Elevated levels of serum,but not salivary,brain-derived neurotrophic factor following mild traumatic brain injury in collegiate athletes post return-to-play

Abstract

Brain-derived neurotrophic factor (BDNF) helps restore neuronal function following mild traumatic brain injury. BDNF levels can be obtained in blood serum and more recently in saliva. However, the relationship between serum and salivary BDNF is poorly understood—especially in relation to alterations in BDNF levels following mild traumatic brain injury. In this study, serum and salivary BDNF were collected from a sample of 42 collegiate student athletes. Half of the participants were recently cleared by a physician and/or an athletic trainer to return-to-play after experiencing a sports-related concussion. The other half had not experienced a concussion within the past year and were matched by age, sex, sport, and time of sample. Results suggest that incidences of depression, anxiety, and stress were all elevated in the concussion group, relative to the control participants. When controlling for stress-related negative affect, serum BDNF was elevated in the concussion group. However, there was no difference in salivary BDNF. Serum and salivary BDNF were uncorrelated across the entire sample. Yet, these measures of BDNF were correlated in the concussion group, but not the control group. In sum, serum BDNF is elevated in concussion post return-to-play; however, further research is needed to explore the utility of salivary BDNF following concussion.

Keywords

Brain-derived neurotrophic factor traumatic brain injury concussion mild traumatic brain injury

Introduction

In the United States, roughly 1.6 to 3.8 million sports-related concussions occur each year.^1,2 Concussion is a mild traumatic brain injury (mTBI) defined as a complex pathophysiological process affecting the brain, induced by traumatic biomechanical forces.^3,4 Brain-derived neurotrophic factor (BDNF) is a protein in the neurotrophin family of growth factors that plays an important role in neuronal survival, synaptic plasticity, and neurogenesis.⁵ In animal models of traumatic brain injury (TBI), BDNF helps restore synaptic connectivity and provides a neuroprotective role by reducing secondary brain injury.^6,7 In human participants, acute BDNF levels are lower in TBI patients relative to controls,^8,9 but higher in mTBI relative to moderate or severe TBI.⁹ However, beyond these immediate effects of BDNF, there is limited information on the relationship between BDNF and TBI—particularly in mTBI or concussion, which is the most common form of TBI.^1,2 There is a great deal of recent interest in establishing reliable biological correlates of concussion—especially sports-related concussion/mTBI.¹⁰ Yet, the potential relationship between BDNF and concussion in this population is unclear.

BDNF is also associated with stress-related factors such as depression and anxiety,¹¹ which are additionally elevated following sports-related concussions.^12,13 In particular, post-concussive symptoms of depression and anxiety are common and can take one to three months or more to normalize depending on the individual.¹⁴ In some cases, it has been hypothesized that post-concussive symptoms may be undiagnosed anxiety or mood disorders caused by concussions. Thus, there is a need for research to determine the relationship between concussion and variation in BDNF levels, which may account for the effects of stress-related negative affect.

In humans, neural BDNF levels are typically indirectly measured in blood serum.^9,15 Although serum-based measures are effective in detecting BDNF, they are invasive and require a trained phlebotomist. This procedure is commonly stressful and inconvenient. However, within the past decade, the detection of BDNF in human saliva^16,17 has provided an easy to obtain, noninvasive, and relatively stress-free method of collecting BDNF.^15,18 Indeed, differences in salivary BDNF have been linked to morning vs. evening personality type¹⁹ and exercise-related training.²⁰ Yet, to the best of our knowledge, the relationship between salivary BDNF and concussion has not been assessed. Furthermore, the extent to which salivary-based measures of BDNF are a reliable substitute for serum-based measures is questionable, as several studies have shown that the two measures are unrelated.^19,21 As mentioned above, there is much interest in establishing biomarkers of concussion,¹⁰ if salivary BDNF is reliable, it would be an ideal biomarker given its ease of collection and accessibility. Accordingly, the current study aimed to assess the extent to which serum and salivary-based measures of BDNF (1) are altered in the later stages of concussion after athletes have been cleared to return-to-play and (2) are correlated with each other.

