Model Of Ischemic Preconditioning Research Articles

Genomic analysis of ischemic preconditioning in adult rat hippocampal slice cultures

Understanding endogenous mechanisms of neuroprotection may have important clinical applications. It is well established that brain tissue becomes more resistant to ischemic injury following a sublethal ischemic insult. This process, called ischemic preconditioning (IPC), can be induced in adult rat hippocampal slice cultures by a brief oxygen–glucose deprivation (OGD) [Hassen, G.W., Tian, D., Ding, D., Bergold, P.J., 2004. A new model of ischemic preconditioning using young adult hippocampal slice cultures. Brain Res. Brain Res. Protoc. 13, 135–143]. We have analyzed the changes in gene expression brought about by IPC in this model in order to understand the mechanisms involved. Total RNA was isolated at different time points following a brief OGD (3, 6 and 12 h) and used to probe genome-wide expression microarrays. Genes were identified that were significantly up- or down-regulated relative to controls. We placed genes that were differentially expressed into statistically significant groups based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and gene ontology (GO) terms. Genes involved in signal transduction, transcription, and oxidative phosphorylation are differentially expressed at each time point. The analysis demonstrates that alterations in signaling pathways (TGF-β, Wnt, MAPK, ErbB, Toll-like receptor, JAK-STAT, VEGF) consistently accompany IPC. RT-PCR was used to confirm that members of these signaling pathways are regulated as predicted by the microarray analysis. We verified that protein translation following OGD is necessary for IPC. We also found that blocking the NMDA receptor during OGD does not significantly inhibit IPC in this model or produce large changes in gene expression. Our data thus suggests that changes in signaling pathways and their down-stream targets play an important role in triggering endogenous neuroprotection.

The Hsp70.3 isoform of Hsp70, but not Hsp70.1, contributes to NF‐kappaB‐dependent cardioprotection of late ischemic preconditioning

It has been shown by our lab and others that the transcription factor NF‐κB is necessary for the development of the late window of protection after ischemic preconditioning (IPC) in the heart. However, the specific mechanisms and downstream gene expression profiles that underlie the cardioprotection of NF‐κB in late IPC remain unknown. To investigate NF‐κB‐dependent gene expression in IPC, we conducted a directed microarray analysis to compare the gene expression patterns of wild‐type and NF‐κB dominant negative mice 3.5 hrs after an IPC stimulus. Among the potential cardioprotective candidates identified by gene array analysis as being regulated in an NF‐κB‐dependent manner after IPC were both isoforms of heat shock protein 70 (Hsp70.1 and Hsp70.3). An in vivo mouse model of IPC followed 24 hours later by a 30 minute ischemia and reperfusion was used to assess the impact of Hsp70 isoform expression on late IPC induced cardioprotection. As previously shown by others, an Hsp70.1/70.3 double knockout mouse model failed to exhibit a cardioprotective phenotype during the late window of IPC. However, the Hsp70.1 single isoform knockout exhibited late IPC cardioprotection similar to that of wild‐type mice. In conclusion, this work demonstrates that while both Hsp70.1 and Hsp70.3 expression are induced by NF‐κB after an IPC stimulus, only the Hsp70.3 isoform contributes to cardioprotection in the late phase of IPC. This work was supported by NIH grant R01 HL63034 (WKJ) and NSF award number 0333377.

What Should Be the Ideal Time for Ischemic Preconditioning in a Laparoscopic Rat Model?

Pneumoperitoneum (Pp) induces an ischemia and reperfusion (I/R) injury as a result of released oxidative stress markers. Ischemic preconditioning (IP) is one of the used methods to reduce the harmful effects of Pp, which is a mechanism for reducing organ I/R injury by a brief period of organ ischemia. The aim of this study was to investigate the ideal time for IP in the laparoscopic model. Thirty-two rats were assigned into four groups: group 1 (control, n = 8) was subjected to a sham operation. Group 2 (5-minutes IP, n = 8) was subjected to 5 minutes of Pp with 15 mm Hg of pressure followed immediately by 5 minutes of deflation, and after that, 60 minutes of Pp with 15 mm Hg, followed by 60 minutes of deflation. Group 3 (10-minutes IP, n = 8) was subjected to 10 minutes of Pp and 10 minutes of deflation. Group 4 (Pp only, n = 8) was subjected to 60 minutes of Pp with 15 mm Hg of pressure, followed by 60 minutes of deflation. At the end of the experiment, plasma malondialdehyde (MDA) values, the oxidative stress marker, and plasma-reduced glutathione (GSH) levels, the marker showing antioxidant activity, were determined. Highest plasma MDA values were in group 4 (Pp only), followed by groups 2 and 3 and group 1 (P = 0.181). In addition, IP groups had almost the same values for MDA. Plasma GSH levels in the control group were significantly higher than those in the IP groups and the Pp-only group (P < 0.001). Similarly, as in MDA levels, no difference was found between plasma GSH levels of the IP 5-minutes and IP 10-minutes groups. Five minutes of the IP model may be as reliable as 10 minutes of the IP model. In that case, 5 minutes of IP can be more suitable in reducing I/R injury in laparoscopy.

