CD200 Change Is Involved in Neuronal Death in Gerbil Hippocampal CA1 Field Following Transient Forebrain Ischemia and Postischemic Treatment with Risperidone Displays Neuroprotection without CD200 Change.

It has been reported that CD200 (Cluster of Differentiation 200), expressed in neurons, regulates microglial activation in the central nervous system, and a decrease in CD200 expression causes an increase in microglial activation and neuronal loss. The aim of this study was to investigate time-dependent changes in CD200 expression in the hippocampus proper (CA1, 2, and 3 fields) after transient forebrain ischemia for 5 min in gerbils. In this study, 5-min ischemia evoked neuronal death (loss) of pyramidal neurons in the CA1 field, but not in the CA2/3 fields, at 5 days postischemia. In the sham group, CD200 expression was found in pyramidal neurons of the CA1 field, and the immunoreactivity in the group with ischemia was decreased at 6 h postischemia, dramatically increased at 12 h postischemia, decreased (to level found at 6 h postischemia) at 1 and 2 days postischemia, and significantly increased again at 5 days postischemia. At 5 days postischemia, CD200 immunoreactivity was strongly expressed in microglia and GABAergic neurons. However, in the CA3 field, the change in CD200 immunoreactivity in pyramidal neurons was markedly weaker than that in the CA1 field, showing there was no expression of CD 200 in microglia and GABAergic neurons. In addition, treatment of 10 mg/kg risperidone (an atypical antipsychotic drug) after the ischemia hardly changed CD200 immunoreactivity in the CA1 field, showing that CA1 pyramidal neurons were protected from the ischemic injury. These results indicate that the transient ischemia-induced change in CD200 expression may be associated with specific and selective neuronal death in the hippocampal CA1 field following transient forebrain ischemia.

Characterization of a novel model of global forebrain ischaemia-reperfusion injury in mice and comparison with focal ischaemic and haemorrhagic stroke.

Stroke is caused by obstructed blood flow (ischaemia) or unrestricted bleeding in the brain (haemorrhage). Global brain ischaemia occurs after restricted cerebral blood flow e.g. during cardiac arrest. Following ischaemic injury, restoration of blood flow causes ischaemia-reperfusion (I/R) injury which worsens outcome. Secondary injury mechanisms after any stroke are similar, and encompass inflammation, endothelial dysfunction, blood-brain barrier (BBB) damage and apoptosis. We developed a new model of transient global forebrain I/R injury (dual carotid artery ligation; DCAL) and compared the manifestations of this injury with those in a conventional I/R injury model (middle-cerebral artery occlusion; MCAo) and with intracerebral haemorrhage (ICH; collagenase model). MRI revealed that DCAL produced smaller bilateral lesions predominantly localised to the striatum, whereas MCAo produced larger focal corticostriatal lesions. After global forebrain ischaemia mice had worse overall neurological scores, although quantitative locomotor assessment showed MCAo and ICH had significantly worsened mobility. BBB breakdown was highest in the DCAL model while apoptotic activity was highest after ICH. VCAM-1 upregulation was specific to ischaemic models only. Differential transcriptional upregulation of pro-inflammatory chemokines and cytokines and TLRs was seen in the three models. Our findings offer a unique insight into the similarities and differences in how biological processes are regulated after different types of stroke. They also establish a platform for analysis of therapies such as endothelial protective and anti-inflammatory agents that can be applied to all types of stroke.

Glial type specific regulation of CNS angiogenesis by HIFα-activated different signaling pathways.

