Cell-Free DNA as a Marker for Prediction of Brain Damage in Traumatic Brain Injury in Rats

Abstract

Traumatic brain injury (TBI) is a major cause of morbidity and mortality, and early predictors of neurological outcomes are of great clinical importance. Cell free DNA (CFD), a biomarker used for the diagnosis and monitoring of several diseases, has been implicated as a possible prognostic indicator after TBI. The purpose of this study was to determine the pattern and timing of CFD levels after TBI, and whether a relationship exists between the level of CFD and brain edema and neurological outcomes. Thirty-nine Sprague–Dawley rats were randomly assigned to two groups: rats in group 1 (sham group) were anesthetized and had a scalp incision without TBI, and rats in group 2 were anesthetized and had a scalp incision with TBI, which was induced by using a weight drop model that causes diffuse brain injury. A neurological severity score (NSS) was assessed at 1, 24, and 48 h after TBI. CFD was measured via blood samples drawn at t=0 (baseline), 12, 24, 48, 72, and 120 h after TBI. At 48 h after TBI, brain edema was determined in a subgroup of 11 rats by calculating the difference between rats' wet and dry brain weight. The significance of comparisons between and within groups (CFD levels, brain water content, and NSS) were determined using the Kruskal–Wallis, Mann–Whitney and Student t test. The correlation between CFD levels and the NSS, as well as between CFD levels and the extent of brain edema, was calculated using the Spearman and Pearson tests, respectively. Compared with baseline levels, the CFD levels in rats subjected to TBI were significantly increased at 24 and 48 h after TBI (p<0.01 and p<0.05, respectively). A positive correlation was demonstrated between CFD levels 24 h following TBI and the extent of brain edema (r=0.63, p<0.05), as well as between CFD levels and the NSS (r=0.79, p<0.005). In this study, we demonstrated an increase in CFD levels after TBI, as well as a correlation between CFD levels and brain edema and NSS. CFD levels may provide a quick, reliable, and simple prognostic indicator of neurological outcome in animals after TBI. Its role in humans has not been clearly elucidated, but has potentially significant clinical implications.

Introduction

T raumatic brain injury (TBI) is a major cause of morbidity and mortality of young adults in industrialized countries (Krug et al., 2000, Waxweiler et al., 1995). TBI often leads to a poor neurological outcome and may result in death. The Glasgow Coma Scale (GCS) is the most frequently used tool to assess neurological status following TBI. There are limitations in using the GCS to assess neurological status in patients with communication or language barriers, including those who are deaf; intubated; in severe pain; or under the influence of alcohol, drugs, or pain medications (Sbordone and Ruff, 2010).

Early predictors of neurological outcomes are of great clinical importance. Finding an optimal marker would be a valuable tool for noninvasive evaluation of TBI and for the assessment of possible treatments. Cell-free DNA (CFD), a biomarker used for the diagnosis and monitoring of several diseases (Swarup and Rajeswari, 2007), has been implicated as a possible noninvasive prognostic indicator after TBI. Previous studies suggest that the timing of extracting plasma DNA samples relative to the TBI is important when considering its predictive value (Campello Yurgel et al., 2007). High plasma DNA concentrations measured at an average of 35.7 h after injury were found to be a predictor of mortality. However, early measurements of plasma DNA concentrations at an average of 11.7 h after injury were not found to be predictive.

Until now, there have been no studies measuring CFD in a rat model of TBI. A better understanding of its properties in TBI may help prompt its clinical use. In this study, we used a simple, inexpensive, and accurate test for CFD, as recently described by Goldshtein and colleges (Goldshtein et al., 2009).

Many experimental models have been developed to investigate the mechanisms of TBI and to test different neuroprotective strategies. Examples of experimental models include the fluid percussion model, rigid indentation injury, rotational acceleration, the impact acceleration model, and weight drop closed head injury (Laurer et al., 2000).

The purpose of this study was to determine the pattern and timing of CFD levels after TBI, and whether a relationship exists between the level of CFD and brain edema and neurological outcomes.

Methods

Animals

The experiments were conducted in accordance with the recommendations of the Declarations of Helsinki and Tokyo and to the Guidelines for the Use of Experimental Animals of the European Community. The experiments were approved by the Animal Care Committee of Ben-Gurion University of the Negev, Israel.

