24-h Reperfusion Research Articles

Caspase recruitment domain-containing protein 9 (CARD9) knockout reduces regional ischemia/reperfusion injury through an attenuated inflammatory response.

Ischemic heart disease remains a leading cause of morbidity and mortality in the United States. Interventional reperfusion induces further damage to the ischemic myocardium through neutrophil infiltration and acute inflammation. As caspase recruitment domain-containing protein 9 (CARD9) plays a critical role in innate immune response and inflammation, we hypothesized that CARD9 knockout would provide protection against ischemia and reperfusion (I/R) injury through attenuation of acute inflammatory responses. C57BL/6 wild-type (WT) and CARD9-/- mice were subjected to 45 min left anterior descending (LAD) coronary artery occlusion followed by 24-h reperfusion. Area at risk (AAR) and infarct size were measured by Evans blue and triphenyltetrazolium chloride (TTC) staining. Frozen heart sections were stained with anti-mouse GR-1 antibody to detect infiltrated neutrophils. Concentrations of cytokines/chemokines TNF-α, IL-6, CXCL-1 and MCP-1 were determined in heart tissue homogenate and serum by ELISA assay. Western immunoblotting analyses were performed to measure the phosphorylation of p38 MAPK. Our results indicate that following I/R, infarct size was significantly smaller in CARD9-/- mice compared to WT. The number of infiltrated neutrophils was significantly lower in CARD9-/- mice compared to WT. Levels of TNF-α, IL-6, CXCL-1 and MCP-1 were significantly reduced in heart tissue and serum from CARD9-/- mice compared to WT. CARD9-/- mice also exhibited significantly lower levels of phosphorylated p38 MAPK. Taken together, our results suggest that CARD9 knockout protects the heart from ischemia/reperfusion (I/R) injury, possibly through reduction of neutrophil infiltration and attenuation of CARD9-associated acute inflammatory signaling.

Antioxidant-mediated neuroprotection by Allium schoenoprasum L. leaf extract against ischemia reperfusion-induced cerebral injury in mice

Background Oxidative stress is strongly implicated in ischemia reperfusion (IR)-mediated functional and neuronal impairment. Therefore, strategies employing antioxidants to reverse the damage due to IR are being investigated. Allium schoenoprasum L. is a culinary medicine whose antioxidant properties are well documented but whose neuroprotective potential has not been examined. Hence, the present study was designed to evaluate the effect of A. schoenoprasum leaf extract (ASLE) on functional deficit against IR-induced cerebral injury in mice. Methods Acute toxicity studies of ASLE were performed following the Organisation for Economic Co-operation and Development Guideline 423. IR injury was induced by bilateral common carotid artery occlusion (BCCAO) for 15 min followed by 24-h reperfusion. Animals were treated for 7 days with ASLE (200 and 400 mg/kg, p.o. once daily) after IR injury. Functional outcomes (memory and sensorimotor functions) were measured using Morris water maze and neurological severity score, respectively. Cerebral infarct size and oxidative stress (thiobarbituric acid reactive species (TBARS), reduced glutathione (GSH), and superoxide dismutase (SOD) activity) were measured in order to elucidate the neuroprotective mechanism of ASLE. Results No toxic effects of ASLE were observed in mice. Oral treatment with ASLE for 7 days significantly attenuated IR-mediated memory and sensorimotor function deficit in the animals. The extract also reduced the cerebral infarct size and rise in brain TBARS levels, and restored the GSH levels and SOD activity. Conclusions The results of the present study suggest that ASLE is safe and effective in improving functional outcomes. It demonstrates neuroprotective effect by enhancing the antioxidant defence against IR injury.

Hepatorenal protection during renal ischemia by quercetin and remote ischemic perconditioning

