Limb Ischemic Postconditioning Research Articles

Remote limb ischemic postconditioning inhibits microglia pyroptosis by modulating HGF after acute ischemia stroke.

The repetitive inflation-deflation of a blood pressure cuff on a limb is known as remote limb ischemic postconditioning (RIPostC). It prevents brain damage induced by acute ischemia stroke (AIS). Pyroptosis, executed by the pore-forming protein gasdermin D (GSDMD), is a type of regulated cell death triggered by proinflammatory signals. It contributes to the pathogenesis of ischemic brain injury. However, the effects of RIPostC on pyroptosis following AIS remain largely unknown. In our study, linear correlation analysis confirmed that serum GSDMD levels in AIS patients upon admission were positively correlated with NIHSS scores. RIPostC treatment significantly reduced GSDMD level compared with patients without RIPostC at 3 days post-treatment. Besides, middle cerebral artery occlusion (MCAO) surgery was performed on C57BL/6 male mice and RIPostC was induced immediately after MCAO. We found that RIPostC suppressed the activation of NLRP3 inflammasome to reduce the maturation of GSDMD, leading to decreased pyroptosis in microglia after AIS. Hepatocyte growth factor (HGF) was identified using the high throughput screening. Importantly, HGF siRNA, exogenous HGF, and ISG15 siRNA were used to reveal that HGF/ISG15 is a possible mechanism of RIPostC regulation in vivo and in vitro.

Remote limb ischemic postconditioning attenuates myocardial dysfunction induced by testicular torsion/detorsion in rats.

Torsion of the spermatic cord is a urological emergency that must be treated immediately with surgery, yet detorsion of the testis can cause testicular tissue damage because of ischemia‒reperfusion (I/R) injury. I/R injury is a complex pathophysiological process that may affect the functions of distant organs. Here, we examined whether testicular torsion/detorsion causes myocardial dysfunction. We next investigated the potential beneficial effect and underlying mechanisms of remote ischemic postconditioning (RIPost) on cardiac function after testicular torsion/detorsion. Male Sprague Dawley rats were assigned to three different sets of experimental groups. Testicular I/R was induced by rotating the right testis to 1080 degrees clockwise for 3 hours followed by 3 h of detorsion. RIPost was induced at the onset of testicular detorsion by four cycles of 5-min bilateral femoral artery occlusion with 5-min reperfusion. Cardiac function was determined postdetorsion, and the cardioprotective effect of RIPost was examined. Testicular torsion/detorsion-treated rats had reduced serum testosterone levels, impaired systemic hemodynamics, elevated systemic inflammatory responses and increased serum levels of LDH, CK-MB, α-HBDH and cTnI. Healthy rats transfused with testicular torsion/detorsion-treated plasma presented impaired cardiac function. However, RIPost attenuated remote heart dysfunction induced by testicular torsion/detorsion. Furthermore, RIPost enhanced the phosphorylation of ventricular STAT-3, which is a key component of the SAFE signaling pathways. Inhibition of STAT-3 with Ag490 abolished the RIPost-induced cardioprotection and STAT-3 phosphorylation. Testicular torsion followed by detorsion resulted in impairment of cardiac function. RIPost effectively attenuates this remote cardiac dysfunction. RIPost-induced protective effects may be mediated by the STAT-3 signaling pathway.

Protective effect of remote ischemic postconditioning in rat testes after testicular torsion/detorsion.

Testicular torsion/detorsion can lead to severe testicular damage. The organ-protective effect of remote ischemic postconditioning (RLIPost) against ischemia/reperfusion injury has been characterized; however, it remains unknown whether RLIPost has a testicular protective effect. Here, we tested the hypothesis that RLIPost can protect the testes in a rat model of testicular torsion/detorsion in vivo. Male Sprague-Dawley rats were assigned to sham-operated, control, or remote liver and limb ischemic postconditioning-treated groups. Testicular torsion/detorsion was performed by 3h of testicular torsion (720° clockwise unilateral spermatic cord torsion), followed by 3h of detorsion. For liver and limb ischemic postconditioning, four cycles of 5 min of liver or limb ischemia with 5-min intermittent reperfusion stimuli were conducted at the onset of testicular reperfusion. Liver and limb ischemic postconditioning significantly ameliorated ipsilateral and contralateral testicular swelling responses, preserved morphological integrity and spermatogenesis and inhibited testicular apoptosis. In addition, RLIPost enhanced the phosphorylation of AKT/ERK1/2/GSK-3β/STAT-3 in the ipsilateral testis while suppressing JNK activation in the ipsilateral and contralateral testes. To the best of our knowledge, this study is the first to demonstrate the involvement of RLIPost in an animal model of testicular torsion/detorsion. We showed that RLIPost protects both ipsilateral and contralateral testes against testicular torsion/detorsion in vivo, via at least in part, the RISK and SAFE-mediated signaling pathways.

