependence and the molecular background of apoptotic mechanisms in the retina.     

N-methyl-D-aspartate (NMDA)--induced apoptosis in rat retina.

PURPOSE: The involvement of apoptosis in N-methyl-D-aspartate (NMDA)-induced excitotoxicity in adult rat retinas was examined. METHODS: Excitotoxic loss of inner retinal elements was induced by intravitreal injections of various concentrations of neutralized NMDA in adult albino Lewis rats. Tissue responses were quantified by measuring the inner retinal thickness (IRT) in plastic sections of the retinas and cell counts in the retinal ganglion cell layer in flatmount preparations of the whole retinas. Internucleosomal DNA fragmentation, a hallmark of apoptosis, was assayed with agarose DNA gel electrophoresis. The in situ TdT-mediated biotin-dUTP nick end labeling (TUNEL) method was used to locate nicked DNA in paraffin sections of the retinas. Ultrastructural changes of the degenerating cells were examined by electron microscopy. The efficacy of Ac-Tyr-Val-Ala-Asp-CMK (YVAD-CMK), a peptidyl caspase inhibitor, and 3-aminobenzamide (ABA), an inhibitor of poly(ADP-ribose) polymerase (PARP), in ameliorating the loss of inner retinal elements was evaluated using morphometry to examine the apoptotic pathways. RESULTS. Intravitreal injection of NMDA induced a dose-dependent loss of inner retinal elements as evidenced by the measurements of IRT and RGCCs. There were time- and dose-related appearances of internucleosomal fragmentation of retinal DNA and a time-related appearance of TUNEL-positive nuclei in the inner retinas after intravitreal NMDA injection. Ultrastructural features consistent with classic apoptotic changes were noted in degenerating cells in the retinal ganglion cell layer and the inner nuclear layer. Control retinas given vehicle, N-methyl-L-aspartate (the L-isomer of NMDA), or NMDA plus MK-801, a specific antagonist, did not show these changes. Simultaneous administration of NMDA and YVAD-CMK or ABA abolished or attenuated the loss of RGCCs in the posterior retinas. CONCLUSIONS. NMDA-induced excitotoxicity involved apoptosis and caspases and PARP may play important roles in the pathways.     

Neuroprotective effects of eliprodil in retinal excitotoxicity and ischemia

PURPOSE: To evaluate whether eliprodil (SL82.0715), a NR2B-selective N-methyl-D-aspartate (NMDA) antagonist, is protective of retina subjected to an excitotoxic or ischemic insult. METHODS: To evaluate protection against retinal excitotoxicity, eliprodil was administered intraperitoneally before and after the injection of NMDA (5 microl, 20 nmol) into the vitreous of rats. Integrity of the retina was assessed by counting cells in the retinal ganglion cell layer (GCL) and measuring choline acetyltransferase (ChAT) activity. In a subsequent experiment, total retinal ischemia, as measured by a cessation of electroretinographic (ERG) activity, was induced in anesthetized rabbits by elevating intraocular pressure above systolic blood pressure for 65 minutes. After ischemia, recovery of ERG activity was assessed at 24 and 48 hours in animals treated with vehicle or eliprodil (1.0-10.0 mg/kg). RESULTS: Intravitreal NMDA injection resulted in a dose-related decrease in cells of the GCL and in ChAT activity. Eliprodil administered intraperitoneally at 10 mg/kg completely prevented the loss of ChAT and the loss of cells in the GCL. Twenty-four hours after retinal ischemia, A and B waves of vehicle-treated animals were suppressed by 60% to 70%. Eliprodil administered intraperitoneally at 10 mg/kg ameliorated the A- and B-wave depression throughout the 48-hour experiment. CONCLUSIONS: Eliprodil is neuroprotective of retinae subjected to either an excitotoxic or ischemic challenge and may be useful for treating a variety of retinal and optic nerve head disorders.     

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.     

