Skeletal Muscle Ischemia-reperfusion Research Articles

The Role of Extracellular Superoxide Dismutase (EC-SOD) in Skeletal Muscle Ischemia-reperfusion Injury

Purpose: This study investigated the role of extracellular superoxide dismutase (EC-SOD), which is a major extracellular antioxidant enzyme in skeletal muscle ischemia and reperfusion (I/R) injury. Materials and Methods: The pedicled cremaster muscle flaps from homozygous EC-SOD knockout (), heterozygous CuZn-SOD knockout () and wild-type (WT) mice were subjected to 4.5 hour ischemia followed by 90 min reperfusion. The pedicled cremaster muscle flaps were examined by functional analysis during the reperfusion. The mRNA and protein expressions of each SOD after I/R were evaluated using quantitative real-time PCR and western blot. Results: The results showed that the mice had a more profound I/R injury than the or WT mice. In particular, there was a delayed and incomplete recovery of the arterial diameter and blood flow during reperfusion and as well as there being more severe inflammation. After 90 min reperfusion, the EC-SOD mRNA levels increased more in the mice than in the WT mice. However, the CuZn-SOD and Mn-SOD mRNA levels decreased similarly in all 3 groups. The CuZn-SOD protein levels decreased in all groups. The EC-SOD protein levels decreased in the and WT mice, but the Mn-SOD protein levels were unchanged or slightly increased in all groups. The histological results showed diffuse edema and inflammatory cell infiltration around the muscle fibers and these changes were more severe in the mice. Conclusion: EC-SOD plays an important role in protecting the skeletal muscle from I/R injury caused by the excessive generation of reactive oxygen species.

IgM binding to injured tissue precedes complement activation during skeletal muscle ischemia-reperfusion

Skeletal muscle reperfusion injury is mediated by IgM natural antibodies and by complement activation, as shown by the attenuation of reperfusion injury seen in mice with no natural IgM [] and in mice deficient in complement C3 and C4 []. We postulate that tissue, when ischemic, expresses neoantigens to which preformed natural IgM antibodies bind, in turn producing harmful complement activation and reperfusion injury. C57Bl/6 mice were subjected to 2 h of tourniquet-induced hind limb ischemia followed by variable periods of reperfusion. Two hours of ischemia and 3 h of reperfusion produced severe muscle necrosis and edema. Deposition of IgM and C3 in tissue was assessed using immunohistochemistry on both frozen and Formalin-fixed tissue samples. IgM binding to the endothelium and muscle bundles of the hind limb began during the ischemic period and continued throughout reperfusion up to 6 h. C3 deposition was not present during ischemia and, in contrast, began to appear at 1 h of reperfusion and increased progressively thereafter. These data demonstrate that IgM binding to ischemic tissues precedes the damaging complement activation by a significant period of time. This has important therapeutic implications when considering anti-inflammatory therapy for reperfusion injury.

Reperfusion injury to skeletal muscle affects primarily type II muscle fibers 1

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. 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. 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). 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.

Complement deposition from reperfusion injury to skeletal muscle occurs primarily on type II muscle fibers

Introduction: The injury caused by reperfusion of ischemic skeletal muscle is mediated by the membrane attack complex of complement (C) (Am J Phys 1999; 277:1263). This C activation results from local classical pathway activation after deposition of IgM in injured muscle, in analogy to gut reperfusion injury (J Appl Phys 1999; 86:938). This study was performed to elucidate the exact site of C and IgM deposition in tissue injured by ischemia and reperfusion. Methods: C57Bl/6 mice were subjected to 2 hours of tourniquet-induced hindlimb ischemia followed by reperfusion for 0–6 hours. Three muscle groups (vastus, gastrocnemius, 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 C3 and IgM bind, exclusively. Immunostaining for slow twitch (Type I) or fast twitch (Type II) fibers reveals that injury and C3 deposition is confined to Type II fibers with lower myosin content. A correlation of Type II fiber content and degree of muscle injury showed that the predominantly fast-twitch vastus muscle had greatest number of damaged fibers per 10x field (28.2 ± 12.4) when compared to the mixed fiber-type gastronemius 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 II 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 the complement activation may be revealed in the comparison of the two muscle fiber types.

