In view of the high rate of intubation and difficulties in the proper evaluation of the eye opening, Stocchetti et al. concluded that motor GCS score was more important than eye opening or verbal responses to predict the severity of the neurological injury. Other recent research has provided evidence that the use of sedative drugs avoids the accurate assessment of GCS during the first 24 h [5].

Other challenges for diagnosis are presented by the progressive nature of some brain injuries, which can lead to further neurological deterioration. In addition, neurological responses after TBI may vary over time for reasons unrelated to the injury. For example, trauma is frequently associated with alcohol and drug intoxication [6]. These factors together place the GCS in a position full of limitations that diminish its reliability as a highly sensitive test in specific and not infrequent circumstances such as those already mentioned.

On the other hand, neuroimaging techniques are used to provide objective information about the injury and its location [7] and are not influenced by the aforementioned confounding factors. However, the CT scan has a low sensitivity for diffuse brain injury, when the TBI is mild [8] and the availability and usefulness of MRI in the acute stage is limited. These facts, among others, have led to the search for alternative methods to assess the damage, being of special interest, the search for biomarkers, which are more reliable indicators of neuronal injury, due to its molecular context and its early expression.

Research in this field has increased exponentially in the last 20 years, with most publications on the subject in the last 10 years. Most markers are associated with cell damage. **Table 1** presents a summary of the TBI biomarkers most studied to date, including information about their nature, tissue location, molecular weight, half-life, basal levels, and physiopathological significance.

The main physiopathological mechanisms reflected by the glial or neuronal biomarkers are the disruption of the blood-brain barrier (BBBD) and neuronal injury, respectively. Taking into account this basis, it would be advantageous to have a panel of complementary biomarkers that show different temporal profiles and that reflect different physiopathological conditions subsequent to TBI. In a parallel manner, Papa et al. [9] propose that an ideal biomarker should have the following characteristics:

**69**

**Biomarker**

UCH-L1

NSE αIIespectrina

SBDP S-100B

MBP GFAP

**Table 1.**

*Main biomarkers in TBI and their properties.*

Glial (oligodendrocytes and

18.5 [50]

Myelin sheath component

protein

Cytoskeleton component

—

<0.03 ng/mL [30]

BBBD and neuronal

injury

protein

Schwann cells)

Glial (astrocytes)

40–53 [30]

Glial (astrocytes)

Neuronal

280 [41] 120 [41] 145 [41] 150 [41]

21 [50]

Calcium binding protein

Cytoskeleton component protein

2.9 h [48]

1.5 days [49]

1 day [49]

1 day [49]

97 minutes [47]

0.328–0.01 pg/mL [11]

BBBD

112 minutes [43]

12 h [43]

<0.3 ng/mL [50]

White matter injury

Neuronal

90 [45] 78 [46]

Enzyme

24 h [46] 48 h [41]

<12.5 ng/mL [47]

≤15 ng/mL [46]

—

Apoptosis

**Location** Neuronal

**Molecular mass [KDa]**

20 [41] 24 [42]

**Nature** Ubiquitination enzyme

20 minutes [43]

0.12 ng/mL [44]

**Half life**

**Basal concentrations**

**Significance** Neuronal injury

*Neuronal and Glial Biomarkers Research for Traumatic Brain Injury*

*DOI: http://dx.doi.org/10.5772/intechopen.85555*

Neuronal injury

*Main biomarkers in TBI and their properties.*

#### *Neuronal and Glial Biomarkers Research for Traumatic Brain Injury DOI: http://dx.doi.org/10.5772/intechopen.85555*

*Traumatic Brain Injury - Neurobiology, Diagnosis and Treatment*

avoids the accurate assessment of GCS during the first 24 h [5].

circumstances such as those already mentioned.

molecular context and its early expression.

half-life, basal levels, and physiopathological significance.

1.demonstrate high sensitivity and specificity for brain injury;

2. stratify the patients according to the severity of the injury;

3.have rapid appearance in the accessible biological liquid;

8. easily measured by simple techniques widely available.

6.monitor the progress of the disease and the response to the treatment;

4.provide information about injury mechanisms;

5.have biokinetic properties;

7.predict the functional result; and

frequent circumstances [4].

and/or alcohol, and in those who are intubated or sedated will have limited linguistic capacities [3]. Given that the severity of the neurological injury may be underestimated in some cases and overestimated in others, attention has been focused on early assessment strategies in patients with TBI and their inaccuracy in special and

In view of the high rate of intubation and difficulties in the proper evaluation of the eye opening, Stocchetti et al. concluded that motor GCS score was more important than eye opening or verbal responses to predict the severity of the neurological injury. Other recent research has provided evidence that the use of sedative drugs

Other challenges for diagnosis are presented by the progressive nature of some brain injuries, which can lead to further neurological deterioration. In addition, neurological responses after TBI may vary over time for reasons unrelated to the injury. For example, trauma is frequently associated with alcohol and drug intoxication [6]. These factors together place the GCS in a position full of limitations that diminish its reliability as a highly sensitive test in specific and not infrequent

On the other hand, neuroimaging techniques are used to provide objective information about the injury and its location [7] and are not influenced by the aforementioned confounding factors. However, the CT scan has a low sensitivity for diffuse brain injury, when the TBI is mild [8] and the availability and usefulness of MRI in the acute stage is limited. These facts, among others, have led to the search for alternative methods to assess the damage, being of special interest, the search for biomarkers, which are more reliable indicators of neuronal injury, due to its

Research in this field has increased exponentially in the last 20 years, with most publications on the subject in the last 10 years. Most markers are associated with cell damage. **Table 1** presents a summary of the TBI biomarkers most studied to date, including information about their nature, tissue location, molecular weight,

The main physiopathological mechanisms reflected by the glial or neuronal biomarkers are the disruption of the blood-brain barrier (BBBD) and neuronal injury, respectively. Taking into account this basis, it would be advantageous to have a panel of complementary biomarkers that show different temporal profiles and that reflect different physiopathological conditions subsequent to TBI. In a parallel manner, Papa et al. [9] propose that an ideal biomarker should have the following

**68**

characteristics:

**69**

In this chapter, we present a compendium of the most studied biomarkers in the TBI, its possible applications, and the current techniques for its detection.

#### **2. Most studied biomarkers in TBI**

As explained in previous paragraphs, there is no single biomarker that is sufficiently sensitive and specific to study the physiopathological mechanisms that derive from head trauma. Next, we will mention some of the most studied biomarkers given its rapid elevation after trauma and its relationship with the mechanism of injury. One of the most studied biomarkers is the Ca binder protein S-100β, a glial protein at the astrocyte level that is related to alterations in the blood-brain barrier [10]. Its rapid elevation and its considerable concentration release in the serum facilitate the study of the protein and its correlation with the severity of the injury. Due to the type of cells found in the central nervous system, it is necessary to study biomarkers that allow us to demonstrate not only glial injury but also neuronal. One of the most studied biomarkers in this sense is the C-terminal hydrolase of ubiquitin-L1, which is a highly specific cytoplasmic neuronal enzyme [11, 12]. Finally, we will delve into glial fibrillary acidic protein (GFAP), which is also a glial protein and is part of the cytoskeleton of astrocytes and is also related to the disruption of the blood-brain barrier [11, 13].

#### **2.1 The Ca binder protein S-100β**

S-100 β is a central nervous system (CNS) protein found predominantly in astrocytes and is the most studied peripheral biomarker of BBBD. This calcium binding protein (CBP) S-100β increases initially after the accident and then decreases rapidly after the traumatic injuries. In cell models, their release has been demonstrated from the first 15 seconds after the trauma. In humans, the earliest that has been detected is 30 minutes posttrauma. The approximate half-life of this protein is 97 minutes [10], the peak occurs on day 0, and the concentrations decrease toward the sixth day in both CSF and serum.

Goyal et al. [14] reported basal levels of S-100β in healthy CSF controls of 0.0754–0.0034 ng/mL and in serum of 0.328–0.101 pg/mL. This protein has been studied extensively in mild TBI (mTBI), so that high levels in serum are associated with an increase in the incidence of post-concussion syndrome [15] and neurological dysfunction. There are also several studies that have reported a correlation between serum levels of S-100β and the presence of pathological findings in cerebral magnetic resonance imaging (MRI), as well as abnormalities in neuropsychological exploration after mTBI [16].

Most studies show that the S-100β measurement can distinguish injured patients from noninjured patients with an uncertain degree of utility in predicting mortality either acutely or at several points in time (**Table 2**) [17–19]. In general terms, S-100β is a sensitive but not specific predictor of CT abnormalities. Using low serum cut-off values, the sensitivity oscillates between 90 and 100% with a specificity between 4 and 65%.

Müller et al. [17] reported a sensitivity of 0.95 (95% CI 0.76–1.0) for S-100β measured within the first 12 h with a specificity of 31% (95% CI 0.25–0.38) relative to abnormal findings on skull CT scan in a study of 226 adult patients admitted to the hospital with a diagnosis of mild TBI (GCS 13–15). Biberthaler et al. [19] found similar results using a cut-off level of S-100β of 0.1 ng/mL, measured within the first 3 h posttrauma in 1309 patients with mTBI and correlating them to head CT findings. The sensitivity was 99% (95% CI 0.96–1.0), and the specificity was 30% (95% CI 0.29–0.31).

**71**

0.41–0.59) [20].

cut-off value of 0.08 ng/mL [21].

polytraumatized patient.

*Neuronal and Glial Biomarkers Research for Traumatic Brain Injury*

**Reference Detection method Sample Findings**

ELISA CSF and

serum

Increase in CSF and serum first 6 days

Correlation between serum and CSF levels

Level in CSF is a potential predictor of GOS

Mean and peak are predictors of mortality in

It cannot replace the clinical examination or

It can serve as support for the selection of

using the two measurement techniques; cut-off value calculated: 0.18 ng/mL.

post-trauma

and DRS

severe TBI

decreased over time

Serum Significantly elevated in intracranial injury

Serum Increase was related to findings in the CT

the use of CT in mTBI

patients for TC S: 90–100%, E: 4–65%

S: 100%, E: 46%

scan S: 99%, E: 30%

Long term and Rapid test Serum Concentrations were significantly correlated

The usefulness of S-100β as a marker does not seem to be affected by the concomitant consumption of alcohol. Mussack et al. conducted a study in which they included patients with mild TBI with demonstrated blood alcohol levels (mean = 182 mg/dL), and found that the sensitivity of S-100β in the first 3 h posttrauma was 100% (95% CI 0.83 a 1.0) and the specificity was 50% (95% CI

ELISA Serum S: 80%, E: 40%

On the other hand, Bazarian et al. studied 96 patients with TBI, GCS 13–15 who also presented trauma of extracranial localization, and found a sensitivity of 80% (95% CI 0.36–0.96) and a specificity of 40% (95% CI 0.01–0.09) for S-100β with a

From the studies described above, it can be deduced that the sensitivity increases as the time elapsed between the trauma and the sample taking (window) decreases, as well as an increase in specificity is observed when the cut-off value increases. In contrast, the limitations of the use of S-100β as a marker are due to the marked decrease in sensitivity and specificity in the context of the polytraumatized patient, since the presence of concomitant extracranial trauma also causes the release and plasma elevation of this protein. The presence of S-100β has been reported in tissues other than the nervous one, mainly in adipose tissue [22]. From this observation, a negative effect on the specificity of this marker is expected, due to the increase that would occur in the context of extracranial lesions, as occurs in the

Pham et al. [22] characterized the tissue specificity of S-100β and evaluated the extracranial sources of this marker and how they affect serum levels of this marker. For this purpose, they performed the extraction of proteins from nine different human tissues (liver, bladder, kidney, colon, lung, muscle, pancreas, adipose tissue, brain, tonsils, stomach, and skin) and their subsequent analysis through ELISA

*DOI: http://dx.doi.org/10.5772/intechopen.85555*

Automated LIAISON system [AB DiaSorin, Bromma, Sweden]

Elecsys S100 [Roche Diagnostics, Mannheim,

*Summary of the evidence reported in the literature on biomarkers in S-100B.*

Germany]

Goyal et al. [14]

Berger et al [51]

Biberthaler et al [19]

Biberthaler et al [52]

Bazarian, et al [21]

**Table 2.**

*Neuronal and Glial Biomarkers Research for Traumatic Brain Injury DOI: http://dx.doi.org/10.5772/intechopen.85555*

#### **Table 2.**

*Traumatic Brain Injury - Neurobiology, Diagnosis and Treatment*

**2. Most studied biomarkers in TBI**

**2.1 The Ca binder protein S-100β**

the sixth day in both CSF and serum.

chological exploration after mTBI [16].

ficity between 4 and 65%.

