Lethal Ischemia Research Articles

BAS/BSCR15 The role of PINK1, a mitochondrial pro-survival kinase, in myocardial ischaemia-reperfusion

PINK1, a kinase localised mainly in mitochondria, can protect neurons against oxidative damage and apoptosis by maintaining mitochondrial structure and function. Although PINK1 is highly expressed in the myocardium, there...

Critical hematocrit and oxygen partial pressure in the beating heart of pigs

In cardiac surgery the substitution of lost blood volume by plasma substitutes is a common therapeutical approach. None of the currently available blood substitutes has a sufficient oxygen transport capacity. This can limit the functional integrity of the myocardium known as highly oxygen consumptive. The study was aimed to get information about the minimal hematocrit, also known as critical hematocrit (cHct), which guarantees a stable and adequate oxygen partial pressure in the myocardium (pO2). In adult female pigs (n = 7) the hematocrit was reduced by isovolemic blood dilution with an intravenous infusion of isotonic 4% gelatine polysuccinate solution, The substituted blood volume ranged between 3000 ml and 7780 ml (mean: 5254 ± 1672 ml). In all animals the pO2 of the myocardium of the beating heart and of the resting skeletal muscle increased until blood dilution resulted in a Hct decrease down to 15%. Further blood dilution resulted in a decrease of the pO2. Only after the Hct was < 10% the pO2 was lower than before blood dilution and accompanied by a lethal ischemia of the myocardium. These data indicate a cHct of about 10% in the pig animal model.

Ischemic preconditioning attenuates of ischemia-induced degradation of spectrin and tau: implications for ischemic tolerance

Global ischemia selectively induces CA1 neuronal death in the hippocampus. Pretreatment with non-lethal ischemia (i.e. ischemic preconditioning) prevents CA1 neuronal death induced by lethal ischemia. While ischemic tolerance is a well-known phenomenon, the underlying molecular mechanisms are not fully understood. Cytoskeletal proteins including α-spectrin, tau, and microtubule-associated protein 2 (MAP-2) are indispensable for the maintenance of neuronal homeostasis. Here, we report the effects of ischemic preconditioning on the ischemia-induced degradation of cytoskeletal proteins α-spectrin, tau, and MAP-2 in the rat CA1 region. We found that most neurons of the CA1 region had died after 5 min of ischemia. However, exposing the brain to 3 min of ischemic preconditioning 3 days earlier significantly reduced the number of neuronal death. A significant degradation of α-spectrin and tau, but not of MAP-2, was found in the CA1 region after 5 min of ischemia. Ischemic preconditioning attenuated the ischemia-induced massive degradation of α-spectrin and tau. Our results suggest that the attenuation of ischemia-induced degradation of α-spectrin and tau by ischemic preconditioning may be associated with the neuroprotective mechanism of the ischemic tolerance.

Effects of Acute and Chronic Administration of Erythropoietin on Arrhythmias Induced by Myocardial Ischemia and Preconditioning in Anesthetized Rats

Erythropoietin is a cytokine that is commonly associated with its role as a hormonal stimulator of erythropoiesis. Recombinant human erythropoietin is used to correct anemia in patients undergoing chronic hemodialysis. Ischemic preconditioning is a powerful endogenous protective phenomenon in which brief episodes of ischemia induce protection against future, lethal ischemia. Despite the intensive investigation, the exact molecular mechanisms underlying ischemic preconditioning are not well known. Erythropoietin has been suggested as another endogenous mediator of ischemic preconditioning since it is produced after ischemic or hypoxic insults. In order to determine its cardioprotective effects, we examined the effects of acute and chronic administration of erythropoietin on occlusion-induced arrhythmias in anesthetized rats in the present study. Erythropoietin produced marked antiarrhythmic effects with reduction in ventricular tachycardia, abolition of ventricular fibrillation and attenuation of arrhythmia scores. üur results showed that erythropoietin itself was able to induce preconditioning and preserved the cardioprotective effects of ischemic and carbachol-induced pharmacological preconditioning. Chronic erythropoietin administration generated hypertension and this effect was markedly augmented in the presence of Nü synthase inhibitor L-NAME. However, L-NAME did not inhibit the cardioprotective effects of erythropoietin in anesthetized rats. Although plasma Nü levels was markedly reduced with L-NAME, chronic erythropoietin led to an increased in Nü levels. Erythropoietin could also act protectively by upregulating enzymes that scavenge oxygen radicals. Therefore, erythrocyte superoxide dismutase enzyme levels were measured in the present study, but no changes were observed. There were marked inhibitions in malondialdehyde and lactate levels in all groups when compared to control. These results suggest that erythropoietin is able to protect heart against ventricular arrhythmias and can produce cardiac preconditioning.

