Postischemic Treatment Research Articles

Rho‐kinase activation in endothelial cells contributes to expansion of infarction after focal cerebral ischemia

Microcirculatory disturbances contribute to the expansion of infarct lesions after focal cerebral ischemia. Recently, it was shown that Rho-kinase involves in endothelial dysfunction via down-regulation of endothelial nitric oxide synthase function in a rodent stroke model. However, it is not clear whether endothelial Rho-kinase is activated in vivo or Rho-kinase activation contributes to microcirculatory disturbances after cerebral ischemia. In this study, we assessed the temporal and spatial profiles of Rho-kianse activity and the effect of the Rho-kinase inhibitor fasudil on microcirculatory disturbances in the focal brain ischemia. Rho-kinase activation was evaluated by analyzing the phosphorylation of adducin, a substrate of Rho-kinase, by immunohistochemistry. Staining for p-adducin was found in endothelia in the ischemic area 6 hr after induction of ischemia. Microcirculatory disturbances and increased endothelial cell staining for von Willebrand factor (vWF) were observed in the same area. Postischemic treatment with fasudil suppressed endothelial Rho-kinase activation, preserved microcirculation, and inhibited endothelial cell vWF staining. These effects resulted in inhibition of infarct expansion and improvement of neurologic deficits. These findings indicate that Rho-kinase is activated in the endothelial cells and contributes to microcirculatory disturbances in cerebral ischemia. The vascular protective effect of Rho-kinase inhibitors may be useful in the treatment of the acute phase of ischemic stroke.

Cytoprotective effect of bis(1-oxy-2-pyridinethiolato)oxovanadiun(IV) on myocardial ischemia/reperfusion injury elicits inhibition of Fas ligand and Bim expression and elevation of FLIP expression

VO(OPT), bis(1-oxy-2-pyridinethiolato)oxovanadium(IV), has been shown to increase tyrosine phosphorylation of proteins and promote the insulin receptor signaling, thereby elicit anti-diabetic action. We here investigated the cytoprotective action of VO(OPT) on myocardial infarction and cardiac functional recovery in rats subjected to myocardial ischemia/reperfusion and defined mechanisms underlying its cytoprotective action. Rats underwent 30 min myocardial ischemia by left anterior descending coronary artery occlusion followed by 24 h reperfusion. Post-ischemic treatment with VO(OPT) significantly reduced infarct size and improved cardiac function (left ventricular developed pressure and +/-dP/dt) after 72 h reperfusion and in a dose-dependent manner. Moreover, VO(OPT) treatment also dose-dependently significantly inhibited caspases-3, -9 and -7 processing, thereby elicited the anti-apoptotic effect. The cytoprotective effect of VO(OPT) was closely associated with restoration of Akt activity. The recovered Akt activity correlated with increased phosphorylation of Bad and forkhead transcription proteins, thereby inhibiting apoptotic signaling. Furthermore, treatment with VO(OPT) significantly increased FLIP expression, and decreased expression of Fas ligand and Bim in cardiomyocytes. Taken together, cardiomyocytes rescue following post-treatment with VO(OPT) from ischemia/reperfusion injury was mediated by increased FLIP expression and decreased Fas ligand and Bim expression via activation of Akt. These results demonstrate that treatment with VO(OPT) exerts significant cytoprotective effects along with improvement of cardiac functional recovery.

Pyrrolidine Dithiocarbamate Reduces Lung Injury Caused by Mesenteric Ischemia/Reperfusion in a Rat Model