(95% CI 0.29–0.31).

In this chapter, we present a compendium of the most studied biomarkers in the

As explained in previous paragraphs, there is no single biomarker that is sufficiently sensitive and specific to study the physiopathological mechanisms that derive from head trauma. Next, we will mention some of the most studied biomarkers given its rapid elevation after trauma and its relationship with the mechanism of injury. One of the most studied biomarkers is the Ca binder protein S-100β, a glial protein at the astrocyte level that is related to alterations in the blood-brain barrier [10]. Its rapid elevation and its considerable concentration release in the serum facilitate the study of the protein and its correlation with the severity of the injury. Due to the type of cells found in the central nervous system, it is necessary to study biomarkers that allow us to demonstrate not only glial injury but also neuronal. One of the most studied biomarkers in this sense is the C-terminal hydrolase of ubiquitin-L1, which is a highly specific cytoplasmic neuronal enzyme [11, 12]. Finally, we will delve into glial fibrillary acidic protein (GFAP), which is also a glial protein and is part of the cytoskeleton of astrocytes and is also related to the disruption of the blood-brain barrier [11, 13].

S-100 β is a central nervous system (CNS) protein found predominantly in astrocytes and is the most studied peripheral biomarker of BBBD. This calcium binding protein (CBP) S-100β increases initially after the accident and then decreases rapidly after the traumatic injuries. In cell models, their release has been demonstrated from the first 15 seconds after the trauma. In humans, the earliest that has been detected is 30 minutes posttrauma. The approximate half-life of this protein is 97 minutes [10], the peak occurs on day 0, and the concentrations decrease toward

Goyal et al. [14] reported basal levels of S-100β in healthy CSF controls of 0.0754–0.0034 ng/mL and in serum of 0.328–0.101 pg/mL. This protein has been studied extensively in mild TBI (mTBI), so that high levels in serum are associated with an increase in the incidence of post-concussion syndrome [15] and neurological dysfunction. There are also several studies that have reported a correlation between serum levels of S-100β and the presence of pathological findings in cerebral magnetic resonance imaging (MRI), as well as abnormalities in neuropsy-

Most studies show that the S-100β measurement can distinguish injured patients from noninjured patients with an uncertain degree of utility in predicting mortality either acutely or at several points in time (**Table 2**) [17–19]. In general terms, S-100β is a sensitive but not specific predictor of CT abnormalities. Using low serum cut-off values, the sensitivity oscillates between 90 and 100% with a speci-

Müller et al. [17] reported a sensitivity of 0.95 (95% CI 0.76–1.0) for S-100β measured within the first 12 h with a specificity of 31% (95% CI 0.25–0.38) relative to abnormal findings on skull CT scan in a study of 226 adult patients admitted to the hospital with a diagnosis of mild TBI (GCS 13–15). Biberthaler et al. [19] found similar results using a cut-off level of S-100β of 0.1 ng/mL, measured within the first 3 h posttrauma in 1309 patients with mTBI and correlating them to head CT findings. The sensitivity was 99% (95% CI 0.96–1.0), and the specificity was 30%

TBI, its possible applications, and the current techniques for its detection.

**70**

*Summary of the evidence reported in the literature on biomarkers in S-100B.*

The usefulness of S-100β as a marker does not seem to be affected by the concomitant consumption of alcohol. Mussack et al. conducted a study in which they included patients with mild TBI with demonstrated blood alcohol levels (mean = 182 mg/dL), and found that the sensitivity of S-100β in the first 3 h posttrauma was 100% (95% CI 0.83 a 1.0) and the specificity was 50% (95% CI 0.41–0.59) [20].

On the other hand, Bazarian et al. studied 96 patients with TBI, GCS 13–15 who also presented trauma of extracranial localization, and found a sensitivity of 80% (95% CI 0.36–0.96) and a specificity of 40% (95% CI 0.01–0.09) for S-100β with a cut-off value of 0.08 ng/mL [21].

From the studies described above, it can be deduced that the sensitivity increases as the time elapsed between the trauma and the sample taking (window) decreases, as well as an increase in specificity is observed when the cut-off value increases. In contrast, the limitations of the use of S-100β as a marker are due to the marked decrease in sensitivity and specificity in the context of the polytraumatized patient, since the presence of concomitant extracranial trauma also causes the release and plasma elevation of this protein. The presence of S-100β has been reported in tissues other than the nervous one, mainly in adipose tissue [22]. From this observation, a negative effect on the specificity of this marker is expected, due to the increase that would occur in the context of extracranial lesions, as occurs in the polytraumatized patient.

Pham et al. [22] characterized the tissue specificity of S-100β and evaluated the extracranial sources of this marker and how they affect serum levels of this marker. For this purpose, they performed the extraction of proteins from nine different human tissues (liver, bladder, kidney, colon, lung, muscle, pancreas, adipose tissue, brain, tonsils, stomach, and skin) and their subsequent analysis through ELISA

and Western blot in 200 subjects receiving chemotherapy for the management of CNS lymphomas. A dose of mannitol (1.4M) was administered intra-arterially in the carotid or vertebral artery, subsequently confirming the presence of BBBD by a head CT performed immediately after chemotherapy.

The results presented in that study showed that extracranial sources of S-100β do not affect serum levels. Therefore, the diagnostic value and the negative predictive value of S-100β are not compromised in the context of patients with neurological diseases, but without traumatic lesions, whether cerebral or extracranial.

Goyal et al. [14] also evaluated S-100β as a prognostic biomarker in adult subjects with severe TBI (sTBI) by comparing the results with the S-100β temporal profiles in both CSF (n = 138 subjects) and serum (n = 80 subjects) for 6 days. The variables used to evaluate the extracerebral sources of S-100β in serum were: long bone fracture, Injury Severity Score (ISS), and isolated skull trauma. After TBI, levels of S-100β in CSF and serum were increased compared to healthy controls during the first 6 days after TBI (p ≤ 0.005 and p ≤ 0.031). Although levels in CSF and serum had a high correlation at the early post-TCE time points, this association decreased with time. The bivariate analysis showed that subjects who had temporary CSF profiles with higher concentrations of S-100B had higher acute mortality (p < 0.001) and worse Glasgow Outcome Scale (GOS; p = 0.002) and disability scores (DRS) (p = 0.039) 6 months after the injury. Temporary profiles in serum were associated with acute mortality (p = 0.015), possibly as a result of the extracerebral sources of S-100β in the serum, represented by high ISSs (p = 0.032).

Due to its temporal elevation profile, and the pathophysiological mechanisms that cause its release toward serum, S-100β constitutes an excellent candidate as an early biomarker of TBI, with the possible limitation in patients with concomitant trauma in other sites that leads to the serum elevation of S-100β from extracranial sources.

#### **2.2 Ubiquitin C-terminal hydrolase-L1 (UCH-L1)**

The C-terminal hydrolase of ubiquitin-L1 (ubiquitin C-terminal hydrolase-L1, UCH-L1) is an E2 conjugation enzyme present in the cytoplasm of almost all neurons [13] and has previously been used as a neuronal histological marker due to its great abundance and specific expression in these cells [11]. Its location has also been shown in neurons of the peripheral nervous system, particularly in the neuromuscular junction [12], as well as in cells of the neuroendocrine system. In addition, the presence of UCH-L1 has been demonstrated in aortic endothelial cells and in smooth muscle and tumor cells [23]. This enzyme accomplishes the function of adding and removing ubiquitin from proteins in order to promote its degradation via the proteasome-dependent pathway [24].

UCH-L1 is one of the most recent biomarkers proposed for TBI, and for this reason, there are still limited data that demonstrate its usefulness (**Table 3**).

Three isoenzymes of UCH (UCH-L1, UCH-L2, and UCH-L3) have been identified, being UCH-L1, the only one present in high concentrations in the central nervous system [24]. In a prospective case-control study with 66 patients, Papa et al. [24] obtained ventricular CSF samples for each patient after 6, 12, 24, 48, 72, 96, 120, 144, and 168 h after TBI for the UCH-L1 detection by ELISA. The severity was determined by the Glasgow Scale (GCS) and CT findings. Mortality and neurological sequelae were evaluated at 6 months. This study showed that patients with TBI had a significant elevation of CSF UCH-L1 levels at each point in time compared to controls, with total mean in TBI patients = 44.2 ng/mL (±7.9) vs. 2.7 ng/mL (±0.7) in controls (p < 0.001). Significantly elevated levels of UCH-L1 were found in

**73**

required to validate these findings.

*Summary of the evidence reported in the literature on UCH-L1.*

*Neuronal and Glial Biomarkers Research for Traumatic Brain Injury*

**Reference Detection method Sample Findings**

ELISA CSF

Sandwich ELISA CSF

Electrochemiluminescence immunoassay (ECL-IA)

ELISA CSF Increase at 6, 12, 24, 48, 72, 96, 120, 144,

and serum

and serum 6 months.

ELISA Serum Elevated in GCS 15 vs. controls without trauma

Sandwich ELISA Serum Measurement <24 h posttrauma distinguished

ELISA Serum There were no significant differences between

received.

and 168 h post-trauma, X = 44.2 ng/mL (±7.9), versus controls X = 2.7 ng/mL (±0.7) (p < 0.001). Also elevated when it exists: lower GCS at 24 h, post-trauma complications, deaths in the first 6 weeks, or serious sequelae at

Significant correlation between biokinetics and means of (UCH-L1) in CSF and serum in severe TBI (rs = 0.59, p < 0.001) (AUC, rs = 0.3, p = 0.027). Increased levels <24 h posttrauma, statistically significant in Cmax (0–24 h) in CSF

It remains elevated up to 7 days after TBI, serum AUC and statistically significant CSF at all-time points up to 24 h (p < 0.001). Levels in <12 h in GCS 3–5 > GCS 6–8 (p = 0.07 and p = 0.02, Mann-Whitney test, respectively). Significantly higher and prolonged serum and CSF levels in non-survivors. A level of >5.22 ng/mL was a

(AUC 0.8) and controls with trauma. Higher elevation in GCS 15 plus TAC or neurosurgical intervention requirement. It provides evidence

injury. Significantly elevated levels in patients in

presence and absence of intracranial lesions (AUC of 0.713). No correlation between levels in mild TCE and recovery at 6 months. Significant increase in levels in moderate/ severe TCE compared with mild TBI. Good sensitivity to discriminate between TCE and controls (AUC 0.87). Combination with GFAP showed greater sensitivity and specificity for

the levels of negative controls and TCE <6 h posttrauma, independent of the CT. The levels were high after each game but without correlation with the number of hits

and serum in those who died.

predictor of mortality (OR 4.8).

as a potential marker of mild TBI.