Preconditioning by sub‐lethal ischemia/reperfusion or glibenclamide ameliorates lactate dehydrogenase activity to different extends in rat hippocampus and cerebral cortex (brain) subjected to ischemia/reperfusion injury

Sub‐lethal short term ischemic exert adaptive responses which may protect from lethal ischemia, a phenomenon referred to as ischemia preconditioning (IPC). In the CNS, IPC causes increased tolerance to lethal ischemia reperfusion (I/R) injury; however, the precise mechanism is not fully understood. Similarly, glibenclamide, a selective blocker of K+(ATP) channels, was reported to protect against I/R injury via a preconditioning mechanism. Hence, the present study aimed to investigate the role of free radicals as well as pro‐ and anti‐inflammatory cytokines in rats exposed to a I/R injury. To this end, 32 male Wister Rats were divided into 4 groups, group I served as sham operated control, group II was subjected to 15 min ischemia by occlusion of both carotid arteries, followed by 60 min reperfusion, group III injected glibenclamide (1 mg/kg, i.p) 10 min before ischemia (15 min) followed by reperfusion (60 min), and in group IV, preconditioned (IPC) rats were subjected to 3 episodes of ischemia (3 min) followed by reperfusion (10 min) afterwards animals underwent ischemia (15 min) followed by reperfusion (60 min). I/R increased LDH activity, as well as lipid peroxides level, and decreased total antioxidant capacity (TAC) and IL‐10 level in the hippocampus; however, just cortical LDH and TCA levels were deranged. IPC and glibenclamide before I/R improved only LDH activity in the cerebral cortex and partly in the hippocampus. Hence, the study shows that both IPC and glibenclamide impart neuroprotection independent of free radical scavenging nor cytokine production in rats subjected to I/R injury.

The activation of p38alpha, and not p38beta, mitogen-activated protein kinase is required for ischemic preconditioning

Numerous studies show that pharmacological inhibition of p38 mitogen-activated protein kinases (p38s) before lethal ischemia prevents conditioning. However, these inhibitors have off-target effects and do not discriminate between the alpha and beta isoforms; the activation of which is thought to have diverse and perhaps opposing actions with p38α aggravating, and p38β reducing, myocardial injury. We adopted a chemical genetic approach using mice in which either the p38α (DRα) or p38β (DRβ) alleles were targeted to substitute the “gatekeeper” threonine residue for methionine, thereby preventing the binding of a pharmacological inhibitor, SB203580. Isolated, perfused wild-type (WT), DRα and DRβ mouse hearts underwent ischemic preconditioning with 4cycles of 4min ischemia/6min reperfusion, with or without SB203580 (10µM), followed by 30min of global ischemia and 120min of reperfusion. In WT and DRβ hearts, SB203580 completely abolished the reduction in myocardial infarction seen with preconditioning and also the phosphorylation of downstream substrates of p38. These effects of SB203580 were not seen in DRα hearts. Furthermore ischemic preconditioning occurred unaltered in p38β null hearts. Contrary to expectation the activation of p38α, and not p38β, is necessary for ischemic preconditioning. Since p38α is also the isoform that leads to lethal myocardial injury, it is unlikely that targeted therapeutic strategies to achieve isoform-selective inhibition will only prevent the harmful consequences of activation.