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.

CREB activation in the rapid, intermediate, and delayed ischemic preconditioning against hypoxic–ischemia in neonatal rat

Ischemic preconditioning (IP) is a defense program in which exposure to sublethal ischemia followed by a period of reperfusion results in subsequent resistance to severe ischemic insults. Very few in vivo IP models have been established for neonatal brain. We examined whether rapid, intermediate, and delayed IP against hypoxic-ischemia (HI) could be induced in neonatal brain, and if so, whether the IP involved phosphorylation of cAMP response element-binding protein (pCREB) after HI. Postnatal day 7 rat pups were subjected to HI at 2 h (2-h IP), 6 h (6-h IP), or 22 h (22-h IP) after IP. We found all three IP groups had significantly reduced neuronal damage and TUNEL-(+) cells 24 h post-HI than no-IP group. Compared with control, the no-IP group had significant decreases of pCREB and mitochondria Bcl-2 levels in the ipsilateral cortex 24 h post-HI. In contrast, the three IP groups had increased pCREB and mitochondria Bcl-2 levels, and significant differences were found between three IP and no-IP groups. The increases of cleavage of caspase-3 and poly (ADP-ribose) polymerase and of cells with nuclear apoptosis inducing factor post-HI in no-IP group were all significantly reduced in three IP groups. The increases of caspase-3 and calpain-mediated proteolysis of a-spectrin post-HI were significantly reduced only in 22-h IP group. Furthermore, all three IP groups had long-term neuroprotection at behavioral and pathological levels compared with no-IP group. In conclusion, IP, rapid, intermediate, or delayed, in neonatal rat brain activates CREB, up-regulates Bcl-2, induces extensive brakes on caspase-dependent and -independent apoptosis after HI, and provides long-term neuroprotection.

Involvement of mitoK ATP channel in protective mechanisms of cerebral ischemic tolerance

Little work has been performed to determine roles of mitochondrial ATP-sensitive potassium channels (mitoK ATP) in ischemic preconditioning (IPC) in brain. To investigate the role on cerebral IPC, we examined effect of 5-hydroxydecanoate (5-HD), a selective mitoK ATP blocker, and diazoxide (DZX), a selective mitoK ATP opener on various IPC models. An IPC model with gerbil: 2 min bilateral common carotid arteries occlusion (BLCO) + 24 h recovery + 5 min BLCO. 5-HD, DZX, vehicle was administered 30 min before 5 min BLCO. Seven days later, surviving CA1 neurons were counted. A focal IPC model with rat: 15 min middle cerebral artery occlusion (MCAO) + 48 h recovery + 90 min MCAO. Twenty-four hours before 90 min MCAO, 5-HD, DZX, or vehicle was administered. One day after 90 min MCAO, neurological symptoms and infarct volumes were evaluated. An in vitro IPC model with primary neuronal cultures: 8 min oxygen–glucose deprivation (OGD) + 24 h recovery + 70 min OGD. Thirty minutes before 70 min OGD, 5-HD or DZX were added. One day later, surviving neurons were counted. Mitochondrial membrane potential was also monitored. 5-HD significantly attenuated the protective effect of IPC in gerbil model, rat model, and in vitro OGD model. DZX significantly facilitated the protective effect of IPC in gerbil and rat model. The mitochondrial membranes were depolarized with IPC, and 5-HD treatment significantly reduced this effect. These results strongly suggest that mitoK ATP channel activation plays a key role in development of a protective mechanism of cerebral IPC.

