Our previous study has reported that superoxide mediates ischemia-reperfusion (IR)-induced necrosis in mouse skeletal muscle. However, it remains poorly understood whether IR induces apoptosis and what factors are involved in IR-induced apoptosis in skeletal muscle. Using a murine model of tourniquet-induced hindlimb IR, we investigated the relationship between mitochondrial dysfunction and apoptosis in skeletal muscle. Hindlimbs of C57/BL6 mice were subjected to 3 h ischemia and 4 h reperfusion via placement and release of a rubber tourniquet at the greater trochanter. Compared to sham treatment, tourniquet-induced IR significantly elevated mitochondria-derived superoxide production, activated opening of mitochondrial permeability transition pore (mPTP), and caused apoptosis in the gastrocnemius muscles. Pretreatment with a superoxide dismutase mimetic (tempol, 50 mg/kg) or a mitochondrial antioxidant (co-enzyme Q10, 50 mg/kg) not only decreased mitochondria-derived superoxide production, but also inhibited mPTP opening and apoptosis in the IR gastrocnemius muscles. Additionally, an inhibitor of mPTP (cyclosporine A, 50 mg/kg) also inhibited both mPTP opening and apoptosis in the IR gastrocnemius muscles. These results suggest that mitochondria-derived superoxide overproduction triggers the mPTP opening and subsequently causes apoptosis in tourniquet-induced hindlimb IR.
Ischemia-reperfusion (I/R) injury contributes to organ dysfunction in a variety of clinical disorders, including myocardial infarction, stroke, organ transplantation, and hemorrhagic shock. Recent investigations have demonstrated that apoptosis as an important mechanism of cell death leading to organ dysfunction following I/R. Intracellular danger-associated molecular patterns (DAMPs) released during cell death can activate cytoprotective responses by engaging receptors of the innate immune system.
Ischemia was induced in the mouse hind limb by tourniquet or in the heart by coronary artery ligation. Reperfusion injury of skeletal or cardiac muscle was markedly reduced by intraperitoneal or subcutaneous injection of recombinant human (rh)BCL2 protein or rhBCL2-related protein A1 (BCL2A1) (50 ng/g) given prior to ischemia or at the time of reperfusion. The cytoprotective activity of extracellular rhBCL2 or rhBCL2A1 protein was mapped to the BH4 domain, as treatment with a mutant BCL2 protein lacking the BH4 domain was not protective, whereas peptides derived from the BH4 domain of BCL2 or the BH4-like domain of BCL2A1 were. Protection by extracellular rhBCL2 or rhBCL2A1 was associated with a reduction in apoptosis in skeletal and cardiac muscle following I/R, concomitant with increased expression of endogenous mouse BCL2 (mBCL2) protein. Notably, treatment with rhBCL2A1 protein did not protect mice deficient in toll-like receptor-2 (TLR2) or the adaptor protein, myeloid differentiation factor-88 (MyD88).
Treatment with cytokine-like doses of rhBCL2 or rhBCL2A1 protein or BH4-domain peptides reduces apoptosis and tissue injury following I/R by a TLR2-MyD88-dependent mechanism. These findings establish a novel extracellular cytoprotective activity of BCL2 BH4-domain proteins as potent cytoprotective DAMPs.
Salvia leriifolia have been shown to decrease ischemia-reperfusion (I/R) injury in brain tissues. In this study, the effects of S. leriifolia aqueous and ethanolic extracts were evaluated on an animal model of I/R injury in the rat hind limb.
Ischemia was induced using free-flap surgery in skeletal muscle. The aqueous and ethanolic extracts of S. leriifolia (100, 200 and 400 mg/kg) root and normal saline (10 ml/kg) were administered intraperitoneally 1 h prior reperfusion. During preischemia, ischemia and reperfusion conditions the electromyographic (EMG) potentials in the muscles were recorded. The markers of oxidative stress including thiobarbituric acid reactive substances (TBARS), total sulfhydryl (SH) groups and antioxidant capacity of muscle (using FRAP assay) were measured.
In peripheral ischemia, the average peak-to-peak amplitude during ischemic-reperfusion was found to be significantly larger in extracts groups in comparison with control group. Following extracts administration, the total SH contents and antioxidant capacity were elevated in muscle flap. The MDA level was also declined significantly in test groups.
It is concluded that S. leriifolia root extracts have some protective effects on different markers of oxidative damage in muscle tissue injury caused by lower limb ischemia-reperfusion.
Ischemia reperfusion injury is partly responsible for the high mortality associated with induced myocardial injury and the reduction in the full benefit of myocardial reperfusion. Remote ischemic preconditioning, perconditioning, and postconditioning have all been shown to be cardioprotective. However, it is still unknown which one is the most beneficial. To examine this issue, we used adult male Wistar rat ischemia reperfusion models to compare the cardioprotective effect of these three approaches applied on double-sided hind limbs.
