Different Time Points Of Reperfusion Research Articles

Development of a new cerebral ischemia reperfusion model of Mongolian gerbils and standardized evaluation system.

The Mongolian gerbil is an excellent laboratory animal for preparing the cerebral ischemia model due to its inherent deficiency in the circle of Willis. However, the low incidence and unpredictability of symptoms are caused by numerous complex variant types of the circle. Additionally, the lack of an evaluation system for the cerebral ischemia/reperfusion (I/R) model of gerbils has shackled the application of this model. We created a symptom-oriented principle and detailed neurobehavioral scoring criteria. At different time points of reperfusion, we analyzed the alteration in locomotion by rotarod test and grip force score, infarct volume by triphenyltetrazolium chloride (TTC) staining, neuron loss using Nissl staining, and histological characteristics using hematoxylin-eosin (H&E) straining. With a successful model rate of 56%, 32 of the 57 gerbils operated by our method harbored typical features of cerebral I/R injury, and the mortality rate in the male gerbils was significantly higher than that in the female gerbils. The successfully prepared I/R gerbils demonstrated a significant reduction in motility and grip strength at 1 day after reperfusion; formed obvious infarction; exhibited typical pathological features, such as tissue edema, neuronal atrophy and death, and vacuolated structures; and were partially recovered with the extension of reperfusion time. This study developed a new method for the unilateral common carotid artery ligation I/R model of gerbil and established a standardized evaluation system for this model, which could provide a new cerebral I/R model of gerbils with more practical applications.

R-(+)-WIN55212-2 protects pericytes from ischemic damage and restores retinal microcirculatory patency after ischemia/reperfusion injury

Background and purposeCannabinoids are vasoactive substances that act as key regulators of arterial tone in the blood vessels supplying peripheral tissues and the central nervous system. This study aimed to investigate the potential of R-(+)-WIN55212-2 (WIN), a cannabinoid receptor 1 agonist (CB1), as a treatment for retinal ischemia/reperfusion (I/R) injury. Experimental approachMale Wistar rats were subjected to retinal I/R injury by increasing intraocular pressure in the anterior chamber. The rats were randomly divided into four groups: normal control, I/R, vehicle (pre-treated with dimethyl sulfoxide [DMSO] via intraperitoneal injection), and experimental (pre-treated with WIN at a dose of 1 ml/kg via intraperitoneal injection). The rats were sacrificed at different time points of reperfusion (1 hour, 3 hours, 6 hours, and 1 day) after inducing retinal I/R injury, and their retinas were collected for analysis. Oxygen-glucose deprived/reperfusion (OGD/R) was performed by initially perfusing the retinas with oxygenated artificial cerebrospinal fluid (ACSF), then switching to an OGD solution to simulate ischemia, followed by another perfusion with ACSF. Pericyte contraction and the "no-reflow" phenomenon were observed using infrared differential interference contrast (IR-DIC) microscopy and immunohistochemistry. Western blot, enzyme-linked immunosorbent assay (ELISA), and nitric oxide (NO) detection were used to explore the potential mechanism. Key resultsIn both the OGD/R and I/R models, retinal pericytes exhibited persistent contraction even after reperfusion. The ability of WIN to regulate the tone of retinal pericytes and capillaries was specifically blocked by the BKCa inhibitor iberiotoxin (100 nM). WIN demonstrated a protective effect against retinal I/R injury by preserving blood flow in vessels containing pericytes. Pretreatment with WIN alleviated the persistent contraction and apoptosis of retinal pericytes in I/R-induced rats, accompanied by a reduction in intracellular calcium ion (Ca2+) concentration. The expression of CB1 decreased in a time-dependent manner in the I/R group. After I/R injury, endothelium-derived nitric oxide (eNOS) levels were reduced at all time points, which was successfully reversed by WIN therapy except for the 1 day group. Additionally, the downregulation of cyclic guanosine monophosphate (cGMP) and BKCa expression at 3 hours, 6 hours, and 1 day after I/R injury was restored by pretreatment of WIN. Conclusions & implicationsWIN exerted its protective effects on retinal I/R injury by inhibiting the contraction and apoptosis of pericytes through the CB1-eNOS-cGMP-BKCa signaling pathway, thus ameliorated the occlusion of retinal capillaries.

