Brain biomarkers and pre-injury cognition are associated with long-term cognitive outcome in children with traumatic brain injury

Following traumatic brain injury (TBI), children and adolescents experience changes in both cognitive and behavioural functioning [1]. These deficits are frequently associated with damage to the frontal and temporal regions of the brain [2]. The frontal lobes are known to have protracted development throughout childhood and adolescence [3], thus are particularly vulnerable to insult, such as TBI, experienced during development. Two cognitive areas that are dependent on intact functioning of frontal networks, and thus frequently reported as impaired following TBI, are executive functioning and attention [4].

Executive functioning is an umbrella term used to describe a variety of abilities allowing purposeful, goal-directed, problem-solving behaviour, including behavioural regulation, planning and organizational skills, and self-monitoring [5]. Deficits in executive functioning have been seen within the first year [6, 7] and up to five [8] and 10 years [4] following childhood TBI. At 10 years post-injury, 26% of those who sustained a moderate TBI and 42% of those who sustained a severe TBI had clinically significant impairments on measures of executive functioning [4]. Deficits in executive functioning have been variously related to injury severity [4, 6‐8], age at injury [6] and socioeconomic status (SES) [6].

Behavioural symptoms of attention difficulties are observed as inattention, hyperactivity and impulsivity, which are the primary symptoms associated with attention deficit hyperactivity disorder (ADHD) [9]. These symptoms are over-represented in children with TBI, with 20% of children meeting ADHD criteria prior to the injury [10] and an additional 18–20% of children experiencing de novo attention difficulties by two years post-injury [11, 12]. A relation between injury severity and the development of attention problems has been reported in some studies [11‐14], but not others [10, 15, 16]. Thus, standard measures of injury severity do not seem to capture the true biological impact of the trauma. Other associated variables, such as SES, age at injury and sex, are also not reliable predictors of attention difficulties across studies [10, 11, 14‐16].

These attention and executive functioning deficits are particularly relevant to children and adolescents as they impact progress in academics and psychosocial development, and may have a negative influence on family functioning [6, 8, 11]. If clinicians were able to identify patients at high risk of long-term deficits in attention and executive functioning, management strategies could be applied in the early stages of recovery to improve functioning and quality of life for the children with TBI and their families.

Due to the poor reliability of clinical factors, recent research in childhood TBI has started to focus on the use of serum biomarkers as predictors of outcome. A few serum biomarkers, such as neuron specific enolase (NSE), S100 calcium binding protein B (S100B), interleukin-6 (IL-6) and interleukin-8 (IL-8), have previously been shown to be associated with global neurological function and cognitive outcomes in children with TBI [17‐21]. These studies did not allow for a comprehensive understanding of post-injury cognitive and behavioural deficits that may occur in those who appear to have recovered physically and yet still have serious cognitive sequelae [22].

We conducted a proof of principle study to determine which combination of injury (Glasgow Coma Scale; GCS) and child (age at injury, sex, SES, and pre-injury functioning) variables and six brain-injury and inflammatory serum protein biomarkers relate to long-term outcome in attention and executive functioning, as rated by parents, following childhood TBI. Serum biomarkers S100B, NSE, Il-6 and IL-8 were chosen due to their association with outcome following paediatric TBI. Soluble neuron cell adhesion molecule (sNCAM) and soluble vascular cell adhesion molecule (sVCAM-1) were also measured as potential biomarkers of microvascular injury and inflammation that may be associated with outcome following TBI. This study identifies the characteristics that may be most closely associated with long-term cognitive and behavioural outcome in attention and executive functioning following paediatric TBI. We hypothesized a combination of serum biomarkers [21, 22] will be more strongly associated with long-term outcome measured at least a year and a half post-injury than other child and injury related variables, which have been inconsistently related to cognitive and behavioural outcome in the past [4, 6‐8, 10, 11, 14‐16].

Twenty-nine of the 85 children and adolescents who were recruited into the original study completed an extended follow-up assessment at more than one year post-injury. Of these participants, 23 had serum biomarkers measured at time of injury and returned completed questionnaires at a long-term follow-up time point, and thus were analyzed in the present study. The patient demographics, injury severity, injury mechanisms and associated injuries are shown in Table 1. Participants included in the follow-up did not differ significantly in terms of demographics and injury characteristics from those in the larger cohort of 85 patients with TBI, who did not participate in this study. However, there were differences between groups for mechanism of injury.