A total of 39 male Sprague–Dawley rats (Harlan Laboratories, Israel) with no overt pathology were used in this experiment, each weighing 230–280 g. Rats were housed in cages, three rats per cage for at least 3 days after their arrival, to allow for adaptation. Unrestricted access to water and food was given throughout the entire experiment.

The rats were randomly divided into two groups. The first group, which included 20 animals, was used as a control (sham) group. These rats were anesthetized and had a scalp incision without TBI. CFD was measured via blood samples drawn at t=0, 12, 24, 48, 72, and 120 h. The second group, which contained 19 animals, was anesthetized and had a scalp incision with TBI. The TBI group was divided into two subgroups: The first subgroup contained seven rats in which CFD measurements were measured at t=0, 12, 24, 48, 72, and 120 h and neurological performance was measured at 1, 24, and 48 h after TBI. The second subgroup included 11 rats in which CFD was measured via blood samples drawn at 0, 12, 24, and 48 h, and were euthanized for the determination of brain water content at 48 h after TBI.

Experimental design

Using general anesthesia with isoflurane 2% in the respiratory mixture on spontaneous ventilation, the tail vein was cannulated, and then 250 μL blood samples were collected. TBI was applied at t=0 min, immediately after collection of the first baseline blood sample. The rats were allowed to recover after anesthesia and surgery, so that their neurological performance at 1, 24, and 48 h after TBI could be examined. Blood samples for the determination of CFD were collected before TBI as a baseline level and then at 12, 24, 48, 72, and 120 h after TBI. A subgroup of 11 rats was euthanized after 48 h for determination of brain water content. The decision to collect blood samples at the mentioned time points was based on a small preliminary study showing that the first elevation of CFD was not seen at 8 h but appeared 24 h after TBI (Campello Yurgel et al., 2007). In light of this observation, we eliminated any superfluous blood sample collections in order to prevent unnecessary rat exsanguinations. While the rats were anesthetized, a BD Neoflon™ 24 G catheter was introduced into the tail vein for blood analysis at the times mentioned above. After each blood sample collection rats received 750 μL of saline for replacement of circulated blood volume and the catheter was removed immediately after each blood collection. Rats were re-anesthetized with 2% isoflurane for a few minutes while collecting each blood sample.

TBI

It is a common practice to distinguish “focal” from “diffuse” injuries following TBI (Graham et al., 1995; Povlishock et al., 1994). Focal injuries present as lacerations and contusions that are usually accompanied with hematomas (Gennarelli, 1994). Diffuse injuries present as diffused brain swelling, diffused axonal injury, and ischemic brain damage (Adams et al., 1989; Blumbergs et al., 1989, 1994, 1995; Graham et al., 1995; Maxwell et al., 1997; Mittl et al., 1994). Moreover, the mechanisms of damage are divided into primary and secondary causes. Primary mechanisms include focal mechanical damage to the brain tissue at the moment of the traumatic event. Secondary mechanisms include the delayed damage caused by low perfusion, ionic imbalance, diffuse edema, release of autotoxins, high intracranial pressure, malfunction of the blood–brain barrier (BBB), and inflammation (Graham et al., 1995). Unlike primary damage, secondary damage is considered reversible and is therefore the main focus of the current study (McIntosh et al., 1998; Povlishock et al., 1999).

Nevertheless, it should be stressed that human TBI usually results in a combination of primary and secondary brain injury. It is often difficult in observational studies to identify the specific mechanism and form of injury that results in irreversible morbidity and mortality. Therefore, animal models are highly valuable in examining therapeutic strategies and clinical approaches. There are various models of TBI that represent different types of brain injury: fluid percussion, rigid indentation, inertial acceleration, impact acceleration, and weight drop (Laurer et al., 1999; Morganti-Kossmann et al., 2010; Prieto et al., 2009; Schneider et al., 2002). In this study, we used the weight drop TBI with low injury severity. This has been shown to produce concussive-like TBI without local lesions, and thus represents diffuse brain injury (Laurer et al., 2000; Tang et al., 1997a).

The weight drop closed TBI model used in our study has several advantages to other methodologies, and closely reflects clinical situations. Rodent studies using this model demonstrated short-term neurological impairment and neuronal loss both in the cortex and in the hippocampus (Chen et al., 1996; Tang et al., 1997a,b). Nevertheless, the absence of the trephination and the vertex skull of rodents lead to a wide variability in the extent of damage to the rats. This is in contrast to the fluid percussion model, which produces a focused reproducible lesion. Moreover, studies have found long-term neurological dysfunction when using the latter model (Pierce et al., 1998).

