Kudret Türeyen

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 acidu2003×u2003receptor 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, pu2003<u20030.05) and the SVZ (by twofold, pu2003<u20030.05). 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 3u2003weeks 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 1u2003week of reperfusion, MCAO‐induced progenitor cell proliferation in the ipsilateral DG was significantly increased by i.c.v. infusion of IGF‐1 (by 127u2003±u200314%, pu2003<u20030.05) and GDNF (by 91u2003±u20035%, pu2003<u20030.05), 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.

Infarct volume quantification in mouse focal cerebral ischemia: a comparison of triphenyltetrazolium chloride and cresyl violet staining techniques

Triphenyltetrazolium chloride (TTC) and cresyl violet (CV) staining are routinely used methods to determine cerebral infarct volume and area. In this study, we compared these staining techniques using the mouse middle cerebral artery occlusion (MCAO) model of focal ischemia. Male C57BL6 mice were subjected to a 90 min transient MCAO and sacrificed at 24 h reperfusion. Sham operated mice served as controls. Two millimeters coronal brain slices were cut at +1.3, -0.7, -2.7 and -4.7 mm from bregma. The sections were stained with 2% TTC for 20 min and the caudal face of each slice was scanned with a flatbed scanner. The sections were kept in 4% paraformaldehyde solution for 4 weeks (the solution was changed every week). The slices were cryosectioned (40 microm thick), mounted on slides and stained with CV and scanned. The infarct volume and area were measured by the image-J program for both the staining techniques. There was no significant difference in either infarct area or volume between the TTC and CV stained sections (P > 0.05). TTC and CV staining showed a high degree of correlation in infarct area and volume indicating that both methods are suitable for producing accurate measurements of cerebral experimental infarcts.

Decreased brain damage and curtailed inflammation in transcription factor CCAAT/enhancer binding protein β knockout mice following transient focal cerebral ischemia

CCAAT/enhancer binding protein β (C/EBPβ) 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/EBPβ. We report that focal cerebral ischemia induced by transient middle cerebral artery occlusion (MCAO) significantly increases C/EBPβ gene expression in mouse brain at between 6 and 72u2003h of reperfusion. To understand the functional significance of C/EBPβ in postischemic inflammation and brain damage, we induced transient MCAO in cohorts of adult C/EBPβ null mice and their wild‐type littermates. At 3u2003days of reperfusion following transient MCAO, C/EBPβ 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/EBPβ–/– mice compared with C/EBPβ+/+ mice. These results suggest a significant role for C/EBPβ in postischemic inflammation and brain damage.

EGF and FGF-2 infusion increases post-ischemic neural progenitor cell proliferation in the adult rat brain.

OBJECTIVE: Epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) play a critical role in neurogenesis. In the present study, we evaluated the additive effect of administering these two factors on post-ischemic progenitor cell proliferation, survival, and phenotypic maturation in the hippocampal dentate gyrus (DG) and the subventricular zone (SVZ) in the adult rat brain after transient middle cerebral artery occlusion. METHODS: A combination of EGF+FGF-2 (each 1.44 ng/d) was continuously administered into the lateral ventricles for 3 days, 5-bromodeoxyuridine (BrdUrd) was injected (50 mg/Kg) twice daily for 3 days starting on Day 1 of reperfusion, and cohorts of rats were sacrificed on Day 5 and Day 21 of reperfusion. RESULTS: Compared with sham controls, ischemic rats showed a significantly higher number of newly proliferated cells in both the DG (by 766 ± 37%, P < 0.05) and the SVZ (by 650 ± 43%, P < 0.05). Of the progenitor cells proliferated on Day 5 after ischemia, 41 ± 6% in the DG and 28 ± 5% in the SVZ survived to 3 weeks. Compared with vehicle control, the EGF + FGF-2 infusion significantly increased the post-ischemic progenitor cell proliferation (by 319 ± 40%, P < 0.05 in the DG and by 366 ± 32%, P < 0.05 in the SVZ) and survival (by 40 ± 12%, P < 0.05 in the DG and by 522 ± 47%, P < 0.05 in the SVZ) studied at 5 and 21 days, respectively. Furthermore, of the newly proliferated cells survived to 3 weeks after ischemia, EGF + FGF-2 infusion caused a significantly higher number of neuronal nuclear protein-BrdUrd double-positive mature neurons in the DG (46 ± 9%, P < 0.05) compared with vehicle control. Neuronal nuclear protein and BrdUrd double-positive mature neurons were also found in the DG. Glial fibrillary acidic protein-positive astrocytes did not show double-positive staining in either region. CONCLUSION: Specific growth factor infusion enhances post-ischemic progenitor cell proliferation by 5 days of reperfusion and neuronal maturation by 21 days of reperfusion in both the DG and SVZ in the adult rat brain.

