Induction Of Ischemic Preconditioning Research Articles

Ischemic Preconditioning Protects Astrocytes against Oxygen Glucose Deprivation Via the Nuclear Erythroid 2-Related Factor 2 Pathway.

Induction of ischemic preconditioning (IPC) represents a potential therapy against cerebral ischemia by activation of adaptive pathways and modulation of mitochondria to induce ischemic tolerance to various cells and tissues. Mitochondrial dysfunction has been ascribed to contribute to numerous neurodegenerative conditions and cerebral ischemia. Nuclear erythroid 2-related factor 2 (Nrf2) is a transcription factor that has traditionally been involved in upregulating cellular antioxidant systems to combat oxidative stress in the brain; however, the association of Nrf2 with mitochondria in the brain remains unclear. In the present study, we investigated the effects of Nrf2 on (i) IPC-induced protection of astrocytes; (ii) OXPHOS protein expression; and (iii) mitochondrial supercomplex formation.Oxygen-glucose deprivation (OGD) was used as an in vitro model of cerebral ischemia and IPC in cultured rodent astrocytes derived from WT C57Bl/6J and Nrf2-/- mice. OXPHOS proteins were probed via western blotting, and supercomplexes were determined by blue native gel electrophoresis.IPC-induced cytoprotection in wild-type, but not Nrf2-/- mouse astrocyte cultures following a lethal duration of OGD. In addition, our results suggest that Nrf2 localizes to the outer membrane in non-synaptic brain mitochondria, and that a lack of Nrf2 in vivo produces altered supercomplex formation in mitochondria.Our findings support a role of Nrf2 in mediating IPC-induced protection in astrocytes, which can profoundly impact the ischemic tolerance of neurons. In addition, we provide novel evidence for the association of Nrf2 to brain mitochondria and supercomplex formation. These studies offer new targets and pathways of Nrf2, which may be heavily implicated following cerebral ischemia.

Protection of retinal ganglion cells against optic nerve injury by induction of ischemic preconditioning.

To explore if ischemic preconditioning (IPC) can enhance the survival of retinal ganglion cells (RGCs) after optic nerve axotomy. Twenty-four hours prior to retinal ischemia 60min or axotomy, IPC was applied for ten minutes in groups of (n=72) animals. The survival of RGCs, the cellular expression of heat shock protein 27 (HSP27) and heat shock protein 70 (HSP70) and the numbers of retinal microglia in the different groups were quantified at 7 and 14d post-injury. The cellular expression of HSP27 and HSP70 and changes in the numbers of retinal microglia were quantified to detect the possible mechanism of the protection of the IPC. Ten minutes of IPC promoted RGC survival in both the optic nerve injury (IPC-ONT) and the retinal ischemia 60min (IPC-IR60) groups, examined at 7d and 14d post-injury. Microglial proliferation showed little correlation with the extent of benefit effects of IPC on the rescue of RGCs. The number of HSP27-positive RGCs was significantly higher in the IPC-ONT group than in the sham IPC-ONT group, although the percentage of HSP27-positive RGCs did not significantly differ between groups. For the IPC-IR60 group, neither the number nor the percentage of the HSP27-positive RGCs differed significantly between the IPC and the sham-operated groups. The number of HSP70-positive RGCs was significantly higher for both the IPC-ONT and the IPC-IR60 experimental groups, but the percentages did not differ. The induction of IPC enhances the survival of RGCs against both axotomy and retinal ischemia.

Natural Health Products as Modulators of Adenosine and ATP Metabolism for Cardiovascular Protection

Adenosine is an important endogenous purine nucleoside and an essential component of the molecular energy generated from adenosine 5’-triphosphate (ATP). It acts as both a precursor and metabolite of adenine nucleotides. As every cell utilizes the energy generated from catabolism of ATP, adenosine is found ubiquitously in the body. It is also a signaling molecule in the cardiovascular system, and its role for cardioprotection and cardiovascular homeostasis has been studied for over 80 years [1-3]. Adenosine is also known as a “homeostatic metabolite in cardiac energy metabolism” [4] owing to its wide range of beneficial effects on the cardiovascular system [5] which include negative chronotropic and dromotropic effect in cardiac tissue, vasodilatation, inhibition of platelet aggregation, modulation of vascular smooth muscles and endothelium and induction of ischemic preconditioning [6]. Adenosine modulates these actions by interacting with adenosine receptors (AR), which are widely distributed throughout the body. It is believed that metabolic condition of the myocardium may be assessed quantitatively by the level of adenosine production which maintains a healthy balance between energy supply and demand in the cardiovascular system [7].

