A poly(ADP-ribose) synthetase inhibitor, benzamide protects smooth muscle cells but not endothelium against ischemia/reperfusion injury in isolated guinea-pig heart

Activation of the nuclear enzyme poly(ADP-ribose) synthetase (PARS) is important in the cellular response to oxidative stress. During ischemia and reperfusion (I/R) increased free radical production leads to DNA breakage that stimulates PARS which in turn results in an energy-consuming metabolic cycle and initiation of the apoptotic process. Previous studies have reported that PARS inhibition confers protection in various models of I/R-induced cardiovascular damage. The purpose of this study was to determine the role of PARS inhibition in I/R-induced injury of smooth muscle cells and endothelium in the coronary circulation of the isolated guinea-pig heart. Control hearts and those treated with a PARS inhibitor--benzamide (100 micromol L(-1)), were subjected to 30 min of subglobal ischemia and subsequent reperfusion (90 min). To analyze the functional integrity of smooth muscle cells and endothelium, one-minute intracoronary infusions of endothelium-independent (sodium nitroprusside, NaNP; 3 micromol L(-1)) and endothelium-dependent (substance P, SP; 10 nmol L(-1)) vasodilators were used before ischemia and at the reperfusion time. The degree of the injury of coronary smooth muscle and endothelial cells induced by I/R was estimated in terms of diminished vasodilator responses to NaNP (at 55 min and 85 min of reperfusion) and to SP (at 70 min of reperfusion), respectively, and expressed as the percentage of preischemic response. I/R reduced vasorelaxant responses to both vasodilators by half (to 54.1 +/- 5.1% and to 53.6 +/- 4.9% of preischemic value for NaNP at 55 min and 85 min of reperfusion, respectively and to 45.9 +/- 6.5% for SP at 70 min of reperfusion). PARS inhibition provided complete restoration of vasorelaxation induced by NaNP (107.6 +/- 13.3% and 104 +/- 14.4% of preischemic response at the two time points of reperfusion, respectively). However, there was no effect on the SP-induced response (48+12.1% of preischemic response). We conclude that pharmacological PARS inhibition with benzamide protects coronary smooth muscle cells but not endothelium against I/R-induced reperfusion injury in the coronary circulation of the guinea-pig heart.     

Poly(ADP-Ribose) Synthetase Activation: An Early Indicator of Neurotoxic DNA Damage

Abstract: DNA damage activates a nuclear enzyme poly(ADP-ribose) synthetase (PARS) that facilitates DNA repair by adding multiple ADP-ribose groups to nuclear proteins such as histones and PARS itself. N -Methyl- d -aspartate neurotoxicity may involve DNA damage excessively activating PARS to deplete its substrate NAD, as PARS inhibitors prevent this toxicity. We now show that PARS is rapidly and markedly activated in PC12 cells following treatment with neurotoxic agents, including the amyloid β-protein, hydrogen peroxide, N -methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and its active metabolite N -methyl-4-phenylpyridine (MPP⁺). With MPP⁺, PARS activity is increased fivefold in 1 h and 20-fold by 3 h. By contrast, direct measurement of DNA damage by the terminal-deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assay shows no significant increase by 3 h and less than fourfold by 24 h. These findings indicate that PARS activity can provide a simple, sensitive, and early index of DNA damage following neurotoxic insults.     

Possible role of nicaraven in neuroprotective effect on hippocampal slice culture

Nicaraven is an agent that is especially beneficial in vasospasm or brain damage caused by subarachnoid hemorrhage. It ameliorates neurological deficits of patients and protects the central nervous system from ischemia. We investigated the neuroprotective effect of nicaraven against oxygen-glucose deprivation (OGD) induced or N-methyl-D-aspartic acid (NMDA) induced hippocampal neuronal cell death in organotypic brain slice cultures. The effect of nicaraven on hippocampal neuronal injury was evaluated by inhibition of uptake of propidium iodide (PI) into dead cells. The results demonstrated that nicaraven protected neuronal cells from both OGD- and NMDA-induced cell death. While nicaraven has a strong hydroxyl radical scavenging effect, another radical scavenger, N-acetyl-L-cysteine (NAC), inhibited cell death only caused by OGD. In contrast, the poly(ADP-ribose) synthetase (PARS) inhibitors 3-aminobenzamide (3-AB) and theophylline protected cells from both OGD- and NMDA-induced cell death. Since nicaraven has an inhibitory effect in PARS, as well as a radical scavenging effect, these results suggest that inhibition of hippocampal cell death caused by NMDA may be attributable to PARS inhibition by nicaraven.     