Ischemia-reperfusion causes exudative detachment of the rabbit retina

PURPOSE: To characterize the activation of macroglial (Müller) and microglial cells, as well as neuronal cell degeneration, during ischemia-reperfusion in rabbit retina and to test the possible effect of triamcinolone acetonide on gliosis. METHODS: Transient retinal ischemia was produced by increasing intraocular pressure for 60 minutes. Triamcinolone (8 mg) was intravitreally applied immediately after the cessation of ischemia. At 3 and 8 days after reperfusion, the K+ currents of acutely isolated Müller cells were recorded, and the Ca2+ responses of Müller cells on stimulation of P2Y receptors were recorded fluorometrically in retinal wholemounts. Microglial/immune cells in the nerve fiber layer of retinal wholemounts were labeled with isolectin. To evaluate neuronal and Müller cell loss, the numbers of cells were counted in retinal slices. RESULTS: Transient ischemia caused exudative detachment of the central retina that was characterized by disruption of the pigment epithelial monolayer, the presence of scattered pigment epithelial and immune cells in the expanded subretinal space, and retinal folds. A significant loss of photoreceptor cells was observed at 8 days after reperfusion. At 3 and 8 days after reperfusion, Müller cell gliosis was apparent, as indicated by cellular hypertrophy, downregulation of K+ channel expression, and an increased number of cells that displayed P2Y receptor-mediated Ca2+ responses. The number of microglial/immune cells increased strongly after reperfusion. Intravitreal triamcinolone did not affect the parameters of Müller cell gliosis but decreased the number of microglial/immune cells. CONCLUSIONS: Ischemia-reperfusion of the rabbit retina causes exudative retinal detachment that is characterized by a loss of photoreceptor cells, whereas the inner retina remains largely preserved. Micro- and macroglial cells are activated early during reperfusion, even before dropout of the photoreceptor cells. Intravitreal triamcinolone may decrease the degree of microglial/immune cell activation.     

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.     

Kainic acid-mediated upregulation of matrix metalloproteinase-9 promotes retinal degeneration

PURPOSE: Excitotoxicity has been proposed to play a pivotal role in retinal damage, but the mechanisms that underlie retinal damage are not clearly understood. In this study, the role of matrix metalloproteinases in excitotoxin-mediated retinal damage was investigated. METHODS: KA, CNQX (6-cyano-7-nitroquinoxaline-2,3,-dione), NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline), MK801, or PBS was injected into the vitreous of CD-1 mice. MMP expression in the retina was analyzed by zymography, Western blot, and immunohistochemistry. Retinal ganglion cells (RGCs) were retrogradely labeled with aminostilbamidine methanesulfonate (Molecular Probes, Eugene, OR), and loss of fluorescently labeled RGCs in retinal flatmounts was quantified. Apoptotic cell death was assessed by TUNEL staining. Astrocyte activation was determined by immunohistochemistry, and laminin decrease was determined by immunohistochemistry and Western blot analysis. RESULTS: Intravitreal injection of KA caused time- and dose-related MMP-9 upregulation in the retina. Increased MMP-9 activity and protein levels were associated with activation of astrocytes. Astrocyte-associated MMP-9 correlated with a decrease in laminin immunoreactivity in the ganglion cell layer and significant loss of retinal ganglion cells. KA-mediated upregulation of MMP-9 activity was associated with apoptosis of cells in the ganglion cell layer as early as 6 hours after injection, followed by apoptosis in cells in the inner nuclear layer by day 1. Intravitreal injection of the non-NMDA receptor antagonists, CNQX and NBQX decreased KA-induced MMP-9 activity and protein levels in the retina and attenuated retinal degeneration, whereas the NMDA receptor antagonist MK801 failed to offer protection. Further, a synthetic MMP inhibitor GM6001 decreased KA-mediated MMP-9 activity and offered significant protection against ganglion cell loss in the retina. CONCLUSIONS: These results indicate that KA-mediated upregulation of MMP-9 activity promotes retinal degeneration and suggest that inhibition of KA-mediated MMP activity may offer protection against excitotoxin-induced retinal damage.     

Quantitative ex vivo detection of rodent retinal ganglion cells by immunolabeling Brn-3b

To evaluate the neuroprotective potential of drug candidates to treat human glaucoma, a short-term rodent model of retinal ganglion cell death was employed. Transient ischemia applied to the rodent retina, with subsequent reperfusion for 1-4 weeks, produces an experimental retinal ganglion cell death that is quantifiable. A widely used method to detect viable retinal ganglion cells involves surgical injection of labeling compounds into the superior colliculus of the rodent brain, the retrograde transport of the compounds along the axons to the retina, and subsequent microscopic evaluation of the retina. In order to circumvent the labor intensive and invasive surgery of this method, we sought an alternative means of assessing retinal ganglion cell survival that would be more suitable for high-throughput analysis. We therefore developed a method of immunolabeling whole retinas ex vivo with an antibody to Brn-3b, an antigen expressed in a subpopulation of retinal ganglion cells, that allows for detection of a representative retinal ganglion cell population. Fluorescently tagged Brn-3b immunolabeled retinas were flat-mounted, digitally imaged, and assessed using image analysis software. We determined that 60 min of ischemia caused a 49% and a 32% decrease in Brn-3b positive retinal ganglion cells in Lewis rats after 4 weeks reperfusion, and Sprague-Dawley rats after 2 weeks reperfusion, respectively. In Swiss Webster ND4 mouse retinas subjected to 45 min ischemia and 7 days reperfusion, we found a 70% decrease in Brn-3b positive cells. Thus, ex vivo immunolabeling of retinal ganglion cells using antibody to Brn-3b provides an alternative to other methods of quantifying retinal ganglion cells.     