The mechanisms by which oligodendroglia modulate CNS angiogenesis remain elusive. Previous in vitro data suggest that oligodendroglia regulate CNS endothelial cell proliferation and blood vessel formation through hypoxia inducible factor alpha (HIFα)-activated Wnt (but not VEGF) signaling. Using in vivo genetic models, we show that HIFα in oligodendroglia is necessary and sufficient for angiogenesis independent of CNS regions. At the molecular level, HIFα stabilization in oligodendroglia does not perturb Wnt signaling but rather activates VEGF. At the functional level, genetically blocking oligodendroglia-derived VEGF but not Wnt significantly decreases oligodendroglial HIFα-regulated CNS angiogenesis. Blocking astroglia-derived Wnt signaling reduces astroglial HIFα-regulated CNS angiogenesis. Together, our in vivo data demonstrate that oligodendroglial HIFα regulates CNS angiogenesis through Wnt-independent and VEGF-dependent signaling. These findings suggest an alternative mechanistic understanding of CNS angiogenesis by postnatal glial cells and unveil a glial cell type-dependent HIFα-Wnt axis in regulating CNS vessel formation.

Transient Forebrain Ischemia Induces Differential Bdnf Transcript Expression and Histone Acetylation Patterns in the Rat Hippocampus.

Forebrain ischemia induces delayed, selective neuronal death in hippocampal CA1. It has been established that BDNF (brain-derived neurotrophic factor) is an important factor in ischemic injury. However, the exact mechanism of BDNF expression in the hippocampus after ischemia is unclear. We found that the decrease of BDNF protein expression in hippocampal CA1 was associated with a decrease of Bdnf transcript IV expression in the same region of the rats after ischemia/reperfusion (I/R). In hippocampal CA3 and DG, the results showed increased expression of BDNF protein and transcripts I, IIc, III, IV, VI, and X1. Furthermore, at the Bdnf promoters, I/R led to decreased H3K27ac, increased H3K9ac, and H3K14ac in CA1; increased H3K9ac, H3K14ac, and H3K27ac in CA3; no significant change of H3K9ac, H3K14ac, and H3K27ac in DG. HDAC inhibitor SAHA increased the expression of Bdnf transcripts IV, VI, and X1 in CA1. These findings suggest a potential of modulation Bdnf transcript expression to resolve ischemic brain injury through histone acetylation patterns at the Bdnf promoters.

Chitosan nanoparticles release nimodipine in response to tissue acidosis to attenuate spreading depolarization evoked during forebrain ischemia.

Stroke is an important cause of mortality and disability. Treatment options are limited, therefore the progress in this regard is urgently needed. Nimodipine, an L-type voltage-gated calcium channel antagonist dilates cerebral arterioles, but its systemic administration may cause potential side effects. We have previously constructed chitosan nanoparticles as drug carriers, which release nimodipine in response to decreasing pH typical of cerebral ischemia. Here we have set out to evaluate this nanomedical approach to deliver nimodipine selectively to acidic ischemic brain tissue. After washing a nanoparticle suspension with or without nimodipine (100 µM) on the exposed brain surface of anesthetized rats (n = 18), both common carotid arteries were occluded to create forebrain ischemia. Spreading depolarizations (SDs) were elicited by 1M KCl to deepen the ischemic insult. Local field potential, cerebral blood flow (CBF) and tissue pH were recorded from the cerebral cortex. Microglia activation and neuronal survival were evaluated in brain sections by immunocytochemistry. Ischemia-induced tissue acidosis initiated nimodipine release from nanoparticles, confirmed by the significant elevation of baseline CBF (47.8 ±â€¯23.7 vs. 29.3 ±â€¯6.96%). Nimodipine shortened the duration of both SD itself (48.07 ±â€¯23.29 vs. 76.25 ±â€¯17.2 s), and the associated tissue acidosis (65.46 ±â€¯20.2 vs. 138.3 ±â€¯66.07 s), moreover it enhanced the SD-related hyperemia (48.15 ±â€¯42.04 vs. 17.29 ±â€¯11.03%). Chitosan nanoparticles did not activate microglia. The data support the concept that tissue acidosis linked to cerebral ischemia can be employed as a trigger for targeted drug delivery. Nimodipine-mediated vasodilation and SD inhibition can be achieved by pH-responsive chitosan nanoparticles applied directly to the brain surface.