A summary of serum biomarker levels and the time they were collected can be found in Table 2. Seven of the 23 participants had only one blood draw. The first blood draw for these seven participants took place at a mean of 16.51 h following injury. Figure 2 depicts the change in serum levels of NSE (Fig. 2a) and sNCAM (Fig. 2b) over days following injury. Mean T-scores for the 23 participants included in the long-term follow-up portion of the study for the eight clinical scale scores of the BRIEF can be seen in Table 3 and the six content scales of the Conners-3 can be seen in Table 4. Tables 3 and 4 also report the percentage of participants with borderline or clinically significant T-scores on each scale. Children were seen at follow-up between the ages 6.0 and 18.8 years [Mean age = 14.1 years, Standard Deviation (SD) = 4.0 years] and at 1.5 to 5.7 years (Mean time = 3.1 years, SD = 1.1 years) post-injury.

We identified associations between time of injury variables and cognitive and behavioural outcomes as measured by parent questionnaires. PLS analyses revealed similar patterns of associations between the predictors and all three sets of outcome variables. In a sample of 23 children who had experienced a TBI and were seen at follow-up an average of 3.1 years post-injury, we found that the combination of highest levels of NSE and the lowest measured levels of sNCAM were associated with greater difficulties with inattention, hyperactivity/impulsivity and executive functioning as measured by the Conners-3. We also found that a combination of these two biomarkers combined with a high PIFOS Cognition score was associated with the initiate and working memory scales of the BRIEF MI. The highest levels of NSE were also associated with the inhibit scale of the BRIEF BRI. This profile of predictor variables measured at the time of injury reflect those who are at risk for these specific aspects of attention and executive functioning issues following childhood TBI.

The use of PLS is well suited for this study, as this technique allows the evaluation of large numbers of variables with high multicollinearity, in a relatively small sample and provides insight into the multivariate relations among predictors and outcome variables [26]. In addition to the associations we found between serum biomarkers and outcome, we found that not all pre-injury risk factors and injury characteristics were predictive of cognitive and behavioural outcomes. This is an important consideration as the variables included in this study are routinely used when studying outcome. Injury severity (as measured by GCS), age at injury, SES and sex have been related to cognitive outcome in various studies [1, 4, 6‐8, 10, 11, 14‐16]. Here, none of those variables were significantly associated; instead we showed a set of specific variables measured at the time of injury were related to long-term attention and executive functioning outcome.

Predicting individual outcomes following TBI based upon pre-injury variables and injury characteristics is an important undertaking. To our knowledge, this is the first study to report on serum biomarkers and long-term cognitive outcomes. Interestingly, the same two biomarkers were consistently part of the clinical profiles associated with outcome. Serum NSE was a significant predictor in all three PLS analyses and serum sNCAM was a significant predictor in two of the analyses. It has been hypothesized that a combination of serum biomarkers or a combination of biomarkers and injury severity would most likely be the best predictors of outcome [21]. NSE has been shown to relate to global outcome using the Glasgow Outcome Scale (a five point scale assessing physical disability) [18‐20], but sNCAM has not been previously studied in relation to outcome following paediatric TBI. In a related study, we investigated nine serum biomarkers and their relations to the inattention content scale of the Conners-3 measured 12 months after TBI. While sNCAM was found to be a moderate predictor of inattention at 12 months post-injury, we found the combination of NSE with pre-injury estimates of inattention was the strongest predictor of inattention at 12 months post-injury [28]. The present study has also shown a relation between NSE and sNCAM with three of the Conners-3 content scales at a longer follow-up time, including inattention.

The two content scales of the Conners-3, inattention and hyperactivity/impulsivity, that were part of the outcome profile of the first PLS analysis are the primary symptoms of ADHD [9, 25], which we know is frequently seen in children following TBI [10‐12]. These ADHD symptoms have been seen in the chronic stages following injury in previous studies [9, 14], and we have now shown a relation with these symptoms measured in the chronic stages following injury and serum biomarkers measured in the acute period following injury. The executive functioning content scale from the Conners-3 was also part of the outcome profile found in the first PLS analysis. The BRIEF allowed for a more in-depth evaluation of executive functioning outcome in the second and third PLS analyses. To our knowledge, this is the first study to report the relations between serum biomarkers and parent-reported executive functioning following TBI, although other investigators have found long-term deficits in executive functioning post TBI [4, 7, 8]. These studies reported significant declines on the two index scores and the composite score of the BRIEF in children with TBI, but Sesma and colleagues [7] reported significant differences in all of the clinical scales except organization of materials, between children with TBI at a year following injury when compared to both baseline scores and a control group. Working memory was the only clinical scale of the BRIEF that was consistently significantly different following TBI regardless of the severity of TBI or time over the first year following injury. In this current study, working memory was one of the three clinical scales of the BRIEF significantly related to predictors in the two PLS models. A variety of executive functions are evidently impaired following TBI.

