Hind Limb Ischemia-reperfusion Injury Research Articles

Detecting viscosity changes in the limb ischemia-reperfusion in mice with a near-infrared fluorescence probe

BackgroundLimb ischemia-reperfusion is a common phenomenon in clinical surgery, which disrupts the balanced physiological response process and ultimately leads to changes in intracellular viscosity. Intracellular viscosity is an important microenvironmental parameter that affects the normal function of organisms, and its level is closely related to many diseases. In addition, oxidative stress in the lower limbs can impair body function, and changes in pressure can lead to changes in the viscosity of limb tissues. Therefore, it is necessary to develop effective tools to detect changes in intracellular viscosity and visualize the progression of hind limb ischemia-reperfusion injury. ResultsIn order to solve this problem, a near infrared viscometry sensitive fluorescence probe (PH-XQ) with long emission wavelength and stable luminescence performance was designed and synthesized by using oxanthracene derivatives and malononitrile. The fluorescence probe (PH-XQ) has excellent selectivity, high sensitivity, low toxicity, high biocompatibility and excellent detection performance. The fluorescence intensity of the PH-XQ probe at 667 nm is highly sensitive to the change of viscosity. With the increase of viscosity, the fluorescence intensity of probe PH-XQ was significantly enhanced, and the fluorescence enhancement ratio was about 14-fold. In addition, PH-XQ can detect not only changes in viscosity between normal cells and drug-induced inflammatory cells, but also changes in the viscosity of the hind limbs of normal mice and mice after ischemia reperfusion. SignificanceIn particular, we are the first to successfully detect changes in handlimb viscosity after ischemia-reperfusion in mice using a probe. This study clearly elucidates changes in viscosity during ischemia-reperfusion of mouse limbs, providing favorable support for the relationship between viscosity and related diseases, and further providing a potential tool for the diagnosis of viscosity-related diseases.

Piezo1 mediates abnormal type 2 immune response to aggravate ischemia–reperfusion acute lung injury which by regulating alveolar macrophages derived IL-33: A hypothesis

The pathogenesis of ischemia–reperfusion acute lung injury (IR-ALI) is not yet fully understood. We previously reported that unwonted increased type 2 immunity plays a key role in the pathogenesis and the pathophysiological process in acute haemorrhagic inducedlung injury. Progressive lung edema after ischemia reperfusion is accompanied by the change of lung cyclical hydrostatic force. The changed lung mechanical force could activate the mechanosensitive Piezo1 channel in alveolar macrophages. Furthermore, our group had elucidated that microenvironmental stiffness exacerbated hindlimb ischemia reperfusion injury by activating Piezo1 in bone marrow-derived macrophages. Considering early findings that IL-33 is a potent driver of pulmonary type 2 immunity, that Piezo1 plays an important role in regulating macrophages biological activity and that alveolar macrophages are involved in orchestrating the early abnormal detrimental type 2 inflammatory response in lung ischemia–reperfusion injury, we herein hypothesize that the change of mechanical force in lung after ischemia–reperfusion might activate Piezo1/IL-33/type 2 immunity axis and aggravate lung ischemic injury. Mechanistically, Piezo1 mediates extracellular Ca2+influx and activates Ca2+-dependent Calcineurin-NFAT signaling in alveolar macrophages, leading to transcriptional activation of IL-33, thereby promoting abnormal type 2 immune response and participating in the occurrence and development of IR-ALI. Future validation of these mechanisms could provide a deeper explanation of the new pathogenesis of IR-ALI and ultimately serve for clinical treatment.

