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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.

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.

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.

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.

Ischemic postconditioning refers to several transient reperfusion and ischemia cycles after an ischemic event and before a long duration of reperfusion. The procedure produces neuroprotective effects. The mechanisms underlying these neuroprotective effects are poorly understood. In this study, we found that most neurons in the CA1 region died after 10 minutes of ischemia and is followed by 72 hours of reperfusion. However, brain ischemic postconditioning (six cycles of 10 s/10 s reperfusion/re-occlusion) significantly reduced neuronal death. Significant up-regulation of Glutamate transporter-1 was found after 3, 6, 24, 72 hours of reperfusion. The present study showed that ischemic postconditioning decreases cell death and that upregulation of GLT-1 expression may play an important role on this effect.

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.

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.

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.

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.

Background

Ischaemic preconditioning results in a reduction in ischaemic‐reperfusion injury to the heart. This beneficial effect is seen both with direct local preconditioning of the myocardium and with remote preconditioning of easily accessible distant non‐vital limb tissue. Ischaemic postconditioning with a comparable sequence of brief periods of local ischaemia, when applied immediately after the ischaemic insult, confers benefits similar to preconditioning.

Objective

To test the hypothesis that limb ischaemia induces remote postconditioning and hence reduces experimental myocardial infarct size in a validated swine model of acute myocardial infarction.

Methods

Acute myocardial infarction was induced in 24 pigs with 90 min balloon inflations of the left anterior descending coronary artery. Remote ischaemic postconditioning was induced in 12 of the pigs by four 5 min cycles of blood pressure cuff inflation applied to the lower limb immediately after the balloon deflation. Infarct size was assessed by measuring 72 h creatinine kinase release, MRI scan and immunohistochemical analysis.

Results

Area under the curve of creatinine kinase release was significantly reduced in the postconditioning group compared with the control group with a 26% reduction in the infarct size (p<0.05). This was confirmed by MRI scanning and immunohistochemical analysis that revealed a 22% (p<0.05) and a 47.52% (p<0.01) relative reduction in the infarct size, respectively.

Conclusion

Remote ischaemic postconditioning is a simple technique to reduce infarct size without the hazards and logistics of multiple coronary artery balloon inflations. This type of conditioning promises clear clinical potential.

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.

During the past few decades, a large number of animal studies demonstrated that commonly used opioids could provide cardioprotection against ischemia-reperfusion (I/R) injury. Opioid-induced preconditioning or postconditioning mimics ischemic preconditioning (I-Pre) or ischemic postconditioning (I-Post). Both δ- and κ-opioid receptors (OPRs) play a crucial role in opioid-induced cardioprotection (OIC). Down stream signaling effectors of OIC include ATP-sensitive potassium (KATP) channels, protein kinase C (PKC), tyrosine kinase, phosphatidylinositol-3-kinase (PI3-kinase), extracellular signal regulated kinase1/2 (ERK1/2), glycogen synthase kinase-3β (GSK-3β), and mitochondrial permeability transition pore (MPTP), among others. Recently, various reports also suggest that opioids could provide cardioprotection in humans. This review will discuss OIC using mostly morphine and remifentanil which are widely used during cardiac anesthesia in addition to the clinical implications of OIC.

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.