IgM binding precedes complement activation during skeletal muscle ischemia-reperfusion

Introduction: Skeletal muscle reperfusion injury is mediated by IgM natural antibodies and by complement activation, as shown by the attenuation of reperfusion injury seen in mice with no natural IgM (J Immunology 2002; 168:3433) and in mice deficient in complement C3 and C4 (J Exp Med 1996; 183:2343). We postulate that tissue, when ischemic, expresses neo-antigens to which pre-formed natural IgM antibodies bind, in turn producing harmful complement activation and reperfusion injury. Methods: C57Bl/6 mice were subjected to 2 hours of tourniquet-induced hind limb ischemia followed by variable periods of reperfusion. Two hours of ischemia and 3 hours of reperfusion produced severe muscle necrosis and edema. Deposition of IgM and C3 in tissue was assessed using immunohistochemistry on both frozen and formalin-fixed tissue samples. Results: IgM binding to the endothelium and muscle bundles of the hind limb began during the ischemic period and continued throughout reperfusion up to 6 h. C3 deposition was not present during ischemia, and in contrast, began to appear at 1h of reperfusion and increased progressively thereafter. Conclusion: These data demonstrate that IgM binding to ischemic tissues precedes the damaging complement activation by a significant period of time. This has important therapeutic implications when considering anti-inflammatory therapy for reperfusion injury.

Activated protein C attenuates skeletal muscle ischaemia reperfusion injury.

Aims: Pharmacological modulation of skeletal muscle reperfusion injury after an ischaemic insult may improve limb salvage rates and prevent the associated systemic sequelae. Activated Protein C (APC) is an endogenous anti-coagulant with anti-inflammatory properties that has been extensively studied in the setting of sepsis. The purpose of our study was to evaluate the effects of APC on skeletal muscle ischaemia reperfusion injury. Methods: Adult male Sprague Dawley rats (n = 24) were randomised into three groups: control group, I/R group treated with normal saline and I/R group treated with APC. Bilateral hind-limb ischaemia was induced by rubber band application proximal to the level of the greater trochanters for two hours. Treatment groups received either normal saline or APC prior to tourniquet release. Following twelve hours reperfusion, the tibialis anterior was dissected and muscle function assessed electrophysiologically by electrical field stimulation. The animals were then sacrificed and skeletal muscle harvested for evaluation. Skeletal muscle injury was assessed based on myeloperoxidase content as a measure of neutrophil infiltration and wet to dry ratio as a measure of oedema formation. Histological analysis was also performed on the muscle. Results: APC significantly attenuated skeletal muscle reperfusion injury as shown by reduced myeloperoxidase content, wet to dry ratio and electrical properties of skeletal muscle (Table). TABLE—ABSTRACT 28Skel muscle wet:dry ratioSkel muscle MPO (U/g)Max twitch contraction (N/g)Tetanic contraction at 50 HZ (N/g)Control group3.17 ± 0.100.74 ± 0.032.45 ± 0.264.21 ± 0.26I/R saline3.40 ± 0.140.95 ± 0.131.52 ± 0.202.88 ± 0.53APC3.21 ± 0.110.82 ± 0.071.86 ± 0.183.52 ± 0.36P valueP < 0.03P < 0.02P < 0.01P < 0.01 These findings were supported by our histological findings. Statistical significance was determined using variance analysis. Conclusion: Activated Protein C may have a protective role in the setting of skeletal muscle ischaemia reperfusion injury.