The rats were randomly distributed to the following five groups: sham, ischemia reperfusion, remote preconditioning, remote perconditioning, and remote post-conditioning. The ischemia/reperfusion model was established by sternotomy followed by a 30-min ligation of the left coronary artery and a subsequent 3-h reperfusion. Remote conditioning was induced with three 5-min ischemia/5-min reperfusion cycles of the double-sided hind limbs using a tourniquet.
A lower early reperfusion arrhythmia score (1.50±0.97) was found in the rats treated with remote perconditioning compared to those in the ischemia reperfusion group (2.33±0.71). Meanwhile, reduced infarct size was also observed (15.27±5.19% in remote perconditioning, 14.53±3.45% in remote preconditioning, and 19.84±5.85% in remote post-conditioning vs. 34.47±7.13% in ischemia reperfusion, p<0.05), as well as higher expression levels of the apoptosis-relevant protein Bcl-2/Bax following global (ischemia/reperfusion) injury in in vivo rat heart models (1.255±0.053 in remote perconditioning, 1.463±0.290 in remote preconditioning, and 1.461±0.541 in remote post-conditioning vs. 1.003±0.159 in ischemia reperfusion, p<0.05).
Three remote conditioning strategies implemented with episodes of double-sided hind limb ischemia/reperfusion have similar therapeutic potential for cardiac ischemia/reperfusion injury, and remote perconditioning has a greater ability to prevent reperfusion arrhythmia.
Cardioprotective Property; Ischemia/Reperfusion Injury; Models
Cellular studies have demonstrated a protective role of mitochondrial hexokinase against oxidative insults. It is unknown whether HK protective effects translate to the in vivo condition. In the present study, we hypothesize that HK affects acute ischemia–reperfusion injury in skeletal muscle of the intact animal. Male and female heterozygote knockout HKII (HK+/-), heterozygote overexpressed HKII (HKtg), and their wild-type (WT) C57Bl/6 littermates mice were examined. In anesthetized animals, the left gastrocnemius medialis (GM) muscle was connected to a force transducer and continuously stimulated (1-Hz twitches) during 60 min ischemia and 90 min reperfusion. Cell survival (%LDH) was defined by the amount of cytosolic lactate dehydrogenase (LDH) activity still present in the reperfused GM relative to the contralateral (non-ischemic) GM. Mitochondrial HK activity was 72.6 ± 7.5, 15.7 ± 1.7, and 8.8 ± 0.9 mU/mg protein in male mice, and 72.7 ± 3.7, 11.2 ± 1.4, and 5.9 ± 1.1 mU/mg in female mice for HKtg, WT, and HK+/-, respectively. Tetanic force recovery amounted to 33 ± 7% for male and 17 ± 4% for female mice and was similar for HKtg, WT, and HK+/-. However, cell survival was decreased (p = 0.014) in male HK+/- (82 ± 4%LDH) as compared with WT (98 ± 5%LDH) and HKtg (97 ± 4%LDH). No effects of HKII on cell survival was observed in female mice (92 ± 2% LDH). In conclusion, in this mild model of acute in vivo ischemia–reperfusion injury, a partial knockout of HKII was associated with increased cell death in male mice. The data suggest for the first time that HKII mediates skeletal muscle ischemia–reperfusion injury in the intact male animal.
Mitochondria; Cell death; Ischemia; Muscle; Muscle ischemia
The purpose of this study was to determine if inhaled carbon monoxide (CO) can ameliorate skeletal muscle injury, modulate endogenous heme oxygenase-1 (HO) expression, improve indices of tissue integrity and inflammation following hind limb ischemia reperfusion(IR).
C57BL6 mice inhaling CO (250ppm) or room air were subjected to 1.5 hrs of ischemia followed by limb reperfusion for either 3 or 6 hours (total treatment time of 4.5 or 7.5 hrs). After the initial period of reperfusion, all mice breathed only room air until 24 hours after the onset of ischemia. Mice were sacrificed at either the end of CO treatment or at 24 hours reperfusion. Skeletal muscle was subjected to histologic and biochemical analysis.
CO treatment for 7.5 hours protected skeletal muscle from histologic and structural evidence of skeletal muscle injury. Serum and tissue cytokines were significantly reduced (p<0.05) in mice treated with CO for 7.5 hours. Tubulin, Heme Oxygenase, and ATP levels were higher in CO treated mice.
Inhaled CO protected muscle from structural injury and energy depletion following IR.
Carbon Monoxide; Reperfusion Injury; Heme Oxygenase; Skeletal Muscle
AIM: To investigate the protective effect of penehyclidine hydrochloride post-conditioning in the damage to the barrier function of the small intestinal mucosa caused by limb ischemia-reperfusion (LIR) injury.
