tMCAo induces activation of caspase-6 in neuronal processes and soma. A, Schematic of core and penumbra region in frontocorticostriatal region. B–E, Images were taken from cortical layers III-IV of the cingulate, primary motor, primary + secondary somatosensory, and granular insular cortices in the penumbra. The MCA to the ipsilateral hemisphere has been transiently occluded, whereas the contralateral hemisphere has not been manipulated. B, Caspase-6 is activated in cell bodies and processes in stroke penumbra in rat tMCAo. Rats were subjected to 2 h tMCAo followed by reperfusion for the indicated duration. Rodent tissue was immunostained for cl-C6 (green), and nuclei were stained with Hoechst (blue). cl-C6 appears in cell bodies and processes at 12 hpr. Cell body and process staining is observed through 3 dpr. By 7 dpr, nuclei and cell structures that resemble apoptotic bodies are positive for cl-C6. Scale bars, 50 μm. C, Caspase-6 is activated in cell bodies and processes in stroke penumbra in mouse tMCAo. Mice were subjected to 1 h tMCAo followed by reperfusion for the indicated duration. Tissue was immunostained for cl-C6 (green), and nuclei were stained with Hoechst (blue). cl-C6 appears in processes. Epifluorescence microscopy was used. Scale bars, 50 μm. D, cl-C6 is neuron specific. Cortical penumbra sections from stroked rats (24 hpr) were immunostained for cl-C6 (green), NeuN (red), a neuronal marker, and Hoechst (blue). Top left, cl-C6; top right, NeuN; bottom, the merge of cl-c6, NeuN, and Hoechst. cl-C6 does not colocalize with the astrocyte marker GFAP (data not shown). Epifluorescence microscopy was used. Scale bars, 50 μm. E, cl-C6 is present in axons. Top, cortical penumbra sections from stroked rats (12 hpr) were immunostained for cl-C6 (red) and Tuj1 (green), an axonal marker. Left, cl-C6; center,Tuj1; right, a merge of both. Single processes that contain cl-C6 are apparent. Regions of axons with nonfragmented Tuj1 staining do not have cl-C6 staining. In contrast, regions with cl-C6 exhibited fragmented Tuj1 staining. Bottom, brain sections were immunostained for cl-C6 (red) and NF-L (green), another axon marker. Left, cl-C6; center, NF-L; right, a merge of both. The staining pattern is similar to cl-C6 and Tuj1: regions of axons with nonfragmented NF-L staining do not have cl-C6 staining. In contrast, regions with cl-C6 exhibited fragmented NF-L staining. Confocal microscopy was used. Scale bars, 50 μm.