Figure 6.

Bicarbonate or GPR68 overexpression offers protection in vivo. A, A model illustrating the protective and injurious pathways of acidosis on ischemic injury. B, Effect of bicarbonate (2 mg/kg) on ischemic injury. i.c.v. injection was performed at 1 h after transient middle cerebral artery occlusion (MCAO) (tMCAO). C, Adenoassociated virus (AAV)-mediated targeting of brain neurons. Top diagram illustrates the design of the AAV-GPR68. Left panel shows a brain injected with trypan blue; arrows indicate positions for the 2-site injection. Middle panel shows a typical set of GFP images at second week after AAV-CMV-eGFP injection. To help visualization, boundaries of the slices are traced with dashed lines. Right: Blots shown were from AAV-infected organotypic slices, blotted with a custom made GPR68 antibody (upper blot) and tubulin (lower blot). Confocal images show immunofluorescence of cryosections prepared from an AAV-GPR68 infected mouse brain. The majority of GFP-positive cells were positive for NeuN. D, GPR68 overexpression on MCAO-induced injury. Top diagram illustrates the experimental design: at 3 to 5 wks after AAV injection, 60 min MCAO was performed followed by TTC staining 24 h later. Lower panel shows representative images for TTC staining and quantification of % infarct. P were from Mann-Whitney U test. ASIC indicates acid-sensing ion channel; CMV, cytomegalovirus promoter; eGFP, enhanced GFP; GFP, green fluorescent protein; i.c.v., intracerebroventricular; IRES, internal ribosome entry site; ITR, inverted terminal repeat; PAC, proton-activated chloride channel; PKC, protein kinase C; and TTC, 2,3,5-triphenyltetrazolium chloride.