Intestinal preconditioning prevents systemic inflammatory response in hemorrhagic shock. Role of HO-1

Intestinal ischemia-reperfusion has been implicated in the systemic inflammatory response and organ injury in hemorrhagic shock, but the exact role of the intestine has never been directly demonstrated. Preconditioning (PC) with brief periods of intermittent ischemia is a known potent anti-ischemic intervention and thus can be used as a tool to assess the role of local intestinal ischemia-reperfusion injury in systemic inflammatory response. Thus rats were first subjected to sham surgery or intestinal preconditioning with four cycles of 1-min ischemia and 10 min of reperfusion 24 h before hemorrhagic shock followed by resuscitation. PC reduced fluid requirements, lung edema, and lactate and tumor necrosis factor-alpha production. These effects were abolished by the heme-oxygenase-1 (HO-1) inhibitor tin protoporphyrin (Sn-PP). PC induced more than fivefold in intestinal HO-1 expression. These results suggest that intestinal ischemia-reperfusion is a major trigger for inflammatory response and organ injury in nonseptic shock. HO-1 appears to play an important role in the protective effect of intestinal preconditioning.     

Brief small intestinal ischemia lessens renal ischemia-reperfusion injury in rats

Ischemic preconditioning (IPC) not only reduces local tissue injury caused by subsequent ischemia-reperfusion (IR) but may also have a beneficial effect on IR injury of tissues remote from those undergoing preconditioning. In this study, we investigated the effect of small intestinal IPC on renal IR injury in rats. Renal IR injury was induced by a 45-min renal artery occlusion and reperfusion for 2 or 24 h in rats with a previous contralateral nephrectomy, and ischemic preconditioning was induced by 3 cycles of 8-min ischemia and 5-min reperfusion of the small intestine. We then measured the concentrations of plasma creatinine (Cr) and blood urine nitrogen (BUN) and the level of malondialdehyde (MDA) and activities of superoxide dismutase (SOD) and catalase (CAT) in the renal cortex. Renal histopathology also was evaluated. Pretreatment with intestinal ischemic preconditioning significantly alleviated renal IR injury, as shown by decreases in the levels of Cr, BUN, and MDA, decreased renal morphologic change, and improved preservation of SOD and CAT activities. These results suggest that remote ischemic preconditioning of the small intestine protects against renal IR injury by inhibition of lipid peroxidation and preservation of antioxidant enzyme activities.     

Effect of age on alpha-calcitonin gene-related peptide-mediated delayed cardioprotection induced by intestinal preconditioning in rats

In the present study, we examined whether age-related reduction in cardioprotection of intestinal ischemic preconditioning is related to stimulation of the release and synthesis of calcitonin gene-related peptide (CGRP) in rats. Ischemia-reperfusion injury was induced by a 45-min coronary artery occlusion and 180-min reperfusion, and ischemic preconditioning was induced by six cycles of 4-min ischemia and 4-min reperfusion of the small intestine. The serum concentration of creatine kinase, infarct size, the expression of CGRP isoforms (alpha- and beta-CGRP) mRNA in lumbar dorsal root ganglia and CGRP concentration in plasma were measured. Pretreatment with intestinal ischemic preconditioning for 24 h significantly reduced infarct size and creatine kinase release concomitantly with a significant increase in the expression of alpha-CGRP mRNA, but not beta-CGRP mRNA, and plasma concentrations of CGRP at 6 months of age but not at 24 months of age. These results suggest that the delayed cardioprotective effect of intestinal ischemic preconditioning is decreased in senescent rats, and the age-related change is related to reduction of the synthesis and release of alpha-CGRP.     

beta1-Adrenoreceptor activation contributes to ischemia-reperfusion damage as well as playing a role in ischemic preconditioning

