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Interleukin-6 (IL-6) is a proinflammatory cytokine known to modulate neurogenesis. We presently evaluated neural progenitor proliferation, survival, and phenotypic maturation in the hippocampal dentate gyrus, subventricular zone, and the posterior periventricle in the brains of IL-6 knockout mice and their wild-type littermates. In all the three neurogenic regions of the IL-6 mice there was a significant decrease in the number of 5-bromo-2-deoxyuridine positive (BrdU) proliferating progenitors compared with the IL-6 mice. The IL-6 mice also showed a significantly lower progenitor cell survival in the dentate gyrus and subventricular zone compared with the IL-6 mice. In conclusion, a complete lack of IL-6 might be detrimental to neurogenesis in the adult brain.

J. Neurochem. (2011) 116, 499–507. Abstract One of the limiting factors in stroke therapeutic development is the use of animal models that do not well represent the underlying medical conditions of patients. In humans, diabetes increases the risk of stroke incidence as well as post-stroke mortality. To understand the mechanisms that render diabetics to increased brain damage, we evaluated the effect of transient middle cerebral artery occlusion in adult db/db mice. The db/db mouse is a model of type-2 diabetes with four times higher blood sugar than its normoglycemic genetic control(db/+ mouse). Following transient middle cerebral artery occlusion, the db/db mice showed significantly higher mortality, bigger infarcts, increased cerebral edema, worsened neurological status compared to db/+ mice. The db/db mice also showed significantly higher post-ischemic inflammatory markers (ICAM1+ capillaries, extravasated macrophages/neutrophils and exacerbated proinflammatory gene expression) compared to db/+ mice. In addition, the post-ischemic neuroprotective heat-shock chaperone gene expression was curtailed in the db/db compared to db/+ mice.

Genetic changes due to dietary intervention in the form of either calorie restriction (CR) or intermittent fasting (IF) are not reported in detail until now. However, it is well established that both CR and IF extend the lifespan and protect against neurodegenerative diseases and stroke. The current research aims were first to describe the transcriptomic changes in brains of IF mice and, second, to determine whether IF induces extensive transcriptomic changes following ischemic stroke to protect the brain from injury. Mice were randomly assigned to ad libitum feeding (AL), 12 (IF12) or 16 (IF16) h daily fasting. Each diet group was then subjected to sham surgery or middle cerebral artery occlusion and consecutive reperfusion. Mid-coronal sections of ipsilateral cerebral tissue were harvested at the end of the 1 h ischemic period or at 3, 12, 24 or 72 h of reperfusion, and genome-wide mRNA expression was quantified by RNA sequencing. The cerebral transcriptome of mice in AL group exhibited robust, sustained up-regulation of detrimental genetic pathways under ischemic stroke, but activation of these pathways was suppressed in IF16 group. Interestingly, the cerebral transcriptome of AL mice was largely unchanged during the 1 h of ischemia, whereas mice in IF16 group exhibited extensive up-regulation of genetic pathways involved in neuroplasticity and down-regulation of protein synthesis. Our data provide a genetic molecular framework for understanding how IF protects brain cells against damage caused by ischemic stroke, and reveal cellular signaling and bioenergetic pathways to target in the development of clinical interventions.

We and others have shown that focal cerebral ischemia induces lateral migration of neuroblasts from the ipsilateral subventricular zone (SVZ) to the ischemic striatum. The signaling pathways underlying this phenomenon are not fully understood. The present study examined the role of osteopontin (OPN) in post-ischemic lateral migration of neuroblasts. Focal ischemia was induced by transient middle cerebral artery occlusion in adult spontaneous hypertensive rats. The expression of OPN in the ischemic brain was evaluated by immunohistochemistry, which showed that an up-regulation of OPN expression in the ipsilateral striatum at day 3, 7, 14 and 1 month of reperfusion with a peak at day 7. Double staining showed co-localization of OPN with ED1(+) macrophages/microglia in the ischemic regions. Inhibition of OPN activity by infusing a neutralizing antibody against OPN into the ischemic striatum significantly decreased the area covered with doublecortin(+) neuroblasts in the ipsilateral striatum. In vitro, OPN treatment did not affect the proliferation of neural progenitors, but induced an increased trans-well and radial migration of neural progenitors. The cultured neural progenitors expressed the OPN receptors CD44 and integrin beta(1). Blockade of the CD44 receptor had no effects on OPN mediated trans-well and radial migration of neural progenitors. However, blockade of integrin beta(1) receptor abolished the migration of neural progenitors in the absence or the presence of OPN. These results suggest that up-regulated expression of OPN produced by macrophages/microglia in the ischemic brain is an attractant and inducer for the lateral migration of neuroblasts from the SVZ to the injured region.

A brief ischemic episode induces protection against a subsequent severe ischemic insult. This phenomenon, known as ischemic preconditioning (PC) might be mediated by the activation of several genes of the second messenger signalling pathways. A 60-min transient middle cerebral artery occlusion (MCAO) in adult, SHR rats results in an infarct encompassing cerebral cortex and striatum by 24 h reperfusion. Present study observed that a 10-min PC, 3 days before the 60 min transient MCAO, results in a 30% reduction in the infarct volume. To understand the molecular mechanisms of the PC induced neuroprotection, we analyzed the mRNA expression profiles of rat cerebral cortex at 6, 24 and 72 h after a 10-min transient MCAO by using Affymetrix GeneChip and real-time PCR. Expression of the members of TGF-β signalling (TGF-β1, p 38 MAP kinase, Smad 1 and Smad 7) is observed to be up-regulated after PC. This pathway plays an essential role in tissue repair and immune homeostasis, but not hitherto implicated in PC induced neuroprotection. TGF-β1 enhances Smad transcriptional activity through activation of P38 gene expression to induce neuroprotection. Consistent with previous studies, expression of several neuroprotective genes including HSP70, HSP27, HO-1, MT-1/MT-2 and IL-6, were also observed to be up-regulated after PC. These studies shows that TGF-β1/p38 MAP-kinase signalling pathway is a promising target for developing drugs to limit ischemic neuronal damage. Acknowledgements: Funded by AHA.

Object Neurogenesis continues throughout the life of mammals in the subventricular zone (SVZ) of the lateral ventricles and the dentate gyrus (DG) of the hippocampus. The authors tested the potential of the neuropeptide substance P (SP) acting via the neurokinin-1 receptor (NK1R) in promoting the proliferation of adult rat neural progenitor cells (NPCs). Methods Focal ischemia was induced in spontaneously hypertensive rats by transient middle cerebral artery occlusion. Substance P and the NK1R antagonist L-703,606 were infused into the lateral ventricles of rats by using Alzet osmotic minipumps. Progenitor cell proliferation was evaluated with immunostaining for bromodeoxyuridine (BrdU) and immature neural marker doublecortin (DCX). Neurospheres were cultured from the SVZ of adult rats. Results Under in vitro conditions, SP (0.01–10 μmol/L) increased the proliferation of cultured NPCs, with a peak increase of 52 ± 7% at 0.1 μmol/L. Substance P (0.1 μmol/L) continuously increased NPC proliferation from 6 hours to 5 days, which was prevented by L-703,606 (by 69–98%). The cultured NPCs expressed both SP and NK1R proteins, indicating that these effects are receptor specific. Continuous infusion of SP (1 μmol/L) into the lateral ventricles for 5 days significantly increased the number of proliferating NPCs (cells positive for both BrdU and DCX) in both the SVZ (by 173 ± 24%, p < 0.05) and DG (by 82 ± 12%, p < 0.05) in adult rats; however, infusion of L-703,606 (10 μmol/L) significantly prevented the postischemic induction of NPC proliferation in both the SVZ (by 84 ± 6%, p < 0.05) and DG (by 63 ± 7%, p < 0.05). Conclusions Data in these studies indicated that SP plays a role in normal and ischemia-induced neurogenesis in the adult brain and thus could help central nervous system plasticity following injury.