Mexican tea (Dysphania ambrosioides (L.) Mosyakin & Clemants) seeds attenuate tourniquet-induced hind limb ischemia–reperfusion injury by modulating ROS and NLRP3 inflammasome pathways

The main pathophysiological mechanisms of hind limb ischemia–reperfusion injury (HLIRI) are increased oxidative stress and inflammation. The present study was designed to characterize the phytocontents of Dysphania ambrosioides (DA) seeds extract via LC-MS/MS and investigate its effect on left hindlimb IR injury and the underlying mechanisms. Thirty adult rats (n = 6 per group) were divided into five groups: Control group; HLIRI group, HLIRI + DA (100 mg/kg) group, HLIRI + DA (200 mg/kg) group, and HLIRI + cilostazol (30 mg/kg) group. HE staining’s of the left gastrocnemius muscle and left kidney was done. Renal function, and both total and muscle creatine kinases were measured in serum. Oxidative stress markers (MDA, SOD, Nrf2 and HO-1), inflammatory markers (NLRP3, IL-1β and TNF-α) and caspase-3 as an apoptotic marker were measured in left gastrocnemius muscle. We found that DA extract contains 60 metabolites and its pretreatment prevented left kidney and left gastrocnemius muscle damage and decreased oxidative stress markers, inflammatory markers, and caspase-3 levels, compared to the control group. Moreover, the effect of the extract was dose-dependent and better than that of the reference drug, cilostazol. This study suggested that D. ambrosioides seeds have a protective effect on HLIRI, which may be related to the antioxidant, anti-inflammatory and antiapoptotic mechanisms.

ProBDNF Upregulation in Murine Hind Limb Ischemia Reperfusion Injury: A Driver of Inflammation.

Brain-derived neurotropic factor (BDNF) has been shown to be expressed in many nonneuronal tissues including skeletal muscle. Skeletal muscle BDNF has been studied regarding its function in metabolism and exercise; however, less is known about its role in skeletal muscle injury. The precursor to BDNF, proBDNF, has an unknown role in skeletal muscle. The levels of proBDNF, mature BDNF, and their receptors were compared in the skeletal muscle and brain tissues of C57BL/6J mice. Tourniquet-induced hind limb ischemia-reperfusion injury was used to assess the function of skeletal muscle-derived proBDNF in skeletal muscle injury. Skeletal muscle-specific knockout of BDNF and pharmacological inhibition of p75NTR, the proBDNF receptor, were used to determine the role of proBDNF-p75NTR signaling. We show for the first time that proBDNF is the predominantly expressed form of BDNF in skeletal muscle and that proBDNF is significantly upregulated in skeletal muscle following hind limb ischemia-reperfusion injury. Skeletal muscle-specific knockout of BDNF blunted the inflammatory response in the injured tissue and appears to be mediated by the proBDNF-p75NTR pathway, as shown by the pharmacological inhibition of p75NTR. These findings suggest that skeletal muscle proBDNF plays a critical role in driving the inflammatory response following skeletal muscle injury.

Lyophilized Extracellular Vesicles from Adipose-Derived Stem Cells Increase Muscle Reperfusion but Degrade Muscle Structural Proteins in a Mouse Model of Hindlimb Ischemia-Reperfusion Injury.

Ischemia-reperfusion (I/R) injury is a complication impacting multiple organs and tissues in clinical conditions ranging from peripheral arterial disease to musculoskeletal trauma and myocardial infarction. Stem cell-derived extracellular vesicles (EVs) may represent one therapeutic resource for preventing the tissue damage associated with I/R injury. Here we tested the hypothesis that lyophilized extracellular vesicles derived from adipose stem cells could serve as an "off-the-shelf" treatment modality for I/R injury in a mouse hindlimb ischemia model. Ischemia was induced for 90 min using a rubber band tourniquet and extracellular vesicles (0, 50, or 100 µg) administered via tail vein injection immediately prior to reperfusion. Perfusion was measured prior to, during, and after ischemia using laser Doppler imaging. Serum and tissue were collected 24 h after reperfusion. Mass spectrometry (MS)-based proteomics was used to characterize the EV cargo and proteins from the ischemic and non-ischemic hindlimb. Inflammatory cytokines were measured in muscle and serum using a multiplex array. Results indicate that EVs significantly increase reperfusion and significantly increase expression of the anti-inflammatory factor annexin a1 in skeletal muscle; however, the increased reperfusion was also associated with a marked decrease in muscle structural proteins such as dystrophin, plectin, and obscurin. Circulating inflammatory cytokines TNF-alpha and IL-6 were increased with EV treatment, and serum TNF-alpha showed a significant, positive correlation with reperfusion level. These findings suggest that, while EVs may enhance reperfusion, the increased reperfusion can negatively impact muscle tissue and possibly remote organs. Alternative approaches, such as targeting mitochondrial permeability, may be more effective at mitigating I/R injury.

