Min Of Reperfusion Research Articles

Protective Effect of Hydrogen Sulphide in Cardioplegia on Global Myocardium Ischemia‐reperfusion Injury

ObjectiveThe endogenous gaseous transmitter hydrogen sulfide (H2S) is an important regulator of the cardiovascular system and is reported to have cardioprotective activity against ischemia/reperfusion (I/R) injury. We investigated the cardioprotective effect of H2S added in cardioplegia against I/R injury in global cardioplegic arrest.MethodsIsolated Wistar rat hearts were perfused in Langendorff apparatus and underwent 30 min perfusion, 120 min cold ischemia, and 90 min reperfusion. The rats were randomly divided into 5 groups: Control group, ischemic and reperfusion (I/R), St. Thomas cardioplegia (St. Thomas +I/R), St. Thomas plus H2S donor NaHS (NaHS+St.Thomas+I/R), St.Thomas plus NaHS and glibenclamide (an antagonist of KATP channel; NaHS+Glib+St.Thomas+I/R). The following indices were compared among the groups: the change of left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), heart rate, heart rhythm, and coronary blood flow. The difference in left ventricular developed pressure (LVDP), the maximum left ventricular pressure rise rate (+dp/dtmax), and the maximum left ventricular pressure drop rate (‐dp/dtmax) were also compared. Western blot was used to detect the changes of related myocardial enzymes among different groups.ResultsNaHS+St.Thomas+I/R treatment significantly improved cardiac function (LVSP, LVDP, +dp/dtmax and ‐dp/dtmax) compared to other groups (p<0.05), although no significant differences on coronary flow and heart rate were observed. After reperfusion, the frequency of ventricular arrhythmia in I/R group and St.Thomas+I/R group was significantly higher than that in NaHS+St.Thomas+I/R group. Further, the expression of cardiac Troponin T, troponin I, and creatine kinase in the NaHS+St.Thomas+I/R group was significantly lower than that in other groups (p<0.05).ConclusionCardioplegia containing H2S confers significant protection against global I/R injury during cold cardioplegic arrest. This effect is associated with opening of the myocardial KATP channels. Therefore, H2S may be a useful adjunct to cardioplegic solutions in heart surgery.

Loss of Mitochondrial Dynamics Proteins Mitofusin‐2 and Drp‐1 in Myocardial Ischemia‐Reperfusion Injury Is Prevented by Matrix Metalloproteinase‐2 Preferring Inhibitors

ObjectiveMatrix metalloproteinase‐2 (MMP‐2) is a ubiquitous protease that cleaves several extracellular and intracellular proteins. MMP‐2 is activated intracellularly in response to enhanced oxidative stress resulting from myocardial ischemia/reperfusion (I/R) injury. Oxidative stress impairs mitochondrial function which is regulated by different proteins including mitofusin‐2 (Mfn‐2) and dynamin‐related protein‐1 (Drp‐1) which control mitochondrial dynamics involving fusion and fission, respectively. As both proteins are localized at the mitochondrial outer membrane and MMP‐2 is localized at the mitochondrial‐endoplasmic reticulum associated membrane we hypothesized that MMP‐2 may proteolyze Mfn‐2 and Drp‐1. We therefore investigated whether inhibition of MMP‐2 could protect against the loss of mitochondrial dynamics proteins during I/R injury.MethodsHearts isolated from 3 month old C57BL/6J mice were perfused according to Langendorff at constant pressure and subjected to I/R injury (30 min ischemia and 40 min reperfusion) in absence or presence of MMP‐2 preferring inhibitors ARP‐100 (10 µM) or ONO‐4817 (50 µM) or their vehicle (n=5 hearts per group). Effects on mechanical function were recorded. At the end of reperfusion, the hearts were homogenized and subcellular fractions were prepared. The degradation of troponin I (TnI), an intracellular MMP‐2 target, was measured in the cytosolic fraction as a marker of MMP‐2 activity. Levels of Mfn‐2 and Drp‐1 in the mitochondrial fraction were also measured using western blot. In silico analysis of potential MMP‐2 cleavage sites was performed using Procleave.ResultsARP‐100 or ONO‐4817 significantly increased left ventricular developed pressure compared to vehicle‐treated I/R hearts (% of pre‐ischemic baseline value at R40: IR+vehicle 25.5±3.2, IR+ARP 50.1±3.2, IR+ONO 57.3±1.6%, p<0.05). Similarly, both inhibitors significantly improved the rates of contraction and relaxation (+/‐dP/dt). TnI loss was increased in I/R hearts as shown by a significant reduction in TnI (~30 kDa) and the appearance of an ~22 kDa band. ARP‐100 or ONO‐4817 attenuated TnI loss, indicating MMP‐2 activation. Levels of Mfn‐2 and Drp‐1 in the mitochondrial fraction were significantly reduced in the I/R group and ARP‐100 or ONO‐4817 attenuated this reduction (p<0.05). Similar results were obtained using a different Mfn‐2 antibody that binds to its C‐terminus. In silico analysis of both mouse Mfn‐2 and Drp‐1 sequences showed several potential sites that could be targeted by MMP‐2.ConclusionsDuring myocardial I/R injury, MMP‐2 may affect mitochondrial dynamics by proteolysis of Mfn‐2 and Drp‐1. Inhibition of MMP‐2 activity could protect against cardiac contractile dysfunction in part by preserving these proteins affecting mitochondrial fusion and fission.

