Cell Free DNA as a Potential Biomarker of Blast-Wave Mild Traumatic Brain Injury and Posttraumatic Stress Disorder.


 BackgroundIndividuals being within non-lethal distance from explosion may present with blast induced mild traumatic brain injury, post traumatic stress disorder, or combination of the two conditions. Early diagnosis to enable interventions is important. This study tested the possible role of cell free DNA in the diagnosis of blast related head injuries in a rat model.MethodsA rat controlled model of a blast. Cell free DNA concentrations were determined in the serum by a direct fluorescence method. Cognitive and behavioral tests were used to diagnose affected rats. ResultsMean cell free DNA concentration increased significantly at 2 hours following the blast compared to baseline level and remained high throughout the follow-up period (665.43±159.15 ng/ml vs. 344.20±69.62 ng/ml, p<0.0001). The rate of affected rats among the blast exposed animals was 42.5%. A significant increase in mean cell free DNA concentration was found at 2 hours after exposure in the affected group (741.40±47.18 ng/ml) compared to both the baseline concentration (372.42±149.11 ng/ml), p<0.0001 and to the well-adapted group (517.47 ng/ml), p<0.0045. ConclusionThis rat model of blast demonstrated that the incidence of mild brain injury and or PTSD is significant and that affected animals demonstrated increased serum concentrations of cell free DNA. Cell free DNA may potentially serve as a biomarker to diagnose brain psychopathology early in individuals exposed to blast.

344.20±69.62 ng/ml, p<0.0001). The rate of affected rats among the blast exposed animals was 42.5%. A signi cant increase in mean cell free DNA concentration was found at 2 hours after exposure in the affected group (741.40±47.18 ng/ml) compared to both the baseline concentration (372.42±149.11 ng/ml), p<0.0001 and to the well-adapted group (517.47 ng/ml), p<0.0045.

Conclusion
This rat model of blast demonstrated that the incidence of mild brain injury and or PTSD is signi cant and that affected animals demonstrated increased serum concentrations of cell free DNA. Cell free DNA may potentially serve as a biomarker to diagnose brain psychopathology early in individuals exposed to blast.

