Monophosphoryl lipid A induces pharmacologic ‘preconditioning’ in rabbit hearts without concomitant expression of 70-kDa heat shock protein

The purpose of this study was to evaluate the protective effect of a new endotoxin analogue, monophosphoryl lipid A (MLA) in a rabbit model of myocardial ischemia/reperfusion and to show if this protection was mediated via synthesis of 70 kDa heat shock protein (HSP 70). Three groups of New Zealand White rabbits underwent 30 min coronary occlusion, followed by 4 hours reperfusion. First group of rabbits (n = 6) were treated with 0.35 ml vehicle (40 % propylene glycol, 10 % ethanol in water). The second and third group of rabbits (n = 6–8) were treated with MLA (35 g/kg, i.v.) 12 and 24 hours prior to ischemia and reperfusion. MLA treatment either 12 or 24 h prior to ischemia/reperfusion demonstrated significantly reduced infarct size (12.5 ± 1.7 and 14.7 ± 2.1% for 12 and 24 h) when compared with vehicle control (40.4 ± 8.6%, mean ± S.E.M, p < 0.05). No significant differences in the infarct size was observed between the 12 and 24 h MLA treated groups. The area at risk was not significantly different between the three groups. Baseline values of heart rate, systolic and diastolic blood pressure were not significantly different between the control and MLA treated groups. However, the systolic as well as diastolic blood pressure during reperfusion were significantly lower in rabbits treated with MLA. Western blot analysis of the protein extracts of the hearts (n = 2/group) demonstrated no increase in the expression of the inducible form of HSP 70 following treatment with MLA. We conclude that MLA has significant anti-infarct effect in rabbit which is not mediated by the cardioprotective protein HSP 70. The anti-infarct effect of this drug is superior to the reported protective effects of delayed ischemic or heat stress preconditioning. We hypothesize that the pharmacologic preconditioning afforded by MLA is accomplished via a unique pathway that bypasses the usual intracellular signaling pathways which lead to the myocardial protection with the expression of heat shock proteins.A preliminary form of this paper was presented at the XV World Congress of the International Society for Heart Research held in Prague, Czech Republic [17].This work was supported in part by grants HL 46763 and 51045 from Natinal Institute of Health and a grant from RIBI ImmunoChem Research, Hamilton, Montana. JBS was supported from a training grant HL 07537 from National Institutes of Health.This article was published in Molecular and Cellular Biochemistry 156: 1–8, 1996. Kluwer Academic Publishers regret the publication of the incorrect version.     

Cerebral Blood Flow Threshold and Regional Heterogeneity of Heat Shock Protein 72 Induction Following Transient Forebrain Ischemia in Rats

Heat shock proteins (HSPs) induced by brain ischemia may play an important role in neuroprotection from neuronal degeneration. In this study, we examined the cerebral blood flow (CBF) threshold to produce regional differences in HSP72 induction after transient forebrain ischemia in spontaneously hypertensive rats (SHRs). Female SHRs were subjected to 20 min of cerebral ischemia induced by bilateral carotid artery occlusion. The CBF was measured by laser Doppler flowmetry. At forty-eight hours after cerebral ischemia and reperfusion, the rats were decapitated and the brains were removed. Specific areas (hippocampal CA1, CA2-3, dentate gyrus, dorsolateral and ventromedial striatum, and parietal cortex) were thereafter dissected from the brain. The amounts of HSP72 in these samples were determined using Western blot analysis. In the hippocampus, HSP72 was induced when the CBF decreased to less than 18–25% of the resting level. The mean values of HSP72 produced in the CA1 area, CA2-3 area, and the dentate gyrus following ischemia and reperfusion treatment were 4.44 ± 1.43 (±SD) ng/g prtein, 3.51 ± 0.72 ng/g protein and 3.77 ± 1.05 ng/g protein, respectively. In the parietal cortex, the amount of HSP72 induction was less pronounced (2.55 ± 0.40 ng/g protein), while HSP72 was hardly detected at all in the striatum, even under conditions of very severe CBF reduction and reperfusion. We demonstrated the existence of both a CBF threshold (i.e., approximately 20% of the resting level) for HSP72 induction and regional heterogeneity for the induction of HSP72 protein.     

