Unmasking the bias: Can diffusion-weighted imaging reliably assess glymphatic function in awake and anesthetized brain?

• Spectral ADC shows anesthesia-specific interstitial and microvascular space changes • K/X increases, ISO decreases CSF tracer influx compared with awake condition • ADC and IVIM do not correlate with CSF tracer transport measured by DCE-MRI • IVIM modeling cannot reliably separate slow and fast MR diffusivities across states • DWI alone cannot quantify state-related interstitial volume and transport changes This study investigated whether magnetic resonance (MR) diffusivity parameters derived from diffusion-weighted imaging (DWI) can serve as biomarkers of glymphatic function in awake and anesthetized mice. Spectral apparent diffusion coefficient (ADC) analysis, obtained using an inverse Laplace transform, revealed that both Isoflurane (ISO) and Ketamine/Xylazine (K/X) anesthesia reduced the magnitude and range of diffusivities associated with interstitial fluid space (≤1 µm²/ms) compared with the awake state. Perfusion-related diffusivities (20–80 µm²/ms) increased under ISO but decreased under K/X. Monoexponential ADC and biexponential intravoxel incoherent motion (IVIM) modeling showed that ISO dose-dependently elevated, while K/X reduced, both slow and fast diffusivities across the brain. Dynamic contrast-enhanced MRI (DCE-MRI) indicated intermediate glymphatic influx in awake mice relative to both anesthetic conditions, without regional correlation to DWI-derived parameters. Perfusion micro–computed tomography (µCT) further demonstrated that ADC and IVIM metrics correlated regionally with mean transit time, suggesting confounding by cerebral blood flow (CBF). Two-photon microscopy confirmed anesthesia-induced changes in cortical microvessel diameters consistent with perfusion alterations. Collectively, these findings indicate that MR diffusivity measures are strongly influenced by state-dependent physiological changes, and in particular perfusion. This represents an important limitation that warrants caution when using DWI to compare extracellular space, interstitial fluid flow, or glymphatic activity across different physiological or anesthetic states. Therefore, although technically challenging, we suggest that DWI studies aimed at assessing glymphatic or interstitial dynamics should be performed in awake conditions to minimize variability and anesthesia-related perfusion confounds across studies.