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
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
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.
Delayed postconditioning reduced ischemic injury after focal ischemia, which opens a new research avenue for stroke therapy and its underlying protective mechanisms.
We recently demonstrated that limb remote preconditioning (LRP) protects against focal ischemia measured 2 days post-stroke. Here, we studied whether LRP provides long-term protection and improves neurological function. We also investigated whether LRP transmits its protective signaling via the afferent nerve pathways from the preconditioned limb to the ischemic brain and whether inflammatory factors are involved in LRP, including the novel galectin-9/Tim-3 inflammatory cell signaling pathway, which induces cell death in lymphocytes. LRP in the left hind femoral artery was performed immediately before stroke. LRP reduced brain injury size both at 2 days and 60 days post-stroke and improved behavioral outcomes for up to 2 months. The sensory nerve inhibitors capsaicin and hexamethonium, a ganglion blocker, abolished the protective effects of LRP. In addition, LRP inhibited edema formation and blood-brain barrier (BBB) permeability measured 2 days post-stroke. Western blot and immunostaining analysis showed that LRP inhibited protein expression of both galectin-9 and T-cell immunoglobulin domain and mucin domain 3 (Tim-3), which were increased after stroke. In addition, LRP decreased iNOS and nitrotyrosine protein expression after stroke. In conclusion, LRP executes long-term protective effects against stroke and may block brain injury by inhibiting activities of the galectin-9/Tim-3 pathway, iNOS, and nitrotyrosine.
Protection of remote ischemic preconditioning on neurocognitive function caused by bilateral common carotid artery occlusion has been investigated in rats.
Thirty-six male Sprague-Dawley rats were divided into 3 groups – control group (Group C, n=12), bilateral carotid arteries occlusion group (Group B, n=12) and remote ischemic precondition group (Group P, n=12). In Group P, remote ischemic preconditioning (RIPC) was performed on the right femoral artery with 3 cycles (10 min) of occlusion/perfusion. After 3 cycles of preconditioning, bilateral carotid arteries were occluded immediately for 60 min. In Group B, ischemic insults were conducted without RIPC. Sham surgeries were performed in Group C. Evaluation of memory and learning capacity was performed on days 5–8 after surgery by Morris water maze testing of spatial learning capacity (n=6 for each group). Apoptosis of cells in the hippocampus region was determined by TUNEL tests and Bcl-2 at this region was determined by ELISA 24 h and 9 days after vessel occlusion (n=6 for each group).
Neurocognitive tests showed that latency time was significantly longer in Group B than in Group P on day 7 (p=0.016) and day 8 (p=0.036). Moreover, frequency of platform crossings was significant less in group B than in the other 2 groups on day 9. Bcl-2 level was significantly increased in the hippocampal region of rats in Group P on days 1 and 9 after vessel occlusion. TUNEL test showed that apoptosis could be observed at 24 h after occlusion in Group B, but not in Group P and Group C. No apoptosis was observed on day 9.
Our results suggest that RIPC can protect neurocognitive function of rats after bilateral carotid occlusions, and that Bcl-2 may play an important role in this protective effect.
remote ischemic preconditioning; neurocognition; water maze; Bcl-2
The aim of our study was to investigate whether remote preconditioning (RPC) improves myocardial function after ischemia/reperfusion injury in both normal and hypertrophic isolated rat hearts. This is the first time in world literature that cardioprotection by RPC in hypertrophic myocardium is investigated.
Four groups of 7 male Wistar rats each, were used: Normal control, normal preconditioned, hypertrophic control and hypertrophic preconditioned groups. Moderate cardiac hypertrophy was induced by fludrocortisone acetate and salt administration for 30 days. Remote preconditioning of the rat heart was achieved by 20 minutes transient right hind limb ischemia and 10 minutes reperfusion of the anaesthetized animal. Isolated Langendorff-perfused animal hearts were then subjected to 30 minutes of global ischemia and reperfusion for 60 minutes. Contractile function and heart rhythm were monitored. Preconditioned groups were compared to control groups.
Left ventricular developed pressure (LVDP) and the product LVDP × heart rate (HR) were significantly higher in the hypertrophic preconditioned group than the hypertrophic control group while left ventricular end diastolic pressure (LVEDP) and severe arrhythmia episodes did not differ. Variances between the normal heart groups were not significantly different except for the values of the LVEDP in the beginning of reperfusion.
Remote preconditioning seems to protect myocardial contractile function in hypertrophic myocardium, while it has no beneficial effect in normal myocardium.
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.
