Min Of Occlusion Research Articles

β2-Adrenergic Receptor Agonist Clenbuterol Protects Against Acute Ischemia/Reperfusion-Induced Arrhythmia by Regulation of Akt/eNOS/NO/Cx43 Signaling Pathway.

Ventricular arrhythmias induced by ischemia/reperfusion injury limits the therapeutic effect of early reperfusion therapy for acute myocardial infarction. This study investigated the protective effects of the β2-adrenergic receptor (β2-AR) agonist clenbuterol against ischemia/reperfusion-induced arrhythmias and the underlying mechanism. Anesthetized rats were subjected to 10-min left coronary artery occlusion and 10-min reperfusion invivo. Langendorff-perfused mice hearts were exposed to 10-min global ischemia and 10-min reperfusion. Arrhythmic events were recorded during early reperfusion. Hearts were collected for measuring nitric oxide (NO) concentration and immunoblotting of Connexin 43 (Cx43), endothelial nitric oxide synthase (eNOS), and protein kinase B (Akt). After the ischemia/reperfusion injury in anesthesia rats, clenbuterol markedly reduced the duration and incidence of ventricular tachycardia and ventricular fibrillation, and arrhythmia score, which was abrogated by selective β2-AR antagonist or Cx43 inhibitor. Furthermore, a marked increase in dephosphorylated Cx43 expression and a decrease in the ratio of phosphorylated Cx43 to total Cx43 were observed after the ischemia/reperfusion injury. Mechanistically, clenbuterol increased the phosphorylation of e-NOS and NO concentration, while L-NAME abolished Cx43 phosphorylation and the protective effect of clenbuterol. Clenbuterol also promoted Akt phosphorylation, and blockade of Akt inhibited eNOS phosphorylation and NO production, as well as Cx43 phosphorylation and protective effect of clenbuterol. The present study elucidates that β2-AR stimulation activates the Akt/eNOS signaling pathway, augments NO bioavailability, maintains Cx43 phosphorylation, and prevents Cx43 remodeling, ultimately attenuating arrhythmia induced by ischemia/reperfusion.

Sex and age-related day-to-day variability in the skin microcirculation during post-occlusive reactive hyperemia.

Little is known about the day-to-day variability of different skin microcirculation parameters, and how this variability is influenced by age and sex. The aim was to examine the day-to-day variability of microcirculatory parameters in relation to age and sex. The cutaneous microcirculation was measured using a fiber optic probe integrating laser Doppler flowmetry (LDF) and diffuse reflectance spectroscopy (DRS) to measure oxygen saturation, red blood cell (RBC) tissue fraction, speed-resolved and conventional perfusion. Measurements at two separate days were compared during baseline, a 5-min occlusion and during the following post-occlusive reactive hyperemia (PORH) period on the volar forearm and dorsal foot in totally 48 men and women aged 20-30 and 50-60 years, respectively. Variability was expressed as the coefficient of variation CV and repeatability as the intraclass correlation coefficient ICC. Peak oxygen saturation during PORH had the lowest day-to-day variability for the forearm (CV = 2.1 %) and the foot (CV = 3.8 %) as well as an excellent repeatability (ICC = 0.80 and ICC = 0.82, respectively). Older women had a higher day-to-day variability in baseline conventional perfusion compared to younger women on the forearm (p = 0.007). On the foot, older women had a lower day-to-day variability than younger women for baseline oxygen saturation (p = 0.006) and peak RBC tissue concentration (p = 0.008). Older men had a lower day-to-day variability than younger men for baseline oxygen saturation (p = 0.012) but a higher variability for baseline and peak RBC tissue concentration (p = 0.008 and p = 0.002) on the foot. Peak oxygen saturation had the lowest day-to-day variability of the measured parameters. A lower value of peak oxygen saturation has previously been associated with increasing systematic coronary risk implying that this is a suitable parameter for measuring microcirculatory dysfunction. Sex and age only affected the day-to-day variability of very few parameters.

Chronic high fat diet-induced cerebrovascular remodeling impairs recovery of blood flow after cerebral ischemia in mice.

