Bifunctional staining for ex vivo determination of area at risk in rabbits with reperfused myocardial infarction

AIM: To develop a method for studying myocardial area at risk(AAR) in ischemic heart disease in correlation with cardiac magnetic resonance imaging(c MRI). METHODS: Nine rabbits were anesthetized, intubated and subjected to occlusion and reperfusion of the left circumflex coronary artery(LCx) to induce myocardial infarction(MI). ECG-triggered c MRI with delayed en-hancement was performed at 3.0 T. After euthanasia, the heart was excised with the LCx re-ligated. Bifunctional staining was performed by perfusing the aorta with a homemade red-iodized-oil(RIO) dye. The heart was then agar-embedded for ex vivo magnetic resonance imaging and sliced into 3 mm-sections. The AAR was defined by RIO-staining and digital radiography(DR). The perfusion density rate(PDR) was derived from DR for the AAR and normal myocardium. The MI was measured by in vivo delayed enhancement(i DE) and ex vivo delayed enhancement(e DE) c MRI. The AAR and MI were compared to validate the bifunctional straining for cardiac imaging research. Linear regression with Bland-Altman agreement, one way-ANOVA with Bonferroni's multiple comparison, and paired t tests were applied for statistics.RESULTS: All rabbits tolerated well the surgical procedure and subsequent c MRI sessions. The openchest occlusion and close-chest reperfusion of the LCx, double suture method and bifunctional staining were successfully applied in all animals. The percentage MI volumes globally(n = 6) and by slice(n = 25) were 36.59% ± 13.68% and 32.88% ± 12.38% on i DE, and 35.41% ± 12.25% and 32.40% ± 12.34% on e DE. There were no significant differences for MI determination with excellent linear regression correspondence(r global = 0.89; r slice = 0.9) between i DE and e DE. The percentage AAR volumes globally(n = 6) and by slice(n = 25) were 44.82% ± 15.18% and 40.04% ± 13.64% with RIO-staining, and 44.74% ± 15.98% and 40.48% ± 13.26% by DR showing high correlation in linear regression analysis(r global = 0.99; r slice = 1.0). The mean differences of the two AAR measurements on BlandAltman were almost zero, indicating RIO-staining and DR were essentially equivalent or inter-replaceable. The AAR was significantly larger than MI both globally and slice-by-slice(P < 0.01). After correction with the background and the blank heart without bifunctional staining(n = 3), the PDR for the AAR and normal myocardium was 32% ± 15% and 35.5% ± 35%, respectively,which is significantly different(P < 0.001), suggesting that blood perfusion to the AAR probably by collateral circulation was only less than 10% of that in the normal myocardium.CONCLUSION: The myocardial area at risk in ischemic heart disease could be accurately determined postmortem by this novel bifunctional staining, which may substantially contribute to translational cardiac imaging research.

Inhibitory Effect of Allopurinol on the Generation of Free Radicals in Reperfused Rat Brain

Free radicals in ischemic and reperfused rat brain were measured by electron spinresonance(ESR) spectrometer.The inhibitory effect of allopurinol on the free radical genera-tion in reperfused rat brain was observed.The experimental results revealed that the free radi-cal content of the brain in the ischemia group was markedly higher than that in the control group(P【0.01),that in the reperfusion group was markedly higher than that in the ischemia group(P【0.01)and that in the allopurinol group was markedly lower than that in the reperfusiongroup(P【0.01).These results suggest that free radicals increase.greatly after cerebralischemia-reperfusion and that allopurinol plays a certain inhibitory role in the free radical genera-tion in the reperfused rat brain.