Pyrrolidine dithiocarbamate (PDTC) is a low-molecular thiol antioxidant and potent inhibitor of nuclear factor-kappaB (NF-kappaB) activation. It has been shown to attenuate local harmful effects of ischemia/reperfusion (I/R) injury in many organs. In this study, we aimed to study the effect of PDTC on lung reperfusion injury induced by superior mesenteric occlusion. Male Wistar-albino rats randomized into three groups: (1) sham-operated control group (n = 12), laparotomy without I/R injury; (2) intestinal ischemia/reperfusion (I/R) group (n = 12), 60 min of ischemia by superior mesenteric occlusion followed by 2 h of reperfusion; and (3) I/R+PDTC-treated group (n = 12), 100 mg/kg injection of PDTC intravenously, 30 min after the commencement of reperfusion. Evans blue dye was injected to half of rats in all groups before the induction of I/R. We assessed the degree of pulmonary tissue injury biochemically by measuring malondialdehyde (MDA), glutathione (GSH), and nitric oxide (NO) levels, and histopathologically by establishing pulmonary neutrophil sequestration and acute lung injury scoring. Pulmonary edema was evaluated by Evans blue dye extravasation, as well as lung tissue wet/dry weight ratios. Pyrrolidine dithiocarbamate treatment significantly reduced the MDA and NO levels, and increased the GSH levels in the lung parenchyma, biochemically (p < 0.05), and atteneuated the pulmonary parenchymal damage, histopathologically (p < 0.05). However, pulmonary neutrophil sequestration was not affected by postischemic treatment with PDTC (p > 0.05). Pyrrolidine dithiocarbamate administration also significantly alleviated the formation of pulmonary edema, as evidenced by the decreased Evans blue dye extravasation and organ wet/dry weight ratios (p < 0.05). This study showed that postischemic treatment with PDTC significantly attenuated the lung reperfusion injury. Further clinical studies are needed for better understanding of the specific mechanisms of PDTC protection against I/R-related organ injury and to clarify whether PDTC may be a useful therapeutic agent during particular operations where remote organ I/R injury occurs.

Delayed treatment with cannabidiol has a cerebroprotective action via a cannabinoid receptor‐independent myeloperoxidase‐inhibiting mechanism

We examined the neuroprotective mechanism of cannabidiol, non-psychoactive component of marijuana, on the infarction in a 4 h mouse middle cerebral artery (MCA) occlusion model in comparison with Delta(9)-tetrahydrocannabinol (Delta(9)-THC). Release of glutamate in the cortex was measured at 2 h after MCA occlusion. Myeloperoxidase (MPO) and cerebral blood flow were measured at 1 h after reperfusion. In addition, infarct size and MPO were determined at 24 and 72 h after MCA occlusion. The neuroprotective effect of cannabidiol was not inhibited by either SR141716 or AM630. Both pre- and post-ischemic treatment with cannabidiol resulted in potent and long-lasting neuroprotection, whereas only pre-ischemic treatment with Delta(9)-THC reduced the infarction. Unlike Delta(9)-THC, cannabidiol did not affect the excess release of glutamate in the cortex after occlusion. Cannabidiol suppressed the decrease in cerebral blood flow by the failure of cerebral microcirculation after reperfusion and inhibited MPO activity in neutrophils. Furthermore, the number of MPO-immunopositive cells was reduced in the ipsilateral hemisphere in cannabidiol-treated group. Cannabidiol provides potent and long-lasting neuroprotection through an anti-inflammatory CB(1) receptor-independent mechanism, suggesting that cannabidiol will have a palliative action and open new therapeutic possibilities for treating cerebrovascular disorders.

Role of Na+/H+ Exchanger in the Pathogenesis of Ischemic Acute Renal Failure in Mice

We evaluated the effects of 5-(N-ethyl-N-isopropyl) amiloride (EIPA), a Na+/H+ exchanger (NHE) inhibitor, on ischemia/reperfusion (I/R)-induced acute renal failure (ARF) in mice. Ischemic ARF was induced by clamping the left renal artery and vein for 40 minutes followed by reperfusion 2 weeks after the contralateral nephrectomy. Preischemic treatment with EIPA attenuated the I/R-induced renal dysfunction. Histopathological examination of the kidney of ARF mice revealed severe renal damage such as tubular necrosis and proteinaceous casts in the tubuli. Histologically evident damage was also improved by preischemic treatment with EIPA. In addition, the I/R-induced increase in renal endothelin-1 (ET-1) content was suppressed by preischemic treatment with EIPA, reflecting the difference in immunohistochemical ET-1 localization in necrotic tubular epithelium. However, the postischemic treatment with EIPA failed to improve the I/R-induced renal dysfunction and ET-1 overproduction. These findings suggest that NHE activation, followed by renal ET-1 overproduction, plays an important role in the pathogenesis of I/R-induced renal injury. The inhibition of NHE by EIPA may be considered as a therapeutic approach to protect the postischemic ARF.

