Regulation of enhanced cerebrovascular expression of proinflammatory mediators in experimental subarachnoid hemorrhage via the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway

Background: Subarachnoid hemorrhage (SAH) is associated with high morbidity and mortality. It is suggested that the associated inflammation is mediated through activation of the mitogen-activated protein kinase (MAPK) pathway which plays a crucial role in the pathogenesis of delayed cerebral ischemia after SAH. The aim of this study was first to investigate the timecourse of altered expression of proinflammatory cytokines and matrix metalloproteinase in the cerebral arteries walls following SAH. Secondly, we investigated whether administration of a specific mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, U0126, given at 6 h after SAH prevents activation of the MEK/extracellular signal-regulated kinase 1/2 pathway and the upregulation of cerebrovascular inflammatory mediators and improves neurological function. Methods: SAH was induced in rats by injection of 250 mu l of autologous blood into basal cisterns. U0126 was given intracisternally using two treatment regimens: (A) treatments at 6, 12, 24 and 36 h after SAH and experiments terminated at 48 h after SAH, or (B) treatments at 6, 12, and 24 h after SAH and terminated at 72 h after SAH. Cerebral arteries were harvested and interleukin (IL)-6, IL-1 beta, tumor necrosis factor alpha (TNF)alpha, matrix metalloproteinase (MMP)-9 and phosphorylated ERK1/2 (pERK1/2) levels investigated by immunohistochemistry. Early activation of pERK1/2 was measured by western blot. Functional neurological outcome after SAH was also analyzed. Results: Expression levels of IL-1 beta, IL-6, MMP-9 and pERK1/2 proteins were elevated over time with an early increase at around 6 h and a late peak at 48 to 72 h post-SAH in cerebral arteries. Enhanced expression of TNF alpha in cerebral arteries started at 24 h and increased until 96 h. In addition, SAH induced sensorimotor and spontaneous behavior deficits in the animals. Treatment with U0126 starting at 6 h after SAH prevented activation of MEK-ERK1/2 signaling. Further, U0126 significantly decreased the upregulation of inflammation proteins at 48 and 72 h following SAH and improved neurological function. We found no differences between treatment regimens A and B. Conclusions: These results show that SAH induces early activation of the MEK-ERK1/2 pathway in cerebral artery walls, which is associated with upregulation of proinflammatory cytokines and MMP-9. Inhibition of the MEK-ERK1/2 pathway by U0126 starting at 6 h post-SAH prevented upregulation of cytokines and MMP-9 in cerebral vessels, and improved neurological outcome

Expressional Changes in Cerebrovascular Receptors after Experimental Transient Forebrain Ischemia

Background: Global ischemic stroke is one of the most prominent consequences of cardiac arrest, since the diminished blood flow to the brain results in cell damage and sometimes permanently impaired neurological function. The post-arrest period is often characterised by cerebral hypoperfusion due to subacute hemodynamic disturbances, the pathophysiology of which are poorly understood. In two other types of stroke, focal ischemic stroke and subarachnoid hemorrhage, it has earlier been demonstrated that the expression of certain vasoconstrictor receptors is increased in cerebral arteries several days after the insult, a phenomenon that leads to increased contraction of cerebral arteries, reduced perfusion of the affected area and worsened ischemic damage. Based on these findings, the aim of the present study was to investigate if transient global cerebral ischemia is associated with upregulation of vasoconstrictive endothelin and 5-hydroxytryptamine receptors in cerebral arteries. Experimental transient forebrain ischemia of varying durations was induced in male wistar rats, followed by reperfusion for 48 hours. Neurological function was assessed daily by three different tests and cerebrovascular expression and contractile function of endothelin and 5-hydroxytryptamine receptors were evaluated by wire myography, immunohistochemistry and western blotting. Results: Transient forebrain ischemia induced neurological deficits as well as functional upregulation of vasoconstrictive ETB and 5-HT1B receptors in cerebral arteries supplying mid-and forebrain regions. No receptor upregulation was seen in arteries supplying the hindbrain. Immunohistochemical stainings and western blotting demonstrated expressional upregulation of these receptor subtypes in the mid-and forebrain arteries and confirmed that the receptors were located in the smooth muscle layer of the cerebral arteries. Conclusions: This study reveals a new pathophysiological aspect of global ischemic stroke, namely expressional upregulation of vasoconstrictor receptors in cerebral arteries two days after the insult, which might contribute to cerebral hypoperfusion and delayed neuronal damage after cardiac arrest

