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Temporal and spatial profile of caspase 8 expression and proteolysis after experimental traumatic brain injury
1Department of Neurology, University Hospital Innsbruck, Austria.
Abstract:
Recent studies have demonstrated that the downstream caspases, such as caspase 3, act as executors of the apoptotic cascade after traumatic brain injury (TBI) in vivo. However, little is known about the involvement of caspases in the initiation phase of apoptosis, and the interaction between these initiator 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 caspase 8 p20 from 1 h to 14 days after cortical impact-induced TBI in rats. Caspase 8 messenger RNA levels, estimated by semiquantitaive RT-PCR, were elevated 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 injury, with a peak at 24 h after TBI. Similar to our previous studies, immunoreactivity 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 examinations revealed increased expression of caspase 8 in neurons, astrocytes and oligodendrocytes. Assessment of DNA damage using TUNEL identified caspase 8- and caspase 3-immunopositive cells with apoptotic-like morphology in the cortex ipsilateral to the injury site, and immunohistochemical investigations of caspase 8 and activated caspase 3 revealed expression of both proteases in cortical layers 2-5 after TBI. Quantitative analysis revealed that the number of caspase 8 positive cells exceeds the number of caspase 3 expressing cells up to 24 h after impact injury. In contrast, no evidence of caspase 8 and caspase 3 activation was seen in the ipsilateral hippocampus, contralateral cortex and hippocampus up to 14 days after the impact. Our results provide the first evidence of caspase 8 activation after experimental TBI and suggest that this may occur in neurons, astrocytes and oligodendrocytes. Our findings also suggest a contributory role of caspase 8 activation to caspase 3 mediated apoptotic cell death after experimental TBI in vivo.
Insights
This study shows caspase 8 activation in neurons, astrocytes, and oligodendrocytes after traumatic brain injury (TBI). This suggests caspase 8 contributes to caspase 3-mediated cell death following TBI.
Area of Science:
- Neuroscience
- Cell Biology
- Apoptosis Research
Background:
- Downstream caspases, like caspase 3, execute apoptosis post-traumatic brain injury (TBI).
- The role of initiator caspases, such as caspase 8, in the early stages of apoptosis after TBI remains largely unknown.
- Understanding caspase interactions is crucial for TBI-induced cell death mechanisms.
Purpose of the Study:
- To investigate the temporal expression and cell-specific distribution of procaspase 8 and cleaved caspase 8 p20 following experimental TBI.
- To explore the interplay between caspase 8 activation and caspase 3 execution in the apoptotic cascade after TBI.
- To determine the cellular localization and time course of caspase 8 activation in different brain regions post-TBI.
Main Methods:
- Induction of cortical impact TBI in a rat model.
- Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) to measure caspase 8 mRNA levels.
- Western blotting to assess procaspase 8 and cleaved caspase 8 p20 protein expression.
- Immunohistochemistry to identify caspase 8 and caspase 3 localization in various cell types and brain regions.
- Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for DNA damage assessment.
Main Results:
- Caspase 8 mRNA levels increased from 1 to 72 hours post-TBI, peaking within the first 72 hours.
- Procaspase 8 and cleaved caspase 8 p20 protein levels elevated from 6 to 72 hours, with a peak at 24 hours post-TBI.
- Caspase 8 activation was observed in neurons, astrocytes, and oligodendrocytes in the injured cortex, but not in the hippocampus or contralateral hemisphere.
- Caspase 8 positive cells outnumbered caspase 3 positive cells up to 24 hours post-injury.
- Caspase 3 activation followed a similar temporal pattern but peaked later than caspase 8 activation.
Conclusions:
- This study provides the first evidence of caspase 8 activation in the brain following experimental TBI.
- Caspase 8 activation occurs in multiple cell types, including neurons, astrocytes, and oligodendrocytes, in the injured cortex.
- Caspase 8 activation likely contributes to caspase 3-mediated apoptotic cell death in the context of TBI.