The Reno-Vascular A2B Adenosine Receptor Protects the Kidney from Ischemia

BackgroundAcute renal failure from ischemia significantly contributes to morbidity and mortality in clinical settings, and strategies to improve renal resistance to ischemia are urgently needed. Here, we identified a novel pathway of renal protection from ischemia using ischemic preconditioning (IP).Methods and FindingsFor this purpose, we utilized a recently developed model of renal ischemia and IP via a hanging weight system that allows repeated and atraumatic occlusion of the renal artery in mice, followed by measurements of specific parameters or renal functions. Studies in gene-targeted mice for each individual adenosine receptor (AR) confirmed renal protection by IP in A1−/−, A2A−/−, or A3AR−/− mice. In contrast, protection from ischemia was abolished in A2BAR−/− mice. This protection was associated with corresponding changes in tissue inflammation and nitric oxide production. In accordance, the A2BAR-antagonist PSB1115 blocked renal protection by IP, while treatment with the selective A2BAR-agonist BAY 60–6583 dramatically improved renal function and histology following ischemia alone. Using an A2BAR-reporter model, we found exclusive expression of A2BARs within the reno-vasculature. Studies using A2BAR bone-marrow chimera conferred kidney protection selectively to renal A2BARs.ConclusionsThese results identify the A2BAR as a novel therapeutic target for providing potent protection from renal ischemia.

Use of a hanging-weight system for liver ischemic preconditioning in mice

Ischemic preconditioning (IP) represents a powerful experimental strategy to identify novel molecular targets to attenuate hepatic injury during ischemia. As a result, murine studies of hepatic IP have become an important field of research. However, murine IP is technically challenging, and experimental details can alter the results. Therefore, we systematically tested a novel model of hepatic IP by using a hanging-weight system for portal triad occlusion. This system has the benefit of applying intermittent hepatic ischemia and reperfusion without manipulation of a surgical clamp or suture, thus minimizing surgical trauma. Systematic evaluation of this model revealed a close correlation of hepatic ischemia time with liver damage as measured by alanine (ALT) and aspartate (AST) aminotransferase serum levels. Using different numbers of IP cycles and times intervals, we found optimal liver protection with four cycles of 3 min ischemia/3 min reperfusion as measured by ALT, AST, lactate dehydrogenase, and interleukin-6. Similarly, ischemia-associated increases in hepatic infarct size, neutrophil infiltration, and histological injury were maximally attenuated with the above regimen. To demonstrate transcriptional consequences of liver IP, we isolated RNA from preconditioned liver and confirmed transcriptional modulation of known target genes (equilibrative nucleoside transporters, acute-phase complement genes). Taken together, these studies confirm highly reproducible liver injury and protection by IP when using the hanging-weight system for hepatic ischemia and intermittent reperfusion. Further studies of murine IP may consider this technique.

Ischemic Preconditioning of Skeletal Muscle Mitigates Remote Injury and Mortality

Ischemic preconditioning (IPC) mitigates ischemia-reperfusion (I/R) injury in experimental models. However, the clinical significance of this protection has been unclear and a mortality reduction has not been previously reported in noncardiac models. This study examined the local and remote protection afforded by skeletal muscle IPC and sought to determine the significance of this protection on mortality. Mice subjected to 2 h hindlimb ischemia/24 h reperfusion (standard I/R injury) were compared with those undergoing a regimen of two 20-min cycles of IPC followed by standard I/R injury. Local injury was assessed via gastrocnemius histology, and remote injury was evaluated via intestinal histology and pulmonary neutrophil infiltration (n = 7). Mortality was compared in parallel groups for 1 week (n = 6). Groups were analyzed using an unpaired Student's t-test for gastrocnemius and pulmonary injury, and a Mann-Whitney rank sum test for intestinal injury. Mortality differences were interpreted through a hazard ratio. Significant protection was observed in preconditioned animals. There was a 35% local injury reduction in skeletal muscle (71.2% versus 46.0%, P < 0.01), a 50% reduction in remote intestinal injury (2.3 versus 1.1, P < 0.01), and a 43% reduction in remote pulmonary injury (14.9 versus 8.5, P < 0.01) compared with standard injury controls. Preconditioned animals were also significantly protected from mortality, demonstrating a 66.7% survival at 1 wk compared with 0% survival after standard injury alone (hazard ratio 0.20, 95% CI: 0.02-0.59). We have developed a murine model of IPC that demonstrates local and remote protection against I/R injury, and exhibits significant mortality reduction. This model demonstrates the powerful effect of IPC on local and remote tissues and will facilitate further study of potential mechanisms and therapies.