Locus Coeruleus Degeneration Induces Forebrain Vascular Pathology in a Transgenic Rat Model of Alzheimer's Disease.

Noradrenergic locus coeruleus (LC) neuron loss is a significant feature of mild cognitive impairment and Alzheimer's disease (AD). The LC is the primary source of norepinephrine in the forebrain, where it modulates attention and memory in vulnerable cognitive regions such as prefrontal cortex (PFC) and hippocampus. Furthermore, LC-mediated norepinephrine signaling is thought to play a role in blood-brain barrier (BBB) maintenance and neurovascular coupling, suggesting that LC degeneration may impact the high comorbidity of cerebrovascular disease and AD. However, the extent to which LC projection system degeneration influences vascular pathology is not fully understood. To address this question in vivo, we stereotactically lesioned LC projection neurons innervating the PFC of six-month-old Tg344-19 AD rats using the noradrenergic immunotoxin, dopamine-ß-hydroxylase IgG-saporin (DBH-sap), or an untargeted control IgG-saporin (IgG-sap). DBH-sap-lesioned animals performed significantly worse than IgG-sap animals on the Barnes maze task in measures of both spatial and working memory. DBH-sap-lesioned rats also displayed increased amyloid and inflammation pathology compared to IgG-sap controls. However, we also discovered prominent parenchymal albumin extravasation with DBH-sap lesions indicative of BBB breakdown. Moreover, microvessel wall-to-lumen ratios were increased in the PFC of DBH-sap compared to IgG-sap rats, suggesting that LC deafferentation results in vascular remodeling. Finally, we noted an early emergence of amyloid angiopathy in the DBH-sap-lesioned Tg344-19 AD rats. Taken together, these data indicate that LC projection system degeneration is a nexus lesion that compromises both vascular and neuronal function in cognitive brain areas during the prodromal stages of AD.

Disruption of the Extracellular Matrix Progressively Impairs Central Nervous System Vascular Maturation Downstream of ß-Catenin Signaling.

Objective- The Wnt/ß-catenin pathway orchestrates development of the blood-brain barrier, but the downstream mechanisms involved at different developmental windows and in different central nervous system (CNS) tissues have remained elusive. Approach and Results- Here, we create a new mouse model allowing spatiotemporal investigations of Wnt/ß-catenin signaling by induced overexpression of Axin1, an inhibitor of ß-catenin signaling, specifically in endothelial cells ( Axin1 iEC- OE). AOE (Axin1 overexpression) in Axin1 iEC- OE mice at stages following the initial vascular invasion of the CNS did not impair angiogenesis but led to premature vascular regression followed by progressive dilation and inhibition of vascular maturation resulting in forebrain-specific hemorrhage 4 days post-AOE. Analysis of the temporal Wnt/ß-catenin driven CNS vascular development in zebrafish also suggested that Axin1 iEC- OE led to CNS vascular regression and impaired maturation but not inhibition of ongoing angiogenesis within the CNS. Transcriptomic profiling of isolated, ß-catenin signaling-deficient endothelial cells during early blood-brain barrier-development (E11.5) revealed ECM (extracellular matrix) proteins as one of the most severely deregulated clusters. Among the 20 genes constituting the forebrain endothelial cell-specific response signature, 8 ( Adamtsl2, Apod, Ctsw, Htra3, Pglyrp1, Spock2, Ttyh2, and Wfdc1) encoded bona fide ECM proteins. This specific ß-catenin-responsive ECM signature was also repressed in Axin1 iEC- OE and endothelial cell-specific ß-catenin-knockout mice ( Ctnnb1-KOiEC) during initial blood-brain barrier maturation (E14.5), consistent with an important role of Wnt/ß-catenin signaling in orchestrating the development of the forebrain vascular ECM. Conclusions- These results suggest a novel mechanism of establishing a CNS endothelium-specific ECM signature downstream of Wnt-ß-catenin that impact spatiotemporally on blood-brain barrier differentiation during forebrain vessel development. Visual Overview- An online visual overview is available for this article.

