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The author reviews the protective effects of ischemic postconditioning, a recently emerging strategy with broad implications in the search for new treatments in stroke and myocardial ischemic injury. Ischemic postconditioning, which refers to a series of brief ischemia and reperfusion cycles applied immediately at the site of the ischemic organ after reperfusion, results in reduced infarction in both cerebral and myocardial ischemia. Conventional postconditioning induced within a few minutes after reperfusion is arbitrarily defined as rapid postconditioning. In contrast, postconditioning performed hours to days after stroke is defined as delayed postconditioning. In addition, postconditioning can be mimicked using anesthetics or other pharmacological agents as stimuli to protect against ischemia/reperfusion injury or performed in a distant organ, which is known as remote postconditioning. In this article, the author discusses the conceptual origin of classical rapid ischemic postconditioning and its evolution into a term that represents a broad range of stimuli or triggers, including delayed postconditioning, pharmacological postconditioning, and remote postconditioning. Thereafter, various in vivo and in vitro models of postconditioning and its potential protective mechanisms are discussed. Since the concept of postconditioning is so closely associated with that of preconditioning and both share some common protective mechanisms, whether a combination of preconditioning and postconditioning offers greater protection than preconditioning or postconditioning alone is also discussed.

Background

We and others have reported that rapid ischemic postconditioning, interrupting early reperfusion after stroke, reduces infarction in rats. However, its extremely short therapeutic time windows, from a few seconds to minutes after reperfusion, may hinder its clinical translation. Thus, in this study we explored if delayed postconditioning, which is conducted a few hours after reperfusion, offers protection against stroke.

Methods and Results

Focal ischemia was generated by 30 min occlusion of bilateral common carotid artery (CCA) combined with permanent occlusion of middle cerebral artery (MCA); delayed postconditioning was performed by repetitive, brief occlusion and release of the bilateral CCAs, or of the ipsilateral CCA alone. As a result, delayed postconditioning performed at 3h and 6h after stroke robustly reduced infarct size, with the strongest protection achieved by delayed postconditioning with 6 cycles of 15 min occlusion/15 min release of the ipsilateral CCA executed from 6h. We found that this delayed postconditioning provided long-term protection for up to two months by reducing infarction and improving outcomes of the behavioral tests; it also attenuated reduction in 2-[18F]-fluoro-2-deoxy-D-glucose (FDG)-uptake therefore improving metabolism, and reduced edema and blood brain barrier leakage. Reperfusion in ischemic stroke patients is usually achieved by tissue plasminogen activator (tPA) application, however, t-PA's side effect may worsen ischemic injury. Thus, we tested whether delayed postconditioning counteracts the exacerbating effect of t-PA. The results showed that delayed postconditioning mitigated the worsening effect of t-PA on infarction.

Conclusion

Delayed postconditioning reduced ischemic injury after focal ischemia, which opens a new research avenue for stroke therapy and its underlying protective mechanisms.

Transient forebrain or global ischemia induces neuronal death in vulnerable CA1 pyramidal cells with many features. A brief period of ischemia, i.e., ischemic preconditioning, or a modified reperfusion such as ischemic postconditioning, can afford robust protection of CA1 neurons against ischemic challenge. Therefore, we investigated the effect of ischemic preconditioning and postconditioning on neural cell apoptosis in rats. The result showed that both ischemic preconditioning and postconditioning may attenuate the neural cell death and DNA fragment in the hippocampal CA1 region. Further western blot study suggested that ischemic preconditioning and postconditioning down-regulates the protein of cleaved caspase-3, caspase-6, caspase-9 and Bax, but up-regulates the protein Bcl-2. These findings suggest that ischemic preconditioning and postconditioning have a neuroprotective role on global brain ischemia in rats through the same effect on inhibition of apoptosis.

Ischemic postconditioning is a concept originally defined to contrast with that of ischemic preconditioning. While both preconditioning and postconditioning confer a neuroprotective effect on brain ischemia, preconditioning is a sublethal insult performed in advance of brain ischemia, and postconditioning, which conventionally refers to a series of brief occlusions and reperfusions of the blood vessels, is conducted after ischemia/reperfusion. In this article, we first briefly review the history of preconditioning, including the experimentation that initially uncovered its neuroprotective effects and later revealed its underlying mechanisms-of-action. We then discuss how preconditioning research evolved into that of postconditioning – a concept that now represents a broad range of stimuli or triggers, including delayed postconditioning, pharmacological postconditioning, remote postconditioning – and its underlying protective mechanisms involving the Akt, MAPK, PKC and KATP channel cell-signaling pathways. Because the concept of postconditioning is so closely associated with that of preconditioning, and both share some common protective mechanisms, we also discuss whether a combination of preconditioning and postconditioning offers greater protection than preconditioning or postconditioning alone.