Inflammation is a known precipitator of neuronal death after cerebral ischemia. The mechanisms that promote or curtail the start and spread of inflammation in brain are still being debated. By virtue of their capability to modulate gene expression, several transcription factors induced in the ischemic brain can modulate the post-ischemic inflammation. While the induction of transcription factors such as IRF1, NF-kappaB, ATF-2, STAT3, Egr1 and C/EBPbeta is thought to promote post-ischemic inflammation, activation of transcription factors such as HIF-1, CREB, c-fos, PPARalpha, PPARgamma and p53 is thought to prevent post-ischemic inflammation and neuronal damage. Of these, PPARgamma which is a ligand-activated transcription factor was recently shown to prevent inflammatory gene expression in several animal models CNS disorders. This review article discusses some of the molecular mechanisms of PPARgamma induction by its agonists following focal cerebral ischemia.

Insulin-like growth factor I (IGF-I) is involved in the proliferation and differentiation of adult neural progenitor cells; however, the underlying mechanism is not clear. We analysed the involvement of the phosphatidylinositol 3-kinase/Akt and MEK/extracellular signal-regulated kinase (ERK) pathways in the IGF-I-mediated proliferation of rat neural progenitor cells. Stimulation of neural progenitor cells with IGF-I enhanced the phosphorylation of Akt but not ERK. Cell proliferation assay demonstrated that 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (phosphoinositide 3-kinase inhibitor) but not 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)-butadiene (U0126) (ERK inhibitor) inhibited the IGF-I-induced survival of cells, whereas fibroblast growth factor 2 (FGF-2) enhanced the IGF-I-mediated survival of cells. Consistent with the cell proliferation assay, 5'bromo-2-deoxy-uridine incorporation studies established a negative role for IGF-I in proliferation. However, FGF-2 (ERK activator) in the presence of IGF-I (Akt activator) increased the proliferation of cells. Accordingly, stimulation of the ERK pathway by FGF-2 induced the expression of cyclin D1, which is essential for the entry of cells into cell cycle, and IGF-I in the presence of FGF-2 up-regulated the expression of cyclin D1. IGF-I in the absence or presence of FGF-2 increased the phosphorylation of glycogen synthase kinase, thus supporting its role in the survival of neural progenitor cells. To further confirm the role of ERK activation in the proliferation, we cultured cells in FGF-2 + IGF-I-containing medium in the presence and absence of U0126 (ERK inhibitor), and showed the inhibition of nestin expression in U0126-treated cells. The decrease in the cyclin D1 content in conjunction with the inhibition of nestin expression by ERK inhibitor confirms the role of ERK in the proliferation of cells.

MicroRNAs (miRNAs) are small non-coding RNAs that are known to control mRNA translation. Most miRNAs are transcribed from specific genes with well-defined promoters located throughout the genome. The mechanisms that control miRNA expression under normal and pathological conditions are not yet understood clearly. Peroxisome proliferator-activated receptor (PPAR) γ is a ligand-activated transcription factor that is extensively distributed in the CNS. PPARγ activation induces neuroprotection by modulating genes that contain peroxisome proliferator response elements (PPREs) in their promoters. We presently evaluated if PPARγ modulates miRNA expression. When adult rats were treated with PPARγ agonist rosiglitazone, expression of 28 miRNAs altered significantly (12 up- and 16 down-regulated; 3-119 fold) in the cerebral cortex compared to vehicle-treated controls. In silico analysis showed 1-5 PPREs in the putative promoter regions (within 1 Kb upstream of the transcription start site) of these miRNA genes. Cotransfection with a PPARγ constitutively expressing vector significantly induced the miR-145 and miR-329 promoter vectors (each have four PPREs), which was curtailed by point mutations of PPREs in their promoters. Interestingly, the PPARγ promoter has binding sites for both these miRNAs and transfection with miR-329 mimic and miR-145 mimic induced the PPARγ expression. Thus, these studies show a cyclical induction of miRNAs and PPARγ, indicating that the pleiotropic beneficial effects of PPARγ agonists might be modulated in part by miRNAs and their down-stream mRNAs. We proposed that promoters of many microRNAs contain the binding sites for the transcription factor PPARγ. Activation of PPARγ modulates the expression of these microRNAs. Two such PPARγ-responsive microRNAs (miR-145 and miR-329) bind to PPARγ promoter to induce its expression. This indicates the presence of a feedback loop by which transcription factors and microRNAs can modulate each other.

Male neonate brains are more susceptible to the effects of perinatal asphyxia resulting in hypoxia and ischemia (HI)-related brain injury. The relative resistance of female neonatal brains to adverse consequences of HI suggests that there are sex-specific mechanisms that afford females greater neuroprotection and/or facilitates recovery post-HI. We hypothesized that HI preferentially induces estrogen receptor α (ERα) expression in female neonatal hippocampi and that ERα is coupled to Src family kinase (SFK) activation that in turn augments phosphorylation of the TrkB and thereby results in decreased apoptosis. After inducing the Vannucci’s HI model on P9 (C57BL/6J) mice, female and male ERα wild-type (ERα+/+) or ERα null mutant (ERα−/−) mice received vehicle control or the selective TrkB agonist 7,8-dihydroxyflavone (7,8-DHF). Hippocampi were collected for analysis of mRNA of ERα and BDNF, protein levels of ERα, p-TrkB, p-src, and cleaved caspase 3 (c-caspase-3) post-HI. Our results demonstrate that: (1) HI differentially induces ERα expression in the hippocampus of the female versus male neonate, (2) src and TrkB phosphorylation post-HI is greater in females than in males after 7,8-DHF therapy, (3) src and TrkB phosphorylation post-HI depend on the presence of ERα, and (4) TrkB agonist therapy decreases the c-caspase-3 only in ERα+/+ female mice hippocampus. Together, these observations provide evidence that female-specific induction of ERα expression confers neuroprotection with TrkB agonist therapy via SFK activation and account for improved functional outcomes in female neonates post-HI.

Transient focal ischemia is known to induce proliferation of neural progenitors in adult rodent brain. We presently report that doublecortin positive neuroblasts formed in the subventricular zone (SVZ) and the posterior peri-ventricle region migrate towards the cortical and striatal penumbra after transient middle cerebral artery occlusion (MCAO) in adult rodents. Cultured neural progenitor cells grafted into the non-infarcted area of the ipsilateral cortex migrated preferentially towards the infarct. As chemokines are known to induce cell migration, we investigated if monocyte chemoattractant protein-1 (MCP-1) has a role in post-ischemic neuroblast migration. Transient MCAO induced an increased expression of MCP-1 mRNA in the ipsilateral cortex and striatum. Immunostaining showed that the expression of MCP-1 was localized in the activated microglia and astrocytes present in the ischemic areas between days 1 and 3 of reperfusion. Furthermore, infusion of MCP-1 into the normal striatum induced neuroblast migration to the infusion site. The migrating neuroblasts expressed the MCP-1 receptor CCR2. In knockout mice that lacked either MCP-1 or its receptor CCR2, there was a significant decrease in the number of migrating neuroblasts from the ipsilateral SVZ to the ischemic striatum. These results show that MCP-1 is one of the factors that attract the migration of newly formed neuroblasts from neurogenic regions to the damaged regions of brain after focal ischemia.

