Cho Y. Pang

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.

Pharmacological Augmentation of Skin Flap Viability: A Hypothesis to Mimic the Surgical Delay Phenomenon or a Wishful Thought

The importance of the research in skin flap pharmacology is two-fold. First, observations made from the vasoactive drug actions in the vasculature of skin flaps can provide insight into the regulatory mechanism of cutaneous circulation and the pathophysiology of ischemic necrosis in skin flap surgery. Second, there is the possibility that the aforementioned information may eventually contribute to the development of a drug treatment for the augmentation of skin blood flow and viability in acute skin flaps (i.e., to mimic the surgical delay mechanism). To this end, the objectives of this article are: (1) to present a brief review of the recent research progress in the pharmacological treatment of ischemic skin necrosis in experimental flap surgery, and (2) to attempt to identify the future directions and important areas of research to be pursued in the pharmacology of skin flaps.

Preconditioning: A New Technique for Improved Muscle Flap Survival

Musculocutaneous regional and distal flaps have become an important tool available to the head and neck surgeon. Vascularized autogenous muscle transplants allow single-stage reconstruction of complex defects. Ischemic muscle necrosis is a well-recognized complication with serious potential morbidity. It has been shown that myocardial muscle is protected from ischemic damage by brief periods of coronary artery occlusion and reperfusion subsequent to prolonged ischemia. This is called preconditioning. To our knowledge, this technique has never been extrapolated to skeletal muscle. This article presents a discussion of preconditioning and the potential benefits of this new technique as a means to enhance skeletal muscle survival to sustained normothermic global ischemia. Theories behind ischemic muscle injury are presented. A review of the development of preconditioning in myocardial muscle is discussed. Experimental models used to investigate this phenomenon are also presented. In addition, results of our laboratory investigations using the latissimus dorsi porcine model are discussed. Preconditioning is a new, nonpharmacologic means to improve muscle flap survival. This simple technique may have great clinical application in reducing ischemic muscle necrosis in regional and distal muscle transplantation.

Augmentation of blood flow in delayed random skin flaps in the pig: effect of length of delay period and angiogenesis.

Skin capillary blood flow and angiogenesis were studied by radioactive microsphere and morphometry technique, respectively, in delayed random skin flaps in the pig. Skin flaps were delayed for 2, 3, 4, 6, or 14 days. Blood flow was measured 6 hours after complete raising of acute and delayed random skin flaps on the opposite flanks of the same pig. It was observed that the capillary blood flow increased significantly (p less than 0.05) within 2 days of delay compared to the acute skin flaps. This capillary blood flow further increased by about 100 percent between days 2 and 3, started to plateau after day 3, and remained unchanged between days 4 and 14 of delay. This increase in capillary blood flow was mainly in the distal portion of the delayed skin flaps. There was no indication of an increase in the density of arteries in all delay periods studied. Our observations did not support the hypotheses that the delay phenomenon involves angiogenesis or long-term adaptation to ischemia, as have been hypothesized previously. The possible mechanism of delay is discussed.

Dose and time effects of nicotine treatment on the capillary blood flow and viability of random pattern skin flaps in the rat

The deleterious effects of nicotine treatment on skin haemodynamics and survival of 4 X 10 cm acute random pattern skin flaps constructed on the dorsum of the rat were studied. Rats were injected subcutaneously with 0.2 ml of saline containing varying doses (0, 1, 2, 4 or 8 mg kg-1; bid) of nicotine for 5 weeks, starting 4 weeks before flap surgery. It was observed that nicotine treatment at the dose of 2 mg kg-1 (bid), or higher, significantly (p less than 0.05) decreased the length and area of skin flap survival compared with the control. This dose of nicotine treatment also significantly (p less than 0.05) decreased the capillary blood flow and distal perfusion in the skin flaps compared with the control. However, the detrimental effect of nicotine treatment on the survival of acute random pattern skin flaps was not seen if the treatment was started 2 instead of 4 weeks preoperatively. It is concluded that nicotine may cause hypoperfusion and necrosis in acute random pattern skin flap surgery, and the deleterious effects are time-dependent.