DNase I and Sivelestat Ameliorate Experimental Hindlimb Ischemia-Reperfusion Injury by Eliminating Neutrophil Extracellular Traps.

Neutrophil extracellular traps (NETs) play an important role in ischemia-reperfusion injury (IRI) of the hindlimb. The aim of this study was to investigate the effect of recombinant DNase I and sivelestat in eliminating NETs and their effects on IRI limbs. An air pump was used to apply a pressure of 300 mmHg to the root of the right hindlimb of the rat for 2 h and then deflated to replicate the IRI model. The formation of NETs was determined by the detection of myeloperoxidase (MPO), neutrophil elastase (NE), and histone H3 in the skeletal muscles of the hindlimbs. Animals were administered 2.5 mg/kg bw/d DNase I, 15 or 60 mg/kg bw/d sivelestat by injection into the tail vein or intramuscularly into the ischemic area for 7d. Elimination of NETs, hindlimb perfusion, muscle fibrosis, angiogenesis and motor function were assessed. DNase I reduced NETs, attenuated muscle fibrosis, promoted angiogenesis in IRI area and improved limb motor function. Local administration of DNase I improved hindlimb perfusion more than intravenous administration. Sivelestat at a dose of 15 mg/kg bw/d increased perfusion, counteracted skeletal muscle fibrosis, promoted angiogenesis and enhanced motor function. However, sivelestat at a dosage of 60 mg/kg bw/d had an adverse effect on tissue repair, especially when injected locally. Both DNase I and moderate doses of sivelestat can eliminate IRI-derived NETs. They improve hindlimb function by improving perfusion and angiogenesis, preventing muscle fibrosis. Appropriate administration mode and dosage is the key to prevent IRI by elimination of NETs. DNase I is more valid when administered topically and sivelestat is more effective when administered intravenously. These results will provide a better strategy for the treatment of IRI in clinical.

Oridonin attenuates hind limb ischemia–reperfusion injury by modulating Nrf2-mediated oxidative stress and NLRP3-mediated inflammation

Ethnopharmacological relevanceOridonin (Ori), extracted from Isodon rubescens (Hemsl.) H.Hara, is a well-known traditional Chinese herbal medicinal product that possesses antioxidant and anti-inflammatory activities. Oxidative stress and inflammation are the main pathophysiological mechanisms in hindlimb IR injury. However, whether Ori has a protective effect on hind limb IR injury is unknown. Aim of the studyThe present study was designed to determine the effect of Ori on hindlimb IR injury and its relationship with oxidative stress and inflammation. Materials and methodsThe hind limb IR injury model in mice was used to evaluate the protective effect and related mechanisms of Ori. Forty-eight C57BL/6 mice (n = 12 per group) were randomly divided into four groups: Sham group; IR group; IR + Ori (10 mg/kg) group and IR + Ori (20 mg/kg) group. Mice in the IR and IR + Ori groups were subjected to hindlimb IR injury, while mice in the Sham group were subjected to no hindlimb IR injury. HE staining, Masson's staining, TTC staining, DHE staining, TUNEL staining, western blotting analysis and quantitative real-time PCR were employed to explore the mechanisms by which Ori exerts a protective effect on a classical hindlimb IR model in mice. ResultsWe found that Ori pretreatment prevented muscle damage and decreased cell apoptosis levels compared with the vehicle control. Moreover, the SOD2, CAT, MDA and ROS levels in muscle showed that Ori could significantly reduce oxidative stress in hindlimb IR mice, while the IL-1β and TNF-α levels in muscle showed that Ori could significantly attenuate IR-induced inflammation. We also found that Ori could increase the expression of Nrf2 and its downstream protein HO-1 and inhibit the expression levels of NLRP3-related proteins (NLRP3, ASC and Caspase-1) in vivo. ConclusionsOur study suggested that Ori has a protective effect on hindlimb IR injury, which may be related to Nrf2-mediated oxidative stress and NLRP3-mediated inflammasome activation.

