Initial biological qualification of SBDP-145 as a biomarker of compound-induced neurodegeneration in the rat.

Detection of compound-related neurodegeneration is currently limited to brain histopathology in veterinary species and functional measurements such as electroencephalography and observation of clinical signs in patients. The objective of these studies was to investigate whether concentrations of spectrin breakdown product 145 (SBDP-145) in cerebrospinal fluid (CSF) correlate with the severity of neurodegeneration in rats administered neurotoxic agents, as part of a longer term objective of developing in vivo biomarkers of neurotoxicity for use in non-clinical and clinical safety studies. Non-erythroid alpha-II spectrin is a cytoskeletal protein cleaved by the protease calpain when this enzyme is activated by dysregulation of calcium in injured cells. Calcium dysregulation is also associated with some toxicological responses in animals, and may be sufficient to activate neuronal calpain and produce SBDPs that can be released into CSF. Neurotoxicants (kainic acid, 2-chloropropionic acid, bromethalin, and pentylenetetrazole) known to affect different portions of the brain were administered to rats in dose-response and time-course studies in which neurodegeneration was measured by histopathology and SBDP-145 concentrations in CSF were measured by ELISA. We consistently observed >3-fold increases in SBDP-145 concentration in rats with minimal to slight neurodegenerative lesions, and 20 to 150-fold increases in animals with more severe lesions. In contrast, compounds that caused non-degenerative changes in central nervous system (CNS) did not increase SBDP-145 in CSF. These data support expanded use of SBDP-145 as a biomarker for monitoring compound-induced neurodegeneration in pre-clinical studies, and support the investigation of clinical applications of this biomarker to promote safe dosing of patients with compounds that have potential to cause neurodegeneration.

Different expression of ubiquitin C-terminal hydrolase-L1 and αII-spectrin in ischemic and hemorrhagic stroke: Potential biomarkers in diagnosis.

The two primary categories of stroke, ischemic and hemorrhagic, both have fundamentally different mechanisms and thus different treatment options. These two stroke categories were applied to rat models to identify potential biomarkers that can distinguish between them. Ischemic stroke was induced by middle cerebral artery occlusion (MCAO) without reperfusion while hemorrhagic stroke was induced by injecting collagenase IV into the striatum. Brain hemispheres and biofluids were collected at two time points: 3 and 6h after stroke. Known molecules were tested on the rat samples via quantitative immunoblotting (injured brain, CSF) and Banyan's proprietary ELISA assays (CSF, serum). The injured brain quantitative analyses revealed that αII-spectrin breakdown products (SBDP150, SBDP145) were strongly increased after 6h ischemia. In CSF, SBDP145 and ubiquitin C-terminal hydrolase-L1 (UCH-L1) levels were elevated after 6h ischemic stroke detected by Western blot and ELISA. In serum UCH-L1 levels were increased after 3 and 6h of ischemia detected by ELISA. However, levels of those proteins in hemorrhagic stroke remain normal. In summary, in both the brain and the biofluids, SBDPs and UCH-L1 were elevated after ischemic but not hemorrhagic stroke. These molecules behaved differently in the two stroke models and thus may be capable of being differentiated.

Cerebrospinal fluid protein biomarker panel for assessment of neurotoxicity induced by kainic acid in rats.