Surge of corticocardiac coupling in SHRSP rats exposed to forebrain cerebral ischemia.

Sudden death is an important but underrecognized consequence of stroke. Acute stroke can disturb central control of autonomic function and result in cardiac dysfunction and sudden death. Previous study showed that bilateral common carotid artery ligation (BCCAL) in the spontaneously hypertensive stroke-prone rat strain (SHRSP) is a well-established model for forebrain ischemic sudden death. This study aims to investigate the temporal dynamic changes in electrical activities of the brain and heart and functional interactions between the two vital organs following forebrain ischemia. EEG and ECG signals were simultaneously collected from nine SHRSP and eight Wistar-Kyoto (WKY) rats. RR interval was analyzed to investigate the cardiac response to brain ischemia. EEG power and coherence (CCoh) analysis were conducted to study the cortical response. Corticocardiac coherence (CCCoh) and directional connectivity (CCCon) were analyzed to determine brain-heart connection. Heart rate variability (HRV) was analyzed to evaluate autonomic functionality. BCCAL resulted in 100% mortality in SHRSP within 14 h, whereas no mortality was observed in WKY rats. The functionality of both the brain and the heart were significantly altered in SHRSP compared with WKY rats after BCCAL. SHRSP, but not WKY rats, exhibited intermittent surge of CCCoh, which paralleled the elevated CCCon and reduced HRV, following the onset of ischemia until sudden death. Elevated brain-heart coupling invariably associated with the disruption of the autonomic nervous system and the risk of sudden death. This study may improve our understanding of the mechanism of forebrain ischemia-induced sudden death. NEW & NOTEWORTHY This study demonstrates a marked surge of corticocardiac coupling in rats dying from focal cerebral ischemia, consistent with our earlier data in rats exposed to fatal asphyxia. Since the bidirectional electrical signal coupling (corticocardiac coherence) and communication (corticocardiac connectivity) between the brain and the heart are only identified in dying animals, they could be used as potential biomarkers to predict the risk of sudden death.

Amiodarone does not affect brain injury in a rat model of transient forebrain ischemia.

OBJECTIVES: Although amiodarone may cause neurotoxicity that can affect patient outcomes when used during cardiopulmonary resuscitation (CPR), it has been commonly prescribed during CPR. This study investigated the possible neurotoxic effects of amiodarone in a rat model of transient forebrain ischemia. DESIGN: A prospective laboratory animal study was carried out. SETTING: Animal laboratory. MATERIALS: Male Sprague-Dawley rats. INTERVENTION: Eight minutes of forebrain ischemia was induced in rats by bilateral carotid occlusion and hypotension (mean arterial pressure=35mmHg) under isoflurane (1.5%) anesthesia. Amiodarone (0, 50, 100 and 150mg/kg) with saline was injected intraperitoneally 10min after ischemia. Rats given 0mg/kg of amiodarone were used as saline-treated controls. Sham operated rats received no treatment. VARIABLES OF INTEREST: Animals were evaluated neurologically on postoperative days 4-7, and histologically after a one-week recovery period. RESULTS: The greatest improvement in water maze test performance corresponded to the sham operated group (p=0.015 vs. saline-treated controls). No differences in performance were seen in amiodarone-treated rats compared with saline-treated controls. In the control group, 45% of the CA1 hippocampal neurons survived, compared with 78% in the sham operated group (p=0.009). Neuron survival after ischemia in the amiodarone treatment groups (50, 100 and 150mg/kg) (58%, 40% and 36%, respectively) and in the control rats did not differ significantly. CONCLUSIONS: The administration of amiodarone immediately after transient forebrain ischemia did not worsen spatial cognitive function or neuronal survival in the hippocampal CA1 region in rats. The current results must be applied with caution in humans. However, they indicate that the potential neurotoxicity induced by amiodarone during resuscitation after cardiac arrest may be negligible.

