Ischemic Rat Brain Research Articles

EP 2 receptor activation by prostaglandin E 2 leads to induction of HO-1 via PKA and PI3K pathways in C6 cells

Recently we proposed that COX-2 induction precedes expression of HO-1 in ischemic preconditioned rat brain. In the current study, we investigated the molecular mechanism by which prostaglandin E 2, one of COX-2 metabolites, induces HO-1 in rat C6 brain cells. We demonstrated that concentration of PGE 2 increased HO-1 expression in C6 cells in vitro. The effects of PGE 2 were mimicked by PGE 2 receptor EP 2 agonists, 11-deoxy PGE 2, and cAMP analog, dibutyl-cAMP. HO-1 expression by PGE 2 was inhibited by LY294002, PI3K inhibitor and H89, PKA inhibitor. The EP 2-specific antagonist, AH8006 also inhibited PGE 2-mediated HO-1 expression in a concentration-dependent manner. Finally, PGE 2 inhibited GOX-induced apoptosis as assayed by FACS analysis or DNA strand breaks assay, and this cell death was reversed by ZnPPIX, HO-1 inhibitor. In addition to HO-1 induction, PGE 2 also increased phosphorylation of Bad by PKA- and PI3K-depednent manner. Taken together, we conclude that PGE 2 induces HO-1 protein expression through PKA and PI3K signaling pathways via EP 2 receptor in C6 cells. The induction of HO-1 along with increase of p-Bad by PGE 2 is responsible for anti-apoptosis against oxidant stress.

Upregulation of Macrophage Migration Inhibitory Factor Gene Expression in Stroke

MIF has been implicated to function in many inflammatory processes. This study examined whether MIF expression was affected in stroke and its underlying molecular mechanism. ELISA and qRT-PCR were used to detect MIF protein and mRNA in PBMCs from stroke patients, the ischemic rat brains, and controls. A MIF promoter assay under hypoxia was performed. MIF protein and mRNA were significantly increased in stroke patients. Increasing levels of MIF were correlated to the severity of stroke and peaked 24 hours after stroke. MIF was significantly upregulated in focal ischemic rat brains. The activity of the human MIF promoter was significantly increased under hypoxia compared to normoxia. MIF gene expression is upregulated after stroke, and hypoxia signaling plays an important role in upregulation of MIF expression under stroke.

Panax notoginseng Attenuates the Infarct Volume in Rat Ischemic Brain and the Inflammatory Response of Microglia

The roots of Panax notoginseng (PN) are commonly used as a therapeutic agent to stop hemorrhage and as a tonic to promote health in traditional Korean medicine. Stroke triggers an inflammatory response that not only plays a central role in the pathogenesis of cerebral ischemia, but also induces secondary damage. This study was designed to investigate the neuroprotective effects of the methanol extract of PN on the infarct volume induced by middle cerebral artery occlusion (MCAO) (90-min occlusion and 24-h reperfusion) in rat brains. The PN extract (50 mg/kg, i.p.) was administered 2 h after the onset of MCAO. The PN-treated groups had a reduction in infarct volume by 23.82 ± 8.9%. In the PN extract–treated groups, the microglial density was significantly decreased in the peri-infarct region; the underlying mechanism was inhibition of inflammatory mediators, such as inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2, via blocking of the NF-κB pathway. Furthermore, in vitro studies showed that the PN extract significantly reduced the production of iNOS-derived NO and COX-2– derived prostaglandin E2 through the regulation of gene transcription levels in primary microglia and BV-2 cells. These results suggest that anti-inflammatory and microglial activation inhibitory effects of the PN extract may contribute to its neuroprotective effects in brain ischemia.

