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- Materials and methods
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 αII-spectrin is a cytoskeletal protein that is a substrate of both calpain and caspase-3 cysteine proteases. As we have previously demonstrated, cleavage of αII-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 αII-spectrin and αII-SBDPs in CSF after TBI has never been examined. Following a moderate level (2.0 mm) of controlled cortical impact TBI in rodents, native αII-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 αII-spectrin and αII-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.
The incidence of traumatic brain injury (TBI) in the United States of America is conservatively estimated to be more than 2 million persons annually with approximately 500 000 hospitalizations (Goldstein 1990). Of these, about 70 000–90 000 head injury survivors are permanently disabled. The annual economic cost to society for care of head-injured patients is estimated at $25 billion (Goldstein 1990). Thus, accurate and reliable measurement of outcome following head injury is of great interest to both head injury survivors and clinicians. Assessment of pathology and neurological impairment immediately after TBI is crucial for determination of appropriate clinical management and for predicting long-term outcome. The outcome measures most often used in head-injured patients are the Glasgow coma scale (GCS), the Glasgow outcome scale (GOS), and computed tomography (CT) scans to detect intracranial pathology. However, despite dramatically improved emergency triage systems based on these outcome measures, most TBI survivors suffer long-term (for a number of years) impairment, and a large number of TBI survivors are severely affected by TBI despite predictions of ‘good recovery’ on the GOS (Marion 1996). Because of the limitations of current clinical assessments of TBI severity, there has been an increased interest in the development of neurochemical markers for determining injury severity and for clinical evaluation of pathophysiological mechanisms operative in traumatized brain.
For example, TBI results in neuronal tissue death that can cause a variety of neurochemicals such as amino acids, ions and lactate, as well as a number of cellular proteins and enzymes, to be released into the blood and CSF (Goodman and Simpson 1996). Although assessment of cardiac and liver protein levels in the blood has routinely been used in medical practice for years (e.g. creatine kinase MB or troponin-T), assessment of CNS proteins in blood or CSF is far less developed. Thus, recent studies have measured a variety of neurochemical substances in the CSF or blood in attempt to identify specific surrogate markers of cellular damage and outcome after TBI and other CNS disorders (Haber and Grossman 1980; Inao et al. 1988; Robinson et al. 1990; Lyeth et al. 1993; Raabe and Seifert 1999; Raabe et al. 1999; Zemlan et al. 1999; Clark et al. 2000a; Tapiola et al. 2000). For example, creatine kinase BB, lactate dehydrogenase, myelin basic protein, and neuron-specific enolase have been measured in blood or CSF in various CNS disorders including TBI. However, these proteins are non-specific to the brain, offer no insight as to mechanism of injury, and/or prediction of outcome utilizing these proteins has not proven reliable (Goodman and Simpson 1996). Other proteins detected in CSF after brain injury such as S-100B are highly specific to the CNS and have been more robustly correlated with outcome (Raabe and Seifert 1999; Raabe et al. 1999). Although brain-specific surrogate biomarkers like S-100B may be useful indicators of outcome after brain injury, detection of these proteins in blood or CSF offers no insight into neurochemical alterations that mediate brain damage after TBI. Thus, identification of neurochemical markers that are specific to the CNS and that provide information about specific ongoing neurochemical events would prove immensely beneficial for both prediction of outcome and for guidance of targeted therapeutic delivery.
