Recent Advances in the Development of Biofluid-Based Prognostic Biomarkers of Diffuse Axonal Injury

Abstract

Even though head injury is a silent pandemic of the century producing immense social and economic impact, predictive models have not been established to develop strategies promoting the development of reliable diagnostic tools and effective therapeutics capable of improving the prognosis. Diffuse axonal injury (DAI) is a type of traumatic brain injury (TBI) that results from a blunt injury to the brain. Discovering biomarkers for DAI have been a matter of debate and research. A number of studies have reported biomarkers that are correlated with severity of TBI but no conclusive and reproducible clinical evidence regarding the same has been put forward till now. Additionally, many DAI biomarkers have limitations so that they cannot be generalized for universal applications. The properties of these biomarkers should be extensively researched along with the development of novel biomarkers to aid important clinical decisions for the benefit of the society. This chapter summarizes the existing biofluid-based biomarkers, critically examines their limitations and highlights the possibilities of a few novel biomolecules as prognostic biomarkers of DAI.

1. Introduction

Central nervous system (CNS) trauma including traumatic brain injury (TBI) is a major cause of long-term injury, disability and death among young adults worldwide [1, 2]. Around 1.6 million individuals suffer from TBI every year in India with 200,000 deaths [3]. In the USA, there were more than 223,000 TBI-related hospitalizations in 2018 and about 166 Americans died from TBI-related injury each day in 2019 [4]. These estimates do not include the many TBIs that are only treated in the emergency department, primary care, urgent care, or those that go untreated [5]. Since the affected individuals are disabled and out of work during the most productive period of their life, these devastating conditions have an enormous physical, mental and economic burden to the country.

TBI is a heterogeneous neurological condition, ranging from single or repetitive concussion /mild TBI to penetrating head injury, focal contusion, different forms of hematoma (subdural and epidural) and diffuse injury. Depending on the motor, verbal, and eye-opening responses of the affected individuals, the Glasgow coma scale (GCS) was designed to access the disability. The GCS measures the following three functions: Motor response (scores: 6-normal, 5-localized to pain, 4-withdraws to pain, 3-flexion response to pain, 2-extension response to pain, 1-no motor response), Verbal response (scores: 5-normal conversation, 4-oriented conversation, 3-words, but not coherent, 2-no words, only sounds, 1-none), and Eye-opening response (scores: 4-spontaneous, 3-to voice, 2-to pain, 1-none). Based on the GCS score, TBI is classified as mild, moderate, or severe. TBI patients with GCS of 13 to 15 are classified to be mild, which includes the majority of these patients. Patients with a GCS of 9 to 12 are considered to have a moderate TBI, while patients with a GCS below eight are classified as having a severe TBI [6].

The heterogeneous nature of TBI with respect to severity of the injury and comorbidities make patient outcome difficult to predict. While mild TBI or concussion may affect neural cells temporarily, severe TBI is associated with substantial axonal injury and physical damage to the brain, which can result in blood-brain barrier disruption and neuroinflammatory changes [7]. Moderate to severe TBI can usually be visible as structural abnormalities using radiological examinations such as computed tomography (CT) or magnetic resonance imaging (MRI). However, more subtle neural alterations characteristic of mild TBI are not easily detected by these imaging techniques.

Although TBI is an extremely complex condition, there have been many advances in recent years in relation to the diagnosis, monitoring and treatment of the affected patients. Shortcomings in our knowledge of the physiopathology of TBI and the development of reliable predictive models capable of offering an early orientation as to the patient outcome, will improve the diagnostic and therapeutic strategies on an individualized basis. Likewise, we need valid predictive models in severe TBI in order to define efficacy endpoints in the evaluation of new drugs or treatment strategies–since the usual primary endpoints (death and disability) are widely recognized as being inadequate and could explain the discouraging results obtained with certain promising drugs.

2. Pathogenesis of DAI

Diffuse axonal injury (DAI) is a type of TBI that results from a blunt injury to the brain, which happens when the brain rapidly shifts inside the skull as an injury is occurring. It is one of the most common but devastating types of TBI. Neuronal injuries associated with DAI fall into two categories: (i) primary injury, which is directly caused by mechanical forces during the initial insult; and (ii) secondary injury, which is caused as a consequence of primary injury to further tissue and cellular damages.