BackgroundPathogenesis of renal ischemia/reperfusion injury (IRI) involves oxidative stress response in the kidney and remote organs. Both quercetin and remote ischemic perconditioning (RIPerC) can protect partially against IRI. This study determined whether combined quercetin and RIPerC could provide an augmented hepatorenal protection against renal IRI. Materials and methodsI/R was induced by clamping renal arteries for 45 min followed by 24-h reperfusion. RIPerC consisted of four cycles of 2 min of left femoral artery ischemia followed by 3 min of reperfusion administered at the beginning of renal ischemia. Rats were divided into five groups: sham, I/R, RIPerC, quercetin (Q + I/R), and combined quercetin and RIPerC (Q + RIPerC). At the end of reperfusion period, blood, urine, and tissue samples were collected. ResultsI/R caused kidney dysfunction, as proved by significant decrease in creatinine clearance, and a significant increase in liver functional indicators as evidenced by increased plasma alanine aminotransferase and aspartate aminotransferase activity. This was accompanied by a decrease of glutathione peroxidase and catalase activities with an increase of malondialdehyde levels and histological damages in renal and hepatic tissues. Treatment with RIPerC and quercetin reduced all these changes. However, the measure of improvements was enhanced by combined quercetin and RIPerC treatment. ConclusionsThis study demonstrated protective effects of quercetin and RIPerC strategy on the both kidney and liver after renal I/R. The results suggest that combined quercetin and RIPerC provides an enhanced protection against renal IRI by reduction of lipid peroxidation and augmentation of antioxidant systems.

Modulation of brain ACE and ACE2 may be a promising protective strategy against cerebral ischemia/reperfusion injury: an experimental trial in rats.

The brain renin-angiotensin system (RAS) is considered a crucial regulator for physiological homeostasis and disease progression. We evaluated the protective effects of the angiotensin receptor blocker (ARB) telmisartan and the angiotensin-converting enzyme 2 (ACE2) activator xanthenone on experimental cerebral ischemia/reperfusion (I/R) injury. Rats were divided into a sham control, a cerebral I/R control, a standard treatment (nimodipine, 10mg/kg/day, 15days, p.o.), three telmisartan treatments (1, 3, and 10mg/kg/day, 15days, p.o.), and three xanthenone treatments (0.5, 1, and 2mg/kg/day, 15days, s.c.) groups. One hour after the last dose, all rats except the sham control group were exposed to 30-min cerebral ischemia followed by 24-h reperfusion. Brain ACE and ACE2 activities and the apoptotic marker caspase-3 levels were assessed. Glutathione (GSH), malondialdehyde (MDA), and nitric oxide end products (NOx) as oxidative markers and tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and IL-10 as immunological markers were assessed. Histopathological examination and immunohistochemical evaluation of glial fibrillary acidic protein (GFAP) were performed in cerebral cortex and hippocampus sections. Telmisartan and xanthenone in the higher doses restored MDA, NOx, TNF-α, IL-6, caspase-3, ACE, and GFAP back to normal levels and significantly increased GSH, IL-10, and ACE2 compared to I/R control values. Histopathologically, both agents showed mild degenerative changes and necrosis of neurons in cerebral cortex and hippocampus compared with I/R control group. Modulation of brain RAS, either through suppression of the classic ACE pathway or stimulation of its antagonist pathway ACE2, may be a promising strategy against cerebral I/R damage.

Neuroprotection of Cytisine Against Cerebral Ischemia-Reperfusion Injury in Mice by Regulating NR2B-ERK/CREB Signal Pathway.

The aim of the study was to elucidate the therapeutic effects of Cytisine (CYT) on cerebral ischemia-reperfusion injury in mice. Male ICR mice were pretreated with reagents (drug), and then subjected to 2h focal cerebral ischemia and 24h reperfusion. Morphologically, the histopathological impairment were estimated by the TTC, HE and TUNEL staining. The expression of GluN2B-containing NMDA receptor, phosphorylation of extracellular regulated protein kinases, total ERK, phosphorylation of cAMP-response element binding protein and total CREB were determined by immunofluorescence and Western blot assay, respectively. The mRNA expression of NR2B, ERK and CREB were quantified by the real-time RT-PCR. CYT significantly diminished the infarct size and neuronal apoptosis. Additionally, it ameliorated histopathological lesion dramatically. CYT promoted the phosphorylation of ERK, CREB and their mRNA expression. In contrast, the expression of NR2B was suppressed in concomitant with the down-regulation of genes. The overall results thus far suggest that CYT confers the neuroprotection against cerebral I/R injury by regulating the NR2B-ERK/CREB signal pathway.

