Current Neurovascular Research (v.12, #4)

Sodium Butyrate, a Histone Deacetylase Inhibitor, Reverses Behavioral and Mitochondrial Alterations in Animal Models of Depression Induced by Early- or Late-life Stress by Samira S. Valvassori, Wilson R. Resende, Josiane Budni, Gustavo C. Dal-Pont, Daniela V. Bavaresco, Gislaine Z. Reus, Andre F. Carvalho, Cinara L. Goncalves, Camila B. Furlanetto, Emilio L. Streck, Joao Quevedo (312-320).
The aim of the present study was to evaluate the effects of sodium butyrate on depressive-like behavior and mitochondrial alteration parameters in animal models of depression induced by maternal deprivation or chronic mild stress in Wistar rats. maternal deprivation was established by separating pups from their mothers for 3 h daily from postnatal day 1 to day 10. Chronic mild stress was established by water deprivation, food deprivation, restraint stress, isolation and flashing lights. Sodium butyrate or saline was administered twice a day for 7 days before the behavioral tests. Depressive behavior was evaluated using the forced swim test. The activity of tricarboxylic acid cycle enzymes (succinate dehydrogenase and malate dehydrogenase) and of mitochondrial chain complexes (I, II, II-III and IV) was measured in the striatum of rats. From these analyses it can be observed that sodium butyrate reversed the depressive-like behavior observed in both animal models of depression. Additionally, maternal deprivation and chronic mild stress inhibited mitochondrial respiratory chain complexes and increased the activity of tricarboxylic acid cycle enzymes. Sodium butyrate treatment reversed -maternal deprivation and chronic mild stress- induced dysfunction in the striatum of rats. In conclusion, sodium butyrate showed antidepressant effects in maternal deprivation and chronic mild stress-treated rats, and this effect can be attributed to its action on the neurochemical pathways related to depression.

Epigenetic Changes in the Methylation Patterns of KCNQ1 and WT1 after a Weight Loss Intervention Program in Obese Stroke Patients by Itziar Abete, Ana M. Gomez-Uriz, Maria L. Mansego, Ana De Arce, Estibaliz Goyenechea, Vanessa Blazquez, Maria T. Martinez-Zabaleta, Pedro Gonzalez-Muniesa, Adolfo Lopez De Munain, J. Alfredo Martinez, Javier Campion, Fermin I. Milagro (321-333).
Ischemic stroke patients often show high concentrations of circulating inflammatory markers that are associated with increased risk of recurrence. Epigenetic mechanisms could be involved in obesity, inflammation and stroke. The objective of this research was to investigate, in obese patients suffering a previous stroke, the effects of a nutritional program on anthropometric and biochemical variables, and on the methylation patterns of two stroke-related genes (KCNQ1: potassium channel, voltage gated KQT-like subfamily Q, member 1; and WT1: Wilms tumor 1). Twenty-two ischemic stroke patients were compared with a control group composed of eighteen obese subjects with similar age and body mass index ranges. Both groups followed a 20-week nutritional program based on an energy-restricted balanced diet with high adherence to the Mediterranean dietary pattern. The intervention significantly improved anthropometric and metabolic variables, such as the Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) and C-reactive protein concentration, in ischemic stroke patients, and was accompanied by changes in the methylation patterns of both stroke-related genes, which correlated with anthropometric and biochemical variables.

Prognostic Significance of Homocysteine Levels in Acute Ischemic Stroke: A Prospective Cohort Study by Yan Ji, Bo Song, Yuming Xu, Hui Fang, Jun Wu, Shilei Sun, Lu Zhao, Changhe Shi, Yuan Gao, Yongli Tao, Yapeng Li (334-340).
Associations between hyperhomocysteinemia and prognosis of stroke were seldom explored and always indefinite. We therefore performed a study to elucidate the relationship between homocysteine levels and stroke prognosis. Between 2008 and 2013, baseline data and blood samples of acute ischemic stroke patients were collected from the Henan Province Stroke Registry. Using a prospective cohort, scheduled follow-up, and multivariable logistic regression analysis, associations among the blood homocysteine level and acute neurological impairment and outcomes, stroke recurrence, and all-cause death were investigated. Relevant cutoff homocysteine levels were determined using the area under the receiver operating characteristics curve. Of 1,460 patients, 1,342 completed the 12-month follow-up. We observed higher homocysteine levels in males, those with an advanced age, concomitant hyperlipidemia, a smoking habit, and excessive alcohol consumption. The homocysteine level was an independent risk factor for severe neurological impairment (adjusted relative risk [RR]: 1.021, 95% confidence interval [CI]: 1.004-1.037), a poor functional outcome (adjusted RR with 95% CI: 3-month, 1.029, 1.018-1.039; 6-month, 1.029, 1.018-1.039; and 12-month, 1.038, 1.027-1.049), and stroke recurrence in the large artery atherosclerosis subtype (adjusted RR: 1.025, 1.006-1.045). The optimal cutoff for severe neurological impairment was 17.64 µmol/L, and the cutoffs for poor functional outcomes were 17.28 µmol/L, 17.28 µmol/L, and 14.78 µmol/L at 3, 6, and 12 months, respectively. We found an elevated homocysteine level independently predicted severe neurological impairment, a poor functional outcome, and stroke recurrence in the large artery atherosclerosis stroke subtype. The relevant cutoff homocysteine levels also provide a reference for future clinical work.