Thiazolidinediones (TZDs) are potent synthetic agonists of the ligand-activated transcription factor peroxisome proliferator-activated receptor-gamma (PPARgamma). TZDs were shown to induce neuroprotection after cerebral ischemia by blocking inflammation. As spinal cord injury (SCI) induces massive inflammation that precipitates secondary neuronal death, we currently analyzed the therapeutic efficacy of TZDs pioglitazone and rosiglitazone after SCI in adult rats. Both pioglitazone and rosiglitazone (1.5 mg/kg i.p.; four doses at 5 min and 12, 24, and 48 h) significantly decreased the lesion size (by 57 to 68%, p < 0.05), motor neuron loss (by 3- to 10-fold, p < 0.05), myelin loss (by 66 to 75%, p < 0.05), astrogliosis (by 46 to 61%, p < 0.05), and microglial activation (by 59 to 78%, p < 0.05) after SCI. TZDs significantly enhanced the motor function recovery (at 7 days after SCI, the motor scores were 37 to 45% higher in the TZD groups over the vehicle group; p < 0.05), but the treatment was effective only when the first injection was given by 2 h after SCI. At 28 days after SCI, chronic thermal hyperalgesia was decreased significantly (by 31 to 39%; p < 0.05) in the pioglitazone group compared with the vehicle group. At 6 h after SCI, the pioglitazone group showed significantly less induction of inflammatory genes [interleukin (IL)-6 by 83%, IL-1beta by 87%, monocyte chemoattractant protein-1 by 75%, intracellular adhesion molecule-1 by 84%, and early growth response-1 by 67%] compared with the vehicle group (p < 0.05 in all cases). Pioglitazone also significantly enhanced the post-SCI induction of neuroprotective heat shock proteins and antioxidant enzymes. Pretreatment with a PPARgamma antagonist, 2-chloro-5-nitro-N-phenyl-benzamide (GW9662), prevented the neuroprotection induced by pioglitazone.

Thiazolidinediones (TZDs) are synthetic agonists of the ligand-activated transcription factor peroxisome proliferator-activated receptor-gamma (PPARgamma). TZDs are known to curtail inflammation associated with peripheral organ ischemia. As inflammation precipitates the neuronal death after stroke, we tested the efficacy of TZDs in preventing brain damage following transient middle cerebral artery occlusion (MCAO) in adult rodents. As hypertension and diabetes complicate the stroke outcome, we also evaluated the efficacy of TZDs in hypertensive rats and type-2 diabetic mice subjected to transient MCAO. Pre-treatment as well as post-treatment with TZDs rosiglitazone and pioglitazone significantly decreased the infarct volume and neurological deficits in normotensive, normoglycemic, hypertensive and hyperglycemic rodents. Rosiglitazone neuroprotection was not enhanced by retinoic acid x receptor agonist 9-cis-retinoic acid, but was prevented by PPARgamma antagonist GW9662. Rosiglitazone significantly decreased the post-ischemic intercellular adhesion molecule-1 expression and extravasation of macrophages and neutrophils into brain. Rosiglitazone treatment curtailed the post-ischemic expression of the pro-inflammatory genes interleukin-1beta, interleukin-6, macrophage inflammatory protein-1alpha, monocyte chemoattractant protein-1, cyclooxygenase-2, inducible nitric oxide synthase, early growth response-1, CCAAT/enhancer binding protein-beta and nuclear factor-kappa B, and increased the expression of the anti-oxidant enzymes catalase and copper/zinc-superoxide dismutase. Rosiglitazone also increased the expression of the anti-inflammatory gene suppressor of cytokine signaling-3 and prevented the phosphorylation of the transcription factor signal transducer and activator of transcription-3 after focal ischemia. Thus, PPARgamma activation with TZDs might be a potent therapeutic option for preventing inflammation and neuronal damage after stroke with promise in diabetic and hypertensive subjects.

CCAAT/enhancer binding protein beta (C/EBPbeta) is a leucine-zipper transcription factor that regulates cell growth and differentiation in mammals. Expression of many pro-inflammatory genes including the cytokine interleukin-6 is known to be controlled by C/EBPbeta. We report that focal cerebral ischemia induced by transient middle cerebral artery occlusion (MCAO) significantly increases C/EBPbeta gene expression in mouse brain at between 6 and 72 h of reperfusion. To understand the functional significance of C/EBPbeta in postischemic inflammation and brain damage, we induced transient MCAO in cohorts of adult C/EBPbeta null mice and their wild-type littermates. At 3 days of reperfusion following transient MCAO, C/EBPbeta null mice showed significantly smaller infarcts, reduced neurological deficits, decreased terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells, decreased intercellular adhesion molecule 1 (ICAM1) immunopositive vessels, decreased extravasated neutrophils and fewer activated microglia/macrophages, compared with their wild-type littermates. Furthermore, GeneChip analysis showed that postischemic induction of many transcripts known to promote inflammation and neuronal damage was less pronounced in the brains of C/EBPbeta-/- mice compared with C/EBPbeta+/+ mice. These results suggest a significant role for C/EBPbeta in postischemic inflammation and brain damage.

The molecular mechanisms that render a carotid atherosclerotic plaque symptomatic have not yet been identified. Using an Affymetrix Human GeneChip set, we analyzed the gene expression patterns of 44 862 mRNA transcripts in surgically removed carotid artery plaques from six patients with symptomatic stroke and four non-symptomatic patients. The age, body mass index and the degree of stenosis were similar in the two groups. Some 236 transcripts (approximately 0.5% of the total transcripts analyzed) were expressed more abundantly in the symptomatic than the asymptomatic group. Of these, 61 transcripts are those that participate in ionic homeostasis, signal transduction and metabolism. The other groups of transcripts up-regulated in the symptomatic plaques include oncogenes, growth factors, tumor markers, angiogenesis promoters, transcription factors, and RNA splicing and processing factors. This study indicates that the higher metabolic activity in some atherosclerotic plaques leads to their faster growth and precipitation of stroke symptoms. The implications of these findings are that both diagnosis and prevention of stroke symptoms may become possible at the genetic level.

In nerve terminals, vesicular transporters pack neurotransmitters into synaptic vesicles, which is an essential prerequisite for transmitter release. To date, three distinct families of vesicular transporters have been identified which are specific for (a) excitatory amino acids (glutamate and aspartate), (b) inhibitory amino acids (GABA and glycine) and (c) acetylcholine and monoamines. The present study evaluated the effect of transient focal cerebral ischemia on the expression of these vesicular transporters in adult rat brain. Ischemia was induced by a 1 h transient middle cerebral artery occlusion (MCAO) in spontaneously hypertensive rats. At various reperfusion periods (3-72 h), mRNA levels of the vesicular transporters were estimated in the contralateral and the ipsilateral cerebral cortex by real-time PCR analysis. Following transient focal ischemia, mRNA expression of the vesicular GABA transporter (VGAT) decreased significantly by 3 h of reperfusion and remained at a significantly lower level than sham until at least 72 h of reperfusion. Western blotting showed a significant decrease in the VGAT immunoreactive protein levels in the ipsilateral cortex of rats subjected to focal ischemia and 24 h reperfusion. Immunohistochemistry demonstrated many VGAT immunopositive puncta in the contralateral cortex, which were significantly decreased in the ipsilateral cortex at 24 h reperfusion. Focal ischemia had no effect on the mRNA levels of the vesicular transporters specific for glutamate/aspartate, acetylcholine and monoamines at either 6 h or 24 h of reperfusion.

