VIRAL-MEDIATED FLUORESCENT LABELING OF HYALURONAN REVEALS EXTRACELLULAR MATRIX DYNAMICS IN THE MOUSE BRAIN IN VIVO

The extracellular matrix (ECM) of the brain is primarily composed of the glycan polymer hyaluronan (HA), a core scaffold that nucleates proteoglycans into a self-assembled matrix that functions as structural framework and signaling hub. Since most of the neural matrix is composed of sugars, development of genetically encoded tags has been limited. Consequently, while several protocols exist for ECM staining in fixed tissue, reliable tools for live ECM imaging are lacking. Here we report a viral-mediated fluorescent probe that binds to HA and labels the brain ECM in mice. The vector encodes the HA binding domain from Neurocan fused to GFP and an externalization tag (AAV-Ncan-GFP), enabling transduced cells to secrete the fluorescent probe into the extracellular space and selectively label HA. We demonstrate stable ECM labeling in organotypic and acute rodent brain slices, as well as in vivo in the mouse cortex, visualizing both perineuronal nets and interstitial matrix. HA specificity is validated by colocalization with HABP and sensitivity to hyaluronidase, confirming extracellular localization by shadow imaging. As a proof of concept, we combine AAV-Ncan-GFP with dendritic spine imaging ex vivo and calcium imaging in vivo, providing a real-time map of local ECM alongside neural function. The probe further enables time-lapse imaging of ECM dynamics in live mice following brain ischemia, allowing longitudinal observation of matrix remodeling during pathology. Together, these results establish AAV-Ncan-GFP as a tool for real-time, in vivo investigation of brain ECM dynamics in parallel with neural structure and function.