The Need for Biomarkers for Brain Trauma

There have been significant advances in our understanding of the pathobiology and biochemical pathways relevant to the areas of traumatic brain injury (TBI). At the same period, numerous experimental drugs have been tested and shown to be neuroprotective in experimental animal model of brain injury; however, these efforts failed to translate successfully into TBI clinical trials [1]. These unsuccessful outcomes have led researchers and pharmaceutical companies to modify their approach for TBI clinical trials by switching into utilizing drug intervention-tracking biomarkers’ changes [2]. For this purpose, the areas of proteomic/genomic methods have been the key instruments in the discovery of novel brain injury biomarker candidates [3–6]. As discussed below, several brain injury biomarkers have the potential to revolutionize medical practice and biomedical research in the area of TBI [7]. However, we and others strongly believe that there is an unmet medical need for designing a simple biofluid-based rapid diagnostic test for both the management of severe and moderate TBI patients in the intense care unit as well as for triaging mild TBI patients arriving in the emergency room.

Diagnostic tests based on protein biomarkers have already demonstrated proven clinical diagnostic utility in acute care environments. For example, in the area of cardiac injury, cardiac troponin proteins (T and I) and various forms of brain natriuretic peptide (BNP), often in combination with other biomarkers, are routinely used to facilitate accurate diagnosis of congestive heart failure and myocardial infarction in patients presenting with chest pain [8–10]. With the growing recognition of the importance of biomarkers, a Biomarkers Consortium was launched in the USA in October 2006 as a public-pharmaceutical industry partnership including the National Institutes of Health (NIH), the Food and Drug Administration (FDA) as a part of the FDA’s critical path initiative, the Centers for Medicare and Medicaid Services, as well as pharmaceutical industry representatives and nonprofit organizations and advocacy groups. An NIH workshop on improving diagnosis of TBI for targeting therapies highlighted the need for biomarker identification [11].

Current and Novel Brain Injury Protein Biomarkers

Several brain injury biomarkers have been cited in literature. Table 1 describes a number of studied traumatic and ischemic brain injury biomarkers documented in literature. Among these, neuron-specific enolase (NSE) [12], glial protein S-100β [13], glial fibrillary acidic protein (GFAP), and myelin basic protein (MBP) have been shown to have great utility in TBI specifically [5, 7]. Different studies illustrated the diagnostic potential of these brain injury biomarkers; however, other studies have provided conflicting results. NSE, for example, initially held great potential as a specific brain injury biomarker since it was originally believed to be strictly neuronal. Of interest, assays of serum NSE together with S100β proteins have been valuable in predicting TBI outcomes [13, 14]. However, additional research found that NSE was also present in red blood cells and platelets, decreasing its diagnostic utility as a marker due to possible cross contamination from blood [15]. After multiple traumas, increase in NSE levels has been observed; however, this increase was not specific to the occurrence of TBI, limiting its ability to be a discriminator of brain injury magnitude [12, 16, 17].

Table 1 Current and novel biomarkers for ischemic and traumatic brain injury
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Along the same lines, our group and many others have characterized αII-spectrin protein and its breakdown products SBDPs. αII-spectrin generates a number of BDPs; SBDP150 and SBDP145 are produced by calpain activation in acute necrosis phase, while SBDP120 is produced by caspase-3 activation during delayed apoptosis phase. These specific SBDPs serve as potential biomarkers for excitotoxic, traumatic, and ischemic brain injury in rat as well as in human brain trauma [5, 7, 18]. Others have proposed that the cleaved tau protein (c-tau) and a fragmented form of the glutamate-N-methyl-d-aspartate (NMDA) receptor (NR2A/2B subtype) [19] might have similar utility as potential biomarkers in brain trauma. In addition, studies by Petzold and Shaw have identified neurofilament-H as a promising axonal injury biomarker for various forms of acute brain damage [20, 21].

Using differential neuroproteomic methods, a systematic assessment was made to identify additional unidentified protein biomarkers for TBI, ischemic and penetrating brain injury with relevant animal models [22–26]. As a follow-up, proteomics-based approach was applied to select top-down candidate markers that represent distinct pathways and hot spots [4]. One such candidate biomarker identified was ubiquitin C-terminal hydrolase-L1 (UCH-L1). It has recently published by us that UCH-L1 is released into both the CSF and blood following both experimental TBI (controlled cortical impact) and ischemic stroke (transient middle cerebral artery occlusion); [18, 27, 28] and in a relevant model of blast overpressure wave-induced brain injury in rats [29]. Similarly, Papa et al. and Brophy et al. have also reported that UCH-L1 was released into the CSF and blood almost immediately following a severe TBI incident [18, 27]. Siman and his colleagues described that UCH-L1 (in addition to phosphorylated neurofilament-H, αII-spectrin breakdown product, and 14-3-3 proteins) is elevated in human CSF following surgically induced circulation arrest [30, 31], as well as in a small severe TBI cohort [30–32]. Independently, UCH-L1 was found to be released into the CSF following aneurysmal subarachnoid hemorrhage in humans [28]. Table 1 summarizes the major identified biomarker candidates in TBI.

