Combined l-citrulline and glutathione administration prevents neuronal cell death following transient brain ischemia

- Combined l-Cit and GSH administration protects neurons from brain ischemia.
- Combined l-Cit and GSH administration prevents eNOS S-glutathionylation in ischemic hippocampus.
- Combined l-Cit and GSH administration rescues decreased NOS activity following BCCAO ischemia.

We previously reported that oral l-citrulline (l-Cit) administration antagonizes neuronal cell death in hippocampus following transient brain ischemia and that oral glutathione (GSH) administration prevents neuronal death through antioxidant activity. Here, we tested potential synergy of combined l-Cit and GSH administration in protection against neuronal death following cerebral ischemia. One day after a 20-min bilateral common carotid artery occlusion (BCCAO), mice were orally administered l-Cit or GSH alone (at 40 or 100 mg/kg p.o.) or both (at 40 mg/kg p.o. each) daily for 10 days. The combination, but not l-Cit or GSH alone at 40 mg/kg p.o., significantly prevented neuronal death in the hippocampal CA1 region in BCCAO mice. Consistently, combined l-Cit and GSH administration improved memory-related behavioral deficits observed in BCCAO mice. Combination treatment also significantly rescued reduced endothelial nitric oxide synthase (eNOS) protein levels and antagonized eNOS S-glutathionylation seen following BCCAO ischemia. Recovery of eNOS activity was confirmed by in vivo NO production in hippocampus of BCCAO mice. Taken together, combined administration of l-Cit with GSH rescues eNOS function, thereby inhibiting delayed neuronal death in hippocampus.