Influence of Hyperglycemia and Diabetes on Cardioprotection by Humoral Factors Released after Remote Ischemic Preconditioning (RIPC)

Remote ischemic preconditioning (RIPC) protects hearts from ischemia–reperfusion (I/R) injury in experimental studies; however, clinical RIPC trials were unsatisfactory. This discrepancy could be caused by a loss of cardioprotection due to comorbidities in patients, including diabetes mellitus (DM) and hyperglycemia (HG). RIPC is discussed to confer protective properties by release of different humoral factors activating cardioprotective signaling cascades. Therefore, we investigated whether DM type 1 and/or HG (1) inhibit the release of humoral factors after RIPC and/or (2) block the cardioprotective effect directly at the myocardium. Experiments were performed on male Wistar rats. Animals in part 1 of the study were either healthy normoglycemic (NG), type 1 diabetic (DM1), or hyperglycemic (HG). RIPC was implemented by four cycles of 5 min bilateral hind-limb ischemia/reperfusion. Control (Con) animals were not treated. Blood plasma taken in vivo was further investigated in isolated rat hearts in vitro. Plasma from diseased animals (DM1 or HG) was administered onto healthy (NG) hearts for 10 min before 33 min of global ischemia and 60 min of reperfusion. Part 2 of the study was performed vice versa—plasma taken in vivo, with or without RIPC, from healthy rats was transferred to DM1 and HG hearts in vitro. Infarct size was determined by TTC staining. Part 1: RIPC plasma from NG (NG Con: 49 ± 8% vs. NG RIPC 29 ± 6%; p < 0.05) and DM1 animals (DM1 Con: 47 ± 7% vs. DM1 RIPC: 38 ± 7%; p < 0.05) reduced infarct size. Interestingly, transfer of HG plasma showed comparable infarct sizes independent of prior treatment (HG Con: 34 ± 9% vs. HG RIPC 35 ± 9%; ns). Part 2: No infarct size reduction was detectable when transferring RIPC plasma from healthy rats to DM1 (DM1 Con: 54 ± 13% vs. DM1 RIPC 53 ± 10%; ns) or HG hearts (HG Con: 60 ± 16% vs. HG RIPC 53 ± 14%; ns). These results suggest that: (1) RIPC under NG and DM1 induces the release of humoral factors with cardioprotective impact, (2) HG plasma might own cardioprotective properties, and (3) RIPC does not confer cardioprotection in DM1 and HG myocardium.     

Immune Cells Invade the Collateral Circulation during Human Stroke: Prospective Replication and Extension

It remains unclear if principal components of the local cerebral stroke immune response can be reliably and reproducibly observed in patients with acute large-vessel-occlusion (LVO) stroke. We prospectively studied a large independent cohort of n = 318 consecutive LVO stroke patients undergoing mechanical thrombectomy during which cerebral blood samples from within the occluded anterior circulation and systemic control samples from the ipsilateral cervical internal carotid artery were obtained. An extensive protocol was applied to homogenize the patient cohort and to standardize the procedural steps of endovascular sample collection, sample processing, and laboratory analyses. N = 58 patients met all inclusion criteria. (1) Mean total leukocyte counts were significantly higher within the occluded ischemic cerebral vasculature (I) vs. intraindividual systemic controls (S): +9.6%, I: 8114/µL ± 529 vs. S: 7406/µL ± 468, p = 0.0125. (2) This increase was driven by neutrophils: +12.1%, I: 7197/µL ± 510 vs. S: 6420/µL ± 438, p = 0.0022. Leukocyte influx was associated with (3) reduced retrograde collateral flow (R² = 0.09696, p = 0.0373) and (4) greater infarct extent (R² = 0.08382, p = 0.032). Despite LVO, leukocytes invade the occluded territory via retrograde collateral pathways early during ischemia, likely compromising cerebral hemodynamics and tissue integrity. This inflammatory response can be reliably observed in human stroke by harvesting immune cells from the occluded cerebral vascular compartment.     