Protein kinase A (PKA) activation has been implicated in early-phase ischemic preconditioning. We recently found that during ischemia PKA activation causes inactivation of cytochrome-c oxidase (CcO) and contributes to myocardial damage due to ischemia-reperfusion. It may be that beta-adrenergic stimulation during ischemia via endogenous catecholamine release activates PKA. Thus beta-adrenergic stimulation may mediate both myocardial protection and damage during ischemia. The present studies were designed to determine the role of the beta(1)-adrenergic receptor (beta(1)-AR) in myocardial ischemic damage and ischemic preconditioning. Langendorff-perfused rabbit hearts underwent 30-min ischemia by anterior coronary artery ligation followed by 2-h reperfusion. Occlusion-reperfusion damage was evaluated by delineating the nonperfused volume of myocardium at risk and volume of myocardial necrosis after 2-h reperfusion. In some hearts ischemic preconditioning was accomplished by two 5-min episodes of global low-flow ischemia separated by 10 min before coronary occlusion-reperfusion. Orthogonal electrocardiograms were recorded, and coronary flow was monitored by a drip count. Three hearts from each experimental group were used to determine mitochondrial CcO and aconitase activities. Two-hour reperfusion after occlusion caused an additional decrease in CcO activity vs. that after 30-min occlusion alone. Blocking the beta(1)-AR during occlusion-reperfusion reversed CcO activity depression and preserved myocardium at risk for necrosis. Similarly, mitochondrial aconitase activity exhibited a parallel response after occlusion-reperfusion as well as for the other interventions. Furthermore, classic ischemic preconditioning had no effect on CcO depression. However, blocking the beta(1)-AR during preconditioning eliminated the cardioprotection. If the beta(1)-AR was blocked after preconditioning, the myocardium was preserved. Interestingly, in both of the latter cases the depression in CcO activity was reversed. Thus the beta(1)-AR plays a dual role in myocardial ischemic damage. Our findings may lead to therapeutic strategies for preserving myocardium at risk for infarction, especially in coronary reperfusion intervention.     

Ischemic preconditioning attenuates ischemia-reperfusion-induced mucosal apoptosis by inhibiting the mitochondria-dependent pathway in rat small intestine

Ischemic preconditioning provides a way of protecting organs from damage inflicted with prolonged ischemia-reperfusion. In this study, we investigated the mechanism of ischemic preconditioning involved in inhibition of prolonged ischemia-reperfusion-induced mucosal apoptosis in rat small intestine. Ischemic preconditioning was triggered by a transient occlusion of the superior mesenteric artery followed by reperfusion. Ischemia-reperfusion was induced by 60-min occlusion of the superior mesenteric artery followed by 60-min reperfusion in the small intestine. Ischemia-reperfusion alone induced mucosal apoptosis and mitochondrial respiratory dysfunction via promoted reactive oxygen species generation, reduced mitochondrial glutathione oxidation, increased mitochondrial lipid peroxidation, reduced mitochondrial membrane potential, and enhanced release of cytochrome c from mitochondria to activate caspase-9 and caspase-6 in the small intestine. Pretreatment with 20-min ischemia followed by 5-min reperfusion significantly inhibited the prolonged ischemia-reperfusion-induced mucosal apoptosis by 30%. Ischemic preconditioning ameliorated mitochondrial respiratory dysfunction by 50%, reduced reactive oxygen species generation by 38%, and suppressed mitochondrial lipid peroxidation by 36%, resulting in improvement of the mitochondrial membrane potential and prevention of cytochrome c release as well as caspase-6 activation. Results suggest that ischemic preconditioning attenuated ischemia-reperfusion-induced mucosal apoptosis partly by inhibiting the reactive oxygen species-mediated mitochondria-dependent pathway in the rat small intestine.     