Glutamate excitotoxicity plays a key role in the etiology of a variety of neurological, psychiatric, and neurodegenerative disorders. The goal of this study was to investigate spatiotemporal distribution in the brain and cerebrospinal fluid (CSF) concentrations of ubiquitin C-terminal hydrolase-1 (UCH-L1), glial fibrillary acidic protein (GFAP), αII-spectrin breakdown products (SBDP150, SBDP145, and SBDP120), and their relationship to neuropathology in an animal model of kainic acid (KA) excitotoxicity. Triple fluorescent labeling and Fluoro-Jade C staining revealed a reactive gliosis in brain and specific localization of degenerating neurons in hippocampus and entorhinal cortex of KA-treated rats. Immunohistochemistry showed upregulation of GFAP expression in hippocampus and cortex beginning 24h post KA injection and peaking at 48h. At these time points concurrent with extensive neurodegeneration all SBDPs were observed throughout the brain. At 24h post KA injection, a loss of structural integrity was observed in cellular distribution of UCH-L1 that correlated with an increase in immunopositive material in the extracellular matrix. CSF levels of UCH-L1, GFAP, and SBDPs were significantly increased in KA-treated animals compared with controls. The temporal increase in CSF biomarkers correlated with brain tissue distribution and neurodegeneration. This study provided evidence supporting the use of CSF levels of glial and neuronal protein biomarkers to assess neurotoxic damage in preclinical animal models that could prove potentially translational to the clinic. The molecular nature of these biomarkers can provide critical information on the underlying mechanisms of neurotoxicity that might facilitate the development of novel drugs and allow physicians to monitor drug safety.

Comparing levels of biochemical markers in CSF from cannulated and non-cannulated rats.

Cerebrospinal fluid (CSF) is commonly used for assessing biomarkers of drug efficacy or disease progression in the central nervous system. Studies of CSF from pre-clinical species can characterize biomarkers for use in clinical trials. However, obtaining CSF from pre-clinical species, particularly rodents, can be challenging due to small body sizes, and consequently, low volumes of CSF. Surgical cannulation of rats is commonly used to allow for CSF withdrawal from the cisterna magna. However, cannulae do not remain patent over multiple days, making chronic studies on the same rats difficult. Moreover, CSF biomarkers may be affected by cannulation. Thus cannulation may contribute confounding factors to the understanding of CSF biomarkers. To determine the potential impact on biomarkers, CSF was analyzed from cannulated rats, surgically implanted with catheters as well as from non-cannulated rats. Brain protein biomarkers (αII-spectrin SBDP150 and total tau) and albumin, were measured in the CSF using ELISA assays. Overall, cannulated rat CSF had elevated levels of the biomarkers examined compared to non-cannulated rat CSF. Additionally, the variation in biomarker levels observed among CSF from cannulated rats was greater than that observed for non-cannulated rat CSF. These results demonstrate that in some cases, biomarker assessment using CSF from cannulated rats may differ from that of non-cannulated animals and may contribute confounding factors to biomarker measurements and assay development for clinical use.

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 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.

Ubiquitin C-terminal hydrolase-L1 as a biomarker for ischemic and traumatic brain injury in rats.

Ubiquitin C-terminal hydrolase-L1 (UCH-L1), also called neuronal-specific protein gene product 9.5, is a highly abundant protein in the neuronal cell body and has been identified as a possible biomarker on the basis of a recent proteomic study. In this study, we examined whether UCH-L1 was significantly elevated in cerebrospinal fluid (CSF) following controlled cortical impact (CCI) and middle cerebral artery occlusion (MCAO; model of ischemic stroke) in rats. Quantitative immunoblots of rat CSF revealed a dramatic elevation of UCH-L1 protein 48 h after severe CCI and as early as 6 h after mild (30 min) and severe (2 h) MCAO. A sandwich enzyme-linked immunosorbent assay constructed to measure UCH-L1 sensitively and quantitatively showed that CSF UCH-L1 levels were significantly elevated as early as 2 h and up to 48 h after CCI. Similarly, UCH-L1 levels were also significantly elevated in CSF from 6 to 72 h after 30 min of MCAO and from 6 to 120 h after 2 h of MCAO. These data are comparable to the profile of the calpain-produced alphaII-spectrin breakdown product of 145 kDa biomarker. Importantly, serum UCH-L1 biomarker levels were also significantly elevated after CCI. Similarly, serum UCH-L1 levels in the 2-h MCAO group were significantly higher than those in the 30-min group. Taken together, these data from two rat models of acute brain injury strongly suggest that UCH-L1 is a candidate brain injury biomarker detectable in biofluid compartments (CSF and serum).