Serum Complements brain MRI in the detection of

the diagnosis of TBI (AUC 0.94).

the acute state of mild TBI.

patients with a lower score in the GCS at 24 h, in those who had presented posttrauma complications, in those who died within the first 6 weeks, and in those with severe sequelae at 6 months. These data suggest that this marker would be useful in determining severity in patients with TBI. Similar studies with larger samples are

Additional studies have confirmed the positive correlation between the concentrations of UCH-L1 at the CSF level and serum samples [25]. Similarly, Mondello

*DOI: http://dx.doi.org/10.5772/intechopen.85555*

Papa et al. [24]

Brophy et al. [53]

Mondello et al. [26]

Papa et al. [11]

Kou et al. [27]

Diaz-Arrastia et al. [28]

Puvenna et al. [15]

**Table 3.**

*Neuronal and Glial Biomarkers Research for Traumatic Brain Injury DOI: http://dx.doi.org/10.5772/intechopen.85555*

#### **Table 3.**

*Traumatic Brain Injury - Neurobiology, Diagnosis and Treatment*

head CT performed immediately after chemotherapy.

**2.2 Ubiquitin C-terminal hydrolase-L1 (UCH-L1)**

via the proteasome-dependent pathway [24].

and Western blot in 200 subjects receiving chemotherapy for the management of CNS lymphomas. A dose of mannitol (1.4M) was administered intra-arterially in the carotid or vertebral artery, subsequently confirming the presence of BBBD by a

The results presented in that study showed that extracranial sources of S-100β do not affect serum levels. Therefore, the diagnostic value and the negative predictive value of S-100β are not compromised in the context of patients with neurological diseases, but without traumatic lesions, whether cerebral or extracranial. Goyal et al. [14] also evaluated S-100β as a prognostic biomarker in adult subjects with severe TBI (sTBI) by comparing the results with the S-100β temporal profiles in both CSF (n = 138 subjects) and serum (n = 80 subjects) for 6 days. The variables used to evaluate the extracerebral sources of S-100β in serum were: long bone fracture, Injury Severity Score (ISS), and isolated skull trauma. After TBI, levels of S-100β in CSF and serum were increased compared to healthy controls during the first 6 days after TBI (p ≤ 0.005 and p ≤ 0.031). Although levels in CSF and serum had a high correlation at the early post-TCE time points, this association decreased with time. The bivariate analysis showed that subjects who had temporary CSF profiles with higher concentrations of S-100B had higher acute mortality (p < 0.001) and worse Glasgow Outcome Scale (GOS; p = 0.002) and disability scores (DRS) (p = 0.039) 6 months after the injury. Temporary profiles in serum were associated with acute mortality (p = 0.015), possibly as a result of the extracerebral sources of S-100β in the serum, represented by high ISSs

Due to its temporal elevation profile, and the pathophysiological mechanisms that cause its release toward serum, S-100β constitutes an excellent candidate as an early biomarker of TBI, with the possible limitation in patients with concomitant trauma in other sites that leads to the serum elevation of S-100β from extracranial

The C-terminal hydrolase of ubiquitin-L1 (ubiquitin C-terminal hydrolase-L1, UCH-L1) is an E2 conjugation enzyme present in the cytoplasm of almost all neurons [13] and has previously been used as a neuronal histological marker due to its great abundance and specific expression in these cells [11]. Its location has also been shown in neurons of the peripheral nervous system, particularly in the neuromuscular junction [12], as well as in cells of the neuroendocrine system. In addition, the presence of UCH-L1 has been demonstrated in aortic endothelial cells and in smooth muscle and tumor cells [23]. This enzyme accomplishes the function of adding and removing ubiquitin from proteins in order to promote its degradation

UCH-L1 is one of the most recent biomarkers proposed for TBI, and for this reason, there are still limited data that demonstrate its usefulness (**Table 3**).

Three isoenzymes of UCH (UCH-L1, UCH-L2, and UCH-L3) have been identified, being UCH-L1, the only one present in high concentrations in the central nervous system [24]. In a prospective case-control study with 66 patients, Papa et al. [24] obtained ventricular CSF samples for each patient after 6, 12, 24, 48, 72, 96, 120, 144, and 168 h after TBI for the UCH-L1 detection by ELISA. The severity was determined by the Glasgow Scale (GCS) and CT findings. Mortality and neurological sequelae were evaluated at 6 months. This study showed that patients with TBI had a significant elevation of CSF UCH-L1 levels at each point in time compared to controls, with total mean in TBI patients = 44.2 ng/mL (±7.9) vs. 2.7 ng/mL (±0.7) in controls (p < 0.001). Significantly elevated levels of UCH-L1 were found in

**72**

(p = 0.032).

sources.

*Summary of the evidence reported in the literature on UCH-L1.*

patients with a lower score in the GCS at 24 h, in those who had presented posttrauma complications, in those who died within the first 6 weeks, and in those with severe sequelae at 6 months. These data suggest that this marker would be useful in determining severity in patients with TBI. Similar studies with larger samples are required to validate these findings.

Additional studies have confirmed the positive correlation between the concentrations of UCH-L1 at the CSF level and serum samples [25]. Similarly, Mondello

et al. [26] conducted a case-control study with 95 patients with severe TBI in order to evaluate the CSF and serum concentrations of UCH-L1 by sandwich ELISA and its association with clinical results. The temporal profile of the marker in both CSF and serum was studied during the first 7 days following the trauma and compared with controls showing significantly higher levels compared to the controls throughout the 7-day period, also confirming a high sensitivity and specificity for the diagnosis of TBI versus controls, with statistically significant serum AUC and CSF values at all-time points up to 24 h (p < 0.001).

The levels of UCH-L1 in the first 12 h in both CSF and serum in patients with GCS 3–5 were also significantly higher than in those with GCS 6–8. In addition, UCH-L1 levels in CSF and serum appeared to distinguish between patients with severe TBI survivors and nonsurvivors within the study, such that those who died had significantly higher CSF and serum UCHL1 levels, as well as greater permanence of these levels over time. In this study, a serum level of UCH-L1 > 5.22 ng/mL was a predictor of mortality (OR 4.8).

Papa et al. [11] also analyzed UCH-L1 in serum taken in the first 4 h posttrauma in patients with mild (n = 86) and moderate (n = 10) TBI, as well as in controls with trauma and controls without trauma. For patients with a GCS of 15, serum UCH-L1 was significantly elevated compared to controls without trauma, with an AUC of 0.87, and was also compared with controls with trauma, and was even higher in those patients with GCS of 15 who also had positive findings on the CT scan or required some neurosurgical intervention, suggesting that UCH-L1 may be a potential marker of mild TBI. Additionally, 5% of patients with GCS of 15 (4/77) required neurosurgical intervention, which was higher than the 1% found in patients with GCS 14–15 reported in the study by Jagoda et al., in which the samples were taken within the first 24 h posttrauma [10].

It is inferred from these data that the earlier it is detected posttrauma, the sensitivity of this marker increases. In a smaller study (n = 9), serum UCHL1 (taken <6 h posttraumatic) was found to be significantly elevated in patients with mild TBI [27].

In another study focused on all levels of severity of TBI, serum UCH-1 measured before 24 h posttrauma could distinguish patients with intracranial lesions from those without intracranial lesions with an AUC = 0.713 [28]. However, there was no correlation between UCH-L1 levels in patients with mTBI and recovery at 6 months as measured by the GOSE scale. While there was a significant increase in UCH-L1 levels in patients with moderate/severe TBI compared to mild TBI, patients with mild TBI were not compared with controls.

In a research carried out in a secondary school, Puvenna et al. [15] selected 15 American football players; they obtained serum samples before and after each of two different games. They did not observe significant differences between the levels of UCH-L1 between the negative controls and the positive individuals for mild TBI within the first 6 h posttrauma, regardless of whether or not positive CT findings existed. In addition to this, there was no correlation between the serum levels of UCH-L1 and the number of impacts received. The levels of UCH-L1 and S-100β, markers of neuronal injury and BBBD, respectively, were both elevated after each game. However, only S-100β, unlike UCH-L1, was correlated with the number of hits received and the UCH-L1 elevation did not correlate with the S-100β increments. The authors suggest that elevated postgame UCH-L1 levels may be due to the release of this protein from the neuromuscular junction.

It can be concluded that there are very divergent data regarding the use of UCH-L1 as a serum biomarker of mild TBI. Some studies suggest that it is a promising marker, while others do not find a correlation with the lesion. Release from sources other than the central nervous system could contribute to elevated serum levels.

**75**

*Neuronal and Glial Biomarkers Research for Traumatic Brain Injury*

sample does not allow the conclusions to be validated.

median of 0.85 and correlated positively with age.

than when it was done late (**Table 4**).

was 0.82 for GFAP and 0.77 for S-100β.

Glial fibrillary acidic protein (GFAP) is a protein derived from glial cells, which is a part of the intermediate filament of the cytoskeleton of astrocytes, where it is the most abundant protein. It is considered a specific marker of CNS diseases, and is also related to several neuronal processes' harmful agents that compromise the integrity of the blood-brain barrier [29], and has been shown to be a potentially useful biomarker for predicting clinical outcomes in TBI. Its normal level in serum is <0.03 ng/mL [30], so any elevation thereof will indicate BBBD (**Table 4**).

Due to its great immunoreactivity, GAFP has been used as an indicator of brain injury in experimental models of mTCE [31]. The first successful measurement of GFAP in human blood was made in 1999 in 12 of 25 patients with severe TBI [32]. Using a weight drop model with mice [33] to evaluate two levels of mTBI, one with hemorrhage (complicated mTBI) and another without bleeding (uncomplicated mTBI), Yang et al. [34] found that serum GFAP was significantly elevated in both injury models at 90 minutes and 6 h after injury, but had returned to normal at 24 h. In the study of Kou et al. [27], significantly elevated serum levels of GFAP in the first 24 h posttrauma in 9 mTBI patients was also reported; this elevation being even more significant in those with hemorrhagic lesions; however, the small size of the

In another study, Mondello et al. [35] evaluated whether the relationship between a neuronal marker (UCH-L1) and a glial marker (GFAP) correlates with the presence of different intracranial pathologies after brain trauma. They obtained serum samples from 59 patients with sTBI on admission to the hospital and measured levels of UCH-L1 and GFAP. The glial/neuronal ratio (GNR) was measured as the quotient between the concentrations of GFAP and UCH-L1. Logistic regression analysis identified variables associated with the type of injury. The increase in GNR was associated independently with the type of injury, but not with the age, gender, GCS, or trauma mechanism. This quotient was significantly higher in the patients who died, but it was not an independent predictor of mortality. The GNR had a

When evaluating the CT scan of the skull on admission, 29 patients presented a diffuse lesion and 30 localized lesions. The GNR was significantly higher in the group with focal lesions compared to the group with diffuse lesions. The receiver operating characteristic (ROC) analysis showed that the GNR discriminated between the two types of injury. GNR was more accurate when performed early

These data indicate that the GNR provides valuable information about the different types of injury, which is of great clinical utility. In addition, the GNR can help to identify the pathophysiological mechanisms subsequent to the different types of TBI. This is very useful when implementing therapeutic measures.

In an investigation carried out by Papa et al. [36], the capacity of the GFAP taken <4 h posttrauma was compared to predict intracranial lesions in the CT compared to S-100β. Although patients had GCS 9–15, only 3 of 209 patients had GCS <13 and only 10% had intracranial lesions, both S-100β and GFAP were significantly elevated in all patients, and even more so in those with intracranial lesions. For those patients with GCS 14–15, the AUC for the identification of intracranial lesions

In the presence of extracranial lesions and using a cut-off value of 0.067 ng/mL, GFAP was 100% sensitive and 55% specific in the prediction of intracranial lesions. With a cut-off value of 0.20 ng/mL, S-100β also had 100% sensitivity but only 5% specificity. This study concludes that GFAP exceeds S-100β in the identification of intracranial lesions in mild and moderate TBI, even in the presence of extracranial lesions.