Ischemic preconditioning of the whole heart confers protection on subsequently isolated ventricular myocytes

Current cellular models of ischemic preconditioning (IPC) rely on inducing preconditioning in vitro and may not accurately represent complex pathways triggered by IPC in the intact heart. Here, we show that it is possible to precondition the intact heart and to subsequently isolate individual ventricular myocytes that retain the protection triggered by IPC. Myocytes isolated from Langendorff-perfused hearts preconditioned with three cycles of ischemia-reperfusion were exposed to metabolic inhibition and reenergization. Injury was assessed from induction of hypercontracture and loss of Ca(2+) homeostasis and contractile function. IPC induced an immediate window of protection in isolated myocytes, with 64.3 +/- 7.6% of IPC myocytes recovering Ca(2+) homeostasis compared with 16.9 +/- 2.4% of control myocytes (P < 0.01). Similarly, 64.1 +/- 5.9% of IPC myocytes recovered contractile function compared with 15.3 +/- 2.2% of control myocytes (P < 0.01). Protection was prevented by the presence of 0.5 mM 5-hydroxydecanoate during the preconditioning stimulus. This early protection disappeared after 6 h, but a second window of protection developed 24 h after preconditioning, with 54.9 +/- 4.7% of preconditioned myocytes recovering Ca(2+) homeostasis compared with 12.6 +/- 2.9% of control myocytes (P < 0.01). These data show that "true" IPC of the heart confers both windows of protection in the isolated myocytes, with a similar temporal relationship to in vivo preconditioning of the whole heart. The model should allow future studies in isolated cells of the protective mechanisms induced by true ischemia.

In Vivo and In Vitro Characterization of a Novel Neuroprotective Strategy for Stroke: Ischemic Postconditioning

As clinical trials of pharmacological neuroprotective strategies in stroke have been disappointing, attention has turned to the brain's own endogenous strategies for neuroprotection. Recently, a hypothesis has been offered that modified reperfusion subsequent to a prolonged ischemic episode may also confer ischemic neuroprotection, a phenomenon termed 'postconditioning'. Here we characterize both in vivo and in vitro models of postconditioning in the brain and offer data suggesting a biological mechanism for protection. Postconditioning treatment reduced infarct volume by up to 50% in vivo and by approximately 30% in vitro. A duration of 10 mins of postconditioning ischemia after 10 mins of reperfusion produced the most effective postconditioning condition both in vivo and in vitro. The degree of neuroprotection after postconditioning was equivalent to that observed in models of ischemic preconditioning. However, subjecting the brain to both preconditioning as well as postconditioning did not cause greater protection than each treatment alone. The prosurvival protein kinases extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and Akt show prolonged phosphorylation in the cortex of postconditioned rats. Neuroprotection after postconditioning was inhibited only in the presence of LY294002, which blocks Akt activation, but not U0126 or SB203580, which block ERK and P38 MAP kinase activity. In contrast, preconditioning-induced protection was blocked by LY294002, U0126, and SB203580. Our data suggest that postconditioning may represent a novel neuroprotective approach for focal ischemia/reperfusion, and one that is mediated, at least in part, by the activation of the protein kinase Akt.

Ischemic preconditioning induces XRCC1, DNA polymerase-β, and DNA ligase III and correlates with enhanced base excision repair