Remote Limb Ischemic Postconditioning Protects Against Ischemic Stroke by Promoting Regulatory T Cells Thriving.

BackgroundRemote limb ischemic postconditioning (RLIPoC) has been demonstrated to protect against ischemic stroke. However, the underlying mechanisms of RLIPoC mediating cross‐organ protection remain to be fully elucidated.Methods and ResultsIschemic stroke was induced by middle cerebral artery occlusion for 60 minutes. RLIPoC was performed with 3 cycles of 10‐minute ischemia followed by 10‐minute reperfusion of the bilateral femoral arteries immediately after middle cerebral artery reperfusion. The percentage of regulatory T cells (Tregs) in the spleen, blood, and brain was detected using flow cytometry, and the number of Tregs in the ischemic hemisphere was counted using transgenic mice with an enhanced green fluorescent protein‐tagged Foxp3. Furthermore, the metabolic status was monitored dynamically using a multispectral optical imaging system. The Tregs were conditionally depleted in the depletion of Treg transgenic mice after the injection of the diphtheria toxin. The inflammatory response and neuronal apoptosis were investigated using immunofluorescent staining. Infarct volume and neurological deficits were evaluated using magnetic resonance imaging and the modified neurological severity score, respectively. The results showed that RLIPoC substantially reduced infarct volume, improved neurological function, and significantly increased Tregs in the spleen, blood, and ischemic hemisphere after middle cerebral artery occlusion. RLIPoC was followed by subsequent alteration in metabolites, such as flavin adenine dinucleotide and nicotinamide adenine dinucleotide hydrate, both in RLIPoC‐conducted local tissues and circulating blood. Furthermore, nicotinamide adenine dinucleotide hydrate can mimic RLIPoC in increasing Tregs. Conversely, the depletion of Tregs using depletion of Treg mice compromised the neuroprotective effects conferred by RLIPoC.ConclusionsRLIPoC protects against ischemic brain injury, at least in part by activating and maintaining the Tregs through the nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide hydrate pathway.

Ischemic postconditioning reduces spinal cord ischemia-reperfusion injury through ATP-sensitive potassium channel.

Animal study. Explore the neuroprotective effect of remote limb ischemic postconditioning (Post C) in spinal cord ischemic reperfusion injury (SCII) and related mechanisms. Anesthesiology Laboratory of Southwest Medical University. We established a rabbit SCII model and processed it with Post C. To evaluate the neural function, spinal cord tissue was taken 48 h later, normal neurons were evaluated by HE staining, and the expression of ATP-sensitive potassium channel (KATP) marker molecule Kir6.2 was detected by Western blot. Immunofluorescence detection of spinal cord Iba-1 expression, ELISA detection of M1 type microglia marker iNOS and M2 type microglia marker Arg, and Western blot detection of NF-κB and IL-1β expression. Through these experiments, we will explore the protective effect of Post C in SCII, observe the changes in the protective effect after using KATP blockers, and verify that Post C can play a neuroprotective effect in SCII by activating KATP. We observed that Post C significantly improved exercise ability and the number of spinal motor neurons in the SCII model. Microglia are activated and expression of M1 microglia in the spinal cord was decreased, while M2 was increased. This neuroprotective effect was reversed by the nonspecific KATP inhibitor. Post C has a neuroprotective effect on SCII, and maybe a protective effect produced by activating KATP to regulate spinal microglia polarization and improve neuroinflammation.

The hypoxia sensitive metal transcription factor MTF-1 activates NCX1 brain promoter and participates in remote postconditioning neuroprotection in stroke