Oridonin Attenuates Hind Limb Ischemia–Reperfusion Injury by Modulating Nrf2-Mediated Oxidative Stress and NLRP3-Mediated Inflammation

Oridonin Attenuates Hind Limb Ischemia–Reperfusion Injury by Modulating Nrf2-Mediated Oxidative Stress and NLRP3-Mediated Inflammation

Microenergy acoustic pulses promotes muscle regeneration through in situ activation of muscle stem cells.

Microenergy acoustic pulses (MAP) is a modified low-intensity extracorporeal shock wave therapy that currently used for treating musculoskeletal disorders. However, its function on muscle regeneration after ischemia-reperfusion injury (IRI) remains unknown. This study aimed to explore the effect of MAP on muscle injury after IRI and its underlying mechanisms. Ten-week-old C57BL/6J mice underwent unilateral hindlimb IRI followed with or without MAP treatment. Wet weight of tibialis anterior muscles at both injury and contralateral sides were measured followed with histology analysis at 3 weeks after IRI. In in vitro study, the myoblasts, endothelial cells and fibro-adipogenic progenitors (FAP) were treated with MAP. Cell proliferation and differentiation were assessed, and related gene expressions were measured by real-time PCR. Our results showed that MAP significantly increased the muscle weight and centrally nucleated regenerating muscle fiber size along with a trend in activating satellite cells. In vitro data indicated that MAP promoted myoblast proliferation and differentiation and endothelial cells migration. MAP also induced FAP brown/beige adipogenesis, a promyogenic phenotype of FAPs. Our findings demonstrate the beneficial function of MAP in promoting muscle regeneration after IR injury by inducing muscle stem cells proliferation and differentiation.

Peripheral arterial disease: Effects of ethanolic extracts of seed kernels of mango (Mangifera indica .L) on acute hind limb ischemia-reperfusion injury in diabetic rats

Background and aimHind limb ischemia is one of the peripheral arterial diseases affecting majority of the people with atherosclerosis, diabetes and chronic cigarette smokers. Hind limb ischemic-reperfusion injury is also one of the exacerbating events in these peoples, resulting in hind limb dysfunction. The aim of this study was to identify the effects of ethanolic extracts of mangifera indica (EEMI) on reversing hind limb dysfunction in diabetic rats with acute hind limb ischemia-reperfusion injury. Experimental procedureUnilateral femoral artery ligated diabetic rats were orally fed with EEMI (0.2 and 0.4 g/kg) for 14 days. At the end of the study, plasma levels of pro-inflammatory cytokines and relevant biochemical parameters were measured. The isolated gastrocnemius muscles were used for gene expression and histopathological studies. ResultsThere was a significant reduction (p < 0.05) in the plasma levels of pro-inflammatory cytokines, nitric oxide, malondialdehyde; and the expression levels of mRNA of induced nitric oxide synthase and intercellular adhesion molecule −1; and increase in anti-inflammatory cytokine, in isolated gastrocnemius muscles of animals treated with 0.2 and 0.4 g/kg of EEMI in comparison to disease control. In addition, histopathological study of gastrocnemius muscle and hind limb function test indicated the recovery of tissue damage from ischemic reperfusion at 0.2 and 0.4 g/kg of EEMI in comparison to disease control. ConclusionWe conclude that 14-day EEMI treatment of rats with acute hind limb ischemia/reperfusion in diabetic rats recovered from ischemic/reperfusion injury by modulating (decreasing) oxidative stress and inflammation.