Combination of Cyclosporine A and Levosimendan Induces Cardioprotection under Acute Hyperglycemia

Prognosis of patients with myocardial infarction is detrimentally affected by comorbidities like diabetes mellitus. In the experimental setting, not only diabetes mellitus but also acute hyperglycemia is shown to hamper cardioprotective properties by multiple pharmacological agents. For Levosimendan-induced postconditioning, a strong infarct size reducing effect is demonstrated in healthy myocardium. However, acute hyperglycemia is suggested to block this protective effect. In the present study, we investigated whether (1) Levosimendan-induced postconditioning exerts a concentration-dependent effect under hyperglycemic conditions and (2) whether a combination with the mitochondrial permeability transition pore (mPTP) blocker cyclosporine A (CsA) restores the cardioprotective properties of Levosimendan under hyperglycemia. For this experimental investigation, hearts of male Wistar rats were randomized and mounted onto a Langendorff system, perfused with Krebs-Henseleit buffer with a constant pressure of 80 mmHg. All isolated hearts were subjected to 33 min of global ischemia and 60 min of reperfusion under hyperglycemic conditions. (1) Hearts were perfused with various concentrations of Levosimendan (Lev) (0.3–10 μM) for 10 min at the onset of reperfusion, in order to investigate a concentration–response relationship. In the second set of experiments (2), 0.3 μM Levosimendan was administered in combination with the mPTP blocker CsA, to elucidate the underlying mechanism of blocked cardioprotection under hyperglycemia. Infarct size was determined by tetrazolium chloride (TTC) staining. (1) Control (Con) hearts showed an infarct size of 52 ± 12%. None of the administered Levosimendan concentrations reduced the infarct size (Lev0.3: 49 ± 9%; Lev1: 57 ± 9%; Lev3: 47 ± 11%; Lev10: 50 ± 7%; all ns vs. Con). (2) Infarct size of Con and Lev0.3 hearts were 53 ± 4% and 56 ± 2%, respectively. CsA alone had no effect on infarct size (CsA: 50 ± 10%; ns vs. Con). The combination of Lev0.3 and CsA (Lev0.3 ± CsA) induced a significant infarct size reduction compared to Lev0.3 (Lev0.3+CsA: 35 ± 4%; p < 0.05 vs. Lev0.3). We demonstrated that (1) hyperglycemia blocks the infarct size reducing effects of Levosimendan-induced postconditioning and cannot be overcome by an increased concentration. (2) Furthermore, cardioprotection under hyperglycemia can be restored by combining Levosimendan and the mPTP blocker CsA.     

Inhibition of Soluble Epoxide Hydrolase Ameliorates Phenotype and Cognitive Abilities in a Murine Model of Niemann Pick Type C Disease

Niemann–Pick type C (NPC) disease is a rare autosomal recessive inherited childhood neurodegenerative disease characterized by the accumulation of cholesterol and glycosphingolipids, involving the autophagy-lysosome system. Inhibition of soluble epoxide hydrolase (sEH), an enzyme that metabolizes epoxy fatty acids (EpFAs) to 12-diols, exerts beneficial effects in modulating inflammation and autophagy, critical features of the NPC disease. This study aims to evaluate the effects of UB-EV-52, an sEH inhibitor (sEHi), in an NPC mouse model (Npc) by administering it for 4 weeks (5 mg/kg/day). Behavioral and cognitive tests (open-field test (OF)), elevated plus maze (EPM), novel object recognition test (NORT) and object location test (OLT) demonstrated that the treatment produced an improvement in short- and long-term memory as well as in spatial memory. Furthermore, UB-EV-52 treatment increased body weight and lifespan by 25% and reduced gene expression of the inflammatory markers (i.e., Il-1β and Mcp1) and enhanced oxidative stress (OS) markers (iNOS and Hmox1) in the treated Npc mice group. As for autophagic markers, surprisingly, we found significantly reduced levels of LC3B-II/LC3B-I ratio and significantly reduced brain protein levels of lysosomal-associated membrane protein-1 (LAMP-1) in treated Npc mice group compared to untreated ones in hippocampal tissue. Lipid profile analysis showed a significant reduction of lipid storage in the liver and some slight changes in homogenated brain tissue in the treated NPC mice compared to the untreated groups. Therefore, our results suggest that pharmacological inhibition of sEH ameliorates most of the characteristic features of NPC mice, demonstrating that sEH can be considered a potential therapeutic target for this disease.     