METHODS: Male Wistar rats were randomly divided into three groups (36 rats each): the sham-operation group (group S), lower limb ischemia-reperfusion group (group LIR), and penehyclidine hydrochloride post-conditioning group (group PHC). Each group was divided into subgroups (n = 6 in each group) according to ischemic-reperfusion time, i.e. immediately 0 h (T1), 1 h (T2), 3 h (T3), 6 h (T4), 12 h (T5), and 24 h (T6). Bilateral hind-limb ischemia was induced by rubber band application proximal to the level of the greater trochanter for 3 h. In group PHC, 0.15 mg/kg of penehyclidine hydrochloride was injected into the tail vein immediately after 3 h of bilateral hind-limb ischemia. The designated rats were sacrificed at different time-points of reperfusion; diamine oxidase (DAO), superoxide dismutase (SOD) activity, myeloperoxidase (MPO) of small intestinal tissue, plasma endotoxin, DAO, tumor necrosis factor-α (TNF-α), and interleukin (IL)-10 in serum were detected in the rats.
RESULTS: The pathological changes in the small intestine were observed under light microscope. The levels of MPO, endotoxin, serum DAO, and IL-10 at T1-T6, and TNF-α level at T1-T4 increased in groups LIR and PHC (P < 0.05) compared with those in group S, but tissue DAO and SOD activity at T1-T6 decreased (P < 0.05). In group PHC, the tissue DAO and SOD activity at T2-T6, and IL-10 at T2-T5 increased to higher levels than those in group LIR (P < 0.05); however, the levels of MPO, endotoxin, and DAO in the blood at T2-T6, and TNF-α at T2 and T4 decreased (P < 0.05).
CONCLUSION: Penehyclidine hydrochloride post-conditioning may reduce the permeability of the small intestines after LIR. Its protection mechanisms may be related to inhibiting oxygen free radicals and inflammatory cytokines for organ damage.
Penehyelidine hydrochloride; Post-conditioning; Limb ischemia-reperfusion injury; Small intestine; Protection
Saffron and its constituents have been shown to decrease ischemia-reperfusion (I/R) injury in kidney or brain tissues. In this study, the effects of saffron ethanolic extract and its constituents, crocin and safranal, were evaluated in skeletal muscle during I/R injury. Hind limb ischemia was induced using clamping the common femoral artery and vein. After 2 h ischemia, the clamp of the femoral vessels of animals was taken off and the animal underwent 1h reperfusion. Muscle injuries were evaluated by recording of the electromyographic (EMG) potentials and performing some biochemical analysis including thiobarbituric acid reactive substances (TBARS), total sulfhydryl (SH) groups and antioxidant capacity of muscle (using FRAP assay). The ethanolic extract of saffron (5, 20 and 80 mg kg−1), crocin (50, 200 and 400 mg kg−1), safranal (0.1, 0.25 and 0.5 ml kg−1) and normal saline (10 ml kg−1) were administered intraperitoneally 1 h prior reperfusion. The average peak-to-peak amplitude during I/R was significantly increased in extract, crocin and safranal groups in comparison with control-ischemic group. Following saffron, crocin and safranal administration, the total SH contents and antioxidant capacity were elevated in muscle flap. The MDA level was declined significantly in test groups. It is concluded that saffron extract and its constituents show a protective effect against lower limb I/R in rat.
Saffron (Crocus sativus L.); Crocin; Safranal; Oxidative stress; Lower limb ischemia; Reperfusion
Since flavonoids scavenge reactive oxygen species, they may potentially protect against ischemia/reperfusion injury. This study compared the scavenging capacity of specific flavonoids towards different reactive oxygen species. Whether the differential oxidant scavenging capacity correlated with their protective efficacy in ischemia/reperfusion injury of cardiomyocytes was determined. The free radical scavenging capacity of five flavonoids (wogonin, baicalin, baicalein, catechin and procyanidin B2) was analyzed using electron spin resonance spectrometry for 3 radicals: 1,1-diphenyl-2picrylhydrazyl (DPPH), superoxide and hydroxyl radical. A well-established chick cardiomyocyte model of ischemia (1 h)/reperfusion (3 h) was used to evaluate flavonoid-induced protection against ischemia/reperfusion injury in chronic treatment (pretreated 72 h and treated through ischemia/reperfusion) and acute treatment protocols (during ischemia/reperfusion or only at reperfusion). The cell viability was assessed by propidium iodide. The DPPH scavenging was most significant with catechin, followed by procyanidin B2, baicalein, baicalin, and wogonin. The superoxide scavenging was, similarly, most significant with catechin, followed by baicalein, procyanidin B2, and baicalin. For hydroxyl radical, only baicalein showed a significant scavenging capacity (> 50% reduction in ESR signal). For the cardiomyocyte studies, all flavonoids but wogonin showed protection against ischemia/reperfusion injury in the chronic treatment protocol. When flavonoids were administered only during ischemia/reperfusion, baicalein, procyanidin B2, and catechin significantly reduced cell death. If flavonoids were administered just at reperfusion, only baicalein and procyanidin B2 had protective effects, and the efficacy was less. Flavonoids possess specific but differential radical scavenging capacity, which, in conjunction with the timing of treatment, affects their protective efficacy in cardiomyocytes exposed to ischemia/reperfusion.
