Ischemic postconditioning, including early and delayed ischemic postconditioning, has been recognized as a simple and promising strategy in the treatment of stroke. However, the effects of the combination of early and delayed ischemic postconditioning, and the mechanisms underlying these effects, remain unclear. The aim of the present study was to determine whether the combination of early and delayed ischemic postconditioning offers greater protection against stroke, and enhances the production of brain-derived neurotrophic factor (BDNF). A combination of early and delayed ischemic postconditioning was established by repeated, transient occlusion and reperfusion of the ipsilateral common carotid artery in a rat model of middle cerebral artery occlusion. Infarct size, motor function, cerebral blood flow and brain edema were then evaluated, in order to confirm the effects of combinative ischemic postconditioning. TUNEL staining was used to analyze the rate of apoptosis of cells in the penumbral area. BDNF, extracellular signal-regulated kinases 1/2 (ERK1/2) and cAMP response element-binding protein (CREB) expression was detected using immunofluorescence staining and western blot analysis. The results of the present study indicated that the combination of early and delayed ischemic postconditioning further reduced the infarct volume, stabilized cerebral blood disturbance and attenuated neuronal apoptosis, compared with either alone. However, combinative postconditioning exerted the same effect on neurological function and brain edema, compared with early or delayed ischemic postconditioning alone. Further investigation indicated that combinative ischemic postconditioning increased the expression of BDNF, and a significantly higher number of BDNF-positive cells was observed in neurons and astrocytes from the combined group than in the early or delayed groups. Combinative ischemic postconditioning also induced the phosphorylation of ERK1/2 and CREB in the cortex, following focal ischemia. The results of the present study suggest that the combination of early and delayed ischemic postconditioning may further reduce brain ischemic reperfusion injury following focal ischemia, compared with either treatment alone. In addition, it induces the production of BDNF in neurons and astrocytes. Furthermore, the effects of combinative ischemic postconditioning may be mediated by the activation of ERK1/2 and CREB.
combination; neuroprotection; brain-derived neurotrophic factor; ischemic postconditioning; focal brain ischemia
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.
Postconditioning; preconditioning; stroke; cerebral ischemia; focal ischemia; neuroprotection
Objective(s): It has been reported that ischemic postconditioning, conducted by a series of brief occlusion and release of the bilateral common carotid arteries, confers neuroprotection in permanent or transient models of stroke. However, consequences of postconditioning on embolic stroke have not yet been investigated.
Materials and Methods: In the present study, rats were subjected to embolic stroke (n=30) or sham stroke (n=5). Stroke animals were divided into control (n=10) or three different patterns of postconditioning treatments (n=20). In the first pattern of postconditioning (PC10, n=10), the common carotid arteries (CCA) were occluded and reopened 10 and 30 sec, respectively for 5 cycles. Both occluding and releasing times in pattern 2 (PC30, n=5) and 3 (PC60, N=5) of postconditionings, were five cycles of 30 or 60 sec, respectively. Postconditioning was induced at 30 min following the stroke. Subsequently, cerebral blood flow (CBF) was measured from 5 min before to 60 min following to stroke induction. Infarct size, brain edema and neurological deficits and reactive oxygen species (ROS) level was measured two days later.
Results: While PC10 (P<0.001), PC30 and PC60 (P<0.05) significantly decreased infarct volume, only PC10 decreased brain edema and neurological deficits (P<0.05). Correspondingly, PC10 prevented the hyperemia of brain at 35, 40, 50 and 60 min after the embolic stroke (P<0.005). No significant difference in ROS level was observed between PC10 and control group.
Conclusion: Ischemic postconditioning reduces infarct volume and brain edema, decreases hyperemia following to injury and improves neurological functions after the embolic model of stroke.
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.
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.
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.
These results suggest that ischemic postconditioning may not influence the early brain damage induced by focal cerebral ischemia in rats.
