Perineuronal nets (PNNs) are specialized, mesh-like structures of the brain's extracellular matrix (ECM) that enwrap parvalbumin-positive (PV+) interneurons in the cortex. Despite their known role in neuroplasticity regulation, the limited resolution of conventional microscopy hindered quantitative analysis of PNN morphology. This study aimed to quantify morphological rearrangements in PNNs after experimental stroke in mice and evaluate their role in post-stroke brain remodeling.

Stroke was induced by transient medial cerebral artery occlusion (tMCAO) in male mice. PNN morphology and glutamatergic synapse density (GSD) of motor cortical layer 5 (L5) interneurons were analyzed during the post-acute stroke phase at 7, 14, and 28 days postischemia (dpi). Glutamatergic synapses were detected by immunohistochemical labeling of vesicular glutamate transporter-1 (Vglut-1) in axonal terminals. PNNs were labeled with the Wisteria floribunda agglutinin binding to proteoglycans enriched in PNNs. With super-resolution structured illumination microscopy (SR-SIM) and 3D image reconstruction we analyzed PNN topology beyond optical systems' resolution limit. Starting at 7 dpi, we observed a significant increase in the average internode distance (AID) and PNN node count in the ipsilesional and contralesional motor cortex, signifying a loosened PNN-mesh. This persisted at 14 and 28 dpi. PNN-adjacent GSD increased at 14 dpi in both hemispheres and persisted at 28 dpi. These data suggest that PNN reorganization precedes and potentially facilitates glutamatergic synapse formation in the vicinity of PNNs, highlighting their role in post-stroke neuroplasticity regulation. Noteworthy, the sequential PNN and synapse density changes were observed in regions distant from the stroke lesion during the post-acute stroke phase, indicating a novel poststroke neuroplasticity and brain remodeling mechanism.

The synapse formation facilitated by post-stroke PNN remodeling may be driven by the enrichment of chondroitin sulfate proteoglycans (CSPGs) on the PNN surface and their impact on axonal growth cones and synapses. Alterations in PNN morphology are likely mediated by extracellular enzymes such as matrix metalloproteinases released by reactive glia. In future research, precise modulation of PNNs and ECM degrading systems after stroke can help to promote stroke recovery in patients.