Kellie K. Bowen

Transient Focal Ischemia Induces Extensive Temporal Changes in Rat Cerebral MicroRNAome

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.

Thiazolidinedione Class of Peroxisome Proliferator-Activated Receptor γ Agonists Prevents Neuronal Damage, Motor Dysfunction, Myelin Loss, Neuropathic Pain, and Inflammation after Spinal Cord Injury in Adult Rats

Thiazolidinediones (TZDs) are potent synthetic agonists of the ligand-activated transcription factor peroxisome proliferator-activated receptor-γ (PPARγ). 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-1β 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 PPARγ antagonist, 2-chloro-5-nitro-N-phenyl-benzamide (GW9662), prevented the neuroprotection induced by pioglitazone.

Peroxisome proliferator-activated receptor-γ agonists induce neuroprotection following transient focal ischemia in normotensive, normoglycemic as well as hypertensive and type-2 diabetic rodents

Thiazolidinediones (TZDs) are synthetic agonists of the ligand‐activated transcription factor peroxisome proliferator‐activated receptor‐γ (PPARγ). 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 × receptor agonist 9‐cis‐retinoic acid, but was prevented by PPARγ 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‐1β, interleukin‐6, macrophage inflammatory protein‐1α, monocyte chemoattractant protein‐1, cyclooxygenase‐2, inducible nitric oxide synthase, early growth response‐1, CCAAT/enhancer binding protein‐β 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, PPARγ activation with TZDs might be a potent therapeutic option for preventing inflammation and neuronal damage after stroke with promise in diabetic and hypertensive subjects.

Gene expression analysis of spontaneously hypertensive rat cerebral cortex following transient focal cerebral ischemia

Identification of novel modulators of ischemic neuronal death helps in developing new strategies to prevent the stroke‐induced neurological dysfunction. Hence, the present study evaluated the gene expression changes in rat cerebral cortex at 6 and 24 h of reperfusion following transient middle cerebral artery occlusion (MCAO) by GeneChip® analysis. Transient MCAO resulted in selective increased mRNA levels of genes involved in stress, inflammation, transcription and plasticity, and decreased mRNA levels of genes which control neurotransmitter function and ionic balance. In addition to a number of established ischemia‐related genes, many genes not previously implicated in transient focal ischemia‐induced brain damage [suppressor of cytokine signaling (SOCS)‐3, cAMP responsive element modulator (CREM), cytosolic retinol binding protein (CRBP), silencer factor‐B, survival motor neuron (SMN), interferon‐γ regulatory factor‐1 (IRF‐1), galanin, neurotrimin, proteasome subunit RC8, synaptosomal‐associated protein (SNAP)‐25 A and B, synapsin 1a, neurexin 1‐β, ras‐related rab3, vesicular GABA transporter (VGAT), digoxin carrier protein, neuronal calcium sensor‐1 and neurodap] were observed to be altered in the ischemic cortex. Real‐time PCR confirmed the GeneChip® results for several of these transcripts. SOCS‐3 is a gene up‐regulated after ischemia which modulates inflammation by controlling cytokine levels. Antisense knockdown of ischemia‐induced SOCS‐3 protein expression exacerbated transient MCAO‐induced infarct volume assigning a neuroprotective role to SOCS‐3, a gene not heretofore implicated in ischemic neuronal damage.

Stroke-induced progenitor cell proliferation in adult spontaneously hypertensive rat brain: effect of exogenous IGF-1 and GDNF.

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 < 0.05) and the SVZ (by twofold, p < 0.05). DG showed increased proliferation only in the first week of reperfusion and 49% of the cells formed in this period survived to the end of third week. Whereas, SVZ showed a continuous proliferation up to 3 weeks after MCAO, but the cells formed survived for less than a week. In both DG and SVZ, at the end of the first week of reperfusion, majority of the BrdU‐positive (BrdU+) cells were immature neurons (DCX positive). In the DG, 28% of the cells formed in the first week after MCAO mature into neurons (NeuN positive). The ischemic cortex and striatum showed several BrdU+ cells which were ED‐1 positive microglia/macrophages. At 1 week of reperfusion, MCAO‐induced progenitor cell proliferation in the ipsilateral DG was significantly increased by i.c.v. infusion of IGF‐1 (by 127 ± 14%, p < 0.05) and GDNF (by 91 ± 5%, p < 0.05), compared to vehicle. In the growth factor treated rats subjected to transient MCAO, several BrdU+ cells formed in the first week survived up to the third week.

Traumatic brain injury leads to increased expression of peripheral-type benzodiazepine receptors, neuronal death, and activation of astrocytes and microglia in rat thalamus

