András Büki

All roads lead to disconnection? - Traumatic axonal injury revisited

SummaryTraumatic brain injury (TBI) evokes widespread/diffuse axonal injury (TAI) significantly contributing to its morbidity and mortality. While classic theories suggest that traumatically injured axons are mechanically torn at the moment of injury, studies in the last two decades have not supported this premise in the majority of injured axons. Rather, current thought considers TAI a progressive process evoked by the tensile forces of injury, gradually evolving from focal axonal alteration to ultimate disconnection. Recent observations have demonstrated that traumatically induced focal axolemmal permeability leads to local influx of Ca2+ with the subsequent activation of the cysteine proteases, calpain and caspase, that then play a pivotal role in the ensuing pathogenesis of TAI via proteolytic digestion of brain spectrin, a major constituent of the subaxolemmal cytoskeletal network, the “membrane skeleton”. In this pathological progression this local Ca2+ overloading with the activation of calpains also initiates mitochondrial injury that results in the release of cytochrome-c, with the activation of caspase. Both the activated calpain and caspases then participate in the degradation of the local axonal cytoskeleton causing local axonal failure and disconnection. In this review, we summarize contemporary thought on the pathogenesis of TAI, while discussing the potential diversity of pathological processes observed within various injured fiber types. The anterograde and retrograde consequences of TAI are also considered together with a discussion of various experimental therapeutic approaches capable of attenuating TAI.

Cytochrome c release and caspase activation in traumatic axonal injury.

Axonal injury is a feature of traumatic brain injury (TBI) contributing to both morbidity and mortality. The traumatic axon injury (TAI) results from focal perturbations of the axolemma, allowing for calcium influx triggering local intraaxonal cytoskeletal and mitochondrial damage. This mitochondrial damage has been posited to cause local bioenergetic failure, leading to axonal failure and disconnection; however, this mitochondrial damage may also lead to the release of cytochrome c (cyto-c), which then activates caspases with significant adverse intraaxonal consequences. In the current communication, we examine this possibility. Rats were subjected to TBI, perfused with aldehydes at 15–360 min after injury, and processed for light microscopic (LM) and electron microscopic (EM) single-labeling immunohistochemistry to detect extramitochondrially localized cytochrome c (cyto-c) and the signature protein of caspase-3 activation (120 kDa breakdown product of α-spectrin) in TAI. Combinations of double-labeling fluorescent immunohistochemistry (D-FIHC) were also used to demonstrate colocalization of calpain activation with cyto-c release and caspase-3-induction. In foci of TAI qualitative–quantitative LM demonstrated a parallel, significant increase in cyto-c release and caspase-3 activation over time after injury. EM analysis demonstrated that cyto-c and caspase-3 immunoreactivity were associated with mitochondrial swelling–disruption in sites of TAI. Furthermore, D-IFHC revealed a colocalization of calpain activation, cyto-c release, and caspase-3 induction in these foci, which also revealed progressive TAI. The results demonstrate that cyto-c and caspase-3 participate in the terminal processes of TAI. This suggests that those factors that play a role in the apoptosis in the neuronal soma are also major contributors to the demise of the axonal appendage.

The role of calpain-mediated spectrin proteolysis in traumatically induced axonal injury.

In animals and man, traumatic brain injury (TBI) results in axonal injury (AI) that contributes to morbidity and mortality. Such injured axons show progressive change leading to axonal disconnection. Although several theories implicate calcium in the pathogenesis of AI, experimental studies have failed to confirm its pivotal role. To explore the contribution of Ca2+-induced proteolysis to axonal injury, this study was undertaken in an animal model of TBI employing antibodies targeting both calpain-mediated spectrin proteolysis (CMSP) and focal neurofilament compaction (NFC), a marker of intra-axonal cytoskeletal perturbation, at 15-120 minutes (min) postinjury. Light microscopy (LM) revealed that TBI consistently evoked focal, intra-axonal CMSP that was spatially and temporally correlated with NFC. These changes were seen at 15 min postinjury with significantly increasing number of axons demonstrating CMSP immunoreactivity over time postinjury. Electron microscopy (EM) demonstrated that at 15 min postinjury CMSP was confined primarily to the subaxolemmal network. With increasing survival (30-120 min) CMSP filled the axoplasm proper. These findings provide the first direct evidence for focal CMSP in the pathogenesis of generalized/diffuse AI. Importantly, they also reveal an initial subaxolemmal involvement prior to induction of a more widespread axoplasmic change indicating a spatial-temporal compartmentalization of the calcium-induced proteolytic process that may be amenable to rapid therapeutic intervention.

