Therapeutic interventions against reperfusion injury in skeletal muscle

Ischemia/reperfusion (I/R) injury in the skeletal muscle is inevitable in many vascular and musculoskeletal traumas, diseases, free tissue transfers, and during time-consuming reconstructive and transplantation surgeries. Although skeletal muscle has a higher tolerance to ischemia than other organs, prolonged ischemia can still result in significant complications, including muscle necrosis and apoptosis. One of the major goals in the treatment of ischemia is early restoration of blood flow (reperfusion) to the area at risk. However, reperfusion has led to a new pathophysiologic condition called "reperfusion injury," a phenomenon which actually provokes a distinct degree of tissue injury. The purpose of this review is to examine the current state of understanding of I/R injury as well as to highlight recent developments on I/R interventions including our own experience in this particular field. We expect, as our acquired experience and the increased knowledge of underlying mechanisms of I/R injury, more effective interventions aimed to reduce I/R injury will be developed to interfere with or modulate this particular pathophysiologic processes.     

Ischemia-reperfusion injury is more severe in older versus young rat livers

BACKGROUND: Hepatic warm ischemia during surgery remains a significant problem, particularly in organs with possible baseline dysfunction. The objective of this study was to investigate whether age influences the degree of warm ischemia-reperfusion injury in rat livers. MATERIALS AND METHODS: The left and median lobes of young (3 months) and adult (9 months) male rats were exposed to 75 min of ischemia followed by reperfusion. Each age group was divided into two sub-groups. One sub-group was observed for 8 h, whereas the other was allowed to survive. Animals in the 8-h groups (young and adult) were sacrificed, and blood and tissue were taken to determine liver enzymes, neutrophil accumulation, and blood metabolic profiles and to examine the histology. RESULTS: Hepatocellular injury was significantly greater in adult rats after 8 h of reperfusion, as determined by hepatic enzyme levels and histology. Liver enzyme levels were massively elevated in adult rats and were significantly higher compared with those of young rats. The degree of necrosis and neutrophil accumulation was significantly higher in adult rats. After 8 h of reperfusion, the metabolic profiling of the blood revealed elevated levels of creatine, creatinine, allantoin, and amino acids (tyrosine, methionine) in the adult rats. At 24 h of reperfusion, all adult rats died, in contrast to young rats, which all survived. CONCLUSIONS: Aging in rats is associated with greater hepatocellular injury and poor survival rate after 75 min of warm hepatic ischemia.     

Ischemic preconditioning of skeletal muscle mitigates remote injury and mortality

BACKGROUND: Ischemic preconditioning (IPC) mitigates ischemia-reperfusion (I/R) injury in experimental models. However, the clinical significance of this protection has been unclear and a mortality reduction has not been previously reported in noncardiac models. This study examined the local and remote protection afforded by skeletal muscle IPC and sought to determine the significance of this protection on mortality. METHODS: Mice subjected to 2 h hindlimb ischemia/24 h reperfusion (standard I/R injury) were compared with those undergoing a regimen of two 20-min cycles of IPC followed by standard I/R injury. Local injury was assessed via gastrocnemius histology, and remote injury was evaluated via intestinal histology and pulmonary neutrophil infiltration (n = 7). Mortality was compared in parallel groups for 1 week (n = 6). Groups were analyzed using an unpaired Student's t-test for gastrocnemius and pulmonary injury, and a Mann-Whitney rank sum test for intestinal injury. Mortality differences were interpreted through a hazard ratio. RESULTS: Significant protection was observed in preconditioned animals. There was a 35% local injury reduction in skeletal muscle (71.2% versus 46.0%, P < 0.01), a 50% reduction in remote intestinal injury (2.3 versus 1.1, P < 0.01), and a 43% reduction in remote pulmonary injury (14.9 versus 8.5, P < 0.01) compared with standard injury controls. Preconditioned animals were also significantly protected from mortality, demonstrating a 66.7% survival at 1 wk compared with 0% survival after standard injury alone (hazard ratio 0.20, 95% CI: 0.02-0.59). CONCLUSIONS: We have developed a murine model of IPC that demonstrates local and remote protection against I/R injury, and exhibits significant mortality reduction. This model demonstrates the powerful effect of IPC on local and remote tissues and will facilitate further study of potential mechanisms and therapies.     

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.     