Delayed remote ischemic postconditioning (DRIPost) has been shown to protect the rat brain from ischemic injury. However, extremely short therapeutic time windows hinder its translational use and the mechanism of action remains elusive. Because opening of the mitochondria KATP channel is crucial for cell apoptosis, we hypothesized that the neuroprotective effect of DRIPost may be associated with KATP channels. In the present study, the neuroprotective effects of DRIPost were investigated using adult male Sprague-Dawley rats. Rats were exposed to 90 minutes of middle cerebral artery occlusion followed by 72 hours of reperfusion. Delayed remote ischemic postconditioning was performed with three cycles of bilateral femoral artery occlusion/reperfusion for 5 minutes at 3 or 6 hours after reperfusion. Neurologic deficit scores and infarct volumes were assessed, and cellular apoptosis was monitored by terminal deoxynucleotidyl transferase nick-end labeling. Our results showed that DRIPost applied at 6 hours after reperfusion exerted neuroprotective effects. The KATP opener, diazoxide, protected rat brains from ischemic injury, while the KATP blocker, 5-hydroxydecanote, reversed the neuroprotective effects of DRIPost. These findings indicate that DRIPost reduces focal cerebral ischemic injury and that the neuroprotective effects of DRIPost may be achieved through opening of KATP channels.

Remote ischemic postconditioning (RIP) refers to an ischemia conducted in a distant organ that protects against a prior ischemia in another organ. We tested whether RIP protects against focal ischemia in the rat brain. Stroke was generated by a permanent occlusion of the left distal middle cerebral artery combined with a 30 min occlusion of the bilateral common carotid arteries (CCA) in male rats. After CCA release, RIP was generated by 3 cycles of 15 min occlusion/15 min release of the left hind femoral artery. The results showed that rapid RIP performed immediately after CCA release reduced infarction by 67% measured at 2 d after stroke. In addition, delayed RIP initiated as late as 3 h, but not 6 h, still robustly reduced infarction by 43% 2 d after stroke. RIP's protective effect was abolished by injecting the protein synthesis inhibitor, cycloheximide, and the afferent nerve blocker, capsaicin, suggesting that RIP blocks ischemic injury by modulating protein synthesis and nerve activity. Nevertheless, rapid RIP did not reduce infarction size 2 months after stroke while it ameliorated the outcome of the behavioral test. In conclusion, RIP attenuates brain injury after focal ischemia.

Background

Experimental studies have shown that ischemic postconditioning can reduce neuronal injury in the setting of cerebral ischemia, but the mechanisms are not yet clearly elucidated. This study was conducted to determine whether ischemic postconditioning can alter expression of heat shock protein 70 and reduce acute phase neuronal injury in rats subjected to transient focal cerebral ischemia/reperfusion.

Methods

Focal cerebral ischemia was induced by intraluminal middle cerebral artery occlusion for 60 min in twenty male Sprague-Dawley rats (250-300 g). Rats were randomized into control group and an ischemic postconditioning group (10 rats per group). The animals of control group had no intervention either before or after MCA occlusion. Ischemic postconditioning was elicited by 3 cycles of 30 s reperfusion interspersed by 10 s ischemia immediately after onset of reperfusion. The infarct ratios, brain edema ratios and motor behavior deficits were analyzed 24 hrs after ischemic insult. Caspase-3 reactive cells and cells showing heat shock protein 70 activity were counted in the caudoputamen and frontoparietal cortex.

Results

Ischemic postconditiong did not reduce infarct size and brain edema ratios compared to control group. Neurologic scores were not significantly different between groups. The number of caspase-3 reactive cells in the ischemic postconditioning group was not significantly different than the value of the control group in the caudoputamen and frontoparietal cortex. The number of cells showing heat shock protein 70 activity was not significantly different than the control group, as well.

Conclusions

These results suggest that ischemic postconditioning may not influence the early brain damage induced by focal cerebral ischemia in rats.

Background

Ischemic pre- and postconditioning protects the liver against ischemia/reperfusion injuries. The aim of the present study was to examine how ischemic pre- and postconditioning affects gene expression of hypoxia inducible factor 1α (HIF-1α), vascular endothelial growth factor A (VEGF-A) and transforming growth factor β (TGF-β) in liver tissue.

Methods

28 rats were randomized into five groups: control; ischemia/reperfusion; ischemic preconditioning (IPC); ischemic postconditioning (IPO); combined IPC and IPO. IPC consisted of 10 min of ischemia and 10 min of reperfusion. IPO consisted of three cycles of 30 sec. reperfusion and 30 sec. of ischemia.