Remote limb ischemic postconditioning (RLIP) is an experimental strategy in which short femoral artery ischemia reduces brain damage induced by a previous harmful ischemic insult. Ionic homeostasis maintenance in the CNS seems to play a relevant role in mediating RLIP neuroprotection and among the effectors, the sodium-calcium exchanger 1 (NCX1) may give an important contribution, being expressed in all CNS cells involved in brain ischemic pathophysiology. The aim of this work was to investigate whether the metal responsive transcription factor 1 (MTF-1), an important hypoxia sensitive transcription factor, may (i) interact and regulate NCX1, and (ii) play a role in the neuroprotective effect mediated by RLIP through NCX1 activation. Here we demonstrated that in brain ischemia induced by transient middle cerebral occlusion (tMCAO), MTF-1 is triggered by a subsequent temporary femoral artery occlusion (FAO) and represents a mediator of endogenous neuroprotection. More importantly, we showed that MTF-1 translocates to the nucleus where it binds the metal responsive element (MRE) located at −23/−17 bp of Ncx1 brain promoter thus activating its transcription and inducing an upregulation of NCX1 that has been demonstrated to be neuroprotective. Furthermore, RLIP restored MTF-1 and NCX1 protein levels in the ischemic rat brain cortex and the silencing of MTF-1 prevented the increase of NCX1 observed in RLIP protected rats, thus demonstrating a direct regulation of NCX1 by MTF-1 in the ischemic cortex of rat exposed to tMCAO followed by FAO. Moreover, silencing of MTF-1 significantly reduced the neuroprotective effect elicited by RLIP as demonstrated by the enlargement of brain infarct volume observed in rats subjected to RLIP and treated with MTF-1 silencing. Overall, MTF-dependent activation of NCX1 and their upregulation elicited by RLIP, besides unraveling a new molecular pathway of neuroprotection during brain ischemia, might represent an additional mechanism to intervene in stroke pathophysiology.

Remote Limb Ischemic Postconditioning Protects against Ischemic Stroke via Modulating Microglia/Macrophage Polarization in Mice.

Aim The protection against ischemia/reperfusion injury mediated by remote limb ischemic postconditioning (RIPC) shows great clinical value in ischemic stroke therapy, but the particular mechanism of RIPC remains unclear. Methods We carried out middle cerebral artery occlusion/reperfusion (MCAO/R) surgery on C57BL/6 male mice. RIPC was generated by 10-minute occlusion followed by the same period of reperfusion of the bilateral hind limb femoral artery and repeated for 3 cycles. Infarct size and neurological score were performed to assess stroke outcomes. Ly6Chi monocytes were quantified in the blood and brain by flow cytometry. Real-time PCR, ELISA, and immunofluorescence were utilized to detect phenotype of proinflammatory M1 and anti-inflammatory M2 microglia/macrophage. Nuclear factor κB (NF-κB) and peroxisome proliferator-activated receptor γ (PPARγ) levels were detected using Western blot. Results At 24 and 72 h after MCAO, RIPC drastically attenuated infarct size and ameliorated the neurological deficits of mice and facilitated transmigration of Ly6Chi monocytes to the brain postischemia reperfusion. Furthermore, RIPC contributed to increased M2 and reduced M1 microglia/macrophage through inhibiting NF-κB and promoting PPARγ activation. Conclusion Our results reveal pharmacological effect of RIPC in promoting microglia/macrophage transferring from M1 to M2 phenotype after MCAO/R in mice, which provides theoretical support for the therapeutic effect of RIPC in ischemic stroke.

The neuroprotective role of SIRT1/PGC-1α signaling in limb postconditioning in cerebral ischemia/reperfusion injury

Limb ischemic postconditioning (LPostC) is an innovative treatment for ischemia/reperfusion injury (IRI). However, its mechanisms have not yet been elucidated. Herein, we assessed the importance of SIRT1/PGC-1α signaling in LPostC neuroprotection following cerebral I/R injury in rats. In this study, we used 40 male SD rats that were separated into sham, I/R, LPostC, and LPostC + EX-527 (SIRT1 inhibitor) groups (each with 10 rats), with a middle cerebral artery occlusion (MCAO) model used to induce IRI. LPostC was induced via three cycles of bilateral femoral artery occlusion and non-occlusion. At 24 h, we examined SIRT1 and PGC-1α protein levels by western blotting in ischemic areas. The mRNA levels of SIRT1, PGC-1α, NRF-1 and CytoC1 in the ischemic area were assessed by qRT-PCR. We also quantified neurological deficit scores and evaluated cerebral infarct volume by TTC staining. TUNEL staining was used to evaluate the apoptotic rates in neurons. In addition, antioxidant SOD activity and MDA levels were measured by the Microplate Reader. Our findings indicated that LPostC increased the protein and mRNA levels of SIRT1 and PGC-1α in cerebral ischemic tissue, then up-regulated the downstream protein NRF-1, down-regulated CytoC1, and improved mitochondrial function, thereby reducing brain damage. LPostC relieved cerebral IRI via reducing the size of the cerebral infarct, neuronal apoptosis, and neurological deficits. Meanwhile LPostC increased SOD activity and reduced MDA content in brain tissue. Treatment with EX-527 reversed the protection of LPostC after IRI (all P < 0.05). This suggests that LPosC may protect against cerebral IRI at least in part via up-regulating the SIRT1/PGC-1α signaling pathway, thereby increasing the individual’s ability to resist oxidative stress.