Background
Blast injuries are common among military personnel involved in the modern battle elds. However, nowadays civilians all over the globe are also exposed to blast incidents due to terrorist acts involving suicide bombers, car bombs, rockets and other artillery attacks on civilian communities.
The American and NATO forces have gained a vast experience in modern warfare in Iraq and Afghanistan during the last two decades. This experience added a lot to our understanding of the nature of blast injuries. 1,2 According to these reports, blast exposure accounts for 78% of injuries, with an incident of blast traumatic brain injury (bTBI) being as high as 60% in this group of patients. 3 Blast-related brain injury can be caused by several mechanisms and to present itself in a variety of pathologies. The blast-wave that hits the skull and is transferred to the brain, can force it to move inside the skull and hit it, resulting in a concussion and hemorrhagic contusions. This movement can also tear super cial veins that connect the brain surface to the dural venous sinus to cause subdural hemorrhage.
Shear and stress waves from the primary blast could lead to diffuse axonal injury, hemorrhages, and edema. 4 While a blast can cause mild TBI (mTBI) in many victims who are exposed to explosion, the management of such patients can be delayed, or even worse, not delivered to undiagnosed patients. For many years there was no consensus over the de nition of mTBI. We adopt the de nition of the World Health Organization (WHO) task force on mTBI that is an acute brain injury resulting from mechanical energy to the head from external physical forces with one or more of the following clinical presentations: (i) confusion or disorientation, loss of consciousness for 30 minutes or less, post-traumatic amnesia for less than 24 hours, and/or other transient neurological abnormalities such as focal signs, seizure, and intracranial lesion not requiring surgery; (ii) Glasgow Coma Scale score of 13-15 after 30 minutes postinjury or later upon presentation for healthcare. 5 Recently, more than 100 U.S. troops have been diagnosed with TBI following an Iranian missile attack on a military compound at al-Asad airbase in Iraq. This was a typical presentation of mild bTBI which started with an announcement of no wounded individuals in the attack. Later, more and more service personnel were diagnosed as having symptoms of mTBI. This incident demonstrated clearly the problematic situation of sorting out the affected individuals from hundreds of people who were at the vicinity of an explosion without a MRI machine available to accurately diagnose such victims. 6 The diagnosis of TBI in the acute setting is based on neurological examination and neuro-imaging tools such as CT scan and MRI. However, CT scanning has low sensitivity to diffuse mild brain damage and exposes the patient to radiation. On the other hand, MRI can provide information on the extent of diffuse injuries but its widespread application is restricted by cost and the limited availability of MRI in many centers. Under the circumstances of military eld hospitals that lack such imaging modalities and in mass casualty incidents the utility of these is very low or absent. The clinical symptoms of mTBI are less clear-cut than those of TBI and may overlap with those of post-traumatic stress disorder (PTSD) and other mental syndromes. The traumatizing events associated with explosive detonations during combat or terrorist attacks often make it di cult for clinicians to distinguish between PTSD and mTBI, but they are often comorbid. The impact of the comorbid condition on treatment and rehabilitation is vast. 7 It is reasonable to assume that a biomarker may serve as a rapid reliable diagnostic aid to diagnose TBI and especially mTBI. Despite extensive research which explored a wide range of biomarkers speci c to brain injury, there is still no proved biomarker in routine clinical use. 8 One of the major drawbacks of the studied biomarkers is that they originate in speci c neuroanatomical locations and so using one of them may lead to false negative diagnosis of TBI. In a previous study we determined the levels of cell free DNA (cfDNA) in the serum of isolated brain injury patients. We used a validated, simple, rapid and cheap direct uorescence method which we developed in our laboratory. The results showed that severely injured patients had high cfDNA concentrations on admission compared to patients with mild injuries that had low concentrations. In addition, patients who recovered fully from their injury had lower cfDNA concentrations compared to the high concentrations found in patients who were discharged with disabilities. Thus, the conclusion of the study was that cfDNA may be used as a marker to assess the severity of TBI and to predict the prognosis. 9 This study aimed to test cfDNA as a potential marker for fast identi cation of blast-wave obscured injuries and the evaluation of the resultant psychopathology in an innovative rat model. The main goal of the study was to explore the changes of cfDNA concentrations in the serum of rats exposed to non-lethal blast wave. We evaluated the pattern and timing of cfDNA concentration changes after blast-wave exposure in different intensities. The secondary goal was to learn whether a relationship exists between the concentration of cfDNA and behavioral responses. We hypothesized that the low pressure blast-wave that causes psychopathology (mTBI, PTSD, or comorbid mTBI-PTSD phenotypes) but no other internal injuries would result in a detectable increase in cfDNA that may be useful in the future to detect early patients with blast induced mTBI. Experimental design The rats were exposed to 2 different intensities of blast-wave (see subsequent description). Fourty rats were exposed to a low-pressure blast-wave and 47 were exposed to a highpressure blast-wave. The animals were awake through the entire procedure and received no drugs. All animals underwent neurological assessment using the Neurological Severity Score (NSS) which was performed 1.5-2 hours following the blast and daily thereafter. Behavior measures were conducted on day 7. The rats were initially assessed in the elevated plus maze (EPM) followed 1 h later by the acoustic startle response (ASR) paradigm. Spatial memory performance using the Morris water maze (MWM) test was assessed 8 days post-exposure for 7 consecutive days. The prevalence rate of rats exhibiting post traumatic stress disorder (PTSD) phenotype or mTBI phenotype responses (see details described subsequently) were calculated from this data and compared with a sham-exposed group (n = 5) (see details described subsequently) and with those of unexposed controls (n = 5). In all experiments, the rats were tested in batches of 10-15 individuals.

