Alterations in the morphology of skeletal myofibres after 90 minutes of ischaemia and 3 hours of reperfusion

Morphometric, light and electron microscopic methods were employed to determine whether skeletal myofibres were damaged by 90 minutes of tourniquet-mediated ischaemia. Open biceps muscle biopsies were obtained before 90 minutes of upper limb tourniquet ischaemia in 5 Chacma baboons. Further biopsies were obtained just before tourniquet release in 2 animals and after 3 hours' reperfusion in the remaining 3 animals. Other than a slight reduction in myofibre diameter and the anaerobic depletion of intermyofibrillar glycogen, no pathological changes were noted in skeletal myofibres after ischaemia. However, after reperfusion there was myofibre enlargement, intermyofibrillar oedema, internalisation of nuclei, myofibrillar and mitochondrial disorganisation and dissolution, and Z-band streaming. These results show that reperfusion injury affects skeletal myofibres after 90 minutes of tourniquet-mediated ischaemia.     

Apoptotic cell death makes a minor contribution to reperfusion injury in skeletal muscle in the rat.

OBJECTIVE: to determine if apoptotic cell death contributes to skeletal muscle reperfusion injury. METHODS: leg ischaemia was induced in rats with a tourniquet and maintained for 4 h before reperfusion for 24 or 72 h. Apoptosis was assessed by morphology, in situ end labelling of DNA fragments, DNA laddering, expression of p53 mRNA and detection of caspase-3-like proteolytic activity. RESULTS: increased caspase-3-like activity was detected in muscle following ischaemia and zero, 24 h or 72 h of reperfusion. Levels remained relatively low but with a highly significant difference in enzyme activity between the ischaemic and non-ischaemic legs (p <0.0001, Repeated Measures Analysis of Variance). Morphological examination showed considerable oedema, disruption of muscle fibres and infiltration of white cells into tissues. Muscle nuclei did not show any morphological evidence of apoptosis and were negative for DNA fragmentation, while occasional neutrophils contained fragmented DNA. Expression of p53 was not induced by ischaemia and reperfusion and DNA ladders were not detected. CONCLUSIONS: the cells undergoing apoptosis were infiltrating neutrophils rather than muscle cells and reperfused muscle was damaged largely by an inflammatory process involving considerable oedema.     

Skeletal muscle blood flow after prolonged tourniquet ischaemia and reperfusion with and without intervening reoxygenation: an experimental study in rats using laser Doppler perfusion imaging.

The total, safe, time available for operating during tourniquet ischaemia is thought to be prolonged by short, intervening episodes of reperfusion. However, animal experiments have suggested that this may cause a reduction of the postischaemic reperfusion injury. The purpose of the present study was to investigate the effect on final postischaemic reperfusion of intermittent, short periods of reperfusion compared with that of prolonged, continuous tourniquet ischaemia. A rat tourniquet model of total limb ischaemia and laser Doppler imaging to measure postischaemic microvascular perfusion in skeletal muscle was used in a total of 25 anaesthetised rats. Four were non-ischaemic controls. In 21 rats one hind leg was made totally ischaemic by a tourniquet. Fourteen were exposed to uninterrupted periods of either 1.5 hours or three hours, and seven to interrupted total ischaemia of three hours, with a 20 minutes reperfusion interval after 1.5 hours of ischaemia. The postischaemic blood flow was monitored 20 minutes after release of the tourniquet. Postischaemic skeletal muscle blood flow was significantly reduced after three hours of continuous ischaemia compared with the group that had an intervening short period of reoxygenation. The postischaemic reperfusion after three hours of ischaemia with a short reperfusion interval was not reduced after ischaemia compared with either that in controls or to that in the group exposed to only 1.5 hours of tourniquet ischaemia. These findings support the clinical practice of using intervening periods of reperfusion to prolong the total tourniquet time that can safely be used. There were no adverse effects on postischaemic reperfusion from intermittent reoxygenation.     

