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.     

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。     

A new method and device to induce transient retinal ischemia in the rat

PURPOSE: To describe and characterize a novel, non-invasive method to induce retinal ischemia in the rat. METHODS: Retinal ischemia was produced by applying a compression force with a suture thread anchored about 2mm behind the limbus of the eye in order to increase intraocular pressure and reduce ocular blood flow. Both ends of the suture thread were connected to weights via a pulley system. The effect of various weights (20, 25, or 35 g) on retinal damage was tested in animals of different body weights (350 and 574 g). Scotopic ERG measurements and histological sections of retinal tissue were used to evaluate possible functional and morphological changes to the retina as a consequence of ischemia followed by reperfusion. RESULTS: Retinal ischemia was achieved by this method with good reproducibility. The degree of retinal damage, as quantified by ERG measurement, correlated with the amount of weight connected to the suture thread. Seven days after 60 minutes of retinal ischemia, the amplitudes of A- and B-waves of eyes compressed with a 20 g weight were 84 +/- 11% and 86 +/- 16% (mean +/- SEM, n = 4, amplitudes before ischemia defined 100%), respectively. The A- and B-wave amplitudes of eyes compressed with 25 g were 69 +/- 7% and 76 +/- 6% (n = 11), respectively. A 35 g treatment produced A- and B-wave amplitudes of 55 +/- 3.9% and 52 +/- 4% (n = 35), respectively. There was no significant difference in the level of ischemic damage in the two groups of animals with different body weights evaluated in this study. At two weeks after 60-minute retinal ischemia, there was a significant decrease (by 31.1%) in the cell density in the retinal ganglion cell layer. CONCLUSIONS: The pulley method is an easy, non-invasive, and highly reproducible technique for inducing retinal ischemia in rats. This procedure may provide a useful animal model of ischemic retinopathy.     

Activation of the aldosterone/mineralocorticoid receptor system and protective effects of mineralocorticoid receptor antagonism in retinal ischemia-reperfusion injury

The purpose of this project was to investigate the effects of the mineralocorticoid receptor antagonist against retinal ischemia-reperfusion injury and identify the aldosterone/mineralocorticoid receptor (MR) system in the rat retina. Retinal ischemia was induced by increasing intraocular pressure to 130?mmHg. Rats were treated with the angiotensin II type 1 receptor (AT1-R) antagonist (candesartan), MR antagonist (spironolactone), or aldosterone. Retinal damage was evaluated at 7 days after the ischemia by measuring the retinal thickness and the number of retinal ganglion cells. Pretreatment with candesartan, spironolactone, or candesartan and spironolactone significantly inhibited retinal ischemic injury. However, there was no protective effect against retinal ischemia-reperfusion injury provided by the combined aldosterone with candesartan treatment. Additionally, pretreatment with aldosterone alone also did not provide any neuroprotective effects against retinal ischemia-reperfusion injury. When rats were treated via local administration of aldosterone in the absence of ischemia, the number of retinal ganglion cells decreased while the retinal thickness remained unchanged. The present findings demonstrated the existence of a local aldosterone/MR system in the retina. Our results also demonstrated that an MR antagonist can attenuate subsequent ischemic damage in the rat retina.     

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.     

Neuroprotective effects of D-allose against retinal ischemia-reperfusion injury

PURPOSE: To investigate the effect of D-allose, a rare sugar, against ischemia reperfusion injury in the rat retina. METHODS: Retinal ischemia was induced by increasing intraocular pressure to 130 mm Hg and maintaining that level for 45 minutes. Morphometric studies were performed to study the effect of D-allose on the histologic changes induced by ischemia in the rat retina. Glutamate release from the rat retina and intravitreal P(O2) profiles were monitored during and after ischemia with a microdialysis biosensor and oxygen-sensitive microelectrodes. The release of hydrogen peroxide stained with diaminobenzidine hydrochloride was monitored by an in vitro retinal ischemia model. RESULTS: Seven days after the ischemia, significant reductions in both the number of ganglion cells and the thickness of the inner plexiform layer were observed. Pretreatment with D-allose significantly inhibited the ischemic injury of the inner retina. A large release of glutamate occurred during the ischemia. After the recirculation, glutamate levels were increased again and reached a maximum in approximately 20 minutes. The increases in extracellular glutamate during and after ischemia tend to be suppressed by administration of d-allose. d-Allose attenuated the increase in intravitreal P(O2) during reperfusion. After the ischemia, production of hydrogen peroxide was detected within approximately 30 minutes. D-allose suppressed the production of hydrogen peroxide. CONCLUSIONS: These results suggest that D-allose may protect neurons by decreasing extracellular glutamate and attenuating oxidative stress in ischemic insult.     

