However, it is possible that the SND scoring system may not be sensitive enough to detect subtle differences in the chronic phase. In the late reperfusion period, rCBF was higher in the AGL-treated group (Fig. 3). We speculate that the brain damage during ischemia was more severe in the vehicle group, which brought on more severe cerebral edema during reperfusion,

and reduced the rCBF, as demonstrated in our previous studies (Yanamoto et al., 2008 and Yamamoto et al., 2011). Although INK 128 treatment of mice with AGL may upregulate endothelial nitric oxide synthase (eNOS) (Ban et al., 2008), rCBF was not increased during ischemia. In DM-2 rats, treatment with Ex-4 (0.1, 1 or 5 μg/kg, via intraperitoneal injections, twice a day), before (for four weeks) and after (for two or four weeks) the induction of focal ischemia, reduced hyperglycemia and the volumes of infarcted lesions in a dose-dependent manner (Darsalia et al., 2012). In normal rats, prophylactic treatment with Ex-4 (0.5 μg/kg, via intraperitoneal injections, twice a day) for seven days reduced volumes of

infarct lesion, the extent of neurological deficits, and also markers of oxidative stress (Briyal et al., 2012). Recently, intravenous injection of Ex-4 (0.5 or 2.5 mg/kg, immediately, or 1 h after the induction of reperfusion) reduced the volumes of infarcted lesions and the extent of functional deficits, without altering plasma insulin or glucose levels, in non-diabetic C57BL/6 mice (Teramoto et al., 2011). The conflict between the finding with post hoc Ex-4 (Teramoto et al., 2011) and NU7441 post hoc GLP treatment of focal ischemia may be explained by the different conditions present in the two sets of experiments: (1) A 100–1000 fold larger dose was used than was the case with effective prophylaxis against ischemia using Ex-4 (Darsalia et al., 2012 and Briyal et al., 2012), with the same dose used for effective prophylaxis against ischemia with AGL; (2) Ex-4 acts as a long-acting analog of GLP-1, while AGL increases intrinsic GLP-1; (3) Ex-4 was given intravenously, in contrast to the intragastric

5-Fluoracil in vitro gavage used to administer AGL; (4) the intraluminal thread insertion (ITI) method (a 60-min focal ischemia) was used to assess volumes of infarcted lesions with Ex-4, but the three-vessel occlusion (3-VO) method (a 15-min focal ischemia limited in the cortex) was used with AGL. Considering the difference in biological time, 15-min delay (plus the delay for the transfer into the brain) after the onset in mice could be translated into more than 3-h delay in humans. Further investigations are needed, in which AGL is administered immediately, or within 15 min after the onset of ischemia. Ischemia induces abnormal release, from 5- to 50-fold elevations, of glutamate and gamma-aminobutyric acid (GABA) in the brain (Matsumoto et al., 1996).