Cholinergic action of spleen‐derived cardioprotective factors

IntroductionThere is an ambivalent relationship between the spleen and the heart in myocardial infarction (Circ Res 124; 2019; 26‐8). Sympathetic activation of the spleen initially increases myocardial inflammatory infiltration but resolves it later by initiating a myocardial healing response. Vagal activation of the spleen induces the release of cardioprotective factors: Perfusion of an isolated rat spleen with the vagomimetic carbachol resulted in the release of factors which then reduced infarct size (IS) in an isolated perfused rat heart.AimTo characterize cardioprotection of spleen‐derived factors, with a focus on their cholinergic action.MethodsRat spleens and hearts were isolated and buffer‐perfused at constant pressure. Isolated spleens were either challenged by infusion of saline (as control) or the vagomimetic carbachol (500 pmol/L), and splenic effluate (SEff; 100 mL) was sampled after saline infusion (SEffsaline) or carbachol infusion (SEffcarbachol), respectively. Cell‐free SEff was prepared by centrifugation and filtration. SEffsaline or SEffcarbachol was infused into isolated hearts which were then subjected to 30 min global ischemia and 120 min reperfusion (I/R). In subsets of experiments, the hearts were pretreated with the nicotinic receptor antagonist hexamethonium (50 µmol/L) or the muscarinic receptor antagonist atropine (100 pmol/L). IS was demarcated by triphenyltetrazolium chloride staining and calculated as percent of ventricular mass. To study the relevance of spleen‐derived protection on the cardiomyocyte level, adult rat ventricular cardiomyocytes were prepared. Cells were incubated for 30 minutes with SEffsaline or SEffcarbachol, respectively, and then subjected to 30 min hypoxia/ 5 min reoxygenation (H/R) or exposed to SEffsaline or SEffcarbachol for 35 minutes as time control (TC). Since nicotinic and muscarinic receptors are also expressed on cardiomyocytes, all experiments were repeated after pretreatment with hexamethonium (1 µmol/L) or atropine (100 nmol/L). Cardiomyocyte viability was quantified before (baseline) and after H/R or TC, as the percent fraction of rod‐shaped, unstained (5% trypan blue) cells over all cells. The estimated final concentration of carbachol in the SEffcarbachol of ≤10 pmol/L and the SEffsaline did neither reduce IS in isolated perfused rat hearts nor improve cardiomyocyte viability in isolated rat cardiomyocytes.ResultsWith SEffsaline, IS was 34±7%. With SEffcarbachol, IS was reduced to 19±8%. Hexamethonium or atropine had no impact on IS per se in hearts with SEffsaline (33±8%; 34±6%), but attenuated the cardioprotection by SEffcarbachol (27±10%; 26±10%; Figure A). The viability of cardiomyocytes subjected to H/R was better preserved with SEffcarbachol than with SEffsaline or saline alone. Again, this protection was attenuated by hexamethonium or atropine (Figure B, insert).ConclusionThe cardioprotection by spleen‐derived factors, which were released in response to the vagomimetic carbachol, is mediated through nicotinic and muscarinic receptors within the heart. Apparently, this protection does not need to involve neuronal structures within the myocardium – nicotinic and muscarinic receptors on cardiomyocytes per se mediate protection.

What Doesn't Kill You Makes You Stronger—Even Heart Radiation?

Radiation therapy (RT) is a commonly used treatment for thoracic cancers. However, the risk of coronary events increase by 4‐16% per Gy of heart dose. Our prior studies show that localized heart RT leads to radiation‐induced heart disease (RIHD) and increased mortality in Dahl Salt Sensitive (SS) rats starting at 3mo post‐RT. We have also shown that whole thoracic RT increases susceptibility to ischemia‐reperfusion (IR) injury in Wistar rats. Taken together, we hypothesized that 10weeks after targeted cardiac RT (prior to RIHD) SS rat hearts would have worsened cardiac function as measured by echocardiography and be more susceptible to IR injury. Echocardiograms were performed 10weeks after 24Gy dose of CT‐guided localized cardiac RT and in age‐matched controls (C). Next, hearts were isolated and perfused ex vivo using the Langendorf method ‐ without ischemia reperfusion (time controls; TC; n=3 in C and RT) or exposed to 25min of global ischemia followed by 60min reperfusion (IR; n=6 in C and RT). During TC and IR, cardiac function, mitochondrial redox state, and vascular reactivity were assessed; the hearts were then sectioned and stained to assess infarct size. Echocardiography of the RT group when compared to C showed an increase in interventricular septal thickness (1.32±0.1 vs 0.78±0.07 cm, p<0.0001; mean±SD) and left ventricular posterior wall thickness (1.03±0.08 vs 0.75±0.06 cm, p<0.005). Accordingly, a decrease in end diastolic (1.79±0.46 vs 2.38±0.35 ml/kg, p<0.05) and end systolic (0.10±0.04 vs 0.23±0.08 ml/kg, p<0.05) volume was also seen, with no significant differences in ejection fraction (94.5±2.1 vs 90.3±3.9 %) or stroke volume (1.69±0.44 vs 2.15±0.27 ml; p>0.05). Interestingly, in the ex vivo perfused hearts, TC RT had a 2 times higher rate‐pressure product – a measure of cardiac work – compared to C (p<0.05), and following IR, the RT group recovered to 2.5x higher levels to C (p<0.0001). Following IR, the RT group compared to C also showed a greater recovery of coronary flow rate (1.6x, p<0.05) and rates of contractility (2.25x) and relaxation (lusitropy; 2.05x) (p<0.0001). No difference in mitochondrial redox state, infarct size, and endothelium‐dependent and independent vascular reactivity were seen between the groups. In conclusion, our results show for the first time that 10weeks post‐targeted cardiac RT, ex vivo hearts show better recovery and cardiac function compared to C after IR through mechanisms other than differences in redox state and vascular reactivity.