Protective effects of poly (ADP-ribose) synthase inhibitors in zymosan-activated plasma induced paw edema.

The aim of the present study was to investigate the role of poly (ADP-ribose) synthetase (PARS) in a model of acute local inflammation (zymosan-activated plasma (ZAP)-induced paw edema), in which the oxyradicals, nitric oxide and peroxynitrite, are known to play a crucial role. Injection of zymosan-activated plasma (ZAP) into the rat paw induced edema formation. The maximal increase in paw volume was observed at three hours after administration (maximal in paw volume: 1.29+/-0.09 ml). At this time point, there was a marked increase in neutrophil infiltration in the paw, as measured by an increase in myeloperoxidase (MPO) activity in the paw tissue (260+/-25 mU/100 mg wet tissue). However, ZAP-induced paw edema was significantly reduced in a dose-dependent manner by treatment with 3-aminobenzamide (3-AB) or nicotinamide (NIC), two inhibitors of PARS, at 1, 2, 3, 4 hours after ZAP injection. PARS inhibition also caused a significant reduction of MPO activity. The paw tissues were also examined immunohistochemically for the presence of nitrotyrosine (a footprint for peroxynitrite formation). At 3 h following ZAP injection, staining for nitrotyrosine were also found to be localised within discrete cells in the inflamed paw tissue. Treatment with PARS inhibitor prevented the appearance of nitrotyrosine in the tissues. Our results suggest that in paw edema induced by ZAP, inhibition of PARS exert potent anti-inflammatory effects.     

Inhibitors of poly (ADP-ribose) synthetase reduce renal ischemia-reperfusion injury in the anesthetized rat in vivo.

The activation of poly (ADP-ribose) synthetase (PARS) subsequent to DNA damage caused by reactive oxygen or nitrogen species has been implicated in several pathophysiological conditions, including ischemia-reperfusion injury and shock. The aim of this study was to investigate whether PARS inhibitors could provide protection against renal ischemia-reperfusion injury in the rat in vivo. Male Wistar rats were subjected to 45 min bilateral clamping of the renal pedicles, followed by 6 h reperfusion (control animals). Animals were administered the PARS inhibitors 3-aminobenzamide, 1, 5-dihydroxyisoquinoline, or nicotinamide during the reperfusion period. Ischemia, followed by reperfusion, produced significant increases in plasma concentrations of urea, creatinine, and fractional excretion of Na(+) (FE(Na)) and produced a significant reduction in glomerular filtration rate (GFR). However, administration of the PARS inhibitors significantly reduced urea and creatinine concentrations, suggesting improved renal function. The PARS inhibitors also significantly increased GFR and reduced FE(Na), suggesting the recovery of both glomerular and tubular function, respectively, with a more pronounced recovery of tubular function. In kidneys from control animals, histological examination revealed severe renal damage and immunohistochemical localization demonstrated PARS activation in the proximal tubule. Both renal damage and PARS activation were attenuated by administration of PARS inhibitors during reperfusion. Therefore, we propose that PARS activation contributes to renal reperfusion injury and that PARS inhibitors may be beneficial in renal disorders associated with oxidative stress-mediated injury.     

Myocardial ischemic preconditioning in rodents is dependent on poly (ADP-ribose) synthetase

BACKGROUND: Activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS) in response to oxidant-mediated DNA injury has been shown to play an important role in the pathogenesis of reperfusion injury. Here we investigated the role of PARS in myocardial ischemic preconditioning (IPC). MATERIALS AND METHODS: Mice with or without genetic disruption of PARS and rats in the absence or presence of the PARS inhibitor 3-aminobenzamide underwent coronary occlusion and reperfusion with or without IPC. RESULTS: Both poly(ADP-ribose) synthetase (PARS) deficiency and ischemic preconditioning (IPC) induced protection from reperfusion injury, attenuated inflammatory mediator production, and reduced neutrophil infiltration when compared to the response in wild-type mice. Surprisingly, the protective effect of IPC not only disappeared in PARS-/- mice, but the degree of myocardial injury and inflammatory response was similar to the one seen in wild-type animals. Similarly, in the rat model of IPC, 3-aminobenzamide pretreatment blocked the beneficial effect of IPC. Myocardial NAD+ levels were maintained in the PARS-deficient mice during reperfusion, while depleted in the wild-type mice. The protection against reperfusion injury by IPC was also associated with partially preserved myocardial NAD+ levels, indicating that PARS activation is attenuated by IPC. This conclusion was further strengthened by poly(ADP-ribose) immunohistochemical measurements, demonstrating that IPC markedly inhibits PARS activation during reperfusion. CONCLUSIONS: The mode of IPC's action is related, at least in part, to an inhibition of PARS. This process may occur either by self-auto-ribosylation of PARS during IPC, and/or via the release of endogenous purines during IPC that inhibit PARS activation during reperfusion.     