Early assessment of skeletal muscle damage after ischaemia-reperfusion injury using Tc-99m-glucarate

PURPOSE: After acute arterial obstruction of the lower extremity, muscle damage is the critical determinant for clinical outcome. The extent of muscle damage and limb viability are currently assessed by clinical examination, which is inaccurate. Tc-99m-pyrophosphate (PYP) has been applied for imaging ischaemia-reperfusion damage. More recently, a new imaging agent, Tc-99m-glucarate (GLUC), was introduced for delineating early myocardial infarction after ischaemia-reperfusion. The aim of this study was to determine if GLUC could delineate early skeletal muscle damage after ischaemia-reperfusion. Both tracers were used in a novel murine model of hindlimb ischaemia-reperfusion. METHODS: In anaesthetised mice, ischaemia of one hindlimb was maintained for 2, 3 and 4h using a tourniquet, followed by a reperfusion period of 1h. Additionally, reperfusion periods of 3, 24 and 96h were studied after 3h of ischaemia. PYP or GLUC was injected 45min before end of reperfusion. Concentrations of both agents were determined in blood, reperfused and contralateral muscle. Reperfused-to-contralateral muscle ratios were calculated. In separate experiments, muscle biopsies were obtained for histologic examination. RESULTS: Ischaemia and reperfusion damage was demonstrated histologically. Using scintigraphy GLUC depicted reperfusion significantly better than PYP. After 2, 3 and 4h of ischaemia, the reperfused-to-contralateral ratios for GLUC were 10.7+/-0.9, 8.9+/-0.9 and 8.6+/-1.1, as compared to 4.5+/-0.7, 4.9+/-0.4 and 4.5+/-0.4 for PYP (P<0.05 at all points). For longer periods of reperfusion, the ratios for GLUC decreased to similar levels as observed for PYP. CONCLUSION: The present study indicates that GLUC is a specific early marker of myocyte necrosis after ischaemia-reperfusion. GLUC may become an useful agent for clinical, early, non-invasive monitoring of skeletal muscle damage after ischaemia-reperfusion.     

Carvedilol reduces ischaemic skeletal muscle necrosis.

Carvedilol is an alpha1 and nonselective beta-adrenergic receptor antagonist with antioxidative properties known to protect against reperfusion injury in the heart, brain, and kidneys. The aim of this study was to test the hypothesis that carvedilol improves postischaemic reperfusion and tissue survival in skeletal muscle. Sixteen Wistar rats underwent tourniquet ischaemia of the left hindlimb for 3 hours and 15 minutes at 27 degrees C. Single-fiber laser Doppler probes were inserted in the left and right anterior tibial muscles, and microvascular perfusion was measured until 2 hours after removal of the tourniquet. Perfusion indices for each 15-minute interval were calculated for the left hindlimb (tourniquet ischaemia) by dividing the postischaemic by the pre-ischaemic laser Doppler flowmetry values, and the geometrical areas under the curves representing a plot of perfusion index relative to time, measured in arbitrary units, were compared. Laser Doppler flowmetry values for the right anterior tibial muscle were compared. Tissue damage was measured by histomorphometry of necrotic areas and no-reflow zones in cross sections from the anterior tibial muscle 72 hours after ischaemia. Neutrophils were counted in the same sections. The treatment group received 1 mg carvedilol/kg body weight before ischaemia and 1 mg/kg immediately before removal of the tourniquets. The areas under the curves representing the plot of perfusion index relative to time were larger for the rats treated with carvedilol: 9.5 compared with 3.0 arbitrary units (p = 0.0003). Treatment did not change the laser Doppler flowmetry values for the right hindlimbs. The histomorphometric areas of necrosis in cross sections from the muscles were reduced from 88% (38-96%) in the control animals to 41% (7-85%) in those treated with carvedilol (p = 0.01), and the area of no-reflow was reduced from 20% (2-52%) to 0% (0-7%) (p = 0.006). The number of neutrophils did not differ between groups. The study supports the hypothesis that carvedilol improves early reperfusion and protects skeletal muscle subjected to 3 hours and 15 minutes of ischaemia.     

