Effect of superoxide dismutase for improvement of survival of ischemic reperfused island skin flap]

Using a rat model, we evaluated the effect of SOD on the survival of ischemic reperfused island skin flaps. In experiment 1, the oxygen free radical concentration in the flaps was measured by the technique of ESR. The results showed that the oxygen free radical concentration in ischemic reperfused flaps was significantly higher than in the corresponding control flaps (P less than 0.001). In experiment 2, the flaps were perfused with SOD (2000 U in 1 ml saline) before reperfusion after 8 hours of ischemia. Seven days after operation, the area of flap survived in the test group was significantly larger than in the control group (P less than 0.0005). The obtained data demonstrated that the generation of oxygen free radical increases with time during ischemia reperfusion in island skin flap and the role of oxygen free radical in tissue injury following ischemia and reperfusion. The use of SOD can enhance the survival of ischemic island skin flap.     

Effect of intrathecal superoxide dismutase and catalase on oxyhemoglobin-induced vasospasm in monkeys.

A gel consisting of agarose and oxyhemoglobin (OxyHb) was developed so that, when placed in the subarachnoid space, OxyHb would be slowly released, simulating lysis of erythocytes after subarachnoid hemorrhage. The system was used to investigate the importance of reactions mediated by free radicals in the genesis of OxyHb-induced vasospasm in monkeys. Seventeen monkeys were randomly assigned to have subarachnoid placement, on Day 0, of one of the following: 1) agarose gel alone (n = 2); 2) agarose plus OxyHb (n = 3); 3) agarose plus OxyHb plus intrathecal administration of superoxide dismutase and catalase (n = 6); and 4) agarose plus OxyHb plus intrathecal administration of placebo (n = 6). Vasospasm was assessed by comparison of angiograms performed on Day 0 and 7 days after subarachnoid placement of compounds, and by electron microscopy. OxyHb alone caused significant reduction in the diameter of the middle cerebral artery (40 +/- 8%, P less than 0.005, paired t test), which was associated with ultrastructural damage to smooth muscle. Treatment with superoxide dismutase plus catalase or with placebo attenuated vasospasm of the middle cerebral artery, although significant narrowing persisted in both groups (27 +/- 12% and 26 +/- 13%, respectively, P less than 0.05, paired t test). Analysis of variance showed no difference in the degree of vasospasm between groups exposed to subarachnoid placement of OxyHb. Cerebrospinal fluid aspirated from the cisterna magna on Day 7 contained elevated activity of superoxide dismutase in animals that received treatment. Malondialdehyde was undetectable in cerebrospinal fluid after subarachnoid placement of agarose alone, although it was present in similar amounts in all groups that received subarachnoid placement of OxyHb. Since intrathecal superoxide dismutase and catalase failed to protect against OxyHb-induced vasospasm, mechanisms mediated by free radicals may not be important in its genesis. As only one combination of doses of superoxide dismutase and catalase was administered, however, it may be that other dosage schedules might be efficacious.     

Effects of supplementing hypothermic crystalloid cardioplegic solution with catalase, superoxide dismutase, allopurinol, or deferoxamine on functional recovery of globally ischemic and reperfused isolated hearts