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.
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).
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.
Cardioprotective Property; Ischemia/Reperfusion Injury; Models
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.
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.
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.
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.
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.
A brief ischemic insult induces significant protection against subsequent massive ischemic events. The molecular mechanisms known as preconditioning (PC)-induced ischemic tolerance are not completely understood. We investigated whether kinetic changes of cyclooxygenase (COX)-2 during reperfusion time-periods after PC were related to ischemic tolerance. Rats were given PC by occlusion of middle cerebral artery (MCAO) for 10 min and sacrificed after the indicated time-periods of reperfusion (1, 2, 4, 8, 12, 18 or 24 h). In PC-treated rats, focal ischemia was induced by occlusion of MCA for 24 h and brain infarct volume was then studied to determine whether different reperfusion time influenced the damage. We report that the most significant protection against focal ischemia was obtained in rats with 8 h reperfusion after PC. Administration of indomethacin (10 mg/kg, oral) or rofecoxib (5 mg/kg, oral) 48 h prior to PC counteracted the effect of PC. Immunohistochemical analysis showed that COX-2 and HO-1 protein were induced in PC-treated rat brain, which was significantly inhibited by rofecoxib. Taken together, we concluded that the kinetic changes of COX-2 expression during the reperfusion period after PC might be partly responsible for ischemic tolerance.
Ischemic preconditioning; Stroke; Heme oxygenase; Cyclooxygenase
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
Ischaemia–reperfusion (IR) injury causes endothelium-dependent vasomotor dysfunction that can be prevented by ischaemic preconditioning. The effects of IR injury and preconditioning on endothelium-dependent tissue plasminogen activator (t-PA) release, an important mediator of endogenous fibrinolysis, remain unknown.
Methods and results
Ischaemia–reperfusion injury (limb occlusion at 200 mmHg for 20 min) was induced in 22 healthy subjects. In 12 subjects, IR injury was preceded by local or remote ischaemic preconditioning (three 5 min episodes of ipsilateral or contralateral limb occlusion, respectively) or sham in a randomized, cross-over trial. Forearm blood flow (FBF) and endothelial t-PA release were assessed using venous occlusion plethysmography and venous blood sampling during intra-arterial infusion of acetylcholine (5–20 µg/min) or substance P (2–8 pmol/min). Acetylcholine and substance P caused dose-dependent increases in FBF (P<0.05 for all). Substance P caused a dose-dependent increase in t-PA release (P<0.05 for all). Acetylcholine and substanceP-mediated vasodilatation and substanceP-mediated t-PA release were impaired following IR injury (P<0.05 for all). Neither local nor remote ischaemic preconditioning protected against the impairment of substance P-mediated vasodilatation or t-PA release.
Ischaemia–reperfusion injury induced substanceP-mediated, endothelium-dependent vasomotor and fibrinolytic dysfunction in man that could not be prevented by ischaemic preconditioning.
Clinical Trial Registration Information: Reference number: NCT00789243, URL: http://clinicaltrials.gov/ct2/show/NCT00789243?term=NCT00789243&rank=1
Endogenous fibrinolysis; Endothelium; Ischaemia–reperfusion; Preconditioning; Substance P
Reactive oxygen species (ROS) mediate the effects of anesthetic precondition to protect against ischemia and reperfusion injury, but the mechanisms of ROS generation remain unclear. In this study, we investigated if mitochondria-targeted antioxidant (mitotempol) abolishes the cardioprotective effects of anesthetic preconditioning. Further, we investigated the mechanism by which isoflurane alters ROS generation in isolated mitochondria and submitochondrial particles.
Rats were pretreated with 0.9% saline, 3.0 mg/kg mitotempol in the absence or presence of 30 min exposure to isoflurane. Myocardial infarction was induced by left anterior descending artery occlusion for 30 min followed by reperfusion for 2h and infarct size measurements. Mitochondrial ROS production was determined spectrofluorometrically. The effect of isoflurane on enzymatic activity of mitochondrial respiratory complexes was also determined.
Isoflurane reduced myocardial infarct size (40±9 % = mean±SD) compared to control experiments (60±4 %). Mitotempol abolished the cardioprotective effects of anesthetic preconditioning (60±9%). Isoflurane enhanced ROS generation in submitochondrial particles with NADH, but not with succinate, as substrate. In intact mitochondria, isoflurane enhanced ROS production in the presence of rotenone, antimycin A, or ubiquinone when pyruvate and malate were substrates, but isoflurane attenuated ROS production when succinate was substrate. Mitochondrial respiratory experiments and electron transport chain complex assays revealed that isoflurane inhibited only complex I activity.