Cardiac Alpha1-Adrenergic Receptors: Novel Aspects of Expression, Signaling Mechanisms, Physiologic Function, and Clinical Importance

Adrenergic receptors (AR) are G-protein-coupled receptors (GPCRs) that have a crucial role in cardiac physiology in health and disease. Alpha1-ARs signal through Gαq, and signaling through Gq, for example, by endothelin and angiotensin receptors, is thought to be detrimental to the heart. In contrast, cardiac alpha1-ARs mediate important protective and adaptive functions in the heart, although alpha1-ARs are only a minor fraction of total cardiac ARs. Cardiac alpha1-ARs activate pleiotropic downstream signaling to prevent pathologic remodeling in heart failure. Mechanisms defined in animal and cell models include activation of adaptive hypertrophy, prevention of cardiac myocyte death, augmentation of contractility, and induction of ischemic preconditioning. Surprisingly, at the molecular level, alpha1-ARs localize to and signal at the nucleus in cardiac myocytes, and, unlike most GPCRs, activate "inside-out" signaling to cause cardioprotection. Contrary to past opinion, human cardiac alpha1-AR expression is similar to that in the mouse, where alpha1-AR effects are seen most convincingly in knockout models. Human clinical studies show that alpha1-blockade worsens heart failure in hypertension and does not improve outcomes in heart failure, implying a cardioprotective role for human alpha1-ARs. In summary, these findings identify novel functional and mechanistic aspects of cardiac alpha1-AR function and suggest that activation of cardiac alpha1-AR might be a viable therapeutic strategy in heart failure.

Adiponectin and resistin protect steatotic livers undergoing transplantation

Numerous steatotic livers are discarded for transplantation because of their poor tolerance to ischemia-reperfusion. Controversial roles for adiponectin and related adipocytokines visfatin and resistin have been described in different liver pathologies, nevertheless it is unknown their possible implication in ischemia-reperfusion injury associated with liver transplantation. Our study aimed at characterizing the role of the adiponectin-derived molecular pathway in transplantation with steatotic and non-steatotic liver grafts. Steatotic and non-steatotic liver transplantation was carried out and the hepatic levels of adiponectin, visfatin and resistin were measured and modulated either pharmacologically or surgically. Steatotic liver grafts exhibited downregulation of both adiponectin and resistin when subjected to transplantation. Adiponectin pre-treatment only protected steatotic grafts and did it so through a visfatin-independent and resistin-dependent mechanism. Adiponectin-derived resistin accumulation activated the PI3K/Akt pathway, unravelling AMPK as an upstream mediator of adiponectin's actions in steatotic grafts. Strategies aimed at increasing adiponectin including either AMPK activators or the induction of ischemic preconditioning (which activates AMPK) increased resistin accumulation, prevented the downregulation of PI3K/Akt pathway and protected steatotic liver grafts. Conversely, PI3K/Akt pathway upregulation and hepatic protection induced by adiponectin were abolished when resistin action was inhibited. Our findings reveal a new protective pathway in steatotic liver transplantation, namely AMPK-adiponectin-resistin-PI3K/Akt, which may help develop new strategies aimed at increasing either adiponectin or resistin in the steatotic liver undergoing transplant to ultimately increase organ donor pool and reduce waiting list.

Consequences of Ischemic Preconditioning of Kidney: Comparing between Male and Female Rats.