Results

HIF-1α mRNA expression was significantly increased after liver ischemia compared to controls (p = 0.010). HIF-1α mRNA expression was significantly lower in groups subjected to IPC or combined IPC and IPO when compared to the ischemia/reperfusion group (p = 0.002). VEGF-A mRNA expression increased in the ischemia/reperfusion or combined IPC and IPO groups when compared to the control group (p < 0.05).

Conclusion

Ischemic conditioning seems to prevent HIF-1α mRNA induction in the rat liver after ischemia and reperfusion. This suggests that the protective effects of ischemic conditioning do not involve the HIF-1 system. On the other hand, the magnitude of the HIF-1α response might be a marker for the degree of I/R injuries after liver ischemia. Further studies are needed to clarify this issue.

We have shown that isoflurane application at the onset of reperfusion (postconditioning) reduces brain ischemic injury in rats. This study was designed to determine whether this protection involved activation of prosurvival protein kinases and maintenance of normal mitochondrial membrane permeability. Two-month old male rats were subjected to a 90-min middle cerebral arterial occlusion. They then were exposed or were not exposed to 2% isoflurane for 1 h. Ischemic penumbral cerebral cortex was harvested immediately and separated into the mitochondrial and cytosolic fractions. We showed that the mitochondrial nicotinamide adenine dinucleotide content in the ischemic penumbral cortex was significantly reduced, suggesting an increased mitochondrial membrane permeability. This increase was partly attenuated by isoflurane postconditioning. The mitochondrial adenosine diphosphate content in the penumbral cortex was reduced no matter whether the animals were postconditioned with isoflurane. The mitochondrial adenosine triphosphate concentration was not different among various experimental conditions. The phospho-Akt in the cytosolic and mitochondrial fractions of the ischemic penumbral cortex was higher than that in the control cortex. This increase trended to be higher in animals with isoflurane postconditioning. A similar change pattern was observed in the mitochondrial phospho-glycogen synthase kinase 3β, an Akt substrate that can regulate the mitochondrial membrane permeability. Isoflurane postconditioning reduced oxygen-glucose deprivation-induced injury of rat cortical neuronal cultures and increased phospho-Akt in these cells. The isoflurane postconditioning-induced protection in the neuronal cultures was decreased by the Akt inhibitor LY294002. These results suggest that isoflurane postconditioning effects may be mediated by Akt and involve reduced mitochondrial membrane permeability.

Ischemic postconditioning initially referred to a stuttering reperfusion performed immediately after reperfusion, for preventing ischemia/reperfusion injury in both myocardial and cerebral infarction. It has evolved into a concept that can be induced by a broad range of stimuli or triggers, and may even be performed as late as 6 h after focal ischemia and 2 days after transient global ischemia. The concept is thought to be derived from ischemic preconditioning or partial/gradual reperfusion, but in fact the first experiment for postconditioning was carried out much earlier than that of preconditioning or partial/gradual reperfusion, in the research on myocardial ischemia. This review first examines the protective effects and parameters of postconditioning in various cerebral ischemic models. Thereafter, it provides insights into the protective mechanisms of postconditioning associated with reperfusion injury and the Akt, mitogen-activated protein kinase (MAPK), protein kinase C (PKC), and ATP-sensitive K+ (KATP) channel cell signaling pathways. Finally, some open issues and future challenges regarding clinical translation of postconditioning are discussed.

We previously reported that ischemic postconditioning with a series of mechanical interruptions of reperfusion reduced infarct volume 2 days after focal ischemia in rats. Here, we extend this data by examining long-term protection and exploring underlying mechanisms involving the Akt, mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) signaling pathways. Post-conditioning reduced infarct and improved behavioral function assessed 30 days after stroke. Additionally, postconditioning increased levels of phosphorylated Akt (Ser473) as measured by western blot and Akt activity as measured by an in vitro kinase assay. Inhibiting Akt activity by a phosphoinositide 3-kinase inhibitor, LY294002, enlarged infarct in postconditioned rats. Postconditioning did not affect protein levels of phosphorylated-phosphatase and tensin homologue deleted on chromosome 10 or -phosphoinositide-dependent protein kinase-1 (molecules upstream of Akt) but did inhibit an increase in phosphorylated-glycogen synthase kinase 3β, an Akt effector. In addition, postconditioning blocked β-catenin phosphorylation subsequent to glycogen synthase kinase, but had no effect on total or non-phosphorylated active β-catenin protein levels. Furthermore, postconditioning inhibited increases in the amount of phosphorylated-c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 in the MAPK pathway. Finally, postconditioning blocked death-promoting δPKC cleavage and attenuated reduction in phosphorylation of survival-promoting εPKC. In conclusion, our data suggest that postconditioning provides long-term protection against stroke in rats. Additionally, we found that Akt activity contributes to postconditioning’s protection; furthermore, increases in εPKC activity, a survival-promoting pathway, and reductions in MAPK and δPKC activity; two putative death-promoting pathways correlate with postconditioning’s protection.