Ruscogenin Attenuates Cerebral Ischemia-Induced Blood-Brain Barrier Dysfunction by Suppressing TXNIP/NLRP3 Inflammasome Activation and the MAPK Pathway

Ruscogenin, an important steroid sapogenin derived from Ophiopogon japonicus, has been shown to inhibit cerebral ischemic injury. However, its potential molecular action on blood-brain barrier (BBB) dysfunction after stroke remains unclear. This study aimed to investigate the effects of ruscogenin on BBB dysfunction and the underlying mechanisms in middle cerebral artery occlusion/reperfusion (MCAO/R)-injured mice and oxygen–glucose deprivation/reoxygenation (OGD/R)-injured mouse brain microvascular endothelial cells (bEnd.3). The results demonstrated that administration of ruscogenin (10 mg/kg) decreased the brain infarction and edema, improved neurological deficits, increased cerebral brain flow (CBF), ameliorated histopathological damage, reduced evans blue (EB) leakage and upregulated the expression of tight junctions (TJs) in MCAO/R-injured mice. Meanwhile, ruscogenin (0.1–10 µM) treatment increased cell viability and trans-endothelial electrical resistance (TEER) value, decreased sodium fluorescein leakage, and modulated the TJs expression in OGD/R-induced bEnd.3 cells. Moreover, ruscogenin also inhibited the expression of interleukin-1β (IL-1β) and caspase-1, and markedly suppressed the expression of Nucleotide-binding domain (NOD)-like receptor family, pyrin domain containing 3 (NLRP3) and thiredoxin-interactive protein (TXNIP) in vivo and in vitro. Furthermore, ruscogenin decreased reactive oxygen species (ROS) generation and inhibited the mitogen-activated protein kinase (MAPK) pathway in OGD/R-induced bEnd.3 cells. Our findings provide some new insights into its potential application for the prevention and treatment of ischemic stroke.     

Cardioprotective Properties of Mannitol—Involvement of Mitochondrial Potassium Channels

Cardiac preconditioning (PC) and postconditioning (PoC) are powerful measures against the consequences of myocardial ischemia and reperfusion (I/R) injury. Mannitol—a hyperosmolar solution—is clinically used for treatment of intracranial and intraocular pressure or promotion of diuresis in renal failure. Next to these clinical indications, different organ-protective properties—e.g., perioperative neuroprotection—are described. However, whether Mannitol also confers cardioprotection via a pre- and/or postconditioning stimulus, possibly reducing consequences of I/R injury, remains to be seen. Therefore, in the present study we investigated whether (1) Mannitol-induced pre- and/or postconditioning induces myocardial infarct size reduction and (2) activation of mitochondrial ATP-sensitive potassium (mK_ATP) channels is involved in cardioprotection by Mannitol. Experiments were performed on isolated hearts of male Wistar rats via a pressure controlled Langendorff system, randomized into 7 groups. Each heart underwent 33 min of global ischemia and 60 min of reperfusion. Control hearts (Con) received Krebs–Henseleit buffer as vehicle only. Pre- and postconditioning was achieved by administration of 11 mmol/L Mannitol for 10 min before ischemia (Man-PC) or immediately at the onset of reperfusion (Man-PoC), respectively. In further groups, the mK_ATP channel blocker 5HD, was applied with and without Mannitol, to determine the potential underlying cardioprotective mechanisms. Primary endpoint was infarct size, determined by triphenyltetrazolium chloride staining. Mannitol significantly reduced infarct size both as a pre- (Man-PC) and postconditioning (Man-PoC) stimulus compared to control hearts (Man-PC: 31 ± 4%; Man-PoC: 35 ± 6%, each p < 0.05 vs. Con: 57 ± 9%). The mK_ATP channel inhibitor completely abrogated the cardioprotective effect of Mannitol-induced pre- (5HD-PC-Man-PC: 59 ± 8%, p < 0.05 vs. Man-PC) and postconditioning (5HD-PoC-Man-PoC: 59 ± 10% vs. p < 0.05 Man-PoC). Infarct size was not influenced by 5HD itself (5HD-PC: 60 ± 14%; 5HD-PoC: 54 ± 14%, each ns vs. Con). This study demonstrates that Mannitol (1) induces myocardial pre- and postconditioning and (2) confers cardioprotection via activation of mK_ATP channels.     

