缺血后处理对大鼠骨骼肌缺血再灌注损伤的影响

目的 探讨缺血后处理对大鼠骨骼肌缺血再灌注损伤的影响以及应用缺血后处理的时机.方法 将32只大鼠随机分成四组,采用切断患肢全部皮肤、肌肉和神经,保留患肢股动静脉的动物模型,通过夹闭和开放股动静脉造成骨骼肌缺血和再灌注损伤.采用测定骨骼肌缺血4 h.再灌注1 h后血清丙二醛(MDA)、骨骼肌髓过氧化物酶(MPO),再灌注6 h后骨骼肌的死亡程度来观察缺血后处理对大鼠骨骼肌缺血再灌注损伤的影响,以及再灌注5 min后应用缺血后处理是否对骨骼肌缺血再灌注损伤有保护作用.结果 对骨骼肌缺血4 h再灌注6 h的损伤,再灌注开始后即刻应用30 s缺血、30 s再通,三次循环的缺血后处理对骨骼肌的缺血再灌注损伤即有保护作用,不仅减少了骨骼肌再灌注区域中性粒细胞浸润(MPO)和血清氧自由基水平(MDA)水平,而且减少了骨骼肌的死亡程度;再灌注5 min后应用缺血后处理并没有降低骨骼肌缺血再灌注区域的MPO和血清MDA水平,也没有降低骨骼肌缺血再灌注后的死亡程度,与直接缺血再灌注组相同,对骨骼肌缺血再灌注损伤并没有保护作用.结论 骨骼肌缺血后再灌注开始前立刻应用缺血后处理对大鼠骨骼肌缺血再灌注损伤有一定的保护效果,可以减少骨骼肌缺血再灌注损伤后的死亡程度;缺血后处理应用时机非常重要,再灌注5 min后应用缺血后处理则失去对骨骼肌缺血再灌注损伤的保护作用.     

川芎嗪在骨骼肌缺血再灌注损伤中的作用

目的　探讨中药川芎嗪在骨骼肌缺血再灌注损伤中有无保护作用。方法　健康成年家兔14只 ,随机分对照组、实验组 ,每组 7只。应用家兔肢体缺血再灌注损伤动物模型 ,在恢复血流再灌注当时 ,实验组静脉输注川芎嗪注射液 ,对照组静脉输注 0 9%生理盐水。测定缺血前、缺血后、再灌注后血浆丙二醛 (MDA)、乳酸脱氢酶 (LDH)及超氧化物歧化酶 (SOD)的含量。制备骨骼肌标本进行光镜及透射电镜观察。结果　实验组在恢复血流并注射川芎嗪后 1小时 ,其血浆MDA、LDH的含量较对照组明显降低 (P <0 0 1) ,而SOD较对照组明显升高 (P <0 0 1)。光镜及电镜下观察可见实验组骨骼肌损害轻于对照组。结论　实验表明中药川芎嗪对骨骼肌缺血再灌注损伤有保护作用     

The role of oxygen-derived free radicals and the effect of free radical scavengers on skeletal muscle ischemia/reperfusion injury

The aim of this study was to clarify the role of oxygen-derived free radicals and the effect of free radical scavengers on skeletal muscle ischemia/reperfusion injury. Male Wistar rats were divided into a complete ischemia group (C-group) and an incomplete ischemia group (IC-group) and each animal was subjected to 2h of ischemia and 1h of reperfusion. In an attempt to decrease reperfusion injury, the rats were given free radical scavengers either as allopurinol 50 mg/kg for 2 days or as superoxide dismutase 60,000 units/kg plus catalase 500,000 units/kg. Tissue malondialdehyde, a product of lipid peroxidation, was measured as an indicator of free radicals, with higher levels indicating higher concentrations of free radicals. The malondialdehyde level in the gastrocnemius muscle after 1h of reperfusion increased significantly in both groups when compared to the levels before and 2h after ischemia, although there was no significant difference between the two groups. The water content of the gastrocnemius muscle and serum creatinine phosphokinase MM isoenzyme (CPK-MM) in both groups, and GOT in the C-group, increased significantly after 1h of reperfusion when compared the values before and 2h after ischemia. In the C-group, these values were significantly higher than in the IC-group. The administration of free radical scavengers suppressed the increase in malondialdehyde in the gastrocnemius muscle after reperfusion in both groups. The increase in water content and CPK-MM after reperfusion was also suppressed by free radical scavengers in the IC-group, but not in the C-group. These findings suggest that ischemic damage predominates in complete severe ischemia/reperfusion injury, whereas reperfusion injury predominates in incomplete mild ischemia/reperfusion injury.     