Neuronal protection induced by ischemic preconditioning has an important role in the reduction of stroke volume and attenuation of neuronal cell death. Ischemic injury is associated with increased oxidative DNA damage, and failure to efficiently repair these oxidatively damaged lesions results in the accumulation of mutations and neuronal cell death. Although the effects of ischemic tolerance can have profound implications, the precise mechanisms mediating this phenomenon remain unclear. The base excision repair (BER) pathway has a major role in the repair of oxidative DNA base damage after ischemic injury. Using a rat model of ischemic preconditioning, we now report that the neuronal protection observed after induction of ischemic tolerance is associated with increased BER. In situ detection of single-strand breaks and apurinic/apyrimidinic sites reduced to baseline levels after reperfusion following ischemic preconditioning. By contrast, no change was seen in the quantity of in situ lesions after reperfusion in non-ischemic preconditioned brain. Induction of the BER proteins XRCC1, DNA polymerase-β, and DNA ligase III was seen after reperfusion in ischemically conditioned brain. Moreover, an increase in binding between XRCC1 and DNA polymerase-β was seen under these conditions, as might be expected during formation of functional BER complexes. Using in vitro BER oligonucleotides, we directly demonstrated an increase in total BER capacity of nuclear extracts prepared from ischemic-conditioned brain after reperfusion compared with sham-operated brain. These findings provide direct evidence that increased BER is associated with the neuroprotection induced after ischemic preconditioning, and provides important new mechanistic insight into the important biologic pathways that protect neurons against irreversible ischemic injury.

Is 15-min Ischemia too Lethal to Be an Ischemic Preconditioning Stimulus?

HomeCirculation ResearchVol. 99, No. 10Is 15-min Ischemia too Lethal to Be an Ischemic Preconditioning Stimulus? Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBIs 15-min Ischemia too Lethal to Be an Ischemic Preconditioning Stimulus? Zheqing Cai and Gregg L. Semenza Zheqing CaiZheqing Cai Vascular Biology Program, Institute for Cell Engineering;, Departments of Pediatrics,, Medicine, Oncology, and Radiation Oncology;, and McKusick-Nathans Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, Md Search for more papers by this author and Gregg L. SemenzaGregg L. Semenza Vascular Biology Program, Institute for Cell Engineering;, Departments of Pediatrics,, Medicine, Oncology, and Radiation Oncology;, and McKusick-Nathans Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, Md Search for more papers by this author Originally published10 Nov 2006https://doi.org/10.1161/01.RES.0000249066.23049.70Circulation Research. 2006;99:e86To the Editor:We would like to take this opportunity to respond to comments by Hausenloy et al1 regarding our recent study.2 In our study, we demonstrated that PTEN activity is modulated during cardiac ischemia/reperfusion, suggesting critical involvement of PTEN in both the induction and decay of ischemic preconditioning (IPC).2 Hausenloy et al appear more intent on promulgating a “standard accepted IPC protocol” than on understanding mechanisms of protection against ischemia/reperfusion injury. Without providing any supporting data, they characterize 15 minutes of ischemia as a “prolonged episode of lethal ischemia.”1 In contrast, Kloner and Jennings wrote in the first sentence of their highly cited review of IPC that “total proximal coronary artery occlusions of up to 15 minutes result in reversible injury, meaning that the myocytes survive this insult.”3Our data2 show that 15 minutes of ischemia followed by 5 minutes of reperfusion induces higher levels of phosphorylated (activated) Akt in the heart, as compared with hearts subjected to 5 or 10 minutes of ischemia followed by 5 minutes of reperfusion. This ischemic stimulus protects the heart against subsequent prolonged ischemia (30 minutes) and reperfusion (thus fulfilling the criteria for IPC), as demonstrated by significantly improved recovery of left ventricular developed pressure, reduced caspase-3 activation, and reduced PARP cleavage. Finally, we demonstrated that reactive oxygen species (ROS), which are generated after hearts are subjected to 15 minutes of ischemia and then reperfused, oxidize and inactivate PTEN, thus leading to increased Akt activity. Our data provide for the first time a direct molecular mechanism for transduction of the protective IPC signal that is based on the inherent sensitivity of this phosphatase to oxidative inactivation because of the presence of a cysteine residue in the active site.4 Because ROS are produced in every IPC model and PTEN is expressed in every tissue, this pathway may underlie IPC in all organs.Practicing scientists appreciate that every experimental model has its strengths and limitations. We have presented a paradigm-shifting hypothesis that can now be tested in other experimental models of ischemia/reperfusion injury. Whereas we recognize the potential value of comments in the editorial pages, we hope that our novel findings will stimulate research at the laboratory bench, which will advance the field beyond what is “standard” and “accepted”.1 Hausenloy DJ, Mocanu MM, Yellon DM. Is this truly ischemic preconditioning? Circ Res. 2006; 99: e11.LinkGoogle Scholar2 Cai Z, Semenza GL. PTEN activity is modulated during ischemia and reperfusion: involvement in the induction and decay of preconditioning. Circ Res. 2005; 97: 1351–1359.LinkGoogle Scholar3 Kloner RA, Jennings RB. Consequences of brief ischemia: stunning, preconditioning, and their clinical implications: part 1. Circulation. 2001; 104: 2981–2989.CrossrefMedlineGoogle Scholar4 Rhee SG, Bae YS, Lee SR, Kwon J. Hydrogen peroxide: a key messenger that modulates protein phosphorylation through cysteine oxidation. Sci STKE. 2000; (53): PE1.Google Scholar Previous Back to top Next FiguresReferencesRelatedDetailsCited By Wang H, Li Z and Liu X (2008) Effects of various protocols of ischemic preconditioning on rat tram flaps, Microsurgery, 10.1002/micr.20436, 28:1, (37-43), Online publication date: 1-Jan-2008. November 10, 2006Vol 99, Issue 10 Advertisement Article InformationMetrics https://doi.org/10.1161/01.RES.0000249066.23049.70PMID: 17095727 Originally publishedNovember 10, 2006 PDF download Advertisement