In the area of stroke research, Allard and colleagues (2005) have identified PARK7 (DJ-1) protein and nucleotide diphosphate kinase A protein as potential ischemic stroke markers, but their brain specificity and distribution have not been well characterized [33]. Endothelial monocyte-activating polypeptide II precursor (EMAP-II) is another potential microglia biomarker identified by differential neuroproteomics [24, 25], which is unregulated in the CSF and plasma after experimental TBI (Table 1). S100β, UCH-L1, and GFAP proteins are also useful markers for stroke therapeutic development [32]. GFAP might have the ability to distinguish hemorrhagic from ischemic stroke [34], allowing for the use of TPA, which is contraindicated for hemorrhagic stroke patients. These biomarkers could represent different pathways that can be at play at various time points after the initial injury.

Finally, neuroinflammatory-linked cytokines [interleukin (IL)-6, IL-8, tumor necrosis factor-α (TNF-α), and MMP9] have been also studied in the area of stroke biomarkers. It is also appropriate to think of TBI biomarkers as a continuum of biomarkers that might be released at different time points following the initial brain injury event (Fig. 1) [35].

Fig. 1
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Biomarkers for monitoring various temporally and biochemically distinct events following brain injury

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Neurosystems Biology Analysis in Brain Trauma

In the area of stroke, one major hurdle arises from the fact that the diagnosis and management of stroke patients are limited by the lack of rapid diagnostic assays for use in emergency settings as will be discussed later. Thus, the hunt for specific and sensitive stroke biomarkers has been recently initiated since these signature proteins would aid in discriminating among different stroke phenotypes and help in designing rapid diagnostic assays [36]. In our work in the area of neurotrauma, we have utilized different “Omics” approaches coupled with systems biology (SB) analysis to identify novel key brain injury markers that can be used as a clinical end point in neurotrauma [37, 38].

Coupled to current genomics and proteomics analysis utilized in biomarker identification, SB represents a mathematical model capable of predicting the altered processes or functions of a complex system under normal and perturbed conditions. It combines experimental, basic science data sets, proteomic and genetic data sets, literature, and text mining. When constructed properly, SB databases can provide a context or framework for understanding biological responses within physiological networks at the organism level, rather than in isolation [39].

In the area of brain trauma, neurosystems biology platform harnesses data sets that, by themselves, would be overwhelming, into an organized, interlinked database that can be queried to identify non-redundant brain injury pathways or convert hot spots. These can be exploited to determine their utilities as diagnostic biomarkers and/or therapeutic targets. The ultimate goals of system biology are: first by exploring the systems component (gene, protein, small molecule, metabolite, etc.), help biologists, pharmaceutical companies, and doctors to better understand the mechanisms underlying the disease components [40]. In the field of neurotrauma, identifying and analyzing brain injury-related networks play important and practical clues relating to biological pathways relevant to disease processes. However, the more important underlying goal is to provide important cues that may suggest radically new approaches to therapeutics. In the brain injury for example, it has been shown that calpain and caspase proteases are major components in cell death pathways taking part in two destructive proteolytic pathways that not only contribute to key forms of cell death (necrosis and apoptosis) but also in the destruction of important structural components of the axons [alpha II-spectrin breakdown products (SBDPs) and tau], dendrites (MAP2), and myelin (MBP; Fig. 2). Interestingly, two different forms of SBDPs reflect either neuronal necrosis (SBDP150 and SBDP145 cleaved by calpain) or neuronal apoptosis (SBDP120 cleaved by caspase-3) [5]. These SBDPs and other similar neural protein breakdown products can serve as target pathway specific biomarkers as illustrated in Fig. 2.

Fig. 2
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System biology based on TBI biomarker study

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For this purpose, rather than focusing on individual molecular components, systems biology seeks to understand the system dynamics that govern protein networks, the functional set of proteins that regulate cellular decisions related to TBI. From the perspectives of drug discovery and diagnostics, systems biology gives important and practical clues concerning the pathways relevant to TBI and the effects that drugs might have on them. Therefore, it enhances the entire biomarker and therapeutic drug discovery, development, and commercialization process.