Methods

Participants

Forty-two collegiate athletes between the ages of 18 and 23 (Females = 16, M = 19.67, SD = 1.24) from Northern Michigan University’s athletic programs participated in the study. Half (n = 21, M_age = 19.67) were recently cleared by an athletic trainer and/or physician to return-to-play after sustaining a concussion. Three of the concussed athletes did lose consciousness and two experienced back to back concussions prior to participating in the study. The other half (n = 21, M_age = 19.67) of participants were controls without a history of concussion in the 12 months preceding the study. Controls were matched based on age, sex, sport, and time of sample collection (see Table 1). All participation was completely voluntary and written consent was obtained by every participant; all were compensated for their time.

Table 1.

Comparison of participant groups.

	Concussion	Control	Comparison
N	21	21
Age	M = 19.67, SD = 1.39	M = 19.67, SD = 1.11
Sex	Female = 8, Male = 13	Female = 8, Male = 13
DASS D	M = 8.05, SD = 7.45	M = 2.90, SD = 5.06	t(40) = 2.62, p = .01
DASS A	M = 8.95, SD = 8.05	M = 2.62, SD = 2.91	t(40) = 3.39, p < .01
DASS S	M = 11.19, SD = 8.91	M = 4.48, SD = 5.06	t(40) = 3.00, p < .01
DASS T	M = 28.19, SD = 22.20	M = 10.00, SD = 10.70	t(40) = 3.38, p < .01
Saliva	M = 0.155, SD = 0.13	M = .102, SD = 0.13
Serum	M = 2.00, SD = 0.43	M = 1.70, SD = 0.43
Intake prior to data collection	Nicotine = 2, OTC = 1,Caffeine = 5	Nicotine = 2, OTC = 5,Caffeine = 7
Time of sample	M = 13.40, SD = 3.42	M = 13.15, SD = 2.64	t(40) = .265, p = .792
Sport	USA Wrestling = 2	USA Wrestling = 2
	Football = 6	Football = 6
	Diving = 2	Diving = 2
	Soccer = 6	Soccer = 6
	Club Hockey = 2	Club Hockey = 2
	Club Volleyball = 1	Club Volleyball = 1
	Swimming = 2	Swimming = 2

DASS: Depression Anxiety Stress Scales. OTC: over the counter medication.

DASS questionnaire

The Depression Anxiety Stress Scales (DASS) was given prior to sample collection. The DASS is composed of three self-report scales aimed to measure states of depression, anxiety, and stress.²² Each scale is comprised of 14 statements, for a total of 42 items, participants are to answer each statement using a four-point severity scale to the degree in which they have experienced each statement over the past week. The maximum score for each scale is 42, a composite score for DASS ranges from 0 to 126.

Collection of BDNF samples

Fasting

All participants did not consume food or drink besides water at least one hour prior to data collection.²¹ Usage of alcohol, nicotine, prescription, and over-the-counter medication was collected (see Table 1). Participants were scheduled based on their availability, ranging from 8:00 AM and 8:00 PM. Although time of sample varied across participants, time of sample was balanced across groups (see Table 1).

Serum

Blood samples were collected in 5 ml vacutainer serum separator tubes (Becton Dickinson, Franklin Lakes, NJ). Following collection, the serum separator tubes were immediately inverted four times and allowed to clot for 30 min at room temperature. Serum was separated via centrifugation at 3500 r/min for 10 min and 1 ml aliquots were transferred to sterile 2 ml cryovials (Nalgene, Rochester, NY). Samples were stored at −80°C until use.

Saliva

Saliva was collected by the passive drool method, because it is known to detect higher levels of BDNF when compared to the salivette method.²¹ After pooling saliva in their mouth, participants used the saliva collection aid to force the saliva into a vial. At least 2.0 ml of saliva was collected from each participant. Samples were placed on ice immediately until further handling. All samples were centrifuged for 15 min at 4000 r/min to 4°C. Consistent with serum samples, saliva samples were stored at –80°C until analysis.