Equal reduction in infarct size by ethylisopropyl-amiloride pretreatment and ischemic preconditioning in the in situ rabbit heart

Inhibition of Na⁺/H⁺ exchange with amiloride analogues has been shown to provide functional protection during ischemia and reperfusion and to reduce infarct size in isolated rat hearts. In rat hearts, treatment with ethylisopropyl-amiloride (EIPA, a selective Na⁺/H⁺ exchange inhibitor) was additive to the protection afforded by ischemic preconditioning. In addition, such compounds have been demonstrated to reduce infarct size in in situ rabbit hearts. The aim of the present study was to determine to what extent preischemic treatment with EIPA could reduce infarct size in an in situ rabbit model of regional ischemia and reperfusion. We also wished to determine if this effect was additive to the infarct reducing effect of ischemic preconditioning. Anaesthetized, open chest rabbits, were subjected to 45 min of regional ischemia and 150 min of reperfusion. The risk zone was determined by fluorescent particles and infarct size was determined by TTC staining. Four groups were investigated: control, ischemic preconditioned (IP) (5 min of ischemia followed by 10 min reperfusion), EIPA (0.65 mg/kg iv given preischemically) and EIPA + IP. The main results expressed as percent infarction of the risk zone ± S.E.M. for the different groups were: control 59.2 ± 3.3% (n = 6), IP 16.3 ± 2.1% (n = 6) (p < 0.001 vs. control), EIPA 16.9 ± 4.1% (n = 5) (p < 0.001 vs. control), EIPA + IP 22.5 ± 9.5% (n = 6) (p < 0.001 vs. control). In conclusion: EIPA, when administered prior to ischemia, caused a reduction in infarct size in the in situ rabbit heart which was similar to that seen with ischemic preconditioning, however, the effect was not additive to ischemic preconditioning.     

Altered ischemic induction of immediate early gene and heat shock protein 70 mRNAs after preconditioning in rat hearts

Immediate early genes and heat shock protein (HSP) 70s, which may play a role in adaptation and cellular protection, respectively, are induced by ischemia in hearts. We examined if the induction of immediate early gene (c-fos, c-myc, c-jun, and junB) and HSP70 mRNAs by ischemia is affected by ischemic preconditioning. Transient ischemia (5 or 10 minute) was applied to Wistar rat (n=75) hearts, by tightening a snare placed around left coronary arterial branches 7 days before applying ischemia. Rats without earlier ischemia (control group, C) and rats with 5-minute ischemia 12 or 24 hours earlier (EI12 or 24 group) were given 10-minute ischemia and sacrificed at 0, 0.5, 1, 2, and 4 hour. RNA was extracted from the ischemic region and Northern blot analysis was performed. The induction of c-fos and c-myc mRNAs was significantly increased in EI12 but not in EI24 compared with that in C. The induction of c-jun and junB mRNAs showed no change in both EI12 and EI24 compared with that in C. The induction of HSP72 and 73 mRNAs was decreased in EI12 and decreased further in EI24. Thus, ischemic preconditioning altered the induction of immediate early gene and HSP70 mRNAs by ischemia. The effect of preconditioning differed among genes studied and changed with time after preconditioning. Ischemic preconditioning alters protective and adaptive responses to ischemia at the gene level.     

Hypoxic preconditioning reinforces HIF-alpha-dependent HSP70 signaling to reduce ischemic renal failure-induced renal tubular apoptosis and autophagy

