The time course of plasma DNA levels in patients with acute cervical SCI produced was examined and yielded the following major findings. First, both plasma nDNA and mtDNA levels at admission were significantly higher in SCI patients compared to controls. Patients with severe JOA score at admission had significantly elevated plasma nDNA levels when compared patients with moderate and mild JOA score (53.2 vs. 15.2 vs. 24.4 ng/ml,
p = 0.007), but no significant difference in plasma mtDNA levels (45.0 vs. 23.3 vs. 36.8 ng/ml,
p = 0.647). Second, plasma nDNA levels at admission in acute cervical SCI patients were significantly correlated with the JOA score (r = − 0.447,
p = 0.003), AST level (r = 0.414,
p = 0.019), and length of intensive care unit stay (r = 0.336,
p = 0.028). Third, plasma mtDNA levels at admission were only significantly correlated with AST levels (r = 0.410,
p = 0.020). Fourth, plasma nDNA increased significantly from day 1 to 30 in patients with a poor outcome (Fig.
2a). Moreover, the JOA score at admission and plasma nDNA level on day 14 were independently associated with outcome. Lastly, a cut-off value of 45.6 ng/ml of plasma nDNA level on day 14 is associated with poor outcome in acute cervical SCI patients.
Increases in plasma DNA in SCI patients are associated with severity of trauma and predict outcomes
Both our previous study [
12] and that of Filho et al. [
13] reported that the plasma cell-free DNA level at admission was an independent predictor of poor outcome and mortality in patients with acute traumatic brain injury (TBI). In our previous study [
12], higher plasma nDNA levels (> 72.95 ng/ml) were associated with poorer outcomes. Filho et al. [
13] reported that a higher plasma DNA level (> 171,381 kilogenome-equivalents/l; equal to 1131.1 ng/ml) predicted mortality with 90% specificity and 43% sensitivity in severe TBI patients.
Similar findings have been reported by other studies; [
8‐
13] all individuals who have suffered a severe trauma have elevated plasma concentrations of cell-free DNA at admission compared to healthy individuals. These elevated DNA concentrations correlate with trauma severity and may be useful for predicting outcome [
8‐
13]. It is possible that acute trauma results in tissue damage and the release of DNA into the circulation via necrosis [
42,
43] and apoptosis [
44,
45]. Macher et al. [
10] revealed that plasma DNA levels at 24 h after admission were positively correlated with the ISS and Acute Physiology and Chronic Health Enquiry (APACHE II) score in patients with TBI, but there was no association with the presence of extracranial injury. Yurgel et al. [
9] found no significant difference in plasma DNA concentration between patients with isolated TBI versus those with TBI and extracranial injuries.
In the current study, plasma nDNA levels at admission in acute cervical SCI patients were significantly correlated with the JOA score at admission (r = − 0.447,
p = 0.003). The extent of injury on admission, as evaluated by the ISS, predicted the clinical outcome (
p = 0.022, Table
3). However, plasma nDNA and mtDNA levels at admission were not correlated with the ISS at admission (r = 0.184,
p = 0.237, and r = 0.160,
p = 0.306, respectively). Because we had excluded patients having severe multiple trauma with unstable hemodynamic status and there was no patient had complete spinal cord injury in our study, the injury severity is relative mild in this study (median ISS = 17.5, Table
1). The ISS majorly came from Head/Neck region (spinal cord injury). Although the ISS was higher in poor outcome group (Table
3), the ISS was not significant correlated with nuclear and mitochondrial DNA plasma levels. In Lo et al. study, significant correlations were observed between plasma DNA concentrations and the AIS values for the head and neck region (Spearman rank-order correlation, p < 0.0001; r = 0.440), the thorax (Spearman rank-order correlation, p < 0.001; r = 0.520), and the abdomen (Spearman rank-order correlation, p = 0.0002; r = 0.418). No significant correlation was observed between plasma DNA concentrations and the AIS values for the extremities (Spearman rank-order correlation, p = 0.136; r = 0.165) [
11]. The elevated plasma cell-free DNA level seen in SCI patients may be due to its direct release from damaged extraspinal cord tissues and damaged spinal cord tissue through a disrupted brain-blood barrier. Plasma nDNA levels at admission, and on days 14 and 30 (
p = 0.018,
p = 0.001 and
p = 0.014, respectively), were more powerful predictors of outcome than the ISS score at admission (
p = 0.022). Therefore, in the current study, nuclear and mitochondrial DNA from extraspinal cord injury did not confuse our main results.
Plasma DNA level is predictive of complications and mortality for different diseases
Previous studies have shown that the plasma DNA level is significantly higher in patients with a severe injury who eventually develop severe complications [
8], and is also correlated with mortality [
9,
10,
13]. However, both plasma nDNA and mtDNA levels were much lower in our SCI patients (40.3 and 43.6 ng/ml, respectively) compared to cohorts in other studies of severe trauma (560–2418 ng/ml) [
8‐
10,
13], and sepsis patients (436 and 149 ng/ml, respectively [median values]) [
15]. Moreover, plasma nDNA and mtDNA levels were similar at admission in our SCI patients (40.3 and 43.6 ng/ml, respectively) to those reported for other brain diseases, such as ischemic stroke [
21] (44 and 25 ng/ml), spontaneous intracranial hemorrhage [
19] (20 and 10 ng/ml), aneurysmal subarachnoid hemorrhage [
20] (56 and 17 ng/ml), bacterial meningitis (100 and 22 ng/ml), and aseptic meningitis [
14] (34 and 17 ng/ml).
