## 4 Discussion  
Our study did not support a dose-dependent relationship between t-PBM and changes in neurometabolite levels. While an increase in NAA levels was observed, this finding was independent of the dose (irradiance level) of t-PBM therapy. There were correlations between neurometabolites and psychometric measures, including MADRS and SDQ scores, suggesting potential neurometabolic changes associated with MDD severity. However, it is important to note that these correlations do not establish causality and may reflect broader physiological or temporal fluctuations unrelated to specific treatment effects. The increase in NAA levels in both the one-visit and three-visit analyses warrants further investigation. NAA, a marker of neuronal health and mitochondrial function, is synthesized by the mitochondrial enzyme aspartate N-acetyltransferase, directly linking its levels to mitochondrial metabolism [38]. Reduced NAA levels have been frequently reported in patients with MDD, reflecting impaired mitochondrial function and neuronal integrity [39]. Although t-PBM is hypothesized to enhance mitochondrial function by interacting with cytochrome c oxidase, thereby boosting adenosine triphosphate (ATP) production and reducing oxidative stress [5], the increase in NAA levels after a single t-PBM session was not statistically significantly associated with any of the t-PBM doses or sham and could be attributable to natural fluctuations or time-related changes. In contrast, other neuromodulatory interventions, such as repetitive transcranial magnetic stimulation (rTMS), have shown dose-dependent increases in NAA levels within the dlPFC after multiple sessions, which were associated with improvements in depressive symptoms [40]. Similarly, increases in NAA have been reported in a study investigating repeated t-PBM administration for chronic traumatic encephalopathy [41]. However, that study targeted the anterior cingulate cortex (ACC) rather than the dlPFC and employed different parameters, including longer session durations (22-40 min), higher treatment frequency (three times per week over six weeks), and NIR wavelengths ranging from 810 to 870 nm [41]. These differences in stimulation protocols and target regions could explain the discrepancies in findings, as the full biological and clinical effects of t-PBM may require repeated administration over several weeks. The correlation between Cho levels and depression severity outcomes may offer insights into the pathophysiology of MDD, even though the observed changes in Cho levels were not dependent on t-PBM dose. While overall Cho levels remained stable post-therapy, we found a significant positive association between changes in Cho levels and changes in MADRS scores in the two-visit analysis (r = 0.59, \(p\) = 0.02). This finding suggests that increases in Cho levels compared to baseline were associated with less improvement in depressive symptoms, as higher MADRS scores indicate greater severity [34]. Brain Cho levels are considered markers of phospholipid synthesis and degradation, reflecting cell membrane turnover and integrity [42]. Alterations in choline metabolism have been implicated in psychiatric conditions, including MDD [43]. Our findings are consistent with prior work, such as [PERSON] et al. (2016), who observed a positive association between Cho levels in the left dlPFC of adolescents with MDD and Beck Depression Inventory scores, indicating that Cho levels rise with the severity of MDD symptoms [44]. However, readers should be aware that this is a correlation analysis, and it should not be interpreted as a cause-and-effect relationship. We observed a significant negative correlation between changes in Cr levels and changes in SDQ scores at medium t-PBM dose in the one-visit analysis (r = \(-\)0.91, \(p\) = 0.011), suggesting that higher Cr levels were associated with improved depression outcomes. However, it is important to emphasize that this association does not establish causality, as other factors may contribute to these observed changes.