Noninvasive remote ischemic preconditioning for global protection of skeletal muscle against infarction

The aim of this study was to investigate the efficacy and mechanism of action of a noninvasive remote ischemic preconditioning (IPC) technique for the protection of multiple distant skeletal muscles against ischemic necrosis (infarction). It was observed in the pig that three cycles of 10-min occlusion and reperfusion in a hindlimb by tourniquet application reduced the infarction of latissimus dorsi (LD), gracilis (GC), and rectus abdominis (RA) muscle flaps by 55%, 60%, and 55%, respectively, compared with their corresponding control (n = 6, P < 0.01) when they were subsequently subjected to 4 h of ischemia and 48 h of reperfusion. This infarct-protective effect of remote IPC in LD muscle flaps was abolished by an intravenous bolus injection of the nonselective opioid receptor antagonist naloxone (3 mg/kg) 10 min before remote IPC and a continuous intravenous infusion (3 mg/kg) during remote IPC and by an intravenous bolus injection of the selective delta 1-opioid receptor antagonist 7-benzylidenealtrexone maleate (3 mg/kg). However, this infarct-protective effect of remote IPC was not affected by an intravenous bolus injection of the ganglionic blocker hexamethonium chloride (20 mg/kg) or the nonspecific adenosine receptor antagonist 8-(p-sulfophenyl)theophylline (10 mg/kg) or by a local intra-arterial injection of the adenosine1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (3 mg/muscle flap) given 10 min before remote IPC. It was also observed that this remote IPC of skeletal muscle against infarction was associated with a slower rate of muscle ATP depletion during the 4 h of sustained ischemia and a reduced muscle neutrophilic myeloperoxidase activity after 1.5 h of reperfusion. These observations led us to speculate that noninvasive remote IPC by brief cycles of occlusion and reperfusion in a pig hindlimb is effective in global protection of skeletal muscle against infarction. This infarct-protective effect is most likely triggered by the activation of opioid receptors in the skeletal muscle, and remote IPC is associated with an energy-sparing effect during sustained ischemia and attenuation of neutrophil accumulation during reperfusion.     

Role and mechanism of PKC in ischemic preconditioning of pig skeletal muscle against infarction

Protein kinase C (PKC) inhibitors, chelerythrine (Chel, 0.6 mg) and polymyxin B (Poly B, 1.0 mg), and PKC activators, phorbol 12-myristate 13-acetate (PMA, 0.05 mg) and 1-oleoyl-2-acetyl glycerol (OAG, 0.1 mg), were used as probes to investigate the role of PKC in mediation of ischemic preconditioning (IPC) of noncontracting pig latissimus dorsi (LD) muscles against infarction in vivo. These drugs were delivered to each LD muscle flap (8 x 12 cm) by 10 min of local intra-arterial infusion. It was observed that LD muscle flaps sustained 43 +/- 5% infarction when subjected to 4 h of global ischemia and 24 h of reperfusion. IPC with three cycles of 10 min ischemia-reperfusion reduced muscle infarction to 25 +/- 3% (P < 0.05). This anti-infarction effect of IPC was blocked by Chel (42 +/- 7%) and Poly B (37 +/- 2%) and mimicked by PMA (19 +/- 10%) and OAG (14 +/- 5%) treatments (P < 0.05), given 10 min before 4 h of ischemia. In addition, the ATP-sensitive K(+) (K(ATP)) channel antagonist sodium 5-hydroxydecanoate attenuated (P < 0.05) the anti-infarction effect of IPC (37 +/- 2%), PMA (44 +/- 17%), and OAG (46 +/- 9%). IPC, OAG, and Chel treatment alone did not affect mean arterial blood pressure or muscle blood flow assessed by 15-microm radioactive microspheres. Western blot analysis of muscle biopsies obtained before (baseline) and after IPC demonstrated seven cytosol-associated isoforms, with nPKCepsilon alone demonstrating progressive cytosol-to-membrane translocation within 10 min after the final ischemia period of IPC. Using differential fractionation, it was observed that nPKCepsilon translocated to a membrane compartment other than the sarcolemma and/or sarcoplasmic reticulum. Furthermore, IPC and preischemic OAG but not postischemic OAG treatment reduced (P < 0.05) muscle myeloperoxidase activity compared with time-matched ischemic controls during 16 h of reperfusion after 4 h of ischemia. Taken together, these observations indicate that PKC plays a central role in the anti-infarction effect of IPC in pig LD muscles, most likely through a PKC-K(ATP) channel-linked signal-transduction pathway.     