Protective effect of melatonin on zymosan-induced cellular damage

We investigated whether in vivo melatonin treatment inhibits cellular injury induced by peroxynitrite production and PARS activation in macrophages collected from rats subjected to zymosan-induced shock. Macrophages harvested from the peritoneal cavity exhibited a significant production of peroxynitrite, as measured by the oxidation of the fluorescent dye dihydrorhodamine 123. Furthermore, zymosan-induced shock suppressed macrophage mitochondrial respiration, DNA strand breakage, activation of the nuclear enzyme poly(ADP-ribose)synthetase (PARS) and reduction of cellular levels of NAD+. In vivo treatment with melatonin (25 and 50 mg/kg, i.p., 1 h after zymosan injection) significantly and dose-dependently reduced peroxynitrite formation and prevented the appearance of DNA damage, decrease in mitochondrial respiration, loss of cellular levels of NAD+ and PARS activation. Our study supports the view that the antioxidant and anti-inflammatoy effect of melatonin is also correlated with the inhibition of peroxynitrite production and PARS activation. In conclusion, melatonin may be a novel pharmacological approach to prevent cell injury in inflammation.     

Role of poly(ADP-ribose) synthetase in pulmonary leukocyte recruitment

During systemic inflammation, recruitment and activation of leukocytes in the pulmonary microcirculation may result in a potentially life-threatening acute lung injury. We elucidated the role of the poly(ADP-ribose) synthetase (PARS), a nucleotide-polymerizing enzyme, in the regulation of leukocyte recruitment within the lung with regard to the localization in the pulmonary microcirculation and in correlation to hemodynamics in the respective vascular segments and expression of intercellular adhesion molecule 1 during endotoxemia. Inhibition of PARS by 3-aminobenzamide reduced the endotoxin-induced leukocyte recruitment within pulmonary arterioles, capillaries, and venules in rabbits as quantified by in vivo fluorescence microscopy. Microhemodynamics and thus shear rates in all pulmonary microvascular segments remained constant. Simultaneously, inhibition of PARS with 3-aminobenzamide suppressed the endotoxin-induced adhesion molecules expression as demonstrated for intercellular adhesion molecule 1 by immunohistochemistry and Western blot analysis. We confirmed this result with the use of PARS knockout mice. The inhibitory effect of 3-aminobenzamide on leukocyte recruitment was associated with a reduction of pulmonary capillary leakage and edema formation. We first provide evidence that PARS activation mediates the leukocyte sequestration in pulmonary microvessels through upregulation of adhesion molecules. As reactive oxygen species released from leukocyte are supposed to cause an upregulation of adhesion molecules we conclude that PARS inhibition contributes to termination of this vicious cycle and inhibits the inflammatory process.     

PARP-1 gene disruption in mice preferentially protects males from perinatal brain injury

Poly(ADP-ribose) polymerase-1 is over-activated in the adult brain in response to ischemia and contributes to neuronal death, but its role in perinatal brain injury remains uncertain. To address this issue, 7-day-old wild-type (wt) and PARP-1 gene deficient (parp+/- and parp-/-) Sv129/CD-1 hybrid mice were subjected to unilateral hypoxia-ischemia and histologic damage was assessed 10 days later by two evaluators. Poly(ADP-ribose) polymerase-1 knockout produced moderate but significant (p < 0.05) protection in the total group of animals, but analysis by sex revealed that males were strongly protected (p < 0.05) in contrast to females in which there was no significant effect. Separate experiments demonstrated that PARP-1 was activated over 1-24 h in both females and males after the insult in neonatal wt mice and rats using immnocytochemistry and western blotting for poly(ADP-ribose). Brain levels of NAD+ were also significantly reduced, but the decrease of NAD+ during the early post-hypoxia-ischemia (HI) phase was only seen in males. The results indicate that hypoxia-ischemia activates Poly(ADP-ribose) polymerase-1 in the neonatal brain and that the sex of the animal strongly influences its role in the pathogenesis of brain injury.     