Flavonoids; Reperfusion injury; Scavenging capacity; Superoxide; Hydroxyl radical
Experiments were designed to investigate the effects of ethyl pyruvate (EP) in a murine model of hind-limb ischemia-reperfusion (IR) injury.
C57BL6 mice underwent 90 minutes of unilateral ischemia followed by 24 hours of reperfusion using two treatment protocols. For the preischemic treatment (pre-I) protocol, mice (n = 6) were given 300 mg/kg EP before ischemia, followed by 150 mg/kg of EP just before reperfusion and at 6 hours and 12 hours after reperfusion. In a postischemic treatment (post-I) protocol, mice (n = 7) were treated with 300 mg/kg EP at the end of the ischemic period, then 15 minutes later, and 2 hours after reperfusion and 150 mg/kg of EP at 4 hours, 6 hours, 10 hours, 16 hours, and 22 hours after reperfusion. Controls mice for both protocols were treated with lactated Ringers alone at time intervals identical to EP. Skeletal muscle levels of adenosine triphosphate (ATP), interleukin-1β, keratinocyte chemoattractant protein, and thrombin antithrombin-3 complex were measured. Skeletal muscle architectural integrity was assessed microscopically.
ATP levels were higher in mice treated with EP compared with controls under the both treatment protocols (p = 0.02). Interleukin-1β, keratinocyte chemoattractant protein, thrombin antithrombin-3 complex (p < 0.05), and the percentage of injured fibers (p < 0.0001) were significantly decreased in treated versus control mice under the both protocols.
Muscle fiber injury and markers of tissue thrombosis and inflammation were reduced, and ATP was preserved with EP in pre-I and post-I protocols. Further investigation of the efficacy of EP to modulate IR injury in a larger animal model of IR injury is warranted.
Ischemia-reperfusion; Inflammation; Skeletal muscle; cytokines
Activation of the endothelium, complement activation and generation of cytokines are known events during ischemia-reperfusion (I/R) that mediate tissue injury. Our aim was to elucidate their respective participation at the onset of the reperfusion phase. Tourniquet application in hand surgery causes short-term ischemia, followed by reperfusion and was therefore used as the model in this study.
Ten patients were included in the study after obtaining informed consent. A tourniquet was placed on the upper arm and inflated to 250 mmHg for 116 ± 16 min, during which the surgery was performed. Venous blood and tissue samples from the surgical area were taken at baseline as well as 0, 2, and 10 min after reperfusion and analyzed for the following parameters: Endothelial integrity and/or activation were analyzed by measuring heparan sulfate and syndecan-1 in serum, and vWF, heparan sulfate proteoglycan as well as CD31on tissue. Complement activation was determined by C3a and C4d levels in plasma, levels of C1-inhibitor in serum, and IgG, IgM, C3b/c, and C4b/c deposition on tissue. Cytokines and growth factors IL-5, IL-6, IL-7, IL-8, IL-10, IL-17, G-CSF, GM-CSF, MCP-1, TNFα, VEGF, and PDGF bb were measured in the serum. Finally, CK-MM levels were determined in plasma as a measure for muscle necrosis.
Markers for endothelial activation and/or integrity as well as complement activation showed no significant changes until 10 min reperfusion. Among the measured cytokines, IL-6, IL-7, IL-17, TNFα, GM-CSF, VEGF, and PDGF bb were significantly increased at 10 min reperfusion with respect to baseline. CK-MM showed a rise from baseline at the onset of reperfusion (p < 0.001) and dropped again at 2 min (p < 0.01) reperfusion, suggesting ischemic muscle damage.
In this clinical model of I/R injury no damage to the endothelium, antibody deposition or complement activation were observed during early reperfusion. However, an increase of pro-inflammatory cytokines and growth factors was shown, suggesting a contribution of these molecules in the early stages of I/R injury.
Tourniquet; Hand surgery; Ischemia; Reperfusion injury; Cytokines; Complement; Endothelium; Glycocalyx
In mouse models of familial amyotrophic lateral sclerosis (fALS) motor neurons are especially vulnerable to oxidative stresses in vitro. To determine whether this increased vulnerability also extends to motor nerve terminals in vivo, we assayed the effect of tourniquet-induced ischemia/reperfusion (I/R) injury on motor terminals innervating fast and slow hindlimb muscles in male G93A-SOD1 mice and their wild-type littermates. These mice also expressed yellow fluorescent protein (YFP) in motor neurons. We report that in SOD1-G93A/YFP mice the motor terminals innervating two predominantly fast muscles, extensor digitorum longus (EDL) and plantaris, were more vulnerable to I/R injury than motor terminals innervating the predominantly slow soleus muscle. The mean duration of EDL ischemia required to produce a 50% reduction in endplate innervation in SOD1-G93A/YFP mice was 26 min, compared to 45 min in YFP-only mice. The post-I/R destruction of EDL terminals in SOD1-G93A mice was rapid (< 2 hr) and was not duplicated by cutting the sciatic nerve at the tourniquet site. The increased sensitivity to I/R injury was evident in EDL muscles of SOD1-G93A/YFP mice as young as 31 days, well before the onset of motor neuron death at ~90 days. This early vulnerability to I/R injury may correlate with the finding (confirmed here) that in fALS mice motor nerve terminals innervating fast hindlimb muscles degenerate before those innervating slow muscles, at ages that precede motor neuron death. Early vulnerability of fast motor terminals to I/R injury thus may signal, and possibly contribute to, early events involved in motor neuron death.