A High Burden of Ischemic Stroke in Regions of Eastern/Central Europe is Largely Due to Modifiable Risk Factors by Slaven Pikija, Vladimir Trkulja, Branko Malojcic, J. Sebastian Mutzenbach, Johann Sellner (341-352).
Incidence and mortality of ischemic stroke in Croatia is significantly higher than in many other developed European countries. Here, we aimed to evaluate underlying conditions of this unacceptably high disease burden. An observational prospective cohort study of first-ever ischemic stroke patients identified in a population-based incidence study (N=751) (study 1, S1) and a concurrent case-control trial (215 patients, 125 controls, S2) were conducted in the county of Varazdin from 2007-2010. Atrial fibrillation (AF) was common (36.1% in S1, 40.9% in S2) and mostly (>50%) unrecognized before the index event. In a multivariate analysis, odds of stroke increased with tobacco smoking (OR=3.95, 95%CI 1.33-10.8), unhealthy diet (OR=2.12, 1.12-4.01) and AF (OR=9.40, 4.01-22.0), and decreased with higher education (OR=0.33, 0.11-0.98), moderate alcohol consumption (OR=0.48, 0.25-0.93), higher fasting HDL (OR=0.14, 0.07-0.30) and pre-stroke use of anticoagulants (OR=0.09, 0.01-0.59), antihypertensive drugs (OR=0.52, 0.27-1.00) and statins (OR=0.29, 0.12-0.69). Fourteen-day case fatality was 10.8% (S1) and 6.1% (S2) and the risk was higher with AF, cardioembolic stroke, older age, higher fasting serum glucose, and lower with acute aspirin. Among the acute phase survivors, 30.9% died over the subsequent 1-3 years (p<0.05). We conclude that the incidence of ischemic stroke in Croatia is related to conventional risk factors and largely due to a high rate of unawareness and inadequate treatment of predisposing morbidity.

Normobaric Hyperoxia Preconditioning Induces Changes in the Brain Lipidome by Masoumeh Faezi, Mohammad Reza Bigdeli, Fateme Mirzajani, Alireza Ghasempour (353-362).
Recent investigations have demonstrated that normobaric hyperoxia induces neuroprotection against ischemic injury. The aim of study was to determine the survey of HO (hyperoxia) preconditioning on brain lipidome.The animals were assigned into three groups, the first experimental group was exposed to 95% inspired HO for 4 h /day for six consecutive days. The second experimental group considered as the control group and was exposed to 21% oxygen as room air (RA) in the same chamber. The third group acted as sham, which was under the stress of surgery condition without ischemia. The first two groups were divided into 2 subgroups, intact (without any surgery) and middle cerebral artery occlusion- operated (MCAO). Twenty-four hours after exposure to hyperoxia, MCAO subgroups were subjected to 60 min of right middle cerebral artery occlussion. After 24 h reperfusion, infarct volume (IV) and neurological deficit score (NDS) were assessed in MCAO subgroup. Brain lipidomics were measured in the intact subgroup. Preconditioning with HO significantly reduced NDS and IV and elevated the level of phosphatidylethanolamine (PE), sphingomyelin (SM), cholesterol ester (CE), cholesterol (Chol), phosphatidylcholine (PC), triglyceride (TG) and cerebroside (CB) in the brain as compared with the control (sham and RA). HO preconditioning, significantly decreased the brain ceramide (Cer) and lyso- phosphatidylcholine (Lyso-PC or LPC) levels. Preconditioning with HO decreases brain ischemia injury via changes in brain lipidomics and significantly decreases the brain ceramides (CER).Although more studies are required to explain the mechanisms of time course of neuroprotection, HO preconditioning partly decreases brain ischemia injury via changes in brain lipidome.