Obesity and associated metabolic disturbances worsen brain ischemia outcome. High fat diet (HFD)-fed mice are obese and have cerebrovascular remodeling and worsened brain ischemia outcome. We determined whether HFD-induced cerebrovascular remodeling impaired reperfusion to the ischemic penumbra. Six-week-old C57BL/6J or matrix metalloprotease-9 knockout (MMP-9-/-) mice were on HFD or regular diet (RD) for 12 to 14 months before a 60-min left middle cerebral arterial occlusion (MCAO). Photoacoustic microscopy was performed at left cerebral frontal cortex. HFD increased cerebrovascular density and tortuosity in C57BL/6J mice but not in MMP-9-/- mice. Blood flow to the ischemic penumbra slowly recovered but did not reach the baseline 2 h after MCAO in RD-fed mice. Oxygen extraction fraction was increased to maintain cerebral metabolic rate of oxygen (CMRO2) throughout brain ischemia and reperfusion period. This blood flow recovery was worsened in HFD-fed mice, leading to decreased CMRO2. MMP-9-/- attenuated these HFD effects. HFD increased MMP-9 activity and interleukin 1β. Pyrrolidine dithiocarbamate, an anti-inflammatory agent, abolished the HFD effects. Interleukin 1β increased MMP-9 activity. In summary, HFD induces cerebrovascular remodeling, leading to worsened recovery of blood supply to the ischemic penumbra to contribute to poor outcome after brain ischemia. Neuroinflammation may activate MMP-9 in HFD-fed mice.

Cardiomyocyte STIM1 downregulation exacerbates post-Myocardial Infarction remodeling by dysregulating mitochondrial ultrastructure and metabolic signaling.

Loss of stromal interaction molecule 1 (STIM1) expression in smooth muscle cells protects against ischemia-reperfusion (I/R) injury. Whether and how decreased STIM1 expression in cardiomyocytes (CM) impacts cardiac remodeling in response to I/R injury remains unknown. To examine mechanisms by which decreased CM-STIM1 expression in the adult heart modulates cardiac function before and after I/R injury. 8-week old mice underwent cardiotropic AAV9-mediated gene transfer of shRNA directed against STIM1 (shSTIM1). Control (Ctrl) mice underwent shRNA luciferase or PBS injections. Ctrl and shSTIM1 mice were then challenged by 30-min coronary occlusion to induce MI, in-vivo . Mechanical, structural and electrophysiological (EP) properties were compared 1-week following MI. In a second cohort of mice, the impact of CM-STIM1 knockdown per se on upstream metabolic signaling, mitochondrial ultrastructure, and electrophysiological properties were studied. CM-STIM1 expression was markedly decreased in shSTIM1 vs Ctrl hearts. Challenge with in-vivo I/R injury resulted in more pronounced (p<0.0001) LV dysfunction indexed by % drop in fractional shortening in shSTIM1 (44.3%) vs Ctrl (12.2%) hearts 1-week post-MI. Similarly, post-MI structural remodeling and the extent of fibrosis were more severe in shSTIM1 vs Ctrl despite comparable infarct size (p=0.514). Consistently, shSTIM1 exhibited greater impairment in post-MI EP function including predisposition to spatially-discordant action potential alternans. To understand mechanisms underlying this differential remodeling, we examined the impact of CM-STIM1 downregulation on mitochondrial ultrastructure and regulation by metabolic signaling. Quantification of mitochondrial morphology revealed smaller, more rounded mitochondria caused by CM-STIM1 downregulation per se . Underlying these changes was a marked (by 55%, p=0.0057) increase in phosphorylated (p)DRP1 at S616 along with reduced OPA1 expression. Mitochondrial alterations were associated with significant decreases in AMPK downstream signaling with loss of phosphorylated-to-total Raptor and ACC expression in shSTIM1-vs-Ctrl hearts consistent with impaired fatty acid oxidation. These MI-independent metabolic alterations coincided with higher pro-arrhythmic vulnerability under conditions of elevated heart rate. Our findings reveal that decreased CM-STIM1 expression exacerbates post-MI remodeling likely by altering metabolic processes and mitochondrial network dynamics.Functionally, STIM1-dependent mitochondrial alterations impact EP function during conditions of elevated heart rate even without the confounding influence of MI.

Understanding metabolic responses to forearm arterial occlusion measured with two-channel broadband near-infrared spectroscopy.

