3D imaging and reconstruction of cortical perineuronal nets in focal cerebral ischemia and mild hypoperfusion
Description
Perineuronal nets are predominantly expressed around the fast spiking parvalbumin expressing interneurons. In accordance with previous observations, the significant loss of cortical perineuronal nets (PNNs) was observed in the ipsilesional hemisphere following temporary occlusion of middle cerebral artery (MCAO). Importantly, the reduced expression of PNNs following MCAO and hypoperfusion was not associated with neuronal cell death in the ipsilesional cortex, but correlated with reactive gliosis in the ipsilesional striatum. However, the expression of PNNs was not completely abolished following ischemic stroke. Two major components of PNNs were immunolabeled using anti-aggrecan antibody (detecting the core protein of aggrecan, one of the most abundant proteoglycans within PNNs) and Wisteria floribunda agglutinin (WFA), binds to the disaccharide LacdiNAc chondroitin sulfate chains of CSPGs within the proteoglycans of PNNs. The ultrastructural analysis of PNN morphology was performed using superresolution structured illumination imaging, which revealed the subtle organization of these meshes. At all depths of the sample, imaging resolution comprised at least 200 nm in spatial directions and 400 nm in axial direction, as indicated by sub-resolution microsphere size analysis. To provide the quantitative analysis of the 3D organization of PNNs, a novel graph computation algorithm was developed, provided as MatLab code accompanying this article. The topological analysis of 3D meshes of single PNNs was conducted separately for aggrecan and WFA-labelled nets and the four key metrics were derived: i) percentage of nodes with non-zero degree; ii) maximal internode distance, at which 95% of the nodes have non-zero degree (this metric will be further abbreviated as R95); iii) average internode distance; iv) average degree of a node.