2.1 Primary brain injuries

Primary brain injuries refer to the sudden and profound injury to the brain that occurs at the time of the motor vehicle accident, gunshot wound, or accidental fall. The immediate impact of different mechanical insults to the brain can cause two types of primary TBI: focal and diffuse brain injuries. Focal TBI generally results from a blow to the head that produces cerebral contusions or hematomas. Epidural hematomas, subdural hematomas, and cerebral contusions are the results of focal brain injuries [8]. In contrast, diffuse lesions (also known as DAI) are seen more commonly in lesions that involve rapid acceleration, deceleration, or rotational forces. DAI accounts for about 70% of TBI cases. The sites that are most prone to DAI are the reticular formation, basal ganglia, superior cerebellar peduncles, limbic fornices, hypothalamus and corpus callosum [8]. Interestingly, both types of injuries may co-exist in patients who suffered from moderate to severe TBI [9].

Brain injuries may occur in one of two ways: closed brain TBI and penetrating TBI. Closed brain TBIs occur when there is a non-penetrating injury to the brain with no break in the skull. A closed brain injury is caused by a rapid forward or backward movement and shaking of the brain inside the bony skull that result in bruising and tearing of brain tissue and blood vessels. In contrast, Penetrating, or open head injuries occur when there is a break in the skull, caused by hitting with a sharp object such as a bullet. These injuries exhibit focal brain damage due to lacerations, compression and concussion forces with evidence of skull fracture and localized contusion (Figure 1) at the core of injury site, known as the ‘coup’ area [10, 11]. Compromised blood supply at the coup area due to epidural, subdural and intracerebral hemorrhages and hematomas might result in necrosis of neuronal and glial cells at confined layers of the brain. Secondary contusion may develop in brain tissues opposite to or surrounding the coup area due to secondary impact when the brain rebounds and strikes the skull [11].

Figure 1.

Schematic representation of the pathogenesis of TBI. TBI may be divided into primary injury and secondary injury. Primary TBI results from mechanical injury at the time of insult, while secondary injury is caused by the physiologic responses to the initial injury. Primary brain injury comprises the direct physical injury to the brain such as compression, deformation, displacement, stretching, shearing, tearing, and crushing of brain which results in damage to vasculature, neural, and glial tissues. Most of the neurological damage from TBI is due to the secondary injury which evolves over the ensuing hours and days after the initial injury or impact. The mechanisms by which TBI trigger neurodegeneration are areas of active research. Previous investigations found roles of excitotoxicity, loss of cerebral autoregulation, blood-brain barrier compromise, mitochondrial dysfunction, oxidative stress, lipid peroxidation, neuroinflammation, axon degeneration, impaired autophagy and apoptotic cell death in the development of neurodegeneration following brain injury. TBI produces both acute and chronic consequences that lead to permanent disabilities that increase long-term mortality and reduced life expectancy. The direct consequences of a single or repetitive TBI can result in various secondary pathological conditions, including seizures, sleep disorders, neurodegenerative diseases, neuroendocrine dysregulation, and psychiatric problems. Changes initiated by TBI can persist for weeks to months or even years after injury and significantly affect quality-of-life of the affected victims.

2.2 Secondary brain injuries

Secondary brain injuries refer to the changes that evolve over a period of time after the primary brain injury. The biochemical, cellular and physiological events that occur during primary injury often progress into delayed and prolonged secondary damaging cascade of cellular, chemical, tissue, or blood vessel changes in the brain that contribute to further destruction of brain tissue. Secondary brain injuries can last from hours to days and even weeks and may be caused by impairment or local declines in cerebral blood flow resulting in local edema, hemorrhage, or increased intracranial pressure and even brain herniation. Other types of secondary injury due to TBI include hypercapnia, acidosis, meningitis and brain abscess [12]. Mechanistically, a number of factors contribute to these changes, which include excitotoxicity, loss of cerebral autoregulation, blood-brain barrier compromise, mitochondrial dysfunction, oxidative stress, lipid peroxidation, neuroinflammation, axon degeneration, impaired autophagy and apoptotic cell death (Figure 1) [10, 13].

The hallmark feature of DAI is extensive damage of axons predominantly in subcortical and deep white matter tissue, which leads to impairment of axonal transport and degradation of axonal cytoskeleton. The strong tensile forces generated during primary injury by rapid deceleration and acceleration of the brain due to multiple non-contact forces causing shearing and stretching injury in cerebral brain tissues damage neuronal axons, oligodendrocytes and blood vasculature, leading to brain edema and ischemic brain damage [14]. These axonal damages can persist for months after DAI.

The degree of axonal injury and neuronal degeneration determines the severity of TBI. While explosive blast TBI is a result of shock waves instead of inertial forces, it displays the characteristics of a typical DAI. Depending on the severity of the injury, patients may later develop cognitive deficits, behavioral changes and hemiparesis (Figure 1).