Activation of NR2A Receptors Induces Ischemic Tolerance through CREB Signaling

Previous exposure to a nonlethal ischemic insult protects the brain against subsequent harmful ischemia. N-methyl-D-aspartate (NMDA) receptors are a highly studied target of neuroprotection after ischemia. Recently, NMDA receptor subtypes were implicated in neuronal survival and death. We focused on the contribution of NR2A and cyclic-AMP response element (CRE)-binding protein (CREB) signaling to ischemic tolerance using primary cortical neurons. Ischemia in vitro was modeled by oxygen-glucose deprivation (OGD). Ischemic tolerance was induced by applying 45-mins OGD 24 h before 180-mins OGD. Sublethal OGD also induced cross-tolerance against lethal glutamate and hydrogen peroxide. After sublethal OGD, expression of phosphorylated CREB and CRE transcriptional activity were significantly increased. When CRE activity was inhibited by CREB-S133A, a mutant CREB, ischemic tolerance was abolished. Inhibiting NR2A using NVP-AAM077 attenuated preconditioning-induced neuroprotection and correlated with decreased CRE activity levels. Activating NR2A using bicuculline and 4-aminopiridine induced resistance to lethal ischemia accompanied by elevated CRE activity levels, and this effect was abolished by NVP-AAM077. Elevated brain-derived neurotrophic factor (BDNF) transcriptional activities were observed after sublethal OGD and administration of bicuculline and 4-aminopiridine. NR2A-containing NMDA receptors and CREB signaling have important functions in the induction of ischemic tolerance. This may provide potential novel therapeutic strategies to treat ischemic stroke.

Preconditioning-induced ischemic tolerance: a window into endogenous gearing for cerebroprotection

Ischemic tolerance defines transient resistance to lethal ischemia gained by a prior sublethal noxious stimulus (i.e., preconditioning). This adaptive response is thought to be an evolutionarily conserved defense mechanism, observed in a wide variety of species. Preconditioning confers ischemic tolerance if not in all, in most organ systems, including the heart, kidney, liver, and small intestine. Since the first landmark experimental demonstration of ischemic tolerance in the gerbil brain in early 1990's, basic scientific knowledge on the mechanisms of cerebral ischemic tolerance increased substantially. Various noxious stimuli can precondition the brain, presumably through a common mechanism, genomic reprogramming. Ischemic tolerance occurs in two temporally distinct windows. Early tolerance can be achieved within minutes, but wanes also rapidly, within hours. Delayed tolerance develops in hours and lasts for days. The main mechanism involved in early tolerance is adaptation of membrane receptors, whereas gene activation with subsequent de novo protein synthesis dominates delayed tolerance. Ischemic preconditioning is associated with robust cerebroprotection in animals. In humans, transient ischemic attacks may be the clinical correlate of preconditioning leading to ischemic tolerance. Mimicking the mechanisms of this unique endogenous protection process is therefore a potential strategy for stroke prevention. Perhaps new remedies for stroke are very close, right in our cells.

Neuroprotective Effects of Ischemic Preconditioning on Global Brain Ischemia through Up-Regulation of Acid-Sensing Ion Channel 2a

Transient forebrain or global ischemia induces cell death in vulnerable CA1 pyramidal neurons. A brief period of ischemia, i.e., ischemic preconditioning, affords CA1 neurons robust protection against a subsequent, more prolonged ischemic challenge. Using the four-vessel occlusion model, we established an ischemic preconditioning model in which rodents were subjected to 3 min of sublethal ischemia 48 h before a 15 min lethal ischemia. We showed that preconditioning attenuated the ischemia-induced neural cell death and DNA fragmentation in the hippocampal CA1 region. RT-PCR and western blot analysis showed that preconditioning prior to an ischemic insult significantly increased ASIC 2a mRNA and protein expression in comparison to the ischemic insult alone (p < 0.01). These findings implicate a new role of ASIC 2a on endogenous neuroprotection from ischemic insult.

Preconditioning and Stem Cell Survival

The harsh ischemic and cytokine-rich microenvironment in the infarcted myocardium, infiltrated by the inflammatory and immune cells, offers a significant challenge to the transplanted donor stem cells. Massive cell death occurs during transplantation as well as following engraftment which significantly lowers the effectiveness of the heart cell therapy. Various approaches have been adopted to overcome this problem nevertheless with multiple limitations with each of these current approaches. Cellular preconditioning and reprogramming by physical, chemical, genetic, and pharmacological manipulation of the cells has shown promise and "prime" the cells to the "state of readiness" to withstand the rigors of lethal ischemia in vitro as well as posttransplantation. This review summarizes the past and present novel approaches of ischemic preconditioning, pharmacological and genetic manipulation using preconditioning mimetics, recombinant growth factor protein treatment, and reprogramming of stem cells to overexpress survival signaling molecules, microRNAs, and trophic factors for intracrine, autocrine, and paracrine effects on cytoprotection.

