Treatment with β-sitosterol ameliorates the effects of cerebral ischemia/reperfusion injury by suppressing cholesterol overload, endoplasmic reticulum stress, and apoptosis

β-Sitosterol is a type of phytosterol that occurs naturally in plants.Previous studies have shown that it has anti-oxidant,anti-hyperlipidemic,anti-inflammatory,immunomodulatory,and anti-tumor effects,but it is unknown whetherβ-sitosterol treatment reduces the effects of ischemic stroke.Here we found that,in a mouse model of ischemic stroke induced by middle cerebral artery occlusion,β-sitosterol reduced the volume of cerebral infarction and brain edema,reduced neuronal apoptosis in brain tissue,and alleviated neurological dysfunction;moreover,β-sitosterol increased the activity of oxygen-and glucose-deprived cerebral cortex neurons and reduced apoptosis.Further investigation showed that the neuroprotective effects ofβ-sitosterol may be related to inhibition of endoplasmic reticulum stress caused by intracellular cholesterol accumulation after ischemic stroke.In addition,β-sitosterol showed high affinity for NPC1L1,a key transporter of cholesterol,and antagonized its activity.In conclusion,β-sitosterol may help treat ischemic stroke by inhibiting neuronal intracellular cholesterol overload/endoplasmic reticulum stress/apoptosis signaling pathways.

The miR-9-5p/CXCL11 pathway is a key target of hydrogen sulfide-mediated inhibition of neuroinflammation in hypoxic ischemic brain injury

We previously showed that hydrogen sulfide(H2S)has a neuroprotective effect in the context of hypoxic ischemic brain injury in neonatal mice.However,the precise mechanism underlying the role of H2S in this situation remains unclear.In this study,we used a neonatal mouse model of hypoxic ischemic brain injury and a lipopolysaccharide-stimulated BV2 cell model and found that treatment with L-cysteine,a H2S precursor,attenuated the cerebral infarction and cerebral atrophy induced by hypoxia and ischemia and increased the expression of miR-9-5p and cystathionineβsynthase(a major H2S synthetase in the brain)in the prefrontal cortex.We also found that an miR-9-5p inhibitor blocked the expression of cystathionineβsynthase in the prefrontal cortex in mice with brain injury caused by hypoxia and ischemia.Furthermore,miR-9-5p overexpression increased cystathionine-β-synthase and H2S expression in the injured prefrontal cortex of mice with hypoxic ischemic brain injury.L-cysteine decreased the expression of CXCL11,an miR-9-5p target gene,in the prefrontal cortex of the mouse model and in lipopolysaccharide-stimulated BV-2 cells and increased the levels of proinflammatory cytokines BNIP3,FSTL1,SOCS2 and SOCS5,while treatment with an miR-9-5p inhibitor reversed these changes.These findings suggest that H2S can reduce neuroinflammation in a neonatal mouse model of hypoxic ischemic brain injury through regulating the miR-9-5p/CXCL11 axis and restoringβ-synthase expression,thereby playing a role in reducing neuroinflammation in hypoxic ischemic brain injury.

Overexpression of low-density lipoprotein receptor prevents neurotoxic polarization of astrocytes via inhibiting NLRP3 inflammasome activation in experimental ischemic stroke

Neurotoxic astrocytes are a promising therapeutic target for the attenuation of cerebral ischemia/reperfusion injury.Low-density lipoprotein receptor,a classic cholesterol regulatory receptor,has been found to inhibit NLR family pyrin domain containing protein 3(NLRP3)inflammasome activation in neurons following ischemic stroke and to suppress the activation of microglia and astrocytes in individuals with Alzheimer’s disease.However,little is known about the effects of low-density lipoprotein receptor on astrocytic activation in ischemic stroke.To address this issue in the present study,we examined the mechanisms by which low-density lipoprotein receptor regulates astrocytic polarization in ischemic stroke models.First,we examined low-density lipoprotein receptor expression in astrocytes via immunofluorescence staining and western blotting analysis.We observed significant downregulation of low-density lipoprotein receptor following middle cerebral artery occlusion reperfusion and oxygen-glucose deprivation/reoxygenation.Second,we induced the astrocyte-specific overexpression of low-density lipoprotein receptor using astrocyte-specific adeno-associated virus.Low-density lipoprotein receptor overexpression in astrocytes improved neurological outcomes in middle cerebral artery occlusion mice and reversed neurotoxic astrocytes to create a neuroprotective phenotype.Finally,we found that the overexpression of low-density lipoprotein receptor inhibited NLRP3 inflammasome activation in oxygen-glucose deprivation/reoxygenation injured astrocytes and that the addition of nigericin,an NLRP3 agonist,restored the neurotoxic astrocyte phenotype.These findings suggest that low-density lipoprotein receptor could inhibit the NLRP3-meidiated neurotoxic polarization of astrocytes and that increasing low-density lipoprotein receptor in astrocytes might represent a novel strategy for treating cerebral ischemic stroke.