Adenosine-enhanced ischemic preconditioning: adenosine receptor involvement during ischemia and reperfusion

Adenosine-enhanced ischemic preconditioning (APC) extends the cardioprotection of ischemic preconditioning (IPC) by both significantly decreasing myocardial infarct size and significantly enhancing postischemic functional recovery. In this study, the role of adenosine receptors during ischemia-reperfusion was determined. Rabbit hearts (n = 92) were used for Langendorff perfusion. Control hearts were perfused for 180 min, global ischemia hearts received 30-min ischemia and 120-min reperfusion, and IPC hearts received 5-min ischemia and 5-min reperfusion before ischemia. APC hearts received a bolus injection of adenosine coincident with IPC. Adenosine receptor (A(1), A(2), and A(3)) antagonists were used with APC before ischemia and/or during reperfusion. GR-69019X (A(1)/A(3)) and MRS-1191/MRS-1220 (A(3)) significantly increased infarct size in APC hearts when administered before ischemia and significantly decreased functional recovery when administered during both ischemia and reperfusion (P < 0.05 vs. APC). DPCPX (A(1)) administered either before ischemia and/or during reperfusion had no effect on APC cardioprotection. APC-enhanced infarct size reduction is modulated by adenosine receptors primarily during ischemia, whereas APC-enhanced postischemic functional recovery is modulated by adenosine receptors during both ischemia and reperfusion.     

Ischemic postconditioning during reperfusion activates Akt and ERK without protecting against lethal myocardial ischemia-reperfusion injury in pigs

Transient episodes of ischemic preconditioning (PC) render myocardium protected against subsequent lethal injury after ischemia and reperfusion. Recent studies indicate that application of short, repetitive ischemia only during the onset of reperfusion after the lethal ischemic event may obtain equivalent protection. We assessed whether such ischemic postconditioning (Postcon) is cardioprotective in pigs by limiting lethal injury. Pentobarbital sodium-anesthetized, open-chest pigs underwent 30 min of complete occlusion of the left anterior descending coronary artery and 3-h reflow. PC was elicited by two cycles of 5-min occlusion plus 10-min reperfusion before the 30-min occlusion period. Postcon was elicited by three cycles of 30-s reperfusion, followed by 30-s reocclusion, after the 30-min occlusion period and before the 3-h reflow. Infarct size (%area-at-risk using triphenyltetrazolium chloride macrochemistry; means +/- SE) after 30 min of ischemia was 26.5 +/- 5.2% (n = 7 hearts/treatment group). PC markedly limited myocardial infarct size (2.8 +/- 1.2%, n = 7 hearts/treatment group, P < 0.05 vs. controls). However, Postcon had no effect on infarct size (37.8 +/- 5.1%, n = 7 hearts/treatment group). Within the subendocardium, Postcon increased phosphorylation of Akt (74 +/- 12%) and ERK1/2 (56 +/- 10%) compared with control hearts subjected only to 30-min occlusion and 15-min reperfusion (P < or = 0.05), and these changes were not different from the response triggered by PC (n = 5 hearts/treatment group). Phosphorylation of downstream p70S6K was also equivalent in PC and Postcon groups. These data do not support the hypothesis that application of 30-s cycles of repetitive ischemia during reperfusion exerts a protective effect on pig hearts subjected to lethal ischemia, but this is not due to a failure to phosphorylate ERK and Akt during early reperfusion.     

Lovastatin interferes with the infarct size-limiting effect of ischemic preconditioning and postconditioning in rat hearts

Statins have been shown to be cardioprotective; however, their interaction with endogenous cardioprotection by ischemic preconditioning and postconditioning is not known. In the present study, we examined if acute and chronic administration of the 3-hydroxy-3-methylglutaryl CoA reductase inhibitor lovastatin affected the infarct size-limiting effect of ischemic preconditioning and postconditioning in rat hearts. Wistar rats were randomly assigned to the following three groups: 1) vehicle (1% methylcellulose per os for 12 days), 2) chronic lovastatin (15 mg.kg(-1).day(-1) per os for 12 days), and 3) acute lovastatin (1% methylcellulose per os for 12 days and 50 micromol/l lovastatin in the perfusate). Hearts isolated from the three groups were either subjected to a nonconditioning (aerobic perfusion followed by 30-min coronary occlusion and 120-min reperfusion, i.e., test ischemia-reperfusion), preconditioning (three intermittent periods of 5-min ischemia-reperfusion cycles before test ischemia-reperfusion), or postconditioning (six cycles of 10-s ischemia-reperfusion after test ischemia) perfusion protocol. Preconditioning and postconditioning significantly decreased infarct size in vehicle-treated hearts. However, preconditioning failed to decrease infarct size in acute lovastatin-treated hearts, but the effect of postconditioning remained unchanged. Chronic lovastatin treatment abolished postconditioning but not preconditioning; however, it decreased infarct size in the nonconditioned group. Myocardial levels of coenzyme Q9 were decreased in both acute and chronic lovastatin-treated rats. Western blot analysis revealed that both acute and chronic lovastatin treatment attenuated the phoshorylation of Akt; however, acute but not chronic lovastatin treatment increased the phosphorylation of p42 MAPK/ERK. We conclude that, although lovastatin may lead to cardioprotection, it interferes with the mechanisms of cardiac adaptation to ischemic stress.     