Riluzole improves functional recovery after ischemia in the rat retina

PURPOSE: Retinal ischemia leads to neuronal death. The effects of riluzole, a drug that protects against the deleterious effect of cerebral ischemia by acting on several types of ion channels and blocking glutamatergic neurotransmission, were investigated in a rat model of retinal ischemic injury. METHODS: Retinal ischemia was induced by increasing intraocular pressure above systolic blood pressure for 30 minutes. Electroretinograms were recorded before ischemia and at different periods of reperfusion. Riluzole was injected or topically applied to the eye before or after ischemia and twice daily during the reperfusion period. Retinas were harvested for histopathology (toluidine blue and silver-impregnation stainings, Tdt-dUTP terminal nick-end labeling [TUNEL] method) and immunohistochemistry for cytoskeletal glial fibrillary acid protein and c-jun NH2-terminal kinase (p-JNK). RESULTS: Ischemia for 30 minutes caused a reduction of a- and b-waves of the electroretinogram. Systemic and topical treatments with riluzole significantly enhanced the recovery of the reduced a- and b-waves after defined reperfusion times. Riluzole also prevented or attenuated ischemia-induced retinal cell death (necrosis and apoptosis) and reduced the activation of p-JNK, c-jun phosphorylation, and the increase of cytoskeletal proteins induced by ischemic injury. CONCLUSIONS: Riluzole acted in vivo as a potent neuroprotective agent against pressure-induced ischemia. Therefore, riluzole may be a major drug for use in protection against retinal injury.     

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.     

盐酸氟桂嗪对家兔视网膜缺血—再灌注损伤保护作用的ERG观察

目的 动态观察盐酸氟桂嗪对家兔视网膜缺血－再灌注损伤的保护作用。方法 应用前房灌注加压法使前房内压力升高至16kPa。维持１h后再灌注,再灌注后第2、7、14天分别记录其ERG,与缺血前ERG相比,观察b波的变化。药物治疗组在造成缺血前12h,缺血－再灌注即刻及再灌注后12h静脉给予药物治疗。结果 药物治疗组ERG的b波与正常眼相比无明显差异。结论 盐酸氟桂嗪对视网膜缺血－再灌注损伤有保护作用。     

Inducible nitric oxide synthase inhibitors abolished histological protection by late ischemic preconditioning in rat retina

Brief ischemia was reported to protect retinal cells against injury induced by subsequent ischemia-reperfusion with de novo protein synthesis, and this phenomenon is known as late ischemic preconditioning. The aims of the present study were to determine whether nitric oxide synthase (NOS) was involved in the mechanism of late ischemic preconditioning in rat retina using pharmacological tools. Under anesthesia with pentobarbital sodium, male Sprague-Dawley rats were subjected to 60 min of retinal ischemia by raising intraocular pressure to 130 mm Hg. Ischemic preconditioning was achieved by applying 5 min of ischemia 24 hrs before 60 min of ischemia. Retinal sections sliced into 5 microm thick were examined 7 days after ischemia. Additional groups of rats received NG-nitro-L-arginine and NG-monomethyl-L-arginin, non-selective NO synthase inhibitors, 7-nitroindazole, a neuronal NOS inhibitor, and aminoguanidine and L-N6-(1-iminoethyl) lysine, inducible NO synthase (iNOS) inhibitors before preconditioning, and were subjected to 60 min of ischemia. In the non-preconditioned group, cell loss in the ganglion cell layer and thinning of the inner plexiform and inner nuclear layer were observed 7 days after 60 min of ischemia. Ischemic preconditioning for 5 min completely protected against the histological damage induced by 60 min of ischemia applied 24 hrs thereafter. Treatment of rats with aminoguanidine and L-N6-(1-iminoethyl) lysine, but not NG-nitro-L-arginine, NG-monomethyl-L-arginine or 7-nitroindazole, wiped off the protective effect of ischemic preconditioning. The inhibitory effect of aminoguanidine was abolished by L-arginine, but not D-arginine. The results in the present study suggest that NO synthesized by iNOS is involved in the histological protection by late ischemic preconditioning in rat retina.     

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     

Effect of trimetazidine on retinal ischemia/reperfusion injury in rats

PURPOSE: To investigate the effect of trimetazidine (TMZ), an antioxidant agent, on the ischemia/reperfusion (I/R) injury in rat retina histopathologically. METHODS: The retinal I/R model was carried out by the 4-vessel occlusion method on Wistar albino rats. Twenty-one rats were divided into 7 groups, each comprising 3 rats. The animals in groups 1, 2 and 3 underwent 30 min of ischemia + 4 h of reperfusion and were treated by the administration of saline, TMZ before reperfusion and TMZ before ischemia, respectively. The animals in groups 4, 5 and 6 underwent 90 min of ischemia + 4 h of reperfusion and were treated in the same way as those in groups 1, 2 and 3, respectively. The 7th group was sham operated. RESULTS: Thirty and 90 min of ischemia followed by 4 h of reperfusion induced retinal injury in the rat retina. Histopathologically, the inner plexiform and inner nuclear layers were the most affected parts. TMZ was able to reduce almost all retinal I/R damage when administered before ischemia. A cytoprotective effect of TMZ was partly observed in those animals which were treated before reperfusion. CONCLUSION: TMZ seemed to have a protective effect against retinal I/R injury in rats.     