Quercetin Protects Against Oxidative Stress Associated Damages in a Rat Model of Transient Focal Cerebral Ischemia and Reperfusion

Experimental studies have demonstrated that oxidative stress and apoptosis play an important role in cerebral ischemic pathogenesis and may represent a target for treatment. The purpose of this study was to determine whether the quercetin dihydrate (Q) protects against cerebral ischemia neuronal damage. Male Wistar rats were subjected to transient middle cerebral artery occlusion (MCAO) for 2?h and reperfused for 72?h. Quercetin (30?mg/kg, i.p) was administrated 30?min before the onset of ischemia and after the ischemia at interval of 0, 24, 48, and 72?h. The administration of Q showed marked reduction in infarct size, reduced the neurological deficits in terms of behaviors, suppressed neuronal loss and diminished the p53 expression in MCAO rats. Q was found to be successful in upregulating the antioxidant status and lowering the TBARS level. Conversely, the elevated activity of poly (ADP-ribose) polymerase (PARP), and activity of caspase-3 in MCAO group was attenuated significantly in Q treated group when compared with MCAO group. Our study reveals that Q, as a powerful antioxidant, could prevent free radicals associated oxidative damage and morphological changes in the MCAO rats. Thus, it may have a therapeutic value for the treatment of stroke.     

TLR2 has a detrimental role in mouse transient focal cerebral ischemia

A significant up-regulation of Toll-like-receptor (TLR) mRNAs between 3 and 48 h reperfusion time after induction of transient focal cerebral ischemia for 1h was revealed by applying global gene expression profiling in postischemic mouse brains. Compared to TLR4 and TLR9, TLR2 proved to be the most significantly up-regulated TLR in the ipsilateral brain hemisphere. TLR2-protein was found to be expressed mainly in microglia in the postischemic brain tissue, but also in selected endothelial cells, neurons, and astrocytes. Additionally, TLR2-related genes with pro-inflammatory and pro-apoptotic capabilities were induced. Therefore we hypothesized that TLR2-signaling could exacerbate the primary brain damage after ischemia. Two days after induction of transient focal cerebral ischemia (1h), we found a significant decrease of the infarct volume in TLR2 deficient mice compared to wild type mice (75+/-5 vs. 42+/-7 mm(3)). We conclude that TLR2 up-regulation and TLR2-signaling are important events in focal cerebral ischemia and contribute to the deterioration of ischemic damage.     

Repetitive intraperitoneal caspase-3 inhibitor and anesthesia reduces neuronal damage

Caspase inhibitors are usually administered intracranially. There’s very limited evidence showing that they can be used intraperitoneally, and still have a beneficial effect. We tested the hypothesis that, during focal cerebral ischemia, caspase inhibitors when used in combination with an anesthetic agent results in a significantly reduction in the neuronal damage. Male Sprague Dawley rats were randomly divided into six different groups: control, Isoflurane, Propofol, Isoflurane and Caspase-3 inhibitor intraperitoneally (IP), propofol and Caspase-3 inhibitor IP and only caspase-3 inhibitor, during post-ischemia. Neurological evaluation and histochemical analysis was assessed post-ischemia. The treatment proposed, resulted in a significant decrease in the cerebral infarction volume. Combination of treatments, and caspase-3 inhibitor alone significantly decreased the number of TUNEL and cleaved caspase-3 positive cells in the boundary area of cortical infarction. IP administration appears to reach cerebral targets similarly to intracerebral model. This combination reduces the neurological damage caused by focal cerebral ischemia.     

Preconditioning with cortical spreading depression decreases intraischemic cerebral glutamate levels and down-regulates excitatory amino acid transporters EAAT1 and EAAT2 from rat cerebal cortex plasma membranes

