Cell-specific upregulation of survivin after experimental traumatic brain injury in rats.

In this study, we examined the expression and cellular localization of survivin and proliferating cell nuclear antigen (PCNA) after controlled cortical impact traumatic brain injury (TBI) in rats. There was a remarkable and sustained induction of survivin mRNA and protein in the ipsilateral cortex and hippocampus of rats after TBI, peaking at five days post injury. In contrast, both survivin mRNA and protein were virtually undetectable in craniotomy control animals. Concomitantly, expression of PCNA was also significantly enhanced in the ipsilateral cortex and hippocampus of these rats with similar temporal and spatial patterns. Immunohistochemistry revealed that survivin and PCNA were co-expressed in the same cells and had a focal distribution within the injured brain. Further analysis revealed a frequent co-localization of survivin and GFAP, an astrocytic marker, in both the ipsilateral cortex and hippocampus, while a much smaller subset of cells showed co-localization of survivin and NeuN, a mature neuronal marker. Neuronal localization of survivin was observed predominantly in the ipsilateral cortex and contralateral hippocampus after TBI. PCNA protein expression was detected in both astrocytes and neurons of the ipsilateral cortex and hippocampus after TBI. Collectively these data demonstrate that the anti-apoptotic protein survivin, previously characterized in cancer cells, is abundantly expressed in brain tissues of adult rats subjected to TBI. We found survivin expression in both astrocytes and a sub-set of neurons. In addition, the expression of survivin was co-incident with PCNA, a cell cycle protein. This suggests that survivin may be involved in regulation of neural cell proliferative responses after traumatic brain injury.

Increased expression and processing of caspase-12 after traumatic brain injury in rats.

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 (approximately 60 kDa) and processed form (approximately 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.

Accumulation of calpain and caspase-3 proteolytic fragments of brain-derived alphaII-spectrin in cerebral spinal fluid after middle cerebral artery occlusion in rats.

Preclinical studies have identified numerous neuroprotective drugs that attenuate brain damage and improve functional outcome after cerebral ischemia. Despite this success in animal models, neuroprotective therapies in the clinical setting have been unsuccessful. Identification of biochemical markers common to preclinical and clinical cerebral ischemia will provide a more sensitive and objective measure of injury severity and outcome to facilitate clinical management and treatment. However, there are currently no effective biomarkers available for assessment of stroke. Nonerythroid alphaII-spectrin is a cytoskeletal protein that is cleaved by calpain and caspase-3 proteases to signature alphaII-spectrin breakdown products (alphaII-SBDPs) after cerebral ischemia in rodents. This investigation examined accumulation of calpain- and caspase-3-cleaved alphaII-SBDPs in cerebrospinal fluid (CSF) of rodents subjected to 2 hours of transient focal cerebral ischemia produced by middle cerebral artery occlusion (MCAO) followed by reperfusion. After MCAO injury, full-length alphaII-spectrin protein was decreased in brain tissue and increased in CSF from 24 to 72 hours after injury. Whereas alphaII-SBDPs were undetectable in sham-injured control animals, calpain but not caspase-3 specific alphaII-SBDPs were significantly increased in CSF after injury. However, caspase-3 alphaII-SBDPS were observed in CSF of some injured animals. These results indicate that alphaII-SBDPs detected in CSF after injury, particularly those mediated by calpain, may be useful diagnostic indicators of cerebral infarction that can provide important information about specific neurochemical events that have occurred in the brain after acute stroke.

Diffusion magnetic resonance imaging study of a rat hippocampal slice model for acute brain injury.

Diffusion magnetic resonance imaging (MRI) provides a surrogate marker of acute brain pathology, yet few studies have resolved the evolution of water diffusion changes during the first 8 hours after acute injury, a critical period for therapeutic intervention. To characterize this early period, this study used a 17.6-T wide-bore magnet to measure multicomponent water diffusion at high b-values (7 to 8,080 s/mm(2)) for rat hippocampal slices at baseline and serially for 8 hours after treatment with the calcium ionophore A23187. The mean fast diffusing water fraction (Ffast) progressively decreased for slices treated with 10-microM/L A23187 (-20.9 +/- 6.3% at 8 hours). Slices treated with 50-micromol/L A23187 had significantly reduced Ffast 80 minutes earlier than slices treated with 10-microM/L A23187 (P < 0.05), but otherwise, the two doses had equivalent effects on the diffusion properties of tissue water. Correlative histologic analysis showed dose-related selective vulnerability of hippocampal pyramidal neurons (CA1 > CA3) to pathologic swelling induced by A23187, confirming that particular intravoxel cell populations may contribute disproportionately to water diffusion changes observed by MRI after acute brain injury. These data suggest diffusion-weighted images at high b-values and the diffusion parameter Ffast may be highly sensitive correlates of cell swelling in nervous issue after acute injury.

Temporal and spatial profile of Bid cleavage after experimental traumatic brain injury.

Apoptosis plays an essential role in the cascade of CNS cell degeneration after traumatic brain injury. However, the underlying mechanisms are poorly understood. The authors examined the temporal profile and cell subtype distribution of the proapoptotic protein Bid from 6 hours to 7 days after cortical impact injury in the rat. Increased protein levels of tBid were seen in the cortex ipsilateral to the injury site from 6 hours to 3 days after trauma. Immunohistologic examinations revealed expression of tBid in neurons, astrocytes, and oligodendrocytes from 6 hours to 3 days after impact injury, and concurrent assessment of DNA damage using TUNEL identified tBid-immunopositive cells with apoptoticlike morphology in the traumatized cortex. Moreover, Bid cleavage and activation of caspase-8 and caspase-9 occurred at similar time points and in similar brain regions (i.e., cortical layers 2 to 5) after impact injury. In contrast, there was no evidence of caspase-8 or caspase-9 processing or Bid cleavage in the ipsilateral hippocampus, contralateral cortex, and hippocampus up to 7 days after the injury. The results provide the first evidence of Bid cleavage in the traumatized cortex after experimental traumatic brain injury in vivo, and demonstrate that tBid is expressed in neurons and glial cells. Further, findings indicate that cleavage of Bid may be associated with the activation of the initiator caspase-8 and caspase-9. Finally, these data support the hypothesis that cleavage of Bid contributes to the apoptotic degeneration of different CNS cells in the injured cortex.

Up-regulation of tissue-type transglutaminase after traumatic brain injury.

Tissue-type transglutaminase (tTG, EC 2.3.2.13) has been implicated in various disease paradigms including neurodegenerative disease. In these studies, tTG induction after traumatic brain injury was studied using a rat cortical impact model. Using western blots, two forms of tTG protein expression were identified--an approximately 79-kDa primary form (tTG-L) and a less abundant approximately 70-kDa form (tTG-S). Both forms of tTG protein were elevated after injury. In ipsilateral cortex, peak induction of tTG-L protein [561% +/- 80% of control (n = 5)] was observed five days after injury, with expression remaining elevated after two weeks. Peak induction of tTG-S protein [302% +/- 81% of control (n = 5)] was observed three days after injury. Lesser tTG protein induction was observed in hippocampus. Northern blot analysis demonstrated two tTG transcripts in the ipsilateral cortex with peak induction of tTG-L mRNA three days after injury. However, tTG-S mRNA was not identified in control samples and only faintly detected in injured tissue. To facilitate analysis of low abundance transcripts in smaller tissue samples, a semiquantitative real-time PCR strategy was used. Semi-quantitative PCR analysis of tTG-L mRNA induction in ipsilateral cortex (peak after three days; 414% +/- 21% of control, n = 3) confirmed tTG-L mRNA induction determined by northern blot (410% of control).