IL-35 is a Protective Immunomodulator in Brain Ischemic Injury in Mice

IL-35 has been identified as a novel anti-inflammatory cytokine that belongs to the IL-12 cytokine family and has been verified to play a protective role in autoimmune diseases. In this study, we investigated the protective effects of IL-35 on cerebral ischemia/reperfusion (I/R) injury in a middle cerebral artery occlusion mouse model. We determined that the expression of IL-35 was initially decreased and subsequently increased in I/R injury. Moreover, IL-35 (i.c.v.) pre- and posttreatment significantly reduced the infarct volume and improved neurological deficits after 45min of ischemia and 24h of reperfusion. Importantly, IL-35 treatment improved neurological function recovery, particularly in balance ability, at 14days after treatment. Finally, our results showed that IL-35 treatment reduced the expression of IL-6 and IL-1β, which are confirmed proinflammatory cytokines, thus indicating that these cytokines have both been linked to the anti-inflammatory mechanisms of IL-35. Therefore, IL-35 may be a key immune mediator in brain ischemic injury and appears to have promising potential for clinical trials.

Deep Sequencing Reveals Uncharted Isoform Heterogeneity of the Protein-Coding Transcriptome in Cerebral Ischemia.

Gene expression in cerebral ischemia has been a subject of intense investigations for several years. Studies utilizing probe-based high-throughput methodologies such as microarrays have contributed significantly to our existing knowledge but lacked the capacity to dissect the transcriptome in detail. Genome-wide RNA-sequencing (RNA-seq) enables comprehensive examinations of transcriptomes for attributes such as strandedness, alternative splicing, alternative transcription start/stop sites, and sequence composition, thus providing a very detailed account of gene expression. Leveraging this capability, we conducted an in-depth, genome-wide evaluation of the protein-coding transcriptome of the adult mouse cortex after transient focal ischemia at 6, 12, or 24h of reperfusion using RNA-seq. We identified a total of 1007 transcripts at 6h, 1878 transcripts at 12h, and 1618 transcripts at 24h of reperfusion that were significantly altered as compared to sham controls. With isoform-level resolution, we identified 23 splice variants arising from 23 genes that were novel mRNA isoforms. For a subset of genes, we detected reperfusion time-point-dependent splice isoform switching, indicating an expression and/or functional switch for these genes. Finally, for 286 genes across all three reperfusion time-points, we discovered multiple, distinct, simultaneously expressed and differentially altered isoforms per gene that were generated via alternative transcription start/stop sites. Of these, 165 isoforms derived from 109 genes were novel mRNAs. Together, our data unravel the protein-coding transcriptome of the cerebral cortex at an unprecedented depth to provide several new insights into the flexibility and complexity of stroke-related gene transcription and transcript organization.

Low-Dose 4-Hydroxy-2-Nonenal (HNE) Reperfusion Therapy Displays Cardioprotective Effects in Mice After Myocardial Infarction That Are Abrogated by Genipin.

BackgroundRevascularization is a successful therapeutic strategy for myocardial infarction. However, restoring coronary blood flow can lead to ischemia-reperfusion (I/R) injury. Low-dose 4-hydroxy-2-nonenal (HNE) therapy appears to play a key role in myocardial tolerance to I/R injury. We hypothesized that the positive effects of HNE on myocardial I/R injury may be UCP3-dependent.Material/MethodsAdult male wild-type (WT) or UCP3 knockout (UCP3−/−) mice were pre-treated with the UCP inhibitor genipin or saline 1 h before ischemia and underwent 30-min coronary artery ligation followed by 24-h reperfusion. Mice were treated with intravenous HNE (4 mg/kg) or saline 5 min before reperfusion. Echocardiography was conducted to measure left ventricular end-diastolic posterior wall thickness (LVPWd), end-diastolic diameter (LVEDD), and fractional shortening (FS). Infarct size was measured by TTC staining. qRT-PCR and Western blotting were used to assess the expression of UCP3, UCP2, and the apoptosis markers cytochrome C and cleaved caspase-3.ResultsHNE improved survival at 24 h post-MI in wild-type mice (p<0.05) but not in UCP3−/− mice. HNE preserved LVEDD and FS in WT mice (p<0.05) but not in UCP3−/− mice. HNE reduced infarct size in WT mice (p<0.05) but not in UCP3−/− mice. HNE upregulated UCP3 expression (p<0.05) but did not affect UCP2 expression. HNE reduced apoptosis marker expression in WT mice (p<0.05) but not in UCP3−/− mice. HNE’s positive effects were abrogated by genipin in an UCP3-dependent manner.ConclusionsLow-dose HNE reperfusion therapy attenuates murine myocardial I/R injury in an UCP3-dependent manner. These effects are abrogated by genipin in an UCP3-dependent manner.