Decreased muscle speed, strength and fatigability following two hours of tourniquet-induced ischaemia.

Contractile and morphological properties of the rabbit tibialis anterior muscle were measured 48 hours following a two-hour ischaemic episode. Ischaemia was induced using a specially-designed pneumatic tourniquet placed on the rabbit thigh. Maximum tetanic tension of muscle subjected to ischaemia (381 +/- 77 g) was only about 30% of the tension generated by control muscles (1,212 +/- 67 g). The rate of rise of tetanic tension of muscles subjected to ischaemia (15.9 +/- 3 g/ms) was only 33% of control values (44.5 +/- 5.9 g/ms). Muscle fatigue index increased significantly from 0.22 +/- 0.7 in control muscles to 0.55 +/- 0.09 in ischaemic muscles suggesting that muscles subjected to ischaemia had a greater endurance capacity than control muscles. Morphologically, focal necrotic regions and inflammatory cells were observed in ischaemic muscle fibers. Taken together, these data are consistent with selective damage to the fast glycolytic muscle fibers within the ischaemic tibialis anterior muscles. Thus, ischaemia results in overall decreased muscle speed, strength and fatigability.     

Energy metabolism during microsurgical transfer of human skeletal muscle assessed by high-pressure liquid chromatography and by 31P-nuclear magnetic resonance.

The effect of ischaemia and reperfusion on human skeletal muscle was studied during free vascularised muscle transfer. Muscle biopsy specimens were taken from patients having microsurgical muscle transfer, 18 cases (17 patients; 12 men, 5 women). The biopsies were taken three times: before transfer of the muscle (control), at maximum ischaemic time, and one hour after revascularisation. The biopsy specimens were analysed for purine nucleotides, by high-pressure liquid chromatography (HPLC), and by nuclear magnetic resonance (NMR) at 500 MHz. Phosphocreatine (PCr) recovered only partially (79%) and adenosine triphosphate (ATP) did not differ significantly from normal control after revascularisation and a mean ischaemic time of 114 minutes. NMR measurements showed an accumulation of glucose-6-phosphate (G-6-P) during the ischaemic period, indicating anaerobic metabolism. After three hours of ischaemia and one hour of reperfusion the PCr recovery was less than 60% (r = 0.7). The results confirm those of previous animal studies, which set three hours normothermic ischaemia as a safe limit for tissue preservation when transferring skeletal muscle. Longer ischaemic times may cause serious postoperative healing problems and reduced muscle function.     

Up-regulation of MMP-2 and MMP-9 leads to degradation of type IV collagen during skeletal muscle reperfusion injury; protection by the MMP inhibitor, doxycycline.

OBJECTIVES: to determine the role of matrix metalloproteinases, MMP-2 and MMP-9, in reperfusion injury following skeletal muscle ischaemia and whether inhibition of MMPs by doxycycline protects against tissue damage. METHODS: rats were anaesthetised and a tourniquet applied to the proximal thigh to occlude blood flow. Four hours of ischaemia was followed by reperfusion for 0, 4, 24 or 72 h. Two further groups received doxycycline for 7 days prior to bilateral ischaemia and 24 h reperfusion. Skeletal muscle from both limbs, kidneys and lungs were harvested for zymography and immunohistochemical staining for type IV collagen. RESULTS: upregulation of MMP-2 and MMP-9 was detected by zymography in the ischaemic leg and lung but not in the kidney. Quantitative immunohistochemical analysis showed marked degradation of type IV collagen in reperfused muscle, lung and kidney. Doxycycline-treated rats showed significant preservation of type IV collagen in skeletal muscle and a trend towards preservation in kidney and lung. CONCLUSIONS: MMP-2 and MMP-9 are strongly upregulated in skeletal muscle ischaemia/reperfusion injury and are also upregulated in remote organs, leading to degradation of basement membranes. Inhibition of MMP activity may therefore be potentially therapeutically useful in reducing the severity of reperfusion injury.     