Technetium 99m pyrophosphate quantitation of skeletal muscle ischemia and reperfusion injury

The study of ischemia and reperfusion injury in the extremity has been hampered by lack of an accurate method of measuring skeletal muscle injury. We used a bilateral isolated in vivo canine gracilis muscle model in 15 anesthetized dogs. The experimental muscles had 4, 6, or 8 hours of ischemia and 1 hour of reperfusion. The contralateral gracilis muscle served as a control. Technetium 99m pyrophosphate (99mTc-PYP), an agent which localizes in injured muscle cells, was used to quantitate canine skeletal muscle damage. After 6 hours of ischemia and 1 hour of reperfusion, there was a significant increase of 215% of 99mTc-PYP uptake in the experimental vs the control muscle. Experimental muscle uptake was 8% greater than control after 4 hours and 405% more after 8 hours of ischemia and reperfusion. Segmental distribution of 99mTc-PYP uptake showed localization to be greatest in the middle of the muscle at the entry site of the gracilis artery. Electron microscopic evaluation also documented this area to have undergone the most severe injury. Distal portions of the muscle did not show increased damage. Our results show that 99mTc-PYP effectively quantitates skeletal muscle ischemia and reperfusion injury. The pattern of 99mTc-PYP uptake suggests that considerable injury is caused during reperfusion.     

冬虫夏草对缺血再灌注肢体能量代谢和线粒体酶活性的影响

目的 观察冬虫草醇提取液对肢体缺血再灌注损伤的影响。方法 制作兔肢体缺血再灌注损伤动物模型，实验分对照组、再灌注组和治疗组。取骨骼肌测定三磷酸腺苷、二磷酸腺苷、一磷酸腺苷、磷酸肌酸含量和线粒体ATP酶活性。结果 再灌注组与对照组比较，骨骼肌能量代谢障碍及其线粒体ATP酶活性降低。使用冬虫夏草后，骨骼肌各项测定指标较再灌注组相比明显改善。结论 冬虫夏草对缺血再灌注损伤骨骼肌有保护作用。     

Biochemical metabolism and oxygen free radical changes following ischemic and reperfused injured limbs. An experimental study

Oxygen free radicals may contribute to skeletal muscle damage associated with ischemia and reperfusion. This study utilized rabbit right hindlimb ischemic model to investigate the biochemical metabolism and oxygen free radicals change in limbs subjected to periods of ischemia and reperfusion. The results showed that the levels of malondialdehyde (MDA) as the oxygen free radical product in the plasma from ischemic and reperfused limb was significantly higher than that from the single ischemic limb, and varied with intracellular enzymes (CPK, LDH, GOT) which reflected the injury of skeletal muscle cells after ischemia. It Suggests that increased free radical product might be one of the important causes of ischemic and reperfused injury in limbs.     

Effect of phosphoenolpyruvate and adenosine triphosphate on rabbit skeletal muscle after ischaemia: preliminary biochemical study

The present study examined the effect of phosphoenolpyruvate (PEP) and adenosine triphosphate (ATP) on rabbit skeletal muscle flap survival after warm ischaemia. Two muscle flap models, rectus femoris pedicle flap and latissimus dorsi free flap, were subjected to a total ischaemia of 4 hours at 37 degrees C and 20 degrees C, respectively. Immediately prior to revascularisation, the muscles were infused with either Hanks' balanced salt solution (BSS) or Hanks' BSS containing 200 mumol PEP and 6.6 mumol ATP. Quantification of muscle damage was determined by measuring the plasma levels of creatinine kinase (CK), lactate dehydrogenase (LDH), lactate, potassium, and phosphate at 0, 2, 24, and 96 hours after revascularisation. Infusion of PEP/ATP compared with Hanks' BSS alone significantly decreased the efflux of CK in both rectus femoris (P less than 0.025) and latissimus dorsi muscles (P less than 0.05) and of LDH in the rectus femoris muscle (P less than 0.01). No significant changes were observed, however, for the plasma levels of lactate, potassium, and phosphate. From this study it was concluded that PEP and ATP partially protect skeletal muscle from ischaemia and reperfusion injury.     