BDNF ELISA assays

Both saliva and serum BDNF levels were analyzed using an enzyme-linked immunosorbent assay (ELISA) kit (CYT306, Millipore Sigma). Assay of saliva samples were not diluted, while serum samples were diluted to a 1:32 ratio. Each assay was performed in duplicate. ELISA plate readings were performed using a microplate reader (Epoch2; BioTek Instruments, Inc., Winooski, VT, USA). Analysis was performed using Gen5 software (BioTek Instruments, Inc., Winooski, VT, USA). Standard curves were obtained for saliva (R² = .992) and serum (R² = .995).

Statistical analyses

Of the 42 participants, 39 yielded data for the serum analysis, 36 for the saliva analysis, and 35 participants had both serum and saliva data. To assess the relationship between groups (concussion vs. control) and DASS measures, independent samples t-tests were run on each DASS scale and the composite score. Separate analyses of covariance (ANCOVA) were used to test the effect of concussion group on serum and salivary BDNF while controlling for negative affect (i.e. DASS). Single-tailed Pearson correlations were used to test for a (positive) relationship between serum and salivary BDNF measures across the entire sample as well as separately for each group. Pearson correlations were also used to test for a relationship between BDNF levels and days since concussion and days since return-to-play.

Results

Days since return-to-play varied from 1 to 41 days (M = 9.71, SD = ±12.08). Days since concussion varied from 1 to 50 days (M = 26.82, SD = ±13.10). There was no significant relationship between days since concussion and days since return-to-play in the concussion participants, r = .30, p = .20.

DASS questionnaire

The concussion group exhibited significantly higher levels of depression, anxiety, stress, and total DASS measures compared to the control group (see Table 1). Thus, the concussion group displayed a non-specific increase in stress-related negative affect, relative to the control group.

Serum

There was an effect of group on serum BDNF levels, F(1, 36) = 4.13, p < .05, η_p² = .10, where concussion participants (M = 2.00, SE = .099) had greater levels of BDNF than controls (M = 1.70, SE = .097). The total DASS score was not related to serum BDNF levels, F(1, 36) = 1.00, p = .32, η_p² = .03 (Figure 1(a)). For concussion participants, the relationships between serum-based BDNF and days since return-to-play (r = .18, p = .46) as well as days since concussion (r = .25, p = .30) were not statistically significant.

Figure 1.

(a) The concussion group had significantly higher levels of serum BDNF compared to the control group. (b) Salivary BDNF was also higher in the concussion group, but not significantly different than the control group. (c) Salivary and serum BDNF levels were not correlated.

Saliva

Observations of box and whisker plots indicated three outliers (2 concussion and 1 control) that were > three SDs above the respective group M. These outliers were excluded from all analyses involving saliva. There was no effect of group on salivary BDNF levels, F(1, 30) = .36, p = .55, η_p² = .01. BDNF levels in concussion participants (M = .11, SE = .02) did not differ from control participants (M = .09, SE = .02, see Figure 1(b)). The total DASS score was not related to salivary BDNF levels, F(1, 30) = .16, p = .70, η_p² = .00. In the concussion group, a strong positive correlation was found between salivary BDNF and days since return-to-play, r = .69, p = .004. However, no such correlation was found between salivary BDNF and days since concussion, r = .05, p = .87.

Relationship between salivary and serum-based measures of BDNF

Across all participants, the relationship between serum and salivary BDNF was weak and did not reach statistical significance, r = .05, p = .40 (Figure 1(c)). However, a significant relationship was found between salivary and serum BDNF in the concussion (r = .49, p = .03) group, but not the control (r = –.25, p = .16) group. When controlling for time of sample collection and days since concussion, the relationship remained significant, r = .61, p = .01.

Discussion

Elevated serum BDNF was found in collegiate athletes after being cleared to return-to-play following concussion in comparison to controls (see Figure 1(a)). Conversely, there was no difference between groups in salivary BDNF (see Figure 1(b)). In addition, serum and salivary BDNF were not correlated with each other across groups. However, within the concussion group, salivary and serum BDNF were strongly correlated (see Figure 1(c)). Thus, serum-based BDNF appears to be elevated in concussion post return-to-play, but salivary BDNF was not different between groups.

