Postischemic Reperfusion Research Articles

Effectiveness of remote ischaemic conditioning is not affected by hyper-inflammation in a rat model of stroke

The inflammation and coagulopathy during coronavirus disease (COVID-19) impairs the efficiency of the current stroke treatments. Remote ischaemic conditioning (RIC) has shown potential in recent years to protect the brain and other organs against pathological conditions. This study aimed to evaluate the efficiency of RIC in brain infarct size using TTC staining and lung injury reduction by H&E staining during the hyper-inflammatory response in rats. The inflammation and coagulopathy were assessed by sedimentation rate, haematocrit, systemic oxidative stress and clotting time. Moreover, we observed changes in the cytokine profile. The results of the first part of the experiment showed that the inflammation and lung injury are fully developed after 24 h of intratracheal LPS administration. At this time, we induced focal brain ischaemia and examined the effect of RIC pre- and post-treatment. Our results showed that RIPre-C reduced the infarct size by about 23%, while RIPost-C by about 30%. The lung injury was also reduced following both treatments. Moreover, RIC modulated systemic inflammation. The level of chemokines CINC-1, LIX and RANTES decreased after 24 h of post-ischaemic reperfusion in treated animals compared to non-treated. The RIC-mediated decrease of inflammation was reflected in improved sedimentation rate and hematocrit, as well as reduced systemic oxidative stress. The results of this work showed neuroprotective and lung protective effects of RIC with a decrease in inflammation response. On the basis of our results, we assume that immunomodulation through the chemokines CINC-1, LIX, and RANTES play a role in RIC-mediated protection.

Mitigation of cisplatin-induced acute kidney injury through oral administration of FAAH Inhibitor PF-04457845.

Fatty acid amide hydrolase (FAAH) serves as the primary enzyme responsible for degrading the endocannabinoid anandamide (AEA). Inhibition of FAAH, either through pharmacological means or genetic manipulation, can effectively reduce inflammation in various organs, including the brain, colon, heart, and kidneys. Infusion of a FAAH inhibitor into the kidney medulla has been shown to induce diuretic and natriuretic effects. FAAH knockout mice have shown protection against both post-ischemia reperfusion injury and cisplatin-induced acute kidney injury (AKI), although through distinct mechanisms. The present study was based on the hypothesis that pharmacological inhibition of FAAH activity could mitigate cisplatin-induced AKI, exploring potential renoprotective mechanism. Male wild type C57BL/6 were administered an oral gavage of a FAAH inhibitor (PF-04457845, 5mg/kg) or vehicle (10% PEG200+5% Tween80+normal saline) at 72, 48, 24, and 2 hours before and 24 and 48 hours after a single intraperitoneal injection of cisplatin (Cis, 25 mg/kg). Mice were euthanatized 72 hours after cisplatin treatment. Compared to vehicle-treated mice, PF-04457845-treated mice showed a decrease of cisplatin-induced plasma creatinine, blood urea nitrogen levels, kidney injury biomarkers (NGAL and KIM-1) and renal tubular damage. The renal protection from oral gavage of PF-04457845 against cisplatin-induced nephrotoxicity was associated with an enhanced AEA tone and reduced levels of DNA damage response biomarkers p53 and p21. Our work demonstrates that PF-04457845 effectively alleviates cisplatin-induced nephrotoxicity in mice, underscoring the potential of orally targeting FAAH as a novel strategy to prevent cisplatin nephrotoxicity. Significance Statement Oral administration of FAAH inhibitor, can reduce cisplatin-induced DNA damage response, tubular damages, and kidney dysfunction. Inactivation of FAAH could be a potential strategy to prevent cisplatin-induced nephrotoxicity.

Comparison of the effects of axillary brachial plexus block, inhalation anesthesia, and total intravenous anesthesia on tourniquet-induced ischemia-reperfusion injury in upper extremity surgery.