Exacerbated Brain Damage, Edema and Inflammation in Type-2 Diabetic Mice Subjected to Focal Ischemia

J. Neurochem. (2011) 116, 499–507.

B1 and TRPV-1 receptor genes and their relationship to hyperalgesia following spinal cord injury.

Study Design. Laboratory investigation of pain behavior following spinal cord injury. Objective. To explore changes in the spinal cord expression of nociceptive genes following spinal cord injury (SCI) as they relate to the manifestation of pain behavior in rats. Summary of Background Data. Neuropathic pain following SCI is common, disabling, and largely untreatable. In peripheral nerve injury models, bradykinin B1 and vanilloid 1 (TRPV-1) receptor activity is associated with neuropathic pain behavior. We sought to examine the role of these gene products in SCI-mediated pain. Methods. Rats were subjected to SCI using the MASCIS impactor. Animals were tested preinjury and at regular intervals postinjury for the appearance of thermal hyperalgesia using a hind limb withdrawal latency test. The expression of B1 and TRPV-1 genes was assessed using real-time polymerase chain reaction. Immunohistochemistry was used to localize the B1 and TRPV-1 receptors within the spinal cord. Results. Greater than twofold increases in the expression of the B1 and TRPV-1 genes were detected in the injured region of the spinal cord in animals exhibiting hyperalgesia compared with animals with SCI that did not display hyperalgesia. Immunohistochemical staining revealed that both receptor types were largely localized to the dorsal horn. Staining for TRPV-1 receptors decreased while that for B1 receptors increased in all of the injured animals when compared with sham-operated controls. Conclusion. B1 and TRPV-1 receptor genes are overexpressed in the injured spinal cord of animals manifesting thermal hyperalgesia following SCI compared with similarly injured animals without hyperalgesia. This finding is consistent with past work regarding the role of these receptors in nociception and indicates that ongoing modifiable processes are occurring in the spinal cord that lead to clinical pain syndromes.

Increased angiogenesis and angiogenic gene expression in carotid artery plaques from symptomatic stroke patients.

OBJECTIVE:Carotid plaque rupture is one of the main causes of stroke by creating cerebral emboli. The biochemical, molecular, and structural factors that promote carotid plaque rupture are not yet understood in detail. We hypothesize that increased microvascular blood flow within a carotid plaque might fissure the plaque, elevate local pressure, and promote plaque rupture. The aim of this study is to determine the role of angiogenesis and angiogenesis-related gene expression in symptomatic carotid plaque. METHODS:The present study evaluated the new vessel formation (using hematoxylin-eosin staining and CD34 immunohistochemistry) and angiogenic gene expression (using microarray and real-time polymerase chain reaction analysis) in carotid plaque specimens obtained during endarterectomy from 13 symptomatic stroke patients in comparison with eight asymptomatic patients. RESULTS:Symptomatic plaques showed significantly higher new vessel density in the fibrous cap (by 347%, P < 0.05) as well as in the plaque proper (by 196%, P < 0.05) compared with the asymptomatic plaques. The fibrous caps of the plaques were threefold thinner in the symptomatic patients when compared with the asymptomatic patients. In symptomatic plaque, gene expression analysis showed increased abundance of 31 transcripts known to promote angiogenesis and cell division compared with plaques of asymptomatic patients. CONCLUSION:This study suggests that angiogenic gene expression and the ensuing angiogenesis in the plaques might contribute to their destabilization and resulting symptoms.