Biomarker-Based Management of TBI and Stroke Patients Assessing Therapeutic Intervention

There have been more than 200 unsuccessful clinical trials assessing potential therapies for traumatic brain injury (TBI), and currently, there are no FDA-approved therapies. As early as 2002, an NIH workshop recognized the need for development of more refined surrogate measures to improve design and execution of clinical trials in the field of head injury [41]. There are a number of areas in which incorporation of biomarkers could significantly improve clinical trial design and execution. Injury magnitude is an important criterion for determining patient eligibility for TBI clinical trials. Obviously, it is important to have patients with similar magnitudes of injury in different treatment groups. At present, the Glasgow Coma Scale (GCS) is the primary, if not the exclusive, entry criterion assessment tool for injury magnitude. Given the difficulties associated with accurate GCS assessment outlined above, biomarkers could provide an objective, quantitative assessment of injury magnitudes [41].

Secondary brain insults worsen neurologic outcome after TBI. In an effort to prevent occurrences of these insults, physiologic vital signs (e.g., intracranial pressure, mean arterial blood pressure, and tissue oxygenation) are routinely assessed in intensive care environments, although usually recorded only intermittently in the medical record. Conventional manual recording of vital signs can underestimate the total number of secondary insults. Undetected occurrence of secondary insults and increased neurologic damage in different treatment groups can significantly enhance variability in clinical trials. Detection of elevated biomarker levels, in conjunction with physiological assessment and management, could provide critical information to reduce the number of undetected secondary insults and allow for stratification of patients by occurrence of these insults [32].

As discussed previously, management of severe TBI patients can importantly influence clinical outcome, potentially by altering the number of secondary insults. In spite of various educational programs, the American Association of Neurological Surgeon Guidelines for Management of Severe TBI Patients is not uniformly followed. Moreover, even when efforts are made, failure to rigorously standardize clinical management in different centers could contribute to outcome variability in severe TBI clinical trials. For example, in a recent trial assessing the effects of moderate hypothermia on severe TBI, treatment effects for the five largest centers varied between 14% positive and 20% negative. Although there were significant differences in cerebral perfusion pressure management among the centers, investigators did not detect a correlation with treatment effect and attributed center differences to other baseline variables. In any case, a systematic assessment of biomarkers on admission and during the course of management could provide critical insights into potential differences between patient cohorts among centers [41].

Accurate prediction of outcome is a critical component of the design for the clinical TBI trials. In severe TBI, the Glasgow Outcome Score (GOS) or an extended version of this scale (GOS-E) is typically used. These scales provide broad distinctions and include categories such as “good outcome,” “severely disabled,” “vegetative,” or “dead.” New more powerful clinical trial designs for phase III trials in TBI and stroke employ statistical techniques based on outcome predictions as better as or worse than expected, taking into account each individual patient’s baseline prognosis. Thus, the use of biomarkers to enhance the accuracy of early predictions of outcome could importantly improve the power of phase III trial designs.

The recent Workshop on Classification of TBI emphasized the need for surrogate markers based on pathophysiological mechanisms of TBI in humans in clinical trials assessing targeted TBI therapies [1]. For example, recent work by us has confirmed the potential utility of biochemical markers to assess pathophysiological mechanisms of necrosis and apoptosis, and specific forms of cell death occurring after severe TBI. The biochemical markers identify the activity of specific destructive proteases (e.g., calpain and caspases) related to these pathological processes. Appropriate drug therapies targeting proteolytic by-products could use these biochemical markers as indices of therapeutic efficacy. This therapeutic marker would be available to investigators during the acute phase of injury in advance of 6 months’ outcome assessments typically used in such studies. Obviously, these biochemical markers are more closely linked to therapeutic effects on brain tissue compared to the neurological outcome measures such as the GOS or GOS-E.

Acute ischemic stroke is a significant international health concern, representing a potentially catastrophic debilitating medical emergency with poor prognosis for long-term disability. Stroke is the second leading cause of death worldwide [1] and the third leading cause of death in the USA [42]. Stroke affects more than 700,000 Americans annually [43]; every 45 s, someone in America has a stroke, and every 3 min, someone dies of a stroke [42]. In the USA, this type of injury is the primary cause of serious, long-term disability and the main reason for nursing home admissions [42, 43]. In 2007, the estimated direct and indirect cost of stroke is $62.7 billion [42]. This number will continue to escalate as the aging US population grows.