*DOI: http://dx.doi.org/10.5772/intechopen.85555*

**2.3 The fibrillary acid glial protein (GFAP)**

#### **2.3 The fibrillary acid glial protein (GFAP)**

*Traumatic Brain Injury - Neurobiology, Diagnosis and Treatment*

values at all-time points up to 24 h (p < 0.001).

were taken within the first 24 h posttrauma [10].

mild TBI were not compared with controls.

release of this protein from the neuromuscular junction.

was a predictor of mortality (OR 4.8).

et al. [26] conducted a case-control study with 95 patients with severe TBI in order to evaluate the CSF and serum concentrations of UCH-L1 by sandwich ELISA and its association with clinical results. The temporal profile of the marker in both CSF and serum was studied during the first 7 days following the trauma and compared with controls showing significantly higher levels compared to the controls throughout the 7-day period, also confirming a high sensitivity and specificity for the diagnosis of TBI versus controls, with statistically significant serum AUC and CSF

The levels of UCH-L1 in the first 12 h in both CSF and serum in patients with GCS 3–5 were also significantly higher than in those with GCS 6–8. In addition, UCH-L1 levels in CSF and serum appeared to distinguish between patients with severe TBI survivors and nonsurvivors within the study, such that those who died had significantly higher CSF and serum UCHL1 levels, as well as greater permanence of these levels over time. In this study, a serum level of UCH-L1 > 5.22 ng/mL

Papa et al. [11] also analyzed UCH-L1 in serum taken in the first 4 h posttrauma

It is inferred from these data that the earlier it is detected posttrauma, the sensitivity of this marker increases. In a smaller study (n = 9), serum UCHL1 (taken <6 h posttraumatic) was found to be significantly elevated in patients with mild TBI [27]. In another study focused on all levels of severity of TBI, serum UCH-1 measured before 24 h posttrauma could distinguish patients with intracranial lesions from those without intracranial lesions with an AUC = 0.713 [28]. However, there was no correlation between UCH-L1 levels in patients with mTBI and recovery at 6 months as measured by the GOSE scale. While there was a significant increase in UCH-L1 levels in patients with moderate/severe TBI compared to mild TBI, patients with

In a research carried out in a secondary school, Puvenna et al. [15] selected 15 American football players; they obtained serum samples before and after each of two different games. They did not observe significant differences between the levels of UCH-L1 between the negative controls and the positive individuals for mild TBI within the first 6 h posttrauma, regardless of whether or not positive CT findings existed. In addition to this, there was no correlation between the serum levels of UCH-L1 and the number of impacts received. The levels of UCH-L1 and S-100β, markers of neuronal injury and BBBD, respectively, were both elevated after each game. However, only S-100β, unlike UCH-L1, was correlated with the number of hits received and the UCH-L1 elevation did not correlate with the S-100β increments. The authors suggest that elevated postgame UCH-L1 levels may be due to the

It can be concluded that there are very divergent data regarding the use of UCH-L1 as a serum biomarker of mild TBI. Some studies suggest that it is a promising marker, while others do not find a correlation with the lesion. Release from sources other than the central nervous system could contribute to elevated

in patients with mild (n = 86) and moderate (n = 10) TBI, as well as in controls with trauma and controls without trauma. For patients with a GCS of 15, serum UCH-L1 was significantly elevated compared to controls without trauma, with an AUC of 0.87, and was also compared with controls with trauma, and was even higher in those patients with GCS of 15 who also had positive findings on the CT scan or required some neurosurgical intervention, suggesting that UCH-L1 may be a potential marker of mild TBI. Additionally, 5% of patients with GCS of 15 (4/77) required neurosurgical intervention, which was higher than the 1% found in patients with GCS 14–15 reported in the study by Jagoda et al., in which the samples

**74**

serum levels.

Glial fibrillary acidic protein (GFAP) is a protein derived from glial cells, which is a part of the intermediate filament of the cytoskeleton of astrocytes, where it is the most abundant protein. It is considered a specific marker of CNS diseases, and is also related to several neuronal processes' harmful agents that compromise the integrity of the blood-brain barrier [29], and has been shown to be a potentially useful biomarker for predicting clinical outcomes in TBI. Its normal level in serum is <0.03 ng/mL [30], so any elevation thereof will indicate BBBD (**Table 4**).

Due to its great immunoreactivity, GAFP has been used as an indicator of brain injury in experimental models of mTCE [31]. The first successful measurement of GFAP in human blood was made in 1999 in 12 of 25 patients with severe TBI [32]. Using a weight drop model with mice [33] to evaluate two levels of mTBI, one with hemorrhage (complicated mTBI) and another without bleeding (uncomplicated mTBI), Yang et al. [34] found that serum GFAP was significantly elevated in both injury models at 90 minutes and 6 h after injury, but had returned to normal at 24 h.

In the study of Kou et al. [27], significantly elevated serum levels of GFAP in the first 24 h posttrauma in 9 mTBI patients was also reported; this elevation being even more significant in those with hemorrhagic lesions; however, the small size of the sample does not allow the conclusions to be validated.

In another study, Mondello et al. [35] evaluated whether the relationship between a neuronal marker (UCH-L1) and a glial marker (GFAP) correlates with the presence of different intracranial pathologies after brain trauma. They obtained serum samples from 59 patients with sTBI on admission to the hospital and measured levels of UCH-L1 and GFAP. The glial/neuronal ratio (GNR) was measured as the quotient between the concentrations of GFAP and UCH-L1. Logistic regression analysis identified variables associated with the type of injury. The increase in GNR was associated independently with the type of injury, but not with the age, gender, GCS, or trauma mechanism. This quotient was significantly higher in the patients who died, but it was not an independent predictor of mortality. The GNR had a median of 0.85 and correlated positively with age.

When evaluating the CT scan of the skull on admission, 29 patients presented a diffuse lesion and 30 localized lesions. The GNR was significantly higher in the group with focal lesions compared to the group with diffuse lesions. The receiver operating characteristic (ROC) analysis showed that the GNR discriminated between the two types of injury. GNR was more accurate when performed early than when it was done late (**Table 4**).

These data indicate that the GNR provides valuable information about the different types of injury, which is of great clinical utility. In addition, the GNR can help to identify the pathophysiological mechanisms subsequent to the different types of TBI. This is very useful when implementing therapeutic measures.

In an investigation carried out by Papa et al. [36], the capacity of the GFAP taken <4 h posttrauma was compared to predict intracranial lesions in the CT compared to S-100β. Although patients had GCS 9–15, only 3 of 209 patients had GCS <13 and only 10% had intracranial lesions, both S-100β and GFAP were significantly elevated in all patients, and even more so in those with intracranial lesions. For those patients with GCS 14–15, the AUC for the identification of intracranial lesions was 0.82 for GFAP and 0.77 for S-100β.

In the presence of extracranial lesions and using a cut-off value of 0.067 ng/mL, GFAP was 100% sensitive and 55% specific in the prediction of intracranial lesions. With a cut-off value of 0.20 ng/mL, S-100β also had 100% sensitivity but only 5% specificity. This study concludes that GFAP exceeds S-100β in the identification of intracranial lesions in mild and moderate TBI, even in the presence of extracranial lesions.

#### **Table 4.**

*Summary of the evidence reported in the literature on GFAP in TBI.*

In general, GFAP seems to increase in TBI and could represent a more sensitive marker than S-100β for the identification of intracranial lesions. However, for further validation, more studies are needed that focus specifically on mTBI (GCS 13–15), which include appropriate controls and adequate statistical comparisons.

#### **3. Discussions and conclusions**

One of the main purposes of the search for potential biomarkers in the TBI is to predict the presence of pathological findings in head CT and brain MRI; however, the studies published in this regard are inconclusive, and the evidence favors the use of S-100β over other markers in mTBI, as a predictor of negative-CT.

For example, Posti et al. [37] showed that patients with orthopedic trauma had higher levels of GFAP at admission, than those with mTBI and negative-CT (p = 0.026), and did not show that UCH-L1 levels presented significant differences in both groups, performing measurements at different time points, which suggest that these markers are not useful for distinguishing patients with negative-CT mTBI from patients with orthopedic trauma, and that high levels of UCH-L1 or GFAP can

**77**

*Neuronal and Glial Biomarkers Research for Traumatic Brain Injury*

sum of Rotterdam CT score and Stockholm CT score [54].

lead to a false diagnosis of mTBI in polytraumatized patients, leading to the unnec-

On the other hand, the use of the S-100β marker has been recommended in the Scandinavian guidelines for the initial management of minimal, mild, and moderate head injuries in adults [38] as an alternative to reduce the number of CT in the subgroup of mTBI with low risk of intracranial complications or surgical interventions. More studies are needed that show the usefulness of S-100β as a predictor of

The use of neuroimaging is necessary to improve the accuracy of biomarkers in the diagnosis and prognosis of patients who have suffered a TBI, with CT being the first option and the one with the most studies in relation to the release and correlation of biomarkers. Some reviews report higher serum S-100β levels in more severe, focal lesions, compared to diffuse lesions using Marshall scale, and a strong correlation between S100B increasing and the severity of the CT finding when using the

Olivecrona et al. reported how S-100β and neuronal specific enolase (NSE) levels correlate with CT findings using the aforementioned scales. Specifically, S-100β levels, but not to the NSE levels, correlates with Morris-Marshall score for the classification of traumatic subarachnoid hemorrhage (tSAH). This is probably associated with the physiopathological pathways described by each of these biomarkers after a neurotrauma. Likewise, the volume of the parenchymal contusions is also associated with the S-100β levels. Furthermore, in mild TBI, initial low levels of S-100β can be used as a predictor of a stationary injury, suggesting that the CT

Diagnosis of severity and prognosis of CT findings cannot be performed by a single biomarker test. Instead, a combination of biomarkers of diverse origins and pathways displays a better performance. Thereby, the joint use of GFAP, heart fatty acid binding protein (H-FABP), S-100β, and IL-10 results in a more efficient diagnostic tool with a 46% specificity and 100% sensitivity for predicting CT injuries. This biomarker panel increases specificity by 14% compared to the best

The ALERT-TBI study, developed in 22 centers in USA and Europe, validated the ability of the combination of UCH-L1 and GFAP to predict CT injuries within 12 h of mTBI, resulting in a sensitivity of 97.6%, a negative predictive value (NPV) of 99.6%, and a specificity of 36.4%. Therefore, when indicating CT only in those patients with a positive GFAP and UCH-L1 test, the CT use could be reduced by approximately onethird. The extent of these findings to patients with moderate TBI is uncertain [57]. The study of the available evidence on the different serum markers in TBI presented in this chapter allows us to conclude that, currently, there is not a single biomarker capable of predicting the clinical deterioration of patients with high sensitivity and specificity. However, the pathophysiological mechanisms of TBI suggest that instead, a panel of markers that reflect different aspects of traumatic

The literature has shown that the joint use of S-100β and GFAP or UCH-L1 would represent a valuable early prognostic and follow-up tool in TBI in addition to the GCS and the CT, thus guiding the decisions of initial management and aggres-

Likewise, given that the kinetic profile of these markers is different, since it presents peaks of appearance earlier than others and different times of permanence in serum, its usefulness would also be correlated with different post-traumatic stages, so that S-100β and UCH-L1 are better early markers [24, 25], whereas GFAP is a better predictor of CT lesions and surgical interventions in the first 7 days post-

injury should be available, including BBBD and neuronal injury.

*DOI: http://dx.doi.org/10.5772/intechopen.85555*

neurodeterioration in moderate TBI.

classification does not evolve [55].

single biomarker [56].

sive interventions.

trauma in mild and moderate TBI [27].

essary use of neuroimaging.