NMR metabolomic study of blood plasma in ischemic and ischemically preconditioned rats: an increased level of ketone bodies and decreased content of glycolytic products 24h after global cerebral ischemia.

Cardiac arrest is one of the leading causes of death among adults in older age. Understanding mechanisms how organism responds to ischemia at global level is essential for the prevention and ischemic patient's treatment. In this study, we used a global cerebral ischemia induced by four-vessel occlusion as an established animal model for ischemic stroke to investigate metabolic changes after 24h reperfusion, when transitions occur due to the onset of delayed neuronal death. We also focused on the endogenous phenomenon known as ischemic tolerance by the pre-ischemic treatment. The experiments were carried out on blood plasma samples as easily available and metabolically reflecting the overall changes in injured organism. Our results imply that disturbed glycolysis pathway, as a consequence of ischemic injury, leads to the increased level of ketone bodies (acetone, acetoacetate and β-hydroxybutyrate) along with increased utilization of triacylglycerols in plasma of ischemic and ischemically preconditioned rats. Complementary to, a decreased level of glycolytic intermediates (lactate, pyruvate, acetate) with increased level of glucose was found in ischemic and preconditioned animals. The protective effect of ischemic preconditioning on metabolome recovery was demonstrated by significantly increased level of creatine compared to ischemic, non-preconditioned rats. We also document that acetoacetate, pyruvate, lactate, and leucine have the best discriminatory power between ischemic and control plasma. Conclusively, our results provide evidence that NMR spectra analysis can identify specific group of metabolites present in plasma with the capability for discrimination between individual groups of animals. In addition, an excellent feasibility for the statistical discrimination among ischemic, preconditioned, and control rats can be applied regardless of native or deproteinated plasma and also regardless of noesy or cpmg NMR acquisition.

Artesunate Inhibits Renal Ischemia-Reperfusion-Mediated Remote Lung Inflammation Through Attenuating ROS-Induced Activation of NLRP3 Inflammasome

The molecular mechanisms of acute lung injury (ALI) are closely associated with nucleotide-binding domains and leucine-rich repeat (NLR) pyrin domains containing 3 (NLRP3) inflammasome, in which alveolar macrophages (AMs) exert an essential function. Our study has been proved that artesunate (AS) inhibits ALI. Nevertheless, the inhibition actions of AS on activation of NLRP3 in renal ischemia-reperfusion (RIR)-mediated ALI remain to be further discussed. Male Sprague-Dawley rats were randomly assigned into four groups: sham + NS, sham + AS, RIR + NS, and RIR + AS. RIR-mediated ALI was performed through bilateral renal pedicle occlusion for 60min followed by reperfusion for 24h. AS (15mg/kg) or NS was injected intraperitoneal to rat 1h before RIR treatment. AMs were rendered hypoxic (0.5%) for 2h and reoxygenated for 24h. Lung injury index and histology, and inflammatory cells and cytokine release in the BALF and AMs were examined. The protein and mRNA levels of NLRP3, ASC, and caspase-1 in the lung and AMs were evaluated via Western blot and real-time RCR. In this research, we indicated that AS preconditioning inhibited RIR-mediated lung damage, vascular permeability, and edema in rats. AS reduced RIR-mediated ALI, as characterized by abatement in the count of inflammatory cells, and the production of inflammatory cytokines in the BALF. AS administration inhibited the number of F4/80-positive cells, the activity of myeloperoxidase, and the fiery cytokines mRNA expression in lung samples of RIR-stimulated rats. Furthermore, AS alleviated the activity of caspase-1 and activation of NLRP3 through depending on reactive oxygen species (ROS). An in vitro finding that AS mitigated hypoxia/reoxygenation-mediated activation of AMs partially supported in vivo study. In a word, these findings demonstrate that AS pretreatment attenuated RIR-mediated ALI potentially through reducing ROS-induced activation of the NLRP3 inflammasome.

Oxidized phosphatidylcholines are produced in renal ischemia reperfusion injury.

