2. Current Guidelines
There is no uniformity concerning the guidelines to be followed nationally and internationally. There are significant between-center variations in policies for diagnostics, admission, and discharge decisions in patients with TBI in the emergency department and hospital ward
[14].
Commonalities between the different guidelines include the focus of evaluation mainly on assessing the patient’s mental status, cranial nerves, sensory awareness, motor functions, and reflexes. Patients receiving antiplatelet/anticoagulant therapy should have treatment suspended for the entire duration of the observation
[14]. Neurological imaging is essential to identify a patient with head trauma caused by acute injury or persistent symptoms, and computed tomography is the primary method of radiological examination
[4].
Each country has its directives for CT use; for instance, in Europe and Canada, CT for minor head injury cases is used very selectively. In Italy in particular, CT is only recommended if a fracture has been shown by skull radiography; in Denmark, it is rarely ordered and then only by a neurosurgeon; in the UK and Spain, CT is only recommended for cases with a documented skull fracture, focal neurological deficit, or deterioration in mental status
[15].
The most frequently used guidelines are the Canadian CT Head Rule, the Scandinavian Guidelines, the New Orleans Criteria for TC scan in mild head injury, the guidelines of the National Institute for Health and Care Excellence (NICE 2014), and those of the Neurotramatology Committee of the World Federation of Neurosurgical Societies (NCWFS)
[14]. These guidelines differ in terms of the parameters taken into consideration, as shown in
Table 1: The Canadian CT rules are based on five high-risk and two medium-risk criteria
[15]; the Scandinavian guidelines also consider the serum levels of S100 calcium-binding protein B (S100B)
[16][17]; the New Orleans Criteria for TC scan (NOC) included seven items and were only developed for use in patients with a GCS score of 15
[18]; the guidelines of the National Institute for Health and Care Excellence (NICE 2014) are based upon the Canadian CT head rule and lead to more CT scans being performed, but fewer skull radiographs and admissions
[19][20][21]; and lastly, the Neurotraumatology Committee of the World Federation of Neurosurgical Societies (NCWFS) protocol is similar to the NICE guidelines. However, it is less strict and leads to more CTs
[22][23].
Table 1. Comparison between the already-in-use prediction rules for traumatic brain injury. NICE: National Institute for Health and Care Excellence; NCWFS: Neurotraumatology Committee of the World Federation of Neurosurgical Societies. High risk: risk factor is present in the prediction rule as a major criterion; medium risk: risk factor is present in the prediction rule as a minor criterion; blank: the variable is not a risk factor in the model.
3. Markers
An ideal biomarker should be easy to measure in accessible bodily fluids such as cerebrospinal fluid or blood (serum/plasma); it should allow repeated detection for monitoring the initial brain injury in the hours that follow, and its elevated levels should correlate directly with the presence of brain trauma and the degree of severity of traumatic brain injury. Therefore, all the substances that can be released because of neuronal cell injury, glial cell injury, axonal injury, and inflammation are potential biomarkers for TBI.
Research into blood-based TBI biomarkers has accelerated rapidly in the past decade, leading to the identification of proteins resulting from axonal, neuronal, or glial cell injuries and released into the interstitial fluid (ISF), cerebrospinal fluid (CSF), and blood circulation due to altered function of the blood–brain barrier (BBB) after TBI
[12].
Neuroimaging techniques such as computed tomography (CT) and magnetic resonance imaging (MRI) can identify gross head injuries but not minute neural and structural changes typical of mild TBI. Conversely, fluid biomarkers are accurate tools that can be used to assess mild TBI pathophysiology
[11]: increased understanding of individual biomarker trajectories in the hours, days, and weeks post-injury will enable a greater understanding of diagnostic windows from acute and chronic perspectives. It will furthermore allow studies to investigate the best potential biomarkers for predicting outcomes and tracking pathophysiological recovery or measuring response to treatment in mTBI clinical trials
[12].
The following paragraphs report the current understanding of the cellular sources, temporal profile, and potential utility of leading and emerging blood-based protein biomarkers of TBI (Figure 1). Attention is focused on the characteristics that may favor use of these markers as surrogates for imaging techniques in the case of sports-related traumatic brain injuries.
3.1. Markers of Neuronal Cell Body Injury
Neuron-specific enolase (NSE) is a neuronal cytoplasmatic enzyme necessary for the glycolytic pathway. NSE serum concentration rises in the first 12 h after TBI and declines within hours or days. The main drawback of using NSE as a TBI diagnostic tool is its high erythrocyte concentration. Therefore, it can also be elevated without TBI, for instance, in hemolysis or multi-trauma conditions
[11]. Hemolysis of blood samples, extracranial injury, and physical exercise may generate false positives
[12].