It has been recently shown that a short sublethal brain ischemia subsequent to a prolonged harmful ischemic episode may confer ischemic neuroprotection, a phenomenon termed ischemic postconditioning. Na+/Ca2+ exchanger (NCX) isoforms, NCX1, NCX2, and NCX3, are plasma membrane ionic transporters widely distributed in the brain and involved in the control of Na+ and Ca2+ homeostasis and in the progression of stroke damage. The objective of this study was to evaluate the role of these three proteins in the postconditioning-induced neuroprotection. The NCX protein and mRNA expression was evaluated at different time points in the ischemic temporoparietal cortex of rats subjected to tMCAO alone or to tMCAO plus ischemic postconditioning. The results of this study showed that NCX3 protein and ncx3 mRNA were upregulated in those brain regions protected by postconditioning treatment. These changes in NCX3 expression were mediated by the phosphorylated form of the ubiquitously expressed serine/threonine protein kinase p-AKT, as the p-AKT inhibition prevented NCX3 upregulation. The relevant role of NCX3 during postconditioning was further confirmed by results showing that NCX3 silencing, induced by intracerebroventricular infusion of small interfering RNA (siRNA), partially reverted the postconditioning-induced neuroprotection. The results of this study support the idea that the enhancement of NCX3 expression and activity might represent a reasonable strategy to reduce the infarct extension after stroke.

AIM: To investigate the protective effect and mechanisms of ghrelin postconditioning against hypoxia/reoxygenation (H/R)-induced injury in human gastric epithelial cells.

METHODS: The model of H/R injury was established in gastric epithelial cell line (GES-1) human gastric epithelial cells. Cells were divided into seven groups: normal control group (N); H/R postconditioning group; DMSO postconditioning group (DM); ghrelin postconditioning group (GH); D-Lys3-GHRP-6 + ghrelin postconditioning group (D + GH); capsazepine + ghrelin postconditioning group (C + GH); and LY294002 + ghrelin postconditioning group (L + GH). 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to detect GES-1 cell viability. Hoechst 33258 fluorochrome staining and flow cytometry were conducted to determine apoptosis of GES-1 cells. Spectrophotometry was performed to determine release of lactate dehydrogenate (LDH). Protein expression of Bcl-2, Bax, Akt, and glycogen synthase kinase (GSK)-3β was determined by western blotting. Expression of vanilloid receptor subtype 1 (VR1), Akt and GSK-3β was observed by immunocytochemistry.

RESULTS: Compared with the H/R group, cell viability of the GH group was significantly increased in a dose-dependent manner (55.9% ± 10.0% vs 69.6% ± 9.6%, 71.9% ± 17.4%, and 76.3% ± 13.3%). Compared with the H/R group, the percentage of apoptotic cells in the GH group significantly decreased (12.38% ± 1.51% vs 6.88% ± 0.87%). Compared with the GH group, the percentage of apoptotic cells in the D + GH group, C + GH group and L + GH groups significantly increased (11.70% ± 0.88%, 11.93% ± 0.96%, 10.20% ± 1.05% vs 6.88% ± 0.87%). There were no significant differences in the percentage of apoptotic cells between the H/R and DM groups (12.38% ± 1.51% vs13.00% ± 1.13%). There was a significant decrease in LDH release following ghrelin postconditioning compared with the H/R group (561.58 ± 64.01 U/L vs 1062.45 ± 105.29 U/L). There was a significant increase in LDH release in the D + GH, C + GH and L + GH groups compared with the GH group (816.89 ± 94.87 U/L, 870.95 ± 64.06 U/L, 838.62 ± 118.45 U/L vs 561.58 ± 64.01 U/L). There were no significant differences in LDH release between the H/R and DM groups (1062.45 ± 105.29 U/L vs 1017.65 ± 68.90 U/L). Compared with the H/R group, expression of Bcl-2 and Akt increased in the GH group, whereas expression of Bax and GSK-3β decreased. Compared with the GH group, expression of Bcl-2 decreased and Bax increased in the D + GH, C + GH and L + GH groups, and Akt decreased and GSK-3β increased in the L + GH group. The H/R group also upregulated expression of VR1 and GSK-3β and downregulated Akt. The number of VR1-positive and Akt-positive cells in the GH group significantly increased, whereas the number of GSK-3β-positive cells significantly decreased. These effects of ghrelin were reversed by capsazepine and LY294002.

CONCLUSION: Ghrelin postconditioning protected against H/R-induced injury in human gastric epithelial cells, which indicated that this protection might be associated with GHS-R, VR1 and the PI3K/Akt signaling pathway.

