3D imaging and reconstruction of cortical perineuronal nets in focal cerebral ischemia and mild hypoperfusion

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.

神经元周围网状结构（Perineuronal nets, PNNs）主要表达于快速放电的小白蛋白阳性中间神经元周围。与既往研究结果一致，在临时大脑中动脉闭塞（middle cerebral artery occlusion, MCAO）后的患侧大脑半球皮层中，观察到PNNs出现显著丢失。值得注意的是，MCAO及低灌注后PNNs的表达下调，与患侧皮层的神经元死亡并无关联，而是与患侧纹状体的反应性胶质增生呈正相关。但在缺血性脑卒中后，PNNs的表达并未完全消失。本研究采用抗聚集蛋白聚糖抗体（anti-aggrecan antibody，用于识别PNNs中丰度最高的蛋白聚糖之一——聚集蛋白聚糖的核心蛋白）以及紫藤凝集素（Wisteria floribunda agglutinin, WFA）对PNNs的两种主要组分进行免疫标记，后者可结合PNNs蛋白聚糖中含二糖LacdiNAc的硫酸软骨素链。采用超分辨率结构照明显微镜对PNNs的形态进行超微结构分析，揭示了这类网状结构的精细组织结构。通过亚分辨率微球尺寸分析验证，在样本的所有深度层面，成像分辨率在空间方向上至少可达200 nm，轴向方向可达400 nm。为实现PNNs三维组织结构的定量分析，本研究开发了一种新型图计算算法，并以配套MatLab代码的形式随本文一同发布。针对单个PNNs的三维网状结构，分别对聚集蛋白聚糖标记与WFA标记的网状结构进行拓扑分析，并得到四项核心指标：① 非零连接度节点占比；② 95%节点均具有非零连接度时的最大节点间距（该指标后续将缩写为R95）；③ 平均节点间距；④ 节点平均连接度。