The Impact of Overtraining on Anxiety-Like Behavior and Tight Junction Proteins of the Blood-Brain Barrier in Mice.

PURPOSE: Overtraining Syndrome (OTS) is a chronic maladaptive condition characterized by persistent performance decline due to excessive training and inadequate recovery. OTS is associated with mood disturbances, but the underlying neurobiological mechanisms remain unclear. This study investigated whether a 6-week overtraining (OT) protocol induces anxiety-like behavior and changes in hippocampal BBB-related tight junction proteins in mice.

METHODS

Eight-week-old male C57BL/6J mice were assigned to sedentary (SED, n = 5), moderate exercise (ME, n = 5), or overtraining (OT; downhill treadmill running, initially n = 10) groups and completed a 6-week training protocol. The final OT sample size was n = 4 after 6 of the 10 mice were excluded due to tail or hindlimb injuries. Endurance capacity was assessed using an incremental loading test (ILT). Anxiety-like behaviors were evaluated using the nest building test (NBT), open-field test (OFT), and elevated plus maze (EPM). Hippocampal tight junction (TJ) proteins zonula occludens-1 (ZO-1), occludin, claudin-5, as well as tumor necrosis factor-alpha (TNF-α) were quantified by Western blot analysis.

RESULTS

After 6 weeks, endurance performance in the OT group significantly decreased from baseline (p < 0.01) and was lower than that of the ME group at Week 6 (p < 0.0001). In the quadriceps, TNF-α protein levels were significantly elevated in the OT group compared with both the SED (p < 0.05) and ME groups (p < 0.01). In the OFT, OT mice exhibited fewer center entries and reduced time spent in the center zone versus the SED and ME groups (p < 0.05), indicating increased anxiety related behavioral change. In the hippocampus, zonula occludens-1 (ZO-1) levels were reduced and TNF-α levels were elevated in OT compared with SED (p < 0.01 and p < 0.05, respectively), whereas claudin-5 levels were higher in ME than SED (p < 0.05).

CONCLUSION

OT was associated with anxiety-like behavioral alterations and changes in hippocampal BBB-related tight junction proteins. Reduced expression of TJ proteins and increased neuroinflammation may contribute to affective symptoms observed in OTS. However, given the small final sample size in the OT group, these findings should be interpreted with caution.