ENERGY METABOLISM DURING MICROSURGICAL TRANSFER OF HUMAN SKELETAL MUSCLE ASSESSED BY HIGH-PRESSURE LIQUID CHROMATOGRAPHY AND BY 31 P-NUCLEAR MAGNETIC RESONANCE

The effect of ischaemia and reperfusion on human skeletal muscle was studied during free vascularised muscle transfer. Muscle biopsy specimens were taken from patients having microsurgical muscle transfer, 18 cases (17 patients; 12 men, 5 women). The biopsies were taken three times: before transfer of the muscle (control), at maximum ischaemic time, and one hour after revascularisation. The biopsy specimens were analysed for purine nucleotides, by high-pressure liquid chromatography (HPLC), and by nuclear magnetic resonance (NMR) at 500 MHz. Phosphocreatine (PCr) recovered only partially (79%) and adenosine triphosphate (ATP) did not differ significantly from normal control after revascularisation and a mean ischaemic time of 114 minutes. NMR measurements showed an accumulation of glucose-6-phosphate (G-6-P) during the ischaemic period, indicating anaerobic metabolism. After three hours of ischaemia and one hour of reperfusion the PCr recovery was less than 60% ( r = 0.7). The results confirm those of previous animal studies, which set three hours normothermic ischaemia as a safe limit for tissue preservation when transferring skeletal muscle. Longer ischaemic times may cause serious postoperative healing problems and reduced muscle function.     

The effect of surgery, performed under ischemia, on the fine structure of the hand muscles]

The ultrastructural picture of the intact and injured hand muscles has been examined by the authors during and after operations in tourniquet ischaemia of 12 patients, in connection with tendon and nerve reconstruction. Muscle specimina were taken partly in the beginning of the ischaemization, and partly at the end ot it, every 15--20 minutes, and the muscle specimina have been examined by means of electron microscope. It has been found that in the first 2 hours of ischaemia neither the intact muscles, nor those previously injured suffer demonstrable damage.     

Skeletal muscle tissue oxygen pressure distribution during early reperfusion after prolonged ischaemia.

OBJECTIVES: The aim of this study was to investigate the skeletal muscle tissue oxygen pressure (PtO2) distributions during early reperfusion (10-45 min) after prolonged ischaemia in a rat animal model. MATERIAL AND METHODS: Skeletal muscle ischaemia was induced in anaesthetised rats by applying a tourniquet on the left thigh for 3 h (group I) or 4 h (group II), and tissue oxygen pressure measurements were made after 10-45 min of reperfusion. Assessment of PtO2 was made by a multiwire Clark-type oxygen microelectrode, placed on the surface of the left tibialis anterior muscle. RESULTS: During reperfusion a similar PtO2 pattern was evaluated after both 3 and 4 h of total ischaemia, where the sum PtO2 distributions were shifted to the left associated with low tissue oxygen pressure values. After 10 min of reperfusion the median PtO2 was 0.28 kPa and 0.18 kPa, in groups I and II, respectively; after 45 min of reperfusion 0.61 kPa and 0.60 kPa, respectively. The median PtO2 in the non-ischaemic muscle in groups I and II were 2.19 and 2.17 Pa. CONCLUSION: The results show that local skeletal muscle oxygenation is severely impaired during the initial 45 min of reperfusion after both 3 and 4 h of total muscle ischaemia with a slow-reflow phenomenon generally present, despite pronounced needs.     

Effect of tourniquet ischaemia on carbohydrate metabolism of dog skeletal muscle

Metabolic changes in blood and skeletal muscle of dogs before, during and after tourniquet ischaemia were investigated to obtain further information on cellular metabolic abnormalities and restitution during and following long-lasting blood flow interruptions. Total carbohydrate and glycogen contents in the muscle tissue fell during ischaemia and remained significantly decreased even 1 h after recirculation due to inhibition of glycogen synthetase activity. Muscle glucose concentration remained stable during ischaemia and was significantly elevated 1 h after tourniquet release. In contrast, muscle lactate concentration was elevated during ischaemia and normal after recirculation. Blood lactate, pyruvate and serum inorganic phosphate concentrations increased markedly after tourniquet release and were still significantly elevated 1 h after recirculation, whereas ketone bodies and citric acid cycle intermediates remained unchanged. Tourniquet ischaemia had no effect on muscle phosphate concentration or on the activities of proteases, protease inhibitors or hydrolases in the blood. Nevertheless, our results clearly indicate metabolic abnormalities in the blood and skeletal muscle during 5 h of tourniquet ischaemia and even after 1 h of recirculation.     

