The current standard for acute diagnosis of traumatic brain injury (TBI) is cranial computed tomography, unfortunately, not all the TBIs are visible [1]. An early, reliable and more importantly non-invasive biomarker of TBI could lower the number of scans, reducing the cost of healthcare, lives and also cancer risk [2]. The severity of head injuries varies, and it is a key goal of the TBI research to discover a biomarker able to estimate the prognosis. The study of dynamics of the biomarkers in clinics is often limited, especially in the early time period after TBI, by approximation of the time of TBI. In contrast to clinical studies, animal models provide the possibility to assess early dynamics of a tested biomarker on a relatively homogeneous cohort and with the precisely stated time of the TBI. An ideal clinically relevant biomarker should be informative and predictive even early after the TBI occurs. In severe cases of TBI, the need for medical attention is often obvious, importance of a biomarker is in the middle ground in cases, where TBI is mild, and there are no other indicators for necessary treatment. Therefore, we focused on mild TBI.

There are several biomarkers for TBI currently used and studied. First biomarkers approved in the United States of America were ubiquitin C-terminal hydrolase 1 [3], [4] and glial fibrillary acidic protein (GFAP) [1], [2], [3], [4], [5], [6], [7] however, there are many more such as S100 calcium-binding protein B (S100B) [2], [8]. This topic has been extensively reviewed [9], [10], the GFAP seemed to have the best predictive ability of survival and severity of the TBI.

In this study, we analyzed GFAP in plasma [1], [2], [3], [4], [5], [6], [7]. GFAP is an intermediate filament protein produced in activated astrocyte cells [11]. GFAP is widely used in animal studies as a marker of brain damage in tissue homogenates [11]. Serum or plasma GFAP has been studied as a biomarker in human TBI [1], [2], [3], [12], in severe TBI in rats [13], [14] and in pigs [5]. However, there are no studies analyzing the amount of plasma GFAP in the most used mouse model of closed head TBI. The dynamics of GFAP have been studied in pigs [5] and in humans [3]. In both, the differences in serum levels of GFAP are not detectable until 8 h after the TBI [3], [5].

Extracellular DNA (ecDNA) in plasma is a very sensitive marker of tissue damage, analyzed in heterogeneous diseases including sepsis [15], [16] liver failure [17], colitis [18], hypertension [19] and also trauma [20]. Higher ecDNA concentrations are associated with the severity of the studied diseases [20], [21], [22]. Several studies have shown that ecDNA might be a potential predictor of mortality in patients with TBI [4], [23], [24], [25], [26], [27] and also in a TBI model in rats [28]. Furthermore, patients who died in the ICU were shown to have significantly higher ecDNA than survivors [25], [29]. There are no studies focusing on ecDNA levels in plasma in early time points after TBI. Moreover, there has been a global effort to develop rapid for ecDNA detection, possibly usable in emergency rooms for quick diagnosis [30], [31].

With different protein biomarkers discovered in animal models, a problem often withstands with applicability to human studies. This problem could be avoided by using non-specific biomarkers like ecDNA. While GFAP [1], [2] and many other potential biomarkers are investigated in TBI [13], [14], the difference in its concentration in patients’ serum was not detected until 8 h after TBI [3], which could be an important time period for diagnosis. The early dynamics of ecDNA after TBI, has not been studied yet. For ecDNA the earliest time point was 12 h after the TBI–which could be too late for many patients. We hypothesized that changes in ecDNA could be observable earlier after TBI. Therefore, the aim of our study was to examine the early dynamics of GFAP and ecDNA in plasma after closed head weight-drop TBI in mice.