## 3 Results  
### Demonstration of Oxidative Stress Effect on Ex Vivo Models  
A gene expression analysis was performed after repeated induction of oxidative stress on skin scalp explants. To explore the global effect of oxidative stress, we selected genes potentially responsible for canities development and involved in these three major functions: antioxidant activities, renewal, melanogenesis and related functions. In the category of renewal, we introduced hair cycle-related genes and genes involved in apoptosis and autophagy. The main objective of this study was to understand the impact of oxidative stress in the greying process that we hypothesised to be a key inducer. After the oxidative stress, the results in Figure 1 demonstrated a significant down-regulation of genes involved in autophagy (ATG7, MAP1 LC3A). The significant down-regulation of KRT19, COX2, FST and BAX might describe a reduction of proliferation and the activation of stem cell quiescence. Moreover, the oxidative stress showed a clear effect on melanogenesis by down-regulating the expression of various genes directly linked to this synthesis (MITF, TYR, DKK1, EDN1) but also genes encoding for melanogenesis-related functions, such as melanosome formation and transfer (CTNS, HPS5, MLANA, MLPH, MYO5A, SLC24A5). The expression of the melanocyte precursor cell marker gene (DCT) was also significantly down-regulated. Finally, there was a significant negative impact on the expression of genes involved in antioxidant activities (FTL, GLRX, HMOX1, MGST1, PRDX1 and SOD2) even though the expression of GPX1 was activated. Overall, these results suggest that oxidative stress was able to induce a strong impact on renewal, pigmentation and antioxidant defence, which are the three main causes of canities development. Pursuing our investigation further, we attempted to induce oxidative stress in microdissecreted hair follicles in order to confirm the hypothesis proposed following the gene expression results. After the stress, we observed a significant production of reactive oxygen species (ROS) inside the hair follicles, 264% in comparison with the untreated condition (Figure 2). Moreover, we evaluated the number of NKI/beteb-positive cells to observe the effect of oxidative stress on the melanocyte reservoirs. The counting of the red fluorescent cells showed a significant decrease by 78% of the number of NKI/beteb-positive cells compared with the untreated condition (Figure 3). Figure 1: Gene expression analysis in full scalp skin explants after oxidative stress. The results (mean \(\pm\) SEM) are expressed as relative expression versus the untreated condition. Statistical analysis was performed using one-way ANOVA followed by [PERSON]’s \(t\)-test in comparison with the untreated condition, with \({}^{*}p<0.05\), \({}^{**}p<0.01\) and \({}^{***}p<0.001\). These results confirmed that oxidative stress triggers the redox balance, as observed with the reduction of gene expressions coding for antioxidant enzymes but also by the ROS release. As a consequence, it induced stem cell quiescence and the depletion of the melanocyte reservoirs, as demonstrated at the transcriptomic level and by the strong reduction of melanocyte detection. Finally, it also reduced the ability to synthesise melanin. In this first part of this work, we evidenced that oxidative stress triggered biological dysregulations which are the main causes of canities and, therefore, demonstrated the key role of oxidative stress in its development. We used this canities representative model for further investigations. Figure 2: Reactive oxygen species (ROS) production assessment following oxidative stress (cumene hydroperoxide at 50 \(\upmu\)m) in hair follicles. (**A**) Representative images. Magnification: 20\(\times\). (**B**) Quantification of the mean fluorescence intensity (\(\pm\) SEM) of dichlorofluoorescein diacetate (DCFH-DA) probe. Statistical analysis was performed using one-way ANOVA with a permutation test followed by [PERSON]’s test, with **_p_ < 0.01.