Post-ischemia reperfusion can lead to oxidative stress and an increase in oxidative markers. Employing preventive strategies and antioxidant agents may help mitigate ischemia-reperfusion injury (IRI). The use of a tourniquet in extremity surgery has been associated with IRI. This study aims to investigate the impact of three different approaches- brachial plexus block, total intravenous anesthesia (TIVA), and inhalation anesthesia-on IRI during upper extremity surgery using a tourniquet. Patients aged 18 to 45 with American Society of Anesthesiologists (ASA) I-II scores were randomly assigned to one of three groups: Group A received an axillary block with bupivacaine; Group I underwent inhalation anesthesia with sevoflurane; and Group T received TIVA with propofol and remifentanil infusion. Blood samples were collected to measure glucose, lactate, total anti-oxidant status (TAS), total oxidant status (TOS), and ischemia-modified albumin (IMA) levels at various time points: before anesthesia (t1), 1 minute before tourniquet release (t2), 20 minutes after tourniquet release (t3), and 4 hours after tourniquet release (t4). In Group I, lactate levels at t3, and glucose levels at t2 and t3, were higher compared to the other groups. Group A exhibited lower IMA levels at t2, t3, and t4 than the other groups. Additionally, Group I had lower IMA levels at t2, t3, and t4 compared to Group T. TAS levels were higher in Group I at t2, t3, and t4 compared to the other groups. TOS levels at t2 and t3 were lower in Group A than in Group I. Axillary anesthesia results in a sympathetic block, promoting better perfusion of the upper extremity. This study demonstrated lower levels of oxidative stress markers with axillary plexus block. Therefore, these results suggest that the axillary block has the potential to mitigate IRI.

Neuroprotective effect of ischemic postconditioning against hyperperfusion and its mechanisms of neuroprotection.

In recent years, stroke and ischemia-reperfusion injury has motivated researchers to find new ways to reduce the complications. Although reperfusion is essential for brain survival, it is like a double-edged sword that may cause further damage to the brain. Ischemic postconditioning (IPostC) refers to the control of blood flow in postischemia-reperfusion that can reduce ischemia-reperfusion injuries. Articles were collected by searching for the terms: Ischemic postconditioning and neuroprotective and ischemic postconditioning and hyperperfusion. Suitable articles were collected from electronic databases, including ISI Web of Knowledge, Medline/PubMed, ScienceDirect, Embase, Scopus, Biological Abstract, Chemical Abstract, and Google Scholar. New investigations show that IPostC has protection against hyperperfusion by reducing the amount of blood flow during reperfusion and thus reducing infarction volume, preventing the blood-brain barrier damage, and reducing the rate of apoptosis through the activation of innate protective systems. Numerous mechanisms have been suggested for IPostC, which include reduction of free radical production, apoptosis, inflammatory factors, and activation of endogenous protective pathways. It seems that postconditioning can prevent damage to the brain by reducing the flow and blood pressure caused by hyperperfusion. It can protect the brain against damages such as stroke and hyperperfusion by activating various endogenous protection systems. In the present review article, we tried to evaluate both useful aspects of IPostC, neuroprotective effects, and fight against hyperperfusion.

Autotaxin-Lysophosphatidic Acid Axis: Key Regulator of Blood-Brain Barrier Integrity and Stroke Outcomes during Cerebral Ischemia-Reperfusion

Background: Endothelial hyperpermeability subsequent to ischemic stroke poses severe ramifications for patient outcomes, with the preservation of blood-brain barrier (BBB) integrity being paramount. The pathological role of the autotaxin-lysophosphatidic acid (ATX-LPA) axis in various disorders is established. While LPA's detrimental effects in stroke pathology have been documented, the mechanistic role of its enzymatic precursor, autotaxin (ATX), especially in the context of BBB disruption and mitochondrial dysfunction, necessitates deeper exploration.Objective: Our research aimed to dissect the intricate interplay of the ATX-LPA axis in modulating BBB integrity and mitochondrial bioenergetics during cerebral ischemia-reperfusion injury.Methods: Employing well-characterized murine models, ischemic strokes were meticulously induced, succeeded by reperfusion. A gamut of advanced assays—including the electrical cell-substrate impedance sensor, Seahorse XF24 analyzer, and AR-2 probe fluorescence—were systematically deployed. Moreover, to attain a nuanced understanding, we genetically engineered a mouse model exhibiting endothelial-specific ATX deletion, facilitating a direct interrogation of ATX's endothelial role.Results: Post-ischemic reperfusion culminated in a statistically significant upregulation of ATX mRNA (1.7-fold, p<0.05) and a quadrupled surge in its enzymatic activity (p<0.01). Disturbingly, the I/R cohort manifested augmented LPA concentrations, concomitant with a pronounced mitochondrial bioenergetic collapse, evidenced by metrics such as the oxygen consumption rate and ATP synthesis (both p<0.01). BBB integrity was critically compromised, a derangement that ATX inhibitors ameliorated (p<0.05). Intriguingly, mice with endothelial-specific ATX ablation exhibited remarkably attenuated permeability (p<0.05) and infarct volumetric reductions (p<0.01), while preserving cerebral perfusion (p<0.05).Conclusion: Our findings underscore the ATX-LPA axis as a linchpin in the pathophysiological cascade following ischemic stroke, specifically impinging on BBB and mitochondrial health. Deciphering and subsequently targeting this nexus could herald a paradigm shift in therapeutic strategies for ischemic stroke. This research was supported by National Institutes of Health grants HL141998, HL141998-01S1, AA025744, AA026708, and AA025744-02S1. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Selective cardiac myosin inhibitors protect the heart following ischemia-reperfusion injury

