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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.

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

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.

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.

Background

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.

Methods

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.

Results

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.

Conclusions

Remote preconditioning seems to protect myocardial contractile function in hypertrophic myocardium, while it has no beneficial effect in normal myocardium.

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.

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.

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.

Aims

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.

Conclusion

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

Backround

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.

Methods

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.

Results

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.

Conclusions

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.

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.

Aims

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.

Conclusion

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.

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.

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.

OBJECTIVE

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.

METHODS

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.

RESULTS

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.

INTERPRETATION

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.

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.

Methods

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.

Results

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.

Conclusions

We found limb preconditioning to be safe and well tolerated, even at ischemia times of 10 minutes, in critically ill patients with subarachnoid hemorrhage.

Background:

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.

Objective:

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.

Results:

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.

Conclusion:

These data provide the evidence that α-1-AR activation involved in cardioprotective effect of RAPC-mediated trough opening of KATP channels.

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.

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.

A short episode of ischemia induced by coronary artery occlusion can precondition the myocardium against arrhythmia. The factors that have the potential to protect the myocardium from subsequent ischemia and reperfusion are controversial. In this study, the preconditioning-like effects of adrenaline were investigated in both anesthetized and conscious rats. Adrenaline 0.1 and 0.5 mg/kg or saline was administered 10 min before coronary occlusion in conscious and anesthetized rats. The 0.5 mg/kg dose of adrenaline decreased the total duration of arrhythmia in both models. The incidence of ventricular fibrillation decreased and survival rate increased only in conscious rats administered 0.5 mg/kg adrenaline. As a result, it is suggested that exogenous administration of adrenaline before coronary ligation may precondition and protect the heart against arrhythmia.

Background

The hypothesis of the present study was that molecular mechanisms differ markedly when mediating ischemic preconditioning induced by repetitive episodes of ischemia versus classic first- or second-window preconditioning.

Methods and Results

To test this, chronically instrumented conscious pigs were subjected to either repetitive coronary stenosis (RCS) or a traditional protocol of second-window ischemic preconditioning (SWIPC). Lethal ischemia, induced by 60 minutes of coronary artery occlusion followed by reperfusion, resulted in an infarct size/area at risk of 6±3% after RCS and 16±3% after SWIPC (both groups P<0.05, less than shams 42±4%). Two molecular signatures of SWIPC, the increased expression of the inducible isoform of nitric oxide synthase and the translocation of protein kinase Cε to the plasma membrane, were observed with SWIPC but not with RCS. Confirming this, pretreatment with a nitric oxide synthase inhibitor prevented the protection conferred by SWIPC but not by RCS. Microarray analysis revealed a qualitatively different genomic profile of cardioprotection between ischemic preconditioning induced by RCS and that induced by SWIPC. The number of genes significantly regulated was greater in RCS (5739) than in SWIPC (2394) animals. Of the 5739 genes regulated in RCS, only 31% were also regulated in SWIPC. Broad categories of genes induced by RCS but not SWIPC included those involved in autophagy, endoplasmic reticulum stress, and mitochondrial oxidative metabolism. The upregulation of these pathways was confirmed by Western blotting.

Conclusions

RCS induces cardioprotection against lethal myocardial ischemia that is at least as powerful as traditional ischemic preconditioning but is mediated through radically different mechanisms.

