n-hydroxy-n--(4-butyl-2-methylphenyl)formamidine and Brain-Ischemia

N-hydroxy-N-(4-butyl-2-methylphenyl) formamidine (HET0016) is a specific 20-hydroxyeicosatetraenoic acid (20-HETE) inhibitor which was first synthesized in 2001. It has been demonstrated that HET0016 reduces cerebral infarction volume in rat middle cerebral artery occlusion (MCAO) models. However, little is known about the role of HET0016 in the blood-brain barrier (BBB) dysfunction after cerebral ischemia/reperfusion (I/R) injury. The present study was designed to examine the effect of HET0016 in a MCAO and reperfusion rat model to determine whether it protects against brain edema and BBB disruption. Rats were subjected to 90 min MCAO, followed by 4, 24, 48, and 72 h reperfusion. Brain edema was measured according to the wet and dry weight method. BBB permeability based on the extravasation of Evans blue and sodium fluorescein was detected. BBB ultrastructure alterations were presented through transmission electron microscope. Superoxide production in ischemic tissue was also measured by dihydroethidium fluorescent probe. Western blot was used to analyze the expression of Claudin-5, ZO-1, MMP-9, and JNK pathway. At 24h after reperfusion, HET0016 reduced brain edema and BBB leakage. Ultrastructural damage of BBB and the increase of superoxide production were attenuated by HET0016 treatment. Western blot showed that HET0016 suppressed the activation of MMP-9 and JNK pathway but restored the expression of Claudin-5 and ZO-1. In conclusion, these results suggest that HET0016 protects BBB dysfunction after I/R by regulating the expression of MMP-9 and tight junction proteins. Furthermore, inhibition of oxidative stress and JNK pathway may be involved in this protecting effect.

Topics: Amidines; Animals; Blood-Brain Barrier; Brain Edema; Brain Ischemia; Claudin-5; Male; MAP Kinase Signaling System; Matrix Metalloproteinase 9; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Zonula Occludens-1 Protein

20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P450 metabolite of arachidonic acid that that contributes to infarct size following focal cerebral ischemia. However, little is known about the role of 20-HETE in global cerebral ischemia or neonatal hypoxia-ischemia (H-I). The present study examined the effects of blockade of the synthesis of 20-HETE with N-hydroxy-N'-(4-n-butyl-2-methylphenyl) formamidine (HET0016) in neonatal piglets after H-I to determine if it protects highly vulnerable striatal neurons. Administration of HET0016 after H-I improved early neurological recovery and protected neurons in putamen after 4 days of recovery. HET0016 had no significant effect on cerebral blood flow. cytochrome P450 4A immunoreactivity was detected in putamen neurons, and direct infusion of 20-HETE in the putamen increased phosphorylation of Na(+), K(+) -ATPase and NMDA receptor NR1 subunit selectively at protein kinase C-sensitive sites but not at protein kinase A-sensitive sites. HET0016 selectively inhibited the H-I induced phosphorylation at these same sites at 3 h of recovery and improved Na(+), K(+) -ATPase activity. At 3 h, HET0016 also suppressed H-I induced extracellular signal-regulated kinase 1/2 activation and protein markers of nitrosative and oxidative stress. Thus, 20-HETE can exert direct effects on key proteins involved in neuronal excitotoxicity in vivo and contributes to neurodegeneration after global cerebral ischemia in immature brain.

Topics: Amidines; Animals; Animals, Newborn; Brain Ischemia; Hydroxyeicosatetraenoic Acids; Infusions, Intraventricular; Male; Swine

Partial exchange transfusion with a cell-free hemoglobin (Hb) polymer during transient middle cerebral artery occlusion (MCAO) reduces infarct volume but fails to increase blood flow, as might be expected with the induced decrease in hematocrit. In ischemic brain, endothelin antagonists are known to produce vasodilation. In nonischemic brain, pial arterioles constrict after Hb exchange transfusion, and the constriction is blocked by an inhibitor of 20-HETE synthesis. We tested the hypothesis that a 20-HETE synthesis inhibitor and an endothelin A receptor antagonist increase pial arteriolar dilation after Hb exchange transfusion during MCAO. Pial arteriolar diameter was measured in the ischemic border region of the distal MCA border region through closed cranial windows in anesthetized rats subjected to the filament model of MCAO. During 2 h of MCAO, pial arteriolar dilation gradually subsided from 37 +/- 3 to 7 +/- 5% (+/-SE). Compared with residual dilation at 2 h of MCAO with vehicle superfusion (14 +/- 3%), loss of dilation was not prevented by superfusion of a 20-HETE synthesis inhibitor (21 +/- 5%), partial Hb exchange transfusion (7 +/- 5%) that decreased hematocrit to 23%, or a combination of the two (5 +/- 5%). However, loss of dilation was prevented by superfusion of an endothelin A receptor antagonist with (35 +/- 4%) or without (32 +/- 5%) Hb transfusion. Pial artery constriction during reperfusion was attenuated by HET0016 alone and by BQ610 with or without Hb transfusion. Systemic administration of the endothelin antagonist during prolonged MCAO increased blood flow in the border region. Thus loss of pial arteriolar dilation in the ischemic border region during prolonged MCAO depends on endothelin A receptor activation, and this effect was independent of the presence of cell-free Hb polymers in the plasma. In contrast to previous work in nonischemic brain, inhibition of oxygen-dependent 20-HETE synthesis does not significantly influence the pial arteriolar response to polymeric Hb exchange transfusion during focal ischemia.