Age-Related Differences of Neutrophil Activation in a Skeletal Muscle Ischemia–Reperfusion Model

Free tissue transfers and replantation of amputated limbs are better tolerated by young adolescents than mature adults. The authors hypothesized that this observation may be, in part, because of an attenuated ischemia-reperfusion (IR) injury in younger patients. Because neutrophils have been identified as a critical cell line responsible for IR injury, the authors investigated the effects of animal age on the degree of neutrophil activation in a rat model. Activation was evaluated by monitoring expression of integrin surface markers (mean fluorescence intensity [MFI] of CD11b) and oxidative burst potential (MFI of dihydrorhodamine [DHR] oxidation) by flow cytometry in neutrophils analyzed after 4 hours of ischemia and 1, 4, and 16 hours of reperfusion in a gracilis muscle flap model in mature adult and young adolescent rats. Neutrophil activation was also evaluated in control sham-operated animals, which underwent elevation of gracilis muscle flaps without exposure to an ischemic insult. Muscle edema, determined by wet-to-dry muscle weight ratio, and muscle viability, determined by nitro blue tetrazolium (NBT) staining, were completed for gracilis muscles exposed to ischemia after 24 hours of reperfusion for each of the groups. Integrin expression, assessed by MFI of CD11b, was increased significantly in ischemic muscles of mature adult rats at 4 hours of reperfusion (71.10+/-3.53 MFI vs. 54.88+/-12.73 MFI, p=0.025). Neutrophil oxidative potential, assessed by MFI of DHR oxidation, was increased significantly in ischemic muscles of mature adult rats compared with young adolescent rats at 1 hour of reperfusion (78.10+/-9.53 MFI vs. 51.78+/-16.91 MFI, p=0.035) and 4 hours of reperfusion (83.69+/-15.29 MFI vs. 46.55+/-8.09 MFI, p=0.005). Increased edema formation was observed in the ischemic muscles of mature adult rats when compared with young adolescent rats (1.25+/-0.04 vs. 1.12+/-0.05, p=0.031) after 24 hours of reperfusion. A trend toward decreased muscle viability was observed in the mature adult rats when compared with young adolescent rats (23.7+/-3.1% NBT staining vs. 32.3+/-13.7% NBT staining, p=0.189) after 24 hours of reperfusion. The authors present evidence of an attenuated IR injury in young adolescent animals when compared with mature adult rats. These findings emphasize the importance that studies involving IR injury should be performed with consideration of animal age.

Activation time course of activator protein-1 and effect of proline dithiocarbamate during ischemia-reperfusion in rat skeletal muscle.

Activator protein 1 (AP-1) is thought to play an important role in the expression of genes expressed in response to ischemia-reperfusion injury. In this report, the activation of AP-1 in rat skeletal muscle during reperfusion after a 4-hour ischemic period was studied. AP-1 activation displayed a biphasic pattern, showing peak activities at 1 hour after perfusion and from 4 hours to 12 hours after perfusion. Inhibition of AP-1 activation was investigated using a potent nuclear factor kappa B inhibitor, proline dithiocarbamate (Pro-DTC). AP-1 binding activity at 1 hour of reperfusion was significantly reduced (29.0 +/- 10.1% SEM; p < 0.05) after intravenous administration of Pro-DTC (n = 7 animals in each group). Further elucidation of the role of AP-1 is warranted in hopes of developing strategies to reduce the deleterious effects of ischemia-reperfusion injury.

Ischemia–Reperfusion Does Not Cause Significant Hyaluronan Depolymerization in Skeletal Muscle

The size of hyaluronan in lymph and gastrocnemius muscle was measured to test the hypothesis that reperfusion of ischemic skeletal muscle leads to hyaluronan depolymerization. Prenodal lymph was collected from the gastrocnemius muscle in anesthetized rabbits. Hyaluronan size was measured using a combination of agarose gel electrophoresis and a radiometric assay. In control legs, muscle contained primarily high-molecular-weight hyaluronan (greater then 4 × 10 6), while lymph contained primarily low-molecular-weight hyaluronan (less than 0.79 × 10 6) which was absent from tissue. Following 3 h of ischemia and 8 h of reperfusion, the lymph flux for high-molecular-weight hyaluronan was 25 times the value from the control leg. Neither the size nor the content of hyaluronan in tissue decreased. Muscle albumin content from the reperfused leg was 2.3 times the value from the control leg, while lymph albumin flux was 4 times the control value. Measurements following 24 h of reperfusion confirmed the absence of changes in the size or content of hyaluronan in tissue although an increased albumin content and wet weight-to-dry weight ratio indicated sustained edema. The daily removal of hyaluronan by lymph was calculated to be 2–3% of the tissue content. Since the lymph drainage of hyaluronan represented only a very small fraction of tissue hyaluronan, the amount of depolymerization was too small to produce significant changes in the tissue.

