Hindlimb Ischemia-reperfusion Research Articles

Effects of fish oil-based lipid emulsion on inflammation and kidney injury in mice subjected to unilateral hind limb ischemia/reperfusion

This study investigated the effects of a fish oil-based lipid emulsion (FO) on local skeletal muscle and remote renal damage at 72 h post-reperfusion in a murine model of hind limb ischemia-reperfusion (IR) injury. Mice were assigned to 1 sham group and 3 IR groups. The IR groups were treated daily with either saline or FO from 3 days prior to limb ischemia till 3 days after reperfusion. Limb IR was induced by applying a 4.5-oz orthodontic rubber band above the left greater trochanter for 120 min followed by band-released reperfusion for 72 h. Mice were then sacrificed to harvest blood, muscle, and kidney for analysis. The results showed that IR injury led to upregulation of pro-inflammatory monocytes in blood, infiltration of leukocytes into injured muscle, and over-expression of pro-inflammatory genes in muscle and kidney tissues. Supplementing FO either before or after IR injury alleviated IR-induced inflammatory gene expressions in muscle and kidney tissues. Furthermore, FO given after IR injury reduced circulating pro-inflammatory monocytes, limited muscle leukocytic infiltration, and improved renal histology. These results suggest that FO may protect the muscles from IR injury. FO given after IR injury can better downregulate the inflammation seen in IR-induced remote kidney injury.

Ginsenoside Rg1 protects against hind-limb ischemia reperfusion induced lung injury via NF-κB/COX-2 signaling pathway

Background/aimsGinsenoside Rg1 is regarded as the primary bioactive ingredient in Panax notoginseng that has been well recognized for its protective effects against ischemia/reperfusion (IR) injury. However, the mechanisms still remain elusive. Our study aims to investigate the effects of Rg1 against lung injury induced by hind-limb IR in rats. MethodsTwenty-four Sprague Dawley rats were randomly submitted to sham operation (SM group), hind-limb IR (IR group), hind-limb IR + Rg1 (Rg1 group), and hind-limb IR + Pro-DTC group (PD group). All the rats except those in SM group were subjected to 3 h of ischemia followed by 6 h of reperfusion, and extra intravenous Rg1 and pyrrolidine dithiocarbamate (Pro-DTC), a selective inhibitor of nuclear factor kappa B (NF-κB), was administered intravenously before ischemia in the Rg1 and PD group, respectively. The activities of myeloperoxidase (MPO), superoxide dismutase (SOD) and catalase (CAT), as well as protein expressions of NF-κB p65 and cyclooxygenases-2 (COX-2) in lung tissue, and thromboxane B2 (TXB2) and 6-keto-ProstaglandinF1α (6-keto-PGF1α) levels in bronchoalveolar lavage (BAL) fluid were detected. Morphological changes, index of quantitative assessment of histologic lung injury (IQA), apoptosis index (AI) and lung Wet/Dry ratio were also evaluated. ResultsThe levels of Wet/Dry ratio, IQA, AI, activities of MPO and 6-keto-PGF1α/TXB2 ratio were increased, and NF-κB p65 and COX-2 protein expression were upregulated, while SOD and CAT levels were decreased in lung tissue in IR group as compared with SM group (p < 0.05), all the alterations could be significantly reversed by Rg1 or Pro-DTC pretreatment (p < 0.05). And Rg1 and Pro-DTC also significantly attenuated the pulmonary histological abnormalities induced by IR. ConclusionGinsenoside Rg1 potentially attenuated lung injury induced by hind-limb IR by regulating NF-κB/COX-2 signaling pathway.

Protective effects of mitochondrion-targeted peptide SS-31 against hind limb ischemia-reperfusion injury.

Hind limb ischemia-reperfusion injury is an important pathology in vascular surgery. Reactive oxygen species are thought to be involved in the pathogenesis of hind limb ischemia-reperfusion injury. SS-31, which belongs to a family of mitochondrion-targeted peptide antioxidants, was shown to reduce mitochondrial reactive oxygen species production. In this study, we investigated whether the treatment of SS-31 could protect hind limb from ischemia-reperfusion injury in a mouse model. The results showed that SS-31 treatment either before or after ischemia exhibited similar protective effects. Histopathologically, SS-31 treatment prevented the IR-induced histological deterioration compared with the corresponding vehicle control. SS-31 treatment diminished oxidative stress revealed by the reduced malondialdehyde level and increased activities and protein levels of Sod and catalase. Cellular ATP contents and mitochondrial membrane potential increased and the level of cytosolic cytC was decreased after SS-31 treatment in this IR model, demonstrating that mitochondria were protected. The IR-induced increase of levels of inflammatory factors, such as Tnf-α and Il-1β, was prevented by SS-31 treatment. In agreement with the reduced cytosolic cytC, cleaved-caspase 3 was kept at a very low level after SS-31 treatment. Overall, the effect of SS-31 treatment before ischemia is mildly more effective than that after ischemia. In conclusion, our results demonstrate that SS-31 confers a protective effect in the mouse model of hind limb ischemia-reperfusion injury preventatively and therapeutically.