We evaluated whether supplemental pharmacologic interventions that altered formation or degradation of reactive oxygen metabolites, when added to hypothermic crystalloid cardioplegic solution (procaine-free St. Thomas' Hospital solution), alter postischemic function of isolated rabbit hearts. Hypoxic, substrate-free cardioplegic solutions cooled to 27 degrees C were perfused through isolated rabbit hearts for 5 minutes before and after an uninterrupted 2 hour period of global ischemia at 27 degrees C. Hearts were then reperfused with standard buffer for 1 hour at 37 degrees C. In some experiments, the cardioplegic solution was supplemented with the following: superoxide dismutase (30 micrograms/ml; degrades superoxide anion); catalase (1.7 micrograms/ml; degrades hydrogen peroxide); allopurinol (1 mmol/L; inhibits xanthine oxidase); or deferoxamine (Desferal, 0.5 mmol/L; selectively chelates ferric iron). Postreperfusion contractile parameters of supplemented hearts, including left ventricular pressure development and its first derivative, left ventricular compliance, spontaneous heart rate, and coronary vascular resistance, were statistically compared to data obtained from hearts arrested with unsupplemented cardioplegic solution. Catalase supplementation provided statistically significant improvement of most functional parameters; somewhat less protection was obtained with allopurinol. Deferoxamine provided little added protection except for the ability to prevent ischemia-induced increases of coronary vascular resistance. There was no evidence of added protection by superoxide dismutase. The data suggest that an important component of ischemia-induced cardiac cell damage in an asanguineous setting is hydrogen peroxide-dependent, and interventions that either inhibit production of superoxide anion or degrade hydrogen peroxide offer best protection. They may be clinically efficacious additives to crystalloid cardioplegic solutions.     

Effect of prostaglandin I2 and superoxide dismutase on reperfusion injury of warm ischemic lung.

A prostaglandin I2 (PGI2) analogue and superoxide dismutase (SOD) were administered to dogs with pulmonary denervation, and their effects on warm ischemic damage to the lung were studied. Twenty-seven adult mongrel dogs were divided into a control group (6 dogs), a PGI2 group (7 dogs), an SOD group (6 dogs), and a heparin group (8 dogs). The left pulmonary hilum was dissected, with PGI2 (1 microgram/kg) being administered to the PGI2 group and heparin (100 U/kg) to the heparin group. Then the left lung was placed in a warm ischemic state for 1 hour. The SOD group also received 20 mg/kg of SOD intravenously 1 minute before reperfusion. Before warm ischemia, immediately after reperfusion, and 1 hour and 2 hours afterward, the blood gases, left pulmonary vascular resistance, and other data were measured under right pulmonary artery clamping. Arterial oxygen tension showed significantly better values in the SOD and PGI2 groups than in the control and heparin groups. The left pulmonary vascular resistance increased with time in the control group but did not increase in the PGI2 group. Pulmonary microangiography showed that dilatation of the pulmonary arterioles was prominent in the PGI2 group. The quantity of pulmonary extravascular fluid was significantly less in the PGI2 and SOD groups than in the control and heparin groups. Histological examination showed marked collapse of capillaries, intraalveolar hemorrhage, and edema in the control and heparin groups, whereas these changes were only slight in the PGI2 and SOD groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of iNOS attenuates skeletal muscle reperfusion injury in extracellular superoxide dismutase knockout mice

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are closely involved in the mechanism of skeletal muscle ischemia/reperfusion (I/R) injury. This study was designed to determine the effects of inducible nitric oxide synthase (iNOS) inhibitor 1400 W on the reperfused cremaster muscle in extracellular super-oxide dismutase knockout (EC-SOD(-/-)) mice. The muscle was exposed to 4.5 h of ischemia, followed by 90 min of reperfusion. Mice received either 3 mg/kg of 1400 W or the same amount of phosphate-buffered saline (PBS, as a control) subcutaneously at 10 min before the start of reperfusion. 1400 W treatment markedly improved the recovery speed of vessel diameter and blood flow in the reperfused cremaster muscle of EC-SOD(-/-) mice compared to controls. Histological examination showed reduced edema in the interstitial space and muscle fiber, and reduced density of nitrotyrosine (a marker of total peroxi-nitrate (ONOO(-)) level) in 1400 W-treated muscles compared to controls. Our results suggest that iNOS and ONOO(-) products are involved in skeletal muscle I/R injury. Reduced I/R injury by using selective inhibition of iNOS perhaps works by limiting cytotoxic ONOO(-) generation, a reaction product of nitric oxide (NO) and super-oxide anion (O(2) (-)). Thus, inhibition of iNOS appears to be a treatment strategy for reducing clinical I/R injury.     