Ischemic tolerance in the cochlea

Ischemic tolerance in the cochlea was investigated in a gerbil model of cochlear ischemia. Transient cochlear ischemia was produced by extracranial occlusion of the bilateral vertebral arteries. The gerbils were divided into two groups; single ischemia group and double ischemia group. In the single ischemia group, animals were subjected to lethal cochlear ischemia for 15min. In the double ischemia group, animals were subjected to sublethal cochlear ischemia for 2min at 2 days before lethal ischemia for 15min. Consequently, sublethal ischemia prevented lethal ischemia-induced hair cell degeneration and ameliorated hearing impairment, suggesting ischemic tolerance in the cochlea.

Small Interfering RNA Targeting RelB Protects Against Renal Ischemia-reperfusion Injury

Nuclear factor kappaB (NF-kappaB) has been found to be critical to the pathogenesis of renal ischemia-reperfusion injury (IRI). Using small interfering RNA (siRNA) to silence the expression of RelB, a component of the transcription factors Rel/nuclear factor kappaB, may protect renal IRI. Here, we report an siRNA-based treatment of preventing IRI. Renal IRI was induced in mice by clamping the left renal pedicle for 25 or 35 min. The therapeutic effects of siRNA were evaluated in renal function, histologic examination, and overall survival after lethal IRI. A single injection of RelB siRNA resulted in knockdown of renal RelB expression. In comparison with control mice, levels of blood urea nitrogen and serum creatinine were significantly decreased in mice treated with siRNA. Pathologic examination demonstrated that tissue injury caused by IRI was markedly reduced as a result of RelB siRNA treatment. Additionally, with RelB siRNA treatment, immunohistochemistry showed a significant attenuation of tumor necrosis factor-alpha expression. Furthermore, survival experiments revealed that more than 90% of control mice died from lethal IRI, whereas 80% of siRNA-pretreated mice survived until the end of the 8-day observation period. Silencing RelB, using siRNA, can significantly attenuate IRI-induced renal dysfunction and protect mice against lethal kidney ischemia, highlighting the potential for siRNA-based clinical therapy.

Alterations induced by ischemic preconditioning on secretory pathways Ca2+-ATPase (SPCA) gene expression and oxidative damage after global cerebral ischemia/reperfusion in rats.

Ischemic preconditioning (IPC) represents the phenomenon of CNC adaptation, which results in increased tolerance of CNS to lethal ischemia. Brain ischemia/reperfusion (IRI) initiates a catastrophic cascade in which many subcellular organelles play an important role. The Golgi apparatus, which is a part of secretory pathways (SP), represents the Ca(2+) store and regulates secretion of proteins for growth/reorganization of neuronal circuit by secretory Ca(2+)ATPases (SPCA1). The purpose of this study is to evaluate the effect of IRI and preconditioning on SPCA1 gene expression and oxidative damage after 4-vessel occlusion for 15 min and after being exposed to different reperfusion periods. Rats were preconditioned by 5 min of sub-lethal ischemia and 2 days later, 15 min of lethal ischemia was induced. Our experiments conclusively showed IRI-induced depression of SPCA activity and lipo- and protein oxidation in rat hippocampal membranes. IRI also activates the induction of SPCA1 gene expression in later reperfusion periods. IPC partially suppresses lipo- and protein oxidation in hippocampal membranes and leads to partiall rovery of the ischemic-induced depression of SPCA activity. In addition, IPC initiates earlier cellular response to the injury by the significant elevation of mRNA expression to 142% comparing to 1 h of corresponding reperfusion and to 11% comparing to 24 h of corresponding reperfusion, respectively. Similar patterns were observed on the translational level by Western blot analysis. Our results indicate the specific SPCA1 expression pattern in ischemic hippocampus. It also shows that the SPCA expression and the post-translational changes induced by ischemia are modulated by the IPC. This might serve to understand the molecular mechanisms involved in the structural integrity and function of the SP after ischemic challenge. It also suggests that there is a correlation of SPCA function with the role of SP in the response to pre-ischemic challenge.