Ischemia-reperfusion-induced muscle damage: Protective effect of corticosteroids and antioxidants in rabbits

We examined the potential protective effect of pretreatment with corticosteroids or antioxidants (ascorbic acid or allopurinol) in rabbits with reper-fusion-induced damage to skeletal muscle after ischemia.

4 hours of limb ischemia induced by a pneumatic tourniquet, followed by reperfusion for 1 hour, caused a considerable amount of ultrastructural damage to the anterior tibialis muscles accompanied by a rise in circulating creatine kinase activity. Pretreatment of animals with depomedrone by a single 8 mg bolus injection led to a preservation of the anterior tibialis structure on both light and electron microscopy. High-dose continuous intravenous infusion with ascorbic acid (80 mg/hr) throughout the period of ischemia and reperfusion also preserved skeletal muscle structure, although allopurinol in various doses had no protective effect.

These data are fully compatible with a mechanism of ischemia/reperfusion-induced injury to skeletal muscle, involving generation of oxygen radicals and neutrophil sequestration and activation. They also indicate that damage to human skeletal muscle caused by prolonged use of a tourniquet is likely to be reduced by simple pharmacological interventions.     

The effects of dichloroacetate in a rabbit model of acute hind-limb ischemia and reperfusion

BACKGROUND: Dichloroacetate (DCA) is a drug that allows pyruvate dehydrogenase to remain active under anaerobic conditions by inhibiting the inactivating enzyme, pyruvate dehydrogenase kinase. We hypothesize that the administration of DCA during acute limb ischemia may have a beneficial effect by reducing the severity of anaerobic metabolism and lessening the irreversible injury. STUDY DESIGN: We studied a rabbit model using unilateral ligation of the iliac artery or femoral artery to evaluate two degrees of ischemia. After 2 hours of hind-limb ischemia, the animals were administered IV DCA (15 mg/kg) or an equivalent volume of saline. RESULTS: Higher serum lactate levels were seen after high compared to low ligation in control animals consistent with more severe ischemia. DCA treatment significantly reduced serum lactate levels after both high and low ligation. Similarly, the rise in percentage end-tidal CO(2) after reperfusion was less after DCA. All animals regained hind-limb function after the procedure, but ischemia or reperfusion resulted in appreciable muscle necrosis (> 10% area) in 50% of high- and 22% of low-ligation control animals. DCA treatment eliminated significant muscle necrosis in 100% of high-ligation animals. Muscle histology was similar in control and DCA-treated low-ligation animals. CONCLUSIONS: Treatment with DCA during acute arterial occlusion did significantly lower markers of anaerobic metabolism and reduced muscle necrosis in a rabbit model of acute hind-limb ischemia. DCA delivery through collateral blood flow may prolong the ischemia time interval before the onset of irreversible muscle injury and potential limb loss.     

The temporal relationship between endothelial cell dysfunction and skeletal muscle damage after ischemia and reperfusion.