In mammalian CNS, the peripheral-type benzodiazepine receptor (PTBR) is localized on the outer mitochondrial membrane within the astrocytes and microglia. PTBR transports cholesterol to the site of neurosteroid biosynthesis. Several neurodegenerative disorders were reported to be associated with increased densities of PTBR. In the present study, we evaluated the changes in the PTBR density and gene expression in the brains of rats as a function of time (6 h to 14 days) after traumatic brain injury (TBI). Sham-operated rats served as control. Between 3 and 14 days after TBI, there was a significant increased in the binding of PTBR antagonist [(3)H]PK11195 (by 106 to 185%, P < 0.01, as assessed by quantitative autoradiography and in vitro filtration binding) and PTBR mRNA expression (by 2- to 3. 4-fold, P < 0.01, as assessed by RT-PCR) in the ipsilateral thalamus. At 14 days after the injury, the neuronal number decreased significantly (by 85 to 90%, P < 0.01) in the ipsilateral thalamus. At the same time point, the ipsilateral thalamus also showed increased numbers of the glial fibrillary acidic protein positive cells (astrocytes, by approximately 3.5-fold) and the ED-1 positive cells (microglia/macrophages, by approximately 36-fold), the two cell types known to be associated with PTBR. Increased PTBR expression following TBI seems to be associated with microglia/macrophages than astrocytes as PTBR density at different periods after TBI correlated better with the number of ED-1 positive cells (r(2) = 0.95) than the GFAP positive cells (r(2) = 0.56). TBI-induced increased PTBR expression is possibly an adaptive response to cellular injury and may play a role in the pathophysiology of TBI.

Putative endogenous mediators of preconditioning-induced ischemic tolerance in rat brain identified by genomic and proteomic analysis

In brain, a brief ischemic episode induces protection against a subsequent severe ischemic insult. This phenomenon is known as preconditioning‐induced neural ischemic tolerance. An understanding of the molecular mechanisms leading to preconditioning helps in identifying potential therapeutic targets for preventing the post‐stroke brain damage. The present study conducted the genomic and proteomic analysis of adult rat brain as a function of time following preconditioning induced by a 10‐min transient middle cerebral artery (MCA) occlusion. GeneChip analysis showed induction of 40 putative neuroprotective transcripts between 3 to 72 h after preconditioning. These included heat‐shock proteins, heme oxygenases, metallothioneins, signal transduction mediators, transcription factors, ion channels and apoptosis/plasticity‐related transcripts. Real‐time PCR confirmed the GeneChip data for the transcripts up‐regulated after preconditioning. Two‐dimensional gel electrophoresis combined with MALDI‐TOF analysis showed increased expression of HSP70, HSP27, HSP90, guanylyl cyclase, muskelin, platelet activating factor receptor and β‐actin at 24 h after preconditioning. HSP70 protein induction after preconditioning was localized in the cortical pyramidal neurons. The infarct volume induced by focal ischemia (1‐h MCA occlusion) was significantly smaller (by 38 ± 7%, p < 0.05) in rats subjected to preconditioning 3 days before the insult. Preconditioning also prevented several gene expression changes induced by focal ischemia.

JAK2 and STAT3 activation contributes to neuronal damage following transient focal cerebral ischemia.

Increased levels of interleukin‐6 (IL‐6) play a role in post‐ischemic cerebral inflammation. IL‐6 binding to its receptors induces phosphorylation of the receptor associated janus kinases (JAKs), and the down‐stream signal transducer and activator of transcription (STAT) family of transcription factors, which amplify the IL‐6 signal transduction. We evaluated the functional significance of JAK2 and STAT3 activation in focal ischemia‐induced neuronal damage. Transient middle cerebral artery occlusion in adult rats led to increased JAK2 and STAT3 phosphorylation in the ipsilateral cortex and striatum after 6–72 h of reperfusion. Fluorescent immunohistochemistry with cell specific markers (NeuN for neurons, glial fibrillary acidic protein for reactive astrocytes and ED1/OX42 for activated macrophages/microglia) showed that both pJAK2 and pSTAT3 staining is predominantly localized in the macrophages/microglia in the post‐ischemic brain. Intracerebroventricular infusion of rats with AG490 (a JAK2 phosphorylation inhibitor) prevented the post‐ischemic JAK2 and STAT3 phosphorylation and significantly decreased the infarct volume, number of apoptotic cells and neurological deficits, compared to vehicle control. Furthermore, intracerebral injection of siRNA specific for STAT3 led to curtailed STAT3 mRNA expression and phosphorylation, decreased infarct volume, fewer apoptotic cells and improved neurological function following transient middle cerebral artery occlusion. These studies show that JAK2‐STAT3 activation plays a role in post‐ischemic brain damage.

Inhibition of Intercellular Adhesion Molecule-1 Protein Expression by Antisense Oligonucleotides Is Neuroprotective After Transient Middle Cerebral Artery Occlusion in Rat

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 <0.05; 226±35 mm3 in control ODN group and 104±27 mm3 in antisense ODN group; n=8 each) and mean neurological deficit score (by 44%; P <0.05; 2.4 in control ODN group and 1.3 in antisense ODN group; n=8 each). Neither control nor antisense ODN had any effect on mean arterial blood pressure and the physiological parameters monitored during MCAO. Compared with noninfused control, intracerebroventricular infusion of artificial cerebrospinal fluid or antisense or sense ODN had no significant effect on the regional cerebral blood flow changes that accompanied ischemia and reperfusion. 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.

Neuroprotection by memantine, a non-competitive NMDA receptor antagonist after traumatic brain injury in rats.

This study investigated whether memantine, a non-competitive NMDA receptor antagonist is neuroprotective after traumatic brain injury (TBI) induced in adult rats with a controlled cortical impact device. TBI led to significant neuronal death in the hippocampal CA2 and CA3 regions (by 50 and 59%, respectively), by 7 days after the injury. Treatment of rats with memantine (10 and 20 mg/Kg, i.p.) immediately after the injury significantly prevented the neuronal loss in both CA2 and CA3 regions. This is the first study showing the neuroprotective potential of memantine to prevent the TBI-induced neuronal damage.