Current Neurovascular Research - Volume 9, Issue 3, 2012

Chronic cerebral hypoperfusion (CCH) is common in the pathogenesis of cognitive impairment, in which oxidative stress plays an important role. Here we describe an alternative rat model for CCH that involves two-stage, threevessel occlusion (2s-3VO) and compare its effects with those of permanent bilateral occlusion (2VO) of the common carotid arteries. Real-time cerebral blood flow (CBF) during the surgery was monitored. Spatial learning and memory were tested with the Morris water maze, and oxidative damage was evaluated by measuring malondialdehyde (MDA) levels in both the hippocampus and cortex. We found that the CBF drop in the early stage of the 2s-3VO model was more modest than that in the 2VO model. Like 2VO rats, 2s-3VO rats showed impaired spatial learning and memory and increased MDA levels 8 weeks after surgery. Interestingly, when pooling observations from previous studies, we confirmed that oxidative damage appeared later than spatial learning and memory deficits but lasted longer than did cerebral hypoperfusion. Thus, the 2s-3VO model appears to be a suitable model for the study of CCH. Moreover, data support the notion that cognitive impairment in CCH rat models may be induced early by cerebral hypoperfusion early and in a later phase by oxidative stress.

In the subarachnoid hemorrhage (SAH) blood mixes with cerebrospinal fluid, what starts immunoinflammatory processes - increased synthesis of proinflammatory cytokines, and formation of reactive oxygen species (ROS), resulting in pre-activation of different populations of peripheral leukocytes. Migration of leukocytes to the brain parenchyma through broken blood brain barrier may produce extra brain tissue injury besides of that resulting from SAH. We examined in adult rats the effect of interleukin-1β (IL-1β) neutralization on secretion of cytokines as well as production of ROS in the course of SAH. SAH was produced by injection of 150 μL of autologous arterial blood into cisterna magna. In 50% of animals, IL-1beta activity was inhibited by intracerebroventricular administration of anti-rat IL-1β antibodies. Ninety minutes or 24 hrs following surgery, blood samples were drawn from the extraorbital plexus and centrifuged to obtain two leukocyte subpopulations – polymorphonuclear (PMN) and mononuclear (MN). The chemiluminescence, a hallmark of ROS synthesis, was measured in PMNs. In supernatants from MNs cultures, concentrations endothelin-1 (ET-1), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were assessed. SAH caused the increase ofn PMNs chemiluminescence as well as the increase of production of ET-1 and TNF-α by MNs but had no influence on IL-6 concentration. Neutralization of IL-1β resulted in significant decrease of chemiluminescence as well as concentration of both ET-1 and TNF-α, while IL-6 concentration was increased. These revealed an important role of IL-1β in the activation of peripheral leukocytes in the course of subarachnoid hemorrhage.

DL-3-n-Butylphthalide (NBP) is a synthetic compound based on L-3-n-Butylphthalide which was isolated from seeds of Apium graveolens. The present study aims at evaluating the outcome of NBP given prior to and after the onset of ischemic stroke in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). Stroke was induced by the middle cerebral artery occlusion (MCAO) in SHR and WKY. For pre-treatment, NBP was administered to SHR and WKY daily for two months prior to MCAO. For post-treatment, NBP was given daily for seven consecutive days after MCAO. Seven days post-surgery, rats were tested for the presence of neurological deficits. Magnetic resonance imaging (MRI) and 2,3,5-triphenyltetrazolium chloride (TTC) staining were employed to calculate the infarct volume. The cerebral cortex and corpus striatum in the ischemic penumbra area were examined microscopically for pathological changes. In SHR, NBP pre- and post-treatment significantly lowered neurological deficit scores, reduced infarct volume, and minimized pathological changes in the penumbra area when compared to oil-vehicle treated controls. In WKY, these beneficial effects were observed only in the post-treatment group. The beneficial effects of NBP post-treatment were greater in WKY than in SHR. Results indicated that NBP could exert both preventive and therapeutic effects on ischemic stroke in SHR, but only exerted therapeutic effect in WKY.

