Sustained Ischemia Research Articles

Mitochondrial KATP channels in hindlimb remote ischemic preconditioning of skeletal muscle against infarction.

We previously demonstrated in the pig that instigation of three cycles of 10 min of occlusion and reperfusion in a hindlimb by tourniquet application (approximately 300 mmHg) elicited protection against ischemia-reperfusion injury (infarction) in multiple distant skeletal muscles subsequently subjected to 4 h of ischemia and 48 h of reperfusion, but the mechanism was not studied. The aim of this project was to test our hypothesis that mitochondrial ATP-sensitive potassium (KATP) (mKATP) channels play a central role in the trigger and mediator mechanisms of hindlimb remote ischemic preconditioning (IPC) of skeletal muscle against infarction in the pig. We observed in the pig that hindlimb remote IPC reduced the infarct size of latissimus dorsi (LD) muscle flaps (8 x 13 cm) from 45 +/- 2% to 22 +/- 3% (n = 10; P < 0.05). The nonselective KATP channel inhibitor glibenclamide (0.3 mg/kg) or the selective mKATP channel inhibitor 5-hydroxydecanoate (5-HD, 5 mg/kg), but not the selective sarcolemmal KATP (sKATP) channel inhibitor HMR-1098 (3 mg/kg), abolished the infarct-protective effect of hindlimb remote IPC in LD muscle flaps (n = 10, P < 0.05) when these drugs were injected intravenously at 10 min before remote IPC. In addition, intravenous bolus injection of glibenclamide (1 mg/kg) or 5-HD (10 mg/kg) at the end of hindlimb remote IPC also abolished the infarct protection in LD muscle flaps (n = 10; P < 0.05). Furthermore, intravenous injection of the specific mKATPchannel opener BMS-191095 (2 mg/kg) at 10 min before 4 h of ischemia protected the LD muscle flap against infarction to a similar extent as hindlimb remote IPC, and this infarct-protective effect of BMS-191095 was abolished by intravenous bolus injection of 5-HD (5 mg/kg) at 10 min before or after intravenous injection of BMS-191095 (n = 10; P < 0.05). The infarct protective effect of BMS-191095 was associated with a higher muscle content of ATP at the end of 4 h of ischemia and a decrease in muscle neutrophilic myeloperoxidase activity at the end of 1.5 h of reperfusion compared with the time-matched control (n = 10, P < 0.05). These observations led us to conclude that mKATP channels play a central role in the trigger and mediator mechanisms of hindlimb remote IPC of skeletal muscle against infarction in the pig, and the opening of mKATP channels in ischemic skeletal muscle is associated with an ATP-sparing effect during sustained ischemia and attenuation of neutrophil accumulation during reperfusion.

Ischemic preconditioning protects by activating prosurvival kinases at reperfusion.

Pharmacological activation of the prosurvival kinases Akt and ERK-1/2 at reperfusion, after a period of lethal ischemia, protects the heart against ischemia-reperfusion injury. We hypothesized that ischemic preconditioning (IPC) protects the heart by phosphorylating the prosurvival kinases Akt and ERK-1/2 at reperfusion. In isolated perfused Sprague-Dawley rat hearts subjected to 35 min of lethal ischemia, the phosphorylation states of Akt, ERK-1/2, and p70 S6 kinase (p70S6K) were determined after 15 min of reperfusion, and infarct size was measured after 120 min of reperfusion. IPC induced a biphasic response in Akt and ERK-1/2 phosphorylation during the preconditioning and reperfusion phases after the period of lethal ischemia. IPC induced a fourfold increase in Akt, ERK-1/2, and p70S6K phosphorylation at reperfusion and reduced the infarct risk-to-volume ratio (56.9 +/- 5.7 and 20.9 +/- 3.6% for control and IPC, respectively, P < 0.01). Inhibiting the IPC-induced phosphorylation of Akt, ERK-1/2, and p70S6K at reperfusion with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002 or the MEK-1/2 inhibitor PD-98059 abrogated IPC-induced protection (46.3 +/- 5.8, 49.2 +/- 4.0, and 20.9 +/- 3.6% for IPC + LY-294002, IPC + PD-98059, and IPC, respectively, P < 0.01), demonstrating that the phosphorylation of these kinases at reperfusion is required for IPC-induced protection. In conclusion, we demonstrate that the reperfusion phase following sustained ischemia plays an essential role in mediating IPC-induced protection. Specifically, we demonstrate that IPC protects the heart by phosphorylating the prosurvival kinases Akt and ERK-1/2 at reperfusion.

