Immunohistochemical Study of Calpain-Mediated Breakdown Products to α-Spectrin Following Controlled Cortical Impact Injury in the Rat

Abstract

This study examined the effect of unilateral controlled cortical impact on the appearance of calpain-mediated alpha-spectrin breakdown products (BDPs) in the rat cortex and hippocampus at various times following injury. Coronal sections were taken from animals at 15 min, 1 h, 3 h, 6 h, and 24 h after injury and immunolabeled with an antibody that recognizes calpain-mediated BDPs to alpha-spectrin (Roberts-Lewis et al., 1994). Sections from a separate group of rats were also taken at the same times and stained with hematoxylin and eosin. Analyses of early time points (15 min, 1 h, 3 h, and 6 h following injury) revealed alpha-spectrin BDPs in structurally intact neuronal soma and dendrites in cortex ipsilateral to site of injury that was not present in tissue from sham-injured control rats. By 24 h after injury labeling was not restricted to clearly defined neuronal structures in ipsilateral cortex, although there was an increased extent of diffuse labeling. BDPs to alpha-spectrin in axons were not detected until 24 h after injury, in contrast to the more rapid accumulation of BDPs observed in neuronal soma and dendrites. The presence of BDPs to alpha-spectrin in the cortex at the site of impact, and in the rostral and contralateral cortex, coincided with morphopathology detected by hematoxylin and eosin. alpha-Spectrin BDPs were also observed in the hippocampus ipsilateral to the injury in the absence of overt cell death. This investigation provides further evidence that calpain is activated after controlled cortical impact and could contribute to necrosis at the site of injury. The appearance of calpain-mediated BDPs at sites distal to the contusion site and in the hippocampus also suggests that calpain activation may precede and/or occur in the absence of extensive morphopathological changes.

Intracranial biomechanics following cortical contusion in live rats

“…Given that the elastic behavior of brain tissue is largely a function of the cell structure, 6,26 this change corresponds well to the timing of proteolytic tissue destruction following contusion, which begins at 24 hours. 20 We also observed increased gradual deformation of intracranial contents in the hold-force and multicycle tests. Reduced viscosity beginning 2-6 hours after contusion and reaching a minimum at 3 days correlates well with the temporal changes in edema and tissue degeneration.…”

The Cysteine Protease Cathepsin B Is a Key Drug Target and Cysteine Protease Inhibitors Are Potential Therapeutics for Traumatic Brain Injury

“…A likely offtarget candidate may be inhibition of the calpains, which are known TBI drug targets, 43 because calpain-1 KO studies have validated as such. 44 Brain calpain activity spikes within 24 h of trauma, [45][46][47] and E64d administration has been shown to reduce calpain activity and provide neuroprotection after trauma. 48,49 Thus, whereas the cathepsin B KO studies here show that E64d acts primarily by inhibition of cathepsin B, some additional benefits may occur in TBI treatment through E64d inhibition of other proteases, especially calpains.…”

αII-Spectrin Breakdown Products (SBDPs): Diagnosis and Outcome in Severe Traumatic Brain Injury Patients

“…Thus SBDPs can be used to monitor different temporal characteristics of protease activation. Previous investigations into calpain activation and subsequent spectrin proteolysis in the injured brain (including both contusional and non-contusional domains) have largely associated these proteolytic processes with overt somatic damage and/or necrotic cell death of neurons in the contusional and peri-contusional regions (Hall et al, 2005;Newcomb et al, 1997;Saatman et al, 1996). Our data are in agreement with previous findings; furthermore, our results are consistent with a growing body of experimental literature in which emphasis has been placed on the relationship between nerve fiber involvement and SBDPs McGinn et al, 2009;Saatman et al, 1996Saatman et al, , 2003Serbest et al, 2007).…”