Muscarinic M5 receptors trigger acetylcholine-induced Ca2+ signals and nitric oxide release in human brain microvascular endothelial cells.

Basal forebrain neurons control cerebral blood flow (CBF) by releasing acetylcholine (Ach), which binds to endothelial muscarinic receptors to induce nitric (NO) release and vasodilation in intraparenchymal arterioles. Nevertheless, the mechanism whereby Ach stimulates human brain microvascular endothelial cells to produce NO is still unknown. Herein, we sought to assess whether Ach stimulates NO production in a Ca2+ -dependent manner in hCMEC/D3 cells, a widespread model of human brain microvascular endothelial cells. Ach induced a dose-dependent increase in intracellular Ca2+ concentration ([Ca2+ ]i ) that was prevented by the genetic blockade of M5 muscarinic receptors (M5-mAchRs), which was the only mAchR isoform coupled to phospholipase Cß (PLCß) present in hCMEC/D3 cells. A comprehensive real-time polymerase chain reaction analysis revealed the expression of the transcripts encoding for type 3 inositol-1,4,5-trisphosphate receptors (InsP3 R3), two-pore channels 1 and 2 (TPC1-2), Stim2, Orai1-3. Pharmacological manipulation showed that the Ca2+ response to Ach was mediated by InsP3 R3, TPC1-2, and store-operated Ca2+ entry (SOCE). Ach-induced NO release, in turn, was inhibited in cells deficient of M5-mAchRs. Likewise, Ach failed to increase NO levels in the presence of l-NAME, a selective NOS inhibitor, or BAPTA, a membrane-permeant intracellular Ca2+ buffer. Moreover, the pharmacological blockade of the Ca2+ response to Ach also inhibited the accompanying NO production. These data demonstrate for the first time that synaptically released Ach may trigger NO release in human brain microvascular endothelial cells by stimulating a Ca2+ signal via M5-mAchRs.

Metabolomic study of altered energy metabolism during global forebrain ischemia and ischemic precoditioning in blood plasma in homocysteine treated rats.

Elevated homocysteine (Hcy) level is a well known risk factor for cardiovascular and neuropsychiatric diseases. In this study, we investigated metabolic changes in blood plasma in Hcy-treated rats. In combination with Hcy injections to induce hyperhomocysteinemia-like state, we used an animal model of global cerebral ischemia to investigate metabolic changes after 24 h reperfusion in rats. We also focused on the endogenous phenomenon known as ischemic tolerance induced by the preischemic treatment. The experiments were carried out on blood plasma samples as they are easily available and metabolically reflect the overall changes in injured organism. We observed significant changes in plasma metabolite levels of: pyruvate, citrate, acetate implicating alterations in energy metabolism, and increase in triacylglycerols, arginine and lysine, in Hcy-treated rats compared with naive animals. Ischemic insult with 24 reperfusion in Hcy-treated rats led to increase in plasma lactate, and decrease in plasma glucose, pyruvate, citrate and acetate. Complementary, an increase in ketone body 3-hydroxybutyrate was observed. The plasma metabolites: alanine, lactate, valine, glucose, leucine, isoleucine, acetate, citrate and 3-hydroxybutyrate were considered to reflect the response induced by ischemic preconditioning in Hcy rats, where the extent of postischemic damage was not as high as in the non-preconditioned rats. Our results provide evidence that nuclear magnetic resonance (NMR) spectra analysis can identify a specific group of metabolites present in plasma with the capability of discriminating between individual groups of animals. Regarding the effect of elevated Hcy level on plasma metabolome, we showed, that acetate, pyruvate and glucose had the excellent discriminatory power between Hcy-treated and naive rats plasma. Concerning ischemic insult in Hcy-treated animals, we also document the ideal discrimination of ischemic from non-ischemic rats by various groups of metabolites, that can be considered as a potential plasma biomarkers.