#### *Neuronal and Glial Biomarkers Research for Traumatic Brain Injury DOI: http://dx.doi.org/10.5772/intechopen.85555*

*Traumatic Brain Injury - Neurobiology, Diagnosis and Treatment*

Electrochemiluminescence immunoassay (ECL-IA)

Kou et al. [27]

Mondello et al. [35]

Papa et al. [36]

Papa et al. [54]

Okonkwo et al. [55]

**Table 4.**

**Reference Detection method Sample Findings**

Serum Significantly elevated in all cases of intracranial

validate the conclusions.

extracranial lesions.

of severity at the time.

Sandwich ELISA Serum Evaluation of GNR (GFAP/UCH-L1):

ELISA Serum S-100B and GFAP significantly elevated in all

ELISA Serum GFAP-BDP significantly elevated in mild

ELISA Serum GFAP-BDP <24 h posttrauma distinguished

hemorrhage, with potential screening capacity. Small size of the sample does not allow to

Median = 0.85, positive correlation with age (R = 0.45, p = 0.003). Greater in focalized lesions vs. diffuse lesion (1.77 vs. 0.48, respectively, p = 0.003). Different type of lesions (AUC = 0.72, p = 0.003). More precise early measurement (<12 h posttrauma) vs. late (AUC = 0.80, p = 0.002). Independent association with the type of injury, but not with the GCS. Independent predictor of mortality.

patients, especially in intracranial injuries. For GCS 14–15, AUC = 0.82 in identification of intracranial lesions for GFAP (0.77 for S-100B). With extracranial lesions and cut-off 0.067 ng/ mL, GFAP: S = 100% and E = 55% to predict intracranial lesions. GFAP outperforms S-100B in the identification of intracranial lesions in mild and moderate TBI, even in the presence of

TCE vs. controls with or without trauma. AUC = 0.88 to identify brain injury in GCS 15. Higher levels in GCS 15 with positive CT.

between mild and moderate/severe TBI (AUC of 0.87). Controls were not included, mild to moderate TCE was not compared, and most of the statistical analysis was made with all levels

In general, GFAP seems to increase in TBI and could represent a more sensitive

One of the main purposes of the search for potential biomarkers in the TBI is to predict the presence of pathological findings in head CT and brain MRI; however, the studies published in this regard are inconclusive, and the evidence favors the use

For example, Posti et al. [37] showed that patients with orthopedic trauma had higher levels of GFAP at admission, than those with mTBI and negative-CT (p = 0.026), and did not show that UCH-L1 levels presented significant differences in both groups, performing measurements at different time points, which suggest that these markers are not useful for distinguishing patients with negative-CT mTBI from patients with orthopedic trauma, and that high levels of UCH-L1 or GFAP can

marker than S-100β for the identification of intracranial lesions. However, for further validation, more studies are needed that focus specifically on mTBI (GCS 13–15), which include appropriate controls and adequate statistical comparisons.

of S-100β over other markers in mTBI, as a predictor of negative-CT.

**3. Discussions and conclusions**

*Summary of the evidence reported in the literature on GFAP in TBI.*

**76**

lead to a false diagnosis of mTBI in polytraumatized patients, leading to the unnecessary use of neuroimaging.

On the other hand, the use of the S-100β marker has been recommended in the Scandinavian guidelines for the initial management of minimal, mild, and moderate head injuries in adults [38] as an alternative to reduce the number of CT in the subgroup of mTBI with low risk of intracranial complications or surgical interventions. More studies are needed that show the usefulness of S-100β as a predictor of neurodeterioration in moderate TBI.

The use of neuroimaging is necessary to improve the accuracy of biomarkers in the diagnosis and prognosis of patients who have suffered a TBI, with CT being the first option and the one with the most studies in relation to the release and correlation of biomarkers. Some reviews report higher serum S-100β levels in more severe, focal lesions, compared to diffuse lesions using Marshall scale, and a strong correlation between S100B increasing and the severity of the CT finding when using the sum of Rotterdam CT score and Stockholm CT score [54].

Olivecrona et al. reported how S-100β and neuronal specific enolase (NSE) levels correlate with CT findings using the aforementioned scales. Specifically, S-100β levels, but not to the NSE levels, correlates with Morris-Marshall score for the classification of traumatic subarachnoid hemorrhage (tSAH). This is probably associated with the physiopathological pathways described by each of these biomarkers after a neurotrauma. Likewise, the volume of the parenchymal contusions is also associated with the S-100β levels. Furthermore, in mild TBI, initial low levels of S-100β can be used as a predictor of a stationary injury, suggesting that the CT classification does not evolve [55].

Diagnosis of severity and prognosis of CT findings cannot be performed by a single biomarker test. Instead, a combination of biomarkers of diverse origins and pathways displays a better performance. Thereby, the joint use of GFAP, heart fatty acid binding protein (H-FABP), S-100β, and IL-10 results in a more efficient diagnostic tool with a 46% specificity and 100% sensitivity for predicting CT injuries. This biomarker panel increases specificity by 14% compared to the best single biomarker [56].

The ALERT-TBI study, developed in 22 centers in USA and Europe, validated the ability of the combination of UCH-L1 and GFAP to predict CT injuries within 12 h of mTBI, resulting in a sensitivity of 97.6%, a negative predictive value (NPV) of 99.6%, and a specificity of 36.4%. Therefore, when indicating CT only in those patients with a positive GFAP and UCH-L1 test, the CT use could be reduced by approximately onethird. The extent of these findings to patients with moderate TBI is uncertain [57].

The study of the available evidence on the different serum markers in TBI presented in this chapter allows us to conclude that, currently, there is not a single biomarker capable of predicting the clinical deterioration of patients with high sensitivity and specificity. However, the pathophysiological mechanisms of TBI suggest that instead, a panel of markers that reflect different aspects of traumatic injury should be available, including BBBD and neuronal injury.

The literature has shown that the joint use of S-100β and GFAP or UCH-L1 would represent a valuable early prognostic and follow-up tool in TBI in addition to the GCS and the CT, thus guiding the decisions of initial management and aggressive interventions.

Likewise, given that the kinetic profile of these markers is different, since it presents peaks of appearance earlier than others and different times of permanence in serum, its usefulness would also be correlated with different post-traumatic stages, so that S-100β and UCH-L1 are better early markers [24, 25], whereas GFAP is a better predictor of CT lesions and surgical interventions in the first 7 days posttrauma in mild and moderate TBI [27].

In addition to the above, the literature also shows that these biomarkers are being measured with techniques that demand the use of complex equipment and procedures (such as ELISA) in which the use of labels is necessary [6, 39], displaying the need for the development of rapid and cost-effective techniques that allow the implementation of biomarkers in the clinical setting.

### **Acknowledgements**

We thank Universidad del Norte and Colciencias for the financial support of the research project in which the development of this work is framed.

### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Alexander Rodríguez\*, Eliana Cervera and Pedro Villalba Department of Medicine, Universidad del Norte, Barranquilla, Colombia

\*Address all correspondence to<EMAIL_ADDRESS>
© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**79**

*Neuronal and Glial Biomarkers Research for Traumatic Brain Injury*

Press/Taylor and Francis Group; 2016. pp. 263-276. DOI: 10.1201/b18959-13

[9] Papa L et al. Use of biomarkers for diagnosis and management of traumatic brain injury patients. Expert Opinion on Medical Diagnostics. 2008;**2**(8):937-945.

DOI: 10.1517/17530<IP_ADDRESS>7

[10] Jagoda A et al. Clinical policy: Neuroimaging and decisionmaking in adult mild traumatic brain injury in the acute setting. Annals of Emergency Medicine. 2008;**52**(6):714-748. DOI: 10.1016/j.annemergmed.2008.08.021

[11] Papa L et al. Serum levels of ubiquitin C-terminal hydrolase

TA.0b013e3182491e3d

pnas.0911516107

BCJ20160082

neu.2012.2579

distinguish mild traumatic brain injury from trauma controls and are elevated in mild and moderate traumatic brain injury patients with intracranial lesions and neurosurgical intervention. Journal of Trauma and Acute Care Surgery. 2012;**72**(5):1335-1344. DOI: 10.1097/

[12] Chen F, Sugiura Y, Myers K, Liu Y, Lin W. Ubiquitin carboxyl-terminal hydrolase L1 is required for maintaining the structure and function of the neuromuscular junction. Proceedings of the National Academy of Sciences of the United States of America. 2010;**107**(4):1636-1641. DOI: 10.1073/

[13] Bishop P, Rocca D, Henley J. Ubiquitin C-terminal hydrolase L1 (UCH-L1): Structure , distribution and roles in brain function and

dysfunction. The Biochemical Journal. 2016;**473**(16):2453-2462. DOI: 10.1042/

[14] Goyal A et al. S100b as a prognostic biomarker in outcome prediction for patients with severe traumatic brain injury. Journal of Neurotrauma. 2013;**30**(11):946-957. DOI: 10.1089/

*DOI: http://dx.doi.org/10.5772/intechopen.85555*

[1] Corrigan J, Selassie A, Orman J. The epidemiology of traumatic brain injury. The Journal of Head Trauma Rehabilitation. 2010;**2**(2):72-80. DOI: 10.1097/HTR.0b013e3181ccc8b4

[2] Matis G, Birbilis T. The Glasgow coma scale—A brief review past, present, future. Acta Neurologica Belgica. 2008;**108**(3):75-89

[3] Forastero P, Echevarria C, Barrera J. Traumatismos craneoencefálicos. Escalas de valoración para la medida de resultados en rehabilitación. Rehabilitación. 2002;**36**(6):408-417. DOI: 10.1016/S0048-7120(02)73314-8

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[5] Livingston B, Mackenzie S, MacKirdy F, Howie J. Should the presedation glasgow coma scale value be used when calculating acute physiology and chronic health evaluation scores for sedated patients? Critical Care Medicine.

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**References**

*Neuronal and Glial Biomarkers Research for Traumatic Brain Injury DOI: http://dx.doi.org/10.5772/intechopen.85555*

#### **References**

*Traumatic Brain Injury - Neurobiology, Diagnosis and Treatment*

the implementation of biomarkers in the clinical setting.

The authors declare no conflict of interest.

research project in which the development of this work is framed.

**Acknowledgements**

**Conflict of interest**

In addition to the above, the literature also shows that these biomarkers are being measured with techniques that demand the use of complex equipment and procedures (such as ELISA) in which the use of labels is necessary [6, 39], displaying the need for the development of rapid and cost-effective techniques that allow

We thank Universidad del Norte and Colciencias for the financial support of the

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Department of Medicine, Universidad del Norte, Barranquilla, Colombia

\*Address all correspondence to<EMAIL_ADDRESS>
**78**

**Author details**

provided the original work is properly cited.