A panel of neuron-enriched proteins as markers for traumatic brain injury in humans.

Surrogate markers have enormous potential for contributing to the diagnosis, prognosis, and therapeutic evaluation of acute brain damage, but extensive prior study of individual candidates has not yielded a biomarker in widespread clinical practice. We hypothesize that a panel of neuron-enriched proteins measurable in cerebrospinal fluid (CSF) and blood should vastly improve clinical evaluation and therapeutic management of acute brain injuries. Previously, we developed such a panel based initially on the study of protein release from degenerating cultured neurons, and subsequently on rodent models of traumatic brain injury (TBI) and ischemia, consisting of 14-3-3beta, 14-3-3zeta, three distinct phosphoforms of neurofilament H, ubiquitin hydrolase L1, neuron-specific enolase, alpha-spectrin, and three calpain- and caspase-derived fragments of alpha-spectrin. In the present study, this panel of 11 proteins was evaluated as CSF and serum biomarkers for severe TBI in humans. By quantitative Western blotting and sandwich immunoassays, the CSF protein levels were near or below the limit of detection in pre-surgical and most normal pressure hydrocephalus (NPH) controls, but following TBI nine of the 11 were routinely elevated in CSF. Whereas different markers peaked coordinately, the time to peak varied across TBI cases from 24-96 h post-injury. In serum, TBI increased all four members of the marker panel for which sandwich immunoassays are currently available: a calpain-derived NH(2)-terminal alpha-spectrin fragment and the three neurofilament H phosphoforms. Our results identify neuron-enriched proteins that may serve as a panel of CSF and blood surrogate markers for the minimally invasive detection, management, mechanistic, and therapeutic evaluation of human TBI.

Alpha II-spectrin breakdown products serve as novel markers of brain injury severity in a canine model of hypothermic circulatory arrest.

BACKGROUND: The development of specific biomarkers to aid in the diagnosis and prognosis of neuronal injury is of paramount importance in cardiac surgery. Alpha II-spectrin is a structural protein abundant in neurons of the central nervous system and cleaved into signature fragments by proteases involved in necrotic and apoptotic cell death. We measured cerebrospinal fluid alpha II-spectrin breakdown products (alphaII-SBDPs) in a canine model of hypothermic circulatory arrest (HCA) and cardiopulmonary bypass. METHODS: Canine subjects were exposed to either 1 hour of HCA (n = 8; mean lowest tympanic temperature 18.0 +/- 1.2 degrees C) or standard cardiopulmonary bypass (n = 7). Cerebrospinal fluid samples were collected before treatment and 8 and 24 hours after treatment. Using polyacrylamide gel electrophoresis and immunoblotting, SBDPs were isolated and compared between groups using computer-assisted densitometric scanning. Necrotic versus apoptotic cell death was indexed by measuring calpain and caspase-3 cleaved alphaII-SBDPs (SBDP 145+150 and SBDP 120, respectively). RESULTS: Animals undergoing HCA demonstrated mild patterns of histologic cellular injury and clinically detectable neurologic dysfunction. Calpain-produced alphaII-SBDPs (150 kDa+145 kDa bands-necrosis) 8 hours after HCA were significantly increased (p = 0.02) as compared with levels before HCA, and remained elevated at 24 hours after HCA. In contrast, caspase-3 alphaII-SBDP (120 kDa band-apoptosis) was not significantly increased. Animals receiving cardiopulmonary bypass did not demonstrate clinical or histologic evidence of injury, with no increases in necrotic or apoptotic cellular markers. CONCLUSIONS: We report the use of alphaII-SBDPs as markers of neurologic injury after cardiac surgery. Our analysis demonstrates that calpain- and caspase-produced alphaII-SBDPs may be an important and novel marker of neurologic injury after HCA.