Humoral transfer and intramyocardial signal transduction of protection by remote ischemic perconditioning in pigs, rats, and mice

Remote ischemic perconditioning (RPER) during ongoing myocardial ischemia reduces infarct size. The signal transduction of RPER's cardioprotection is still largely unknown. Anesthetized pigs were therefore subjected to RPER by 4 × 5 min/5 min of hindlimb ischemia-reperfusion during 60 min of coronary occlusion before 3 h of reperfusion. Pigs without RPER served as placebo (PLA). The phosphorylation of Akt and ERK [reperfusion injury salvage kinase (RISK) pathway] and STAT3 [survivor activating factor enhancement (SAFE) pathway] in the area at risk was determined by Western blot analysis. Wortmannin/U0126 or AG490 was used for pharmacological RISK or SAFE blockade, respectively. Pig plasma/plasma dialysate sampled after RPER or PLA, respectively, was transferred to isolated rat and mouse hearts subjected to 30 min/120 min of global ischemia-reperfusion. Mitochondria were isolated from rat hearts at early reperfusion. Isolated mouse cardiomyocytes were subjected to 1 h of hypoxia/5 min of reoxygenation without and with prior plasma dialysate incubation. RPER reduced infarct size in pigs to 21 ± 15% versus 44 ± 9% in PLA (percentage of the area at risk, mean ± SD, P < 0.05) and increased STAT3 phosphorylation at early reperfusion. AG490 but not RISK blockade abolished the protection. RPER plasma/plasma dialysate reduced infarct size in rat (22 ± 3% of ventricular mass vs. 40 ± 11% with PLA plasma, P < 0.05) and mouse (29 ± 4% vs. 63 ± 8% with PLA plasma dialysate, P < 0.05) hearts and improved mitochondrial function (e.g., increased respiration, ATP formation, and calcium retention capacity and decreased reactive oxygen species formation). RPER dialysate also improved the viability of mouse cardiomyocytes after hypoxia/reoxygenation. RISK or SAFE blockade each abrogated these beneficial effects. NEW & NOTEWORTHY Remote ischemic perconditioning salvages the myocardium in patients with acute infarction. We identified a signal transduction with humoral transfer and STAT3 activation in pigs and an involvement of reperfusion injury salvage kinases and STAT3 in rat and mouse hearts, along with better cardiomyocyte viability and mitochondrial function.

Murine remote preconditioning increases glucose uptake and suppresses gluconeogenesis in hepatocytes via a brain-liver neurocircuit, leading to counteracting glucose intolerance

AimsOur previous study revealed that cyclic hindlimb ischaemia–reperfusion (IR) activates cardiac acetylcholine (ACh) synthesis through the cholinergic nervous system and cell-derived ACh accelerates glucose uptake. However, the mechanisms regulating glucose metabolism in vivo remain unknown. We investigated the effects and mechanisms of IR in mice under pathophysiological conditions. MethodsUsing IR-subjected male C57BL/6J mice, the effects of IR on blood sugar (BS), glucose uptake, central parasympathetic nervous system (PNS) activity, hepatic gluconeogenic enzyme expression and those of ACh on hepatocellular glucose uptake were assessed. ResultsIR decreased BS levels by 20% and increased c-fos immunoreactivity in the center of the PNS (the solitary tract and the dorsal motor vagal nucleus). IR specifically downregulated hepatic gluconeogenic enzyme expression and activities (glucose-6-phosphatase and phosphoenolpyruvate carboxykinase) and accelerated hepatic glucose uptake. Transection of a hepatic vagus nerve branch decreased this uptake and reversed BS decrease. Suppressed gluconeogenic enzyme expression was reversed by intra-cerebroventricular administration of a choline acetyltransferase inhibitor. Moreover, IR significantly attenuated hyperglycaemia in murine model of type I and II diabetes mellitus. ConclusionsIR provides another insight into a therapeutic modality for diabetes mellitus due to regulating gluconeogenesis and glucose-uptake and advocates an adjunctive mode rectifying disturbed glucose metabolism.

