Cardiac tolerance to ischemia in neonatal spontaneously hypertensive rats

Hypertension is the risk factor of serious cardiovascular diseases, such as ischemic heart disease and atherosclerosis. The aim of the present study was to analyze the development of cardiac tolerance to ischemia in neonatal spontaneously hypertensive rats (SHR) and possible protective effect of ischemic preconditioning (IP) or adaptation to intermittent high-altitude hypoxia (IHAH). For this purpose we used 1- and 10-day-old pups of SHR and their normotensive control Wistar Kyoto rats (WKY). Isolated hearts were perfused in the Langendorff mode with Krebs-Henseleit solution at constant pressure, temperature and rate. Cardiac tolerance to ischemia was expressed as a percentage of baseline values of developed force (DF) after global ischemia. IP was induced by three 3-min periods of global ischemia, each separated by 5-min periods of reperfusion. IHAH was simulated in barochamber (8 h/day, 5000 m) from postnatal day 1 to 10. Cardiac tolerance to ischemia in 1-day-old SHR was higher than in WKY. In both strains tolerance decreased after birth, and the difference disappeared. The high cardiac resistance in 1- and 10-day-old SHR and WKY could not be further increased by both IP and adaptation to IHAH. It may be concluded that hearts from newborn SHR are more tolerant to ischemia/reperfusion injury as compared to age-matched WKY; cardiac resistance decreased in both strains during the first ten days, similarly as in Wistar rats.     

Intermittent Hypobaric Hypoxia Preconditioning Induced Brain Ischemic Tolerance by Up-Regulating Glial Glutamate Transporter-1 in Rats

Several studies showed that the up-regulation of glial glutamate transporter-1 (GLT-1) participates in the acquisition of brain ischemic tolerance induced by cerebral ischemic preconditioning or ceftriaxone pretreatment in rats. To explore whether GLT-1 plays a role in the acquisition of brain ischemic tolerance induced by intermittent hypobaric hypoxia (IH) preconditioning (mimicking 5,000?m high-altitude, 6?h per day, once daily for 28?days), immunohistochemistry and western blot were used to observe the changes in the expression of GLT-1 protein in hippocampal CA1 subfield during the induction of brain ischemic tolerance by IH preconditioning, and the effect of dihydrokainate (DHK), an inhibitor of GLT-1, on the acquisition of brain ischemic tolerance in rats. The basal expression of GLT-1 protein in hippocampal CA1 subfield was significantly up-regulated by IH preconditioning, and at the same time astrocytes were activated by IH preconditioning, which appeared normal soma and aplenty slender processes. The GLT-1 expression was decreased at 7?days after 8-min global brain ischemia. When the rats were pretreated with the IH preconditioning before the global brain ischemia, the down-regulation of GLT-1 protein was prevented clearly. Neuropathological evaluation by thionin staining showed that 200?nmol DHK blocked the protective role of IH preconditioning against delayed neuronal death induced normally by 8-min global brain ischemia. Taken together, the up-regulation of GLT-1 protein participates in the acquisition of brain ischemic tolerance induced by IH preconditioning in rats.     

Evidence for calcitonin gene-related peptide-mediated ischemic preconditioning in the rat heart.

Previous studies have suggested that calcitonin gene-related peptide (CGRP) may play an important role in the mediation of ischemic preconditioning. In the present study, we examined the release of CGRP during ischemic preconditioning and the effect of preconditioning frequency on this effect in the isolated rat heart. Thirty minutes of global ischemia and 40 min of reperfusion caused a significant cardiac dysfunction and an increase in the release of creatine kinase (CK) during reperfusion. Preconditioning with one, two or three cycles of 5-min ischemia and 5-min reperfusion caused a marked improvement of cardiac function and a decrease in the release of CK, and there was no difference in the degree of improvement among groups. The protective effects of ischemic preconditioning were abolished by the CGRP receptor antagonist CGRP(8-37). A single preconditioning cycle induced a significant increase in the release of CGRP in the coronary effluent. In the hearts treated with two or three preconditioning cycles, the level of CGRP was highest in the first cycle, and was gradually decreased with increasing number of cycles of preconditioning. These results suggest that the protective effects of ischemic preconditioning are mediated by endogenous CGRP in the isolated rat heart.     