Reperfusion-induced vascular endothelial cell dysfunction may exacerbate skeletal muscle damage after an ischemic insult. Although concurrent endothelial and skeletal muscle injury has been documented after ischemia and reperfusion, their temporal relationship has not been well characterized. An isolated rat hindlimb model was used to measure the effect of progressive ischemia and reperfusion on both endothelial cell function and skeletal muscle damage. Endothelial cell dysfunction as reflected by changes in permeability was measured by protein clearance techniques with use of albumin labeled with iodine 125 (125I-albumin). Skeletal muscle damage was assessed by tissue uptake of technetium 99m pyrophosphate (99mTc-pyrophosphate). The soleus muscle was used for evaluation of endothelial and skeletal muscle damage throughout the study. Significant increases in vascular permeability preceded skeletal muscle damage. The protein leak index increased after 60 minutes of ischemia and reperfusion (7.5 +/- 1.2 vs 4.1 +/- 0.9 control), whereas the muscle injury index did not change until 120 minutes of ischemia and 60 minutes of reperfusion (10.5 +/- 0.6 vs 4.5 +/- 0.5 control). Significant graded increases in both indexes were noted with longer intervals of ischemia. Electron microscopy revealed ultrastructural evidence of endothelial and skeletal muscle damage after 120 minutes of ischemia and 60 minutes of reperfusion but not after 60 minutes of ischemia and reperfusion. These studies indicate that microvascular injury precedes skeletal muscle damage after ischemia and reperfusion. This temporal relationship may have important implications in designing strategies to minimize ischemia-reperfusion injury.     

Protective effect of anisodamine on cultured bovine pulmonary endothelial cell injury induced by oxygen-free radicals.

Anisodamine, a Chinese traditional medicine herb, has been used for treatment of adult respiratory distress syndrome effectively, but little is known about its mechanism. We attempted to investigate if anisodamine could protect bovine pulmonary endothelial cell injury induced by exogenous oxygen-free radicals that were generated by xanthine/xanthine oxidase or opsonized zymosan-stimulated polymorphonuclear leukocytes. Results showed that with the addition of xanthine/xanthine oxidase into cultured bovine pulmonary endothelial cells, production of malondialdehyde and release of lactate dehydrogenase in supernatant increased, and synthesis of prostacyclin decreased. Damaged cellular membranes were revealed by scanning electron microscopy. The same was true for the addition of opsonized zymosan-stimulated polymorphonuclear leukocytes. While treatment with anisodamine greatly attenuated all of the above-mentioned parameters, results showed that (1) cultured bovine pulmonary endothelial cells could be damaged by oxygen-free radicals, (2) anisodamine had a protective effect on this injury as effective as that of superoxide dismutase and catalase, and (3) the membrane-stable action might contribute to the mechanism of protective effect against this injury.     

术中预缺血对猪骨骼肌保护作用的实验研究

OBJECTIVE: The aim of this study was to investigate metabolism of ischemic muscle and the efficacy of acute ischemic preconditioning for protection of skeletal muscles against infarction. METHODS: The efficacy of preconditioning was tested by subjecting pig latissimus dorsi muscle to 3 cycles of ischemia reperfusion, each for 10 min, before 4 h of global ischemia. Infarction was assessed at 48 h reperfusion using nitroblue tetrazolium dye. Muscle biopsies were taken from the latissimus dorsi before ischemia, at the end of 2 and 4 h of ischemia and 1.5 h of reperfusion. RESULTS: Preconditioning reduced the total infarct size by 44% in the latissimus dorsi. The muscle contents of ATP were maintained higher and the lactate lower (P < 0.05) in the preconditioned than in the non-preconditioned muscle at the end of 2 h, 4 h of ischemia and 1.5 h of reperfusion. CONCLUSION: Preconditioning of pig skeletal muscle is associated with a lower energy metabolism during sustained ischemia. At the present time, it is not known if this energy sparing effect is a major mechanism of ischemia preconditioning against infarction in the skeletal muscle.     

Reduced ischemia-reperfusion injury in muscle Experiments in rats with EPC-K1, a new radical scavenger

L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1 -benzopyran-6-yl hydrogen phosphate] potassium salt (EPC-K1), a phosphate diester of alpha-tocopherol and ascorbic acid, is a potent antioxidant. We examined the effects of EPC-K1 on ischemia-reperfusion injury in the skeletal muscle of rats, using an ischemic revas-cularized hind limb model. Warm ischemia (25 C), produced by vascular pedicle clamping, was sustained for 4 hours. After 24 hours of reperfusion, skeletal muscle injury was evaluated in 2 groups: one group treated by intravenous injection of EPC-K1 (10 mg/kg) prior to ischemia, and a group of controls. The EPC-K1 -treated group showed a statistically significant amelioration in the reduction of the isometric muscle contraction, inhibition of the elevation of the muscle wet- to dry-weight ratio, limitation of the muscle level of thiobarbituric acid reactive substances and the serum levels of creatine phos-phokinase, lactate dehydrogenase and mitochondrial glutamic oxaloacetic transaminase, and reduction of the extent of muscle injury according to the histological findings. These observations indicate that EPC-K1 acted effectively on ischemia-reperfusion injury in the rat skeletal muscle and thereby improved muscle function.     