NO produced by endothelial NO synthase is a mediator of delayed preconditioning-induced endothelial protection

Preconditioning with brief periods of ischemia-reperfusion (I/R) induces a delayed protection of coronary endothelial cells against reperfusion injury. We assessed the possible role of nitric oxide (NO) produced during prolonged I/R as a mediator of this endothelial protection. Anesthetized rats were subjected to 20-min cardiac ischemia/60-min reperfusion, 24 h after sham surgery or cardiac preconditioning (1 x 2-min ischemia/5-min reperfusion and 2 x 5-min ischemia/5-min reperfusion). The nonselective NO synthase (NOS) inhibitor l-NAME, the selective inhibitors of neuronal (7-nitroindazole) or inducible (1400W) NOS, or the peroxynitrite scavenger seleno-l-methionine were administered 10 min before prolonged ischemia. Preconditioning prevented the reperfusion-induced impairment of coronary endothelium-dependent relaxations to acetylcholine (maximal relaxation: sham 77 +/- 3; I/R 44 +/- 6; PC 74 +/- 5%). This protective effect was abolished by l-NAME (41 +/- 7%), whereas 7-NI, 1400W or seleno-l-methionine had no effect. The abolition of preconditioning by l-NAME, but not by selective nNOS or iNOS inhibition, suggests that NO produced by eNOS is a mediator of delayed endothelial preconditioning.     

Inhibition of phosphatase activity enhances preconditioning and limits cell death in the ischemic/reperfused aged rat heart

Brief, nonlethal episodes of ischemia in the mammalian heart provide cardioprotection against the detrimental effects of a longer duration ischemia. The manifestation of this preconditioning (PC) phenomenon is initiated by the enhanced phosphorylation state of signal transduction proteins. We reported previously that PC is decreased in the aged rat myocardium. Although the mechanism responsible for this loss is not understood, a reduction in the phosphorylation of critical proteins associated with PC may be postulated. Experiments were conducted to investigate whether PC in the aged heart can be restored with the inhibition of endogenous protein phosphatases thereby enhancing phosphorylation of signaling proteins. Levels of phosphatase activities were also assessed with adult heart aging. Hearts from young adult (3-4 mo.) and aged (21-22 mo.) Fischer-344 rats were perfused in the presence or absence of okadaic acid (OKA; 0.1 microM). Aged adult hearts were either not preconditioned or were preconditioned with two PC cycles (5 min ischemia/5 min reperfusion). Myocardial cellular death that developed with a subsequent ischemia was determined with triphenyltetrazolium. With PC, 55% of the aged heart after ischemia was no longer viable. OKA administered before or after ischemia reduced this ischemia-induced cellular death by 29%. Without PC, OKA reduced viability 18% only when present before and after the ischemic episode. OKA in the ischemic young heart during reperfusion reduced the loss of viability 31%. The Protein Phosphatase 2A (PP2A) activity was found to be up to 82% greater in ventricular myocardium of aged rats. In conclusion, aging-induced changes in protein dephosphorylation may be one mechanism reducing the manifestation of preconditioning in the aged heart.     