Magnetic susceptibility of Dy-DTPA-BMA to reperfused myocardial infarction in an excised dog heart model: evidence of viable myocardium

Objective To assess the effects of Dy DTPA BMA (sprodiamide) on ex vivo MR imaging of reperfused acute myocardial infarction Methods Eighteen dogs were subjected to 2 hour coronary artery occlusion followed by 24 hour reperfusion Dysprosium chelate (Dy DTPA BMA) was injected into 16 dogs Twenty minutes before their sacrifice Two dogs did not receive the contrast medium and were used as controls Excised hearts were imaged on T2 weighted spin echo sequence (T2W SE) and T2 * weighted gradient recalled echo sequence (T2 *W GRE), then sectioned and double perfused for planimetric comparison Results Dy DTPA BMA induced myocardial signal loss was detected on T2W SE and on T2 *W GRE images The signal loss was observed at the subendocardial location of the myocardial wall inducing an apparent enlargement of the left ventricle cavity and a thinning appearance of the anterior myocardial wall Conclusions Myocyte necrosis diminishes the potency of dysprosium to cause MR imaging signal intensity loss in reperfused myocardial infarction Pre infarcted myocardium with potentially reversible viability may be responsible for the effect of the contrast medium

Effects of high-dose glucose-insulin-potassium on acute coronary syndrome patients receiving reperfusion therapy:a meta-analysis

BACKGROUND:This meta-analysis aimed to assess the efficacy of high-dose glucose-insulinpotassium(GIK) therapy on clinical outcomes in acute coronary syndrome(ACS) patients receiving reperfusion therapy.METHODS:We searched the PubMed,Web of Science,MEDLINE,Embase,and Cochrane Library databases from inception to April 26,2022,for randomized controlled trials(RCTs) that compared high-dose GIK and placebos in ACS patients receiving reperfusion therapy.The primary endpoint was major adverse cardiovascular events(MACEs).RESULTS:Eleven RCTs with 884 patients were ultimately included.Compared with placebos,high-dose GIK markedly reduced MACEs(risk ratio [RR] 0.57,95% confidence interval [95% CI]:0.35 to 0.94,P=0.03) and the risk of heart failure(RR 0.48,95% CI:0.25 to 0.95,P=0.04) and improved the left ventricular ejection fraction(LVEF)(mean difference [MD] 2.12,95% CI:0.40 to 3.92,P=0.02) at 6 months.However,no difference was observed in all-cause mortality at 30 d or 1 year.Additionally,high-dose GIK was significantly associated with increased incidences of phlebitis(RR 4.78,95% CI:1.36 to 16.76,P=0.01),hyperglycemia(RR 9.06,95% CI:1.74 to 47.29,P=0.009) and hypoglycemia(RR 6.50,95% CI:1.28 to 33.01,P=0.02) but not reinfarction,hyperkalemia or secondary reperfusion.In terms of oxidative stress-lowering function,high-dose GIK markedly reduced superoxide dismutase(SOD) activity but not glutathione peroxidase(GSH-Px) or catalase(CAT) activity.CONCLUSION:Patients with ACS receiving reperfusion therapy exhibited a reduction in MACEs and good oxidative stress-lowering eflcacy in response to high-dose GIK.Moreover,with a higher incidence of complications such as phlebitis,hyperglycemia,and hypoglycemia.Furthermore,there were no observed survival benefits associated with high-dose GIK.More trials with long-term follow-up are still needed.

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.

Maintaining moderate levels of hypochlorous acid promotes neural stem cell proliferation and differentiation in the recovery phase of stroke

It has been shown clinically that continuous removal of ischemia/reperfusion-induced reactive oxygen species is not conducive to the recovery of late stroke.Indeed,previous studies have shown that excessive increases in hypochlorous acid after stroke can cause severe damage to brain tissue.Our previous studies have found that a small amount of hypochlorous acid still exists in the later stage of stroke,but its specific role and mechanism are currently unclear.To simulate stroke in vivo,a middle cerebral artery occlusion rat model was established,with an oxygen-glucose deprivation/reoxygenation model established in vitro to mimic stroke.We found that in the early stage(within 24 hours)of ischemic stroke,neutrophils produced a large amount of hypochlorous acid,while in the recovery phase(10 days after stroke),microglia were activated and produced a small amount of hypochlorous acid.Further,in acute stroke in rats,hypochlorous acid production was prevented using a hypochlorous acid scavenger,taurine,or myeloperoxidase inhibitor,4-aminobenzoic acid hydrazide.Our results showed that high levels of hypochlorous acid(200μM)induced neuronal apoptosis after oxygen/glucose deprivation/reoxygenation.However,in the recovery phase of the middle cerebral artery occlusion model,a moderate level of hypochlorous acid promoted the proliferation and differentiation of neural stem cells into neurons and astrocytes.This suggests that hypochlorous acid plays different roles at different phases of cerebral ischemia/reperfusion injury.Lower levels of hypochlorous acid(5 and 100μM)promoted nuclear translocation ofβ-catenin.By transfection of single-site mutation plasmids,we found that hypochlorous acid induced chlorination of theβ-catenin tyrosine 30 residue,which promoted nuclear translocation.Altogether,our study indicates that maintaining low levels of hypochlorous acid plays a key role in the recovery of neurological function.