Postconditioning for Salvage of Ischemic Skeletal Muscle from Reperfusion Injury: Efficacy and Mechanism

We tested our hypothesis that postischemic conditioning (PostC) is effective in salvage of ischemic skeletal muscle from reperfusion injury and the mechanism involves inhibition of opening of the mitochondrial permeability transition pore (mPTP). In bilateral 8x13 cm pig latissimus dorsi muscle flaps subjected to 4 h ischemia, muscle infarction increased from 22+/-4 to 41+/-1% between 2 and 24 h reperfusion and remained unchanged at 48 (38+/-6%) and 72 (40+/-1%) h reperfusion (P<0.05; n=4 pigs). PostC induced by four cycles of 30-s reperfusion/reocclusion at the onset of reperfusion after 4 h ischemia reduced muscle infarction from 44+/-2 to 22+/-2% at 48 h reperfusion. This infarct protective effect of PostC was mimicked by intravenous injection of the mPTP opening inhibitor cyclosporin A or NIM-811 (10 mg/kg) at 5 min before the end of 4 h ischemia and was abolished by intravenous injection of the mPTP opener atractyloside (10 mg/kg) at 5 min before PostC (P<0.05; n=4-5 pigs). PostC or intravenous cyclosporin A injection at 5 min before reperfusion caused a decrease in muscle myeloperoxidase activity and mitochondrial free Ca2+ concentration and an increase in muscle ATP content after 4 h ischemia and 2 h reperfusion compared with the time-matched controls. These effects of PostC were abolished by intravenous injection of atractyloside at 5 min before PostC (P<0.05; n=6 pigs). These observations support our hypothesis that PostC is effective in salvage of ischemic skeletal muscle from reperfusion injury and the mechanism involves inhibition of opening of the mPTP.

Pathogenesis of Ischemic Necrosis in Random-pattern Skin Flaps Induced by Long-term Low-dose Nicotine Treatment in the Rat

The objectives of the present experiments were to study the effects of long-term low-dose nicotine treatment on skin hemodynamics, viability, and microvascular morphology in 4 × 10 cm dorsally based acute random-pattern skin flaps in the rat. In addition, the reversibility of the nicotine-induced detrimental effects on skin-flap viability following cessation of nicotine treatment also was investigated. Low-dose nicotine (0.6 mg/kg) administered twice daily and subcutaneously for 24 weeks significantly (p < 0.05) decreased skin-flap capillary blood flow, distal perfusion, and length and area of skin viability compared with the saline-treated control (n = 15). However, these same parameters in rats (n = 15) whose nicotine treatment had been withheld for 2 weeks prior to skin-flap surgery were not significantly different from the control, thus indicating that the detrimental effects of this long-term, low-dose nicotine treatment were reversible. The mean plasma level of nicotine in the nicotine-treated rats was 8.1 ± 0.4 μg/dl and was within the range of plasma nicotine levels reported for human heavy cigarette smokers. Light and electron microscopic studies did not show evidence of histologic damage to the cutaneous microvasculature in acute random-pattern skin flaps and samples of normal (nonoperated) skin in nicotine-treated rats. It is concluded that long-term plasma levels of nicotine similar to those of heavy cigarette smokers are detrimental to the capillary blood flow and viability of random-pattern skin flaps in the rat. These deleterious effects can be avoided if skin flaps are raised 2 weeks after cessation of nicotine treatment. This low-dose nicotine treatment does not cause histologic damage to the microvasculature. Other pathogenic mechanisms of nicotine-induced skin flap ischemia are discussed.

