**5. Current biomarker research**

As should be evident from the previous sections from this chapter, both the ability to diagnose PTSD comorbid with mTBI and the ability to effectively monitor treatment of the concurrent conditions would benefit from the identification of biomarkers. For this discussion we adapt the definition of a biomarker using a conceptual framework that is useful for clinical research and treatment purposes. This may include any information that can be used as an objective indication of a relevant medical state observed from outside the patient. Importantly, these signs must be able to be measured accurately and have high levels of replicability. This is captured in the WHO's definition of a biomarker as "any substance, structure, or process that can be measured in the body or its products and influence or predict the incidence of outcome or disease" [77] and can be expanded to "… almost any measurement reflecting an interaction between a biological system and a potential hazard…[and] may be functional and physiological, biochemical at the cellular level, or a molecular interaction" [78]. In alignment with these requirements, our discussion will focus on the relevance and validity of the suggested biomarkers, allowing for it to be used as a surrogate endpoint [79]. There are a wide range of biomarkers and targets currently being researched for roles in both mTBI and PTSD. A summary of biomarkers currently undergoing research that meet the criteria previously discussed can be seen in **Table 2**, with in-depth discussion of each following.


*Current Understanding of Biomarkers in Post Traumatic Stress Disorder and Mild Traumatic… DOI: http://dx.doi.org/10.5772/intechopen.102766*


#### **Table 2.**

*Summary of current biomarker research.*

#### **5.1 PTSD**

Most of the promising biomarkers for the presence of PTSD are related to either dysfunction of the HPA axis, monoamine systems, heightened inflammation, genetic and epigenetic changes thought to be a result of methylation brought about through exposure to prolonged stress, or functional and structural neuroimaging. There has also been growing interest and research in the examination of psychophysical biomarkers of PTSD, such as indicators of hyperarousal (heart rate, blood pressure, skin conductance, etc.). However, examination of these forms of hyperactivity through psychological testing is beyond the scope of this chapter.

*HPA Axis Dysregulation.* Cortisol, a circulating adrenal glucocorticoid hormone that modulates the HPA axis is known to be involved in anxiety responses and sleep regulation [80]. Research has shown that within a PTSD population, lower salivary cortisol levels were found when compared to control participants, especially when measurements were taken in the morning [80]. Typically, there is a diurnal cycle of salivary cortisol with peak concentration observed shortly after waking, and then drops across the waking hours. In addition to a lower morning level of cortisol, PTSD patients have also demonstrated a blunted cortisol response throughout the day [81]. This blunted cortisol reactivity in response to exposure to acute stress may offer more promise as it removes confounds associated with the measurement of baseline cortisol, such as sex differences and time of day effects. Therefore, although not specific to the presence of PTSD, measurements of circulating cortisol levels may form part of a panel of assays designed to detect the presence of PTSD in a clinical population due to its non-invasive status when measured from a saliva sample.

*Monoamine Dysfunction.* As PTSD includes increased sympathetic nervous system tone, it is not surprising that levels of norepinephrine (NE) are also heightened [82]. In a prospective study of motor vehicle accident survivors, urinary levels of NE were positively correlated with the likelihood of development of PTSD in the month following trauma, but only in males [83]. Changes in the serotonergic (5-HT) system have also been observed in PTSD. Specifically, 5-HT transporter binding within the amygdala is reduced in PTSD and correlated with both anxiety and depression within PTSD patients [84].

*Inflammatory and Immune Function.* Stemming from the high comorbidity between PTSD and general physical illnesses [85], there has been extensive examination of the potential role of markers of inflammation as a proxy for PTSD and PTSD symptomology. In all instances examined, there is a positive correlation between inflammatory markers and PTSD symptomology. This includes interleukin (IL) -6 [86], IL-2 [87], IL-1β [88]. Additionally, increased C-reactive protein (CRP) levels are shown to be elevated in individuals with PTSD [89–91], but also has been shown to be predictive of post-deployment PTSD when examined in a prospective study [92]. Continuing this trend, PTSD is also positively correlated with higher levels of TNF-α and IFN-γ when compared to healthy controls, likely as a result of the persistent stress experienced [93]. In general, data concerning the relationship between inflammatory responses and PTSD confirm that PTSD is likely associated with chronic inflammation. Although this may lead to inflammation as a viable therapeutic target to alleviate at least some of the symptoms associated with PTSD, they do not serve well as a general biomarker of PTSD presence or prognosis due to its status as a hallmark finding in many other diseased states, including those that are often comorbid with PTSD [85, 94–97].