BackgroundThe aim of this study was to determine the individual oxidized phosphatidylcholine (OxPC) molecules generated during renal ischemia/ reperfusion (I/R) injury.MethodsKidney ischemia was induced in male Sprague–Dawley rats by clamping the left renal pedicle for 45 min followed by reperfusion for either 6h or 24h. Kidney tissue was subjected to lipid extraction. Phospholipids and OxPC species were identified and quantitated using liquid chromatography coupled to electrospray ionization tandem mass spectrometry using internal standards.ResultWe identified fifty-five distinct OxPC in rat kidney following I/R injury. These included a variety of fragmented (aldehyde and carboxylic acid containing species) and non-fragmented products. 1-stearoyl-2-linoleoyl-phosphatidylcholine (SLPC-OH), which is a non-fragmented OxPC and 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PAzPC), which is a fragmented OxPC, were the most abundant OxPC species after 6h and 24 h I/R respectively. Total fragmented aldehyde OxPC were significantly higher in 6h and 24h I/R groups compared to sham operated groups (P = 0.03, 0.001 respectively). Moreover, levels of aldehyde OxPC at 24h I/R were significantly greater than those in 6h I/R (P = 0.007). Fragmented carboxylic acid increased significantly in 24h I/R group compared with sham and 6h I/R groups (P = 0.001, 0.001). Moreover, levels of fragmented OxPC were significantly correlated with creatinine levels (r = 0.885, P = 0.001). Among non-fragmented OxPC, only isoprostanes were elevated significantly in 6h I/R group compared with sham group but not in 24h I/R group (P = 0.01). No significant changes were observed in other non-fragmented OxPC including long chain products and terminal furans.ConclusionWe have shown for the first time that bioactive OxPC species are produced in renal I/R and their levels increase with increasing time of reperfusion in a kidney model of I/R and correlate with severity of I/R injury. Given the pathological activity of fragmented OxPCs, therapies focused on their reduction may be a mechanism to attenuate renal I/R injury.

SIRT2 Inhibition Confers Neuroprotection by Downregulation of FOXO3a and MAPK Signaling Pathways in Ischemic Stroke.

Sirtuin 2 (SIRT2) is a family member of nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases which appears to have detrimental roles in an array of neurological disorders such as Parkinson's disease (PD) and Huntington's disease (HD). In light of the recently emerging roles of sirtuins in normal physiology and pathological conditions such as ischemic stroke, we investigated the role of SIRT2 in ischemic stroke-induced neuronal cell death. Primary cortical neurons were subjected to oxygen-glucose deprivation (OGD) under in vitro ischemic conditions, and subsequently tested for the efficacy of SIRT2 inhibitors AK1 and AGK2 in attenuating apoptotic cell death caused by OGD. We have also evaluated the effect of SIRT2 inhibition in C57BL/6 mice subjected to 1h middle cerebral artery occlusion (MCAO) followed by 24h reperfusion, which is a model for ischemic reperfusion injury in vivo. Significant reductions in apoptotic cell death were noted in neurons treated with AK1 or AGK2, as evidenced by reduced cleaved caspase-3 and other apoptotic markers such as Bim and Bad. In addition, downregulation of phosphorylated-AKT and FOXO3a proteins of the AKT/FOXO3a pathway, as well as a marked reduction of JNK activity and its downstream target c-Jun, were also observed. When tested in animals subjected to MCAO, the neuroprotective effects of AGK2 in vivo were evidenced by a substantial reduction in ipsilateral infarct area and a significant improvement in neurological outcomes. A similar reduction in the levels of pro-apoptotic proteins in the infarct tissue, as well as downregulation of AKT/FOXO3a and JNK pathway, were also noted. In summary, the current study demonstrated the neuroprotective effects of SIRT2 inhibition in ischemic stroke, and identified the downregulation of AKT/FOXO3a and MAPK pathways as intermediary mechanisms which may contribute to the reduction in apoptotic cell death by SIRT2 inhibition.

Sirtuin 5 as a novel target to blunt blood–brain barrier damage induced by cerebral ischemia/reperfusion injury