Cyclosporin A limits calcium-induced axonal damage following traumatic brain injury.

In traumatic axonal injury, Ca2+ influx across a focally damaged axolemma precipitates local mitochondrial failure, degradation of the subaxolemmal spectrin network and compaction of neurofilaments, which collectively contribute to axonal failure. In previous studies, cyclosporin A pretreatment preserved mitochondrial integrity and attenuated axonal failure following trauma. Here we investigate whether this CsA-linked protection was related to the concomitant blunting of intra-axonal, Ca2+-induced cytoskeletal changes in traumatic axonal injury, assessed with antibodies targeting spectrin proteolysis and neurofilament compaction. CsA pretreatment dramatically reduced Ca2+-induced cytoskeletal damage following injury; CsA-treated rats, compared with vehicle-treated rats, displayed a 70% decrease in immunoreactive/damaged profiles. We suggest that CsA-mediated preservation of mitochondrial integrity enables the restoration of ionic and metabolic homeostasis thereby short-circuiting Ca2+-induced proteolysis in injured axons.

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

In this study we assessed the clinical utility of quantitative assessments of alphaII-spectrin breakdown products (SBDP145 produced by calpain, and SBDP120 produced by caspase-3) in cerebrospinal fluid (CSF) as markers of brain damage and outcome after severe traumatic brain injury (TBI). We analyzed 40 adult patients with severe TBI (Glasgow Coma Scale [GCS] score <or=8) who underwent ventriculostomy. Patients requiring CSF drainage for other medical reasons served as controls. CSF samples were taken at admission and every 6 h thereafter for a maximum of 7 days and assessed using novel quantitative fragment-specific ELISAs for SBDPs. Outcome was assessed using the 3-month Glasgow Outcome Scale. Mean CSF levels of SBDPs were significantly higher in TBI patients than in controls at all time points examined. Different temporal release patterns of CSF SBDP145 and SBDP120 were observed. SBDP145 provided accurate diagnoses at all time points examined, while SBDP120 release was more accurate 24 h after injury. Within 24 h after injury, SBDP145 CSF concentrations significantly correlated with GCS scores, while SBDP120 levels correlated with age. SBDP levels were significantly higher in patients who died than in those who survived. SBDP145 levels (>6 ng/mL) and SBDP120 levels (>17.55 ng/mL) strongly predicted death (odds ratio 5.9 for SBDP145, and 18.34 for SBDP120). The time course of SBDPs in nonsurvivors also differed from that of survivors. These results suggest that CSF SBDP levels can predict injury severity and mortality after severe TBI, and can be useful complements to clinical assessment.

Neuronal and glial markers are differently associated with computed tomography findings and outcome in patients with severe traumatic brain injury: A case control study