3. Need for biofluid-based brain damage biomarkers

While mild TBI or concussion may affect neural cells temporarily, severe TBI is associated with substantial axonal injury and physical damage to the brain, which can result in blood-brain barrier disruption and neuroinflammatory changes [7]. Although, moderate to extensive brain injury may be visible as structural abnormalities using CT scan or MRI, more subtle neural alterations characteristic of mild TBI are not easily detected by these imaging techniques. Moreover, changes due to DAI in the brain are often microscopic and may not be visualized on CT scan or MRI scans.

Mild TBI is highly prevalent in military populations, with many service members suffering from long-term symptoms [15]. It is also very common among road accident victims. The condition results from etiologies of neural contusion and axonal injury, which subsequently results in biochemical, metabolic, and cellular changes that may be responsible for some of the long-term problems observed in patients who develop post-concussion syndrome [16]. Moreover, moderate to severe TBI remains another important public health problem, due to the large percentage of unfavorable outcomes involved such as death and disabling sequelae. The huge treatment costs, associated compensations, disability pensions and years of income from work lost in affected individuals are the major financially devastating turns in the affected families. Therefore, identifying critical markers of neural injury in biofluids of these patients would be crucial for predicting long-term functional outcome and for taking rehabilitation decisions. Encouragingly, significant scientific advances on the TBI biomarker research in the last decade have increased our understanding of the complex and heterogeneous pathophysiological processes associated with this condition. Emerging evidence from multiple research teams suggests that biofluid-based TBI biomarkers may have the potential to diagnose the presence of TBI of different severities, and to predict outcome.

4. Biomarkers for TBI/DAI

A biomarker is defined as a quantifiable biological indicator specific for a given physiological or pathological condition. Based on clinical utility, biomarkers may be categorized as: 1) diagnostic biomarkers, which identify the presence or absence of TBI, 2) prognostic biomarkers, which inform the clinicians about expected outcomes in injured individuals, and 3) predictive biomarkers, which predict response to a specific intervention and can be used to monitor response to therapy. Identification of biological markers of TBI could offer a more precise indication of the extent and severity of TBI, independently of the prior biological substrate and of other circumstances that accompany severe TBI–thereby contributing to homogeneously define different patient categories and risk stratify the head injury. These can also serve to screen and identify patients who may expect an altered or complicated recovery or might develop neurobehavioral deficits during the latter part of their life. Such markers would facilitate

• individualization of the intensity of TBI/DAI

• improve knowledge of the physiopathology of brain damage

• afford essential complementary information for the diagnosis

• predict the outcome of these patients

• timing of patient management

• development of strategies for preventing or minimizing secondary damage

• evaluation of neuroprotective effects of novel biomolecules.

4.1 Which is an ideal biomarker?

An ideal TBI/DAI biomarker should have

• high specificity and sensitivity for the brain tissue

• rapid appearance in accessible biofluids such as cerebrospinal fluid (CSF), plasma and/or whole blood immediately after irreversible brain tissue damage.

• must be elevated in various forms and/or severities of brain damage in the acute phase (3 h to 24 h post-injury).

• must reflect the extent and severity of the damage, as defined by GCS, CT abnormality, in due course of time.

• variations between age and gender groups must be minimal.

• must have low basal biofluid levels in non-injured healthy control population

• should be responsive to therapeutic treatments

• the tools for analysis and immediate detection of the marker must be available and reproducible.

• determination of the marker must be clinically relevant.

The literature given below is a concise description of the principal investigational brain damage biomarkers with a description of the tissues in which they originate, the compartment from which samples were collected, their pathological serum concentrations, and the main prognostic features (Table 1).

Biomarker	Localization	Role in prognosis	Drawbacks	References
Tau/c-tau	Neuronal axons and astrocytes	May predict outcomes after severe TBI	Not fully characterized	[17, 18, 19]
Aβ	Extracellular space	Altered levels in CSF	Contradictory findings	[20, 21, 22]
MBP	Axonal myelin sheath, oligodendroglial cells	Elevated levels in serum and predict severity and outcome	Limited sensitivity	[23, 24]
CK-BB	astrocytes	May predict outcomes after severe TBI	Low sensitivity and specificity in polytrauma	[25, 26, 27]
NSE	Neuron	Elevated serum levels and specific to neuronal tissue than glial cell	Long half-life and are expressed in red blood cells	[28, 29, 30, 31, 32]
S100B	Astrocytes	Predict severity and act as adjuvant marker	Reduced specificity; not suitable for children under 2 years	[33, 34]
GFAP	Astrocytes	May predict neurological outcomes	Also expressed by the Leydig cells of the testes	[35, 36, 37]
UCH-L1	Neuronal cell body	Increased serum levels in brain damage	Not fully characterized	[38, 39, 40]
SBDPs	Axons and presynaptic neuronal endings	May predict neurological outcomes	Not brain specific and difficult to quantify	[41, 42, 43, 44]
NfL	Neuronal cytoplasm	Released in response to CNS neuronal damage	Released in response to neurodegeneration and neuroinflammation	[45]

Table 1.

