Defining the role of NG2-expressing cells in experimental models of multiple sclerosis. A biofunctional analysis of the neurovascular unit in wild type and NG2 null mice

Abstract

During experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis associated with blood-brain barrier (BBB) disruption, oligodendrocyte precursor cells (OPCs) overexpress proteoglycan nerve/glial antigen 2 (NG2), proliferate, and make contacts with the microvessel wall. To explore whether OPCs may actually be recruited within the neurovascular unit (NVU), de facto intervening in its cellular and molecular composition, we quantified by immunoconfocal morphometry the presence of OPCs in contact with brain microvessels, during postnatal cerebral cortex vascularization at postnatal day 6, in wild-type (WT) and NG2 knock-out (NG2KO) mice, and in the cortex of adult naïve and EAE-affected WT and NG2KO mice. As observed in WT mice during postnatal development, a higher number of juxtavascular and perivascular OPCs was revealed in adult WT mice during EAE compared to adult naïve WT mice. In EAE-affected mice, OPCs were mostly associated with microvessels that showed altered claudin-5 and occludin tight junction (TJ) staining patterns and barrier leakage. In contrast, EAE-affected NG2KO mice, which did not show any significant increase in vessel-associated OPCs, seemed to retain better preserved TJs and BBB integrity. As expected, absence of NG2, in both OPCs and pericytes, led to a reduced content of vessel basal lamina molecules, laminin, collagen VI, and collagen IV. In addition, analysis of the major ligand/receptor systems known to promote OPC proliferation and migration indicated that vascular endothelial growth factor A (VEGF-A), platelet-derived growth factor-AA (PDGF-AA), and the transforming growth factor-β (TGF-β) were the molecules most likely involved in proliferation and recruitment of vascular OPCs during EAE. These results were confirmed by real time-PCR that showed Fgf2, Pdgfa and Tgfb expression on isolated cerebral cortex microvessels and by dual RNAscope-immunohistochemistry/in situ hybridization (IHC/ISH), which detected Vegfa and Vegfr2 transcripts on cerebral cortex sections. Overall, this study suggests that vascular OPCs, in virtue of their developmental arrangement and response to neuroinflammation and growth factors, could be integrated among the classical NVU cell components. Moreover, the synchronized activation of vascular OPCs and pericytes during both BBB development and dysfunction, points to NG2 as a key regulator of vascular interactions.

Mesenchymal stem cells instruct a beneficial phenotype in reactive astrocytes

“…Using a vibrating microtome (Leica Microsystem), serial sagittal sections (30–35‐μm thick) evenly spaced at 200 μm intervals, were cut from each hemisphere. The sections were submitted to immunostaining for laser scanning confocal microscopy, according to the previously described protocol (Girolamo et al, 2019). Briefly, after permeabilization (0.5% Triton X‐100 in PBS), free‐floating sections were incubated with single or combined primary antibodies—rabbit anti‐GFAP (26 μg/ml; Immunostar, cat.…”

The Evolving Concept of the Blood Brain Barrier (BBB): From a Single Static Barrier to a Heterogeneous and Dynamic Relay Center

“…Oligodendrocyte progenitors can modulate BBB integrity via secretion of TGFβ-1, resulting in the upregulation of junctional proteins in CECs (Seo et al, 2014). Pericytes also influence progenitor development and neuronal myelination via Lama2 and VEGF signaling and by regulating the bioavailability of PDGF and TGFβ (De La Fuente et al, 2017; Girolamo et al, 2019). Due to the high oligodendrocyte prevalence in WM, CECs in WM are likely to have differential cellular interactions than CECs residing in GM.…”

The Lactate Receptor HCA1 Is Present in the Choroid Plexus, the Tela Choroidea, and the Neuroepithelial Lining of the Dorsal Part of the Third Ventricle

“…Pericytes are known to have an important role in controlling cerebral blood flow [36,37], and in the CP, this would regulate the rate of the CSF production. Given the co-localization of mRFP-HCA 1 with PDGFR-β, which may suggest the presence of HCA 1 in immature pericytes, a new labeling experiment was performed with mRFP-HCA 1 and a marker for immature cells, including pericytes, such as neuron-glial antigen 2 (NG2) [38]. The NG2 antibody produced a strong cellular labeling along the outline of the CP.…”