The main findings 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 significant 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 (mTBI-phenotype, PTSD-phenotype and comorbid mTBI-PTSD-phenotype) selectively displayed significantly 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 deficits. NSS assesses somatomotor and somatosensory function by evaluating the animals' activities in motor, sensory, reflexes, beam walking, and beam balancing tasks. Taken together, these findings 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 significant 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 identified by the presence of the S100B, and glial fibrillary acidic protein (GFAP) proteins in the blood. Damaged neurons release neuron-specific proteins including neuron-specific enolase (NSE), ubiquitin carboxyl-terminal hydrolase isoenzyme L1 (UCHL1), and Cellular prion protein (PrPc). Axonal injury is associated with increased concentrations of hyperphosphorylated tau protein (p-tau), and Neurofilaments (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, inflammation, infection and sepsis.19–23 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 finding that only the 2 hour point in our study showed a significant 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 significantly above baseline levels in humans.