The significant association between scales from both questionnaires and serum biomarkers NSE and sNCAM suggests that these serum biomarkers reflect significant brain injury that has long-term sequelae. NSE is a glycolytic enzyme found primarily in the cytoplasm of neurons and released into extracellular space as a result of neuron damage [29]. sNCAM is a binding glycoprotein expressed on the surface of neurons which helps promote neurite outgrowth and is passively released upon cell destruction [30]. Future studies should further characterize the predictive power of these two serum biomarkers in predicting cognitive outcome following paediatric and adult TBI.

For the current study, we did not have pre-injury Conners-3 or BRIEF questionnaires available on all of our participants to study the impact of pre-injury functioning on outcome. We did, however, have the PIFOS as a proxy of pre-injury functioning. The PIFOS Cognition score included questions on judgement/safety, memory, attention, speed of processing, academic placement and executive function, and specifically inquires about impulses, planning and organizing activities and problem solving [23]. Many of these questions tap into the indices of the BRIEF and the PIFOS was originally validated by showing relations with the BRIEF [23]. The questions that make up the PIFOS Cognition score appear to be measuring comparable functions to the clinical scales of the MI rather than the BRI. Thus, this could explain why the predictor profile for the MI outcome included the serum biomarkers as well as the PIFOS Cognition score. This study was designed prior to 2009, and thus prior to the publication of the current National Institute of Health (NIH) TBI common data elements (CDEs) [31]. In accordance with these recommendations, we are currently also collecting the GOS-Extended Pediatric Revision (GOS-E Peds) and other NIH TBI CDEs in ongoing studies.

Parent questionnaires provide ecologically valid assessments of a range of children’s behaviour [8]. The investigation of the relations between serum biomarkers and cognition through neuropsychological measures would allow, however, greater insight into these deficits and would be the obvious next step in the research of serum biomarkers and long-term cognitive sequelae of TBI. Cognitive interventions have not been widely researched in childhood TBI, associations between serum biomarkers and neurocognitive outcome could contribute to planning for early interventions during the acute time period following TBI. Some early studies have shown improvements on the BRIEF and measures of attention and executive functioning following cognitive intervention programs including the Attention Improvement and Management program [32] and a family-centred, online-counselor-assisted program intervention [33, 34].

This proof of principle revealed promising results, but before clinical decision making can be directed the results need to be replicated in a prospective study with a larger sample size. We acknowledge that we were limited by the number of blood sample draws available for each patient, with some patients only having one blood sample drawn, in particular those with mild TBI. In the current study, we required informed consent prior to collecting blood samples, and this may have led to missing the highest serum levels for biomarkers that peak early, such as S100B. In subsequent studies, Research Ethics Boards at hospitals in Canada and Australia have provided permission to use a deferred consent model, allowing the collection of daily blood samples up to 48 h prior to obtaining informed consent.

Additionally, further understanding of these serum biomarkers in both injured and non-injured children is needed. Collecting medically unnecessary blood samples from typically developing controls is a difficult task in the paediatric population, both from a participant and a Research Ethics Board standpoint. One previous study found no significant difference in NSE and S100B levels between two control groups recruited from those undergoing routine blood work or with isolated fractures, but did see significant differences in biomarker levels between these controls and those with head injury [35]. Some of the biomarkers that we investigated in this study are released as a result of injuries to other parts of the body, as outlined in Table 1. Thus, another excellent control group could be those undergoing care for serious injuries without head injury. Future studies should carefully consider the addition of injured and non-injured control groups, as well as the impact of activity to the serum biomarker levels of the patients prior to incurring the injury.

Following TBI, children are at risk of persistent attention and executive functioning problems. In this proof of principle study, we showed a relation between the serum biomarkers, NSE and sNCAM, with long-term outcome on the Conners-3 and BRIEF. For the MI from the BRIEF, this relationship included a combination of these biomarkers with the PIFOS Cognition score, while the BRI from the BRIEF was only associated with NSE. When validated in future research studies, these associations could improve clinical decisions, prediction of long-term outcome and planning during the acute time period following TBI.