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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.sub.ATP] channels play a pivotal role in the trigger and mediator mechanisms. We observed that pig bilateral latissimus dorsi (LD) muscle flaps sustained 46 [+ or -] 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 [+ or -] 3, 26 [+ or -] 1, 23 [+ or -] 2, 24 [+ or -] 2 and 24 [+ or -] 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.sub.ATP] (S[K.sub.ATP]) channel opener P-1075 (2 [micro]g/kg) at 24 h before 4 h of sustained ischemia (i.e., late preconditioning) reduced muscle infarction from 43 [+ or -] 4% (ischemic control) to 24 [+ or -] 2 and 19 [+ or -] 3%, respectively (P < 0.05, n = 8). Intravenous injection of the S[K.sub.ATP] channel inhibitor HMR 1098 (6 mg/kg) or the nonspecific [K.sub.ATP] channel inhibitor glibenclamide (Glib; 1 mg/kg) at 10 min before remote IPC completely blocked the infarctprotective effect of remote [PC in LD muscle flaps subjected to 4 h of sustained ischemia at 24 h after remote IPC. Intravenous bolus injection of the mitochondrial [K.sub.ATP] (N[K.sub.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 S[K.sub.ATP] and m[K.sub.ATP] channels play a central role in the trigger and mediator mechanism, respectively. late phase protection; adenosine 5'-triphosphate; myeloperoxidase; muscle ischemia/reperfusion injury

We previously demonstrated in the pig that instigation of three cycles of 10 min of occlusion and reperfusion in a hindlimb by tourniquet application (~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 ([K.sub.ATP]) (m[K.sub.ATP]) 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 [+ or -] 2% to 22 [+ or -] 3% (n = 10; P < 0.05). The nonselective [K.sub.ATP], channel inhibitor glibenclamide (0.3 mg/kg) or the selective m[K.sub.ATP] channel inhibitor 5-hydroxydecanoate (5-HD, 5 mg/kg), but not the selective sarcolemmal [K.sub.ATP] (s[K.sub.ATP]) 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 m[K.sub.ATP] channel 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 m[K.sub.ATP] 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 m[K.sub.ATP] channels in ischemic skeletal muscle is associated with an ATP-sparing effect during sustained ischemia and attenuation of neutrophil accumulation during reperfusion. pig skeletal muscle; remote ischemic preconditioning; trigger and mediator mechanism

Various laboratories have reported that local subcutaneous or subdermal injection of VEG[F.sub.165] at the time of surgery effectively attenuated ischemic necrosis in rat skin flaps, but the mechanism was not studied and enhanced angiogenesis was implicated. In the present study, we used the clinically relevant isolated perfused 6 x 16-cm pig buttock skin flap model to 1) test our hypothesis that VEG[F.sub.165] is a potent vasodilator and acute VEG[F.sub.165] treatment increases skin perfusion; and 2) investigate the mechanism of VEG[F.sub.165]-induced skin vasorelaxation. We observed that VEG[F.sub.165] (5 x [10.sup.16] -5 x [10.sup.-11] M) elicited a concentration-dependent decrease in perfusion pressure (i.e., vasorelaxation) in skin flaps preconstricted with a submaximal concentration of norepinephrine (NE), endothelin-1, or U-46619. The VEG[F.sub.165]-induced skin vasorelaxation was confirmed using a dermofluorometry technique for assessment of skin perfusion. The vasorelaxation potency of VEG[F.sub.165] in NE-preconstricted skin flaps (p[D.sub.2] = 13.57 [+ or -] 0.31) was higher (P < 0.05) than that of acetylcholine (p[D.sub.2] = 7.08 [+ or -] 0.24). Human placental factor, a specific VEGF receptor-1 agonist, did not elicit any vasorelaxation effect. However, a specific antibody to VEGF receptor-2 (1 [micro]g/ml) or a specific VEGF receptor-2 inhibitor (5 x [10.sup.-6] M SU-1498) blocked the vasorelaxation effect of VEG[F.sub.165] in NE-preconstricted skin flaps. These observations indicate that the potent vasorelaxation effect of VEG[F.sub.165] in the skin vasculature is initiated by the activation of VEGF receptor-2. Furthermore, using pharmacological probes, we observed that the postreceptor signaling pathways of VEG[F.sub.165]-induced skin vasorelaxation involved activation of phospholipase C and protein kinase C, an increase in inositol 1,4,5-trisphosphate activity, release of the intracellular [Ca.sub.2+] store, and synthesis/release of endothelial nitric oxide, which predominantly triggered the effector mechanism of VEG[F.sub.165]-induced vasorelaxation. This information provides, for the first time, an important insight into the mechanism of VEG[F.sub.165] protein or gene therapy in the prevention/treatment of ischemia in skin flap surgery and skin ischemic diseases. VEG[F.sub.165] receptors; signaling pathways; nitric oxide; prostacyclin; skin flap

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].sub.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 [adenosine.sub.1] 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. ischemic preconditioning; humoral mediator; ATP; lactate; myeloperoxidase

We previously demonstrated in the pig that instigation of three cycles of 10 min of occlusion and reperfusion in a hindlimb by tourniquet application (∼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 × 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 mKATP channel 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. [ABSTRACT FROM AUTHOR]