The Kynurenic Acid Analog SZR72 Enhances Neuronal Activity after Asphyxia but Is Not Neuroprotective in a Translational Model of Neonatal Hypoxic Ischemic Encephalopathy

Hypoxic–ischemic encephalopathy (HIE) remains to be a major cause of long-term neurodevelopmental deficits in term neonates. Hypothermia offers partial neuroprotection warranting research for additional therapies. Kynurenic acid (KYNA), an endogenous product of tryptophan metabolism, was previously shown to be beneficial in rat HIE models. We sought to determine if the KYNA analog SZR72 would afford neuroprotection in piglets. After severe asphyxia (pHa = 6.83 ± 0.02, ΔBE = −17.6 ± 1.2 mmol/L, mean ± SEM), anesthetized piglets were assigned to vehicle-treated (VEH), SZR72-treated (SZR72), or hypothermia-treated (HT) groups (n = 6, 6, 6; Tcore = 38.5, 38.5, 33.5 °C, respectively). Compared to VEH, serum KYNA levels were elevated, recovery of EEG was faster, and EEG power spectral density values were higher at 24 h in the SZR72 group. However, instantaneous entropy indicating EEG signal complexity, depression of the visual evoked potential (VEP), and the significant neuronal damage observed in the neocortex, the putamen, and the CA1 hippocampal field were similar in these groups. In the caudate nucleus and the CA3 hippocampal field, neuronal damage was even more severe in the SZR72 group. The HT group showed the best preservation of EEG complexity, VEP, and neuronal integrity in all examined brain regions. In summary, SZR72 appears to enhance neuronal activity after asphyxia but does not ameliorate early neuronal damage in this HIE model.     

High-Fat Diet Aggravates Cerebral Infarct,Hemorrhagic Transformation and Neuroinflammation in a Mouse Stroke Model

Background: Stroke in context of type 2 diabetes (T2D) is associated with a poorer outcome than in non-diabetic conditions. We aimed at creating a new reproducible mouse model of stroke in impaired glucose tolerance conditions induced by high-fat diet. Methods: Adult C57BL6 mice were fed for 2 months with either normal diet (ND) or high-fat diet (HFD). We used a model of Middle Cerebral Artery Occlusion (MCAO) for 90 min. Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT) were used to assess pre-diabetic status. Brain infarct volume, hemorrhagic transformation (HT) as well as systemic and cerebral inflammatory markers were evaluated. Results: HFD was associated with an increased body weight and glycemia following OGTT. The HFD group presented a significant increase in brain infarct volume (38.7 (IQR 30–46.7%) vs. 28.45 (IQR 21–30%); p = 0.016) and HT (HFD: 2 (IQR 1–5) vs. ND: 0 (IQR 0–1); p = 0.012) and higher levels of IL-6 and MCP-1 in infarcted hemisphere compared to the ND group. Conclusion: Two months of HFD in adult mice were sufficient to alter the lipid profile and the control of hyperglycemia. These metabolic perturbations were significantly associated with increased infarct volume and hemorrhagic complications.     

Statin Therapy and the Development of Cerebral Amyloid Angiopathy—A Rodent in Vivo Approach

Background: Cerebral amyloid angiopathy (CAA) is characterized by vascular deposition of amyloid β (Aβ) with a higher incidence of cerebral microbleeds (cMBs) and spontaneous hemorrhage. Since statins are known for their benefit in vascular disease we tested for the effect on CAA. Methods: APP23-transgenic mice received atorvastatin-supplemented food starting at the age of eight months (n = 13), 12 months (n = 7), and 16 months (n = 6), respectively. Controls (n = 16) received standard food only. At 24 months of age cMBs were determined with T2*-weighted 9.4T magnetic resonance imaging and graded by size. Results: Control mice displayed an average of 35 ± 18.5 cMBs (mean ± standard deviation), compared to 29.3 ± 9.8 in mice with eight months (p = 0.49), 24.9 ± 21.3 with 12 months (p = 0.26), and 27.8 ± 15.4 with 16 months of atorvastatin treatment (p = 0.27). In combined analysis treated mice showed lower absolute numbers (27.4 ± 15.6, p = 0.16) compared to controls and also after adjustment for cMB size (p = 0.13). Conclusion: Despite to a non-significant trend towards fewer cMBs our results failed to provide evidence for beneficial effects of long-term atorvastatin treatment in the APP23-transgenic mouse model of CAA. A higher risk for bleeding complications was not observed.     