In this study, spontaneously breathing, male Sprague–Dawley rats weighing 230–280 g were anesthetized with a mixture of isoflurane (initial inspired concentration 2%) in 100% oxygen (1l/min). The rectal temperature was maintained at 37°C using a heating pad, and anesthesia was considered as being sufficient for surgery when the tail reflex was abolished. Under general anesthesia, scalp was infiltrated with 0.5% bupivacaine. The scalp was incised and reflected laterally, and a cranial impact of 0.5 J was delivered by a silicone-coated rod, protruding from the center of a free-falling plate as previously described (Shapira et al., 1988; Zlotnik et al., 2007; Zlotnik et al., 2008). The impact point was 1–2 mm lateral to the midline of the skull's convexity. Following TBI, the incision was sutured and the rats were laid on their left side for recovery from anesthesia.

Determination of neurological performance

Rats' neurological severity score (NSS) was determined by a blinded observer (Menzies et al., 1992; Shapira et al., 1988). Points were assigned for alterations of motor functions and behavior, such that the maximal score of 25 represented greatest neurological dysfunction whereas a score of 0 indicated an intact neurological condition. Specifically, the following were assessed: ability to exit from a circle (3- point scale), gait on a wide surface (3-point scale), gait on a narrow surface (4-point scale), effort to remain on a narrow surface (2-point scale), reflexes (5-point scale), seeking behavior (2-point scale), beam walking (3-point scale), and beam balance (3- point scale).

Determination of serum CFD

We used a simple, inexpensive and accurate test for CFD, described lately by Goldshtein and colleges (Goldshtein et al., 2009). CFD was detected with the present assay directly in serum. SYBR Gold Nucleic Acid Gel Stain, (Invitrogen, Paisley, U.K.) was diluted first at 1:1000 in dimethyl sulphoxide (DMSO, Sigma-Aldrich, Rehovot, Israel) and then at 1:8 in phosphate-buffered saline (PBS, Biological Industries, Beth Haemek, Israel). Ten microliters of DNA solutions were applied to black 96-well plates (Greiner Bio-One, Frickenhausen, Germany). Forty microliters of diluted SYBR Gold was added to each well (final dilution 1:10,000) and fluorescence was measured with a 96-well fluorometer (Spectrafluor Plus, Tecan, Durham, NC) at an emission wavelength of 535 nm and an excitation wavelength of 485 nm.

Brain water content

Brain hemispheres were removed at 48 h after TBI in 11 of 19 rats. The rats were euthanized by exposure to CO₂ and then decapitated. Brain tissue samples of ∼50 mg were excised from a location immediately adjacent to the area of macroscopic damage in the injured hemisphere. These tissue samples were used for the determination of water content. Water content was determined from the difference between wet weight (WW) and dry weight (DW). Specifically, after WW of fresh brain tissue samples was obtained, samples were dried in a desiccating oven at 120°C for 48 h and weighed again to obtain DW. Tissue water content (%) was calculated as (WW–DW)×100/WW.

Statistical analysis

Statistical evaluation of the results was performed using the SPSS 14 package (SPSS Inc., Chicago, IL), including descriptive statistics. Data of infarcted brain volume and brain edema are presented as mean±SD. NSS data are presented as median±range using a 25-point scale. The significance of comparisons between groups (CFD levels, brain water content and, NSS) was determined using the Kruskal–Wallis, Mann–Whitney and Student t test. The correlation between CFD levels and the NSS, as well as between CFD levels and the extent of brain edema was calculated using the Spearman and Pearson tests, respectively. Differences in data were considered significant when p<0.05 and highly significant when p<0.01.

Results

A total of 39 rats were used for this study. The mortality rate was 0%. Twenty rats underwent a sham procedure and 19 rats were subjected to TBI. NSS was assessed at 1, 24, and 48 h after TBI. CFD was measured via blood samples drawn at t=0 (baseline), 12, 24, 48, 72, and 120 h after TBI. A subgroup of 11 rats was euthanized after 48 h for the determination of brain water content.