Broadband near-infrared spectroscopy (bbNIRS) is useful for the quantification of cerebral metabolism. However, its usefulness has not been explored for broad biomedical applications. We aimed to quantify the dynamic responses of oxidized cytochrome c oxidase ( ) within the mitochondria to arterial occlusion and the dynamic correlations between hemodynamic ( ) and responses during and after occlusion in forearm tissues. We recruited 14 healthy participants with two-channel bbNIRS measurements in response to a 5-min forearm arterial occlusion. The bbNIRS system consisted of one shared white-light source and two spectrometers. The modified Beer-Lambert law was applied to determine the occlusion-induced changes in and in the shallow- and deep-tissue layers. During the 5-min occlusion, dynamic responses in hemodynamics exhibited the expected changes, but remained constant, as observed in the 1- and 3-cm channels. A linear correlation between and was observed only during the recovery phase, with a stronger correlation in deeper tissues. The observation of a constant during the cuff period was consistent with two previous reports. The interpretation of this observation is based on the literature that the oxygen metabolism of the skeletal muscle during arterial occlusion remains unchanged before all oxy-hemoglobin (and oxy-myoglobin) resources are completely depleted. Because a 5-min arterial occlusion is not adequate to exhaust all oxygen supply in the vascular bed of the forearm, the local oxygen supply to the muscle mitochondria maintains redox metabolism uninterrupted by occlusion. We provide a better understanding of the mitochondrial responses to forearm arterial occlusion and demonstrate the usefulness of bbNIRS.

DP/dtmax: An underestimated prognostic factor in large animal infarction model.

The present study aims to establish a reproducible large animal experimental unit using a minipig model to monitor cardiac function changes. A 90-min closed-chest balloon occlusion of the left anterior descending branch of the coronary artery was used to induce myocardial infarction in Pannon minipigs. To monitor the cardiac function, measurements were made by cardiac magnetic resonance imaging (cMRI), invasive pressure monitoring, and a Pulse index Continuous Cardiac Output (PiCCO) hemodynamic system at 0, 72, and 720 h during the follow-up period. End-diastolic and end-systolic volumes (EDV, ESV), left ventricular ejection fraction (LVEF) obtained by cMRI evaluation, global ejection fraction and aortic dP/dtmax obtained by the invasive method, were recorded and compared. The 72- and 720-h EDV data showed a significant increase (p = 0.012, <0.001) compared to baseline, and the Day 30 data showed a significant increase compared to Day 3 (p = 0.022). The ESV 72 h after the infarction showed a significant increase (p = 0.001) compared to baseline, which did not change significantly by Day 30 (p = 0.781) compared to Day 3. EDV and ESV were significantly negatively correlated with aortic dpmax, and ESV was significantly correlated with LVEF. For LVEF and dPmax, a significant (p < 0.001 and p = 0.002) worsening was demonstrated at Day 3 compared to baseline, which was no longer statistically detectable for LVEF at Day 30 (p = 0.141), while the difference for dPmax was maintained (p = 0.002). The complementary use of PiCCO hemodynamic measurements in large animal models makes the previously used methodologies more robust and reliable.

Septal wall synthesis is sufficient to change ameba-like cells into uniform oval-shaped cells in Escherichia coli L-forms

A cell wall is required to control cell shape and size to maintain growth and division. However, some bacterial species maintain their morphology and size without a cell wall, calling into question the importance of the cell wall to maintain shape and size. It has been very difficult to examine the dispensability of cell wall synthesis in rod-shaped bacteria such as Escherichia coli for maintenance of their shape and size because they lyse without cell walls under normal culture conditions. Here, we show that wall-less E. coli L-form cells, which have a heterogeneous cell morphology, can be converted to a mostly uniform oval shape solely by FtsZ-dependent division, even in the absence of cylindrical cell wall synthesis. This FtsZ-dependent control of cell shape and size in the absence of a cell wall requires at least either the Min or nucleoid occlusion systems for positioning FtsZ at mid cell division sites.

Sevoflurane Affects Myocardial Autophagy Levels After Myocardial Ischemia Reperfusion Injury via the microRNA-542-3p/ADAM9 Axis.