Abstract Background/Introduction Following an acute myocardial infarction, reperfusion of an occluded coronary artery is often accompanied by cardiomyocyte injury, leading to a worse long-term prognosis. Cardiomyocyte hypercontracture in the first few minutes of post-ischemic reperfusion (IR) is a critical determinant of reperfusion-induced cell death. Inhibition of hypercontracture is highly cardioprotective, but the therapies available are non-specific and unsuitable for clinical translation. Recently, cardiac myosin-targeted inhibitors (CMIs) of contraction, Mavacamten and Aficamten, have been developed to treat patients with hypertrophic cardiomyopathy. These CMIs are well tolerated and safe in clinical trials, however they have not been investigated in the setting of IR injury. Purpose To investigate whether these CMIs could be repurposed to reduce reperfusion-induced hypercontracture following IR, which in turn would limit infarct size. Methods Male Sprague Dawley rats underwent in-vivo myocardial infarction with 30 min ischemia followed by 120 min reperfusion. 15 min prior to reperfusion, Mavacamten or vehicle (10 % DMSO with 90 % corn oil) were administered intraperitoneally. In addition, in vitro experiments using primary adult rat cardiomyocytes (ARVC) were undertaken to confirm the ability of both CMIs to inhibit hypercontracture. Isolated ARVC were pre-treated with either Mavacamten or Aficamten for 30 min before incubating with carbonyl cyanide m-chlorophenylhydrazone (cCCP) to induce cell contracture. Results Mavacamten (2mg/kg) vs control significantly reduced regional infarct size (38.4 ± 4.6 % vs 60.3 ± 5 %, p < 0.05, n= 7-8). Notably, treatment with both Mavacamten (10mM) and Aficamten (1mM) were also inhibited the ability of cCCP to induce ARVC contracture in vitro, with significant reduction in cell contracture (59.7 ± 10.1 % and 49.8 ± 18.5 %, respectively vs control, 99.1 ± 0.6 %, p < 0.05, n= 7). Conclusion These findings provide additional information for further studies into the potential of these CMIs to reduce reperfusion-induced hypercontracture clinically.

Splenic monocytes mediate inflammatory response and exacerbate myocardial ischemia/reperfusion injury in a mitochondrial cell-free DNA-TLR9-NLRP3-dependent fashion.

The spleen contributes importantly to myocardial ischemia/reperfusion (MI/R) injury. Nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) recruits inflammasomes, initiating inflammatory responses and mediating tissue injury. We hypothesize that myocardial cell-free DNA (cfDNA) activates the splenic NLRP3 inflammasome during early reperfusion, increases systemic inflammatory response, and exacerbates myocardial infarct. Mice were subjected to 40min of ischemia followed by 0, 1, 5, or 15min, or 24h of reperfusion. Splenic leukocyte adoptive transfer was performed by injecting isolated splenocytes to mice with splenectomy performed prior to left coronary artery occlusion. CY-09 (4mg/kg) was administered 5min before reperfusion. During post-ischemic reperfusion, splenic protein levels of NLRP3, cleaved caspase-1, and interleukin-1β (IL-1β) were significantly elevated and peaked (2.1 ± 0.2-, 3.4 ± 0.4-, and 3.2 ± 0.2-fold increase respectively, p < 0.05) within 5min of reperfusion. In myocardial tissue, NLRP3 was not upregulated until 24h after reperfusion. Suppression by CY09, a specific NLRP3 inflammasome inhibitor, or deficiency of NLRP3 significantly reduced myocardial infarct size (17.3% ± 4.2% and 33.2% ± 1.8% decrease respectively, p < 0.01). Adoptive transfer of NLRP3-/- splenocytes to WT mice significantly decreased infarct size compared to transfer of WT splenocytes (19.1% ± 2.8% decrease, p < 0.0001). NLRP3 was mainly activated at 5min after reperfusion in CD11b+ and LY6G- splenocytes, which significantly increased during reperfusion (24.8% ± 0.7% vs.14.3% ± 0.6%, p < 0.0001). The circulating cfDNA level significantly increased in patients undergoing cardiopulmonary bypass (CPB) (43.3 ± 5.3ng/mL, compared to pre-CPB 23.8 ± 3.5ng/mL, p < 0.01). Mitochondrial cfDNA (mt-cfDNA) contributed to NLRP3 activation in macrophages (2.1 ± 0.2-fold increase, p < 0.01), which was inhibited by a Toll-like receptor 9(TLR9) inhibitor. The NLRP3 inflammasome in splenic monocytes is activated and mediates the inflammatory response shortly after reperfusion onset, exacerbating MI/R injury in mt-cfDNA/TLR9-dependent fashion. The schema reveals splenic NLRP3 mediates the inflammatory response in macrophages and exacerbates MI/R in a mitochondrial cfDNA/ TLR9-dependent fashion.