Non-erythroid αII-spectrin is the major structural component of the cortical membrane cytoskeleton, is particularly abundant in axons and presynaptic terminals (Riederer et al. 1986; Goodman et al. 1995), and is a major substrate for both calpain and caspase-3 cysteine proteases (Wang et al. 1998). The calpain-mediated cleavage of αII-spectrin occurs between Tyr1176 and Gly1177 resulting in the formation of calpain-signature spectrin breakdown products (SBDPs) of 150 and 145 kDa (Harris et al. 1988). The caspase-3-mediated cleavages of αII-spectrin occur at Asp1185, Ser1186, Asp1478 and Ser1479 resulting in the formation of caspase-3-signature SBDPs of 150 and 120 kDa, respectively (Wang et al. 1998). Importantly, numerous investigations have documented increased pathological activation of calpain and/or caspase-3 proteases after TBI (Saatman et al. 1996a, 2000; Kampfl et al. 1997; Newcomb et al. 1997; Posmantur et al. 1997; Yakovlev et al. 1997; Pike et al. 1998a; Clark et al. 1999, 2000b; LaPlaca et al. 1999; Okonkwo et al. 1999; Zhang et al. 1999; Beer et al. 2000; Buki et al. 2000). In addition, our laboratory and others have provided extensive evidence that αII-spectrin is processed by calpains and/or caspase-3 to signature cleavage products in vivo after TBI (Beer et al. 2000; Newcomb et al. 1997; Pike et al. 1998a; Buki et al. 2000) and in in vitro models of mechanical stretch injury (Pike et al. 2000b), necrosis (Zhao et al. 1999), apoptosis (Nath et al. 1996a, 1996b; Pike et al. 1998b), glutamate or NMDA excitotoxicity (Nath et al. 2000; Zhao et al. 2000), and oxygen-glucose deprivation (Nath et al. 1998; Newcomb et al. 1998). Moreover, use of selective αII-SBDP antibodies has been used to demonstrate that brain regions with the highest accumulation of SBDPs also have the highest levels of neuronal cell death (Roberts-Lewis et al. 1994; Newcomb et al. 1997). Thus, the ubiquitous distribution of αII-spectrin in the brain coupled with the ability to utilize signature αII-spectrin proteolytic fragments generated by pathological activation of calpain and/or caspase-3 after TBI makes αII-spectrin a potentially important biomarker of brain damage. To test this hypothesis, the present investigation examined alterations in brain levels of αII-spectrin and αII-SBDPs after controlled cortical impact TBI in rodents, and compared these changes to accumulation of αII-spectrin and αII-SBDPs in CSF in the same animals.
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- Materials and methods
This paper provides the first evidence for accumulation of non-erythroid αII-spectrin protein and calpain-mediated αII-SBDPs in CSF after TBI. Detection of calpain-specific proteolytic fragments to αII-spectrin were confirmed with two antibodies, one that recognizes both intact αII-spectrin and calpain-specific SBDPs (FG 6090 Ab), and one that recognizes only the N-terminal region of calpain-cleaved 150 kDa SBDP (SBDP150 Ab). Results of this investigation indicate that CSF detection of αII-spectrin and αII-SBDPs can provide both a sensitive surrogate biochemical measure of TBI pathology and provide important information about specific neurochemical events that have occurred in the brain after TBI. To our knowledge, this is the first investigation of any CNS pathology to indicate that identification of accumulated CSF proteins or protein metabolic products can be used to infer specific neurochemical events (i.e. calpain activation) in the brain. Thus, use of αII-SBDPs as surrogate biochemical markers of TBI has important clinical ramifications for assessment of outcome after injury and for determination of specific pathological proteolytic cascades known to occur after TBI. Although other CNS proteins have been detected in CSF after brain injury (e.g. S-100B) and have been correlated with outcome, these proteins offer no insight into pathological mechanisms that have occurred in the brain after TBI. Obviously, identification of metabolic products with known neurochemical etiology will be beneficial for appropriate application of targeted therapeutics (such as calpain inhibitors) after TBI.