Alexander Rodríguez\*, Eliana Cervera and Pedro Villalba

[1] Corrigan J, Selassie A, Orman J. The epidemiology of traumatic brain injury. The Journal of Head Trauma Rehabilitation. 2010;**2**(2):72-80. DOI: 10.1097/HTR.0b013e3181ccc8b4

[2] Matis G, Birbilis T. The Glasgow coma scale—A brief review past, present, future. Acta Neurologica Belgica. 2008;**108**(3):75-89

[3] Forastero P, Echevarria C, Barrera J. Traumatismos craneoencefálicos. Escalas de valoración para la medida de resultados en rehabilitación. Rehabilitación. 2002;**36**(6):408-417. DOI: 10.1016/S0048-7120(02)73314-8

[4] Stocchetti N et al. Inaccurate early assessment of neurological severity in head injury. Journal of Neurotrauma. 2004;**21**(9):1131-1140. DOI: 10.1089/ neu.2004.21.1131

[5] Livingston B, Mackenzie S, MacKirdy F, Howie J. Should the presedation glasgow coma scale value be used when calculating acute physiology and chronic health evaluation scores for sedated patients?  Expert Review of Molecular Diagnostics. 2011;**11**(1):65-78. DOI: 10.1586/erm.10.104

[7] Carney N et al. Guidelines for the management of severe traumatic brain injury, fourth edition. Journal of Neurotrauma. 2017;**80**(1):6-15. DOI: 10.1227/NEU.0000000000001432

[8] Zhang J, Puvenna V, Janigro D. Biomarkers of traumatic brain injury and their relationship to pathology. In: Laskowitz D, Grant G, editors. Translational Research in Traumatic Brain Injury. Boca Raton, FL: CRC

Press/Taylor and Francis Group; 2016. pp. 263-276. DOI: 10.1201/b18959-13

[9] Papa L et al. Use of biomarkers for diagnosis and management of traumatic brain injury patients. Expert Opinion on Medical Diagnostics. 2008;**2**(8):937-945. DOI: 10.1517/17530<IP_ADDRESS>7

[10] Jagoda A et al. Clinical policy: Neuroimaging and decisionmaking in adult mild traumatic brain injury in the acute setting. Annals of Emergency Medicine. 2008;**52**(6):714-748. DOI: 10.1016/j.annemergmed.2008.08.021

[11] Papa L et al. Serum levels of ubiquitin C-terminal hydrolase distinguish mild traumatic brain injury from trauma controls and are elevated in mild and moderate traumatic brain injury patients with intracranial lesions and neurosurgical intervention. Journal of Trauma and Acute Care Surgery. 2012;**72**(5):1335-1344. DOI: 10.1097/ TA.0b013e3182491e3d

[12] Chen F, Sugiura Y, Myers K, Liu Y, Lin W. Ubiquitin carboxyl-terminal hydrolase L1 is required for maintaining the structure and function of the neuromuscular junction. Proceedings of the National Academy of Sciences of the United States of America. 2010;**107**(4):1636-1641. DOI: 10.1073/ pnas.0911516107

[13] Bishop P, Rocca D, Henley J. Ubiquitin C-terminal hydrolase L1 (UCH-L1): Structure , distribution and roles in brain function and dysfunction. The Biochemical Journal. 2016;**473**(16):2453-2462. DOI: 10.1042/ BCJ20160082

[14] Goyal A et al. S100b as a prognostic biomarker in outcome prediction for patients with severe traumatic brain injury. Journal of Neurotrauma. 2013;**30**(11):946-957. DOI: 10.1089/ neu.2012.2579

[15] Puvenna V et al. Significance of ubiquitin carboxy terminal hydrolase L1 elevations in athletes after subconcussive head hits. PLoS One. 2014;**9**(5):e96296. DOI: 10.1371/journal. pone.0096296

[16] Adrian H et al. Biomarkers of traumatic brain injury: Temporal changes in body fluids. eNeuro. 2016;**3**(6):ENEURO.0294-16.2016. DOI: 10.1523/ENEURO.0294-16.2016

[17] Müller K et al. S100B serum level predicts computed tomography findings after minor head injury. The Journal of Trauma. 2007;**62**(6):1452-1456. DOI: 10.1097/TA.0b013e318047bfaa

[18] Ingebrigtsen T et al. The clinical value of serum S-100 protein measurements in minor head injury: A Scandinavian multicentre study. Brain Injury. 2000;**14**(12):1047-1055

[19] Biberthaler P et al. Serum S100B concentration provides additional information for the indication of computed tomography in patients after minor head injury: A prospective multicenter study. Shock. 2006;**25**(5):446-453. DOI: 10.1097/01. shk.0000209534.61058.35

[20] Mussack T et al. Immediate S-100B and neuron-specific enolase plasma measurements for rapid evaluation of primary brain damage in alcoholintoxicated, minor head-injured patients. Shock. 2002;**18**(5):395-400

[21] Bazarian J, Beck C, Blyth B, Von Ahsen N, Hasselblatt M. Impact of creatine kinase correction on the predictive value of S-100B after mild traumatic brain injury. Restorative Neurology and Neuroscience. 2010;**24**(3):163-172

[22] Pham et al. Extracranial sources of S100B do not affect serum levels. PLoS One. 2010;**5**(9):e12691. DOI: 10.1371/ journal.pone.0012691

[23] Campbell L, Thomas J, Lamps L, Smoller B, Folpe A. Protein gene product 9.5 (PGP 9.5) is not a specific marker of neural and nerve sheath tumors: An immunohistochemical study of 95 mesenchymal neoplasms. Modern Pathology. 2003;**16**(10):963-969. DOI: 10.1097/01.MP.0000087088.88280.B0

[24] Papa et al. Ubiquitin C-terminal hydrolase is a novel biomarker in humans for severe traumatic brain injury. Critical Care Medicine. 2010;**38**(1):138-144. DOI: 10.1097/ CCM.0b013e3181b788a

[25] Brophy G et al. Biokinetic analysis of ubiquitin C-terminal hydrolase-L1 (UCH-L1) in severe traumatic brain injury patient biofluids. Journal of Neurotrauma. 2001;**28**(6):861-870. DOI: 10.1089/neu.2010.156

[26] Mondello S et al. Clinical utility of serum levels of ubiquitin C-terminal hydrolase as a biomarker for severe traumatic brain injury. Neurosurgery. 2012;**70**(3):666-675. DOI: 10.1227/ NEU.0b013e318236a809

[27] Kou Z et al. Combining biochemical and imaging markers to improve diagnosis and characterization of mild traumatic brain injury in the acute setting: Results from a pilot study. PLoS One. 2013;**19**(8):11, e80296. DOI: 10.1371/journal.pone.0080296

[28] Diaz-Arrastia et al. Acute biomarkers of traumatic brain injury: Relationship between plasma levels of ubiquitin C-terminal hydrolase-L1 and glial fibrillary acidic protein. Journal of Neurotrauma. 2014;**31**(1):19-25. DOI: 10.1089/neu.2013.3040

[29] Eng L, Ghirnikar R, Lee Y. Glial fibrillary acidic protein: GFAP-thirtyone years (1969-2000). Neurochemical Research. 2000;**25**(9):1439-1451

**81**

*Neuronal and Glial Biomarkers Research for Traumatic Brain Injury*

[38] Undén L et al. Validation of the Scandinavian guidelines for initial management ofminimal, mild and moderate traumatic brain injury in adults. BMC Medicine. 2015;**13**:292. DOI: 10.1186/s12916-015-0533-y

[39] Strathmann FG et al. Blood-based biomarkers for traumatic brain injury: Evaluation of research approaches, available methods and potential utility from the clinician and clinical laboratory perspectives. Clinical Biochemistry. 2014;**47**(10-11):876-888. DOI: 10.1016/j.clinbiochem.2014.01.028

[40] Papa L, Brophy G, Welch R, Lewis L, Braga C, Tan C, et al. Time course and diagnostic accuracy of glial and neuronal blood biomarkers GFAP and UCH-L1 in a large cohort of trauma patients with and without mild traumatic brain injury. JAMA Neurology. 2016;**73**(5):551-560. DOI: 10.1001/jamaneurol.2016.0039

[41] Kövesdi E, Lückl J, Bukovics P, Farkas O, Pál J, Czeiter E, et al. Update on protein biomarkers in traumatic brain injury with emphasis on clinical use in adults and pediatrics. Acta Neurochirurgica. 2010;**152**:1-17. DOI:

10.1007/s00701-009-0463-6

[42] North S, Shriver-Lake L, Taitt C, Ligler F. Rapid analytical methods for on-site triage for traumatic brain injury. Annual Review of Analytical Chemistry. 2012;**5**(1):35-56. DOI: 10.1146/ annurev-anchem-062011-143105

[43] Mondello S, Akinyi L, Buki A, Robicsek S, Gabrielli A, Tepas J, et al. NIH public access. 2013;**70**(3):666-675

Determinación radioinmunométrica de la enolasa neuronal específica en humor acuoso y suero, en pacientes con retinoblastoma. Revista Española de Medicina Nuclear e Imagen Molecular.

[44] Benito I, Rodríguez J.

2000;**7**:472-478

*DOI: http://dx.doi.org/10.5772/intechopen.85555*

traumatic brain injury. The Journal of

[31] Saatman K, Bolton A. Regional neurodegeneration and gliosis are amplified by mild traumatic brain injury repeated at {24-hour} intervals. Journal of Neuropathology and Experimental Neurology. 2014;**73**(10):933-947. DOI: 10.1097/NEN.0000000000000115

Trauma. 2002;**52**(4):798-808

[32] Missler U, Wiesmann M, Wittmann G, Magerkurth O,

1999;**45**(1):138-141

Hagenström H. Measurement of glial fibrillary acidic protein in human blood: Analytical method and preliminary clinical results. Clinical Chemistry.

[33] Albert-Weissenberger C, Sirén AL. Experimental traumatic brain injury. Experimental & Translational

Stroke Medicine. 2010;**2**(1):16

10.1186/2040-7378-2-16

DOI: 10.1089/neu.2011.2092

DOI: 10.1089/neu.2013.3245

DOI: 10.1089/neu.2016.4442

[34] Yang S et al. A murine model of mild traumatic brain injury exhibiting cognitive and motor deficits. The Journal of Surgical Research. 2013;**184**(2):981-988. DOI:

[35] Mondello S et al. Glial neuronal ratio: A novel index for differentiating injury type in patients with severe traumatic brain injury. Journal of Neurotrauma. 2012;**29**(6):1096-1104.

[36] Papa L et al. GFAP out-performs S100beta in detecting traumatic intracranial lesions on computed tomography in trauma patients with mild traumatic brain injury and those with extracranial lesions. Journal of Neurotrauma. 2014;**31**(22):1815-1822.

[37] Posti JP et al. Glial fibrillary acidic protein and ubiquitin C-terminal hydrolase-L1 are not specific biomarkers for mild CT-negative traumatic brain injury. Journal of Neurotrauma. 2017.

[30] Ingebrigtsen T, Romner B. Biochemical serum markers of *Neuronal and Glial Biomarkers Research for Traumatic Brain Injury DOI: http://dx.doi.org/10.5772/intechopen.85555*

traumatic brain injury. The Journal of Trauma. 2002;**52**(4):798-808

*Traumatic Brain Injury - Neurobiology, Diagnosis and Treatment*

[23] Campbell L, Thomas J, Lamps L, Smoller B, Folpe A. Protein gene product 9.5 (PGP 9.5) is not a specific marker of neural and nerve sheath tumors: An immunohistochemical study of 95 mesenchymal neoplasms. Modern Pathology. 2003;**16**(10):963-969. DOI: 10.1097/01.MP.0000087088.88280.B0

[24] Papa et al. Ubiquitin C-terminal hydrolase is a novel biomarker in humans for severe traumatic brain injury. Critical Care Medicine. 2010;**38**(1):138-144. DOI: 10.1097/

[25] Brophy G et al. Biokinetic analysis of ubiquitin C-terminal hydrolase-L1 (UCH-L1) in severe traumatic brain injury patient biofluids. Journal of Neurotrauma. 2001;**28**(6):861-870. DOI:

[26] Mondello S et al. Clinical utility of serum levels of ubiquitin C-terminal hydrolase as a biomarker for severe traumatic brain injury. Neurosurgery. 2012;**70**(3):666-675. DOI: 10.1227/

[27] Kou Z et al. Combining biochemical

and imaging markers to improve diagnosis and characterization of mild traumatic brain injury in the acute setting: Results from a pilot study. PLoS One. 2013;**19**(8):11, e80296. DOI:

10.1371/journal.pone.0080296

[28] Diaz-Arrastia et al. Acute

10.1089/neu.2013.3040

biomarkers of traumatic brain injury: Relationship between plasma levels of ubiquitin C-terminal hydrolase-L1 and glial fibrillary acidic protein. Journal of Neurotrauma. 2014;**31**(1):19-25. DOI:

[29] Eng L, Ghirnikar R, Lee Y. Glial fibrillary acidic protein: GFAP-thirtyone years (1969-2000). Neurochemical

Research. 2000;**25**(9):1439-1451

[30] Ingebrigtsen T, Romner B. Biochemical serum markers of

CCM.0b013e3181b788a

10.1089/neu.2010.156

NEU.0b013e318236a809

[15] Puvenna V et al. Significance of ubiquitin carboxy terminal hydrolase

L1 elevations in athletes after subconcussive head hits. PLoS One. 2014;**9**(5):e96296. DOI: 10.1371/journal.

