Src Kinase Inhibition Improves Acute Outcomes After Experimental Intracerebral Hemorrhage

Abstract

Background and Purpose-The mechanisms by which intracerebral hemorrhages produce changes of blood flow and metabolism, cell death, and behavioral abnormalities are complex. In this study, we begin to test the hypothesis that intracerebral hemorrhage activates Src kinases that phosphorylate other molecules to produce cell injury and behavioral deficits after intracerebral hemorrhage (ICH). Methods-ICH was produced in adult Sprague Dawley rats by direct injection of autologous blood (50 L) into striatum.Src kinase activity, glucose hypermetabolic areas around the ICH, TUNEL-stained cells, and apomorphine-induced rotational behaviors were assessed in animals with ICH pretreated with the Src kinase inhibitor, PP1, or with vehicle. Results-PP1 (3 mg/kg) blocked increases of Src kinase activity (5-fold) at 3 hours after ICH. PP1 also blocked the areas of glucose hypermetabolism and decreased the numbers of TUNEL-stained cells surrounding the ICH at 24 hours. Finally, apomorphine-induced (1 mg/kg) rotation at 24 hours after ICH was markedly attenuated by previous treatment with PP1 (3 mg/kg intraperitoneal). Conclusions-PP1 decreases Src kinase activation, glucose metabolic activation, cell death, and behavioral abnormalities after ICH in striatum of adult rats. It is hypothesized that intracerebral hemorrhage, possibly via thrombin activation of protease-activated receptors, activates Src that phosphorylates NMDA receptors, matrix metalloproteinases, and other proteins that mediate injury after ICH.

Src kinase inhibition decreases thrombin-induced injury and cell cycle re-entry in striatal neurons

“…This protection is roughly equivalent to that produced by MK801. The findings are consistent with our previous report that the Src kinase inhibitor PP1 improves outcomes following whole blood injections, and the protection was similar to that afforded by MK801 (Ardizzone et al, 2004, Ardizzone et al, 2007.…”

“…This led us to test the role of Src kinase inhibitors in ICH. Src kinase inhibitors, which are less likely to affect coagulation (Thomas and Brugge, 1997), decreased glucose hypermetabolism and cell death around ICH and improved behavioral deficits following ICH (Ardizzone et al, 2007). These results led to the current experiments that determined whether the improvement observed following ICH treated with Src kinase inhibitors was due, at least in part, to Src kinase inhibitors blocking thrombin and thrombin signaling.…”

“…After closure of the operative sites, rats were allowed to recover in an incubator maintained at 37°C, and then returned to their home cages with free access to food and water. Using our previously described methods with minor modifications (Ardizzone et al, 2007), Src kinase activity was assessed at 4h, motor deficits at 23.5h and histological damage 24h after the injection of thrombin or control BSA. All experimental procedures were performed in accordance with National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committee, University of California at Davis.…”

“…Four hours after the injection of thrombin or control BSA, rats were euthanized with chloral hydrate (350 mg/kg, i.p., Sigma, St. Louis, MO) and the ipsilateral and contralateral striatum were dissected, homogenized, and protein isolated in lysis buffer (Ardizzone et al, 2007). Src protein was immunoprecipitated with a c-Src antibody (Delta Biolabs, Gilroy, CA), washed three times in lysis buffer, twice in kinase buffer, and resuspended in the latter, optimized to preserve the Src activity.…”

lschémie cérébrale

“…Nous avons montré que rendre inactif Src avant une ischémie transitoire empêche à la fois la potentialisation des récepteurs NMDA et la mort neuronale à long terme des neurones CA1 dans l'hippocampe [15]. Enfin, les inhibiteurs de SKF suppriment l'excitotoxicité dépendante de la suractivation des NMDAR in vitro [15,31] mais diminuent également la surface de lésion cérébrale post-ischémique in vivo [32,33]. Ces résultats suggèrent une implication forte des modulations SKF-NMDAR dans le mécanisme physiopathologique de la mort neuronale induite par l'ischémie [29].…”