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

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.

alphaII-Spectrin breakdown product cerebrospinal fluid exposure metrics suggest differences in cellular injury mechanisms after severe traumatic brain injury.

Traumatic brain injury (TBI) produces alphaII-spectrin breakdown products (SBDPs) that are potential biomarkers for TBI. To further understand these biomarkers, the present study examined (1) the exposure and kinetic characteristics of SBDPs in cerebrospinal fluid (CSF) of adults with severe TBI, and (2) the relationship between these exposure and kinetic metrics and severity of injury. This clinical database study analyzed CSF concentrations of 150-, 145-, and 120-kDa SBDPs in 38 severe TBI patients. Area under the curve (AUC), mean residence time (MRT), maximum concentration (C(max)), time to maximum concentration (T(max)), and half-life (t(1/2)) were determined for each SBDP. Markers of calpain proteolysis (SBDP150 and SBDP145) had a greater median AUC and C(max) and a shorter MRT than SBDP120, produced by caspase-3 proteolysis in the CSF in TBI patients ( p < 0.001). AUC and MRT for SBDP150 and SBDP15 were significantly greater in patients with worse Glasgow Coma Scale (GCS) scores at 24 h after injury compared to those whose GCS scores improved (AUC p=0.013, MRT p=0.001; AUC p=0.009, MRT p=0.021, respectively). A positive correlation was found between patients with longer elevations in intracranial pressure (ICP) measurements of 25mmHg or higher and those with a greater AUC and MRT for all three biomarkers. This is the first study to show that the biomarkers of proteolysis differentially associated with calpain and caspase-3 activity have distinct CSF exposure profiles following TBI that suggest a prominent role for calpain activity. Further studies are being conducted to determine if exposure and kinetic metrics for biofluid-based biomarkers can predict clinical outcome.

Alpha-II spectrin breakdown products in aneurysmal subarachnoid hemorrhage: a novel biomarker of proteolytic injury.

OBJECT: Aneurysmal subarachnoid hemorrhage (ASAH) is a serious event with grave consequences. Delayed ischemic neurological deficits caused by cerebral arterial vasospasm contribute significantly to death and disability. Biomarkers may reflect brain injury and provide an early warning of impending neurological decline and stroke from ASAH-induced vasospasm. Alpha-II spectrin is a cytoskeletal protein whose breakdown products are candidate surrogate markers of injury magnitude, treatment efficacy, and outcome. In addition, all spectrin breakdown products (SBDPs) can provide information on the proteolytic mechanisms of injury. METHODS: Twenty patients who received a diagnosis of Fisher Grade 3 ASAH were enrolled in this study to examine the clinical utility of SBDPs in the detection of cerebral vasospasm in patients with ASAH. All patients underwent placement of a ventriculostomy for continual cerebrospinal fluid drainage within 72 hours of ASAH onset. Cerebrospinal fluid samples were collected every 6 hours and analyzed using Western Blotting for SBDPs. Onset of vasospasm was defined as an acute onset of a focal neurological deficit or a change in Glasgow Coma Scale score of two or more points. All suspected cases of vasospasm were confirmed on imaging studies. RESULTS: Both calpain- and caspase-mediated SBDP levels are significantly increased in patients suffering ASAH. The concentration of SBDPs was found to increase significantly over baseline level up to 12 hours before the onset of cerebral arterial vasospasm. CONCLUSIONS: Differential expression of SBDPs suggests oncotic necrotic proteolysis may be predominant in acute brain injury after ASAH and cerebral arterial vasospasm.

Clinical significance of alphaII-spectrin breakdown products in cerebrospinal fluid after severe traumatic brain injury.