There was no significant difference in baseline plasma CFD levels between the groups (CFD_ctrl=417.87 ng/mL; CFD_TBI=498.305 ng/mL; p>0.05). It should be noted that in our previous studies, we calculated the baseline plasma CFD levels in 129 naïve uninjured male Sprague–Dawley rats weighing 230–280 g. The average CFD level measured 458.11 ng/mL, with SD 147.985 ng/mL. There were no significant differences in baseline levels between this “normal population” and the two other groups in our current study. In the control group, plasma CFD levels appeared to rise 24 h after the procedure, but this rise was statistically insignificant compared to baseline (p>0.05). Compared with baseline levels, the CFD levels in rats subjected to TBI were significantly increased at 24 and 48 h after TBI (p<0.01, p<0.05, respectively; Fig. 1). Plasma CFD levels were significantly higher in the TBI group in comparison to the control group throughout the experiment (p<0.05, p<0.01, and p<0.05 at 12, 24, and 48 h respectively). It's important to note that it was the decline in CFD levels in the control group that resulted in a difference between the control and TBI groups at 12 h after TBI. This difference was not the result of an increase in CFD levels in the TBI group. Plasma CFD in the TBI group reached peak values at 24 h, partially decreased by 48 h, and returned to baseline by 72 h.

FIG. 1.

Blood circulating cell free DNA (CFD) levels following TBI. Compared with baseline levels, the CFD levels in rats subjected to TBI were significantly increased at 24 and 48 h after TBI (p<0.01 and p<0.05, respectively). Significant differences between TBI and control groups was seen at 12, 24, and 48 h after TBI (p<0.05, p<0.01 and p<0.05, respectively). Data are presented as mean±SEM.

Neurological performance was examined using the delta NSS in the TBI group (difference between NSS values at 1 h and 48 h following the procedure). A positive correlation was demonstrated between CFD levels the NSS (r=0.79, p<0.005; Fig. 2a). Brain edema was estimated by measuring brain water percentage 48 h after TBI was performed. A positive correlation was demonstrated between CFD levels and extent of brain edema (r=0.63, p<0.05; Fig. 2b).

FIG. 2.

(A) The correlation between plasma cell free DNA (CFD) levels 24 h after the procedure and the Neurological Severity Score (NSS) (r=0.79, p<0.005). (B) The correlation between CFD levels 24 h after the procedure and the estimation of brain edema. A positive correlation was demonstrated between CFD levels and extent of brain edema (r=0.63, p<0.05). Data are presented as mean±SEM.

Discussion

The purpose of this study was to assess the relation between plasma levels of CFD and the severity of brain damage caused by TBI in rats. The principal findings of the present study were as follows. TBI in rats induced by a weight drop model led to a significant elevation of CFD levels in plasma within 24 h following TBI. Plasma CFD levels remained elevated 48 h after TBI. Compared to controls, rats subjected to TBI had significantly higher levels of plasma CFD from 12 to 48 h following TBI. In addition, we found a strong correlation between plasma CFD levels at 24 h and the severity of neurological impairment assessed at 48 h after TBI. These time points were chosen because plasma CFD in the TBI group reached peak values at 24 h and brain edema is maximal at 48 h. We intended to follow CFD levels and NSS at least during this period of time using a maximal number of rats. We further found a strong correlation between levels of plasma CFD and the extent of brain edema in the injured hemisphere.

Although new techniques enable researchers to better understand the pathological mechanisms resulting in tissue damaged following TBI, there is currently no available neuroprotective treatment and TBI is still one of the leading causes of morbidity and mortality in young adults in developed countries (Waxweiler et al., 1995). The annual incidence is estimated to be ∼100 per 100,000 persons (Kelly, 1999) and currently TBI is considered to be a hidden epidemic (Gordon et al., 1998). Therefore, there is a strong demand for further research, in order to ultimately yield better therapeutic outcomes.

The main goal of this study was to locate a new biological marker that provides a quick, reliable, and simple prognostic indicator of neurological outcome after TBI. Such a marker is of great importance both clinically and in further research of animal models of TBI. Plasma CFD may serve to be such a marker. Circulated CFD levels were shown to correlate with tissue injury (Lo et al., 2000), and it was reported that plasma CFD levels might have a prognostic value in patients following ischemic stroke (Rainer et al., 2003) and a diagnostic value in patients with myocardial infarction (Chang et al., 2003; Shimony et al., 2010).

Circulated CFD levels are expected to elevate in response to the release of DNA from cellular lysis, necrosis, apoptosis, and disrupters of the BBB (Swarup and Rajeswari, 2007), which follows mechanical and ischemic brain insults (Shojo et al., 2010). Therefore, we expected to find a similar elevation following TBI. Unfortunately, however, this approach seems to be more valuable in the context of laboratory studies rather than in clinical practice. The process of neuronal and glial injury extends beyond the first few hours after TBI. Secondary brain injury contributes to further cell death. This sequence includes multiple pathological mechanisms, including brain edema, hypoperfusion, and cell hypoxia. Secondary brain injury often contributes to more significant damage than does the primary injury. This process may take several hours and even days to develop. Therefore, one may expect that the elevation of markers such as CFD may take a long time before it is detectable in the bloodstream.