The results demonstrated that isoflurane produces ROS at complex I and III of the respiratory chain via the attenuation of complex I activity. The action on complex I decreases unfavorable reverse electron flow and ROS release in myocardium during reperfusion.
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
We hypothesized that activation of the central histaminergic system is required for neuroprotection induced by hypoxic preconditioning. Wild-type (WT) and histidine decarboxylase knockout (HDC-KO) mice were preconditioned by 3 hours of hypoxia (8% O2) and, 48 hours later, subjected to 30 minutes of middle cerebral artery (MCA) occlusion, followed by 24 hours of reperfusion. Hypoxic preconditioning improved neurologic function and decreased infarct volume in WT or HDC-KO mice treated with histamine, but not in HDC-KO or WT mice treated with α-fluoromethylhistidine (α-FMH, an inhibitor of HDC). Laser-Doppler flowmetry analysis showed that hypoxic preconditioning ameliorated cerebral blood flow (CBF) in the periphery of the MCA territory during ischemia in WT mice but not in HDC-KO mice. Histamine decreased in the cortex of WT mice after 2, 3, and 4 hours of hypoxia, and HDC activity increased after 3 hours of hypoxia. Vascular endothelial growth factor (VEGF) mRNA and protein expressions showed a greater increase after hypoxia than those in HDC-KO or α-FMH-treated WT mice. In addition, the VEGF receptor-2 antagonist SU1498 prevented the protective effect of hypoxic preconditioning in infarct volume and reversed increased peripheral CBF in WT mice. Therefore, endogenous histamine is an essential mediator of hypoxic preconditioning. It may function by enhancing hypoxia-induced VEGF expression.
histamine; hypoxic preconditioning; vascular endothelial growth factor
Although a major mechanism for cardioprotection is altered metabolism, little is known regarding metabolic changes in ischaemic preconditioning and subsequent ischaemia. Our objective was to examine the effects of the second window of preconditioning (SWOP), the delayed phase of preconditioning against infarction and stunning, on long-chain free fatty acid (LCFA) oxidation during ischaemia in chronically instrumented, conscious pigs.
Methods and results
We studied three groups: (i) normal baseline perfusion (n = 5); (ii) coronary artery stenosis (CAS; n = 5); (iii) CAS 24 h following 2 × 10 min coronary occlusions and 10 min reperfusion (n = 7). Ischaemia was induced by a left anterior descending (LAD) stenosis (40% flow reduction) for 90 min, dropping systolic wall thickening by 72%. LCFA oxidation was assessed following LAD infusion of 13C palmitate, i.e. during control or stenosis, by in vitro nuclear magnetic resonance of the sampled myocardium. Stenosis reduced subendocardial blood flow subendocardially, but not subepicardial, yet induced transmural reductions in LCFA oxidation and increased non-oxidative glycolysis. During stenosis, preconditioned hearts showed normalized contributions of LCFA to oxidative ATP synthesis, despite increased lactate accumulation. SWOP induced a shift towards LCFA oxidation during stenosis, despite increased malonyl-CoA, and marked protection of contractile function with a significant improvement in systolic wall thickening.
Thus, the second window of preconditioning normalized oxidative metabolism of LCFA during subsequent ischaemia despite elevated non-oxidative glycolysis and malonyl-CoA and was linked to protection of regional contractile function resulting in improved mechanical performance. Interestingly, the metabolic responses occurred transmurally while ischaemia was restricted solely to the subendocardium.
Mitochondria; Coronary stenosis; Long-chain fatty acids; β-Oxidation; Ischaemic preconditioning
Remote ischaemic preconditioning (RIPC) gained attention as a possibility to reduce myocardial injury after a subsequent sustained episode of myocardial ischaemia. This prospective randomized study was carried out to assess whether RIPC reduces myocardial injury in coronary artery bypass grafting patients. Eighty patients were assigned to remote preconditioning or control treatment. Ischaemic preconditioning was induced by three 5-min cycles of upper limb ischaemia and reperfusion after anaesthesia induction. Haemodynamic and markers of myocardial damage were analysed preoperatively and over 48 h postoperatively. The cardiac index was higher immediately after remote preconditioning in the main group. There were no differences in other haemodynamic, troponin I and creatine kinase-MB concentrations at any time point between groups. Thus, short-term remote preconditioning improves haemodynamics and does not reduce myocardial injury after coronary artery bypass surgery. Further study of high-risk patients may be needed to fully evaluate the clinical effect of RIPC.