Focal cerebral ischemia; Neuroproctection; Postconditioning; Rat
The lack of efficient neuroprotective strategies for neonatal stroke could be ascribed to pathogenic ischemic processes differentiating adults and neonates. We explored this hypothesis using a rat model of neonatal ischemia induced by permanent occlusion of the left distal middle cerebral artery combined with 50 min of occlusion of both common carotid arteries (CCA). Postconditioning was performed by repetitive brief release and occlusion (30 s, 1 and/or 5 min) of CCA after 50 min of CCA occlusion. Alternative reperfusion was generated by controlled release of the bilateral CCA occlusion. Blood-flow velocities in the left internal carotid artery were measured using color-coded pulsed Doppler ultrasound imaging. Cortical perfusion was measured using laser Doppler. Cerebrovascular vasoreactivity was evaluated after inhalation with the hypercapnic gas or inhaled nitric oxide (NO). Whatever the type of serial mechanical interruptions of blood flow at reperfusion, postconditioning did not reduce infarct volume after 72 hours. A gradual perfusion was found during early re-flow both in the left internal carotid artery and in the cortical penumbra. The absence of acute hyperemia during early CCA re-flow, and the lack of NO-dependent vasoreactivity in P7 rat brain could in part explain the inefficiency of ischemic postconditioning after ischemia-reperfusion.
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.
stroke; cerebral ischemia; preconditioning; remote postconditioning
Application of isoflurane, a volatile anesthetic, after brain ischemia can reduce ischemic brain injury in rodents (isoflurane postconditioning). This study is designed to determine whether isoflurane postconditioning improves long-term neurological outcome after focal brain ischemia and whether this protection is mediated by attenuating neuroinflammation. Adult male Sprague–Dawley rats were subjected to a 90-min middle cerebral arterial occlusion (MCAO). Isoflurane postconditioning was performed by exposing rats to 2% isoflurane for 60 min immediately after the MCAO. Isoflurane postconditioning reduced brain infarct volumes, apoptotic cells in the ischemic penumbral brain tissues and neurological deficits of rats at 4 weeks after the MCAO. Isoflurane postconditioning reduced brain ischemia/reperfusion-induced nuclear transcription factor (NF)-κB (NF-κB) activation as well as interleukin 1β (IL-1β) and interleukin-6 production in the ischemic penumbral brain tissues at 24 h after the MCAO. IL-1β deficient mice had smaller brain infarct volumes and better neurological functions than wild-type mice at 24 h after a 90-min focal brain ischemia. Isoflurane posttreatment failed to induce neuroprotection in the IL-1β deficient mice. Our results suggest that isoflurane postconditioning improved long-term neurological outcome after transient focal brain ischemia. This protection may be mediated by inhibiting NF-κB activation and the production of the proinflammatory cytokine IL-1β.
interleukin 1β; isoflurane; neuroprotection; nuclear factor-κB; postconditioning
Background and Purpose
Postconditioning may be a clinically feasible way to protect the brain after a stroke. However, its effects during the recovery phase post-stroke remain to be fully elucidated. Here, we examine the hypothesis that ischemic postconditioning amplifies neurogenesis and angiogenesis during stroke recovery.
Male Sprague-Dawley rats were subjected to 100min transient middle cerebral artery occlusion (MCAO) or postconditioning (100min MCAO plus 10min reperfusion plus 10 min reocclusion). After 2 weeks, infarct volumes, behavioral outcomes and immunohistochemical markers of neurogenesis and angiogenesis were quantified.
Postconditioning significantly reduced infarction and improved neurologic outcomes. Concomitantly, brains subjected to postconditioning showed an increase in doublecortin/BrdU and collagen-IV/Ki67 positive cells.
These results suggest that therapeutic effects of postconditioning may involve the promotion of neurogenesis and angiogenic remodeling during the recovery phase after focal cerebral ischemia.
Focal ischemia; postconditioning; neurogenesis; angiogenesis; recovery
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.
Akt; cerebral ischemia; mitogen-activated protein kinase; postconditioning; protein kinase C; β-catenin
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.
rapid preconditioning; ischemic tolerance; cerebral ischemia; focal ischemia; neuroprotection; Akt
Brief episodes of ischemia and reperfusion after a lethal ischemic insult confer cardioprotection, a phenomenon termed “ischemic postconditioning.” However, all studies reported to date have been conducted in open-chest animal models. We sought to determine whether postconditioning occurs in conscious animals and whether it protects against severe myocardial injury.
Chronically instrumented rats were assigned to a 30- (Subset 1), 45- (Subset 2), or 60-min (Subset 3) coronary occlusion followed by 24 h of reperfusion. In each subset, rats received no further intervention (control), were preconditioned with 12 cycles of 2-min occlusion/2-min reperfusion immediately (early preconditioning; EPC) or 24 h (late preconditioning; LPC) before myocardial infarction, or were postconditioned with 20 cycles of 10-s occlusion/10-s reperfusion immediately after myocardial infarction (20-10 PostC).