Effect of free radical scavenging on skeletal muscle blood flow during postischaemic reperfusion.

After 6-h tourniquet ischaemia of one hindlimb in male Sprague-Dawley rats, gastrocnemius muscle blood flow was measured following 10, 120 and 240 min of reperfusion using radiolabelled microspheres. A perfusion index was calculated (experimental limb: contralateral limb) for each of these times. Comparison of perfusion indices in ten control animals (6 h ischaemia, 4 h reperfusion) with similar measurements in ten normal rats with no ischaemia and in ten ischaemic animals with the tourniquet in situ demonstrated low median (interquartile range (i.q.r.)) reflow after 10 min (control 0.12 (0.02-0.43), ischaemia 0.04 (0.00-0.07), normal 1.05 (0.68-1.18); control versus ischaemia, P not significant; control versus normal, P < 0.01). Relative reperfusion occurred at 120 min (control 0.48 (0.11-0.70), ischaemia 0.02 (0.01-0.07), normal 0.97 (0.79-1.13); control versus ischaemia, P < 0.05; control versus normal, P < 0.05) and reperfusion injury after 240 min of revascularization, with muscle blood flow being little different from that in the ischaemic group (control 0.05 (0.01-0.38), ischaemia 0.03 (0.00-0.07), normal 1.01 (0.73-1.16); control versus ischaemia, P not significant; control versus normal, P < 0.01). Two groups of 12 rats were given either intravenous superoxide dismutase and catalase or dimethylthiourea 30 min before tourniquet release, continuing throughout the period of reperfusion. Superoxide dismutase and catalase reversed low reflow, producing a median (i.q.r.) perfusion index of 0.94 (0.54-1.12) (P < 0.01 versus control, P not significant versus normal), but had no effect on relative reperfusion (0.66 (0.42-1.01), P not significant versus control) or on reperfusion injury (0.27 (0.01-0.35), P not significant versus control). In contrast, dimethylthiourea had no effect on perfusion at either 10 min (0.10 (0.03-0.15), P not significant versus control) or 240 min (0.04 (0.00-0.11), P not significant versus control), but abolished the phase of relative reperfusion at 120 min (0.04 (0.02-0.21), P < 0.01 versus control). These results indicate that, although superoxide radicals are harmful during postischaemic reperfusion, hydroxyl radicals may be beneficial.     

Vascular function in the cadaver up to six hours after cardiac arrest.

BACKGROUND: The aim of the study was to evaluate how well vascular function is retained in a cadaver kept in a room with a temperature of 21 degrees C. METHODS: The aorta and pulmonary artery of rats were investigated in organ baths as fresh controls and after 1, 2, 3, or 6 hours' storage in the cadaver. Six-hour-old cadaver aortas were transplanted and investigated after 24 hours and 60 days. RESULTS: After 3 hours' storage there was no significant decrease in smooth muscle contractile function in either aorta or pulmonary artery. After 6 hours' storage both the aorta and the pulmonary artery demonstrated a significant decrease in smooth muscle contractile function, 30% (p < 0.05) and 44% (p < 0.001), respectively, compared to fresh controls. Storing the aorta for 2 hours and the pulmonary artery for 6 hours caused no significant decrease in endothelium-dependent relaxing function. In aorta segments investigated after 3 and 6 hours there was a significant decrease in endothelium-dependent relaxation, 12% (p < 0.05) and 29% (p < 0.001), respectively. Six-hour-old cadaver aortas transplanted and investigated after 24 hours or 60 days demonstrated no significant changes in endothelium-dependent relaxation and smooth muscle function compared to fresh controls. CONCLUSION: The pulmonary artery can tolerate 3 hours of warm ischemia in the nonheart-beating cadaver without loss of endothelium-dependent relaxation and smooth muscle function. The dysfunction seen in 6-hour-old cadaver aortas was normalized after transplantation and 24 hours of reperfusion.     