X-Linked Inhibitor of Apoptosis Protein Expression After Ischemic Injury in the Human and Rat Developing Brain

X-linked inhibitor of apoptosis protein (XIAP) is a potent suppressor of neuronal death. The aim of this study was to investigate the expression of XIAP after ischemia in the human and rat developing brain. Autopsy specimens from 19 children with neuropathologic diagnosis of focal cerebral ischemic infarct were processed immunohistochemically for XIAP expression. XIAP positive cells were compared in pathologically classified acute (1-4 d), subacute (5-30 d), and chronic (months) strokes vs. age-matched controls with normal brain histology. For the animal studies, ischemia was induced in 1-wk-old rats by unilateral carotid artery occlusion and transient hypoxia. XIAP expression was quantified at four time points after ischemia in the infarct core and peri-infarct area. Neuronal XIAP expression was higher in the penumbra of subacute human infarcts compared with controls (p < 0.05). XIAP expression in the peri-infarct of rat pup was highest at 7 d postischemic injury (p < 0.05). The increase in XIAP expression was associated with a reduction in activated caspase-3 in ischemic neonatal rat brain. Our results demonstrate that XIAP expression postischemic injury is delayed in both species and may continue for several days. Therefore, potentiation of XIAP expression may be neuroprotective in the developing brain.

Investigation of relationships between transverse relaxation rate, diffusion coefficient, and labeled cell concentration in ischemic rat brain using MRI

MRI has been used to evaluate labeled cell migration and distribution. However, quantitative determination of labeled cell concentration using MRI has not been systematically investigated. In the current study, we investigated the relationships between labeled cell concentration and MRI parameters of transverse relaxation rate, R(2), and apparent diffusion coefficient (ADC), in vitro in phantoms and in vivo in rats after stroke. Significant correlations were detected between iron concentration or labeled cell concentration and MRI measurements of R(2), ADC, and ADC x R(2) in vitro. In contrast, in vivo labeled cell concentration did not significantly correlate with R(2), ADC, and ADC x R(2). A major factor for the absence of a significant correlation between labeled cell concentration and MRI measurements in vivo may be attributed to background effects of ischemic tissue. By correcting the background effects caused by ischemic damage, DeltaR(2) (difference in R(2) values in the ischemic tissue with and without labeled cells) exhibited a significant correlation to labeled cell concentration. Our study suggests that MRI parameters have the potential to quantitatively determine labeled cell concentration in vivo.

Edaravone, a Free Radical Scavenger, Inhibits MMP-9–Related Brain Hemorrhage in Rats Treated With Tissue Plasminogen Activator

Intracerebral hemorrhage, induced by recombinant tissue plasminogen activator (rtPA) in ischemic stroke, is attributable to the increased activity of matrix metalloproteinase-9 (MMP-9). Patients with acute infarct benefit from the neuroprotective drug edaravone, a free radical scavenger. We examined the mechanisms by which edaravone may help to suppress rtPA-induced brain hemorrhage. Male Wistar rats weighing 250 to 280 g were subjected to 3-hour transient middle cerebral artery occlusion (MCAO) and divided randomly into 3 groups. Immediately after reperfusion, 1 group was intravenously injected with 10 mg/kg rtPA, another with rtPA plus 3 mg/kg edaravone, and the 3rd group received no treatment. We assessed the hemorrhage volume and the activity of MMP-9 in the brain 24 hours postischemia. We also studied the activity of MMP-9, its mRNA expression, and nuclear factor-kappa B (NF-kappaB) activity in rtPA-stimulated human microvascular endothelial cells (HBECs). The degree of hemorrhage and the level of endothelial cell-derived MMP-9 were elevated in rats treated with rtPA alone and attenuated in rats treated with rtPA plus edaravone. In rtPA-stimulated HBECs, edaravone suppressed the activity and mRNA expression of MMP-9 in a dose-dependent manner. Edaravone also inhibited NF-kappaB activation. We demonstrate that edaravone inhibits rtPA-induced cerebral hemorrhage in the ischemic brain of rats via the inhibition of MMP-9 expression in vivo, which is substantiated by inhibition of MMP-9 expression and NF-kappaB activation in HBECs. Edaravone may render thrombolytic therapy safer for the administration of rtPA in patients with ischemic stroke.