Remote ischaemic preconditioning; Myocardial protection; Troponin I; Coronary artery bypass grafting
The present study further identified factors involved in the cardioprotective phenomenon of remote preconditioning of trauma (RPCT) with special emphasis on the role of the epoxyeicosatrienoic acids (EETs) in mediating this phenomenon. RPCT was produced by an abdominal incision only through the skin. Subsequently, all rats were subjected to 30 min of left coronary artery occlusion followed by 2 h of reperfusion and infarct size was determined. RPCT produced a reduction in infarct size expressed as a percent of the area-at-risk from 63.0±1.1 to 44.7±1.4%, P<0.01 vs. control. To test the 3 major triggers of classical preconditioning in mediating RPCT, blockers of the bradykinin B2 receptor (B2BK), WIN 64338 (1 mg/kg, iv), or HOE 140 (50 µg/kg, iv), the nonselective opioid receptor blocker, naloxone (3mg/kg, iv), or the adenosine A1 receptor blocker, DPCPX (1mg/kg, iv) were administered 10 min prior to RPCT. Only the two B2BK selective antagonists blocked RPCT (60.2±1.1%, WIN 64338; 62.3±2.0%, HOE 140). To test EETs in RPCT, we administered the EET receptor antagonist 14,15-EEZE (2.5 mg/kg, iv) or the EET synthesis inhibitor, MSPPOH (3.0 mg/kg, iv) 10 min prior to RPCT. In both groups, EET antagonists completely blocked RPCT (62.0±0.8%, 14,15-EEZE; 61.8±1.0%, MSPPOH). EET antagonists also blocked the effect of B2BK activation. We also determined if the sarcolemmal KATP or the mitochondrial KATP channel mediate RPCT by pretreating rats with HMR 1098, or 5-HD, respectively. Interestingly, 5-HD blocked RPCT (64.7±1.3%), whereas, HMR 1098 did not (50.3±1.3%). The two EET antagonists completely blocked capsaicin-induced cardioprotection. These results clearly suggest that EETs mediate RPCT-, bradykinin- and capsaicin-induced cardioprotection in rat hearts.
Background and Purpose
Remote ischemic postconditoning, a phenomenon in which brief ischemic stimuli of 1 organ protect another organ against an ischemic insult, has been demonstrated to protect the myocardium and adult brain in animal models. However, mediators of the protection and underlying mechanisms remain to be elucidated. In the present study, we tested the hypothesis that remote limb ischemic postconditioning applied immediately after hypoxia provides neuroprotection in a rat model of neonatal hypoxia–ischemia (HI) by mechanisms involving activation of the opioid receptor/phosphatidylinositol-3-kinase/Akt signaling pathway.
HI was induced in postnatal Day 10 rat pups by unilateral carotid ligation and 2 hours of hypoxia. Limb ischemic postconditioning was induced by 4 conditioning cycles of 10 minutes of ischemia and reperfusion on both hind limbs immediately after HI. The opioid antagonist naloxone, phosphatidylinositol-3-kinase inhibitor wortmannin, or opioid agonist morphine was administered to determine underlying mechanisms. Infarct volume, brain atrophy, and neurological outcomes after HI were evaluated. Expression of phosphorylated Akt, Bax, and phosphorylated ERK1/2 was determined by Western blotting.
Limb ischemic postconditioning significantly reduced infarct volume at 48 hours and improved functional outcomes at 4 weeks after HI. Naloxone and wortmannin abrogated the postconditioning-mediated infarct-limiting effect. Morphine given immediately after hypoxia also decreased infarct volume. Furthermore, limb ischemic postconditioning recovered Akt activity and decreased Bax expression, whereas no differences in phosphorylated ERK1/2expression were observed.
Limb ischemic postconditioning protects against neonatal HI brain injury in rats by activating the opioid receptor/phosphatidylinositol-3-kinase/Akt signaling pathway.