Intravenous atenolol and esmolol maintain the protective effect of ischemic preconditioning in vivo

Catecholamines bind to α- and β-adrenoreceptors and are capable of preconditioning ischemic myocardium. Our purpose was to investigate the effect of acute either short or prolonged i.v. administration of β-adrenoreceptor antagonists on ischemic preconditioning in vivo. Fifty-five anesthetized rabbits were divided into 10 groups ( n=5–7 per group) and were subjected to 30-min regional ischemia of the heart after ligation of a prominent left coronary artery and 3-h reperfusion after releasing the snare. Ischemic preconditioning was obtained by three cycles of 5-min ischemia separated by 10-min reperfusion. β-Adrenoreceptor blockade was obtained by the long acting β-adrenoreceptor antagonist atenolol or by the short acting esmolol, which were given as a short 5-min infusion or as a prolonged 45-min infusion, starting respectively 20 min before and ending 15 min before the beginning of sustained ischemia, or starting 45 min before and ending immediately before the beginning of sustained ischemia. Atenolol was given at a rate of 0.2 mg min −1 during 5 min or at a rate of 0.088 mg min −1 as a 45-min infusion. Esmolol was given as an initial dose of 500 μg kg −1 within 1 min, followed by a 4-min infusion at a rate of 50 μg kg −1 min −1 or as an initial dose of 3.4 mg within 1 min, followed by a 44-min infusion at a rate of 0.15 mg min −1. Blood pressure and heart rate were continuously monitored. The infarcted and risk areas were delineated with the aid of tetrazolium chloride staining and fluorescent Zn–Cd particles. Infarct size was expressed in percent of the area at risk. All the animals without preconditioning developed an infarct size ranging between 36.3±2.4% and 49.6±7.6% ( P=NS) and all the preconditioning groups developed an infarct size ranging between 14.9±1.2% and 21.0±2.2% ( P=NS). All the preconditioning groups, independently of the use of β-adrenoreceptor antagonists, had a smaller infarct size than the control group, which developed an infarct size of 47.3±2.5% ( P<0.01). Intravenous atenolol and esmolol, independent of timing and mode of administration, does not seem to interfere with protection afforded by ischemic preconditioning in vivo.

Coronary microembolization does not induce acute preconditioning against infarction in pigs-the role of adenosine.

After coronary microembolization (ME) adenosine is released from ischemic areas of the microembolized myocardium. This adenosine dilates vessels in adjacent nonembolized myocardium and increases coronary blood flow. For ischemic preconditioning (IP) to protect the myocardium against infarction, an increase in the interstitial adenosine concentration (iADO) prior to the subsequent ischemia/reperfusion is necessary. We hypothesized that the adenosine release after ME is sufficient to increase iADO and protect the myocardium against infarction from subsequent ischemia/reperfusion. We have therefore compared myocardial protection by either coronary microembolization or ischemic preconditioning prior to ischemia/reperfusion. In anesthetized pigs, the left anterior descending (LAD) was cannulated and perfused from an extracorporeal circuit. In 11 pigs, sustained ischemia was induced by 85% inflow reduction for 90 min (controls). Two other groups of pigs were subjected either to IP (n = 8; 10-min ischemia/15-min reperfusion) or coronary ME (n = 9; i.c. microspheres; 42 microm Ø; 3000 x ml(-1) x min inflow) prior to sustained ischemia. Coronary venous adenosine concentration (vADO) and iADO (microdialysis) were measured. Infarct size was determined after 2-h reperfusion by triphenyl tetrazolium chloride staining. In pigs subjected to IP, infarct size was reduced to 2.6 +/- 1.1% (mean +/- S.E.M.) vs. 17.0 +/- 3.2% in controls. iADO was increased from 2.4 +/- 1.3 to 13.1 +/- 5.8 micromol x l(-1) during the reperfusion following IP. In pigs subjected to ME, at 10 min after ME, coronary blood flow (38.6 +/- 3.6 to 53.6 +/- 4.3 ml x min(-1)) and vADO (0.25 +/- 0.04 to 0.48 +/- 0.07 micromol x l(-1)) were increased. However, iADO (2.0 +/- 0.5 at baseline vs. 2.3 +/- 0.6 micromol x l(-1) at 10 min after ME) did not increase. Infarct size induced by sustained ischemia following ME (22.5 +/- 5.2%) was above that of controls for any given subendocardial blood flow. ME released adenosine into the vasculature and increased coronary blood flow. The failure of iADO to increase with ME possibly explains the lack of protection against infarction after ME.