Effects of nitric oxide donor and inhibitor on prostaglandin E2-like activity, malondialdehyde and reduced glutathione levels after skeletal muscle ischemia-reperfusion

Oxygen free radicals are implicated in the pathophysiology of ischemia-reperfusion (I/R) injury in skeletal muscle. Nitric oxide (NO) and prostaglandin E2 (PGE2) are important regulators of the microcirculation in skeletal muscle. The effects of L-arginine, substrate for NO, and NG-nitro L-arginine methyl ester (L-NAME) on PGE2 synthesis, lipid peroxidation and reduced glutathione (GSH) levels was investigated in the rat gastrocnemius muscle after 3 h of reperfusion following 2 h of ischemia.Lipid peroxidation and GSH levels showed a non-significant changes in the I/R groups compared to the control group. According to these results, it can be assumed that skeletal muscle can resist 2 h of ischemia followed by 3 h of reperfusion-induced oxidative stress.PGE2-like activity in the gastrocnemius muscle increased in the L-NAME treated and I/R groups. L-arginine administration reversed the increase in PGE2-like activity of reperfused skeletal muscle. These findings support the conclusion that endothelium-derived PGE2 synthesis increases during reperfusion and suggest that PGE2 may have a protective role in the maintenance of endothelial function.

Recombinant soluble P-selectin glycoprotein ligand 1 moderates local and remote injuries following experimental lower-torso ischaemia.

A central role for the polymorphonuclear leucocyte (PMN) in skeletal muscle ischaemia-reperfusion has been demonstrated by the observation that PMN depletion reduced local and remote pulmonary vascular permeability. This study investigated the role of recombinant soluble P-selectin glycoprotein ligand-immunoglobulin fusion protein (rPSGL-Ig), a P- and E-selectin antagonist, in moderating injury. Mice underwent 2 h of hindlimb ischaemia and 3 h of reperfusion. Muscle and lung vascular permeability index (PI) was assessed by extravasation of (125)I-radiolabelled albumin. Lung myelo peroxidase (MPO) activity was also measured. In mice treated with rPSGL-Ig 1 mg/kg before reperfusion (n = 12) muscle PI was reduced by 40 per cent, whereas it was moderated by 20 per cent in animals treated 30 min after reperfusion (n = 15). Lung PI in mice treated with rPSGL-Ig before (n = 12) and 30 min after (n = 15) reperfusion was reduced by over 99 and 98 per cent respectively. Lung MPO activity in mice treated with rPSGL-Ig before (n = 10) and 30 min after (n = 12) reperfusion was reduced by 68 and 58 per cent respectively. Treatment with rPSGL-Ig 1 h after reperfusion, or with m20ek.Fc 1 mg/kg (n = 9; negative control for rPSGL-Ig which is inactive for selectin binding) before reperfusion failed significantly to moderate local or remote organ injury. Selectin blockade moderated local skeletal muscle and remote lung injury following hindlimb ischaemia--reperfusion. Significantly, delayed antiselectin therapy also decreased injury.

Limited use of cyclosporin A in skeletal muscle ischemia--reperfusion injury.