Ischemia-reperfusion injury (IRI) is a leading cause of kidney transplantation failure, and ischemic-preconditioning (IPC) is a protective method against the IRI. In the present study, the defensive effect of IPC on rats' kidney was investigated and more importantly the differences between two genders were appraised. THIRTY TWO WISTAR RATS WERE RANDOMLY ALLOCATED TO FOUR GROUPS: group A (8 male IR), B (8 female IR), C (8 male IPC) and D (8 female IPC). Ischemia was induced by clamping of left renal arteries for 45 min in groups A and B. Rats in groups C and D experienced four cycles of 4 min arterial clamping followed by 11 min of de-clamping prior to the final 45 min of ischemia. 24 hr later, serum was provided to assess the blood urea nitrogen (BUN) and creatinine values. Also, renal tissues were obtained for histological measurements. Induction of IPC in both male and female rats led to significant decrease in creatinine levels in comparison with sham groups (P<0.01). The same results were seen in BUN levels (P<0.01). However, there were no significant difference between two genders. Besides, histological protective effects of IPC was proved especially in female rats (P<0.01). Findings of our study confirmed that renal IPC reduces the damages in both genders especially females. Thus, the IPC procedure seems to be a useful method mainly in females.

Platelet activating factor: the good and the bad in the ischemic/reperfused heart

The present review is focused on the dual role played by platelet-activating factor (PAF) in ischemia and reperfusion (I/R) injury of the heart. Although the involvement of PAF in the pathogenesis of myocardial reperfusion injury is well established, in the last few years it has emerged that very low concentrations of PAF exert cardioprotective effects, comparable to that afforded by ischemic preconditioning (IP). PAF is a potent phosphoglyceride involved in different pathophysiological conditions affecting the cardiovascular system, including the development of myocardial I/R injury. PAF is released from the I/R myocardium in concentrations (1-10 nmol/L) high enough to negatively modulate coronary circulation as well as electrical and contractile activities. PAF may act either directly, via generation of secondary mediators, or through the activation of inflammatory cells like platelets and polymorphonuclear neutrophils, which exacerbate postischemic myocardial injury. The effects of PAF are mediated through specific receptors (PAFRs) that belong to the superfamily of G protein-coupled receptors. Since cardiomyocytes not only produce PAF but also possess PAFRs, it is likely that PAF acts as an autocrine/paracrine mediator. Although the negative effects exerted by high concentrations of PAF are well established, several recent findings from our and other laboratories have demonstrated that very low concentrations (pmol/L) of PAF infused before ischemia induce cardioprotective effects similar to those afforded by IP, and that endogenous PAF production participates in the induction of IP itself. The IP-like action exerted by low concentrations of PAF is due to the activation/phosphorylation of kinases included in the reperfusion injury salvage kinase (RISK) pathway, such as protein kinase C, Akt/PkB and nitric oxide synthase. Together with the activation of mitochondrial K(ATP) channels, these events may allow prevention of mitochondrial permeability transition pores opening at reperfusion. Moreover, the nitric oxide-dependent S-nitrosylation of L-type Ca(2+) channels induced by PAF reduces intracellular Ca(2+) overload.

Increased Mitochondrial Sensitivity To Proton Leak Is Responsible For Decreased Reactive Oxygen Species Production In Ischemic Preconditioning