Methods
Blast-wave exposure An exploding wire technique was used to generate small-scale cylindrical and spherical blast waves. This technique has been previously demonstrated to simulate the effects of air blast exposure under experimental conditions, and permits safe operation with high repeatability. 10 Pilot tests have shown that this model is non-lethal for the rats and does not result in any external, limbs, thoracic, or intra abdominal injuries (data not shown).
Exploding wires The experimental system is based on the rapid discharge of high voltage through 0.4 mm thickness copper wire. When electrical currency passes through the copper wire, Joule heating is created. This heating vaporizes and lique es the copper in a very short time (5 µsec) at 1084°C, with virtually no change in the wire volume. The high current continues to pass through the liquid copper and continues to heat it to the point at which it transitioned to a gaseous state beyond the temperature of 2927°C. As the copper gas begins to expand in space, it forms a blast-wave. This method of explosion produces a cylindrical blast-wave that simulates a blast-wave pro le similar to that seen from an explosive device common to the battle eld. In an actual explosion, the blast-wave causes an acute, short duration elevation in pressure followed by a negative phase. The exploding wire system has been shown to be capable of duplicating this overpressure and negative pressure blast-wave pro le.
Procedure Each rat was restrained in a custom exible harness located on a tray, which was then placed in the blast-wave generator system at a distance of 20 cm from the wire (high-intensity blast group), or a distance of 35 cm (low-intensity blast group). Minimal movement of the animals was allowed during the blast exposure. Pressure values were recorded using a Kistler 211B3 piezoelectric pressure transducer mounted on a perpendicular wall. Rats (two at the same time) were subjected to a single blast-wave with the head facing the blast without any body shielding, resulting in a full body exposure to the blast wave.
A high-speed video was taken during the exposure by a PHANTOM V12.1 high-speed camera. Following the blast rats were returned to their home cage.
Sham exposure (control) Sham-exposed animals were treated identically, except that they were not exposed to the blast. The rats were in the same room where the explosion occurred, but were placed 150 cm below the blast wave. This enabled them to experience the bright ash of light and smell of the explosion while shielding them from any potential physical injury. This enabled us to focus on the psychological components without the compounding effects of physical brain injuries.
Cell free DNA Blood samples were obtained at baseline (just before the blast) and 1 hour, 2 hours, 3 hours, 5 hours, 24 hours, day 10, and day 15 after the blast in Flex-Tube ® , Eppendorf blood collection tubes.
Blood samples were immediately centrifuged at 2000 G for 10 minutes at 4°C and serum was transferred to collection tubes and stored in −20°C. cfDNA levels were quanti ed by a direct rapid uorometric assay, the uorochrome SYBR Gold which does not require prior processing of samples, that is, DNA extraction and ampli cation. Brie y, SYBR Gold Nucleic Acid Gel Stain (Invitrogen Paisley, UK) was diluted 1 : 1000 in dimethyl sulphoxide and then 1 : 8 in phosphate-buffered saline. Ten microliters of serum or DNA standard was applied to a 96-well plate and forty microliters of diluted SYBR Gold was applied to each well. Fluorescence was measured with a 96-well uorometer (Spectra uor Plus, Tecan, Durham, NC) at an emission wavelength of 535 nm and an excitation wavelength of 485 nm. The method was tested in comparison with the gold standard, QPCR, and was found to be in good correlation of R 2 0.9987 (p<0.0001) as previously described. 11 Neurological Severity Score (NSS) To ensure that any damage to the central nervous system (CNS) caused by the blast-wave did not result in vast neurological de cits, we employed the NSS. The NSS was performed 1 h following the initial blast-wave exposure and served as a baseline for comparison with later evaluations throughout the study. The NSS assesses somatomotor and somatosensory function by evaluating the animals' activities as was described before. 12 An observer who was blind to the different treatment groups tested the animals.
Behavioral assessments All rats underwent a number of different behavioral assessments. All behavioral tests were performed in standardized conditions. Rats underwent more than one behavioral test; therefore, tests were performed with a break of at least 24 h between sessions. Also, no animals underwent the same test twice. All behavioral tests were video recorded for future analysis using the ETHO-VISION program (Noldus), by an investigator blinded to the experimental protocol. The behavioral tests included the EPM and ASR for anxiety phenotype/PTSD phenotype responses, and the MWM for cognitive performance.

Statistical analysis
Cell free DNA concentrations and behavioral data were analyzed using a one way analysis of variance (ANOVA) or two way ANOVA. In the event of a signi cant F ratio, a Bonferroni post-hoc test was used for multiple comparisons. Repeated measures ANOVA (RM-ANOVA) was used to analyze MWM data. The prevalences of the affected and well adapted rats are expressed in ratios and percentages. All data are reported as mean ± SE. An α level of p < 0.05 was used to determine statistical signi cance.

