Fig. 3. Single bolus injection of methylene blue induces apoptosis in the brain. (  A ) Representative Hoechst counterstained photomicrograph of the cerebral cortex in the control group 24 h after single bolus saline injection. (  B and  C ) Fluoro-Jade B staining of this same section demonstrates the absence of dying neurons in the control group. (  D ) Hoechst counterstaining of cortical sections from methylene blue–treated rats (50 mg/kg). In contrast to the control group, Fluoro-Jade B staining (  E and  F ) reveals numerous dying neurons in the cerebral cortex. (  G –  I ) Representative photomicrographs of cleaved caspase-3 immunohistochemistry from the control (  G ), 5 mg/kg methylene blue (MB 5;  H ), and 50 mg/kg methylene blue (MB 50;  I ) groups 24 h after bolus injections revealing the dose-dependent effect of methylene blue on neuronal apoptosis. (  J ) Quantitative analysis of methylene blue–induced apoptosis in the cerebral cortex in the concomitant presence (isoflurane+) or absence (isoflurane−) of anesthesia. Correction bar  A –  B ,  D –  E : 1 mm;  C ,  F : 35 μm;  G –  I : 30 μm. In  J , results are presented as mean ± SEM; n = 3 animals for each treatment expressed. Values are expressed as the number of neurons/mm². *  P < 0.05 compared with the untreated control group.