Introduction: Mitochondrial reactive oxygen species (mROS) cause significant damage to myocardium after an episode of ischemia-reperfusion (IR). Ischemic preconditioning (IPC) decreases mROS formation and protects myocardium from IR. The mechanism of decreased mROS in IPC is unknown. Proton leak (H+ leak) dissipates mitochondria membrane potential (mΔΨ) and may regulate mROS production by changing the redox status of mitochondria. We hypothesize that increased H+ leak is responsible for decreased mROS production during IPC. Methods: Rat hearts were assigned to CONTROL (30 min of equilibration (EQ), 30 min ischemia (I), and 30 min of reperfusion (RP)) or IPC (10 min EQ, two 5 min episodes of IR, 30 min I, and 30 min RP). Rate pressure product (RPP) was measured throughout. Mitochondria were isolated at end equilibration (end EQ), end ischemia (end I), and end reperfusion (end RP). Respiration was measured by polarography, and mΔΨ with a TPP electrode. H+ leak is the respiratory rate required to maintain mΔΨ of -130mV in the presence of oligomycin-A, and was titrated with FCCP (0-160nM). Complex III mROS production was measured by fluorometry using Amplex-Red at end EQ and by electron paramagnetic resonance at end I and end RP. Results: Induction of IPC increased mROS production at end EQ (59±2 vs. 43±2 nmoles H2O2/mg protein/min, p<0.05, Fig.1) but did not change H+ leak (37±9 vs. 30±10 nmoles O/mg protein/min) when compared to CONTROL. IPC attenuated the increase in H+ leak at end I seen in CONTROL (44±11 vs. 140±11 nmoles O/mg protein/min, p<0.001). At end RP, IPC decreased mROS production (2115±146 vs. 3277±333 arbitrary units/mg protein, p<0.05). There was no difference in H+ leak between IPC and CONTROL at end RP (34±9 vs. 45±9 nmoles O/mg protein/min). IPC mitochondria responded to depolarization with greater reduction in mROS production than CONTROL (Fig.1). Further depolarization led to a trend of increased mROS in both CONTROL and IPC. The inflection point occurred at a higher mΔΨ in IPC mitochondria (-160 vs. -138 mV; Fig. 1). Conclusions: Increased rate of H+ leak is not the mechanism of decreased mROS after IR. While IPC did not lead to increased H+ leak, the induction of IPC increases the sensitivity of mitochondria to changes in mΔΨ leading to greater reduction in mROS per unit of change in mΔΨ. This increased sensitivity explains the greater reduction in mROS at end RP despite a lower H+ leak. Reducing mROS formation after ischemia-reperfusion is important to improving outcomes in patients with acute coronary syndrome.

Remote ischemic preconditioning improves the survival of rat random-pattern skin flaps

Random-pattern flap transfer is one of the most popular procedures for covering soft tissue defects. Ischemic preconditioning is a protective endogenous mechanism capable of reducing ischemia-reperfusion injury, and it has been shown that preconditioning by proximal pedicle clamping can improve flap survival. However, the method is not suitable for random-pattern flap transfer in the clinical setting. The present study evaluates the effect of ischemic preconditioning of Wistar rat hind limbs upon dorsal random-pattern skin flap survival. Ischemic preconditioning was induced by ischemia of the right hind limb during 10 min, followed by 30 min of reperfusion. Thirty-two animals were divided in two groups. In group 1, a dorsal random-pattern skin flap measuring 2 × 7 cm was raised immediately after the induction of ischemic preconditioning. The animals in group 2 (controls) received the same treatment, but without ischemic preconditioning. The survival area was defined as the surface of the viable tissue (square centimeter) on the fifth postoperative day. The average survival area was 6.57 ± 0.18 cm2 in group 1 and 4.44 ± 0.21 cm2 in group 2. All preconditioned animals presented significantly higher flap survival areas than the controls (p ≤ 0.01, Student’s t test). Our findings show that flap necrosis was reduced by the induction of ischemic preconditioning in a body area distant from the flap harvest site, and that ischemic preconditioning has a systemic protective effect on dorsal random-pattern skin flaps and increase survival. A better understanding of the mechanism involved may improve the clinical condition of patients requiring random-pattern skin flap transfer, especially in high-risk groups.

Glycogen‐Synthase Kinase‐3beta/beta‐Catenin Axis Promotes Myocardial Angiogenesis: Role of VEGF

