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     

家兔视网膜缺血-再灌注损伤后形态学的动态变化

目的　观察家兔视网膜缺血 -再灌注后视网膜结构的动态变化。方法　通过前房灌注加压至 16 .7k Pa,维持 1h,建立缺血模型 ,观察再灌注 2～ 14d内其结构变化及内层视网膜平均厚度的变化。结果　家兔视网膜在缺血 -再灌注后 2～ 14d内表现为神经细胞持续丢失 ,视网膜层次逐渐不清 ,萎缩、变薄。其中神经节细胞、神经纤维及视锥、视杆对缺血最敏感 ,外颗粒层次之 ,内颗粒层最能耐受缺血 -再灌注后的损伤。结论　视网膜缺血 -再灌注损伤是个持续性、进行性的损伤过程 ,与功能变化相一致     

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.     

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。     

Glutamate elevation in rabbit vitreous during transient ischemia-reperfusion

PURPOSE: To investigate the response of the retina to an ischemic insult, we measured the levels of glutamate in the vitreous of rabbit eyes. METHODS: Ocular ischemia was induced in the vitreous of rabbit eyes by simultaneous ligature of the optic nerve, ciliary arteries, and extraocular muscles for 45 minutes. Contralateral eyes were subjected to a conjunctival peritomy to serve as sham-operated controls. Reperfusion was achieved by releasing the ligature. Eyes were enucleated at the end of the ischemic period or 15, 45, or 75 minutes after reperfusion. RESULTS: Analysis of the vitreous free amino acids showed a significant increase in glutamate levels in the operated eyes at the end of the ischemic period (P <.001) and after 15 minutes of reperfusion (P <.05) as compared with the contralateral, sham-operated eyes. Glutamine levels in the vitreous were unchanged throughout the study. CONCLUSIONS: These results show that glutamate, which is considered to be derived from synaptic release of the retinal neurons or accumulation due to a deterioration of glutamate uptake or a degradation system in the retina, was transiently elevated in the vitreous.     

Anti-excitotoxic activity of trimetazidine in the retina.

The purpose of this study was to investigate the mechanism of the neuroprotective activity of trimetazidine in animal retina stressed by ischemia or kainate. Flash electroretinograms were recorded in guinea pigs after ischemia, induced by an acute increase in the intraocular pressure (IOP), or after an intravitreal injection of kainate. Treatment with trimetazidine per os afforded a significant protection of the electroretinogram against the ischemic as well as the excitotoxic insult as an antioxidant (dimethylthiourea) and a nitric oxide synthase inhibitor (nitroarginine) did. The effect of the drug on the extracellular accumulation of glutamate induced by chemical ischemia was studied by incubating rat retina in vitro. Trimetazidine was able to inhibit the extracellular glutamate accumulation, which represents the first step of the excitotoxic phenomenon. Then the compound activity on the glial uptake of glutamate was studied in a rat Müller cell line (rMC-1) in culture. Chemical ischemia inhibited the active 3H-glutamate transport, an effect that was reversed by trimetazidine, at micromolar concentrations. These results demonstrate that trimetazidine which is recognized as an efficient drug against ischemic injuries, is also capable of protecting the retina against excitotoxicity by reducing ischemia-induced accumulation of glutamate due in particular to glial transporter inhibition.     

Changes of GABA metabolic enzymes in acute retinal ischemia.

It is reported that GABA accumulates in Müller cells in ischemic and diabetic rat retina. To investigate the mechanism of GABA accumulation in Müller cells, we localized GABA and glutamate in ischemic rat retina and measured the activity of GAD and GABA-T, enzymes involved in GABA metabolism. Using general anesthesia, we incised the bulbar conjunctiva of the rat around the limbus and clamped the left optic nerve. A sham operation was performed on the right eyes. Ocular ischemia was sustained for 30, 60 and 90 minutes. Rat eyes were enucleated immediately after ischemia and prepared for immunohistochemistry and enzyme activity measurement. Glutamate-like immunoreactivity (Glu-IR) in the sham-operated rat retina was observed in all retinal layers, showing intense staining in the nerve fiber layer (NFL), ganglion cell layer (GCL), and inner plexiform layer (IPL). Glu-IR increased in the outer plexiform layer (OPL) and outer nuclear layer (ONL) in an ischemic time-dependent manner. GABA-like immunoreactivity (GABA-IR) in sham-operated rat retina was observed in NFL, GCL, IPL and inner nuclear layer (INL). When the ischemic time was extended, GABA-IR intensely stained Müller cells. GAD activity was not changed in ischemic rat retina as compared to normal rat retina, but GABA-T activity was significantly decreased in ischemic rat retina. These results suggested that glutamate was induced by ischemia and was converted to GABA by GAD activity. Increased GABA was not metabolized because GABA-T activity was decreased. GABA accumulation in Müller cells progressed during the change in activity of these metabolic enzymes.     