Naltrindole Pretreatment Exhibits Robust Cardioprotection in an Acute In Vivo Model of Ischemia/Reperfusion

Myocardial infarctions (MI), remain prevalent in the U.S. with over one million individuals experiencing this cardiac event every year. Delta opioid receptor activation is known to mimic pharmacologic preconditioning to reduce infarct size post‐MI. Surprisingly, results from our laboratory have shown that naltrindole (NTI), a selective delta opioid receptor antagonist, elicits unexpected cardioprotective effects ex vivo when given prior to 30 min global ischemia (I) followed by 45 min reperfusion (R). Whether NTI’s cardioprotective effects can be translated in vivo with intact neurohormonal compensatory mechanisms, has not yet been elucidated. In this study, we aim to determine the cardioprotective effects of NTI in an in vivo model that more closely mimics acute MI clinically.Female Sprague‐Dawley rats (~250g) were assigned into three cohorts: non‐treated control (n=15), high dose (HD, 4.0‐8.0 mg/kg, n=5) NTI, and low dose (LD, 0.2‐1.0 mg/kg, n=18) treatment groups. All three cohorts were anesthetized with ketamine‐xylazine (90 mg/kg, 9mg/kg). Control rats received normal saline (0.9% NaCl), while treatment groups received either HD or LD NTI. NTI was infused through the jugular vein (0.4mL/min) over 5 mins before inducing regional ischemia (pretreatment). The proximal left coronary artery was occluded for 30 min followed by 3 hr reperfusion. At the end of the experiment, blue dye was administered to determine the area at risk (AR). Planimetered photographs of heart slices were then stained with 1% triphenyltetrazolium chloride to determine area of necrosis (AN) and infarct size (AN/AR). Cardiac function was measured via a pressure transducer placed into the left ventricle (LV) in a small cohort of the LD NTI (0.4‐1.0 mg/kg, n=3) and HD NTI groups (n=5). Data were analyzed via ANOVA Student Newman Keuls post‐hoc analysis (infarct size) and unpaired t‐test (cardiac function), respectively, with p<0.05 considered statistically significant.HD NTI exhibited a robust ~60% reduction in infarct size (21±7 %; n=4, p<0.01) compared to control (57±2 %; n=15) and LD NTI (55±4 %; n=18) hearts. HD NTI (n=5) also exerted a significant depression of the maximal and minimal rate of LV pressure change on dP/dt max (6167±489 mmHg/s vs. 3714±645 mmHg/s) and dP/dt min (‐7015±942 mmHg/s vs. ‐4034±776 mmHg/s) respectively and heart rate (261±20 BPM vs. 208±3 BPM) at the end of pretreatment compared to LD NTI (0.4‐1.0 mg/kg, n=3 all p<0.05). This preconditioning effect was correlated with the robust decrease in infarct size compared to LD NTI and control groups. These results were similar to our ex‐vivo studies, although a 20x dose was needed in vivo to presumably overcome compensatory neurohormonal responses (i.e. 4mg/kg vs 0.2mg/kg). Results suggest that NTI pretreatment can protect against I/R injury. Future studies will examine the effects of drug infusion at different ischemic time points for optimal cardioprotection.

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

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

No sex-related differences in infarct size, no-reflow, and protection by ischaemic pre-conditioning in Göttingen minipigs.