Background

Ischemic postconditioning (IPOC), or relief of ischemia in a stuttered manner, has emerged as an innovative treatment strategy to reduce programmed cell death, attenuate ischemic injuries, and improve neurological outcomes. However, the mechanisms involved have not been completely elucidated. Recent studies indicate that autophagy is a type of programmed cell death that plays elusive roles in controlling neuronal damage and metabolic homeostasis. This study aims to determine the role of autophagy in IPOC-induced neuroprotection against focal cerebral ischemia in rats.

Methodology/Principal Findings

A focal cerebral ischemic model with permanent middle cerebral artery (MCA) occlusion plus transient common carotid artery (CCA) occlusion was established. The autophagosomes and the expressions of LC3/Beclin 1/p62 were evaluated for their contribution to the activation of autophagy. We found that autophagy was markedly induced with the upregulation of LC3/Beclin 1 and downregulation of p62 in the penumbra at various time intervals following ischemia. IPOC, performed at the onset of reperfusion, reduced infarct size, mitigated brain edema, inhibited the induction of LC3/Beclin 1 and reversed the reduction of p62 simultaneously. Rapamycin, an inducer of autophagy, partially reversed all the aforementioned effects induced by IPOC. Conversely, autophagy inhibitor 3-methyladenine (3-MA) attenuated the ischemic insults, inhibited the activation of autophagy, and elevated the expression of anti-apoptotic protein Bcl-2, to an extent comparable to IPOC.

Conclusions/Significance

The present study suggests that inhibition of the autophagic pathway plays a key role in IPOC-induced neuroprotection against focal cerebral ischemia. Thus, pharmacological inhibition of autophagy may provide a novel therapeutic strategy for the treatment of stroke.

Background and Purpose

Complications due to brain edema and breakdown of blood brain barrier are an important factor affecting the treatment effects of patients with severe carotid stenosis. In this study, we investigated the protective effects of ischemic postconditioning on brain edema and disruption of blood brain barrier via establishing rat model of hypoperfusion due to severe carotid stenosis.

Methods

Wistar rat model of hypoperfusion due to severe carotid stenosis was established by binding a stainless microtube to both carotid arteries. Ischemic postconditioning procedure consisted of three cycles of 30 seconds ischemia and 30 seconds reperfusion. Brain edema was evaluated by measuring cerebral water content, and blood brain barrier permeability was assayed by examining cerebral concentration of Evans' Blue (EB) and fluorescein sodium (NaF). ELISA was used to analyze the expression of MMP-9, claudin-5 and occludin. The activity and location of MMP-9 was analyzed by gelatin zymography and in situ zymography, respectively. The distribution of tight junction proteins claudin-5 and occludin was observed by immunohistochemistry.

Results

The increased brain water content and cerebral concentration of EB and NaF were suppressed by administration of ischemic postconditioning prior to relief of carotid stenosis. Zymographic studies showed that MMP-9 was mainly located in the cortex and its activity was significantly improved by relief of carotid stenosis and, but the elevated MMP-9 activity was inhibited markedly by ischemic postconditioning. Immunohistochemistry revealed that ischemic postconditioning improved the discontinuous distribution of claudin-5 and occludin. ELISA detected that the expression of up-regulated MMP-9 and down-regulated claudin-5 and occludin caused by carotid relief were all attenuated by ischemic postconditioning.

Conclusions

Ischemic postconditioning is an effective method to prevent brain edema and improve BBB permeability and could be used during relief of severe carotid stenosis.

Hepatic Ischemia and Reperfusion Injury (IRI) is a major cause of liver damage during liver surgery and transplantation. Ischemic preconditioning and postconditioning are strategies that can reduce IRI. In this study, different combined types of pre- and postconditioning procedures were tested in a murine warm hepatic IRI model to evaluate their protective effects. Proanthocyanidins derived from grape seed was used before ischemia process as pharmacological preconditioning to combine with technical preconditioning and postconditioning. Three pathways related to IRI, including reactive oxygen species (ROS) generation, pro-inflammatory cytokines release and hypoxia responses were examined in hepatic IRI model. Individual and combined pre- and postconditioning protocols significantly reduce liver injury by decreasing the liver ROS and cytokine levels, as well as enhancing the hypoxia tolerance response. Our data also suggested that in addition to individual preconditioning or postconditioning, the combination of these two treatments could reduce liver ischemia/reperfusion injury more effectively by increasing the activity of ROS scavengers and antioxidants. The utilization of grape seed proanthocyanidins (GSP) could improve the oxidation resistance in combined pre- and postconditioning groups. The combined protocol also further increased the liver HIF-1 alpha protein level, but had no effect on pro-inflammatory cytokines release compared to solo treatment.