Limb Ischemic Postconditioning Alleviates Postcardiac Arrest Syndrome through the Inhibition of Mitochondrial Permeability Transition Pore Opening in a Porcine Model.

Objective Previously, the opening of mitochondrial permeability transition pore (mPTP) was confirmed to play a key role in the pathophysiology of postcardiac arrest syndrome (PCAS). Recently, we demonstrated that limb ischemic postconditioning (LIpostC) alleviated cardiac and cerebral injuries after cardiac arrest and resuscitation. In this study, we investigated whether LIpostC would alleviate the severity of PCAS through inhibiting mPTP opening. Methods Twenty-four male domestic pigs weighing 37 ± 2 kg were randomly divided into three groups: control, LIpostC, and LIpostC+atractyloside (Atr, the mPTP opener). Atr (10 mg/kg) was intravenously injected 30 mins prior to the induction of cardiac arrest. The animals were subjected to 10 mins of untreated ventricular fibrillation and 5 mins of cardiopulmonary resuscitation. Coincident with the beginning of cardiopulmonary resuscitation, LIpostC was induced by four cycles of 5 mins of limb ischemia and then 5 mins of reperfusion. The resuscitated animals were monitored for 4 hrs and observed for an additional 68 hrs. Results After resuscitation, systemic inflammation and multiple organ injuries were observed in all resuscitated animals. However, postresuscitation systemic inflammation was significantly milder in the LIpostC group than in the control group. Myocardial, lung, and brain injuries after resuscitation were significantly improved in the LIpostC group compared to the control group. Nevertheless, pretreatment with Atr abolished all the protective effects induced by LIpostC. Conclusion LIpostC significantly alleviated the severity of PCAS, in which the protective mechanism was associated with the inhibition of mPTP opening.

Remote postconditioning ameliorates stroke damage by preventing let-7a and miR-143 up-regulation.

Remote limb ischemic postconditioning (RLIP) is a well-established neuroprotective strategy able to protect the brain from a previous harmful ischemic insult through a sub-lethal occlusion of the femoral artery. Neural and humoral mechanisms have been proposed as mediators required to transmit the peripheral signal from limb to brain. Moreover, different studies suggest that protection observed at brain level is associated to a general genetic reprogramming involving also microRNAs (miRNAs) intervention.Methods: Brain ischemia was induced in male rats by transient occlusion of the middle cerebral artery (tMCAO), whereas RLIP was achieved by one cycle of temporary occlusion of the ipsilateral femoral artery after tMCAO. The expression profile of 810 miRNAs was evaluated in ischemic brain samples from rats subjected either to tMCAO or to RLIP. Among all analyzed miRNAs, there were four whose expression were upregulated after stroke and returned to basal level after RLIP, thus suggesting a possible involvement in RLIP-induced neuroprotection. These selected miRNAs were intracerebroventricularly infused in rats subjected to remote ischemic postconditioning, and their effect was evaluated in terms of brain damage, neurological deficit scores and expression of putative targets.Results: Twenty-one miRNAs, whose expression was significantly affected by tMCAO and by tMCAO plus RLIP, were selected based on microarray microfluidic profiling. Our data showed that: (1) stroke induced an up-regulation of let-7a and miR-143 (2) these two miRNAs were involved in the protective effects induced by RLIP and (3) HIF1-α contributes to their protective effect. Indeed, their expression was reduced after RLIP and the exogenous intracerebroventricularly infusion of let-7a and miR-143 mimics prevented neuroprotection and HIF1-α overexpression induced by RLIP.Conclusions: Prevention of cerebral let-7a and miR-143 overexpression induced by brain ischemia emerges as new potential strategy in stroke intervention.

