Piezo1 induces Wnt7b ⁺ astrocytes transformation to modulate glial scar stiffness and neuro-regeneration after stroke

<b>Background:</b> Reactive astrocytes form a chemical and mechanical glial scar that inhibits neuro-regeneration after stroke. Astrocyte heterogeneity is accompanied by changes in morphology and mechanical properties altering during scar formation after injury. This work aimed to elucidate the relationship between glial scar stiffness and astrocyte subtype transformation. <b>Methods:</b> Astrocyte-specific archaerhodopsin-3 and channelrhodopsin-2 knock-in C57BL/6J mice underwent distal MCAO. Atomic force microscopy, ultrasound elastography and synchrotron radiation were used to determine changes in glial scar stiffness. A proteomic analysis of astrocyte subtypes was performed <i>ex vitro</i> using single-cell laser capture microdissection-MS. Furthermore, optogenetics was employed <i>in vivo</i> to reduce the glial scar stiffness, thereby facilitating neural regeneration following brain injury. <b>Results:</b> Glial scar stiffness systematically increases following stroke and correlates with an increased number of Wnt7b⁺ fibrotic astrocytes. Furthermore, these results indicate that Piezo1 is the key regulator of astrocytic stiffness and anisotropy, which contributes to the glial scar stiffness in the peri-infarct area. The downregulation of Piezo1 expression promotes activation of the Wnt7b-Ca²⁺ nonclassical signaling pathway to modulate cytoskeletal reorganization. Finally, the specific optogenetic inhibition of Ca²⁺ signaling in astrocytes can effectively reduce glial scar stiffness by decreasing the proportion of Wn7b⁺ astrocytes, which further promotes neuro-regeneration and improves the recovery of motor function after ischemic stroke. <b>Conclusions:</b> This study successfully revealed astrocyte subtype transformation as a key determinant of glial scar physical barrier formation after stroke and highlighted Piezo1 as a potential therapeutic target for modulating the mechanical microenvironment post-injury.