Chronic cerebral hypoperfusion (CCH) has been gradually prevalent in the patients over middle age, especially the old over 60 years. It has been proved that CCH is highly related with cognitive impairment. CCH emerges not only in vascular dementia (VaD), but also in Alzheimer’s disease (AD), which regarded as a critical causative for cognitive impairment in these diseases. Nevertheless, the mechanisms underlying cognitive deficit remain elusive. Moreover, there are no dramatically effective preventions. In the present study, by employing a recognized CCH rat model, we found that CCH induced spatial learning/memory deficits with simultaneously increasing tau hyperphosphorylation at multiple Alzheimer-related phosphorylation sites with activation of glycogen synthase kinase-3β (GSK-3β), Cyclin-dependent kinase (Cdk5), Calcium/calmodulin-dependent protein kinase II (CaMKII), and protein kinase B (Akt), and inhibition of protein phosphatase (PP) 2A (PP-2A). Interestingly, enriched environment (EE) treatment, an effect environment stimuli filled with various novel objects, could prevent rats from the EE-induced memory deficits and alterations of tau hyperphosphorylation. Our data suggested that EE might be potentially used for attenuating the detrimental cognition induced by CCH through regulating tau hyperphosphorylation.

Activated platelets shed microparticles, which contain a variety of growth factors central to angiogenesis and neurogenesis. The aim of this study was to explore whether platelet derived microparticles (PMP) can boost endogenous neural stem cells dependent repair mechanisms following stroke in a rat model. To examine the effects of PMP therapy in-vivo, we delivered PMP or vehicle via a biodegradable polymer to the brain surface after permanent middle cerebral artery occlusion (PMCAO) in rats. Rats were tested with the neurological severity score and infarct volumes were measured at 90 days post-ischemia. Immunohistochemistry was used to determine the fate of newborn cells and to count blood vessels in the ischemic brain. The results show that PMP led to a dose dependent increase in cell proliferation, neurogenesis and angiogenesis at the infarct boundary zone and significantly improved behavioral deficits.

Studies investigating cognitive impairment in stroke-free patients with carotid artery stenosis have led to inconsistent results. Furthermore, the pathophysiological mechanism leading to cognitive impairment remains unclear. Cerebral hypoperfusion and arterio-arterial microembolization are discussed. The aims of our study were (1) to delineate patterns of cognitive impairment in stroke-free patients with carotid artery stenosis and (2) to investigate if cognitive impairment is independent of white matter lesion load in brain MRI. We identified 212 (93 women, mean age 70.2) stroke free, non-demented patients, who were referred for carotid artery stenting or because of subjective cognitive impairment. All patients completed a neurocognitive test battery measuring verbal fluency, constructional praxis, figural memory, verbal short-term- and long-term-memory, verbal recognition memory, semantic processing, speed of cognitive processing and divided attention. Grade of maximum carotid artery stenosis was categorized into three groups (mild, moderate, or severe). White matter lesion load was graded using a visual rating scale. Cognitive test scores of groups with different grades of carotid artery stenosis were compared. Univariate regression analysis was used to measure the predictive value of carotid artery stenosis. Multivariate logistic regression analysis was performed when integrating carotid artery stenosis and white matter lesion load. Carotid artery stenosis negatively correlated with measures of verbal fluency, constructional praxis, verbal short-termmemory, semantic processing, speed of cognitive processing, and divided attention. After adjustment for white matter lesions, carotid artery stenosis did not independently predict divided attention. Significance persisted in all other cognitive domains. In our selected group of patients, a higher grade of carotid artery stenosis is associated with cognitive decline. This process is independent of white matter lesion load. Possible pathophysiological implications are discussed.

The low-density lipoprotein receptor (LDLR) gene has been reported to be associated with cerebral infarction. This study aimed to explore 2 genetic LDLR variants, rs688 and rs5925, for their potential roles in cerebral infarction. This genetic association study was conducted within an isolated Taiwanese population; 815 ischemic stroke patients (431 with atherothrombotic stroke and 384 with lacunar infarction) and 430 normal controls were enrolled. There was no significant difference in the genetic frequency of rs688 and rs5925 between the control group and overall ischemic stroke, atherothrombotic stroke, or lacunar infarct groups. However, when analyzing the association between the haplotypes related to rs688 and rs5925 and cerebral ischemic stroke, the most common haplotype allele CT was used as the reference allele, and the haplotype TC associated with a 65% increased risk of overall ischemic stroke, 72% increased risk of atherothrombotic stroke, and 70% increased risk of lacunar infarction; this indicated a synergistic effect between these 2 single-nucleotide polymorphisms. The LDLR analysis based on the haplotypes rs688 and rs5925 was conducted in a Taiwanese population and provided preliminary evidence suggesting that genetic polymorphisms of LDLR are associated with cerebral infarction.