Ischemic preconditioning attenuates apoptotic cell death in the rat retina

PURPOSE: Ischemic preconditioning (IPC) protects the rat retina against the injury that ordinarily follows prolonged ischemia. It has been shown that release of adenosine, de novo protein synthesis, and mediators, such as protein kinase C and K(ATP) channels, is required for IPC protection. However, the molecular mechanisms of neuroprotection by IPC are unknown. Retinal cells die after ischemia by necrosis and apoptosis. This study was undertaken to investigate the effect of IPC on apoptosis after ischemia and some of the key proteins involved in the apoptotic cascade. METHODS: Retinal ischemia or IPC was produced in anesthetized Sprague-Dawley rats by increasing intraocular pressure above systolic arterial pressure. Retinal ischemia was induced 24 hours after either IPC or sham IPC. TUNEL staining was used to quantitate the number of cells with DNA fragmentation. The authors examined expression of cleaved forms of caspases-2 and -3, bax, and poly-adenosine diphosphate-ribose-polymerase (PARP) by Western blot analysis for evidence of apoptosis-related gene expression. To examine possible mechanisms of apoptosis after ischemia, the authors studied the expression of mitogen-activated protein kinases (MAP kinases). Functional recovery after ischemia was measured using electroretinography, and retinal histology was examined and quantitated by light microscopy. RESULTS: Positive TUNEL staining, increases in caspase-2 and -3 cleavage, expression of bax and PARP, and activation of MAP kinases were found with ischemia. IPC attenuated these changes, but paradoxically, IPC itself triggered increased expression of MAP kinases. CONCLUSIONS: IPC protects against ischemic injury, in part, by diminishing apoptosis-related gene expression and by altering protein phosphorylation.     

Mechanism of the pathogenesis of glutamate neurotoxicity in retinal ischemia

· Purpose: This study was carried out to examine the involvement of glutamate and nitric oxide neurotoxicity in ischemia/reperfusion-induced retinal injury in vivo. · Methods: We monitored glutamate release from in vivo cat retina during and after pressure-induced ischemia using a microdialysis technique. Morphometric studies were performed to study the effects of MK-801 (dizocilpine), L-NAME (N ^ω-nitro-l-arginine methyl ester), and D-NAME (N ^ω-nitro-d-arginine methyl ester) on the histological changes in the rat retina induced by ischemia or intravitreal injection of NMDA (N-methyl-d-aspartate; 200 nmol). · Results: A large release of glutamate occurred during ischemia, followed by a marked release after reperfusion. Histological changes occurred selectively in the inner part of the retina after ischemia as well as intravitreal injection of NMDA. Pretreatment with intravenous injection of MK-801 or L-NAME significantly inhibited the ischemic injury of the inner retina. Intravitreal injection of L-NAME inhibited NMDA-induced neurotoxicity in the retina. · Conclusion: These findings indicate that nitric oxide mediates neurotoxic actions of glutamate which are responsible for ischemic injury in the retina. Received: 17 November 1997 Revised version received: 3 February 1998 Accepted: 5 February 1998     

Functional and morphological effects of β-estradiol in eyes with N-methyl-D-Aspartate-induced retinal neurotoxicity in rats

