Major depressive disorder (MDD) is a prevalent and debilitating psychiatric condition that affects millions of individuals worldwide [1-3]. Despite extensive research, the etiology of MDD remains complex and multifaceted, involving genetic, environmental, and neurobiological factors [4]. Recent studies have highlighted the potential role of glymphatic system dysfunction in the pathophysiology of MDD, suggesting a novel avenue for understanding its etiology [5-7].

The glymphatic system is a brain-wide network of perivascular channels that facilitates the flow of cerebrospinal fluid (CSF) and the clearance of metabolic waste products from the brain parenchyma. This system plays a crucial role in maintaining brain homeostasis by removing potentially toxic proteins and metabolites [8]. Glymphatic system dysfunction has been implicated in various neurological conditions, such as Alzheimer’s disease, traumatic brain injury, and even normal aging [9-11]. Depression is a common comorbidity in Alzheimer’s disease, and glymphatic dysfunction plays a role in this association [7].

Gu et al. [5] showed that glymphatic dysfunction induces oxidative stress and neuroinflammation in patients with MDD, suggesting a direct contribution of these pathological processes to depressive symptoms. Further supporting this notion, the regulation of stress-induced depression by ependymal cells and CSF flow emphasizes the critical role of CSF dynamics in mood regulation [12]. Loureiro-Campos et al. [13] discussed how disruptions in CSF circulation can influence depression, reinforcing the idea that glymphatic dysfunction may be intricately related to MDD pathogenesis. Additionally, polyunsaturated fatty acid supplementation alleviates depression-induced cognitive dysfunction by protecting the cerebrovascular and glymphatic systems, indicating that enhancing glymphatic function may have therapeutic benefits in depression [6]. Collectively, these findings suggest that the impaired glymphatic system may play a central role in the neurobiological foundations of MDD.

Several structural MRI findings suggest a possible connection between MDD and impaired glymphatic system. Studies have consistently reported lateral ventricular enlargement and increased CSF volumes in patients with MDD compared to healthy controls (HCs) [14,15]. Patients with MDD also show reduced volumes of several brain regions, including the hippocampus and basal ganglia, which are influenced by or contribute to impaired waste clearance in the brain [16,17]. Furthermore, recent studies have supported these findings by showing that enlargement of the brain ventricles and choroid plexus size can predict poor treatment response in patients with MDD [18]. Despite these observations, direct evidence linking glymphatic dysfunction to MDD in structural MRI studies remains limited. Diffusion tensor image analysis along the perivascular space (DTI-ALPS) index, calculated from diffusion tensor imaging (DTI) data, has been proposed as a measure of perivascular clearance activity in the human brain [19]. Importantly, as an MRI-derived surrogate, the DTI-ALPS index can be influenced by white-matter microstructure, crossing-fiber geometry, and scanner variability, and should not be interpreted as a direct measure of glymphatic clearance. Although not specifically studied in MDD, this index has shown associations with cognitive impairment in other neurological disorders and could potentially be applied to investigate glymphatic function in depression [20].

Synthesizing this literature, prior work has prioritized structures directly linked to glymphatic function, including the ventricles and choroid plexus. Accordingly, the present study aimed to investigate glymphatic system function in patients with MDD using advanced structural MRI techniques. Furthermore, we explored the relationships between glymphatic function impairment, depressive symptoms, glymphatic-related brain structural changes, and inflammatory cytokine levels. The first hypothesis was that patients with MDD show reduced glymphatic function compared to HCs. The second hypothesis is that glymphatic dysfunction is associated with enlargement of the ventricle and choroid plexus and levels of inflammatory cytokines. Based on the results of our second hypothesis, if factors are correlated with glymphatic function, we intend to verify through mediation analysis whether neuroinflammation accompanying the onset of depression affects glymphatic function and, consequently, can induce changes in brain volume. Understanding the role of glymphatic dysfunction in MDD could provide valuable insight into the neurobiology of the disorder and potentially lead to the development of novel therapeutic approaches targeting the glymphatic system. Identifying biomarkers associated with glymphatic function can also improve diagnosis and inform personalized treatment strategies for patients with MDD.