Cardiomyocytes can resist ischemia/reperfusion (I/R) injury through ischemic postconditioning (IPoC) which is repetitive ischemia induced during the onset of reperfusion. Myocardial ischemic preconditioning up-regulated protein 2 (MIP2) is a member of the WD-40 family proteins, we previously showed that MIP2 was up-regulated during ischemic preconditioning (IPC). As IPC and IPoC engaged similar molecular mechanisms in cardioprotection, this study aimed to elucidate whether MIP2 was up-regulated during IPoC and contributed to IPoC-mediated protection against I/R injury. The experiment was conducted on two models, an in vivo open chest rat coronary artery occlusion model and an in vitro model with H9c2 myogenic cells. In both models, 3 groups were constituted and randomly designated as the sham, I/R and IPoC/hypoxia postconditioning (HPoC) groups. In the IPoC group, after 45 min of ischemia, hearts were allowed three cycles of reperfusion/ischemia phases (each of 30 s duration) followed by reperfusion. In the HPoC group, after 6 h of hypoxia, H9c2 cells were subjected to three cycles of 10 minute reoxygenation and 10 minute hypoxia followed by reoxygenation. IPoC significantly reduced the infarct size, plasma level of Lactate dehydrogenase and creatine kinase MB in rats. 12 h after the reperfusion, MIP2 mRNA levels in the IPoC group were 10 folds that of the sham group and 1.4 folds that of the I/R group. Increased expression of MIP2 mRNA and attenuation of apoptosis were similarly observed in the HPoC group in the in vitro model. These effects were blunted by transfection with MIP2 siRNA in the H9c2 cells. This study demonstrated that IPoC induced protection was associated with increased expression of MIP2. Both MIP2 overexpression and MIP2 suppression can influence the IPoC induced protection.

OBJECTIVE

Ischemic preconditioning (PR) protects hearts from ischemia-reperfusion injury. The purpose of the present study was to examine the protective effect of PR and postconditioning (PT) against hypoxia-reoxygenation injury and H2O2-induced damage in isolated rat hearts.

METHODS AND RESULTS

Hearts from male Sprague-Dawley rats were perfused with Krebs-Henseleit solution by Langendorff methods and subjected to two protocols. In protocol A, control hearts underwent 45 min of hypoxia and 30 min of reoxygenation. Three PT cycles of 10 s of ischemia and 10 s of reperfusion after 45 min of hypoxia increased the recovery of the pressure-rate product. Three PR cycles of 3 min of ischemia and 5 min of reperfusion before hypoxia were also protective, and decreased the release of glutamic oxaloacetic transaminase. A combination of PR and PT resulted in greater protection than either alone. In protocol B, control hearts underwent perfusion with H2O2 (120 μM) until the left ventricular end-diastolic pressure was elevated to 50 mmHg, and then H2O2 was washed out for 30 min. Three PT cycles of 30 s of ischemia and 30 s of reperfusion before the 30 min washout increased the level of recovery of the pressure-rate product and decreased left ventricular end-diastolic pressure to baseline levels.

CONCLUSIONS

The results of the present study indicate that PT protects hearts from hypoxia-reoxygenation injury and H2O2-induced damage. In addition, PR combined with PT offers more effective protection than PR or PT alone.

Myocardial injury is increased in the aged heart following ischemia-reperfusion (ISC-REP) compared to adult hearts. Intervention at REP with ischemic postconditioning decreases injury in the adult heart by attenuating mitochondrial driven cell injury. Unfortunately, postconditioning is ineffective in aged hearts. Blockade of electron transport at the onset of REP with the reversible inhibitor amobarbital (AMO) decreases injury in adult hearts. We tested if AMO treatment at REP protects the aged heart via preservation of mitochondrial integrity. Buffer-perfused elderly Fischer 344 24 mo. rat hearts underwent 25 min global ISC and 30 min REP. AMO (2.5 mM) or vehicle was given for 3 min at the onset of REP. Subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria were isolated after REP. Oxidative phosphorylation (OXPHOS) and mitochondrial inner membrane potential were measured. AMO treatment at REP decreased cardiac injury. Compared to untreated ISC-REP, AMO improved inner membrane potential in SSM and IFM during REP, indicating preserved inner membrane integrity. Thus, direct pharmacologic modulation of electron transport at REP protects mitochondria and decreases cardiac injury in the aged heart, even when signaling-induced pathways of postconditioning that are upstream of mitochondria are ineffective.

Aging hearts are known to have diminished capacity to be protected against reoxygenation ischemia/reperfusion (IR) injury provided by various cardioprotective regimens. In search of a more successful regimen, we have studied the response of aged hearts to preconditioning (PC) and postconditioning (POST) elicited by sphingosine or sphingosine 1-phosphate treatment.