Recombinant human manganese superoxide dismutase attenuates early but not delayed skeletal muscle dysfunction following reperfusion injury.

OBJECTIVES: to assess the efficacy of recombinant human manganese superoxide dismutase (rhMnSOD) in prevention of early and late skeletal muscle ischaemia-reperfusion injury mediated by superoxide (O2-). Design : randomised controlled trial. MATERIALS: seventy-two Sprague-Dawley rats (250-350 g) randomised to receive either 7.5 mg/kg of rhMnSOD or saline. Four hours of ischaemia was induced in the cremaster muscle by dissecting free and clamping its vascular supply. Cremaster muscle contractile function was assessed following 90 minutes, 24, 48 hours and one week of reperfusion. Electrophysiological muscle function was assessed using electrical field stimulation in an organ bath system. RESULTS: muscle function in the untreated groups following ischaemia reperfusion was significantly reduced at 90 minutes, 24, 48 hours and one week of reperfusion (p <0.05). rhMnSOD significantly protected and maintained normal muscle function at 24 and 48 hours (p <0.001). However at one week of reperfusion there was a reduction in function of the treated muscle, such that there was no significant difference between treated and untreated muscle at this point in time. CONCLUSIONS: these data demonstrate that skeletal muscle dysfunction after ischaemia reperfusion injury is attenuated at 24 and 48 hrs of reperfusion by the superoxide scavenger rhMnSOD. This protective effect is not maintained after seven days of reperfusion.     

Tourniquet ischemia: ultrastructural and histochemical observations of ischemic human muscle and of monkey muscle and nerve.

Biopsies of muscle from ten patients and of muscle and nerve from three monkeys were obtained during periods of continuous ischemia and studied by light and electron microscopy and by histochemical means. Between the third and fourth hours of ischemia, swelling of the mitochondria was noted in monkey muscle. One week later the appearance was normal. In the monkey nerves degenerated myelin was seen one week later but appeared to be normal at 2 weeks. If after 3 hours the tourniquet was deflated for 30 minutes and then reapplied for 2 additional hours, no changes were found. Succinic dehyrogenase analysis was used to identify swelling of the mitochondria. No mitochondrial changes were seen in human muscle made ischemic by a tourniquet for 2 hours.     

Effect of low-dose N-acetyl-cysteine infusion on tourniquet-induced ischaemia-reperfusion injury in arthroscopic knee surgery

BACKGROUND: Temporary occlusion of blood flow is used during arthroscopic knee surgery in order to provide a bloodless surgical field. The resulting ischaemia-reperfusion causes lipid peroxidation, which contributes to tissue injury. The aim of the study was to investigate the effect of low-dose n-acetyl cysteine (NAC) infusion on oxidative stress by determining malondialdehyde (MDA) levels during arthroscopic knee surgery. METHODS: Thirty patients, ASA I - II, undergoing arthroscopic knee debridement under a tourniquet were divided into NAC and control groups. Anaesthesia was induced with propofol, fentanyl and vecuronium bromide and maintained with desflurane in an equal parts O(2)-N(2)O mixture. In the NAC group, an infusion of NAC, 5 mg kg(-1).h(-1), was started after intubation, and continued until extubation. An equal volume of saline was infused to the control group. Duration of ischaemia, anaesthesia time, total dose of NAC infused were also recorded. Venous blood and synovial membrane tissue samples were obtained 10 min after the onset of NAC infusion but before tourniquet inflation (t1), after 30 min of ischaemia (t2), and after 5 min of reperfusion following tourniquet release (t3). RESULTS: Plasma MDA levels were significantly lower in the NAC group on reperfusion. There were no differences between the groups in tissue MDA levels at ischaemia and reperfusion times. CONCLUSION: Low-dose n-acetyl cysteine infusion attenuates lipid peroxidation and ischaemia-reperfusion injury in arthroscopic knee surgery requiring tourniquet application.     