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.     

碱性成纤维细胞生长因子对鼠视网膜缺血再灌注损伤的治疗作用

目的探讨玻璃体腔注射碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)对实验性视网膜缺血再灌注损伤的治疗作用.方法采用升高眼内压的方法,制作实验性视网膜缺血再灌注损伤大鼠模型.将Wistar大鼠随机分为正常组、缺血组及治疗组.再灌注开始时,缺血组大鼠玻璃体腔内注入平衡盐溶液,治疗组注入bFGF 2 μg.观察再灌注后不同时间段各组鼠视网膜组织学及超微结构变化,光镜下计数视网膜神经节细胞(retinal ganglion cells, RGCs),应用图像分析系统测量视网膜内层厚度.结果视网膜缺血再灌注早期,治疗组大鼠视网膜水肿较缺血组轻,各时间段治疗组大鼠视网膜内层厚度均较缺血组厚,治疗组大鼠RGCs数目多于缺血组.再灌注后168 h,缺血组大鼠神经纤维层厚度及RGCs数目明显低于正常组,而治疗组大鼠神经纤维层厚度及RGCs数目与正常组比较,差异无显著意义(P<0.05).再灌注后24 h,缺血组大鼠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;而治疗组仅部分核膜轻度肿胀,胞浆内细胞器丰富,线粒体及微管结构较清楚.结论大鼠玻璃体腔注射bFGF对实验性视网膜缺血再灌注损伤具有治疗作用.     

Morphine pretreatment provides histologic protection against ischemia-reperfusion injury in rabbit retina

PURPOSE: Pharmacologic preconditioning with morphine has been shown to protect several kinds of tissues against ischemia-reperfusion injury. The aim of the present study was to investigate whether intravitreal administration of morphine induces structural protection against ischemic damage in a rabbit model of ischemic retinopathy. METHODS: Twenty-eight male white New Zealand rabbits were used. Animals in saline control group received 0.1 mL of phosphate-buffered saline (PBS) intravitreally with no postinjection ischemia. In the saline-control ischemia group, 15 minutes after injection of PBS, retinal ischemia was induced by raising intraocular pressure to 150 mmHg for 60 minutes. In three treatment-ischemia groups, morphine (1, 5, and 10 micromol/L) was administered intravitreally 15 minutes before induction of ischemia. In another experiment, naloxone (40 micromol/L) was administered 5 minutes before intravitreal administration of morphine (10 micromol/L) followed by 60 minutes of ischemia to investigate the role of opioid receptors in mediating the possible protective effect of morphine. Toxicity controls were performed with morphine (10 micromol/L) and naloxone (40 micromol/L) without ischemia. Histologic evaluation was performed for all groups on the seventh postoperative day. RESULTS: Sixty minutes of ischemia led to severe cell loss in ganglion cell layer and thinning of the inner nuclear layer in saline-control ischemia compared to that of the nonischemia control group (P < 0.001). Thickness of the inner plexiform layer to the inner limiting membrane was significantly increased due to edema (P < 0.001). Administration of morphine in higher doses (5 and 10 micromol/L) significantly improved all of the above mentioned indices (P < 0.05). Administration of naloxone 15 minutes before morphine reversed most of the morphine protective effects. CONCLUSIONS: Morphine pretreatment provides significant histologic protection against ischemic injury in rabbit retina. Pharmacologic evidence suggests that this protective phenomenon may be mediated in part by opiate receptors.     

Betaxolol,a beta1-adrenoceptor antagonist,protects a transient ischemic injury of the retina

In the present study, we investigated the protective effects of the topical beta-adrenoceptor antagonist Betoptic((R)) (0.25% betaxolol) in the rat retina following the ischemic injury induced by a transient increase of intraocular pressure (IOP). Like other areas of the central nervous system, the retina is highly vulnerable to ischemic-induced injury. Ischemia was induced in the rat retina by raising the IOP above the systolic blood pressure for 60min. After an ischemia/reperfusion, the thickness of the retinal layers and the immunoreactivities of choline acetyltransferase (ChAT), gamma-amino butyric acid (GABA) and tyrosine hydroxylase (TH) were examined. After a reperfusion period of 7 days, the thickness of both the inner plexiform layer and inner nuclear layer was much decreased. After a reperfusion period of 14-28 days, the thickness of the outer nuclear layer decreased markedly. Moreover, the ChAT and TH immunoreactivity had almost completely disappeared in the retinas after 7 days, while GABA immunoreactivity remained for 28 days. These results suggest that the inner retinal layers are more susceptible to ischemic-induced injury than the outer retinal layer.Histological examination demonstrated protective effects of betaxolol on ischemic-induced retinal damage, which was more substantial in the inner retinal layer. When two drops of betaxolol, once before ischemic injury and twice daily for 28 days after ischemia, were continuously administered, the reductions in the retinal ChAT, GABA and TH immunoreactivities were significantly attenuated. The present study suggests that topically applied betaxolol is an efficient neuroprotective agent and prevents the retinal cell damage induced by ischemic injury in rats.     

