Although a number of increased CSF proteins have been correlated with brain damage and outcome after traumatic brain injury (TBI), a major limitation of currently tested biomarkers is a lack of speci®city for de®ning neuropathological cascades. Identi®cation of surrogate biomarkers that are elevated in CSF in response to brain injury and that offer insight into one or more pathological neurochemical events will provide critical information for appropriate administration of therapeutic compounds for treatment of TBI patients. Nonerythroid aII-spectrin is a cytoskeletal protein that is a substrate of both calpain and caspase-3 cysteine proteases. As we have previously demonstrated, cleavage of aII-spectrin by calpain and caspase-3 results in accumulation of proteasespeci®c spectrin breakdown products (SBDPs) that can be used to monitor the magnitude and temporal duration of protease activation. However, accumulation of aII-spectrin and aII-SBDPs in CSF after TBI has never been examined. Following a moderate level (2.0 mm) of controlled cortical impact TBI in rodents, native aII-spectrin protein was decreased in brain tissue and increased in CSF from 24 h to 72 h after injury. In addition, calpain-speci®c SBDPs were observed to increase in both brain and CSF after injury. Increases in the calpain-speci®c 145 kDa SBDP in CSF were 244%, 530% and 665% of sham-injured control animals at 24 h, 48 h and 72 h after TBI, respectively. The caspase-3-speci®c SBDP was observed to increase in CSF in some animals but to a lesser degree. Importantly, levels of these proteins were undetectable in CSF of uninjured control rats. These results indicate that detection of aII-spectrin and aIISBDPs is a powerful discriminator of outcome and protease activation after TBI. In accord with our previous studies, results also indicate that calpain may be a more important effector of cell death after moderate TBI than caspase-3.
Activation of calpain results in the breakdown of alpha II spectrin (alpha-fodrin), a neuronal cytoskeleton protein, which has previously been detected in various in vitro and in vivo neuronal injury models. In this study, a 150 kDa spectrin breakdown product (SBDP150) was found to be released into the cell-conditioned media from SH-SY5Y cells treated with the calcium channel opener maitotoxin (MTX). SBDP150 release can be readily quantified on immunoblot using an SBDP150-specific polyclonal antibody. Increase of SBDP150 also correlated with cell death in a time-dependent manner. MDL28170, a selective calpain inhibitor, was the only protease inhibitor tested that significantly reduced MTX-induced SBDP150 release. The cell-conditioned media of cerebellar granule neurons challenged with excitotoxins (NMDA and kainate) also exhibited a significant increase of SBDP150 that was attenuated by pretreatment with an NMDA receptor antagonist, R(-)-3-(2-carbopiperazine-4-yl)-propyl-1-phosphonic acid (CPP), and MDL28170. In addition, hypoxic/hypoglycemic challenge of cerebrocortical cultures also resulted in SBDP150 liberation into the media. These results support the theory that an antibody-based detection of SBDP150 in the cell-conditioned media can be utilized to quantify injury to neural cells. Furthermore, SBDP150 may potentially be used as a surrogate biomarker for acute neuronal injury in clinical settings.
The authors present two studies that investigate the biochemical and histologic effects of the nonimmunosuppressive neuroimmunophilin (NIMM) ligand V-10,367 in a mouse model of traumatic brain injury (TBI). In study 1, the authors examined the effect of V-10,367 (50 mg/kg x 2 per day, by mouth) on neurofilament M (NFM) protein levels and on alpha-spectrin breakdown products (SBDPs) when dosed for 2 days, starting 24 hours after TBI and killed on day 3. In study 2, V-10,367 was given for 10 days, starting 24 hours after TBI and the mice killed 6 weeks after TBI, to measure the extent of neurodegeneration (amino CuAg stain). The results in study 1 revealed that V-10,367-treatment significantly increased NFM protein levels in both sham and TBI mice. In addition, V-10,367 attenuated SBDP 150 levels in the cortex, striatum, and hippocampus. The results of study 2 indicated that TBI mice treated with V-10,367 demonstrated significantly less neurodegeneration compared to injured, vehicle-treated mice. In summary, these results suggest that NIMMs may be neuroprotective indirectly through inhibition of calpain-mediated cytoskeletal damage and perhaps via maintenance of neuronal plasticity. In the context of this mouse model of TBI, the therapeutic window for V-10,367's positive effects is at least 24 hours after injury, which, in the case of TBI models, is largely unprecedented for a neuroprotective compound.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.