Reperfusion injury is propagated by an inflammatory-mediated tissue edema and damage after reestablishment of vascular flow following an initial ischemic insult. In the field of transplantation, cyclosporin A(CsA) provides protection against chronic graft rejection through lymphocyte immunosuppression. Evidence for an independent protective effect of CsA against ischemia-reperfusion (IR) injury during organ transfer has prompted studies showing the benefit of CsA in various ischemia-exposed visceral organs. The authors hypothesized that CsA administration may similarly benefit IR injury after skeletal muscle amputations. To determine the effects of CsA on IR injury the authors induced 4 hours of ischemia on the gracilis muscle in a rat model. CsA (15 mg per kilogram orally) was administered in two experimental groups: (1) preischemic (N = 6): 48, 24, and 3 hours before ischemia; and (2) postischemic (N = 6): 30 minutes after induction of ischemia. The effects of CsA on IR muscle injury were observed in each of the experimental groups as well as a control group (N = 6) exposed to similar ischemia and administered a saline vehicle. Muscle viability (nitro blue tetrazolium staining) and muscle edema (wet-to-dry weight ratio) were assessed 24 hours after reperfusion. The preischemic CsA-treated gracilis muscle group demonstrated improved muscle viability (39.1 +/- 4.8%) when compared with the ischemic control muscle group (23.8 +/- 7.1%; p = 0.039). Furthermore, the preischemic CsA-treated muscle group demonstrated decreased edema (1.137 +/- 0.095 times the contralateral nonischemic muscle) when compared with the control ischemic muscle group (1.248 +/- 0.045 times the contralateral nonischemic muscle; p = 0.011). Although a trend toward improved muscle viability (32.1 +/- 4.2%) and decreased edema formation (1.200 +/- 0.062 times the contralateral nonischemic muscle) was observed in the peri-ischemic CsA-treated group when compared with the control ischemic muscle group, these differences were not significant. These observations confirm the beneficial effects of preischemic CsA therapy observed in organ transplantation research and suggest limited clinical use of peri-ischemic CsA therapy for patients with musculoskeletal amputations.

Role of priming stresses and Hsp70 in protection from ischemia-reperfusion injury in cardiac and skeletal muscle.

Ischemia-reperfusion injury limits the survival of muscle involved in tissue trauma or transfers during microsurgical reconstruction. Priming stresses such as ischemic preconditioning or mild hyperthermia have frequently been associated with improved survival of ischemic-reperfused cardiac muscle, such protection coinciding with induction of the stress-related heat shock protein 70 (Hsp70). Little is known about the response of skeletal muscle to priming stresses. This review summarizes the current knowledge on the use of priming stresses as protective strategies against the consequences of ischemia-reperfusion in cardiac and skeletal muscle and the potential role of Hsp70.

Role of leukocytes and tissue-derived oxidants in short-term skeletal muscle ischemia-reperfusion injury.

The relative contribution of xanthine oxidase (XO) and leukocytes to tissue injury after short-term ischemia is unknown. In this study, we subjected three groups of rat spinotrapezius muscles to 30-min ischemia and 1-h reperfusion: 1) ischemia-reperfusion (I/R) + 0.9% saline, 2) I/R + superoxide dismutase, and 3) I/R + oxypurinol. A fourth group served as nonischemic control. We quantified the increase in resistance (%DeltaR) caused by leukocyte-capillary plugging concurrently with myocyte uptake of propidium iodide (PI) [expressed as no. of PI spots per total volume of perfused tissue (N(PI)/V)] and performed assays to quantify XO activity, thiobarbituric acid-reactive substances (TBARS), and myeloperoxidase (MPO). Groups 2 and 3 exhibited significant decreases in N(PI)/V relative to group 1. MPO levels and TBARS were similar among all groups, and mean %DeltaR was significantly reduced in groups 2 and 3 relative to group 1. However, elevated XO was observed in groups 1 and 2 relative to group 3 and nonischemic controls. These data are consistent with the hypothesis that XO, rather than toxic species produced by plugging or venule-adherent leukocytes, is responsible for postischemic damage in this model.