CircR-ZC3HC1 mediates MiR-384-5p/SIRT1 axis to promote neuronal autophagy and relieves ischemic stroke

Objective:Circular RNAs(circRNAs)have been shown to involve in pathological processes of ischemic stroke(IS),including autophagy.This study was designed to explore the effect of circR-ZC3HC1 on neuronal autophagy in IS and the related mechanisms.Methods:Expression of circR-ZC3HC1 in blood samples of IS patients and healthy controls was detected.Hippocampal neurons were treated with oxygen and glucose deprivation(OGD)to establish IS in vitro model.The expression of LC3 and p62 and the number of autophagosomes were examined to evaluate the autophagy level of OGD induced neurons using western blotting and transmission electron microscope.Cell apoptosis rate and the expression of cleaved caspase-3,Bax,and Bcl-2 were assessed byflow cytometry and western blotting.The binding relationships among circR-ZC3HC1,miR-384-5p,and SIRT1 were predicted and verified.Results:Low expression of circR-ZC3HC1 was found in blood samples of IS patients and OGD-treated neurons.Overexpressed circR-ZC3HC1 or inhibited miR-384-5p expression promoted autophagy and inhibited apoptosis of OGD-treated neurons,which could be reversed by further 3-MA treatment.Mechanistically,circR-ZC3HC1 targeted miR-384-5p to mediate SIRT1 expression.miR-384-5p overexpression or SIRT1 knockdown in the presence of circR-ZC3HC1 overexpression in OGD-treated neurons lead to reduced autophagy and enhanced apoptosis.Conclusion:Collectively,circR-ZC3HC1 promoted neuronal autophagy to attenuate IS via miR-384-5p/SIRT1 axis.

A molecular probe carrying anti-tropomyosin 4 for early diagnosis of cerebral ischemia/reperfusion injury

In vivo imaging of cerebral ischemia/reperfusion injury remains an important challenge.We injected porous Ag/Au@SiO_(2) bimetallic hollow nanoshells carrying anti-tropomyosin 4 as a molecular probe into mice with cerebral ischemia/reperfusion injury and observed microvascular changes in the brain using photoacoustic imaging with ultrasonography.At each measured time point,the total photoacoustic signal was significantly higher on the affected side than on the healthy side.Twelve hours after reperfusion,cerebral perfusion on the affected side increased,cerebrovascular injury worsened,and anti-tropomyosin 4 expression increased.Twenty-four hours after reperfusion and later,perfusion on the affected side declined slowly and stabilized after 1 week;brain injury was also alleviated.Histopathological and immunohistochemical examinations confirmed the brain injury tissue changes.The nanoshell molecular probe carrying anti-tropomyosin 4 has potential for use in early diagnosis of cerebral ischemia/reperfusion injury and evaluating its progression.

Remote ischemic perconditioning prevents liver transplantation-induced ischemia/reperfusion injury in rats: Role of ROS/RNS and e NOS

