Since 1997, a multidimensional approach has been recommended to diagnose and manage athletes suspected of having a sport concussion (SC) (1, 2). This recommended multidimensional assessment consists of clinical measures of balance, neurocognitive function, and symptoms and has been demonstrated to have a sensitivity of up to 100% within 24 h of a diagnosed SC in collegiate athletes (3, 4). Despite the established clinical utility of this multifaceted approach, a growing body of evidence suggests that physiological recovery from a SC may persist well beyond clinical recovery (5, 6). Although the majority of athletes diagnosed with a SC make an unrestricted return-to-play (uRTP) within 2 weeks of their injury (2), persistent cognitive (7) and motor deficits (8, 9), heightened mood states (10, 11), and other concussion-related symptoms (12) have been observed several months and or years following an unrestricted RTP or retirement as observed in surveyed athletes (11, 13). The etiology of these persisting symptoms remains unclear.

Physiological deficits observed beyond clinical recovery have been primarily reported in high school and collegiate athletes. Wang et al. investigated high school and collegiate football players using standard-of-care clinical measures of SC (6). Participants completed a battery of clinical measures prior to the start of their respective seasons, within 24 h of a diagnosed SC, and 8 days following the injury. Participants also completed a magnetic resonance imaging (MRI) Arterial Spin Labeling protocol to measure cerebral blood flow perfusion. Injured participants were compared to healthy, non-injured football players. Injured participants had significantly lower cerebral blood flow perfusion than the healthy control group at each time point. Despite this finding, the injured group scored within normal limits when compared to pre-injury values on all clinical measures (6). Similarly, Churchill et al. compared collegiate athletes diagnosed with a SC to a healthy collegiate athlete control group using the SCAT3, global functional connectivity, arterial spin labeling, fractional anisotropy, and mean diffusivity. Despite the injured athletes returning to pre-injury levels on the SCAT3 and making an unrestricted RTP, significantly decreased cerebral blood flow and white matter diffusivity were observed up to 1 year following injury (5). Additional research is needed with more sophisticated clinical measures to substantiate the authors' findings (6, 14–16).

The underlying mechanism of the “metabolic crisis” associated with SC has been refined to include a neuroinflammatory response (17). More specifically, the role of microglia following a SC has been questioned largely based on findings in animal models. Microglia play an essential role in surveillance of brain parenchyma and homeostasis (18). Approximately 5–10% of adult brain cells are microglia and are present in all areas of the brain; however, their population density is highly variable (whether under physiological or stress conditions) up to 10-fold in the human brain (19, 20). Microglia function to serve and protect the brain and are wholly dedicated to maintaining homeostasis. Microglia phagocytose apoptotic neurons and secrete inflammatory factors to attract other immune cells to the site of injury. The resolution of this series of events is a tightly regulated process that occurs when injury has been resolved (20, 21). However, if left unresolved, such as the case of chronic or pathologic inflammation, excessive release of cytotoxic factors (reactive oxygen species, pro-inflammatory cytokines, TNF-α, etc.) result in neuronal damage and loss of brain function, which ultimately impede recovery. The persistent over-activation of microglia has been implicated in a number of central nervous system disorders, such as stroke, traumatic brain injury (TBI), mild traumatic brain injury (mTBI), epilepsy, Alzheimer's disease, Parkinson's disease and others (20, 22–24).

Several studies have investigated central neuroinflammation following SC using peripheral fluid biomarkers (25–31). This body of research suggests that neuroinflammation may be short-lived (i.e., < 48 h) and recovery of fluid biomarker levels may occur before clinical recovery as measured by a symptom inventory (4, 32). More recent human-based studies have investigated central inflammation using positron emission tomography (PET) and single-photon emission computerized tomography (SPECT) following a SC (33–35). Originally coined the peripheral benzodiazepine receptor, the translocator protein (TSPO) is an 18 kDa outer mitochondrial membrane protein implicated in a number of neurodegenerative functions including redox homeostasis, modulating immune response, cholesterol transport, and steroidogenesis (36, 37). TSPO expression has been demonstrated to be significantly elevated in microglia as a result of neuronal insult. In addition, the basal expression of TSPO is relatively low (38), thus, TSPO has generated significant interest as an imaging biomarker for microglial activation. Pyrazolopyrimidines DPA-713 and DPA-714 are small molecules with characterized high affinity (Ki = 4.7 nM and Ki = 7.0 nM, respectively) for the TSPO receptor (39, 40).