Anaesthetic Management of Planned or Emergency Endovascular Stent Graft Surgery for Thoracic Aneurysms and Dissections

We reviewed the intraoperative anaesthetic management and outcome of seven consecutive cases of endovascular stent graft surgery for thoracic aortic aneurysms or dissections over a period of 20 months in our institution. Seven males (median age 63) underwent endovascular stent graft surgery of the thoracic aorta under general anaesthesia. Four were emergency procedures for acute dissection or leaks of thoracic aneurysms. The duration of the procedures ranged from 120 to 300 minutes. Intraoperative stent migration occurred in one patient. Induced hypotension and immobilization were critical during stent deployment. Six out of the seven patients were discharged home between three to 20 days postoperatively. There was no 30-day mortality. One patient died sixty days postoperatively. Anaemia and respiratory complications were the most common postoperative problems encountered. None of the patients sustained spinal cord ischaemia, acute myocardial infarction or renal impairment. Endovascular stent graft surgery of the thoracic aorta is a relatively new alternative to conventional open surgery. Our experience suggests satisfactory short-term outcome even for patients with acute dissection. However, long-term survival and results require further evaluation.

Histological protection against ischemia–reperfusion injury by early ischemic preconditioning in rat retina

Brief ischemia was reported to protect various cells against injury induced by subsequent ischemia–reperfusion, and this phenomenon is known as ischemic preconditioning. The aims of the present study were to clarify whether early ischemic preconditioning could be observed in the rat retina by histological examination. Male Sprague–Dawley rats were subjected to 60 min of retinal ischemia by raising intraocular pressure to 130 mm Hg. Ischemic preconditioning was achieved by applying 5 min of ischemia 5–60 min before 60 min of ischemia. Additional groups of rats received 10 mg/kg 8-phenyltheophiline and 4.5 mg/kg 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), adenosine A 1 receptor antagonists, 5 mg/kg 5-hydroxydecanoate and 1 mg/kg glibenclamide, ATP-sensitive K + channel blockers, or 2.5 mg/kg chelerythrine and 0.1 mg/kg bisindolylmaleimide I, protein kinase C inhibitors, 15 or 30 min before preconditioning. In the non-preconditioned group, cell loss in the ganglion cell layer and thinning of the inner plexiform and inner nuclear layer were observed 7 days after 60 min of ischemia. Five minutes of preconditioning ischemia 20–40 min before 60 min of sustained ischemia completely prevented the retinal tissue damage induced by the sustained ischemia. Treatment with 8-phenyltheophylline, DPCPX, 5-hydroxydecanoate, glibenclamide, chelerythrine and bisindolylmaleimide I almost completely reduced the protective effect of early ischemic preconditioning. The results in the present study indicated that early ischemic preconditioning was demonstrated in the rat retina. Stimulation of adenosine receptors, opening of ATP-sensitive K + channels and activation of protein kinase C might be involved in the underlying protective mechanisms.

Protein kinase A as another mediator of ischemic preconditioning independent of protein kinase C.

