Traumatic brain injury (TBI) disrupts tissue homeostasis resulting in pathological apoptotic activation. Recently, caspase-12 was reported to be induced and activated by the unfolded protein response following excess endoplasmic reticulum (ER) stress. This study examined rat caspase-12 expression using the controlled cortical impact TBI model. Immunoblots of fractionalized cell lysates found elevated caspase-12 proform (60 kDa) and processed form (12 kDa), with peak induction observed within 24 h post-injury in the cortex (418% and 503%, respectively). Hippocampus caspase-12 proform induction peaked at 24 h post-injury (641%), while processed form induction peaked at 6 h (620%). Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis confirmed elevated caspase-12 mRNA levels after TBI. Injury severity (1.0, 1.2 or 1.6 mm compression) was associated with increased caspase-12 mRNA expression, peaking at 5 days in the cortex (657%, 651% and 1259%, respectively) and 6 h in the hippocampus (435%, 451% and 460%, respectively). Immunohistochemical analysis revealed caspase-12 induction in neurons in both the cortex and hippocampus as well as in astrocytes at the contusion site. This is the first report of increased expression of caspase-12 following TBI. Our results suggest that the caspase-12-mediated ER apoptotic pathway may play a role in rat TBI pathology independent of the receptor-or mitochondria-mediated apoptotic pathways.

Caspases, a cysteine proteinase family, are required for the initiation and execution phases of apoptosis. It has been suggested that caspase 7, an apoptosis executioner implicated in cell death proteolysis, is redundant to the main executioner caspase 3 and it is generally believed that it is not present in the brain or present in only minute amounts with highly restricted activity. Here we report evidence that caspase 7 is up-regulated and activated after traumatic brain injury (TBI) in rats. TBI disrupts homeostasis resulting in pathological apoptotic activation. After controlled cortical impact TBI of adult male rats we observed, by semiquantitative real-time PCR, increased mRNA levels within the traumatized cortex and hippocampus peaking in the former about 5 days post-injury and in the latter within 6-24 h of trauma. The activation of caspase 7 protein after TBI, demonstrated by immunoblot by the increase of the active form of caspase 7 peaking 5 days post-injury in the cortex and hippocampus, was found to be up-regulated in both neurons and astrocytes by immunohistochemistry. These findings, the first to document the up-regulation of caspase 7 in the brain after acute brain injury in rats, suggest that caspase 7 activation could contribute to neuronal cell death on a scale not previously recognized.

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Despite a preponderance of studies demonstrating gene expression and/or enzymatic activation of calpain and caspase proteases after traumatic brain injury (TBI), no studies have examined the effects of injury magnitude on expression levels of these cell death effectors after TBI. Determination of the degree to which injury severity affects specific expression profiles is critical to understanding the relevant pathways contributing to post-trauma pathology and for developing targeted therapeutics. This investigation tested the hypothesis that different injury magnitudes (1.0, 1.2, and 1.6 mm) cause alterations in the regional and temporal patterns of mRNA expression of calpain-related (calpain-1 and -2, calpastatin) and caspase-related (caspases -3, -8, -9, BID) gene products after cortical impact in rats. Quantitative RT-PCR was used to compare effects of injury severity on mRNA levels in ipsilateral (injured) cortex and hippocampus, 6 h to 5 days post-injury. TBI caused increases in mRNA expression of all proteins examined, with the highest expression detected in the cortex. Generally, injury magnitude and levels of gene expression were positively correlated. High levels of gene induction were observed with BID, caspase-3, and -8, while caspase-9 mRNA had the lowest level of induction. Interestingly, although calpains are activated within minutes of TBI, calpain mRNA expression was highest 72 h to 5 days post-TBI. This study is the first analysis of the regional and temporal expression of calpains and caspases after TBI. These data provide insight into the inter-relationship of these two protease families and on the distinct but overlapping cascades of cell death after TBI.