The changes of systemic immune responses during the neuroprotection induced by remote ischemic postconditioning against focal cerebral ischemia in mice

ABSTRACTObjective: Remote limb ischemic postconditioning (RIPostC) protects the brain from damage induced by transient focal ischemia/reperfusion. However, the underlying mechanism remains unclear.Methods: RIPostC induced by 10 min of occlusion and another 10 min releasing of blood flow for three cycles in the hind limbs was performed immediately after the reperfusion in a focal ischemia mice model. Neurological scores, immune cell population in the blood, spleen and lymph node, and inflammatory factors in the blood and brain were analyzed 2 days after the reperfusion.Results: Our results demonstrate that RIPostC reduced cerebral injuries and improved neurological functions 2 days after reperfusion. RIPostC significantly inhibited the reduction in the percentage of CD4 T cells in the spleen and lymph node, CD8 T cells in the blood and lymph node, and natural killer T (NKT) cells in the spleen by flow cytometry analysis. RIPostC attenuated the increase of B cells and NK cells in the spleen and noninflammatory monocytes in the blood. The cytokine assay showed that RIPostC decreased the elevation of IL-10, IL-6, and TNF-α in the blood after ischemia. The quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) results indicated that the mRNA level of IL-4 in the brain increased in the middle cerebral artery occlusion mice after RIPostC treatment.Conclusions: The present study indicates that there were significant changes of inflammatory responses during the neuroprotection induced by RIPostC in stroke mice.

Upper Limb Ischemic Postconditioning as Adjunct Therapy in Acute Stroke Patients: A Randomized Pilot

ObjectiveThis study aims to observe the clinical effect of upper limb ischemic postconditioning (LIPostC) as an adjunct to treatment with acute stroke patients, possibly due to increased cerebral perfusion. MethodsWe perform a randomized blinded placebo controlled trial in nonthrombolysis patients with acute ischemic stroke, within 72hours of ictus, divided into the LIPostC group and control group. The LIPostC group is induced by 4 cycles of intermittent repeated limb ischemia: alternating 5 minutes inflation (20mm Hg above systolic blood pressure) and 5 minutes deflation performed manually using a standard upper arm blood pressure cuff in the nonparetic arm. The control group receives a sham procedure (cuff inflation to 30mm Hg). Patients underwent the intervention from the time of enrollment to Day 14. Comparison of National Institutes of Health Stroke Scale (NIHSS) score, cerebral infarction volume, relative Perfusion weighted imaging (PWI) parameters (regional relative cerebral blood flow, regional relative mean transit time; preintervention [day 0], day 14, day 90), modified Rankin Scale (mRS; the preintervention score [day 0], the curative ratio at day 90 [we define 0-1 score as close to recovery or full recovery]). ResultsSixty eligible patients with acute stroke (29 LIPostC and 31 control) are recruited age 65years (SD 12.22), blood pressure 156/74mm Hg (SD 14/10), and NIHSS score 5.98 (SD 3.35), mRS score 2.25 (SD .79). Only 1 in the LIPostC group is intolerant the first cycle to give up. All patients tolerate the sham procedure. Two patients experience recurrent stroke versus none in the LIPostC group. Day 90, compared with the control group, there is a significant decrease the NIHSS score, regional relative mean transit time (P < .05) and increase the curative ratio of mRS, regional relative cerebral blood flow(P < .05) in the LIPostC group, which infarct volume decreased by 31.3% (P < .05). ConclusionsLIPostC after acute stroke is well tolerated and appears safe and feasible. LIPostC may improve neurological outcome, and protective mechanisms may be increased cerebral blood flow to improve cerebral perfusion. A larger trial is warranted.

Remote limb ischemic postconditioning protects against cerebral ischemia-reperfusion injury by activating AMPK-dependent autophagy

Remote limb ischemic postconditioning (RIPoC) is a promising adjunct treatment for cerebral ischemia-reperfusion (IR) injury. However, the underlying mechanisms have not been fully elucidated yet. The present study aims to investigate potential involvement and regulatory mechanisms of autophagy in RIPoC treatment against cerebral IR injury in mice. Mice were subjected to 2 h middle cerebral artery occlusion (MCAO) then treated with vehicle, 3-methyladenine (3-MA, an autophagy inhibitor), or compound C (an AMPK inhibitor) at the onset of reperfusion. RIPoC was carried out by 3 cycles of 10-min occlusion-reperfusion of bilateral femoral artery at the beginning of the reperfusion. Infarct volume, neurological score, and brain water content of the mice were assessed after 12 h reperfusion. Autophagy markers, cell apoptosis markers, and AMPK pathway activity were also evaluated. Our results indicated that RIPoC treatment reduced neurological deficits, brain water content, and infarct volume after IR. Meanwhile, RIPoC was proved to induce autophagy and activate AMPK pathway. Furthermore, the RIPoC-induced autophagy and neuroprotection were abolished by 3-MA and partially blocked by compound C. In conclusion, the present study suggests that RIPoC attenuates cerebral IR injury by activating AMPK-dependent autophagy.