Effect of nitric oxide on the contractile function of rat reperfused skeletal muscle

BACKGROUND: The involvement of nitric oxide (NO) in ischemia-reperfusion injury remains controversial and has been reported to be both beneficial and deleterious. The purpose of this study was to examine the contribution of NO and superoxide to skeletal muscle function using an ischemic revascularized hind limb model in rats. PATIENTS AND MATERIALS: Warm ischemia produced by vascular pedicle clamping was sustained for 3 h. The animals were divided into four groups according to the solution administrated: (1) saline, (2) N-methyl-L-arginine acetate (L-NMMA), (3) L-NMMA + N-(N-L-g-glutamyl-S-nitroso-l-cysteinyl)glycine (S-nitrosoglutathione), or (4) superoxide dismutase (SOD). Saline, L-NMMA, or L-NMMA + S-nitrosoglutathione was infused for the first 2 h of reperfusion. The SOD was administered as an intravenous bolus 5 min before the onset of reperfusion. Postischemic blood flow was measured by a Doppler flow meter. Muscle contractile function was determined after 24 h of reperfusion. RESULTS: Postischemic blood flow was significantly decreased by the L-NMMA infusion compared with that in the saline-treated group. No significant difference in postischemic blood flow was noted in the saline-, L-NMMA + S-nitrosoglutathione-, and SOD-treated groups. Contractile function of the gastrocnemius muscle in the L-NMMA-and SOD-treated groups, but not in the L-NMMA + S-nitrosoglutathione group, was significantly better than that in the saline-treated group. CONCLUSION: Limiting postischemic blood flow and SOD infusion are both beneficial in decreasing the ischemia-reperfusion injury of skeletal muscle. S-Nitrosoglutathione infusion following suppression of endogenous NO production does not reduce ischemia-reperfusion injury.     

Effects of melatonin and superoxide dismutase on free radical formation in the postischemic reperfused heart

Purpose. Melatonin has been reported to protect against oxygen free radicals. We investigated whether melatonin or superoxide dismutase (SOD) would decrease hydroxyl radical concentration in the postischemic reperfused heart. Methods. An isolated rat heart-lung preparation was used. Eighty-one male Wistar rats were allocated into control (no drug), S1 (SOD 400 U·ml⁻¹), S2 (SOD 2000 U·ml⁻¹), M1 (melatonin 0.1 μg·ml⁻¹), M2 (melatonin 1.0 μg·ml⁻¹), M3 (melatonin 10 μg·ml⁻¹), SM (SOD 400 U·ml⁻¹ and melatonin 1.0 μg·ml⁻¹) groups. The heart was perfused initially at the cardiac output of 30 ml·min⁻¹ and the mean arterial pressure of 70 mmHg. Drugs were administered into the reservoir 7 min after the start of perfusion. Ten minutes after the start of perfusion, the heart was rendered globally ischemic for 10 min by reducing the preload and afterload to zero and then reperfused for 10 min. At the end of reperfusion, the heart was freeze-dried for 6 days. The perfusate blood was collected just before and after ischemia and at the end of reperfusion. The formation of hydroxyl radicals in perfusate blood and heart was measured with high-performance liquid chromatography using salicylic acid. Hydroxyl radicals react with salicylic acid, yielding 2,3-, 2,4-, 2,5-, and 3,4-dihydroxybenzoic acid (DHBA). Results. Before and after ischemia, there were no significant differences among the groups in cardiac output, systolic pressure, heart rate, and right atrial pressure. The concentrations of DHBAs in the perfusate blood and heart after ischemia and reperfusion in all groups were significantly higher than those before ischemia. DHBAs in the heart of all drug-administered groups were significantly lower than those in the control group. In the perfusate blood, DHBAs in the S2 group were significantly lower than those in the control group. Conclusions. SOD and melatonin decrease hydroxyl radical concentration in the postischemic reperfused heart. Received for publication on January 26, 1998; accepted on August 5, 1998     

