Preliminary Study On The Drug Combinations For The Treatment Of Ischemic Stroke Based On NMDA Receptors

Cerebral ischemia is a common disease characterized by high death and disability rate.Over-activation of NMDA receptors after cerebral ischemia is the key factor resulting in neuronal death.Although the researchers have developed a variety of NMDA receptor antagonists,they have to suspend the clinical trials due to unidentified efficacy or serious adverse reactions.This research mainly focuses on the combination of two types of antagonists: the combination of non-competitive antagonists(GluN2A and GluN2 B antagonists)and the combination of competitive antagonist(glutamate and glycine site antagonists),and their effects on cerebral ischemia,aiming at determining whether the combination of antagonists can be used to improve the therapeutic effect of cerebral ischemia.Firstly,we studied the slicing process and established a set of methods for obtaining complete hippocampal slices.Secondly,we explored the concentration of NMDA,and acute hippocampal slices,organotypic hippocampal slice cultures and hippocampal neuron excitotoxicity models were established.Finally,LDH assay and PI staining were employed to test the inhibiting effects of the combination of non-competitive antagonists(TCN-201+Ro25-6981,TCN-201+ifenprodil)and competitive antagonist combination(D-APV+CGP 78608)on NMDA excitotoxic damage.The results showed that the combination of non-competitive antagonists significantly reduced the level of NMDA-induced LDH and PI staining in acute rat hippocampal slices of P 7 SD rats(P<0.05),but it did not hold true for rat hippocampal slices of P 14 and P 21 rats(P>0.05).However,this protective effect can also be seen for the combination of non-competitive antagonists in the experiments of organotypic hippocampal slice cultures and hippocampal neurons.That means this combination can greatly decrease the NMDA-induced damage in cultured 7-day and 14-day hippocampal slices and 7-day hippocampal neurons(P<0.05),but not for the 21-day hippocampal slices and 14-day neurons(P>0.05).The experimental results of the competitive antagonist combination indicated that the effectiveness of the antagonists alone on acute hippocampal slices,hippocampal slices cultures and hippocampal neuronal NMDA excitotoxicity was comparable to that of combination of antagonists,both significantly reducing neuronal damage(P<0.05).From the above experimental results,it is known that the antagonist combinations in this research do not show a therapeutic effect superior to that of a single antagonist.Although the ideal efficacy was not achieved,we can come to the conclusion:(1)TCN-201 is a GluN2 A antagonist,which can attenuate the protective effect of GluN2 B antagonists.GluN2 A may play a pro-survival role during acute injury stage of cerebral ischemia;(2)Blocking metabotropic pro-death signal of the NMDA receptor is possible with an agonist site antagonist alone.The research has certain guiding significance for the development of anti-cerebral ischemia drugs based on NMDA receptors.