“Lifting Yang to Dredging Du Meridian Manipulation” acupuncture alleviates cerebral ischemia-reperfusion injury by mediating the NF-κB pathway

BackgroundIschemic stroke is a permanent neurological impairment resulting from the narrowing or blockage of blood vessels in the brain. The effectiveness of “Lifting Yang to Dredging Du Meridian Manipulation” (LYDD) acupuncture in clinical treatment of ischemic stroke patients has been well-established. Nevertheless, its mechanism is still uncertain. MethodsMCAO/R rat models at different time points of reperfusion (24, 36, 48 and 72 h) were constructed, and LYDD acupuncture treatment was performed. Zea-Longa score and TTC staining were used for assessing neurological impairment and cerebral infarct in rats, respectively. The pathological changes of cerebral tissue in each group were observed by HE and Nissl's staining. Cerebral tissue from each group was subjected to RNA-seq, and differentially expressed genes (DEGs) were performed for GO and KEGG enrichment analysis, and hub gene was identified based on the String database and MCODE algorithm. ResultsLYDD acupuncture treatment significantly reduced Zea-Longa score, dry-wet weight ratio, infarct area, inflammatory factor levels (IL-1β and TNF-α), cerebral lesions, number of Nissl body and neuronal apoptosis in the MCAO/R model at different time points of reperfusion. A total of 3518 DEGs were identified in the MCAO/R model compared to the control group, and 3461 DEGs were present in the treatment group compared to the MCAO/R model, and they may be implicated in neurotransmitter transmission, synaptic membrane potential, cell junctions, inflammatory response, immune response, cell cycle, and ECM. The expression trends of BIRC3, LTBR, PLCG2, TLR4 and TRADD mRNAs in the Hub gene were consistent with the RNA-seq results, and LYDD acupuncture treatment significantly inhibited MCAO/R-induced p65 nuclear translocation. ConclusionsLYDD acupuncture ameliorates cerebral ischemia-reperfusion injury by inhibiting NF-κB pathway activity.

Effects of lysophosphatidic acid receptor 5 on NLRC4 inflammasome in brain tissues of transient cerebral ischemia/reperfusion rat.

To explore whether LPA5 was involved in the inflammatory responses in CI/R injury by regulation of NLRC4. The cerebral I/R model in rats was constructed with ischemia of 2h and different time points of reperfusion. After that, western blot was used to determine protein expression (LPA5, NLRC4, AIM2, caspase-1, cleaved-caspase-1, mature IL-1β, and precursor IL-1β). And LPA5 and NLRC4 expression were also detected by using immunofluorescence experiment. Afterward, two sequence of LPA5-siRNA were injected into rats via intracerebroventricular administration. TTC staining and HE staining were performed. As the reperfusion time was prolonged, LPA5 content was continuously increased, and the highest expression of NLRC4 was found at 4h of reperfusion. And protein expression of AIM2, cleaved-caspase-1, and mature IL-1β was also at highest level at 4h. And after reperfusion of 4h, LPA5 siRNA1# or 2# was injected into lateral ventricles. LPA5 silence markedly reduced the infract volume and improved the histological change of ischemic zone. And LPA5 silence significantly downregulated NLRC4, AIM2, and the ratio of cleaved-caspase-1/caspase-1 and mature IL-1β/precursor IL-1β. And compared with LPA5-siRNA2#, LPA5-siRNA1# exerted a more significant effect. Low expression of LPA5 can protect against the inflammatory responses in CI/R model of rats through inhibiting NLRC4 inflammasomes.

γ-Tocotrienol Protects against Mitochondrial Dysfunction, Energy Deficits, Morphological Damage, and Decreases in Renal Functions after Renal Ischemia.

Ischemia-induced mitochondrial dysfunction and ATP depletion in the kidney result in disruption of primary functions and acute injury of the kidney. This study tested whether γ-tocotrienol (GTT), a member of the vitamin E family, protects mitochondrial function, reduces ATP deficits, and improves renal functions and survival after ischemia/reperfusion injury. Vehicle or GTT (200 mg/kg) were administered to mice 12 h before bilateral kidney ischemia, and endpoints were assessed at different timepoints of reperfusion. GTT treatment reduced decreases in state 3 respiration and accelerated recovery of this function after ischemia. GTT prevented decreases in activities of complexes I and III of the respiratory chain, and blocked ischemia-induced decreases in F0F1-ATPase activity and ATP content in renal cortical tissue. GTT improved renal morphology at 72 h after ischemia, reduced numbers of necrotic proximal tubular and inflammatory cells, and enhanced tubular regeneration. GTT treatment ameliorated increases in plasma creatinine levels and accelerated recovery of creatinine levels after ischemia. Lastly, 89% of mice receiving GTT and 70% of those receiving vehicle survived ischemia. Conclusions: Our data show novel observations that GTT administration improves mitochondrial respiration, prevents ATP deficits, promotes tubular regeneration, ameliorates decreases in renal functions, and increases survival after acute kidney injury in mice.