Following traumatic brain injury (TBI), the cytoskeletal protein alpha-II-spectrin is proteolyzed by calpain and caspase-3 to signature breakdown products. To determine whether alpha -II-spectrin proteolysis is a potentially reliable biomarker for TBI in humans, the present study (1) examined levels of spectrin breakdown products (SBDPs) in cerebrospinal fluid (CSF) from adults with severe TBI and (2) examined the relationship between these levels, severity of injury, and clinical outcome. This prospective case control study enrolled 41 patients with severe TBI, defined by a Glasgow Coma Scale (GCS) score of < or =8, who underwent intraventricular intracranial pressure monitoring. Patients without TBI requiring CSF drainage for other medical reasons served as controls. Ventricular CSF was sampled from each patient at 6, 12, 24, 48, 72, 96, and 120 h following TBI and analyzed for SBDPs. Outcome was assessed using the Glasgow Outcome Score (GOS) 6 months after injury. Calpain and caspase-3 mediated SBDP levels in CSF were significantly increased in TBI patients at several time points after injury, compared to control subjects. The time course of calpain mediated SBDP150 and SBDP145 differed from that of caspase-3 mediated SBDP120 during the post-injury period examined. Mean SBDP densitometry values measured early after injury correlated with severity of injury, computed tomography (CT) scan findings, and outcome at 6 months post-injury. Taken together, these results support that alpha -II-spectrin breakdown products are potentially useful biomarker of severe TBI in humans. Our data further suggests that both necrotic/oncotic and apoptotic cell death mechanisms are activated in humans following severe TBI, but with a different time course after injury.

Detection of alphaII-spectrin and breakdown products in humans after severe traumatic brain injury.

AIM: alphaII-Spectrin is the major structural component of the cortical membrane cytoskeleton. It is a major substrate for the calpain and caspase-3 cysteine proteases there are considerable evidence that alfaII-spectrin is processed by the calpains and caspase-3 to signature cleavage products in vivo after experimental traumatic brain injury (TBI). We sought to determine whether aII-spectrin proteolysis is a potentially reliable biomarker for TBI in humans measuring the levels of spectrin and spectrin breakdown products (SBDPs) in cerebrospinal fluid (CSF) from adults with severe TBI, and studying the relationship between these levels and clinical outcome. METHODS: This prospective case control study enrolled 8 patients with severe TBI, defined by a Glasgow Coma Score (GCS) of <8, and requiring intraventricular pressure monitoring. Patients without TBI requiring CSF drainage served as controls. Ventricular CSF was drained from each patient at 6, 12, 24, 48, 72, and 96 h following TBI and measured for spectrin and SBDPs. Outcome was assessed using the Glasgow Outcome Score (GOS) 6 months after injury. RESULTS: CSF alphaII-spectrin and calpain and caspase-3 mediated SBDP levels were significantly increased compared to control patients at all time points examined (P<0.001). In patients with a better outcome, CSF spectrin and SBDPs significantly decreased from 6 to 96 h. Patients whose spectrin and SBDP levels remained elevated or failed to decline had a worse outcome (P<0.019). CONCLUSIONS: The present work provides the first evidence that protein degradation of alphaII-spectrin is a reliable marker of severe TBI in humans and that both necrotic and apoptotic cell death mechanisms are activated in humans following a severe TBI. Moreover, the temporal profile of degradation may be an important indicator of clinical outcome.

Spectrin breakdown products in the cerebrospinal fluid in severe head injury--preliminary observations.