Neural progenitor/stem cells reside in the subventricular zone and dentate gyrus of the adult rat brain, which proliferate in response to several growth factors. Here we show that the neurospheres generated from the adult dentate gyrus and subventricular zone proliferate in vitro in response to fibroblast growth factor-2, but not epidermal growth factor. Likewise, extracellular signal-regulated kinase phosphorylation was stimulated by fibroblast growth factor-2, but not epidermal growth factor. Immunoblotting demonstrated negligible epidermal growth factor receptor content in these neurospheres, indicating that neural progenitor cells isolated from the adult rat brain are not responsive to epidermal growth factor in vitro, because of the lack of epidermal growth factor receptors and/or their signaling. However, the lack of response by epidermal growth factor in our study could be because of the culture conditions, and may not reflect the physiological condition.

Background and Purpose— The present study was performed to determine whether antisense inhibition of intercellular adhesion molecule-1 (ICAM-1) protein expression decreases focal ischemic brain damage. Methods— Male spontaneously hypertensive rats underwent 1-hour middle cerebral artery occlusion (MCAO) and 24-hour reperfusion. Rats were infused with ICAM-1 antisense or control oligodeoxynucleotides (ODNs) (48 nmol/d ICV) or vehicle, starting 24 hours before MCAO and continuing until the time of death. ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1) mRNA levels were measured by real-time polymerase chain reaction. ICAM-1 protein knockdown was confirmed by Western blotting. Infarct volume was quantified by the use of cresyl violet–stained brain sections. Neurological deficits were evaluated. Mean arterial blood pressure was recorded by laser Doppler. Tissue penetration of antisense was confirmed by the use of fluorescent ODNs. Results— Transient MCAO upregulated ICAM-1, but not VCAM-1, mRNA expression in the ipsilateral cortex between 3 and 72 hours of reperfusion. ICAM-1 antisense infusion prevented ischemia-induced ICAM-1 protein expression and reduced total infarct volume (by 53%; P 3 in control ODN group and 104±27 mm 3 in antisense ODN group; n=8 each) and mean neurological deficit score (by 44%; P Conclusions— Increased ICAM-1 expression is implicated in the pathogenesis of focal ischemia since ICAM-1 protein knockdown decreased ischemic brain damage. The mechanism by which ICAM-1 inhibition offers neuroprotection is independent of blood pressure modulation.

Progenitor cells in the dentate gyrus of hippocampus (DG) and the subventricular zone of lateral ventricles (SVZ) generate new neurons throughout the life of mammals. Cerebral ischemia increases this basal progenitor cell proliferation. The present study evaluated the time frame of proliferation, length of survival and the phenotypes of the new cells formed after transient middle cerebral artery occlusion (MCAO) in adult spontaneously hypertensive rats. Compared to sham controls, ischemic rats showed a significantly higher number of newly proliferated cells (as defined by BrdU immunostaining) in both the DG (by fourfold, p

The mechanisms that promote brain dysfunction after acute liver failure (ALF) are not clearly understood. The small noncoding RNAs known as microRNAs (miRNAs) significantly control mRNA translation and thus normal and pathological functions in the mammalian body. To understand their significance in ALF, we currently profiled the expression of miRNAs in the cerebral cortex of mice sacrificed at coma stage following treatment with azoxymethane. Of the 470 miRNAs profiled using microarrays, 37 were significantly altered (20 up- and 17 down-regulated) in their expression in the ALF group compared to sham group. In silico analysis showed that the ALF-responsive miRNAs target on average 231 mRNAs/miRNA (range: 3 to 840 targets). Pathways analysis showed that many miRNAs altered after ALF target multiple mRNAs that are part of various biological and molecular pathways. Glutamatergic synapse, Wnt signaling, MAP-kinase signaling, axon guidance, PI3-kinase-AKT signaling, T-cell receptor signaling and ubiquitin-mediated proteolysis are the top pathways targeted by the ALF-sensitive miRNAs. At least 28 ALF-responsive miRNAs target each of the above pathways. We hypothesize that alterations in miRNAs and their down-stream mRNAs of signaling pathways might play a role in the induction and progression of neurological dysfunction observed during ALF.

The mammalian genome is replete with various classes of non-coding (nc) RNA genes. Many of them actively transcribe, and their relevance to CNS diseases is just beginning to be understood. CNS is one of the organs in the body that shows very high ncRNAs activity. Recent studies demonstrated that cerebral ischemia rapidly changes the expression profiles of different classes of ncRNAs: including microRNA, long noncoding RNA and piwi-interacting RNA. Several studies further showed that post-ischemic neuronal death and/or plasticity/regeneration can be altered by modulating specific microRNAs. These studies are of interest for therapeutic development as they may contribute to identifying new ncRNA targets that can be modulated to prevent secondary brain damage after stroke.

Introduction: Redox imbalance during ischemia/reperfusion Injury in the brain following focal ischemic stroke is associated with impaired neurological functions. Following ischemia/reperfusion, there is a burst of oxidative stress/reactive oxygen species (ROS). ROS physiologically serve as signaling molecules but excessive amounts of ROS contribute to destruction of endothelial integrity, blood brain barrier disruption, and neuronal cell death. NADPH oxidases (NOX) are important sources of ROS during ischemic stroke. Dual oxidases (Duox) are new members of NOX family and are known for directly generating hydrogen peroxide/ROS. Role of Duox following brain ischemic injury has been little investigated. Methods: Ischemia was induced by middle cerebral artery occlusion (MCAO) and reperfusion. In vitro ischemia was generated by oxygen-glucose deprivation (OGD). Expression was analysed by western blot analysis, RT-PCR, Immunofluorescence staining. ROS was measured by dichloro-dihydrofluorescein staining. Results: Our preliminary studies for the first time have identified that expression of Duox are spatially and temporally regulated in ipsilateral hemisphere of spontaneously hyertensive rat brains following MCAO and reperfusion. Expression of Duox 1 was up-regulated about 2 folds after 3 days, and16 folds after 7 days of reperfusion. Its expression was further increased to about 30 folds at day 21 after reperfusion. The expression of Duox 1 was specifically up-regulated in activated astrocytes and VWF and CD31 positive endothelial cells indicating its presence in blood vessels. In support of these in vivo observations, Duox1 expression was significantly increased in astrocytes and endothelial cell cultures exposed to either ATP or OGD. Duox1 expression was associated with increased ROS production and decreased cell viability. However, pre-treatment with Duox1 specific siRNA decreased the ROS production and increased the cell viability by about 30 percent. Summary: Up-regulation of Duox under ischemic/reperfusion conditions suggests that this enzyme potentially contributes to excessive ROS production. Further studies targeting these novel oxidases may open up a promising approach to reduce brain injury after stroke.

MicroRNAs (miRNAs) are known to repress translation by binding to the 3'UTRs of mRNAs. Using bioinformatics, we recently reported that several miRNAs also have target sites in DNA particularly in the promoters of the protein-coding genes. To understand the functional significance of this phenomenon, we tested the effects of miR-324-3p binding to RelA promoter. In PC12 cells, co-transfection with premiR-324-3p induced a RelA promoter plasmid in a dose-dependent manner and this effect was lost when the miR-324-3p binding site in the promoter was mutated. PremiR-324-3p transfection also significantly induced the endogenous RelA mRNA and protein expression in PC12 cells. Furthermore, transfection with premiR-324-3p increased the levels of cleaved caspase-3 which is a marker of apoptosis. Importantly, the miR-324-3p effects were Ago2 mediated as Ago2 knockdown prevented RelA expression and cleavage of caspase-3. Thus, our studies show that miRNA-mediated transcriptional activation can be seen in PC12 cells which are neural in origin.