AIM: To investigate the relation of reactive oxygen species (ROS) to hypoxia induced factor 1α (HIF-1α) in gastric ischemia.
METHODS:The animal model of gastric ischemia reperfusion was established by placing an elastic rubber band on the proximal part of the bilateral lower limb for ligature for 3 h and reperfusion for 0, 1, 3, 6, 12 or 24 h. Ischemic post-conditioning, three cycles of 30-s reperfusion and 30-s femoral aortic reocclusion were conducted before reperfusion. Histological and immunohistochemical methods were used to assess the gastric oxidative damageand the expression of HIF1-α in gastric ischemia. The malondialdehyde (MDA) content and superoxide dismutase (SOD), xanthine oxidase (XOD) and myeloperoxidase (MPO) activities were determined by colorimetric assays.
RESULTS: Ischemic post-conditioning can reduce post-ischemic oxidativestressand the expression of HIF-1α of gastric tissue resulting from limb ischemia reperfusion injury. MDA, SOD, XOD and MPO were regarded as indexes for mucosal injuries from ROS, and ROS was found to affect the expression of HIF-1α under gastric ischemic conditions.
CONCLUSION: ROS affects HIF-1α expression under gastric ischemic conditions induced by limb ischemia reperfusion injury. Therefore, ROS can regulate HIF-1α expression in gastric ischemia.
Oxidative stress; Reactive oxygen species; HIF-1α expression; Gastric ischemia; Limb ischemia reperfusion
These experiments were designed to determine whether systemic post ischemic administration of PJ34, a Poly ADP-ribose polymerase inhibitor, decreased tissue injury and inflammation following hind limb ischemia reperfusion (I/R).
C57BL6 mouse limbs were subjected to 1.5 hrs ischemia followed by 24 hours reperfusion. The treatment group (PJ) received intraperitoneal PJ34 (30 mg/Kg) immediately before, 15 minutes and 2 hours into reperfusion. Control group (CG) received Lactated Ringers alone at the same time intervals as PJ34 administration. Skeletal muscle levels of ATP, Macrophage Inflammatory Protein-2 (MIP-2), Keratinocyte Derived Chemokine (KC) and Myeloperoxidase (MPO) were measured. Quantitative measurement of skeletal muscle tissue injury was assessed by microscopic analysis of fiber injury.
ATP levels were higher in limbs of PJ vs. CG (Absolute ATP: 4.7 ± 0.35 vs. 2.3 ± 0.15 ng/mg tissue, p=0.002). Levels of MIP-2, KC and MPO were lower in PJ vs. CG (MIP-2: 1.4±0.34 vs. 3.67±0.67 pg/mg protein, p=0.014; KC: 4.97±0.97 vs. 12.65±3.05 pg/mg protein, p=0.037, MPO: 46.27±10.53 vs. 107.34±13.58 ng/mg protein, p=0.008). Muscle fiber injury was markedly reduced in PJ vs. CG (4.25±1.9% vs 22.68±3.0% total fibers, p=0.0004).
Systemic post ischemic administration of PJ34 preserved skeletal muscle energy levels, decreased inflammatory markers and preserved tissue viability post I/R. These results support PARP inhibition as a viable treatment for skeletal muscle I/R in a clinically relevant “post-hoc” scenario.
Basic science; skeletal muscle; cytokines; inflammation; vascular disease
Tourniquets are compressive devices that occlude venous and arterial blood flow to limbs and are commonly used in upper limb surgery. With the potential risk of complications, there is some debate as to whether tourniquets should continue to be routinely used. In this review, we first look at the different designs, principles, and practical considerations associated with the use of tourniquets in the upper limb. The modern pneumatic tourniquet has many design features that enhance its safety profile. Current literature suggests that the risk of tourniquet-related complications can be significantly reduced by selecting cuff inflation pressures based on the limb occlusion pressure, and by a better understanding of the actual level of pressure within the soft tissue, and the effects of cuff width and contour. The evidence behind tourniquet time, placement, and limb exsanguination is also discussed as well as special considerations in patients with diabetes mellitus, hypertension, vascular calcification, sickle cell disease and obesity. We also provide an evidence-based review of the variety of local and systemic complications that may arise from the use of upper limb tourniquets including pain, leakage, and nerve, muscle, and skin injuries. The evidence in the literature suggests that upper limb tourniquets are beneficial in promoting optimum surgical conditions and modern tourniquet use is associated with a low rate of adverse events. With the improvement in knowledge and technology, the incidence of adverse events should continue to decrease. We recommend the use of tourniquets in upper limb surgery where no contraindications exist.