Retinal ischemia and reperfusion causes capillary degeneration: similarities to diabetes

PURPOSE: Retinal neurons and vasculature interact with each other under normal conditions, and occlusion of the retinal vasculature can result in damage to retinal neurons. Whether damage to the neural retina will damage the vasculature, however, is less clear. This study was conducted to explore the relationship between vascular and nonvascular cells of the retina. The response of the retinal vasculature to an injury (ischemia and reperfusion; I/R) that is known to cause neuronal degeneration was studied. METHODS: I/R injury to the retinas was induced in Lewis rats and C57BL/6J mice by elevating intraocular pressure (IOP), and reperfusion was established immediately afterward. Some rats were pretreated with aminoguanidine (AMG, 50 mg/Kg BW in drinking water) before the procedure. Poly(ADP-ribose) polymerase (PARP) activity and expression of inducible nitric oxide synthase (iNOS), and cycloxygenase-2 (COX-2) were measured by Western blot analysis, and levels of TNF-alpha and intercellular adhesion molecule (ICAM)-1 mRNA were measured by qPCR at 2 and 7 days after the procedure. Also at 2 and 7 days after the I/R injury, apoptosis of retinal neural cells (demonstrated by TUNEL assay), density of cells in the ganglion cell layer, and thickness of retinas were quantitated, and the number of TUNEL-positive capillary cells and degenerated capillaries were assessed. Retinal neurodegeneration and capillary degeneration were also examined in C57BL/6J mice 2, 5, 8, and 14 days after I/R injury. RESULTS: As expected, loss of cells in the retinal ganglion cell layer was apparent 2 days after I/R injury in the rat and mouse models. In contrast, the retinal vasculature had essentially no pathology at this time in either model. Surprisingly, the number of degenerated capillaries increased greatly by 7 to 8 days after the injury. Administration of aminoguanidine significantly inhibited the I/R-induced capillary degeneration as well as neurodegeneration in the rat model. Retinal I/R caused increased PARP activity (detected by poly(ADP-ribosy)lated proteins), as well as upregulation of iNOS, COX-2, TNF-alpha, and ICAM-1 levels in rats, consistent with an inflammatory process. CONCLUSIONS: Capillary degeneration is an unrecognized component of acutely elevated IOP and develops only after neurodegeneration is severe. Thus, this finding raises the possibility that damage to the neural retina contributes to capillary degeneration. Aminoguanidine, a nonspecific inhibitor of iNOS, inhibited I/R-induced degeneration of both neuronal and vascular cells of the retina. The model of retinal ischemia and reperfusion will be a useful tool for investigating the relationship between neuronal damage and vascular damage in glaucoma and other diseases such as diabetic retinopathy.     

Neuroprotective effect of small interfering RNA targeted to caspase-3 on rat retinal ganglion cell loss induced by ischemia and reperfusion injury

Purpose: To investigate neuroprotective effects of siRNA targeted to caspase-3 against ischemia and reperfusion (I/R) injury in rat eyes. Methods: Retinal ischemia was induced in Wistar rats by increasing the intraocular pressure (IOP) to 110 mmHg for 120 min. To examine the effect of siRNA on rat caspase-3, siRNA was injected into the vitreous cavity 24 h prior to induction of retinal ischemia. Eyes were removed at 2, 7 or 14 days later, and then analyzed for the number of retinal ganglion cells (RGCs), the retinal thickness and the amount of apoptosis of the retinal neural cells (as demonstrated by the TUNEL assay). The amount of caspase-3 mRNA was analyzed by rt-PCR. Differences between groups were evaluated by an unpaired t test. Results: The numbers of RGCs in the saline and non-silencing siRNA controls were reduced significantly at 2 and 7 days after the I/R injury. RGCs were significantly retained in eyes pretreated with siRNA targeted to caspase-3 as compared to the control eyes at 2 days after the I/R injury. Inner retinal thickness in the control eyes was significantly thinner as compared to the treated eyes at 2 and 7 days after the I/R injury. After siRNA treatment, the amount of caspase-3 mRNA was significantly lower when compared to the saline control group. Conclusions: The injection of siRNA targeted to caspase-3 into the vitreous cavity of rat eyes may block caspase-3, and may thus be able to prevent retinal cell death associated with ischemic injury. As inhibition of the apoptosis pathway may provide a neuroprotective effect, examination of new strategies for treating these disorders needs to be undertaken.     