Ischemic preconditioning: Lack of delayed protection against skeletal muscle ischemia-reperfusion

We investigated the ability of ischemic preconditioning to induce expression of heat shock protein 70 (Hsp 70) and/or to increase muscle survival after ischemia-reperfusion in the rat hind limb. Ischemic preconditioning regimens tested were; 1 x 5 min of ischemia, 4 x 5 min of ischemia interrupted by 10 min of reperfusion, 1 x 10 min of ischemia or 2 x 10 min of ischemia interrupted by 15 min of reperfusion. Western blot analysis revealed only a modest induction of Hsp 70 at 24 h after preconditioning using the latter two protocols of 1 x 10 min of ischemia or 2 x 10 min. Used at 24 h prior to prolonged ischemia, neither protocol improved muscle survival measured at 24 h after reperfusion. In conclusion, ischemic preconditioning did not produce delayed protection from ischemia-reperfusion in this model and the study suggests that ischemic preconditioning is not a useful protective strategy against skeletal muscle necrosis in the long-term.

Do Prostaglandins Have a Salutary Role in Skeletal Muscle Ischaemia–reperfusion Injury?

Objectives: the effects of prostaglandins (PG) E1, E2, and the prostacyclin analogue iloprost with and without the addition of free-radical scavengers catalase and superoxide dismutase on gastrocnemius blood flow and oedema were studied in a rodent model of hindlimb ischaemia–reperfusion. Methods: male Sprague–Dawley rats underwent 6-h hindlimb ischaemia with 4-h reperfusion. Prostaglandins were infused prior to reperfusion and their effects on limb blood flow and oedema examined. Results: control animals exhibited a triphasic pattern of muscle blood flow during reperfusion compared to normal animals. PGE1 did not abolish low reflow at 10 min, relative reperfusion was preserved but reperfusion injury was abolished at 120 min. Muscle blood flow was increased at 240 min compared to controls. Increased limb swelling was also seen. Addition of free-radical scavengers caused the abolition of low reflow. Similar results were seen with iloprost. PGE2 abolished low reflow at 10 min and increased perfusion at 120 min but did not prevent reperfusion injury at 240 min. Conclusions: PGE1 and iloprost enhance muscle blood flow at 4-h reperfusion, though neither abolishes low reflow; PGE2 improved flow at 10 and 120 min but not after 240 min. This study demonstrates a potentially beneficial role for prostaglandins in improving muscle blood flow in skeletal muscle ischaemia–reperfusion injury.

L-selectin and leukocyte function in skeletal muscle reperfusion injury.

Treatment with anti-L-selectin monoclonal antibody will reduce venular neutrophil-endothelial rolling (flux and velocity) and adhesion associated with ischemia reperfusion injury in rat skeletal muscle. Prospective, randomized experimental trials. Basic science research laboratory. Male Wistar rats weighing 109 +/- 5 g (mean +/- SEM). Gracilis pedicle muscle flaps were elevated and microcirculation was observed by intravital microscopy. Two groups were evaluated: (1) the control group, which received 4 hours of global ischemia, and (2) the experimental group, which received 4 hours of global ischemia, plus treatment with anti-L-selectin monoclonal antibody 30 minutes before reperfusion. The number of rolling and adherent leukocytes in postcapillary venules were counted in the 2 groups at baseline and at 1 through 5, 10, 15, 20, 30, 45, and 60 minutes of reperfusion. Treatment with the monoclonal antibody to L-selectin significantly reduced the number of rolling leukocytes (flux) at 2 through 5, 20, 30, 45, and 60 minutes of reperfusion compared with controls (P<.05). Use of the monoclonal antibody significantly reduced the number of adherent neutrophils at 5, 10, 15, 20, 30, 45, and 60 minutes of reperfusion (P<.05). There was no significant difference in leukocyte velocity. L-Selectin plays a significant role in leukocyte rolling and adherence to venular endothelium in rat skeletal muscle ischemia reperfusion injury.