Opioid receptor contributes to ischemic preconditioning through protein kinase C activation in rabbits

Recent studies have reported that protection from ischemic preconditioning (PC) is blocked by the opioid receptor antagonist naloxone (NAL). We tested whether an opioid agonist could mimic PC in the rabbit heart, whether that protection involved protein kinase C (PKC) activation, and whether opioid receptors act in concert with other PKC-coupled receptors. Rabbit hearts were subjected to 30min coronary occlusions and were reperfused for either 3 (in situ) or 2 (in vitro) h. Infarct size was determined by staining with triphenyltetrazolium chloride. In untreated in situ hearts 38.5 ± 1.6% of the risk zone infarcted. PC with 5 min ischemia/10 min reperfusion significantly limited infarction to 12.7 ± 2.9% (p < 0.01). NAL infusion did not modify infarction (39.6 ± 1.6%) in non-PC hearts, but blocked the effect of one cycle of PC (34.4 ± 3.6% infarction). NAL, however, could not block cardioprotection when PC was amplified with 3 cycles of ischemia/reperfusion (9.9 ± 1.4% infarction, p < 0.01 vs. control). Morphine could also mimic ischemic preconditioning, but only at a dose much higher than would be used clinically (3 mg/kg). In isolated hearts pretreatment with morphine (0.3 M) significantly limited infarction to 9.3 ± 1.2% (p < 0.01 vs. 32.0 ± 3.1% in controls). This cardioprotective effect of morphine could be blocked by either the PKC inhibitor chelerythrine (30.4 ± 2.6% infarction) or NAL (34.0 ± 2.6% infarction). Neither chelerythrine nor NAL by itself modified infarction in non-PC hearts. NAL could not block protection from one cycle of PC in isolated hearts indicating that an intact innervation may be required for endogenous opioid production. Thus, opioid receptors, like other PKC-coupled receptors, participate in the triggering PC in the rabbit heart.     

Intestinal preconditioning prevents inflammatory response by modulating heme oxygenase-1 expression in endotoxic shock model

Gut mucosal injury observed during ischemia-reperfusion is believed to trigger a systemic inflammatory response leading to multiple organ failure. It should be interesting to demonstrate this relationship between gut and multiple organ failure in a sepsis model. Intestinal preconditioning (PC) can be used as a tool to assess the effect of intestinal ischemia in inflammatory response after LPS challenge. The aim of this study was to investigate the protective effect of PC against LPS-induced systemic inflammatory and intestinal heme oxygenase-1 (HO-1) expression. ES was performed with LPS (10 mg/kg iv) with or without PC, which was done before LPS. Rats were first subjected to sham surgery or PC with four cycles of 1 min ischemia and 4 min of reperfusion 24 h before LPS challenge or saline administration. PC significantly reduced fluid requirements, lung edema, intestinal lactate production, and intestinal injury. Inflammatory mRNA expressions for intestine and lung ICAM and TNF were significantly reduced after PC, and these effects were significantly abolished by zinc-protoporphyrin (a specific HO-1 activity inhibitor) and mimicked by bilirubin administration. Intestinal PC selectively increased HO-1 mRNA expression in intestine, but we have observed no expression in lungs. These findings demonstrate that intestinal injury is a important event for inflammatory response and multiple organ injury after LPS challenge. Intestinal HO-1 expression attenuates LPS-induced multiple organ failure by modulating intestine injury and its consequences on inflammatory response. Identification of the exact mechanisms responsible for intestine HO-1 induction may lead to the development of new pharmacological interventions.     