Ubiquitin C-terminal hydrolase-L1 (UCH-L1) is a neuronal cytoplasmatic deubiquitinating enzyme needed to remove abnormal neuronal proteins in physiological and pathological conditions. The increased serum concentration of UCH-L1 increases within the first 6–24 h after TBI and correlates with injury severity and clinical outcomes, including GCS score at admission and CT lesions
[11]. In CT-positive patients, serum levels 6–12 h post-injury were greater in those with unfavorable neurological outcomes
[12]. It is, therefore, a potential prognostic and diagnostic biomarker for mild, moderate, and severe TBIs.
3.2. Markers of Glial Cell Injury
S100 calcium-binding protein B (S100B) is a calcium-binding protein within astroglial cells, which can be released in the extracellular space following trauma and ischemic events. Studies of moderate-to-severe TBI show peaks of S100B serum around 24–48 h after injury; however, a recent study found elevated levels at 1 h but not at 12 or 36 h post-concussion
[12]. High levels of S100B are related to injury severity and predict the occurrence of post-concussion syndrome after mild TBI, poor clinical outcomes, and increased mortality. S100B protein is currently used in the early control of minimal, mild, and moderate TBI in Scandinavia, according to their head injury management guidelines (2007), to predict normal CT after mTBI, reducing unnecessary CT scans when S100B < 0.1 μg/L
[11]. A limitation in using S100B as a prognostic biomarker after TBI is the extra-neural injury release from cardiac muscle, adipose tissue, and skeletal muscles. S100B is also present in melanocytes, and patients with darker skin show higher levels of the biomarker. Nonetheless, strenuous exercise and extracranial injury can increase blood S100B levels, thus potentially reducing their utility as a biomarker in sports-related concussions and polytrauma. Another pitfall relating to S100B is its short half-life of 90 min, making it difficult to use as a biomarker for brain injury
[12][24][25].
Glial fibrillary acidic protein (GFAP) is an intermediate filament within astroglial cells that is needed to maintain their structure and to activate glial cells. After TBI, astroglial cells are activated and induce gliosis or glial scar formation, increasing the expression of GFAP
[11]. There is a positive correlation between GFAP levels and TBI severity; therefore, GFAP can be used to assess mTBI severity and evaluate the need for neuroimaging with CT and MRI, predicting poor outcomes and the risk of developing cognitive and psychiatric disabilities
[11]. Blood levels of GFAP peak within the first 24–48 h after mTBI, and the acute measures of blood GFAP in isolation or combined with UCH-L1 are susceptible to intracranial lesions in mTBI patients: this combination was recently approved by the FDA to reduce radiation exposure by CT
[26]. Elevated UCH-L1 and GFAP measured within 12 h of injury indicate intracranial lesions requiring CT
[12].
3.3. Markers of Axonal Injury
Neurofilament proteins (NFs) are the primary components of the neuronal cytoskeleton. Phosphorylated filaments interact with each other in order to increase neuronal stability. However, after TBI, there is an increase in intracellular calcium levels that activate various calcium-dependent enzymes such as proteases, calpains, and phosphatase calcineurin, leading to NFs dephosphorylation, proteolysis, dissociation, and release in the extracellular space, then to CSF and blood
[11]. NFs are formed by three different polypeptide subunits: light (NF-L, 68 kDa), medium (NF-M, 160 kDa), and heavy (NF-H, 200 kDa)
[27]. Evidence suggests that NF levels, and in particular NF-L levels, rise throughout the first few weeks (12 d) post-injury, accurately distinguishing patients with TBI from controls up to six months post-injury and, even more impressively, between patients with mild, moderate, and severe TBI at 30 d post-injury
[12][24]. NFs are specific for neurons and axons and are not affected by body trauma or strenuous physical activity; therefore, their extra-neural detection indicates neural death and axonal disintegration and lasts for days after the trauma, predicting poor outcomes, CT lesions, and the occurrence of chronic morbidities and cognitive disability
[11][25].
Myelin basic protein (MBP) is a oligodendroglial protein released in the blood following axonal damage; it is not specific for CNS injury, because peripheral nerve injury also increases MBP blood levels. Its release is delayed (1–3 days after injury), and the initial levels do not correlate with the GCS. MBP would be an inaccurate diagnostic and prognostic biomarker, unsuitable for emergency room screenings
[11][25].
