The present study explored the profile of CSF inflammatory biomarkers in athletes with post-concussive symptoms persisting more than 6 months following their most recent sports-related concussion (SRC). Compared to age- and sex- matched athletic controls, we identified increased levels in eight out of the 25 inflammatory biomarkers. This implies an ongoing inflammatory process in the brain, as reflected in the CSF, several months or years following the injury that potentially contributes to the persistent symptoms.
Axonal stretching and shearing may occur at time of the head impact that results in an SRC, accompanied by a neuroinflammatory cascade that is triggered within minutes post-injury. This inflammatory response can continue for several months to years, and lead to a chronic inflammatory phase that may be maladaptive and exacerbate the initial injury(21). These long-lasting inflammatory processes may then lead to secondary brain injury, reactive pathological processes such as astrogliosis, microglial activation, axonal beading, DNA damage and neurodegeneration may be a result of (3, 4, 22–24).
The inflammatory response that occurs following an SRC appears less marked, and more characterized by gliosis(21), than that observed in severe TBI(12, 26). The neuroinflammatory cascade after TBI is complex and involves the activation of resident microglia, increased permeability of the blood–brain barrier, recruitment of peripheral immune cells, tissue damage and cytokine release(27). While direct evidence of an inflammatory response in humans is rare, when four young athletes who died from other causes shortly after SRCs were investigated clusters of activated perivascular microglia were observed in subcortical white matter tracts indicating an inflammatory response (25).
In a mouse model of mild TBI (mTBI), an early increase in serum brain cytokines, associated with an impaired motor function, was observed (28, 29). One single mTBI in mice was found to elicit parenchymal neuroinflammation in the cortex and hippocampus up to three weeks post-injury on histology and micro-PET imaging(30). In both single and repeated mTBI, mimicking SRC, axonal injury and neuroinflammation play significant roles in the neuropathological events that include ongoing white matter degradation up to 12 months post-injury and chronic functional impairments(31, 32).
In mTBI patients, there is a distinct inflammatory signature that correlates with functional and cognitive outcome (2, 33), and inflammatory markers may predict recovery following the brain injury(34). A large longitudinal study showed that mTBI patients, injured outside of a sports context, had a prolonged increase in serum cytokines for one year post-injury, reflecting a low-level and persistent systemic inflammation following mTBI(35). These studies were, however, conducted on peripheral blood samples and may thus partly represent also a systemic inflammatory response. Plasma cytokine levels may poorly reflect the intracerebral inflammation, with large differences between brain parenchymal and systemic cytokine concentrations as shown in severe TBI(36).
To date, and to the best of our knowledge, there are no previous studies investigating inflammatory mediators in the CSF of SRC athletes in the chronic phase.
CSF sampling is invasive and not entirely without risks, and other indirect methods have included neuroimaging studies such as PET. PET imaging using a tracer binding to translocator proteins can be used as a marker of microglial activation and astrocytosis. In former professional American football players, increased neuroinflammation and hippocampal atrophy was observed 24–42 years after retirement (37), and at a mean of 7 years following the most recent SRC(38). Moreover, glial fibrillary acidic protein (GFAP), a biomarker of astrogliosis(39), was found to increase acutely and remain elevated several years following SRC(40, 41). Finally, we recently observed by PET imaging increased neuroinflammation in the hippocampus of young SRC athletes > 6 months following their last SRC(12), of relevance in view of the observed cognitive impairment observed here. Taken together, these studies and our present work find evidence of both an acute and chronic neuroinflammatory process triggered by SRC. Since there is much evidence linking axonal injury, neuroinflammation and neurodegeneration (42–44), the persistent neuroinflammation observed in our present study may be detrimental.
The immune response following TBI is complex and time dependent, and it involves both pro- and anti-inflammatory mechanisms(21). While cytokines are often classified as either pro- or anti-inflammatory, this may be an oversimplification since many cytokines can have a dual role determined by the activation signal, the timing, and the target cell(45). The levels of cytokines vary also depending on the analyzed timepoint following TBI(2, 28, 35, 36), suggesting that the temporal aspect is of importance in the interpretation of these results. In the present study, we observed a significant elevation of many cytokines in the CSF of SRC athletes at a mean of 17 months post-injury, of which several may have both pro- and anti-inflammatory roles. However, based on the evidence presented in the previous paragraphs it is plausible that the increased neuroinflammation exacerbates the brain injury in chronic SRC.
We did not find any convincing correlations between cytokines and neither the cognitive impairment nor the post-concussion symptoms, which may be explained by the homogeneity of the included SRC athletes. Most of the SRC athletes reported many and severe post-concussion symptoms and scored low on cognitive function, with a low dispersal within the group. Inclusion of asymptomatic SRC athletes may have altered these results. Previously a correlation between blood cytokines and post-concussion symptoms, depression, and post-traumatic stress were found(2). Although the time course of inflammation, functional impairment and symptoms has not been established, a PET-imaging study of American football players suggested that neuroinflammation may precede cognitive symptoms(38). Our cohort of athletes had persisting symptoms since their last SRC, without a symptom free period, and we hypothesize that these symptoms are associated with an ongoing neuroinflammation. To establish this association, further studies including athletes with diverging symptom burden, including those who recover fully from SRC-induced symptoms, and a longitudinal follow-up design is warranted.
Our present study has limitations. We aimed for including young athletes with long-term persisting symptoms following SRC, thus this selected cohort may not be representative of asymptomatic SRC athletes. This presumably influenced the correlation analyses, where a more diverged group may have produced other results. Biomarkers of inflammation was analyzed at one time-point, and therefore the temporal dynamics of the inflammatory process following SRC was not established. Most of our athletes, with one exception, also had previous SRCs throughout their career and we cannot establish whether the observed increase in the inflammatory markers were caused by their most recent SRC, or an cumulative effect of the previous SRCs. The sample size is rather small, reflected by the strict inclusion criteria used here. We also used athletic controls (runners) without any previous SRC, and we cannot exclude that their inflammatory biomarker profile is different from non-athletic controls. Finally, this was an explorative study analyzing many inflammatory markers in a relatively small cohort. To avoid the risk of type II errors, multiple comparisons were not performed(46). However, with an alpha set to 0.05, 10% of tests would be expected to be false positives. Thus, if more than 10% of analyses were different between groups at the 0.05 level, it is unlikely that this was explained merely by statistical chance. Despite the difficulties recruiting for CSF sampling, a study using a larger cohort of concussed athletes may be needed to confirm the present results.