We and others have reported that transient accumulation of cyclic AMP (cAMP) in the myocardium during ischemic preconditioning (IP) limits infarct size independent of protein kinase C (PKC). Accumulation of cAMP activates protein kinase A (PKA), which has been demonstrated to cause reversible inhibition of RhoA and Rho-kinase. We investigated the involvement of PKA and Rho-kinase in the infarct limitation by IP. Dogs were subjected to 90-minute ischemia and 6-hour reperfusion. We examined the effect on Rho-kinase activity during sustained ischemia and infarct size of (1) preischemic transient coronary occlusion (IP), (2) preischemic activation of PKA/PKC, (3) inhibition of PKA/PKC during IP, and (4) inhibition of Rho-kinase or actin cytoskeletal deactivation during myocardial ischemia. Either IP or dibutyryl-cAMP treatment activated PKA, which was dose-dependently inhibited by 2 PKA inhibitors (H89 and Rp-cAMP). IP and preischemic PKA activation substantially reduced infarct size, which was blunted by preischemic PKA inhibition. IP and preischemic PKA activation, but not PKC activation, caused a substantial decrease of Rho-kinase activation during sustained ischemia. These changes were cancelled by preischemic inhibition of PKA but not PKC. Furthermore, either Rho-kinase inhibition (hydroxyfasudil or Y27632) or actin cytoskeletal deactivation (cytochalasin-D) during sustained ischemia achieved the same infarct limitation as preischemic PKA activation without affecting systemic hemodynamic parameters, the area at risk, or collateral blood flow. Transient preischemic activation of PKA reduces infarct size through Rho-kinase inhibition and actin cytoskeletal deactivation during sustained ischemia, implicating a novel mechanism for cardioprotection by ischemic preconditioning independent of PKC and a potential new therapeutic target.

Melatonin does not prevent the protection of ischemic preconditioning in vivo despite its antioxidant effect against oxidative stress

Free radicals are involved in the protective mechanism of preconditioning (PC), whereas antioxidant compounds abolish this benefit. Melatonin is a hormone with antioxidant properties. The aim of our study was to evaluate the effect of melatonin on infarct size in ischemic preconditioning in vivo. We randomly divided 33 male rabbits into four groups and subjected them to 30 min of myocardial ischemia and 3 h of reperfusion with the following prior interventions: (i) no intervention, (ii) iv melatonin at a total dose of 50 mg/kg, (iii) PC with two cycles of 5 min ischemia and 10 min reperfusion, and (iv) combined melatonin and PC. In a second series of experiments, another antioxidant agent N-acetylcysteine (NAC) was used in a control and in a PC group. Myocardial infarct size was determined and blood samples were drawn at different time points for the determination of lipid peroxidation products, total superoxide dismutase (SOD) activity, and 1H-NMR spectra to evaluate the changes in the metabolic profile. Melatonin showed no effect on myocardial infarct size in the group of sustained ischemia (42.9 ± 3.6% vs 47.4 ± 4.9%) and it did not attenuate the reduction of myocardial infarct size in the PC group (13.6 ± 2.4% vs 14.0 ± 1.7%). A similar effect was found in NAC-treated groups (44.8 ± 3.4% vs 14.3 ± 1.3%). Lipid peroxidation product levels were significantly elevated in the control and PC groups, whereas melatonin decreased them in both groups. The SOD activity was enhanced in the PC group compared to controls; melatonin kept SOD activity unchanged during ischemia/reperfusion and enhanced its activity when it was combined with PC. Melatonin did not change the metabolic profile of the control and PC groups. Melatonin does not prevent the beneficial effect of ischemic PC on infarct size despite its antioxidant properties.

Hepatocyte growth factor attenuates cerebral ischemia-induced learning dysfunction

Hepatocyte growth factor (HGF) acts as an organotropic factor for regeneration and protection in various organs and has the ability to attenuate cerebral ischemia-induced cell death. However, the effect of HGF on learning and memory function after a cerebral ischemic event is unknown. We demonstrate here that administration of human recombinant HGF (hrHGF) into the ventricle reduced the prolongation of the escape latency in the acquisition and retention tests in the water maze task on days 12–28 after microsphere embolism-induced cerebral ischemia. In addition, disruption of the blood–brain barrier at the early stage after microsphere embolism, which was determined by FITC–albumin leakage, was markedly reduced by treatment with hrHGF. We demonstrated that this effect of hrHGF on the blood–brain barrier was associated with protection against the apoptotic death of the cerebral endothelial cells at the early stage after the ischemia. These results suggest that hrHGF can prevent the learning and memory dysfunction soon after sustained cerebral ischemia by protecting against injury to the endothelial cells. The use of HGF may be a potent strategy for the treatment of cerebrovascular diseases, including cerebral infarct and vascular dementia.

Sublethal simulated ischemia promotes delayed resistance against ischemia via ATP-sensitive (K+) channels in murine myocytes: role of PKC and iNOS.