Is This Truly Ischemic Preconditioning?

To the Editor: In a recent article, Cai and Semenza1 claim to have demonstrated a role for modulation of PTEN activity in the setting of ischemic preconditioning (IPC), using an isolated perfused rat heart. We believe the findings of this study are potentially of great significance as this study is the first to implicate PTEN in myocardial ischemia–reperfusion injury. However, we question the model of IPC used in this study. Specifically we question whether 15 minutes of myocardial ischemia followed by 30 minutes of myocardial reperfusion actually …

Inhibition of Myocardial Apoptosis by Ischaemic and Beta-Adrenergic Preconditioning is Dependent on p38 MAPK

Apoptosis occurring during ischaemia /reperfusion contributes independently to tissue damage, and involves activation of the stress-kinase, p38 MAPK during reperfusion. Ischaemic preconditioning (IPC) protects against ischaemia/reperfusion mediated necrosis and apoptosis. The role of p38 MAPK in the protective effect of preconditioning against apoptosis is unknown. Pharmacologic preconditioning with isoproterenol (beta-PC) also protects against necrosis, but it is not known whether it protects against apoptosis. The aim of the study was to investigate whether the protective effect of IPC against apoptosis is related to activation of p38 MAPK and whether beta-PC also protects against apoptosis. Isolated perfused rat hearts were used to study the effect of ischaemia and reperfusion on apoptosis and infarct size. Ischaemic preconditioning was elicited by 3 x 5 min global ischaemia, and beta-PC by 5 min isoproterenol 10(-7) M. For infarct size hearts were subjected to regional ischaemia for 35 min followed by 120 min reperfusion. Infarct size was determined by the tetrazolium staining technique, and expressed as percentage of area at risk. For markers of apoptosis hearts were subjected to global ischaemia of 25 min plus 30 min reperfusion. Apoptosis was determined by Western blot using antibodies against caspase-3 and PARP. p38 MAPK activation was inhibited by SB203580 (1 microM) administration 10 min prior to commencing ischaemia, and bracketing the IPC and beta-PC preconditioning protocols. p38 MAPK was activated by administration of anisomycin (5 microM) 10 min prior to index ischaemia in one protocol, and 10 min during reperfusion in non-preconditioned as well as IPC and beta-PC hearts. Results were analysed using ANOVA and a Newman-Keuls post-hoc test. In the apoptosis model using global ischaemia, IPC and beta-PC both resulted in a significant decrease in p38 MAPK activation at the end of reperfusion when compared to non-preconditioned hearts. This was accompanied by a significant decrease in apoptosis as measured with both caspase-3 activation and PARP cleavage. Inhibiting p38 MAPK by administration of SB203580 10 min prior to ischaemia resulted in a significant reduction in both markers of apoptosis. Bracketing the triggering phase of either IPC or beta-PC with SB203580 resulted in attenuated p38 MAPK activation during reperfusion and did not abolish the protective effect of IPC or beta-PC against apoptosis. Activating p38 MAPK with anisomycin prior to ischaemia resulted in a reduction of markers of apoptosis, whereas activation of p38 MAPK with anisomycin during reperfusion did not exacerbate apoptosis in any groups, exept for an increase in PARP cleavage in IPC hearts. In the model of regional ischaemia, IPC and beta-PC reduced infarct size significantly, and to the same extent as inhibition of p38 MAPK by administration of SB203580 10 min prior to ischaemia. Bracketing the triggering phase of either IPC or beta-PC did not abolish the reduction in infarct size. Activating p38 MAPK during reperfusion was accompanied by an increase in infarct size only in IPC hearts, but not in beta-PC hearts. These results indicate that (1) Both IPC and beta-PC elicit protection against apoptosis and necrosis, (2) activation of p38 MAPK is not a trigger of preconditioning against apoptosis and necrosis and (3) activation of p38 MAPK during reperfusion increases necrosis only if ischaemia is used to precondition hearts, but not with pharmacologic preconditioning with isoproterenol.