AIM To investigate the underlying mechanisms of the protective role of remote ischemic perconditioning(RIPerC) in rat liver transplantation. METHODS Sprague-Dawley rats were subjected to sham, orthotopic liver transplantation(OLT), ischemic postconditioning(IPostC) or RIPerC. After 3 h reperfusion, blood samples were taken for measurement of alanine aminotransferase, aspartate aminotransferase, creatinine(Cr) and creatinine kinase-myocardial band(CK-MB). The liver lobes were harvested for the following measurements: reactive oxygen species(ROS), H2O2, mitochondrial membrane potential(ΔΨm) and total nitric oxide(NO). These measurements were determined using an ROS/H2O2, JC1 and Total NOx Assay Kit, respectively. Endothelial NO synthase(e NOS) was analyzed by reverse transcription-polymerase chain reaction(RTPCR) and western blotting, and peroxynitrite was semiquantified by western blotting of 3-nitrotyrosine. RESULTS Compared with the OLT group, the grafts subjected to RIPerC showed significantly improved liver and remote organ functions(P < 0.05). ROS(P < 0.001) including H2O2(P < 0.05) were largely elevated in the OLT group as compared with the sham group, and RIPerC(P < 0.05) reversed this trend. The collapse of ΔΨm induced by OLT ischemia/reperfusion(I/R) injury was significantly attenuated in the RIPerC group(P < 0.001). A marked increase of NO content and phosphoserine eN OS, both in protein and mR NA levels, was observed in liver graft of the RIPer C group as compared with the OLT group(P < 0.05). I/R-induced 3-nitrotyrosine content was significantly reduced in the RIPerC group as compared with the OLT group(P < 0.05). There were no significant differences between the RIPerC and IPostC groups for all the results except Cr. The Cr level was lower in the RIPerC group than in the IPostC group(P < 0.01).CONCLUSION Liver graft protection by RIPerC is similar to or better than that of IPostC, and involves inhibition of oxidative stress and up-regulation of the PI3K/Akt/e NOS/NO pathway.

Remote ischemic preconditioning as treatment for non-ischemic gastrointestinal disorders: Beyond ischemia-reperfusion injury

Common gastrointestinal diseases such as radiation enteritis(RE),acute pancreatitis,inflammatory bowel diseases(IBD)and drug-induced hepatotoxicity share pathophysiological mechanisms at the molecular level,mostly involving the activation of many pathways of the immune response,ultimately leading to tissue injury.Increased oxidative stress,inflammatory cytokine release,inflammatory cell infiltration and activation and the up-regulation of inflammatory transcription factors participate in the pathophysiology of these complex entities.Treatment varies in each specific disease,but at least in the cases of RE and IBD immunosuppressors are effective.However,full therapeutic responses are not always achieved.The pathophysiology of ischemiareperfusion(IR)injury shares many of these mechanisms.Brief and repetitive periods of ischemia in an organ or limb have been shown to protect against subsequent major IR injury in distant organs,a phenomenon called remote ischemic preconditioning(RIP).This procedure has been shown to protect the gut,pancreas and liver by modulating many of the same inflammatory mechanisms.Since RIP is safe and tolerable,and has shown to be effective in some recent clinical trials,I suggest that RIP could be used as a physiologicallyrelevant adjunct treatment for non-ischemic gastrointestinal inflammatory conditions.

Ischemic post-conditioning to counteract intestinal ischemia/reperfusion injury

Intestinal ischemia is a severe disorder with a variety of causes.Reperfusion is a common occurrence during treatment of acute intestinal ischemia but the injury resulting from ischemia/reperfusion(IR)may lead toeven more serious complications from intestinal atrophy to multiple organ failure and death.The susceptibility of the intestine to IR-induced injury(IRI)appears from various experimental studies and clinical settings such as cardiac and major vascular surgery and organ transplantation.Where as oxygen free radicals,activation of leukocytes,failure of microvascular perfusion,cellular acidosis and disturbance of intracellular homeo-stasis have been implicated as important factors inthe pathogenesis of intestinal IRI,the mechanisms underlying this disorder are not well known.To date,increasing attention is being paid in animal studies to potential pre-and post-ischemia treatments that protect against intestinal IRI such as drug interference with IR-induced apoptosis and inflammation processes and ischemic pre-conditioning.However,better insight is needed into the molecular and cellular events associated with reperfusion-induced damage to develop effective clinical protection protocols to combat this disorder.In this respect,the use of ischemic post-conditioning in combination with experimentally prolonged acidosis blocking deleterious reperfusion actions may turn out to have particular clinical relevance.

Ischemic postconditioning enhances glycogen synthase kinase-3β expression and alleviates cerebral ischemia/reperfusion injury

The present study established global brain ischemia using the four-vessel occlusion method. Following three rounds of reperfusion for 30 seconds, and occlusion for 10 seconds, followed by reperfusion for 48 hours, infarct area, the number of TUNEL-positive cells and Bcl-2 expression were significantly reduced. However, glycogen synthase kinase-3β activity, cortical Bax and caspase-3 expression significantly increased, similar to results following ischemic postconditioning. Our results indicated that ischemic postconditioning may enhance glycogen synthase kinase-3β activity, a downstream molecule of the phosphatase and tensin homolog deleted on chromosome 10/phosphatidylinositol 3-kinase/protein kinase B signaling pathway, which reduces caspase-3 expression to protect the brain against ischemic injury.