Reduced Functional Deficits, Neuroinflammation, and Secondary Tissue Damage after Treatment of Stroke by Nonerythropoietic Erythropoietin Derivatives

Carbamylerythropoietin (CEPO) does not bind to the classical erythropoietin (EPO) receptor. Nevertheless, similarly to EPO, CEPO promotes neuroprotection on the histologic level in short-term stroke models. In the present study, we investigated whether CEPO and other nonerythropoietic EPO analogs could enhance functional recovery and promote long-term histologic protection after experimental focal cerebral ischemia. Rats were treated with the compounds after focal cerebral ischemia. Animals survived 1, 7, or 60 days and underwent behavioral testing (sensorimotor and foot-fault tests). Brain sections were stained and analyzed for Iba-1, myeloperoxidase, Tau-1, CD68 (ED1), glial fibrillary acidic protein (GFAP), Fluoro-Jade B staining, and overall infarct volumes. Treatment with CEPO reduced perifocal microglial activation (P<0.05), polymorphomonuclear cell infiltration (P<0.05), and white matter damage (P<0.01) at 1 day after occlusion. Carbamylerythropoietin-treated rats showed better functional recovery relative to vehicle-treated animals as assessed 1, 7, 14, 28, and 50 days after stroke. Both GFAP and CD68 were decreased within the ipsilateral thalamus of CEPO-treated animals 60 days postoperatively (P<0.01 and P<0.05, respectively). Furthermore, behavioral analysis showed efficacy of CEPO treatment even if administered 24 h after the stroke. Other nonerythropoietic derivatives such as carbamylated darbepoetin alfa and the mutant EPO-S100E were also found to protect against ischemic damage and to improve postischemic neurologic function. In conclusion, these results show that postischemic intravenous treatment with nonerythropoietic EPO derivatives leads to improved functional recovery, which may be linked to their long-term effects against neuroinflammation and secondary tissue damage.

Dose dependence and therapeutic window for the neuroprotective effects of 17β-estradiol when administered after cerebral ischemia

The present study was undertaken to determine if the neuroprotective effect of 17β-estradiol (E 2) when administrated after ischemia is dose-dependent and if the therapeutic window for estrogen can be prolonged. Ischemic injury was induced by permanent middle cerebral artery occlusion (p-MCAO). Administration of E 2 at 30 min after ischemia resulted in a reduction in lesion volume. A higher dose of E 2 extended the therapeutic window to 6 h after cerebral ischemia in 33% of the rats. These findings suggest that postischemic treatment with estrogen affords protection against ischemic damage and that it acts within a clinically useful therapeutic window.

Post-ischemic treatment of pentoxifyline reduces cortical not striatal infarct volume in transient model of focal cerebral ischemia in rat

Pervious studies reported that pentoxifylline (PTX) have a neuroprotective effect in the brain trauma and the global cerebral ischemia in the experimental models. However, the effect of PTX in transient model of focal cerebral ischemia has not been investigated yet. Therefore, this study was designed to investigate the effect of post-ischemic treatment of PTX on ischemic injuries in focal cerebral ischemic. Male Wistar rats ( n = 32) were assigned to control or PTX- (60 mg/kg i.p.) treated groups. PTX at dose 60 mg/kg i.p. administered at the beginning, or 1, or 3 h after ischemia. Focal cerebral ischemia was induced by middle cerebral artery occlusion, followed by 24-h reperfusion. At the end of 24 h ischemia, neurological dysfunction score was tested and infarct volumes were determined using triphenyltetrazolium chloride staining. Administration of PTX (60 mg/kg) at the beginning of ischemia, or 1, or 3 h after ischemia significantly reduces cortical infarct volumes by 43%, 40% and 41%, respectively. However, PTX did not significantly affect striatal infarct volumes and neurological dysfunction. The findings of the present study indicate that administration of PTX at least 3 h post-transient focal stroke reduces cortical brain ischemic damage in the rat model of transient focal cerebral ischemia.