In this study, we examined whether sublethal simulated ischemia (SSI) induces delayed cellular protection in mouse cardiac myocytes, and whether the delayed cellular protection depends on the activation of protein kinase C-epsilon (PKC-epsilon), inducible nitric oxide synthase (iNOS), and ATP-sensitive K(+) (K(ATP)) channels against subsequent sustained simulated ischemia (SI). The following groups of mouse cardiac myocytes were studied: (a) SI: incubation with SI buffer for 1 h; (b) SSI: incubation with SSI buffer for 30 min; (c) SSI + PKC inhibitor, chelerythrine chloride (CCl): SSI and 1 micro M CCl; (d) SSI + iNOS inhibitor, S-methylthiourea (SMT): SSI and 100 nM SMT; (e) SSI + K(ATP) channel blocker, glibenclamide (Glb): SSI and 50 micro M Glb; (f) SSI + mitochondrial K(ATP) channel blocker, 5-hydroxydecanoate (5-HD): SSI and 50 micro M 5-HD. The release of lactate dehydrogenase into the medium and the amount remaining in the cells was measured, and A(1) adenosine receptor, PKC-epsilon, and iNOS were detected through western blot analysis. The delayed cellular protection acquired due to SSI showed a decreased release of lactate dehydrogenase (%) from 46.51 +/- 1.60 to 37.00 +/- 1.34 (p < 0.001) and was blocked by CCl (47.08 +/- 0.95), SMT (48.08 +/- 1.18), Glb (45.88 +/- 1.31), and 5-HD (47.20 +/- 1.56). Simultaneously, SSI-induced up-regulation of A(1) adenosine receptor, PKC-epsilon, iNOS, and opening of both membrane and mitochondrial K(ATP) channels also was observed compared with controls.

Propyl paraben inhibits voltage-dependent sodium channels and protects cardiomyocytes from ischemia-reperfusion injury

The effects of propyl paraben, an antimicrobial preservative, on voltage-dependent sodium current and myocardial ischemia-reperfusion injury were investigated in isolated adult rat cardiomyocytes. Whole cell voltage-clamp recording showed that propyl paraben reversibly blocked the voltage-gated sodium channel both in concentration- and voltage-dependent manners. Propyl paraben (500 μM but not 100 μM) significantly shifted the steady-state inactivation of the sodium channel toward the hyperpolarizing direction at the V 1/2 point. Consistent with the above result, the propidium iodide (PI) uptake test revealed that pretreatment with 500 μM but not 100 μM of propyl paraben significantly reduced cell death induced by 45 min of sustained ischemia followed by 15 h of reperfusion (42.37 ± 7.01% of cell viability in control and 71.05 ± 7.06% in the propyl paraben group), suggesting that propyl paraben can protect myocytes from ischemia-reperfusion injury. These results indicate a possible correlation between the inhibition of sodium current and cardioprotection against ischemia-reperfusion injury.

Ischemic preconditioning prevents reperfusion heart injury in cardiac hypertrophy by activation of mitochondrial K ATP channels

Background: Cardiac hypertrophy has been demonstrated to decreases the ATP-sensitive potassium channels (K ATP), the major protective mechanism following the energy depletion, a common condition seen during the reperfusion after open heart surgery. In this study we have demonstrated the role of ischemic preconditioning (IP) in preventing the reperfusion injury of the hypertrophied heart by activation of the depleted K ATP channels. Methods: Pressure overload left ventricular hypertrophy was induced in 6 weeks old male Wistar rats by supra renal transverse abdominal aortic constriction and the study was conducted 10–12 weeks later. Hypertrophied rats were subjected to IP protocols by four episodes of 3 min ischemia each being separated by 10 min reperfusion, followed by 30 min of sustained ischemia and 120 min of reperfusion with or without treating the rats with K ATP channel antagonists 5-hydroxydecanoic acid (10 mg/kg per i.v.) or glibenclamide (1 mg/kg per i.v.), 10 min before the sustained ischemia. Results: IP resulted in (a) less incidence of ventricular arrhythmias (b) less area of myocardial infarction (9.3% vs. 48.1%, IP to control) (c) less tissue water content (76.5% vs. 94.8%, IP to control) (d) well preserved myocardial ATP content ( P<0.001 from control) content and (e) much fewer apoptotic cells (4.7% vs. 13.2%, IP to control). Pre treating the rats with the K ATP channel inhibitors before sustained ischemia resulted in inhibition of these protective effects of IP on cardiac hypertrophy. Conclusion: The above results, therefore, suggest to us that IP by activation of K ATP channels can afford protection against the ischemia–reperfusion injury in the hypertrophied heart.