[16] Adrian H et al. Biomarkers of traumatic brain injury: Temporal changes in body fluids. eNeuro.

10.1523/ENEURO.0294-16.2016

2016;**3**(6):ENEURO.0294-16.2016. DOI:

[17] Müller K et al. S100B serum level predicts computed tomography findings after minor head injury. The Journal of Trauma. 2007;**62**(6):1452-1456. DOI: 10.1097/TA.0b013e318047bfaa

[18] Ingebrigtsen T et al. The clinical value of serum S-100 protein

Injury. 2000;**14**(12):1047-1055

shk.0000209534.61058.35

2010;**24**(3):163-172

journal.pone.0012691

measurements in minor head injury: A Scandinavian multicentre study. Brain

[19] Biberthaler P et al. Serum S100B concentration provides additional information for the indication of computed tomography in patients after minor head injury: A prospective multicenter study. Shock. 2006;**25**(5):446-453. DOI: 10.1097/01.

[20] Mussack T et al. Immediate S-100B and neuron-specific enolase plasma measurements for rapid evaluation of primary brain damage in alcoholintoxicated, minor head-injured patients. Shock. 2002;**18**(5):395-400

[21] Bazarian J, Beck C, Blyth B, Von Ahsen N, Hasselblatt M. Impact of creatine kinase correction on the predictive value of S-100B after mild traumatic brain injury. Restorative Neurology and Neuroscience.

[22] Pham et al. Extracranial sources of S100B do not affect serum levels. PLoS One. 2010;**5**(9):e12691. DOI: 10.1371/

pone.0096296

**80**

[31] Saatman K, Bolton A. Regional neurodegeneration and gliosis are amplified by mild traumatic brain injury repeated at {24-hour} intervals. Journal of Neuropathology and Experimental Neurology. 2014;**73**(10):933-947. DOI: 10.1097/NEN.0000000000000115

[32] Missler U, Wiesmann M, Wittmann G, Magerkurth O, Hagenström H. Measurement of glial fibrillary acidic protein in human blood: Analytical method and preliminary clinical results. Clinical Chemistry. 1999;**45**(1):138-141

[33] Albert-Weissenberger C, Sirén AL. Experimental traumatic brain injury. Experimental & Translational Stroke Medicine. 2010;**2**(1):16

[34] Yang S et al. A murine model of mild traumatic brain injury exhibiting cognitive and motor deficits. The Journal of Surgical Research. 2013;**184**(2):981-988. DOI: 10.1186/2040-7378-2-16

[35] Mondello S et al. Glial neuronal ratio: A novel index for differentiating injury type in patients with severe traumatic brain injury. Journal of Neurotrauma. 2012;**29**(6):1096-1104. DOI: 10.1089/neu.2011.2092

[36] Papa L et al. GFAP out-performs S100beta in detecting traumatic intracranial lesions on computed tomography in trauma patients with mild traumatic brain injury and those with extracranial lesions. Journal of Neurotrauma. 2014;**31**(22):1815-1822. DOI: 10.1089/neu.2013.3245

[37] Posti JP et al. Glial fibrillary acidic protein and ubiquitin C-terminal hydrolase-L1 are not specific biomarkers for mild CT-negative traumatic brain injury. Journal of Neurotrauma. 2017. DOI: 10.1089/neu.2016.4442

[38] Undén L et al. Validation of the Scandinavian guidelines for initial management ofminimal, mild and moderate traumatic brain injury in adults. BMC Medicine. 2015;**13**:292. DOI: 10.1186/s12916-015-0533-y

[39] Strathmann FG et al. Blood-based biomarkers for traumatic brain injury: Evaluation of research approaches, available methods and potential utility from the clinician and clinical laboratory perspectives. Clinical Biochemistry. 2014;**47**(10-11):876-888. DOI: 10.1016/j.clinbiochem.2014.01.028

[40] Papa L, Brophy G, Welch R, Lewis L, Braga C, Tan C, et al. Time course and diagnostic accuracy of glial and neuronal blood biomarkers GFAP and UCH-L1 in a large cohort of trauma patients with and without mild traumatic brain injury. JAMA Neurology. 2016;**73**(5):551-560. DOI: 10.1001/jamaneurol.2016.0039

[41] Kövesdi E, Lückl J, Bukovics P, Farkas O, Pál J, Czeiter E, et al. Update on protein biomarkers in traumatic brain injury with emphasis on clinical use in adults and pediatrics. Acta Neurochirurgica. 2010;**152**:1-17. DOI: 10.1007/s00701-009-0463-6

[42] North S, Shriver-Lake L, Taitt C, Ligler F. Rapid analytical methods for on-site triage for traumatic brain injury. Annual Review of Analytical Chemistry. 2012;**5**(1):35-56. DOI: 10.1146/ annurev-anchem-062011-143105

[43] Mondello S, Akinyi L, Buki A, Robicsek S, Gabrielli A, Tepas J, et al. NIH public access. 2013;**70**(3):666-675

[44] Benito I, Rodríguez J. Determinación radioinmunométrica de la enolasa neuronal específica en humor acuoso y suero, en pacientes con retinoblastoma. Revista Española de Medicina Nuclear e Imagen Molecular. 2000;**7**:472-478

[45] Test ID: NSESF. Neuron-Specific Enolase (NSE), Spinal Fluid [Internet]. 2018. Available from: https://www. mayocliniclabs.com/test-catalog/ pod/MayoTestCatalog-Rochester-LaboratoryReferenceEdition-SortedByTestName-duplex.pdf [Accessed: 7 December 2018]

[46] Dash P, Zhao J, Hergenroeder G, Moore A. Biomarkers for the diagnosis, prognosis, and evaluation of treatment efficacy for traumatic brain injury. Neurotherapeutics. 2010;**7**(1):100-114. DOI: 10.1016/j.nurt.2009.10.019

[47] Wang K, Posmantur R, Nath R, McGinnis K, Whitton M, Talanian R, et al. Simultaneous degradation of alphaII and betaII-spectrin by caspase 3 (CPP32) in apoptotic cells. The Journal of Biological Chemistry. 1998;**273**(35):22490-22497

[48] Brophy G, Pineda J, Papa L, Lewis S, Valadka A, Hannay H, et al. alphaII-Spectrin breakdown product cerebrospinal fluid exposure metrics suggest differences in cellular injury mechanisms after severe traumatic brain injury. Journal of Neurotrauma. 2009;**26**(4):471-479. DOI: 10.1089/ neu.2008.0657

[49] Berger R, Hymel K, Gao W. The use of biomarkers after inflicted traumatic brain injury: Insight into Etiology, pathophysiology, and biochemistry. Clinical Pediatric Emergency Medicine. 2006;**7**(3):186-193. DOI: 10.1016/j. cpem.2006.06.001

[50] Biberthaler P, Mussack T, Wiedemann E, Kanz K-G, Mutschler W, Linsenmaier U, et al. Rapid identification of high-risk patients after minor head trauma (MHT) by assessment of S-100β: Ascertainment of a cut-off level. European Journal of Medical Research. 2002;**7**(4):164-170

[51] Brophy G, Mondello S, Papa L, Robicsek S, Gabrielli A, Tepas J,

et al. Biokinetic analysis of ubiquitin C-terminal hydrolase-L1 (UCH-L1) in severe traumatic brain injury pa¬tient biofluids. Journal of Neurotrauma. 2011;**28**(6):861-870. DOI: 10.1089/ neu.2010.1564

[52] Papa L, Lewis L, Falk J, Zhang SS, Giordano P, et al. Elevated levels of serum glial fibrillary acidic protein breakdown products in mild and moderate traumatic brain injury are associated with intracranial lesions and neurosurgical intervention. Annals of Emergency Medicine. 2012;**59**:471-483. DOI: 10.1016/j. annemergmed.2011.08.021

[53] Okonkwo D, Yue J, Puccio A, Panczykowski D, Inoue T, Mcmahon P, et al. GFAP-BDP as an acute diagnostic marker in traumatic brain injury: Results from the pros¬pective transforming research and clinical knowledge in traumatic brain injury study. Journal of Neurotrauma. 2013;**30**(17):1490-1497. DOI: 10.1089/ neu.2013.2883

[54] Thelin E et al. A review of the clinical utility of serum S100B protein levels in the assessment of traumatic brain injury. Acta Neurochirurgica. 2017;**159**(2):209-225. DOI: 10.1007/ s00701-016-3046-3

[55] Olivecrona Z et al. Association of ICP, CPP, CT findings and S-100B and NSE in severe traumatic head injury. Prognostic value of the biomarkers. Brain Injury. 2014:29. DOI: 10.3109/02699052.2014.989403

[56] Lagerstedt L, Egea-Guerrero JJ, Bustamante A, Rodríguez-Rodríguez A, El Rahal A, Quintana-Diaz M, et al. Combining H-FABP and GFAP increases the capacity to differentiate between CT-positive and CT-negative patients with mild traumatic brain injury. PLoS One. 2018;**13**(7):e0200394. DOI: 10.1371/journal.pone.0200394

**83**

*Neuronal and Glial Biomarkers Research for Traumatic Brain Injury*

*DOI: http://dx.doi.org/10.5772/intechopen.85555*

[57] Bazarian JJ, Biberthaler P, Welch RD, Lewis LM, Barzo P, Bogner-Flatz V, et al. Serum GFAP and UCH-L1 for prediction of absence of intracranial injuries on head CT (ALERT-TBI): A multicentre observational study. Lancet Neurology. 2018;**17**(9):782-789. DOI: 10.1016/S1474-4422(18)30231-X

*Neuronal and Glial Biomarkers Research for Traumatic Brain Injury DOI: http://dx.doi.org/10.5772/intechopen.85555*

[57] Bazarian JJ, Biberthaler P, Welch RD, Lewis LM, Barzo P, Bogner-Flatz V, et al. Serum GFAP and UCH-L1 for prediction of absence of intracranial injuries on head CT (ALERT-TBI): A multicentre observational study. Lancet Neurology. 2018;**17**(9):782-789. DOI: 10.1016/S1474-4422(18)30231-X

*Traumatic Brain Injury - Neurobiology, Diagnosis and Treatment*

et al. Biokinetic analysis of ubiquitin C-terminal hydrolase-L1 (UCH-L1) in severe traumatic brain injury pa¬tient biofluids. Journal of Neurotrauma. 2011;**28**(6):861-870. DOI: 10.1089/

[52] Papa L, Lewis L, Falk J, Zhang SS, Giordano P, et al. Elevated levels of serum glial fibrillary acidic protein breakdown products in mild and moderate traumatic brain injury are associated with intracranial lesions and neurosurgical intervention. Annals of Emergency Medicine. 2012;**59**:471-483. DOI: 10.1016/j. annemergmed.2011.08.021

[53] Okonkwo D, Yue J, Puccio A, Panczykowski D, Inoue T, Mcmahon P, et al. GFAP-BDP as an acute diagnostic marker in traumatic brain injury: Results from the pros¬pective transforming research and clinical knowledge in traumatic brain injury study. Journal of Neurotrauma. 2013;**30**(17):1490-1497. DOI: 10.1089/

[54] Thelin E et al. A review of the clinical utility of serum S100B protein levels in the assessment of traumatic brain injury. Acta Neurochirurgica. 2017;**159**(2):209-225. DOI: 10.1007/

[55] Olivecrona Z et al. Association of ICP, CPP, CT findings and S-100B and NSE in severe traumatic head injury. Prognostic value of the

10.3109/02699052.2014.989403

Combining H-FABP and GFAP increases the capacity to differentiate between CT-positive and CT-negative patients with mild traumatic brain injury. PLoS One. 2018;**13**(7):e0200394. DOI: 10.1371/journal.pone.0200394

[56] Lagerstedt L, Egea-Guerrero JJ, Bustamante A, Rodríguez-Rodríguez A, El Rahal A, Quintana-Diaz M, et al.

biomarkers. Brain Injury. 2014:29. DOI:

neu.2010.1564

neu.2013.2883

s00701-016-3046-3

[45] Test ID: NSESF. Neuron-Specific Enolase (NSE), Spinal Fluid [Internet]. 2018. Available from: https://www. mayocliniclabs.com/test-catalog/ pod/MayoTestCatalog-Rochester-LaboratoryReferenceEdition-SortedByTestName-duplex.pdf [Accessed: 7 December 2018]

[46] Dash P, Zhao J, Hergenroeder G, Moore A. Biomarkers for the diagnosis, prognosis, and evaluation of treatment efficacy for traumatic brain injury. Neurotherapeutics. 2010;**7**(1):100-114. DOI: 10.1016/j.nurt.2009.10.019

[47] Wang K, Posmantur R, Nath R, McGinnis K, Whitton M, Talanian R, et al. Simultaneous degradation of alphaII and betaII-spectrin by caspase 3 (CPP32) in apoptotic cells. The Journal of Biological Chemistry.