The effect of preconditioning on the iron deposition after transient forebrain ischemia in rat brain

In this study we investigated iron deposition in the hippocampus CA1 area and the corpus striatum pars dorsolateralis in a rat model of cerebral ischemia and ischemic tolerance. Forebrain ischemia was induced by four-vessel occlusion for 5-min as ischemic preconditioning. Two days after the preconditioning or the sham operation, a second ischemia was induced for 20-min. With the use of iron histochemistry, regional changes were examined after 2 to 8 weeks of recirculation following the 20-min ischemia with or without preconditioning. Perl's reaction with DAB intensification demonstrated iron deposits in the CA1 area and in the corpus striatum pars dorsolateralis after 2 weeks of recirculation. These iron deposits gradually increased in density and formed clusters by the 8th week. When the rats were exposed to 5-min ischemia 2 days before lethal 20-min ischemia, the deposition of iron in the CA1 region of the hippocampus and also in the corpus striatum pars dorsolateralis was decreased and produced a minimal number of iron-containing cells between the second and the 8th week of recirculation. Preconditioning with sublethal 5-min ischemia followed by 2 days of reperfusion also prevented the neuronal destruction of the hippocampal CA1 region induced by 20-min ischemia.     

Role of endogenous opioid peptides in protection of ischemic preconditioning in rat small intestine

This study investigated the protective effects of ischemic preconditioning on intestinal ischemic injury and the role of endogenous opioid peptides (EOP) in these effects. Ischemia-reperfusion (I/R) induced by 30-min of ischemia and 60-min of reperfusion significantly increased the levels of malondialdehyde (MDA) and lactate dehydrogenase (LDH) and resulted in serious intestinal edema (wet weight/dry weight). The ischemic preconditioning (PC) elicited by three 8-min occlusion periods interspersed with 10-min reperfusion markedly attenuated intestinal injury caused by ischemia-reperfusion. Pretreatment with morphine (300 microg x kg(-1), i.v.) 10-min before ischemia and reperfusion mimicked the protection produced by PC. Naloxone (3 mg x kg(-1), i.v.) abolished the protection of morphine-induced preconditioning and ischemic preconditioning in rat intestine. However, there were no changes between naloxone alone and control groups. Treatment with naloxone before ischemia-reperfusion had no effect on animals compared with the I/R group. In addition, we also measured the content of endogenous opioid peptides (Leu-enkephalin) in the effluent which was collected before and during preconditioning. It was shown that the release of leu-enkephalin was markedly increased during preconditioning. These results suggested that EOP might play an important role in PC in rat small intestine.     

Influence of ischemic preconditioning on intracellular sodium,pH, and cellular energy status in isolated perfused heart

The possible relationships between intracellular Na(+) (Na(i)(+)), bioenergetic status and intracellular pH (pH(i)) in the mechanism for ischemic preconditioning were studied using (23)Na and (31)P magnetic resonance spectroscopy in isolated Langendorff perfused rat heart. The ischemic preconditioning (three 5-min ischemic episodes followed by two 5-min and one 10-min period of reperfusion) prior to prolonged ischemia (20 min stop-flow) resulted in a decrease in ischemic acidosis and faster and complete recovery of cardiac function (ventricular developed pressure and heart rate) after 30 min of reperfusion. The response of Na(i) during ischemia in the preconditioned hearts was characterized by an increase in Na(i)(+) at the end of preconditioning and an accelerated decrease during the first few minutes of reperfusion. During post-ischemic reperfusion, bioenergetic parameters (PCr/P(i) and betaATP/P(i) ratios) were partly recovered without any significant difference between control and preconditioned hearts. The reduced acidosis during prolonged ischemia and the accelerated decrease in Na(i)(+) during reperfusion in the preconditioned hearts suggest activation of Na(+)/H(+) exchanger and other ion transport systems during preconditioning, which may protect the heart from intracellular acidosis during prolonged ischemia, and result in better recovery of mechanical function (LVDP and heart rate) during post-ischemic reperfusion.     

大鼠肢体缺血预处理对肝缺血/再灌注损伤的保护作用

目的：研究肢体缺血预处理对大鼠肝缺血/再灌注损伤是否具有保护作用。方法：雄性SD大鼠32只,随机分为对照组（S组）;缺血/再灌注组（I/R组）;经典缺血预处理组（IPC组）;肢体缺血预处理组（远端缺血预处理组,RPC组）。S组仅行开腹,不作其他处理;IPC组以肝缺血5min作预处理;RPC组以双后肢缺血5min,反复3次作预处理,2个预处理组及I/R组均行肝缺血1h再灌注3h。取血用于血清谷丙转氨酶（ALT）与血清谷草转氨酶（AST）检测。切取肝组织用于测定湿干比（W/D）、中性粒细胞（PMN）计数及观察显微、超微结构的变化。结果：与I/R组比较,IPC组,RPC组ALT,AST,W/D值,及PMN计数均明显降低（P〈0.01）,肝脏的显微及超微结构损伤减轻。结论：肢体缺血预处理对大鼠肝脏I/R损伤有明显的保护作用,强度与经典缺血预处理相当,其机制可能与抑制肝脏炎症反应、减轻肝脏水肿、改善肝组织微循环有关。     

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.     