The GSK‐3β/β‐catenin axis is one of most interesting pathways involved in the molecular mechanism of angiogenesis. Recently we have shown the critical role of GSK‐3β/β‐catenin in ischemic preconditioning (IP) mediated angiogenesis. We have observed for the first time that IP induces the β‐catenin driven expression of vascular endothelial growth factor (VEGF), Bcl‐2, survivin in chronic rat myocardial infarction (MI) model increasing the vessel density improving myocardial function. β‐catenin is found to be a critical mediator during angiogenesis &amp; it is phosphorylated by GSK‐3β. During IP induction of the PI‐3 kinase pathway leads to phosphorylation of GSK‐3β, leading to the release, accumulation of β‐catenin in cytosol &amp; the subsequent translocation of β‐catenin into the nucleus drives the expression of its target genes (VEGF, Bcl‐2, survivin) through activation of the T‐cell transcription factors/lymphoid‐ enhancer binding factor transcription factors. These astounding results paved the way for the induction of angiogenesis in the ischemic heart by administration of β‐catenin after an MI which depicted cardioprotection as was observed with IP. The efficacy of β‐catenin overexpression in the induction of collateral vessel formation remodeling was further ascertained by Adeno –shRNA‐β‐catenin to silence the gene expression of β‐catenin thereby abolishing the IP mediated cardioprotection.

Ischemic preconditioning mediates cyclooxygenase-2 expression via nuclear factor-kappa B activation in mixed cortical neuronal cultures.

Nuclear factor-kappaB (NF-κB) activation occurs following ischemic preconditioning (IPC) in brain. However, the upstream signaling messengers and down-stream targets of NF-κB required for induction of IPC remain undefined. In a previous study, we demonstrated that epsilon protein kinase c (εPKC) was a key mediator of IPC in brain. Activation of εPKC induced cyclooygenase-2 (COX-2) expression and conferred ischemic tolerance in the neuronal and hippocampal slice models. Here, we hypothesized that IPC-mediated COX-2 expression was mediated by NF-κB. We tested this hypothesis in mixed cortical neuron/astrocyte cell cultures. To simulate IPC or ischemia, cell cultures were exposed to 1 or 4 h of oxygen-glucose deprivation, respectively. Our results demonstrated translocation of p65 and p50 subunits of NF-κB into nucleus following IPC or εPKC activation. NF-κB inhibition with pyrrolidine dithiocarbamate (10 μM) abolished IPC or εPKC activator-mediated neuroprotection indicating that NF-κB activation was involved in ischemic tolerance. In parallel studies, inhibition of either εPKC or the extracellular signal-regulated kinase (ERK 1/2) pathway reduced IPC-induced NF-κB activation. Finally, inhibition of NF-κB blocked IPC-induced COX-2 expression. In conclusion, we demonstrated that IPC-signaling cascade comprises εPKC activation→ERK1/2 activation→NF-κB translocation to nucleus→COX-2 expression resulting in neuroprotection in mixed neuronal culture.

Ischemic preconditioning-induced neuroprotection is associated with differential expression of IL-1β and IL-1 receptor antagonist in the ischemic cortex

Ischemic preconditioning (IP) is a phenomenon that organs develop a tolerance toward subsequent lethal ischemic insults. Among the factors that are involved in IP, IL-1β and its endogenous receptor antagonist IL-1ra have been identified as important players in the induction of IP. The present study investigated whether IP affects the levels of these two antagonistic proteins during tolerance and reperfusion periods after ischemic stroke. The IP 24 h prior to ischemic stroke resulted in neuroprotection in the cortex. IP-induced protection is accompanied by increased IL-1β gene and IL-1ra gene and protein levels during the tolerance period. In the post-ischemic cortex, IP resulted in the suppression of IL-1β mRNA and protein levels at 6 h without affecting IL-1ra expression and the up-regulation of IL-1ra protein at 24 h. These findings demonstrate that IP differentially regulates cortical IL-1β and IL-1ra expression before and after ischemic stroke and suggest that the shift toward an anti-inflammatory state in the post-ischemic cortex may contribute to IP-induced neuroprotection.