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

Background: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 cer-ebral ischemia/reperfusion(I/R)model of gerbils has shackled the application of this model.Methods: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 triphenyltetrazo-lium chloride(TTC)staining,neuron loss using Nissl staining,and histological charac-teristics using hematoxylin-eosin(H&E)straining.Results: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 suc-cessfully prepared I/R gerbils demonstrated a significant reduction in motility and grip strength at 1 day after reperfusion;formed obvious infarction;exhibited typi-cal pathological features,such as tissue edema,neuronal atrophy and death,and vacuolated structures;and were partially recovered with the extension of reperfu-sion time.Conclusion: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.

The action mechanism by which C1q/tumor necrosis factor-related protein-6 alleviates cerebral ischemia/reperfusion injury in diabetic mice

Studies have shown that C1q/tumor necrosis factor-related protein-6 (CTRP6) can alleviate renal ischemia/reperfusion injury in mice. However, its role in the brain remains poorly understood. To investigate the role of CTRP6 in cerebral ischemia/reperfusion injury associated with diabetes mellitus, a diabetes mellitus mouse model of cerebral ischemia/reperfusion injury was established by occlusion of the middle cerebral artery. To overexpress CTRP6 in the brain, an adeno-associated virus carrying CTRP6 was injected into the lateral ventricle. The result was that oxygen injury and inflammation in brain tissue were clearly attenuated, and the number of neurons was greatly reduced. In vitro experiments showed that CTRP6 knockout exacerbated oxidative damage, inflammatory reaction, and apoptosis in cerebral cortical neurons in high glucose hypoxia-simulated diabetic cerebral ischemia/reperfusion injury. CTRP6 overexpression enhanced the sirtuin-1 signaling pathway in diabetic brains after ischemia/reperfusion injury. To investigate the mechanism underlying these effects, we examined mice with depletion of brain tissue-specific sirtuin-1. CTRP6-like protection was achieved by activating the sirtuin-1 signaling pathway. Taken together, these results indicate that CTRP6 likely attenuates cerebral ischemia/reperfusion injury through activation of the sirtuin-1 signaling pathway.

Lactiplantibacillus plantarum AR113 alleviates microbiota dysbiosis of tongue coating and cerebral ischemia/reperfusion injury in rat

Stroke is one of the leading causes of death and disability worldwide.However,information on stroke-related tongue coating microbiome(TCM)is limited,and whether TCM modulation could benefit for stroke prevention and rehabilitation is unknown.Here,TCM from stroke patients(SP)was characterized using molecular techniques.The occurrence of stroke resulted in TCM dysbiosis with significantly reduced species richness and diversity.The abundance of Prevotella,Leptotrichia,Actinomyces,Alloprevotella,Haemophilus,and TM7_[G-1]were greatly reduced,but common infection Streptococcus and Pseudomonas were remarkably increased.Furthermore,an antioxidative probiotic Lactiplantibacillus plantarum AR113 was used for TCM intervention in stroke rats with cerebral ischemia/reperfusion(I/R).AR113 partly restored I/R induced change of TCM and gut microbiota with significantly improved neurological deficit,relieved histopathologic change,increased activities of antioxidant enzymes,and decreased contents of oxidative stress biomarkers.Moreover,the gene expression of antioxidant-related proteins and apoptosis-related factors heme oxygenase-1(HO-1),superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),nuclear factor erythroid 2-related factor 2(Nrf2),NAD(P)H:quinone oxidoreductase-1(NQO-1),and Bcl-2 was significantly increased,but cytochrome C,cleaved caspase-3,and Bax were markedly decreased in the brain by AR113 treatment.The results suggested that AR113 could ameliorate cerebral I/R injury through antioxidation and anti-apoptosis pathways,and AR113 intervention of TCM may have the application potential for stroke prevention and control.