Ischemia-induced mitochondrial apoptosis is significantly attenuated by ischemic preconditioning.

Ischemic preconditioning (IPC) represents an important adaptation of CNS to sub-lethal ischemia, which results in increased tolerance of CNS to the lethal ischemia. Ischemia-induced mitochondrial apoptosis is considered to be an important event leading to neuronal cell death after cerebral blood flow arrest. In presented study, we have determined the effect of IPC on ischemia/reperfusion-induced mitochondrial apoptosis. Global brain ischemia was induced by permanent occlusion of vertebral arteries and temporal occlusion of carotid arteries for 15 min. Rats were preconditioned by 5 min of sub-lethal ischemia and 2 days later 15 min of lethal ischemia was induced. With respect to mitochondrial apoptosis initiation, translocation of p53 to mitochondria was observed in hippocampus but not in cerebral cortex. However, level of both apoptotic bax and anti-apoptotic bcl-xl in both hippocampal and cortical mitochondria was unchanged after global brain ischemia. Detection of genomic DNA fragmentation as well as Fluoro-Jade C staining showed that ischemia induces apoptosis in vulnerable CA1 layer of rat hippocampus. IPC abolished completely ischemia-induced translocation of p53 to mitochondria and had significant protective effect on ischemia-induced DNA fragmentation. In addition, significant decrease of Fluoro-Jade C positive cells was observed as well. Our results indicate that IPC abolished almost completely both initiation and execution of mitochondrial apoptosis induced by global brain ischemia.

Influence of an increased intracranial pressure on cerebral and systemic haemodynamics during endoscopic neurosurgery: an animal model

During endoscopic neurosurgery, direct mechanical stimulation of the brain by the endoscope and increased intracranial pressure (ICP) caused by the continuous rinsing can induce potentially lethal haemodynamic reflexes, brain ischaemia, and excessive fluid resorption. In a newly presented rat model of endoscopic neurosurgery, stereotactic access to the cerebrospinal fluid was secured and the ICP was increased by controlled infusion until complete suppression of the cerebral perfusion pressure (CPP). The haematocrit (Hct) level was determined before and after the procedure. During the whole procedure, invasive arterial pressure, ICP, and heart rate were continuously recorded and evaluated in a subsequent offline analysis. After the procedure, the animals were allowed to recover and 7 days later they were killed for histological examination. Suppression of the CPP resulted in a severe hypertension combined with tachycardia or mild bradycardia. The Hct decreased from 41 to 35 over the minutes of CPP suppression. After cessation of the infusion, the ICP decreased to 37% of the plateau pressure within 2.5 s. In the first few minutes after restoration of normal ICP, five animals died because of pulmonary oedema. Upon complete suppression of the CPP, an obvious hypertension developed, often together with tachycardia, but no severe bradycardia. At high ICP levels, we observed an important translocation of irrigation fluid to the vascular space. Fatality was not caused by ischaemia or arrhythmia but due to pulmonary oedema.

J013 Inhibition of mTOR survival pathway by an embryonic developmental Wnt pathway suppresses preconditioning induced cardioprotection