Tourniquet; Upper limb; Limb occlusion pressure; Complications
With the advancement of age, skeletal muscle undergoes a progressive decline in mass, function, and regenerative capacity. Previously, our laboratory has reported an age-reduction in recovery and local induction of IGF-I gene expression with age following tourniquet (TK)-induced skeletal muscle ischemia/reperfusion (I/R). In this study, young (6 mo) and old (24–28 mo) mice were subjected to 2 hours of TK-induced ischemia of the hindlimb followed by 1, 3, 5, or 7 days of reperfusion. Real time-PCR analysis revealed clear age-related reductions and temporal alterations in the expression of IGF-I and individual IGF-I Ea and Eb splice variants. ELISA verified a reduction of IGF-I peptide with age following 7 days recovery from TK. Western blotting showed that the phosphorylation of Akt, mTOR, and FoxO3, all indicators of anabolic activity, were reduced in the muscles of old mice. These data indicate an age-related impairment of IGF-I expression and intracellular signaling does exist following injury, and potentially has a role in the impaired recovery of skeletal muscle with age.
Tourniquet; sarcopenia; muscle regeneration; mTOR; FoxO
Tourniquets are an effective means of arresting life‐threatening external haemorrhage from limb injury. Their use has not previously been accepted practice for pre‐hospital civilian trauma care because of significant concerns regarding the potential complications. However, in a few rare situations tourniquet application will be necessary and life‐saving. This review explores the potential problems and mistrust of tourniquet use; explains the reasons why civilian pre‐hospital tourniquet use may be necessary; defines the clear indications for tourniquet use in external haemorrhage control; and provides practical information on tourniquet application and removal. Practitioners need to familiarise themselves with commercial pre‐hospital tourniquets and be prepared to use one without irrational fear of complications in the appropriate cases.
The clinical significance of ischemia/reperfusion of the lower extremities demands further investigation to enable the development of more effective therapeutic alternatives. This study investigated the changes in the vascular reactivity of the rabbit femoral artery and nitric oxide metabolites under partial ischemia/reperfusion conditions following cilostazol administration.
Ischemia was induced using infrarenal aortic clamping. The animals were randomly divided into seven groups: Control 90 minutes, Ischemia/Reperfusion 90/60 minutes, Control 120 minutes, Ischemia/Reperfusion 120/90 minutes, Cilostazol, Cilostazol before Ischemia/Reperfusion 120/90 minutes, and Ischemia 120 minutes/Cilostazol/Reperfusion 90 minutes. Dose-response curves for sodium nitroprusside, acetylcholine, and the calcium ionophore A23187 were obtained in isolated femoral arteries. The levels of nitrites and nitrates in the plasma and skeletal muscle were determined using chemiluminescence.
Acetylcholine- and A23187-induced relaxation was reduced in the Ischemia/Reperfusion 120/90 group, and treatment with cilostazol partially prevented this ischemia/reperfusion-induced endothelium impairment. Only cilostazol treatment increased plasma levels of nitrites and nitrates. An elevation in the levels of nitrites and nitrates was observed in muscle tissues in the Ischemia/Reperfusion 120/90, Cilostazol/Ischemia/Reperfusion, and Ischemia/Cilostazol/Reperfusion groups.
Hind limb ischemia/reperfusion yielded an impaired endothelium-dependent relaxation of the femoral artery. Furthermore, cilostazol administration prior to ischemia exerted a protective effect on endothelium-dependent vascular reactivity under ischemia/reperfusion conditions.
Cilostazol; Nitric oxide; Ischemia/reperfusion; Femoral artery; Skeletal muscle
Suitable and reproducible experimental models of translational research in reconstructive surgery that allow in-vivo investigation of diverse molecular and cellular mechanisms are still limited. To this end we created a novel murine model of acute hindlimb ischemia-reperfusion to mimic a microsurgical free flap procedure. Thirty-six C57BL6 mice (n = 6/group) were assigned to one control and five experimental groups (subject to 6, 12, 96, 120 hours and 14 days of reperfusion, respectively) following 4 hours of complete hindlimb ischemia. Ischemia and reperfusion were monitored using Laser-Doppler Flowmetry. Hindlimb tissue components (skin and muscle) were investigated using histopathology, quantitative immunohistochemistry and immunofluorescence. Despite massive initial tissue damage induced by ischemia-reperfusion injury, the structure of the skin component was restored after 96 hours. During the same time, muscle cells were replaced by young myotubes. In addition, initial neuromuscular dysfunction, edema and swelling resolved by day 4. After two weeks, no functional or neuromuscular deficits were detectable. Furthermore, upregulation of VEGF and tissue infiltration with CD34-positive stem cells led to new capillary formation, which peaked with significantly higher values after two weeks. These data indicate that our model is suitable to investigate cellular and molecular tissue alterations from ischemia-reperfusion such as occur during free flap procedures.
