DTI-ALPS REFLECTS WHITE MATTER DIFFUSION ANISOTROPY AND MICROSTRUCTURAL HETEROGENEITY RATHER THAN GLYMPHATIC FLOW: INSIGHTS FROM MULTI-MODAL MRI

The glymphatic system plays a critical role in the clearance of metabolic waste and pathological proteins such as amyloid-β and tau from the brain, and its dysfunction has been increasingly implicated in the pathogenesis of neurodegenerative diseases, making its in vivo characterization a major research priority. The diffusion tensor imaging–analysis along the perivascular space (DTI-ALPS) has been widely adopted as a putative non- invasive MRI biomarker of glymphatic flow. However, the biological specificity of the DTI- ALPS index remains unclear, and recent evidence has raised concerns that it may primarily reflect white-matter (WM) microstructure rather than perivascular fluid transport. In this study, we sought to clarify the determinants of DTI-ALPS and to assess whether this metric primarily reflects WM microstructural damage rather than glymphatic activity. A total of 100 participants were recruited from individuals referred to the Neurodegenerative Diseases Unit of the Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico in Milan (Italy) for suspected dementia. All the participants underwent the same 3T MRI protocol comprising high-resolution T1-weighted and FLAIR imaging, double-shell diffusion-weighted imaging (b=1000 and 2000 s/mm2), and multi-echo gradient-echo sequences for quantitative susceptibility mapping. Within standard centrum semiovale regions of interest, we computed DTI-ALPS together with fractional anisotropy (FA), mean diffusivity (MD), mode of anisotropy (MA), and neurite orientation and density imaging (NODDI) metrics, including the orientation dispersion index (ODI), neurite density index (NDI), and isotropic volume fraction (FISO). We also computed the T1/FLAIR signal ratio and the diamagnetic component of susceptibility (DCS) derived from DECOMPOSE within the same ROIs, as complimentary structural metrics of WM damage. Lower DTI-ALPS values were associated with higher FA and MD (|r|=0.40–0.60), with stronger effects observed at b=2000 than at b=1000. The DTI-ALPS index correlated strongly and inversely with MA (r=–0.84 at b=1000; r=–0.86 at b=2000) and positively with ODI (r=0.73), and both metrics remained strong independent predictors after correction for age and sex. Moderate associations were observed with structural markers—direct with the T1/FLAIR ratio (r 0.33) and inverse with DCS standard deviation (r -0.50) —consistent with altered myelin integrity and increased microstructural heterogeneity. Exploratory factor analysis demonstrated that DTI-ALPS did not form a separate latent component but clustered with MA and ODI, indicating shared variance related to axonal geometry rather than to perivascular flow. Collectively, these findings demonstrate that DTI-ALPS predominantly reflects WM architecture—specifically, loss of crossing fibers, increased diffusion coherence, and reduced orientation dispersion—rather than the dynamics of glymphatic transport. Although DTI-ALPS may still capture secondary effects of glymphatic impairment arising from WM injury, the present results suggest that it cannot be considered a direct biomarker of glymphatic function. Its use as a surrogate measure of glymphatic flow should therefore be approached with caution and re-evaluated through direct validation against intrathecal contrast-enhanced MRI or other physiological reference methods.