TNFR1 mediates increased neuronal membrane EAAT3 expression after in vivo cerebral ischemic preconditioning

A short ischemic event (ischemic preconditioning) can result in subsequent resistance to severe ischemic injury (ischemic tolerance). Glutamate is released after ischemia and produces cell death. It has been described that after ischemic preconditioning, the release of glutamate is reduced. We have shown that an in vitro model of ischemic preconditioning produces upregulation of glutamate transporters which mediates brain tolerance. We have now decided to investigate whether ischemic preconditioning-induced glutamate transporter upregulation takes also place in vivo, its cellular localization and the mechanisms by which this upregulation is controlled. A period of 10 min of temporary middle cerebral artery occlusion was used as a model of ischemic preconditioning in rat. EAAT1, EAAT2 and EAAT3 glutamate transporters were found in brain from control animals. Ischemic preconditioning produced an up-regulation of EAAT2 and EAAT3 but not of EAAT1 expression. Ischemic preconditioning-induced increase in EAAT3 expression was reduced by the TNF-α converting enzyme inhibitor BB1101. Intracerebral administration of either anti-TNF-α antibody or of a TNFR1 antisense oligodeoxynucleotide also inhibited ischemic preconditioning-induced EAAT3 up-regulation. Immunohistochemical studies suggest that, whereas the expression of EAAT3 is located in both neuronal cytoplasm and plasma membrane, ischemic preconditioning-induced up-regulation of EAAT3 is mainly localized at the plasma membrane level. In summary, these results demonstrate that in vivo ischemic preconditioning increases the expression of EAAT2 and EAAT3 glutamate transporters the upregulation of the latter being at least partly mediated by TNF-α converting enzyme/TNF-α/TNFR1 pathway.

Lithium Microdialysis and Its Use for Monitoring of Stomach and Colon Submucosal Blood Perfusion – A Pilot Study Using Ischemic Preconditioning in Rats

During shock, exposure of gut to ischemia determines patient's survival. Ischemic preconditioning (ISP) elevates nitric oxide and blood perfusion, whereby it protects organs against subsequent severe ischemia/reperfusion. Using appropriate flow marker, microdialysis may serve to monitor interstitial microcirculation. Hence, our aim was to test the reliability of lithium as a flow marker (lithium microdialysis, LM) on an ISP model. Rats were divided into three groups. Two (ischemic and preconditioned) groups underwent 30 min celiac artery occlusion (CAO) with 2.5 h reperfusion. 25 min before CAO, the latter experienced 5 min ischemia. Sham-operated animals served as controls. LM in stomach and colon submucosa, serum nitric oxide, hepatic and pancreatic enzymes were measured. In stomach, LM indicated a decrease in blood perfusion evoked by CAO (p < 0.01) in both experimental groups. During reperfusion, the ischemic animals showed a restoration of microcirculation, unlike the preconditioned ones, whose blood perfusion failed to regenerate (p < 0.001). For any group, LM showed no microcirculation modification in colon. Serum analytes remained unchanged. We conclude that LM appears to be a potentially suitable indicator of gastrointestinal interstitial microcirculation. However, we failed to demonstrate any beneficial effect of ISP on pancreas, systemic nitric oxide and local/remote microcirculation within studied organs.