The volatile anesthetic isoflurane is capable of inducing preconditioning and postconditioning effects in the brain. However, the mechanisms for these neuroprotective effects are not fully understood. Here, we showed that rat hippocampal neuronal cultures exposed to 2% isoflurane for 30 min at 24 h before a 1-h oxygen-glucose deprivation (OGD) and a 24-h simulated reperfusion had a reduced lactate dehydrogenase release. Similarly, this OGD and simulated reperfusion-induced lactate dehydrogenase release was attenuated by exposing the neuronal cultures to 2% isoflurane for 1 h at various times after the onset of the simulated reperfusion (isoflurane postconditioning). The combination of isoflurane preconditioning and postconditioning induced a better neuroprotection than either alone. Inhibition of the calcium/calmodulin-dependent protein kinase II (CaMKII), inhibition of N-methyl D-aspartate (NMDA) receptors, or activation of adenosine A2A receptors resulted in reduction of the OGD and simulated reperfusion-induced cell injury. The combination of CaMKII inhibition and isoflurane preconditioning or postconditioning did not provide better protection than CaMKII inhibition, isoflurane preconditioning, or isoflurane postconditioning alone. The combination of NMDA receptor inhibition and isoflurane postconditioning was not better than NMDA receptor inhibition or isoflurane postconditioning alone for neuroprotection. However, the combination of adenosine A2A receptor activation with either isoflurane preconditioning or isoflurane postconditioning induced a better neuroprotective effect than adenosine A2A receptor activation, isoflurane preconditioning, or isoflurane postconditioning alone. The combination of NMDA receptor inhibition and isoflurane preconditioning caused a better neuroprotective effect than NMDA receptor inhibition or isoflurane preconditioning alone. These results suggest that isoflurane preconditioning- and postconditioning-induced neuroprotection can be additive. Isoflurane preconditioning and isoflurane postconditioning may involve CaMKII inhibition, but may not involve adenosine A2A receptor activation. Inhibition of NMDA receptors may mediate the effects of isoflurane postconditioning, but not isoflurane preconditioning.

Reperfusion therapy must be applied as soon as possible to attenuate the ischemic insult of acute myocardial infarction (AMI). However reperfusion is responsible for additional myocardial damage, which likely involves opening of the mitochondrial permeability transition pore (mPTP). In reperfusion injury, mitochondrial damage is a determining factor in causing loss of cardiomyocyte function and viability. Major mechanisms of mitochondrial dysfunction include the long lasting opening of mPTPs and the oxidative stress resulting from formation of reactive oxygen species (ROS). Several signaling cardioprotective pathways are activated by stimuli such as preconditioning and postconditioning, obtained with brief intermittent ischemia or with pharmacological agents. These pathways converge on a common target, the mitochondria, to preserve their function after ischemia/reperfusion. The present review discusses the role of mitochondria in cardioprotection, especially the involvement of adenosine triphosphate-dependent potassium channels, ROS signaling, and the mPTP. Ischemic postconditioning has emerged as a new way to target the mitochondria, and to drastically reduce lethal reperfusion injury. Several clinical studies using ischemic postconditioning during angioplasty now support its protective effects, and an interesting alternative is pharmacological postconditioning. In fact ischemic postconditioning and the mPTP desensitizer, cyclosporine A, have been shown to induce comparable protection in AMI patients.

Short non-lethal ischemic episodes administered to hearts prior to (ischemic preconditioning, IPC) or directly after (ischemic postconditioning, IPost) ischemic events facilitate myocardial protection. Transferring coronary effluent collected during IPC treatment to un-preconditioned recipient hearts protects from lethal ischemic insults. We propose that coronary IPC effluent contains hydrophobic cytoprotective mediators acting via PI3K/Akt-dependent pro-survival signaling at ischemic reperfusion. Ex vivo rat hearts were subjected to 30 min of regional ischemia and 120 min of reperfusion. IPC effluent administered for 10 min prior to index ischemia attenuated infarct size by ≥55% versus control hearts (P < 0.05). Effluent administration for 10 min at immediate reperfusion (reperfusion therapy) or as a mimetic of pharmacological postconditioning (remote postconditioning, RIPost) significantly reduced infarct size compared to control (P < 0.05). The IPC effluent significantly increased Akt phosphorylation in un-preconditioned hearts when administered before ischemia or at reperfusion, while pharmacological inhibition of PI3K/Akt-signaling at reperfusion completely abrogated the cardioprotection offered by effluent administration. Fractionation of coronary IPC effluent revealed that cytoprotective humoral mediator(s) released during the conditioning phase were of hydrophobic nature as all hydrophobic fractions with molecules under 30 kDa significantly reduced infarct size versus the control and hydrophilic fraction-treated hearts (P < 0.05). The total hydrophobic effluent fraction significantly reduced infarct size independently of temporal administration (before ischemia, at reperfusion or as remote postconditioning). In conclusion, the IPC effluent retains strong cardioprotective properties, containing hydrophobic mediator(s) < 30 kDa offering cytoprotection via PI3K/Akt-dependent signaling at ischemic reperfusion.