BDNF rs6265 Polymorphism and Its Methylation in Patients with Stroke Undergoing Rehabilitation

Brain-Derived Neurotrophic Factor (BDNF) and its rs6265 single nucleotide polymorphism (SNP) play an important role in post-stroke recovery. We investigated the correlation between BDNF rs6265 SNP and recovery outcome, measured by the modified Barthel index, in 49 patients with stroke hospitalized in our rehabilitation center at baseline (T0) and after 30 sessions of rehabilitation treatment (T1); moreover, we analyzed the methylation level of the CpG site created or abolished into BDNF rs6265 SNP. In total, 11 patients (22.4%) were heterozygous GA, and 32 (65.3%) and 6 (12.2%) patients were homozygous GG and AA, respectively. The univariate analysis showed a significant relationship between the BDNF rs6265 SNP and the modified Barthel index cut-off (χ²(1, N = 48) = 3.86, p = 0.049), considering patients divided for carrying (A+) or not carrying (A−) the A allele. A higher percentage of A− patients obtained a favorable outcome, as showed by the logistic regression model corrected by age and time since the stroke onset, compared with the A+ patients (OR: 5.59). At baseline (T0), the percentage of BDNF methylation was significantly different between GG (44.6 ± 1.1%), GA (39.5 ± 2.8%) and AA (28.5 ± 1.7%) alleles (p < 0.001). After rehabilitation (T1), only patients A− showed a significant increase in methylation percentages (mean change = 1.3, CI: 0.4–2.2, p = 0.007). This preliminary study deserves more investigation to confirm if BDNF rs6265 SNP and its methylation could be used as a biological marker of recovery in patients with stroke undergoing rehabilitation treatment.     

Hepatocyte-Specific Deficiency of BAP31 Amplified Acetaminophen-Induced Hepatotoxicity via Attenuating Nrf2 Signaling Activation in Mice

Liver-specific deficiency of B-cell receptor-associated protein 31 knockout mice (BAP31-LKO) and the littermates were injected with acetaminophen (APAP), markers of liver injury, and the potential molecular mechanisms were determined. In response to APAP overdose, serum aspartate aminotransferase and alanine aminotransferase levels were increased in BAP31-LKO mice than in wild-type controls, accompanied by enhanced liver necrosis. APAP-induced apoptosis and mortality were increased. Hepatic glutathione was decreased (1.60 ± 0.31 μmol/g tissue in WT mice vs. 0.85 ± 0.14 μmol/g tissue in BAP31-LKO mice at 6 h, p < 0.05), along with reduced glutathione reductase activity and superoxide dismutase; while malondialdehyde was significantly induced (0.41 ± 0.03 nmol/mg tissue in WT mice vs. 0.50 ± 0.05 nmol/mg tissue in BAP31-LKO mice for 6 h, p < 0.05). JNK signaling activation and APAP-induced hepatic inflammation were increased in BAP31-LKO mice. The mechanism research revealed that BAP31-deficiency decreased Nrf2 mRNA stability (half-life of Nrf2 mRNA decreased from ~1.3 h to ~40 min) and miR-223 expression, led to reduced nuclear factor erythroid 2-related factor 2 (Nrf2) signaling activation and antioxidant genes induction. BAP31-deficiency decreased mitochondrial membrane potentials, reduced mitochondria-related genes expression, and resulted in mitochondrial dysfunction in the liver. Conclusions: BAP31-deficiency reduced the antioxidant response and Nrf2 signaling activation via reducing Nrf2 mRNA stabilization, enhanced JNK signaling activation, hepatic inflammation, and apoptosis, amplified APAP-induced hepatotoxicity in mice.     

Soluble Epoxide Hydrolase in Aged Female Mice and Human Explanted Hearts Following Ischemic Injury

Myocardial infarction (MI) accounts for a significant proportion of death and morbidity in aged individuals. The risk for MI in females increases as they enter the peri-menopausal period, generally occurring in middle-age. Cytochrome (CYP) 450 metabolizes N-3 and N-6 polyunsaturated fatty acids (PUFA) into numerous lipid mediators, oxylipids, which are further metabolised by soluble epoxide hydrolase (sEH), reducing their activity. The objective of this study was to characterize oxylipid metabolism in the left ventricle (LV) following ischemic injury in females. Human LV specimens were procured from female patients with ischemic cardiomyopathy (ICM) or non-failing controls (NFC). Female C57BL6 (WT) and sEH null mice averaging 13–16 months old underwent permanent occlusion of the left anterior descending coronary artery (LAD) to induce myocardial infarction. WT (wild type) mice received vehicle or sEH inhibitor, trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (tAUCB), in their drinking water ad libitum for 28 days. Cardiac function was assessed using echocardiography and electrocardiogram. Protein expression was determined using immunoblotting, mitochondrial activity by spectrophotometry, and cardiac fibre respiration was measured using a Clark-type electrode. A full metabolite profile was determined by LC–MS/MS. sEH was significantly elevated in ischemic LV specimens from patients, associated with fundamental changes in oxylipid metabolite formation and significant decreases in mitochondrial enzymatic function. In mice, pre-treatment with tAUCB or genetic deletion of sEH significantly improved survival, preserved cardiac function, and maintained mitochondrial quality following MI in female mice. These data indicate that sEH may be a relevant pharmacologic target for women with MI. Although future studies are needed to determine the mechanisms, in this pilot study we suggest targeting sEH may be an effective strategy for reducing ischemic injury and mortality in middle-aged females.     