Expression of S100 protein and protective effect of arundic acid on the rat brain in chronic cerebral hypoperfusion

S100 protein is expressed primarily by astroglia in the brain, and accumulates in and around the ischemic lesions. Arundic acid, a novel astroglia-modulating agent, is neuroprotective in acute cerebral infarction, whereas the protective effects remain unknown during chronic cerebral hypoperfusion. Rats undergoing chronic cerebral hypoperfusion were subjected to a bilateral ligation of the common carotid arteries, and were allowed to survive for 3, 7 and 14 days. The animals received a daily intraperitoneal injection of 5.0, 10.0 or 20.0 mg/kg of arundic acid, or vehicle, for 14 days. Alternatively, other groups of rats received a delayed intraperitoneal injection of 20.0 mg/kg of arundic acid or vehicle, which started from 1, 3 or 7 days after ligation and continued to 14 days. The degree of white matter (WM) lesions and the numerical density of S100 protein-immunoreactive astroglia were estimated. In the WM of rats with vehicle injections, the number of S100 protein-immunoreactive astroglia increased significantly after chronic cerebral hypoperfusion as compared to the sham-operation. A dosage of 10.0 and 20.0 mg/kg of arundic acid suppressed the numerical increase in S100 protein-immunoreactive astroglia and the WM lesions. These pathological changes were suppressed with delayed treatment up to 7 days in terms of astroglial activation, and up to 3 days in terms of the WM lesions. The protective effects of arundic acid against WM lesions were demonstrated in a dose-dependent manner, and even after postischemic treatments. These results suggest the potential usefulness of arundic acid in the treatment of cerebrovascular WM lesions.

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.

Post‐ischaemic treatment with the cyclooxygenase‐2 inhibitor nimesulide reduces blood–brain barrier disruption and leukocyte infiltration following transient focal cerebral ischaemia in rats

Several studies suggest that cyclooxygenase (COX)-2 plays a pivotal role in the progression of ischaemic brain damage. In the present study, we investigated the effects of selective inhibition of COX-2 with nimesulide (12 mg/kg) and selective inhibition of COX-1 with valeryl salicylate (VAS, 12-120 mg/kg) on prostaglandin E(2) (PGE(2)) levels, myeloperoxidase (MPO) activity, Evans blue (EB) extravasation and infarct volume in a standardized model of transient focal cerebral ischaemia in the rat. Post-ischaemic treatment with nimesulide markedly reduced the increase in PGE(2) levels in the ischaemic cerebral cortex 24 h after stroke and diminished infarct size by 48% with respect to vehicle-treated animals after 3 days of reperfusion. Furthermore, nimesulide significantly attenuated the blood-brain barrier (BBB) damage and leukocyte infiltration (as measured by EB leakage and MPO activity, respectively) seen at 48 h after the initial ischaemic episode. These studies provide the first experimental evidence that COX-2 inhibition with nimesulide is able to limit BBB disruption and leukocyte infiltration following transient focal cerebral ischaemia. Neuroprotection afforded by nimesulide is observed even when the treatment is delayed until 6 h after the onset of ischaemia, confirming a wide therapeutic window of COX-2 inhibitors in experimental stroke. On the contrary, selective inhibition of COX-1 with VAS had no significant effect on the evaluated parameters. These data suggest that COX-2 activity, but not COX-1 activity, contributes to the progression of focal ischaemic brain injury, and that the beneficial effects observed with non-selective COX inhibitors are probably associated to COX-2 rather than to COX-1 inhibition.

Inhibition of integrin αvβ3 reduces blood–brain barrier breakdown in focal ischemia in rats

Ischemic stroke is a major cause of morbidity and mortality in industrialized nations. We tested the effect of postischemic treatment of cyclo-RGDfV (cRGDfV), a selective inhibitor of integrin alphavbeta3, in the middle cerebral artery occlusion (MCAO) model of ischemic stroke in rats. Rats were randomly divided into three groups: sham operation (n = 13), MCAO with no treatment (n = 18), and MCAO with cRGDfV treatment (n = 28). Focal ischemia was induced with the suture occlusion method for 2 hr, and treatment was given 1 hr after reperfusion (3 hr after ischemia). All animals were sacrificed 24 hr after reperfusion. Assessment included neurological scores, infarction volumes, brain water content, Evans blue exudation, IgG exudation, histology, immunohistochemistry, and Western blotting. Treatment with cRGDfV ameliorated neurological deficits, reduced brain edema, and reduced exudation of Evans blue dye and IgG, but failed to reduce infarction volumes. Western blotting showed a reduction in phosphorylation of one subset of vascular endothelial growth factor (VEGF) receptors in the cRGDfV treatment group. Western blotting also demonstrated a significant reduction of fibrinogen in the cRGDfV treatment group. We conclude that poststroke treatment with cRGDfV reduces blood-brain barrier breakdown in focal ischemia, possibly through inhibition of VEGF-mediated vascular breakdown.