Our finding of elevated serum BDNF in concussion post return-to-play adds to the understanding of the brain’s response to minimize secondary injury following neurotrauma. Recently, reductions in day-of-injury⁹ serum BDNF have been observed in concussion. However, it has been hypothesized that although BDNF levels are initially lower, they later increase, as higher levels of day-of-injury BDNF are predictive of more successful recovery.⁹ All of the concussion participants in the present study were beyond the acute phase and already cleared to return-to-play. Taken together with data from this previous study, our results suggest that the time-course for serum-based BDNF is dynamic with initial levels being relatively lower and later levels being relatively higher. This notion is consistent with animal models where BDNF plays a neuroprotective role in reducing secondary brain injury.^6,7 That is, secondary brain injury can arise from various ischemic, inflammatory, cytotoxic, and apoptotic functions resulting from the initial injury, and evidence suggests increases in BDNF following secondary brain injury minimizes these effects.¹⁰ In addition, BDNF is thought to repair synaptic connectivity of damaged brain areas during recovery.^6,7 Given that our samples were collected after participants were cleared to return-to-play, it is likely that our BDNF levels represent secondary brain injury and/or recovery phases. In summary, our findings indicate that serum-based BDNF levels are higher post return-to-play following concussion—suggesting that the recovery process is ongoing even after acute effects have subsided and athletes are cleared to return-to-play.

On the other hand, salivary BDNF measures were not significantly different between groups. This relationship was absent even though we were able to detect BDNF, which has previously been inconsistent.¹⁸ We found that serum and salivary BDNF measures are unrelated across the entire sample. This finding is consistent with previous research comparing salivary and serum BDNF.^19,21 However, a strong correlation was found in the concussion group between saliva and serum-based BDNF, indicating there is convergence across salivary and serum-based measures of BDNF in concussion. In short, our results add to previous research suggesting that saliva is not a universally reliable measure for BDNF²¹ and does not appear to distinguish between concussion and control groups. Yet, according to our findings, saliva may reliably measure BDNF under certain circumstances—namely after a recent concussion. Thus, the utility of salivary measures shows some promise in the ability to detect BDNF that correlates with serum-based BDNF. Future research is needed to further assess the potential of salivary BDNF following concussion.

The present study had several limitations that should be considered when interpreting our results and when designing future research. First, there was variability in the time of our samples. However, this factor was matched across participant groups and therefore unlikely to have affected our group comparisons. Nevertheless, future research should aim to collect samples at a consistent time of day. Second, diurnal variation was not accounted for in our study and has previously been related to BDNF levels in plasma samples of men, but not women (or serum samples of either sex).²³ Therefore, time of sample collection may more appropriately be set relative to the participant’s average wake time rather than an absolute time of day. Third, there was also variability in the time course of concussion recovery. Future research should aim to collect samples during a smaller window within the time course of recovery. In addition, the collection of multiple samples in each individual across the time course of recovery would strengthen our understanding of the dynamics in BDNF levels during recovery. Fourth, we had a relatively small sample size. Future research should replicate our effects in a larger sample. In sum, we used a highly matched sample, controlled for food and liquid consumption prior to sample collection, followed the standardized passive drool collection method, and controlled for stress-related negative affect. Yet, further standardization of BDNF collection should be implemented in future research. In particular, the time of sample acquisition relative to injury and chronobiology should be standardized.

Conclusion

Depression, anxiety, and stress levels were elevated in athletes with concussion. Based on our findings, no relationship exists between salivary and serum BDNF measures in the control group, yet this relationship was significant in the concussion group. Serum-based measures of BDNF may be useful in measuring the effects of concussion post return-to-play, but salivary measures are likely not effective at detecting group differences at this timeframe.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by the internal Northern Michigan University faculty research grants and a PRIME grant awarded to MTM and JMC as well as a Spooner grant awarded to TRS.

ORCID iD

Joshua M Carlson

References

Thurman

Branche

Sniezek

JE.

The epidemiology of sports-related traumatic brain injuries in the United States: recent developments. J Head Trauma Rehabil 1998; 13: 1–8.

Langlois

Rutland-Brown

Wald

MM.

The epidemiology and impact of traumatic brain injury: a brief overview. J Head Trauma Rehabil 2006; 21: 375–378.