Early effects of high intensity x-radiation on skeletal muscle

Comparative studies of x-radiation effects on both isolated cold blooded (frog) and intact warm blooded (rabbit) muscles were performed. Frog muscles irradiated with doses above 50 kr showed early fatigue, contracture, prolongation of relaxation time, decreased contraction amplitude for heavy loads, and histologic changes noticeable 8 hours after exposure. Rabbit muscles exposed to 72 kr exhibited a gradually progressing impairment of function. Complete abolishment of function was reached within 24 hours following irradiation and was accompanied by severe histologic alterations.     

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

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

Inducing late phase of infarct protection in skeletal muscle by remote preconditioning: efficacy and mechanism

We have previously demonstrated that remote ischemic preconditioning (IPC) by instigation of three cycles of 10-min occlusion/reperfusion in a hindlimb of the pig elicits an early phase of infarct protection in local and distant skeletal muscles subjected to 4 h of ischemia immediately after remote IPC. The aim of this project was to test our hypothesis that hindlimb remote IPC also induces a late phase of infarct protection in skeletal muscle and that K(ATP) channels play a pivotal role in the trigger and mediator mechanisms. We observed that pig bilateral latissimus dorsi (LD) muscle flaps sustained 46 +/- 2% infarction when subjected to 4 h of ischemia/48 h of reperfusion. The late phase of infarct protection appeared at 24 h and lasted up to 72 h after hindlimb remote IPC. The LD muscle infarction was reduced to 28 +/- 3, 26 +/- 1, 23 +/- 2, 24 +/- 2 and 24 +/- 4% at 24, 28, 36, 48 and 72 h after remote IPC, respectively (P < 0.05; n = 8). In subsequent studies, hindlimb remote IPC or intravenous injection of the sarcolemmal K(ATP) (sK(ATP)) channel opener P-1075 (2 microg/kg) at 24 h before 4 h of sustained ischemia (i.e., late preconditioning) reduced muscle infarction from 43 +/- 4% (ischemic control) to 24 +/- 2 and 19 +/- 3%, respectively (P < 0.05, n = 8). Intravenous injection of the sK(ATP) channel inhibitor HMR 1098 (6 mg/kg) or the nonspecific K(ATP) channel inhibitor glibenclamide (Glib; 1 mg/kg) at 10 min before remote IPC completely blocked the infarct- protective effect of remote IPC in LD muscle flaps subjected to 4 h of sustained ischemia at 24 h after remote IPC. Intravenous bolus injection of the mitochondrial K(ATP) (mK(ATP)) channel inhibitor 5-hydroxydecanoate (5-HD; 5 mg/kg) immediately before remote IPC and 30-min intravenous infusion of 5-HD (5 mg/kg) during remote IPC did not affect the infarct-protective effect of remote IPC in LD muscle flaps. However, intravenous Glib or 5-HD, but not HMR 1098, given 24 h after remote IPC completely blocked the late infarct-protective effect of remote IPC in LD muscle flaps. None of these drug treatments affected the infarct size of control LD muscle flaps. The late phase of infarct protection was associated with a higher (P < 0.05) muscle content of ATP at the end of 4 h of ischemia and 1.5 h of reperfusion and a lower (P < 0.05) neutrophilic activity at the end of 1.5 h of reperfusion compared with the time-matched control. In conclusion, these findings support our hypothesis that hindlimb remote IPC induces an uninterrupted long (48 h) late phase of infarct protection, and sK(ATP) and mK(ATP) channels play a central role in the trigger and mediator mechanism, respectively.     