Current and emerging DAI biomarkers.

4.2 Tau protein

Tau protein is an axonal cytoskeleton-stabilizing protein (Figure 2) of molecular weight of 30–50 kDa that provides structural elements of the cytoskeleton that are crucial for neuronal protein flow [46, 47]. There are 6 different tau protein isomers, which is phosphorylated at many sites by kinases such as casein kinase II, tau tubulin kinases, glycogen synthase kinase 3β, and cyclin dependent kinase 5 [48, 49, 50]. While these are present in a stable, unfolded and monomeric morphology in a healthy brain, tau proteins exist in hyperphosphorylated state in several neurodegenerative diseases including Alzheimer’s disease (AD) [46, 51]. Interestingly, TBI has been indicated as a risk factor for later development of AD and other neurodegenerative conditions [52, 53, 54, 55, 56] .

Figure 2.

Origin of biomarkers of DAI. In normal brain, NSE and UCH-L1 are localized in neuronal cytoplasm, while tau/c-tau are restricted mainly to axons and its terminals. Although tau is abundant in the neurons of the CNS, astrocytes express very low levels of this protein and it can be secreted into the brain interstitial fluid. APP is a type I transmembrane protein expressed in many cell types, including neurons. Aβ is derived from APP by enzymatic cleavage and is released to extracellular space. MBP is a constituent of neuronal myelin sheath, which is produced by oligodendrocytes. Spectrins (precursors of SBDPs), specifically βIV-spectrin is concentrated at axon initial segments and nodes of Ranvier. GFAP, CK-BB and S100B are normally present in astrocytes. DAI not only injures pre- and post-synaptic neurons but also damages their synapses, axons, myelin sheaths and neighboring astrocytes, oligodendrocytes, blood microvasculature and even the extracellular matrix network. Damages to specific cells and cellular components during DAI enable release of various molecules contained in those cells into the extracellular space. The released molecules, including those present in extracellular space like Aβ enter the damaged blood vessels and may be detected in the circulation.

Physical trauma causes activation of a number of proteases, which cause release of tau protein fragments in cleaved tau (c-tau) into the blood and CSF [57, 58]. Studies showed that the c-tau levels in CSF increase in the first 24 h after severe TBI [17, 59, 60]. Plasma phosphorylated tau (p-tau) and p-tau/t-tau ratios have been demonstrated to distinguish patients with acute and chronic TBI from healthy controls [18]. Smith and colleagues (1999) have shown deposition of p-tau following TBI [61]. C-tau in CSF is shown to be a predictor of clinical outcomes in severe TBI subjects [60, 62]. Moreover, elevated c-tau could be a chronic manifestation in DAI, since tau is localized to the axons [59]. However, the practical role of this molecule in DAI has not been fully established.

4.3 Amyloid-β (Aβ) protein

Amyloid-β (Aβ) is a 4 kDa extracellular protein derived from amyloid precursor protein (APP), which is cleaved by secretase enzymes [63]. APP is a membrane protein expressed in both CNS (APP 695) and peripheral organs and tissues (APP 751 and APP 770) [64]. APP with 695 amino acids is present as glycosylated receptors on cell surface and is hydrolysed by α-secretase followed by γ-secretase under normal conditions to produce soluble Aβ through non-amyloidogenic pathway [63, 65]. In amyloidogenic pathway, the mutations in APP and components of α-secretase, presinillin 1 (PSEN1) and presinillin 2 (PSEN2) leads to the cleavage by β amyloid cleaving enzyme-1 (BACE1) and γ-secretase to form insoluble Aβ, Aβ_1–40 and Aβ_1–42. This amyloidogenic cleavage leads to extracellular accumulation of Aβ plaques, a pathological hallmark in AD [20, 65, 66]. APP plays an important role in cell adhesion processes and thus high concentrations are found at neuronal synaptic junction. Certain type of caspase breaks it down into a series of products which accumulate in cell bodies and axons. There are discordant evidence on the use of this protein as biomarker of TBI. One study of 29 patients with severe TBI revealed low levels of this protein in CSF probably due to reabsorption of the protein in the form of amyloid plaques [21]. As a contradiction to the above, Emmerling et al. (2020) found increasing levels in CSF after trauma and suggested that this could be a result of secondary axonal damage or loss of integrity of the BBB [22]. These contradictory findings, have caused Aβ-protein to be regarded as a non-reproducible biomarker and its potential role remains unclear requiring further research.