We previously reported a 50% reduction in cortical infarct volume following transient focal cerebral ischemia in rats preconditioned 3 days earlier with cortical spreading depression (CSD). The mechanism of the protective effect of prior CSD remains unknown. Recent studies demonstrate reversal of excitatory amino acid transporters (EAATs) to be a principal cause for elevated extracellular glutamate levels during cerebral ischemia. The present study measured the effect of CSD preconditioning on (a) intraischemic glutamate levels and (b) regulation of glutamate transporters within the ischemic cortex of the rat. Three days following either CSD or sham preconditioning, rats were subjected to 200 min of focal cerebral ischemia, and extracellular glutamate concentration was measured by in vivo microdialysis. Cortical glutamate exposure decreased 70% from 1,772.4 +/- 1,469.2 microM-min in sham-treated (n = 8) to 569.0 +/- 707.8 microM-min in CSD-treated (n = 13) rats (p <0.05). The effect of CSD preconditioning on glutamate transporter levels in plasma membranes (PMs) prepared from rat cerebral cortex was assessed by western blot analysis. Down-regulation of the glial glutamate transporter isoforms EAAT2 and EAAT1 from the PM fraction was observed at 1, 3, and 7 days but not at 0 or 21 days after CSD. Semiquantitative lane analysis showed a maximal decrease of 90% for EAAT2 and 50% for EAAT1 at 3 days post-CSD. The neuronal isoform EAAT3 was unaffected by CSD. This period of down-regulation coincides with the time frame reported for induced ischemic tolerance. These data are consistent with reversal of glutamate transporter function contributing to glutamate release during ischemia and suggest that down-regulation of these transporters may contribute to ischemic tolerance induced by CSD.     

Postconditioning fails to prevent radial artery endothelial dysfunction induced by ischemia and reperfusion: evidence from a human in vivo study

Animal studies have shown that, as compared with unrestricted reperfusion, exposure to brief periods of controlled ischemia (postconditioning) at the end of a prolonged ischemia reduces the extent of tissue damage. We set out to test whether postconditioning can prevent endothelial dysfunction induced by ischemia and reperfusion in a human in vivo model. Ten healthy young non-smoking volunteers were enrolled in this cross-over, controlled, investigator-blinded study. Subjects were exposed to 15 min of forearm ischemia followed by either unrestricted reperfusion or postconditioning (3 periods of 20 s of ischemia separated by 10 s of reperfusion). Endothelium-dependent flow-mediated dilation (FMD) was measured at the level of the radial artery before and after ischemia (with or without postconditioning). Forearm ischemia blunted FMD in both study visits (unrestricted reperfusion visit: before ischemia, 7.7% +/- 1.3%; after ischemia, 2.5% +/- 1.4%; and postconditioning visit: before, 7.3% +/- 1.2%; after, 2.6 +/- 1.6%; P < 0.05 for both, P = not significant (NS) between visits). In contrast with data from animal studies, postconditioning (20 s ischemia-- 10 s reperfusion repeated 3 times) does not limit post-ischemic endothelial dysfunction in this human in vivo model. Further human studies are necessary to evaluate other reperfusion protocols in an attempt to limit post-ischemic tissue damage.     

Neuroprotective Effects of Poly(ADP-ribose)polymerase Inhibitor Olaparib in Transient Cerebral Ischemia

Olaparib was the first poly(ADP-ribose)polymerase inhibitor approved by Food and Drug Administration for oncology treatment. However, its neuroprotective effects have not been elucidated. This study aimed to evaluate the effects of olaparib in transient cerebral ischemia. A mouse model of transient middle cerebral artery occlusion was used. Reperfusion was performed at 2 h after ischemia. Different doses of olaparib (1, 3, 5, 10 and 25 mg/kg) were administered intraperitoneally immediately after reperfusion. Twenty-four hours after ischemia, the neurological score was assessed, and grip and string tests were performed to evaluate the behavioral deficits in the mice. Cresyl violet staining was used to assess cerebral edema and the lesion volume. Immunohistochemistry was performed to evaluate the expression of blood–brain barrier proteins collagen IV and claudin-5, as well as extravasation of IgG. Ischemia induced a neurological deficit, which was significantly ameliorated by olaparib at 3 and 5 mg/kg. However, this neuroprotective effect was not observed in mice treated with either low-dose or high-dose olaparib. Both 3 and 5 mg/kg olaparib markedly reduced cerebral infarction volume, but not cerebral edema. The expression of collagen IV decreased after cerebral ischemia, which was improved by olaparib at 3 and 5 mg/kg. These results were confirmed by the reduction of IgG extravasation with olaparib. Olaparib showed clear neuroprotective effects in transient ischemic mice mainly through the reduction of cerebral infarction and blood–brain barrier damage.     

