FIGURE 8.

Reduction of (−)-PA level exacerbates brain injury in MCAO mice. Mice were subjected to 1 h of MCAO and 24 h of reperfusion as described under “Experimental Procedures.” An osmotic pump was preimplanted before MCAO surgery to deliver PA monoclonal antibody into the ischemic side of the brain ventricle as described under “Experimental Procedures.” TTC staining of brain slices is shown in A. The infarct areas are highlighted with dotted lines (A). The infarct volume at 24 h of reperfusion was quantified and is shown in B. Infusion of PA monoclonal antibody significantly increased infarct volume compared with vehicle (saline)-infused ischemic brain. The neurological deficit scores (C) and forepaw pulling strength (D) were also measured and compared among the three groups (▴, PA without MCAO; ○, PA Ab with MCAO; ■, no PA Ab with MCAO). E, a sample of ischemic brain coronal section highlighting areas of interests. Fluorescence double immunohistochemical staining of ischemic mouse brain infused with PA antibody showed reduced PA immunofluorescence (red color) both in the ischemic core (F) and the surrounding penumbra (G). Blood vessels were stained with lectin (green color). For comparison with a control group lacking PA antibody treatment, please see Fig. 5, D, E, and F. To confirm this, brain tissues were laser-microdissected out from areas 1 and 2 and the contralateral side (labeled as right) for UPLC/MS/MS quantification of (−)-PA levels (H). The (−)-PA level was significantly reduced in the ischemic side of the brain surrounding the core. Error bars represent the mean ± S.D. ** indicates p < 0.01 by paired t test (n = 5). n.s., non-significant with p = 0.2, one-way ANOVA with Tukey's post hoc analysis. d, days. Scale bars, 100 μm.