With a 30-min occlusion, infarct size (54.4 ± 2.3% of risk region in control-30) was significantly reduced in EPC-30, LPC-30, and 20-10 PostC-30 groups (by 72, 70, and 47%, respectively; all P < 0.05 vs. control-30). With a 45-min occlusion, infarct size (62.2 ± 2.4% in control-45) was reduced in EPC-45 and LPC-45 groups (by 47 and 41%, respectively; all P < 0.05 vs. control-45) but not in the 20-10 PostC-45 group [55.4 ± 2.3%, P = not significant (NS) vs. control-45]. With a 60-min occlusion, infarct size (72.7 ± 2.2% in control-60) was reduced in the EPC-60 (by 20%, P < 0.05) but not in the LPC-60 (63.6 ± 2.5%, P = NS) or in the 20-20 PostC group (71.5 ± 3.4%, P = NS).
Both early and late ischemic preconditioning as well as ischemic postconditioning confer protection in conscious rats; however, unlike early preconditioning, postconditioning protects only against coronary occlusions <45 min. In the conscious rat, the cardioprotection afforded by postconditioning is limited to mild to moderate myocardial injury.
myocardium; ischemia; infarct size; preconditioning
Background and Purpose
Accumulating evidences have demonstrated that nuclear factor κB/p65 plays a protective role in the protection of ischemic preconditioning and detrimental role in lethal ischemia-induced programmed cell death including apoptosis and autophagic death. However, its role in the protection of ischemic postconditioning is still unclear.
Rat MCAO model was used to produce transient focal ischemia. The procedure of ischemic postconditioning consisted of three cycles of 30 seconds reperfusion/reocclusion of MCA. The volume of cerebral infarction was measured by TTC staining and neuronal apoptosis was detected by TUNEL staining. Western blotting was used to analyze the changes in protein levels of Caspase-3, NF-κB/p65, phosphor- NF-κB/p65, IκBα, phosphor- IκBα, Noxa, Bim and Bax between rats treated with and without ischemic postconditioning. Laser scanning confocal microscopy was used to examine the distribution of NF-κB/p65 and Noxa.
Ischemic postconditioning made transient focal ischemia-induced infarct volume decrease obviously from 38.6%±5.8% to 23.5%±4.3%, and apoptosis rate reduce significantly from 46.5%±6.2 to 29.6%±5.3% at reperfusion 24 h following 2 h focal cerebral ischemia. Western blotting analysis showed that ischemic postconditioning suppressed markedly the reduction of NF-κB/p65 in cytoplasm, but elevated its content in nucleus either at reperfusion 6 h or 24 h. Moreover, the decrease of IκBα and the increase of phosphorylated IκBα and phosphorylated NF-κB/p65 at indicated reperfusion time were reversed by ischemic postconditioning. Correspondingly, proapoptotic proteins Caspase-3, cleaved Caspase-3, Noxa, Bim and Bax were all mitigated significantly by ischemic postconditioning. Confocal microscopy revealed that ischemic postconditioning not only attenuated ischemia-induced translocation of NF-κB/p65 from neuronal cytoplasm to nucleus, but also inhibited the abnormal expression of proapoptotic protein Noxa within neurons.
We demonstrated in this study that the protection of ischemic postconditioning on neuronal apoptosis caused by transient focal ischemia is associated with attenuation of the activation of NF-κB/p65 in neurons.
Background and purpose
Ischemic postconditioning has been demonstrated to be a protective procedure to brain damage caused by transient focal ischemia/reperfusion. However, it is elusive whether the protection of postconditioning against brain damage and neuroinflammation is via regulating TLR2 and TLR4 pathways. In the present study, we examined the protection of ischemic postconditioning performed immediately prior to the recovery of cerebral blood supply on brain damage caused by various duration of ischemia and tested the hypothesis that its protection is via inhibition of neuroinflammation by modulating TLR2/TLR4 pathways.
Brain damage in rats was induced by using the middle cerebral artery occlusion (MCAO) model. Ischemic postconditioning consisting of fivecycles of ten seconds of ischemia and reperfusion was performed immediately following theischemic episode Theduration of administration of ischemic postconditioning was examined by comparing its effects on infarction volume, cerebral edema and neurological function in 2, 3, 4, 4.5and 6 hour ischemia groups. The protective mechanism of ischemic postconditioning was investigated by comparing its effects on apoptosis, production of the neurotoxic cytokine IL-1β and the transcription and expression of TLR2, TLR4 and IRAK4 in the 2 and 4.5 hour ischemia groups.