Spatiotemporal Progression of Microcalcification in the Hippocampal CA1 Region following Transient Forebrain Ischemia in Rats: An Ultrastructural Study.

Calcification in areas of neuronal degeneration is a common finding in several neuropathological disorders including ischemic insults. Here, we performed a detailed examination of the onset and spatiotemporal profile of calcification in the CA1 region of the hippocampus, where neuronal death has been observed after transient forebrain ischemia. Histopathological examinations showed very little alizarin red staining in the CA1 pyramidal cell layer until day 28 after reperfusion, while prominent alizarin red staining was detected in CA1 dendritic subfields, particularly in the stratum radiatum, by 14 days after reperfusion. Electron microscopy using the osmium/potassium dichromate method and electron probe microanalysis revealed selective calcium deposits within the mitochondria of degenerating dendrites at as early as 7 days after reperfusion, with subsequent complete mineralization occurring throughout the dendrites, which then coalesced to form larger mineral conglomerates with the adjacent calcifying neurites by 14 days after reperfusion. Large calcifying deposits were frequently observed at 28 days after reperfusion, when they were closely associated with or completely engulfed by astrocytes. In contrast, no prominent calcification was observed in the somata of CA1 pyramidal neurons showing the characteristic features of necrotic cell death after ischemia, although what appeared to be calcified mitochondria were noted in some degenerated neurons that became dark and condensed. Thus, our data indicate that intrahippocampal calcification after ischemic insults initially occurs within the mitochondria of degenerating dendrites, which leads to the extensive calcification that is associated with ischemic injuries. These findings suggest that in degenerating neurons, the calcified mitochondria in the dendrites, rather than in the somata, may serve as the nidus for further calcium precipitation in the ischemic hippocampus.

Homeostatic changes in neuronal network oscillations in response to continuous hypoperfusion in the mouse forebrain.

Neuronal activity is highly sensitive to changes in oxygen tension. In this study, we examined the impact of hypoxic/ischemic conditions on neuronal ensemble activity patterns in the mouse brain using in vivo extracellular electrophysiological recordings from up to 8 sites in the thalamus, dorsal hippocampus, and neocortex, while cerebral hypoperfusion was induced by unilateral carotid artery occlusion. After a few minutes, the occlusion triggered a rapid change in the power of the local field oscillations. In the hippocampus, but not in the neocortex, the absolute power changes at all frequency ranges (relative to the baseline) became less pronounced with time, and no significant changes were observed 30min after the occlusion-induced hypoperfusion. We also tested whether continuous hypoperfusion induced by the occlusion for up to 1 week alters neuronal activity. In the hippocampus and the thalamus, the chronic occlusion did not lead to a reduction in the power of the local field oscillations. These results indicate that certain neuronal populations have the ability to maintain internal neurophysiological homeostasis against continuous hypoperfusion.

Metformin pretreatment enhanced learning and memory in cerebral forebrain ischaemia: the role of the AMPK/BDNF/P70SK signalling pathway.

Context Metformin induced AMP-activated protein kinase (AMPK) and protected neurons in cerebral ischaemia. Objective This study examined pretreatment with metformin and activation of AMPK in molecular and behavioral levels associated with memory. Materials and methods Rats were pretreated with metformin (200 mg/kg) for 2 weeks and 4-vessels occlusion global cerebral ischaemia was induced. Three days after ischaemia, memory improvement was done by passive avoidance task and neurological scores were evaluated. The amount of Brain-Derived Neurotropic Factor (BDNF) and phosphorylated and total P70S6 kinase (P70S6K) were measured. Results Pretreatment with metformin (met) in the met + ischaemia/reperfusion (I/R) group reduced latency time for enter to dark chamber compared with the sham group (p < 0.001) and increased latency time compared with the I/R group (p < 0.001). Injection of Compound C (CC) (as an AMPK inhibitor) concomitant with metformin reduced latency time in I/R rats compared with the I/R + met group (p < 0.05). Neurological scores were reduced in met treated rats compared with the sham group. Pretreatment with metformin in I/R animals reduced levels of pro-BDNF compared with the I/R group (p < 0.001) but increased that compared with the sham group (p < 0.001). The level of pro-BDNF decreased in the met + CC + I/R group compared with the met + I/R group (p < 0.01). Pretreatment with metformin in I/R animals significantly increased P70S6K compared with the I/R group (p < 0.001). Conclusion Short-term memory in ischaemic rats treated with metformin increased step-through latency; sensory-motor evaluation was applied and a group of ischaemia rats that were pretreated with metformin showed high levels of BDNF, P70S6K that seemed to be due to increasing AMPK.