4.4 Myelin basic protein (MBP)

MBP (molecular weight of 18.5 kDa), found in oligodendroglial cells, is a key structural component of the multi-layered myelin sheath covering nerve fibers. The myelin sheath on neuronal axons serves as an insulator to increase the velocity of axonal impulse conduction. Due to the extended length of axonal fiber tracks, axons are particularly vulnerable to physical trauma to the brain. Thus, axonal injury is a common occurrence in both focal as well as diffuse brain trauma and can be found in TBI of all severities [14, 67]. As MBP maintains myelin structure by interacting with the lipids in the myelin membrane [68], axonal injury causes breakdown of the myelin sheath and release of MBP. This myelin-specific protein is also released into the bloodstream in cases of demyelinating diseases such as multiple sclerosis, or degradation by proteases, such as calpain [69, 70].

MBP is found to be elevated in serum after severe TBI in children [23, 24] and after mild TBI in adults [24]. Even though, it takes around 1–2 days to appear in the serum after TBI, the peak levels of MBP can persist for up to 2 weeks and can be a specific indicator for future intracranial hemorrhage [71]. However, as per present literature there is contradicting evidence for its role in TBI/DAI [72, 73, 74]. There was no difference in initial levels of serum MBP in a pediatric population with mild TBI when compared with controls, but there was a significant difference in the peak MBP levels between patients and controls [72]. MBP is also expressed on the myelin of peripheral nerves and its transcripts are present in the bone marrow and immune system and therefore it is not specific to the CNS. Even though serum levels are correlated with patient severity and outcome [71, 75] it has limited sensitivity as a marker for predicting severity of TBI [76].

4.5 Cerebral creatine kinase (CK) isoenzyme

CK isoenzymes are of three types: CK-1 (also known as CK-BB) is predominantly expressed in brain, lung, thyroid and prostate glands, gastrointestinal tract, urinary bladder, uterus and placenta. CK-2 (CK-MB) and CK-3 (CK-MM) are expressed in cardiac and skeletal muscles [77]. Brain tissue-specific CK-1 (CK-BB), with a molecular weight of 40–53 kDa, is found in astrocytes [25, 26]. A peak in serum cerebral CK concentration is observed in the first few hours after severe TBI and then gradually decrease and the marker remains high for days [78, 79, 80, 81]. In polytrauma, it remains persistently high without an initial dip [82, 83] . Its levels have been shown to rise significantly in CSF following hypoxic brain injury in cardiac arrest, which suggests that CK-BB release may be secondary to cerebral hypoperfusion due to systemic trauma [84]. The major limiting factor is that it has low sensitivity and specificity especially in cases of polytrauma [27, 85].

4.6 Neuron-specific enolase (NSE)

NSE, also known as γ -enolase or enolase 2, is a glycolytic enzyme with a molecular weight of 78 kDa and a half-life of 48 h. It exists as a homodimer (γ–γ) in mature neurons and neuroendocrine cells. The normal concentration of NSE in blood is <10 ng/ml. NSE elevations in blood compartment has been documented in severe as well as mild TBI [28, 29, 30, 31, 86]. Experimental models of trauma have correlated serum NSE to the severity of damage in TBI [87]. Major limitation of using NSE as specific biomarker for TBI is that it is also abundantly expressed in red blood cells [88]. Moreover, increased levels of NSE was also recorded previously in the serum of patients following non-traumatic brain damage such as ischemic events, intracerebral hemorrhage, cardiopulmonary resuscitation, secondary cerebral hypoxia etc. [89]. Some studies had correlated the biomarker to the development of DAI, though its behavior has not been clearly established in prospective trials.

NSE was initially suggested to be a very promising TBI severity marker due to its specificity to neuronal tissue than of glial cells. However, the results published till date has been contradictory on its role in predicting prognosis of patients with severe TBI. Long half-life is a major limiting factor for its use in trauma setting. Also, extracranial origin of NSE was demonstrated in hemorrhagic shock, long bone fracture, hemolysis, heart surgery, ischemia–reperfusion injury and malignant lung tumors making it a poor marker for TBI [90, 91, 92, 93, 94].