This research focused on investigating the effects of sevoflurane (Sev) on myocardial autophagy levels after myocardial ischemia reperfusion (I/R) injury via the microRNA-542-3p (miR-542-3p)/ADAM9 axis. Mice underwent 30min occlusion of the left anterior descending coronary (LAD) followed by 2h reperfusion. Cardiac infarction was determined by 2,3,5-triphenyltetrazolium chloride triazole (TTC) staining. Cardiac function was examined by echocardiography. Cardiac markers and oxidative stress factors were evaluated by ELISA. Autophagy-associated factors were detected by western blot. Relationship between miR-542-3p and ADAM9 was tested by dual-luciferase reporter gene assay, RT-qPCR, and western blot. Sev treatment ameliorated cardiac dysfunction, myocardial oxidative stress, and histopathological damages, decreased myocardial infarction size and myocardial apoptotic cells after myocardial I/R injury. Sev treatment elevated miR-542-3p expression and decreased ADAM9 expression in myocardial tissues after myocardial I/R injury. miR-542-3p overexpression could enhance the ameliorative effects of Sev on myocardial injury and myocardial autophagy in I/R mice. miR-542-3p targeted and negatively regulated ADAM9 expression. ADAM9 overexpression reversed the ameliorative effects of miR-542-3p up-regulation on myocardial injury and myocardial autophagy in Sev-treated I/R mice. Sev treatment could ameliorate myocardial injury and myocardial autophagy in I/R mice, mediated by mechanisms that include miR-542-3p up-regulation and ADAM9 down-regulation.

Flow-mediated dilation facilitates radial artery cannulation in patients undergoing intravenous general anesthesia: A prospective randomized controlled trial.

Flow-mediated dilation (FMD) is commonly used as a diagnostic tool to assess endothelial function, and compared with other methods for stimulating radial artery dilation, FMD offers several advantages such as non-invasiveness, ease of execution, minimal equipment requirements, and negligible risk. The study aimed to investigate the effect of FMD in facilitating radial arterial cannulation in the context of intravenous general anesthesia. Eighty patients undergoing intravenous general anesthesia and requiring radial artery cannulation were randomized 1:1 to the FMD group and control group. Patients in the FMD group received an upper arm occlusion for 5 min after anesthesia induction, and the cuff was placed without inflation for the equivalent duration in the control group. The primary outcome was first-attempt success rate. Secondary outcomes were the diameter and percentage of dilation of radial artery, overall success rate, total number of attempts, cannulation time, and occurrence of procedure-related complications. Intravenous anesthetic agents significantly dilated the radial artery (p < 0.05), which was further increased by FMD. An increase in both the first-attempt and overall success rate of radial artery cannulation was demonstrated with the use of FMD (67.5% vs 42.5%, p < 0.05). The total number of attempts needed to cannulate the radial artery was reduced in the FMD group as compared with the control group (p < 0.05), but no differences in cannulation time and procedure-related complications were found between the two groups (p > 0.05). FMD induced by a 5-min upper arm occlusion may facilitate radial artery cannulation in patients undergoing intravenous general anesthesia.

Peripheral venous dilation using flow-mediated dilation response: A randomized crossover study.

Venodilation is crucial in enhancing the success rate of peripheral intravenous cannulation. Flow-mediated dilation (FMD) is a vasodilatory response initiated by temporary ischemia followed by reperfusion. This crossover study aimed to test the hypothesis that FMD induces dilation of the peripheral veins of the forearm. Fifteen healthy volunteers underwent the FMD and control conditions in a randomized order. FMD involved a 5-min occlusion of blood flow in the brachial artery, followed by reperfusion, achieved by inflating and deflating a cuff placed on the upper arm. The control condition involved participants remaining at rest. The primary outcome measure was a change in the cross-sectional area of the cephalic vein post-intervention. The secondary outcomes included changes in venous diameter and perfusion index (PI). FMD significantly increased the cross-sectional area of the cephalic vein compared with the control condition (relative change to baseline: 37.7% (31.4) vs 2.2% (11.7)), with a mean difference of 35.4% (95% confidence interval (CI): 16.4-54.5, p = 0.001). Both longitudinal and transverse diameters were significantly expanded with FMD compared to the control (relative change to baseline: 15.7% (15.4) vs 2.6% (3.6), p = 0.004; 18.9% (15.6) vs -0.0 (10.2), p = 0.003, respectively). Additionally, PI significantly increased with FMD compared with the control (relative change to baseline: 77.8% (56.9) vs 14.6% (36.0)), with a mean difference of 63.2% (95% CI: 31.2-95.2, p = 0.001). FMD application induced dilation of the cephalic vein of the forearm. The findings suggest that FMD is an effective technique for dilating the venous area and potentially improving the success rate of peripheral intravenous cannulation.

Skin Microvascular Dysfunction in Type 2 Diabetes Mellitus Using Laser Speckle Contrast Analysis and Association with Carotid Intima-Media Thickness.