Ischemic postconditioning significantly attenuated cerebral infarction, cerebral edema and neurological dysfunction in ischemia groups of up to 4 hours duration, but not in 4.5and 6 hour ischemia groups. It also inhibited apoptosis, production of IL-1β, abnormal transcription and expression of TLR2, TLR4 and IRAK4 in the 2 hour ischemia group, but not in the 4.5 hour ischemia group.
Ischemic postconditioning protected brain damage caused by 2, 3 and 4 hours of ischemia, but not by 4.5 and 6 hours of ischemia. The protection of ischemic postconditioning is associated with its inhibition of neuroinflammation via inhibition of TLR2 and TLR4 pathways.
Ischemic postconditioning; Cerebral ischemia/reperfusion; TLR2; TLR4; Neuroinflammation
The present study sought to determine whether the combination of late preconditioning (PC) with postconditioning enhances the reduction in infarct size. Chronically instrumented rats were assigned to a 45-min (subset 1) or 60-min (subset 2) coronary occlusion followed by 24 h of reperfusion. In each subset, rats received no further intervention (control) or were preconditioned 24 h before occlusion (PC), post-conditioned at the onset of reperfusion following occlusion, or pre-conditioned and postconditioned without (PC + postconditioning) or with the COX-2 inhibitor celecoxib (3 mg/kg ip; PC + postconditioning + celecoxib) 10 min before postconditioning. Myocardial cyclooxygenase-2 (COX-2) protein expression and COX-2 activity (assessed as myocardial levels of PGE2) were measured 6 min after reperfusion in an additional five groups (control, PC, postconditioning, PC + postconditioning, and PC + postconditioning + celecoxib) subjected to a 45-min occlusion. PC alone reduced infarct size after a 45-min occlusion but not after a 60-min occlusion. Postconditioning alone did not reduce infarct size in either setting. However, the combination of late PC and postconditioning resulted in a robust infarct-sparing effect in both settings, suggesting additive cardioprotection. Celecoxib completely abrogated the infarct-sparing effect of the combined interventions in both settings. Late PC increased COX-2 protein expression and PGE2 content. PGE2 content (but not COX-2 protein) was further increased by the combination of both interventions, suggesting that postconditioning increases the activity of COX-2 induced by late PC. In conclusion, the combination of late PC and postconditioning produces additive protection, likely due to a postconditioning-induced enhancement of COX-2 activity.
myocardium; ischemia; infarct size; cyclooxygenase-2
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.
brain ischemia; KATP; remote ischemic postconditioning; reperfusion injury
Results of previous reports on ischemic postconditioning in animals and humans were very encouraging. Although ischemic postconditioning possessed a wide prospect of clinical application, debates on the precise ischemic postconditioning algorithm to use in clinical settings were ongoing. In this regard, pharmacological strategies were possible alternative methods. Accumulating data demonstrated that pharmacological postconditioning with morphine conferred cardioprotection in animals. This trial aimed to evaluate the effect of morphine-induced postconditioning on protection against myocardial ischemia/reperfusion injury in patients undergoing corrections of Tetralogy of Fallot (TOF).
Eight-nine consecutive children scheduled for corrections of TOF were enrolled and randomly assigned to either a postconditioning group (patients received a dose of morphine (0.1 mg/kg) injected via a cardioplegia needle into the aortic root for direct and focused delivery to the heart within 1 minute starting at 3 min before aorta cross-clamp removal, n=44) or a control group (the same protocol was performed as in the postconditioning group except that patients received the same volume of saline instead, n=45). The peri-operative relevant data were investigated and analyzed, and the cardiac troponin I (cTnI) was assayed preoperatively, and then 4 h, 8 h, 12 h, 24 h and 48 h after reperfusion.