4.7 Glial fibrillary acidic protein (GFAP)

GFAP is a monomeric intermediate filament protein (molecular weight 52 kDa), present in the cytoskeleton of astrocytes in the brain. An increase in blood level of this biomarker suggests injury to the astrocytes and the BBB. Plasma concentrations >0.033 μg/l are regarded as pathological. Missler et al. (2002) were the first to propose the possible use of GFAP as an identifier of brain damage in serial serum measurements [95]. Later studies also confirmed that the serum concentration of this protein is not affected by extracranial injuries thus making it an effective biomarker for predicting poor outcome in the acute phase of severe TBI as well as for advocating the need for urgent neurosurgical procedures [41, 96]. GFAP (52 kDa) or its breakdown products (44–38 kDa) are released from injured brain tissue into biofluids such as CSF and enter the bloodstream after crossing the BBB with an early plasma peak (within 3 to 34 h) following brain injury [97, 98]. The blood levels then decrease gradually over the first week, starting from third day of injury.

Previous studies demonstrated that GFAP levels show an unfavorable outcome in patients with moderate or severe TBI [41, 96, 99, 100]. However, this may not be the case in patients with mild TBI due to the contamination from other sources [101, 102, 103]. However, Serum GFAP levels were also significantly higher in patients who died or had an unfavorable outcome [104]. Moreover, GFAP levels have correctly predicted neurological outcome at 6 months [35, 36, 104]. Furthermore, serum GFAP measured on day 1 of injury in pediatric TBI cases significantly correlated with functional outcomes at 6 months [105]. Thus, GFAP can be considered as an ideal biomarker of brain damage when combined with clinical variables though multicenter studies are needed for further validation.

4.8 S100-calcium binding protein B (S100B)

S100B, the most widely studied brain damage biomarker, is a low molecular weight (11 kDa) calcium binding protein of astroglial origin [33]. The homodimeric beta-subtype of S100 proteins (S100B) is synthesized in astrocytes of the CNS and in Schwann cells of the peripheral nerves, where it regulates intracellular calcium levels [106, 107, 108]. S100B localizes to the nucleus and cytoplasm associating with endomembranes, the centrosomes, microtubules and type III intermediate filaments [109]. The protein is naturally secreted by astrocytes into the extracellular space. Low amounts of S100B can cross the BBB and enter the microvasculature. Elevated levels of S100B in the serum were observed in TBI patients as well as in patients suffering from neurodegenerative diseases [110]. The serum levels of the protein have been associated with clinical severity, radiological severity, and an unfavorable outcome [111, 112, 113, 114] .

The biological function of this protein has not been fully established till date, though it is known to participate in neurogenesis, astrocytosis and axonal elongation. However, the molecule can also be produced and found outside the CNS, e.g., in kidney epithelial cells, ependymocytes, chondrocytes, adipocytes, melanocytes, Langerhans cells, dendritic cells, certain lymphocyte subpopulations, skeletal myofibers, myoblasts and muscle satellite cells [109]. Metabolism takes place in the kidneys, followed by excretion in urine, with an approximate half-life of 30–113 min, and is not affected by hemolytic phenomena [115]. Its role in urine level also needs further validated study. Since S100B can also be released from adipose tissue and cardiac/skeletal muscles, its levels are also elevated in orthopedic trauma without head injury [116]. Despite these confounders, S100B is actually a sensitive TBI biomarker for predicting CT abnormality and post-concussive syndrome development [117, 118, 119]. A number of previous studies have shown that S100B can actually differentiate between mild and severe TBI [120, 121].

The maximum serum concentration of S100B is reached 20 min after brain damage. The normal upper limit for this protein in relation to the detection of intracranial damage was defined as 0.1 μg/l based on a multicenter study in patients with mild TBI [122]. The measurement of S100B-protein can be influenced by patient age and gender in CSF samples but not in serum samples thus making it a practically feasible biomarker.

Some studies have determined its usefulness as a predictor of mortality, establishing orientative serum cut-off points for predicting a course leading to death or an unfavorable outcome [123, 124, 125]. On the other hand, S100B level has been correlated to the presence of secondary lesions, the extent of diffuse brain damage, and to modifications in intracranial pressure following different release patterns [126, 127]. S100B levels can also detect brain death development or mortality after severe TBI [128, 129]. Interestingly, serum levels of S100B > 0.7 ng/mL were reported to correlate with 100% mortality [130].

Another possible application of this protein refers to its time course according to the severity of the patient condition. A number of studies have documented persistently elevated serum levels in patients in those with poor GCS, while the plasma levels have been seen to decrease after 36 h among survivors [82, 131, 132, 133]. On the other hand, S100B protein has been suggested as a tool for monitoring management efficacy [134, 135], since it has been seen that the blood concentrations of the protein decrease after effective neurosurgical treatment thus making its role more relevant. A high level of S100B during the initial TBI can predict a poor outcome, especially if it is accompanied by a second increase in levels of serum S100B that occurs during the subacute phase [131, 136]. This second peak during the subacute phase may be due to secondary injury to the astroglial cells exhibiting excitotoxicity and neuroinflammation. In addition, elevation in serum levels of S100B and GFAP in TBI patients has been correlated with unfavorable neurological outcomes [137, 138, 139]. On the other hand, an initial lower level of S100B and the lack of second peak might suggest the occurrence of a mild TBI and a good functional recovery [140, 141].