Failed Neuroprotection of Combined Inhibition of L-Type and ASIC1a Calcium Channels with Nimodipine and Amiloride

Effective pharmacological neuroprotection is one of the most desired aims in modern medicine. We postulated that a combination of two clinically used drugs—nimodipine (L-Type voltage-gated calcium channel blocker) and amiloride (acid-sensing ion channel inhibitor)—might act synergistically in an experimental model of ischaemia, targeting the intracellular rise in calcium as a pathway in neuronal cell death. We used organotypic hippocampal slices of mice pups and a well-established regimen of oxygen-glucose deprivation (OGD) to assess a possible neuroprotective effect. Neither nimodipine (at 10 or 20 µM) alone or in combination with amiloride (at 100 µM) showed any amelioration. Dissolved at 2.0 Vol.% dimethyl-sulfoxide (DMSO), the combination of both components even increased cell damage (p = 0.0001), an effect not observed with amiloride alone. We conclude that neither amiloride nor nimodipine do offer neuroprotection in an in vitro ischaemia model. On a technical note, the use of DMSO should be carefully evaluated in neuroprotective experiments, since it possibly alters cell damage.     

Retrospective Study of Metastatic Melanoma and Renal Cell Carcinoma to the Brain with Multivariate Analysis of Prognostic Pre-Treatment Clinical Factors

Patients with brain metastasis from renal cell carcinoma (RCC) or melanoma have historically had very poor prognoses of less than one year. Stereotactic radiosurgery (SRS) can be an effective treatment for patients with these tumors. This study analyzes the effect of pretreatment prognostic factors on overall survival (OS) for RCC and melanoma patients with metastasis to the brain treated with SRS. A total of 122 patients with brain metastases from either RCC or melanoma were grouped by age at brain metastasis diagnosis, whether they received whole brain radiation therapy (WBRT) in addition to SRS, or they underwent surgical resection, Karnofsky Performance Score (KPS), number of brain metastases, and primary tumor. Median survival times for melanoma patients and RCC patients were 8.20 ± 3.06 and 12.70 ± 2.63 months, respectively. Patients with >5 metastases had a significantly shorter median survival time (6.60 ± 2.45 months) than the reference group (1 metastasis, 10.70 ± 13.40 months, p = 0.024). Patients with KPS ≤ 60 experienced significantly shorter survival than the reference group (KPS = 90–100), with median survival times of 5.80 ± 2.46 months (p < 0.001) and 45.20 ± 43.52 months, respectively. We found a median overall survival time of 12.7 and 8.2 months for RCC and melanoma, respectively. Our study determined that a higher number of brain metastases (>5) and lower KPS were statistically significant predictors of a lower OS prognosis.     

VLDL from Metabolic Syndrome Individuals Enhanced Lipid Accumulation in Atria with Association of Susceptibility to Atrial Fibrillation

Metabolic syndrome (MetS) represents a cluster of metabolic derangements. Dyslipidemia is an important factor in MetS and is related to atrial fibrillation (AF). We hypothesized that very low density lipoproteins (VLDL) in MetS (MetS-VLDL) may induce atrial dilatation and vulnerability to AF. VLDL was therefore separated from normal (normal-VLDL) and MetS individuals. Wild type C57BL/6 male mice were divided into control, normal-VLDL (nVLDL), and MetS-VLDL (msVLDL) groups. VLDL (15 µg/g) and equivalent volumes of saline were injected via tail vein three times a week for six consecutive weeks. Cardiac chamber size and function were measured by echocardiography. MetS-VLDL significantly caused left atrial dilation (control, n = 10, 1.64 ± 0.23 mm; nVLDL, n = 7, 1.84 ± 0.13 mm; msVLDL, n = 10, 2.18 ± 0.24 mm; p < 0.0001) at week 6, associated with decreased ejection fraction (control, n = 10, 62.5% ± 7.7%, vs. msVLDL, n = 10, 52.9% ± 9.6%; p < 0.05). Isoproterenol-challenge experiment resulted in AF in young msVLDL mice. Unprovoked AF occurred only in elderly msVLDL mice. Immunohistochemistry showed excess lipid accumulation and apoptosis in msVLDL mice atria. These findings suggest a pivotal role of VLDL in AF pathogenesis for MetS individuals.     

