Temporal and spatial profile of caspase 8 expression and proteolysis afterexperimental traumatic bra in injury

Recent studies have demonstrated that the downstream caspases, such as casp ase 3, act as executors of the apoptotic cascade after traumatic brain Inju ry (TBI) in vivo. However, little is known about the involvement of caspase s in the initiation phase of apoptosis, and the interaction between these i nitiator caspases (e.g. caspase 8) and executor caspases after experimental brain injuries in vitro and In vivo. This study investigated the temporal expression and cell subtype distribution of procaspase 8 and-cleaved caspas e 8 p20 from 1 h to 14 days after cortical impact-induced TBI in rats. Casp ase 8 messenger RNA levels, estimated by semi-quantitaive RT-PCR, were elev ated from 1 h to 72 h in the traumatized cortex. Western blotting revealed increased immunoreactivity for procaspase 8 and the proteolytically active subunit of caspase 8, p20, in the ipsilateral cortex from 6 to 72 h after i njury, with a peak at 24 h after TBI. Similar to our previous studies, immu noreactivity for the p18 fragment of activated caspase 3 also increased in the current study from 6 to 72 h after TBI, but peaked at a later timepoint (48 h) as compared with proteolyzed caspase 8 p20. Immunohistologic examin ations revealed increased expression of caspase 8 In neurons, astrocytes an d oligodendrocytes. Assessment of DNA damage using TUNEL identified caspase 8- and caspase 3-immunopositive cells with apoptotic-like morphology in th e cortex ipsilateral to the injury site, and immunohistochemical investigat ions of caspase 8 and activated caspase 3 revealed expression of both prote ases in cortical layers 2-5 after TBI. Quantitative analysis revealed that the number of caspase 8 positive cells exceeds the number of caspase 3 expr essing cells up to 24 h after impact injury. In contrast, no evidence of ca spase 8 and caspase 3 activation was seen in the ipsilateral hippocampus, c ontralateral cortex and hippocampus up to 14 days after the impact. Our res ults provide the first evidence of caspase 8 activation after experimental TBI and suggest that this may occur in neurons, astrocytes and oligodendroc ytes. Our findings also suggest a contributory role of caspase 8 activation to caspase 3 mediated apoptotic cell death after experimental TBI In vivo.