BackgroundIn acute ischemic stroke (AIS) patients, impaired blood–brain barrier (BBB) integrity is associated with hemorrhagic transformation and worsened outcome. Yet, the mechanisms underlying these relationships are poorly understood and consequently therapeutic strategies are lacking. This study sought to determine whether SIRT5 contributes to BBB damage following I/R brain injury. Methods and resultsSIRT5 knockout (SIRT5−/−) and wild type (WT) mice underwent transient middle cerebral artery (MCA) occlusion (tMCAO) followed by 48h of reperfusion. Genetic deletion of SIRT5 decreased infarct size, improved neurological function and blunted systemic inflammation following stroke. Similar effects were also achieved by in vivo SIRT5 silencing. Immunohistochemical analysis revealed decreased BBB leakage and degradation of the tight junction protein occludin in SIRT5−/− mice exposed to tMCAO as compared to WT. In primary human brain microvascular endothelial cells (HBMVECs) exposed to hypoxia/reoxygenation (H/R), SIRT5 silencing decreased endothelial permeability and upregulated occludin and claudin-5; this effect was prevented by the PI3K inhibitor wortmannin. Lastly, SIRT5 gene expression was increased in peripheral blood monocytes (PBMCs) of AIS patients at 6h after onset of stroke compared to sex- and age-matched healthy controls. ConclusionSIRT5 is upregulated in PBMCs of AIS patients and in the MCA of WT mice exposed to tMCAO; SIRT5 mediates I/R-induced brain damage by increasing BBB permeability through degradation of occludin. This effect was reproduced in HBMVECs exposed to H/R, mediated by the PI3K/Akt pathway. Our findings shed new light on the mechanisms of I/R-dependent brain damage and suggest SIRT5 as a novel therapeutic target.

Pentose phosphate pathway activation via HSP27 phosphorylation by ATM kinase: A putative endogenous antioxidant defense mechanism during cerebral ischemia-reperfusion

Molecular mechanism underlying ischemic stroke remains poorly understood. We previously reported glucose 6-phosphate dehydrogenase (G6PD) activity in pentose phosphate pathway (PPP) is activated via heat shock protein 27 (HSP27) phosphorylation at serine 85 (S85) by ataxia telangiectasia mutated (ATM) kinase during cerebral ischemia. This mechanism seems to be endogenous antioxidative system. To determine whether this system also works during reperfusion, we performed comparative metabolic analysis of reperfusion effect on metabolism in rat cortex using middle cerebral artery occlusion (MCAO). Metabolic profiling using gas-chromatography/mass-spectrometry analysis showed changes in metabolic state that depended on reperfusion time. Enrichment analysis showed PPP was significantly upregulated during ischemia-reperfusion. Significant increases in fructose 6-phosphate and ribulose 5-phosphate after reperfusion also suggested enhancement of PPP. In relation to PPP, ischemia-reperfusion induced an increase of up to 69-fold in HSP27 transcripts after 24-h reperfusion. Immunoblotting showed gradual increase in HSP27 protein and marked increase in HSP27 phosphorylation (S85) that were time-dependent (4.5-fold after 24-h reperfusion). G6PD activity was significantly elevated after 1-h MCAO (20%), reduced after 1-h reperfusion, increased gradually thereafter and significantly elevated after 24-h reperfusion. The NADPH/NAD+ ratio displayed similar increasing pattern. Intracerebroventricular injection of ATM kinase inhibitor (KU-55933) significantly reduced HSP27 phosphorylation and G6PD activity, significantly increased protein carbonyl, and resulted in increase in infarct size (100%) 24-h after reperfusion following 90-min MCAO. Consequently, G6PD activation via HSP27 phosphorylation by ATM kinase may be part of endogenous antioxidant defense neuroprotection mechanism that is activated during ischemia-reperfusion. These findings have important implications for treatment of stroke.

Salvianolic acid A attenuates ischemia reperfusion induced rat brain damage by protecting the blood brain barrier through MMP-9 inhibition and anti-inflammation

Salvianolic acid A (SAA) is a water-soluble component from the root of Salvia Miltiorrhiza Bge, a traditional Chinese medicine, which has been used for the treatment of cerebrovascular diseases for centuries. The present study aimed to determine the brain protective effects of SAA against cerebral ischemia reperfusion injury in rats, and to figure out whether SAA could protect the blood brain barrier (BBB) through matrix metallopeptidase 9 (MMP-9) inhibition. A focal cerebral ischemia reperfusion model was induced by middle cerebral artery occlusion (MCAO) for 1.5-h followed by 24-h reperfusion. SAA was administered intravenously at doses of 5, 10, and 20 mg·kg−1. SAA significantly reduced the infarct volumes and neurological deficit scores. Immunohistochemical analyses showed that SAA treatments could also improve the morphology of neurons in hippocampus CA1 and CA3 regions and increase the number of neurons. Western blotting analyses showed that SAA downregulated the levels of MMP-9 and upregulated the levels of tissue inhibitor of metalloproteinase 1 (TIMP-1) to attenuate BBB injury. SAA treatment significantly prevented MMP-9-induced degradation of ZO-1, claudin-5 and occludin proteins. SAA also prevented cerebral NF-κB p65 activation and reduced inflammation response. Our results suggested that SAA could be a promising agent to attenuate cerebral ischemia reperfusion injury through MMP-9 inhibition and anti-inflammation activities.

