The GluN1/GluN2B NMDA receptor and metabotropic glutamate receptor 1 negative allosteric modulator has enhanced neuroprotection in a rat subarachnoid hemorrhage model

Excessive glutamate in cerebrospinal fluid after subarachnoid hemorrhage (SAH) causes excitotoxic damage through calcium overloading and a subsequent apoptotic cascade. GluN1/GluN2B containing N-methyl-Daspartate (NMDA) receptor and metabotropic glutamate receptor 1 (mGluR1) can play a leading role in glutamate-mediated excitotoxicity. Here we report that Ifenprodil (100 μM), a negative allosteric modulator (NAM) of GluN1/GluN2B NMDA receptors, and JNJ16259685 (10 μM), a NAM of mGluR1, have an additive efficacy against glutamate (100 μM)-induced Ca^{2 +} release and cell apoptosis in primary cortical, hippocampal, and cerebellar granule neurons. Compared with intraperitoneal injection of Ifenprodil (10 mg/kg) and JNJ16259685 (1 mg/kg) separately, the combination therapy of Ifenprodil plus JNJ16259685 significantly improves the neurological deficit at 24 h and 72 h after experimental SAH. It reduces the number of TUNEL/DAPI-positive and activated caspase-3/NeuN-positive cells in cortical and hippocampal CA1 regions at 72 h, decreases levels of glutamate in cerebrospinal fluid at 72 h, and reduces the mitochondrial Ca^{2 +} concentration. Meanwhile, the combination therapy attenuates apoptosis as shown by an increased Bcl-2 expression, decreased Bax expression and release of cytochrome c, and reduction of cleaved caspase-9 and caspase-3 at 24 h after SAH. These findings indicate that targeting both the intracellular Ca^{2 +} overloading and neuronal apoptosis using the Ifenprodil and JNJ16259685 is a promising new therapy for SAH.