Memantine reduces alterations to the mammalian retina, in situ, induced by ischemia

The aim of the study was to determine whether memantine could slow down the changes seen in the rabbit and rat retina following ischemia/reperfusion. A "suction cup procedure," which raises the intraocular pressure, was used to give an ischemic insult to the rabbit retina. The electroretinogram was recorded before ischemia and after 2 days of reperfusion. Memantine or saline (10 microl) was injected into the eye before ischemia. Immunohistochemistry was used to study the effect of ischemia/reperfusion on the GABA, ChAT, and alphaPKC immunoreactivities. Ischemia/reperfusion injury to the rat retina was induced by raising the intraocular pressure above the systolic blood pressure for 60 min, followed by reperfusion of 3-14 days. Memantine (5 mg/kg) or saline was injected i.p. at the onset of ischemia or reperfusion. Immunohistochemistry was used to study the effect of ischemia/reperfusion on the ChAT, alphaPKC, and Thy-1 immunoreactivities. In addition, morphometric analysis was carried out to determine the effects of ischemia/reperfusion on the thickness of the retina. Ischemia for 75 min caused a change in the nature of the normal GABA and ChAT immunoreactivities in the rabbit retina and a reduction in the b-wave of the electroretinogram. When memantine was injected into the vitreous humour at the onset of an ischemic insult, the changes in the GABA and ChAT immunoreactivities were reduced and the recovery of the reduced b-wave of the electroretinogram after 2 days reperfusion was enhanced significantly. Ischemia for 60 min followed by 3 days reperfusion showed a clear change in ChAT immunoreactivity in the rat retina. The Thy-1 immunoreactivity was only clearly altered after a reperfusion period of 7 days. Moreover, a measurable change in the thickness of the inner retinal layers was detected after 14 days of reperfusion. When given at the onset of ischemia, memantine counteracted the effect of ischemia/reperfusion to varying degrees. However, when memantine was given at the onset of the reperfusion this was not the case. The combined data show that a single injection of memantine given i.p. or intravitreally will protect the retina from a subsequent ischemic insult.     

Role of extracellular signal-regulated kinase in glutamate-stimulated apoptosis of rat retinal ganglion cells

PURPOSE: To investigate the involvement of the extracellular signal-regulated kinase (ERK) signaling pathway after intravitrevous injection of glutamate in rat retina. METHODS: Three groups of five Sprague-Dawley rats each were studied. Group I was a normal control group, intravitreal saline injections. In Group II, one eye received an intravitreal glutamate injection (375 nmol, dissolved in saline) while the contralateral eye served as control. In Group III, intravitreal PD98059 (100 micro mol, an inhibitor of ERK) injections were administered 1 hr before glutamate injections. Seven days after injections, phosphorylated (activated) ERK in retina was localized by immunohistochemistry and fluorescent double labeling of retinal cryosections. Specific ERK blockade was documented to assess the functional significance of activated ERK. TUNEL staining was performed to assess apoptotic cell death. RESULTS: Expression of phosphorylated ERK in rat retina was observed in the inner nuclear layer, the outer nuclear layer, and the nerve fiber layer after 3 days intravitreous injection of glutamate, increasing significantly after 7 days. Double immunofluorescence labling demonstrated that the increased retinal immunostaining for phospho-ERK was predominantly localized to the retinal Müller cells after 7 days intravitreous injection of glutamate. Moreover, blocking activation of ERK significantly improved the number of TUNEL-positive cells in the eyes receiving intravitreal PD98059 injections compared with the eyes receiving glutamate injections. CONCLUSIONS: The ERK pathway is involved in signal transduction in the retina after excessive stimulation by glutamate, which may contribute to the antiapoptotic role in retinal ganglion cell death induced by glutamate.     

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.     