Expression of N-cadherin by skeletal muscle in the degenerationand the degeneration/regeneration processes after nerve injury

We investigated the expression of N-cadherin by skeletal muscle during the degeneration and degeneration/regeneration processes using the rat sciatic nerve and gastrocnemius muscle model. The right sciatic nerve was exposed in the mid-thigh region, and the nerve was transsected with small scissors. After then, nerve was sutured (sutured group), or both edges of the resected nerve were turned and sutured to the muscle of each side (unsutured group). At various periods up to 24 weeks after the operation the middle portion of the gastrocnemius muscle of the treated hindlimbs was removed. Expression of N-cadherin was detected by western blot analysis and immunofluorescent staining with an anti N-cadherin antibody. In the both groups, the degree of expression had already increased by the end of the first postoperative week, but there were no significant differences between the first and second postoperative weeks between the two groups. However, the values recorded at the fourth, sixth, ninth, and twelfth postoperative weeks were significantly higher in the unsutured group than in the sutured group. Immunofluorescent staining was present around the muscular membrane in all specimens including the control. These results indicated that there was a difference in the kinetics of expression of N-cadherin in skeletal muscle between the degeneration and degeneration/regeneration processes of the muscle after injury to the nerve. It was also clear that N-cadherin has a role at the surface of the muscle cell in skeletal muscle, not in the satellite and inflammatory cells, in both groups.     

Longitudinal evaluation of local muscle conditions in a rat model of gastrocnemius muscle injury using an in vivo imaging system

This study aimed to evaluate the time course of local changes during the acute phase of gastrocnemius muscle strain, in a rat model, using an in vivo imaging system. Thirty‐eight, 8‐week‐old Sprague‐Dawley male rats were used in our study. Experimental injury of the right gastrocnemius muscle was achieved using the drop‐mass method. After inducing muscle injury, a liposomally formulated indocyanine green derivative (LP‐iDOPE, 7 mg/kg) was injected intraperitoneally. We evaluated the muscle injuries using in vivo imaging, histological examinations, and enzyme‐linked immunosorbent assays. The fluorescence peaked approximately 18 h after the injury, and decreased thereafter. Histological examinations revealed that repair of the injured tissue occurred between 18 and 24 h after injury. Quantitative analyses for various cytokines demonstrated significant elevations of interleukin‐6 and tumor necrosis factor‐α at 3 and 18 h post‐injury, respectively. The time course of fluorescence intensity, measured using in vivo imaging, demonstrated that the changes in cytokine levels and histopathologic characteristics were consistent. Specifically, these changes reached peaked 18 h post‐injury, followed by trends toward recovery. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1034–1038, 2015.     

缺血预处理减轻骨骼肌缺血再灌注损伤

目的 观察缺血预处理对骨骼肌缺血再灌注损伤的保护作用。方法 选择２４只健康兔,随机等分为实验组和对照组。实验组先进行缺血预处理,再持续阻断后肢血流４ｈ;对照组直接阻断后肢血流４ｈ,制作骨骼肌缺血再灌注损伤模型。测定再灌注期血清中肌酸磷酸激酶（ＣＰＫ）和天门冬氨酸氨基转移酶（ＡＳＴ）,镜下观察骨骼肌变化。结果 实验组血清中ＣＰＫ和ＡＳＴ的含量均明显低于对照组（Ｐ〈０．０５）。实验组骨骼肌线粒体空泡变     

Reperfusion injury to skeletal muscle affects primarily type II muscle fibers

INTRODUCTION: The injury caused by reperfusion of ischemic skeletal muscle is mediated by the membrane attack complex of complement (C) . This C activation results from local classical pathway activation after deposition of IgM in injured muscle, an event analogous to C deposition in the mucosa of the gut during reperfusion . Our past analysis has indicated that the injury is not uniform even within a single microscopic section. This study was performed to elucidate the exact site of IgM and C deposition on muscle injured by ischemia and reperfusion. MATERIALS AND METHODS: C57Bl/6 mice were subjected to 2 h of tourniquet-induced hindlimb ischemia followed by reperfusion for 0-6 h. Three muscle groups (vastus, gastrocnemius, and soleus) of varying fast-myosin content were compared for muscle fiber damage and C deposition. Adjacent paraffin-embedded cross-sections were immunostained to correlate C3 deposition with muscle fiber type as defined by monoclonal antibodies. RESULTS: Muscle injury after ischemia and reperfusion is not uniform and not all fibers in the same microscopic field are affected. Damaged fibers are also those to which IgM and C bind. Immunostaining for slow-twitch (Type 1) or fast-twitch (Type 2) fibers reveals that injury and C3 deposition is confined to Type 2 fibers with lower myosin content. A correlation of Type 2 fiber content and degree of muscle injury showed that the predominantly fast-twitch vastus muscle had the greatest number of damaged fibers per x10 field (28.2 +/- 12.4) when compared to the mixed fiber-type gastrocnemius muscle (20.5 +/- 5.3) and the mixed, but slow-twitch enriched soleus muscle (17.3 +/- 11.8). CONCLUSION: Complement activation and skeletal muscle reperfusion injury occurs predominantly on Type 2 fibers with low myosin content. This suggests that attempts to control the post-reperfusion inflammation will likely produce substantial muscle recovery. Furthermore, the basis of IgM deposition and complement activation may be revealed in the comparison of the two muscle fiber types.     