CaMKII inhibition with KN93 attenuates endothelin and serotonin receptor-mediated vasoconstriction and prevents subarachnoid hemorrhage-induced deficits in sensorimotor function

It has been suggested that transcriptional upregulation of cerebral artery contractile endothelin (ETB) and 5-hydroxytryptamine (5-HT1B) receptors play an important role in the development of late cerebral ischemia and increased vasoconstriction after subarachnoid hemorrhage (SAH). We tested the hypothesis that inhibition of calcium calmodulin-dependent protein kinase II (CaMKII) may reduce cerebral vasoconstriction mediated by endothelin and serotonin receptors and improve neurological outcome after experimental SAH

ET_B and 5-HT_1B receptor expression determined by western blotting.

<p>Representative western blots and band intensity quantifications showing endothelin type B (ET_B) protein expression in basilar artery (BA) (<b>A</b>) and in pooled middle cerebral artery (MCA) and anterior cerebral artery (ACA) tissue (<b>B</b>), and 5-hydroxotryptamin type 1B (5-HT_1B) protein expression in BA (<b>C</b>) and in pooled MCA and ACA tissue (<b>D</b>) from control-operated (sham) rats and rats subjected to 15 minutes of transient forebrain ischemia. The band recognised by the ET_B receptor antibody was approximately 48 kDa and the band recognised by the 5-HT1B receptor antibody was approximately 41 kDa. Actin was used as a loading control. Data are expressed as mean ± SEM percentages of the mean band intensity in sham-operated animals and student’s t-test was used for statistical comparison. n = 5–13. *p<0.05.</p

Summary of data for neurological function, contractile function and protein expression levels of cerebrovascular ET_B and 5-HT_1B receptors as a function of the duration of the ischemic insult.

<p>Data for endothelin type B (ET_B) and 5-hydroxotryptamin type 1B (5-HT_1B) receptor-mediated contractile function represent E_max values for the first phase (E_max-1) of the biphasic concentration-contraction curves for anterior cerebral arteries (ACA) stimulated with endothelin-1 (ET-1) and 5-carboxamidotryptamine (5-CT), respectively. Data for ET_B and 5-HT_1B protein expression levels represent receptor subtype-specific immunohistochemical staining intensities in ACAs. Neurology score data represents pooled data from grip strength and rotating pole. All data are values for ischemia-induced rats (2, 10 or 15 minutes) terminated 48 hours after ischemia in percentages of corresponding values for control-operated (sham) rats. All data are expressed as mean ± SEM.</p

5-HT receptor immunohistochemitry.

<p>Photomicrographs showing immunohistochemical stainings of anterior cerebral artery (ACA) sections with antibodies against 5-hydroxotryptamin type 1B (5-HT_1B) (<b>A and B</b>) or 5-hydroxotryptamin type 2A (5-HT_2A) (<b>D and E</b>) receptors. Bar graphs show quantifications of staining intensities for 5-HT_1B (<b>C</b>) and 5-HT_2A (<b>F</b>) receptors. Two sections from each of 6 to 12 animals were analyzed. Data are presented as mean ± SEM in percentages of the mean staining intensity in control-operated (sham) animals. Significant differences between sham-operated and 15 minutes ischemia rats were determined using student’s t-test.</p

Physiological Parameters.

<p>Mean arterial blood pressure (MABP), pH, CO₂ and O₂ pressure (p) body temperature (T) and cranial temperature of animals subjected or not (sham) to 2, 10 or 15 minutes of transient forebrain ischemia. Values are means ± SEM, n = 7–17 rats in each group.</p