A matrix metalloproteinase-responsive hydrogel system controls angiogenic peptide release for repair of cerebral ischemia/reperfusion injury

Vascular endothelial growth factor and its mimic peptide KLTWQELYQLKYKGI(QK)are widely used as the most potent angiogenic factors for the treatment of multiple ischemic diseases.However,conventional topical drug delivery often results in a burst release of the drug,leading to transient retention(inefficacy)and undesirable diffusion(toxicity)in vivo.Therefore,a drug delivery system that responds to changes in the microenvironment of tissue regeneration and controls vascular endothelial growth factor release is crucial to improve the treatment of ischemic stroke.Matrix metalloproteinase-2(MMP-2)is gradually upregulated after cerebral ischemia.Herein,vascular endothelial growth factor mimic peptide QK was self-assembled with MMP-2-cleaved peptide PLGLAG(TIMP)and customizable peptide amphiphilic(PA)molecules to construct nanofiber hydrogel PA-TIMP-QK.PA-TIMP-QK was found to control the delivery of QK by MMP-2 upregulation after cerebral ischemia/reperfusion and had a similar biological activity with vascular endothelial growth factor in vitro.The results indicated that PA-TIMP-QK promoted neuronal survival,restored local blood circulation,reduced blood-brain barrier permeability,and restored motor function.These findings suggest that the self-assembling nanofiber hydrogel PA-TIMP-QK may provide an intelligent drug delivery system that responds to the microenvironment and promotes regeneration and repair after cerebral ischemia/reperfusion injury.

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.

Inhibition of SLC26A4 regulated by electroacupuncture suppresses the progression of myocardial ischemia-reperfusion injury

Introduction:Myocardial ischemia-reperfusion(IR)injury has received widespread attention due to its damaging effects.Electroacupuncture(EA)pretreatment has preventive effects on myocardial IR injury.SLC26A4 is a Na+independent anion reverse transporter and has not been reported in myocardial IR injury.Objectives:Tofind potential genes that may be regulated by EA and explore the role of this gene in myocardial IR injury.Methods:RNA sequencing and bioinformatics analysis were performed to obtain the differentially expressed genes in the myocardial tissue of IR rats with EA pretreatment.Myocardial infarction size was detected by TTC staining.Serum CK,creatinine kinase-myocardial band,Cardiac troponin I,and lactate dehydrogenase levels were determined by ELISA.The effect of SLC26A4 on cardiomyocyte apoptosis was explored by TUNEL staining and western blotting.The effects of SLC26A4 on inflammation were determined by HE staining,ELISA,and real-time PCR.The effect of SLC26A4 on the NF-κB pathway was determined by western blotting.Results:SLC26A4 was up-regulated in IR rats but downregulated in IR rats with EA pretreatment.Compared with IR rats,those with SLC26A4 knockdown exhibited improved cardiac function according to decreased myocardial infarction size,reduced serum LDH/CK/CK-MB/cTnI levels,and elevated left ventricular ejection fraction and fractional shortening.SLC26A4 silencing inhibited myocardial inflammation,cell apoptosis,phosphorylation,and nuclear translocation of NF-κB p65.Conclusion:SLC26A4 exhibited promoting effects on myocardial IR injury,while the SLC26A4 knockdown had an inhibitory effect on the NF-κB pathway.These results further unveil the role of SLC26A4 in IR injury.