Trigger-dependent gene expression profiles in cardiac preconditioning: evidence for distinct genetic programs in ischemic and anesthetic preconditioning.

DNA chips facilitate genomic-wide exploration of gene expression. The authors hypothesized that ischemic (IPC) and anesthetic preconditioning (APC) would differentially modulate gene expression in hearts. Affymetrix rat U34A gene chips were used to explore the transcriptional response to IPC and APC, sustained ischemia (110 min) without reperfusion, and time-matched perfusion in isolated rat hearts. IPC was induced by three cycles of 5 min of ischemia, and APC was induced by 1.5 minimum alveolar concentration isoflurane (110 min). For each heart, a separate chip was used for hybridization. Data were analyzed for significant > or = 2.0-fold changes in gene expression. Microarray results were confirmed by quantitative real-time reverse-transcription polymerase chain reaction. Of the 8,799 genes represented on U34A, 217 transcripts in the APC group, 234 in the IPC group, and 29 in the ischemia group displayed significant > or = 2.0-fold up-regulation in messenger RNA levels, and 185 transcripts in the APC group, 55 in the IPC group, and 49 in the ischemia group displayed significant > or = 2.0-fold down-regulation. Many of these transcripts were unknown genes. A high number of commonly regulated genes were found in IPC and APC (39 up-regulated, 17 down-regulated). Genes commonly regulated included those associated with cell defense (heat shock protein 10, aldose reductase, Bcl-xS). Conversely, a pool of protective and antiprotective genes was differentially regulated in APC versus IPC (heat shock protein 27/70, programmed cell death 8), suggesting trigger-dependent transcriptome variability. The novel microarray technology provides evidence for distinct cardioprotective phenotypes in IPC and APC. The observed transcriptional changes raise the possibility of a second window of protection by volatile anesthetics. The authors' molecular portraits are the first global genomic comparison between IPC and APC.

DWI abnormalities and clinical characteristics in TIA patients

To determine the clinical characteristics of diffusion-weighted images (DWIs) in patients with TIA. The authors performed prospectively MRI including DWI in 129 consecutively studied patients with TIA (mean age, 67 years; 68% men) within 14 days after TIA onset. They examined the frequency of TIA-related DWI abnormalities and compared the clinical background of patients with and without DWI abnormalities. Using multivariate statistical methods, the authors investigated the independent factors associated with detection of DWI abnormalities. Fifty-seven patients with TIA (44%) had DWI abnormalities (positive group), whereas 72 (56%) had no abnormalities (negative group). Prolonged TIA duration (TIA duration > or = 30 minutes), hemiparesis, monoparesis, disturbance of higher brain function, history of either stroke or TIA, diabetes mellitus, and atrial fibrillation were observed more frequently in the positive group than in the negative group. No difference in other symptoms, vascular risk factors, or emboligenic cardiac and arterial disease was observed between the two groups. A multiple logistic regression model demonstrated that prolonged TIA duration (odds ratio [OR], 3.7; 95% CI, 1.3 to 10.8) and disturbance of higher brain function (OR, 10.2; 95% CI, 2.2 to 46.9) are significant and independent factors in correlating with DWI abnormalities. TIA-related DWI abnormalities are associated with prolonged duration of TIA and disturbance of higher brain function. More sustained and extensive ischemia may contribute to DWI abnormalities in patients with TIA.

Effects of monochlorobimane on cerebral ischemia-induced damage to mitochondria.

A possible involvement of inhibitory effects of monochlorobimane (MCB) on the opening of mitochondrial permeability transition (MPT) pore in the cerebroprotection against the ischemic brain injury was examined. MCB (1 mM) inhibited the opening of MPT pore in vitro. Sustained cerebral ischemia was induced by injecting 900 microspheres (48 microm in diameter) into the right hemisphere of rats. At 12 to 72 h after microsphere embolism (ME), the mitochondrial activity was determined histochemically by staining cytochrome c oxidase (COX) and succinate dehydrogenase (SDH) of the brain sections. The COX and SDH stainings in the hippocampus were observed intensively in the pyramidal neurons in the CA2-3 and dentate gyrus rather than those in the CA-1 region. The staining was decreased with time after the embolism. Pretreatment with 10 microg/animal MCB 30 min prior to the embolism significantly attenuated the ME-induced reduction in the staining of COX and SDH in the hippocampus, but not in the pariatal cortex. The results suggest that prevention of the opening of MPT pore by MCB may play an important role in the cerebroprotection against cerebral ischemic injury.