Ischaemia reperfusion (IR) injury of skeletal muscle, is a significant cause of morbidity following trauma and surgical procedures, in which muscle fibre types exhibit different susceptibilities. The relative degree of mast cell mediated injury, within different muscle types, is not known.
In this study we compared susceptibility of the fast-twitch, extensor digitorum longus (EDL), mixed fast/slow-twitch gastrocnemius and the predominately slow-twitch soleus, muscles to ischemia reperfusion (IR) injury in four groups of mice that harbour different mast cell densities; C57/DBA mast cell depleted (Wf/Wf), their heterozygous (Wf/+) and normal littermates (+/+) and control C57BL/6 mice. We determined whether susceptibility to IR injury is associated with mast cell content and/or fibre type and/or mouse strain. In experimental groups, the hind limbs of mice were subjected to 70 minutes warm tourniquet ischemia, followed by 24 h reperfusion, and the muscle viability was assessed on fresh whole-mount slices by the nitroblue tetrazolium (NBT) histochemical assay.
Viability was remarkably higher in the Wf/Wf strain irrespective of muscle type. With respect to muscle type, the predominately slow-twitch soleus muscle was significantly more resistant to IR injury than gastrocnemius and the EDL muscles in all groups. Mast cell density was inversely correlated to muscle viability in all types of muscle.
These results show that in skeletal muscle, IR injury is dependent upon both the presence of mast cells and on fibre type and suggest that a combination of preventative therapies may need to be implemented to optimally protect muscles from IR injury.
Ischemia and reperfusion (I/R) of tissue provokes an inflammatory process that is highly dependent on circulating natural immunoglobulin M (IgM) and the complement cascade. In mice, a single IgM specificity produced by peritoneal B cells can initiate reperfusion injury. It is unknown whether humans express natural IgM with a similar specificity. It is also unknown whether pathogenic IgM is produced solely from peritoneal B cells or can also be made by circulating B cells.
Immunodeficient mice lacking endogenous immunoglobulin were used. Mice were reconstituted with normal saline, human serum, or xenografted human peripheral blood lymphocytes (PBLs) and then subjected to tourniquet induced hindlimb ischemia and reperfusion. Serum human IgM and IgG were measured by ELISA. Skeletal muscle was harvested for injury assessment by histology and for immunohistochemistry.
Immunodeficient mice are protected from skeletal muscle injury following hindlimb I/R. Transfer of human serum restores skeletal muscle damage. Rag2/γR -/- mice engrafted with human PBL (huPBL-SCID) have high levels of human IgM. huPBL-SCID mice develop significantly more skeletal muscle injury than control saline treated (p ≤ 0.01) and human serum reconstituted Rag2/γR -/- mice (p ≤ 0.01). Sham treated huPBL-SCID mice have no muscle injury, demonstrating that human lymphocyte engraftment does not cause injury in the absence of ischemia. Deposition of human IgM is seen on injured but not sham injured muscle.
Human serum can initiate murine skeletal muscle ischemia reperfusion injury. Circulating human PBL may be a source of pathogenic IgM. The huPBL-SCID mouse may be a useful model to define the specificity of pathogenic human IgM and to test therapeutics for ischemia-reperfusion injury.
Background and purpose CHOP is a C/EBP family transcription factor involved in endoplasmic reticulum (ER) stress-mediated apoptosis. Several studies have demonstrated that ischemia reperfusion results in apoptosis. Oxidative stress is central to ischemia reperfusion-induced apoptosis. Taurine protects against lung injury after limb ischemia reperfusion (LIR) through antioxidation. The effects of taurine on ER stress-induced apoptosis have not been well explored, however. We studied the effects of taurine in ER stress-induced apoptosis following LIR.
Methods Adult male Sprague-Dawley rats (n = 40) were randomized into 4 groups: (1) a control group, (2) an LIR group, (3) an LIR group treated with taurine, and (4) an LIR group treated with saline. Bilateral hindlimb ischemia was induced by application of a rubber band proximal to the level of the greater trochanters for 4 h. The treatment groups received either taurine (200 mg/kg as a 4% solution in 0.9% saline) or saline alone prior to reperfusion. Following 4h of reperfusion, blood oxygen was analyzed. The animals were killed and plasma and lung tissue were harvested for evaluation.
Results Taurine statistically significantly attenuated lung injury following LIR, as shown by reduced malondialdehyde content, reduced cell apoptosis, and expression of activating transcription factor 4 (ATF4), X-box binding protein 1 (XBP1), and transcriptional activators of the CHOP gene. Furthermore, partial pressure values of oxygen in arterial blood and the activities of superoxide dismutase and catalase were higher in the taurine pretreatment group than in the group of rats that underwent LIR alone.