A1AR-mediated renal protection against ischemia/reperfusion injury is dependent on HSP27 induction.

A1 adenosine receptor (AR) activation has been demonstrated to attenuate renal ischemia/reperfusion injury (IRI), but the exact mechanism of this protection remains to be well elucidated. Male C57BL/6 mice were used in the present study. Expression of heat shock protein (HSP) 27 and HSF-1 were detected using western blot analysis. An RNA interference with adenovirus vector using short hairpin RNA targeting HSP27 was developed. Together with renal IRI model, indicators of renal function, acute tubular necrosis, inflammation and apoptosis were measured in kidneys after 24-h reperfusion. We found activation of A1AR stimulated induction of HSP27 and its major transcriptional factor HSF-1. It was observed that renal inhibition of HSP27 abolished the renoprotective effects afforded by A1AR activation indicated by worse renal function, severer acute tubular necrosis and pro-inflammatory reaction. In addition, HSP27 induction by A1AR activation protects the kidney from IRI via suppressing cell apoptosis, proved by decreased caspase-3 activation and DNA fragmentation, which was also removed by inhibition of HSP27. Activation of A1AR produces renoprotective effects via HSP27 induction. It suggests that preconditional HSP27 activation might have a great potential for the treatment of renal IRI.

Recovery of the Cholangiocytes After Ischemia and Reperfusion Injury: Ultra-Structural, Hystological and Molecular Assessment in Rats

Ischemia-reperfusion (I/R) injury of the liver is a common area of interest to transplant and hepatic surgery. Nevertheless, most of the current knowledge of I/R of the liver derives from the hepatocyte and little is known of what happens to the cholangiocytes. Herein, we assess the sequence of early events involved in the I/R injury of the cholangiocytes. Sixty Wistar rats were randomized in a SHAM group and I/R group. Serum biochemistry, histopathology, immunohistochemistry, transmission electron microscopy (TEM) and laser capture microdissection (LCM) were used for group comparison. There was peak of alkaline phosphatase 24h after IR injury, and an increase of aspartate aminotransferase and alanine aminotransferase after 6h of reperfusion, followed by a return to normal levels 24h after injury. The I/R group presented the liver parenchyma with hepatocellular degeneration up to 6h, followed by hepatocellular necrosis at 24h. TEM showed cholangiocyte injury, including a progressive nuclear degeneration and cell membrane rupture, beginning at 6h and peaking at 24h after reperfusion. Cytokeratin-18 and caspase-3-positive areas were observed in the I/R group, peaking at 24-h reperfusion. Anti-apoptotic genes Bcl-2 and Bcl-xl activity were expressed from 6 through 24h after reperfusion. BAX expression showed an increase for 24h. I/R injury to the cholangiocyte occurs from 6 through 24h after reperfusion and a combination of TEM, immunohistochemistry and LCM allows a better isolation of the cholangiocyte and a proper investigation of the events related to the I/R injury. Apoptosis is certainly involved in the I/R process, particularly mediated by BAX.

Etanercept restores vasocontractile sensitivity affected by mesenteric ischemia reperfusion