Ca2+ uptake and Ca2+ release by skeletal muscle sarcoplasmic reticulum: differing sensitivity to inhalational anesthetics

The effects of halothane, enflurane, and isoflurane were measured on two different mechanisms of Ca2+ regulation by isolated skeletal muscle sarcoplasmic reticulum (SR) membranes. A 100,000-dalton Ca2+-ATPase protein transports Ca2+ from outside to inside the SR membrane. At concentration ranges representing anesthetic levels of 0.06 to 2.3 times MAC, halothane, enflurane, and isoflurane each increased rate of Ca2+ uptake by SR. Each concentration of isoflurane produced a greater rate of Ca2+ uptake, whereas halothane and enflurane produced maximum stimulation of Ca2+ uptake at 1 and 1.6 times MAC, respectively. The second Ca2+ regulation mechanism studied was a Ca2+ release channel in the SR membrane. The release of Ca2+ via this mechanism requires a critical threshold Ca2+ load (nmol Ca2+/mg SR protein) for Ca2+-induced Ca2+ release to occur. Each anesthetic tested effectively lowered the critical Ca2+ load threshold for Ca2+ release, i.e., the Ca2+ channel was more readily induced to an open state in the presence of anesthetic. The concentrations of anesthetics having this effect on the putative Ca2+ channel were between 0.0026 and 0.078 MAC equivalents for each agent, and these concentrations are much lower than the anesthetic concentrations affecting Ca2+ uptake. These data show that in isolated skeletal muscle SR membranes a Ca2+ channel release function is altered at anesthetic concentrations far below those that change Ca2+ uptake function by a Ca2+-ATPase and below concentrations of the volatile agents producing clinical anesthesia. The Ca2+ channel effect may represent protein-anesthetic interaction, whereas the Ca2+-ATPase effect may occur by a generalized SR membrane perturbation by the anesthetics.     

Effect of hypothermia on the ischemic and reperfused rat skeletal muscle, monitored by in vivo (31)P-magnetic resonance spectroscopy.

The aim of this study was to compare the ischemic and postischemic energetic changes of rat skeletal muscle in response to hypothermia or room temperature, monitored noninvasively and continuously by in vivo (31)P-magnetic resonance spectroscopy ((31)P-MRS). A model of pedicled rat rectus femoris muscle was developed and analyzed by in vivo (31)P-MRS at a magnetic field strength of 2.35 T. Measurements were performed at three time points: before ischemia, after 4 hours of ischemia, and after 1 hour of reperfusion. Three groups were studied: (1) sham-operated rats (n = 6); (2) rats subjected to room temperature (24-26 degrees C, n = 6); and (3) rats subjected to hypothermia (9-12 degrees C, n = 6). Blood perfusion was measured by laser Doppler flowmetry (LDF). In the hypothermic group, phosphocreatine (PCr) recovered to 75% and adenosine triphosphate (ATP) to 86%; in the room temperature group, the recovery was 53% and 51%, respectively (P < 0.05). Skeletal muscle subjected to hypothermia (9-12 degrees C) was found to recover to a higher postischemic energetic level compared with skeletal muscle subjected to room temperature. Hypothermia appears to be a simple and effective method with which to reduce the damage related to ischemia and reperfusion.     