Ischemic limb preconditioning downregulates systemic inflammatory activation

We examined local and systemic antiinflammatory consequences of ischemic preconditioning (IPC) in a rat model of limb ischemia-reperfusion (I-R) by characterizing the leukocyte-endothelial interactions in the periosteum and the expression of adhesion molecules playing a role in leukocyte-mediated inflammatory processes. IPC induction (2 cycles of 10 min of complete limb ischemia and 10 min of reperfusion) was followed by 60 min of ischemia/180 min of reperfusion or sham-operation. Data were compared with those on animals subjected to I-R and sham-operation. Neutrophil leukocyte-endothelial cell interactions (intravital videomicroscopy), intravascular neutrophil activation (CD11b expression changes by flow cytometry), and soluble and tissue intercellular adhesion molecule-1 (ICAM-1; ELISA and immunohistochemistry, respectively) expressions were assessed. I-R induced enhanced leukocyte rolling and adherence in the periosteal postcapillary venules after 120 and 180 min of reperfusion. This was associated with a significantly enhanced CD11b expression (by approximately 80% and 72%, respectively) and moderately increased soluble and periosteal ICAM-1 expressions. IPC prevented the I-R-induced increases in leukocyte adherence and CD11b expression without influencing the soluble and tissue ICAM-1 levels. The results show that limb IPC exerts not only local, but distant antiinflammatory effects through significant modulation of neutrophil recruitment.

Ischemic preconditioning‐induced cardioprotection is lost in mice with immunoproteasome subunit low molecular mass polypeptide‐2 deficiency

The ubiquitin-proteasome system plays an important role in many cellular processes through degradation of specific proteins. Low molecular mass polypeptide 2 (LMP-2 or beta(1i)) is one important subunit of the immunoproteasome. Ischemic preconditioning (IPC) activates cell signaling pathways and generates cardioprotection but has not been linked to LMP-2 function previously. LMP-2 knockout mice (C57BL6 background) and wild-type C57BL6 mice were subjected to 30 min of ischemia (I-30) and 120 min of reperfusion (R-120) with or without preceding IPC (10 min of infusion and 5 min of reperfusion). IPC significantly increased left ventricular developed pressure and decreased infarct size in wild-type mice, but this protective effect of IPC was lost in LMP-2 knockout mice. IPC-mediated degradation of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and activation of the downstream protein kinase Akt were impaired in LMP-2 knockout hearts. The impairment of PTEN degradation was associated with defective immunoproteasomes and decreased proteolytic activities. When LMP-2 knockout mice were pretreated with the PTEN inhibitor bpV(HOpic), cardiac function was significantly improved, and myocardial infarct size was significantly reduced after I-30/R-120. In conclusion, LMP-2 is required for normal proteasomal function and IPC induction in the heart. Its action may be related to PTEN protein degradation.

ɛPKC phosphorylates the mitochondrial K+ATP channel during induction of ischemic preconditioning in the rat hippocampus

Neuroprotection against cerebral ischemia conferred by ischemic preconditioning (IPC) requires translocation of epsilon protein kinase C (epsilonPKC). A major goal in our laboratory is to define the cellular targets by which epsilonPKC confers protection. We tested the hypothesis that epsilonPKC targets the mitochondrial K(+)(ATP) channel (mtK(+)(ATP)) after IPC. Our results demonstrated a rapid translocation of epsilonPKC to rat hippocampal mitochondria after IPC. Because in other tissues epsilonPKC targets mtK(+)(ATP) channels, but its presence in brain mitochondria is controversial, we determined the presence of the K(+)(ATP) channel-specific subunits (Kir6.1 and Kir6.2) in mitochondria isolated from rat hippocampus. Next, we determined whether mtK(+)(ATP) channels play a role in the IPC induction. In hippocampal organotypic slice cultures, IPC and lethal ischemia were induced by oxygen-glucose deprivation. Subsequent cell death in the CA1 region was quantified using propidium iodide staining. Treatment with the K(+)(ATP) channel openers diazoxide or pinacidil 48 h prior to lethal ischemia protected hippocampal CA1 neurons, mimicking the induction of neuroprotection conferred by either IPC or epsilonPKC agonist-induced preconditioning. Blockade of mtK(+)(ATP) channels using 5-hydroxydecanoic acid abolished the neuroprotection due to either IPC or epsilonPKC preconditioning. Both ischemic and epsilonPKC agonist-mediated preconditioning resulted in phosphorylation of the mtK(+)(ATP) channel subunit Kir6.2. After IPC, selective inhibition of epsilonPKC activation prevented Kir6.2 phosphorylation, consistent with Kir6.2 as a phosphorylation target of epsilonPKC or its downstream effectors. Our results support the hypothesis that the brain mtK(+)(ATP) channel is an important target of IPC and the signal transduction pathways initiated by epsilonPKC.