Protective Effect of Hydrogen Gas on Mouse Hind Limb Ischemia-Reperfusion Injury

The aim of this study was to investigate the mechanism of hydrogen gas on hind limb IR injury. Male C57BL/6 mice were randomly divided into three groups: sham group (Sham), ischemia-reperfusion group (IR), IR plus H2 inhalation group (IR+H2). IR was induced by interrupting hind limb blood flow for 3h, followed by 4h of reperfusion, and H2 was administered by inhalation throughout the reperfusion process. Our data show that H2 inhalation could significantly decrease the infarct-affected tissue volume (P < 0.05), attenuate the degree of morphological injury (P < 0.05), and suppress the level of oxidative stress damage (P < 0.05), compared with the IR group. In exploring the underlying mechanisms, we found that hydrogen could markedly mitigate the degree of IR-induced ER stress and apoptosis (P < 0.05). Additionally, hydrogen could markedly inhibit the IR injury by modulating the phosphorylated c-Jun N-terminal kinase (JNK) signaling pathway (P < 0.05). Taken together, these results revealed the protective effect of hydrogen gas on hind limb ischemia reperfusion injury on mice by attenuating oxidative stress, impairing ER stress and apoptosis, and its ability to modulate JNK signaling pathway.

Preconditioning improves muscle regeneration after ischemia-reperfusion injury.

Ischemia-reperfusion injury (IRI) is a critical condition associated with serious clinical manifestations. Extensive research has focused on the strategies increasing organ tolerance to IRI. Preconditioning (PC) has been shown to provide protection to various organs toward IRI. However, the underlying mechanisms remain unknown. This study aimed to evaluate the role of PC on muscle regeneration after IRI and the potential underlying mechanisms. Three-month-old male UCP-1 reporter mice underwent unilateral hindlimb IRI with or without PC, the tissue viability and injury index were measured at 24 h after IRI. Hindlimb gait, muscle contractility, muscle histology were analyzed at 2 weeks after IRI. In another group of animals, β3 adrenergic receptor (β3AR) agonist amibegron and β3AR antagonist SR-59230A were administrated before PC/IRI, the hindlimb function and muscle regeneration were evaluated at 2 weeks after IRI. Our results showed that PC has little effect on improving the tissue viability at the acute phase of IRI, but it showed a long-term beneficial role of improving hindlimb function and muscle regeneration as evidenced by increased central nuclei regenerating myofibers. The effects of PC are related to inducing muscle fibro-adipogenic progenitor (FAP) brown/beige-like adipocyte (BAT) differentiation. Amibegron treatment displayed a similar role of PC while SR-59230A abolished the effect of PC. This study suggests PC has a beneficial role in promoting muscle regeneration after IRI through β3AR signaling pathway-stimulated FAP-BAT differentiation.

The role of neutrophil extracellular traps and TLR signaling in skeletal muscle ischemia reperfusion injury.

Ischemia reperfusion (IR) injury results in devastating skeletal muscle fibrosis. Here, we recapitulate this injury with a mouse model of hindlimb IR injury which leads to skeletal muscle fibrosis. Injury resulted in extensive immune infiltration with robust neutrophil extracellular trap (NET) formation in the skeletal muscle, however, direct targeting of NETs via the peptidylarginine deiminase 4 (PAD4) mechanism was insufficient to reduce muscle fibrosis. Circulating levels of IL-10 and TNFα were significantly elevated post injury, indicating toll-like receptor (TLR) signaling may be involved in muscle injury. Administration of hydroxychloroquine (HCQ), a small molecule inhibitor of TLR7/8/9, following injury reduced NET formation, IL-10, and TNFα levels and ultimately mitigated muscle fibrosis and improved myofiber regeneration following IR injury. HCQ treatment decreased fibroadipogenic progenitor cell proliferation and partially inhibited ERK1/2 phosphorylation in the injured tissue, suggesting it may act through a combination of TLR7/8/9 and ERK signaling mechanisms. We demonstrate that treatment with FDA-approved HCQ leads to decreased muscle fibrosis and increased myofiber regeneration following IR injury, suggesting short-term HCQ treatment may be a viable treatment to prevent muscle fibrosis in ischemia reperfusion and traumatic extremity injury.