超氧化物歧化酶灌注对断肢血管平滑肌和骨骼肌的保护作用

目的：探讨超氧化物歧化酶对缺血骨骼肌和血管平滑肌的保护作用。方法：应用大鼠离体肢体分别用超氧化物歧化酶（ＳＯＤ）和生理盐水溶液灌注，保存于０～４℃，经２、４、８、１６、２４、４８、７２小时取小腿三头肌进行Ｎａ＋－Ｋ＋－ＡＴＰ酶和Ｃａ２＋－ＡＴＰ酶活性检测，并对此骨骼肌和平滑肌进行超微结构观察。结果：ＳＯＤ灌注对缺血骨骼肌、股动脉平滑肌的酶活性及细胞形态都优于生理盐水组（Ｐ＜０．０１）。结论：ＳＯＤ灌注对离断肢体的存活有保护作用     

The role of Ca2+ influx for insulin-mediated glucose uptake in skeletal muscle

The involvement of Ca(2+) in insulin-mediated glucose uptake is uncertain. We measured Ca(2+) influx (as Mn(2+) quenching or Ba(2+) influx) and 2-deoxyglucose (2-DG) uptake in single muscle fibers isolated from limbs of adult mice; 2-DG uptake was also measured in isolated whole muscles. Exposure to insulin increased the Ca(2+) influx in single muscle cells. Ca(2+) influx in the presence of insulin was decreased by 2-aminoethoxydiphenyl borate (2-APB) and increased by the membrane-permeable diacylglycerol analog 1-oleyl-2-acetyl-sn-glycerol (OAG), agents frequently used to block and activate, respectively, nonselective cation channels. Maneuvers that decreased Ca(2+) influx in the presence of insulin also decreased 2-DG uptake, whereas increased Ca(2+) influx was associated with increased insulin-mediated glucose uptake in isolated single cells and whole muscles from both normal and insulin-resistant obese ob/ob mice. 2-APB and OAG affected neither basal nor hypoxia- or contraction-mediated 2-DG uptake. 2-APB did not inhibit the insulin-mediated activation of protein kinase B or extracellular signal-related kinase 1/2 in whole muscles. In conclusion, alterations in Ca(2+) influx specifically modulate insulin-mediated glucose uptake in both normal and insulin-resistant skeletal muscle. Moreover, the present results indicate that Ca(2+) acts late in the insulin signaling pathway, for instance, in the GLUT4 translocation to the plasma membrane.     

Effect of cimetidine,hydrocortisone, superoxide dismutase and catalase on the development of oedema after thermal injury

A new hypothesis for the pathophysiological mechanism underlying the development of oedema after a thermal injury has been tested in an experimental burn model. Support was given to the suggestion that oxygen-derived free radicals produced by invading leucocytes which upon activation release the superoxide radical (O₂^?), may be partly responsible for the increase in microvascular permeability seen after thermal injury. By removal of oxygen-derived free radicals with radical scavengers (superoxide dismutase and catalases) it was possible to reduce significantly the post-burn oedema formation. For comparison, one series of rats was pretreated with hydrocortisone and another with the histamine H₂-blocker cimetidine. Hydrocortisone reduced the very early post-burn oedema formation which might partly be due to its membrane-stabilizing influence and partly to a direct effect on the microvasculature, causing a reduction of the vasodilatation observed post-burn. The inhibition of post-burn oedema formation by cimetidine, earlier demonstrated in animal burn models, was confirmed in the present study. The mean arterial blood pressure (MAP) in the cimetidine-treated rats decreased, however, after treatment. It is therefore difficult to determine to what extent the concentration of oedema is attributable to histamine H₂-receptor blockade and to what extent to the reduced blood pressure. The influence of MAP on post-burn oedema formation was further illustrated in two series of rats anaesthetized with Inactin and Hypnorm/Valium respectively. The results underline the importance of using the same anaesthetic throughout the experimental series.     

Effect of ropivacaine on Ca function of skinned skeletal muscle

It is well know that the amide-linked local anesthetics such as lidocaine accelerate Ca induced Ca release (CICR) rate. Since ropivacaine is a new amide-linked local anesthetic, effects of ropivacaine on Ca functions were studied using skinned skeletal muscle. The extensor digitorum longus muscle of male Hartley guinea pigs of about 500 g was prepared for this study. According to Endo's method, CICR rates were measured using chemically skinned fibers. Ropivacaine accelerated the (CICR) rate only at concentrations of 3 mM and 10 mM with pCa 5.0. Ropivacaine at a concentration of 10 mM inhibited initial rate of Ca uptake by sarcoplasmic reticulum. Ca sensitivity of the contractile system was not affected with 10 mM of ropivacaine. These results suggest that ropivacaine can be used safely in patients susceptible to malignant hyperthermia since ropivacaine 3 mM is not a concentration for clinical use.     