Protective effects of remote ischemic preconditioning in rat hindlimb on ischemia- reperfusion injury

Three cycles of remote ischemic pre-conditioning induced by temporarily occluding the bilateral femoral arteries （10 minutes） prior to 10 minutes of reperfusion were given once a day for 3 days before the animal received middle artery occlusion and reperfusion surgery. The results showed that brain infarct volume was significantly reduced after remote ischemic pre-conditioning. Scores in the forelimb placing test and the postural reflex test were significantly lower in rats having undergone remote ischemic pre-conditioning compared with those who did not receive remote ischemic pre-conditioning. Thus, neurological function was better in rats having undergone remote ischemic pre-conditioning compared with those who did not receive remote ischemic pre-conditioning. These results indicate that remote ischemic pre-conditioning in rat hindlimb exerts protective effects in ischemia-reperfusion injury.

Ischemic preconditioning ameliorates intestinal injury induced by ischemia-reperfusion in rats

AIM: To evaluate preventative effects of ischemic preconditioning(IP) in a rat model of intestinal injury induced by ischemia-reperfusion(IR).METHODS: Male Sprague-Dawley rats(250-300 g) were fasted for 24 h with free access to water prior to the operation.Eighteen rats were randomly divided into three experimental groups: S group(n = 6),rats were subjected to isolation of the superior mesenteric artery(SMA) for 40 min,then the abdomen was closed; IRgroup(n = 6),rats were subjected to clamping the SMA 40 min,and the abdomen was closed followed by a 4-h reperfusion; IP group(n = 6) rats underwent three cycles of 5 min ischemia and 5 min reperfusion,then clamping of the SMA for 40 min,then the abdomen was closed and a 4-h reperfusion followed.All animals were euthanized by barbiturate overdose(150 mg/kg pentobarbital sodium,i.v.) for tissue collection,and the SMA was isolated via median abdominal incision.Intestinal histologic injury was observed.Malondialdehyde(MDA),myeloperoxidase(MPO) and tumor necrosis factor(TNF)-a concentrations in intestinal tissue were measured.Intercellular adhesion molecule(ICAM)-1 and vascular cell adhesion molecule(VCAM)-1 expression,as well as nuclear factor(NF)-κB activity and expression in intestinal tissue were also determined.RESULTS: Compared with the IR group,IP reduced IR-induced histologic injury of the intestine in rats(2.00 ± 0.71 vs 3.60 ± 0.84,P < 0.05).IP significantly inhibited the increase in MDA content(5.6 ± 0.15 μmol/L vs 6.84 ± 0.18 μmol/L,P < 0.01),MPO activity(0.13 ± 0.01 U/L vs 0.24 ± 0.01 U/L,P < 0.01),and TNF-a levels(7.79 ± 2.35 pg/m L vs 10.87 ± 2.48 pg/m L,P < 0.05) in the intestinal tissue of rats.IP also markedly ameliorated the increase in ICAM-1(204.67 ± 53.27 vs 353.33 ± 45.19,P < 0.05) and VCAM-1(256.67 ± 58.59 vs 377.33 ± 41.42,P < 0.05) protein expression in the intestinal tissues.Additionally,IP remarkably decreased NF-κB activity(0.48 ± 0.16 vs 0.76 ± 0.22,P < 0.05) and protein expression(320.23 ± 38.16 vs 520.76 ± 40.53,P < 0.01) in rat intestinal tissue.CONCLUSION: IP may protect against IR-induced intestinal injury by attenuation of the neutrophilendothelial adhesion cascade via reducing ICAM-1 and VCAM-1 expression and TNF-a-induced NF-κB signaling pathway activity.