BACKGROUND: Calcium-induced proteolytic processes are considered key players in the progressive pathobiology of traumatic brain injury (TBI). Activation of calpain and caspases after TBI leads to the cleavage of cytoskeletal proteins such as non-erythroid alpha II-spectrin. Recent reports demonstrate that the levels of spectrin and spectrin breakdown products (SBDPs) are elevated in vitro after mechanical injury, in the cerebrospinal fluid (CSF) and brain tissue following experimental TBI, and in human brain tissue after TBI. METHODS: This study was initiated to detect spectrin and SBDP accumulation in the ventricular CSF of 12 severe TBI-patients with raised intracranial pressure (ICP). Nine patients with non-traumatically elevated ICP and 5 undergoing diagnostic lumbar puncture (LP) served as controls. Intact spectrin and calpain and caspase specific SBDPs in CSF collected once a day over a several day period were assessed via Western blot analysis. Parameters of severity and outcome such as ICP, Glasgow Coma Scale and Glasgow Outcome Scale were also monitored in order to reveal a potential correlation between these CSF markers and clinical parameters. RESULTS: In control patients undergone LP no immunoreactivity was detected. Non-erythroid alpha-II-spectrin and SBDP occurred more frequently and their level was significantly higher in the CSF of TBI patients than in other pathological conditions associated with raised ICP. Those TBI patients followed for several days post-injury revealed a consistent temporal pattern for protein accumulation with the highest level achieved on the 2(nd) -3(rd) days after TBI. CONCLUSION: Elevation of calpain and caspase specific SBDPs is a significant finding in TBI patients indicating that intact brain spectrin- and SBDP-levels are closely associated with the specific neurochemical processes evoked by TBI. The results strongly support the potential utility of these surrogate markers in the clinical monitoring of patients with severe TBI and provide further evidence of the role of calcium-induced, calpain- and caspase-mediated structural proteolysis in TBI.

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.

A novel marker for traumatic brain injury: CSF alphaII-spectrin breakdown product levels.

Currently, there is no definitive diagnostic test for traumatic brain injury (TBI) to help physicians determine the seriousness of injury or the extent of cellular pathology. Calpain cleaves alphaII-spectrin into breakdown products (SBDP) after TBI and ischemia. Mean levels of both ipsilateral cortex (IC) and cerebral spinal fluid (CSF) SBDP at 2, 6, and 24 h after two levels of controlled cortical impact (1.0 mm and 1.6 mm of cortical deformation) in rats were significantly elevated by injury. CSF and IC SBDP levels were significantly higher after severe (1.6 mm) injury than mild (1.0 mm) injury over time. The correlation between CSF SBDP levels and lesion size from T2-weighted magnetic resonance images 24 hours after TBI as well as correlation of tau and S100beta was assessed. Mean levels of CSF SBDP (r = 0.833) and tau (r = 0.693) significantly correlated with lesion size while levels of CSF S100beta did not (r = 0.188). Although levels of CSF and IC SBDP and lesion size are all significantly higher after 1.6 mm than 1.0 mm injury, the correlation between CSF SBDP and lesion size was not significant following the removal of controls from the analysis. This indicates CSF SBDP is a reliable marker of the presence or absence of injury. Furthermore, larger lesion sizes 24 h after TBI were negatively correlated with motor performance on days 1-5 after TBI (r = -0.708). Based on these data, evaluation of CSF SBDP levels as a biomarker of TBI is warranted in clinical studies.

Accumulation of non-erythroid alpha II-spectrin and calpain-cleaved alpha II-spectrin breakdown products in cerebrospinal fluid after traumatic brain injury in rats.

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 specificity for defining neuropathological cascades. Identification 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. Non-erythroid alpha II-spectrin is a cytoskeletal protein that is a substrate of both calpain and caspase-3 cysteine proteases. As we have previously demonstrated, cleavage of alpha II-spectrin by calpain and caspase-3 results in accumulation of protease-specific spectrin breakdown products (SBDPs) that can be used to monitor the magnitude and temporal duration of protease activation. However, accumulation of alpha II-spectrin and alpha II-SBDPs in CSF after TBI has never been examined. Following a moderate level (2.0 mm) of controlled cortical impact TBI in rodents, native alpha II-spectrin protein was decreased in brain tissue and increased in CSF from 24 h to 72 h after injury. In addition, calpain-specific SBDPs were observed to increase in both brain and CSF after injury. Increases in the calpain-specific 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-specific 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 alpha II-spectrin and alpha II-SBDPs 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.