Morphine-induced postconditioning reduced postoperative peak cTnI release as compared to the control group (0.57 ± 0.15 versus 0.75 ± 0.20 ng/mL, p<0.0001). Morphine-induced postconditioned patients had lower peak inotropic score (5.7 ± 2.4 versus 8.4 ± 3.6, p<0.0001) and shorter duration of mechanical ventilation as well as ICU stay (20.6 ± 6.8 versus 28.5 ± 8.3 hours, p<0.0001 and 40.4 ± 10.3 versus 57.8 ± 15.2 hours, p<0.0001, respectively), while higher left ventricular ejection fraction as well as cardiac output (0.57±0.15 versus 0.51±0.13, p=0.0467 and 1.39 ± 0.25 versus 1.24 ± 0.21 L/min, p=0.0029, respectively) as compared to the control group during the first postoperative 24 hours.
Morphine-induced postconditioning may provide enhanced cardioprotection against ischemia/reperfusion injury in children undergoing corrections of TOF.
Pharmacological postconditioning; Morphine; Ischemia reperfusion injury; Pediatric cardiac surgery; Trials
Postconditioning is a novel reperfusion technique to reduce ischemia-reperfusion injuries. The aim of the study was to investigate this method in an animal model of lower limb revascularization for purpose of preventing postoperative renal failure.
Bilateral lower limb ischemia was induced in male Wistar rats for 3 hours by infrarenal aorta clamping under narcosis. Revascularization was allowed by declamping the aorta. Postconditioning (additional 10 sec reocclusion, 10 sec reperfusion in 6 cycles) was induced at the onset of revascularization. Myocyte injury and renal function changes were assessed 4, 24 and 72 hours postoperatively. Hemodynamic monitoring was performed by invasive arterial blood pressure registering and a kidney surface laser Doppler flowmeter.
Muscle viability studies showed no significant improvement with the use of postconditioning in terms of ischemic rhabdomyolysis (4 h: ischemia-reperfusion (IR) group: 42.93 ± 19.20% vs. postconditioned (PostC) group: 43.27 ± 27.13%). At the same time, renal functional laboratory tests and kidney myoglobin immunohistochemistry demonstrated significantly less expressed kidney injury in postconditioned animals (renal failure index: 4 h: IR: 2.37 ± 1.43 mM vs. PostC: 0.92 ± 0.32 mM; 24 h: IR: 1.53 ± 0.45 mM vs. PostC: 0.77 ± 0.34 mM; 72 h: IR: 1.51 ± 0.36 mM vs. PostC: 0.43 ± 0.28 mM), while systemic hemodynamics and kidney microcirculation significantly improved (calculated reperfusion area: IR: 82.31 ± 12.23% vs. PostC: 99.01 ± 2.76%), and arterial blood gas analysis showed a lesser extent systemic acidic load after revascularization (a defined relative base excess parameter: 1st s: IR: 2.25 ± 1.14 vs. PostC: 1.80 ± 0.66; 2nd s: IR: 2.14 ± 1.44 vs. PostC: 2.44 ± 1.14, 3rd s: IR: 3.99 ± 3.09 vs. PostC: 2.07 ± 0.82; 4th s: IR: 3.28 ± 0.32 vs. PostC: 2.05 ± 0.56).
The results suggest a protective role for postconditioning in major vascular surgeries against renal complications through a possible alternative release of nephrotoxic agents and exerting a positive effect on hemodynamic stability.
Myoglobinuria; Postconditioning; Renal microcirculation; HSP72
Ischemic postconditioning (IPostC) has been shown to attenuate brain injury in rat stroke models, but a mouse model has not been reported. This study establishes an IPostC model in mice and investigates how IPostC affects infiltration of leukocytes in the ischemic brain and lymphopenia associated with stroke-induced immunodepression.
Material and Methods
A total of 125 mice were used. IPostC was performed by a repeated series of brief occlusions of the middle cerebral artery (MCA) after reperfusion, in a focal ischemia model in mice. Infarct sizes, neurological scores, inflammatory brain cells and immune cell populations in lymph nodes, spleen and bone marrow were analyzed with FACS.
IPostC performed immediately, 2 min and 3 hr after reperfusion significantly reduced infarct sizes and attenuated neurological scores as measured up to 3 days post-stroke. In the group with strongest protection, infarct sizes were reduced from 49.6 ± 2.8% (n=16) to 27.9 ± 2.9% (n=10, P<.001). The spared infarct areas were seen in the ischemic penumbra or ischemic margins, i.e., the border zones between the cortical territories of the anterior cerebral artery (ACA) and those of the MCA, as well as in the ventromedial and dorsolateral striatum. FACS analyses showed that IPostC significantly blocked increases in the numbers of microglia (CD45intCD11b+), macrophages (CD45hiCD68+), CD4 T cells (CD45+CD4+) and CD8 T cells (CD45+CD8+) as well as B lymphocytes (CD45+CD19+) in the ischemic brain (n=5/group). Reduced-immune cell numbers in the peripheral blood and spleen were increased by IPostC while immune cell populations in the bone marrow were not altered by IPostC.