Background: It is established that diabetes mellitus (DM) is characterized by increased cardiovascular risk associated with subclinical atherosclerosis as well as microvascular alterations. Laser speckle contrast analysis (LASCA) is an innovative, non-invasive method for assessing skin microvascular function. Objectives: We sought to assess skin microvascular function in patients with type 2 DM and matched controls. Methods: Consecutive patients with DM and individuals matched for age, sex and BMI were included in the study. Skin microvascular perfusion was assessed, using LASCA, during baseline, a 5 min occlusion period and a 5 min reperfusion period. Carotid intima-media thickness (cIMT) was measured as a surrogate marker of macrocirculation. Results: In total, 18 patients with DM and 22 in the control group were enrolled. No statistically significant differences were observed in baseline flux, peak flux and percentage decrease during arterial occlusion. During reperfusion, individuals with DM exhibited a smaller peak magnitude compared to controls (147.0 ± 64.7% vs. 189.4 ± 46.0%, respectively; p < 0.05). Moreover, cIMT was higher in patients with DM compared to controls (0.68 ± 0.09 mm vs. 0.60 ± 0.08 mm, respectively, p < 0.01) and was negatively correlated with skin microvascular reactivity in the univariate analysis. In the multivariate analysis, glucose and office systolic blood pressure levels remained significant predictors of microvascular reactivity. Conclusions: Our study shows that patients with type 2 DM exhibit impaired skin microvascular function compared to controls. Furthermore, glucose levels and blood pressure play a key role in microvascular dysfunction. However, additional studies are needed to address the clinical significance of early microvascular changes in DM.

Tissue desaturation is not a major determinant of aging-related impairment in skeletal muscle reactive hyperemia: A retrospective analysis.

Near-infrared spectroscopy (NIRS) combined with vascular occlusion test (NIRS-VOT) is a reactive hyperemia technique for in vivo evaluation of skeletal muscle microvascular reactivity. Previous studies using NIRS-VOT have been shown to be able to detect impairments in microvascular function in high-risk cardiovascular disease (CVD) populations such as older individuals. It has been demonstrated that older individuals have slower reactive hyperemia compared to young individuals. Importantly, older individuals also show less desaturation during ischemia compared to young. Based on these findings, it has been suggested that the slower reactive hyperemia observed in older individuals is explained by the lower desaturation during blood flow occlusion (reduced ischemic stimulus). This retrospective analysis compared reactive hyperemia in 36 young and 47 older tissue desaturation-matched individuals that underwent 5-min blood flow occlusion. Overall, we showed that older individuals have impaired reactive hyperemia compared to young when matching for the degree of desaturation and blood flow occlusion time. These findings provide evidence that lower tissue desaturation during ischemia is not a major determinant of impaired reactive hyperemia in older individuals.

Reduction of left ventricular diastolic pressure as a key regulator of infarct coronary flow under mechanical left ventricular support.

Restoring ischaemic myocardial tissue perfusion is crucial for minimizing infarct size. Acute mechanical left ventricular (LV) support has been suggested to improve infarct tissue perfusion. However, its regulatory mechanism remains unclear. We investigated the physiological mechanisms in six Yorkshire pigs, which were subjected to 90-min balloon occlusion of the left anterior descending artery. During the acute reperfusion phase, LV support using an Impella heart pump was initiated. LV pressure, coronary flow and pressure of the infarct artery were simultaneously recorded to evaluate the impact of LV support on coronary physiology. Coronary wave intensity was calculated to understand the forces regulating coronary flow. Significant increases in coronary flow velocity and its area under the curve were found after mechanical LV support. Among the coronary flow-regulating factors, coronary pressure was increased mainly during the late diastolic phase with less pulsatility. Meanwhile, LV pressure was reduced throughout diastole resulting in significant and consistent elevation of coronary driving pressure. Interestingly, the duration of diastole was prolonged with LV support. In the wave intensity analysis, the duration between backward suction and pushing waves was extended, indicating that earlier myocardial relaxation and delayed contraction contributed to the extension of diastole. In conclusion, mechanical LV support increases infarct coronary flow by extending diastole and augmenting coronary driving pressure. These changes were mainly driven by reduced LV diastolic pressure, indicatingthat the key regulator of coronary flow under mechanical LV support is downstream of the coronary artery, rather than upstream. Our study highlights the importance of LV diastolic pressure in infarct coronary flow regulation. KEY POINTS: Restoring ischaemic myocardial tissue perfusion is crucial for minimizing infarct size. Although mechanical left ventricular (LV) support has been suggested to improve infarct coronary flow, its specific mechanism remains to be clarified. LV support reduced LV pressure, and elevated coronary pressure during the late diastolic phase, resulting in high coronary driving pressure. This study demonstrated for the first time that mechanical LV support extends diastolic phase, leading to increased infarct coronary flow. Future studies should evaluate the correlation between improved infarct coronary flow and resulting infarct size.