*Genetic Variation.* Most genetic and epigenetic findings have clustered around modulators of HPA axis function either before or following trauma. Perhaps the most cited modulator is FKBP5, a protein encoding gene involved in immunoregulation [98]. Polymorphisms on FKBP5, specifically Val66Met, have been associated PTSD [99]. Met-allele carriers are also reported to have greater severity in PTSD symptoms amongst veterans compared to Val/Val genotypes [100]. However, FKBP5 is also associated with depression [101], a condition known to often co-occur with PTSD therefore making its use as a solitary differential marker of PTSD unlikely. The serotonin transporter gene linked polymorphic region (5-HTTLPR short and long) has also been linked to trauma exposure and depression. Individuals with the LL genotype exhibit lower intrusion and avoidance symptoms compared to those with the S-allele, though no differences were found in other PTSD symptoms [102]. Increased methylation levels at 4 promotor sites on BDNF were found in PTSD patients that experienced high combat exposure compared to those without PTSD [103]. Reduced glucocorticoid receptor NR3C1-1F promotor methylation was found in combat veterans that developed PTSD when compared to those that did not [104]. Hypermethylation at NR3C1 gene promoters were associated with lower risk of PTSD in male genocide survivors, but not female [105].

As has become apparent, many (if not all) of these genetic regions have been associated with other psychiatric conditions and may therefore be a better marker of stress-induced psychopathology in general rather than PTSD specifically, and there has yet to be a single genetic or epigenetic factor that reliably predicts the presence or severity of PTSD in isolation of other psychiatric conditions.

*Functional and Structural Neuroimaging.* One of the most consistent findings regarding neuroimaging of PTSD is the presence of increased amygdala activation when compared to controls when patients have been exposed to fear inducing *Current Understanding of Biomarkers in Post Traumatic Stress Disorder and Mild Traumatic… DOI: http://dx.doi.org/10.5772/intechopen.102766*

stimuli [106]. For example, there have now been a number of studies that demonstrate hyperactivity of the amygdala when PTSD participants have been exposed to trauma-relevant words when compared to amygdala activity of control participants [107–110]. Further studies have shown that this increased activity may be a result of weakened inhibitory control of the amygdala by the medial prefrontal cortex [106, 108, 110]. Furthering these findings, a recent meta-analysis of imaging studies during emotional tasks for individuals with PTSD, anxiety, and phobia revealed that only the PTSD patients demonstrated decreased activity within the rostral anterior cingulate cortex, offering a potential mechanism to distinguish between aberrant functional activity observed in PTSD and not in other anxiety disorders [111].

In addition to functional studies, a number of structural examinations of PTSD have taken place using neuroimaging techniques. Early studies examining structural differences between PTSD and non-PTSD patients demonstrated that smaller hippocampal volume may be associated with an increased risk of developing PTSD [112], though this finding has more recently been questioned with hippocampal volume reductions being acquired with trauma exposure [113]. When examining specific regions of the hippocampus using structural MRI, it appears as though reductions in specific subregions can be associated with PTSD symptoms. Specifically, reductions within the cornu ammonis 3 (CA3) layer of the hippocampus and the dentate gyrus are related to PTSD symptomology [114].

#### **5.2 mTBI**

Currently, mTBI is typically diagnosed based solely on clinical presentation, in comparison to TBI which has prominent and objective neuroimaging findings. This has several implications as to the utility of biomarkers of mTBI. Perhaps of primary concern is the fact that any biomarker that would offer clinical benefits must be correlated with clinical symptom presentation. For example, a marker that elevates with impacts to the head without observable changes in clinical presentation in the patient would be of little clinical use. Potential biomarkers for mTBI are most often related to, or spawned, by the axonal injury that occurs following the much smaller forces related to a mTBI. These can be broadly categorized as those that are related to neuronal and axonal injury, blood brain barrier disturbances, neuroinflammation, cerebral blood flow changes, and genetic variation.

*Neuronal and Axonal Injury*. Disturbances of the cellular environment often occur following the shearing forces that often accompany mTBI [115], and while this usually is not to the extend to the point of axonal disconnection, it can indirectly affect membrane homeostasis which ultimately results in cell damage [53, 116]. There are several potential biomarkers associated with neuronal damage. Tau protein is known to be changed in response to injury [117] including mTBI, at least in animal models [118]. In one of the larger human studies (196 patients), the ratio of phosphorylatedtau to total tau had both a good diagnostic and prognostic marker for acute TBI, including those with a mild severity [119]. Other biomarkers of neuronal and axonal injury that have been explored as potentials include ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), [120–122], neuron-specific enolase (NSE) [123–125], and neutrophil gelatinase-associated lipocalin (NGAL) [126]. However, current research into their utility has not demonstrated sufficient levels of specificity and/or replicability to be discussed in detail, but likely warrant further examination.