Remote limb ischemic postconditioning promotes motor function recovery in a rat model of ischemic stroke via the up-regulation of endogenous tissue kallikrein.

Remote ischemic conditionings, such as pre- and per-conditioning, are known to provide cardioprotection in animal models of ischemia. However, little is known about the neuroprotection effect of postconditioning after cerebral ischemia. In this study, we aim to evaluate the motor function rescuing effect of remote limb ischemic postconditioning (RIPostC) in a rat model of acute cerebral stroke. Left middle cerebral artery occlusion (MCAO) was performed to generate the rat model of ischemic stroke, followed by daily RIPostC treatment for maximum 21days. The motor function after RIPostC was assessed with foot fault test and balance beam test. Local infarct volume was measured through MRI scanning. Neuronal status was evaluated with Nissl's, HE, and MAP2 immunostaining. Lectin immunostaining was performed to evaluate the microvessel density and area. Daily RIPostC for more than 21days promoted motor function recovery and provided long-lasting neuroprotection after MCAO. Reduced infarct volume, rescued neuronal loss, and enhanced microvessel density and size in the injured areas were observed. In addition, the RIPostC effect was associated with the up-regulation of endogenous tissue kallikrein (TK) level in circulating blood and local ischemic brain regions. A TK receptor antagonist HOE-140 partially reversed RIPostC-induced improvements, indicating the specificity of endogenous TK mediating the neuroprotection effect of RIPostC. Our study demonstrates RIPostC treatment as an effective rehabilitation therapy to provide motor function recovery and alleviate brain impairment in a rat model of acute cerebral ischemia. We also for the first time provide evidence showing that the up-regulation of endogenous TK from remote conditioning regions underlies the observed effects of RIPostC.

Mechanism underlying inhibition of inflammatory responses induced by α7nAChR agonist postconditioning alone or in combination with remote limb ischemic postconditioning during myocardial I/R in rats: the relationship with GSK-3β

Objective To evaluate the relationship between the mechanism underlying inhibition of inflammatory responses induced by α7 nicotinic acetylcholine receptor(α7nAChR)agonist postconditioning alone or in combination with remote limb ischemic postconditioning during myocardial ischemia-reperfusion(I/R)and glycogen synthase kinase-3β(GSK-3β)in rats. Methods Eighty adult male Sprague-Dawley rats, aged 8 weeks, weighing 290-320 g, were divided into 4 groups(n=20 each)using a random number table: I/R group, α7nAChR agonist postconditioning group(group P), remote limb ischemic postconditioning group(group L)and α7nAChR agonist postconditioning plus remote limb ischemic postconditioning group(group P+ L). Myocardial I/R was induced by 30 min occlusion of the left anterior descending branch of coronary artery followed by 120 min reperfusion.Specific α7nAChR agonist PNU282987 2 mg/kg was intravenously injected immediately before reperfusion in group P. In group L, limb ischemia was induced by tourniquet occlusion of bilateral hind paws for 10 min starting from 20 min of myocardial ischemia, and the tourniquet was released at the beginning of reperfusion.Combination of intervention measures previously described in P and L groups was performed in group P+ L.Venous blood samples were taken at 120 min of reperfusion for determination of serum troponin I(TnI)and creatine kinase-MB(CK-MB)concentrations, myocardial infarct size(IS)and expression of phosphorylated GSK-3β [p-GSK-3β(Ser536)], NF-κBp65 and phosphorylated nuclear factor-κBp65(p-NF-κBp65)in myocardial tissues(by Western blot). Results Compared with group I/R, myocardial IS and serum cTnI and CK-MB concentrations were significantly decreased, the expression of p-GSK-3β(Ser9)in ischemic area was up-regulated, and the expression of p-NF-κBp65 in ischemic area was down-regulated in P, L and P+ L groups(P<0.05). Compared with group L, myocardial IS and serum cTnI and CK-MB concentrations were significantly decreased, the expression of p-GSK-3β(Ser9)in ischemic area was up-regulated, and the expression of p-NF-κBp65 in ischemic area was down-regulated in group P+ L(P<0.05). Conclusion The mechanism by which α7nAChR agonist postconditioning alone or in combination with remote limb ischemic postconditioning inhibits inflammatory responses during myocardial I/R may be related to inhibiting GSK-3β activity in rats. Key words: Myocardial reperfusion injury; Inflammation; Glycogen synthase kinase 3; Cholinergic agonists; Extremities; Ischemic postconditioning