Abstract WMP115: Rapid Vascular Complement Activation: A Key Driver of Blood-Brain Barrier Disruption in Ischemic Stroke With Acute Hyperglycemia

Background: Acute hyperglycemia, which occurs in over 40% of ischemic stroke patients irrespective of pre-existing diabetes, exacerbates stroke injury. Understanding its mechanisms is vital for innovative treatments. Here we investigate the role of complement in hyperglycemia-exacerbated damage after stroke. Method: Male C57/BL6 mice (10-11 weeks) underwent 30-min suture-induced middle cerebral artery occlusion followed by reperfusion, resembling clinical thrombectomy. Acute hyperglycemia was induced 10 min pre-stroke via glucose injection. Mice were sacrificed at 4.5h and 24h post-stroke to analyze brain swelling, blood-brain barrier (BBB) leakage and hemorrhagic transformation (HT). Mortality, neurological deficits, and motor-sensory functions were assessed at 14 days post-stroke. Complement activation was visualized via C3d immunostaining. Complement C3's role was examined using knock-out mice and the targeted complement inhibitor CR2-Crry fusion protein. Result: Hyperglycemia rapidly worsens BBB leakage (p&lt;0.0001), brain swelling (p&lt;0.05), and HT (p&lt;0.0001) at 4.5 hr after stroke. Hyperglycemic stroke mice exhibit higher mortality (100% vs 25%, p=0.0008), body weight loss, and behavior impairment at the sub-acute phase compared to normoglycemic mice. Notably, acute hyperglycemia rapidly increased plasma C3 levels at 1hr after stroke (p&lt;0.01), accompanied by rapid and time-dependent activation of C3 in ischemic brain vessels, as marked by increased vascular C3d; and this co-localized with autoantibody (IgM/IgG) vessel buildup and vessel leakage. Additionally, C3d levels positively correlated with brain swelling and HT (p&lt;0.01). Vascular C3d and leakage into parenchyma were markedly reduced in the absence of reperfusion. Blocking complement via C3 knock-out or post-reperfusion systemic injection of C3 inhibitor CR2-Crry mitigates hyperglycemia's detrimental effects, including BBB leakage. Conclusion: Rapid vascular activation of complement C3 is a significant driver of hyperglycemic-exacerbated pathology in experimental stroke. Inhibiting C3 activation could be a potential therapeutic approach to improve hyperglycemic stroke outcome.

Hyperglycemia exacerbates cerebral ischemia/reperfusion injury by up-regulating autophagy through p53-Sesn2-AMPK pathway

Hyperglycemia exacerbates ischemic brain injury by up-regulating autophagy. However, the underlying mechanisms are unknown. This study aims to determine whether hyperglycemia activates autophagy through the p53-Sesn2-AMPK signaling pathway. Rats were subjected to 30-min middle cerebral artery occlusion (MCAO) with reperfusion for 1- and 3-day under normo- and hyperglycemic conditions; and HT22 cells were exposed to oxygen deprivation (OG) or oxygen–glucose deprivation and re-oxygenation (OGD/R) with high glucose. Autophagy inhibitors, 3-MA and ARI, were used both in vivo and in vitro. The results showed that, compared with the normoglycemia group (NG), hyperglycemia (HG) increased infarct volume and apoptosis in penumbra area, worsened neurological deficit, and augmented autophagy. after MCAO followed by 1-day reperfusion. Further, HG promoted the conversion of LC-3I to LC-3II, decreased p62, increased protein levels of aldose reductase, p53, P-p53ser15, Sesn2, AMPK and numbers of autophagosomes and autolysosomes, detected by transmission electron microscopy and mRFP-GFP-LC3 molecular probe, in the cerebral cortex after ischemia and reperfusion injury in animals or in cultured HT22 cells exposed to hypoxia with high glucose content. Finally, experiments with autophagy inhibitors 3-MA and aldose reductase inhibitor (ARI) revealed that while both inhibitors reduced the number of TUNEL positive neurons and reversed the effects of hyperglycemic ischemia on LC3 and p62, only ARI decreased the levels of p53, P-p53ser15. These results suggested that hyperglycemia might induce excessive autophagy to aggravate the brain injury resulted from I/R and that hyperglycemia might activate the p53-Sesn2-AMPK signaling pathway, in addition to the classical PI3K/AKT/mTOR autophagy pathway.