Akt; limb ischemic postconditioning; neonatal hypoxia–ischemia; opioid receptor
To test the hypothesis that a critical stenosis prevents delayed preconditioning against stunning, studies were conducted in pigs chronically-instrumented with occluders and segment-shortening crystals. In the setting of a critical stenosis, a preconditioning stimulus of repetitive brief occlusions resulted in infarction. Thereafter, a single 10-minute occlusion was used as the preconditioning stimulus. Delayed preconditioning against stunning was documented on subsequent days by the deficit-of-function following brief repetitive occlusions. In contrast to experiments in the naïve heart, the deficit-of-function improved on the day after a single 10-minute occlusion (from 60±14 to 24±6 arbitrary units, p=0.003), and similar improvement occurred when reperfusion was performed through a critical stenosis (32±6 units, p=0.02 vs. naïve and p=0.34 vs. no stenosis). Delayed preconditioning also reduced the frequency of ventricular fibrillation, and produced a 4-fold increase in both calcium-dependent and calcium-independent NOS activity. Thus, a critical stenosis did not prevent delayed preconditioning against stunning.
Late or Delayed Preconditioning; Preconditioning against Stunning; Ischemia; Arrhythmias
Brief systemic hypoxia protects the rodent brain from subsequent ischemic injury, although the protection wanes within days. We hypothesized that the duration of ischemic tolerance could be extended from days to months by repeated intermittent hypoxia of varying magnitude and duration.
Infarction volumes following a 60-min transient middle cerebral artery occlusion were determined in adult male mice 2 days through 8 wks after completion of a 2-week repetitive hypoxic preconditioning (RHP) protocol. Separate cohorts were studied for the protective effects of RHP on postischemic and cytokine-induced cerebrovascular inflammation, and for potential deleterious effects of the RHP stimulus itself.
RHP protection against transient focal stroke persisted for 8 weeks. Leukocyte adherence to cortical venules was attenuated in response to stroke, as well as following TNF-α administration, indicating that reductions in postischemic inflammation were not secondary to smaller infarct volumes. RHP reduced post-stroke leukocyte diapedesis concomitant with a long-lasting downregulation of endothelial adhesion molecule mRNAs, and also reduced postischemic blood-brain barrier permeability to endogenous IgG. RHP was without effect on hippocampal CA1 pyramidal cell viability, only transiently elevated hematocrit, and did not affect the magnitude of CBF during and after ischemia.
Taken together, our findings reveal a novel form of epigenetic neurovascular plasticity characterized by a prominent anti-inflammatory phenotype that provides protection against stroke many weeks longer than previously established windows of preconditioning-induced tolerance. Translating these endogenous protective mechanisms into therapeutics could afford sustained periods of cerebroprotection in subpopulations of individuals at identified risk for stroke.
Remote preconditioning is a phenomenon in which brief episodes of ischemia and reperfusion to remote organs protect the target organ against sustained ischemia/reperfusion (I/R)-induced injury. Protective effects of remote aortic preconditioning (RAPC) are well established in the heart, but their mechanisms still remain to be elucidated.
This study has been designed to investigate the possible involvement of α-1-adrenergic receptor (AR) and KATP channels in cardio-protective effect of RAPC in isolated rat heart.
Materials and Methods:
Four episodes of ischemia and reperfusion, each comprising of 5 min occlusion and 5 min reperfusion, were used to produce RAPC. Isolated perfused rat heart was subjected to global ischemia for 30 min followed by reperfusion for 120 min. Coronary effluent was analyzed for LDH and CK-MB release to assess the degree of cardiac injury. Myocardial infarct size was estimated macroscopically using TTC staining.
Phenylephrine (20 μ/kg i.p.), as α-1-AR agonist, was noted to produce RAPC-like cardio-protection. However, administration of glibenclamide concomitantly or prior to phenylephrine abolished cardioprotection. Moreover, prazocin (1 mg/kg. i.p), as α-1-AR antagonist and glibenclamide (1 mg/kg i.p), a KATP channel blocker, abolished the cardioprotective effect of RAPC.
These data provide the evidence that α-1-AR activation involved in cardioprotective effect of RAPC-mediated trough opening of KATP channels.
Cardio-protection; ischemic preconditioning; ischemia / reperfusion injury; remote aortic preconditioning
Background and Purpose
Making a limb transiently ischemic has been shown to induce ischemic tolerance in a distant organ. This phenomenon is known as remote ischemic limb preconditioning. We conducted a Phase IB study of remote ischemic limb preconditioning to determine the safety and feasibility of increasing durations of limb ischemia in patients with subarachnoid hemorrhage.
Patients with aneurysmal subarachnoid hemorrhage underwent limb preconditioning every 24 to 48 hours for 14 days. Limb preconditioning consisted of 3 5-minute inflations of a blood pressure cuff to 200 mm Hg around a limb followed by 5 minutes of reperfusion. In the lead-in phase, we preconditioned the upper extremities, but this proved impractical and we began preconditioning the leg in a similar manner. Ischemia times were then escalated to 7.5 and 10 minutes. After each session, a visual analog scale was obtained and the extremity examined for neurovascular complications.