Synthetic Oligodeoxynucleotides Containing Multiple Telemeric TTAGGG Motifs Suppress Inflammasome Activity in Macrophages Subjected to Oxygen and Glucose Deprivation and Reduce Ischemic Brain Injury in Stroke-Prone Spontaneously Hypertensive Rats

The immune system plays a fundamental role in both the development and pathobiology of stroke. Inflammasomes are multiprotein complexes that have come to be recognized as critical players in the inflammation that ultimately contributes to stroke severity. Inflammasomes recognize microbial and host-derived danger signals and activate caspase-1, which in turn controls the production of the pro-inflammatory cytokine IL-1β. We have shown that A151, a synthetic oligodeoxynucleotide containing multiple telemeric TTAGGG motifs, reduces IL-1β production by activated bone marrow derived macrophages that have been subjected to oxygen-glucose deprivation and LPS stimulation. Further, we demonstrate that A151 reduces the maturation of caspase-1 and IL-1β, the levels of both the iNOS and NLRP3 proteins, and the depolarization of mitochondrial membrane potential within such cells. In addition, we have demonstrated that A151 reduces ischemic brain damage and NLRP3 mRNA levels in SHR-SP rats that have undergone permanent middle cerebral artery occlusion. These findings clearly suggest that the modulation of inflammasome activity via A151 may contribute to a reduction in pro-inflammatory cytokine production by macrophages subjected to conditions that model brain ischemia and modulate ischemic brain damage in an animal model of stroke. Therefore, modulation of ischemic pathobiology by A151 may have a role in the development of novel stroke prevention and therapeutic strategies.     

Spironolactone reduces cerebral infarct size and EGF-receptor mRNA in stroke-prone rats

Remodeling of the cerebral vasculature contributes to the pathogenesis of cerebral ischemia. Remodeling is caused by increased smooth muscle proliferation and may be due to an increase in the responsiveness of vascular cells to epidermal growth factor (EGF). Aldosterone is a risk factor for stroke, and the literature suggests it may play a role in increasing the expression of the receptor for EGF (EGFR). We hypothesized that mRNA for the EGF-stimulated pathway would be elevated in the vasculature of stroke-prone spontaneously hypertensive rats (SHRSP) and that this and experimental ischemic cerebral infract size would be reduced by aldosterone inhibition with spironolactone. We found that spironolactone treatment reduced the size of cerebral infarcts after middle cerebral artery occlusion in SHRSP (51.69 +/- 3.60 vs. 22.00 +/- 6.69% of hemisphere-infarcted SHRSP vs. SHRSP + spironolactone P < 0.05). Expression of EGF and EGFR mRNA was higher in cerebral vessels and aorta from adult SHRSP compared with Wistar-Kyoto rats. Only the expression of EGFR mRNA was elevated in the young SHRSP. Spironolactone reduced the EGFR mRNA expression in the aorta (1.09 +/- 0.25 vs. 0.56 +/- 0.11 phosphorimage units SHRSP vs. SHRSP + spironolactone P < 0.05) but had no effect on EGF mRNA. In vitro incubation of aorta with aldosterone +/- spironolactone produced similar results, suggesting a direct effect of aldosterone. Thus spironolactone may reduce the size of cerebral infarcts via a reduction in the expression of the EGFR mRNA, leading to reduced remodeling.     

The role of differential activation of p38-mitogen-activated protein kinase in preconditioned ventricular myocytes.

Activation of protein kinase C (PKC) and more recently mitogen-activated protein kinases (MAPKs) have been associated with the cardioprotective effect of ischemic preconditioning. We examined the interplay between these kinases in a characterized model of ischemic preconditioning in cultured rat neonatal ventricular cardiocytes where ectopic expression of active PKC-delta results in protection. Two members of the MAPK family, p38 and p42/44, were activated transiently during preconditioning by brief simulated ischemia/reoxygenation. Overexpression of active PKC-delta, rather than augmenting, completely abolished this activation. We therefore determined whether a similar process occurred during lethal prolonged simulated ischemia. In contrast to ischemia, brief, lethal-simulated ischemia activated only p38 (2.8+/-0.45 vs. basal, P<0.01), which was attenuated by expression of active PKC-delta or by preconditioning (0.48+/-0.1 vs. ischemia, P<0.01). To determine whether reduced p38 activation was the cause or an effect of protection, we used SB203580, a p38 inhibitor. SB203580 reduced ischemic injury (CK release 38.0+/-3.1%, LDH release 77.3+/-4.0%, and MTT bioreduction 127.1+/-4.8% of control, n=20, P<0.05). To determine whether p38 activation was isoform selective, myocytes were infected with adenoviruses encoding wild-type p38alpha or p38beta. Transfected p38alpha and beta show differential activation (P<0.001) during sustained simulated ischemia, with p38alpha remaining activated (1.48+/-0.36 vs. basal) but p38beta deactivated (0.36+/-0.1 vs. basal, P<0.01). Prior preconditioning prevented the activation of p38alpha (0.65+/-0.11 vs. ischemia, P<0.05). Moreover, cells expressing a dominant negative p38alpha, which prevented ischemic p38 activation, were resistant to lethal simulated ischemia (CK release 82.9+/-3.9% and MTT bioreduction 130.2+/-6.5% of control, n=8, P<0.05). Thus, inhibition of p38alpha activation during ischemia reduces injury and may contribute to preconditioning-induced cardioprotection in this model.     