Protective effect of ischemic preconditioning on hepatic ischemia-reperfusion injury by advancing the expressive phase of survivin in rats

BACKGROUND: Survivin is a new and important gene in the regulation of apoptosis. It is very important to explore the effect of the expression of survivin protein caused by ischemia-reperfusion (IR) injury. The effect of IR injury caused by ischemic preconditioning (IP) on the liver in rats and the relation between the protective effect of IP and the expression of survivin are unclear. METHODS: One hundred and fifty male Wistar rats (weighing 190-210 g, aged 6-7 weeks) were divided into three groups at random: ischemic preconditioning (IP), ischemia-reperfusion (IR) and sham-operation (SO). Sample specimens were collected from each group at 6, 12, 24, 48, and 72 hours after reperfusion. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured by an automatic biochemical analyzer. Malondialdehyde (MDA) in liver tissue was measured. Pathological changes in the liver and immunohistochemical staining for survivin were determined with an optical microscope. RESULTS: The ALT levels in the IP and IR groups after reperfusion at each time were higher than those in the SO group (P<0.05), whereas after reperfusion for 6 and 12 hours, the ALT levels in the IP group were lower than those in the IR group (P<0.05). The AST levels in all IP and IR groups were higher than those in the SO group (P<0.05), whereas after reperfusion for 12, 24, 48 and 72 hours, the AST levels in the IP group were lower than those in the IR group (P<0.05). The MDA concentrations after reperfusion in the IP group were lower than those in the IR group (P<0.05), though the MDA concentrations in the IP and IR groups increased in contrast to those in the SO group after reperfusion at each time (P<0.05). After reperfusion for 12, 24, 48 and 72 hours, the number of survivin-positive cells was larger in the IP and IR groups than in the SO group (P<0.05). After reperfusion for 12, 24, and 48 hours the number of survivin-positive cells in the IP group increased compared with that in the IR group (P<0.05). CONCLUSIONS: IR increases the protein expression of survivin in liver tissue. IP inhibits the accumulation of MDA, advances the expressive phase of survivin protein in hepatic tissue, and improves liver function.

Effect of Minocycline Postconditioning and Ischemic Postconditioning on Myocardial Ischemia-reperfusion Injury in Atherosclerosis Rabbits

This study examined the protective effect of ischemic postconditioning(IPoC) and minocycline postconditioning(MT) on myocardial ischemia-reperfusion(I/R) injury in atherosclerosis(AS) animals and the possible mechanism.Forty male healthy rabbits were injected with bovine serum albumin following feeding on a high fat diet for 6 weeks to establish AS model.AS rabbits were randomly divided into 3 groups:(1) I/R group,the rabbits were subjected to myocardial ischemia for 35 min and then reperfusion for 12 h;(2) IPoC group,the myocardial ischemia lasted for 35 min,and then reperfusion for 20 s and ischemia for 20 s [a total of 3 cycles(R20s/I20s×3)],and then reperfusion was sustained for 12 h;(3) MT group,minocycline was intravenously injected 10 min before reperfusion.The blood lipids,malondialdehyde(MDA),superoxide dismutase(SOD),soluble cell adhesion molecule(sICAM),myeloperoxidase(MPO),and cardiac troponin T(cTnT) were biochemically determined.The myocardial infarction size(IS) and apoptosis index(AI) were measured by pathological examination.The expression of bcl-2 and caspase-3 was detected in the myocardial tissue by using reverse transcription-polymerase chain reaction(RT-PCR).The results showed that the AS models were successfully established.The myocardial IS,the plasma levels of MDA,sICAM,MPO and cTnT,and the enzymatic activity of MPO were significantly decreased,and the plasma SOD activity was significantly increased in IPoC group and MT group as compared with I/R group(P<0.05 for all).The myocardial AI and the caspase-3 mRNA expression were lower and the bcl-2 mRNA expression was higher in IPoC and MT groups than those in I/R group(all P<0.05).It is concluded that the IPoC and MT can effectively reduce the I/R injury in the AS rabbits,and the mechanisms involved anti-oxidation,anti-inflammation,up-regulation of bcl-2 expression and down-regulation of caspase-3 expression.Minocycline can be used as an effective pharmacologic postconditioning drug to protect myocardia from I/R injury.