High-dose hydroxymethylglutaryl coenzyme. A reductase inhibitor (statin) administration reduces neuronal injury and improves outcomes in experimental models of acute ischemic stroke, and has been shown to be safe in a phase 1 dose-escalation study using lovastatin at doses higher than currently approved for daily use. Statins also affect the hemostatic system by upregulating t-PA expression and decreasing plasminogen activator inhibitor (PAI-1) expression, platelet adhesion and thrombus formation in animal models. Since a thrombolytic agent, recombinant tissue plasminogen activator (rt-PA), is currently the only FDA-approved therapy for use in ischemic stroke patients, it is important to ascertain whether high statin doses impact the efficacy of rt-PA. The main goal of this study was to evaluate the effect of a high dose of lovastatin and its active form, lovastatin hydroxy acid, on rt-PA thrombolysis in an in vitro model. Percentage clot lysis was measured in the presence and absence of rt-PA in three different treatment groups: lovastatin, lovastatin hydroxy acid, and ethanol. The effect of ethanol on clot lysis was studied since ethanol was used to disperse the highly hydrophobic lovastatin. The decrease in clot width over time was measured using microscopic imaging of an in vitro human whole blood clot model; an approximately 400 μm diameter clot was formed on suture silk, suspended in human fresh frozen plasma (hFFP) and exposed to treatment. In the absence of rt-PA, clot lysis did not show statistically significant differences in the percentage clot lysis between different treatment groups (p=0.103). In the presence of rt-PA, clot lysis was greater than in the absence of rt-PA for all groups, but there were no statistically significant differences between treatment groups (p=0.385). In this in vitro study, high doses of lovastatin neither impaired nor enhanced the lytic efficacy of rt-PA.

The scope of this study highlights application of an emerging hybrid vibrational nanoimaging techniques based on surface enhanced nanoimaging microscopy in investigating the inflammatory process in the vascular system and neurodegenerative disorder. The experiments were performed on transgenic rat and ApoE knockout mice model, intravenously injected with USPIO based MR contrast agent. Marked signal enhancement were observed in contrast treated specimens in specific pathological regions, which is known to be infiltrated by helper T cells. The findings were subsequently correlated with the results from MR imaging and immunohistochemistry. The focus of this work encompasses the latest innovation of nanoimaging technology in monitoring the inflammatory process in neurodegenerative diseases, and implementing the first observation of intracellular iron uptakes in the endothelial and medial smooth muscle cells based on Coherent anti-Stokes Raman Scattering (SECARS) Microscopy.

The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database creates networks from interrelations between molecular biology and underlying chemical elements. This allows for analysis of biologic networks, genomic information, and higher-order functional information at a systems level. We performed microarray experiments and used the KEGG database, systems biology analysis, and annotation of pathway function to study nerve growth factor (NGF)-induced differentiation of PC12 cells. Cells were cultured to 70%–80% confluence, treated with NGF for 1 or 3 hours (h), and RNA was extracted. Stage 1 data analysis involved analysis of variance (ANOVA), and stage 2 involved cluster analysis and heat map generation. We identified 2020 NGF-induced PC12 genes (1038 at 1 h and 1554 at 3 h). Results showed changes in gene expression over time. We compared these genes with 6035 genes from the KEGG database. Cross-matching resulted in 830 genes. Among these, we identified 395 altered genes (155 at 1 h and 301 at 3 h; 2-fold increase from 1 h to 3 h). We identified 191 biologic pathways in the KEGG database; the top 15 showed correlations with neuronal differentiation (mitogen-activated protein kinase [MAPK] pathway: 35 genes at 1 h, 54 genes at 3 h; genes associated with axonal guidance: 12 at 1 h, 26 at 3 h; Wnt pathway: 16 at 1 h, 25 at 3 h; neurotrophin pathway: 4 at 1 h, 14 at 3 h). Thus, we identified changes in neuronal differentiation pathways with the KEGG database, which were synchronized with NGF-induced differentiation.