Abstract P3086: Aldose Reductase Deletion Protects Ischemic Murine Hearts By Modulating Immune Cell Dynamics

Cardiac injury remains a major cause of death worldwide. Previous studies have demonstrated that chronic unchecked inflammation, during post ischemic reperfusion, adversely affects adaptive repair and augments tissue damage. Nature of the infiltrating immune cells determines the extent of repair vs exacerbation of tissue damage during post myocardial ischemia. Understanding mechanisms by which ischemia-mediated inflammatory responses exacerbate tissue damage and mitigate repair pathways are vital to discover therapeutics for myocardial infarction. Our earlier studies demonstrated that aldose reductase (AR) is a key mediator of myocardial ischemia-reperfusion (I/R) injury. Specifically, it established that cardio myocyte AR drives metabolic and signalling mechanisms leads to injury and impaired function in hearts. In this study we investigated the role of AR in influencing time dependent changes in recruitment of immune cells, during in vivo myocardial I/R injury. AR knockout (ARKO) and littermate controls (WT) mice were subjected to LAD sugery followed by reperfusion ( In vivo I/R) and the temporal changes in recruitment of immune cells were determined at earlier (day 3) and later (day 7) time points. We found ablation of AR leads to significant (p&lt;0.0001) cardiac protection during post I/R in mice. Cardiac function measurements revealed greater recovery of ejection fraction and fractional shortening after I/R in ARKO vs WT hearts (p&lt;0.05). Deletion of AR leads to a significant (p =0.0053) reduction in T cells infiltration post I/R in the heart. We observed 2-fold reduction in CD8+ cells, and 2-fold increase in CD4+ cells in ARKO vs WT mice hearts post I/R injury. Specifically, 2-fold reduction in pro-inflammatory Th1, Th2 and Th17 cells and 3-fold increases in anti-inflammatory Treg cells (p = 0.0091) were observed in ARKO vs WT hearts at post I/R injury. In addition, we observed reduction in infiltration of pro-inflammatory macrophages, dendritic cells, and B cells in ARKO vs WT hearts post I/R. Taken together, our data suggests that deletion of AR protects I/R hearts by promoting increases in anti-inflammatory immune cells and paves the way for addressing immune cell-cardiomyocyte crosstalk mechanisms by which AR deletion protects from I/R injury.

Repurposing of rilpivirine for preventing platelet β3 integrin-dependent thrombosis by targeting c-Src active autophosphorylation