An ex vivo rat heart model was used to study the ability of PC and POST to protect old hearts (27 month) against I/R injury generated by 40 minutes (min) of index ischemia followed by 40 min of reperfusion. The response to ischemic PC was reduced in 27 month old hearts relative to 3–6 month (young) hearts as noted by a poor recovery of left ventricular developed pressure (LVDP) upon reperfusion (45% vs. 74% in young hearts) and a large infarct size after 40 min of reperfusion (37% versus 8% in young hearts). PC with sphingosine 1-phosphate (S1P) was also poor in old hearts yielding only 49% recovery of LVDP and a 27% infarct size. In contrast, PC with sphingosine was unaffected by aging; the 78% recovery of LVDP and 8% infarct size were not different from young hearts. Ischemic POST was less affected by aging than ischemic PC, but the old hearts still experienced infarct sizes of 28%. POST of old hearts with S1P was also associated with a substantial infarct size (24%). However, POST of old hearts with sphingosine was superior to the other forms of POST in that it reduced the infarct size to 12%. S1P levels were found to be lower in old hearts which may contribute to the decreased effectiveness of ischemic PC and POST. Further, phospho-Akt levels and distribution were altered in response to cardioprotection in the old hearts. In conclusion, POST was less affected by aging than PC; and sphingosine is a uniquely effective agent for both PC and POST of aging hearts.

Background

Ischemic postconditioning (IPost) has aroused much attention since 2003 when it was firstly reported. The role of microRNAs (miRNAs or miRs) in IPost has rarely been reported. The present study was undertaken to investigate whether miRNAs were involved in the protective effect of IPost against myocardial ischemia-reperfusion (IR) injury and the probable mechanisms involved.

Methods

Thirty SD rats weighing 250-300 g were equally randomized to three groups: Control group, where the rats were treated with thoracotomy only; IR group, where the rats were treated with ischemia for 60 min and reperfusion for 180 min; and IPost group, where the rats were treated with 3 cycles of transient IR just before reperfusion. The extent of myocardial infarction, LDH and CK activities were measured immediately after treatment. Myocardial apoptosis was detected by TUNEL assay. The myocardial tissue was collected after IR or IPost stimulation to evaluate the miRNAs expression level by miRNA-microarray and quantitative real-time RT-PCR. Real-time PCR was conducted to identify changes in mRNA expression of apoptosis-related genes such as Bcl-2, Bax and Caspase-9 (CASP9), and Western blot was used to compare the protein expression level of CASP9 in the three groups. The miRNA mimics and anti-miRNA oligonucleotides (AMO) were transferred into the cultured neonatal cardiomyocytes and myocardium before they were treated with IR. The effect of miRNAs on apoptosis was determined by flow cytometry and TUNEL assay. CASP9, as one of the candidate target of miR-133a, was compared during IR after the miR-133a mimic or AMO-133a was transferred into the myocardium.

Results

IPost reduced the IR-induced infarct size of the left ventricle, and decreased CK and LDH levels. TUNEL assay showed that myocardial apoptosis was attenuated by IPost compared with IR. MiRNA-microarray and RT-PCR showed that myocardial-specific miR-1 and miR-133a were down-regulated by IR, and up-regulated by IPost compared with IR. Furthermore, IPost up-regulated the mRNA expression of Bcl-2, down-regulated that of Bax and CASP9. Western blot showed that IPost also down-regulated the CASP9 protein expression compared with IR. The results of flow cytometry and TUNEL assay showed that up-regulation of miR-1 and miR-133a decreased apoptosis of cardiomyocytes. MiR-133a mimic down-regulated CASP9 protein expression and attenuated IR-induced apoptosis.

Conclusion

MiRNAs are associated with the protective effect of IPost against myocardial IR injury. IPost can up-regulate miR-1 and miR-133a, and decrease apoptosis of cardiomyocyte. Myocardial-specific miR-1 and miR-133a may play an important role in IPost protection by regulating apoptosis-related genes. MiR-133a may attenuate apoptosis of myocardiocytes by targeting CASP9.

Acid-sensing ion channels, ASICs, are proton-gated cation channels widely expressed in peripheral sensory neurons and in neurons of the central nervous system that play an important role in a variety of physiological and pathological processes. To further confirm the role played by ASIC1a in cerebral ischemia, here we examined the involvement of this channel in two endogenous recently characterized neuroprotective strategies: brain ischemic preconditioning and postconditioning. The main aim of this study was to elucidate whether ASIC1a might take part as effector in the neuroprotection evoked by brain ischemic preconditioning and postconditioning. For this purpose we investigated the effect of ischemic preconditioning and postconditioning on (1) ASIC1a mRNA and protein expression in the temporoparietal cortex of rats at different time intervals; and (2) the effect of p-AKT inhibition on ASIC1a expression during ischemic preconditioning and postconditioning. Ischemic preconditioning and postconditioning were experimentally induced in adult male rats by subjecting them to different protocols of middle cerebral artery occlusion and reperfusion. ASIC1a expression was dramatically reduced in both the neuroprotective processes. These changes in ASIC expression were p-AKT mediated, since LY-294002, a specific p-AKT inhibitor, was able to prevent variations in ASIC1a expression. The results of the present study support the idea that the downregulation of ASIC1a expression and activity might be a reasonable strategy to reduce the infarct extension after stroke.