Left Axis Deviation in Brugada Syndrome: Vectorcardiographic Evaluation during Ajmaline Provocation Testing Reveals Additional Depolarization Abnormalities

Patients with Brugada syndrome (BrS) can show a leftward deviation of the frontal QRS-axis upon provocation with sodium channel blockers. The cause of this axis change is unclear. In this study, we aimed to determine (1) the prevalence of this left axis deviation and (2) to evaluate its cause, using the insights that could be derived from vectorcardiograms. Hence, from a large cohort of patients who underwent ajmaline provocation testing (n = 1430), we selected patients in whom a type-1 BrS-ECG was evoked (n = 345). Depolarization and repolarization parameters were analyzed for reconstructed vectorcardiograms and were compared between patients with and without a >30° leftward axis shift. We found (1) that the prevalence of a left axis deviation during provocation testing was 18% and (2) that this left axis deviation was not explained by terminal conduction slowing in the right ventricular outflow tract (4th QRS-loop quartile: +17 ± 14 ms versus +13 ± 15 ms, nonsignificant) but was associated with a more proximal conduction slowing (1st QRS-loop quartile: +12[8;18] ms versus +8[4;12] ms, p < 0.001 and 3rd QRS-loop quartile: +12 ± 10 ms versus +5 ± 7 ms, p < 0.001). There was no important heterogeneity of the action potential morphology (no difference in the ventricular gradient), but a left axis deviation did result in a discordant repolarization (spatial QRS-T angle: 122[59;147]° versus 44[25;91]°, p < 0.001). Thus, although the development of the type-1 BrS-ECG is characterized by a terminal conduction delay in the right ventricle, BrS-patients with a left axis deviation upon sodium channel blocker provocation have an additional proximal conduction slowing, which is associated with a subsequent discordant repolarization. Whether this has implications for risk stratification is still undetermined.     

Citric Acid Cycle Metabolites Predict Infarct Size in Pigs Submitted to Transient Coronary Artery Occlusion and Treated with Succinate Dehydrogenase Inhibitors or Remote Ischemic Perconditioning

Succinate dehydrogenase (SDH) inhibition with malonate during reperfusion reduced myocardial infarction in animals, whereas its endogenous substrate, succinate, is detected in plasma from STEMI patients. We investigated whether protection by SDH inhibition is additive to that of remote ischemic perconditioning (RIC) in pigs submitted to transient coronary artery occlusion, and whether protective maneuvers influence plasma levels of citric acid cycle metabolites. Forty pigs were submitted to 40 min coronary occlusion and reperfusion, and allocated to four groups (controls, sodium malonate 10 mmol/L, RIC, and malonate + RIC). Plasma was obtained from femoral and great cardiac veins and analyzed by LC-MS/MS. Malonate, RIC, and malonate + RIC reduced infarct size (24.67 ± 5.98, 25.29 ± 3.92 and 29.83 ± 4.62% vs. 46.47 ± 4.49% in controls, p < 0.05), but no additive effects were detected. Enhanced concentrations of succinate, fumarate, malate and citrate were observed in controls during initial reperfusion in the great cardiac vein, and most were reduced by cardioprotective maneuvers. Concentrations of succinate, fumarate, and malate significantly correlated with infarct size. In conclusion, despite the combination of SDH inhibition during reperfusion and RIC did not result in additive protection, plasma concentrations of selected citric acid cycle metabolites are attenuated by protective maneuvers, correlate with irreversible injury, and might become a prognosis tool in STEMI patients.     