Effects of C1 inhibitor on endothelial cell activation in a rat hind limb ischemia-reperfusion injury model

ObjectiveIschemia-reperfusion (I/R) injury is a major clinical problem linked to vascular surgery. Currently, no drugs to prevent or to treat I/R injury are approved for clinical use. C1 inhibitor (C1 INH) is known to reduce activation of the plasma cascade systems that are involved in the pathophysiologic process of I/R injury. The aim of this study was therefore to investigate the effect of C1 INH on complement deposition and endothelial cell activation in a rat model of hind limb I/R injury. MethodsMale Wistar rats (wild type, bred at the central animal facility, University of Bern), weighing 250 to 320 g, were used. The rats underwent 2-hour ischemia and 24-hour reperfusion by unilateral clamping of the femoral artery and additional use of a tourniquet. Five groups were divided according to intravenous treatment 5 minutes before ischemia: 50 IU/kg C1 INH (n = 5); 100 IU/kg C1 INH (n = 7); vehicle control (n = 5); nontreated control (n = 7); and normal, healthy control without intervention (n = 4). At the end, muscle edema, tissue viability, and histologic features were assessed. Deposition of immunoglobulin M, C1r, C4d, and fibrin and expression of plasminogen activator inhibitor 1, heparan sulfate (HS), E-selectin, and vascular cell adhesion molecule 1 were evaluated by fluorescence staining. In addition, high-mobility group box 1 protein was measured in plasma. ResultsEdema formation was reduced by C1 INH at two dosages, mirrored by improved histologic injury scores and preserved muscle viability. Deposition of immunoglobulin M, C4d, and fibrin was significantly decreased by 100 IU/kg C1 INH compared with nontreated controls. Pretreatment with 100 IU/kg C1 INH also significantly reduced HS shedding and expression of plasminogen activator inhibitor 1 as well as plasma levels of high-mobility group box 1 protein. ConclusionsPretreatment with both 50 and 100 IU/kg C1 INH attenuated reperfusion injury of rat hind limbs. Pretreatment with 100 IU/kg also preserved the endothelial HS layer as well as the natural, profibrinolytic phenotype of the endothelium. Prevention of endothelial cell activation by C1 INH may therefore be a promising strategy to prevent I/R injury in the clinical setting of peripheral vascular diseases and elective surgery on extremities.

Methylene Blue Attenuates Lung Injury Induced by Hindlimb Ischemia Reperfusion in Rats.

Objective This study was aimed to investigate the protective effect of methylene blue against lung injury induced by reperfusion of ischemic hindlimb in a rat model. Methods Twenty-four healthy adult male Sprague-Dawley rats were equally randomized into three groups: sham (SM) group, ischemia reperfusion (IR) group, and methylene blue (MB) group. Rats in both IR and MB groups were subjected to 4 h of ischemia by clamping the left femoral artery and then followed by 4 h of reperfusion. Treatment with 1% methylene blue (50 mg/kg) was administrated intraperitoneally at 10 min prior to reperfusion in the MB group. After 4 h of reperfusion, malondialdehyde (MDA) level, myeloperoxidase (MPO), and superoxide dismutase (SOD) activities in lung tissue were detected; inflammatory cytokines, including IL-1β and IL-6, were measured in bronchoalveolar lavage fluid (BALF); correspondingly, the morphological changes and water content in both gastrocnemius muscle and lung samples were evaluated. Results Hindlimb IR caused remarkable morphological abnormalities and edema in both muscle and lung tissues. SOD activity was decreased, both the MPO activity and MDA level in lung tissue, as well as IL-1β and IL-6 levels in BALF, were increased in the IR group (p < 0.05). Compared with the IR group, SOD activity was increased, whereas MPO activity and MDA level in lung tissue and IL-1β and IL-6 levels in BALF were decreased in the MB group (p < 0.05). Also, the histological damage and edema in both lung and muscle tissues were significantly attenuated by the treatment of methylene blue. Conclusion Methylene blue attenuates lung injury induced by hindlimb IR in rats, at least in part, by inhibiting oxidative stress.