Impaired skeletal muscle microcirculation in systemic sclerosis

Introduction

Muscle symptoms in systemic sclerosis (SSc) may originate from altered skeletal muscle microcirculation, which can be investigated by means of blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI).

Methods

After ethics committee approval and written consent, 11 consecutive SSc patients (5 men, mean age 52.6 years, mean SSc disease duration 5.4 years) and 12 healthy volunteers (4 men, mean age 45.1 years) were included. Subjects with peripheral arterial occlusive disease were excluded. BOLD MRI was performed on calf muscles during cuff-induced ischemia and reactive hyperemia, using a 3-T whole-body scanner (Verio, Siemens, Erlangen, Germany) and fat-suppressed single-short multi-echo echo planar imaging (EPI) with four different effective echo times. Muscle BOLD signal time courses were obtained for gastrocnemius and soleus muscles: minimal hemoglobin oxygen saturation (T2*_min) and maximal T2* values (T2*_max), time to T2* peak (TTP), and slopes of oxygen normalization after T2* peaking.

Results

The vast majority of SSc patients lacked skeletal muscle atrophy, weakness or serum creatine kinase elevation. Nevertheless, more intense oxygen desaturation during ischemia was observed in calf muscles of SSc patients (mean T2*_min -15.0%), compared with controls (-9.1%, P = 0.02). SSc patients also had impaired oxygenation during hyperemia (median T2*_max 9.2% vs. 20.1%, respectively, P = 0.007). The slope of muscle oxygen normalization was significantly less steep and prolonged (TTP) in SSc patients (P<0.001 for both). Similar differences were found at a separate analysis of gastrocnemius and soleus muscles, with most pronounced impairment in the gastrocnemius.

Conclusions

BOLD MRI demonstrates a significant impairment of skeletal muscle microcirculation in SSc.     

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.     

Early and late systemic effects of colchicine on muscle permeability to inorganic phosphate

A single systemic injection of 75 micrograms colchicine/100 g body weight in the lumbar muscles increases within 6 h the permeability of the extensor digitorum longus muscle to inorganic phosphate. Twenty four hours after the injection the specific activities and isotope uptakes of both inorganic and organic-bound acid-soluble phosphates are markedly increased. By the third day a maximal four-fold increase in rate of inorganic phosphate exchange is reached. The observed effects are slowly reversible, a near normal situation obtaining after 30 days. They are qualitatively different from those observed after administration of vincristine sulphate and bupivaca?ne (Marcaine).     

Pulsatile pressure and flow in the skeletal muscle microcirculation

Although blood flow in the microcirculation of the rat skeletal muscle has negligible inertia forces with very low Reynolds number and Womersley parameter, time-dependent pressure and flow variations can be observed. Such phenomena include, for example, arterial flow overshoot following a step arterial pressure, a gradual arterial pressure reduction for a step flow, or hysteresis between pressure and flow when a pulsatile pressure is applied. Arterial and venous flows do not follow the same time course during such transients. A theoretical analysis is presented for these phenomena using a microvessel with distensible viscoelastic walls and purely viscous flow subject to time variant arterial pressures. The results indicate that the vessel distensibility plays an important role in such time-dependent microvascular flow and the effects are of central physiological importance during normal muscle perfusion. In-vivo whole organ pressure-flow data in the dilated rat gracilis muscle agree in the time course with the theoretical predictions. Hemodynamic impedances of the skeletal muscle microcirculation are investigated for small arterial and venous pressure amplitudes superimposed on an initial steady flow and pressure drop along the vessel.     