IntroductionAuthors of several studies have studied biomarkers and computed tomography (CT) findings in the acute phase after severe traumatic brain injury (TBI). However, the correlation between structural damage as assessed by neuroimaging and biomarkers has not been elucidated. The aim of this study was to investigate the relationships among neuronal (Ubiquitin carboxy-terminal hydrolase L1 [UCH-L1]) and glial (glial fibrillary acidic protein [GFAP]) biomarker levels in serum, neuroradiological findings and outcomes after severe TBI.MethodsThe study recruited patients from four neurotrauma centers. Serum samples for UCH-L1 and GFAP were obtained at the time of hospital admission and every 6 hours thereafter. CT scans of the brain were obtained within 24hrs of injury. Outcome was assessed by Glasgow Outcome Scale (GOS) at discharge and at 6 months.Results81 severe TBI patients and 167 controls were enrolled. The mean serum levels of UCH-L1 and GFAP were higher (p < 0.001) in TBI patients compared to controls. UCH-L1 and GFAP serum levels correlated significantly with Glasgow Coma Scale (GCS) and CT findings. GFAP levels were higher in patients with mass lesions than in those with diffuse injury (2.95 ± 0.48 ng/ml versus 0.74 ± 0.11 ng/ml) while UCH-L1 levels were higher in patients with diffuse injury (1.55 ± 0.18 ng/ml versus 1.21 ± 0.15 ng/ml, p = 0.0031 and 0.0103, respectively). A multivariate logistic regression showed that UCH-L1 was the only independent predictor of death at discharge [adjusted odds ratios 2.95; 95% confidence interval, 1.46-5.97], but both UCH-L1 and GFAP levels strongly predicted death 6 months post-injury.ConclusionsRelationships between structural changes detected by neuroimaging and biomarkers indicate each biomarker may reflect a different injury pathway. These results suggest that protein biomarkers could provide better characterization of subjects at risk for specific types of cellular damage than that obtained with neuroimaging alone, as well as provide valuable information about injury severity and outcome after severe TBI.

Biokinetic Analysis of Ubiquitin C-Terminal Hydrolase-L1 (UCH-L1) in Severe Traumatic Brain Injury Patient Biofluids

Ubiquitin C-terminal hydrolase-L1 (UCH-L1) is a neuron-specific enzyme that has been identified as a potential biomarker of traumatic brain injury (TBI). The study objectives were to determine UCH-L1 exposure and kinetic metrics, determine correlations between biofluids, and assess outcome correlations in severe TBI patients. Data were analyzed from a prospective, multicenter study of severe TBI (Glasgow Coma Scale [GCS] score ≤ 8). Cerebrospinal fluid (CSF) and serum data from samples taken every 6 h after injury were analyzed by enzyme-linked immunosorbent assay (ELISA). UCH-L1 CSF and serum data from 59 patients were used to determine biofluid correlations. Serum samples from 86 patients and CSF from 59 patients were used to determine outcome correlations. Exposure and kinetic metrics were evaluated acutely and up to 7 days post-injury and compared to mortality at 3 months. There were significant correlations between UCH-L1 CSF and serum median concentrations (r(s)=0.59, p<0.001), AUC (r(s)=0.3, p=0.027), Tmax (r(s)=0.68, p<0.001), and MRT (r(s)=0.65, p<0.001). Outcome analysis showed significant increases in median serum AUC (2016 versus 265 ng/mL*min, p=0.006), and Cmax (2 versus 0.4 ng/mL, p=0.003), and a shorter Tmax (8 versus 19 h, p=0.04) in those who died versus those who survived, respectively. In the first 24 h after injury, there was a statistically significant acute increase in CSF and serum median Cmax((0-24h)) in those who died. This study shows a significant correlation between UCH-L1 CSF and serum median concentrations and biokinetics in severe TBI patients, and relationships with clinical outcome were detected.

Moderate posttraumatic hypothermia decreases early calpain-mediated proteolysis and concomitant cytoskeletal compromise in traumatic axonal injury.

Traumatic brain injury (TBI) in animals and man generates widespread axonal injury characterized by focal axolemmal permeability changes, induction of calpain-mediated proteolysis, and neurofilament side-arm modification associated with neurofilament compaction (NFC) evolving to axonal disconnection. Recent observations have suggested that moderate hypothermia is neuroprotective in several models of TBI. Nevertheless, the pathway by which hypothermia prevents traumatic axonal injury (TAI) is still a matter of debate. The present study was conducted to evaluate the effects of moderate, early posttraumatic hypothermia on calpain-mediated spectrin proteolysis (CMSP), implicated in the pathogenesis of TAI. Using moderate (32 degrees C) hypothermia of 90 min duration without rewarming, the density of CMSP immunoreactive/damaged axons was quantified via LM analysis in vulnerable brain stem fiber tracts of hypothermic and normothermic rats subjected to impact acceleration TBI (90 min postinjury survival). To assess the influence of posthypothermic rewarming, a second group of animals was subjected to 90 min of hypothermia followed by 90 min of rewarming to normothermic levels when CMSP was analyzed to detect if any purported CMSP prevention persisted (180 min postinjury survival). Additionally, to determine if this protection translated into comparable cytoskeletal protection in the same foci showing decreased CMSP, antibodies targeting altered/compacted NF subunits were also employed. Moderate hypothermia applied in the acute postinjury period drastically reduced the number of damaged axons displaying CMSP at both time points and significantly reduced NFC immunoreactivity at 180 min postinjury. These results suggest that the neuroprotective effects of hypothermia in TBI are associated with the inhibition of axonal/cytoskeletal damage.