NO在缺血性视网膜病变机制的研究

目的 :研究一氧化氮在短暂高眼压诱导的视网膜缺血性损伤的作用机制。方法 :48只雄性S -D大鼠随机分为两组 ,每组 2 4只。 1组左眼为正常对照组、右眼为视网膜缺血对照组 ;2组左眼为视网膜缺血溶剂对照组 ,右眼为视网膜缺血L -NAME治疗组。视网膜缺血再灌注后 7、 14、 2 8天分别将各组 2 4只眼随机分入 3个时间点 ,每个时间点 8只眼。制备视网膜病理切片 ,定量测量内网状层 (IPL)、内核层 (INL)、外核层 (ONL)的厚度。结果 :在缺血的早期阶段 (第 1周 ) ,内网状层 (IPL)的厚度即下降 ,在缺血的晚期阶段 (第 2周～第 4周 ) ,内核层 (INL)和外核层 (ONL)的厚度才发生显著下降。与对照组相比差异均有显著性 ( χ2 检验 ,P <0 0 5 )。L -NAME ,一氧化氮合酶抑制剂可明显减轻缺血性损伤。减轻视网膜各层的下降幅度。与缺血对照组相比经统计学处理差异均有显著性 ( χ2 检验 ,P <0 0 5 )。结论 :视网膜缺血性损伤的诱导机制除了兴奋性毒性诱导的损伤以外 ,还有其他的机制存在。NO可介导视网膜缺血性损伤 ,并在短暂视网膜缺血后晚期内核层 (INL)与外核层 (ONL)神经元的延迟死亡起到一个主要的作用。     

AQP-1和VEGF在大鼠视网膜缺血再灌注损伤中的表达

目的:研究大鼠视网膜缺血再灌注损伤后水通道蛋白-1和血管内皮生长因子的表达情况并探讨二者在视网膜缺血再灌注损伤中表达的意义。方法:通过提高眼内压的方法建立大鼠视网膜缺血再灌注模型,于术后6,12,24,48h;3,7d用免疫组化的方法检测水通道蛋白-1和血管内皮生长因子在大鼠视网膜的表达情况,并以正常大鼠视网膜作为对照。结果:正常对照组大鼠视网膜未能检测到血管内皮生长因子的阳性表达,缺血再灌注12h后开始出现表达,48h达高峰,且差异具有统计学意义(P<0.01)。水通道蛋白-1在正常对照组可见到阳性表达,缺血再灌注组随时间增长表达不断增强,7d在外层视网膜也出现表达,差异有统计学意义(P<0.01)。结论:水通道蛋白-1和血管内皮生长因子在视网膜缺血性疾病中起着重要的作用,二者可能共同影响了视网膜的水代谢过程。     

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.     

Protective action of nipradilol against ischemia-induced retinal damage in rats

The purpose of this study was to investigate whether nipradilol, a beta-blocker having both vasodilating and alpha(1)-blocking activities, can protect retinal cells from the injury induced by ischemia and reperfusion. Rats were anesthetized and, after an intravitreal injection of nipradilol, the intraocular pressure was raised for 45 min to induce retinal ischemia and reperfusion. Before, and 3 and 7 days after the ischemia, electroretinograms were recorded. After the ischemia, the mean amplitude of the b-waves in rats receiving 5 microl of 1.0 x 10(-6) M nipradilol was significantly larger than of controls (injected with phosphate-buffered saline). Histologically, the reduction in the number of retinal ganglion cells (1.0 x 10(-6) M), and the thickness of the inner and outer plexiform layers and the inner nuclear layer (1.0 x 10(-6), 10(-7) and 10(-8) M) was suppressed by nipradilol. These results indicate that nipradilol protected the retina against retinal ischemia and reperfusion and should be considered for therapeutic use in cases of transient retinal ischemia.     

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)     

Involvement of glutamate in ischemic neurodegeneration in isolated retina

Retinal ischemia, a major cause of visual loss, is believed to result from overexcitation of glutamate receptors. However, under euglycemic and normoxic conditions, exogenously applied glutamate is not neurotoxic in the retina. Under such conditions, exogenous glutamate typically causes glia swelling and requires very high concentrations to produce neurotoxicity. To determine whether ischemic conditions enhance the neurotoxicity of endogenous and exogenous glutamate, we examined the effects of simulated ischemia (deprivation of both glucose and oxygen) on retinal morphology and lactate dehydrogenase (LDH) release. In an ex vivo rat retinal preparation, glutamate was administered during simulated ischemia in the presence of riluzole, an inhibitor of glutamate release. Deprivation of both glucose and oxygen for 60 min at 30 degrees C produced severe acute neurodegeneration. This neurodegeneration, characterized by bull's eye formation in the inner nuclear layer and spongy appearance in the inner plexiform layer, was prevented by the combination of MK-801 and DNQX, antagonists of N-methyl-D-aspartate (NMDA) and non-NMDA receptors, indicating that the damage results from activation of both glutamate receptors. We also found that administration of glutamate pyruvate transaminase (alanine aminotransaminase) with pyruvate diminished the neurodegeneration during simulated ischemia. Furthermore, riluzole, an inhibitor of glutamate release, attenuated the neurodegeneration, suggesting the importance of endogenous glutamate in ischemic damage. In the presence of riluzole and simulated ischemia, exogenously applied glutamate failed to cause Müller cell swelling but was extremely neurotoxic. These results suggest that simulated ischemia enhances glutamate-mediated neurotoxicity in part by depressing glutamate uptake. When glutamate transport is impaired, sub-millimolar glutamate concentrations become profoundly neurotoxic.