Highly selective non-opioid kappa opioid receptor (KOR) agonist salvinorin A protects against forebrain ischemia-induced brain injury in rats.

OBJECTIVE: To investigate the effect of salvinorin A (SA) on brain injury and neurologic function post-brain ischemia/reperfusion (I/R) using a rat forebrain ischemia model and further explore the effect of kappa opioid receptor (KOR) inhibition by SA on aquaporin-4 (AQP4) expression in the hippocampus, cortex and striatum in the forebrain. METHODS: A forebrain ischemia model was established by colligating the bilateral common carotid arteries of SD rats for 10 min. The rats were randomized to receive dimethyl sulfoxide (DMSO), SA (1 µg/100g body weight) or SA (onset of ischemia) plus SA antagonist nor-BIN (0.2 mg/100g body weight. Rat brain water content was measured. Apoptotic neurons in the hippocampal CA1 region, cortex and striatum were enumerated. AQP4 in CA1, the cortex and the striatum were determined by immunoblotting assays and immunohistochemistry at 24h post-ischemia. Neuromotor tests were performed on day 1, 2 and 5 post-ischemia. Water maze test was carried out on the 5th post-ischemia day. RESULTS: SA significantly attenuated I/R-induced increase in brain water content. Our immunoblotting assays and immunohistochemistry further revealed that SA effectively lessened I/R-induced upregulation of AQP4 expression in the hippocampus, cortex and striatum 24h post-ischemia. SA also significantly reduced the percentage of dead and apoptotic neurons in these regions compared to DMSO. Moreover, SA partially reversed I/R-induced decline in rat motor function and cognition. The neuroprotective effects of SA were partially abolished by nor-BIN. CONCLUSION: SA protects against I/R-induced brain injury by attenuating brain edema formation and inhibiting neuronal death and improves neurologic recovery of rats post-I/R.

Inhibition of mTOR Pathway by Rapamycin Reduces Brain Damage in Rats Subjected to Transient Forebrain Ischemia.

The aims of this study are to clarify the role of mTOR in mediating cerebral ischemic brain damage and the effects of rapamycin on ischemic outcomes. Ten minutes of forebrain ischemia was induced in rats, and their brains were sampled after 3 h, 16 h, and 7 days reperfusion for histology, immunohistochemistry and biochemical analysis. Our data demonstrated that cerebral ischemia resulted in both apoptotic and necrotic neuronal death; cerebral ischemia and reperfusion led to significant increases of mRNA and protein levels of p-mTOR and its downstream p-P70S6K and p-S6; elevation of LC3-II, and release of cytochrome c into the cytoplasm in both the cortex and hippocampus. Inhibition of mTOR by rapamycin markedly reduced ischemia-induced damage; suppressed p-Akt, p-mTOR, p-P70S6K and p-S6 protein levels; decreased LC3-II and Beclin-1; and prevented cytochrome c release in the two structures. All together, these data provide evidence that cerebral ischemia activates mTOR and autophagy pathways. Inhibition of mTOR deactivates the mTOR pathway, suppresses autophagy, prevents cytochrome c release and reduces ischemic brain damage.