Female sex has been proposed to be cardioprotective per se. Studies with myocardial ischaemia/reperfusion and infarct size as endpoint have demonstrated cardioprotection in female, castrated male, and male pigs. These studies are difficult to compare, given the different pig strains, models, durations of ischaemia, and methods of infarct size quantification. The few studies using both female and male pigs reported no differences in infarct size and cardioprotection. We, therefore, prospectively compared infarct size in Göttingen minipigs undergoing ischaemia/reperfusion (I/R) without and with ischaemic pre-conditioning (IPC) between female, castrated male, and male pigs. In a prospective, randomized approach, 28 Göttingen open-chest, anaesthetized minipigs underwent 60 min ischaemia by distal left anterior descending artery (LAD) occlusion and 180 min reperfusion without and with IPC by three cycles of 5 min LAD occlusion/10 min reperfusion. Infarct size with I/R was not different between female, castrated male, and male pigs (45 ± 8 vs. 45 ± 13 vs. 41 ± 9% area at risk), as was the reduction in infarct size with IPC (25 ± 11 vs. 30 ± 8 vs. 19 ± 10% area at risk). In addition, the area of no-reflow was not different between female, castrated male, and male pigs with I/R (57 ± 13 vs. 35 ± 7 vs. 47 ± 26% infarct size) or IPC (4 ± 10 vs.12 ± 20 vs. 0 ± 0% infarct size). Phosphorylation of signal transducer and activator of transcription 3 was increased at 10 min reperfusion by IPC but not by I/R to the same extent in female, castrated male, and male pigs (198 ± 30 vs. 230 ± 165 vs. 179 ± 107% of baseline). Our data do not support the notion of sex- or castration-related differences in infarct size, coronary microvascular injury, and cardioprotection by IPC. The translation of successful preclinical studies on cardioprotection to the benefit of patients with reperfused myocardial infarction has been difficult. The difficulties have been attributed to confounders such as co-morbidities and co-medications which patients typically have but animals don´t, but also to age and sex. Notably, female sex has been considered as protective per se. We have now, using our established and clinically relevant pig model of reperfused acute myocardial infarction and ischaemic preconditioning as the most robust cardioprotective intervention looked for sex-related differences of infarct size, no-reflow and cardioprotection by ischaemic preconditioning in a prospectively powered approach but found none such difference.

Macrophage-Derived Extracellular Vesicles (EVs) Have Functional and Metabolic Effects on Cardiac Grafts in a Preclinical Model of Donation After Circulatory Death (DCD)

<h3>Purpose</h3> Donation after circulatory death (DCD) could substantially increase cardiac graft availability. Cardiac ischemia and reperfusion, as occurs during DCD, can trigger the recruitment of macrophages as well as their polarization from the M0 to M1 state, which may occur in parallel with increased glycogen turnover and pentose phosphate flux. Extracellular vesicles (EVs) can transmit the metabolic status of their parent cell to recipient cells. As post-ischemic glucose metabolism is recognized to affect the cardiac recovery, we investigated the effects of M0 and M1 macrophage-derived EV administration during reperfusion on post-ischemic functional recovery and glucose metabolism using an isolated rat heart model of DCD. <h3>Methods</h3> Hearts isolated from adult, male rats were aerobically perfused for 20 minutes, followed by 27 minutes continued perfusion or 27 minutes of warm, global ischemia, and 60 minutes aerobic reperfusion with or without the intravascular administration of EVs. Four experimental groups were examined: 1) continued perfusion, no EVs (No ISCH); 2) ischemia, no EVs (ISCH); 3) ischemia with EVs from M0 macrophages (ISCH M0); 4) ischemia with EVs from M1 macrophages (ISCH M1). Contractile and metabolic post-ischemic recovery were evaluated. Data are reported as mean ± standard deviation. <h3>Results</h3> Left ventricular (LV) work (heart rate-developed pressure product) at 60 minutes reperfusion was significantly decreased in ISCH (17 ± 3) and ISCH M1 (19 ± 3), but not in ISCH M0 (22 ± 3), compared to No ISCH (26 ± 4 mmHg*beats*min^{−1 *}10³; p<0.05 for both). Rates of glycolysis during reperfusion were not different among groups; however, glycogen content was significantly lower after 60 min reperfusion in ISCH M1 (8 ± 2) compared to ISCH M0 (17 ± 3 μmol/g dry; p<0.05). <h3>Conclusion</h3> The ischemia-induced reduction in left ventricular work was improved with M0-, but not M1-, derived EVs. M0 and M1-derived EVs also differentially affected glucose metabolism; M1-macrophage derived EVs limited glycogen content, possibly by increasing glycogen use or turnover and stimulating recipient cells to mimic parent cell metabolic phenotypes. Circulating EVs in the ex-situ perfusion of DCD hearts may be possible targets for modulating metabolic function and optimizing graft recovery following warm ischemia.

Coronary Sinus Integrated Lactate Decreases After Ischemia and Reperfusion with GGA Induced hsp70

<h3>Purpose: Background</h3> Ischemia reperfusion(IR) increases lactate. No one has examined if cardiac-specific coronary sinus lactate(CSL) can be reduced with prior cytoprotective heat shock protein 70(hsp70i) induction. We previous demonstrated improved IR in vivo with inducted hsp70i. Geranylgeranylacetone(GGA), an hsp70i inducer, has never been administered IV preischemically. Interventions to decrease CSL may improve clinical parameters. <h3>Methods</h3> Rabbit hearts underwent 30 cold cardioplegic ischemia then 60 min reperfusion. One group received IVGGA 24 hours prior(GGA+) and the other vehicle(GGA-). CSlactate was collected prior to ischemia and throughout reperfusion. We aimed to determine IVGGA effects on myocardial hsp70i and lactate. Hsp70 western blot was performed. <h3>Results</h3> Baseline CSlactate was similar between GGA+ and GGA-(Figure 1). Both peaked CSlactate at 1 minute reperfusion. However GGA+ peak was less. At every time point GGA+ was less. GGA+ CSlactate continued to decrease throughout reperfusion however in GGA- CSlactate increased later. Integrated CS lactate area was less for GGA+(Figure 1). <h3>Conclusion</h3> In summary, protective IVGGA resulted in five lactate benefits: lactate was less at 1 minute reperfusion peak,decreased faster in early reperfusion, was reduced at all time points, does not have a second rise and lastly results in overall less integrated lactate production in GGA+. GGA induced hsp70. IVGGA may have clinical applications in endothelial protection in IR and COVID.