AimsRepetitive hypoxic preconditioning (RHP) may provide more efficient protection than single hypoxic preconditioning against renal ischemia/reperfusion-induced injury via hypoxia-induced factor 1α (HIF-1α)-dependent heat shock protein 70 (HSP70) pathways.Main methodsWistar rats were subjected to intermittent hypoxic exposure (15 h/day), whereas controls were kept at sea level. We evaluated renal expression of HIF-1α, HSP70, the endoplasmic reticulum stress protein GRP78, caspase 12, Beclin-1, and poly-(ADP-ribose)-polymerase (PARP) with western blotting. Renal apoptosis determined by terminal transferase dUTP nick end labeling (TUNEL), Beclin-1-dependent autophagy, and monocyte/macrophage (ED-1) infiltration were evaluated by immunocytochemistry. Renal function was determined with blood urea nitrogen (BUN) and plasma creatinine levels. HIF-1α inhibitors and Deoxyribonucleotide (DNA) or Ribonucleotide (RNA) interference of HSP70 were used to evaluate their possible roles in this process.Key findingsRenal HIF-1α and HSP70 expression were enhanced by hypoxic preconditioning and inhibited by the HIF-1α inhibitor, YC-1, as well as phosphatidylinositol 3-kinase (PI3K)/Akt inhibitors. After the return to normoxia, renal HSP70 protein levels were maintained for one week in the RHP group, but they decayed after one day in the single hypoxic preconditioning group. Ischemia/reperfusion significantly increased renal TUNEL-apoptosis, Beclin-1-dependent autophagy, ED-1 infiltration, expression of GRP78, caspase 12, Beclin-1, PARP, and BUN and plasma creatinine levels in control rats. RHP significantly decreased all ischemia/reperfusion-enhanced parameters. Intraperitoneal pretreatment with YC-1 and quercetin (an inhibitor of HSP70 induction) eliminated RHP-induced protection. Anti-sense oligodeoxyribonucleotides or interference RNA targeting HSP70 abrogated the protection against hypoxia/reoxygenation-induced oxidative injury in RHP-treated proximal tubules.SignificanceWe demonstrate that RHP promotes HIF-1α-dependent HSP70 signaling to reduce renal ischemia/reperfusion injury.     

Possible Involvement of Oxidative Stress and Inflammatory Mediators in the Protective Effects of the Early Preconditioning Window Against Transient Global Ischemia in Rats

Ischemic preconditioning (IPC), comprising exposure to sub-lethal short term ischemic events, has been shown to exert adaptive responses in many organs including the brain, thus guarding against exacerbations of ischemia reperfusion (IR). However, the mechanisms involved in the early phase of such a protection remain elusive; hence, the present study aimed to investigate the modulatory effect of preconditioning against IR induced injury on infarct size, free radicals, inflammatory/anti-inflammatory markers, caspase-3 and heat shock protein (HSP)70 in the rat hippocampus. To this end, male Wistar rats were divided into 3 groups, (1) sham operated (SO) control; (2) IPC, animals were subject to 3 episodes of ischemia (5 min) followed by reperfusion (10 min), afterwards rats underwent ischemia (15 min) followed by reperfusion (60 min); (3) IR animals were subjected to 15 min global ischemia followed by 60 min reperfusion. IR produced cerebral infarction accompanied by an imbalance in the hippocampal redox status, neutrophil infiltration, elevation in tumor necrosis factor (TNF)-α and prostaglandin (PG)E₂, besides reduction in interleukin (IL)-10 and nitric oxide (NO) levels. IPC reverted all changes except for PGE₂; however, neither HSP70 nor caspase-3 expression was altered following IR or IPC. The current study points thus towards the activation of the antioxidant system, anti-inflammatory pathway, as well as NO in the early phase of preconditioning protection.     

Loss of myocardial protection from ischemic preconditioning following chronic exposure to R(-)-N6-(2-phenylisopropyl)adenosine is related to defect at the adenosine A1 receptor

Exogenously administered adenosine agonist will protect myocardium against infarction during ischemia. However, long-term exposure to adenosine agonists is associated with loss of this protection. To determine why this protection is lost, isolated, perfused rabbit hearts were studied after administration of R(-)-N⁶-(2-phenylisopropyl)adenosine (PIA), 0.25 mg/h IP, for 3-4 days to intact animals. All hearts experienced 30 min of regional ischemia and 120 min of reperfusion. Control groups 1 and 2 were untreated. In group 1 this ischemia/reperfusion was the only intervention, whereas group 2 hearts were preconditioned with a cycle of 5 min global ischemia/10 min reperfusion preceding the 30 min regional ischemia. Groups 3-5 had been chronically exposed to PIA. Group 3 hearts had 1 preconditioning ischemia/reperfusion cycle before the prolonged ischemia. Group 4 received a 5 min infusion of 0.1 mol/L phenylephrine in lieu of global ischemia, whereas group 5 was instead treated with 1 mol/L carbachol. Infarct size averaged 32% of the risk zone in group 1, whereas ischemic preconditioning limited infarction to 8.2 in group 2. Prolonged exposure of group 3 hearts to PIA resulted in the inability of preconditioning with 5 min global ischemia to protect (28.7 ± 4.4% infarction). However, protection was restored by either phenylephrine, an agonist of ₁-adrenergic receptors which couple to G_q and stimulate PKC, or carbachol, an agonist of M₂-muscarinic receptors which couple instead to G_i as do adenosine A₁ receptors (5.2 ± 1.7% and 9.2 ± 2.1% infarction, resp.). Therefore, cross tolerance to ischemic preconditioning develops after chronic PIA infusion. Since both the G_i and the PKC components of the preconditioning pathway were shown to be intact, tolerance must have been related to downregulation or desensitization of the A₁ adenosine receptor.     