Neuroprotective effect of curcumin in middle cerebral artery occlusion induced focal cerebral ischemia in rats

Free radical induced neuronal damage is implicated in cerebral ischemia reperfusion (IR) injury and antioxidants are reported to have neuroprotective activity. Several in vitro and in vivo studies have proved the antioxidant potential of curcumin and its metabolites. Hence, in the present study the neuroprotective potential of curcumin was investigated in middle cerebral artery occlusion (MCAO) induced focal cerebral IR injury. 2 h of MCAO and 22 h of reperfusion resulted in the infarct volume of 210.39 +/- 31.25 mm3. Administration of curcumin 100 and 300 mg/kg, i.p. 30 min. after MCAO produced 37.23 +/- 5.10% and 46.39 +/- 10.23% (p < 0.05) reduction in infarct volume, respectively. Ischemia induced cerebral edema was reduced in a dose dependent manner. Curcumin at 300 mg/kg, i.p. produced 50.96 +/- 6.04% reduction in edema (p < 0.05) volume. Increase in lipid peroxidation after MCAO in ipsilateral and contralateral hemisphere of brain was observed, which was reduced by curcumin (300 mg/kg, i.p.)-treatment. Decrease in superoxide dismutase and glutathione peroxidase activity was observed in ipsilateral hemisphere of MCAO animal. Curcumin-treatment (300 mg/kg, i.p.) prevented IR injury mediated fall in glutathione peroxide activity. Peroxynitrite measured using rhodamine123 fluorescence and anti-nitrotyrosine immunofluorescence indicated increased peroxynitrite formation after IR insult. Curcumin-treatment reduced peroxynitrite formation and hence the extent of tyrosine nitration in the cytosolic proteins. These results suggest the neuroprotective potential of curcumin in cerebral ischemia and is mediated through its antioxidant activity.     

Neuroprotective effects of NU1025, a PARP inhibitor in cerebral ischemia are mediated through reduction in NAD depletion and DNA fragmentation

Oxidative stress induced cell injury is reported to contribute to the pathogenesis of cerebral ischemia. Reactive oxygen species such as hydrogen peroxide (H2O2) and superoxide radical along with nitric oxide and peroxynitrite generated during ischemia-reperfusion injury, causes the overactivation of poly (ADP-ribose) polymerase (PARP) leading to neuronal cell death. In the present study we have evaluated the effects of PARP inhibitor, 8-hydroxy-2 methyl-quinazolin-4-[3H]one (NU1025) in H2O2 and 3-morphilinosyndonimine (SIN-1) induced cytotoxicity in PC12 cells as well as in middle cerebral artery occlusion (MCAO) induced focal cerebral ischemia in rats. Exposure of PC12 cells to H2O2 (0.4 mM) and SIN-1 (0.8 mM) resulted in a significant decrease in cell viability after 6 h. Pretreatment with NU1025 (0.2 mM) restored cell viability to approximately 73 and 82% in H2O2 and SIN-1 injured cells, respectively. In MCAO studies, NU1025 was administered at different time points (1 h before reperfusion, immediately before reperfusion, 3 h after reperfusion and 6 h after reperfusion). NU1025 at 1 and 3 mg/kg reduced total infarct volume to 25% and 45%, respectively, when administered 1 h before reperfusion. NU1025 also produced significant improvement in neurological deficits. Neuroprotection with NU1025 was associated with reduction in PAR accumulation, reversal of brain NAD depletion and reduction in DNA fragmentation. Results of this study demonstrate the neuroprotective activity of NU1025 and suggest its potential in cerebral ischemia.