IPostC reduced brain infarction and mitigated neurological deficits in mice, likely by blocking infiltration of both innate and adaptive immune cells in the ischemic brain. In addition, IPostC robustly attenuated peripheral lymphopenia and thus improved systemic immunodepression.
cerebral ischemia; postconditioning; infarction size; mouse model
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.
calcium/calmodulin-dependent protein kinase II; isoflurane; neuron; preconditioning; postconditioning
Cardioprotection against ischaemia/reperfusion injury in mice can be achieved by delayed ischaemic postconditioning (IPost) applied as late as 30 min after the onset of reperfusion. We determined the efficacy of delayed IPost in a rat model of myocardial infarction (MI) and investigated potential underlying mechanisms of this phenomenon. Rats were subjected to 20, 30 or 45 min of coronary artery occlusion followed by 120 min of reperfusion (I/R). Immediate and early IPost included six cycles of I/R (10/10 s) applied 10 s or 10 min after reperfusion onset. In the second series of experiments, the rats were subjected to 30 min of coronary occlusion followed by IPost applied 10 s, 10, 30, 45 or 60 min after the onset of reperfusion. Immediate and early IPost (applied 10 s or 10 min of reperfusion) established cardioprotection only when applied after a period of myocardial ischaemia lasting 30 min. Delayed IPost applied after 30 or 45 min of reperfusion reduced infarct sizes by 36 and 41 %, respectively (both P < 0.01). IPost applied 60 min after reperfusion onset was ineffective. Inhibition of RISK pathway (administration of ERK1/2 inhibitor PD-98059 or PI3K inhibitor LY-294002) abolished cardioprotection established by immediate IPost but had no effect on cardioprotection conferred by early IPost. Blockade of SAFE pathway using JAK/STAT inhibitor AG490 had no effect on the immediate or early IPost cardioprotection. Blockade of mitochondrial KATP (mitoKATP) channels (with 5-Hydroxydecanoate) abolished cardioprotection achieved by immediate and early IPost, but had no effect on cardioprotection when IPost was applied 30 or 45 min into the reperfusion period. Immediate IPost increased phosphorylation of PI3K-AKT and ERK1/2. Early or delayed IPost had no effect on phosphorylation of PI3K-AKT, ERK1/2 or STAT3. These data show that in the rat model, delayed IPost confers significant cardioprotection even if applied 45 min after onset of reperfusion. Cardioprotection induced by immediate and early postconditioning involves recruitment of RISK pathway and/or mitoKATP channels, while delayed postconditioning appears to rely on a different mechanism.
Ischaemia and reperfusion injury; MitoKATP channels; Myocardial infarction; Postconditioning; Preconditioning; RISK and SAFE pathways
Quercetin (Que), a plant-derived flavonoid, has multiple benefical actions on the
cardiovascular system. The current study investigated whether Que
postconditioning has any protective effects on myocardial ischemia/reperfusion
(I/R) injury in vivo and its potential cardioprotective
mechanisms. Male Sprague-Dawley rats were randomly allocated to 5 groups (20
animals/group): sham, I/R, Que postconditioning, Que+LY294002 [a
phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway inhibitor], and
LY294002+I/R. I/R was produced by 30-min coronary occlusion followed by 2-h
reperfusion. At the end of reperfusion, myocardial infarct size and biochemical
changes were compared. Apoptosis was evaluated by both TUNEL staining and
measurement of activated caspase-3 immunoreactivity. The phosphorylation of Akt
and protein expression of Bcl-2 and Bax were determined by Western blotting. Que
postconditioning significantly reduced infarct size and serum levels of creatine
kinase and lactate dehydrogenase compared with the I/R group (all P<0.05).
Apoptotic cardiomyocytes and caspase-3 immunoreactivity were also suppressed in
the Que postconditioning group compared with the I/R group (both P<0.05). Akt
phosphorylation and Bcl-2 expression increased after Que postconditioning, but
Bax expression decreased. These effects were inhibited by LY294002. The data
indicate that Que postconditioning can induce cardioprotection by activating the
PI3K/Akt signaling pathway and modulating the expression of Bcl-2 and Bax
Ischemia and reperfusion; Quercetin; Postconditioning; PI3K/Akt
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.