The p2 purinoceptor agonist uridine 5′-triphosphate (utp) can enhance tolerance to ischaemia-reperfusion in mouse heart

Whether P2 purinoceptors modify ischaemic tolerance in mammalian myocardium is not known. We investigated effects of the P2Y receptor agonist UTP on functional tolerance to ischaemia, in Langendorff-perfused C57/Bl6 mouse hearts subjected to 20 min ischaemia/45 min reperfusion. Hearts were untreated or subjected to: i) 5 min pre-ischaemic UTP; ii) 5 min post-ischaemic UTP; or iii) 5 min pre-and 5 min post-ischaemic UTP treatment. Low (250 nM) and high (1 μM) UTP was assessed. Untreated hearts recovered 67±3 mmHg left ventricular developed pressure (LVDP; ∼50% of pre-ischaemia), and exhibited sustained diastolic contracture (31±2 mmHg) after 45 min reperfusion.

A comparison of protective effects between l-histidine and hypothermia against ischemia-induced neuronal damage in gerbil hippocampus

An increase in the histamine concentration in the brain has been demonstrated to provide protective effects against ischemia/reperfusion brain injury. Since hypothermia and barbiturates are also regarded to protect ischemic brains, effects of postischemic treatments were compared in gerbils between mild hypothermia and intraperitoneal administration of l-histidine, a precursor of histamine. Furthermore, effects of thioperamide, a histamine H 3 receptor antagonist, were evaluated in histidine-treated gerbils after 60 days. Transient forebrain ischemia for 4 min at 37 °C provoked severe neuronal damage in the hippocampal CA1 pyramidal cells after 7 days. Postischemic hypothermia (33 °C) for 3 h under pentobarbital anesthesia alleviated neuronal death, and the number of preserved neurons was 77 ± 56/mm (mean ± S.D., n = 14). The effect of l-histidine injected three times, immediately, 6 h, and 24 h after reperfusion (1000 mg/kg, i.p., each), was more prominent than that of hypothermia, and the number of preserved neurons was 142 ± 55/mm ( n = 14). When the histologic outcome was evaluated after 60 days, most neurons were damaged in both the hypothermic and histidine groups. The improvement of the histologic outcome was observed even after 60 days in animals injected with thioperamide, immediately and 6 h after reperfusion (5 mg/kg, s.c., each), with three injections of l-histidine. The number of preserved neurons was 133 ± 88/mm ( n = 10), while that in the hypothermic group was 7 ± 15 ( n = 10). Activation of the central histaminergic system provides beneficial effects against cerebral ischemia.

Retraction：Effects of Pre- and Post-ischemic Treatments with FK409, a Nitric Oxide Donor, on Ischemia/Reperfusion-Induced Renal Injury and Endothelin-1 Production in Rats

We have demonstrated that ischemic acute renal failure (ARF) is attenuated by pre-ischemic treatment with a spontaneous nitric oxide (NO) donor, (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (FK409). In the present study, we evaluated the effect of post-ischemic treatment with FK409 on ARF, compared with the pre-ischemic treatment effect. Ischemic ARF was induced by occlusion of the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after contralateral nephrectomy. At 24 h after reperfusion, renal function in untreated ARF rats markedly decreased. In addition, increases in renal contents of endothelin-1 (ET-1), a deleterious mediator in the pathogenesis of ischemic ARF, were evident in untreated ARF rats at 24 h after reperfusion. Pre-ischemic treatment with FK409 (1 or 3 mg/kg, i.v.) at 5 min before ischemia attenuated ischemia/reperfusion-induced renal dysfunction and increased ET-1 contents after reperfusion. In contrast, post-ischemic treatment with FK409 (3 or 10 mg/kg, i.v.) at 6 h after reperfusion aggravated the renal injury, but did not affect the increased ET-1 content after reperfusion. These results suggest that pre-ischemic treatment with FK409 exerts renoprotective effects on ischemic ARF, probably through the suppression of renal ET-1 overproduction, whereas post-ischemic treatment with the NO donor worsens the ischemia/reperfusion-induced renal injury, through mechanisms unrelated to the ET-1 production after reperfusion.