山莨菪碱对大鼠肝脏缺血再灌注损伤保护作用的实验研究

目的 探索山莨菪碱对大鼠肝脏缺血再灌注损伤的保护作用。方法 选雄性Wistar大鼠160只,分为正常对照组、缺血再灌注组、生理盐水组和山莨菪碱组,观察了肝脏缺血60分钟及再灌注1、3、6、12及24小时后血浆内皮素－1（ET－1）、透明质酸（HA）和谷丙转氨酶（ALT）含量变化及肝组织病理学变化。结果 肝脏缺血再灌注后,血浆ET－1、HA和ALT含量均明显增高,同时肝脏瘀血很明显,肝脏缺血再灌注前用山莨菪碱后,血浆HA和ALT含量明显降低,同时肝组织瘀血减轻。结论 山莨菪碱可改善再灌注后的肝脏微循环障碍,对大鼠肝脏缺血再灌注损伤有保护作用。     

术中预缺血对猪骨骼肌保护作用的实验研究

目的　研究术中预缺血对骨骼肌缺血坏死的保护作用及相关的肌肉代谢变化。方法　１０ 只猪背阔肌瓣在４ｈ 缺血前先进行３ 个循环１０ ｍｉｎ 的术中预缺血,４８ｈ 后用染色法记录肌肉成活率,于肌肉缺血前、缺血后２ ,４ｈ 和再灌流１５ｈ 分别作肌肉活检。结果　４ｈ 缺血后的肌瓣,术中预缺血组成活率高出对照组４４ ％ ,肌肉活检三磷酸腺苷（ Ａ Ｔ Ｐ） 增加和乳酸降低（ Ｐ＜ ００５） 。结论　术中预缺血可增加骨骼肌对缺血坏死的保护作用,这与肌肉中能量代谢的减低相关。     

利多卡因对骨骼肌缺血-再灌注损伤作用的实验研究

目的 阐明利多卡因对骨骼肌缺血-再关注损伤的作用.方法 采用MTT法测定骨骼肌组织活力,分别对缺血后、再灌注后加入利多卡因与对照组进行比较,并结合透射电镜,观察利多卡因对缺血再灌注损伤后的作用.结果 缺血后、再灌注后加入利多卡因与对照组进行比较,P＜0.01,差异有统计学意义;缺血后与再灌注后2组比较,P＞0.05,差...     

丹酚酸B和高氧液对兔肢体缺血再灌注氧化应激损伤的影响

目的探讨高氧液和丹酚酸B对肢体缺血再灌注损伤的影响。方法选用健康新西兰家兔24只,随机分为4组,在缺血前从耳缘静脉推注等量的生理盐水(A组)、高氧液(B组)、丹酚酸B(C组)或高氧液加丹酚酸B(D组),夹阻股动、静脉,建立肢体缺血再灌注损伤模型,在缺血前和再灌注4h抽血检测MDA、SOD,取腓肠肌作病理检测。结果血清丙二醛浓度较前明显升高,高氧液和丹酚酸B可以抑制其升高(P<0.01),两者有协同作用(P<0.01);血清超氧化物歧化酶活性较前明显降低,高氧液和丹酚酸B可以抑制其降低(P<0.01),两者有协同作用(P<0.01)。骨骼肌HE染色见:相对于对照组其他组骨骼肌损伤程度较轻,以联合用药组最轻。结论高氧液和丹酚酸B能抑制肢体缺血再灌注氧化应激损伤,而且两者有协同作用。     