Effect of atorvastatin on oxidative damage and inflammation in experimental hindlimb ischemia–reperfusion model

Ischemia–reperfusion is defined as cellular damage after the reperfusion of ischemic tissue, and it is likely to occur in relation to various diseases and surgical procedures. The purpose of this study was to evaluate the capability of atorvastatin to prevent oxidative damage and modulate the release of proinflammatory cytokines in rat hindlimb during ischemia–reperfusion injury. The animals were divided into 4 groups (ischemia–reperfusion + vehicle, ischemia–reperfusion + atorvastatin, sham, and healthy controls) with 15 rats per group. The animals were exposed to ischemia for 6 h, followed by 24 h, 7 days, and 14 days of reperfusion. Atorvastatin was administered by gavage 14 days before ischemia–reperfusion induction. We then measured the serum concentrations and mRNA transcript levels of TNF-α, IL-1β, IL-6, IL-10, SOD2, and CAT. Hematoxylin and eosin stain were performed for histological analyses. Animals subjected to ischemia–reperfusion showed increased serum and transcript levels of TNF-α, IL-1β, IL-6, and IL-10 expressions with a concurrent increase in mRNA transcripts levels compared with sham and healthy controls. Groups treated with atorvastatin showed a significant CAT increase in the first 24 h, but CAT levels decreased at 7 and 14 days. SOD2 enzyme increased in serum without significant changes in mRNA expression. Histological analysis showed inflammatory infiltrate, microhemorrhages, and distortion of the tissue architecture in the first 7 days. At 14 days, the tissue showed loss and damage to myocytes. However, animals treated with atorvastatin showed few histological changes and a decrease in inflammatory cytokines. No significant changes in NO2, NO3, or 8-OHdG were observed. Atorvastatin showed a protective effect on the inflammation and tissue damage induced by ischemia–reperfusion in the hindlimb. The antioxidant effect of atorvastatin in the hindlimb is already unclear, and further research is needed to elucidate the molecular mechanism of this drug in the extremities.

Conditional MitoTimer reporter mice for assessment of mitochondrial structure, oxidative stress, and mitophagy

Assessment of structural and functional changes of mitochondria is vital for biomedical research as mitochondria are the power plants essential for biological processes and tissue/organ functions. Others and we have developed a novel reporter gene, pMitoTimer, which codes for a redox sensitive mitochondrial targeted protein that switches from green fluorescence protein (GFP) to red fluorescent protein (DsRed) when oxidized. It has been shown in transfected cells, transgenic C. elegans and Drosophila m., as well as somatically transfected adult skeletal muscle that this reporter gene allows quantifiable assessment of mitochondrial structure, oxidative stress, and lysosomal targeting of mitochondria-containing autophagosomes. Here, we generated CAG-CAT-MitoTimer transgenic mice using a transgene containing MitoTimer downstream of LoxP-flanked bacterial chloramphenicol acetyltransferase (CAT) gene with stop codon under the control of the cytomegalovirus (CMV) enhancer fused to the chicken β-actin promoter (CAG). When CAG-CAT-MitoTimer mice were crossbred with various tissue-specific (muscle, adipose tissue, kidney, and pancreatic tumor) or global Cre transgenic mice, the double transgenic offspring showed MitoTimer expression in tissue-specific or global manner. Lastly, we show that hindlimb ischemia-reperfusion caused early, transient increases of mitochondrial oxidative stress, mitochondrial fragmentation and lysosomal targeting of autophagosomes containing mitochondria as well as a later reduction of mitochondrial content in skeletal muscle along with mitochondrial oxidative stress in sciatic nerve. Thus, we have generated conditional MitoTimer mice and provided proof of principle evidence of their utility to simultaneously assess mitochondrial structure, oxidative stress, and mitophagy in vivo in a tissue-specific, controllable fashion.

Pyruvate enhancement of cardiac performance: Cellular mechanisms and clinical application.