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)

Vasoconstrictor effect of endothelin-1 in human skin: role of ETA and ETB receptors

The aim of this project was to investigate the role of ETA and ETB receptors in the mediation of endothelin (ET)-1-induced vasoconstriction in human skin. This information should provide important insights into the design of pharmacological intervention against skin vasospasm induced by ET-1 in peripheral vascular disease or surgical trauma. Vasoconstriction in response to intra-arterial drug infusion in isolated perfused human skin flaps (8 × 18 cm) derived from dermolipectomy specimens was assessed by studying changes in skin perfusion and perfusion pressure under constant flow rate in each drug treatment ( n = 4). It was observed that ET-1 (10-10 to 10-8 M) and norepinephrine (NE, 10-8 to 10-5 M) caused skin vasoconstriction in a concentration-dependent manner, with the vasoconstrictor potency of ET-1 ∼200-fold higher than NE. The ETA-receptor antagonist BQ-123 but not the ETB-receptor antagonist BQ-788 blocked the vasoconstrictor effect of ET-1. This observation was confirmed by studying skin perfusion using the dermofluorometry technique. In addition, ETB-receptor agonists BQ-3020 and sarafotoxin S6c (10-9 to 10-6 M) did not evoke skin vasoconstriction. BQ-3020 also did not elicit skin vasoconstriction even in the presence of 10-5M of N ω-nitro-l-arginine methyl ester and indomethacin. Furthermore, results from saturable and competitive ET-1 radioligand membrane receptor binding assays revealed that high-affinity and capacity binding sites are predominantly the ETA receptor subtype in endothelium-denuded skin arteries and veins of 0.5-1.5 mm diameter, with an ETA-to-ETBreceptor ratio of 83:17 in arteries ( n= 5) and 78:22 in veins ( n = 7). Results from the present functional and radioligand receptor binding studies clearly indicate that ET-1 is a very potent vasoconstrictor in human skin and its vasoconstrictor effect is primarily mediated by ETAreceptors, with no significant participation from ETB receptors.The aim of this project was to investigate the role of ETA and ETB receptors in the mediation of endothelin (ET)-1-induced vasoconstriction in human skin. This information should provide important insights into the design of pharmacological intervention against skin vasospasm induced by ET-1 in peripheral vascular disease or surgical trauma. Vasoconstriction in response to intra-arterial drug infusion in isolated perfused human skin flaps (8 x 18 cm) derived from dermolipectomy specimens was assessed by studying changes in skin perfusion and perfusion pressure under constant flow rate in each drug treatment (n = 4). It was observed that ET-1 (10(-10) to 10(-8) M) and norepinephrine (NE, 10(-8) to 10(-5) M) caused skin vasoconstriction in a concentration-dependent manner, with the vasoconstrictor potency of ET-1 approximately 200-fold higher than NE. The ETA-receptor antagonist BQ-123 but not the ETB-receptor antagonist BQ-788 blocked the vasoconstrictor effect of ET-1. This observation was confirmed by studying skin perfusion using the dermofluorometry technique. In addition, ETB-receptor agonists BQ-3020 and sarafotoxin S6c (10(-9) to 10(-6) M) did not evoke skin vasoconstriction. BQ-3020 also did not elicit skin vasoconstriction even in the presence of 10(-5) M of Nomega-nitro-L-arginine methyl ester and indomethacin. Furthermore, results from saturable and competitive ET-1 radioligand membrane receptor binding assays revealed that high-affinity and capacity binding sites are predominantly the ETA receptor subtype in endothelium-denuded skin arteries and veins of 0.5-1.5 mm diameter, with an ETA-to-ETB receptor ratio of 83:17 in arteries (n = 5) and 78:22 in veins (n = 7). Results from the present functional and radioligand receptor binding studies clearly indicate that ET-1 is a very potent vasoconstrictor in human skin and its vasoconstrictor effect is primarily mediated by ETA receptors, with no significant participation from ETB receptors.

Assessment of microsphere technique for measurement of capillary blood flow in random skin flaps in pigs

In this technical paper, we reviewed the theory and methodology of the radioactive microsphere technique for determination of cardiac output and regional blood flow. Furthermore, we described two experiments conducted to assess this technique for measurement of capillary blood flow in skin-flap research. Our experimental data thus far indicated that the radioactive microsphere technique provided highly reproducible measurements for determination of capillary blood flow in 4 X 10 cm acute and delayed random skin flaps constructed in pigs. The advantages and disadvantages of this laboratory technique were also discussed.