Effect of Ischemic Preconditioning Based on Different Epicardial Branching Patterns of the Left Coronary Artery in the Rabbit Heart

The aim of this study was to characterize the effect of increasing numbers of preconditioning cycles on the different branching patterns (bifurcation/trifurcation) of the coronary artery in the rabbit heart. Fifty-six NZW rabbits were assigned to a bifurcation group and subjected to 0, 1, 3, 5, or 7 (B0, B1, B3, B5, or B7 subgroup, respectively) cycles of preconditioning (PC) (5 min of regional ischemia plus 10 min of reperfusion/cycle) and this was followed by 45 min of sustained ischemia and 72 h of reperfusion; 16 rabbits were assigned to a trifurcation group and subjected to 0, 1, 3, or 5 (T0, T1, T3, or T5 subgroup, respectively) cycles of PC. The ratio of necrotic zone (NZ) to ischemic zone (IZ) was calculated. The bifurcation group showed higher mortality (28.6%) than the trifurcation group (0%). The volume of the ischemic zone (expressed as a percentage of volume of the left ventricle) and the volume of the necrotic zone were larger in the bifurcation group than in the trifurcation group. The ratio of the necrotic zone to the ischemic zone was significantly lower in the B5 and B7 subgroups than in the B0, B1, and B3 subgroups. In the trifurcation group, the ratio of the necrotic zone to the ischemic zone showed a diminishing tendency in the subgroups as the PC cycle number increased, but without statistical significance. Thus, in the trifurcation pattern of the rabbit coronary artery there could be little effect on preconditioning, but in the bifurcation pattern the number of preconditioning is recommended to be 5 to 7 cycles. In this regard, different branching patterns of the epicardial branching of the rabbit coronary artery should be considered when interpreting experimental results on ischemic preconditioning.

Ischemic-preconditioning does not prevent neuromuscular dysfunction after ischemia–reperfusion injury

The primary purpose of this study was to evaluate ischemic-preconditioning (IPC) as a means of improving tolerance to ischemia–reperfusion (IR) stress on neuromuscular function. A secondary objective was to isolate the area of injury within the neuromuscular unit responsible for contractile dysfunction after IR injury. Twenty-eight male rabbits were randomly assigned to four groups (sham, IPC only, sustained ischemia only, IPC and sustained ischemia). The IPC protocol consisted of three cycles of 10 min of tourniquet-induced ischemia (125 mmHg) followed by 10 min of reperfusion. Sustained ischemia was induced by 350 mmHg external compression for 2 h. Peak tetanic tension of the tibialis anterior (TA) muscle was evaluated 48 h after the tourniquet protocol by both peroneal nerve and direct muscle stimulation, with and without pharmacologic neuromuscular junction blockade. Animals subjected to combined IPC and sustained ishemia had an equivalent loss of contractile force to those undergoing sustained ischemia alone. Two hours of tourniquet-induced ischemia resulted in marked dysfunction of the TA neuromuscular unit when evaluated by peroneal nerve stimulation ( p<0.0001). Isolation of the TA muscle from the peroneal nerve with direct muscle stimulation and neuromuscular junction blockade preserved muscle function after 3 h of ischemia. In our model, these results clearly demonstrate that IPC provides no significant protective effect from IR injury to either nerve or muscle function in our model. In addition, nerve or neuromuscular junction injury, rather than muscle damage, is identified as primarily responsible for IR-related muscle dysfunction.

Differential expression of Flk-1 and Flt-1 in rat skeletal muscle in response to chronic ischaemia: favourable effect of muscle activity.