Results
Blast-wave details A 0.4 mm diameter 70 mm in length copper wire and charging voltageof 4.2 kV were used to generate the blast-wave. The discharge current was ~500 kA. The short pulse at t = 0 is associated with the electromagnetic pulse generated by the capacitor discharge. Animals subjected to the blast-wave experienced a mean peak overpressure of 95 kPa (13.77 psi) (rise time of 0.01 ms) sustained for duration of 0.189 ms. The peak pressure was equivalent to 193 dB SPL (sound pressure level). The exposure led to a peak impulse of 10.8 × 10-3 kPa. A negative pressure was sustained for more than 0.659 ms with a peak negative pressure of −40 kPa (−5.8 psi). The light intensity generated by the explosion is signi cant and measured to be ∼5 Mlux. This light intensity is of the same order of magnitude as the M84 stun grenade at a distance of 1.5 m (3.1 Mlux). There was no mortality in any of the blast-exposed rats.
cfDNA: Mean cfDNA concentration increased signi cantly at 2 hours after the blast exposure (665.43±159.15 ng/ml) from mean baseline level (344.20±69.62 ng/ml) and remained high, excluding the exceptional time point of 5 hours, to the end of 15 day follow up (One way ANOVA: F(7, 463)=17.4, p<0.0001). Animals of the sham exposure and unexposed groups did not manifest such unique pattern of change in cfDNA concentrations. Furthermore, there was no difference between these two control groups, but at the 2 hour point there was a signi cant difference (p<0.0001) between the mean concentration of the exposed rats (665.43±159.15 ng/ml) and the concentrations of the control groups [273.80±80 ng/ml in the sham exposure, 286.66±73.03 ng/ml in the unexposed group ( Figure 1A)]. Figure  1B Table 2.  NSS: There were no signi cant differences between the groups in re ex responses, motor coordination, motor strength, or sensory function (data not shown). These ndings indicate that differences in behavioral and cognitive tasks were not related to abnormal motor function required of the animals to complete the behavioral tasks.
mTBI and PTSD: The majority, 50/87 rats (57.5%), of the sample exposed to the blast-wave were unaffected (well-adapted phenotype). In contrast, the prevalence of affected rats among the blast exposed rats was 37/87 (42.5%). Speci cally, the prevalence of mTBI-like pattern among the blast exposed animals was 21/87 (24.1%), whereas the prevalence of PTSD-like phenotype was 4/87 (4.6%). The prevalence of PTSD+mTBI-like among the blast exposed animals was 12/87 (13.8%). None of the sham exposed and unexposed rats exhibited phenomena of mTBI or PTSD characteristics. Figure 3A shows  Table 3.