Effects of acute ischemia and hypoxia in young and adult calsequestrin (CSQ2) knock-out and wild-type mice

Calsequestrin (CSQ2) is the main Ca2+-binding protein in the sarcoplasmic reticulum of the mammalian heart. In order to understand the function of calsequestrin better, we compared two age groups (young: 4–5 months of age versus adult: 18 months of age) of CSQ2 knock-out mice (CSQ2(−/−)) and littermate wild-type mice (CSQ2(+/+)). Using echocardiography, in adult mice, the basal left ventricular ejection fraction and the spontaneous beating rate were lower in CSQ2(−/−) compared to CSQ2(+/+). The increase in ejection fraction by β-adrenergic stimulation (intraperitoneal injection of isoproterenol) was lower in adult CSQ2(−/−) versus adult CSQ2(+/+). After hypoxia in vitro (isolated atrial preparations) by gassing the organ bath buffer with 95% N2, force of contraction in electrically driven left atria increased to lower values in young CSQ2(−/−) than in young CSQ2(+/+). In addition, after global ischemia and reperfusion (buffer-perfused hearts according to Langendorff; 20-min ischemia and 15-min reperfusion), the rate of tension development was higher in young CSQ2(−/−) compared to young CSQ2(+/+). Finally, we evaluated signs of inflammation (immune cells, autoantibodies, and fibrosis). However, whereas no immunological alterations were found between all investigated groups, pronounced fibrosis was found in the ventricles of adult CSQ2(−/−) compared to all other groups. We suggest that in young mice, CSQ2 is important for cardiac performance especially in isolated cardiac preparations under conditions of impaired oxygen supply, but with differences between atrium and ventricle. Lack of CSQ2 leads age dependently to fibrosis and depressed cardiac performance in echocardiographic studies.

Modulation of Oxidative Stress Induced Cerebral Ischemia in Wistar Rats by Hydroalcoholic Extract of Talinum triangulare

Abstract: Background: Neuroprotection is one of considerable interest in search of novel therapies to improve ischemic brain damage in the present scenario. The objective of the present investigation is to study the actions of Talinum triangulare in cerebral ischemia/reperfusion induced oxidative stress in Wistar rats and in-vitro antioxidant actions. Methods: Four different groups of adult Wistar rats are selected for study where group III and group IV are treated for 15 days with TTE (200mg/kg, 400mg/kg) earlier to ischemia, on 16th day rats are dissected through global cerebral ischemia by occlusion of unilateral common carotid artery, prior to the dissection animals are sedated with thiopentone and after ischemia induction that 30 min reperfusion was followed. Following the ischemic injury various parameters such as infarct size, SOD, MDA, CAT and GSH levels were assessed after 48hr of reperfusion. Results: Phytochemical screening of TTE indicated the presence of alkaloids, flavonoids, glycosides, carbohydrates, saponins, tannins and resins. TTE also showed a significant in-vitro antioxidant activity. Pre-treatment of TTE for 15 days prior to the ischemic-reperfusion, the results reported a huge reduction in reduction in infarct size, MDA levels and significant increase in SOD, CAT and GSH levels compared to ischemic control rats which was statistically significant. Conclusion: The present study showed that hydroalcoholic extract of TTE leaves showed a significant in-vitro and in-vivo antioxidant activity. The present investigation reported that Talinum triangulare leaves extract showed dose dependant cerebroprotective effect with the selected dose of TTE 200 mg/kg and 400 mg/kg. Key words: Talinum triangulare, Cerebral ischemia reperfusion, Oxidative stress, Cerebroprotection, Antioxidant acivity.

Delayed Cutaneous Microvascular Responses With Non-consecutive 3 Days of Remote Ischemic Preconditioning.

The optimal frequency and duration of remote ischemic preconditioning (RIPC) that augments microvascular function is unknown. A single bout of RIPC increases cutaneous endothelial function for ∼48 h, whereas 1 week of daily RIPC bouts improves more sustained endothelium-independent function. We hypothesized that 3 days of RIPC separated by rest days (3QOD RIPC) would result in sustained increases in both endothelium-dependent and endothelium-independent functions. Cutaneous microvascular function was assessed in 13 healthy young participants (aged 20.5 ± 3.9 years; 5 males, 8 females) before 3QOD and then 24, 48, and 72 h and a week after 3QOD. RIPC consisted of four repetitions of 5 min of blood flow occlusion separated by 5 min of reperfusion. Skin blood flow responses to local heating (Tloc = 42°C), acetylcholine (Ach), and sodium nitroprusside (SNP) were measured using laser speckle contrast imaging and expressed as cutaneous vascular conductance (CVC = PU⋅mmHg–1). Local heating-mediated vasodilation was increased 72 h after 3QOD and the increased responsivity persisted a week later (1.08 ± 0.24 vs. 1.34 ± 0.46, 1.21 ± 0.36 PU⋅mmHg–1; ΔCVC, pre-RIPC vs. 72 h, a week after 3QOD; P = 0.054). Ach-induced cutaneous vasodilation increased a week after 3QOD (0.73 ± 0.41 vs. 0.95 ± 0.49 PU⋅mmHg–1; ΔCVC, pre-RIPC vs. a week after 3QOD; P < 0.05). SNP-induced cutaneous vasodilation increased 24 h after 3QOD (0.47 ± 0.28 vs. 0.63 ± 0.35 PU⋅mmHg–1; ΔCVC, pre-RIPC vs. 24 h; P < 0.05), but this change did not persist thereafter. Thus, 3QOD induced sustained improvement in endothelium-dependent vasodilation but was not sufficient to sustain increases in endothelium-independent vasodilation.