A total of 33 patients completed the study. Mean age was 53±12 years and mean Hunt Hess score was 2.4±0.9. In the lead-in phase, an average of 7.7±2.4 preconditioning sessions was completed with mean visual analog scale 3.6±3.4. In the dose escalation phase, an average of 8.6±2.1 preconditioning sessions was done with mean visual analog scale 1.8±2.2 and 2.5±2.9 for the 7.5- and 10-minute cohorts, respectively. No session was prematurely terminated due to subject discomfort. No objective signs of neurovascular injury were observed.
We found limb preconditioning to be safe and well tolerated, even at ischemia times of 10 minutes, in critically ill patients with subarachnoid hemorrhage.
delayed cerebral ischemia; limb preconditioning; remote ischemic preconditioning
Although many studies have shown the great potential of induced hypothermia in stroke treatment, we recognize that there are limitations to the protective effects of hypothermia even in the laboratory. Here, we review our experiments on the protective effects of mild-to-moderate hypothermia in rats. Focal ischemia was induced by bilateral common carotid artery (CCA) occlusion for 1 to 2 hours combined with permanent or transient middle cerebral artery (MCA) occlusion. We compared the effects of mild (33°C) and moderate (30°C) hypothermia, evaluated therapeutic time windows, and studied the underlying mechanisms. On review, our findings revealed that the protective effects of induced mild hypothermia (33°C) were limited, and the therapeutic time window of even moderate hypothermia (30°C) was very short in our specific models, although this limitation might be due to the relatively brief periods of hypothermia used. In addition, we found that hypothermia reduced brain injury by preserving Akt activity, PTEN phosphorylation and εPKC activity, while inhibiting ROS production, and δPKC activity.
Ischemic preconditioning (IPC) provides protection against subsequent severe ischemic injury. A recent study found that cerebral IPC prolongs bleeding time. In this study, we examined whether IPC protects against intra-cerebral hemorrhage (ICH)-induced brain edema formation and whether IPC affects blood coagulation. There were three sets of experiments in this study. In the first set, male Sprague–Dawley rats were preconditioned with either 15 min of left middle cerebral artery occlusion, an IPC stimulus, or a sham operation. Three days later, rats received an infusion of autologous whole blood in the ipsilateral or contralateral caudate. Rats were killed 24 h later for brain water content measurement. In the second set, rats underwent 15 min of IPC or a sham operation. Three days later, rats were used for bleeding and thrombin clotting time tests. In the third set, the levels of p44/42 mitogen-activated protein kinases (MAPKs), heme oxygenase-1 (HO-1), transferrin (Tf), and transferrin receptor (TfR) in the brain 24 or 72 h after IPC were examined. We found that IPC reduced ICH-induced brain edema when blood was injected into the ipsilateral caudate but it did not when blood was injected into the contralateral caudate. IPC resulted in prolongation of bleeding time and thrombin clotting time. IPC also induced the activation of p44/42 MAPKs and upregulation of HO-1, Tf, and TfR levels in the ipsilateral caudate. These results suggest that IPC protects against ICH-induced brain edema formation and decreases blood coagulation. The protection of IPC against ICH is mainly due to local factors in the brain and may be related to activation of p44/42 MAPKs and upregulation of HO-1, Tf, and TfR.
Brain edema; Ischemic preconditioning; p44/42 mitogen activated protein kinases; Heme oxygenase-1; Transferrin; Transferrin receptor; Intracerebral hemorrhage
Brief episodes of cerebral hypoxia-ischemia cause transient ischemic tolerance to subsequent ischemic events that are otherwise lethal. This study was conducted to evaluate the protective effect of hypoxic preconditioning on hypoxic-ischemic injury in the neonatal rat and the persistence of a protective window after hypoxic preconditioning. The rats were preconditioned with hypoxia (8% oxygen, 92% nitrogen) for three hours, subjected to ischemia using ligation of the right common carotid artery, and then exposed to another three hours of hypoxia. Using proton magnetic resonance spectroscopy, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining, and morphologic scores, this study shows that hypoxic preconditioning 6-hr to 1-day before hypoxic-ischemic injury increases survival rates and has neuroprotective effects against subsequent hypoxic-ischemic injury. The mechanism of the protective effects of hypoxic preconditioning in the newborn rat brain may involve downregulation of apoptotic cell death.
Hypoxia-Ischemia, Brain; Magnetic Resonance Spectroscopy