Anguo Zhong

Acute ischaemic preconditioning protects against skeletal muscle infarction in the pig

OBJECTIVE The aims were to investigate the efficacy of acute ischaemic preconditioning for protection of skeletal muscles against infarction and its effect on muscle blood flow and ischaemic muscle metabolism. METHODS The efficacy of preconditioning was tested by subjecting pig latissimus dorsi and gracilis muscles to different numbers and durations of ischaemia/reperfusion cycles before 4 h of global ischaemia. Infarction was assessed at 48 h of reperfusion, using nitroblue tetrazolium dye. Blood flow in the latissimus dorsi was measured at the end of preconditioning and 1.5 and 3.0 h of reperfusion, using the radioactive microsphere (15 microns) technique. Muscle biopsies were taken from the latissimus dorsi before ischaemia, at the end of 2 and 4 h of ischaemia, and 1.5 h of reperfusion. RESULTS At least three cycles of 10 min ischaemia and 10 min reperfusion were required for preconditioning of latissimus dorsi and gracilis muscles for protection against infarction. Preconditioning reduced the total infarct size by 44% and 62% in latissimus dorsi and gracilis muscles, respectively. Preconditioning did not affect preischaemia muscle blood flow but it reduced the muscle content (preischaemia reserve) of phosphocreatine and ATP and the muscle energy charge potential (ECP) by 13.5%*, 27.5%*, and 8%* (*P < 0.05), respectively. In spite of a lower preischaemia reserve of phosphocreatine and ATP, the muscle contents of phosphocreatine and ATP and muscle ECP were maintained higher and the lactate lower (*P < or = 0.05) in the preconditioned than in the non-preconditioned (control) muscles at the end of 4 h of ischaemia [phosphocreatine 8.0(SEM 0.4) v 3.2(0.3)*; ATP 9.8(0.7) v 7.8(0.3); ECP 0.72(0.02) v 0.66(0.01)*; lactate 115.4(8.6) v 160.5(11.8)* mumol.g-1 dry muscle]. The level of ATP and ECP also remained significantly higher and the level of lactate significantly lower in the preconditioned than in the non-preconditioned latissimus dorsi muscles at 1.5 h of reperfusion. Hyperaemia was seen in the preconditioned latissimus dorsi muscles at 1.5 h of reperfusion and it subsided by the end of 3h of reperfusion. CONCLUSIONS The protective effect of preconditioning can be induced in pig skeletal muscle but at a higher threshold than reported previously in pig cardiac muscle (one cycle). Preconditioning of pig skeletal muscle is associated with a lower energy metabolism during sustained ischaemia. At the present time, it is not known if this energy sparing effect is a major mechanism of ischaemic preconditioning against infarction in skeletal muscles.

Assessment of ischemia-induced reperfusion injury in the pig latissimus dorsi myocutaneous flap model.

Experiments were conducted to assess ischemia-induced reperfusion injury in the pig latissimus dorsi myocutaneous flap model. Forty Yorkshire pigs (19.5 +/- 0.6 kg) were assigned to groups A, B, C, and D (n = 10 pigs). Bilateral 8 x 13 cm latissimus dorsi myocutaneous flaps were constructed in each pig, and one flap was assigned to ischemic treatment and the contralateral flap served as a nonischemic control. The treatment flaps in groups A, B, C, and D were subjected to 2, 4, 6, and 8 hours of warm global ischemia, respectively. Pigs in groups A, B, C, and D were divided into two subgroups (n = 5 pigs), and extents of skin and muscle necrosis in control and treatment flaps were assessed with the fluorescein and nitroblue tetrazolium dye stain tests, respectively, after 2 and 7 days of reperfusion. Significantly (p < 0.01) greater extents of skin and muscle necrosis were observed in latissimus dorsi myocutaneous flaps subjected to 4, 6, or 8 hours of ischemia compared with their contralateral controls. Extents of skin and muscle necrosis also increased significantly (p < 0.01) with increases in ischemia time in treatment flaps. Of particular importance was the observation that there was no significant difference in the extent of skin or muscle necrosis between 2 and 7 days of reperfusion in all control and treatment groups. This observation indicates that 2 days of reperfusion time is adequate to assess the maximum extent of skin and muscle ischemia-induced reperfusion injury in pig latissimus dorsi myocutaneous flaps. Furthermore, it was observed that 1-cm segments of latissimus dorsi muscle were not too thick to allow the use of the nitroblue tetrazolium dye stain test for assessment of muscle viability, as judged by the highly correlated (r = 0.98, n = 40) linear relationship between assessment of muscle viability from one transverse cut surface of muscle segments and by weighing total viable and nonviable muscles dissected from the flaps according to the nitroblue tetrazolium dye stain on both transverse cut surfaces. It is important to note that the maximum length of the latissimus dorsi myocutaneous flap model for ischemia-induced reperfusion injury research should not exceed the maximum length of skin viability in the nonischemic control in order to avoid the complication of skin necrosis due to excessive length of skin.(ABSTRACT TRUNCATED AT 400 WORDS)