Homer1a reduces inflammatory response after retinal ischemia/reperfusion injury

Elevated intraocular pressure(IOP)is one of the causes of retinal ischemia/reperfusion injury,which results in NRP3 inflammasome activation and leads to visual damage.Homerla is repo rted to play a protective role in neuroinflammation in the cerebrum.However,the effects of Homerla on NLRP3inflammasomes in retinal ischemia/reperfusion injury caused by elevated IOP remain unknown.In our study,animal models we re constructed using C57BL/6J and Homer1^(flox/-)/Homerla^(+/-)/Nestin-Cre^(+/-)mice with elevated IOP-induced retinal ischemia/repe rfusion injury.For in vitro expe riments,the oxygen-glucose deprivation/repe rfusion injury model was constructed with M uller cells.We found that Homerla ove rexpression amelio rated the decreases in retinal thickness and Muller cell viability after ischemia/reperfusion injury.Furthermore,Homerla knockdown promoted NF-κB P65^(Ser536)activation via caspase-8,NF-κB P65 nuclear translocation,NLRP3 inflammasome formation,and the production and processing of interleukin-1βand inte rleukin-18.The opposite results we re observed with Homerla ove rexpression.Finally,the combined administration of Homerla protein and JSH-23 significantly inhibited the reduction in retinal thickness in Homer1^(flox/-)Homer1a^(+/-)/Nestin-Cre^(+/-)mice and apoptosis in M uller cells after ischemia/reperfusion injury.Taken together,these studies demonstrate that Homer1a exerts protective effects on retinal tissue and M uller cells via the caspase-8/NF-KB P65/NLRP3 pathway after I/R injury.

Cav3.2 channel regulates cerebral ischemia/reperfusion injury:a promising target for intervention

Calcium influx into neurons triggers neuronal death during cerebral ischemia/reperfusion injury.Various calcium channels are involved in cerebral ischemia/reperfusion injury.Cav3.2 channel is a main subtype of T-type calcium channels.T-type calcium channel blockers,such as pimozide and mibefradil,have been shown to prevent cerebral ischemia/reperfusion injury-induced brain injury.However,the role of Cav3.2 channels in cerebral ischemia/reperfusion injury remains unclear.Here,in vitro and in vivo models of cerebral ischemia/reperfusion injury were established using middle cerebral artery occlusion in mice and high glucose hypoxia/reoxygenation exposure in primary hippocampal neurons.The results showed that Cav3.2 expression was significantly upregulated in injured hippocampal tissue and primary hippocampal neurons.We further established a Cav3.2 gene-knockout mouse model of cerebral ischemia/reperfusion injury.Cav3.2 knockout markedly reduced infarct volume and brain water content,and alleviated neurological dysfunction after cerebral ischemia/reperfusion injury.Additionally,Cav3.2 knockout attenuated cerebral ischemia/reperfusion injury-induced oxidative stress,inflammatory response,and neuronal apoptosis.In the hippocampus of Cav3.2-knockout mice,calcineurin overexpression offset the beneficial effect of Cav3.2 knockout after cerebral ischemia/reperfusion injury.These findings suggest that the neuroprotective function of Cav3.2 knockout is mediated by calcineurin/nuclear factor of activated T cells 3 signaling.Findings from this study suggest that Cav3.2 could be a promising target for treatment of cerebral ischemia/reperfusion injury.

N-acetylserotonin alleviates retinal ischemia-reperfusion injury via HMGB1/RAGE/NF-κB pathway in rats

AIM:To observe the effects of N-acetylserotonin(NAS)administration on retinal ischemia-reperfusion(RIR)injury in rats and explore the underlying mechanisms involving the high mobility group box 1(HMGB1)/receptor for advanced glycation end-products(RAGE)/nuclear factor-kappa B(NF-κB)signaling pathway.METHODS:A rat model of RIR was developed by increasing the pressure of the anterior chamber of the eye.Eighty male Sprague Dawley were randomly divided into five groups:sham group(n=8),RIR group(n=28),RIR+NAS group(n=28),RIR+FPS-ZM1 group(n=8)and RIR+NAS+FPS-ZM1 group(n=8).The therapeutic effects of NAS were examined by hematoxylin-eosin(H&E)staining,and retinal ganglion cells(RGCs)counting.The expression of interleukin 1 beta(IL-1β),HMGB1,RAGE,and nod-like receptor 3(NLRP3)proteins and the phosphorylation of nuclear factorkappa B(p-NF-κB)were analyzed by immunohistochemistry staining and Western blot analysis.The expression of HMGB1 protein was also detected by enzyme-linked immunosorbent assay(ELISA).RESULTS:H&E staining results showed that NAS significantly reduced retinal edema and increased the number of RGCs in RIR rats.With NAS therapy,the HMGB1 and RAGE expression decreased significantly,and the activation of the NF-κB/NLRP3 pathway was antagonized along with the inhibition of p-NF-κB and NLRP3 protein expression.Additionally,NAS exhibited an anti-inflammatory effect by reducing IL-1βexpression.The inhibitory of RAGE binding to HMGB1 by RAGE inhibitor FPS-ZM1 led to a significant decrease of p-NF-κB and NLRP3 expression,so as to the IL-1βexpression and retinal edema,accompanied by an increase of RGCs in RIR rats.CONCLUSION:NAS may exhibit a neuroprotective effect against RIR via the HMGB1/RAGE/NF-κB signaling pathway,which may be a useful therapeutic target for retinal disease.