Remote limb ischemic post conditioning during early reperfusion alleviates cerebral ischemic reperfusion injury via GSK-3β/CREB/ BDNF pathway

Remote limb ischemic post conditioning (RIPOC) has been reported to attenuate cerebral ischemic reperfusion (I/R) injury, while the molecular mechanisms remain elusive. Various studies have highlighted the involvement of glycogen synthase kinase (GSK-3β) in cerebral I/R injury and cognitive disorders. Hence, the present study was designed to explore the role of GSK-3β and its downstream regulators in RIPOC mediated neuroprotection against cerebral I/R injury and associated cognitive impairment. Male Wistar rats are randomly assigned into four groups: Sham, bilateral common carotid arteries occlusion (BCCAO), RIPOC and BCCAO+RIPOC. BCCAO was achieved by transient occlusion of bilateral common carotid arteries for 20min, followed by reperfusion. Non-invasive RIPOC was induced by 3 cycles each of 10min occlusion and reperfusion of both femoral arteries by using tourniquets, during early reperfusion phase. A battery of behavioral and cognitive tests were performed. Biochemical estimation of oxidative markers, anti-oxidants and pro-inflammatory markers were estimated. Levels of GSK-3β, CREB and BDNF were estimated to confirm the molecular mechanism. Hippocampal structural abnormalities were confirmed by H and E staining. The neurobehavioral analysis revealed that neurological and cognitive deficits caused by BCCAO, were reduced by RIPOC intervention. Meanwhile, the results of biochemical tests suggested that RIPOC attenuates the BCCAO induced oxidative damage, neuroinflammation and hippocampal structural abnormalities. Further, RIPOC prevented the elevation of BCCAO induced GSK-3β. RIPOC exerts neuroprotective effect against I/R injury, putatively by attenuating GSK-3β expression and upregulating the levels of CREB and BDNF.

Role of PI3K/Akt or JAK/STAT-3 signaling pathways in reduction of myocardial ischemia-reperfusion injury by combination of limb ischemic and morphine postconditioning in rats

Objective To evaluate the role of the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)or Janus kinase(JAK)/signal transducer and activator of transcription 3(STAT-3)signaling pathways in reduction of myocardial ischemia-reperfusion(I/R)injury by combination of limb ischemic and morphine postconditioning in rats. Methods Eighty SPF healthy male Sprague-Dawley rats, aged 8 weeks, weighing 280-320 g, were used in the study.Myocardial I/R was induced by occlusion of the left anterior descending branch of the coronary artery for 30 min followed by 120 min reperfusion.The rats were divided into 8 groups(n=10 each)using a random number table: I/R group, limb ischemic postconditioning group(LIP group), morphine postconditioning group(group MP), combination of limb ischemic and morphine postconditioning group(LIP+ MP group)and signaling pathway blocker groups(I/Rb group, LIPb group, MPb group, LIP+ MPb group). In I/R, LIP, MP and LIP+ MP groups, the animals were sacrificed at the end of reperfusion, and myocardial specimens in ischemic and non-ischemic regions were obtained for determination of phosphorylated STAT-3(p-STAT-3), STAT-3, phosphorylated Akt(p-Akt)and Akt expression(by Western blot)and STAT-3 and Akt mRNA expression(by polymerase chain reaction). In I/Rb, LIPb, MPb and LIP+ MPb groups, PI3K/Akt signaling pathway blocker LY294002 0.3 mg/kg was intravenously injected in 5 rats of each group, and JAK/STAT-3 signaling pathway blocker AG490 5 mg/kg was intravenously injected in the other 5 rats of each group.The animals were sacrificed at the end of reperfusion, and myocardial specimens in the ischemic region were obtained for determination of myocardial infarct size. Results Compared with I/R group, the p-STAT-3/STAT-3 ratio in LIP, MP and LIP+ MP groups and p-Akt/Akt ratio in LIP+ MP group were significantly increased, and the expression of STAT-3 and Akt mRNA was up-regulated in LIP+ MP group(P 0.05). When JAK2 inhibitor AG490 was applied, the myocardial infarct size was significantly smaller in LIP+ MPb group than in I/Rb, LIPb and MPb groups(P 0.05). Conclusion Combination of limb ischemic and morphine postconditioning can enhance the activation of PI3K/Akt or JAK/STAT-3 signaling pathways, and the cardioprotection is dependent on the integrity of the PI3K/Akt signaling pathway and partially dependent on the integrity of the JAK/STAT-3 signaling pathway when applied in combination in rats. Key words: 1-Phosphatidylinositol 3-kinase; Protein-tyrosine kinases; Extremities; Morphine; Myocardial reperfusion injury; Postconditioning