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.     

Electron microscopy and microcalorimetry of the postnatal rat heart (Rattus norvegicus)

The interplay of ultrastructure and tissue metabolism was examined in neonatal, infant and adult rat hearts by electron microscopy and microcalorimetry. Morphometry was used to determine parameters of oxygen diffusion capacity (distance between capillaries and mitochondria, capillary surface density) and oxidative metabolic capacity (mitochondrial volume fraction). Thin slices and large samples of living tissue were examined calorimetrically to quantify aerobic metabolism and ischemia tolerance, respectively. After birth, rat hearts grow in parallel to body mass and show characteristics of cellular hypertrophy. Capillary surface density increases from neonatal to infant rats, and decreases to an intermediate value in adult rats. The distance between capillaries and mitochondria shows no significant changes throughout postnatal development. Mitochondrial volume fraction increases continuously until adulthood. The specific aerobic tissue metabolic rate is higher in the neonatal than in the infant and adult rat. However, the ischemic decline in metabolic rate is much slower in the neonatal rat, reflecting an elevated hypoxia tolerance. In conclusion, the neonatal rat heart exhibits a high metabolic rate despite a low mitochondrial volume fraction. The subsequent structural rearrangements can be interpreted as long-term adaptations to the increased postnatal workload and may contribute to the progressive loss of hypoxia tolerance.     

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.     

Myocardium tolerant to an adenosine-dependent ischemic preconditioning stimulus can still be protected by stimuli that employ alternative signaling pathways

Clinical studies on cardioprotection by preinfarct angina are ambiguous, which may involve development of tolerance to repeated episodes of ischemia. Not all preconditioning stimuli use identical signaling pathways, and because patients likely experience varying numbers of episodes of preinfarct angina of different degrees and durations, it is important to know whether myocardium tolerant to a particular preconditioning stimulus can still be protected by stimuli employing alternative signaling pathways. We tested the hypothesis that development of tolerance to a particular stimulus does not affect cardioprotection by stimuli that employ different signaling pathways. Anesthetized rats underwent classical, remote or pharmacological preconditioning. Infarct size (IS), produced by a 60-min coronary artery occlusion (CAO), was determined after 120 min of reperfusion. Preconditioning by two 15-min periods of CAO (2CAO15, an adenosine-dependent stimulus) limited IS from 69 +/- 2% to 37 +/- 6%, but when 2CAO15 was preceded by 4CAO15, protection by 2CAO15 was absent (IS = 68 +/- 1%). This development of tolerance coincided with a loss of cardiac interstitial adenosine release, whereas two 15-min infusions of adenosine (200 microg/min i.v.) still elicited cardioprotection (IS = 40 +/- 4%). Furthermore, cardioprotection was produced when 4CAO15 was followed by the adenosine-independent stimulus 3CAO3 (IS = 50 +/- 8%) or the remote preconditioning stimulus of two 15-min periods of mesenteric artery occlusion (IS = 49 +/- 6%). In conclusion, development of tolerance to cardioprotection by an adenosine-dependent preconditioning stimulus still allows protection by pharmacological or ischemic stimuli intervention employing different signaling pathways.     

Noninvasive delayed limb ischemic preconditioning attenuates myocardial ischemia-reperfusion injury in rats by a mitochondrial K(ATP) channel-dependent mechanism

We previously demonstrated in rats that noninvasive delayed limb ischemic preconditioning (LIPC) induced by three cycles of 5-min occlusion and 5-min reperfusion of the left hind limb per day for three days confers the same cardioprotective effect as local ischemic preconditioning of the heart, but the mechanism has not been studied in depth. The aim of this project was to test the hypothesis that delayed LIPC enhances myocardial antioxidative ability during ischemia-reperfusion by a mitochondrial K(ATP) channel (mito K(ATP))-dependent mechanism. Rats were randomized to five groups: ischemia-reperfusion (IR)-control group, myocardial ischemic preconditioning (MIPC) group, LIPC group, IR-5HD group and LIPC-5HD group. The MIPC group underwent local ischemic preconditioning induced by three cycles of 5-min occlusion and 5-min reperfusion of the left anterior descending coronary arteries. The LIPC and LIPC-5HD groups underwent LIPC induced by three cycles of 5-min occlusion and 5-min reperfusion of the left hind limb using a modified blood pressure aerocyst per day for three days. All rats were subjected to myocardial ischemia-reperfusion injury. The IR-5HD and LIPC-5HD groups received the mito K(ATP) channel blocker 5-hydroxydecanoate Na (5-HD) before and during the myocardial ischemia-reperfusion injury. Compared with the IR-control group, both the LIPC and MIPC groups showed an amelioration of ventricular arrhythmia, reduced myocardial infarct size, increased activities of total superoxide dismutase, manganese-superoxide dismutase (Mn-SOD) and glutathione peroxidase, increased expression of Mn-SOD mRNA and decreased xanthine oxidase activity and malondialdehyde concentration. These beneficial effects of LIPC were prevented by 5-HD. In conclusion, delayed LIPC offers similar cardioprotection as local IPC. These results support the hypothesis that the activation of mito K(ATP) channels enhances myocardial antioxidative ability during ischemia-reperfusion, thereby contributing, at least in part, to the anti-arrhythmic and anti-infarct effects of delayed LIPC.     