P4461Chronic administration of Empagliflozin induces cardioprotection in vivo in absence of diabetes mellitus

Abstract Background/Introduction We have recently shown that empagliflozin (EMPA), a sodium-glucose cotransporter 2 (SGLT2) inhibitor approved for type 2 diabetes mellitus management reduces myocardial infarct size in diabetic mice undergone ischemia/reperfusion (I/R) after chronic administration. However its effect on non-diabetic myocardium remains unspecified. Purpose We aimed to investigate: (i) the effect of EMPA on myocardial function and infarct size after I/R in healthy mice, in the absence of diabetes mellitus, (ii) the underlying signaling pathways, (iii) its effects on cell survival in rat embryonic-heart-derived cardiomyoblasts (H9C2) treated with the inhibitor of STAT3, STATTIC. Methods C57BL/6 mice were initially randomized into two groups, Control and EMPA (n=7 per group) and treated with 5% DMSO in water for injection and EMPA at a dose of 10mg/kg/day with 5% DMSO in water for injection, respectively, for 6 weeks. After this period, the mice were subjected to 30 minutes of I and 2 hours of R and infarct size was evaluated. Body weight, blood pressure, blood glucose levels and left ventricular shortening measurements by echocardiography, were taken at baseline and at the end of the treatment. Furthermore, in order to assess potential differences in the signaling cascades involved at different time points of reperfusion, additional mice were randomized into Control and EMPA groups which were furtherly subdivided into groups (n=4) of 10' and (n=4) of 120' of reperfusion each. The mice were subjected to I/R and myocardial biopsies were obtained for the assessment of the signaling cascade at the 10th and 120th minutes of reperfusion. H9C2 cells subjected to ischemia–reoxygenation were treated with STATIC (0.5, 1, and 10 μM) during the 3 hours of reoxygenation and evaluated for viability. Results Body weight, blood pressure and glucose levels remained unchanged between the groups. We observed no statistically significant change in left ventricular fractional shortening in both groups at baseline (41.0% ± 1.92 vs 40.5% ± 2.7) and after the end of the 6th week (42.7% ± 2.8 vs 40.9% ± 4.1). Infarct size was significantly reduced in EMPA group compared to the Control one (29.5% ± 3.0 vs 45.8% ± 3.2, p<0.05). Phosphorylation of STAT3 was significantly increased at the 10th minute of reperfusion but remained unchanged at the 120th compared to control. The contribution of STAT3 in EMPA-mediated effects was evaluated in H9C2 cells using different doses of STATIC; our results indicate that EMPA completely lost its activity when STAT3 is inhibited. However STATIC per se induces cell death even at low doses. Conclusion(s) EMPA reduces infarct size in healthy mice indicating that its cardioprotective effect is independent of the presence of diabetes mellitus. STAT3 activation can be considered as a cardioprotective mechanism of EMPA, however other signaling pathways could be involved in EMPA mechanism of action and are currently under investigation.

Mitophagy is activated in brain damage induced by cerebral ischemia and reperfusion via the PINK1/Parkin/p62 signalling pathway

This study examined the course of mitophagy following cerebral ischemia with reperfusion and the role of the PTEN-induced kinase 1 (PINK1)/Parkin/p62 signalling pathway. The middle cerebral artery of male Sprague-Dawley rats was occluded for 90 min and was followed by different time-points of reperfusion. Cerebral infarct areas were detected by 2,3,5-triphenyl tetrazolium chloride staining, while brain damage was observed by haematoxylin and eosin staining. Levels of LC3, Beclin1 and LAMP-1 were estimated by western blots. LC3B location was observed in various cells in the neurovascular unit. In addition, PINK1 accumulation in damaged mitochondria and Parkin/p62 mitochondrial translocation were investigated by double immunofluorescence staining. Finally, the levels of PINK1, Parkin and p62 expression in mitochondrial fractions were estimated by western blots. Cerebral ischemia with different time-points of reperfusion resulted in infarct in the territory of the middle cerebral artery accompanied by overall brain damage. In addition, we found up-regulation of LC3B, Beclin1, and LAMP-1, as well as mitophagy activation after reperfusion, with peak expression of these proteins at 24 h after reperfusion. Electron microscopy and immunofluorescence indicated that LC3B was primarily located in neurons, although lower levels of expression were found in astrocytes and even less in vascular endothelial cells. Moreover, significant increases in PINK1 accumulation in the outer membrane of mitochondria and increased Parkin/p62 mitochondrial translocation were shown at 24 h after reperfusion. These findings suggest that the PINK1/Parkin/p62 signalling pathway was involved in the pathophysiological processes following ischemia and reperfusion.