Long non-coding RNA-AK138945 regulates myocardial ischemia-reperfusion injury via the miR-1-GRP94 signaling pathway

Objective:Myocardial ischemia-reperfusion injury(MIRI)is one of the leading causes of death from cardiovascular disease in humans,especially in individuals exposed to cold environments.Long non-coding RNAs(lncRNAs)regulate MIRI through multiple mechanisms.This study explored the regulatory effect of lncRNA-AK138945 on myocardial ischemia-reperfusion injury and its mechanism.Methods:In vivo,8-to 12-weeks-old C57BL/6 male mice underwent ligation of the left anterior descending coronary artery for 50 minutes followed by reperfusion for 48 hours.In vitro,the primary cultured neonatal mouse ventricular cardiomyocytes(NMVCs)were treated with 100μmol/L hydrogen peroxide(H_(2)O_(2)).The knockdown of lncRNA-AK138945 was evaluated to detect cardiomyocyte apoptosis,and a glucose-regulated,endoplasmic reticulum stress-related protein 94(GRP94)inhibitor was used to detect myocardial injury.Results:We found that the expression level of lncRNA-AK138945 was reduced in MIRI mouse heart tissue and H2O2-treated cardiomyocytes.Moreover,the proportion of apoptosis in cardiomyocytes increased after lncRNA-AK138945 was silenced.The expression level of Bcl2 protein was decreased,and the expression level of Bad,Caspase 9 and Caspase 3 protein was increased.Our further study found that miR-1a-3p is a direct target of lncRNA-AK138945,after lncRNA-AK138945 was silenced in cardiomyocytes,the expression level of miR-1a-3p was increased while the expression level of its downstream protein GRP94 was decreased.Interestingly,treatment with a GRP94 inhibitor(PU-WS13)intensified H2O2-induced cardiomyocyte apoptosis.After overexpression of FOXO3,the expression levels of lncRNA-AK138945 and GRP94 were increased,while the expression levels of miR-1a-3p were decreased.Conclusion:LncRNA-AK138945 inhibits GRP94 expression by regulating miR-1a-3p,leading to cardiomyocyte apoptosis.The transcription factor Forkhead Box Protein O3(FOXO3)participates in cardiomyocyte apoptosis induced by endoplasmic reticulum stress through up-regulation of lncRNA-AK138945.

Hyperacute experimental model of rat lung transplantation using a coronary shunt cannula