BackgroundHIV-infected individuals are known to be at higher risk for thrombotic cardiovascular disease (CVD), which may also be differentially affected by components of anti-HIV drugs. To identify the effects of a series of FDA-approved anti-HIV drugs on platelet aggregation in humans, focusing on the novel pharmacological effects of rilpivirine (RPV), a reverse transcriptase inhibitor, on platelet function both in vitro and in vivo and the mechanisms involved. Methods and resultsIn vitro studies showed that RPV was the only anti-HIV reagent that consistently and efficiently inhibited aggregation elicited by different agonists, exocytosis, morphological extension on fibrinogen, and clot retraction. Treatment of mice with RPV significantly prevented thrombus formation in FeCl3-injured mesenteric vessels, postcava with stenosis surgery, and ADP -induced pulmonary embolism models without defects in platelet viability, tail bleeding, and coagulation activities. RPV also improved cardiac performance in mice with post-ischemic reperfusion. A mechanistic study revealed that RPV preferentially attenuated fibrinogen-stimulated Tyr773 phosphorylation of β3-integrin by inhibiting Tyr419 autophosphorylation of c-Src. Molecular docking and surface plasmon resonance analyses showed that RPV can bind directly to c-Src. Further mutational analysis showed that the Phe427 residue of c-Src is critical for RPV interaction, suggesting a novel interaction site for targeting c-Src to block β3-integrin outside-in signaling. ConclusionThese results demonstrated that RPV was able to prevent the progression of thrombotic CVDs by interrupting β3-integrin-mediated outside-in signaling via inhibiting c-Src activation without hemorrhagic side effects, highlighting RPV as a promising reagent for the prevention and therapy of thrombotic CVDs.

Treatment With a High-dose of the Omega-3 Fatty Acid, Eicosapentaenoic Acid (EPA), Results in Accelerated Post-ischaemic Reperfusion in Mice

Treatment With a High-dose of the Omega-3 Fatty Acid, Eicosapentaenoic Acid (EPA), Results in Accelerated Post-ischaemic Reperfusion in Mice

IL-17C neutralization protects the kidney against acute injury and chronic injury.

Interleukin-17C (IL-17C), a member of the IL-17 cytokine family, plays a pathogenic role in kidney diseases. Our previous studies have shown that pre-administration of IL-17C neutralizing antibody attenuated acute kidney injury (AKI, a common acute inflammation associated renal disease). In this study, we explored whether post-ischemia reperfusion (IR) of IL-17C blockade has therapeutic effects on AKI and whether IL-17C is involved in the pathogenesis of diabetic nephropathy (DN), a major type of chronic inflammation-associated kidney disease. 12-week-old male C57BL/6JGpt mice were treated with IL-17C neutralizing antibody or normal IgG control antibody at 3h after reperfusion. Renal injury, inflammation, and oxidative stress were assessed. Additionally, we examined renal IL-17C expression in patients with DN and db/db mice and evaluated albuminuria, mesangial matrix accumulation and podocyte loss in db/db mice with IL-17C neutralization. Knockdown of NF-κB p65 using siRNA, and blocking Hypoxia-inducible factor-1α (HIF-1α) using YC-1 in mice and HIF-1α Decoy in HK2 cells were investigated to explore the possible signaling pathway involved in IL-17C regulation. We found that delayed IL-17C neutralization had similar reno-protective effects on renal ischemia-reperfusion injury (IRI). Additionally, renal IL-17C expression was increased in patients with DN and db/db mice, while IL-17C blockade significantly attenuated DN, accompanied with blunted albuminuria, mesangial matrix accumulation, and podocyte loss. Moreover, IL-17C neutralization significantly repressed the expression of downstream pro-inflammatory cytokines, inflammatory cell infiltration, and Th17/IL-17A activation both in mice with renal IRI and DN. Mechanistical studies demonstrated that hypoxia or high glucose-induced IL-17C up-regulation was predominantly mediated by NF-κB pathway. IL-17C participates in the pathogenesis of AKI and DN and inhibition of IL-17C shows potential as a therapeutic strategy for AKI and DN. The National Natural Science Foundation of China (81770741, 81700601 and 81870504).

HBSP improves kidney ischemia-reperfusion injury and promotes repair in properdin deficient mice via enhancing phagocytosis of tubular epithelial cells.