In this study, we investigated glymphatic dysfunction and related brain structural changes in patients with MDD, with the aim of exploring its association with inflammatory cytokines. Our findings revealed that patients with MDD exhibited significantly reduced glymphatic function, as indirectly estimated by decreased DTI-ALPS indices on the right and left sides, compared to HCs. Additionally, patients with MDD showed significant volumetric differences in the brain regions associated with glymphatic function, including the enlarged left inferior lateral ventricle. In particular, although DTI-ALPS scores were not associated with the severity of depressive symptoms or specific symptoms, such as sleep disturbances and appetite changes, after Bonferroni correction, the right DTI-ALPS index showed a statistically significant association with the duration of depression. Furthermore, decreased glymphatic function was correlated with large ventricular and choroid plexus volumes, and the right DTI-ALPS index showed significant negative correlations with the bilateral lateral ventricle and right inferior lateral ventricle, while the left DTI-ALPS index showed similar negative correlations. Conversely, both DTI-ALPS indices were positively correlated with thalamic volume, suggesting that better glymphatic function is associated with increased thalamic volume in patients with MDD. Furthermore, the inflammatory marker CRP showed significant negative correlations with bilateral DTI-ALPS indices and right choroid plexus volume. The mediation analysis highlighted that higher levels of CRP were associated with reduced DTI-ALPS indices, which, in turn, were associated with a reduced choroid plexus volume. These findings highlighted the impact of inflammation on glymphatic function and its subsequent effects on brain structures involved in CSF dynamics. The absence of significant mediating effects of the duration of illness suggests that the chronicity of the depressive illness itself may not play a direct role in linking inflammation with changes in choroid plexus volume. Instead, the inflammatory response exerts its influence primarily by impairing glymphatic clearance, leading to structural changes.

Our findings are consistent with those of previous studies that reported a decrease in DTI-ALPS indices in patients with depression, suggesting potentially impaired glymphatic function in patients with MDD [56]. Although previous research has shown a proportional relationship between DTI-ALPS and the severity of depressive symptoms, our study did not confirm such an association. In ischemic stroke and cerebral small vessel disease, lower DTI-ALPS values persistently correlate with white-matter lesion burden even after acute recovery, indicating an enduring clearance-impairment trait [57]. Similarly, in MDD, DTI-ALPS was reduced relative to controls yet did not track concurrent HDRS scores, and in the blood-consenting subgroup DTI-ALPS mediated the link between CRP and choroid-plexus volume—findings consistent with a longer-timescale mechanism. Taken together, these observations support DTI-ALPS as a potential trait marker reflecting glymphatic dysfunction in MDD rather than a state marker of momentary symptom severity.

Instead, we found that DTI-ALPS data showed a negative correlation with the duration of illness. This result has not previously been reported in patients with MDD; however, our findings are consistent with those of patients with bipolar disorder, in which the DTI-ALPS index was associated with the duration of illness [58]. In addition, patients with MDD who responded to treatment had significantly smaller volumes of the lateral ventricles and choroid plexus than non-responders, suggesting that ventricular enlargement may be related to treatment resistance and disease severity [18]. Furthermore, one of the interesting findings was that the bilateral decrease in the DTI-ALPS index was associated with an increase in the volume of the thalamus on each side. Numerous neuroimaging studies have reported that patients with MDD often exhibit reduced thalamic volume compared to HCs [59,60]. This thalamic atrophy has been associated with symptoms such as impaired cognitive function, disrupted emotional processing, and difficulties in sensory perception [61-63]. An association between the bilateral analysis DTI-ALPS index and increased thalamic volumes on both sides suggests that reduced perivascular water transport, as estimated by DTI-ALPS, might correlate with decreased function of thalamic structures in patients with MDD, potentially mitigating some cognitive and emotional symptoms associated with thalamic atrophy. Integrating these findings, our study suggests that glymphatic dysfunction is associated with factors that prolong the duration of illness or reduce treatment response in MDD, while the mediation model including illness duration did not indicate a significant indirect effect of DTI-ALPS on right choroid plexus volume. Specifically, this dysfunction, manifested as ventricular enlargement and decreased thalamic volume, can lead to a reduced response to treatment, which may contribute to the chronicity of depressive symptoms. As the glymphatic system plays a crucial role in the removal of metabolic waste from the brain, its dysfunction can lead to the accumulation of neurotoxic substances, exacerbating neuronal damage, and promoting ventricular enlargement. Consequently, glymphatic impairment can not only contribute to the prolonged duration of illness, but can also decrease the efficacy of treatments, highlighting its role in the pathophysiology of MDD and its potential as a therapeutic target. The glymphatic system is most active during sleep, especially during slow-wave sleep [64]. Previous studies in animal models and patients with Alzheimer’s disease have reported that glymphatic dysfunction is associated with decreased sleep quality [65,66]. Contrary to these findings, our study of patients with MDD did not reveal a correlation between glymphatic function and the severity of sleep disturbances. However, since our study did not directly assess specific indicators of sleep quality, we cannot definitively conclude an absence of association between glymphatic function and sleep quality in patients with depression. Further studies are required to determine the association between insomnia symptoms and glymphatic function in patients with depression.