The heart is an organ that requires an important energy input to ensure its contractile function. Myocardial ischemia happens when there is a deficiency of blood flow that is responsible for the passage from an aerobic to anaerobic metabolism. Myocardial ischemia results from an imbalance between inputs and the needs of nutrient and oxygen to the myocardium. The restoration of myocardial perfusion called reperfusion is a way to save the ischemic myocardium. However, although reperfusion is beneficial for the survival of the ischemic myocardium, it also induces a deleterious effect in addition to that of ischemic stress. Three decade ago, while several studies, strived to elucidate the protective effect of preconditioning, a phenomenon performed before ischemia and having a powerful protective effects against ischemia/reperfusion injury, very few have believed in the possibility of protecting the myocardium after ischemia (during reperfusion). Actually, both ischemic and pharmacological postconditioning as well as controlled reperfusion methods to protect the ischemic heart have proved effective in the reduction of damage related to ischemia/reperfusion. The possibility of protecting the myocardium during reperfusion opens a new area in the research against damage caused by ischemia/reperfusion because these methods are easily transferable in a clinic setting.

Ischemic preconditioning and postconditioning distinctly attenuate ventricular arrhythmia after ischemia without affecting the severity of myocardial stunning. Therefore, we report the effects of sevoflurane preconditioning and postconditioning on stunned myocardium in isolated rat hearts. Isolated rat hearts were underwent 20 min of global ischemia and 40 min of reperfusion. After an equilibration period (20 min), the hearts in the preconditioning group were exposed to sevoflurane for 5 min and next washout for 5 min before ischemia. Hearts in the sevoflurane postconditioning group underwent equilibration and ischemia, followed immediately by sevoflurane exposure for the first 5 min of reperfusion. The control group received no treatment before and after ischemia. Left ventricular pressure, heart rate, coronary flow, electrocardiogram, and tissue histology were measured as variables of ventricular function and cellular injury, respectively. There was no significant difference in the duration of reperfusion ventricular arrhythmias between control and sevoflurane preconditioning group (P=0.195). The duration of reperfusion ventricular arrhythmias in the sevoflurane postconditioning group was significantly shorter than that in the other two groups (P<0.05). ±(dP/dt)max in the sevoflurane preconditioning group at 5, 10, 15, 20, and 30 min after reperfusion was significantly higher than that in the control group (P<0.05), and there were no significant differences at 40 min after reperfusion among the three groups (P>0.05). As expected, for a 20-min general ischemia, infarct size in heart slices determined by 2,3,5-triphenyltetrazolium chloride staining among the groups was not obvious. Sevoflurane postconditioning reduces reperfusion arrhythmias without affecting the severity of myocardial stunning. In contrast, sevoflurane preconditioning has no beneficial effects on reperfusion arrhythmias, but it is in favor of improving ventricular function and recovering myocardial stunning. Sevoflurane preconditioning and postconditioning may be useful for correcting the stunned myocardium.

Although the protective mechanisms of delayed ischemic preconditioning have received extensive studies, few have addressed the mechanisms associated with rapid ischemic postconditioning. We investigated whether ischemic tolerance induced by rapid preconditioning is regulated by the Akt survival signaling pathway. Stroke was generated by permanent occlusion of the left distal middle cerebral artery (MCA) plus 30 min or 1 h occlusion of the bilateral common carotid artery (CCA) in male rats. Rapid preconditioning performed 1h before stroke onset reduced infarct size by 69% in rats with 30 min CCA occlusion, but by only 19% with 1 h occlusion. After control ischemia with 30 min CCA occlusion, Western Blot showed that P-Akt was transiently increased while Akt kinase assay showed that Akt activity was decreased. Although preconditioning did not change P-Akt levels at 1h and 5h compared with control ischemia, it attenuated reduction in Akt activity at 5h in the penumbra. However, preconditioning did not change the levels of P-PDK1, P-PTEN, and P-GSK3β in the Akt pathway, all of which were decreased after stroke. At last, the PI3K kinase inhibitor, LY294002, completely reversed the protection from ischemic preconditioning. In conclusion, Akt contributes to the protection of rapid preconditionin against stroke.