Recent studies showed that stroke extensively alters cerebral microRNA (miRNA) expression profiles and several miRNAs play a role in mediating ischemic pathophysiology. We currently evaluated the significance of miR-29c, a highly expressed miRNA in rodent brain that was significantly down-regulated after focal ischemia in adult rats as well as after oxygen-glucose deprivation in PC12 cells. Bioinformatics indicated that DNA methyltransferase 3a (DNMT3a) is a major target of miR-29c and co-transfection with premiR-29c prevented DNMT3a 3'UTR vector expression. In PC12 cells, treatment with premiR-29c prevented OGD-induced cell death (by 58 ± 6%; p

Male neonate brains are more susceptible to the effects of perinatal asphyxia resulting in hypoxia and ischemia (HI)-related brain injury. The relative resistance of female neonatal brains to adverse consequences of HI suggests that there are sex-specific mechanisms that afford females greater neuroprotection and/or facilitates recovery post-HI. We hypothesized that HI preferentially induces estrogen receptor α (ERα) expression in female neonatal hippocampi and that ERα is coupled to Src family kinase (SFK) activation that in turn augments phosphorylation of the TrkB and thereby results in decreased apoptosis. After inducing the Vannucci's HI model on P9 (C57BL/6J) mice, female and male ERα wild-type (ERα(+/+)) or ERα null mutant (ERα(-/-)) mice received vehicle control or the selective TrkB agonist 7,8-dihydroxyflavone (7,8-DHF). Hippocampi were collected for analysis of mRNA of ERα and BDNF, protein levels of ERα, p-TrkB, p-src, and cleaved caspase 3 (c-caspase-3) post-HI. Our results demonstrate that: (1) HI differentially induces ERα expression in the hippocampus of the female versus male neonate, (2) src and TrkB phosphorylation post-HI is greater in females than in males after 7,8-DHF therapy, (3) src and TrkB phosphorylation post-HI depend on the presence of ERα, and (4) TrkB agonist therapy decreases the c-caspase-3 only in ERα(+/+) female mice hippocampus. Together, these observations provide evidence that female-specific induction of ERα expression confers neuroprotection with TrkB agonist therapy via SFK activation and account for improved functional outcomes in female neonates post-HI.

Background and Purpose— Long noncoding RNAs (lncRNAs) play a significant role in cellular physiology. We evaluated the effect of focal ischemia on the expression of 8314 lncRNAs in rat cerebral cortex using microarrays. Methods— Ischemia was induced by transient middle cerebral artery occlusion. Genomic and transcriptomic correlates of the stroke-responsive lncRNAs and the transcription factor binding sties in their promoters were evaluated with bioinformatics. Results— Three hundred fifty-nine lncRNAs were upregulated (>2-fold) and 84 were downregulated (90% sequence homology with exons of protein-coding genes. Promoters of stroke-responsive lncRNA genes and their homologous protein-coding genes showed highly overlapping transcription factor binding sites. Despite presence of open reading frames, lncRNAs did not form any product when subjected to in vitro translation. Conclusions— Stroke significantly alters cerebral lncRNA expression profiles.

Focal cerebral ischemia initiates self-repair mechanisms that include the production of neurotrophic factors and cytokines. Galectin-3 is an important angiogenic cytokine. We have previously demonstrated that expression of galectin 3 (Gal-3), a carbohydrate binding protein is significantly upregulated in activated microglia in the brains of rats subjected to focal ischemia. Further blocking of Gal-3 function with Gal-3 neutralizing antibody decreased the microvessel density in ischemic brain. We currently show that Gal-3 significantly increases the viability of microglia BV2 cells subjected to oxygen glucose deprivation (OGD) and re-oxygenation. Exogenous Gal-3 promoted the formation of pro-angiogenic structures in an in vitro human umbilical vein endothelial (HUVEC) and BV2 cell co-culture model. Gal-3 induced angiogenesis was associated with increased expression of vascular endothelial growth factor. The conditioned medium of BV2 cells exposed to OGD contained increased Gal-3 levels, and promoted the formation of pro-angiogenic structures in an in vitro HUVEC culture model. Gal-3 also augmented the in vitro migratory potential of BV2 microglia. Gal-3 mediated functions were associated with increased levels of integrin-linked kinase (ILK) signaling as demonstrated by the impaired angiogenesis and migration of BV2 cells following targeted silencing of ILK expression by siRNA. Furthermore, we show that ILK levels correlate with the levels of phos-AKT and ERK1/2 that are downstream effectors of ILK pathway. Taken together, our studies indicate that Gal-3 contributes to angiogenesis and microglia migration that may have implications in post stroke repair.

Introduction: As restoration of blood flow is critical for improved functional recovery following ischemic stroke, enhancing cerebral angiogenesis might be immensely helpful. Galectin-3 (gal-3) is a β-galactoside-binding lectin that plays key roles in inflammation, survival, and angiogenesis. Methods: We used transient middle cerebral artery occlusion (MCAO), oxygen-glucose deprivation (OGD) model, western blot analysis, RT-PCR, Immunofluorescence staining. Results: We currently report that intra-cerebroventricular infusion of recombinant gal-3 in rats subjected to transient MCAO increases the post-ischemic functional recovery. We further show that gal-3 mRNA and protein expression is up-regulated in vitro when activated microglial BV2 cells were subjected to either oxygen-glucose deprivation (OGD) or LPS (lipo-polysaccharide) stimulation. Immunofluorescence staining showed increased cytoplasmic gal-3 that is known to have pro-survival function. This increase in gal-3 is associated with increased number of pro-angiogenic structures in a 3D human vein umbilical cord endothelial (HUVEC) and microglia co-culture model. The pro-angiogenic effect of gal-3 was suppressed by treatment with gal-3 siRNA indicating the specific role of gal-3 in promoting angiogenesis. Exogenous gal-3 treatment further augmented the angiogenic potential of BV2 microglia cells. In addition, gal-3 increased survival of microglial BV2 cells, HUVEC, and neuro-progenitor cells. Gal-3 levels were also directly correlated with increased levels of Integrin-linked kinase 1(ILK1), pro-survival factor AKT and their down stream effector pro-angiogenic factors bFGF and MMP2. Conclusion: Taken together, our studies emphasize the importance of gal-3 in enhancing angiogenesis that is critical for neuron survival and neurogenesis in the post-stroke brain.

CNS injuries are associated with extensive spatio-temporal alterations in gene expression that dictate the extent of brain damage and subsequent plasticity. Studies in CNS injury models show that microRNAs (miRNAs) modulate gene expression, which contributes to development of the injury. The expression levels of brain miRNAs show diverse spatio-temporal profiles in the healthy brain and are altered significantly over a sustained period of time in injured states. Apart from the conventional mechanisms of translational inhibition of mRNAs, recent studies have revealed that miRNAs can also target gene promoters to induce or repress their expression, adding another layer to the already complex network of miRNA–gene interactions. The extent of the influence of miRNAs in the CNS is only beginning to be elucidated. miRNAs play a major role in the etiology of neurodegenerative diseases as well as in brain injuries such as traumatic brain injury, spinal cord injury, and ischemia. Studies have shown that they considerably influence the outcome of the ischemic pathophysiology. However, miRNAs have also been observed to play a role in endogenous (preconditioning) and exogenous neuroprotective treatments, indicating their role in inducing ischemic tolerance. Lately, miRNAs have shown promise for serving as clinical biomarkers of stroke and other CNS disorders.