Transcription factor early growth response-1 induction mediates inflammatory gene expression and brain damage following transient focal ischemia

Early growth response‐1 (Egr1) is a sequence‐specific transcription factor (TF) which is induced under hypoxic conditions. We presently report that transient middle cerebral artery occlusion (MCAO) leads to increased expression of Egr1 in the brains of adult mice and rats between 2u2003h and 5u2003days of reperfusion with a peak increase of 8–12‐fold at 1u2003day. When subjected to transient MCAO and 3u2003days of reperfusion, Egr1−/− mice showed significantly smaller infarcts (by 44.9u2003±u20038.4%, pu2003<u20030.05) and improved neurological function than Egr1+/+ littermates. Following transient MCAO, brains of Egr1−/− mice showed less water accumulation and decreased neutrophil infiltration (by 42u2003±u20038%, pu2003<u20030.05) compared to Egr1+/+ mice. The number of activated microglia/macrophages were also significantly lower (OX42+ cells by 53u2003±u20039%, pu2003<u20030.05 and ED1+ cells by 59u2003±u200311%) in the post‐ischemic cortex of Egr1−/− mice compared to Egr1+/+ mice. In addition, post‐ischemic inflammatory gene expression was less pronounced in the brains of Egr1−/− mice compared to Egr1+/+ mice. Preventing cerebral Egr1 protein induction with small interference RNAs that target Egr1 decreased inflammatory gene expression and led to smaller infarcts (by 40.2u2003±u20036.9%, pu2003<u20030.05) and reduced neurological deficits in rats subjected to transient MCAO. Conversely, transient MCAO following adenoviral‐mediated Egr1 over‐expression exacerbated the infarct volume (by 29u2003±u20035.3%, pu2003<u20030.05) and worsened the neurological deficits in rats. These studies indicate Egr1 as a significant contributor of inflammation and neuronal damage after stroke.

Ideal suture diameter is critical for consistent middle cerebral artery occlusion in mice.

OBJECTIVE: The use of transgenic and knockout mice has led to a need for a consistent model of mouse transient focal cerebral ischemia. In a great majority of the published mouse middle cerebral artery (MCA) occlusion studies, the methods indicated the type of intraluminal suture used without indicating the actual suture diameter after modification. We attempted to determine the ideal suture diameter to produce consistent occlusion in the MCA of adult male C57BL/6 mice. METHODS: Suture tips were coated to a depth 4 mm with glue, and 6-0 sutures of eight different, precisely measured diameters were produced. The coated 6-0 sutures in different diameters were introduced 10 mm into the internal carotid artery via the external carotid artery of the mice to produce MCA occlusion (n = 40; five animals for each diameter), and the mice (22–24 g) were transaortically perfused with saline. The base of the brain was exposed, and photographs of the vessels were obtained before and after transaortic injection of Evans blue dye to determine the consistency of MCA occlusion for each suture diameter. Cerebral blood flow was measured 10 minutes before occlusion and 20 minutes after reperfusion, and 2,3,5-triphenyltetrazolium chloride staining was performed to demonstrate the ischemic damage in additional animals with 110-&mgr;m (n = 5) and 180-&mgr;m (n = 8) diameter sutures. RESULTS: Sutures measuring 170 &mgr;m and 180 &mgr;m in diameter consistently occluded the MCA of C57BL/6 mice. In addition, 2,3,5-triphenyltetrazolium chloride staining demonstrated consistent infarction with180-&mgr;m diameter sutures. The infarct volume was 36.3 ± 4.2 mm3. CONCLUSION: Small changes in the diameter of the occlusion suture tip affect consistency in the mouse MCA occlusion model.