In vivo bioluminescence imaging of magnetically targeted bone marrow‐derived mesenchymal stem cells in skeletal muscle injury model

The purpose of this study is to clarify the kinetics of transplanted mesenchymal stem cells (MSCs) in rat skeletal muscle injury model and the contribution of the magnetic cell delivery system to muscle injury repair. A magnetic field generator was used to apply an external magnetic force to the injury site of the tibia anterior muscle, and 1 × 10⁶ MSCs labeled with ferucarbotran–protamine complexes, which were isolated from luciferase transgenic rats, were injected into the injury site. MSCs were injected with and without an external magnetic force (MSC M+ and MSC M? groups, respectively), and phosphate‐buffered saline was injected into injury sites as a control. In vivo bioluminescence imaging was performed immediately after the transplantation and, at 12, 24, and 72 h, and 1 and 4 weeks post‐transplantation. Also, muscle regeneration and function were histologically and electromechanically evaluated. In vivo bioluminescence imaging showed that the photon of the MSC M+ group was significantly higher than that of the MSC M? group throughout the observation period. In addition, muscle regeneration and function in the MSC M+ group was histologically and functionally better than that of the MSC M? group. The results of our study indicated that magnetic cell delivery system may be of use in directing the transplanted MSCs to the injury site to promote skeletal muscle regeneration. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 754–759, 2013     

Artificial oxygen carrier improves fatigue resistance in slow muscle but not in fast muscle in a rat in situ model

The effects of liposome-encapsulated hemoglobin with high O₂ affinity (h-LEH), an artificial O₂ carrier in skeletal muscle, were studied by in situ fatigue resistance test in fast-type plantaris (PLT) and slow-type soleus (SOL) muscles with or without ischemia in the rat. The distal tendons of PLT and SOL muscles were isolated in situ and individually attached to the force transducers to record the developed tension in response to stimuli (80 Hz tetanus train, 1.5 minutes) to the ipsilateral sciatic nerve. The fatigue resistance test (five sets separated by 2-minute rests) was evaluated in terms of tension attenuation (fatigue) from the initial to the last tension (A) during each set, attenuation of the initial (B) or last tension (C) in each set, as compared to the first set in the presence or absence of ischemia or h-LEH (10 mL/kg). While ischemia significantly enhanced fatigue only in PLT, h-LEH showed no effect regardless of the perfusion pattern (normal/ischemia) or muscle-type (PLT/SOL) during each set (A). In parameter (B), set-by-set fatigue development was observed in PLT, whereas h-LEH-SOL showed a trend of advanced fatigue resistance. Such trends became clear in the parameter C (last tension), because h-LEH-SOL exerted, rather than decreased, the tension enhancement regardless of the presence or absence of ischemia, whereas there were no h-LEH effects in PLT. In addition, faster recovery of the nicotinamide adenine dinucleotide content in the muscle after 10 minutes of all fatigue tests was observed in h-LEH-SOL, while saline-SOL still showed a significantly higher value than that of control. These results suggested that additional O₂ supply by h-LEH may accelerate the tricarboxylic acid cycle/electron transport chain in slow-type aerobic SOL muscle containing abundant mitochondria and contribute to the faster removal of muscle fatigue substances such as lactate.     

丹参改善骨骼肌缺血再灌注损伤的超微结构观察

目的:通过透射电镜观察骨骼肌缺血再灌注损伤过程的病理生理变化探讨丹参的干预作用及机制。方法:采用90只清洁级雄性SD大鼠,6只作为健康对照组,84只行左侧提睾肌主滋养动脉夹闭造模,在缺血2.5 h时,丹参组腹腔注射丹参(42只),对照组注射等剂量的生理盐水(42只),两组分别设缺血再灌注10、20、30、40、50、60、90 min观测点(每个观测点6只)。观测结束后,迅速切取提睾肌,放入电镜固定液。电镜下进行组织形态学观察。结果:健康对照组正常情况下,组成肌原纤维的粗、细2种肌丝沿其长轴排列,每条肌原纤维上均可见明暗相间的带,肌浆内见有较多糖原颗粒及少量内质网、线粒体等细胞器,肌浆网纵向分布于肌原纤维周围,线粒体膜及嵴清晰可见,基质着色深,无肿胀、空泡形成等改变,在肌丝之间分布有较多糖原颗粒;骨骼肌缺血再灌注后10、20、30、40、50、60、90 min的2组各观测点,其病理变化较相似,肌丝模糊、凌乱,部分溶解、消失,甚至完全消失,肌浆网不同程度扩张,线粒体变大变圆,基质变淡,嵴变短变少甚至消失,部分线粒体极度肿胀,呈空泡状。丹参组,其病理变化大体相似,肌纤维均有不同程度恢复。结论:骨骼肌缺血再灌注损伤导致线粒体极度肿胀,肌纤维紊乱。丹参能有效地消除线粒体的水肿,恢复肌纤维,减轻骨骼肌缺血再灌注损伤。     