A previous study demonstrated the sensitivity of S100B to predict significant intracranial pathology up to 100% but with specificity of only 28%.Moreover, in pediatric population (specifically for children under the age of 2 years), S100B is not a useful marker due to high normal levels in this group [32, 142]. Thus, S100B may be suggested to be used as an adjuvant marker in patients with TBI, but its diagnostic value is still controversial [76].

5. Limitations of the existing biomarkers

Doctors in the acute hospital settings primarily rely on the patient’s neurological examinations and radiologic imaging to characterize TBI/DAI diagnosis. Depending on the severity of the initial insult, different imaging modalities such as CT scan and MRI are used to obtain the necessary information for patient care and prognosis. However, CT scans, used for assessing cerebrovascular integrity or for determining gross anatomical changes induced to the brain, have low sensitivity to diffuse brain damage, and confers exposure to radiation [143]. In contrast, while MRI can provide information on the extent of diffuse injuries, yet its widespread application is restricted by prohibitive cost, limited availability of MRI in many hospitals, and practical difficulty of performing it in physiologically unstable TBI patients [143]. Thus, diagnostic and prognostic tools for risk stratification of TBI patients are very limited in the early stages after injury.

To fill this gap, research in the field of biofluid-based TBI biomarkers has increased exponentially over the last three decades [116]. Extensive research on fluid biomarkers have demonstrated that a number of brain-specific proteins, as illustrated above, have potential for acting as biomarkers of TBI. These biomolecules are released into the CSF and/or blood, after brain injury due to damage of neural cells [28, 144, 145, 146]. Additionally, neuroimmune activation might have the potential to be novel diagnostic and/or prognostic marker of TBI. A few of these molecules, like S100B have shown promise to be clinically used as biomarkers of TBI [145]. However, this has been disputed in recent studies [147] and till now, there are no rapid, definitive diagnostic blood tests for TBI.

Despite its high sensitivity and negative predictive value, S100B protein is not a specific marker of the CNS damage. Polytraumatized patients without TBI can present S100B protein elevations in blood, though the concentrations return to normal within 6 h after trauma. Patients with brain damage and associated extracranial injuries (hypotension, hypothermia, coagulopathy, inotropic drugs, sedatives, corticosteroids, etc.) can alter the early assessment of S100B protein. Therefore, early determination of this protein is to be avoided in patients with extracranial injuries associated with TBI making it’s role dismal probably in trauma care even though the above features of an ideal biomarker are met.

5.1 Difficulty with interpretation

CNS is very complex and can present a range of different lesions, which in turn can affect different target cells with variable degrees of severity. Brain damage markers must establish differentiations with respect to other alterations. Furthermore, the existence of the blood–brain barrier conditions the structural characteristics of these biomarkers, which must be able to cross the mentioned barrier in order to reach the bloodstream. Biomarkers are dynamic elements that experience changes in response to different inflammatory states, tissue necrosis etc. So serial measurements rather than isolated or point determinations are thus required.

5.2 Controversy

As direct sampling of the damaged brain tissue is not practically feasible there is some controversy regarding the type of biological fluid that should be analyzed. CSF compartment is located closer to the damage site, but frequent collection of CSF samples is unethical. As a result, most biomarkers are studied in peripheral blood as the process is simple, accessible and reproducible. Thus, estimation of blood biomarkers will be the most appropriate option for performing simple and minimally invasive serial measurements. Still more easy will be to estimate biomarkers in fluids that serve as vehicles for their clearance, for example urine.

6. Newly discovered biomarkers of interest.

6.1 Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1)

UCH-L1 mainly resides in the cytoplasm of neuronal cell body representing approximately 5% of all the soluble brain proteins. It is implicated in the elimination of degraded and denatured proteins following oxidative phenomena [148]. Proteomics data first implicate UCH-L1 as promising TBI biomarker candidate [149, 150]. Later studies point to it as a promising brain damage biomarker, since there are data indicating that it can predict the presence of lesions on the CT scan, the need for neurosurgery, and the outcome of patients with TBI [151, 152, 153].