A recent study investigated [¹¹C]DPA-713 binding in former NFL football players whose self-reported number of concussions ranged from 0 to 40 and the span of years since retirement from play ranged from 24 to 42 years. The study revealed significantly higher total distribution volume (VT), as measured by PET, in the hippocampus, amygdala, supramarginal gyrus, and temporal pole compared with healthy age-matched controls (41). A follow up study by Coughlin et al. examined active (n = 4) and former (n = 10) NFL football players with a range of 1–21 years since their last self-reported concussion. The data revealed significant binding of [¹¹C]DPA-713 in the bilateral hippocampus, left entorhinal cortex, bilateral parahippocampal cortices and bilateral supramarginal gyri compared to healthy age-matched controls (33). Higher binding was also independent of age group (active vs. retired play), ethnicity, body mass index, and years of education. Taken together, these studies appear to demonstrate higher microglial activation in NFL players across a wide range of age and participation.

The purpose of this pilot study was to determine the presence of central microglial activation beyond clinical recovery following a diagnosed SC in active, collegiate athletes as compared to healthy collegiate students using TSPO PET. We hypothesized that collegiate athletes diagnosed with a SC would have significantly higher distribution volume (VT) representative of central microglial activation when compared to healthy, physically active, college students. Herein, our findings demonstrate a proof-of-concept that central microglial activation persists beyond clinical recovery in collegiate athletes diagnosed with SC. These findings may be foundational in aligning physiological recovery with clinical recovery.

It is increasingly understood that physiological recovery of SC may persist beyond clinical recovery based on current methods of clinical evaluation (5, 6, 17, 78–83). Our understanding of SC-related neuroinflammation is continually evolving based on these novel physiological measures. Despite these scientific advances, microglial activation is an important, yet poorly understood, physiological indicator of SC (84–89). Microglia are widely accepted as the resident immune cells of the central nervous system and they play an essential role in surveillance of brain parenchyma and homeostasis (18, 90–92). In the case of neuronal insult, such as mTBI, microglia transition from their surveilling state to a ramified state to promote both pro- and anti-inflammatory responses. Activated microglia have also been shown to influence synaptic remodeling and white matter recovery with brain injury (93, 94). Taken together, activated microglia, through PET-targeted TSPO overexpression, may serve as an important neuroimaging biomarker benchmark to inform future clinical measures and to identify therapeutic targets following SC.

In this study, we investigated whether microglial activation, as evidenced by [¹⁸F]DPA-714 binding in the brain, persists in collegiate athletes who reported symptom-free following a diagnosed SC. Importantly, all of the collegiate athletes participating in this study had been cleared for uRTP based on the current gold-standard clinical protocol, a graded exercise protocol, and a clinical examination (3, 4). Compared to healthy, age- and genotype-matched controls, all athletes demonstrated higher [¹⁸F]DPA-714 signal within the brain, particularly in the limbic system and medial temporal lobe structures despite scoring within normal limits on a multidimensional battery of tests in comparison to their baseline performance and a physically active collegiate student sample. Visual interpretation of [¹⁸F]DPA-714 images demonstrated striking differences between collegiate athletes who reported symptom-free following a medically diagnosed SC. Geometric mean comparisons of affected areas are also striking in that some athletes demonstrated higher changes in [¹⁸F]DPA-714 binding in upwards of 115%.

Although no individual region showed statistically significant differences in [¹⁸F]DPA-714 binding between groups, the results were notable in that every region tested trended toward higher binding in concussed athletes. Post-hoc statistical testing determined the chances of this finding were very unlikely (p < 0.001), if under the null hypothesis we assume for any particular brain region that the change for the VT geometric mean ratio to be greater than one is 0.50 and we also assume that the VT geometric mean ratios of 10 evaluated brain regions are statistically independent. Thus, this combination of results most likely reflects our small sample size and spatial variability of the brain regions affected in our concussed athletes. In addition, TSPO is known to be expressed on the vascular epithelium and astrocyte (95); thus, this nonspecific binding is expected to further dilute the subtle changes in VT observed in our limited sample size. Nonetheless, these differences in VT suggest a confirmatory study in a larger sample size is warranted.