Type I Diabetes is characterized by the presence of hyperglycemia due to insulin deficiency and consequent impaired hepatic glucose metabolism. During diabetes, the liver becomes the most important tissue for the regulation of serum glucose. However, elevated glucose causes continuous oxidative damage to the liver, reducing its capacity to ameliorate hyperglycemia, which contributes to macrovascular complications [1]. Numerous epidemiological studies have demonstrated that excess human consumption of diets rich in specific bioflavonoid phytochemicals attenuates the effects of diabetes. Thus, this study was designed to investigate whether a bioflavonoid mixture could : i) attenuate streptozotocin (STZ)-induced hyperglycemia, ii) potentiate antioxidant signaling in the liver, and iii) ameliorate the apoptotic signaling cascade in the liver of STZ-induced hyperglycemic mice. In order to examine our hypothesis, three well-investigated antioxidant phytochemicals, curcumin, hesperidin and rutin, were combined into a mixture (CHR) for this study. Diabetes was induced in 6-month-old female ICR mice by STZ (100 mg/kg, i.p.) administration, and CHR or vehicle control was orally administered (200 mg/kg per body weight of each ingredient) to the hyperglycemic mice (blood glucose levels > 250 mg/dl) for a period of 14 days. Administration of CHR to the hyperglycemic mice significantly reduced blood glucose levels, attenuated STZ-induced lipid peroxidation and total nitrate/nitrite levels, and significantly augmented the expression of superoxide dismutase and glutathione in the liver. STZ-induced hyperglycemia resulted in downregulation of antiapoptotic proteins Bcl-2 by 66% and Bcl-XL by 51%, and upregulation of the pro-apoptotic Bad (69%) with an increase in the ratio of cytosolic/mitochondrial cytochrome c by 81% in hepatic tissue. Administration of CHR significantly ameliorated apoptotic signaling in STZ-induced diabetic mice, significantly increasing Bad/Bcl-2 and Bad/Bcl-XL ratios to 410% and 244% respectively in the hyperglycemic group. This study demonstrated that a bioflavonoid mixture of curcumin, hesperidin and rutin (CHR) ameliorates hepatic oxidative stress caused by STZ-induced hyperglycemia, resulting in improved hepatic function and glucose regulation.

The central nervous system is protected by the blood-brain barrier (BBB). The tight junction (TJ) proteins claudin-5 and zonula occludens-1 (ZO-1) as well as the cytoskeletal component F-actin play key roles in maintaining homeostasis of the BBB. Increases in BBB permeability may be beneficial for the delivery of pharmacological substances into the brain. Therefore, here, we assessed the use of ultrasound to induce transient enhancement of BBB permeability. We used fluorescein isothiocyanate (FITC)-dextran 40 to detect changes in the membrane permeability of bEnd.3 cells during ultrasound treatment. Ultrasound increased FITC-dextran 40 uptake into bEnd.3 cells for 2-6 h after treatment; however, normal levels returned after 24 h. An insignificant increase in lactate dehydrogenase (LDH) leakage also occurred 3 and 6 h after ultrasound treatment, whereas at 24 h, LDH leakage was indistinguishable between the control and treatment groups. Expression of claudin-5, ZO-1, and F-actin at the messenger RNA (mRNA) and protein levels was assessed with real-time polymerase chain reaction and western blotting. Ultrasound induced a transient decrease in claudin-5 mRNA and protein expression within 2 h of treatment; however, no significant changes in ZO-1 and F-actin expression were observed. Claudin-5, ZO-1, and F-actin immunofluorescence demonstrated that the cellular structures incorporating these proteins were transiently impaired by ultrasound. In conclusion, our ultrasound technique can temporarily increase BBB permeability without cytotoxicity to exposed cells, and the method can be exploited in the delivery of drugs to the brain with minimal damage.