Effects of Ischemic Preconditioning and Propofol on Cardiac Function and Coronary Flow following Cold Cardioplegia and Reperfusion in Isolated Rat Heart

Background: The interaction between ischemic preconditioning (IPC) and propofol-induced cardioprotective effects during prolonged cold ischemia has not been studied yet. The purpose of this study is to investigate the effects of ischemic preconditioning and propofol on cardiac function and the development of endothelial injury after 4 hours of cold cardioplegia and reperfusion. Methods: After suspension of the isolated heart on the Langendorff perfusion system, we took a stabilizing period for 15 minutes, perfusion period for 15 minutes, global cold (4) ischemic period for 4 hours, and then reperfusion period for 60 minutes. There were 4 groups: (1) CONTROL group, no intervention; (2) IPC group, two 2-minute total coronary clusions interspaced with 5 minutes of normal reperfusion; (3) PROPOFOL group, propofol (2M) was infused during reperfusion period; (4) BOTH group, ischemic preconditioning and postischemic propofol treatment group. The measurements of cardiac performances, such as left ventricular developed pressure (LVDP), rate of ventricular pressure generation (dp/dt), and heart rate (HR) was obtained at pre- and postischemic periods. For the evaluation of endothelial injury during reperfusion period, coronary flow responses to bradykinin were tested. Infarct size was measured using the triphenyl tetrazolium stain. Results: IPC, PROPOFOL, and BOTH groups showed better outcome of LVDP, dp/dt, HR, and flow responses to bradykinin than CONTROL group did. But there is no statistically significant difference in variables among the three groups. Conclusions: Ischemic preconditioning and postischemic propofol have cardioprotective effect respectively but no additive effect after 4 hours cold cardioplegia and reperfusion.

RETRACTION: Opposite Effects of Pre- and Postischemic Treatments with Nitric Oxide Donor on Ischemia/Reperfusion-Induced Renal Injury

We have demonstrated previously that preischemic treatment with FK409 [(+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide], a spontaneous nitric oxide (NO) donor, markedly improves ischemia/reperfusion-induced renal injury. However, there is conflicting information (renoprotective or cytotoxic) as to the contribution of NO to ischemic acute renal failure (ARF). In the present study, we investigated the effect of postischemic treatment with FK409 (1, 3, and 10 mg/kg i.v.) at 6 h after reperfusion on ischemic ARF, in comparison with the preischemic treatment effect. Ischemic ARF was induced by clamping of the left renal artery and vein for 45 min, followed by reperfusion, 2 weeks after contralateral nephrectomy. Renal function in ARF rats markedly decreased at 24 h after reperfusion. Histopathological examination of the kidney of ARF rats revealed severe renal damage. In contrast to the renoprotective effect by preischemic treatment, postischemic treatment with FK409 aggravated the ischemia/reperfusion-induced renal dysfunction and histological damage. Immunohistochemical analysis of renal sections obtained from ARF rats revealed positive staining for nitrotyrosine, a biomarker of peroxynitrite formation, in injured tubular cells, and more intense staining was observed in renal tissues from the animals that received postischemic treatment with FK409. On the other hand, the formation of nitrotyrosine, neutrophil infiltration into renal tissues, and renal superoxide production, all of which were enhanced in ARF rats, were efficiently attenuated by the preischemic treatment with FK409. These results demonstrate that, although preischemic treatment with an NO donor is renoprotective, postischemic treatment with the same agent aggravates the ischemia/reperfusion-induced renal injury, probably through peroxynitrite overproduction.