Cardiac contractile function is adenosine-5'-triphosphate (ATP)-intensive, and the myocardium's high demand for oxygen and energy substrates leaves it acutely vulnerable to interruptions in its blood supply. The myriad cardioprotective properties of the natural intermediary metabolite pyruvate make it a potentially powerful intervention against the complex injury cascade ignited by myocardial ischemia-reperfusion. A readily oxidized metabolic substrate, pyruvate augments myocardial free energy of ATP hydrolysis to a greater extent than the physiological fuels glucose, lactate and fatty acids, particularly when it is provided at supra-physiological plasma concentrations. Pyruvate also exerts antioxidant effects by detoxifying reactive oxygen and nitrogen intermediates, and by increasing nicotinamide adenine dinucleotide phosphate reduced form (NADPH) production to maintain glutathione redox state. These enhancements of free energy and antioxidant defenses combine to augment sarcoplasmic reticular Ca2+ release and re-uptake central to cardiac mechanical performance and to restore β-adrenergic signaling of ischemically stunned myocardium. By minimizing Ca2+ mismanagement and oxidative stress, pyruvate suppresses inflammation in post-ischemic myocardium. Thus, pyruvate administration stabilized cardiac performance, augmented free energy of ATP hydrolysis and glutathione redox systems, and/or quelled inflammation in a porcine model of cardiopulmonary bypass, a canine model of cardiac arrest-resuscitation, and a caprine model of hypovolemia and hindlimb ischemia-reperfusion. Pyruvate's myriad benefits in preclinical models provide the mechanistic framework for its clinical application as metabolic support for myocardium at risk. Phase one trials have demonstrated pyruvate's safety and efficacy for intravenous resuscitation for septic shock, intracoronary infusion for heart failure and as a component of cardioplegia for cardiopulmonary bypass. The favorable outcomes of these trials, which argue for expanded, phase three investigations of pyruvate therapy, mirror findings in isolated, perfused hearts, underscoring the pivotal role of preclinical research in identifying clinical interventions for cardiovascular diseases. Impact statement This article reviews pyruvate's cardioprotective properties as an energy-yielding metabolic fuel, antioxidant and anti-inflammatory agent in mammalian myocardium. Preclinical research has shown these properties make pyruvate a powerful intervention to curb the complex injury cascade ignited by ischemia and reperfusion. In ischemically stunned isolated hearts and in large mammal models of cardiopulmonary bypass, cardiac arrest-resuscitation and hypovolemia, intracoronary pyruvate supports recovery of myocardial contractile function, intracellular Ca2+ homeostasis and free energy of ATP hydrolysis, and its antioxidant actions restore β-adrenergic signaling and suppress inflammation. The first clinical trials of pyruvate for cardiopulmonary bypass, fluid resuscitation and intracoronary intervention for congestive heart failure have been reported. Receiver operating characteristic analyses show remarkable concordance between pyruvate's beneficial functional and metabolic effects in isolated, perfused hearts and in patients recovering from cardiopulmonary bypass in which they received pyruvate- vs. L-lactate-fortified cardioplegia. This research exemplifies the translation of mechanism-oriented preclinical studies to clinical application and outcomes.

Correction to: Apocynin and dimethyl sulfoxide synergistically protect against ischemia-reperfusion injury in a rat hind limb ischemia-reperfusion model

Correction to: Apocynin and dimethyl sulfoxide synergistically protect against ischemia-reperfusion injury in a rat hind limb ischemia-reperfusion model

Estrogen-dependent efficacy of limb ischemic preconditioning in female rats.

Our aim was to examine the effects of ischemic preconditioning (IPC) on the local periosteal and systemic inflammatory consequences of hindlimb ischemia-reperfusion (IR) in Sprague-Dawley rats with chronic estrogen deficiency (13 weeks after ovariectomy, OVX) in the presence and absence of chronic 17beta-estradiol supplementation (E2, 20 µg kg-1 , 5 days/week for 5 weeks); sham-operated (non-OVX) animals served as controls. As assessed by intravital fluorescence microscopy, rolling and the firm adhesion of polymorphonuclear neutrophil leukocytes (PMNs) gave similar results in the Sham + IR and OVX + IR groups in the tibial periosteal microcirculation during the 3-h reperfusion period after a 60-min tourniquet ischemia. Postischemic increases in periosteal PMN adhesion and PMN-derived adhesion molecule CD11b expressions, however, were significantly reduced by IPC (two cycles of 10'/10') in Sham animals, but not in OVX animals; neither plasma free radical levels (as measured by chemiluminescence), nor TNF-alpha release was affected by IPC. E2 supplementation in OVX animals restored the IPC-related microcirculatory integrity and PMN-derived CD11b levels, and TNF-alpha and free radical levels were reduced by IPC only with E2. An enhanced estrogen receptor beta expression could also be demonstrated after E2 in the periosteum. Overall, the beneficial periosteal microcirculatory effects of limb IPC are lost in chronic estrogen deficiency, but they can be restored by E2 supplementation. This suggests that the presence of endogenous estrogen is a necessary facilitating factor of the anti-inflammatory protection provided by limb IPC in females. The IPC-independent effects of E2 on inflammatory reactions should also be taken into account in this model. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:97-105, 2018.