Remote ischemic preconditioning protects liver ischemia-reperfusion injury by regulating eNOS-NO pathway and liver microRNA expressions in fatty liver rats

BACKGROUND: Ischemic preconditioning (IPC) is a strategy to reduce ischemia-reperfusion (I/R) injury. The protective effect of remote ischemic preconditioning (RIPC) on liver I/R injury is not clear. This study aimed to investigate the roles of RIPC in liver I/R in fatty liver rats and the involvement of endothelial nitric oxide synthase-nitric oxide (eNOS-NO) pathway and microRNA expressions in this process. METHODS: A total of 32 fatty rats were randomly divided into the sham group, I/R group, RIPC group and RIPC+I/R group. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and nitric oxide (NO) were measured. Hematoxylin-eosin staining was used to observe histological changes of liver tissues, TUNEL to detect hepatocyte apoptosis, and immunohistochemistry assay to detect heat shock protein 70 (HSP70) expression. Western blotting was used to detect liver inducible NOS (iNOS) and eNOS protein levels and realtime quantitative polymerase chain reaction to detect miR-34a, miR-122 and miR-27b expressions. RESULTS: Compared with the sham and RIPC groups, serum ALT, AST and iNOS in liver tissue were significantly higher in other two groups, while serum NO and eNOS in liver tissue were lower, and varying degrees of edema, degeneration and inflammatory cell infiltration were found. Cell apoptosis number was slightly lower in the RIPC+I/R group than that in I/R group. Compared with the sham group, HSP70 expressions were significantly increased in other three groups (all P<0.05). Compared with the sham and RIPC groups, elevated miR-34a expressions were found in I/R and RIPC+I/R groups (P<0.05). MiR-122 and miR-27b were found significantly decreased in I/R and RIPC+I/R groups compared with the sham and RIPC groups (all P<0.05). CONCLUSION: RIPC can reduce fatty liver I/R injury by affecting the eNOS-NO pathway and liver microRNA expressions.

Influences of ischemic postconditioning at different positions on oxidative stress of myocardial ischemia-reperfusion injury in rats

Objective:To analyze the influences of locial ischemic postconditioning and remote limb ischemic postconditioning on oxidative stress response with myocardial ischemia reperfusion in rats.Methods:Thirty-two SD rats were randomly divided into Sham group,ischemia/reperfusion(I/R)group,local ischemic postconditioning(LIPC)group,and remote limb ischemic postconditioning(RIPC)group,after 3 hourse reperfusion,the contents of serum creatinine kinase,MB isoenzyme(CK-MB),xanthine oxidase(XOD),superoxide dismutase(SOD),myeloperoxidase(MPO),tumor necrosis factor-α(TNF-α)were measured.The 2,3,5-triphenyltetrazolium chloride(TTC)staining was carried out to evaluate the area of myocardial infarction,cardiac function was evaluated by echocardiography,and HE staining was performed to observe the morphology of myocardial cells.Results:Compared with the Sham group,the SOD contents of the I/R group,LIPC group,RIPC group reduced significantly(P<0.05),the XOD,MPO,TNF-αcontents increased significantly(P<0.05);Compared with the I/R group,the TNF-αcontents of the LIPC group reduced significantly(P<0.05),other oxidative stress indicators of the LIPC group had no significant differences;Compared with the I/R group,the MPO and TNF-αcontents reduced(P<0.05),the SOD and XOD contents of the RIPC group had no significant differences;Compared with the LIPC group,the MPO contents reduced(P<0.05)in the RIPC group,other oxidative stress indicators had no significant differences.Compared with the Sham group,myocardial infarction area,CK-MB contents,LVIDs increased with the reduction of EF in I/R group,LIPC group,RIPC group(P<0.05),HE staining had differences;Compared with the I/R group,myocardial infarction area,CK-MB contents,LVIDd,LVIDs,EF and HE staining results had no significant differences in the LIPC group and the RIPC group;Compared with the RIPC group,the LIPC group had no significant differences.Conclusion:Remote limb ischemic postconditioning and local ischemic postconditioning can partially reduce the oxidative stress response,but does not significantly reduce myocardial infarction area,improve cardiac function.

Expression of insulin-like growth factor-1 mRNA and protein level of corpora striata in ischemic side at the early stage of middle cerebral artery ischemia/reperfusion in rhesus monkeys