PTEN Activity Is Modulated During Ischemia and Reperfusion

Ischemic preconditioning (IPC), a brief period of ischemia and reperfusion (I/R), generates profound but transient protection against a subsequent prolonged ischemic episode. The serine-threonine kinase Akt has been shown to mediate IPC, and Akt activation is negatively regulated by the phosphatase PTEN, but whether PTEN activity is modulated by IPC has not been investigated. When isolated, perfused rat hearts were subjected to an IPC stimulus consisting of 15-minute ischemia and 30-minute reperfusion (I-15/R-30), PTEN protein levels and activity were decreased, and levels of phospho-AKT were increased, relative to nonischemic hearts. Hearts subjected to IPC demonstrated improved recovery of cardiac function when subsequently subjected to I-30/R-45 as compared with hearts subjected to I-30/R-45 without prior IPC. When hearts were subjected to I-15 followed by R-30, R-60, or R-120, PTEN reaccumulated gradually and its activity was restored. Phospho-Akt levels at R-120 were decreased and these hearts were no longer protected against injury when subjected to I-30/R-45. Wortmannin administration during reperfusion blocked Akt activation and PTEN reaccumulation. In ischemic hearts, PTEN was rapidly degraded. Pretreatment with proteasome inhibitor MG132 blocked ischemia-induced degradation of PTEN and blocked IPC. Reperfusion following I-15 induced oxidation of the remaining PTEN, leading to Akt activation. Perfusion of H2(O2) was sufficient to induce Akt activation. Thus, loss of PTEN activity leads to induction of IPC and feedback mechanisms designed to ensure that Akt activation is transient are responsible for decay of IPC.

Ischemic tolerance induction in organotypic hippocampal slices: Role for the GABA A receptor?

Ischemic preconditioning (IPC) refers to sublethal ischemic insults rendering brain tissue tolerant against subsequent ischemic insults. We investigated the role of the GABA A receptor (GABA AR) upon IPC induction. Rat organotypic hippocampal slices were subjected to IPC by 15 min of oxygen–glucose deprivation (OGD) followed by 40 min of OGD 48 h later, resulting in robust cell death reduction as assessed by the propidium iodide fluorescence method (‘late’ or ‘second window’ IPC). Superfusion with the GABA AR antagonist bicuculline during IPC ameliorated propidium iodide uptake at a high but not at low doses indicating that GABA AR activation may be assigned a limited role in neuroprotection. In previous studies, we found that increased neuronal excitability can promote IPC neuroprotection. We, therefore, tested the hypothesis that blockade of inhibitory GABAergic transmission conferred ischemic tolerance. However, temporary administration of bicuculline 48 h prior to ischemic challenge was not neuroprotective. In another approach, we tested whether preconditioning with the GABA AR agonist, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) mediated ischemic tolerance and found no significant neuroprotection. The results are discussed in light of the intrinsic excitatory-inhibitory balance of glutamate and GABA.

Epsilon protein kinase C mediated ischemic tolerance requires activation of the extracellular regulated kinase pathway in the organotypic hippocampal slice.