Inhibition of PTEN protects PC12 cells against oxygen-glucose deprivation induced cell death through mitoprotection

Mitochondria involve in the determination of ischemic neuronal cell fate through regulation of apoptotic and necrotic cell death. Phosphatase and tensin homolog (PTEN) protein negatively regulates Akt/PKB signaling which is the major cell survival pathway. The current study aimed to examine the impact of SF1670, a potent PTEN inhibitor, on mitochondria-mediated cell survival pathways in an in vitro stroke-like model. PC12 cells were exposed to one hour oxygen and glucose deprivation (OGD) followed by different time points of reperfusion (0, 30, 60, 120 and 180 min) and SF1670 treatments. Our findings showed that OGD/R exposure increased reactive oxygen species (ROS) levels, reduced phosphorylated Akt (p-Akt), ratios of Bcl-2/BAX, intracellular ATP, mitochondrial vital activity and mitochondrial membrane potential (Δψm). OGD/R exposure also increased cleaved caspase 3/pro-caspase 3 and cleaved caspase 9/pro-caspase 9 ratios associated with low cell viability, high lactate dehydrogenase (LDH) release, and greater apoptotic cell death in the TUNEL assay. Conversely, inhibition of PTEN by SF1670 were associated with increased expression of p-Akt and anti-apoptotic proteins (Bcl-2), attenuated pro-apoptotic proteins (BAX) and oxidative stress index (ROS), improved mitochondrial function (restored MMP and ATP), and decreased apoptotic cell death. These results strongly suggest that neuroprotective effect of SF1670 against OGD/R-induced cell death at least is partially mediated through mitoprotective properties of SF1670.

Optimal Duration of Coronary Ligation and Reperfusion for Reperfusion Injury Study in a Rat Model.

Reperfusion injury (RI) has an important impact on the clinical prognosis for patients with acute myocardial injury who had their coronary blood flow reestablished. However, no studies to date have investigated the timeframe of coronary occlusion and reperfusion effects on RI. A total of 100 rats were divided into 4 groups based on the coronary ligation period: 30, 60, 120, and 180 min, and each group was further divided into 5 subgroups with different reperfusion periods: 0, 30, 60, 120, and 180 min. R0 was the baseline of each subgroup. All animals received the same protocols for designed ligation and reperfusion periods. Evans blue and 2,3,5-triphenyltetrazolium chloride were used to distinguish different myocardial injury areas: area at risk (AAR) and myocardial necrosis. The differences of the ratios of the necrotic area to AAR between each subgroup and baseline were further averaged to calculate an overall value of each heart. The relative RI percentages showed significant differences (0.8 ± 2.3%, 4.9 ± 3.3%, 10.8 ± 3.1%, and 20.3 ± 3.6% respectively, p < 0.001) at different time points of reperfusion but not at different time points of ligation (p = 0.593). The effects of different time courses in RI showed that the L120R180 group (43.4 ± 2.3%) had the highest RI difference with the baseline group. Maximal RI occurred at the timeframe of L120R180 in our animal model. This result may be utilized to assess the substantial benefits of RI therapies in an experimental rat model setting.

RIP3 S-nitrosylation contributes to cerebral ischemic neuronal injury

Cerebral ischemia–reperfusion is associated with NMDA receptor-mediated calcium influx which activates neuronal nitric oxide synthase (nNOS) and consequently induces NO production. NO S-nitrosylates cellular protein and aggravates neuronal injury. Receptor-interacting protein 3 (RIP3) is a sensor molecule regulating cell apoptosis and necrosis. However, the roles of RIP3 in cerebral ischemic injury remain elusive. In this study, we reported that RIP3 could be S-nitrosylated by the exogenous NO donor GSNO in HEK293 cells and the Cys119 residue was the key nitrosylation site. In addition, we found that cerebral ischemia induced RIP3 S-nitrosylation at different time points of reperfusion, which was coupling with RIP3 phosphorylation (which is associated with its activation) and its interaction with receptor-interacting protein 1 (RIP1), and this process facilitated cerebral ischemic injury. Treatment with NMDA receptor antagonist MK801, or nNOS inhibitor 7NI, diminished RIP3 S-nitrosylation and reduced neuronal damage. Taken together, these data demonstrated that NMDAR-dependent RIP3 S-nitrosylation induced by ischemia facilitated its activation in the early stages of ischemia, blocking this process could reduce the ischemia neuronal injury.