Absence of ischemic preconditioning protection in diabetic sheep hearts: Role of sarcolemmal KATP channel dysfunction

Sarcolemmal ATP-sensitive potassium (KATP) channels have been mentioned to participate in preconditioning protection. Since these channels are altered in diabetes, it would be possible that preconditioning does not develop in diabetic (D) hearts. The purpose of this study was to assess whether early (EP) and late (LP) ischemic preconditioning protect diabetic hearts against stunning in a conscious diabetic sheep model and whether diabetes might have altered KATP channel functioning. Sheep received alloxan monohydrate (1 g) and were ascribed to three experimental groups: control (DC, 12 min of ischemia (I) followed by 2 h of reperfusion (R)), early preconditioning (DEP, six 5 min I – 5 min R periods were performed before the 12 min I) and late preconditioning (DLP, same as DEP except that the preconditioning stimulus was performed 24 h before the 12 min I). Regional mechanics during reperfusion was evaluated as the percent recovery of wall thickening fraction (%WTH) expressed as percentage of basal values (100%) and KATP behaviour was indirectly assessed by monophasic action potential duration (MAPD) and sensitivity to glibenclamide blockade (0.1 and 0.4 mg/Kg). The results were compared to those obtained in normal (N) sheep. EP and LP protected against stunning in normal sheep (%WTH: NC = 63 ± 3.7, NLP = 80 ± 5**, NEP equals; 78 ± 3*, *p < 0.05 and **p < 0.01 against NC) whereas contrary results occurred in diabetic ones, where DLP (%WTH = 60 ± 4) afforded a similar recovery to DC (%WTH = 54 ± 5) and DEP surprisingly worsened instead of improving mechanical function (%WTH = 38 ± 6, p < 0.01 against DC). KATP channel behaviour appeared altered in diabetic hearts as shown by MAPD during ischemia in normal sheep (153 ± 9 msec) compared to diabetic ones (128 ± 11 msec, p < 0.05) and by the sensitivity to glibenclamide (while 0.4 mg/Kg blocked action potential shortening in normal and diabetic animals, 0.1 mg/Kg completely blocked KATP in diabetic but not in normal hearts, p ( 0.05). A sarcolemmal KATP channel dysfunction might afford a primary approach to explain the absence of ischemic preconditioning protection against stunning in diabetic sheep.     

Is stunning prevented by ischemic preconditioning?

In a model of global ischemia in the isolated perfused rat heart, a 20-min ischemic period followed by 30 min of reperfusion induces a decrease in isovolumic developed pressure (LVDP) and +dP/dt_max to 61 ± 6% and 61 ± 7% of baseline, respectively. Left ventricular end-diastolic pressure (LVEDP) increases to 36 ± 4 mmHg at the end of the reperfusion period. No significant necrotic area as assessed by triphenyltetrazolium chloride (TTC) was detected at the end of the reperfusion period. By an immunohistochemical method using antiactin monoclonal antibodies 10.8 ± 1.9% of unstained cells were detected in the stunned hearts and 10.3 ± 1.2% in control hearts. Preceding the ischemic episode with a cycle of 5 min of ischemia followed by 10 min of reperfusion (ischemic preconditioning) protected contractile function. LVDP and +dP/dt_max now stabilized at 89 ± 5% and 94 ± 5% of baseline respectively. LVEDP was 20 ± 2 mmHg at the end of the reperfusion period. The protection of contractile dysfunction after 20 min of ischemia was achieved also by early reperfusion of low Ca²⁺-low pH perfusate. With this intervention LVDP stabilized at 87 ± 5% of baseline. LVEDP was 12 ± 2 mmHg at the end of the reperfusion period. A positive inotropic intervention induced by a modified postextrasystolic potentiation protocol at the end of the reperfusion period increases LVDP to levels higher than baseline in the stunned hearts. However, these values were less than those obtained in control hearts. Ischemic preconditioning significantly increased the maximal inotropic response. Therefore, ischemic preconditioning diminishes the contractile dysfunction of early stunning.     