Discussion
The main ndings of this study were the augmentation of cfDNA concentrations following blast-wave exposure, this rise peaked at 2 hour after the exposure and lasted for 15 days. Sham exposure and unexposed subjects did not demonstrate this alteration of cfDNA concentrations. No signi cant difference was found between the low and high pressure blast-wave groups in cfDNA concentrations. The exposure to the two levels of pressure resulted in the same pattern of surge of cfDNA concentrations in the time point of 2 hours after the blast exposure. Moreover, there was a striking association between the degree of behavioral disruption following blast exposure and the pattern of changes in the cfDNA concentrations 2 hours post exposure: animals whose behavior was extremely disrupted (mTBIphenotype, PTSD-phenotype and comorbid mTBI-PTSD-phenotype) selectively displayed signi cantly higher cfDNA concentrations. In contrast, rats whose behavior was minimally affected or unaffected (well-adapted rats) displayed no cfDNA concentrations changes and were indistinguishable from sham exposed or unexposed controls.
The response patterns of the blast-wave exposure model employed in this study replicate our previous data 13,14 in that they demonstrate that exposure to a single low-pressure blast-wave can produce distinctive long-lasting psycho-neuro-behavioral responses which model PTSD, mTBI, and comorbid PTSD-mTBI sequelae in a proportion of animals. Nevertheless, the NSS was normal in all the animals. This set of tests was taken in order to ensure that any damage to the central nervous system caused by the blast-wave is mild and does not result in vast neurological de cits. NSS assesses somatomotor and somatosensory function by evaluating the animals' activities in motor, sensory, re exes, beam walking, and beam balancing tasks. Taken together, these ndings indicate that cfDNA concentrations may provide a quick, reliable, and simple prognostic indicator of pathology after blast-wave exposure. It is not clear to us why the two intensities of blast resulted in non different cfDNA concentrations. A possible explanation may be that the disparity between the two intensities was not enough to be translated into a detectable signi cant different amount of brain injury. Another series of tests that use greater differences in blast intensities may also demonstrate differences in cfDNA levels.
Recent literature on the utility of imaging modalities such as CT scanning and even MRI empahasizes their low sensitivity to accurately diagnose mTBI. 15 This is in accordance with MRI examinations of rats that underwent a blast-wave exposure at exactly the same model conditions. 14 The brain MRI examinations revealed no lesions, edema, or hemorrhage in any of the rats. This was in agreement with the results of the brain tissue histo-pathological assessments. However, despite the lack of macro and micro tissue changes, brain cellular damage does occur in mTBI. There are multiple molecules that point to pathophysiological changes associated with mTBI including brain cell injury and disruption of the blood-brain barrier. 16 To name some of them, astrocyte injury is identi ed by the presence of the S100B, and glial brillary acidic protein (GFAP) proteins in the blood. Damaged neurons release neuron-speci c proteins including neuron-speci c enolase (NSE), ubiquitin carboxyl-terminal hydrolase isoenzyme L1 (UCHL1), and Cellular prion protein (PrP c ). Axonal injury is associated with increased concentrations of hyperphosphorylated tau protein (p-tau), and Neuro laments (NFs). Very few studies have tested the value of nucleic acids for the diagnosis of TBI. Over-expression of a micro RNA (miRNA), miR-21, has been reported by two groups in severe TBI 17,18 , and down-regulation of miR-425-5p and miR-502 in mTBI was reported in one of these studies. 18 We elected to use cfDNA in this study. cfDNA has been well studied for its potential use in the diagnosis, prognosis, and monitoring of a variety of conditions such as trauma, in ammation, infection and sepsis. [19][20][21][22][23] Previous reports have pointed out the utility of cfDNA in TBI. 9,24,25 In our study on human TBI patients we used the same simple "mix and measure" technique, as described in the present study, to measure cfDNA. The study population was isolated head injury patients. The present study attempted to model the unique condition of mTBI caused by blast. The pattern of cfDNA augmentation after the trauma in our model resembles the description of Lam et al. on the early and late changes in plasma DNA in trauma patients. 26 In their study they observed the same early increase in DNA concentrations that peaked around 2 hours from injury and continued to be over baseline levels for about 3 weeks. Also, they reported higher DNA concentrations in patients with severe injuries and in those who had developed organ failure. This is in line with our observations in mild bTBI regarding affected subjects compared with well-adapted animals. The nding that only the 2 hour point in our study showed a signi cant peak is raising the concern that the test may be limited to a short period of time, although as is obvious from the plots there is a trend of increased cfDNA concentrations through the entire length of follow up. Future studies may give us more information regarding the cutoff concentration and the timing of testing before it is possible to introduce the test for routine use in human patients.
Limitations of the study are: from technical reasons not all subjects were sampled in each of the time points which can cause some bias in the results. In these experiments no attempt was made to rule out other possible sources for cfDNA except for the TBI. We relied upon earlier pilot experiments that showed no torso or extremities injuries. Other serum biomarkers have not been measured. It may be interesting to compare cf DNA to other biomarkers such as S100B, NSE, GFAP in future studies. Caution should be made in the translation of rodent experiments to human. Of special interest is to learn when the peak concentration of cfDNA happens, and for how long it remains signi cantly above baseline levels in humans.
Conclusion cfDNA concentrations increase early after mild bTBI and remain high for at least two weeks following the exposure. Determination cf DNA by a simple, cheap, rapid, and reliable method as we use in our laboratory can be done in areas that lack full modern medical infrastructure, or in cases of mass casualty event. It can help clinicians sort out mTBI patients from other persons who are exposed to a blast especially when no other obvious injury is apparent. It can potentially reduce the number of patients who remain undiagnosed or get late to treatment.

Declarations Author Contribution
Gad Shaked -writing the manuscript, Amitai Zuckerman -data collection, Zeev Kaplan -study design, Oren Sadot -explosion model design and performance, Amos Douvdevani -laboratory tests, Hagit Cohen -data analysis, data interpretation, critical revision.