Fig 1.
Samples and analyses in the present study.
HTH, Hits to the Head; PCA, principal component analysis.
Table 1.
Saliva and serum samples used for miRNA analysis.
Table 2.
Functional outcome measures.
Table 3.
Sample classifications used in analysis, separated by fluid type.
Table 4.
Significant effects on functional data obtained during biofluid sampling.
Fig 2.
Hits to the head increase postural sway post-fight versus pre-fight.
MMA fighters who provided saliva or serum samples were classified into three different TBI likelihood categories (Low, Moderate, Very Likely) based on video recordings. Note that one of the sway measures was obtained during a cognitive task performance (Digit Span Backwards, upper) while the other was obtained during a balance test performed without visual guidance (Two Legs, Eyes Closed, lower). The increase in sway is evident for both sets of measures in the Moderate and Very Likely groups compared with Low TBI likelihood groups.
Fig 3.
Changes in body sway or completion time scores post-fight are less consistent in two different dual-task functional tests.
Subjects are grouped by TBI likelihood. Same conventions as Fig 2. Note slightly elevated scores in the Very Likely group of the TMB_Bal task (upper) when a serum (but not a saliva) sample was taken, and the slight elevation in the TMA_Cog score (lower) in the Moderate (but not Very Likely) group.
Fig 4.
Change in serum UCHL1 post-fight related to hits to the head (HTH).
Note that this regression was largely driven by 4 fighters who received more than 30 HTH. Overall, however, there was no significant difference in the group of fighters post-fight versus pre-fight.
Fig 5.
Principal component analysis (PCA) of miRNA data.
The data were normalized across biofluid types and TBI likelihood prior to statistical analysis. The image at left shows intermixing of the samples, with only a slight suggestion of separation of Very Likely serum samples (green boxes) from the main data cloud. When all the data are collapsed, the change values are distributed in a highly normal fashion (lower right).
Fig 6.
Effects of HTH (mTBI likelihood) on miRNA expression changes in serum and saliva.
A total of 925 miRNAs were tested, with 21 showing a significant main effect of TBI likelihood, of which two also showed a significant main effect of Fluid and two showed a significant Fluid x TBI interaction.
Table 5.
miRNAs with changes related to TBI likelihood.
Fig 7.
Accuracy of predicting highest and lowest TBI likelihood based on changes in miRNA expression from serum or saliva.
Stepwise linear regression was used to pre-select an optimal number of miRNAs for prediction of Hits to the Head (HTH) values, and this set of 13 was subjected to 100-fold Monte Carlo Cross Validation (MCCV) using Random Forest, in order to estimate classification accuracy for distinguishing Very Likely from Low likelihood TBI samples.
Table 6.
Logistic regression model performance for binary HTH classification using 13 miRNAs.
Fig 8.
Changes in miRNA expression levels in saliva and serum post-fight, binned according to HTH levels /mTBI likelihood.
Data displayed are whisker-box plots. Each row represents a different miRNA (three miRNAs are shown), and each dot represents the expression level of that miRNA in a sample. Note that some miRNAs showed a pattern of increase in both biofluids post-fight (30b-5p, top), while others showed a change that was most evident in only a single biofluid type (e.g., 92a-3p and 122-5p).
Table 7.
Biological pathways overrepresented by target genes of HTH-related miRNAs.
Table 8.
Correlations between changes in miRNA levels (post-fight), HTH, and functional measures.
Fig 9.
miRNAs with changes in abundance due to Time, Fluid, and Interaction effects in serum and saliva.
Venn diagram denotes the number of genes with significant effects of Time, Fluid, or the Interaction (Intx) of Time x Fluid.
Table 9.
47 miRNAs with significant effect of time in relation to MMA fight in saliva and serum.
Fig 10.
Detection of acute saliva response (ASR) miRNAs.
12 miRNAs were identified with robust temporal effects (all increases) at the 1 hr Post-fight time point (blue shaded area) in saliva samples (upper) that exceeded those at the non-specific exercise- or event-related timepoint (green shaded area). Note that most of the miRNAs returned to near baseline by 2–3 days Post-fight. The pattern for the same miRNAs was distinctly different in serum (several were unchanged and several had delayed decreases).
Fig 11.
Detection of miRNAs with delayed serum response (DSR).
A subset of miRNAs were identified with predominantly delayed increases (solid lines) and decreases (dashed lines) in serum at 1 week Post-fight (upper, blue shaded area) that exceeded those at the non-specific exercise- or event-related timepoint (green shaded area). Note that these miRNAs were unchanged or showed some evidence for non-specific increases in saliva (lower).
Table 10.
Top biological pathways overrepresented by acute saliva response miRNAs.
Table 11.
Top biological pathways overrepresented by delayed serum response miRNAs.
Fig 12.
Functional measures correlated with acute saliva response miRNAs.
Solid lines show cognitive measures (higher values indicate better performance). Dashed lines show normalized body sway measures (higher values indicate worse performance). Note that cognitive measures showed a trend for drop in performance at the 1 hr post-fight time point, while body sway showed an increase at the same time point. Also note that two of the cognitive measures (TMB_COG and TMB_Dual_COG) showed an apparent learning effect (improved performance across time, other than the immediate post-fight time point). A learning effect was also seen in 1 of the balance measures (TLEOFP), with decreased body sway evidence across time, other than the immediate post-fight time point.
Fig 13.
Functional measures correlated with delayed serum response miRNAs.
Solid line shows a balance measure (TSEO) with apparent learning effects (decreased sway at the No HTH control and 1 hr Post-fight time points) that subsequently showed increased sway at 2–3 days Post-fight. The dashed lines indicate two balance measures with delayed effects (TMB_Dual_Bal) or acute plus delayed effects (DSB_Bal).