Association between respiratory and postural adaptations and self-perception of school-aged children with mouth breathing in relation to their quality of life

Objective: To investigate the respiratory and postural adaptations associated with mouth and nasal breathing and to evaluate the associations of such adaptations in mouth breathers' self-perceived quality of life. Method: Cross-sectional study with mouth breathers (initial n=116 and final n=48) and nasal breathers (initial n=131 and final n=24) from elementary school, aged between 7 and 14 years. Chest expansion, using cirtometry, the breathing pattern and the use of accessory muscles, by means of clinical evaluations and photogrammetry, and flexibility tests were evaluated in both groups. Subsequently, the mouth breathers were asked to complete the quality of life questionnaire. Statistical tests: Chi-square, odds ratio, Mann-Whitney, and binomial tests were first applied followed by logistic regressions. Results: Thoracic breathing (p=0.04), using of accessory muscles (p=0.03) and reductions in flexibility (p=0.001) increased the chances of an individual being a mouth breather when compared to nasal breathers. Subsequently, using of accessory muscles decreased the chances of snoring among mouth breathers (p=0.03); the presence of shoulder asymmetry reduced the chances of experiencing quiet sleep (p=0.05) and increased the chances of coughing or being tired when playing or running (p=0.008). Finally, forward head position reduced the chances of waking up at night (p=0.04) and experiencing shortness of breath (p=0.05). Conclusions: Respiratory and postural adaptations increased the chances of individuals persisting with mouth breathing. Additionally, these adaptations could be associated with mouth breathers' self-perceived quality of life. .

Tat-antioxidant 1 protects against stress-induced hippocampal HT-22 cells death and attenuate ischaemic insult in animal model.

Oxidative stress-induced reactive oxygen species (ROS) are responsible for various neuronal diseases. Antioxidant 1 (Atox1) regulates copper homoeostasis and promotes cellular antioxidant defence against toxins generated by ROS. The roles of Atox1 protein in ischaemia, however, remain unclear. In this study, we generated a protein transduction domain fused Tat-Atox1 and examined the roles of Tat-Atox1 in oxidative stress-induced hippocampal HT-22 cell death and an ischaemic injury animal model. Tat-Atox1 effectively transduced into HT-22 cells and it protected cells against the effects of hydrogen peroxide (H2O2)-induced toxicity including increasing of ROS levels and DNA fragmentation. At the same time, Tat-Atox1 regulated cellular survival signalling such as p53, Bad/Bcl-2, Akt and mitogen-activate protein kinases (MAPKs). In the animal ischaemia model, transduced Tat-Atox1 protected against neuronal cell death in the hippocampal CA1 region. In addition, Tat-Atox1 significantly decreased the activation of astrocytes and microglia as well as lipid peroxidation in the CA1 region after ischaemic insult. Taken together, these results indicate that transduced Tat-Atox1 protects against oxidative stress-induced HT-22 cell death and against neuronal damage in animal ischaemia model. Therefore, we suggest that Tat-Atox1 has potential as a therapeutic agent for the treatment of oxidative stress-induced ischaemic damage.

Ultrasound-microbubble transplantation of bone marrow stromal cells improves neurological function after forebrain ischemia in adult mice.

In this study, bone marrow stromal cells (MSCs) were transplanted into the brain of adult rats after forebrain ischemia induced by 4VO. SD rats (n = 60) were randomly divided into three groups: (I) rats (n = 20) were subjected to 4VO and transplanted with MSCs into the ischemic region using ultrasound-microbubble method, (2) rats (n = 20) were subjected to 4VO and transplanted with MSCs into the ischemic region (n = 20), and (3) 4VO alone (n = 20). Rats were sacrificed 28 days after treatment. Neurological functions of rats were evaluated by Morris Water Maze. The current findings suggest that the ultrasound microbubble transplanted MSCs survived in the ischemic brain and significantly improved functional recovery of adult rats compared to regular transplantation.

Isolation and culture of endothelial cells from the embryonic forebrain.

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1 coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.