Current Neurovascular Research - Volume 7, Issue 4, 2010

An enriched environment (EE) is beneficial in modifying behaviors, particularly in tasks involving complex cognitive functions. However, the impact of EE on cognitive impairment induced by chronic cerebral hypoperfusion (CCH) has not been studied. We investigated the effects of EE on cognitive impairment caused by CCH and examined whether CCH altered the protein levels of brain-derived neurotrophic factor (BDNF) and N-methyl-D-aspartate (NMDA) receptor subunit 1 (NR1) and subunit 2B (NR2B) in the hippocampus of rats and whether EE exposure attenuated the effects. Rats were divided into four groups that received either permanent bilateral ligation of the common carotid arteries (2-vessel occlusion) surgery or sham surgery followed by either EE housing or standard environment housing for 4 weeks. We examined non-spatial recognition memory in the novel object recognition task, spatial learning, and memory ability in the Morris water maze as well as the protein levels of BDNF, NR1, and NR2B in the hippocampus. CCH impaired both spatial and non-spatial cognitive functions, and EE exposure reversed the spatial cognitive performance and improved non-spatial memory performance. CCH resulted in decreased levels of BDNF and NR1 protein in the hippocampus, and EE exposure restored the decreased expression. Our results demonstrate for the first time that EE exposure restores cognitive impairment induced by CCH and up-regulates the decreased protein levels of BDNF and NR1. Inversely, BDNF and NR1 may contribute to the beneficial effects of EE on CCH in rats.

Hsp20 is a chaperone protein that is highly and constitutively expressed in the brain, cardiac tissue and many other organs. Recently, it is well established that Hsp20 can enhance cardiac function and render cardioprotection. However, the potential benefits of Hsp20 and its phosphorylation form action on ischemic stroke and the underlying mechanism(s) are largely unknown. To investigate whether Hsp20 exerts protective effects on in vitro ischemia/ reperfusion (I/R) injury, mouse neuroblastoma cells were subjected to oxygen-glucose deprivation (OGD) and reoxygenation. Expressions of Hsp20 were strongly downregulated in mouse N2A cells at the 0-hour and 6-hour recovery time points following 4 hours of OGD, and returned to basal level 12 and 24 hours after OGD treatment, both at mRNA and protein levels. The ratio of phosphorylated to total Hsp20 protein was not significantly affected at the 0-hour and 6- hour recovery time points following 4 hours of OGD. However, markedly higher serine phosphorylation of Hsp20 was observed 12 and 24 hours after OGD treatment. Furthermore, overexpression of Hsp20 reduced OGD-induced apoptosis by reducing the release of cytochrome c from mitochondria to cytosol. However, blockade of Hsp20 phosphorylation at Ser16 abrogated this anti-apoptotic effect. In conclusion, our data demonstrated that increased Hsp20 expression in mouse N2A neuroblastoma cells protected against I/R injury, resulting in reduced apoptosis with the decrease of the release of cytochrome c from mitochondria to cytosol. Phosphorylation of Ser16 played an important role in the neuroprotective effect of Hsp20. Thus, Hsp20 may constitute a new therapeutic target for cerebral ischemic diseases.

This study was aimed to evaluate and compare the effect of chronic hypoxia and aging in the oxidative status of brain vessels and synaptosomes. For this purpose we isolated brain vessels and synaptosomes from 3- and 12-month-old rats subjected to chronic hypoxia (10% O2 for 7 days) or normoxia (21% O2). Several parameters were evaluated: mitochondrial aconitase activity, hydrogen peroxide (H2O2) and malondialdehyde (MDA) levels and enzymatic [superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and glutathione reductase (GR)] and non-enzymatic [glutathione (GSH), glutathione disulfide (GSSG) and vitamin E] antioxidant defences. Concerning brain vessels, we observed an age-dependent increase in MDA levels and SOD, catalase, GR and GPx activities. In vessels isolated from young animals, chronic hypoxia induced an increase in H2O2, GSSG and vitamin E levels and CuZnSOD and catalase activities and a decrease in GSH levels. In mature animals, hypoxia induced a decrease in GSH/GSSG ratio, vitamin E levels and mitochondrial aconitase, MnSOD and GR activities and an increase in H2O2 levels and CuZnSOD and catalase activities. Concerning synaptosomes we observed an age-dependent increase in MDA levels, CuZnSOD and GPx activities and a decrease in MnSOD activity. In synaptosomes from young animals, chronic hypoxia induced a decrease in mitochondrial aconitase activity and GSH levels and an increase in CuZnSOD activity and GSSG levels. In synaptosomes from mature animals, hypoxia induced a decrease in mitochondrial aconitase activity, GSH/GSSG ratio, GSH and vitamin E levels and an increase in GSSG levels. Our results show that chronic hypoxia promotes and potentiates age-dependent oxidative imbalance predisposing to neurodegeneration. Further, synaptosomes and brain vessels are differently affected by aging and chronic hypoxia supporting the idea of the existence of tissue-specific susceptibilities.