Therapeutic time window and underlying therapeutic mechanism of breviscapine injection against cerebral ischemia/reperfusion injury in rats

Ethnopharmacological relevanceBreviscapine injection is a Chinese herbal medicine standardized product extracted from Erigeron breviscapus (Vant.) Hand.-Mazz. It has been widely used for treating cardiovascular and cerebrovascular diseases. However, the therapeutic time window and the action mechanism of breviscapine are still unclear. The present study was designed to investigate the therapeutic time window and underlying therapeutic mechanism of breviscapine injection against cerebral ischemic/reperfusion injury. Materials and methodsSprague–Dawley rats were subjected to middle cerebral artery occlusion for 2h followed by 24h of reperfusion. Experiment part 1 was used to investigate the therapeutic time window of breviscapine. Rats were injected intravenously with 50mg/kg breviscapine at different time-points of reperfusion. After 24h of reperfusion, neurologic score, infarct volume, brain water content and serum level of neuron specific enolase (NSE) were measured in a masked fashion. Part 2 was used to explore the therapeutic mechanism of breviscapine. 4-Hydroxy-2-nonenal (4-HNE), 8-hydroxyl-2′- deoxyguanosine (8-OHdG) and the antioxidant capacity of ischemia cortex were measured by ELISA and ferric-reducing antioxidant power (FRAP) assay, respectively. Immunofluorescence and western blot analysis were used to analyze the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). ResultsPart 1: breviscapine injection significantly ameliorated neurologic deficit, reduced infarct volume and water content, and suppressed the levels of NSE in a time-dependent manner. Part 2: breviscapine inhibited the increased levels of 4-HNE and 8-OHdG, and enhanced the antioxidant capacity of cortex tissue. Moreover, breviscapine obviously raised the expression of Nrf2 and HO-1 proteins after 24h of reperfusion. ConclusionThe therapeutic time window of breviscapine injection for cerebral ischemia/reperfusion injury seemed to be within 5h after reperfusion. By up-regulating the expression of Nrf2/HO-1 pathway might be involved in the therapeutic mechanism of breviscapine injection.

The receptor for activated complement factor 5 (C5aR) conveys myocardial ischemic damage by mediating neutrophil transmigration

Tissue loss after myocardial ischemia with reperfusion (MI/R) is in part conveyed by neutrophil recruitment to post-ischemic myocardium. Strategies to prevent reperfusion injury would help to limit myocardial damage. The receptor for activated complement factor 5 (C5aR) plays a prominent role in inflammation. We examine the effects of C5aR-deficiency on reperfusion injury after MI/R. C5aR−/−-mice and their C57BL/6- (WT) littermates underwent transient myocardial ischemia followed by different time points of reperfusion. Infarct size and leukocyte infiltration were determined. Expression of C5aR, inflammatory cytokines and adhesion molecules were analyzed by real-time RT-PCR. Leukocyte-endothelial interactions were assessed by low-shear adhesion- and transmigration-assays in vitro.Myocardial C5aR mRNA expression was 2.8-fold increased by ischemia. Infarct size per area-at-risk and leukocyte recruitment into infarctions were reduced in C5aR−/−-compared to WT-mice as well as in WT mice treated with the C5aR-antagonist JPE1375. IL-6, IL-1β, ICAM-1 and VCAM-1 expression were not different, while TNFα expression was reduced in C5aR−/−-mice after MI/R. In vitro, C5aR on leukocytes is required for effective transendothelial migration but not adhesion. Expression of MMP9 and JAM-A, molecules that are involved in leukocyte transmigration, were reduced in C5aR−/− mice in vivo.Genetic C5aR deficiency blunts the inflammatory response in murine MI/R resulting in reduced inflammatory cell recruitment, which is due to a C5aR-dependent effect on leukocyte transmigration across inflamed endothelium into the ischemic myocardium. This effect could be related to MMP9- and JAM-A expression in response to ischemia and reperfusion.

Glycyrrhizin protects rat heart against ischemia-reperfusion injury through blockade of HMGB1-dependent phospho-JNK/Bax pathway

Glycyrrhizin (GL) has been found to inhibit extracellular HMGB1 cytokine's activity, and protect spinal cord, liver and brain against I/R-induced injury in experimental animals. The purpose of this study was to investigate the protective effect of GL in rat myocardial I/R-induced injury and to elucidate the underlying mechanisms. Male adult Sprague-Dawley rats underwent a 30-min left coronary artery occlusion followed by a 24-h reperfusion. The rats were treated with glycyrrhizin or glycyrrhizin plus recombinant HMGB1 after 30 min of ischemia and before reperfusion. Serum HMGB1, TNF-α and IL-6 levels, and hemodynamic parameters were measured at the onset and different time points of reperfusion. At the end of the experiment, the heart was excised, and the infarct size and histological changes were examined. The levels of Bcl2, Bax and cytochrome c, as well as phospho-ERK1/2, phospho-JNK and phospho-P38 in the heart tissue were evaluated using Western blot analysis, and myocardial caspase-3 activity was measured colorimetrically using BD pharmingen caspase 3 assay kit. Intravenous administration of GL (10 mg/kg) significantly reduced the infarct size, but did not change the hemodynamic parameters at different time points during reperfusion. GL significantly decreased the levels of serum HMGB1, TNF-α and IL-6. GL changed the distribution of Bax and cytochrome c expression between the mitochondrial and cytosolic fractions in the heart tissue, resulting in inhibition of myocardial apoptosis. Moreover, expression of phospho-JNK, but not ERK1/2 and P38 was decreased by GL in the heart tissue. All of the effects produced by GL treatment were reversed by co-administration with the recombinant HMGB1 (100 μg). Intravenous administration of SP600125, a selective phospho-JNK inhibitor (0.5 mg/kg), attenuated HMGB1-dependent Bax translocation and the subsequent apoptosis. These results demonstrate that GL alleviates rat myocardial I/R-induced injury via directly inhibiting extracellular HMGB1 cytokine activity and blocking the phospho-JNK/Bax pathway.