Background and Purpose— The PIWI-interacting RNA (piRNA) is the most predominant RNA species in eukaryotes. The piRNA are a class of noncoding RNAs that bind and degrade the RNA formed by the transposons to control the transposon-induced gene mutations. The role of piRNA after focal ischemia is not yet evaluated. Methods— We profiled 39 727 piRNAs in the cerebral cortex of adult rats subjected to transient focal ischemia using microarrays. The RT targets of stroke-responsive piRNAs were identified with bioinformatics. To understand how piRNAs are controlled, we analyzed the transcription factor binding sites in the putative promoters of 10 representative stroke-responsive piRNAs. Results— In the ipsilateral cortex of ischemic rats, 105 piRNAs showed altered expression (54 up- and 51 downregulated; >2.5-fold) compared with shams. Twenty-five of those showed a >5-fold change. A bioinformatics search showed that the transposon targets of the highly stroke-responsive piRNAs are distributed among the 20 autosomal chromosomes and there is a redundancy in the targets between the piRNAs. Furthermore, the transposon targets were observed to be highly repetitious for each piRNA across the chromosome length. Of the 159 transcription factors observed to have binding sites in the piRNA gene promoters, 59% belonged to 20 major families indicating that transcription factors control stroke-responsive piRNAs in a redundant manner. Conclusions— The present study is the first to show that many piRNAs are expressed in adult rodent brain and several of them respond to focal ischemia.

Hepatic encephalopathy (HE) and thiamine deficiency (TD) represent two disorders in which metabolic derangements play a major role in their pathophysiology. They also define the areas of primary focus of Dr. Roger F. Butterworth, Ph.D., D.Sc. who retired from the University of Montreal in the summer of 2013. This special issue of Metabolic Brain Disease commemorates his valuable contribution to the study of these maladies. Although HE is considered a metabolic disorder, new evidence is emerging to show that it has a molecular basis as well. Vemuganti et al reports altered cerebral profiles of microRNAs after acute liver failure (ALF) that likely contribute in a significant way to gene expression changes associated with the ensuing HE. Disrupted ammonia detoxification is considered a major precipitator of neurological dysfunction in HE, and current understanding of the contribution of ammonia in liver diseases is discussed in an article by Ott and Vilstrup, while the effects of hyperammonemia on cerebral metabolic function are focussed on in an article by Schousboe and colleagues. In addition, impaired brain energy metabolism is a serious complicating feature of liver failure, with lactate production being a major consequence of this problem. In relation to this, the role of increased lactate accumulation and its pathogenetic basis in HE is discussed by Bosoi and Rose. Also, a major and often lethal complication of ALF is brain edema, and the significance of this edema and its underlying basis in ALF-induced HE is examined by Rama Rao et al, while the usefulness of magnetic resonance imaging as a tool in both acute and chronic liver failure is considered by Chavarria and Cordoba. Minimal hepatic encephalopathy (MHE) represents the mildest of the spectrum of HE forms in which patients do not display clinical symptoms but have mild cognitive and psychomotor deficits. It is also associated with poor survival rates. Two papers examine aspects of MHE, in which cognitive impairment and the issue of cognitive variability as a contributing factor in these patients is a focus of attention in Bisiacchi et al, and in an experimental model of chronic liver failure in which focal increases in the binding of translocator (18kD) protein in brain is reported by Agusti et al. Increasingly, the phenomenon of acute-on-chronic liver failure, in which acute deterioration of liver function in patients with cirrhosis is a consequence, is being considered a distinct clinical entity. The subject is discussed in Wright et al. In addition, considerable evidence has shown that astrocyte dysfunction plays a major role in HE. Karabara and colleagues explore the potential role of GlcNAcylation in these cells, while the protective effect of inhibition of glutamine synthesis is examined in neurological diseases, including HE, by Jeitner and Cooper. Development of well-defined biomarkers and improved therapeutic targets represents an important subject regarding HE. In this regard, the paper by Cooper and Kuhara shows that α-ketoglutaramate is a good marker of HE, and the paper by Mondal and Trigun demonstrates that pannexin-1 can be a marker as well as a target for HE. Although the mechanism(s) underlying the beneficial effects of rifaximin in HE remain largely unknown, the topic is tackled using multi-modal MRI in Ahluwalia et al. In addition, the subject of microglial proliferation and its significance in alcoholics with HE is explored experimentally in Dennis et al. Dienel and Cruz address the subject of decreased proteolysis as a result of hyperammonemia due to liver failure, and the effects of thioacetamide-induced ALF on citrulline uptake is examined in a study by Zielinska et al. Finally, Roger’s interest in the pathophysiology of TD has been long and enduring. A number of papers in this special issue deal with different aspects of TD. Afadlal et al shows that astrocytes play a significant role in TD-induced encephalopathy, while Bettendorff explores the role of thiamine triphosphate in cells. In addition, since mitochondrial dysfunction is an important aspect of both TD and Alzheimer’s disease in which thiamine-dependent enzyme activity is reduced, Huang et al explores how cellular calcium stores are affected in a fibroblast cell line, an important first step towards the eventual determination of how this process comes about in the AD patient and development of protective/treatment strategies targeting this mechanism, which may also have relevance for the future treatment of TD.