Traumatic spinal cord injury (SCI) disrupts the blood-spinal cord barrier and reduces the blood supply caused by microvascular changes. Vessel regression and neovascularization have been observed in the course of secondary injury contributing to microvascular remodeling after trauma. Spatio-temporal distribution of blood vessels and modulation of gene expression of several angiogenic factors have been investigated in rats after spinal cord compression injury. after 2 and 4 weeks, whereas no changes were observed in the penumbra. Investigation of the temporal expression of angiogenic genes using quantitative RT-PCR disclosed a constant down-regulation of the vascular endothelial growth factor (VEGF), and transient decreases of angiopoietin-1 (Ang-1), platelet-derived growth factor-BB (PDGF-BB), as well as placental growth factor (PlGF), with the lowest values obtained 3 days after injury, when compared to the expression levels obtained in sham-operated rats. Hepatocyte growth factor (HGF) was the only angiogenic factor with a constant increased gene expression when compared with controls, starting at day 3 post-SCI. mRNA levels of transforming growth factor-beta 1 (TGF-β1) were elevated at every time point following SCI, whereas those encoding for the cysteine-rich protein CCN1/CYR61 were upregulated after 2 h, 6 h, and 1 week only. Our data provide an overview of the temporal modulated expression of the major angiogenic factors, hampering revascularization in the lesion during the phase of secondary injury. These findings should be considered in order to improve therapeutic interventions.

In the last decade, the neurovascular effects exerted by endocannabinoids (eCBs) have attracted growing interest, because they hold the promise to open new avenues of therapeutic intervention against major causes of death in Western society. Several actions of eCBs are mediated by type-1 (CB1) or type-2 (CB2) cannabinoid receptors, yet there is no clear evidence of the presence of these proteins in platelets. To demonstrate that CB1 and CB2 are expressed in human platelets, we analyzed their protein level by Western blotting and ELISA, visualized their cellular localization by confocal microscopy, and ascertained their functionality by binding assays. We found that CB1, and to a lesser extent CB2, are expressed in highly purified human platelets. Both receptor subtypes were predominantly localized inside the cell, thus explaining why they might remain undetected in preparations of plasma membranes. The identification of authentic CB1 and CB2 in human platelets supports the potential exploitation of selective agonists or antagonists of these receptors as novel therapeutics to combat neurovascular disorders. It seems remarkable that some of these substances have been already used in humans to treat disease states.

One of the therapeutics for acute cerebral ischemia is tissue plasminogen activator (t-PA). Using t-PA after 3 hour time window increases the chances of hemorrhage, involving multiple mechanisms. In order to show possible mechanisms of t-PA toxicity and the effect of the free radical scavenger edaravone, we administered vehicle, plasmin, and t-PA into intact rat cortex, and edaravone intravenously. Plasmin and t-PA damaged rat brain with the most prominent injury in t-PA group on 4-HNE, HEL, and 8-OHdG immunostainings. Such brain damage was strongly decreased in t-PA plus edaravone group. For the neurovascular unit immunostainings, occludin and collagen IV expression was decreased in single plasmin or t-PA group, which was recovered in t-PA plus edaravone group. In contrast, matrix metalloproteinase-9 intensity was the strongest in t-PA group, less in plasmin, and was the least prominent in t-PA plus edaravone group. In vitro data showed a strong damage to tight junctions for occludin and claudin 5 in both administration groups, while there were no changes for endothelial (NAGO) and perivascular (GFAP) stainings. Such damage to tight junctions was recovered in t-PA plus edaravone group with similar recovery in Sodium-Fluorescein permeability assay. Administration of t-PA caused oxidative stress damage to lipids, proteins and DNA, and led to disruption of outer parts of neurovascular unit, greater than the effect in plasmin administration. Additive edaravone ameliorated such an oxidative damage by t-PA with protecting outer layers of blood-brain barrier (in vivo) and tight junctions (in vitro).