The most important finding of this study was the analysis of inflammatory cytokines, which revealed that inflammation affects brain volume, mediated by glymphatic function. Within the depression group, CRP levels showed a significant negative correlation with the DTI-ALPS index and right choroid plexus volume, and mediation analysis was applied. In HCs, however, only left DTI-ALPS showed a weaker negative association with CRP, whereas right DTI-ALPS did not correlate with any inflammatory cytokines. This pattern suggests that the bilateral CRP-DTI-ALPS coupling observed in MDD is amplified or disorder-specific relative to controls. Whether glymphatic dysfunction precedes neuroinflammation or vice versa in depression has not yet been determined. Therefore, in this study, we conducted mediation analyses for both models: one in which neuroinflammation precedes glymphatic dysfunction and another in which glymphatic dysfunction precedes neuroinflammation. In both cases, we confirmed an indirect effect on brain volume. To date, chronic neuroinflammatory states are known to activate glial cells such as microglia and astrocytes, promoting the secretion of various cytokines [67,68]. Since functional changes in astrocytes can affect the function of the glymphatic system, astrocyte alterations due to neuroinflammation are more likely to induce glymphatic dysfunction [8]. Therefore, it is more probable that Model 1 (CRP→DTI-ALPS→choroid plexus volume model) reflects the changes that occur in depression. Furthermore, the choroid plexus, which showed an association with CRP levels in our study, has been consistently reported in previous research to show an increase in the volume in depression [51]. Our group-level comparisons confirmed this pattern, with MDD patients showing larger choroid plexus volumes than HCs. However, within the MDD group, higher CRP levels were associated with reduced right choroid plexus volume, which appears to contrast with prior findings. This discrepancy may reflect the substantial illness chronicity of our sample: under prolonged systemic inflammation, the choroid plexus may initially enlarge as a compensatory response but subsequently undergo atrophy due to cumulative inflammatory damage. Unlike previous studies that did not find a correlation between choroid plexus volume and CRP levels, our study demonstrated a strong association, possibly because we adjusted for factors such as medication use and applied Bonferroni correction, whereas an acknowledged limitation of previous studies is the lack of clearly defined covariates. In addition, inflammatory markers other than CRP did not show significant correlations in our results. CRP is a downstream acute-phase protein synthesized in the liver in response to upstream cytokines, particularly IL-6, reflecting the integrated output of multiple inflammatory pathways [69]. With a biological half-life of approximately 19 hours and minimal diurnal variation, CRP captures sustained inflammatory burden more reliably than individual cytokines such as IL-6 and IL-8, which are inherently phasic and reflect acute inflammatory responses [70,71]. Because the DTI-ALPS index is a trait-level microstructural measure that likely reflects cumulative changes in perivascular water transport, it is biologically plausible that CRP demonstrates stronger coupling with DTI-ALPS and choroid plexus volume than more transient cytokines [72-74]. Consistent with this, in our MDD cohort characterized by substantial illness chronicity, CRP but not IL-6 or TNF-α correlated with DTI-ALPS, supporting the view that sustained inflammatory tone is most relevant to glymphatic impairment. Therefore, more extensive research is needed to determine whether consistent findings regarding the associations between inflammatory cytokines, such as CRP levels, glymphatic function, and the choroid plexus, will continue.

The first strength of our study is that we recruited a relatively large number of patients with depression and HC, allowing us to evaluate and compare glymphatic function using MRI. Second, although it involved a subgroup, we measured various levels of cytokines that represent neuroinflammation (e.g., CRP, IL-1, IL-6, and TNF-α) and confirmed changes in glymphatic function, as well as in the volumes of associated brain regions. Third, rather than merely identifying simple correlations, we applied a mediation analysis to verify the associations among neuroinflammation, glymphatic function, and brain structural changes, which had been theoretically proposed in previous hypotheses, in actual clinical patients. Finally, even within the patient group analyses, we adjusted for various covariates, including medication use, sex, age, and years of education. We also confirmed our results under rigorous statistical criteria by applying the Bonferroni correction to adjust for statistical errors due to multiple comparisons in the brain structure analyses.