BACKGROUND： Insulin-like growth factor-I（IGF-1）, as one of the important members of growth factor family, participants in the regulation of many physiological functions and behaviors, having very strong neuroprotective effect. However, the expression of IGF-1 following cerebral ischemia/reperfusion is still disputed. OBJECTIVE： To observe the expression of IGF-1 and protein of corpora striata in ischemic side at the early stage of middle cerebral artery ischemia/reperfusion in rhesus monkey. DESIGN ： A completely randomized grouping design, controlled animal experiment SETTING ： Institute of Cerebrovascular Disease, Affiliated Hospital of Medical College of Qingdao University MATERIALS： ① Totally 17 rhesus monkeys , of either gender, aged 4 to 5 years, were enrolled . Seven rhesus monkeys observed with gene chip were randomly divided into 2 groups： sham operation group （n=3） and ischemia/reperfusion group 〈n=4〉. Ten rhesus monkeys observed with in situ hybridization and immunohistochemistry method were randomly divided into 2 groups： sham operation group 〈n=3 〉and ischemia/reperfusion group （n=7）. Rhesus monkeys observed under microscope were divided into 2 groups： sham operation group （n=6） and ischamia/reperfusion group （n=-11）.②Materials used in the experiment： cresyl violet （Sigma Company, America）; immunohistochemical reagent kit （ Huamei Bio-engineering Company）; In situ hybridization reagent kit （Boshide Bio-engineering Co.Ltd, Wuhan）; 12 800 dots chip （Boxing Company, Shanghai）. METHODS ： This experiment was carried out at the Institute of Cerebrovascular Disease, Affiliated Hospital of Medical College of Qingdao University from January 2001 to December 2003.① The onset area of middle cerebral artery was blocked for 2 hours, middle cerebral artery ischemia/reperfusion models were created.② After ischemia/reperfusion for 24 hours, cerebral tissue sections of rhesus monkeys were prepared and stained with cresyl violet. Image analysis was performed with 5001W image analysis software. Morphological change of corpora striata of operative side was observed in the rhesus monkeys between two groups. Total RNA was extracted from cerebral tissue. ③ Detection of gene chip： Cy3-duTP and Cy5-duTP were used to respectively perform reverse transcription labeling. The sample was reversely transcribed into cDNA, then hybridized with cDNA of cerebral tissue. Genes with the separate absolute value of cy3 and cy5〉800, cY3/cy5 〉 2（high expression） or 〈 0.5 （low expression） were found out. Those were genes with differential expression. ④ The expressions of IGF-1 mRNA and protein level of corpora striata in ischemic side of rhe- sus monkeys were detected between sham operation group and ischemia/reperfusion group at 9 and 24 hours after ischemia/reperfusion with in situ hybridization method and immunohistochemical method. Brown granules were IGF-1 protein positive cells. ⑤ Analysis of variance was used in the difference comparison of measurement data among groups. MAIN OUTCOME MEASURES ： ① Change of morphological structure of corpora striata at ischemic side in rhesus monkeys. ② Change of cerebral gene expression profiles at ischemia/reperfusion in rhesus monkeys between two groups.③ Expression of IGF-1 mRNA and protein level of corpora striata at ischemia/reperfu- sion in rhesus monkeys between two groups. RESULTS ： ① Pathological change ： Obvious pathological change of cerebral infarction appeared in the ischemia and reperfusion group, while there was no such pathological change in the sham operation group.② Change of gene expression profile ： There were 4480 genes with difference expression in the ischemia/reperfusion group and sham-operation group, in which, 260 genes had high expression and their absolute value was over 800, and 63 genes had low expression, cy3/cy5 of IGF-1 was 0.379, being relative low ex- pression. ③ IGF-1 mRNA and protein positive cell counts in corpora striata at cerebral ischemic side[IGF-1 mRNA： 〈9.72±1.18）,（9.11 ±0.76）,（14.77±0.60） counts/field：lGF-1 protein： （15.11 ±1.83）,（15.39±0.78）, （34.62±0.97）counts/field, P 〈 0.05-0.01]. CONCLUSION： IGF-1 mRNA and protein are lowly expressed in middle cerebral artery of rhesus monkeys at ischemia/reperfusion.

Stroke:Evolution of newer treatment modalities for acute ischemic stroke

Acute ischemic stroke is one of the leading causes of morbidity and mortality worldwide.Restoration of cerebral blood flow to affected ischemic areas has been the cornerstone of therapy for patients for eligible patients as early diagnosis and treatment have shown improved outcomes.However,there has been a paradigm shift in the management approach over the last decade,and with the emphasis currently directed toward including newer modalities such as neuroprotection,stem cell treatment,magnetic stimulation,anti-apoptotic drugs,delayed recanali-zation,and utilization of artificial intelligence for early diagnosis and suggesting algorithm-based management protocols.