BACKGROUND Lung transplantation is a well-established treatment of end-stage lung disease.A rodent model is an inexpensive way to collect biological data from a living model after lung transplantation.However,mastering the surgical technique takes time owing to the small organ size.AIM To conduct rat lung transplantation using a shunt cannula(SC)or modified cannula(MC)and assess their efficacy.METHODS Rat lung transplantation was performed in 11 animals in the SC group and 12 in the MC group.We devised a method of rat lung transplantation using a coronary SC for coronary artery bypass surgery as an anastomosis of pulmonary arteri-ovenous vessels and bronchioles.The same surgeon performed all surgical proce-dures in the donor and recipient rats without using a magnifying glass.The success rate of lung transplantation,operating time,and PaO2 values were com-pared after 2-h reperfusion after transplantation.RESULTS Ten and 12 lungs were successfully transplanted in the SC and MC groups,respectively.In the SC group,one animal had cardiac arrest within 1 h after reperfusion owing to bleeding during pulmonary vein anastomosis.The opera-ting time for the removal of the heart-lung block from the donor and preparation of the left lung graft was 26.8±2.3 and 25.7±1.3 min in the SC and MC groups,respectively(P=0.21).The time required for left lung transplantation in the recipients was 37.5±2.8 min and 35.9±1.4 min in the SC and MC groups,respectively(P=0.12).PaO2 values at 2 h after reperfusion were 456.2±25.5 and INTRODUCTION Lung transplantation is a well-established treatment of end-stage lung disease.Many immune and non-immune mech-anisms in lung transplantation are highly complex,and post-transplant complications such as infections and primary and chronic lung allograft dysfunction must be reduced to improve survival.Therefore,there is a need for immunological and pathophysiological analyses using animal lung transplantation models.The rat lung transplantation model was first reported in 1971[1],followed by the Mizuta Cuff model[2]in 1989.Since then,various improvements in surgical techniques,cuffs,and instruments have been reported[3-7].The advantage of using a rodent model is that it permits inexpensive collection of biological data from a living model after lung transplantation.Although trained surgeons can perform the transplantation procedure,mastering the surgical technique takes time due to the small size of the organs.The risks associated with this technique include damage to the vulnerable pulmonary artery(PA)and pulmonary vein(PV)vessel walls during anastomosis,as well as stenosis of the anastomotic site.We developed an anastomotic technique using a coronary shunt cannula(SC)for cardiac coronary artery bypass surgery as an alternative to the previously reported cuff method[2-6].This method enables anastomosis by inserting and ligating a cannula into the lumen of the PA,PV,and bronchus(Br),which is simpler and more reliable than conventional methods.This study aimed to determine problems with rat lung transplantation using the SC,develop an improved cannula,and investigate its utility.RESULTS After creating 11 lung transplantation model animals in the SC group and 12 in the MC group,all animals underwent reperfusion.One animal in the SC group had cardiac arrest 1 h after reperfusion due to hemorrhage caused by vessel wall injury during PV anastomosis.Two hours after reperfusion,we visually confirmed the maintenance of recipient hemody-namics and blood flow in the graft pulmonary cannula in 10 animals in the SC group and 12 in the MC group.The operating time for the removal of the heart-lung block from the donor and graft lung creation was 26.8±2.3 min in the SC group and 25.7±1.3 min in the MC group(P=0.21,Table 1).The duration for left lung transplantation into the recipient was 37.5±2.8 min in the SC group and 35.9±1.4 min(P=0.12,Table 1)in the MC group.Although no significant difference was found between the SC and MC groups,animals in the MC group experienced a slightly shorter operating time,smoother surgical technique,and less stressful procedure for the surgeons compared with those in the SC group.The graft lung coloration(Grade 1/2/3)after reperfusion was 0/2/8(SC group)and 0/2/10(MC group),and all grafts were reported to be successful,except in one animal in the SC group that had cardiac arrest(Table 2).The PaO2 values after 2 h of reperfusion were 456.2±25.5 mmHg in the SC group and 461.2±21.5 mmHg in the MC group(P=0.63,Table 3),showing no significant difference between the groups.

Research Progress on Myocardial Protection Strategies

Myocaridial protection aims to salvage myocardium from ischemia and reperfusion injury and to reduce infarct size and its consequences.After more than 30 years of development,the concept of ischemic preconditioning has evolved into"ischemic conditioning",a term that encompasses a number of related endogenous cardioprotective strategies,which can be applied either directly to the heart(ischemic preconditioning or postconditioning)or from afar,for example to a limb(remote ischemic preconditioning,preconditioning,or postconditioning).A variety of cardioprotective therapies have shown promising results in reducing infarct size and improving clinical outcomes in patients with ischemic heart disease.