S-Nitrosylation of Mixed Lineage Kinase 3 Contributes to Its Activation after Cerebral Ischemia

Previous studies in our laboratory have shown that mixed lineage kinase 3 (MLK3) can be activated following global ischemia. In addition, other laboratories have reported that the activation of MLK3 may be linked to the accumulation of free radicals. However, the mechanism of MLK3 activation remains incompletely understood. We report here that MLK3, overexpressed in HEK293 cells, is S-nitrosylated (forming SNO-MLK3) via a reaction with S-nitrosoglutathione, an exogenous nitric oxide (NO) donor, at one critical cysteine residue (Cys-688). We further show that the S-nitrosylation of MLK3 contributes to its dimerization and activation. We also investigated whether the activation of MLK3 is associated with S-nitrosylation following rat brain ischemia/reperfusion. Our results show that the administration of 7-nitroindazole, an inhibitor of neuronal NO synthase (nNOS), or nNOS antisense oligodeoxynucleotides diminished the S-nitrosylation of MLK3 and inhibited its activation induced by cerebral ischemia/reperfusion. In contrast, 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (an inhibitor of inducible NO synthase) or nNOS missense oligodeoxynucleotides did not affect the S-nitrosylation of MLK3. In addition, treatment with sodium nitroprusside (an exogenous NO donor) and S-nitrosoglutathione or MK801, an antagonist of the N-methyl-D-aspartate receptor, also diminished the S-nitrosylation and activation of MLK3 induced by cerebral ischemia/reperfusion. The activation of MLK3 facilitated its downstream protein kinase kinase 4/7 (MKK4/7)-JNK signaling module and both nuclear and non-nuclear apoptosis pathways. These data suggest that the activation of MLK3 during the early stages of ischemia/reperfusion is modulated by S-nitrosylation and provides a potential new approach for stroke therapy whereby the post-translational modification machinery is targeted.

Effects of penehyclidine hydrochloride in small intestinal damage caused by limb ischemia-reperfusion

To investigate the protective effect of penehyclidine hydrochloride post-conditioning in the damage to the barrier function of the small intestinal mucosa caused by limb ischemia-reperfusion (LIR) injury. Male Wistar rats were randomly divided into three groups (36 rats each): the sham-operation group (group S), lower limb ischemia-reperfusion group (group LIR), and penehyclidine hydrochloride post-conditioning group (group PHC). Each group was divided into subgroups (n = 6 in each group) according to ischemic-reperfusion time, i.e. immediately 0 h (T₁), 1 h (T₂), 3 h (T₃), 6 h (T₄), 12 h (T₅), and 24 h (T₆). Bilateral hind-limb ischemia was induced by rubber band application proximal to the level of the greater trochanter for 3 h. In group PHC, 0.15 mg/kg of penehyclidine hydrochloride was injected into the tail vein immediately after 3 h of bilateral hind-limb ischemia. The designated rats were sacrificed at different time-points of reperfusion; diamine oxidase (DAO), superoxide dismutase (SOD) activity, myeloperoxidase (MPO) of small intestinal tissue, plasma endotoxin, DAO, tumor necrosis factor-α (TNF-α), and interleukin (IL)-10 in serum were detected in the rats. The pathological changes in the small intestine were observed under light microscope. The levels of MPO, endotoxin, serum DAO, and IL-10 at T₁-T₆, and TNF-α level at T₁-T₄ increased in groups LIR and PHC (P < 0.05) compared with those in group S, but tissue DAO and SOD activity at T₁-T₆ decreased (P < 0.05). In group PHC, the tissue DAO and SOD activity at T₂-T₆, and IL-10 at T₂-T₅ increased to higher levels than those in group LIR (P < 0.05); however, the levels of MPO, endotoxin, and DAO in the blood at T₂-T₆, and TNF-α at T₂ and T₄ decreased (P < 0.05). Penehyclidine hydrochloride post-conditioning may reduce the permeability of the small intestines after LIR. Its protection mechanisms may be related to inhibiting oxygen free radicals and inflammatory cytokines for organ damage.