Experimental stroke research conducted in the past three decades can be divided into two major categories. The first is the molecular and cellular studies that evaluated the mechanisms of stroke-induced neuronal death and the second is the development and testing of putative therapeutic compounds to prevent post-stroke brain damage and neurologic dysfunction. Both categories of studies focused on evaluating and/or targeting specific proteins or the genes that encode those proteins. However, protein-coding genes represent 20% of the miRNAs alter in the ischemic brain, indicating the possibility that ncRNA genes are more susceptible to stroke than protein-coding genes. As the transcription of mRNA and miRNA genes might be independently influenced by stroke, a higher magnitude of change in miRNAs might serve as an extra layer of control to prevent aberrant protein expression under disease conditions. Many individual stroke-responsive miRNAs were shown to play a role in mediating the secondary brain damage and functional outcome after experimental stroke. It was shown that mir-145 will be upregulated to ~eightfold in rat brain following focal ischemia [10]. A major target of the mir-145 is the antioxidant enzyme superoxide dismutase-2 (SOD-2) mRNA and presumably mir-145 prevents SOD-2 protein translation after stroke. When the ischemic rats were treated with antagomir-145, there was an increased expression of SOD-2 in the cortical neurons and a significantly decreased infarction presumably by curtailing the oxidative stress [10]. Another recent study showed that induction of mir-497 following focal ischemia promotes ischemic neuronal death by negatively regulating antiapoptotic proteins, bcl-2 and bcl-w [16]. Activation of the transcription factor peroxisome proliferator-activated receptor (PPAR)-δ induces vascular endothelial cell protection and this beneficial effect was shown to be mediated by preventing the post-ischemic induction of miR-15a which targets bcl-2 [17]. The miRNAs are also thought to promote neuroprotection. Importantly, miRNAs that target mRNAs which code for transcription factors like MeCP2 play a critical role in inducing ischemic tolerance in mouse brain [18]. Ischemic preconditioning was also shown to modulate miRNAs which are upstream to neuroprotective signaling pathways in rat brain [19]. It is interesting to note that miRNA profiles alter rapidly in blood following stroke and intracerebral hemorrhage in rodents [9, 20]. Altered miRNA profiles were also reported in stroke patients [8]. This study showed that many miRNAs that are expressed at a low level in normal human blood were observed to be highly expressed in stroke samples irrespective of etiology. These authors also showed that different stroke subtypes show distinctly different blood miRNA profiles and they could separate the small artery and large artery stroke samples based on miRNA profiles [20]. In addition, the blood miRNAs of the patients showing good outcome and bad outcome after stroke separated into distinct clusters [20]. These studies indicate that blood miRNAs can be used as genomic biomarkers to quickly identify stroke, stroke subtypes, and possibly the outcome after a therapy. The current view is that miRNAs play a more diverse role than just silencing mRNAs as certain miRNAs can also upregulate translation [21]. The 8-bp seed sequences that miRNAs recognize in the 3′UTR of mRNAs can be found in several genomic locations. The miRNA mir-379 was shown to bind to the E-cadherin promoter, leading to the induction of E-cadherin gene expression [22]. This phenomenon known as RNA-induced gene activation might also play an important role in post-ischemic gene induction. Using in silico analysis, we observed that eight miRNAs upregulated after focal ischemia can bind to the complementary seed sequences in 877 promoters in rat genome [10]. Furthermore, it was shown that, if the levels of a specific mRNAs are increased, that mRNA can instruct the expression of an upstream miRNA to prevent its own translation as a feed-forward mechanism [23]. Recent studies from our laboratory showed that miRNA promoters contain several transcription factor binding sites and treating rats with rosiglitazone (a high-affinity PPARγ agonist) alters the cerebral expression of miRNAs that contain PPAR binding sites [24]. Thus, miRNAs and mRNAs can control each other bi-directionally and hence their relationship is complex. To conclude, there is much excitement in understanding the functional significance of miRNAs to ischemic brain damage and the studies conducted so far is just the tip of the iceberg. Importantly, an altered miRNA expression can have profound consequences on transcription as well as translation. As new functions of miRNAs as well as other ncRNAs are still emerging, in the future some of those can be identified as good therapeutic targets to prevent brain damage in stroke patients. Financial support was provided by NIH grant NS061071.

This review encourages the reader to consider cerebral vascular disease beyond the traditional clinical end points of major motor and speech strokes and to consider the possible impact of embolic cerebral vascular disease on vascular cognitive decline. The paper examines the issue of “silent” strokes in the relationship between the structural stability of atherosclerotic carotid plaque and the development of nonmotor symptomatology, including cognitive decline. It addresses the question of the role of carotid emboli in “silent” stroke and their cognitive sequelae. In a study of endarterectomy patients, we relate plaque elasticity and its development of mechanical strain features and thinning of stabilizing fibrous cap at the point of these mechanical strain features. The possibility that microemboli from such mechanically unstable carotid plaques could contribute to “silent strokes” lead to a study of cognitive function in such patients. A linear relationship between the process of mechanically unstable areas of carotid plaques and cognitive decline suggests a contributory role for such a process in “silent strokes”.

Cerebral gene expression is known to be significantly influenced by a sublethal ischemic event (pre-conditioning; PC) that induces tolerance to future damaging ischemic events. Small non-coding RNAs known as microRNAs (miRNAs) were recently shown to control the mRNA translation. We currently profiled cerebral miRNAs in the cerebral cortex of rats subjected to PC. The miRNAome reacted quickly and by 6 h following PC, levels of 51 miRNAs were altered (26 up- and 25 down-regulated;1.5-fold change). Twenty of these stayed at the altered level even at 3 days after PC. At least nine miRNAs showed5-fold change at one or more time points between 6 h to 3 days after PC compared with sham. Bioinformatics analysis showed 2007 common targets of the miRNAs that were up-regulated and 459 common targets of the miRNAs that were down-regulated after PC. Pathways analysis showed that MAP-kinase and Mammalian target of rapamycin (mTOR) signaling are the top two Kyoto Encyclopedia of Genes and Genomes pathways targeted by the up-regulated miRNAs, and Wnt and GnRH signaling are the top two Kyoto Encyclopedia of Genes and Genomes pathways targeted by the down-regulated miRNAs after PC. We hypothesize that alterations in miRNAs and their down-stream mRNAs of signaling pathways might play a role in the induction of ischemic tolerance.

J. Neurochem. (2010) 113, 1685–1691. Abstract Cerebral gene expression is known to be significantly influenced by a sublethal ischemic event (pre-conditioning; PC) that induces tolerance to future damaging ischemic events. Small non-coding RNAs known as microRNAs (miRNAs) were recently shown to control the mRNA translation. We currently profiled cerebral miRNAs in the cerebral cortex of rats subjected to PC. The miRNAome reacted quickly and by 6 h following PC, levels of 51 miRNAs were altered (26 up- and 25 down-regulated; > 1.5-fold change). Twenty of these stayed at the altered level even at 3 days after PC. At least nine miRNAs showed > 5-fold change at one or more time points between 6 h to 3 days after PC compared with sham. Bioinformatics analysis showed 2007 common targets of the miRNAs that were up-regulated and 459 common targets of the miRNAs that were down-regulated after PC. Pathways analysis showed that MAP-kinase and Mammalian target of rapamycin (mTOR) signaling are the top two Kyoto Encyclopedia of Genes and Genomes pathways targeted by the up-regulated miRNAs, and Wnt and GnRH signaling are the top two Kyoto Encyclopedia of Genes and Genomes pathways targeted by the down-regulated miRNAs after PC. We hypothesize that alterations in miRNAs and their down-stream mRNAs of signaling pathways might play a role in the induction of ischemic tolerance.

We investigated intracerebral hemorrhage (ICH)-induced lateral migration of neuroblasts and the mechanism underlying this migration. ICH model was induced by collagenase injection into the striatum of adult wild-type and osteopontin (OPN) knockout mice. In the wild-type mice, the lateral migration of neuroblasts from the ipsilateral subventricular zone (SVZ) towards the hematoma started at day 3 and continued up to day 28 after ICH. In addition to migrating towards the hematoma, neuroblasts also migrated to the area of ipsilateral striatum remote to the hematoma. The migrating neuroblasts were closely associated with activated astrocytes and blood vessels in the injured striatum. Following ICH, the expression of OPN was up-regulated in the ipsilateral striatum from day 1 to day 28. In vitro, OPN treatment did not affect the proliferation of neural progenitors, but enhanced the trans-well and radial migration of neural progenitors. In vivo, OPN deficiency did not affect the proliferation of neural progenitors in the SVZ. However, following ICH a significant decrease in lateral neuroblast migration was observed in the OPN knockout mice compared with the wild-type mice. These results suggest that increased OPN expression in the injured striatum plays a significant role in the lateral migration of neuroblasts following ICH.

Stroke is common, with a high rate of disability and death, and current therapies are both highly time sensitive and carry some risk, making rapid diagnosis important. Many cases of stroke are difficult to diagnose clinically during the acute phase, and there are limitations to the ability of diagnostic imaging to help with the necessary speed. A reliable and valid biomarker would be invaluable for this common clinical situation, as it has been with myocardial infarction. A large number of molecules have been evaluated for this role, both in the laboratory and in patients, but the results to date have been disappointing. In this article, we review the operative concepts in the search for a diagnostic biomarker of stroke, a few of the promising candidates and the current challenges to validation.