Effect of picroside II on hind limb ischemia reperfusion injury in rats

IntroductionMany structural and functional damages are observed in cells and tissues after reperfusion of previously viable ischemic tissues. Acute ischemia reperfusion (I/R) injury of lower extremities occurs especially when a temporary cross-clamp is applied to the abdominal aorta during aortic surgery. Research regarding the treatment of I/R injury has been increasing day-by-day. In this study, we aimed to investigate the effect of picroside II on skeletal muscle of rats experiencing simulated I/R.Materials and methodsTwenty-four male Wistar albino rats weighing between 210 and 300 g were used in this study. Rats were randomly divided into 4 groups of 6 rats each (control, I/R, control + picroside II, and I/R + picroside II). The infrarenal section of the abdominal aorta was occluded with an atraumatic microvascular clamp in I/R group. The clamp was removed after 120 minutes and reperfusion was provided for a further 120 minutes. Picroside II (10 mg kg−1) was administered intraperitoneally to the animals in control + picroside II and I/R + picroside II groups. At the end of the study, skeletal muscle tissue was obtained for the determination of total oxidant status (TOS) and total antioxidant status (TAS) levels. Apoptosis was evaluated by TUNEL experiment.ResultsTOS levels were significantly higher in I/R group than that of control and I/R + picroside II groups (P=0.014, P=0.005, respectively). TAS levels were significantly higher in I/R group than that of control and I/R + picroside II groups (P=0.007 P=0.005, respectively). TUNEL assay revealed that picroside II reduced cell necrosis.ConclusionThe results of this study demonstrated that picroside II plays a critical role to prevent I/R injury. Even though our results were found to be satisfactory, it should be encouraging to those who want to conduct future research on this topic.

Exploring effects of remote ischemic preconditioning in a pig model of hypothermic circulatory arrest

Objectives. During aortic and cardiac surgery, risks for mortality and morbidity are inevitable. Surgical setups involving deep hypothermic circulatory arrest (DHCA) are effective to achieve organ protection against ischemic injury. The aim of this study was to identify humoural factors mediating additive protective effects of remote ischemic preconditioning (RIPC) in a porcine model of DHCA. Design. Twenty-two pigs were randomized into the RIPC group (n = 11) and the control group (n = 11). The RIPC group underwent four 5-minute hind limb ischemia-reperfusion cycles prior to cardiopulmonary bypass and DHCA. All animals underwent identical surgical procedures including 60 min DHCA at 18 °C. Blood samples were collected from vena cava and sagittal sinus at several time points. After the 8-hour follow-up period, the brain, heart, and kidney tissue samples were collected for tissue analyses. Results. Serum levels of brain damage marker S100B recovered faster in the RIPC group, after 4 hours of the arrest, (p < .05). Systemic lactate levels were lower and cardiac index was higher in the RIPC group postoperatively. Immunohistochemical cerebellum regional scores of antioxidant response regulator Nrf2 were better in the RIPC group (mean: 1.1, IQR: 0.0–2.5) compared with the control group (mean: 0.0, IQR: 0.0–0.0), reaching borderline statistical significance (p = .064). RIPC induced detectable modulations of plasma proteome and metabolites. Conclusions. The faster recovery of S100B, lower systemic lactate levels and favourable regional antioxidant response suggest possible neuronal cellular and mitochondrial protection by RIPC, whereas better cardiac index underlines functional effects of RIPC. The exact humoural factor remains unclear.

Apocynin and dimethyl sulfoxide synergistically protect against ischemia-reperfusion injury in a rat hind limb ischemia-reperfusion model

Acute ischemia and reperfusion in extremities ignite a complicated inflammatory cycle and lead to damage in the extremities and target tissues. Rats were randomly divided into four groups. Each experimental group contained eight rats. Group 1 was the sham surgery group. Group 2 was the control I/R group with intraperitoneal serum physiologic (SP) treatment. Group 3 was the I/R group with vehicle treatment (8% DMSO). Group 4 was the I/R group with 20 mg/kg apocynin (vehicle 8% DMSO). The potential protective effects of apocynin and vehicle (DMSO) in the rat hind limb ischemia-reperfusion model were evaluated by measuring the levels of plasma pro-inflammatory cytokines and oxidative stress markers in muscle tissue. The results revealed that both apocynin and vehicle (DMSO) alone increased the antioxidative capacity of ischemic tissue and reduced the levels of plasma proinflammatory cytokines compared to control I/R group. There was a statically more significant effect in the apocynin (vehicle 8% DMSO)-treated group 4. We observed that apocynin and DMSO synergistically prevent the I/R injury in rat hind limb ischemia-reperfusion injury model and meaningfully decrease the inflammatory parameters. Level of Evidence: Not ratable.