Pharmacologic intervention of skin vasospasm and ischemic necrosis in pigs

Ischemic necrosis resulting from vasospasm is a common complication in skin flap surgery, and serotonin released by traumatized platelets is likely to play an important role in the pathogenesis of skin vasospasm in flap surgery. We studied the pathogenic role of serotonin and its pharmacologic intervention thereof in skin flap ischemic necrosis in pigs. We observed that serotonin caused a concentration-dependent (10-8-10-5 M) increase in perfusion pressure in isolated perfused pig skin flaps. This vasoconstrictive effect of serotonin was blocked by SIC/2-serotonergic receptor antagonists LY53857 (10-5 M) and ketanserin (10-5 M), but not by an α1-adrenoceptor antagonist (prazosin 10-5 M), or a thromboxane A2 (TxA2)/endoperoxide receptor antagonist (SQ30741 10-5 M). The vasoconstrictive effect of serotonin was more pronounced (p < 0.05) in the presence of an endothelium-derived nitric oxide (NO) synthesis inhibitor [N&phis;-monomethyl-L-arginine (L-NA) or NG-nitro-L-arginine (L-NMMA) 10-5 M] but not a cyclooxygena se inhibitor (indomethacin 10-5 M). In in vivo studies, serotonin infusion (5 μg/kg/min intravenously, i.v.) significantly (p < 0.05) decreased pig random pattern skin flap capillary blood flow. This in vivo vascular effect was also completely blocked in pigs pretreated with LY53857 (0.4 mg/kg i.v.). In a separate experiment without serotonin infusion, i.v. prazosin (2–8 μg/kg), dazmegrel (2–6 mg/kg), or SQ30741 (2–4 mg/kg) had no significant effect on skin flap capillary blood flow as compared with control. On the other hand, i.v. sergolexole or LY53857 significantly (p < 0.05) increased skin flap capillary blood flow in a dose-dependent manner. Furthermore, oral sergolexole 2 mg/kg three times daily (t.i.d.) 5 days significantly (p < 0.05) increased the length and area of skin flap viability as compared with control. Experimental evidence indicates that serotonin may play a pathogenic role in skin flap vasospasm, an effect probably mediated by S1C/2-serotonergic receptors. Sergolexole or LY53857 ameliorated vasospasm in skin flap surgery, resulting in augmentation of skin flap blood flow and viability in pigs. We discuss the efficacy of combined S1C/2-serotonergic receptor antagonist and topical nitrovasodilator treatment for compromised skin circulation.