Ischemic preconditioning ameliorates excitotoxicity by shifting glutamate/γ‐aminobutyric acid release and biosynthesis

Excitotoxicity is recognized to play a major role in cerebral ischemia-induced cell death. The main goal of the present study was to define whether our model of ischemic preconditioning (IPC) promotes a shift from excitatory to inhibitory neurotransmission during the test ischemia to diminish metabolic demand during the reperfusion phase. We also determined whether gamma-aminobutyric acid (GABA) played a role in IPC-induced neuroprotection. Ten minutes of cerebral ischemia was produced by tightening the carotid ligatures bilaterally following hypotension. Samples of microdialysis perfusate, representing extracellular fluid, were analyzed for amino acid content by HPLC. IPC promoted a robust release of GABA after lethal ischemia compared with control rats. We also observed that the activity of glutamate decarboxylase (the predominant pathway of GABA synthesis in the brain) was higher in the IPC group compared with control and ischemic groups. Because GABAA receptor up-regulation has been shown to occur following IPC, and GABAA receptor activation has been implicated in neuroprotection against ischemic insults, we tested the hypothesis that GABAA or GABAB receptor activation was neuroprotective during ischemia or early reperfusion by using an in vitro model (organotypic hippocampal slice culture). Administration of the GABAB agonist baclofen during test ischemia and for 1 hr of reperfusion provided significant neuroprotection. We concluded that increased GABA release in preconditioned animals after ischemia might be one of the factors responsible for IPC neuroprotection. Specific activation of GABAB receptor contributes significantly to neuroprotection against ischemia in organotypic hippocampal slices.

Heat stress increases the effectiveness of early ischemic preconditioning in spinal cord protection

This study was aimed to test the hypothesis that the combination of heat stress and early ischemic preconditioning (IP) applied before aortic occlusion would be protective against spinal cord ischemic injury. Thirty Whister-Albino rats were randomly divided into three groups. In group 1 (n=10), aorta was clamped just distal to the left renal artery and above the iliac bifurcation for 45 min. Group 2 (n=10) had 5 min of transient aortic occlusion and 30 min later underwent an additional 45 min. In group 3 (n=10), animals were heated to 41 degrees C and maintained at this temperature for 15 min. Twenty-four hours later, this hyperthermia pretreated group underwent the same early IP model of aortic occlusion. Neurologic status was assessed on postoperative 24 and 48 h by using the 15-point neurologic performance scale. Spinal cords were harvested for histopathological grading (1-4) and evaluated for the presence of heat shock protein-ubiquitin staining. At 24 and 48 h, the mean neurologic performance scores of the group 1 were found to be significantly lower than those of groups 2 and 3. Although the neurologic assessment of rats performed on the 24h did not reveal statistically significant difference between groups 2 and 3 (P=0.069); on 48 h, the mean neurologic scores of the group 3 were significantly higher than those of group 2 (P=0.005). At 48 h, a delayed neurologic deterioration was seen in groups 1 and 2 when compared to the results obtained at 24h. Histologic evaluation correlated well with the neurologic outcome with the least cellular damage in group 3. There were six rats with ubiquitin expression and this was detected only in animals pretreated with sublethal heat stress. An early IP model with a short reperfusion interval does not give the minimal required time for the HSPs expression and is associated with a delayed neurologic deterioration. Neuroprotection provided by heat stress combined with an early IP model lasts up to 48 h and heat shock protein-ubiquitin induction may be responsible in this phenomenon.

Hypoxia/hypoglycemia preconditioning prevents the loss of functional electrical activity in organotypic slice cultures

In cerebral ischemic preconditioning (IPC), a first sublethal ischemia increases the resistance of neurons to a subsequent severe ischemia. Despite numerous studies, the mechanisms are not yet fully understood. Our goal is to develop an in vitro model of IPC on hippocampal organotypic slice cultures. Instead of anoxia, we chose to apply varying degrees of hypoxia that allows us various levels of insult graded from mild to severe. Cultures are exposed to combined oxygen and glucose deprivation (OGD) of varying intensities, ranging from mild to severe, assessing both the electrical activity and cell death. IPC was accomplished by exposure to the mildest ischemia condition (10% of O 2 for 15 min) 24 h before the severe deprivation (5% of O 2 for 30 min). Interestingly, IPC not only prevented delayed ischemic cell death 6 days after insult but also the transient loss of evoked potential response. The major interest and advantage of this system over both the acute slice preparation and primary cell cultures is the ability to simultaneously measure the delayed neuronal damage and neuronal function.