1998;**273**(35):22490-22497

neu.2008.0657

cpem.2006.06.001

[50] Biberthaler P, Mussack T, Wiedemann E, Kanz K-G, Mutschler W, Linsenmaier U, et al. Rapid identification of high-risk patients after minor head trauma (MHT) by assessment of S-100β: Ascertainment of a cut-off level. European Journal of Medical Research. 2002;**7**(4):164-170

[51] Brophy G, Mondello S, Papa L, Robicsek S, Gabrielli A, Tepas J,

[48] Brophy G, Pineda J, Papa L, Lewis S, Valadka A, Hannay H, et al. alphaII-Spectrin breakdown product cerebrospinal fluid exposure metrics suggest differences in cellular injury mechanisms after severe traumatic brain injury. Journal of Neurotrauma. 2009;**26**(4):471-479. DOI: 10.1089/

[49] Berger R, Hymel K, Gao W. The use of biomarkers after inflicted traumatic brain injury: Insight into Etiology, pathophysiology, and biochemistry. Clinical Pediatric Emergency Medicine. 2006;**7**(3):186-193. DOI: 10.1016/j.

**82**

Section 3

Treatment and Multiple

Therapeutic Strategies

85

Section 3


**3. The biliary duct epithelium**

a collapsed PCL, concentration of mucins within the mucus layer, and adhesion of mucus

**•** *The high salt hypothesis* suggests that the ASL normally is hypotonic [84] and provides an optimal environment for defensins. According to this view the ASL in CF patients would have a higher salt concentration than normal because the absorbing function of ENaC depends on the state of CFTR and cannot be activated when CFTR is defective or absent [84].

**•** *The low pH hypothesis* focuses on the interactions between CFTR and the SLC26 and proposes an acidic ASL. This may compromise the function of airway immune cells and increase toxic oxidant species. Lowering the pH may also eliminate electrostatic repulsive charges between organisms and facilitate "tighter" biofilm formation as well as reduce electrorepul‐ sive forces between bacteria and negatively charged mucins. Furthermore, ciliary beat frequency in bronchial epithelium is reduced when external pH falls [85]. All the above factors may inhibit mucociliary clearance (MCC) and thus elimination of bacteria from the

**•** *The low oxygenation hypothesis* postulates that the oxygen content of the ASL is low, due to build-up of mucus plugs, resulting in enhanced growth of the facultative anaerobic

**•** *The defect gland function hypothesis* suggests that the primary defect in CF is reduced fluid secretion by airway submucosal glands and possibly altered secretion of mucous glycopro‐

**•** *The soluble mediator hypothesis* proposes that signalling molecules within the ASL itself are controlling ASL volume [89]. These molecules are ATP, which is breathing- or shear-stress induced [90], and adenosine. ATP interacts with receptors such as the purinergic P2Y2 receptors and adenosine reacts with the adenosine A2b receptors, that mediate inhibition of ENaC and activation of both CFTR and CaCC [91, 92]. This mechanism is also supposed

An interesting question is what the role of aquaporins (AQP) is in the production of ASL, compared to paracellular water flow and CFTR. In the epididymis, CFTR appears to regulate AQP-mediated water permeability [93]. In this tissue, CFTR is co-localized with AQP9 in the apical membrane, and this association promotes the activation of AQP9 by cAMP [94]. In a heavily debated study, concerning the clinical benefit of nebulized hypertonic saline in cystic fibrosis, an important role of amiloride-inhibitable AQP water channels in the generation of ASL was proposed [95]. However, although the positive effect of hypertonic saline as such is not disputed, the question whether this effect is mediated by AQP has received conflicting answers [96, 97] and is still open. Recently, it has been found that interleukin (IL)-13 enhances the expression of CFTR but abolishes the expression of AQP in airway epithelial cells [98]. In

The differences in the proposed hypotheses are due to difficulties in determining the accurate composition of the ASL because of the very small depth of the layer. Among the problems encountered there are difficulties to collect an adequate amount of ASL without disturbing the

to include PDZ interactions and cytoskeletal elements [1].

conclusion, the relation between CFTR and AQP needs further study.

to the airway surface [83].

8 Regenerative Medicine and Tissue Engineering

airways [86].

*P.aeruginosa* [87].

teins [88].

The biliary tree is a complex network of conduits within the liver that begins with the canals of Hering and progressively merges into a system of ducts, which finally deliver bile to the gallbladder and to the intestine. Cholangiocytes are the epithelial cells forming the biliary epithelium which shows a morphological heterogeneity that is strictly associated with a variety of functions performed at the different levels of the biliary tree [102]. Thus, the canals of Hering, located at the ductular-hepatocellular junction, constitute the physiologic link of the biliary tree with the hepatocyte canalicular system and they are the site where a facultative progenitor cell compartment resides; these liver progenitor cells are variably elicited only after liver injury. Given the strong capacity of mature hepatocytes to proliferate, cholangiocyte ability to behave as liver progenitor cells becomes evident only when hepatocellular proliferation is hampered as a result of severe liver damage, as that induced by several toxins or drugs, or occurring under certain conditions, *i.e.* viral hepatitis or non alcoholic steatohepatitis [103]. Cells lining the intrahepatic biliary tree have different functional and morphological specializations: the terminal cholangioles (size <15 μm) have some biological properties such as plasticity (i.e., the ability to undergo limited phenotypic changes) and reactivity (*i.e.*, the ability to participate in the inflammatory reaction to liver damage); interlobular (15-100 μm) and large ducts (100 μm to 800 μm) modulates fluidity and alkalinity of the primary hepatocellular bile.

#### **3.1. Ion and water transport in cholangiocytes**

In addition to funnelling bile into the intestine, cholangiocytes are actively involved in bile production. In humans, around 40% of the total bile production is of ductal origin. Cholan‐ giocytes exert a series of reabsorptive and secretory process which dilute and alkalinize the bile during its passage along the biliary tract. Modifications of ductal bile appear to be tightly regulated by the actions of nerves, biliary constituents, and some peptide hormones like secretin [104]. Accordingly to *in vivo* and *in vitro* models, it is possible to distinguish between three different bile flow fractions: 1) the canalicular bile salt-dependent flow that is driven by concentrative secretion of bile acids by the hepatocytes followed by a facilitated efflux of water; 2) the canalicular bile salt-independent flow, which is also created by hepatocytes but through active secretion of both inorganic (bicarbonate) and organic (glutathione) compounds; and 3) the ductal bile flow, that is the bile salt-independent flow contributed by cholangiocytes, mainly through production of a bicarbonate-rich fluid in response to secretin and other regulatory factors. Cl secretion into the ductal lumen is the driving force of a chloride/ bicarbonate exchanger that exports HCO3 into the bile flowing into the biliary tree. Indeed, this AE (anion exchanger) activity is facilitated by the outside to inside transmembrane gradient of Cl at relatively high intracellular concentrations of HCO3- , specially upon secretin stimulation. The AE activity in the liver is operated by AE2/SLC4A2 which is localized not only in the canaliculi but also in the luminal membrane of bile duct cells [105]. Experiments of RNA interference with recombinant adenovirus expressing short/small hairpin RNA have confirmed that AE2/SL4A2 is indeed the main effector of both basal and stimulated Na+ independent Cl- /HCO3 exchange in rat cholangiocytes [106]. Besides acid/base transporters cholangiocytes possess other ion carriers like those for Cl- , Na+ , and K+ , which greatly contrib‐ ute to intracellular pH regulation and bicarbonate secretion. Thus, CFTR had been localized at the apical side, where it plays a role in biliary excretion of bicarbonate [107, 108]. Although bicarbonate permeability through activated CFTR has been shown in several epithelia [109], its main contribution to biliary bicarbonate secretion appears to occur through a coordinated action with AE2/SL4A2 [106, 110, 111]. In addition to CFTR, cholangiocytes possess a dense population of Ca2+-activated Cl- channels. These channels are responsive to interaction of the purinergic-2 (P2) receptors with nucleotides (mainly ATP or UTP) [112, 113]. The apical fluxes of anions results in increased osmotic forces in the bile duct lumen which in the presence of AQPs contributes to water flux. AE2/SLC4A2 and CFTR colocalize with AQP1 in cholangiocyte intracellular vesicles wich coredistribute to the apical cholangiocyte membrane upon both cAMP and secretin stimulations [114].

#### **3.2. The pathogenesis of CF liver disease**

CF is associated with liver disease in almost 30% of all patients. In general, CF-associat‐ ed liver disease develops during the first decades of life and does not progress rapidly. The diagnostic criteria were initially established by Colombo et al. [115]. Hepatobiliary disease in CF encompass a wide variety of complications, including steatosis, focal biliary cirrhosis (FBC), multilobular biliary cirrhosis (MBC), microgallbladder, distended gallblad‐ der, cholelithiasis, intraheapatic sludge or stones, and cholangiocarcinoma [116]. The pathogenesis of steatosis (fatty liver) is not directly ascribed to the CFTR gene defect but has been attributed to malnutrition, essentially fatty acid deficiency, carnitine or choline deficiency, or insulin resistance [117].

With regarding to the pathogenesis of FBC and MBC, various hypotheses have been proposed [118, 119]:

**•** *The low chloride secretion hypothesis* proposes that loss of CFTR function leads to blocked biliary ductules with thick periodic acid-Schiff positive material leading to acute and chronic periductal inflammation, bile duct proliferation and increased fibrosis in scattered portal tracts. Hepatic stellate cells (important drivers of hepatic fibrosis) become activated to produce collagen and stimulate the bile duct epithelium to produce the profibrogenic cytokine TGF-β. The progression of FBC to MBC and portal hypertension, which occurs in up to 8% of patients, may take years to decades, and should be viewed as a continuum [120]. Considering CFTR as a driving force for Cl- /HCO3 exchange, the postulated sequence of CF-associated hepatobiliary complications is that loss of functional CFTR protein in the apical membrane of cholangiocytes presumably initiates a cascade of abnormal Cl- and HCO3 secretion, decreased bile flow, bile duct plugging by thickened secretions, and cholangiocyte/hepatocyte injury [10].