Cardiac extracellular matrix at microvascular obstruction area after reperfused myocardial infarction: a morphometric study

Abstract Background Extracellular matrix (ECM), a structural and biomechanical support to cardiomyocytes, suffers substantial alterations after myocardial infarction (MI). Despite a complete reopening at epicardial level, hypoperfusion within the infarcted myocardium, known as microvascular obstruction (MVO), occurs. Extensive clinical studies and meta-analysis reported the association between the magnitude of MVO and the appearance of adverse remodeling; thus, scrutinizing ECM composition within the infarcted areas with impaired tissue-level reperfusion could provide fundamental insight regarding MVO pathophysiology. Purpose To histologically compare ECM composition within the infarct zone regarding the presence or absence of MVO using an experimental swine model of reperfused MI. Methods MI was induced in swine (n=10) by transitory 90-min coronary occlusion followed by 7 days of reperfusion. Prior to euthanasia, intracoronary thioflavin-S was infused. Within the infarcted myocardium, regions displaying MVO (thioflavin-S-) or no MVO (thioflavin-S+) were isolated and stained to morphometrically compare ECM composition. Results Regarding cellularity, areas with MVO displayed an enlarged presence of neutrophils and lymphocytes, whilst no differences in the amount of macrophages and myofibroblasts were detected in comparison to no MVO-myocardium. Those regions with macroscopic MVO showed lower capillary density than those without MVO. According to the fibers and ground substance, a significant reduction in the extension of type I collagen, laminin, and fibronectin together with an augmentation of glycosaminoglycan were noted in areas showing MVO compared to those without microvascular injury. No changes in the amount of type III collagen was observed. Conclusion The ECM composition in infarcted regions with MVO displays a higher presence of inflammatory infiltrate and glycosaminoglycans as well as a reduced amount of microvessels and fibers compared to those areas without microvascular hypoperfusion. These characteristics might underlie the development of adverse ventricular remodeling in MI patients with extensive MVO.

Intranasal insulin helps overcome brain insulin deficiency and improves survival and post-stroke cognitive impairment in male mice.

Obesity increases the risk for stroke and is associated with worse post-stroke outcomes; however, the mechanisms are poorly understood. Diet-induced obesity leads to insulin resistance and subsequently, brain insulin deficiency. The purpose of this study was to investigate the potential impact of brain insulin deficiency on post-stroke outcomes. To accomplish this, brain insulin levels were assessed in male C57BL/6J (B6) mice placed on either a standard diet or 54% kcal high-fat diet, a known model of insulin resistance. Mice were subjected to either a sham surgery (control) or 30-min middle cerebral artery occlusion to induce an ischemic stroke and administered either intranasal saline (0.9%) or intranasal insulin (1.75 U) twice daily for 5 days beginning on day 1 post-stroke. High-fat diet-induced brain insulin deficiency was associated with increased mortality, neurological and cognitive deficits. On the other hand, increasing brain insulin levels via intranasal insulin improved survival, neurological and cognitive function in high-fat diet mice. Our data suggests that brain insulin deficiency correlates with worse post-stroke outcomes in a diet-induced mouse model of insulin resistance and increasing brain insulin levels may be a therapeutic target to improve stroke recovery.

Abstract P1093: Safety And Feasibility Of Tissue-engineered Biological Ventricular Assist Devices For Ischemic Cardiomyopathy

