Regeneration of the neurogliovascular unit visualized in vivo by transcranial live-cell imaging

Highlights

• Multimodal transcranial in vivo imaging with cellular and temporal precision.

• Longitudinal imaging of multicellular networks overtime to track regeneration.

• Pericyte death and growth at vessel bifurcation impact flow and permeability.

• Functional attributes of newly formed pericytes same as preexisting old pericytes.

• Pericyte morphology and function tailored to the topology of the vasculature.

Abstract

Functional imaging in behaving animals is essential to explore brain functions. Real-time optical imaging of brain functions is limited by light scattering, skull distortion, timing resolution and subcellular precision that altogether, make challenging the rapid acquisition of uncorrupted functional data of cells integrated de novo in the neurogliovascular unit. We report multimodal transcranial in vivo optical imaging for the fast and direct visualization of microcirculation in the perfusion domain where new cells incorporated in the neurogliovascular unit during the progression of a seizure disorder and its treatment. Using this methodology, we explored the performance improvement of cells integrated de novo in the neurogliovascular unit. We report fast transcranial imaging of blood microcirculation at sites of pericyte turnover in the epileptic brain and after treatment with a trophic factor that revealed key features of the regenerating neurogliovascular unit.

Introduction

The neurogliovascular unit is a complex assembly of endothelial cells, perivascular mural cells, extracellular matrix, astrocytes and neurons (Stanimirovic and Friedman, 2012). Each interface between the various cell types assembled in the neurogliovascular unit serves particular functions by way of paracrine signaling with specific receptor systems (Abbott et al., 2006; Sweeney et al., 2019). Mural cells are embedded in the basal membrane of the blood brain barrier (BBB) and the vessel walls connecting with neuronal axons, astrocytes and endothelial cells where it regulates the exchange of nutrients, metabolites and solute between blood and interstitial milieu in the brain, and ensures local energetic supply with spatial and temporal precision (Grutzendler and Nedergaard, 2019; Harris et al., 2012; Iliff et al., 2014). Sensory stimulation, neuronal activity and learning new experience command to the mural cells with a vascular response suited to the local activity of the neuronal network (Hill et al., 2015; Kisler et al., 2020; Whiteus et al., 2014). Mural cells specialize into pericytes and smooth muscle cells (SMC) that cover more than 90 % of the cerebrovasculature (Armulik et al., 2011; Trost et al., 2016; Zhao et al., 2015).

Imaging studies of the living mouse brain over months in adulthood indicated that both the mural cells and microvessels establish very stable interface of contacts (Arango-Lievano et al., 2018; Berthiaume et al., 2018) that contrasts with its extensive dynamic sprouting and pruning during development (Armulik et al., 2010; Coelho-Santos and Shih, 2020; Daneman et al., 2010; Gaengel et al., 2009). However, dynamic remodeling of perivascular mural cells has been reported in the ageing human brain (Montagne et al., 2015) as well as in various vascular pathologies of the adult central nervous system (Sweeney et al., 2019). Neurological diseases associated with pericytosis include Alzheimer’s disease (Sagare et al., 2013), cerebral amyloid angiopathy (Giannoni et al., 2016), stroke (Fernandez-Klett et al., 2013), vascular dementia (Iadecola, 2013), cerebral autosomal dominant arteriopathy with subcortical infarcts leukoencephalopathy (Ghosh et al., 2015), epilepsy (Leal-Campanario et al., 2017), Amytrophic lateral sclerosis (Winkler et al., 2013), traumatic brain injury (Zehendner et al., 2015), neonatal intraventricular hemorrhage (Braun et al., 2007) and others presented in excellent reviews (Lendahl et al., 2019; Rasmussen et al., 2018; Sweeney et al., 2019; Zlokovic, 2008).

Although we know a lot about the individual functions of vessels, pericytes, astrocytes and neurons in the healthy and diseased brains, little is known about their roles as a network organizing the neurogliovascular unit (Iadecola, 2017). Would the damage of one cell type disorganize the structure and function of the neurogliovascular unit? The sole optical ablation of a small number of pericytes in the healthy brain suggested that the neurovasculature tolerates fluctuations of pericytes coverage without damaging consequences in the healthy brain. In fact, pericytes use PDGF-BB and ephrin signaling to regain coverage within days by the extension in non-overlapping territories of neighboring pericytes for bridging the gaps in vascular coverage (Armulik et al., 2010; Berthiaume et al., 2018; Foo et al., 2006; Salvucci et al., 2009). This scenario reads different in the context of CNS diseases as pericytosis, induced either constitutively or conditionally, resulted in neuroinflammatory microbleeds (Ogura et al., 2017; Park et al., 2017; Rustenhoven et al., 2017), circulatory failure and tissue anoxia that precede neurodegeneration and cognitive impairment (Bell et al., 2010; Kisler et al., 2017, 2020; Montagne et al., 2018; Nikolakopoulou et al., 2019). Therefore, pericytosis accelerates CNS diseases progression by degrading barrier and perfusion functions of the neurogliovascular unit (Winkler et al., 2011).

This raises important questions about the repair of the neurogliovascular unit. How would new cells integrate structurally in the topological organization of the damaged neurogliovascular network? Would the new cells be operating isolated or integrated into the damaged neurogliovascular network? Wouldn’t new cells operate as the network, damaged or healthy, depending on the physiological microenvironment and disease state of progression? Would changing the local ratio between individual cell types organizing the neurogliovascular unit modify its function? There are limitations that make answering these questions challenging. First, it requires a longitudinal approach to track the regeneration of the damaged neurogliovascular unit. Second, it requires an in vivo approach to investigate the complex multicellular organization and topology of the neurogliovascular unit in its physiological environment. Third, it requires a non-invasive approach to visualize the newly formed cells and its dynamic assembly in the damaged neurogliovascular unit. Fourth, it requires non-invasive techniques for tracking endophenotypic parameters of disease progression and performance improvement of the neurogliovascular unit in its naturalistic surrounding tissue. Fifth, it requires a treatment that would promote the regeneration of the neurogliovascular unit. In this regard, trophic factors and guidance molecules show promising prospects for the clinic because they promote the assembly, growth and survival of cells organizing the neurogliovascular unit (Maki et al., 2013; Xing and Lo, 2017). Studies that used PDGF-BB as treatment of vascular pathology in models of stroke (Shibahara et al., 2020) and epilepsy (Arango-Lievano et al., 2018) reported regrowth of perivascular mural cells with amelioration of the neuropathology and behavior.

We present a methodology to visualize the regeneration of the neurogliovascular unit with cellular precision and test performance improvement in the physiological environment of the disease state of progression. We report fast transcranial imaging of blood microcirculation at sites of pericyte turnover in the epileptic brain and after treatment with a trophic factor that revealed key features of the regenerating neurogliovascular unit.