Pericyte-mediated constriction of renal capillaries evokes no-reflow and kidney injury following ischaemia.

Acute kidney injury is common, with ~13 million cases and 1.7 million deaths/year worldwide. A major cause is renal ischaemia, typically following cardiac surgery, renal transplant or severe haemorrhage. We examined the cause of the sustained reduction in renal blood flow ('no-reflow'), which exacerbates kidney injury even after an initial cause of compromised blood supply is removed. Adult male Sprague-Dawley rats, or NG2-dsRed male mice were used in this study. After 60 min kidney ischaemia and 30-60 min reperfusion, renal blood flow remained reduced, especially in the medulla, and kidney tubule damage was detected as Kim-1 expression. Constriction of the medullary descending vasa recta and cortical peritubular capillaries occurred near pericyte somata, and led to capillary blockages, yet glomerular arterioles and perfusion were unaffected, implying that the long-lasting decrease of renal blood flow contributing to kidney damage was generated by pericytes. Blocking Rho kinase to decrease pericyte contractility from the start of reperfusion increased the post-ischaemic diameter of the descending vasa recta capillaries at pericytes, reduced the percentage of capillaries that remained blocked, increased medullary blood flow and reduced kidney injury. Thus, post-ischaemic renal no-reflow, contributing to acute kidney injury, reflects pericytes constricting the descending vasa recta and peritubular capillaries. Pericytes are therefore an important therapeutic target for treating acute kidney injury.

Remote ischemic conditioning causes CD4 T cells shift towards reduced cell-mediated inflammation.

Necrotizing enterocolitis (NEC) is a gastrointestinal disease in neonates that is associated with immune-mediated intestinal inflammation. Remote ischemic conditioning (RIC) applied to a limb has been shown to be protective against experimental NEC. In this study, we explore the immune cell-mediated response involved in NEC and the immunomodulatory effects of RIC in an experimental mouse model of the disease. NEC was induced in C57BL/6 mice (ethical approval #58119) pups on postnatal day5 (p5) using gavage hyperosmolar formula, lipopolysaccharide, and hypoxia. RIC consisted of 4 cycles of 5min ischemia followed by 5min reperfusion of the right hindlimb during NEC induction on p6 and p8. Breastfed mice were used as control. The mice were sacrificed on p9, with ileal tissue evaluated for inflammatory cytokines and by characterization of T-cell populations. NEC mice had increased number of CD4+ cells indicating an accumulation of T-cells in the mesenchyme of the NEC ileum. Compared to control, NEC pups had upregulated expression pro-inflammatory cytokines (GATA3, IFNγ, IL1β, IL6, IL17, IL22, and TNFα) and reduced anti-inflammatory cytokine (TGFβ). In NEC, there was also a shift in the balance of Treg/Th17 cells towards Th17. Compared to NEC alone, RIC during the course of NEC resulted in reduction of pro-inflammatory cytokines (GATA3, IFNγ, IL1β, IL6, IL17, IL22, and TNFα), increase in anti-inflammatory cytokine TGFβ and concomitant shift back of Th17 cells towards Treg cells. In experimental NEC, remote ischemic conditioning reduces the production of pro-inflammatory markers and increases the production of anti-inflammatory markers. In addition, during NEC, RIC reverses the imbalance of Treg/Th17 providing support for its effect on cell-mediated inflammation. RIC is a non-invasive physical maneuver that can have a significant beneficial effect in reducing the inflammation seen in NEC.

Post-occlusive reactive hyperemia and skeletal muscle capillary hemodynamics.

In female Sprague-Dawley rats (n=6), the spinotrapezius muscle was exteriorized for evaluation of capillary hemodynamics pre-occlusion, 5min of feed artery occlusion (Occ), and 5min of reperfusion (Post-Occ). There were no differences in mean arterial pressure (MAP) or capillary diameter (Dc) between pre-occlusion and post-occlusion (P>0.05). During 30s of PORH, capillary fRBC was increased (pre: 59±4 vs. 30s-post: 77±2 cells/s; P<0.05) and VRBC was not changed (pre: 300±24 vs. 30s post: 322±25μm/s; P>0.05). Capillary hematocrit (Hctcap) was unchanged across the pre- to post-occlusion conditions (P>0.05). Following occlusion, there was a 20-30% decrease in the number of capillaries supporting RBC flow at 30s and 300s-post occlusion (pre: 92±2%; 30s-post: 66±3%; 300s-post: 72±6%; both P<0.05). Short-term feed artery occlusion (i.e. 5min) resulted in a more heterogeneous capillary flow profile with the presence of capillary no-reflow, decreasing the percentage of capillaries supporting RBC flow. A complex interaction between myogenic and metabolic mechanisms at the arteriolar level may play a role in the capillary no-reflow with PORH. Measurements at the level of the conduit artery mask significant alterations in blood flow distribution in the microcirculation.