Introduction: Advances in material science and cell technology offer new possibilities in myocardial repair. We test the safety and feasibility of implanting epicardial cellularized patches in a porcine ischemic cardiomyopathy model, representative of human disease. Methods: In pigs (n=4) , 90 min occlusion of the circumflex artery induced myocardial infarction (MI) (infarct size at 4w MRI: 16±7% of LV mass with reduced EF 49±12%). To cover the scar, hexagonal multi-layered 4x4cm scaffolds were printed using high-resolution 3D melt electrowriting, and populated with human iPSC-derived cells (90% cardiomyocytes/10% fibroblasts, totaling 90 million cells), loaded in fibrin hydrogel. Four w post-MI and after 2 w immunosuppression, we sutured patches over the infarcted epicardium and monitored arrhythmias with implantable loop recorders for 1 w. Hearts were explanted after a second MRI for histopathology. Results: Pre-implantation cell loss from spontaneously contracting patches was 0.34±0.15% of all iPSC-derived cells. Immunosuppression was well tolerated (normal LFTs and eGFR) and effective and no significant arrhythmias were recorded after patch implantation. MRI showed correct anatomical patch position ( Fig. 1 ). Visual analysis confirmed homogeneous patch attachment and histology evidenced excellent biocompatibility, and marked engraftment of the construct with host vasculature ( Fig. 1 ). Conclusions: We provide proof-of-concept for safety and feasibility of human iPSC-derived cellular constructs of clinically relevant size in porcine ischemic cardiomyopathy. Follow-up placebo-controlled studies are ongoing to evaluate therapeutic efficacy.

Arterial occlusion duration affects the cuff-induced hyperemic response in skeletal muscle BOLD perfusion imaging as shown in young healthy subjects

ObjectiveDynamic BOLD MRI with cuff compression, inducing ischemia and post-occlusive hyperemia in skeletal muscle, has been pointed out as a potential diagnostic tool to assess peripheral limb perfusion. The objective was to explore the robustness of this technique and its sensitivity to the occlusion duration.Materials and methodsBOLD images were acquired at 3 T in 14 healthy volunteers. {mathrm{T}}_{2}^{*}-imaging with 5- and 1.5-min occlusions were acquired and several semi-quantitative BOLD parameters were derived from ROI-based {mathrm{T}}_{2}^{*}-time curves. Differences in parameters from the two different occlusion durations were evaluated in the gastrocnemius and soleus muscles using non-parametrical tests. Intra- and inter-scan repeatability were evaluated with coefficient of variation.ResultsLonger occlusion duration resulted in an increased hyperemic signal effect yielding significantly different values (p < 0.05) in gastrocnemius for all parameters describing the hyperemic response, and in soleus for two of these parameters. Specifically, 5-min occlusion yielded steeper hyperemic upslope in gastrocnemius (41.0%; p < 0.05) and soleus (59.7%; p = 0.03), shorter time to half peak in gastrocnemius (46.9%; p = 0.00008) and soleus (33.5%; p = 0.0003), and shorter time to peak in gastrocnemius (13.5%; p = 0.02). Coefficients of variation were lower than percentage differences that were found significant.DiscussionFindings show that the occlusion duration indeed influences the hyperemic response and thus should play a part in future methodological developments.

No robust reduction of infarct size and no-reflow by metoprolol pretreatment in adult Göttingen minipigs

Whereas prior experiments in juvenile pigs had reported infarct size reduction by intravenous metoprolol early during myocardial ischaemia, two major clinical trials in patients with reperfused acute myocardial infarction were equivocal. We, therefore, went back and tested the translational robustness of infarct size reduction by metoprolol in minipigs. Using a power analysis-based prospective design, we pretreated 20 anaesthetised adult Göttingen minipigs with 1 mg kg−1 metoprolol or placebo and subjected them to 60-min coronary occlusion and 180-min reperfusion. Primary endpoint was infarct size (triphenyl tetrazolium chloride staining) as a fraction of area at risk; no-reflow area (thioflavin-S staining) was a secondary endpoint. There was no significant reduction in infarct size (46 ± 8% of area at risk with metoprolol vs. 42 ± 8% with placebo) or area of no-reflow (19 ± 21% of infarct size with metoprolol vs. 15 ± 23% with placebo). However, the inverse relationship between infarct size and ischaemic regional myocardial blood flow was modestly, but significantly shifted downwards with metoprolol, whereas ischaemic blood flow tended to be reduced by metoprolol. With an additional dose of 1 mg kg−1 metoprolol after 30-min ischaemia in 4 additional pigs, infarct size was also not reduced (54 ± 9% vs. 46 ± 8% in 3 contemporary placebo, n.s.), and area of no-reflow tended to be increased (59 ± 20% vs. 29 ± 12%, n.s.).Infarct size reduction by metoprolol in pigs is not robust, and this result reflects the equivocal clinical trials. The lack of infarct size reduction may be the result of opposite effects of reduced infarct size at any given blood flow and reduced blood flow, possibly through unopposed alpha-adrenergic coronary vasoconstriction.