UCH-L1 can be detected in blood, with early increases in its serum concentration following brain injury [151]. The protein level in the blood is shown to be elevated both in mild and severe cases of TBI [100, 154]. Mondello et al. (2012) have obtained interesting results regarding its possible capacity to distinguish between focal and diffuse brain damage [155]. Additionally, it has been suggested that UCH-L1 together with GFAP form the foundation of a biomarker panel representing the two dominant cell types (neuron and astrocytes) in the brain [38]. Interestingly, serum levels of both of these proteins are elevated in professional breacher trainees who were exposed to repeated explosive discharges as well as athletes who experienced concussions [39, 40]. Further investigations are needed to evaluate the properties of this protein as a promising biomarker of DAI.

7. Spectrin degradation products (SBDPs)

Spectrin is a cytoskeletal protein that lines the intracellular side of the plasma membrane forming a scaffold, which maintains plasma membrane integrity and cytoskeletal structure [156]. It is a heterodimeric protein, composed of two α and two β chains, and contains 106 contiguous amino acid sequence motifs called “spectrin repeats”, which are essential to diverse cell functions such as cell adhesion, cell spreading, and the cell cycle [156].

Necrotic and apoptotic cell death during primary and secondary brain injury respectively, cause overactivation of cysteine proteases, such as calpain and caspase-3. These proteases cleave components of the axonal cytoskeleton [157] including spectrin resulting in generation of SBDPs with characteristic molecular weights [26, 158]. The presence of degradation products of spectrin has been described in the CNS in axons and presynaptic neuronal endings [23, 44, 159, 160]. However, SBDPs are not brain specific and its increased serum levels may reflect multiorgan damage in trauma [42, 161]. Moreover, accurate quantification of brain-derived SBDPs in blood is difficult since some proteins found in erythrocytes are similar to those found in the neuronal cytoskeleton [162], thus reducing the diagnostic value of SBDPs.

8. Neurofilament light chain (NfL)

Neurofilaments, consisting of three chains, light (L), medium (M) and heavy (H), make up part of the axonal cytoskeleton. NfL is 68 kDa subunit of the neurofilaments located on the neuronal cytoplasm which is released in response to CNS neuronal damage due to neuroinflammation, neurodegeneration, and/or traumatic or vascular injury [163, 164]. Following axon damage, the influx of calcium alters the phosphorylation state of NfL and subsequent proteolysis. As a result, there is loss of cytoskeletal structure and NfL is released into both CSF and the bloodstream [165]. A number of investigational studies have underscored the role of NfL as biomarker of axon damage [45, 166, 167]. Serum NfL has been shown to distinguish patients with mild, moderate or severe TBI for months and even years after injury [168]. However, serum detection of NfH is considered a better biomarker candidate [169].

9. Conclusions

Since all brain damage biomarkers have some limitation precluding their universal application in the management of severe TBI/DAI, they do not yet form part of routine clinical practice. Some markers, such as NSE and S100B protein, have shown good correlations to clinical severity, the extent of brain damage, response to treatment, and patient outcome. However, the limitations associated with the clinical yield of the molecule or invasiveness of the technique required to obtain the sample have not allowed their generalized use in this patient population.

On the other hand, further studies are needed to understand the role of these proteins in the physiology of the CNS and in the physiopathology of severe TBI/DAI, as well as to clarify the usefulness of those biomarkers that appear to be promising in this field. In this respect, mention must be made of nervous tissue-specific GFAP, as well as of other biomarkers that are currently the focus of interest, such as ubiquitin carboxy-terminal hydrolase L1, the light neurofilaments and spectrin degradation products.

Since these molecules offer isolated information on some of the many elements implicated in the physiopathology of TBI/DAI, we believe that the best strategy is to analyze them in combination. Rather than seeking a biomarker exclusive for brain damage, this approach would allow us to define a panel of biomarkers which jointly – and considering the characteristics inherent to each of them–could offer information referred to severity, the potential benefits of management, and the evolutive course of patients following severe TBI. Only in this way can we hope to complement the traditional methods with a tool that is simple, non-invasive, reproducible and extraordinarily useful for addressing and managing severe TBI/DAI. Moreover, since TBI/DAI is quite complex heterogeneous conditions, it might be clinically justified to use multi-modal biomarkers to evaluate the status of full clinical endophenotypes by combining a panel of biofluid-based and physiologic biomarkers coupled with advanced neuroimaging that are appropriately obtained at multiple time points during the time course of TBI/DAI.

Conflicts of interest

The authors declare that they have no conflicts of interest.

Financial support

RRM is a senior research fellow of Indian Council of Medical Research (ICMR), Government of India. GC is supported by The Ramalingaswami fellowship from the Department of Biotechnology, and grants from the Department of Health Research and ICMR, Government of India.