Modification of alterations in cardiac function and sarcoplasmic reticulum by vanadate in ischemic-reperfused rat hearts

To study the cardioprotective effects of vanadate on ischemia-reperfusion (I/R) injury, isolated rat hearts perfused at constant flow were subjected to global ischemia for 30 min followed by reperfusion for 30 min. In this experimental model, I/R markedly decreased ventricular developed pressure and increased end-diastolic pressure. Pretreatment of hearts with 4 microM vanadate attenuated I/R-induced cardiac dysfunction. The reduction in sarcoplasmic reticulum (SR) Ca2+ uptake and Ca2+ release, as well as SR protein contents for Ca2+-pump ATPase and Ca2+-release channel, was also prevented by vanadate. Pretreatment of hearts with an antioxidant mixture containing superoxide dismutase + catalase exerted effects similar to those of vanadate in I/R hearts. Postischemic treatment of hearts with vanadate or superoxide dismutase + catalase also had beneficial effects on I/R-induced changes in cardiac performance and SR function. Alterations in cardiac function and SR Ca2+ transport due to an oxyradical-generating system (xanthine + xanthine oxidase) or an oxidant (H2O2) were attenuated by treatment with vanadate. These results suggest that vanadate may exert beneficial effects on cardiac performance and SR function in I/R hearts because of its antioxidant action.

Cinnamophilin reduces oxidative damage and protects against transient focal cerebral ischemia in mice

Acute neuroprotective effects of cinnamophilin (CINN; (8 R, 8′ S)-4, 4′-dihydroxy-3, 3′-dimethoxy-7-oxo-8, 8′-neolignan), a novel antioxidant and free radical scavenger, were studied in a mouse model of transient middle cerebral artery (MCA) occlusion. CINN was administered intraperitoneally either 15 min before (pretreatment) or 2 h after the onset of MCA occlusion (postischemic treatment). Relative to vehicle-treated controls, animals pretreated with CINN, at 20–80 mg/kg, had significant reductions in brain infarction by 33–46% and improvements in neurobehavioral outcome. Postischemic administration with CINN (80 mg/kg) also significantly reduced brain infarction by 43% and ameliorated neurobehavioral deficits. Additionally, CINN administration significantly attenuated in situ accumulation of superoxide anions (O 2 −) in the boundary zones of infarct at 4 h after reperfusion. Consequently, CINN-treated animals exhibited significantly decreased levels of oxidative damage, as assessed by immunopositive reactions for 8-hydroxy-2′-deoxyguanosine (8-OHdG) and 4-hydroxynonenal (4-HNE), and the resultant inflammatory reactions at 24 h postinsult. It is concluded that CINN effectively reduced brain infarction and improved neurobehavioral outcome following a short-term recovery period after severe transient focal cerebral ischemia in mice. The finding of a decreased extent of reactive oxygen species and oxidative damage observed with CINN treatment highlights that its antioxidant and radical scavenging ability is contributory.

Inhibition of T-Type Calcium Channels Protects Neurons from Delayed Ischemia-Induced Damage

Intracellular calcium increase is an early key event triggering ischemic neuronal cell damage. The role of T-type voltage-gated calcium channels in the neuronal response to ischemia, however, has never been studied. Using an in vitro model of ischemia-induced delayed cell death in rat organotypic hippocampal slice cultures, we show that T-type calcium channels inhibitors drastically reduce ischemic cell damage. Immunostaining studies reveal the existence of Ca(V)3.1 and Ca(V)3.2 types of low-voltage-activated calcium channels in rat organotypic hippocampal cultures. Low extracellular calcium (100 nM) or increase of intracellular calcium buffering ability by BAPTA-acetoxymethyl ester significantly reduced ischemia-induced neuronal damage. Pharmacological inhibition of the T-type calcium current by mibefradil, kurtoxin, nickel, zinc, and pimozide during the oxygen-glucose deprivation episode provided a significant protection against delayed neuronal death. Mibefradil and nickel exerted neuroprotective effects, not only if administrated during the oxygen-glucose deprivation episode but also in conditions of postischemic treatment. These data point to a role of T-type calcium currents in ischemia-induced, calcium-mediated neuronal cell damage and suggest a possible new pharmacological approach to stroke treatment.