Nitric Oxide Synthase-Independent Generation of Nitric Oxide in Rat Skeletal Muscle Ischemia-Reperfusion Injury

We have used electron paramagnetic resonance to investigate the time course of nitric oxide (NO) generation and its susceptibility to inhibitors of nitric oxide synthase (NOS) in ischemia–reperfusion (IR) injury to rat skeletal musclein vivo.Significant levels of muscle nitroso-heme complexes were detected 24 h postreperfusion, but not after at 0.05, 3, and 8 h of reperfusion. The levels of muscle nitroso-heme complexes were not decreased by the NOS inhibitorN-nitro-l-arginine methyl ester as a single dose (30 mg/kg) prior to reperfusion or as multiple doses continued throughout the reperfusion (total administered, 120 mg/kg) or by the potent NOS inhibitorS-methylisothiourea (3 mg/kg). In contrast, nitroso-heme levels were reduced by the glucocorticoid dexamethasone (2.5 mg/kg). Muscle necrosisin vitrodid not result in the formation of nitroso-heme complexes. The finding that reperfusion after ischemia is necessary for NO formation suggests that an inflammatory pathway is responsible for NOS-independent NO formation in IR injury to skeletal muscle.

Selective neutrophil depletion with monoclonal antibodies attenuates ischemia/reperfusion injury in skeletal muscle.

The purpose of this study was to determine whether depletion of circulating neutrophils, using an antineutrophil monoclonal antibody (RP3), would attenuate ischemia/reperfusion injury in rat skeletal muscle. A 3- and 5-hr period of ischemia was induced unilaterally into the hindlimbs of rats; the isolated limbs were then reperfused for 24 hr after ischemia. The gastrocnemius muscle was then removed, and blood was taken simultaneously. The hematologic parameters were measured, muscle neutrophil sequestration was assessed by myeloperoxidase (MPO) activity, free radical production was evaluated by the tissue lipid peroxides (LPO) levels, muscle viability was assessed by tissue levels of adenosine triphosphate (ATP) and creatine phosphate (PCr) levels, and muscle wet/dry weights were determined. Treatment with RP3 selectively and sufficiently depleted the circulating neutrophil population, markedly reduced MPO, and significantly attenuated LPO and the tissue water content after both 3- and 5-hr of ischemia. After 3 hr of ischemia, ATP and PCr levels were significantly increased by neutrophil depletion; however, after 5 hr of ischemia, the same effect was not demonstrated. These results suggest that neutrophil depletion after 3 hr of ischemia restrains free radical production and edema formation, and also attenuates skeletal muscle ischemia reperfusion injury; however, after 5 hr of ischemia, ischemic damage was so severe, that neutrophil depletion did not reduce ischemia reperfusion injury.

Role of leukocyte plugging and edema in skeletal muscle ischemia-reperfusion injury.

The purpose of this study was to examine the relationship of increased capillary network resistance due to leukocyte-capillary plugging and tissue edema through macromolecular leakage to tissue injury after ischemia-reperfusion (I/R). After a 3-h complete ischemia in the dorsal skinfold chamber of the awake Syrian hamster, the following parameters were measured: vessel diameter, macromolecular leakage, erythrocyte velocity, adherent leukocytes, rolling leukocytes, freely flowing leukocytes, functional capillary density (FCD), propidium iodide (PI)-positive cell nuclei, and increase in network flow resistance due to leukocyte-capillary plugging. These measurements were made under baseline conditions and after 0.5 and 2 h of reperfusion for I/R alone, I/R with phalloidin (PL) treatment (to block leakage), and I/R with both PL and cytochalasin D (CD) (to block both leakage and plugging). Neither treatment had an effect on the leukocyte adherence or rolling. PL treatment preserved the endothelial barrier, improved FCD, and reduced the amount of PI measured tissue damage. CD treatment eliminated the increase in network resistance due to leukocyte plugging but did not improve FCD or tissue damage. Thus, in this I/R model, macromolecular leakage plays a role in tissue injury, whereas leukocyte plugging does not appear to be an important mechanism.