Augmentation of acute random pattern skin flap viability in the pig

Three experiments were conducted to study the effect of ketanserin and LY53857, S2-serotonergic receptor antagonists, on skin blood flow and viability in acute random pattern skin flaps (4 x 10 cm) in the pig. In experiment 1, the dose-response effect of intravenous ketanserin (0, 0.15, 0.25, 0.35, and 0.50 mg/kg) on skin flap capillary blood flow was studied 6 hr after skin flap surgery, using the radioactive microsphere (15 microns) technique and under pentobarbital anesthesia. Significant (P less than 0.05) increase in skin flap blood flow was seen at the dosages of 0.25 and 0.35 mg/kg compared with the saline-treated control. In experiment 2, the effect of five-day intramuscular ketanserin and LY53857 treatment (0.30 mg/kg/day; in divided doses) on skin flap viability was studied. The drug treatments were started two days preoperatively. It was observed that the length of skin flap viability in ketanserin (6.6 +/- 0.2 cm; n = 40 flaps) and LY53857 (6.8 +/- 0.3 cm; n = 40 flaps) treated flaps were significantly (P less than 0.05) higher than the saline-treated control (5.5 +/- 0.1 cm; n = 48 flaps). Ketanserin treatment started 30 min after flap surgery also significantly (P less than 0.05) increased the length of skin flap viability (6.1 +/- 0.1 cm) compared with the control. There was no significant difference in skin viability between ketanserin and LY53857 treated skin flaps. The preceding study on the effect of ketanserin treatment on random pattern skin flap viability was repeated in experiment 3. Again, it was observed that intramuscular ketanserin treatment significantly (P less than 0.05) increased the skin flap viability. It was concluded that ketanserin and LY53857 treatment resulted in significant augmentation of porcine acute random pattern skin flap viability. This is the first experimental evidence to indicate that S2-serotonergic receptors participate in the pathogenesis of skin flap ischemia.

Role of noradrenaline in the pathogenesis of skin flap ischemic necrosis in the pig

Clinically, ischemic necrosis is one of the most common complications in skin flap surgery, but the etiology is still unclear. The objective of the present experiments was to study the important role of the locally released noradrenaline in the pathogenesis of ischemic necrosis in acute and delayed random pattern skin flaps (4 x 10 cm) raised on both flanks of the pig. In Experiment 1, it was observed that 93, 96, and 94% of the skin contents of noradrenaline were depleted in skin flaps delayed for 2, 4, and 14 days, respectively, compared to the acute skin flaps (n = 8) raised in the same pig. Although the maximum depletion of noradrenaline in the delayed flaps occurred within 2 days of delay, significant (P less than 0.001) increase in the length of dye penetration in the delayed skin flaps was seen after 2 days of delay, compared to the acute skin flaps (n = 12). In Experiments 2 and 3, 5 days of intravenous phenoxybenzamine treatment, starting 2 days preoperatively and at the doses of 0.25, 0.5, 1.0, or 1.5 mg/kg/day, did not have any significant effect on the skin blood flow (n = 24) or viability (1 mg/kg/day; n = 32) in the acute skin flaps compared with the saline-treated control. Similarly, 5 days of intravenous phentolamine treatment (5 mg/kg/day) also did not have any significant effect on the skin blood flow (n = 24) or viability (n = 32) of acute skin flaps compared with the control.(ABSTRACT TRUNCATED AT 250 WORDS)

Role and mechanism of mitochondrial kATP channels in hind-limb ischaemic preconditioning (IPC) of distant muscle flaps against ischaemia/reperfusion (I/R) injury

Abstract Introduction: We reported previously that 3 cycles of 10min occlusion in a hind-limb of the pig by tourniquet application protected multiple distant muscle flaps against I/R injury (infarction) when these muscles are subsequently subjected to 4h ischaemia/48h reperfusion (Am.J.Physiol.285:H1435). There is evidence that kATP channels are involved in remote IPC. Here we used specific pharmacologic probes with known effective dose to investigate the role and mechanism of sarcolemmal kATP (skATP) and mitochondrial kATP (mkATP) channels in hind-limb remote IPC against muscle flap infarction. Methods: Pigs with bilateral lat. dorsi muscle flaps underwent 10min infusion of: (i)saline, (ii)PEG, (iii)PEG with the mkATP channel opener BMS191095(2mg/kg), (iv)saline before remote IPC, (v)saline with the skATP channel blocker HMR1098(3mg/kg) and (vi)saline with the mkATP channel blocker 5-HD(5mg/kg) before remote IPC. All muscle flaps underwent 4h ischaemia/48h reperfusion. Muscle content of ATP and myeloperoxidase activity was studied at 4h ischaemia and 1.5h reperfusion respectively. Results: The muscle infarct size (IS) of remote IPC (24+/−2%) and BMS191095 treatment (20+/−3%) were similar but higher (p Conclusions: mkATP but not skATP channels mediate remote IPC against muscle flap infarction and the mechanism involves energy preservation during ischaemia and anti-neutrophil accumulation during reperfusion to mitigate I/R injury. This information is important for development of pharmacologic preconditioning against infarction.