Topics: Amidines; Animals; Arterioles; Blood Substitutes; Blood Transfusion; Brain Ischemia; Endothelin A Receptor Antagonists; Endothelins; Hemoglobins; Hydroxyeicosatetraenoic Acids; Male; Models, Animal; Oligopeptides; Pia Mater; Polymers; Rats; Rats, Wistar; Regional Blood Flow; Vasodilation

Cytochrome P450 metabolism of arachidonic acid produces the potent vasoconstrictive metabolite, 20-hydroxyeicosatetraenoic acid (20-HETE). Recent studies have implicated 20-HETE as a vasoconstrictive mediator in hemorrhagic stroke. The purpose of this study was to determine the effect of the 20-HETE inhibitor, HET0016, on lesion volume and cerebral blood flow (CBF) after temporary middle cerebral artery occlusion (MCAO) in rats. Plasma pharmacokinetics and tissue concentrations of HET0016 were determined after a 10 mg/kg intraperitoneal dose. Separate rats were treated with HET0016 or vehicle before 90 mins of MCAO. Lesion volume was assessed by 2,3,5-triphenyl-tetrazolium-chloride and cerebral flow was determined using laser Doppler flow. The effect of MCAO on in vitro microsomal formation of mono-oxygenated arachidonic acid metabolites was also determined. Results show that HET0016 has a short biologic half-life, distributes into the brain, and is associated with a 79.6% reduction in 20-HETE concentration in the cortex. Lesion volume was greatly reduced in HET0016-treated (9.1%+/-4.9%) versus vehicle-treated (57.4%+/-9.8%; n=6; P<0.001) rats. An attenuation of the observed decrease in CBF was observed in HET0016-treated (180 mins 89.2%+/-6.2%; 240 mins 88.1%+/-5.7% of baseline flow) versus vehicle control (180 mins 57.6%+/-19.0%; 240 mins 53.8%+/-20.0% of baseline flow; n=6; P<0.05). Brain cortical microsomal formation rate of 20-HETE was also reduced at 24 h in the ipsilateral hemisphere after MCAO. These data support a significant role for 20-HETE in the pathogenesis of ischemic stroke.

Topics: Amidines; Animals; Brain Injuries; Brain Ischemia; Cerebrovascular Circulation; Cytochrome P-450 Enzyme System; Hydroxyeicosatetraenoic Acids; Male; Microsomes; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Stroke; Vasoconstriction

This study examined the interaction between 5-hydroxytryptamine1B (5-HT1B) receptors and 20-hydroxyeiscosatetraenoic acid (20-HETE) in contributing to the acute fall in regional cerebral blood flow (rCBF) after subarachnoid hemorrhage (SAH) in rats.. The effects of intracisternal injection of 0.3 mL of arterial blood, artificial cerebrospinal fluid, and 5-HT on rCBF and the levels of 20-HETE and 5-HT in cerebrospinal fluid were measured in rats pretreated with vehicle, a 5-HT1B receptor antagonist (isamoltane hemifumarate), or an inhibitor of the synthesis of 20-HETE (HET0016). The effects of HET0016 and isamoltane on the vasoconstrictor response and changes in [Ca2+]i to 5-HT were also studied in middle cerebral arteries and vascular smooth muscle cells isolated from these vessels.. 20-HETE and 5-HT levels in cerebrospinal fluid rose from 172+/-10 to 629+/-44 ng/mL and from 6+/-4 to 1163+/-200 nmol/mL, respectively, after SAH. rCBF fell by 30% 10 minutes after SAH, and it remained at this level for the next 2 hours. Blockade of 5-HT1B receptors prevented the sustained fall in rCBF seen after SAH. Intracisternal injection of 5-HT mimicked SAH by increasing 20-HETE levels in cerebrospinal fluid to 475+/-94 ng/mL and reducing rCBF by 30%. Blockade of the synthesis of 20-HETE with HET0016 prevented the fall in rCBF produced by 5-HT. Isamoltane and HET0016 reduced the vasoconstrictor response of isolated MCA to 5-HT by >60% and diminished the rise in [Ca2+]i produced by 5-HT in vascular smooth muscle cells isolated from these arteries.. These results suggest that the release of 5-HT after SAH activates 5-HT1B receptors and the synthesis of 20-HETE and that 20-HETE contributes to the acute fall in rCBF by potentiating the vasoconstrictor response of cerebral vessels to 5-HT.

Topics: Amidines; Animals; Blood; Brain Ischemia; Calcium Signaling; Cerebrospinal Fluid; Cerebrovascular Circulation; Cisterna Magna; Eicosanoic Acids; Enzyme Inhibitors; Hydroxyeicosatetraenoic Acids; Injections; Male; Middle Cerebral Artery; Muscle, Smooth, Vascular; Phospholipases A; Propanolamines; Rats; Rats, Sprague-Dawley; Receptor, Serotonin, 5-HT1B; Receptors, Serotonin; Serotonin; Serotonin Antagonists; Subarachnoid Hemorrhage; Vascular Resistance; Vasoconstriction; Vasospasm, Intracranial