Ischemic preconditioning (IPC) protects against heart prolonged lethal ischemia by activating a cardioprotective signalling cascade involving Akt and GSK3β; however molecular pathways are still incompletely known. In an in vivo model of IPC, we previously demonstrated that the Wnt pathway can inhibit cardioprotection via a direct target, GSK3β. Here we go further in the preconditioning pathway comprehension. On a retrograde isolated heart perfusion model, two models of preconditioning were set up: IPC via four sequences of 5 min 0 flow ischemia followed by 5 min reperfusion ; a Pharmacological Preconditioning (PCP), by 25 min perfusion of diazoxide, a direct activator of mitoKATP. Preconditioning signalling was studied by Western blot analysis, in hearts harvested just after preconditioning, and cardioprotection was analyzed after 40 min 0 flow ischemia followed by 120 min reperfusion, by TTC staining. We verified that PCP, like IPC, induce an inhibition/phosphorylation of GSK3β (ser9) by activation of Akt (ser479). These results were correlated with a significant reduction of infarct size after ischemiareperfusion (10,8 % and 18 % (n=6, p<0.05) of risk area, after PCP and IPC respectively, vs 37,5 % (n=7, p<0.05) in non preconditioned control hearts). We showed then that both IPC and PCP were able to induce the mTOR survival pathway by increasing P-mTOR (ser2448) and its subcellular targets P70S6K (thr389) and 4E-BP1 (ser65) via Akt activation. Wortmanin, an AKT inhibitor, blocked preconditioning induced mTOR activation and infarct size reduction (39,9 % of risk area, n=6, p<0.05 vs control, NS). These effects of preconditioning were also inhibited either by 5-hydroxydecanoate, an antagonist of mitoKATP (44,4 % of risk area, n=6, p<0.05 vs control, NS) or by rapamycin, a specific inhibitor of mTOR (40 % of risk area, n=7, p<0.05 vs control, NS). Hearts of transgenic mouse overexpressing sFRp1 (α-MHC/sFRP1), a Wnt/Frz antagonist, cardioprotections induced by IPC and PCP were inhibited: sFRP1 impaired GSK3β inhibition and mTOR activation (P70S6K and 4E-BP1 phosphorylation), independently of Akt. We evidenced for the first time that cardioprotection involves a crosstalk between an embryonic developmental Wnt pathway and a survival pathway, mTOR/P70S6K.

Action of ERK5 in ischemic tolerance suggests its probable participation in the signaling mechanism

Here we examined the effects of ischemia preconditioning and ketamine, an NMDA receptor antagonist, on the activation and its nucleus translocation of ERK5 in hippocampal CA1 region. Our results showed ERK5 was not activated in rat hippocampus CA1 region. But in cytosol extracts preconditioned with 3 min of sublethal ischaemia, ERK5 activation was enhanced significantly, with two peaks occurring at 3 hr and 3 days, respectively. This activation returned to base level 3 days later. The results lead us to conclude that preconditioning increased the activations of ERK5 during reperfusion after lethal ischemia through NMDA receptor. Preconditioning increased the activation and nucleus translocation of ERK5 during reperfusion after lethal ischemia through the NMDA receptor. These findings might provide some clues to understanding the mechanism underlying ischemia tolerance and to finding clinical therapies for stroke using the endogenous neuroprotection.

Elevated Synaptic Activity Preconditions Neurons against an in Vitro Model of Ischemia

Tolerance to otherwise lethal cerebral ischemia in vivo or to oxygen-glucose deprivation (OGD) in vitro can be induced by prior transient exposure to N-methyl-D-aspartic acid (NMDA): preconditioning in this manner activates extrasynaptic and synaptic NMDA receptors and can require bringing neurons to the "brink of death." We considered if this stressful requirement could be minimized by the stimulation of primarily synaptic NMDA receptors. Subjecting cultured cortical neurons to prolonged elevations in electrical activity induced tolerance to OGD. Specifically, exposing cultures to a K(+)-channel blocker, 4-aminopyridine (20-2500 microm), and a GABA(A) receptor antagonist, bicuculline (50 microm) (4-AP/bic), for 1-2 days resulted in potent tolerance to normally lethal OGD applied up to 3 days later. Preconditioning induced phosphorylation of ERK1/2 and CREB which, along with Ca(2+) spiking and OGD tolerance, was eliminated by tetrodotoxin. Antagonists of NMDA receptors or L-type voltage-gated Ca(2+) channels (L-VGCCs) applied during preconditioning decreased Ca(2+) spiking, phosphorylation of ERK1/2 and CREB, and OGD tolerance more effectively when combined, particularly at the lowest 4-AP concentration. Inhibiting ERK1/2 or Ca(2+)/calmodulin-dependent protein kinases (CaMKs) also reduced Ca(2+) spiking and OGD tolerance. Preconditioning resulted in altered neuronal excitability for up to 3 days following 4-AP/bic washout, based on field potential recordings obtained from neurons cultured on 64-channel multielectrode arrays. Taken together, the data are consistent with action potential-driven co-activation of primarily synaptic NMDA receptors and L-VGCCs, resulting in parallel phosphorylation of ERK1/2 and CREB and involvement of CaMKs, culminating in a potent, prolonged but reversible, OGD-tolerant phenotype.