Differential serotonin responses in the skeletal muscle microcirculation

Closed circuit television microscopy was used to quantitate in vivo responses of small vessels in the rat cremaster muscle to topically applied serotonin. Sprague-Dawley rats were anesthetized with a combination of urethane (800 mg/kg) and alpha-chloralose (60 mg/kg). The cremaster muscle with intact circulation and innervation was suspended in a bath which had controlled pH, pCO2, and pO2. Microvascular diameters of first order arterioles and venules and fourth-order arterioles were measured from the television monitor while serotonin (10(-9)M-10(-4)M) was added to the bath. Fourth-order arterioles (3-11 micron diameter) dilated to a maximum of 267% of their control value with a serotonin concentration of 10(-6)M. Serotonin (10(-4)M) constricted first-order arterioles (78-121 micron) to 61% of their control value. The threshold concentration (10(-8)M) for a serotonin-induced dilation of fourth-order arterioles was 1000 fold less than the threshold concentration (10(-5)M) for serotonin-induced constriction of first-order arterioles. Serotonin (10(-8)M - 10(-4)M) did not alter the diameter of first-order venules (115-195 micron) from the control value. The dose-dependent constriction of first-order arterioles and dose-dependent dilation of fourth-order arterioles by serotonin appear to be independent of each other. In addition, the lack of constriction of first-order venules suggests a heterogenous distribution of serotonin receptors and that the predominate control mechanisms are different at different levels of the arteriolar and venous microcirculation of rat skeletal muscle.     

缓激肽在骨骼肌缺血预适应对猪心肌坏死和凋亡中的作用

目的：研究缓激肽在骨骼肌缺血预适应对心肌坏死和心肌凋亡保护中可能的作用：方法：采用非开胸法建立猪心脏缺血／再灌注（I／R）模型,通过球囊堵塞左股动脉造成骨骼肌短暂缺血,使用缓激肽（BK）的B2受体拮抗剂烟酸已可碱（HOE-140）以及外源BK进行干预。分别观察各组对心肌坏死和凋亡的影响。结果：远端预处理后心肌坏死范围明显缩小,凋亡率明显降低：预处理前使用HOE-140可使对坏死范围的保护作用明显减弱;心肌I／R前使用外源BK注射,可缩小心肌梗死范围。但HOE-140及外源BK对以上凋亡指标无影响．结论：骨骼肌远端预适应可减少心肌坏死和心肌凋亡、BK可能参与对坏死面积的保护．但不参与对凋亡的保护作用。     

蛋白激酶C与缺血预处理心肌保护作用

蛋白激酶Ｃ（ＰＫＣ）是心肌细胞磷脂酰肌醇信号转导系统的重要组成部分,ＰＫＣ在缺血预处理（ＩＰ）过程中的移位和激活在ＩＰ的心肌保护中发挥了关键的作用。ＰＫＣ介导ＩＰ心肌保护作用的机制与其磷酸化底物蛋白有关,包括激活细胞外－５‘－核苷酸酶,激活ＡＴＰ敏感性钾通道以及维持细胞内Ｃａ＾２＋稳态。对ＰＫＣ发挥ＩＰ心肌保护作用机制的探索将为人类心血管疾病的治疗提供新的理论基础和药理手段。     

缺血预适应对肢体缺血/再灌注骨骼肌、小肠和肺损伤的影响

目的:观察肢体缺血/再灌注(LI/R)后骨骼肌、小肠、肺功能损伤变化,并探讨缺血预适应(IPC)的保护效应及机制。方法:实验用雄性Wistar大鼠24只,随机分为3组(n=8):对照(Control)组,缺血/再灌注(I/R)组和缺血预适应(IPC+I/R)组。分别测定血浆乳酸脱氢酶(LDH)、肌酸激酶(CK)、活性氧(ROS)、丙二醛(MDA)、动脉血氧分压(PaO2)和二氧化碳分压(PaCO2),测定血浆血栓素B2(TXB2),6-酮-前列腺素F1α(6-keto-PGF1α)的含量以及TXB2/6-keto-PGF1α比值的变化;测定骨骼肌、小肠、肺组织髓过氧化物酶(MPO)含量,肺湿干比(W/D)及小肠组织DAO含量。观察骨骼肌组织的形态学变化。结果:IPC+I/R组血浆LDH、CK、ROS、MDA、TXB2/6-keto-PGF1α比值明显低于I/R组,PaO2较I/R组明显升高。IPC+I/R组肺湿干比(W/D),骨骼肌、肺、小肠组织MPO含量明显低于I/R组,而小肠DAO活性升高。骨骼肌组织病理学改变减轻。结论:缺血预适应减轻了缺血/再灌注后骨骼肌、小肠、肺功能的损伤,其机制可能与降低氧化损伤、改善TXB2/6-keto-PGF1α的平衡关系有关。     