The role of leukocytes in the pathophysiology of skeletal muscle ischemic injury

Neutrophilic leukocytes have been implicated as important mediators of ischemic myocardial injury. We investigated the role of neutrophils in skeletal muscle ischemia/reperfusion injury by using the rat hindlimb ischemia model. We rendered Wistar rats neutropenic by administering 750 rad of whole-body radiation (mean white blood cell count, 300 +/- 50/mm3; 3 days after radiation). In anesthetized rats (10 neutropenic and 10 control), 3 hours of ischemia were induced in one hindlimb by application of a tourniquet to the proximal thigh; the contralateral limb served as an internal, nonischemic control. After 1 hour of reperfusion the gastrocnemius and soleus muscles were excised bilaterally and evaluated for ischemic injury by means of a quantitative spectrophotometric assay of triphenyltetrazolium chloride (TTC) reduction. In control rats the reduction of TTC by ischemic muscle averaged 27.0% +/- 7.3% of that by nonischemic muscle; whereas in neutropenic rats the value for ischemic muscle was 65.4 +/- 11.6% (p less than 0.05). To determine if the contribution of the neutrophils to ischemic injury is due to oxygen-derived free radical formation, an additional 10 animals were infused with 5000 units of super-oxide dismutase and 10,000 units of catalase at the time reperfusion was restored. After treatment with free radical scavengers, TTC reduction by ischemic limbs was 25.5% +/- 7.0% of that by nonischemic limbs and did not differ from that in control animals (p greater than 0.05). The results show a protective effect of neutropenia and suggest a significant role of the white cell in the pathophysiology of ischemic skeletal muscle injury.     

Photoengineering of tissue repair in skeletal and cardiac muscles

This review discusses the application of He-Ne laser irradiation to injured muscles at optimal power densities and optimal timing, which was found to significantly enhance (twofold) muscle regeneration in rats and, even more, in the cold-blooded toads. Multiple and frequent (daily) application of the laser in the toad model was found to be less effective than irradiation on alternate days. It was found that in the ischemia/reperfusion type of injury in the skeletal leg muscles (3 h of ischemia), infrared Ga-Al-As laser irradiation reduced muscle degeneration, increased the cytoprotective heat shock proteins (HSP-70i) content, and produced a twofold increase in total antioxidants. In vitro studies on myogenic satellite cells (SC) revealed that phototherapy restored their proliferation. Phototherapy induced mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) phosphorylation in these cells, probably by specific receptor phosphorylation. Cell cycle entry and the accumulation of satellite cells around isolated single myofibers cultured in vitro was also stimulated by phototherapy. Phototherapy also had beneficial effects on mouse, rat, dog and pig ischemic heart models. In these models, it was found that phototherapy markedly and significantly reduced (50-70%) the scar tissue formed after induction of myocardial infarction (MI). The phototherapeutic effect was associated with reduction of ventricular dilatation, preservation of mitochondria and elevation of HSP- 70i and ATP in the infarcted zone. It is concluded that phototherapy using the correct parameters and timing has a markedly beneficial effect on repair processes after injury or ischemia in skeletal and heart muscles. This phenomenon may have clinical applications.     

Microcirculatory changes following reperfusion insult in diabetic rat skeletal muscles

We investigated the microcirculatory changes of ischemia/reperfusion injury in the diabetic rat cremaster muscle as well as the therapeutic effect of insulin. Streptozotocin-induced diabetic rats were maintained hyperglycemic for up to 8 weeks or were treated with insulin in the diabetic period. The rat cremaster muscle was prepared as an island flap and subjected to 2-h clamp ischemia followed by 1-h reperfusion. In nonischemic conditions, effective concentrations for 50% response (EC50) of serial orders of arterioles to norepinephrine were higher in diabetic muscles. Ischemia/reperfusion insult significantly decreased the EC50 of arterioles in the normal group, but not in the diabetic group. Light microscopy showed that the diabetic cremasters had more collapsed capillaries and smooth muscle-disarranged arterioles. Insulin therapy showed significant improvement in the diabetes-caused reduction of perfused capillary density, but not in the contractility of the diabetic arterioles. These results indicate that diabetes mellitus may damage the skeletal muscle microvasculature irreversibly and make it less responsive to autonomic regulation. Insulin therapy can improve capillary perfusion, but not the microvascular reactivity of diabetic muscles.