Myocardial ischemic preconditioning and mitochondrial F1F0-ATPase activity

A short period of ischemia followed by reperfusion (ischemic preconditioning) is known to trigger mechanisms that contribute to the prevention of ATP depletion. In ischemic conditions, most of the ATP hydrolysis can be attributed to mitochondrial F₁F₀-ATPase (ATP synthase). The purpose of the present study was to examine the effect of myocardial ischemic preconditioning on the kinetics of ATP hydrolysis by F₁F₀-ATPase. Preconditioning was accomplished by three 3-min periods of global ischemia separated by 3 min of reperfusion. Steady state ATP hydrolysis rates in both control and preconditioned mitochondria were not significantly different. This suggests that a large influence of the enzyme on the preconditioning mechanism may be excluded. However, the time required by the reaction to reach the steady state rate was increased in the preconditioned group before sustained ischemia, and it was even more enhanced in the first 5 min of reperfusion (101 ± 3.0 sec in preconditioned vs. 83.4 ± 4.4 sec in controls, p 0.05). These results suggest that this transient increase in activation time may contribute to the cardioprotection by slowing the ATP depletion in the very critical early phase of post-ischemic reperfusion.     

Insulin improves postischemic recovery of function through PI3K in isolated working rat heart

Insulin improves contractile function after ischemia, but does not increase glucose uptake in the isolated working rat heart. We tested the hypothesis that the positive inotropic effect of insulin is independent of the signaling pathway responsible for insulin-stimulated glucose uptake. We inhibited this pathway at the level of phosphatidyl inositol 3-kinase (PI3K) with wortmannin. Hearts were perfused for 70 min at physiological workload with Krebs-Henseleit buffer containing [2-³H] glucose (5 mM, 0.05 Ci/ml) and oleate (0.4 mM, 1% BSA) in the presence (WM, n = 5) or absence (control, n = 7) of wortmannin (WM, 3 mol/L). After 20 min, hearts were subjected to 15 min of total global ischemia followed by 35 min of reperfusion. Insulin (1 mU/ml) was added at the beginning of reperfusion (WM + insulin n = 8, insulin n = 8). Cardiac power before ischemia was 8.1 ± 0.7 mW. Recovery of contractile function after ischemia was significantly increased in the presence of insulin (73.5 ± 8.9% vs. 38.5 ± 6.7%, p < 0.01). The addition of wortmannin completely abolished the effect of insulin on recovery (32.6 ± 6.4%). Glucose uptake was 1.84 ± 0.32 mol/min/g dry before ischemia and was slightly elevated during reperfusion (2.68 ± 0.35 mol/min/g dry, n.s.). Insulin did not affect postischemic glucose uptake. In the presence of wortmannin, glucose uptake was lowest during reperfusion (n.s.). The results suggest that PI3K is involved in the insulin-induced improvement in postischemic recovery of contractile function. This effect of insulin is independent of its effect on glucose uptake.     

Heat stress pretreatment mitigates postischemic arachidonic acid accumulation in rat heart

Heat stress pretreatment of the heart is known to protect this organ against an ischemic/reperfusion insult 24 h later. Degradation of membrane phospholipids resulting in tissue accumulation of polyunsaturated fatty acids, such as arachidonic acid, is thought to play an important role in the multifactorial process of ischemia/reperfusion-induced damage.The present study was conducted to test the hypothesis that heat stress mitigates the postischemic accumulation of arachidonic acid in myocardial tissue, as a sign of enhanced membrane phospholipid degradation. The experiments were performed on hearts isolated from rats either 24 h after total body heat treatment (42°C for 15 min) or 24 h after sham treatment (control). Hearts were made ischemic for 45 min and reperfused for another 45 min.Heat pretreatment resulted in a significant improvement of postischemic hemodynamic performance of the isolated rat hearts. The release of creatine kinase was reduced from 30 ± 14 (control group) to 17 ± 5 units/g wet wt per 45 min (heat-pretreated group) (p < 0.05). Moreover, the tissue content of the inducible heat stress protein HSP70 was found to be increased 3-fold 24 h after heat treatment. Preischemic tissue levels of arachidonic acid did not differ between heat-pretreated and control hearts. The postischemic ventricular content of arachidonic acid was found to be significantly reduced in heat-pretreated hearts compared to sham-treated controls (6.6 ± 3.3. vs. 17.8 ± 12.0 nmol/g wet wt). The findings suggest that mitigation of membrane phospholipid degradation is a potential mechanism of heat stress-mediated protection against the deleterious effects of ischemia and reperfusion on cardiac cells.     