TSPO expression in microglia, resulting from injury, has been well characterized in several preclinical models (85), which have shown a temporal dependency of PET imaging detection. In murine models of TBI, for example, [¹⁸F]DPA-714 binding is markedly higher between seven and 10 days post-injury, but contrast is lost ~2 weeks post-injury and beyond. The acute onset of microglial activation in athletes, as the result of a SC, in addition to interindividual variability of microglial activation, is not known. All collegiate athletes diagnosed with a SC in our sample underwent our neuroimaging protocol upon reporting symptom free and scoring within normal limits compared to baseline values standard-of-care clinical measures of SC. As previously mentioned, each participant in Group 1 made a full unrestricted return-to-play following a stepwise exercise protocol and clinical examination further indicating clinical recovery (2). The relative timeline for maximizing [¹⁸F]DPA-714 contrast, as a result of temporal changes in TSPO expression, is likely as variable as SC recovery is in each individual. Thus, our sampling timeline for imaging ranged from 8 to 30 days post-injury. Our single participant who underwent imaging at 8 days following injury did show higher [¹⁸F]DPA-714 binding in the temporal pole, however, binding within the medial temporal lobe and limbic system was not higher compared to the control cohort, which is contradictory to our results in the majority of other athletes enrolled in our study. Temporal trends of TSPO status may be more suited to quantify and predict functional recovery of an athlete and should be studied further in a larger cohort of subjects to confirm these changes in microglial activation status. In addition, a larger study with important controls for sub-concussive impacts and a concussion-negative, orthopedic injury are warranted.

This study represents an important first step in understanding central microglial activation associated with SC and the relative timeline these phenotypic changes occur within and beyond clinical recovery. This is an important distinction from previous studies examining TSPO status in athletes participating in collision sports (33, 34, 89). Coughlin et al. reported [¹¹C]DPA-713 binding was significantly higher in active and retired NFL athletes compared to healthy controls. While this was a major finding and spurs many important research questions, these athletes were “late-stage”, i.e.,—career athletes participating in collision sports, compared to the athletes in our study. In fact, many of the participants in Group 1 of our study did not have a prior history of concussion. Thus, it is likely microglial activation in NFL athletes represents a more chronically activated state of microglia (58, 96), which may or may not be related to repetitive head impacts (97). For example, preclinical evidence demonstrates pro-inflammatory and anti-inflammatory microglia are upregulated (higher) following repeated mTBI (one injury every hour for 4 h) within 72 h of injury; however, a predominance of the pro-inflammatory phenotype was observed among the total microglial population (98). In addition, the majority of pro-inflammatory markers were resolved within 72 h following only a single concussive impact, thus, the cumulative pathologic response clearly has a longer temporal resolution course. Furthermore, one must consider ligand-target interactions of the imaging agents, irrespective of pharmacological dose (or lack thereof, in the case of PET) being administered. DPA-714 is an agonist, whereas DPA-713 is an antagonist, and agonists traditionally bind to high affinity states of a given receptor. Affinity state of TSPO has been suggested to be linked to neuroprotective vs. neurodegenerative activity of microglia (99), where chronically activated microglia may contain more TSPO in the high affinity state (99). Thus, if confirmed, [¹⁸F]DPA-714 binding may represent an important early distinction of pro-inflammatory microglial status in an athlete following SC altogether.

An additional aspect of this study that should be stressed is that all injured collegiate athlete participants returned to pre-injury values on clinical measures including balance, neurocognitive function, symptoms, and clinical examination (2, 6). Our findings are similar of those that have demonstrated persisting deficits using global connectivity, cerebral blood flow, fractional anisotropy, and mean diffusivity protocols in high school and collegiate athletes (6, 83). In alignment with this body of research, we observed persisting microglial activation in athletes, despite their return to normal limits on a comprehensive clinical evaluation. Our results cannot and should not be interpreted as indicating the onset of chronic microglial activation as suggested by other research in this area, as our findings may also reflect a normal physiological response to SC. Rather, these data are an important first step in understanding the true physiological response and recovery from SC in collegiate athletes.