Early electrocortical changes consistent with ischemic preconditioning in rat

Ischemic preconditioning (IPC) of the brain describes the neuroprotection induced by a short, conditioning ischemic episode (CIE) to a subsequent severe (test) ischemic episode (TIE). Most of the supporting evidence for IPC is based on histological assessment, several days after TIE. The aim of this study is to investigate if changes induced by IPC can be detected within 30 min of reperfusion following the ischemic episode. A rat model of "four-vessel occlusion" transient global cerebral ischemia and parametric analysis of electrocorticogram were used. A control group was subjected directly to a 10 min TIE, and in a preconditioned group TIE was induced 48 h after a 3 min CIE. Quantitative histology was performed 48 h after TIE. Our key finding is that, 30 min after reperfusion, there is a significant increase in the electrocortical slow activity in the control group but not in the preconditioned group. Moreover the increase inversely correlates with the degree of electrocortical suppression during seconds 10 to 15 after the onset of the ischemic episode.     

Effects of 8-Gy radiation on the microcirculation of muscle flaps in the rat.

Combination of radical excision and radiation has been used as a treatment modality for cancer patients. As a result, in reconstructive surgery there is often a need to harvest flaps in the vicinity of previously irradiated tissues. Radiation has been shown to cause progressive injury to the macrocirculation and microcirculation, often jeopardizing flap survival. The purpose of this study was to examine whether radiation significantly affects the sequence of leukocyte-endothelial interactions or the hemodynamics of the muscle flap in both acute and chronic situations. Male Sprague-Dawley rats (n = 42) were divided into seven groups of six rats each. Rats in group I were not irradiated. Groups II through VII received 8-Gy radiation to the right groin and scrotum. Groups II, III, and IV were examined at 4, 24 and 72 hours, respectively, and groups V, VI, and VII were examined at 1, 2 and 12 weeks. For intravital microscopy, the cremaster muscle was dissected on its neurovascular pedicle. Vessel diameters and red blood cell velocities were measured in the central cremasteric branches and branch arterioles. Capillary perfusion was evaluated in 27 visual fields of each flap. Leukocyte-endothelial interactions were evaluated by numbers of rolling, adhering, and transmigrating leukocytes in post-capillary venules. In the same postcapillary venule, we measured the endothelial edema index (constriction index). The hemodynamics of irradiated flaps did not differ significantly from those of controls. Diameter and red blood cell velocity were increased in the first- and second-order arterioles and were highest at 72 hours and 1 week. After irradiation, third-order arterioles were constricted. Radiation reduced capillary perfusion by 4.3, percent. None of the differences were statistically significant. Neither leukocyte behavior nor the constriction indices differed among the groups. In conclusion, low-dose radiation of 8 Gy does not affect hemodynamics or leukocyte-endothelial interactions of muscle flaps in the rat. Muscle tissue with intact microvasculature can be harvested for reconstructive procedures after low-dose radiation.     

预热处理对缺血心肌的保护作用及蛋白激酶C信号转导通路机制

缺血心肌的保护一直是心血管研究领域的热点问题。目前,除了继续从外源性药物途径进行研究外,有一个崭新的领域越来越受到人们的重视,即通过心肌细胞本身的内源性抗损伤能力,产生自身保护作用来减轻缺血的损害。研究发现,适当的预处理可以有效调动该保护机制。本文就近年来发展较快的预热处理(HP)对缺血心肌的保护作用及其蛋白激酶C(PKC)信号转导通路机制作一简要概述。