Effect Of Montelukast, An Antagonist Of Cysteinyl Leukotriene Receptor 1, On Ischemic Brain Injury

In our laboratory, the protective effect of cysteinyl leukotriene receptor 1 (CysLT1 receptor) antagonists on cerebral ischemia has been demonstrated. The CysLT1 receptor antagonist pranlukast (ONO-1078) can attenuate acute, subacute and chronic brain injuries after focal cerebral ischemia in rats and mice. In the study of chronic brain injury after focal cerebral ischemia in mice, pranlukast not only reduced mortality and promoted recovery of neurological function, but also inhibited glial scar formation. However, the effects of other CysLT1 receptor antagonists on cerebral ischemic injury remains to be further elucidated. At the cellular level, it was reported that transfection of the CysLT1 and CysLT2 receptors affected ischemic injury in PC12 cells, and the CysLT1 receptor mediated the astrocyte proliferation after mild ischemic injury. However, the effect of CysLT1 receptor or its antagonists on ischemic injury in neurons needs investigation.Since pranlukast was used in majority of the experiments, whether its effect is a common effect of CysLT1 receptor antagonists or a specific effect of itself? To illustrate this problem, another CysLT1 receptor antagonist, montelukast, was investigated. Montelukast, [1-([(1(R)-(3-(2-(7-chloro-2-quinoline)-(E)-ethenyl) phenyl)-3-(2-(1-hydroxy-1-methyl ethyl) phenyl) propyl) thiol]-methyl) cyproterone acetate], is a selective CysLT1 receptor antagonist, which is different from pranlukast in molecular structure. We found that intraperitoneal injection of montelukast dose-dependently protected against acute brain injury after focal cerebral ischemia in mice. Montelukast improved neurological symptoms, reduced infarct size and neuron loss, inhibited increase in blood-brain barrier permeability; its therapeutic time window was 0.5 h after ischemia with similar intensity to pranlukast. However, the effect of montelukast on chronic brain injury after focal cerebral ischemia has not been investigated. Therefore, in Part 1 of this study, we investigated the protective effect of montelukast on chronic brain injury after focal cerebral ischemia in rats and mice, and observed its protective effect after oral administration.In addition to the receptor-dependent effect, CysLT1 receptor antagonists still have the receptor-independent effects. Whether montelukast possesses this effect is unknown. On the other hand, neuron injury is regulated by interaction of glial cells. Whether montelukast affects ischemic neuron injury through this interaction needs investigation too. To clarify these problems, in Part 2 of this study, we confirmed the protective effect found in animal experiments in rat primary cortical neurons and the mixed cultures of cortical cells; we also observed the property of its effect, the CysLT1 receptor-independence and the interaction with glial cells.Part 1. Effect of montelukast on chronic brain injury after focal cerebral ischemia in mice and ratsAim:In the present study, we determined the effect of montelukast on chronic brain injury after focal cerebral ischemia in mice and rats. Firstly, in a mouse model of focal cerebral ischemia, we clarified whether montelukast exerts the protective effect on chronic ischemic injury, using pranlukast, another CysLT1 receptor antagonist, as the control. Secondly, in a rat model of focal cerebral ischemia, we determined whether orally administered montelukast has a protective effect on chronic ischemic injury. Edaravone, a free radical scavenger with clinical anti-stroke efficacy, was used as the control in rat experiments.Methods:After transient focal cerebral ischemia was induced by middle cerebral artery occlusion, montelukast (0.01 and 0.1 mg/kg) was intraperitoneally injected in mice for 5 days. In rats, montelukast (0.1 and 0.5 mg/kg) was orally administered for 5 days. During 28 days after ischemia, behavioral dysfunctions [neurological deficit scores, corner test (in mice) and skilled reaching test (in rats)] were assayed to observe impaired neural functions. At the end of 28-day observation, brain lesion volume, brain atrophy and neuron loss were determined to evaluate the changes in brain lesions.Results:Montelukast (0.1 mg/kg) attenuated behavioral dysfunction, brain lesion volume, brain atrophy and neuron loss in mice, which was similar to pranlukast (0.1 mg/kg). Oral montelukast (0.5 mg/kg) was also effective on the chronic injury in rats, and was more effective than edaravone (10 mg/kg). Especially, montelukast increased the success rate in the skilled reaching test more potently in ischemic rats. However, montelukast did not decrease death rate in both mice and rats, indicating that it could not reverse very severe ischemic lesions.Conclusion:(1) In mice, montelukast protects against chronic brain injury after focal cerebral ischemia; it is as effective as pranlukast and its effective dose is about 0.1 mg/kg. (2) In rats, we at first time found that orally administered montelukast exerts a significantly protective effect on the chronic brain injury, especially on the change in exquisite behavior. Its effective oral dose is about 0.5 mg/kg. These findings support the therapeutic potential of CysLT1 receptor antagonists in the treatment of ischemic stroke. Part 2. Effect of mentelukast on ischemic injury of neuronsAim:In the present study, we determined whether montelukast has a protective effect on neuron injury. First, we investigated the effect of montelukast on the ischemic injury in neurons, and the effect on thrombin-induced injury (a secondary injury due to hemorrhage after brain ischemia). Second, we clarified whether the effect of montelukast results from antagonism of the CysLT1 receptor; whether it affects glial cells in the mixed culture of cortical cells.Methods:In rat cortical neurons and mixed cultures, the optimal condition (2 h-OGD and 24 h-recovery, OGD/R) was established to induce ischemic injury. Thrombin (0.1-100 U/ml) was applied to induce neuron injury. Before these injuries, neurons and mixed cultures were pretreated with montelukast and LTD4 (the agonist of CysLT1 receptor) at various concentrations. Neuron injury was evaluated by neuron viability (MTT reduction assay), LDH leakage rate, apoptotic rate (Hoechst 33258 staining), neuron number and morphological changes. In addition, the expression of CysLT1 receptor (immunostaining), production of reactive oxygen species (ROS), and the morphological changes in microglia were observed as well.Results:In neurons, montelukast (0.01-10μM) protected against OGD/R-induced ischemic injury. It attenuated neuron viability reeduction, LDH leakage, apoptotic rate, neuron density reduction and morphological changes. However, montelukast did not exert any protective effect on thrombin-induced injury. The CysLT1 receptor agonist LTD4 (1-100 nM) had similar but somewhat mild protective effect on OGD/R injury. However, no synergic effect of both agents (montelukast 0.01μM+LTD4 1-100 nM) was observed. Montelukast (0.01-10μM) significantly inhibited ROS production after OGD/R, but LTD4 (10-1000 nM) did not affect ROS production. In the mixed cltures of cortical cells, montelukast (0.0001-1μM) significantly changed neuron morphology after OGD/R, but LTD4 (0.1-1000 nM) had no such an effect substantially. Montelukast (0.0001-0.01μM) and LTD4 (1-1000 nM) inhibited OGD/R-induced microglial activation, but they did not have a synergic effect.Conclusion:(1) Montelukast has the protective effect on ischemic injury of neurons, but not on thrombin-induced injury. (2) The protective effect of montelukast results from its antioxidative ability and inhibition of microglial activaton, and does not depend on antagonism of the CysLT1 receptor.