Effects of FTY720 on Lung Injury Induced by Hindlimb Ischemia Reperfusion in Rats

Background Sphingosine-1-phosphate (S1P) is a biologically active lysophospholipid mediator involved in modulating inflammatory process. We investigated the effects of FTY720, a structural analogue of S1P after phosphorylation, on lung injury induced by hindlimb ischemia reperfusion (IR) in rats. Methods Fifty Sprague-Dawley rats were divided into groups SM, IR, F3, F5, and F10. Group SM received sham operation, and bilateral hindlimb IR was established in group IR. The rats in groups F3, F5, and F10 were pretreated with 3, 5, and 10 mg/kg/d FTY720 for 7 days before IR. S1P lyase (S1PL), sphingosine kinase (SphK) 1, and SphK2 mRNA expressions, wet/dry weight (W/D), and polymorphonuclear/alveolus (P/A) in lung tissues were detected, and the lung injury score was evaluated. Results W/D, P/A, and mRNA expressions of S1PL, SphK1, and SphK2 were higher in group IR than in group SM, while these were decreased in both groups F5 and F10 as compared to IR (p < 0.05). The lung tissue presented severe lesions in group IR, which were attenuated in groups F5 and F10 with lower lung injury scores than in group IR (p < 0.05). Conclusions FTY720 pretreatment could attenuate lung injury induced by hindlimb IR by modulating S1P metabolism and decreasing pulmonary neutrophil infiltration.

Effects of intravenous ibuprofen and lornoxicam on erythrocyte deformability in rats undergoing hind limb ischemia reperfusion injury.

Acute hind limb ischemia reperfusion (I/R) injury is a common consequence of abdominal aorta cross‑clamping during aortic surgery. Erythrocyte deformability is affected by I/R process and may lead to increased tissue and organ injury. Lornoxicam and intravenous ibuprofen are becoming commonly used as non-steroidal anti-inflammatory drugs (NSAID) for postoperative analgesia. In this study, we aimed to investigate the effects of lornoxicam (2 mg/kg iv) and intravenous ibuprofen (30 mg/kg iv) on erythrocyte deformability in I/R model in rats. Four study groups, each containing 6 Wistar rats were created. Laparotomy was performed in all groups under general anesthesia with ketamine and xylazine. In all groups except sham group, ischemia and reperfusion were achieved by clamping and declamping the infrarenal abdominal aorta for 120 minutes. Rats in Group IR+L received intravenous infusion of lornoxicam (2 mg/kg) while rats in Group IR+I received intravenous infusion of ibubrofen (30 mg/kg) following 2 hours of ischemic period. At the end of reperfusion period, erythrocyte packs were prepared from heparinized blood samples. Erythrocyte suspensions with hematocrit at a concentration of 5% in a phosphate‑buffered saline (PBS) were used in order to perform deformability measurements. The value of p<0.05 was considered statistically significant. Relative resistance has increased in ischemia reperfusion group when compared to control group (p < 0.0001). Lornoxicam or ibuprofen intravenous treatments did not change the erythrocyte deformability during ischemia reperfusion period in rats (p=0.851, p=0.690). Intravenous ibuprofen or lornoxicam administrations during ischemia reperfusion period in rats have no negative effect on erythrocyte deformability. The findings of the study should be supported with more detailed and extensive clinical/experimental studies in the future (Fig. 1, Ref. 18).

Cardioprotection induced by remote ischemic preconditioning preserves the mitochondrial respiratory function in acute diabetic myocardium.

A 2×2 factorial design was used to evaluate possible preservation of mitochondrial functions in two cardioprotective experimental models, remote ischemic preconditioning and streptozotocin-induced diabetes mellitus, and their interaction during ischemia/reperfusion injury (I/R) of the heart. Male Wistar rats were randomly allocated into four groups: control (C), streptozotocin-induced diabetic (DM), preconditioned (RPC) and preconditioned streptozotocin-induced diabetic (DM+RPC). RPC was conducted by 3 cycles of 5-min hind-limb ischemia and 5-min reperfusion. DM was induced by a single dose of 65 mg/kg streptozotocin. Isolated hearts were exposed to ischemia/reperfusion test according to Langendorff. Thereafter mitochondria were isolated and the mitochondrial respiration was measured. Additionally, the ATP synthase activity measurements on the same preparations were done. Animals of all groups subjected to I/R exhibited a decreased state 3 respiration with the least change noted in DM+RPC group associated with no significant changes in state 2 respiration. In RPC, DM and DM+RPC group, no significant changes in the activity of ATP synthase were observed after I/R injury. These results suggest that the endogenous protective mechanisms of RPC and DM do preserve the mitochondrial function in heart when they act in combination.