Comparison of ischemic and chemical preconditioning in jejunal flaps in the rat

Jejunum is one of the most frequently used free flaps in esophagus reconstruction. However, the sensitivity of intestinal tissue to ischemia decreases the margin of safety of this donor site while increasing the risk of postoperative complications such as fistula formation and stenosis. Ischemic preconditioning can increase the tolerance of jejunal tissue to ischemia. In this study, the authors investigated the effects of chemical preconditioning with adenosine infusion on ischemia reperfusion injury in the rat jejunum, and evaluated the presence of any additive effects of adenosine administration when used together with ischemic preconditioning. Forty Sprague-Dawley rats weighting 200 to 250 mg were used in the study. Rats were randomly divided into five groups. In group I (sham-operated controls), only laparotomy was performed. In group II (ischemia-reperfusion injury), the superior mesenteric artery was clamped for 40 minutes to induce ischemia in the small bowel, followed by 60 minutes of reperfusion. In group III (ischemic preconditioning), two cycles of 5-minute ischemia and 5-minute reperfusion were performed before implementation of the ischemia-reperfusion protocol used in group II. In group IV (chemical preconditioning), adenosine (1000 microg/kg) was infused into the internal jugular vein before the group II ischemia-reperfusion schedule was implemented. In group V (adenosine-enhanced ischemic preconditioning), adenosine (1000 microg/kg) was infused into the internal jugular vein before ischemic preconditioning, followed by 40 minutes of ischemia and 60 minutes of reperfusion. At the end of the reperfusion period, samples from the jejunum were harvested and myeloperoxidase activity was determined as a measure of leukocyte accumulation. Malondialdehyde levels were measured to assess lipid peroxidation. Histopathologic sections stained with hematoxylin-eosin were evaluated for the presence of mucosal damage according to the Chiu scoring method. Immunohistochemical staining by M30 monoclonal antibodies was performed to quantify the number of ischemia-induced apoptotic cells in the intestinal mucosa. The myeloperoxidase and malondialdehyde levels were significantly lower in groups I, III, IV, and V when compared with group II. Although there were no significant differences among myeloperoxidase and malondialdehyde levels in groups III, IV, and V, group I had significantly lower levels of activity compared with the other three groups. Histological scoring reflected significantly less damage in groups I, III, IV, and V compared with group II. Similarly, the number of apoptotic cells was significantly lower in groups I, III, IV, and V when compared with group II. However, no difference was detected among these four groups with regard to either histopathological scoring or apoptosis numbers. This is the first study showing that adenosine administration is as effective as ischemic preconditioning in inducing ischemic tolerance in the rat jejunum. However, there was no enhancement of ischemic preconditioning with prior adenosine infusion.     

Ischemic adaptation of the rat brain as a method for protection of endothelium from ischemic reperfusion injury

This study represents results of investigation carried out to determine the endothelium-protective effect of early and late phases of brain ischemic preconditioning as well as local and remote adaptation. The experiments were performed on adult male rats. Prolonged 30-min four vessels brain ischemia followed by 120-min reperfusion on carotid arteries, was performed (control group). Early and late local ischemic preconditioning was due to both 5-min ischemia and 30-min and 48 h reperfusion respectively on carotid arteries. Remote ischemic preconditioning was caused by 30-min ischemia and also by 15-min and 48 h reperfusion, respectively (early and late phases of adaptation) on femoral artery before prolonged brain ischemia described above. To estimate the role of nitric oxide in ischemic adaptation, mechanisms involved both nonselective blocker of NO-synthesis (N omega-nitro-L-arginine) in the time of early adaptation phase and the relatively selective iNOS inhibitor S-methylisothiourea sulfate, given before sustained brain ischemia, on the late preconditioning. Registration of brain blood flow was made by ultrasonic high-frequency Doppler device. Degree of brain edema was studied and evaluation of desquamated endothelial cells in blood was carried out. Early and late phases of local ischemic preconditioning were found to improve the brain blood flow and level of circulatory endothelial cells as well as to reduce degree of edema. The endothelium-protective effect of remote ischemic preconditioning has been proved in this study only on the late phase. Nitric oxygen was found to be important endothelium-protective factor in ischemic preconditioning.     