Acclimatization to neurological decompression sickness in rabbits

Diving acclimatization refers to a reduced susceptibility to acute decompression sickness (DCS) in individuals undergoing repeated compression-decompression cycles. We demonstrated in a previous study that the mechanism responsible for this acclimatization is similar to that of stress preconditioning. In this study, we investigated the protective effect of prior DCS preconditioning on the severity of neurological DCS in subsequent exposure to high pressure in rabbits. We exposed the rabbits (n = 10) to a pressure cycle of 6 absolute atmospheres (ATA) for 90 min, which induced signs of neurological DCS in 60% of the animals. Twenty-four hours after the pressure cycle, rabbits with DCS expressed more heat-shock protein 70 (HSP70) in the lungs, liver, and heart than rabbits without signs of disease or those in the control group (n = 6). In another group of rabbits (n = 24), 50% of animals presented signs of neurological DCS after exposure to high pressure, with a neurological score of 46.5 (SD 19.5). A course of hyperbaric oxygen therapy alleviated the signs of neurological DCS and ensured the animals' survival for 24 h. Experiencing another pressure cycle of 6 ATA for 90 min, 50% of 12 rabbits with prior DCS preconditioning developed signs of DCS, with a neurological score of 16.3 (SD 28.3), significantly lower than that before hyperbaric oxygen therapy (P = 0.002). In summary, our results show that the occurrence of DCS in rabbits after rapid decompression is associated with increased expression of a stress protein, indicating that the stress response is induced by DCS. This phenomenon was defined as "DCS preconditioning." DCS preconditioning attenuated the severity of neurological DCS caused by subsequent exposure to high pressure. These results suggest that bubble formation in tissues activates the stress response and stress preconditioning attenuates tissue injury on subsequent DCS stress, which may be the mechanism responsible for diving acclimatization.     

Attenuation of ischemic preconditioning in pigs by scavenging of free oxyradicals with ascorbic acid

Free oxyradicals are involved in the signal transduction of ischemic preconditioning in rats and rabbits. Data from larger mammals in which the infarct development is closer to that in humans are lacking. We have therefore investigated the impact of the radical scavenger ascorbic acid on ischemic preconditioning in pigs. In 33 anesthetized pigs, the left anterior descending coronary artery was perfused from an extracorporeal circuit. Infarct size (measured as percent area at risk) was determined by triphenyltetrazolium chloride staining. In placebo-treated animals undergoing 90 min of severe ischemia and 120 min of reperfusion, infarct size averaged 26.9 +/- 3.9% (mean +/- SE; n = 9). Ischemic preconditioning by 10 min of ischemia and 15 min of reperfusion reduced infarct size to 6.4 +/- 2.4% (P < 0.05 vs. placebo; n = 9). Intravenous infusion of ascorbic acid (30 min before ischemic preconditioning or ischemia; 2-g bolus followed by 25 mg/min until the end of ischemia) had no effect on infarct size per se (22.6 +/- 6.5%; n = 6), but largely abolished the infarct size reduction by ischemic preconditioning (19.1 +/- 5.4%; n = 9). Scavenging of free oxyradicals with ascorbic acid largely attenuates the beneficial effect of ischemic preconditioning in pigs.     