Transient global ischemia in rat brain promotes different NMDA receptor regulation depending on the brain structure studied

The mRNA expression of the major subunits of N-methyl- d-aspartate receptors (NR1, NR2A and NR2B) following ischemia–reperfusion was studied in structures with different vulnerabilities to ischemic insult in the rat brain. The study was performed using quantitative real-time PCR on samples from 3-month-old male Sprague–Dawley rats after global transient forebrain ischemia followed by 48 h of reperfusion. Expression of NMDA receptor subunits mRNAs decreased significantly in all structures studied in the injured animals as compared to the sham-operated ones. The hippocampal subfields (CA1, CA3 and dentate gyrus) as well as the caudate-putamen, both reported to be highly ischemic-vulnerable structures, showed outstandingly lower mRNA levels of NMDA receptor subunits than the cerebral cortex, which is considered a more ischemic-resistant structure. The ratios of the mRNA levels of the different subunits were analyzed as a measure of the NMDA receptor expression pattern for each structure studied. Hippocampal areas showed changes in NMDA receptor expression after the insult, with significant decreases in the NR2A with respect to the NR1 and NR2B subunits. Thus, the NR1:NR2A:NR2B (1:1:2) ratios observed in the sham-operated animals became (2:1:4) in insulted animals. This modified expression pattern was similar in CA1, CA3 and the dentate gyrus, in spite of the different vulnerabilities reported for these hippocampal areas. In contrast, no significant differences in the expression pattern were observed in the caudate-putamen or cerebral cortex on comparing the sham-operated animals with the ischemia-reperfused rats. Our results support the notion that the regulation of NMDA receptor gene expression is dependent on the brain structure rather than on the higher or lower vulnerability of the area studied.

Protective Effects of Scutellarin Against Cerebral Ischemia in Rats: Evidence for Inhibition of the Apoptosis-Inducing Factor Pathway

Scutellarin (Scu) is the major active principle (flavonoid) extracted from Erigeron breviscapus (Vant.) Hand-Mazz, a Chinese herbal medicine. In this paper, we investigated the effects of Scu on brain injury through the inhibition of AIF-mediated apoptosis induced by transient focal brain ischemia in rats. Rats were treated with Scu for 7 d and then subjected to cerebral ischemia/reperfusion (I/R) injury induced by a middle cerebral artery occlusion (MCAO). After 2 h of ischemia and 22 h of reperfusion, the infarct volume and the neurological deficit were determined by TTC staining and Longa's score. IN SITU end-labeling of nuclear DNA fragments (TUNEL) was employed to determine the degree of DNA fragmentation. NAD content and PARP activity in brain homogenate were determined. The expression of AIF in the nucleus was analyzed by Western blot. The present study showed that Scu significantly reduced the infarct volume and ameliorated the neurological deficit. An increase in the number of TUNEL-positive cells and a decrease in the NAD level were also observed after 2 h of ischemia and 22 h of reperfusion. At the same time, Scu (50 and 75 mg kg (-1), i. g.) treatment reversed brain NAD depletion and reduced DNA fragmentation. Scu also inhibited PARP overactivation and AIF translocation from the mitochondria to the nucleus following cerebral I/R. These findings suggested that the neuroprotective effects of Scu on brain ischemic injury-induced apoptosis might be associated with inhibition of PARP-dependent mitochondrial dysfunction and subsequent translocation of AIF.

Vagus nerve mediates the protective effects of melanocortins against cerebral and systemic damage after ischemic stroke.