Dynamic change of hydrogen sulfide during global cerebral ischemia–reperfusion and its effect in rats

Hydrogen sulfide (H 2S) is a gaseous messenger and serves as an important neuromodulator in central nervous system. In the current study, we investigated the change of H 2S and cystathionine β-synthase (CBS), an H 2S-synthesizing enzyme at different time points of reperfusion following global cerebral ischemia in rats, and the effect of exogenous H 2S on global cerebral ischemia–reperfusion injury. First, we used global cerebral ischemia–reperfusion model by occlusion of bilateral common carotid arteries and vertebral arteries. Next, we measured H 2S levels in the hippocampus, cortex and plasma, the activity of H 2S-synthesizing enzymes and expression of CBS mRNA and protein in the hippocampus and cortex at 12 h, 24 h, 48 h, 72 h and 7 days of reperfusion following 15 min cerebral ischemia. Second, we pretreated rats with different doses of sodium hydrogen sulfide (NaHS), an H 2S donor and observed its effect on neuronal injury induced by 7 days of reperfusion after 15 min global cerebral ischemia. We found that when compared to sham group the amount of H 2S in the hippocampus was increased significantly at 12 h of reperfusion after cerebral ischemia, markedly decreased at 24 h, restored to the same level as that in sham group at 48 h and maintained at 72 h and 7 days. The same change tendency in the levels of H 2S was found in the cortex as described for the hippocampus. We found a similar change tendency in the activity of H 2S-synthesizing enzymes, CBS mRNA and protein expression to that in the H 2S level at different time points of reperfusion. Furthermore, while 180 μmol/kg NaHS pretreatment deteriorated the neuronal injury after global cerebral ischemia, 25 μmol/kg NaHS attenuated the neuronal injury. We suggest that a decrease of H 2S level at 24 h of reperfusion after global cerebral ischemia may be involved in neuronal injury after cerebral ischemia and lower concentration rather than higher concentration of exogenous H 2S may offer a protection against the neuronal injury induced by global cerebral ischemia–reperfusion.

Neuroprotective effect of grafting GDNF gene-modified neural stem cells on cerebral ischemia in rats

Previous studies indicated the beneficial effects of glial cell line-derived neurotrophic factor (GDNF) and transplanted neural stem cells (NSCs) on stroke. Here, we explored whether transplantation of neural stem cells (NSCs) modified by GDNF gene provides a better therapeutic effect than native NSCs after stroke. Primary rat NSCs were transfected with GDNF plasmid (GDNF/NSCs, labeled by green fluorescent protein from AdEasy-1, GFP). Adult rats were subjected to two-hour middle cerebral artery occlusion and reperfusion, followed by infusion of NSCs (labeled with5-bromo-2′-deoxyuridine before infusion, BrdU), GDNF/NSCs and saline at 3 days after reperfusion (NSCs group, GDNF/NSCs group, control group), respectively. All rats were sacrificed at 1, 2, 3, 5, and 7 weeks after reperfusion. Modified Neurological Severity Scores (mNSS) test and H and E staining were respectively performed to evaluate neurological function and lesion volume. Immunohistochemistry was used to identify implanted cells and observe the expressions of Synaptophysin (Syp) and postsynaptic density-95 (PSD-95) and caspase-3. TdT-mediated dUTP–biotin nick-end labeling (TUNEL) was employed to observe apoptotic cells. Western blotting was used to detect brain-derived neurotrophic factor (BDNF) and NT-3 protein expression. Significant recovery of mNSS was found in GDNF/NSCs rats at 2 and 3 weeks after reperfusion compared with NSCs rats. Lesion volume in the NSCs and GDNF/NSCs groups was reduced significantly compared with control group. The number of NSCs in the GDNF/NSCs group was significantly increased in comparison with NSCs group. Moreover, Syp-immunoreactive product at 2 and 3 weeks after reperfusion and PSD-95 immunoreactive product in the GDNF/NSCs group were significantly increased compared with NSCs group. In contrast, caspase-3 positive cells and TUNEL-positive cells in the GDNF/NSCs group were significantly decreased compared with NSCs group. Significant increase of BDNF protein in the GDNF/NSCs and NSCs groups was observed compared to the control group at different time points of reperfusion, and GDNF/NSCs grafting significantly increased BDNF protein expression compared to NSCs grafting. In addition, significant increase of NT-3 protein in GDNF/NSCs and NSCs groups was detected only at 1 week of reperfusion compared to control group. The results demonstrate that grafting NSCs modified by GDNF gene provides better neuroprotection for stroke than NSCs grafting alone.

