Biochemical, structural, and biomarker evidence for calpain-mediated cytoskeletal change after diffuse brain injury uncomplicated by contusion

Abstract

Calpain-mediated degradation of the cytoskeletal protein alpha-II-spectrin has been implicated in the pathobiology of experimental and human traumatic brain injury (TBI). Spectrin proteolysis after diffuse/widespread TBI uncomplicated by either subtle or overt contusion and/or mass lesions, (i.e. mild to moderate TBI), has not been previously evaluated. To determine the spatiotemporal pattern and cellular localization of calpain-mediated spectrin proteolysis after diffuse/widespread TBI and the extent to which parenchymal changes in calpain-mediated spectrin proteolysis are reflected in the cerebrospinal fluid, adult rats were subjected to a moderate midline fluid percussion injury and allowed to survive for 3 hours to 7 days postinjury. Light and electron microscopic immunocytochemical and Western blot analyses were performed to identify the calpain-specific 145-kDa breakdown product of alpha-II-spectrin (SBDP145). After diffuse TBI, enhanced levels of SBDP145 immunoreactivity were observed in the neocortex, subcortical white matter, thalamus, and hippocampus, peaking between 24 and 48 hours postinjury. Immunoreactivity was localized almost exclusively to damaged axons and axonal terminal debris. Heightened levels of SBDP145 were also observed in the cerebrospinal fluid at 24 hours postinjury. These results confirm the widespread occurrence of calpain-mediated spectrin proteolysis after diffuse TBI without contusion and support the potential utility of SBDPs as biomarkers of a diffusely injured brain.

Figure 1

Fragment-specific SBDP145 Antibody tool characterization. Naïve rat brain lysate (10 μg) was either undigested (1) or digested with rat calpain-2 (1/50 ratio) (2) or with recombinant human caspase-3 (1/50 ratio) (3) for 30 minutes. The samples were subjected to SDS-PAGE, immunoblotting probed with either mouse anti-total alpha-II-spectrin (left) or rabbit anti-SBDP145 (right) and substrate development. In addition to intact alpha-II-spectrin, calpain-generated SBDP150 and SBDP145 fragments and caspase-generated SBDP150i and SBDP120 fragments are demonstrated.

Figure 2

Immunohistochemical detection of calpain-mediated alpha-II-spectrin proteolysis (CMSP) in the neocortex following diffuse brain injury. Using an antibody that targets the 145-kDa calpain-cleavage product of alpha-II-spectrin, there are a marked increases in calpain-mediated spectrin proteolysis is identified in the neocortex following diffuse brain injury. (A) Little to no SBDP145 immunoreactivity is observed in the cortex of sham animals. (B) SBDP145 immunoreactivity is detectable by 3 hours post-injury, particularly at the gray-white matter interface. (C, D) Levels of calpain-mediated spectrin proteolysis appear to peak at 24 hours (C) to 48 hours (D) post-injury; they were diminished to sham levels by 7 days post-injury (not shown). The majority of SBDP145-immunoreactivity appears to be localized to axons and axonal debris in the neocortex and subcortical white matter, but scattered somatic expression is also observed (B, inset). Scale bar = 20 μm.

Figure 3

Prolonged calpain-mediated alpha-II-spectrin proteolysis (CMSP) in the thalamus following diffuse brain injury. Immunocytochemistry demonstrates increased levels of SBD145 in the thalamus. (A) Relatively little SBDP145-immunoreactivity is observed in the thalamus of sham animals. (B, C) There is robust SBDP145 staining throughout the thalamus by 24 hours (B) and 48 hours (C) post-injury. (D) CMSP staining remained elevated albeit at apparently decreased levels from peak at 7 days post-injury. The majority of SBDP145 immunoreactivity appears localized to axons within the thalamus. (E-G) Neuronal somatic immunostaining, with particularly intense somatic staining in neurons of the thalamic reticular nucleus is also observed at 3 hours (E), 24 hours (F) and 7 days (G) post-injury. Scale bar = 50 μm.

Figure 4

Calpain-mediated alpha-II-spectrin (CMSP) proteolysis in the hippocampus following diffuse brain injury. Immunostaining for the 145-kDa breakdown product of alpha-II-spectrin demonstrates enhanced levels of spectrin proteolysis in the hippocampus. (A) Little to no SBDP145 immunoreactivity is observed in the hippocampus of sham animals. (B) Isolated neuronal somatic SBDP145-immunoreactivity is seen in the granule cell layer of the dentate gyrus at 3 hours post-injury. (C) Immunostaining is widespread throughout the hippocampus at 24 hours and later time points post-injury (not shown). Staining is primarily localized to axons and axonal debris. dg = dentate gyrus. Scale bar = 50 μm

Figure 5

Calpain-mediated alpha-II-spectrin proteolysis (CMSP) in white matter tracts following diffuse brain injury. (A-D) SBDP145 immunocytochemistry reveals extensive immunoreactivity localized to axons and axonal debris in the internal capsule (A), external capsule (B), brainstem white matter tracts (C) and cingulum (D) at 24 hours post-injury. Scale bars = 50 μm for (D) and 20 μm for (A-C).

Figure 6

Routine light microscopy (LM) and electron microscopy (EM) confirm axonal localization of calpain-mediated spectrin proteolysis (CMSP). (A, B) High magnification LM images of SBDP145 immunostaining in the thalamus suggest localization of CMSP to damaged axons and associated axonal swellings. The linear orientation of SBDP145 immunoreactivity in B suggests debris fields of degenerating axons. (C-E) SBDP145-immunostained sections processed for EM confirm localization of immunoreactivity in damaged axons and associated axonal swellings. Electron dense SBDP145-immunoreactivity (arrows) in a representative photomicrograph of a damaged thalamic axon exhibits a disrupted myelin sheath and cytoskeletal perturbation (C). Damaged axons display more mature bulb-like axon swellings characterized by pooling of intra-axonal organelles (D, E). Note the presence of SBDP145-immunoreactivity (arrows) within these swellings. Scale bar = 20 μm in A, B, 1 μm in C; 2 μm in D and E.

Figure 7

Spatiotemporal pattern of calpain-mediated alpha-II-spectrin proteolysis (CMSP) following diffuse brain injury. Western blot analyses using an anti = SBDP145 reveal significant increases in calpain-mediated spectrin proteolysis in the neocortex (A, D), thalamus (B, E) and hippocampus (C, F) following diffuse brain injury. Representative immunoblots and densitometric measurements of SBDP145 (normalized to both sham and cyclophilin B load control values) are shown graphically and reveal significantly heightened levels of CMSP in the neocortex at 3 hours, 24 hours and 48 hours post-injury before returning to basal levels by 7 days. Significantly enhanced levels of SBDP145 are also observed at 24 hours and 48 hours post-injury in both the thalamus and hippocampus. In contrast to the cortex, SBDP145 levels remained elevated by 7 days post-injury in the latter two regions. *p < 0.05 ± SEM.

Figure 8

SBDP145 levels are increased in the CSF of diffusely injured animals. An ELISA targeting the 145-kDa calpain-mediated degradation product of alpha-II-spectrin reveals significantly enhanced SBDP145 levels in the CSF of injured animals (6.16 ± 0.92 ng/mL) compared to sham animals (1.44 ± 0.45 ng/mL) at 24 hours post-injury. *p < 0.05 ± SEM.