Preinjury Administration of the Calpain Inhibitor MDL-28170 Attenuates Traumatically Induced Axonal Injury

Traumatic brain injury (TBI) evokes diffuse (traumatic) axonal injury (TAI), which contributes to morbidity and mortality. Damaged axons display progressive alterations gradually evolving to axonal disconnection. In severe TAI, the tensile forces of injury lead to a focal influx of Ca2+, initiating a series of proteolytic processes wherein the cysteine proteases, calpain and caspase modify the axonal cytoskeleton, causing irreversible damage over time postinjury. Although several studies have demonstrated that the systemic administration of calpain inhibitors reduces the extent of ischemic and traumatic contusional injury a direct beneficial effect on TAI has not been established to date. The current study was initiated to address this issue in an impact acceleration rat-TBI model in order to provide further evidence on the contribution of calpain-mediated proteolytic processes in the pathogenesis of TAI, while further supporting the utility of calpain-inhibitors. A single tail vein bolus injection of 30 mg/kg MDL-28170 was administered to Wistar rats 30 min preinjury. After injury the rats were allowed to survive 120 min when they were perfused with aldehydes. Brains were processed for immunohistochemical localization of damaged axonal profiles displaying either amyloid precursor protein (APP)- or RMO-14-immunoreactivity (IR), both considered markers of specific features of TAI. Digital data acquisition and statistical analysis demonstrated that preinjury administration of MDL-28170 significantly reduced the mean number of damaged RMO-14- as well as APP-IR axonal profiles in the brainstem fiber tracts analyzed. These results further underscore the role of calpain-mediated proteolytic processes in the pathogenesis of DAI and support the potential use of cell permeable calpain-inhibitors as a rational therapeutic approach in TBI.

Brain Injury Biomarkers May Improve the Predictive Power of the IMPACT Outcome Calculator

Outcome prediction following severe traumatic brain injury (sTBI) is a widely investigated field of research. A major breakthrough is represented by the IMPACT prognostic calculator based on admission data of more than 8500 patients. A growing body of scientific evidence has shown that clinically meaningful biomarkers, including glial fibrillary acidic protein (GFAP), ubiquitin C-terminal hydrolase-L1 (UCH-L1), and αII-spectrin breakdown product (SBDP145), could also contribute to outcome prediction. The present study was initiated to assess whether the addition of biomarkers to the IMPACT prognostic calculator could improve its predictive power. Forty-five sTBI patients (GCS score≤8) from four different sites were investigated. We utilized the core model of the IMPACT calculator (age, GCS motor score, and reaction of pupils), and measured the level of GFAP, UCH-L1, and SBDP145 in serum and cerebrospinal fluid (CSF). The forecast and actual 6-month outcomes were compared by logistic regression analysis. The results of the core model itself, as well as serum values of GFAP and CSF levels of SBDP145, showed a significant correlation with the 6-month mortality using a univariate analysis. In the core model, the Nagelkerke R(2) value was 0.214. With multivariate analysis we were able to increase this predictive power with one additional biomarker (GFAP in CSF) to R(2)=0.476, while the application of three biomarker levels (GFAP in CSF, GFAP in serum, and SBDP145 in CSF) increased the Nagelkerke R(2) to 0.700. Our preliminary results underline the importance of biomarkers in outcome prediction, and encourage further investigation to expand the predictive power of contemporary outcome calculators and prognostic models in TBI.