Tau protein is a microtubule-associated protein (MAP) expressed mainly in the neurons to stabilize axonal microtubules. Within the context of TBI, microtubules release tau in response to mechanical stress, proteolytic cleavage by calpains and caspases, and calcium-dependent protein kinase activation, resulting in decreased microtubule binding and increased tau phosphorylation
[24]. TBI increases tau release in CSF, and CSF tau concentration positively correlates with TBI severity and poor outcomes. Serum tau protein peaks only two days after TBI, reflecting injury severity and predicting the clinical outcome. Nevertheless, some studies reported that serum tau does not correlate with CT lesions and cannot predict post-concussion syndrome. CSF tau is, therefore, a more accurate diagnostic and prognostic tool than serum tau
[11]. However, the release of tau from extracranial sources and after physical activity may limit the utility of tau in the context of sports-related mTBI. In addition to measures of total tau, quantification of tau in its phosphorylated form (p-tau) has also recently shown encouraging results as an acute marker indicator of mTBI, with elevated plasma levels found within 24 h of injury
[12].
3.4. Markers of Inflammation
Inflammation-associated proteins can function as blood mTBI biomarkers, becuase mTBI pathobiology is characterized by glial activation and release of proinflammatory cytokines
[12]. Circulating cytokine changes appear to be restricted to the first few hours post-mTBI: interleukin-6 (IL-6) levels appear to be elevated within the first 6–8 h but return to control levels by 24–48 h after mTBI. Moreover, IL-6 was associated with CT and MRI findings and longer duration of symptoms after mTBI
[28]. Interestingly, the temporal profile of IL-6 points to a distinctly different inflammatory profile in sports-related concussion (SRC) and military concussion versus the general unselected population with mTBI; athletes show early acute elevation (<8 h) with a return to baseline within 48 h, highlighting an earlier resolution of the inflammatory response in comparison with the general unselected population showing alterations lasting up to six months after injury. An explanation could be that the inflammatory response is milder in the relatively young and healthy athlete population than in the average mTBI patient
[28][29][30]. Moreover, blood IL-1 receptor antagonist levels increase in the first few hours post-mTBI, remaining elevated for 24–48 h
[29]. Other cytokines such as IL-8, IL-10, and TNF-α are excessively produced after TBI, but their correlation with injury severity and outcomes is yet to be confirmed; the best biomarker for mTBI is, therefore, IL-6
[11].
It should be emphasized that the inflammatory response is sensitive to age (immunosenescence leads to a higher basal level of inflammatory markers)
[31], sex (women generally have milder neuroinflammatory responses after TBI compared with males), and prior brain injuries (it is believed that a brain injury might ‘prime’ microglia into a more active state influencing the inflammatory response)
[32].
Studies of mTBI have been limited to measuring inflammatory markers in less invasive fluid compartments such as blood. However, the concentration of blood-based inflammatory markers is much lower than concentrations identified in the cerebrospinal fluid (CSF)
[32].
Importantly, given that inflammatory-associated proteins are produced by cells throughout the body in response to any disease-causing cellular injury, they are not highly specific for TBI. Inflammatory markers may be better suited as part of a multiple biomarker panel, including markers of other pathophysiological processes post-TBI, also having potential for decisions regarding athletes’ return to play or for predicting neuropsychological outcomes following mTBI.
3.5. Other Markers
Extracellular vesicles (EVs) are emerging as biomarkers, as they can be secreted from all types of brain cells and exhibit specific markers on their surface. Intraluminal DNA, RNA, protein, and metabolites are indicators of the state of the cell of origin. The major pitfall in using EVs as biomarkers is their isolation: the presence of other components of biological fluid, including lipoproteins, chylomicrons, and microvesicles, interferes with the isolation process, and this, together with EVs’ nanoscale size and difficulties in separating particular sub-types, makes isolation a very challenging process
[11].
MicroRNA abnormalities are also relevant to many neurodegenerative diseases and brain injuries such as TBI. Dysregulated levels correlate with impaired memory, learning, cognition, and neuropsychiatric disorders
[33]. However, the current limitation to the use of miRNA biomarkers is their variable expression between different individuals; this high heterogeneity makes it difficult to determine the optimal cut-off values for using miRNA biomarkers for TBI diagnosis and prognosis
[11].
Exosomes and miRNAs have recently gained considerable attention as promising biomarkers for TBI. However, despite the current knowledge of their potential, these biomarkers have not yet been optimized for clinical practice.
Figure 1. Diagram of the different types of TBI biomarkers. NSE: neuron-specific enolase
[8]; UCH-L1: ubiquitin C-terminal hydrolase-L1
[8]; S100B: S100 calcium-binding protein B
[9]; GFAP: glial fibrillary acidic protein
[23]; NF: neurofilament proteins
[9][21]; MBP: myelin basic protein
[8][22]; Tau protein
[8]; IL-6: interleukin-6
[25].