Objective

The opening of the mitochondrial permeability transition pore (mPTP) at the time of myocardial reperfusion is a critical determinant of cell death. Emerging studies suggest that suppression of mPTP opening may underlie the cardioprotection elicited by both ischemic preconditioning (IPC) and postconditioning (IPost). To further evaluate the role of the mPTP in cardioprotection, we hypothesized that hearts deficient in cyclophilin-D (CYP-D-/-), a key component of the mPTP, will be resistant to cardioprotection conferred by ischemic and pharmacological preconditioning and postconditioning.

Methods and results

Male/female wild type or CYP-D-/- mice were subjected to 30 min of ischemia and 120 min of reperfusion. In wild type mice subjected to in vivo myocardial ischemia-reperfusion injury, a significant reduction in myocardial infarct size was observed with the following treatments (n ≥ 6/group; P < 0.05): (1) IPC (28±4% vs. 46.2±4% in control); (2) Diazoxide (5 mg/kg) pre-treatment (26.4±3% vs. 54±10% in vehicle control); (3) IPost-1 or IPost-2, three or six 10-s cycles of ischemia-reperfusion (27.2±3% and 32±4%, respectively vs. 46.2±4% in control); (4) Bradykinin (40 μg/kg) (28.3±1% vs. 48±4% in vehicle control); (5) cyclosporin-A (10 mg/kg) (32.3±3% vs. 48±4% in vehicle control) (6) sanglifehrin-A (25 mg/kg) (29.3±3% vs. 48±4% in vehicle control). Interestingly, however, no infarct-limiting effects were demonstrated in CYP-D-/- mice with the same treatment protocols: (27.9±5% in control vs. 31.2±7% with IPC, 30.2±5% with IPost-1, 24.7±8% with IPost-2; 30.1±4% in vehicle control vs. 26.4±7% with diazoxide; 24.6±4% in vehicle control vs. 24.9±5% with bradykinin, 26.8±7% with cyclosporin-A, 32.5±6% with sanglifehrin-A: n ≥ 6/group: P > 0.05).

Conclusion

This study demonstrates that the mPTP plays a critical role in the cardioprotection elicited by ischemic and pharmacological preconditioning and postconditioning.

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.

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.

Ischemic preconditioning (IPC) or postconditioning (Ipost) is proved to efficiently prevent ischemia/reperfusion injuries. Mortality of diabetic patients with acute myocardial infarction was found to be 2–6 folds higher than that of non-diabetic patients with same myocardial infarction, which may be in part due to diabetic inhibition of IPC- and Ipost-mediated protective mechanisms. Both IPC- and Ipost-mediated myocardial protection is predominantly mediated by stimulating PI3K/Akt and associated GSK-3β pathway while diabetes-mediated pathogenic effects are found to be mediated by inhibiting PI3K/Akt and associated GSK-3β pathway. Therefore, this review briefly introduced the general features of IPC- and Ipost-mediated myocardial protection and the general pathogenic effects of diabetes on the myocardium. We have collected experimental evidence that indicates the diabetic inhibition of IPC- and Ipost-mediated myocardial protection. Increasing evidence implies that diabetic inhibition of IPC- and Ipost-mediated myocardial protection may be mediated by inhibiting PI3K/Akt and associated GSK-3β pathway. Therefore any strategy to activate PI3K/Akt and associated GSK-3β pathway to release the diabetic inhibition of both IPC and Ipost-mediated myocardial protection may provide the protective effect against ischemia/reperfusion injuries.

Objective: To investigate the effect of ischemic postconditioning on protein aggregation caused by transient ischemia and reperfusion and to clarify its underlying mechanism.

Methods: Two-vessel-occluded transient global ischemia rat model was used. The rats in ischemic postconditioning group were subjected to three cycles of 30-s/30-s reperfusion/clamping after 15min of ischemia. Neuronal death in the CA1 region was observed by hematoxylin-eosin staining, and number of live neurons was assessed by cell counting under a light microscope. Succinyl-LLVY-AMC was used as substrate to assay proteasome activity in vitro. Protein carbonyl content was spectrophotometrically measured to analyze protein oxidization. Immunochemistry and laser scanning confocal microscopy were used to observe the distribution of ubiquitin in the CA1 neurons. Western blotting was used to analyze the quantitative alterations of protein aggregates, proteasome, hsp70 and hsp40 in cellular fractions under different ischemic conditions.

