Investigating Whether Thymol Pre-Exposure Improves Regeneration Capacity in Hydra vulgaris Following Hydrogen Peroxide (H₂O₂)-Induced Oxidative Stress

Abstract

Regeneration in Hydra vulgaris is driven by the proliferation and differentiation of interstitial stem cells. When exposed to 1 mM hydrogen peroxide (H2O2), a reactive oxygen species (ROS) that induces oxidative stress, Hydra experience DNA damage. This study investigated whether pretreatment with thymol, an antioxidant, is able to preserve the regenerative capacity of Hydra vulgaris under oxidative stress (Tsarkova, Elena, et al, 2023). It was hypothesized that thymol would mitigate oxidative damage and improve regeneration in the presence of H2O2. Hydra were starved for 7 days before experimentation. Five Hydra per trial were trisected around the mid-gastric region to induce regeneration. A toxicity test identified that 10µM thymol was the ideal non-toxic working concentration (Mann-Whitney U Test, p < 0.01, compared with 20µM). Four experimental groups were evaluated: a positive control (thymol), a negative control (H2O2), a control (Hydra Medium), and the treatment (H2O2+ thymol). Regeneration was assessed at 24, 48, and 72 hours using a standardized 0-5 regeneration scale. There was a statistically significant difference in mean regeneration scores between Hydra exposed with H2O2 and Hydra pretreated with H2O2 + thymol (Mann-Whitney U Test, p < 0.01). Hydra pretreated with thymol under oxidative stress conditions demonstrated improved regeneration compared to the 1mM H2O2 only group. These findings suggest that thymol protects regeneration by reducing ROS damage and preserving stem cell-mediated regeneration. Hydra vulgaris provides a cost-effective screening model for evaluating compounds that protect regenerative capacity under oxidative stress and supports further investigation of thymol and related antioxidants in regenerative biology.