A vagus nerve-mediated, efferent cholinergic protective pathway activated by melanocortins is operative in circulatory shock and myocardial ischemia. Moreover, melanocortins have neuroprotective effects against brain damage after ischemic stroke. Here we investigated cerebral and systemic pathophysiologic reactions to focal cerebral ischemia in rats induced by intrastriatal microinjection of endothelin-1, and the possible protective role of the melanocortin-activated vagal cholinergic pathway. In the striatum and liver of saline-treated control rats, the activation of extracellular signal-regulated kinases, c-jun N-terminal kinases, and caspase-3, the increase in tumor necrosis factor-alpha (TNF-alpha) concentration and DNA fragmentation, as well as the increase in TNF-alpha plasma levels, occurred 10 and 20 h after the ischemic insult suggesting an activation of inflammatory and apoptotic responses. Treatment with [Nle(4), D-Phe(7)]alpha-melanocyte-stimulating hormone (NDP-alpha-MSH; 3 or 9 h after stroke) suppressed the inflammatory and apoptotic cascades at central and peripheral level. Bilateral vagotomy and pharmacologic blockade of peripheral nicotinic acetylcholine receptors blunted the protective effect of NDP-alpha-MSH. The present results show that focal brain ischemia in rats causes significant effects not only in the brain, but also in the liver. Moreover, our data support the hypothesis that a protective, melanocortin-activated, vagal cholinergic pathway is likely operative in conditions of ischemic stroke.

The obligatory role of COX-2 expression for induction of HO-1 in ischemic preconditioned rat brain

The molecular mechanisms of preconditioning-induced ischemic tolerance (PCIT) have yet to be elucidated. We investigated whether minimal expression levels of COX-2 induced by preconditioning trigger HO-1, thereby inducing the synthesis of cytoprotective proteins. We show that both COX-2 and HO-1 are induced in rat brains subjected to preconditioning by middle cerebral artery (MCA) occlusion for 10min followed by different amounts of reperfusion time (1–24h). Although preconditioning significantly reduced the brain infarct size against severe ischemia (24h MCA occlusion), pretreatment with the COX-2-selective inhibitor rofecoxib increased infarct size and abolished PCIT-induced COX-2 and HO-1 expression in vivo. We also found that PGE2 increased the phosphorylation of Akt, which was significantly inhibited by the PI3 kinase inhibitor LY294002. Taken together, we conclude that the kinetic changes in COX-2 induction during the reperfusion period following preconditioning may be important for ischemic tolerance.

Effects of Electroacupuncture on Expressions of Angiogenesis Factors and Anti-angiogenesis Factors in Brain of Experimental Cerebral Ischemic Rats after Reperfusion

To explore the mechanism of electroacupuncture (EA) in improving ischemic stroke. The Wistar rat model of focal cerebral ischemia-reperfusion was prepared by the thread embolism method. The rats were randomly divided into a normal group, a model group, and an EA group. EA was given at bilateral "Hegu" (LI 4) in rats of the EA group. Expression of the vascular endothelial growth factor (VEGF) mRNA was detected with hybridization in situ, and expressions of the angiogenin-1 (Ang-1) and endostatin proteins were detected with the immunohistochemical method. As compared with the normal group, the expressions of VEGF mRNA, Ang-1 protein and endostatin protein significantly increased in the model group (all P < 0.05); and when compared with the model group, the EA group showed even more significant increase in expressions of the VEGF mRNA and Ang-1 protein (both P < 0.05), but with an obvious decline in the increase of expression of endostatin protein (P < 0.05). EA can promote angiogenesis in brain of experimental cerebral ischemic rats after reperfusion probably through up-regulating the expression of angiogenesis factors and down-regulating the expression of anti-angiogenesis factors.

Kynurenine diminishes the ischemia-induced histological and electrophysiological deficits in the rat hippocampus

The neuroprotective effect of l-kynurenine sulfate (KYN), a precursor of kynurenic acid (KYNA, a selective N-methyl- d-aspartate receptor antagonist), was studied. KYN (300 mg/kg i.p., applied daily for 5 days) appreciably decreased the number of injured pyramidal cells from 1850 ± 100/mm 2 to 1000 ± 300/mm 2 ( p < 0.001) in the CA1 region of the hippocampus in the four-vessel occlusion (4VO)-induced ischemic adult rat brain. A parallel increase in the number of intact, surviving neurons was demonstrated. Post-treatment with KYN (applied immediately right after reperfusion) proved to be much less effective. In parallel with the histology, a protective effect of KYN on the functioning of the CA1 region was observed: long-term potentiation was abolished in the 4VO animals, but its level and duration were restored by pretreatment with KYN. It is concluded that the administration of KYN elevates the KYNA concentration in the brain to neuroprotective levels, suggesting its potential clinical usefulness for the prevention of neuronal loss in neurodegenerative diseases.