Results: Histological examination showed that the percentage of live neurons in the CA1 region was elevated from 5.21%±1.21% to 55.32%±5.34% after administration of ischemic postconditioning (P=0.0087). Western blotting analysis showed that the protein aggregates in the ischemia group was 32.12±4.87, 41.86±4.71 and 34.51±5.18 times higher than that in the sham group at reperfusion 12h, 24h and 48h, respectively. However, protein aggregates were alleviated significantly by ischemic postconditioning to 2.84±0.97, 13.72±2.13 and 14.37±2.42 times at each indicated time point (P=0.000032, 0.0000051 and 0.0000082). Laser scanning confocal images showed ubiquitin labeled protein aggregates could not be discerned in the ischemic postconditioning group. Further study showed that ischemic postconditioning suppressed the production of carbonyl derivatives, elevated proteasome activity that was damaged by ischemia and reperfusion, increased the expression of chaperone hsp70, and maintained the quantity of chaperone hsp40.

Conclusion: Ischemic postconditioning could rescue significantly neuronal death in the CA1 region caused by transient ischemia and reperfusion, which is closely associated with suppressing the formation of protein aggregation.

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.

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:

Ischemia reperfusion injury is partly responsible for the high mortality associated with induced myocardial injury and the reduction in the full benefit of myocardial reperfusion. Remote ischemic preconditioning, perconditioning, and postconditioning have all been shown to be cardioprotective. However, it is still unknown which one is the most beneficial. To examine this issue, we used adult male Wistar rat ischemia reperfusion models to compare the cardioprotective effect of these three approaches applied on double-sided hind limbs.

METHODS:

The rats were randomly distributed to the following five groups: sham, ischemia reperfusion, remote preconditioning, remote perconditioning, and remote post-conditioning. The ischemia/reperfusion model was established by sternotomy followed by a 30-min ligation of the left coronary artery and a subsequent 3-h reperfusion. Remote conditioning was induced with three 5-min ischemia/5-min reperfusion cycles of the double-sided hind limbs using a tourniquet.

RESULTS:

A lower early reperfusion arrhythmia score (1.50±0.97) was found in the rats treated with remote perconditioning compared to those in the ischemia reperfusion group (2.33±0.71). Meanwhile, reduced infarct size was also observed (15.27±5.19% in remote perconditioning, 14.53±3.45% in remote preconditioning, and 19.84±5.85% in remote post-conditioning vs. 34.47±7.13% in ischemia reperfusion, p<0.05), as well as higher expression levels of the apoptosis-relevant protein Bcl-2/Bax following global (ischemia/reperfusion) injury in in vivo rat heart models (1.255±0.053 in remote perconditioning, 1.463±0.290 in remote preconditioning, and 1.461±0.541 in remote post-conditioning vs. 1.003±0.159 in ischemia reperfusion, p<0.05).

CONCLUSION:

Three remote conditioning strategies implemented with episodes of double-sided hind limb ischemia/reperfusion have similar therapeutic potential for cardiac ischemia/reperfusion injury, and remote perconditioning has a greater ability to prevent reperfusion arrhythmia.

Activated protein C (APC) is known to be beneficial on ischemia reperfusion injury in myocardium. However, the protection mechanism of APC is not fully understood. The purpose of this study was to investigate the effects and possible mechanisms of APC on myocardial ischemic damage. Artificially ventilated anaesthetized Sprague-Dawley rats were subjected to a 30 min of left anterior descending coronary artery occlusion followed by 2 hr of reperfusion. Rats were randomly divided into four groups; Sham, I/R, APC preconditioning and postconditioning group. Myocardial infarct size, apoptosis index, the phosphorylation of ERK1/2, Bcl-2, Bax and cytochrome c genes and proteins were assessed. In APC-administrated rat hearts, regardless of the timing of administration, infarct size was consistently reduced compared to ischemia/reperfusion (I/R) rats. APC improved the expression of ERK1/2 and anti-apoptotic protein Bcl-2 which were significantly reduced in the I/R rats. APC reduced the expression of pro-apoptotic genes, Bax and cytochrome c. These findings suggest that APC produces cardioprotective effect by preserving the expression of proteins and genes involved in anti-apoptotic pathways, regardless of the timing of administration.