Structural changes in temporarily ischaemized and reperfused dog kidneys

The functional and structural study of the ischaemic renal parenchyma has become a central problem from the aspects of kidney transplantation. Reliable data are needed to establish the tolerance of separate or simultaneous cold and warm ischaemia. The changes appearing after 1- and 2-hour ligation of the renal artery (warm ischaemia) followed by autogeneous reperfusion after 3 to 24 hours were analysed on dog kidneys by means of light and electron microscopy. After 1 hour ligation, the changes seem fully reversible as the structures (mitochondria, basal membranes, brush border) suffer no fundamental lesion in spite of a general oedema. In contrast, 2-hour ischaemia caused serious morphological lesions (breaking up of the mitochondrial cristae, decay of the brush border, cell desquamation, necrosis, extensive oedema) leading to no-reflow phenomenon and irreversibility.     

Effect of high-dose of methylprednisolone on tourniquet ischaemia

High doses of corticosteroids have been found to have beneficial effects in various shock states. It has been well recognized that ischaemia is one of the important features in shock states. This prompted us to investigate the effect of high-dose methylprednisolone on tourniquet-induced ischaemia using mongrel dogs. After inflation of tourniquets to 600 mmHg on each thigh of the hind legs, one leg received an intravenous infusion of methyl-prednisolone, 3 mg.kg-1 dissolved in 20 ml of autologous blood. The other leg received the same amount of blood only, as a control. During two hours of tourniquet time and until 30 min after tourniquet deflation, venous blood was sampled five times from both hind legs for measurements of blood gas tensions (PvO2, PvCO2) and pH, lactic acid, creatinine kinase (CK), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH). During tourniquet ischaemia, PvO2 and pH dropped and PvCO2, lactic acid, CK, AST and LDH rose steadily and significantly in both groups of legs, indicating respiratory and metabolic acidosis, and muscle cell damage. However, those changes were significantly smaller in the methylprednisolone-treated legs. The beneficial effect of methylprednisolone could be attributed to its vasodilatory effect, cellular membrane stabilization and direct metabolic effect on skeletal muscle cells. Although the tourniquet-induced ischaemia in our study is slightly different from the clinical paradigm, the results suggest that high-dose methylprednisolone may provide a beneficial effect during tourniquet ischaemia.     

肢体缺血再灌注损伤的新型动物模型制作

[目的]用充气止血带制作肢体缺血再灌注损伤的新型动物模型,研究其对周围神经和骨骼肌损伤的影响.[方法]选择健康新西兰大白兔6个月龄,30只,体重(3.5 ±0.3) kg,雌雄不限,在家兔左侧后肢环扎充气止血带,于不同时间点松开,造成左侧后肢缺血再灌注损伤的模型.随机分为3组,每组10只.A组:对照组,B组:缺血2h,C组:缺血4h.对照组不扎充气止血带,第1、2、3、4、5、6h检测肢体的神经电生理学指标,B组、C组于再灌注(松开止血带,血供恢复后)的1、2、3、4、5、6h检测肢体的神经电生理学指标,A组于第6h观察骨骼肌的形态,B、C组于再灌注(松开止血带,血供恢复后)的第6h观察骨骼肌的形态,每组于术后第5d评估左侧后肢的行走功能.[结果]随着缺血后再灌注时间的延长,B、C和A组相比较,周围神经的潜伏期延长、波幅降低,传导速度降低,三组之间的潜伏期、波幅、传导速度差异均有统计学意义(P＜0.05),光镜观察骨骼肌可见(B、C组):横纹紊乱、肌纤维断裂、间质血管扩张充血、大量中性粒细胞浸润.[结论]经过缺血期和再灌注损伤的交互作用后,肢体的功能性损伤进一步加重,出现了不可逆的病损.该模型制作对动物的损伤较小,更贴近临床.