Curcuma Oil: Reduces Early Accumulation of Oxidative Product and is Anti-apoptogenic in Transient Focal Ischemia in Rat Brain

Curcuma Oil: Reduces Early Accumulation of Oxidative Product and is Anti-apoptogenic in Transient Focal Ischemia in Rat Brain

Down-regulation of neurocan expression in reactive astrocytes promotes axonal regeneration and facilitates the neurorestorative effects of bone marrow stromal cells in the ischemic rat brain.

The glial scar, a primarily astrocytic structure bordering the infarct tissue inhibits axonal regeneration after stroke. Neurocan, an axonal extension inhibitory molecule, is up-regulated in the scar region after stroke. Bone marrow stromal cells (BMSCs) reduce the thickness of glial scar wall and facilitate axonal remodeling in the ischemic boundary zone. To further clarify the role of BMSCs in axonal regeneration and its underlying mechanism, the current study focused on the effect of BMSCs on neurocan expression in the ischemic brain. Thirty-one adult male Wistar rats were subjected to 2 h of middle cerebral artery occlusion followed by an injection of 3 x 10(6) rat BMSCs (n = 16) or phosphate-buffered saline (n = 15) into the tail vein 24 h later. Animals were sacrificed at 8 days after stroke. Immunostaining analysis showed that reactive astrocytes were the primary source of neurocan, and BMSC-treated animals had significantly lower neurocan and higher growth associated protein 43 expression in the penumbral region compared with control rats, which was confirmed by Western blot analysis of the brain tissue. To further investigate the effects of BMSCs on astrocyte neurocan expression, single reactive astrocytes were collected from the ischemic boundary zone using laser capture microdissection. Neurocan gene expression was significantly down-regulated in rats receiving BMSC transplantation (n = 4/group). Primary cultured astrocytes showed similar alterations; BMSC coculture during reoxygenation abolished the up-regulation of neurocan gene in astrocytes undergoing oxygen-glucose deprivation (n = 3/group). Our data suggest that BMSCs promote axonal regeneration by reducing neurocan expression in peri-infarct astrocytes.

Implantation of olfactory ensheathing cells promotes neuroplasticity in murine models of stroke

Murine olfactory ensheathing cells (OECs) promote central nervous system axonal regeneration in models of spinal cord injury. We investigated whether OECs could induce a neuroplastic effect to improve the neurological dysfunction caused by hypoxic/ischemic stress. In this study, human OECs/olfactory nerve fibroblasts (hOECs/ONFs) specifically secreted trophic factors including stromal cell-derived factor-1alpha (SDF-1alpha). Rats with intracerebral hOEC/ONF implantation showed more improvement on behavioral measures of neurological deficit following stroke than control rats. [18F]fluoro-2-deoxyglucose PET (FDG-PET) showed increased glucose metabolic activity in the hOEC/ONF-treated group compared with controls. In mice, transplanted hOECs/ONFs and endogenous homing stem cells including intrinsic neural progenitor cells and bone marrow stem cells colocalized with specific neural and vascular markers, indicating stem cell fusion. Both hOECs/ONFs and endogenous homing stem cells enhanced neuroplasticity in the rat and mouse ischemic brain. Upregulation of SDF-1alpha and CXCR4 in hOECs/ONFs promoted neurite outgrowth of cocultured primary cortical neurons under oxygen glucose deprivation conditions and in stroke animals through upregulation of cellular prion protein (PrP C) expression. Therefore, the upregulation of SDF-1alpha and the enhancement of CXCR4 and PrP C interaction induced by hOEC/ONF implantation mediated neuroplastic signals in response to hypoxia and ischemia.