Early Exercise Protects the Blood-Brain Barrier from Ischemic Brain Injury via the Regulation of MMP-9 and Occludin in Rats

Early exercise within 24 h after stroke can reduce neurological deficits after ischemic brain injury. However, the mechanisms underlying this neuroprotection remain poorly understood. Ischemic brain injury disrupts the blood-brain barrier (BBB) and then triggers a cascade of events, leading to secondary brain injury and poor long-term outcomes. This study verified the hypothesis that early exercise protected the BBB after ischemia. Adult rats were randomly assigned to sham, early exercise (EE) or non-exercise (NE) groups. The EE and NE groups were subjected to ischemia induced by middle cerebral artery occlusion (MCAO). The EE group ran on a treadmill beginning 24 h after ischemia, 30 min per day for three days. After three-days’ exercise, EB extravasation and electron microscopy were used to evaluate the integrity of the BBB. Neurological deficits, cerebral infarct volume and the expression of MMP-9, the tissue inhibitors of metalloproteinase-1 (TIMP-1), and occludin were determined. The data indicated that early exercise significantly inhibited the ischemia-induced reduction of occludin, and an increase in MMP-9 promoted TIMP-1 expression (p < 0.01), attenuated the BBB disruption (p < 0.05) and neurological deficits (p < 0.01) and diminished the infarct volume (p < 0.01). Our results suggest that the neuroprotection conferred by early exercise was likely achieved by improving the function of the BBB via the regulation of MMP-9 and occludin.     

Worsening of the Toxic Effects of (±)Cis-4,4′-DMAR Following Its Co-Administration with (±)Trans-4,4′-DMAR: Neuro-Behavioural,Physiological, Immunohistochemical and Metabolic Studies in Mice

4,4’-Dimethylaminorex (4,4’-DMAR) is a new synthetic stimulant, and only a little information has been made available so far regarding its pharmaco-toxicological effects. The aim of this study was to investigate the effects of the systemic administration of both the single (±)cis (0.1–60 mg/kg) and (±)trans (30 and 60 mg/kg) stereoisomers and their co-administration (e.g., (±)cis at 1, 10 or 60 mg/kg + (±)trans at 30 mg/kg) in mice. Moreover, we investigated the effect of 4,4′-DMAR on the expression of markers of oxidative/nitrosative stress (8-OHdG, iNOS, NT and NOX2), apoptosis (Smac/DIABLO and NF-κB), and heat shock proteins (HSP27, HSP70, HSP90) in the cerebral cortex. Our study demonstrated that the (±)cis stereoisomer dose-dependently induced psychomotor agitation, sweating, salivation, hyperthermia, stimulated aggression, convulsions and death. Conversely, the (±)trans stereoisomer was ineffective whilst the stereoisomers’ co-administration resulted in a worsening of the toxic (±)cis stereoisomer effects. This trend of responses was confirmed by immunohistochemical analysis on the cortex. Finally, we investigated the potentially toxic effects of stereoisomer co-administration by studying urinary excretion. The excretion study showed that the (±)trans stereoisomer reduced the metabolism of the (±)cis form and increased its amount in the urine, possibly reflecting its increased plasma levels and, therefore, the worsening of its toxicity.     

The L10P Polymorphism and Serum Levels of Transforming Growth Factor β1 in Human Breast Cancer

The L10P single nucleotide polymorphism (SNP) is located in the signal sequence of the transforming growth factor β1 (TGFβ1) gene. The proline-encoding (Pro-) allele of this SNP has been associated with an increased breast cancer risk, which has been attributed to the elevated secretion of this TGFβ1 variant observed in vitro and in male subjects. Here we investigated the association of the L10P SNP with serum levels of TGFβ1 in female breast cancer patients and controls. We genotyped the L10P SNP in 276 breast cancer patients and 255 controls. Serum TGFβ1 concentrations were measured by enzyme-linked immunosorbent assay (ELISA) in a subset of the study population (n = 211). We found no evidence for an association of the L10P SNP with breast cancer risk (per-allele odds ratio: 0.91; 95% confidence interval: 0.71–1.16). However, patients with the Pro/Pro genotype exhibited a significantly younger age at breast cancer onset (55.2 ± 14.3 years) than Leu/Leu patients (60.6 ± 13.6 years; p = 0.04), which may reflect the ability of TGFβ to promote tumor progression. Mean TGFβ1 serum levels of Pro-allele carriers were 39.4 ± 7.4 ng/mL, whereas those of Leu/Leu subjects were 37.6 ± 6.0 ng/mL (p = 0.07). Thus, compared to a previous study of male subjects, we observed only a modest increase, if any, in TGFβ1 levels of female Pro-allele carriers.