BackgroundThe aim of the study is to evaluate in vivo and in vitro effects of etanercept, a soluble tumor necrosis factor receptor, on the contractile responses of superior mesenteric artery in an experimental mesenteric ischemia and reperfusion model. Material and methodsAfter obtaining animal ethics committee approval, 24 Sprague–Dawley rats were allocated to three groups. Control group (Gr C, n = 6) underwent a sham operation, whereas ischemia/reperfusion and treatment groups underwent 90 min ischemia and 24-h reperfusion (Gr I/R, n = 12; Gr I/R+E, n = 6). The treatment group received 5 mg/kg etanercept intravenously at the beginning of reperfusion. At the end of reperfusion, all animals were sacrificed, and third branch of superior mesenteric artery was dissected for evaluation of contractile responses. In vitro effects of etanercept on vasocontractile responses were also evaluated. The excised ileums were analyzed under light microscope. Two-way analysis of variance following Bonferroni post hoc test was used for evaluation of contractile responses. ResultsEndothelin-1 and phenylephrine-mediated vasocontractile sensitivity were found increased in Gr I/R when compared with Gr C. Both intravenous administration and organ bath incubation of etanercept decreased the sensitivity of contractile agents for Gr I/R. Mucosal injury, lamina propria disintegration, and denuded villous tips were observed in Gr I/R, whereas the epithelial injury and the subepithelial edema were found to be milder in Gr I/R+E. ConclusionsEtanercept can be a promising agent in mesenteric ischemic reperfusion injury as it does not only inhibit inflammation by blocking tumor necrosis factor-α in circulation but also restores vascular contractility during reflow. These findings support an unexplained recuperative effect of drug beyond its anti-inflammatory effects.

Cardiomyocyte-specific deletion of Sirt1 gene sensitizes myocardium to ischaemia and reperfusion injury.

A longevity gene, Sirtuin 1 (SIRT1) and energy sensor AMP-activated protein kinase (AMPK) have common activators such as caloric restriction, oxidative stress, and exercise. The objective of this study is to characterize the role of cardiomyocyte SIRT1 in age-related impaired ischemic AMPK activation and increased susceptibility to ischemic insults. Mice were subjected to ligation of left anterior descending coronary artery for in vivo ischemic models. The glucose and fatty acid oxidation were measured in a working heart perfusion system. The cardiac functions by echocardiography show no difference in young wild-type C57BL/6 J (WT, 4-6 months), aged WT C57BL/6 J (24-26 months), and young inducible cardiomyocyte-specific SIRT1 knockout (icSIRT1 KO) (4-6 months) mice under physiological conditions. However, after 45 mins ischaemia and 24-h reperfusion, the ejection fraction of aged WT and icSIRT1 KO mice was impaired. The aged WT and icSIRT1 KO hearts vs. young WT hearts also show an impaired post-ischemic contractile function in a Langendorff perfusion system. The infarct size of aged WT and icSIRT1 KO hearts was larger than that of young WT hearts. The immunoblotting data demonstrated that aged WT and icSIRT1 KO hearts vs. young WT hearts had impaired phosphorylation of AMPK and downstream acetyl-CoA carboxylase during ischaemia. Intriguingly, AMPK upstream LKB1 is hyper-acetylated in both aged WT and icSIRT1 KO hearts; this could blunt activation of LKB1, leading to an impaired AMPK activation. The working heart perfusion results demonstrated that SIRT1 deficiency significantly impaired substrate metabolism in the hearts; fatty acid oxidation is augmented and glucose oxidation is blunted during ischaemia and reperfusion. Adeno-associated virus (AAV9)-Sirt1 was delivered into the aged hearts via a coronary delivery approach, which significantly rescued the protein level of SIRT1 and the ischemic tolerance of aged hearts. Furthermore, AMPK agonist can rescue the tolerance of aged heart and icSIRT1 KO heart to ischemic insults. Cardiac SIRT1 mediates AMPK activation via LKB1 deacetylation, and AMPK modulates SIRT1 activity via regulation of NAD+ level during ischaemia. SIRT1 and AMPK agonists have therapeutic potential for treatment of aging-related ischemic heart disease.

Effect of Rabdosia Rubescens Total Flavonoids on Cerebral Ischemic Reperfusion Model Mice

Objective: To study the protective effects of Rabdosia rubescens total flavonoids (RRTF) on the cerebral ischemia reperfusion model(CIRM) of mice. Methods: By blocking bilateral carotid artery blood flow, the mice model of CIRM were established. Before surgery, medicines were given once a day for 7 successive days. The model was constructed after one hour of the last administration, 24h reperfusion after surgery. Measuring LD, LDH and ATP enzyme of mouse brain tissue, and observing morphological changes of brain tissue. Results: Each dose group of RRTF can significantly lower mortality, reduce the content of LD, ease brain tissue damage while can obviously improve the vitality of LDH, enhance the enzyme activity in mice with cerebral ischemia reperfusion injury, such as Na+-K+-ATPase, Mg2+-ATPase, Ca2+-ATPase. Conclusions: Indicators among each dose of the Rabdosia rubescens flavonoids on cerebral ischemia reperfusion model have better improvement, large doses is the best.