Impact of Jugular Vein Valve Function on Cerebral Venous Haemodynamics by Eleuterio F. Toro, Lucas O. Muller, Mariapaola Cristini, Erica Menegatti, Paolo Zamboni (384-397).
We quantify the effect of internal-jugular vein function on intracranial venous haemodynamics, with particular attention paid to venous reflux and intracranial venous hypertension. Haemodynamics in the head and neck is quantified by computing the velocity, flow and pressure fields, and vessel cross-sectional area in all major arteries and veins. For the computations we use a global, closed-loop multi-scale mathematical model for the entire human circulation, recently developed by the first two authors. Validation of the model against in vitro and in vivo Magnetic Resonance Imaging (MRI) measurements have been reported elsewhere. Here, the circulation model is equipped with a sub-model for venous valves. For the study, in addition to a healthy control, we identify two venous-valve related conditions, namely valve incompetence and valve obstruction. A parametric study for subjects in the supine position is carried out for nine cases. It is found that valve function has a visible effect on intracranial venous haemodynamics, including dural sinuses and deep cerebral veins. In particular, valve obstruction causes venous reflux, redirection of flow and intracranial venous hypertension. The clinical implications of the findings are unknown, though they may relate to recent hypotheses linking some neurological conditions to extra-cranial venous anomalies.

The 5-lipoxygenase (5-LOX) Inhibitor Zileuton Reduces Inflammation and Infarct Size with Improvement in Neurological Outcome Following Cerebral Ischemia by Bruno Costa Silva, Aline Silva de Miranda, Flavia Guimaraes Rodrigues, Ana Leticia Malheiros Silveira, Gustavo Henrique de Souza Resende, Marcio Flavio Dutra Moraes, Antonio Carlos Pinheiro de Oliveira, Patricia Martins Parreiras, Luciola da Silva Barcelos, Mauro Martins Teixeira, Fabiana Simao Machado, Antonio Lucio Teixeira, Milene Alvarenga Rachid (398-403).
Stroke is one of the most frequent causes of death and disability worldwide causing a major clinical and socioeconomic impact. Although the pathophysiology of brain ischemia and reperfusion is complex, the inflammatory process plays an important role in pathogenesis, contributing to the expansion of brain injury. The 5-lipoxygenase (5-LOX) is a key enzyme in the biosynthesis of the leukotrienes and has been implicated and in the central nervous system (CNS) disorders such as Alzheimer's disease and acute ischemic stroke. Zileuton, a selective 5-LOX inhibitor, has antiinflammatory properties and exerts an inhibitory effect on inflammatory diseases. The objective of this study was to evaluate the effects of blocking 5-LOX activity in a murine model of transient and global brain ischemia. Zileuton improved neurological deficits and significantly decrease volume and density of lesion, compared to vehicle-ischemic animals measured by magnetic resonance imaging (MRI). In addition, the blockage of 5-LOX reduced infarct area and histopathological changes. Furthermore, by enzyme immunoassay (ELISA) increased brain levels of tumor necrosis factor-alpha (TNFalpha), interferon-gamma (IFN-gamma), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), chemokine (C-X-C motif) ligand 1 (CXCL1), chemokine (C-C motif) ligand 3 (CCL3) and chemokine (C-C motif) ligand 5 (CCL5) were detected in the vehicle-ischemic group, whereas in Zileuton-ischemic group presented reduction of these mediators. The concentration of the antiinflammatory cytokine interleukin-10 (IL-10) was increased after 5-LOX inhibition. Our results suggest that Zileuton decreases brain damage and reduces inflammatory cytokines expression in the CNS which contributes, at least in part, to improve the neurological outcome of brain ischemia.

Mammalian forkhead transcription factors of the O class (FoxO) are exciting targets under consideration for the development of new clinical entities to treat metabolic disorders and diabetes mellitus (DM). DM, a disorder that currently affects greater than 350 million individuals globally, can become a devastating disease that leads to cellular injury through oxidative stress pathways and affects multiple systems of the body. FoxO proteins can regulate insulin signaling, gluconeogenesis, insulin resistance, immune cell migration, and cell senescence. FoxO proteins also control cell fate through oxidative stress and pathways of autophagy and apoptosis that either lead to tissue regeneration or cell demise. Furthermore, FoxO signaling can be dependent upon signal transduction pathways that include silent mating type information regulation 2 homolog 1 (S. cerevisiae) (SIRT1), Wnt, and Wnt1 inducible signaling pathway protein 1 (WISP1). Cellular metabolic pathways driven by FoxO proteins are complex, can lead to variable clinical outcomes, and require in-depth analysis of the epigenetic and post-translation protein modifications that drive FoxO protein activation and degradation.