Ischemic preconditioning (IPC) promotes brain tolerance against subsequent ischemic insults. Using the organotypic hippocampal slice culture, we conducted the present study to investigate (1) the role of adenosine A1 receptor (A1AR) activation in IPC induction, (2) whether epsilon protein kinase C (epsilonPKC) activation after IPC is mediated by the phosphoinositol pathway, and (3) whether epsilonPKC protection is mediated by the extracellular signal-regulated kinase (ERK) pathway. Our results demonstrate that activation of A1AR emulated IPC, whereas blockade of the A1AR during IPC diminished neuroprotection. The neuroprotection promoted by the A1AR was also reduced by the epsilonPKC antagonist. To determine whether epsilonPKC activation in IPC and A1AR preconditioning is mediated by activation of the phosphoinositol pathway, we incubated slices undergoing IPC or adenosine treatment with a phosphoinositol phospholipase C inhibitor. In both cases, preconditioning neuroprotection was significantly attenuated. To further characterize the subsequent signal transduction pathway that ensues after epsilonPKC activation, mitogen-activated protein kinase kinase was blocked during IPC and pharmacologic preconditioning (PPC) (with epsilonPKC, NMDA, or A1AR agonists). This treatment significantly attenuated IPC- and PPC-induced neuroprotection. In conclusion, we demonstrate that epsilonPKC activation after IPC/PPC is essential for neuroprotection against oxygen/glucose deprivation in organotypic slice cultures and that the ERK pathway is downstream to epsilonPKC.

εPKC Is Required for the Induction of Tolerance by Ischemic and NMDA-Mediated Preconditioning in the Organotypic Hippocampal Slice

Glutamate receptors and calcium have been implicated as triggering factors in the induction of tolerance by ischemic preconditioning (IPC) in the brain. However, little is known about the signal transduction pathway that ensues after the IPC induction pathway. The main goals of the present study were to determine whether NMDA induces preconditioning via a calcium pathway and promotes translocation of the protein kinase C epsilon (epsilonPKC) isozyme and whether this PKC isozyme is key in the IPC signal transduction pathway. We corroborate here that IPC and a sublethal dose of NMDA were neuroprotective, whereas blockade of NMDA receptors during IPC diminished IPC-induced neuroprotection. Calcium chelation blocked the protection afforded by both NMDA and ischemic preconditioning significantly, suggesting a significant role of calcium. Pharmacological preconditioning with the nonselective PKC isozyme activator phorbol myristate acetate could not emulate IPC, but blockade of PKC activation with chelerythrine during IPC blocked its neuroprotection. These results suggested that there might be a dual involvement of PKC isozymes during IPC. This was corroborated when neuroprotection was blocked when we inhibited epsilonPKC during IPC and NMDA preconditioning, and IPC neuroprotection was emulated with the activator of epsilonPKC. The possible correlation between NMDA, Ca2+, and epsilonPKC was found when we emulated IPC with the diacylglycerol analog oleoylacetyl glycerol, suggesting an indirect pathway by which Ca2+ could activate the calcium-insensitive epsilonPKC isozyme. These results demonstrated that the epsilonPKC isozyme played a key role in both IPC- and NMDA-induced tolerance.

ROS and NO trigger early preconditioning: relationship to mitochondrial KATP channel.

Reactive oxygen species (ROS) and nitric oxide (NO) are implicated in induction of ischemic preconditioning. However, the relationship between these oxidant signals and opening of the mitochondrial ATP-dependent potassium (K(ATP)) channel during early preconditioning is not fully understood. We observed preconditioning protection by hypoxia, exogenous H(2)O(2), or PKC activator PMA in cardiomyocytes subjected to 1-h ischemia and 3-h reperfusion. Protection was abolished by K(ATP) channel blocker 5-hydroxydecanoate (5-HD) in each case, indicating that these triggers must act upstream from the K(ATP) channel. Inhibitors of NO synthase abolished protection in preconditioned cells, suggesting that NO is also required for protection. DAF-2 fluorescence (NO sensitive) increased during hypoxic triggering. This was amplified by pinacidil and inhibited by 5-HD, indicating that NO is generated subsequent to K(ATP) channel activation. Exogenous NO during the triggering phase conferred protection blocked by 5-HD. Exogenous NO also restored protection abolished by 5-HD or N(omega)-nitro-l-arginine methyl ester in preconditioned cells. Antioxidants given during pinacidil or NO triggering abolished protection, confirming that ROS are generated by K(ATP) channel activation. Coadministration of H(2)O(2) and NO restored PMA-induced protection in 5-HD-treated cells, indicating that ROS and NO are required downstream from the K(ATP) channel. We conclude that ROS can trigger preconditioning by causing activation of the K(ATP) channel, which then induces generation of ROS and NO that are both required for preconditioning protection.