Imaging of the inflammatory response in reperfusion injury after transient cerebral ischemia in rats: correlation of superparamagnetic iron oxide-enhanced magnetic resonance imaging with histopathology

Acute inflammatory responses have been thought to play a central role in ischemia-reperfusion injury after acute ischemic stroke. Superparamagnetic iron oxide (SPIO) particles have been known to enable in-vivo monitoring of macrophage infiltration by magnetic resonance imaging (MRI) in the experimental ischemic rat brain. To determine whether the accumulation of macrophages could be seen in vivo in a reperfusion animal model after focal cerebral ischemia using SPIO-enhanced MRI. Thirty-four adult male rats were enrolled in this study. SPIO particles were injected into the rats at different time points after 1-hour transient occlusion of the middle cerebral artery, and three-dimensional (3D) T2*-weighted magnetic resonance (MR) images with a gradient-echo sequence were performed 24 hours later. Histochemical iron staining was compared with T2* signal abnormalities. At days 3 and 4 post-reperfusion, focal areas of signal loss indicating local accumulation of SPIO particles appeared in a part of the damaged brain. Areas of signal loss corresponded to local accumulation of iron-laden macrophages in histologic sections, and SPIO-induced signal loss indicated active macrophage transmigration into the reperfused brain. SPIO-enhanced MRI demonstrated through in-vivo monitoring that macrophages participate in reperfusion injury at early stages of injury development. SPIO-enhanced MRI could be a useful tool to examine the inflammatory mechanisms involved in reperfusion brain injury.

Comparison between single and combined treatment with candesartan and pioglitazone following transient focal ischemia in rat brain

Angiotensin AT1 receptor blockers (ARBs) and thiazolidinediones (TZDs) have become well established drugs for the treatment of major risk factors of stroke. Since several studies provided evidence that ARBs and TZDs also have additional anti-inflammatory effects, we hypothesized that a combined treatment with the ARB, candesartan, and the TZD, pioglitazone, ameliorates ischemia-induced brain injury and inflammation by synergistic anti-inflammatory actions. Normotensive Wistar rats were pre-treated for 5 days with vehicle (0.9% NaCl), 0.2 mg/kg/day candesartan (s.c.), and/or 2 and/or 20 mg/kg/day pioglitazone (p.o.), respectively and underwent 90 min of middle cerebral artery occlusion (MCAO) with successive reperfusion. Neurological deficits and infarct size were determined 24 h and 48 h after MCAO, respectively, followed by tissue sampling. Animals treated with candesartan, pioglitazone, and the combination of candesartan and pioglitazone had reduced neurological deficits 24 h and 48 h after MCAO, respectively ( P < 0.05–0.01). Infarct size was reduced by treatment of candesartan, pioglitazone, and their respective combination (each P < 0.05) 48 h after stroke compared to vehicle. Treatment with candesartan, pioglitazone, and their combination resulted in significantly reduced mRNA expression of the inflammatory markers CXCL1 and TNFα in vivo ( P < 0.01). The combination of candesartan plus pioglitazone is equally effective compared to their single applications concerning neuroprotection and attenuation of inflammation after MCAO. Therefore, we conclude that a direct synergistic neuroprotective action of parallel ARB and TZD treatment is unlikely.

Identification of neuronal outgrowth cells from peripheral blood of stroke patients

Recent studies have identified a subset of outgrowth cell population with endothelial phenotype in long-term cultures of peripheral blood mononuclear cells. The concept that peripheral blood-derived cells participate in neuronal regeneration remains highly controversial, and no specific cell type has been identified. In this study, we undertook to characterize outgrowth cells in the peripheral blood culture from stroke patients. Mononuclear cells were isolated from the peripheral blood of 30 acute stroke patients, 20 risk factor-only subjects, and 20 healthy volunteers. The isolation frequency of outgrowth cells was measured during the 2 months of culture. The outgrowth cells were characterized by in vitro cultures and in vivo model of transplantation into the ischemic rat brain. Outgrowth cells could be more efficiently isolated from stroke patients (80%) than risk factor-only (30%) and healthy groups (10%). Outgrowth cells were more detected in the patients with greater National Institute of Health Stroke Scale scores (p = 0.023). They exhibited heterogenous populations with different morphologies, for example, cobblestone, palisading, or branching features. Two different types of outgrowth cells were identified: endothelial; neuronal, according to their morphological characteristics; and protein or gene expression profiles. The transplanted neuronal outgrowth cells survived in the ischemic rat brains over 6 months after transplantation. Targeted migration of the transplanted cells was seen in the ischemic brains with phenotypes of neuronal phenotypes. The feasibility of extracting and culturing neuronal outgrowth cells in large numbers suggests that such autologous cells will be useful for applications ranging from basic research to cell-based therapy.