Phagocytosis plays vital roles in injury and repair, while its regulation by properdin and innate repair receptor, a heterodimer receptor of erythropoietin receptor (EPOR)/β common receptor (βcR), in renal ischaemia-reperfusion (IR) remains unclear. Properdin, a pattern recognition molecule, facilitates phagocytosis by opsonizing damaged cells. Our previous study showed that the phagocytic function of tubular epithelial cells isolated from properdin knockout (PKO) mouse kidneys was compromised, with upregulated EPOR in IR kidneys that was further raised by PKO at repair phase. Here, helix B surface peptide (HBSP), derived from EPO only recognizing EPOR/βcR, ameliorated IR-induced functional and structural damage in both PKO and wild-type (WT) mice. In particular, HBSP treatment led to less cell apoptosis and F4/80+ macrophage infiltration in the interstitium of PKO IR kidneys compared to the WT control. In addition, the expression of EPOR/βcR was increased by IR in WT kidneys, and furthered increased in IR PKO kidneys, but greatly reduced by HBSP in the IR kidneys of PKO mice. HBSP also increased PCNA expression in IR kidneys of both genotypes. Moreover, iridium-labelled HBSP (HBSP-Ir) was localized mainly in the tubular epithelia after 17-h renal IR in WT mice. HBSP-Ir also anchored to mouse kidney epithelial (TCMK-1) cells treated by H2O2. Both EPOR and EPOR/βcR were significantly increased by H2O2 treatment, while further increased EPOR was showed in cells transfected with small interfering RNA (siRNA) targeting properdin, but a lower level of EPOR was seen in EPOR siRNA and HBSP-treated cells. The number of early apoptotic cells was increased by EPOR siRNA in H2O2-treated TCMK-1, but markedly reversed by HBSP. The phagocytic function of TCMK-1 cells assessed by uptake fluorescence-labelled E.coli was enhanced by HBSP dose-dependently. Our data demonstrate for the first time that HBSP improves the phagocytic function of tubular epithelial cells and kidney repair post IR injury, via upregulated EPOR/βcR triggered by both IR and properdin deficiency.

Preclinical model to investigate heart failure development following ischemia-reperfusion injury

Heart failure is major public health problem with ischemic cardiomyopathy as the main etiology. Despite therapeutic advances, the high mortality of patients highlights that development of new therapeutic strategies is urgently needed. In this context, the establishment of a pre-clinical model mimicking the clinical pathology would be an asset to characterize the functional mechanisms responsible for the development of heart failure following ischemia-reperfusion injury. In this study, male and female C57Bl/6J mice (8–12 weeks old) underwent left anterior coronary artery ligation for 90 minutes followed by 12 weeks reperfusion (n = 18) or no surgical intervention (CTRL; n = 8). Ejection fraction (EF), left ventricle internal diameter (LVID), E-wave deceleration time (EDT) and the isovolumic relaxation time (IVRT) were assessed by echocardiography during the 12 weeks of post-ischemic reperfusion and cellular Ca2 + (dys)regulation mechanisms in failing isolated cardiomyocytes were investigated at 8 weeks with the imaging Ionoptix system. Our results showed that, despite a significant alteration of EF at day 2 post-infarction (due to ischemic stress by itself), ischemic mice developed a chronical heart dysfunction characterized, on one hand, by a systolic dysfunction with a significant decrease of EF averaging 26% and a LV dilatation around 36% from the day 2 to the week 8 of reperfusion (P < 0.05), and on the other hand, associated with a significant diastolic dysfunction at 12 weeks of reperfusion. Mechanistically, Ca2+ phenotyping showed a significant alteration in excitation/contraction coupling and a contractility defect in failing cardiomyocytes isolated at 8 weeks reperfusion (P < 0.05 vs. CTRL). Our experimental conditions show that 90 minutes ischemia followed by 8 weeks reperfusion seems to be a good algorithm to mimic development of heart failure after an ischemia-reperfusion stress, which is characterized by both systolic and diastolic cardiac dysfunctions.

Age-Associated Resilience Against Ischemic Injury in Mice Exposed to Transient Middle Cerebral Artery Occlusion.

Ischemic stroke is the leading cause of death and disability. Although stroke mainly affects aged individuals, animal research is mostly one on young rodents. Here, we examined the development of ischemic injury in young (9-12-week-old) and adult (72-week-old) C57BL/6 and BALB/c mice exposed to 30 min of intraluminal middle cerebral artery occlusion (MCAo). Post-ischemic reperfusion did not differ between young and adult mice. Ischemic injury assessed by infarct area and blood-brain barrier (BBB) integrity assessed by IgG extravasation analysis was smaller in adult compared with young mice. Microvascular viability and neuronal survival assessed by CD31 and NeuN immunohistochemistry were higher in adult than young mice. Tissue protection was associated with stronger activation of cell survival pathways in adult than young mice. Microglial/macrophage accumulation and activation assessed by F4/80 immunohistochemistry were more restricted in adult than young mice, and pro- and anti-inflammatory cytokine and chemokine responses were reduced by aging. By means of liquid chromatography-mass spectrometry, we identified a hitherto unknown proteome profile comprising the upregulation of glycogen degradation-related pathways and the downregulation of mitochondrial dysfunction-related pathways, which distinguished post-ischemic responses of the aged compared with the young brain. Our study suggests that aging increases the brain's resilience against ischemic injury.