Remote ischemic postconditioning protects against renal ischemia/reperfusion injury by activation of T-LAK-cell-originated protein kinase (TOPK)/PTEN/Akt signaling pathway mediated anti-oxidation and anti-inflammation

BackgroundRecent clinical and animal studies suggested that remote limb ischemic postconditioning (RIPostC) can invoke potent cardioprotection or neuroprotection. However, the effect and mechanism of RIPostC against renal ischemia/reperfusion injury (IRI) are poorly understood. T-LAK-cell-originated protein kinase (TOPK) is crucial for the proliferation and migration of tumor cells. However, the function of TOPK and the molecular mechanism underlying renal protection remain unknown. Therefore, this study aimed to evaluate the role of TOPK in renoprotection induced by RIPostC. Materials and MethodsThe renal IRI model was induced by left renal pedicle clamping for 45min followed by 24h reperfusion and right nephrectomy. All mice were intraperitoneally injected with vehicle, TOPK inhibitor HI-TOPK-032 or Akt inhibitor LY294002. After 24h reperfusion, renal histology, function, and inflammatory cytokines and oxidative stress were assessed. The proteins were measured by Western blotting. ResultsThe results showed that RIPostC significantly protected the kidneys against IRI. The protective effects were accompanied by the attenuation of renal dysfunction, tubular damage, inflammation and oxidative stress. In addition, RIPostC increased the phosphorylation of TOPK, PTEN, Akt, GSK3β and the nuclear translocation of Nrf2 and decreased the nuclear translocation of NF-κB. However, all of the above renoprotective effects of RIPostC were eliminated either by the inhibition of TOPK or Akt with HI-TOPK-032 or LY294002. ConclusionsThe current data reveal that RIPostC protects against renal IRI via activation of TOPK/PTEN/Akt signaling pathway mediated anti-oxidation and anti-inflammation.

36th International Symposium on Intensive Care and Emergency Medicine : Brussels, Belgium. 15-18 March 2016.

36th International Symposium on Intensive Care and Emergency Medicine : Brussels, Belgium. 15-18 March 2016.

Effect of limb ischemic post-conditioning in combination with hypothermia on brain injury after cardiopulmonary resuscitation in swine

Objective To evaluate the effect of limb ischemic post-conditioning (LIPC) in combination with hypothermia on brain injury after cardiopulmonary resuscitation (CPR) in swine. Methods Eighteen adult male white swine, weighing 35-40 kg, were used.The swine model of brain injury was established by inducing ventricular fibrillation for 10 min followed by 5 min of CPR.The animals were randomly divided into 3 groups (n=6 each) using a random number table: cardiac arrest-CPR group (group CA-CPR), group LIPC, and LIPC combined with hypothermia group (group LIPC+ H). In LIPC and LIPC+ H groups, LIPC was performed at the onset of CPR.In addition, the body temperature was cooled down to 32-34 ℃ by using a cooling blanket after successful resuscitation, and then maintained at this level until 4 h after resuscitation, followed by 4 h of re-warming at a rate of 1 ℃/h in group LIPC+ H.At 24, 48 and 72 h after resuscitation (T1-3), neurologic deficit score (NDS) and neurologic alertness score (NAS) were evaluated.Blood samples were collected from the auricular vein for determination of the concentrations of neuron-specific enolase (NSE) and S100B protein in serum by enzyme-linked immunosorbent assay. Results Compared with group CA-CPR, the NDS was significantly decreased, and the NAS was increased at T1-3, and the concentrations of NSE and S100B protein in serum were decreased at T2, 3 in LIPC and LIPC+ H groups (P<0.05). Compared with group LIPC, the NDS was significantly decreased, and the NAS was increased at T1-3, and the concentrations of NSE and S100B protein in serum were decreased at T3 in group LIPC+ H (P<0.05). Conclusion The combination of LIPC and hypothermia provides better efficacy than either alone in reducing brain injury after CPR in swine. Key words: Hypothermia, induced; Ischemic postconditioning; Extremities; Heart arrest; Cardiopulmonary resuscitation; Reperfusion injury; Brain