Increased beneficial outcome of metformin by co-enzyme Q10 against experimentally-induced hind limb ischemia reperfusion injury in normal and diabetic rats

Background: Skeletal muscles are susceptible for ischemic reperfusion injury especially in settings in order to achieve homeostasis in traumatic injury and vascular surgery. This study aimed at investigating the implication of induction of diabetes on generation of ischemic reperfusion injury in rat gastrocnemius muscle. In addition, the possible beneficial effect of metformin and co-enzyme q10 was investigated.Methods: About 80 male adult Sprague Dawley rats divided into 10 groups. Metformin was administrated as continuous oral dose for 28 days. Coenzyme q10 was administrated parenterally 2 and 24 hours before induction of ischemia in diabetic and non-diabetic animals.Results: In diabetic ischemic groups, tested drugs either singly or in combination significantly reduce HB1cA and plasma levels of muscle specific enzyme CPK and muscle myokin IL6, raised natural antioxidant GSH and reduced oxidative stress parameters (SOD and MDA), apoptotic (caspase-3) and inflammatory parameters TNF-α and TGFβ were reduced. Continuous oral metformin for 28 days was more powerful than parenteral short-term coenzyme q10 as regards all tested parameters except for GSH and caspase-3 both drugs were equi-effective. Combined drugs have more powerful ameliorating effect than either drug singly except for HB1cA which was equi-effective with that of metformin. Regarding non diabetic ischemic groups, metformin was more powerful in reduction of caspase-3, IL6 and TNF- α while coenzyme q10 was more powerful in elevating GSH.Conclusions: Co-enzyme q10 can be used as add on therapy with metformin in order to decrease the deleterious effects resulted from hind limb ischemia reperfusion in normal and diabetic rats.

Extracorporeal Filtration of Potassium in a Swine Model of Bilateral Hindlimb Ischemia–Reperfusion Injury With Severe Acute Hyperkalemia

Options for the treatment of hyperkalemia in the pre-hospital setting are limited, particularly in the context of natural disaster or during combat operations. Contemporary interventions require extensive resources and technical expertise. Here we examined the potential for a simple, field deployable bridge-dialysis as a countermeasure for acute hyperkalemia induced by prolonged ischemia-reperfusion. Twenty female swine were randomized into two experimental groups undergoing a 2-hour bilateral hindlimb ischemia-reperfusion injury. Subsequent to injury, hemoperfusion was performed in the presence (Column) and absence (Sham Control) of a high-affinity potassium-binding column (CytoSorbents, Monmouth Junction, NJ, USA). Serial blood gas and chemistries were sampled. Primary endpoint was changed in serum potassium concentrations post-injury and filtration. Serum potassium was significantly elevated following ischemia-reperfusion injury in both groups (149% (12) and 150% (22), p < 0.05 vs respective baseline values). There were no differences observed between groups in respect to physiologic parameters; mean arterial pressure, heart rate, systemic vascular resistance, cardiac output, or central venous oxygenation. Filtration resulted in a significant relative decrease in potassium compared with controls after the first hour as determined by repeated measures two-way ANOVA (p < 0.0001) which continued through end of the study. Significant thrombocytopenia was observed in animals undergoing filtration with a mean reduction in platelets measured at T = 480 minutes (168 × 103μL, p < 0.0001 vs baseline). We demonstrate that serum potassium can be filtered via hemoperfusion utilizing a simple extracorporeal potassium-binding platform, though evolution of this technology will be required to achieve meaningful reduction of potassium in clinically significant hyperkalemia after trauma.

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.

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.

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.

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.

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.