The influence of aprotinin on the mRNA expression of P-selectin and ICAM-1 in the in situ lung tissue after ischemic cold storage and reperfusion

To explore the influence of aprotinin on the mRNA expression of P-selectin and ICAM-1 in lung tissue after ischemic reperfusion.Methods Thirty New Zealand white rabbits were randomly divided into 3 groups:control group,LPD group and aprotinin group.In situ rabbit lung preservation model was established.In control group,the left lower lung lobe was stored at 10℃ in a specially made lung preservation container for 2 hours and reperfused for another 2 hours.In LPD group and aprotinin group,the left lower lobe was perfused with LPD solution or aprotinin containing LPD solution,respectively,after left lung hilus was clamped.The other procedures were the same as those in control group.The lung tissue was harvested at different time intervals including preclamping lung hilus,2 hours after clamping and 2 hours after reperfusion.The mRNA expression of ICAM-1 and P-selectin in the lung tissue was detected with RT-PCR technique.Results The contents of mRNA of P-selectin at 2 hours after reperfusion in control group and LPD group were significant higher than pre-ischemia and 2 hours after champing the left lung hilus.There was no such significant difference in aprotinin group.The mRNA expression of P-selectin in aprotinin group at 2 hours after reperfusion was significantly lower than that in control group and LPD group.The ICAM-1 mRNA expression at 2 hours after Ischemia and 2 hours after reperfusion in control group and LPD group was significantly higher than the pre-ischemia and its was significantly higher than that in aprotinin group.Conclusion Aprotinin can inhibit the upregulation of the mRNA expression of P selectin and ICAM-1 after ischemia reperfusion in the lung tissue,so the addition of aprotinin in LPD solution may reduce the ischemia reperfusion injury in lung tissue.5 refs,1 tab.

Hepatic ischemia-reperfusion injury in liver transplant setting:mechanisms and protective strategies

Hepatic ischemia-reperfusion injury is a major cause of liver transplant failure,and is of increasing significance due to increased use of expanded criteria livers for transplantation.This review summarizes the mechanisms and protective strategies for hepatic ischemia-reperfusion injury in the context of liver transplantation.Pharmacological therapies,the use of pre-and post-conditioning and machine perfusion are discussed as protective strategies.The use of machine perfusion offers significant potential in the reconditioning of liver grafts and the prevention of hepatic ischemia-reperfusion injury,and is an exciting and active area of research,which needs more study clinically.

Puerarin protects rat brain against ischemia/reperfusion injury by suppressing autophagy via the AMPK-mT OR-ULK1 signaling pathway

Puerarin suppresses autophagy to alleviate cerebral ischemia/reperfusion injury, and accumulating evidence indicates that the AMPKm TOR signaling pathway regulates the activation of the autophagy pathway through the coordinated phosphorylation of ULK1. In this study, we investigated the mechanisms underlying the neuroprotective effect of puerarin and its role in modulating autophagy via the AMPK-m TOR-ULK1 signaling pathway in the rat middle cerebral artery occlusion model of cerebral ischemia/reperfusion injury. Rats were intraperitoneally injected with puerarin, 50 or 100 mg/kg, daily for 7 days. Then, 30 minutes after the final administration, rats were subjected to transient middle cerebral artery occlusion for 90 minutes. Then, after 24 hours of reperfusion, the Longa score and infarct volume were evaluated in each group. Autophagosome formation was observed by transmission electron microscopy. LC3, Beclin-1 p62, AMPK, m TOR and ULK1 protein expression levels were examined by immunofluorescence and western blot assay. Puerarin substantially reduced the Longa score and infarct volume, and it lessened autophagosome formation in the hippocampal CA1 area following cerebral ischemia/reperfusion injury in a dose-dependent manner. Pretreatment with puerarin（50 or 100 mg/kg） reduced Beclin-1 expression and the LC3-II/LC3-I ratio, as well as p-AMPK and p S317-ULK1 levels. In comparison, it increased p62 expression. Furthermore, puerarin at 100 mg/kg dramatically increased the levels of p-m TOR and p S757-ULK1 in the hippocampus on the ischemic side. Our findings suggest that puerarin alleviates autophagy by activating the APMK-m TOR-ULK1 signaling pathway. Thus, puerarin might have therapeutic potential for treating cerebral ischemia/reperfusion injury.