Calpain and caspase-3 cysteine proteases are important mediators of cell death and dysfunction in numerous CNS diseases and injuries including TBI. The calpains have historically been associated with necrotic (oncotic) cell death although recent evidence indicates a role in apoptotic cell death as well (Linnik et al. 1996; Nath et al. 1996a,b; Newcomb et al. 1998; Pike et al. 1998b). Numerous investigations have reported calpain activation after TBI (Saatman et al. 1996a, 2000; Kampfl et al. 1997; Newcomb et al. 1997; Posmantur et al. 1997; Pike et al. 1998a) and inhibitors of calpains have been shown to confer neuroprotection after TBI (Posmantur et al. 1997; Saatman et al. 1996b, 2000). Caspase-3 is a critical executioner of apoptosis and caspase-3 activation has been reported in in vitro (Shah et al. 1997; Allen et al. 1999; Pike et al. 2000b) and in vivo (Beer et al. 2000; Yakovlev et al. 1997; Pike et al. 1998a; Clark et al. 2000b) models of TBI. However, it should be noted that at least in our hands, the magnitude of calpain activation after TBI is much greater than that of caspase-3, and that at the moderate level of brain injury employed in the current study, caspase-3 is only transiently elevated in deep, non-cortical brain regions (Pike et al. 1998a). This result most likely accounts for the detection of relatively minimal amounts of the 120 kDa caspase-3-mediated αII-SBDP in CSF after TBI. In contrast to our injury model, Beer et al. (2000) have observed prominent levels of caspase-3 activation in the cortex after cortical impact TBI. However, while our cortical impact model is typically characterized by prominent tissue necrosis and progressive cortical cavitation to the gray–white interface (Kampfl et al. 1996; Newcomb et al. 1997; Dixon et al. 1998; Newcomb et al. 1999; Pike et al. 2000a), the model employed by Beer et al. (2000) was not. Thus, differences in injury magnitude may be important factors affecting calpain and/or caspase-3 activation after TBI, and this hypothesis warrants further investigation. However, it should be pointed out that although caspase-3 activation has not been a prominent feature in our model of cortical impact TBI, we have detected substantial levels of apoptotic cell death in the cortex after TBI (Newcomb et al. 1999). This apparent discrepancy between apoptotic cell death and caspase-3 activation raises the intriguing possibility that apoptosis may occur via a caspase-3-independent pathway after TBI. This observation also warrants further examination.
That different injury magnitudes may result in differential activation of calpain or caspase-3 proteases has important implications for targeted therapeutic intervention after TBI, and importantly, further validates the utility of using surrogate markers of TBI that have known neurochemical etiologies. For example, the current investigation detected CSF accumulation of the calpain-mediated αII-SBDP and not the caspase-3-mediated αII-SBDP. Based on this evidence, administration of calpain but not caspase-3 inhibitors would be predicted to have the most beneficial effect on outcome. However, other injury magnitudes may result in more caspase-3 activation indicating use of caspase-3 inhibitors or a combination of calpain/caspase-3 protease inhibitors. Thus, surrogate measures of TBI will result in selective pharmaceutical therapies based on clinical assessment of neuropathology, and this approach is a superior strategy to promiscuous prophylactic administration of unnecessary and potentially harmful compounds.
The most probable source of peripheral contamination of the CSF after TBI will be blood born. Indeed, we did detect visible red blood cell contamination of CSF after experimental TBI (which was removed by centrifugation). However, our control experiments with brain and whole blood immnuoblots (Fig. 6a,b) clearly demonstrated that the non-erythroid anti-αII-spectrin antibody did not detect any αII-spectrin protein in whole blood samples. Conversly, the erythroid αI-spectrin antibody labeled both brain and blood samples. These results indicate that the major source of potential peripheral CSF contamination after TBI, blood, is not detected by the non-erythroid anti-αII-spectrin antibody. This finding supports the utility of αII-spectrin and αII-SBDPs as surrogate biomarkers of injury after TBI, and importantly, as biomarkers of calpain and/or caspase-3 activation after TBI.
One caveat to the current investigation is the finding that there was more variability in levels of CSF SBDPs than there were in brain levels of SBDPs. This variability is indicated by the larger error bars in Fig. 2 and 4 and can be observed in individual animals in Fig. 1. The reason for the larger variability in CSF protein accumulation is unknown, but may reflect differences in individual animal's CSF circulation after TBI. For example, differences in increased intracranial pressure after TBI may restrict passage of CSF through various foramina that may preclude detection of secreted proteins into the cisterna magna (source of CSF in the present study). Additional studies should examine differences in intraventricular versus intracisternal levels of accumulated SBDPs.
Nonetheless, future studies focused on development of neuron-specific antibodies targeted against calpain-specific and caspase-3-specific αII-SBDPs (such as the SBDP150 Ab) will further strengthen the utility and specificity of αII-SBDPs as surrogate markers of brain injury. In addition, development of enzyme-linked immunosorbent assays (ELISA) will allow greater quantification of calpain and caspase-3 SBDPs and provide a more rapid and practical approach to CSF detection of these proteins.