Unique to our findings are a substantially higher PET signal indicative of persisting microglial activation for two injured participants (Table 3). Of note, these two participants were both female and had a prior history of three concussions. Only three other participants in Group 1 had a history of one or two concussions, but their microglial activation was similar to that of those with no history of concussion. It is important to note that, at this point in the emerging body of research related to central microglial activation using [¹⁸F]DPA-714, it is unknown whether this enhanced binding is associated with biological sex, concussion history, or if this enhanced neuroinflammatory response is neuroprotective or neurotoxic. That said, a history of one or multiple concussions has been associated with increased susceptibility for subsequent injury after uRTP (100, 101), increased risk of depression following retirement from sport (11), and additional general health and neurodegenerative conditions in mid- to late-life (102). However, a causal link for each of these poor outcomes following one or more concussions has yet to be established. For example, three or more self-reported concussions has been associated with less favorable outcomes, however, the underlying mechanism for this increased susceptibility is not understood (100, 102). Caution is warranted when inferring any association between the findings of this study provided its limited sample size and considerable limitations. It is important to note that while [¹⁸F]DPA-714 has been well documented to be useful in other neurodegenerative considerations, the extent of the role of additional mitigating factors (e.g., biological sex, age, physical fitness, and other variables) have yet to be fully understood. Substantial research is needed to confirm and extend the findings of this pilot study.

One limitation to this study is that metabolite corrected input functions were not sampled from the radial artery over the entirety of the scan. While this is the gold standard for calculating the MCIF (and subsequent VT), arterial catheterization and repeated sampling comes with inherent practical recruitment challenges and safety concerns to the study participant. Anecdotally, participants may be hesitant to participate in studies requiring arterial catheterization, particularly athletes. Instead, we computed the MCIF using an image-derived iteration that measures radioactivity per volume within the carotid arteries over the 90-min scan (52). These data were then standardized to a known radioactivity/volume of blood from four late and evenly spaced sampling points (45, 60, 75, and 90 min) as described above for MCIF computation (52). In addition, our calculated parent:metabolite ratios from each venous blood sample and extent of metabolism of [¹⁸F]DPA-714 did not significantly differ among group participants and were also in agreement with previously reported human data (72, 99). Taken together, these differences are not expected to have a meaningful impact in comparing VT differences between each cohort. Another important limitation is that our control group consisted of healthy, active collegiate students who were not part of a formal collegiate athletics team. Though the selection of participants in Groups 1 and 2 are similar to recruitment strategies related research (33, 34), confirmation and extension of our findings with matched, healthy, non-concussed controls from the same sport and non-contact sports is needed. It is possible that the intense physical activity, the time constraints, stress, and the associated negative impact on sleep may have also contributed to the neuroinflammatory response in these collegiate athletes. The research team elected to administer the clinical measures in alignment with the university concussion protocol at limited time points to minimize undue burden on symptomatic athletes.

In summary, our study is the first to demonstrate persistent microglial activation using [¹⁸F]DPA-714 in concussed athletes, following a battery of clinical measures, completing a clinical examination, and progressing through a stepwise exercise protocol allowing uRTP to their respective sport. Our findings further our understanding of the functional recovery following SC in collegiate athletes. However, our results are not conclusive as to whether the observed microglial activation in these subjects is the normal course of pro-inflammatory injury resolution or the onset of an anti-inflammatory chronic pathology (33, 34). As it is increasingly understood that an incongruence exists between clinical and physiological recovery from SC (5, 6, 83), these data are foundational toward understanding the presence of microglial activation in athletes following SC.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by the University of Virginia Institutional Review Board and the University of Virginia Radioactive Drug Research Committee. The patients/participants provided their written informed consent to participate in this study.

KN, DB, BK, and JER conceived the experiments. KN, JD, XT, JR, BK, and JER performed the experiments. KN, VS, JD, JM, BN, XT, JP, BK, and JER analyzed the data. KN, BK, and JER wrote the paper and all authors reviewed and edited. KM contributed to the radiosynthesis of [¹⁸]FDPA-714, as well as conducting sample perpetration and HPLC analysis of the blood samples taken at the various time points. All authors contributed to the article and approved the submitted version.