DOI: 10.6084/m9.figshare.31113075.v1
title: miR-26b-5p responds to aerobic exercise intervention for concussion recovery in adolescent athletes: a pilot trial

description: Guidelines for sport-related concussion (SRC) management recommend aerobic exercise treatment. Salivary microRNAs (miRNAs) associate with exercise and concussion; therefore, we investigated their expression in adolescents with SRC during prescribed exercise. Before and after intervention, athletes with SRC (<i>n</i> = 32, 15.78 ± 1.5 y/o, 66% male, 5.26 ± 3.56 days from injury) and controls (<i>n</i> = 28, 15.96 ± 1.7 y/o, 68% male) provided saliva samples and completed clinical exams. High (5 days/week, 30 min) or low (3 days/week, 20 min) volumes of aerobic exercise were assigned. RT-qPCRs were performed to assess 13 pre-selected miRNAs. miRNAs 1246, 944, 151a-3p, and 26b-5p were reduced (<i>p</i> = 0.0002, 0.0024, 0.0015, 0.0002, CI mean difference: [−4.55, −1.54] [−4.58, −1.08] [−4.77, −1.24] [−5.64, −1.92], respectively) compared to controls. The expression of 182-5p, 532-3p, and 26b-5p increased with exercise volume in concussed participants (<i>p</i> = 0.0035, 0.0035, 0.0020, CI for exercise coefficient: [0.000100, 0.000396] [0.000060, 0.000237] [0.000107, 0.000373], respectively), but not in controls. miRNAs 1246, 944, and 151a-3p may be concussion biomarkers unaffected by aerobic exercise. miR-26b-5p, associated with concussion and exercise volume, may be a useful biomarker to monitor adherence to exercise prescriptions for SRC recovery. This study was pre-registered at Clinicaltrials.gov (NCT05379930). The analysis plan was not formally pre-registered. Power analysis was done using a simulation model and was calculated with pilot miRNA data using 1000 simulations at a p-value of 0.05. This simulation determined that we needed to recruit and retain 30 participants in our experimental group to reach a power of 80%, which was achieved. All participants are accounted for in our CONSORT Diagram in Supplementary File 3. All participants who were randomized provided saliva samples, and all saliva samples were analyzed. No deaths occurred during the study. Incomplete examinations occurred only because of participants who were lost to follow-up (Supplementary File 3). Participants were not randomized and were assigned to groups based on stratification by sex and sequential presentation. Participant blinding was not possible because of the nature of the therapeutic intervention, but participants were blinded to the study hypothesis. Investigator blinding was not fully possible because of the nature of the therapeutic intervention. The treating physician was blinded to intervention assignment. Human participants were blinded to the results of the fluid biomarker measurements. Handling of biofluid samples was performed by team members who were aware of the relevant characteristics of the participants. Fluid biomarker measurements were performed by investigators who were aware of the relevant characteristics of the participants. Fluid biomarker quality control decisions and analyses were performed by investigators who were aware of the relevant characteristics of the participants. Samples were acquired between March 2022 and February 2023. Samples were acquired between 9:00 AM and 3:00 PM. All samples were analyzed at the same time in a single batch. No unexpected events occurred in sample handling and analysis during the study. The number of technical replicates was 3 per sample. Technical replicate results were combined by averaging. No future use of these biofluid samples is possible because of insufficient remaining quantities. All equipment and analytical reagents used to perform measurements on the fluid biomarkers are widely available commercially. The key inclusion criteria2 are established standards in the field. The statistical tests in the linear models were based on independence of observations, correct model selection, and asymptotic normality assumptions for maximum likelihood random variables. Chi-squared tests for categorical variables assumed independence of observations, and two-sample t-tests for continuous variables assumed near normality in each condition. Statistical analysis was performed by Jeffrey C. Miecznikowski, PhD (Statistics). The family-wise error rate across miRNAs at level 0.05 was controlled by the Bonferroni method to correct for multiple comparisons. No replication or external validation studies have been performed or are planned/ongoing at this time to our knowledge. De-identified data from this study are not available in a public archive. De-identified data from this study will be made available (as allowable according to institutional IRB standards) by emailing the corresponding author as of May 20th, 2025. The analytic code used to conduct the analyses presented in this study is not available in a public repository. The authors agree to provide the full content of the manuscript on request by contacting the corresponding author.