Opioid inhibition of luteinizing hormone secretion in the postpartum lactating sow

Twelve lactating sows were used at 22.4 +/- 0.8 days postpartum to determine whether endogenous opioid peptides (EOP) are involved in the suckling-induced inhibition of luteinizing hormone (LH) secretion. Four sows each received either 1, 2, or 4 mg/kg body weight of naloxone (NAL), an opiate antagonist, in saline i.v. Blood was collected at 15-min intervals for 2 h before and 4 h after NAL treatment. All sows were then given 100 micrograms gonadotropin-releasing hormone (GnRH) in saline i.v., and blood samples were collected for an additional h. Pigs were weaned after blood sampling. At 40 h after weaning, sows were treated and blood samples collected as during suckling. Serum concentrations of LH after treatment with NAL were similar for all doses; therefore, the data were pooled across doses. During suckling, serum concentrations of LH were 0.41 +/- 0.04 ng/ml before NAL treatment, increased to 0.65 +/- 0.08 ng/ml at 30 min after NAL treatment, and remained elevated above pretreatment concentrations for 120 min (p less than 0.05). Naloxone failed to alter serum concentrations of LH after weaning. These data indicate that EOP may be involved in the suckling-induced suppression of LH secretion and that weaning may either decrease opioid inhibition of LH secretion or decrease pituitary LH responsiveness to endogenous GnRH released by NAL.     

Preconditioning in the absence or presence of sustained ischemia modulates myocardial Cx43 protein levels and gap junction distribution

In the heart, brief repeated episodes of ischemia prior to a sustained occlusion (ischemic preconditioning; PC) significantly delay the onset of necrosis and arrhythmogenesis. Ischemia has been reported to influence gap junction organization and connexin43 (Cx43) content, but whether PC affects these structures is not known. We investigated the effect of PC (2 cycles of 5-min ischemia plus 10-min reperfusion) followed by prolonged reperfusion without concomitant regional coronary occlusion on the myocardial Cx43 content and its spatial distribution in rabbit hearts. We also compared the effect of sustained ischemia with or without PC on Cx43 spatial distribution. In experiments with PC only, there was an initial decrease in Cx43 levels within the ischemic zone followed by a progressive increase after 48 h reperfusion. End-to-end immunolabeling of Cx43 was augmented in the ischemic region between 24 and 48 h reperfusion; labeling was not uniquely confined to myocyte abutments, but was also dispersed along the sarcolemma. Cx43 immunolabelling was more intense and diffuse in hearts subjected to PC before sustained coronary occlusion (compared to non-PC). These data indicate that gap junctions are significantly altered during brief episodes of ischemia. Reorganization of the gap junction complex could contribute to PC-mediated reductions in cardiac arrhythmias.     

Differential loss of cytochrome-c oxidase subunits in ischemia-reperfusion injury: exacerbation of COI subunit loss by PKC-epsilon inhibition

We have previously described a PKC-epsilon interaction with cytochrome oxidase subunit IV (COIV) that correlates with enhanced CO activity and cardiac ischemic preconditioning (PC). We therefore investigated the effects of PC and ischemia-reperfusion (I/R) injury on CO subunit levels in an anesthetized rat coronary ligation model. Homogenates prepared from the left ventricular regions at risk (RAR) and not at risk (RNAR) for I/R injury were fractionated into cell-soluble (S), 600 g low-speed centrifugation (L), gradient-purified mitochondrial (M), and 100,000 g particulate (P) fractions. In RAR tissue, PC (2 cycles of 5-min ischemia and 5-min reperfusion) decreased the COI in the P fraction ( approximately 29% of total cellular COI), suggesting changes in interfibrillar mitochondria. After 30 min of ischemia and 120 min of reperfusion, total COI levels decreased in the RAR by 72%. Subunit Va was also downregulated by 42% following prolonged I/R in the RAR. PC administered before I/R reduced the loss of COI in the M and P fractions approximately 30% and prevented COVa losses completely. We observed no losses in subunits Vb and VIIa following I/R alone; however, significant losses occurred when PC was administered before prolonged I/R. Delivery of a cell-permeable PKC-epsilon translocation inhibitor (epsilonV1-2) to isolated rat hearts before prolonged I/R dramatically increased COI loss, suggesting that PKC-epsilon protects COI levels. We propose that additional measures to protect CO subunits when coadministered with PC may improve its cardioprotection against I/R injury.     