Ginsenoside-Mc1 reduces ischemia/reperfusion-induced cardiac arrhythmias through activating JAK2/STAT3 pathway and attenuating oxidative/endoplasmic reticulum stress in hyperlipidemic rats

Abstract Objectives Patients suffering from myocardial ischemia-reperfusion (IR) injuries usually have varying degrees of negatively-affecting comorbidities like hyperlipidemia. We evaluated the preconditioning effect of ginsenoside-Mc1 on reperfusion injury-induced myocardial arrhythmias, along with cardiac oxidative stress, endoplasmic reticulum stress protein expression, and histological damage in hyperlipidemic rats, and further, explore the role of JAK2/STAT3 activity. Methods Thirty-five Sprague–Dawley rats fed a high-fat diet for eight weeks. Ginsenoside-Mc1 (10 mg/dL, IP) was administered to hyperlipidemic rats daily for one month before IR injury. IR injury was induced by 35 min LAD coronary artery ligation and subsequent 60-min reperfusion. A selective JAK2 inhibitor (AG490) was injected before IR injury. Electrocardiography was recorded and myocardial arrhythmias (ventricular premature complexes, tachycardia, and fibrillation) were evaluated in the reperfusion phase according to Lambeth convention. Hematoxylin-Eosin staining, spectrophotometry, and Western blotting techniques were employed to measure the endpoints. Results IR injury in hyperlipidemic rats significantly increased the reperfusion-induced myocardial arrhythmias counts, timing, incidence, and severity. The expression of proteins of endoplasmic reticulum stress (p-PERK, p-eIF2α, CHOP), and oxidative stress marker malondialdehyde were significantly upregulated following IR induction, whereas antioxidant superoxide-dismutase and JAK2/STAT3 proteins expression significantly reduced, as compared with untreated-hyperlipidemic rats. Administration of ginsenoside-Mc1 to hyperlipidemic rats significantly corrected the arrhythmogenic feature of IR injury, reduced phosphorylation and expression of PERK, eIF2α, CHOP, and improved oxidative stress and histological changes. Interestingly, inhibition of the JAK2/STAT3 pathway via AG490 significantly abolished ginsenoside-Mc1-induced cardioprotection. Conclusions Taken together, ginsenoside-Mc1 exerts substantial anti-arrhythmogenic influences against myocardial IR injury in hyperlipidemic rats through activation of JAK2/STAT3 pathway and subsequent reduction of oxidative/endoplasmic reticulum stress.

Protective effects of 5(S)-5-carboxystrictosidine on myocardial ischemia-reperfusion injury through activation of mitochondrial KATP channels

5(S)-5-carboxystrictosidine (5-CS) is a compound found in Mappianthus iodoides Hand.-Mazz., root, a traditional Chinese medicine used for the treatment of coronary artery disease. In this study, we investigated whether 5-CS protects heart against I/R injury. Sprague–Dawley rats were treated with 5-CS intraperitoneally for 7 days before the experiment. Hearts were perfused for 20 min global ischemia and 180 min reperfusion. 5-CS significantly inhibited an increase in the post-ischemic left ventricular end-diastolic pressure (LVEDP) and improved the post-ischemic left ventricular developed pressure (LVDP), dP/dt maximum and dP/dt minimum rates of pressure change, and coronary flow as compared with sham group. Pretreatment with 5-hydroxydecanoic acid (5-HD), an inhibitor of mitochondrial KATP channel, for 10 min before ischemia attenuated the improvement of LVEDP, LVDP, dP/dt maximum and dP/dt minimum rates of pressure change, and coronary flow induced by 5-CS. 5-CS markedly decreased the infarct size and attenuated the increased lactate dehydrogenase (LDH) level in effluent during reperfusion. Pretreatment with 5-HD also blocked these protective effects of 5-CS. 5-CS increased Mn-SOD, catalase, and HO-1 levels decreased by I/R injury and pretreatment of 5-HD blocked the 5-CS effects. Increases in Bax, cleaved caspase-3 and cytochrome c levels, caspase-3 and caspase-9 activity, and decrease in Bcl-2 level by I/R injury were attenuated by 5-CS treatment and pretreatment of 5-HD blocked its effects. These results suggest that the protective effects of 5-CS against myocardial I/R injury may be partly related to activating antioxidant enzymes and suppressing apoptosis through opening mitochondrial KATP channels.

Pretreatment with nicotinamide mononucleotide increases the effect of ischaemic postconditioning on cardioprotection and mitochondrial function following ex vivo myocardial reperfusion injury in aged rats.