*Blood Brain Barrier Disturbances.* Although it has been well demonstrated that blood brain barrier (BBB) disruptions are associated with TBI [127], there is growing evidence that there are BBB disruptions following mTBI during both the chronic and the acute phase [128]. There are a number of non-invasive indirect measures of BBB dysfunction that rely on the detection of cerebrospinal fluid (CSF) components within peripheral serum, however, there has been little convincing evidence that suggests it will be a suitable biomarker of mTBI if used in isolation. The CSF/serum albumin ratio is the standard biomarker for BBB integrity [129] but is not sensitive enough to detect the presence of BBB disruption as a result of mTBI [130]. Perhaps the most studied is the astrocyte-specific SNS protein S100B. Research has shown that the detection of this marker approaches the same levels of sensitivity as the CSF/ serum albumin ratio [127], and has been used to rule out mTBI in emergency medicine already, where S100B levels have a high (99 + %) predictive value [131]. However, there is relative non-specificity of elevated S100B (as there are extracerebral sources of S100B in peripheral blood), and it has also shown to be elevated in clinical cases without head trauma [132]. Further dampening enthusiasm, there is still conflicting evidence as to whether S100B levels are positively correlated with mTBI [133]. A less explored, though perhaps more promising marker is the glycoprotein PrPc—cellular prion protein. Since this plasma-soluble prion protein is located within the plasma membrane, is has been suggested that it may be released following an mTBI as a result of BBB dysfunction [134], with animal models showing increased serum levels following blast exposure induced mTBI [135, 136]. Within humans, a small (N = 6) study amongst athletes demonstrated PrPc levels increased and remained elevated following mTBI [134]. More recently, a slightly larger study conducted within a hospital setting (N = 20) confirmed this effect with elevated PrPc levels following TBI, with 8 of the 20 being classified as mild injuries. However, PrPc did levels did not correlate with severity of trauma [137]. A third study confirmed the ability for PrPc levels to differentiate TBI with cognitive symptoms versus TBI in which no cognitive symptoms were present [138]. Although additional study is required, these specific features of PrPc make it a particularly attractive candidate biomarker for mTBI. Specifically, its relative specificity with regard to cognitive dysfunction, and ability to be detected years following trauma, are likely of great utility.

*Neuroinflammation.* Following TBI, including mTBI, there is a cascade of events that ultimately results in the presence of inflammation [139–142], offering an opportunity to examine markers of the neuroinflammatory response as a marker of brain injury. Two promising classes of markers of neuroinflammation are the inflammatory interleukin proteins and the cardiotonic steroid marinobufagenin. There have been many studies demonstrating elevated levels of interleukins including IL-6, IL-8 and IL-10 following brain injury [143–148], as well as studies showing these levels are related to clinical outcome in mTBI [149, 150]. In a small (N = 6) study, marinobufagenin (MBG) levels were initially increased following mTBI, along with symptomology [151]. As MBG levels decreased, symptom scores also decreased, suggesting there may be a relationship between symptoms and MBG. A larger study (N = 110) found MBG levels were elevated following mTBI, and were also correlated with reported symptoms [151] adding further evidence for the potential utility of MBG.

A further drawback to most biofluid based biomarkers of mTBI is the timescale at which they can be detected, necessitating their examination within the acute stage of the injury as they return to baseline levels rather quickly (though PrPc is an exception to this). As an alternative, potentially longer-lasting biomarker, advanced neuroimaging techniques such as diffusion weighted imagery (DWI) and magnetic resonance spectroscopy (MRS) for diagnosing the presence of an mTBI at a timescale that extends beyond the acute stage. Genetic information may offer additional

*Current Understanding of Biomarkers in Post Traumatic Stress Disorder and Mild Traumatic… DOI: http://dx.doi.org/10.5772/intechopen.102766*

information not available through the other methods discussed, such as the susceptibility to mTBI following head trauma, reflected in the likelihood of developing symptoms based on genetic variation.

*Cerebral Blood Flow Changes.* Recent research has shown that following mTBI, there are changes in vasoreactivity that impair smooth muscle response [152], ultimately affecting cerebral blood flow that animal models have shown can persist up to a year after initial damage [153]. Due to the extended period of blood flow changes, this may be an ideal candidate for evaluating whether long-term changes in cognition are a result of a previously acquired mTBI [154]. These changes in blood flow can be detected using modern magnetic resonance imaging techniques as hypoperfusion in many of the anatomical regions previously described as particularly susceptible to mTBI injury including the prefrontal, frontal, and temporal regions of the brain [118].

*Genetic Variation.* The two leading genetic candidates are the genetic mutations in the genes encoding for apolipoprotein E (APOE) and brain-derived neurotrophic factor (BDNF). It is important to note that both of these genes are already being explored as they pertain to the risk of generating various types of neurodegeneration disorders, such as Alzheimer's disease [155]. This finding is not all that unexpected considering the building link between mTBI and subsequent neurodegenerative conditions [156–159]. The APOE ε4 allele has been shown to be a significant risk factor for the development of Alzheimer's disease, but systematic review [160] has shown it is unrelated to mTBI diagnosis. Interestingly, this same allele confers increased risk to some of the cognitive impairment associated with the longer-term symptoms of mTBI [161]. When it comes to studies examining the role of BDNF, a small sample (at least on the scale of genetic studies; N = 110) showed a link between carriers of the minor allele of rs115769 and the memory impairments often associated with mTBI [162], as well as the BDNF Val66Met allele being linked to a higher risk of experiencing an mTBI [163], and increased experience of emotional symptoms following the occurrence of an mTBI [164]. Further, it was been shown that mutations of BDNF rs6265 Val66Met polymorphisms affect neurocognitive performance in patients following mTBI, offering the potential for predicting which patients will go on to develop neurocognitive symptoms following mTBI [165].