The reversible S-nitrosation and inhibition of mitochondrial complex I is a potential mechanism of cardioprotection, recruited by ischemic preconditioning (IPC), S-nitrosothiols, and nitrite. Previously, to exploit this mechanism, the mitochondrial S-nitrosating agent S-nitroso-2-mercaptopropionyl glycine (SNO-MPG) was developed, and protected perfused hearts and isolated cardiomyocytes against ischemia-reperfusion (IR) injury. In the present study, the murine left anterior descending coronary artery (LAD) occlusion model of IR injury was employed, to determine the protective efficacy of SNO-MPG in vivo. Intraperitoneal administration of 1 mg/kg SNO-MPG, 30 min. prior to occlusion, significantly reduced myocardial infarction and improved EKG parameters, following 30 min. occlusion plus 2 or 24 hr. reperfusion. SNO-MPG protected to the same degree as IPC, and notably was also protective when administered at reperfusion. Cardioprotection was accompanied by increased mitochondrial protein S-nitrosothiol content, and inhibition of complex I, both of which were reversed after 2 hr. reperfusion. Finally, hearts from mice harboring a heterozygous mutation in the complex I NDUSF4 subunit were refractory to protection by either SNO-MPG or IPC, suggesting that a fully functional complex I, capable of reversible inhibition is critical for cardioprotection. Overall, these results are consistent with a role for mitochondrial S-nitrosation and complex I inhibition in the cardioprotective mechanism of IPC and SNO-MPG in vivo.

Background and purpose

Cerebral preconditioning provides insights into endogenous mechanisms that protect the brain from ischemic injury. Hypoxia and the anesthetic isoflurane are powerful preconditioning agents. Recent data show that sphingosine 1-phosphate (S1P) receptor stimulation improves outcome in rodent models of stroke. Endogenous S1P levels are controlled by the expression and activity of sphingosine kinases (SPK). We hypothesize that SPK up-regulation mediates preconditioning induced by isoflurane and hypoxia and reduces ischemic injury.

Methods

Male wild-type C57BL/J, SPK1−/− and SPK2−/− mice were exposed to isoflurane (IsoPC) or hypoxia preconditioning (HPC) before transient middle cerebral artery occlusion. Infarct volume and neurological outcome were measured 24 hours later. SPK inhibitors (SKI-II and ABC294640) were used to test the involvement of SPK2. Expressions of SPK1, SPK2 and HIF1α were determined. Primary cultures of mouse cortical neurons were exposed to isoflurane before glutamate- or hydrogen peroxide-induced cell death.

Results

IsoPC and HPC significantly reduced infarct volume and improved neurological outcome in wild-type and SPK1−/− mice, but not in SPK2−/− mice. Pretreatment with SKI-II or ABC294640 abolished the IsoPC-induced tolerance. Western blot showed a rapid and sustained increase in SPK2 level, whereas SPK1 level was similar between preconditioned mice and controls. HIF1α was up-regulated in wild-type IsoPC mice, but not in SPK2−/−. IsoPC protected primary neurons against cell death, which was abolished in ABC294640-treated cells.

Conclusions

Applying genetic and pharmacological approaches, we demonstrate that neuronal SPK2 isoform plays an important role in cerebral preconditioning.

Isoflurane preconditioning (IsoPC) neuroprotection in experimental stroke is male-specific. We determined whether estradiol alters ischemic outcomes in IsoPC brain and examined the role of estrogen receptors (ERs). Seven to 10 days before preconditioning, ovariectomized (OVX) mice were implanted with estradiol, vehicle, or ER subtype agonists. OVX ± estradiol, OVX ± vehicle, OVX ± ER agonists, and ER subtype wild-type (WT) and knockout (KO) mice were preconditioned for 4 h with sham anesthetic preconditioning (sham PC) or 1% IsoPC and recovered for 24 h. Mice then underwent 2 h of middle cerebral artery occlusion followed by 22 h of reperfusion. Infarct volumes were determined by 2,3,5-triphenyltetrazolium chloride staining, with comparisons between IsoPC and corresponding sham PC for each treatment group. Decreased infarct injury was seen in IsoPC OVX ± vehicle, whereas estradiol in IsoPC OVX mice enhanced ischemic damage. In ER studies, increased infarct volumes were seen in IsoPC ERWT mice regardless of ER subtype. IsoPC in ERαKO mice had no effect on infarction volume but reduced only cortical ischemic damage in ERβKO mice. In OVX + ERα agonist, IsoPC had no effect on infarction volume. In OVX + ERβ agonist, IsoPC increased cortical infarct volume. Estradiol depresses the brain’s protective response to IsoPC and may exacerbate cortical ischemic injury mainly through an ERβ-dependent mechanism.