To determine the involvement of vascular endothelial growth factor (VEGF) and its receptors Flk-1 and Flt-1 in capillary growth in ischaemic skeletal muscle, extensor digitorum longus muscles from hindlimbs of Sprague-Dawley rats were studied at 1, 2 and 5 week intervals after iliac artery ligation. Muscle VEGF protein levels (as determined by Western-blot analysis) increased only after 2 (60%) and 5 (80%) weeks, with more capillaries positively immunostained for VEGF than in control muscles. Ischaemia-induced angiogenesis was gradual, with capillary proliferation at 1 and 2 weeks and capillary:fibre ratio increased 20% after 5 weeks. This was associated with an initial doubling of Flk-1 protein after 1 week that declined below control levels by 5 weeks, whereas Flt-1 expression was elevated more than 40% at all time points. During more sustained ischaemia (femoral ligation 3 weeks after iliac ligation), VEGF protein level at 5 weeks was even higher, but Flt-1 and Flk-1 were unchanged from control levels and no capillary growth occurred. Intermittent electrical stimulation (10 Hz, 7x15 min/day) of these ischaemic muscles between weeks 3-5 did not elevate VEGF further, but increased Flk-1 by 32%, decreased Flt-1 by 71%, and led to significant capillary growth. These results demonstrate that during chronic muscle ischaemia Flk-1 and Flt-1 are regulated differentially and that electrical stimulation of ischaemic muscles can promote angiogenesis via Flk-1 up-regulation. Even when ischaemic muscle VEGF levels are high, capillary growth appears to be dependent on the presence of Flk-1.

Ceramide in the antiapoptotic effect of ischemic preconditioning

Although the mechanism by which ischemic preconditioning (PC) inhibits myocardial apoptosis during ischemia-reperfusion is unclear, evidence indicates a role for the secondary messenger ceramide. We investigated in vivo whether PC may affect ceramide and sn-1,2-diacylglycerol (DAG) production, and attenuate apoptosis during ischemia. Rabbits underwent 30 min of ischemia, followed by 4 h of reperfusion. Before this, they received either no intervention (control group) or one episode of 5 min of ischemia, followed by 5 min of reperfusion (PC group), or an intravenous administration of the sphingomyelinase inhibitor D609. Myocardial content of ceramide and DAG was measured using the DAG kinase assay at different time points of the experiment. Apoptosis was detected and quantified by a sandwich enzyme immunoassay. Both AR and infarct size were measured using blue dye injection and triphenyltetrazolium chloride staining. Control hearts exhibited a peak of ceramide production at 5 min of the prolonged ischemia, with a mean value averaging 64 +/- 5 ng/mg tissue (P < 0.05 vs. 48 +/- 4 ng/mg at baseline). In contrast, ischemic PC and D609 prevented ceramide increase during the prolonged ischemia. Myocardial DAG content was increased only in PC hearts at 30 min of ischemia. Preconditioned and D609 groups developed less apoptosis, as well as a limited infarct size, compared with the control group. These results suggest that the antiapoptotic effect of PC may be due to a reduced ceramide production during sustained ischemia in the rabbit heart.

Second window of ischemic preconditioning regulates mitochondrial permeability transition pore by enhancing Bcl-2 expression.

The second window of protection (SWOP) following brief coronary artery occlusion begins at 24 h and may last up to 72 h and occurs via many unknown mechanisms. We investigated the role of the mitochondrial permeability transition pore (PTP), a non specific pore in the inner membrane of the mitochondria in this phenomenon. Ischemic preconditioning (IP) was induced in Wistar rats by left coronary artery occlusion (four, 3-min episodes separated by 10 min of reperfusion) on day 1. On day 2, ischemia was induced with 30 min of ischemia and 120 min of reperfusion in IP and control rats. IP rats showed decreased myocardial infarction (MI) area vs. non-IP control rats (15.32 vs. 45.6%). Furthermore, IP rats had preserved cardiac function (heart rate, rate pressure product, coronary flow and aortic flow) and myocardial ATP (P<0.03), decreased tissue water content (73.2 vs. 90.6%), increased expression of Bcl-2, and less mitochondrial swelling, cytochrome C release and apoptosis (2.6 vs. 12.4%) when compared to sham-operated rats. Activation of the permeability transition pore with PTP activators lonidamine (10 mg/kg body weight) or atractyloside (5 mg/kg body weight) before the sustained ischemia on day 2 resulted in complete abolition of SWOP-mediated cytoprotective effects. These agents had no effect on the cytoprotective effects that took place during the first window of preconditioning. The cytoprotective effects of SWOP are dependent on PTP activation state and may involve upregulation of Bcl-2 expression.