Interleukin-1 and thromboxane release after skeletal muscle ischemia and reperfusion

Interleukin-1 and thromboxane are known to mediate the host response to sepsis, trauma, and myocardial ischemia. A well-established model of canine isolated gracilis muscle was used to evaluate whether cytokine (interleukin-1) played a role in skeletal muscle ischemia-reperfusion injury. Six adult mongrel dogs (25–30 kg) were subjected to six hours of muscle ischemia followed by reperfusion. Gracilis venous samples were collected pre-ischemia and at one hour of reperfusion. Systemic (arterial) blood samples were taken at one hour of reperfusion. Sera were analyzed for interleukin-1 by bioassay and thromboxane (B₂) by radio-immunoassay. The gracilis muscle of the operated limb was harvested in all the animals for assessment of the percentage of muscle necrosis. This was found to be 56.2±14.8% by serial transections, nitroblue tetrazolium staining, and computerized planimetry. Interleukin-1 levels in the gracilis venous effluent increased from 21.88±7.13 units/ml during pre-ischemic baseline to 50.42±9.12 units/ml after six hours of ischemia followed by one hour of reperfusion (p<0.04). Thromboxane B₂ levels were 2983±1083 pg/ml and 9483±2218 pg/ml at pre-ischemia and at one hour of reperfusion respectively (p<0.04). Systemic levels of both interleukin-1 and thromboxane B₂ at one hour of reperfusion were 0 units/ml and 1584±520 pg/ml respectively, which were significantly lower than the one hour reperfusion gracilis venous effluent levels (p<0.04). This is the first report in which cytokines have been implicated in skeletal muscle ischemia-reperfusion injury. Modulation of interleukin-1 may impact positively on muscle necrosis and systemic manifestations of reperfusion injury.Presented at the 16th Annual Meeting of the Peripheral Vascular Surgery Society, June 2, 1991, Boston, Massachusetts.     

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.     

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.     

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...     

缺血后处理对减轻骨骼肌缺血再灌注损伤作用的实验研究

目的：研究缺血后处理对鼠骨骼肌缺血再灌注损伤的保护影响,组织中凋亡和胀亡的存在情况。方法将54只SD大鼠随机分为空白对照组、缺血再灌注组、缺血后处理组,持续缺血4 h,再灌注6 h,24 h,48 h。检测血浆乳酸脱氢酶（LDH）、肌酸磷酸激酶（CPK）活性、肌肉内丙二醛（MDA）含量及总超氧化物歧化酶（SOD）活性,进行组织学、免疫组化、超微结构分析。结果相比缺血再灌注组,后处理组在再灌注6 h时,只SOD活性明显升高,而再灌注24 h,48 h时,在MDA含量下降、SOD活性升高、W/D值下降、组织学改变范围及免疫组化阳性范围方面,均较缺血再灌注组有明显差异。结论再灌注开始时应用后处理对于缺血再灌注损伤有明显的保护作用,主要体现在再灌注的稍后期阶段（再灌注24 h,48 h）。缺血再灌注过程中,凋亡和胀亡是并存的。     

缺血后处理对骨骼肌缺血再灌注后肺损伤保护作用的机制

目的：探讨缺血后处理（I-postC）对大鼠双侧后肢骨骼肌缺血再灌注（I/R）后肺损伤的保护作用及机制。方法：阻断肾下腹主动脉建立大鼠双侧后肢骨骼肌I/R损伤模型。48只大鼠随机分为3组：I/R组、缺血预处理（IPC）组及I-postC组,每组16只。分别于再灌注后12、24h各处死8只,取肺组织标本,观察肺组织形态学、湿/干重比、丙二醛（MDA）及髓过氧化物酶（MPO）的变化。原位杂交和RT-PCR方法检测肺组织中细胞间黏附分子（ICAM）-1mRNA的表达,Western blot检测ICAM-1蛋白表达。结果：再灌注12或24h后I/R组有明显的弥散功能障碍,表现为间质浸润细胞增多并伴有明显水肿。IPC组和I-postC组的各项指标均较I/R组明显降低,差异有统计学意义（P〈0.01）,但2组之间差异无统计学意义（P〉0.05）。结论：I-postC可以减轻大鼠双侧后肢骨骼肌I/R后肺损伤,与IPC可能存在共同的作用机制。