Interpretation Our results suggest that taurine attenuates endoplasmic reticulum stress-induced apoptosis in the lungs of rats after limb ischemia reperfusion.
Mitochondrial reactive oxygen species generation has been implicated in the pathophysiology of ischemia-reperfusion (I/R) injury, however its exact role and its spatial-temporal relationship with inflammation are elusive. Herein we explored the spatial-temporal relationship of oxidative/nitrative stress and inflammatory response during the course of hepatic I/R and the possible therapeutic potential of mitochondrial-targeted antioxidants, using a mouse model of segmental hepatic ischemia-reperfusion injury. Hepatic I/R was characterized by early (at 2 hours of reperfusion) mitochondrial injury, decreased complex I activity, increased oxidant generation in the liver or liver mitochondria, and profound hepatocellular injury/dysfunction with acute pro-inflammatory response (TNF-α, MIP-1αCCL3, MIP-2/CXCL2) without inflammatory cell infiltration, followed by marked neutrophil infiltration and more pronounced secondary wave of oxidative/nitrative stress in the liver (starting from 6 hours of reperfusion and peaking at 24 hours). Mitochondrially-targeted antioxidants, MitoQ or Mito-CP, dose-dependently attenuated I/R-induced liver dysfunction, the early and delayed oxidative and nitrative stress response (HNE/carbonyl adducts, malondialdehyde, 8-OHdG, and 3-nitrotyrosine formation), mitochondrial and histopathological injury/dysfunction, as well as delayed inflammatory cell infiltration and cell death.
Mitochondrially generated oxidants play a central role in triggering the deleterious cascade of events associated with hepatic I/R, which may be targeted by novel antioxidants for therapeutic advantage.
ischemia-reperfusion; antioxidants; oxidative stress; reactive oxygen and nitrogen species
Ischaemic preconditioning (IPC) has emerged as a method of reducing ischaemia-reperfusion injury. However, the complex mechanism through which IPC elicits this protection is not fully understood. The aim of this study was to investigate the genomic response induced by IPC in muscle biopsies taken from the operative leg of total knee arthroplasty patients in order to gain insight into the IPC mechanism.
Twenty patients, undergoing primary total knee arthroplasty, were randomly assigned to IPC (n = 10) and control (n = 10) groups. Patients in the IPC group received ischaemic preconditioning immediately prior to surgery. IPC was induced by three five-minute cycles of tourniquet insufflation interrupted by five-minute cycles of reperfusion. A muscle biopsy was taken from the operative knee of control and IPC-treated patients at the onset of surgery and, again, at one hour into surgery. The gene expression profile of muscle biopsies was determined using the Affymetrix Human U113 2.0 microarray system and validated using real-time polymerase chain reaction (RT-PCR). Measurements of C-reactive protein (CRP), erythrocyte sedimentation (ESR), white cell count (WCC), cytokines and haemoglobin were also made pre- and post-operatively.
Microarray analysis revealed a significant increase in the expression of important oxidative stress defence genes, immediate early response genes and mitochondrial genes. Upregulation of pro-survival genes was also observed and correlated with a downregulation of pro-apoptotic gene expression. CRP, ESR, WCC, cytokine and haemoglobin levels were not significantly different between control and IPC patients.
The findings of this study suggest that IPC of the lower limb in total knee arthroplasty patients induces a protective genomic response, which results in increased expression of immediate early response genes, oxidative stress defence genes and pro-survival genes. These findings indicate that ischaemic preconditioning may be of potential benefit in knee arthroplasty and other musculoskeletal conditions.
In preclinical studies, erythropoietin (EPO) reduces ischemia-reperfusion–associated tissue injury (for example, stroke, myocardial infarction, acute kidney injury, hemorrhagic shock and liver ischemia). It has been proposed that the erythropoietic effects of EPO are mediated by the classic EPO receptor homodimer, whereas the tissue-protective effects are mediated by a hetero-complex between the EPO receptor monomer and the β-common receptor (termed “tissue-protective receptor”). Here, we investigate the effects of a novel, selective-ligand of the tissue-protective receptor (pyroglutamate helix B surface peptide [pHBSP]) in a rodent model of acute kidney injury/dysfunction. Administration of pHBSP (10 μg/kg intraperitoneally [i.p.] 6 h into reperfusion) or EPO (1,000 IU/kg i.p. 4 h into reperfusion) to rats subjected to 30 min ischemia and 48 h reperfusion resulted in significant attenuation of renal and tubular dysfunction. Both pHBSP and EPO enhanced the phosphorylation of Akt (activation) and glycogen synthase kinase 3β (inhibition) in the rat kidney after ischemia-reperfusion, resulting in prevention of the activation of nuclear factor-κB (reduction in nuclear translocation of p65). Interestingly, the phosphorylation of endothelial nitric oxide synthase was enhanced by EPO and, to a much lesser extent, by pHBSP, suggesting that the signaling pathways activated by EPO and pHBSP may not be identical.