Abstract TP207: Effects Of Dietary Genistein And Estrogen On Post-stroke Inflammation: Relation To Age And Length Of Estrogen Deprivation.

Background: Poststroke inflammation plays an important role in the pathophysiology and prognosis of ischemic stroke. Preclinical investigations with the soy-derived phytoestrogen genistein have shown neuroprotective effects under conditions of brain ischemia. However, little is known about how endogenous estrogen deprivation and age influence the neuroprotection of genistein following focal cerebral ischemia. In this study, we investigated the effects of dietary genistein (Gen) on chronic poststroke inflammation and the probable mechanisms underlying its inflammatory responses after hypogonadism. Method: Young adult (~3-4 months) and proven retired breeder middle-aged Sprague-Dawley rats (~9 months) were used in the study. Rats were ovariectomized and randomly assigned to different durations of hypogonadism (2 vs 12 wks) followed by a two-wk pretreatment with Gen or 17β-estradiol (E2) implant before inducing 60 min. middle cerebral artery occlusion. After 21 days post-ischemia reperfusion, brains were harvested and processed for biochemical analyses to determine the effect of Gen and E2 on the Interleukin-6 (IL-6) and Transforming Growth Factor (TGF-β1) as surrogate markers of M1 and M2 activation respectively. GAP43 was further investigated as a marker of functional recovery. Results: Although there were no significant effects in the ischemic hemisphere, Gen treatment reduced IL-6 expression in the contralateral hemisphere in young subjects while E2 did not affect IL-6 across lengths of hypogonadism in the young cohort (p%lt0.05). After short-term estrogen deprivation, E2 and Gen increased TGF-β1 at the contralateral hemisphere in both young rats and middle-aged rats (p%lt0.05). Gen but not E2 increased GAP43 at the contralateral hemisphere of the young following short-term hypogonadism (p%lt0.05). Conclusion: Our results show that Gen reduces contralateral M1 activation in young ovariectomized rat brains independent of the length of estrogen deprivation after focal cerebral ischemia. Both Gen and E2 differentially promote M2 activation as a function of age but may not influence the inflammatory response in the ischemic hemisphere following extended reperfusion poststroke.

Transmembrane BAX inhibitor motif containing 6 suppresses presenilin-2 to preserve mitochondrial integrity after myocardial ischemia-reperfusion injury.

Myocardial ischemia-reperfusion (I/R) damage is characterized by mitochondrial damage in cardiomyocytes. Transmembrane BAX inhibitor motif containing 6 (TMBIM6) and presenilin-2 (PS2) participate in multiple mitochondrial pathways; thus, we investigated the impact of these proteins on mitochondrial homeostasis during an acute reperfusion injury. Myocardial post-ischemic reperfusion stress impaired myocardial function, induced structural abnormalities and promoted cardiomyocyte death by disrupting the mitochondrial integrity in wild-type mice, but not in TMBIM6 transgenic mice. We found that TMBIM6 bound directly to PS2 and promoted its post-transcriptional degradation. Knocking out PS2 in mice reduced I/R injury-induced cardiac dysfunction, inflammatory responses, myocardial swelling and cardiomyocyte death by improving the mitochondrial integrity. These findings demonstrate that sufficient TMBIM6 expression can prevent PS2 accumulation during cardiac I/R injury, thus suppressing reperfusion-induced mitochondrial damage. Therefore, TMBIM6 and PS2 are promising therapeutic targets for the treatment of cardiac reperfusion damage.

A labeling strategy for the three-dimensional recognition and analysis of microvascular obstruction in ischemic stroke.