中医不同治法对骨质疏松症大鼠骨密度及骨骼肌Ca2+-Mg2+-ATP酶影响的比较研究

目的观察中医不同治法对糖皮质激素诱导骨质疏松症大鼠骨密度、骨骼肌Ca2+-Mg2+-ATP酶变化的影响,探讨中医防治骨质疏松症的作用机制。方法将120只雌雄各半的大鼠随机分为正常对照组、模型对照组(模空组)、补肾中药组、健脾中药组、活血化瘀中药组和骨疏康中药组6个组。用地塞米松肌注造模。实验结束后,腹主动脉取血处死大鼠,用酶联免疫法测定大鼠骨骼肌Ca2+-Mg2+-ATP酶,用双能X线骨密度仪测大鼠离体股骨上1/3骨密度。结果①与正常组比较,模空组大鼠离体股骨上1/3骨密度显著降低(P<0.01);与模空组比较,各治疗组大鼠股骨上1/3骨密度均有不同程度的升高,其中以补肾中药组升高程度最为显著(P<0.01),其余各治疗组骨密度较模空组升高程度比较无统计学意义。②与正常组比较,其他各组大鼠骨骼肌的Ca2+-Mg2+-ATP酶显著降低(P<0.01);与模空组比较,各治疗组大鼠骨骼肌的Ca2+-Mg2+-ATP酶均明显升高(P<0.01);补肾组大鼠骨骼肌的Ca2+-Mg2+-ATP酶升高最为明显,明显高于骨疏康组、活血组、健脾组,差异具有非常显著性(P<0.01);活血组大鼠骨骼肌的Ca2+-Mg2+-ATP酶升高程度最低,与补肾组、健脾组、骨疏康组比较具有统计学差异(P<0.01);健脾组和骨疏康组升高程度也比较明显,但二者比较无统计学意义。结论补肾、健脾方法对骨质疏松症大鼠的骨密度和Ca2+-Mg2+-ATP酶具有一定调节作用。     

Ca2+ and AMPK both mediate stimulation of glucose transport by muscle contractions

It is now generally accepted that activation of AMP-activated protein kinase (AMPK) is involved in the stimulation of glucose transport by muscle contractions. However, earlier studies provided evidence that increases in cytosolic Ca(2+) mediate the effect of muscle contractions on glucose transport. The purpose of this study was to test the hypothesis that both the increase in cytosolic Ca(2+) and the activation of AMPK are involved in the stimulation of glucose transport by muscle contractions. Caffeine causes release of Ca(2+) from the sarcoplasmic reticulum. Incubation of rat epitrochlearis muscles with a concentration of caffeine that raises cytosolic Ca(2+) to levels too low to cause contraction resulted in an approximate threefold increase in glucose transport. Caffeine treatment also resulted in increased phosphorylation of calmodulin-dependent protein kinase (CAMK)-II in epitrochlearis muscle. The stimulation of glucose transport by caffeine was blocked by the Ca(2+)-CAMK inhibitors KN62 and KN93. Activation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) also resulted in an approximate threefold increase in glucose transport in the epitrochlearis. The increases in glucose transport induced by AICAR and caffeine were additive, and their combined effect was not significantly different from that induced by maximally effective contractile activity. KN62 and KN93 caused an approximately 50% inhibition of the stimulation of glucose transport by contractile activity. Our results provide evidence that both Ca(2+) and AMPK are involved in the stimulation of glucose transport by muscle contractions. They also suggest that the stimulation of glucose transport by Ca(2+) involves activation of CAMK.