Noradrenaline dynamics in prolonged ischemia after ischemic preconditioning

AIM OF THE STUDY: To determine the effects of two-staged ischemic preconditioning on myocardial noradrenaline in prolonged ischemia and reperfusion. METHODS: Thirty-two male Wistar rats anesthetised with urethane randomly divided into 2 groups: group 1 (ischemic preconditioning group, n = 16), and group 2 (control, n = 16). Myocardial interstitial noradrenaline levels were measured using a microdialysis technique. Ischemic preconditioning was elicited by two episodes: 5 min of ischemia and 10 min of reperfusion. The intermittent occlusions were followed by prolonged occlusion (60 min) and reperfusion (60 min). RESULTS: An increase in interstitial noradrenaline was observed in 10 min of prolonged ischemia in group 2, and in 20 min in group 1. After 20 min of myocardial ischemia there was a significant difference between groups (p < 0.05) in interstitial noradrenaline levels. In control group, it was 60% higher. In reperfusion, noradrenaline levels decreased markedly in group 1. CONCLUSION: We suggest that ischemic preconditioning by two episodes: 5-min ischemia and 10-min reperfusion prevents excessive noradrenaline interstitial accumulation, perhaps, through protection of physiological uptake I carrier.     

VEGFR1 (Flt-1+/-) gene knockout leads to the disruption of VEGF-mediated signaling through the nitric oxide/heme oxygenase pathway in ischemic preconditioned myocardium

This report demonstrates that mice deficient in Flt-1 failed to establish ischemic preconditioning (PC)-mediated cardioprotection in isolated working buffer-perfused ischemic/reperfused (I/R) hearts compared to wild type (WT) subjected to the same PC protocol. WT and Flt-1+/- mice were divided into four groups: (1) WT I/R, (2) WT + PC, (3) Flt-1+/- I/R, and (4) Flt-1+/- + PC. Group 1 and 3 mice were subjected to 30 min of ischemia followed by 2 h of reperfusion and group 2 and 4 mice were subjected to four episodes of 4-min global ischemia followed by 6 min of reperfusion before ischemia/reperfusion. For both wild-type and Flt-1+/- mice, the postischemic functional recovery for the hearts was lower than the baseline, but the recovery for the knockout mice was less compared to the WT mice even in preconditioning. The myocardial infarction and apoptosis were higher in Flt-1+/- compared to wild-type I/R. Flt-1+/- KO mice demonstrated pronounced inhibition of the expression of iNOS, p-AKT & p-eNOS. Significant inhibition of STAT3 & CREB were also observed along with the inhibition of HO-1 mRNA. Results demonstrate that Flt-1+/- mouse hearts are more susceptible to ischemia/reperfusion injury and also document that preconditioning is not as effective as found in WT and therefore suggest the importance of VEGF/Flt-1 signaling in ischemic/reperfused myocardium.     

Sustained exogenous administration of Met5-enkephalin protects against infarction in vivo

The opioid antagonist naloxone abolishes infarct limitation by myocardial ischemic preconditioning, suggesting that one or more endogenous opioid peptides can mediate cardiac protection against ischemic damage. We tested the hypothesis that the naturally occurring opioid peptide Met5-enkephalin (ME) modulates myocardial infarct size in vivo. Experiments were conducted in barbiturate-anesthetized open-chest rabbits subjected to regional myocardial ischemia-reperfusion. ME was administered via osmotic minipump for 24 h. Infarct size was assessed with tetrazolium and is expressed as a percentage of the area at risk. Exogenous ME reduced the amount of the risk zone infarcted by approximately 60% compared with saline-treated controls. ME-induced protection was sensitive to opioid receptor blockade with naloxone [NAL 50 +/- 2% vs. ME + NAL 39 +/- 3%, P = not significant (NS)] and also to blockade of sarcolemmal and mitochondrial ATP-sensitive K+ (KATP) channels [5-hydroxydecanoate (5-HD) 33 +/- 3% vs. ME + 5-HD 43 +/- 8%, P = NS; and HMR-1098 60 +/- 3% vs. ME + HMR-1098 54 +/- 7%, P = NS]. We conclude that ME limits ischemic injury in vivo by an opioid receptor-mediated mechanism that involves both sarcolemmal and mitochondrial KATP channels.