Rationale: Large vessel recanalization in ischemic stroke does not always go along with tissue reperfusion, a phenomenon called "no-reflow". However, knowledge of the mechanism of no-reflow is limited because identifying microvascular obstruction across the cortex and subcortex both in clinical and experimental models is challenging. In this study, we developed a smart three-dimensional recognition pipeline for microvascular obstruction during post-ischemia reperfusion to examine the underlying mechanism of no-reflow. Methods: Transient (60 min) occlusion of the middle cerebral artery (tMCAo) in mice was induced using a filament. Two different fluorophore-conjugated tomato lectins were injected into mice via the tail vein before and after ischemia/reperfusion (I/R), respectively, one to label all blood vessels and the other to label functional blood vessels. Post-I/R microvascular obstruction was visualized using combined iDISCO+-based tissue clearing and optical imaging. Arterioles and capillaries were distinguished using whole-mount immunolabeling with an anti-αSMA antibody. Circulating neutrophils were depleted utilizing an anti-Ly6G antibody. Brain slices were immunostained with the anti-Ly6G antibody to identify co-localized blockage points and neutrophils. MATLAB software was used to quantify the capillary diameters in the ipsilateral brain from the normal and tMCAo mice. Results: Microcirculatory reperfusion deficit worsened over time after I/R. Microvascular obstruction occurred not only in arterioles but also in capillaries, with capillary obstruction associated with local capillary lumen narrowing. In addition, the depletion of circulating neutrophils mitigated reperfusion deficit to a large extent after I/R. The co-localization of blockage points and neutrophils revealed that some neutrophils plugged capillaries with coexisting capillary lumen narrowing and that no neutrophil was trapped in heaps of blockage points. Quantification of the capillary diameter showed that capillary lumen shrunk after I/R but returned to typical measurements when intravascular neutrophils were depleted. Conclusions: According to our findings, both vascular lumen narrowing and neutrophil trapping in cerebral microcirculation are the key causes of microvascular obstruction after I/R. Also, the primary contribution by neutrophils to microvascular obstruction does not occur through microemboli plugging but rather via the exacerbation of capillary lumen narrowing. Our proposed method will help monitor microcirculatory reperfusion deficit, explore the mechanism of no-reflow, and evaluate the curative effect of drugs targeting no-reflow.

Cathepsin-K inhibition improves cardiac function post-Ischemia reperfusion injury

Cathepsin-K inhibition improves cardiac function post-Ischemia reperfusion injury

Aldehyde dehydrogenase 2 and arrhythmogenesis.

Cardiac arrhythmia is a common cardiovascular disease that leads to considerable economic burdens and significant global public health challenges. Despite the remarkable progress made in recent decades, antiarrhythmic therapy remains suboptimal. Aldehyde dehydrogenase 2 (ALDH2), a critical detoxifying enzyme, catalyzes toxic aldehydes and protects individuals from damage caused by oxidative stress. Accumulating evidence has demonstrated that ALDH2 activation has potential antiarrhythmic benefits. The correlation between ALDH2 deficiency and arrhythmogenesis has been widely recognized. In this review, we summarize recent research on the potential role of ALDH2 activation and antiarrhythmic protection, as well as the role played by the ALDH2∗2 polymorphism (rs671) in promoting arrhythmic risk. Additionally, we discuss important new findings illustrating the use of ALDH2 activators, which may prove to be promising antiarrhythmic therapy agents.

Abstract P2097: Hypochloremia Alters Cardiac Physiology After Myocardial Ischemia-Reperfusion Injury

Ischemic heart disease (IHD) is one of the leading causes of death related due to heart failure (HF). It is mainly characterized by impairment of heart contraction-relaxation (rhythmicity) and cardiac output. While a myriad of cellular processes is associated with IHD, one of the major cellular aspects strongly associated is the electrolyte and ionic imbalance. Serum sodium levels have been a well-established adverse prognostic marker for patients with chronic heart failure. Recent evidences have suggested that the lower serum chloride (hypochloremia, &amp;lt90mM) is associated with increased mortality risk in patients with chronic HF independent of serum sodium levels. Serum chloride (Cl - ) is a major anion in physiology but its correlation to IHD has not been studied extensively. Hence, to better understand the role of hypochloremia in HF patients, we reviewed patients who received a left ventricular assist device (LVAD) placement at The Ohio State University Medical Center. Survival analysis demonstrated that patients with hypochloremia before LVAD placement had decreased survival at one year compared to patients with normal serum Cl - (p=0.0055). Moreover, our e x vivo studies with isolated rat hearts indicate hypochloremia aggravates myocardial infarction post ischemia-reperfusion (IR) injury. Hence, to elucidate Cl - mediated signaling mechanism we incorporated highly translational human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). Our cellular studies demonstrate that physiological alteration of extracellular Cl - affects the function of beating hiPSC-CM. Hypochloremia significantly increases the beating rate by reducing the calcium cycling durations in hiPSC-CM. Studies are ongoing to determine the involvement of the chloride channel in maintaining chloride homeostasis. Taken together, our results for the first time establish the novel role and mechanism of chloride homeostasis in IR injury. These results will advance the need to search for effective therapeutic approaches to target novel Cl - channels in IHD.