The authors understand and recognize that the utilization of CFD as a marker may have serious limitations in clinical practice. Moreover, the fact that CFD is a nonspecific marker of tissue damage, means that it is expected to be elevated in cases of multiple trauma regardless of the extent of brain injury. Additionally, other pathological processes existing prior to TBI might significantly affect “baseline CFD levels”. Therefore, the use of CFD for TBI in humans may be limited. However, in animal models of TBI, when baseline levels of CFD may be collected prior to the injury and in the context of isolated trauma, this approach may be very useful. In many studies, the investigators may prefer not to euthanize rats early in the course of the experiment in order to study long-term outcomes or behavioral tests. In this context, CFD, which was shown to correlate well with NSS and the extent of brain edema, may serve as a marker of the degree of brain injury.

The correlation between plasma CFD levels and brain damage was measured by two outcomes, brain edema and NSS. Brain edema leading to an expansion of brain volume has a significant contribution to secondary brain damage caused by impaired cerebral perfusion, and is a well-known important index of severity for diffuse brain injury (Unterberg et al., 2004). Brain edema following trauma is assumed to be vasogenic because of a disruption in the BBB, resulting in extracellular water accumulation and cytotoxicity caused by intracellular water collection (Unterberg et al., 2004). Because of a limited intracranial space, any increase in intracranial volume would compromise the already impaired compliance and contribute to additional ischemic damage caused by reduced perfusion and oxygenation (Unterberg et al., 2004). These events, in addition to the immediate cellular damage, may significantly aggravate the condition and exacerbate the brain damage. Therefore, brain edema has a crucial impact on morbidity and mortality following TBI, and is commonly measured in animal models of TBI (Barzo et al., 1996; Hayasaki et al., 1997; Ishige et al., 1987; Kawamata et al., 1997; Kochanek et al., 1995; Marmarou et al., 1982; Park et al., 1998; Vaz et al., 1998). CFD may be a useful marker for those injuries based on their mechanisms as described previously. Neurological function was assessed by a well-established method of measuring motor and behavioral scores (Shapira et al., 1988; Zlotnik et al., 2007; Zlotnik et al., 2008).

From the first 12 h following TBI, rats had higher plasma CFD levels compared to controls. However, similar to our preliminary study, plasma CFD levels were seen to increase significantly compared with baseline only after 24 h following TBI. This result supports the assumption that plasma CFD levels more accurately represent the delayed secondary damage than does the primary damage following TBI, although further study on the biokinetic properties of CFD should be conducted in order to assess this assumption. We also noticed a slight but insignificant elevation in plasma CFD levels in the control group, which may be a result of minor soft tissue injury from the sham surgical procedure.

There was a correlation found between plasma CFD levels and the neurological damage following TBI as assed by the NSS score, as well as between plasma CFD levels and brain edema. The results support our assumption that CFD levels can be used as a biomarker of brain damage following TBI in rats. This result is further relevant to future animal models of TBI, as measurements of brain edema require histological examination, and, therefore, euthanizing animals, which could now ultimately be avoided.

The main drawback of our study was a limited number of rats, which would increase the chance for a type II error. However, the significant correlations and significant differences between the two groups suggest that a larger number of rats would further increase the significance of the results.

Conclusion

In conclusion, we demonstrated an increase in CFD levels after TBI, as well as a correlation between CFD levels and brain edema and NSS. CFD levels may provide a quick, reliable, and simple prognostic indicator of neurological outcome after TBI. Its role in humans has not been clearly elucidated, but has potentially significant clinical implications. Additional studies can provide a more enhanced understanding of the potential clinical utility of CFD biomarkers in TBI.

Footnotes

Acknowledgments

We gratefully acknowledge the help of Valeria Frishman, laboratory assistant at the Department of Clinical Biochemistry, Soroka Medical Center, Ben-Gurion University of the Negev, for her outstanding help with the biochemical analysis. In addition, we thank the staff at the Critical Care Unit, Soroka Medical Center.

Author Disclosure Statement

No competing financial interests exist.

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