Additive neuroprotection of GABA A and GABA B receptor agonists in cerebral ischemic injury via PI-3K/Akt pathway inhibiting the ASK1-JNK cascade

Co-activation of GABA A and GABA B receptors results in neuroprotection during in vitro ischemia. However, it is unclear whether this mode of action is responsible for its neuroprotective effects in animal models of ischemia in vivo, and the precise mechanisms are also unknown. This study compared the neuroprotective efficacies of muscimol, a GABA A receptor agonist, and a GABA B receptor agonist baclofen in rat brain ischemia. The additive neuroprotection could be obtained in the hippocampal CA1 pyramidal cells prominently when muscimol and baclofen were co-applied. In particular, our study showed that co-activation of GABA A and GABA B receptors could strongly increase Akt activation and inhibit ASK1 activation by phosphorylation of serine 83 of ASK1. PI-3K inhibitor LY294002 reversed the increasing Akt activation and ASK1 (S83) phosphorylation. Moreover, MKK4/MKK7-JNK signaling activation was inhibited during ischemia/reperfusion (I/R) by co-treatment of muscimol with baclofen. JNK substrate, Bcl-2 and c-jun phosphorylation were also attenuated. Our results indicated that co-activation of GABA A receptor and GABA B receptor exerted neuroprotective effect via PI-3K/Akt pathway, which could inhibit the ASK1-c-Jun N-terminal protein kinase (JNK) cascade.

Adherent Self-Renewable Human Embryonic Stem Cell-Derived Neural Stem Cell Line: Functional Engraftment in Experimental Stroke Model

BackgroundHuman embryonic stem cells (hESCs) offer a virtually unlimited source of neural cells for structural repair in neurological disorders, such as stroke. Neural cells can be derived from hESCs either by direct enrichment, or by isolating specific growth factor-responsive and expandable populations of human neural stem cells (hNSCs). Studies have indicated that the direct enrichment method generates a heterogeneous population of cells that may contain residual undifferentiated stem cells that could lead to tumor formation in vivo.Methods/Principal FindingsWe isolated an expandable and homogenous population of hNSCs (named SD56) from hESCs using a defined media supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and leukemia inhibitory growth factor (LIF). These hNSCs grew as an adherent monolayer culture. They were fully neuralized and uniformly expressed molecular features of NSCs, including nestin, vimentin and radial glial markers. These hNSCs did not express the pluripotency markers Oct4 or Nanog, nor did they express markers for the mesoderm or endoderm lineages. The self-renewal property of the hNSCs was characterized by a predominant symmetrical mode of cell division. The SD56 hNSCs differentiated into neurons, astrocytes and oligodendrocytes throughout multiple passages in vitro, as well as after transplantation. Together, these criteria confirm the definitive NSC identity of the SD56 cell line. Importantly, they exhibited no chromosome abnormalities and did not form tumors after implantation into rat ischemic brains and into naïve nude rat brains and flanks. Furthermore, hNSCs isolated under these conditions migrated toward the ischemia-injured adult brain parenchyma and improved the independent use of the stroke-impaired forelimb two months post-transplantation.Conclusions/SignificanceThe SD56 human neural stem cells derived under the reported conditions are stable, do not form tumors in vivo and enable functional recovery after stroke. These properties indicate that this hNSC line may offer a renewable, homogenous source of neural cells that will be valuable for basic and translational research.