Galectin-3 (Gal-3) is a member of a class of carbohydrate-binding proteins and plays a role in a number of cellular functions such as cell proliferation, angiogenesis and differentiation. We observed an up-regulated expression of Gal-3 in the ischemic brain following transient middle cerebral artery occlusion in rats. Compared to the brain of sham-operated rats, the expression of Gal-3 was increased in the ischemic striatum at day 1 of reperfusion. The number of Gal-3+ cells in the ischemic brain was further increased at day 2 and day 3, and peaked at day 7 of reperfusion. The up-regulated expression of Gal-3 persisted from day 14 to 2 months after reperfusion. Double staining showed co-localization of Gal-3 with OX-42+ cells, glial fibrillary acidic protein (GFAP)+ and ED1+ cells, suggesting that activated microglia/infiltrating macrophages and activated astrocytes are the primary source of Gal-3 in the ischemic brain. In the in vitro setting, Gal-3 treatment dose-dependently stimulated the proliferation of endothelial cells and neural progenitors. Blockade of Gal-3 activity by infusing a neutralizing antibody against Gal-3 into the ischemic striatum decreased ischemia-induced angiogenesis and the proliferation of neural progenitors. These results suggest that Gal-3 expressed by activated microglia/infiltrating macrophages and astrocytes in the ischemic brain may play a role in post-ischemic tissue remodeling by enhancing angiogenesis and neurogenesis.

MicroRNAs (miRNAs) are ∼22 nucleotides long, noncoding RNAs that control cellular function by either degrading mRNAs or arresting their translation. To understand their functional significance in ischemic pathophysiology, we profiled miRNAs in adult rat brain as a function of reperfusion time after transient middle cerebral artery occlusion. Of the 238 miRNAs evaluated, 8 showed increased and 12 showed decreased expression at least at 4 out of 5 reperfusion time points studied between 3 h and 3 days compared with sham. Of those, 17 showed > 5 fold change. Bioinformatics analysis indicated a correlation between miRNAs altered to several mRNAs known to mediate inflammation, transcription, neuroprotection, receptors function, and ionic homeostasis. Antagomir-mediated prevention of mir-145 expression led to an increased protein expression of its downstream target superoxide dismutase-2 in the postischemic brain. In silico analysis showed sequence complementarity of eight miRNAs induced after focal ischemia to 877 promoters indicating the possibility of noncoding RNA-induced activation of gene expression. The mRNA expression of the RNases Drosha and Dicer, cofactor Pasha, and the pre-miRNA transporter exportin-5, which modulate miRNA biogenesis, were not altered after transient middle cerebral artery occlusion. Thus, the present studies indicate a critical role of miRNAs in controlling mRNA transcription and translation in the postischemic brain.

Proper CNS function depends on concerted expression of thousands of genes in a controlled and timely manner. Traumatic brain injury (TBI) in humans results in neuronal death and neurological dysfunction, which might be mediated by altered expression of several genes. By employing a CNS-specific GeneChip and real time polymerase chain reaction (PCR), the study analyzed the gene expression changes. The findings of necrosis and apoptosis can help to improve the wound age estimation after TBI in legal medicine.

Acute injuries to CNS such as stroke induce neural progenitor proliferation in adult brain which might be an endogenous attempt to self-repair. This process is known to be altered by several exogenous and endogenous modulators including growth factors that could help to reinforce the post-stroke neurogenesis. Increasing the neurogenesis may be a future therapeutic option to decrease the cognitive and behavioral deficits following stroke. In addition, transplantation of various types of stem cells into the injured brain is currently thought to be an exciting option to replace the neurons lost in the post-ischemic brain. These include immortalized stem cell lines, neural progenitors prepared from embryonic and adult animals and mesenchymal stem cells. Using exogenous stem cells in addition to modulating endogenous neurogenesis, we may be able to repair the injured brain after a devastating stroke. This article reviewed the current literature of these two issues.

Type-2 diabetes is an adult onset condition that affects millions of people worldwide. The ensuing hyperglycemia renders multiple organs to various complications and increases the risk of learning and memory impairment. The Goto-Kakizaki (GK) rat developed from normoglycemic Wistar-Kyoto (WKY) rat is a model for type-2 diabetes, with insulin resistance developing around 12 weeks of age. We presently analyzed the neural progenitor proliferation and survival of the newly generated cells in the dentate gyrus (DG) and the subventricular zone (SVZ) of 6 and 18 week-old GK and WKY rats. At 6 weeks of age, both GK and WKY cohorts showed similar blood glucose levels (112 ± 14 mg/dL) and similar rates of neural progenitor proliferation. At 18 weeks of age, the GK rats showed significantly increased blood glucose levels (by 92 ± 12%; p

Peroxisome proliferator-activated receptor (PPAR)-gamma is a ligand-activated transcription factor of nuclear hormone receptor superfamily. Thiazolidinedione rosiglitazone is a potent agonist of PPARgamma which was shown to induce neuroprotection in animal models of focal ischemia and spinal cord injury. We currently evaluated the therapeutic potential of rosiglitazone (6 mg/kg at 5 min, 6 h and 24 h; i.p.) following controlled cortical impact (CCI)-induced traumatic brain injury (TBI) in adult mice. CCI injury increased the cortical PPARgamma mRNA levels which were further elevated by rosiglitazone treatment. In addition, rosiglitazone treatment significantly decreased the cortical lesion volume measured at 7 days compared to vehicle treatment (by 56+/-7%; p

Stroke (focal cerebral ischemia) is a leading cause of death and disability among adult population. Many pathological events including inflammation and oxidative stress during the acute period contributes to the secondary neuronal death leading the neurological dysfunction after stroke. Transcriptional regulation of genes that promote these pathophysiological mechanisms can be an effective strategy to minimize the poststroke neuronal death. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors known to be upstream to many inflammatory and antioxidant genes. The goal of this review is to discuss the therapeutic potential and putative mechanisms of neuroprotection following PPAR activation after stroke.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors of the nuclear hormone receptor superfamily. The 3 PPAR isoforms (alpha, delta/beta and gamma) are known to control many physiological functions including glucose absorption, lipid balance, and cell growth and differentiation. Of interest, PPAR-gamma activation was recently shown to mitigate the inflammation associated with chronic and acute neurological insults. Particular attention was paid to test the therapeutic potential of PPAR agonists in acute conditions like stroke, spinal cord injury (SCI) and traumatic brain injury (TBI), in which massive inflammation plays a detrimental role. While 15d-prostaglandin J2 (15d PGJ2) is the natural ligand of PPAR-gamma, the thiazolidinediones (TZDs) are potent exogenous agonists. Due to their insulin-sensitizing properties, 2 TZDs rosiglitazone and pioglitazone are currently FDA-approved for type-2 diabetes treatment. Recent studies from our laboratory and other groups have shown that TZDs induce significant neuroprotection in animal models of focal ischemia and SCI by multiple mechanisms. The beneficial actions of TZDs were observed to be both PPAR-gamma-dependent as well as - independent. The major mechanism of TZD-induced neuroprotection seems to be prevention of microglial activation and inflammatory cytokine and chemokine expression. TZDs were also shown to prevent the activation of pro-inflammatory transcription factors at the same time promoting the anti-oxidant mechanisms in the injured CNS. This review article discusses the multiple mechanisms of TZD-induced neuroprotection in various animal models of CNS injury with an emphasis on stroke.