Glutamate-mediated excitotoxicity, mainly induced by N-methyl-d-aspartate (NMDA) receptors, is known to cause retinal ganglion cell death in retinal ischemia, glaucoma, and several other retinal diseases. We evaluated the effects of β-estradiol (E2) against a single intravitreal injection of NMDA using a functional and morphological approach. Male rats were randomly divided into 3 treatment groups: (1) Control; (2) NMDA (intravitreal injection of 5 mM NMDA); and (3) NMDA + E2 (intravitreal injection of 5 mM NMDA and pretreatment with subcutaneous E2 implantation). Seven days after NMDA injection, full-field electroretinograms (ERGs) and quantitative morphological analyses using transverse sections of the retina were conducted. In the NMDA group, full-field ERGs showed reductions in the amplitudes of the negative-scotopic threshold response, rod response b-wave, oscillatory potentials, flicker response second b-wave and cone response b-wave. Morphological evaluations of transverse sections of the retina demonstrated a reduction in the thickness of the inner plexiform layer, increases in the thickness of the outer plexiform and outer nuclear layers, and a loss of cells in the ganglion cell layer. In the NMDA + E2 group, pretreatment with E2 prevented the aggravations in the amplitudes of the ERGs except for oscillatory potential 2 (OP2); however, no morphological differences between the NMDA and NMDA + E2 groups were seen. These findings indicate that E2 can protect retinal function against NMDA-induced neurotoxicity. In addition, these indications suggested that the effect of E2 may have therapeutic benefits in NMDA related diseases, such as retinal ischemia and glaucoma.     

In vivo evaluation of platelet--endothelial interactions after transient retinal ischemia

PURPOSE: Accumulating evidence suggests that platelets play an important role in ischemia-reperfusion injury. To fulfill that role, platelets flowing in the bloodstream would have to interact with retinal endothelial cells and to accumulate in the postischemic retina. This study was designed to investigate quantitatively platelet-endothelial interactions in postischemic retina after transient retinal ischemia. METHODS: Transient retinal ischemia was induced in Long-Evans rats for 60 minutes by temporal ligation of the optic nerve. Isolated platelet samples labeled with carboxyfluorescein diacetate succinimidyl ester were administered intravenously to recipient rats after various reperfusion periods. Platelet-endothelial interactions in postischemic retina were evaluated in vivo with a scanning laser ophthalmoscope. Anti-P-selectin monoclonal antibody (mAb) was administered 5 minutes before the injection of labeled platelets. P-selectin gene expression in the postischemic retina was studied by semiquantitative polymerase chain reaction. RESULTS: Under basal conditions, infused platelets showed minimal interactions with retinal endothelial cells. In contrast, postischemic retinas showed active platelet-endothelial interactions. Many platelets were observed rolling along and adhering to the major retinal veins. The number of rolling and adhering platelets reached a peak (555 +/- 65/mm per min and 25.8 +/- 3.2/mm(2)) 12 hours after reperfusion. However, the interactions between platelets and postischemic retinal endothelial cells were substantially inhibited by neutralizing P-selectin expressed on endothelial cells. In addition, P-selectin gene expression in postischemic retina corresponded with the time course of platelet-endothelial interactions during the reperfusion period. CONCLUSIONS: This study demonstrated that platelets actively interacted with retinal endothelial cells in the postischemic retina through P-selectin expressed on the retinal endothelial cells.     

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.     

Activation of matrix metalloproteinase-9 via neuronal nitric oxide synthase contributes to NMDA-induced retinal ganglion cell death

PURPOSE: Understanding the mechanism of neuronal cell death in retinal diseases like glaucoma is important for devising new treatments. One factor involves excitatory amino acid stimulation of N-methyl-D-aspartate (NMDA)-type glutamate receptors, excessive Ca2+ influx, and formation of nitric oxide (NO) via neuronal NO synthase (nNOS). Another factor is the abnormal activation of matrix metalloproteinases (MMPs), in particular MMP-9, which triggers an extracellular signaling cascade leading to apoptosis. This study was designed to investigate the mechanism of excitotoxic retinal ganglion cell (RGC) death in vivo and its relationship to MMP activation. METHODS: NMDA and glycine were injected into the vitreous of the eye in rats and in nNOS-deficient mice (nNOS-/-) versus control. Gelatinolytic activity of MMP-9 and MMP-2 by zymography and cellular localization by immunohistochemistry were examined, and the effect of MMP inhibition on NMDA-induced RGC death was tested. RESULTS: NMDA was found to upregulate the proform of MMP-9 in the retina and to increase MMP-9 gelatinolytic activity. Retrograde labeling with aminostilbamidine to identify RGCs confirmed that MMP activity occurred only in these retinal neurons and not in glial or other retinal cell types after excitotoxic insult. Deconvolution fluorescence microscopy revealed that MMP activity colocalized with immunoreactive S-nitrosylated protein. NMDA-induced MMP activation was diminished in the retina of nNOS-/- mice, implying that S-nitrosylation of MMP had indeed occurred. In addition, the broad-spectrum MMP inhibitor GM6001 protected RGCs after intravitreal NMDA injection. CONCLUSIONS: These findings suggest that an extracellular proteolytic pathway in the retina contributes to RGC death via NO-activated MMP-9.