Precise diagnosis of stroke patients is typically made by trained clinicians, supported by neuroimaging, usually a brain computed tomogram (CT), in some cases supplemented by diffusion- and perfusion-weighted magnetic resonance imaging (MRI). This allows ready differentiation of ischemic stroke from hemorrhagic ones. However, CT is often normal in the acute phase of stroke and negative in the presence of small ischemic lesions or in certain brain locations (posterior fossae). As a result, a diagnosis of stroke often requires clinical interpretation by highly trained personnel. Emergency Room personnel, likely the first providers to see a patient with potential stroke, are less confident in making a diagnosis in the absence of objective laboratory confirmation. Stroke diagnosis is further complicated by the diversity of presenting symptoms. In the presence of focal deficits (i.e., weakness), stroke is relatively obvious, but with nonlocalizing symptoms such as delirium, seizures, dizziness/vertigo, or transient symptoms, the diagnosis can be more challenging. The presence of aphasia (left hemisphere) or profound neglect (right hemisphere) is often interpreted as confused delirium and is frequently misdiagnosed by nursing or primary care physicians even in hospitalized patients [8]. Stroke mimics include postictal states, hemiplegic migraines, brain tumors, epilepsy, encephalopathies, and at times metabolic derangements (e.g., hypoglycemia), all of which make the early diagnosis of stroke difficult. The diagnosis of transient symptoms, such as a transient ischemic attack (TIA), essentially viewed as a stroke equivalent, is difficult for trained clinicians, with substantial disagreement even between neurologists themselves [10, 13]. On the other hand, MRI can be helpful if ordered, but MRI is not readily available in all facilities and requires cooperation of frequently agitated patients, and may be contraindicated in some patients. In addition, the need to identify patients with acute stroke or TIA is highlighted by the high incidence of early stroke recurrence. Approximately 11% of TIA patients will experience a stroke at 90 days and half of those will occur within 48 h. Identifying such patients early would allow secondary stroke prevention treatment to be implemented more rapidly. The difficulties of stroke diagnosis are greatly increased in the acute settings. In the treatment of acute stroke, intravenous tissue plasminogen activator (i.v. tPA) has to be administered within 3 h of symptoms onset (although this window may be closer to 4.5 h). Pressed for time, a careful clinical diagnosis or complete neuroimaging evaluation may not be possible. Only 4–5% of all stroke patients receive i.v. tPA, in part due to the reluctance of non-neurologically trained emergency room personnel to administer a potentially dangerous treatment to patients with unclear diagnosis [44].

In addition, the presence of intracranial hemorrhage, in which case these agents are contraindicated, must first be ruled out. Therefore, blood biomarker-based diagnostic test in the differential diagnosis of acute stroke will be highly valued in stroke clinical onset. Neuron-specific enolase (NSE), S100β, ischemia modified albumin, matrix metalloproteinase-9 (MMP9), d-dimer, glial fibrillary acidic protein (GFAP), BNP, urea, and creatinine have been reported as biomarkers in the diagnosis of acute stroke. In one recent stroke biomarker study which include 100 stroke patients, it was reported that MMP-9 and d-dimer were found to be effective separately at differential diagnosis of ischemic–hemorrhagic stroke; there was no significance for S100β [45]. However, S100β and BNP have no place in the differentiation of hemorrhagic from ischemic stroke when used individually. However, when combined with BNP, d-dimer, MMP9, and S100β, it has more significance. Thus, it would be better to use a panel of biomarker tests rather than being used individually to differentiate hemorrhagic from ischemic stroke [46].

Another candidate stroke marker is glial fibrillary acidic protein (GFAP) which is highly specific to the brain and is located in glia cell cytoskeleton. GFAP is released when astrocytes become activated (gliosis) after injury. GFAP has been shown to be a sensitive serum biomarker of brain damage in patients with smaller lacunar lesions or minor stroke [47]. In a recent publication, GFAP showed a late upregulation at 24-h post-ischemia/reperfusion injury, indicating the presence of reactive gliosis in the middle cerebral artery territory [48]. Along the same line, our group assessed the glial markers S100β and GFAP and UCH-L1 by swELISA in patient serum 1, 2, 3, 4, and 7 days after stroke onset. Of interest, all biomarkers increased post-stroke indicative to be a good measure to evaluate brain damage [47].

In summary, ideal biomarkers for TBI and stroke that have been identified and validated with preclinical animal models need to be translated and validated in clinical studies as well. They should be tested in terms of their ability to detect injury magnitude as well as drug-based biomarker level reduction. A direct comparison of biomarker occurrence between preclinical models and biomarker data from human clinical studies would allow investigators to gain considerable insight into the validity (or challenges to the validity) of the employed preclinical animal models. Finally, the sensitive and specific cerebral biomarker could impact the delivery of TBI and stroke care in an important manner.