Raghu Vemuganti

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.

Mechanisms of anti-inflammatory and neuroprotective actions of PPAR-gamma agonists.

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.

Monocyte Chemoattractant Protein-1 Plays a Critical Role in Neuroblast Migration after Focal Cerebral Ischemia

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.

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.

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.

Role of transcription factors in mediating post-ischemic cerebral inflammation and brain damage

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.

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.

Repairing brain after stroke : A review on post-ischemic neurogenesis

Stroke is devastating as currently no therapies are available that can prevent stroke-induced neurological dysfunction in humans. With the recent observations that acute insults to adult brain stimulate new neuronal formation in various species of animals, optimism is building for a possible regeneration of stroke-damaged brain. This article reviewed the advances in the understanding of the molecular mechanisms of the various steps of neurogenesis with an emphasis on the endogenous mediators and exogenous promoters of neural progenitor proliferation, migration and survival in the post-ischemic adult brain.

PPARγ agonist rosiglitazone is neuroprotective after traumatic brain injury via anti-inflammatory and anti-oxidative mechanisms

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<0.05; n=6/group). Following TBI, the spared cortex of the rosiglitazone group showed significantly less numbers of GSI-B4(+) activated microglia/macrophages and ICAM1(+) capillaries, and curtailed induction of pro-inflammatory genes IL6, MCP1 and ICAM1 compared to vehicle group. Rosiglitazone-treated mice also showed significantly less number of TUNEL(+) apoptotic neurons and curtailed induction of caspase-3 and Bax, compared to vehicle control. In addition, rosiglitazone significantly enhanced the post-TBI expression of the neuroprotective chaperones HSP27, HSP70 and HSP32/HO1, and the anti-oxidant enzymes catalase, Cu/Zn-SOD and Mn-SOD, compared to vehicle. Treatment with GW9662 (a specific PPARgamma antagonist) prevented all the above PPARgamma-mediated actions. Thus, PPARgamma activation confers neuroprotection after TBI by anti-inflammatory, anti-apoptotic and anti-oxidative mechanisms.