Despite these strengths, our study has several limitations. The most significant limitation is that, although the DTI-ALPS index provides a noninvasive proxy for perivascular water transport, it should be regarded as an indirect surrogate of glymphatic function [19,52]. The metric is derived from directional diffusivity and can be influenced by crossing-fiber geometry, local tissue anisotropy, and scanner variability. Moreover, validation of DTI-ALPS in psychiatric populations remains limited [20,58]. Therefore, interpretations in MDD should remain cautious. Future multimodal studies combining DTI-ALPS with quantitative CSF flow or tracer-based MRI will be essential to confirm its physiological specificity. A second important limitation is insufficient control for potential confounders of inflammation. Although we adjusted for available covariates including medication status, important factors such as body mass index, smoking status, fasting duration, and comorbid metabolic conditions were not accounted for. Furthermore, medication status was treated as a binary variable (medicated vs. medication-free), which does not capture the heterogeneous effects of different psychotropic medication classes on inflammation and brain structure. Although no significant differences in ALPS indices were observed between medicated and medication-free patients, stratified analyses by individual medication class were not feasible due to insufficient subgroup sizes (Supplementary Table 2). Future studies should incorporate comprehensive covariate assessment, standardized protocols, and detailed medication data to enable class-specific stratified analyses. Until such designs are implemented, the generalizability of the current findings should be interpreted with appropriate caution. The third limitation was the relatively small number of participants in the cytokine analysis and mediation analyses. Cytokine data were available only for a subgroup who consented to blood sampling (n=68 MDD, n=54 HC), and the final mediation analysis included only 41 MDD patients with complete data, which limits statistical power. To mitigate this, we employed bootstrap resampling with 5,000 iterations for robust confidence interval estimation. Additionally, this subsample exhibited longer illness duration and higher anxiety severity than the full sample, raising the possibility of selection bias, although key demographic and clinical variables (age, sex, education, ICV, and HDRS) did not differ significantly (Supplementary Tables 2 and 3). The longer illness duration may have enriched for chronic inflammatory profiles, potentially strengthening CRP-ALPS associations while limiting generalizability. Despite these constraints, the observed large effect sizes support the robustness of our findings; however, replication in larger samples with complete cytokine data is warranted. The fourth limitation is that in this study the measurements were limited to the lateral choroid plexus, which introduces several limitations. By excluding the choroid plexus in the third and fourth ventricles, this study may not have fully captured the variations and pathological changes specific to these regions. This restriction could lead to an incomplete understanding of the physiological functions of the choroid plexus and its role in neurological conditions. Consequently, the findings may not accurately reflect the overall dynamics and contributions of the choroid plexus system, potentially skewing interpretations related to brain health and disease. Future studies should consider incorporating all the choroid plexus regions for a more comprehensive analysis. Fifth, regarding statistical considerations, our multiple-comparison control targeted a pre-specified family of 17 glymphatic-relevant structural outcomes using Bonferroni correction, which aligns with common practice but may be less conservative than pooling all variables into a single family. Sensitivity false discovery rate analyses across a broader test set yielded qualitatively similar conclusions, although replication in larger samples remains essential. For the cytokine correlation analyses, we did not apply Bonferroni correction because these analyses were exploratory in nature, and CRP was designated a priori as the primary inflammatory marker based on its established relevance to systemic inflammation. Additionally, given the relatively small cytokine subsample size (n=68 for MDD, n=54 for HC), stringent correction would substantially increase the risk of Type II error. We therefore reported uncorrected p-values while emphasizing the large effect sizes observed for CRP associations to support the robustness of these findings. Finally, our examination of the glymphatic sleep interface was constrained to the sleep disturbance subscale of the HDRS. We did not administer standardized sleep questionnaires (e.g., Pittsburgh Sleep Quality Index) nor collect objective physiological indices such as actigraphy or overnight polysomnography. Future studies that incorporate both subjective and objective sleep metrics will be essential to delineate more precisely how specific aspects of sleep architecture and quality modulate glymphatic clearance in MDD.

This study identified a significant association between glymphatic dysfunction, neuroinflammation, and structural brain changes in patients with MDD. Patients with MDD exhibit reduced glymphatic function, as proven by decreased DTI-ALPS indices and volumetric alterations in the brain regions involved in CSF dynamics, such as enlarged ventricles and altered thalamic volume. In particular, CRP levels showed significant negative correlations with bilateral DTI-ALPS indices and right choroid plexus volume. Mediation analysis suggested that glymphatic function, as indirectly assessed by DTI-ALPS, mediates the relationship between neuroinflammation and brain volume. Although this mediating effect is believed to be associated with prolonged duration of illness or reduced treatment responsiveness in depression, these results should be regarded as preliminary and require replication in larger independent samples and longitudinal designs.