Toll-Like Receptor 4-Mediated Myeloid Differentiation Factor 88-Dependent Signaling Pathway Is Activated by Cerebral Ischemia-Reperfusion in Hippocampal CA1 Region in Mice

The Toll-like receptor 4 (TLR4)-mediated myeloid differentiation factor 88 (MyD88)-dependent signaling pathway plays an essential role in inflammation resulting from invading microbes. However, whether the signaling pathway is activated in the inflammatory reaction of cerebral ischemia-reperfusion and its mechanism is still unclear. In this experiment mice were randomly divided into sham group, ischemia/reperfusion group and TLR4-blocked group with different time points of reperfusion at 12, 24, 48 and 72 h . Mice cerebral ischemia was induced by occlusion of common carotid arteries (CCA) bilaterally. TLR4 signaling pathway was inhibited using specific anti-TLR4 binding protein to prevent TLR4 from interacting with its receptors. We determined the result of TLR4 antibodies-blocking and mice cerebral ischemia-reperfusion injuries by Western blot, and evaluated neuronal damage in the hippocampus. We also determined expression of TLR4 mRNA and MyD88 mRNA by in situ hybridization (ISH), activation of nuclear factor (NF)-kappaB by electrophoretic mobility-shift analysis (EMSA), and expression of interrleukin (IL)-1beta protein by Western blot. The results demonstrated that TLR4-mediated MyD88-dependent signaling pathway activated by ischemia-reperfusion may be involved in the mechanism of ischemia-reperfusion through upregulation of NF-kappaB, IL-1beta.

TLR4-mediated MyD88-dependent signaling pathway is activated by cerebral ischemia–reperfusion in cortex in mice

To study whether the signaling pathway is activated in the inflammatory reaction of cerebral ischemia–reperfusion and its mechanism. The mice were randomly divided into sham group, ischemia–reperfusion group and TLR4-blocked group with different time points of reperfusion 12 h, 24 h, 48 h and 72 h group. We observed the different expression of TLR4 mRNA and MyD88 mRNA, activation of NF-κB and the TNF-α and IL-1β protein levels in each group at different time point after ischemia–reperfusion. Mice cerebral ischemia was induced by occlusion of common carotid arteries (CCA) bilaterally. TLR4 signaling pathway could be inhibited by specific anti-TLR4 binding protein to prevent TLR4 from interacting with its receptors. We determined the result of TLR4 antibodies-blocking and mice cerebral ischemia–reperfusion injuries by Western blot, and evaluated neuronal damage in cortex. We also determined the expression of TLR4 mRNA and MyD88 mRNA by in situ hybridization (ISH), the activation of NF-κB by EMSA, and the expression of TNF-α protein by Western blot. Anti-TLR4 binding TLR4 receptors before reperfusion was effective; There was distinct difference among each group respecting neuronal damage; The expression of TLR4 mRNA and MyD88 mRNA, the activation of NF-κB, and the expression of TNF-α protein showed clear difference as well. LR4-mediated MyD88-dependent signaling pathway activated by ischemia–reperfusion may be involved in the mechanism of ischemia–reperfusion through upregulation of NF-κB and TNF-α.

Delayed administration of alpha-melanocyte-stimulating hormone or combined therapy with BAY 11-7085 protects against gut ischemia-reperfusion injury.

Gut ischemia-reperfusion (I/R) injury is a serious complication of shock. Previously we demonstrated that the administration of alpha-melanocyte-stimulating hormone (MSH) immediately before mesenteric I/R protected against postischemic gut injury. In this report, we tested the therapeutic efficacy of alpha-MSH on gut I/R (60 min ischemia, 6 h reperfusion) injury when given at different time points of reperfusion. Rats underwent sham surgery or were treated with saline or with alpha-MSH that was given 1, 2, or 4 h after superior mesenteric artery clamping. Vehicle-treated I/R rats exhibited severe mucosal injury and increased NF-kappaB DNA binding activity, myeloperoxidase (MPO) activity, and interleukin-6 and heme oxygenase-1 (HO-1) expression. In contrast, rats given alpha-MSH at 1 h of reperfusion, but not 2 h or 4 h, exhibited much less mucosal injury. Rats given alpha-MSH at 1 h or 2 h of reperfusion, but not 4 h, exhibited less MPO activity, NF-kappaB DNA binding activity, and interleukin-6 protein and even higher levels of heme oxygenase-1 than vehicle-treated rats. In addition, we found that combined use of alpha-MSH, a known inhibitor of IkappaBalpha tyrosine phosphorylation, with BAY 11-7085, an inhibitor of IkappaBalpha Ser 32,36 phosphorylation, abrogates gut MPO induction and tissue injury at early and late time points of reperfusion. Thus, alpha-MSH, an endogenous peptide with a favorable side-effect profile, is effective in treating experimental gut I/R injury when given early after the initial ischemia and may represent a candidate therapy for gut I/R in humans in whom recognition and treatment are often delayed.