Daniel Manrique-Castaño

The pathobiology of ischemic stroke is associated with the activation of brain platelet-derived growth factor receptor (PDGFR)β+ perivascular cells. While Krüppel-like factor (KLF)4 is a well-established mediator of peripheral PDGFRβ+ cell activation, its role in brain PDGFRβ+ cells remains elusive. Herein, we aimed to characterize the spatiotemporal reactivity of brain PDGFRβ+ cells, while assessing KLF4 contribution upon experimental ischemic stroke using transgenic mice that enable tracking PDGFRβ+ cells or conditionally depleting KLF4 in PDGFRβ+ cells. Moving beyond the qualitative descriptors, we sought to develop point pattern analysis (PPA) and topological data analysis (TDA) workflows to generate robust and reusable mathematical models to assess spatial cell responses and tissue scar topological changes. First, we report the emergence of phenotypically distinct reactive parenchymal PDGFRβ+ cells, which cannot be causally and exclusively linked, as it is currently proposed, to local cell proliferation or adjacent vascular detachment. Furthermore, we show that KLF4 is barely expressed in brain PDGFRβ+ cells under normal conditions and that it is only marginally induced in reactive PDGFRβ+ cells at the injured brain. Notably, the conditional attenuation of KLF4 expression specifically in reactive PDGFRβ+ cells does not alter spatial cellular distribution nor scar organization. This suggests that, in contrast to peripheral PDGFRβ+ cells upon ischemic insults, KLF4 is not a main regulator of the reactivity of brain cells in response to ischemic stroke. Ultimately, our findings supported by open-source spatiotemporal models indicate that the reactivity of brain PDGFRβ+ perivascular cells is highly heterogeneous and fundamentally distinct from peripheral PDGFRβ+ cells.

Summary CNS injuries are associated with profound changes in cell organization. This protocol presents a stepwise approach to quantitatively describe the spatiotemporal changes in glial cell rearrangement in the injured murine brain, which is applicable to other biological contexts. Herein, we apply common immunolabeling of neurons and glial cells and wide-field microscopy imaging. Then, we employ computational tools for alignment to the Allen Brain Atlas, unbiased/automatic detection of cells, generation of point patterns, and data analysis. For complete details on the use and execution of this protocol, please refer to Manrique-Castano et al.1

Glial scar formation represents a fundamental response to central nervous system (CNS) injury. It is mainly characterized by a well-defined spatial rearrangement of reactive astrocytes and microglia. The mechanisms underlying glial scar formation have been extensively studied, yet quantitative descriptors of the spatial arrangement of reactive glial cells remain limited. Here, we present a novel approach using point pattern analysis (PPA) and topological data analysis (TDA) to quantify spatial patterns of reactive glial cells after experimental ischemic stroke in mice. We provide open and reproducible tools using R and Julia to quantify spatial intensity, cell covariance and conditional distribution, cell-to-cell interactions, and short/long-scale arrangement, which collectively disentangle the arrangement patterns of the glial scar. This approach unravels a substantial divergence in the distribution of reactive astrocytes and microglia after injury that conventional analysis methods cannot fully characterize. PPA and TDA are valuable tools for studying the complex spatial arrangement of reactive glia and other nervous cells following CNS injuries and have potential applications for evaluating glial-targeted restorative therapies.

Despite the crucial role of perineuronal nets (PNNs) in neural plasticity and neurological disorders, their ultrastructural organization remains largely unresolved. We have developed a novel approach combining superresolution structured illumination microscopy (SR-SIM) and mathematical reconstruction that allows for quantitative analysis of PNN topology. Since perineuronal matrix is capable to restrict neural plasticity but at the same time is necessary to maintain synapses, we hypothesized that a beneficial post stroke recovery requires a reversible loosening of PNNs. Our results indicated that focal cerebral ischemia induces partial depletion of PNNs and that mild hypoperfusion not associated with ischemic injury can induce ultra-structural rearrangements in visually intact meshworks. In line with the activation of neural plasticity under mild stress stimuli, we provide evidence that topological conversion of PNNs can support post stroke neural rewiring.

ABSTRACT Tissue scarring upon cerebral ischemia entails a cascade of multifaceted cellular and molecular mechanisms that govern the remodeling of the neurovascular unit, which integrates neuronal, glial, and vascular functions.  The process encompasses inflammation, glial reactivity, vascular reactivity, and neuronal remodeling. In this chapter we cover three major aspects involved in tissue scarring after cerebral ischemia. First, we outline the primary cellular mechanisms underlying glial scar formation, emphasizing on the interactions between astrocytes, microglia, and mural cells, including pericytes and fibroblasts at the injury core and perilesional areas. Next, we address the key routes of extracellular matrix deposition by reactive and fibrogenic cells, including proteoglycans, tenascins, fibronectin, and collagen. Finally, we discuss the promises and challenges of manipulating tissue scarring as a strategy to promote brain structural remodeling and neurological recovery.

As an endogenous activator of toll-like receptor-4 (Tlr4), the extracellular matrix glycoprotein tenascin-C (TnC) regulates chemotaxis, phagocytosis and proinflammatory cytokine production in microglia. The role of TnC for ischemic brain injury, post-ischemic immune responses and stroke recovery has still not been evaluated. By comparing wild type and TnC−/− mice exposed to transient intraluminal middle cerebral artery occlusion (MCAO), we examined the effects of TnC deficiency for ischemic injury, neurological deficits, microglia/macrophage activation and brain leukocyte infiltration using behavioural tests, histochemical studies, Western blot, polymerase chain reaction and flow cytometry. Histochemical studies revealed that TnC was de novo expressed in the ischemic striatum, which contained the infarct core, and overlapped with the area of strongest accumulation of Iba1 + microglia/macrophages. TnC deficiency increased overall Iba1 immunoreactivity in the perilesional cortex, suggesting that TnC might restrict the distribution of microglial cells to the infarct core. By analysing microglial morphology in 3D we found that the post-ischemic loss of microglial cell territory, branching and volume at 3 and 7 days post-ischemia was amplified in the brains of TnC deficient compared with wild type mice. Microglial cell number was not different between genotypes. Hence, TnC deficiency reduced tissue surveillance by microglial cells. Concomitantly, the number of infiltrating leukocytes and, more specifically, T cells was increased in the ischemic brain parenchyma of TnC deficient compared with wild type mice. Ischemic injury and neurological deficits were not affected by TnC deficiency. We propose that the reduced microglia surveillance in TnC deficient mice might favour leukocyte accumulation in the ischemic brain.