Effects of sinomenine on hind limb ischemia-reperfusion injury and expression of Bcl-2 and Bax in skeletal muscle cells of rats

Objective To investigate the effects of sinomenine on hind limb ischemia-reperfusion (I/R) injury and expression of Bcl-2 and Bax in skeletal muscle cells of rats. Methods Fifty-four healthy adult male Wistar rats, aged 6-8 weeks, weighing 180-220 g, were divided into 3 groups (n=18 each) using a random number table: sham operation group (group S), group I/R and sinomenine group (group SIN). The rats were subjected to 4 h of ischemia on the proximal part of the right hind limb using elastic rubber bands followed by reperfusion in I/R and SIN groups.Sinomenine 60 mg/kg was injected intraperitoneally at 30 min before reperfusion in group SIN, and the equal volume of normal saline was given instead of sinomenine at 30 min before reperfusion in S and I/R groups.Immediately after onset of reperfusion and at 4 and 24 h of reperfusion, blood samples were collected from the cardiac apex to measure the concentrations of serum lactate dehydrogenase (LDH) and creatine kinase (CK). The animals were sacrificed immediately after blood sampling, and the gastrocnemius specimens of the hind limb were immediately removed for determination of the wet to dry weight ratio (W/D ratio) and expression of Bcl-2 and Bax in gastrocnemius cells (by immunohistochemistry) and for examination of the pathological changes after haematoxylin and eosin staining.The Bcl-2/Bax ratio was calculated. Results Compared with group S, the gastrocnemius W/D ratio and concentrations of serum LDH and CK were significantly increased, the expression of Bcl-2 was significantly down-regulated, the expression of Bax was significantly up-regulated, and the Bcl-2/Bax ratio was significantly decreased in I/R and SIN groups (P<0.05). Compared with group I/R, the gastrocnemius W/D ratio and concentrations of serum LDH and CK were significantly decreased, the expression of Bcl-2 was significantly up-regulated, the expression of Bax was significantly down-regulated, and the Bcl-2/Bax ratio was significantly increased in group SIN (P<0.05). The pathological changes of the gastrocnemius were significantly attenuated in group SIN as compared with group I/R. Conclusion Sinomenine can attenuate hind limb I/R injury, and the mechanism may be related to maintenance of the balance between Bcl-2 and Bax and to inhibition of apoptosis in skeletal muscle cells of rats. Key words: Sinomenium; Reperfusion injury; Extremities; Proto-oncogene proteins c-bcl-2; bcl-2-associated X protein

Dexamethasone promotes long-term functional recovery of neuromuscular junction in a murine model of tourniquet-induced ischaemia-reperfusion.

Tourniquet-induced ischaemia and subsequent reperfusion cause serious ischaemia-reperfusion (IR) injury in the neuromuscular junction (NMJ) and skeletal muscle. Here, we investigated whether dexamethasone (Dex) promotes long-term functional recovery of the NMJ and skeletal muscle in tourniquet-induced hindlimb IR. Unilateral hindlimb of C57/BL6 mice was subjected to 3h of ischaemia following 6weeks of reperfusion (6-wk IR). Dex treatment began on the day of IR induction and lasted for different periods. Sciatic nerve-stimulated gastrocnemius muscle contraction was detected insitu. Function of the NMJ was measured insitu using electrophysiological recording of the miniature endplate potential (mEPP) and endplate potential (EPP). Western blot was used to detect protein expression of nicotinic acetylcholine receptors (nAChRs) in gastrocnemius muscles. Gastrocnemius muscle contraction in mice with 6-wk IR was about 60% of normal skeletal muscle contraction recorded in age-matched sham mice. The amplitude of the mEPP and EPP was lower in mice with 6-wk IR, compared to sham mice. Dex treatment for 1 or 3days did not restore the function of the NMJ and improve gastrocnemius muscle contraction in mice with 6-wk IR. Dex treatment for 1week exerted a maximum effect on improving the function of the NMJ and skeletal muscle, with the effect of Dex gradually lessening with prolonged Dex treatment. There are no significant differences in protein expression of nAChR-α1 and nAChR-β1 subunits in the gastrocnemius muscle among all groups. Dex promotes repair of the NMJ and subsequently restores skeletal muscle contractile function in tourniquet-induced 6-wk IR.