The present study aims to evaluate the combined effect of ischaemic postconditioning (IPostC) and nicotinamide mononucleotide (NMN) on cardioprotection and mitochondrial function in aged rats subjected to myocardial ischaemia-reperfusion (IR) injury. Sixty aged Wistar rats were randomly divided into fivegroups (n=12), including sham, control, NMN, IPostC, and NMN+IPostC. Regional ischaemia was induced by 30-min occlusion of the left anterior descending coronary artery (LAD) followed by 60-min reperfusion. IPostC was applied at the onset of reperfusion, by six cycles of 10-s reperfusion/ischaemia. NMN (100mg/kg) was intraperitoneally injected every other day for 28days before IR. Myocardial haemodynamics and infarct size (IS) were measured, and the left ventricles samples were harvested to assess cardiac mitochondrial function. The results showed that all treatments reduced lactate dehydrogenase release compared to those of the control group. IPostC alone failed to reduce IS and myocardial function. However, NMN and combined therapy could significantly improve myocardial function and decrease the IS compared to the control animals. Moreover, the effects of combined therapy on the decrease of IS, mitochondrial reactive oxygen species (ROS), and improvement of mitochondrial membrane potential (MMP) were greater than those of stand-alone treatments. These results demonstrated that cardioprotection by combined therapy with NMN+IPostC was superior to individual treatments, and pretreatment of aged rats with NMN was able to correct the failure of IPostC in protecting the hearts of aged rats against IR injury.

Minocycline as heart conditioning agent in experimental type 2 diabetes mellitus - an antibacterial drug in heart protection.

Cardiovascular diseases, and among them certainly myocardial infarction, remain leading cause of death worldwide. Diabetes increases risk of occurrence as well as adverse outcome of myocardial infarction. Conditioning maneuvers are the most attractive method for alleviating both the consequences of ischemia and reperfusion. Minocycline is a tetracycline derivative which exerts antioxidant, anti-inflammatory, and anti-apoptotic effects. The aim of this study was to assess the protective ability of preconditioning and postconditioning of isolated hearts from healthy and rats with experimentally induced type 2 diabetes with minocycline on functional recovery and redox status after ischemia and reperfusion. The hearts from healthy and diabetic rats were excised and retrogradely perfused according to the Langendorff technique. Using sensor in the left ventricle, the cardiodynamic parameters were recorded and in the samples of the coronary venous effluent oxidative stress biomarkers were analyzed. Minocycline was injected directly into the coronary vessels, in preconditioning 5min before global ischemia, and in postconditioning during the first 5min of reperfusion. Results of this study clearly show beneficial effects of minocycline applied both before ischemia and in the first minutes of reperfusion fashion in both healthy and diabetic rat hearts. The most prominent protective effect regarding oxidative stress is related to the decreased production of superoxide anion radical due postconditioning with minocycline in diabetic hearts. Cardiodynamic parameters were significantly improved in minocycline conditioned groups. Superoxide anion radical stands out as the most susceptible to changes induced by minocycline.

Abstract TP232: Post-stroke Suppression Of Matrix Metalloproteinase-12 Attenuates The Expression Of M1 And M2 Markers And Prevents The Elevation Of Other Matrix Metalloproteinases

Introduction: We previously reported a marked elevation of matrix metalloproteinases (MMP-1, MMP-7, MMP-8, MMP-9, MMP-11, MMP-12, and MMP-14) in the ischemic brain on day 3 after ischemic stroke in a rodent model. Also, we showed that MMP-12 suppression after ischemic stroke attenuates blood-brain barrier (BBB) disruption. The aim of the present study was to examine the effects of MMP-12 suppression on the expression of elevated M1 and M2 microglia/macrophage phenotypes and other MMPs in the ischemic brain. We hypothesized that MMP-12 suppression, by attenuating infiltrating leucocytes (especially monocytes), would decrease the expression of both M1 and M2 markers as well as other MMPs, since activated monocytes are the primary source of elevated MMPs in the ischemic brain. Methods: Young adult male Sprague-Dawley rats (n=6/group) were subjected to 2-h transient middle cerebral artery occlusion and received either MMP-12 shRNA (M12sh) expressing plasmids (1 mg/kg, i.v.; formulated as nanoparticles) within 30 min of reperfusion or no treatment. M12sh-treated and untreated ischemia-induced rats along with sham surgery rats were euthanized on day 3 after ischemia and brain tissues were collected for quantitative real-time PCR analysis of specific M1/M2 markers and various MMPs. Results: In untreated ischemia-induced animals, the expression of both M1 markers (CD16, CD68, IL-1β, IL-6, and TNFα) and M2 markers (CD163, CD206, Arg1, TGFβ, and IL-10) was increased approximately 10-150 fold over sham operated animals. Similarly, the expression of all MMPs studied (MMP-1, MMP-7, MMP-8, MMP-9, MMP-11, and MMP-14) was elevated approximately 10-30 fold in untreated ischemia-induced animals. M12sh treatment reduced the expression of most M1/M2 markers, notably CD68, IL-10, Arg1, and TGFβ, compared to no treatment. M12sh treatment also strongly reduced the elevated levels of MMP-7, MMP-9, MMP-11, and MMP-14. Conclusions: Suppression of MMP-12 leads to a general downregulation of M1/M2 markers and MMPs in the ischemic brain. We attribute these effects to reduced monocyte infiltration and subsequent inflammation. Suppression of MMP-12 could prove to be a promising therapy for ischemic stroke.