Repeated simulated ischemia and protection against gap junctional uncoupling

Ischemic preconditioning increases the heart's tolerance to a subsequent longer ischemic period. The aim of this study was to investigate the effect of early and delayed preconditioning on gap junction communication, connexin abundance, and phosphorylation in cultured neonatal rat cardiac myocytes. Prolonged ischemia followed 5 minutes after preconditioning in the early protocol, whereas 20 hours separated preconditioning and prolonged ischemia in the delayed preconditioning protocol. Gap junctional intercellular communication (GJIC) was assessed by Lucifer yellow dye transfer. An initial reduction in communication in response to sublethal ischemia was observed. This may be one mechanism whereby neighboring cells are protected from damaging substances produced during the first phase of subsequent regional ischemia in early preconditioning protocols. With respect to delayed preconditioning, the transient decrease in GJIC disappeared prior to prolonged ischemia, indicating that other mechanisms are responsible for delayed protection. Both early and delayed preconditioning preserved intercellular coupling after prolonged ischemia and this correlated with presence of less connexin43 dephosphorylation assessed by immunoblot.     

Ischemic Preconditioning Protects Against Gap Junctional Uncoupling in Cardiac Myofibroblasts

Ischemic preconditioning increases the heart's tolerance to a subsequent longer ischemic period. The purpose of this study was to investigate the role of gap junction communication in simulated preconditioning in cultured neonatal rat cardiac myofibroblasts. Gap junctional intercellular communication was assessed by Lucifer yellow dye transfer. Preconditioning preserved intercellular coupling after prolonged ischemia. An initial reduction in coupling in response to the preconditioning stimulus was also observed. This may protect neighboring cells from damaging substances produced during subsequent regional ischemia in vivo, and may preserve gap junctional communication required for enhanced functional recovery during subsequent reperfusion.     

Protection of calcitonin gene-related peptide-mediated preconditioning against coronary endothelial dysfunction induced by reperfusion in the isolated rat heart

This study was designed to explore the protective effect of ischemic preconditioning on reperfusion-induced coronary endothelial dysfunction, with a focus on the role of calcitonin gene-related peptide (CGRP) in this effect, in the isolated perfused rat heart. Thirty minutes of global ischemia and 30 min of reperfusion significantly decreased heart rate, left ventricular pressure, and its first derivative and impaired vasodilator responses to acetylcholine. Ischemia-reperfusion did not affect vasodilator responses to sodium nitroprusside. Preconditioning induced by three cycles of 5 min of ischemia and 5 min of reperfusion produced a significant improvement in cardiac function concomitantly with an amelioration of vasodilator responses to acetylcholine. The protective effects of ischemic preconditioning were abolished by CGRP(8-37) (10(-7) M) , the selective CGRP receptor antagonist. After pretreatment with capsaicin (50 mg x kg(-1), s.c.) to deplete endogenous CGRP, the preconditioning effect was absent. Pretreatment with exogenous CGRP (5 x 10(-9) M) for 5 min induced a preconditioning-like protection. The present study suggests that the cardioprotection of ischemic preconditioning is related to the preservation of the coronary endothelial cell, and that the protective effect of preconditioning is mediated by endogenous CGRP in the isolated perfused rat heart.     

Ischemic preconditioning protects against gap junctional uncoupling in cardiac myofibroblasts

Ischemic preconditioning increases the heart's tolerance to a subsequent longer ischemic period. The purpose of this study was to investigate the role of gap junction communication in simulated preconditioning in cultured neonatal rat cardiac myofibroblasts. Gap junctional intercellular communication was assessed by Lucifer yellow dye transfer. Preconditioning preserved intercellular coupling after prolonged ischemia. An initial reduction in coupling in response to the preconditioning stimulus was also observed. This may protect neighboring cells from damaging substances produced during subsequent regional ischemia in vivo, and may preserve gap junctional communication required for enhanced functional recovery during subsequent reperfusion.