Previous studies have shown that plasma DNA decreases rapidly in severe TBI patients who survive and have no major complications [
8,
9]. Plasma DNA concentrations increase early after injury, but then decrease towards reference values within 3 h in patients with less severe injuries [
8]. Yurgel et al. observed a similar pattern, where the plasma DNA concentration (mean concentration 366,485 kilogenomes-equivalents/l; equal to 2419 ng/ml) at study entry fell by 65% after 24 h (mean concentration, 131,708 kilogenomes-equivalents/l; equal to 869 ng/ml) [
9]. In the early stage after a primary injury, plasma DNA concentrations vary markedly among patients, probably due to multiple sources of DNA release. In our previous TBI study [
12], the poor outcome group had a more than a two-fold higher plasma nDNA level than the good outcome group, as early as day 1.
Another interesting finding of the present study was that plasma nDNA and mtDNA levels increased continuously until day 14 after injury, and then dropped quickly thereafter up to day 30 (Fig.
2). The mechanism underlying the appearance of cell-free DNA in the cerebrospinal fluid (CSF) and circulation is unknown. It is possible that the inflammatory response induces further cell damage and causes gradual DNA release into the CSF and circulation. Inflammation can affect the release of DNA from cells undergoing apoptosis or necrosis, although the nature of this effect may vary depending on the inflammatory stimulus and local cellular events [
46]. Previous studies have shown that apoptosis is associated with specific pathological conditions in the CNS, including SCI [
44,
45]. Our previous study also showed that the nDNA and mtDNA levels in the CSF, and of nDNA in the circulation, are significantly higher from day 1–14 in patients with spontaneous aneurysmal subarachnoid hemorrhage compared to controls [
20]. Furthermore, nDNA levels in the CSF and circulation both increased, and reached a peak on day 4. In the brain lateral fluid percussion brain injury rat model of Perez-Polo, inflammatory events and brain–blood barrier dysfunction were evident as early as 3–6 h after injury, and persisted for 18 days post-injury [
47]. Their study implied that injury to the central nervous system can release DNA into the circulation, via a disrupted brain–blood barrier, for up to 18 days post-injury; this is consistent with the present findings.
Another possible reason for DNA release may be that patients undergo treatments, such as cervical spine laminectomy, cervical discectomy and interbody fusion, which can also damage the spinal cord and other tissues. In the present study, 39 patients underwent cervical spine surgery, including 23 emergent cases within 24 h after SCI and 28 elective surgical cases. Of the 28 elective surgical cases, the median (range) number of days elapsed before neurosurgical intervention after SCI was 5 (2–31). The differences in nDNA and mtDNA levels between days 1 and 14 were not significant in patients who did and did not undergo elective surgery (p = 0.545 and p = 0.889, respectively). Neurosurgical interventions may damage tissues and release DNA into the circulation; however, this was not observed in the present study.
The clearance mechanism of DNA from the circulation is not well understood, although previous studies cited the liver and kidneys as prime candidate organs involved in DNA removal [
48]. In the present study, AST levels were significantly correlated with plasma nDNA and mtDNA levels at admission (r = 0.414,
p = 0.019 and r = 0.410,
p = 0.020, respectively). However, the AST level was not an independent predictor of outcome in logistic regression analysis. Changes in plasma nDNA may be partly attributable to impaired liver function, but this did not confound the major results of this study.
In the present study, the plasma nDNA level on day 14 was independently associated with outcome, suggesting that the therapeutic window is within the first 14 days after injury. Medalha et al. [
49] demonstrated that acute DNA damage extends beyond the spinal cord in SCI rats to affect the blood, liver, and kidneys. Bao et al. [
50] administrated a monoclonal antibody against CD11d integrin, an integral leukocyte adhesion protein that reduces oxidative stress-related DNA oxidation after severe compression injury. Their findings provide strong evidence that interventions preventing DNA damage may provide a neurological benefit following SCI.
The current study had several limitations. First, the relatively small sample size and the significant number of analyses performed may have biased the statistical analysis, particularly in terms of inferring causality. Second, although the study showed that higher plasma nDNA levels at admission, and on days 14 and 30, were associated with poor outcome in acute cervical SCI patients, some SCI treatments may damage the spinal cord or other tissues and cause the release of DNA, which may have served as a confounding factor; it is reasonable to postulate that surgical intervention can affect serum DNA levels. Furthermore, when the neurosurgical intervention was included in the multiple logistic regression analysis, it was not independently associated with outcome. Thus, neurosurgical intervention may affect plasma DNA levels but was not a confounder with respect to the major findings of this study, and the maximum likelihood estimates of the coefficients were valid. The sample size for each JOA category was very different [severe (n = 33); moderate (n = 5); mild (n = 6)], which could explain why that nDNA were significantly higher in severe patients when compared to moderate and mild patients. Nonetheless, large-scale prospective studies are warranted to verify the prognostic value of plasma DNA with respect to clinical outcomes.