GABA Synapses Mediate Neuroprotection after Ischemic and εPKC Preconditioning in Rat Hippocampal Slice Cultures

Delayed neuroprotection against ischemic challenges is conferred by both ischemic preconditioning (IPC) and preconditioning by activation of the epsilon-isoform of protein kinase C (epsilonPKC-PC). In vivo, ischemic preconditioning enhances GABA release and ameliorates glutamate release during lethal cerebral ischemia. We tested the hypothesis that IPC and epsilonPKC-PC confer neuroprotection by GABA synapses in rat organotypic hippocampal slices. Ischemic preconditioning or epsilonPKC-PC was induced with 15 mins oxygen-glucose deprivation (OGD) or psiepsilonRACK, a selective epsilonPKC activator; and test ischemia consisted of 40 mins OGD. At the time of peak neuroprotection (48 h after preconditioning), we recorded GABA(A) receptor-mediated miniature postsynaptic currents (GABA mPSCs) in vulnerable CA1 pyramidal neurons using whole-cell voltage clamp techniques. The frequency and amplitude of GABA mPSCs significantly increased 48 h after IPC. In contrast, epsilonPKC-PC enhanced only the amplitude of GABA mPSCs with no effect on frequency. We next asked if neuroprotection depended on these changes in GABA synapses. Weak antagonism of the GABA(A) receptor with bicuculline (100 nmol/L) decreased the amplitude of GABA mPSCs by 20.9+/-6.1%. When applied during test ischemia, 100 nmol/L bicuculline abolished neuroprotection conferred by either IPC or epsilonPKC-PC. We conclude that neuroprotection conferred by preconditioning depends on functional modifications of GABA synapses.

Abstract 1610: Repetitive Ischemia Elicits a Third Window of Ischemic Preconditioning

Ischemic preconditioning (IPC), following 1 or 2 episodes of coronary occlusion (CO) is the most powerful mechanism of cardioprotection, but it remains unclear whether IPC occurs clinically. Since patients with coronary disease experience episodes of repetitive ischemia (RI), we hypothesized that a pattern of RI would provide more clinically relevant cardioprotection. Conscious, chronically instrumented pigs (6/group) were submitted to RI induced by 6 episodes, 12 hrs apart, of coronary stenosis for 90 min (RCS) and compared to pigs submitted to second window IPC (SWIPC) induced by 2 episodes of 10 min CO / 24 hr reperfusion (CR) and to shams. Lethal ischemia, induced by 60 min CO, resulted in an infarct size/area at risk (IS/AAR) of 42±4% in shams, and significantly less, p&lt;0.05, after RCS (6±3%) and after SWIPC (16±4%). SWIPC showed 2–3-fold increases, p&lt;0.05, in translocation of PKCepsilon and the inducible isoform of nitric oxide synthase (NOS), which were not found in RCS. Pretreatment with the NOS inhibitor L-NNA (35 mg/kg) prevented the protection by SWIPC but not by RCS. Microarrays were performed to further elucidate the differences in molecular mechanisms between the SWIPC and RCS models. The number of genes significantly regulated was greater in RCS (5739) than in SWIPC (2394). Of the 5739 genes regulated in RCS, only 31% were also regulated in SWIPC. Since another difference between the two models is that one involves CS and the other complete CO, we examined another group of RI pigs (n=5) with 6 episodes, 12 hrs apart, of 2–10 min periods of CO, which showed reduced IS/AAR with 60 min CO after RI (14±4%); similar to RCS the cardioprotection was still observed after L-NNA. Both RI models, in further contrast to SWIPC, demonstrated downregulation of genes involved in mitochondrial function, and upregulation of genes involving autophagy (such as cathepsins B and D), endoplasmic reticulum stress (Grp78) and cell cycle (E2F1). These results were confirmed at the protein level by western blotting. Therefore, RI, over a time course of several days, provides a NOS-independent “third window” of protection, which is as effective as traditional SWIPC, but is mechanistically distinct. This research has received full or partial funding support from the American Heart Association, AHA National Center.