镁预处理对兔全脑缺血神经保护作用的实验研究

目的探讨硫酸镁预处理对兔全脑缺血神经保护作用及其机制.方法 45只兔随机分为3组:对照组(n=15)、缺血组(n=15)和镁预处理组(n=15).阻断血管,诱导全脑缺血,缺血时间为 6 min.测定缺血再灌注30 min 兔海马谷氨酸、天冬氨酸、γ-氨基丁酸和甘氨酸含量.检测缺血再灌注3 d 海马CA1区神经元密度和凋亡神经元密度.结果 (1)镁预处理组海马天冬氨酸和甘氨酸显著低于缺血组(P<0.01),而γ-氨基丁酸含量显著高于缺血组(P<0.05);(2)镁预处理组正常神经元密度显著高于缺血组 (P<0.01),缺血神经元密度显著低于缺血组(P<0.01);(3)镁预处理组凋亡神经元密度显著低于缺血组(P<0.01).结论 (1)硫酸镁预处理对兔全脑缺血有神经保护作用;(2)该保护作用的机制可能有:1)抑制兔全脑缺血再灌注30 min 海马天冬氨酸和甘氨酸的过度释放以及抑制γ-氨基丁酸的耗竭;2)抑制兔全脑缺血再灌注3 d 海马CA1区神经元凋亡.     

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.     

Protaglandins and the antiarrhythmic effect of preconditioning in the isolated rat heart

Our study evaluated the relationship between the endogenous production of prostacyclin and the antiarrhythmic effect of ischemic preconditioning against ischemic and reperfusion-induced tachyarrhythmia. Langendorff perfused rat hearts underwent 30 min regional ischemia with reperfusion. Preconditioning was induced by a single episode of 5 min ischemia and 15 min reperfusion. Prostaglandin 6-keto F₁ (a stable metabolite of prostacyclin) was determined in the coronary effluent.In the control group the incidence of tachyarrhythmia was 31 % during ischemia and 67% during reperfusion. Preconditioning did not affect ischemic arrhythmias but attenuated arrhythmias a reperfusion (8%, p < 0.01) and was associated with increased release of prostacyclin prior to reperfusion. Aspirin abolished the antiarrhythmic effect of preconditioning against reperfusion tachyarrhythmias. However, no relationship was found between suppression of prostacyclin production and the occurrence of arrhythmia in individual hearts.Thus, our findings suggest that metabolites of arachidonic acid via the cyclooxygenase pathway are involved in the protective effect of ischemic preconditioning against reperfusion-induced tachyarrhythmias. (Mol Cell Biochem 160/161: 249–255, 1996)     

No Effect of Remote Ischemic Conditioning Strategies on Recovery from Renal Ischemia-Reperfusion Injury and Protective Molecular Mediators

Ischemia-reperfusion injury (IRI) is the major cause of acute kidney injury. Remote ischemic conditioning (rIC) performed as brief intermittent sub-lethal ischemia and reperfusion episodes in a distant organ may protect the kidney against IRI. Here we investigated the renal effects of rIC applied either prior to (remote ischemic preconditioning; rIPC) or during (remote ischemic perconditioning; rIPerC) sustained ischemic kidney injury in rats. The effects were evaluated as differences in creatinine clearance (CrCl) rate, tissue tubular damage marker expression, and potential kidney recovery mediators. One week after undergoing right-sided nephrectomy, rats were randomly divided into four groups: sham (n = 7), ischemia and reperfusion (IR; n = 10), IR+rIPC (n = 10), and IR+rIPerC (n = 10). The rIC was performed as four repeated episodes of 5-minute clamping of the infrarenal aorta followed by 5-minute release either before or during 37 minutes of left renal artery clamping representing the IRI. Urine and blood were sampled prior to ischemia as well as 3 and 7 days after reperfusion. The kidney was harvested for mRNA and protein isolation. Seven days after IRI, the CrCl change from baseline values was similar in the IR (δ: 0.74 mL/min/kg [-0.45 to 1.94]), IR+rIPC (δ: 0.21 mL/min/kg [-0.75 to 1.17], p > 0.9999), and IR+rIPerC (δ: 0.41 mL/min/kg [-0.43 to 1.25], p > 0.9999) groups. Kidney function recovery was associated with a significant up-regulation of phosphorylated protein kinase B (pAkt), extracellular regulated kinase 1/2 (pERK1/2), and heat shock proteins (HSPs) pHSP27, HSP32, and HSP70, but rIC was not associated with any significant differences in tubular damage, inflammatory, or fibrosis marker expression. In our study, rIC did not protect the kidney against IRI. However, on days 3–7 after IRI, all groups recovered renal function. This was associated with pAkt and pERK1/2 up-regulation and increased HSP expression at day 7.