McCrory

Traumatic brain injury: revisiting the AAN guidelines on sport-related concussion. Nat Rev Neurol 2013; 9: 361–362.

McCrory

Meeuwisse

Aubry

, et al. Consensus statement on concussion in sport: the 4th International Conference on Concussion in Sport held in Zurich, November 2012. Br J Sports Med 2013; 47: 250–258.

Greenberg

, et al. New insights in the biology of BDNF synthesis and release: implications in CNS function. J Neurosci 2009; 29: 12764–12767.

Zhou

Chen

Zhang

, et al. Protective effect of nerve growth factor on neurons after traumatic brain injury. J Basic Clin Physiol Pharmacol 2003; 14: 217–224.

Sofroniew

Howe

Mobley

WC.

Nerve growth factor signaling, neuroprotection, and neural repair. Ann Rev Neurosci 2001; 24: 1217–1281.

Failla

Conley

Wagner

AK.

Brain-derived neurotrophic factor (BDNF) in traumatic brain injury-related mortality: interrelationships between genetics and acute systemic and central nervous system BDNF profiles. Neurorehabil Neural Repair 2016; 30: 83–93.

Korley

Diaz-Arrastia

, et al. Circulating brain-derived neurotrophic factor has diagnostic and prognostic value in traumatic brain injury. J Neurotrauma 2016; 33: 215–225.

10.

Kim

Tsao

Stanfill

AG.

The current state of biomarkers of mild traumatic brain injury. JCI Insight 2018; 3. https://doi.org/10.1172/jci.insight.97105.

11.

Aydemir

Deveci

Taneli

The effect of chronic antidepressant treatment on serum brain-derived neurotrophic factor levels in depressed patients: a preliminary study. Prog Neuro-Psychopharmacol Biol Psychiatry 2005; 29: 261–265.

12.

Kaplan

Vasterling

Vedak

PC.

Brain-derived neurotrophic factor in traumatic brain injury, post-traumatic stress disorder, and their comorbid conditions: role in pathogenesis and treatment. Behav Pharmacol 2010; 21: 427–437.

13.

Tsai

Wang

, et al. Mood disorders after traumatic brain injury in adolescents and young adults: a nationwide population-based cohort study. J Pediatrics 2014; 164: 136–141.e1.

14.

Belanger

Curtiss

Demery

, et al. Factors moderating neuropsychological outcomes following mild traumatic brain injury: a meta-analysis. J Int Neuropsychol Soc 2005; 11: 215–227.

15.

Segal

SK.

Neuroscience meets salivary bioscience: an integrative perspective. Behav Neurosci 2016; 130: 156–175.

16.

Mandel

Ozdener

Utermohlen

Identification of pro- and mature brain-derived neurotrophic factor in human saliva. Arch Oral Biol 2009; 54: 689–695.

17.

Saruta

Fujino

, et al. Expression and localization of brain-derived neurotrophic factor (BDNF) mRNA and protein in human submandibular gland. Acta Histochem Cytochem 2012; 45: 211–218.

18.

Vrijen

Schenk

Hartman

, et al. Measuring BDNF in saliva using commercial ELISA: results from a small pilot study. Psychiatry Res 2017; 254: 340–346.

19.

Tirassa

Iannitelli

Sornelli

, et al. Daily serum and salivary BDNF levels correlate with morning-evening personality type in women and are affected by light therapy. Riv Psichiatr 2012; 47: 527–534.

20.

McGeown

Sanzo

Zerpa

, et al. The effects of exercise on cognitive function, balance, and salivary brain derived neurotrophic factor in healthy individuals – a pilot study. Res Neurosci 2016; 5: 15–23.

21.

Mandel

Ozdener

Utermohlen

Brain-derived neurotrophic factor in human saliva: ELISA optimization and biological correlates. J Immunoassay Immunochem 2011; 32: 18–30.

22.

Lovibond

Manual for the depression anxiety stress scales. Sydney: Psychology Foundation, 1995.

23.

Piccinni

Marazziti

Del Debbio

, et al. Diurnal variation of plasma brain-derived neurotrophic factor (BDNF) in humans: an analysis of sex differences. Chronobiol Int 2008; 25: 819–826.