Protein kinase A-mediated cardioprotection of Tongxinluo relates to the inhibition of myocardial inflammation, apoptosis, and edema in reperfused swine hearts

Background Our previous studies have demonstrated that Tongxinluo （TXL）, a traditional Chinese medicine, can protect hearts against no-reflow and reperfusion injury in a protein kinase A （PKA）-dependent manner. The present study was to investigate whether the PKA-mediated cardioprotection of TXL against no-reflow and reperfusion injury relates to the inhibition of myocardial inflammation, edema, and apoptosis. Methods In a 90-minute ischemia and 3-hour reperfusion model, minipigs were randomly assigned to sham, control, TXL （0.05 g/kg, gavaged one hour prior to ischemia）, and TXL ＋ H-89 （a PKA inhibitor, intravenously and continuously infused at 1.0 μg/kg per minute） groups. Myocardial no-reflow, necrosis, edema, and apoptosis were determined by pathological and histological studies. Myocardial activity of PKA and myeloperoxidase was measured by colorimetric method. The expression of PKA, phosphorylated cAMP response element-binding protein （p-CREB） （Ser133）, tumor necrosis factor a （TNF-a）, P-selectin, apoptotic proteins, and aquaporins was detected by Western blotting analysis. Results TXL decreased the no-reflow area by 37.4% and reduced the infarct size by 27.0% （P〈0.05）. TXL pretreatment increased the PKA activity and the expression of Ser133 p-CREB in the reflow and no-reflow myocardium （P 〈0.05）. TXL inhibited the ischemia-reperfusion-induced elevation of myeloperoxidase activities and the expression of TNF-a and P-selectin, reduced myocardial edema in the left ventricle and the reflow and no-reflow areas and the expression of aquaporin-4, -8, and -9, and decreased myocytes apoptosis by regulation of apoptotic protein expression in the reflow and no-reflow myocardium. However, addition of the PKA inhibitor H-89 counteracted these beneficial effects of TXL. Conclusion PKA-mediated cardioprotection of TXL against no-reflow and reperfusion injury relates to the inhibition of myocardial inflammation, edema, and apoptosis in the reflow and no-reflow myocardium.

Intracoronary adenosine improves myocardial perfusion in late reperfused myocardial infarction

Background Myocardial perfusion associates with clinical syndromes and prognosis. Adenosine could improve myocardial perfusion of acute myocardial infarction within 6 hours, but few data are available on late perfusion of myocardial infarction （MI）. This study aimed at quantitatively evaluating the value of intracoronary adenosine improving myocardial perfusion in late reperfused MI with myocardial contrast echocardiography （MCE）. Methods Twenty-six patients with anterior wall infarcts were divided randomly into 2 groups： adenosine group （n=12） and normal saline group （n=-14）. Their history of myocardial infarction was about 3-12 weeks. Adenosine or normal saline was given when the guiding wire crossed the lesion through percutaneous coronary intervention （PCI）, then the balloon was dilated and stent （Cypher/Cypher select） was implanted at the lesion. Contrast pulse sequencing MCE with Sonovue contrast via the coronary route was done before PCI and 30 minutes after PCI. Video densitometry and contrast filled-blank area were calculated with the CUSQ off-line software. Heart function and cardiac events were followed up within 30 days. Results Perfusion in the segments of the criminal occlusive coronary artery in the adenosine group was better than that in the saline group （5.71：L-0.29 VS 4.95±1.22, P〈0.05）. Ischemic myocardial segment was deminished significantly after PCI, but the meliorated area was bigger in the adenosine group than in the saline group （（1.56±0.60） cm^2 vs （1.02±0.56） cm^2, P〈0.05）. The video densitometry in cntical segments was also improved significantly in the adenosine group （5.53±0.36 VS 5.26±0.35, P〈0.05）. Left ventricular ejection fraction （LVEF） was improved in all patients after PCI, but EF was not significant between the two groups （（67±6）% vs （62±7）%, P〉0.05）. There was no in-hospital or 30-day major adverse cardiac event （MACE） in the adenosine group but 3 MACE in the saline group in 30 days after PCI. Conclusions Adenosine could improve myocardial microvascular perfusion in the late reopening of an occluded infarct related artery （3 to 12 weeks after AMI） and clinical outcome in the follow-up period, and myocardial microvascular perfusion is a powerful predictor of clinical events.