Postconditioning; Preconditioning; Cardioprotection; Ischemia; Reperfusion; Akt
Sevoflurane postconditioning reduces myocardial infarct size. The objective of this study was to examine the role of the phosphatidylinositol-3-kinase (PI3K)/Akt pathway in anesthetic postconditioning and to determine whether PI3K/Akt signaling modulates the expression of pro- and antiapoptotic proteins in sevoflurane postconditioning. Isolated and perfused rat hearts were prepared first, and then randomly assigned to the following groups: Sham-operation (Sham), ischemia/reperfusion (Con), sevoflurane postconditioning (SPC), Sham plus 100 nmol/L wortmannin (Sham+Wort), Con+Wort, SPC+Wort, and Con+dimethylsulphoxide (DMSO). Sevoflurane postconditioning was induced by administration of sevoflurane (2.5%, v/v) for 10 min from the onset of reperfusion. Left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), maximum increase in rate of LVDP (+dP/dt), maximum decrease in rate of LVDP (−dP/dt), heart rate (HR), and coronary flow (CF) were measured at baseline, R30 min (30 min of reperfusion), R60 min, R90 min, and R120 min. Creatine kinase (CK) and lactate dehydrogenase (LDH) were measured after 5 min and 10 min reperfusion. Infarct size was determined by triphenyltetrazolium chloride staining at the end of reperfusion. Total Akt and phosphorylated Akt (phospho-Akt), Bax, Bcl-2, Bad, and phospho-Bad were determined by Western blot analysis. Analysis of variance (ANOVA) and Student-Newman-Keuls’ test were used to investigate the significance of differences between groups. The LVDP, ±dP/dt, and CF were higher and LVEDP was lower in the SPC group than in the Con group at all points of reperfusion (P<0.05). The SPC group had significantly reduced CK and LDH release and decreased infarct size compared with the Con group [(22.9±8)% vs. (42.4±9.4)%, respectively; P<0.05]. The SPC group also had increased the expression of phosphor-Akt, Bcl-2, and phospho-Bad, and decreased the expression of Bax. Wortmannin abolished the cardioprotection of sevoflurane postconditioning. Sevoflurane postconditioning may protect the isolated rat heart. Activation of PI3K and modulation of the expression of pro- and antiapoptotic proteins may play an important role in sevoflurane-induced myocardial protection.
Sevoflurane; Postconditioning; Cardioprotection; Akt; Bcl-2; Bad
Background and Purpose
Whether the mammalian target of rapamycin (mTOR) pathway is protective against brain injury from stroke or is detrimental is controversial, and whether it is involved in the protective effects of ischemic postconditioning against stroke is unreported. Our study focuses on the protective role of mTOR against neuronal injury after stroke with and without ischemic postconditioning.
We used both an in vitro oxygen-glucose deprivation model with a mixed neuronal culture and hypoxic postconditioning, as well as an in vivo stroke model with ischemic postconditioning. Rapamycin, a specific pharmacological inhibitor of mTOR, and mTOR short hairpin RNA (shRNA) lentiviral vectors were used to inhibit mTOR activity. A lentiviral vector expressing S6K1, a downstream molecule of mTOR, was used to confirm the protective effects of mTOR. Infarct sizes were measured and protein levels were examined by Western blot.
We report that stroke resulted in reduced levels of phosphorylated proteins in the mTOR pathway, including S6K1, S6, and 4EBP1, and that ischemic postconditioning increased these proteins. mTOR inhibition, both by the mTOR inhibitor rapamycin and by mTOR shRNA, worsened ischemic outcomes in vitro and in vivo, and abolished the protective effects of hypoxic postconditioning and ischemic postconditioning on neuronal death in vitro and brain injury size in vivo. Overexpression of S6K1 mediated by lentiviral vectors significantly attenuated brain infarction.
mTOR plays a crucial protective role in brain damage after stroke and contributes to the protective effects of ischemic postconditioning.
ischemic postconditioning; stroke; mTOR; S6K1
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.
Akt; glycogen synthase kinase 3β; isoflurane; neuroprotection; mitochondrial membrane permeability; postconditioning