Cerebrovascular inflammation is triggered by diverse central nervous system (CNS) insults and contributes to disease pathogenesis. The pro-inflammatory cytokine interleukin (IL)-1 is central to this cerebrovascular inflammatory response and understanding the underlying signalling mechanisms of IL-1 actions in brain endothelium may provide therapeutic targets for disease intervention. For the first time, we compare the contributions of p38, JNK and ERK mitogen-activated protein (MAP) kinase and NF-κB pathways to IL-1-induced brain endothelial activation. In cultures of primary mouse brain endothelium and the rat brain endothelial GPNT cell line, interleukin-1β (IL-1β) induced a rapid (within 5 minutes) and transient activation of p38 and JNK (but not ERK) MAP kinases. IL-1β also induced nuclear recruitment of nuclear factor (NF)-κB p65. IL-1β-induced brain endothelial expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 was insensitive to MAP kinase inhibitors. IL-1β-induced brain endothelial expression of ICAM-1 and VCAM-1 was inhibited (80-88 %) by the proteasome inhibitor MG132 or the antioxidant caffeic acid phenethyl ester (CAPE), effects suggested to be NF-κB-dependent. IL-1β-induced brain endothelial CXCL1 expression was partially inhibited by JNK MAP kinase or MG132 (62 or 56 %, respectively). However, CXCL1 secretion from brain endothelium was reduced (65 %) only by MG132, and not MAP kinase inhibitors. Similarly, IL-1β-induced neutrophil transendothelial migration was reduced (77-89 %) by MG132, but not MAP kinase inhibitors. In summary, we show that several key components of IL-1β-induced brain endothelial activation (CAM, CXCL1 expression or release and neutrophil transmigration) are largely independent of MAP kinase activity but are reduced by proteasome inhibition, possibly reflecting a requirement for NF-κB activity. Similar mechanisms may contribute to cerebrovascular inflammation in response to CNS injury.

Previously it has been demonstrated that protein-energy malnutrition (PEM) impairs habituation in the open field test following global ischemia. The present study examined the hypothesis that PEM exerts some of its deleterious effects on functional outcome by altering the post-ischemic expression of the plasticity-associated genes brain-derived neurotrophic factor (BDNF), its receptor tropomyosin-related kinase B (trkB), and growth-associated protein-43 (GAP- 43). Male, Mongolian gerbils (11-12wk) were randomized to either control diet (12.5% protein) or PEM (2% protein) for 4wk, and then underwent 5min bilateral common carotid artery occlusion or sham surgery. Tympanic temperature was maintained at 36.5± 0.5°C during surgery. Brains collected at 1, 3 and 7d post-surgery were processed by in situ hybridization or immunofluorescence. BDNF and trkB mRNA expression was increased in hippocampal CA1 neurons after ischemia at all time points and was not significantly influenced by diet. However, increased trkB protein expression after ischemia was exacerbated by PEM at 7d in the CA1 region. Post-ischemic GAP-43 protein increased at 3 and 7d in the CA1 region, and PEM intensified this response and extended it to the CA3 and hilar regions. PEM exerted these effects without exacerbating CA1 neuron loss caused by global ischemia. The findings suggest that PEM increases the stress response and/or hyper-excitability in the hippocampus after global ischemia. Nutritional care appears to have robust effects on plasticity mechanisms important to recovery after brain ischemia.