Retinal ischemic injury rescued by sodium 4-phenylbutyrate in a rat model

Retinal ischemia is a common cause of visual impairment for humans and animals. Herein, the neuroprotective effects of phenylbutyrate (PBA) upon retinal ischemic injury were investigated using a rat model. Retinal ganglion cells (RGCs) were retrograde labeled with the fluorescent tracer fluorogold (FG) applied to the superior collicoli of test Sprague-Dawley rats. High intraocular pressure and retinal ischemia were induced seven days subsequent to such FG labeling. A dose of either 100 or 400 mg/kg PBA was administered intraperitoneally to test rats at two time points, namely 30 min prior to the induction of retinal ischemia and 1 h subsequent to the cessation of the procedure inducing retinal ischemia. The test-rat retinas were collected seven days subsequent to the induction of retinal ischemia, and densities of surviving RGCs were estimated by counting FG-labeled RGCs within the retina. Histological analysis revealed that ischemic injury caused the loss of retinal RGCs and a net decrease in retinal thickness. For PBA-treated groups, almost 100% of the RGCs were preserved by a pre-ischemia treatment with PBA (at a dose of either 100 or 400 mg/kg), while post-ischemia treatment of RGCs with PBA did not lead to the preservation of RGCs from ischemic injury by PBA as determined by the counting of whole-mount retinas. Pre-ischemia treatment of RGCs with PBA (at a dose of either 100 or 400 mg/kg) significantly reduced the level of ischemia-associated loss of thickness of the total retina, especially the inner retina, and the inner plexiform layer of retina. Besides, PBA treatment significantly reduced the ischemia-induced loss of cells in the ganglion-cell layer of the retina. Taken together, these results suggest that PBA demonstrates a marked neuroprotective effect upon high intraocular pressure-induced retinal ischemia when the PBA is administered prior to ischemia induction.     

Lomerizine, a Ca2+ channel blocker, reduces glutamate-induced neurotoxicity and ischemia/reperfusion damage in rat retina

We examined the effects of a new Ca2+ channel blocker, lomerizine, on the intraocular hypertension-induced ischemia/reperfusion injury in rat retina and on the glutamate-induced neurotoxicity in rat cultured retinal neurons, and compared its effects with those of a Ca2+ channel blocker (flunarizine) and an N-methyl-D-aspartate receptor antagonist (MK-801). Morphometric evaluation at 7 days after ischemia/reperfusion showed that treatment with lomerizine (0.1 and 1 mg kg(-1), i.v.) prior to ischemia and again immediately after reperfusion dose-dependently reduced the retinal damage. Treatment with MK-801 (1 mg kg(-1), i.v.) before ischemia significantly reduced the resulting retinal damage. Flunarizine (0.1 and 1 mg kg(-1), i.v.) tended to reduce the retinal damage, but its effect did not reach statistical significance. In an in vitro study, pretreatment with lomerizine (0.1 and 1 microM) or flunarizine (1 microM) significantly reduced glutamate-induced neurotoxicity, the effects being concentration dependent. Lomerizine (1 microM) also exhibited protective effects against both the N-methyl-D-aspartate and kainate induced types of neurotoxicity. However, lomerizine (1 microM) had little effect on the neurotoxicity induced by ionomycin (1 microM) application. Glutamate-induced neurotoxicity was abolished by removing Ca2+ from the medium. These results indicate that lomerizine protects neuronal cells against retinal neurotoxicity both in vivo and in vitro, and that this Ca2+ channel blocker may be useful as a therapeutic drug against retinal diseases that cause neuronal injury, such as normal tension glaucoma (NTG).     

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.     

实验性急性高眼压对兔视网膜电图的影响

目的：检测家兔实验性急性高眼压不同压力状态下视网膜电图的变化。方法：采用视电生理检测仪测定家兔实验前,30mmHg(1mmHg=0.133kPa),60mmHg,90mmHg和120mmHg前房高压灌注45min及恢复正常眼压4h的视网膜电图（Flash Electroretinogram FERG)和振荡电位（Oscillatory Potentials,OPs)。结果：对照组和30mmHg组视电生理检测在实验前后无差异。60mmHg组在高压持续45min后,b波和OPs波振幅下降,4h后恢复正常。90mmHg和120mmHg组在高压45min后,FERG波形消失。4h后有不同程度恢复。结论：随着实验性高眼压压力的升高,家兔视网膜功能损伤加重,恢复能力减弱。     

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.     

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.