The impact of pericytes on the brain and approaches for their morphological analysis

Accepted Manuscript Title: The impact of pericytes on the brain and approaches for their morphological analysis Authors: Yaroslav Kolinko, Milena Kralickova, Zbynek Tonar PII: DOI: Reference:

S0891-0618(17)30268-5 https://doi.org/10.1016/j.jchemneu.2018.04.003 CHENEU 1568

To appear in: Received date: Revised date: Accepted date:

17-11-2017 10-4-2018 15-4-2018

Please cite this article as: Kolinko Y, Kralickova M, Tonar Z, The impact of pericytes on the brain and approaches for their morphological analysis, Journal of Chemical Neuroanatomy (2010), https://doi.org/10.1016/j.jchemneu.2018.04.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The impact of pericytes on the brain and approaches for their morphological analysis

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Running title: The impact of pericytes on the brain

Yaroslav Kolinko *, Milena Kralickova, Zbynek Tonar.

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Biomedical Centre, Faculty of Medicine in Pilsen, Charles University, Husova 3, 306 05 Pilsen, Czech Republic Corresponding author: Yaroslav Kolinko, Department of Histology and Embryology, Faculty of Medicine in Pilsen, Charles University in Prague, Karlovarská 48, 301 66 Pilsen, Czech Republic; Fax: +420377593149; Phone: +420608170478; e-mail: [email protected]

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The current knowledge regarding brain pericyte function was reviewed and combines. The role of pericytes during neurodegenerative diseases and tumorigenesis is versatile. The approaches for detection and quantification of brain pericytes are proposed.

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Highlights

Abstract:

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The interest in investigating brain pericytes is growing due to their diverse influences on neuronal function. While numerous studies have investigated the particular properties and functions of pericytes, complex insight into their functional histology is often lacking. In this work, we review and combine the current knowledge regarding brain pericyte function in normal physiology and its role in the pathogenesis of neurodegenerative diseases and tumorigenesis. Special attention is paid to the interaction between the components of the neurovascular unit. Finally, approaches used to detect brain pericytes and the methods for generating qualitative and quantitative data to assess pericyte changes are described.

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Keywords: pericyte, brain, neurodegeneration, tumor, histological identify,

1. Introduction: history, concept, and classification The mean length density of blood microvessels in the human brain is approximately 250 mm of microvessels per 1 mm3 (i.e., 250 mm-2) in cortex and approximately 200 mm-2 in the white matter (Kubíková, et al., 2017). The total perfused cerebral vascular length is approximately 600–700 km in an adult human (Zlokovic, 2005). In mice, the total vascular length in only the cerebellum or midbrain

is approximately 8-15 m (Kolinko, et al., 2016), and the average distance from each neuronal cell body to a neighboring capillary is approximately 15 μm (Tsai, et al., 2009; Lovick, et al., 1999). Such a large and variable vascular system cannot reliably perform its functions without local support.

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The first mention of the cells adjacent to small vessels that affected the structural properties and the development of the capillaries came in the 1870s (Stricker & Arnold, 1871; Rouget, 1873). Half a century later, these cells were introduced as “the pericytes” (Zimmermann, 1923). More recently, the terms “mural cell”, “adventitial cell” and even “vascular smooth muscle cell” have been used interchangeably to describe a cell that forms intimate contact with microvessels to provide support (Hall, 2006). The morphology of these cells has been described in detail elsewhere (Dalkara, et al., 2011); therefore, we will only summarize the main points.

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Capillaries lack a tunica media in contrast to pre- and postcapillary vessels. Therefore, their pericytes are separated from endothelial cells by only a single basal lamina (Krueger & Bechmann, 2010). However, in non-capillary vessels, several layers of muscle cells exclude such a direct interaction. The current concepts of pericyte biology suggest that they play critical roles in the maintenance of homeostasis (Krueger & Bechmann, 2010), vascular integrity, angiogenesis, and neovascularization (Dore-Duffy, et al., 2000; Dore-Duffy, et al., 2006).

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Pericytes are extremely important for the formation of the blood–brain barrier (BBB) because their coverage of microvessels determines the relative vascular permeability (Winkler, et al., 2011). Moreover, an increased pericyte density allows for changing the vascular permeability selectively for each segment of the capillary network (Hellström, et al., 2001; Daneman, et al., 2010; Lyle, et al., 2016). As a result, neuronal activity is modulated within a smaller area. Additionally, three major functional roles are ascribed to the pericytes of the central nervous system (CNS): microvasculaturecontractility for the regulation of cerebral blood flow (Krueger & Bechmann, 2010; Blinder, et al., 2013), regulation of endothelial cell activity (Daneman, et al., 2010; Sá-Pereira I., 2012), and macrophage activity (van Deurs, 1976; Thomas, 1999).

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In fact, distinguishing pericytes from the cells of adjacent compartments such as perivascular connective tissue cells, is extremely difficult in standard histological sections but is possible by using immunohistochemistry and analysis at the ultrastructural level (Krueger & Bechmann, 2010).

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Previous studies have shown that neurodegenerative diseases are often accompanied by microvascular changes in the affected tissue (Kolinko, et al., 2015; Kolinko, et al., 2016). These observations have suggested that pericyte deficiency can play a key role in neurodegenerative changes.

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Therefore, we aim to review the current knowledge regarding the roles of pericytes on blood microcirculation in the brain and the approaches used for morphological analysis. We believe that such approaches underpin the understanding of functional imaging and the effect of the vascular network on brain regeneration and viability. Moreover, summarizing the present knowledge on pericytes may contribute to the generation of further hypotheses to be tested.

2. Basic processes in brain-specific pericytes Morphologically, pericytes are cells with small soma that are usually enclosed within the basal lamina (Fig. 1) of the microvasculature (Thomas, 1999) with multiple processes oriented along the axis of the blood vessel, while smaller circumferential arms encircle the vascular wall (Zimmermann, 1923; Rucker, et al., 2000). Pericytes can contract, which results in vasoconstriction, especially in the capillaries, and regulate the properties of the endothelial cells (ECs) (Thomas, 1999). Non-capillary

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microvessel pericytes are often interspersed with other cell types of the vascular wall, the perivascular space, or/and the juxtavascular parenchyma (Krueger & Bechmann, 2010). They perform vessel stabilization, angiogenesis, regulation of vascular tone, and maintenance of local tissue homeostasis (Díaz-Flores, et al., 2009). Their contribution to numerous functions is why these cells are beginning to receive increasing attention in neurobiological studies (Rucker, et al., 2000). Nevertheless, the exact lineage of mature pericytes is still unknown (Hall, 2006).

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Figure 1. Schematic representation of the neurovascular unit. ECs (orange) are separated via the basal lamina (blue) of the capillary. Pericytes (green) are placed between the capillary basal lamina and a dense layer of astrocytic end-foot (brown). Dopamine activity or/and cellular signals from other glial cells (gray) promote the polarization of astrocyte membranes. The potential can be transmitted to the membrane of the pericytes. Pericytes are capable of spreading and changing the intensity of the membrane polarization in different areas. The nuclei of single cells are violet.

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Pericytes together with smooth muscle and ECs, astrocytes and neurons form the neurovascular unit (NVU) (Pombero, et al., 2016; Iadecola, 2017). This functional multicellular structure provides an optimal microenvironment for neural activity provided by different diffusible signals (Winkler, et al., 2011; Dalkara & Alarcon-Martinez, 2015). Furthermore, neuronal activity initiates an increase in blood flow due to the generation of functional signals (Attwell & Iadecola, 2002; Cohen, et al., 1997) and vice versa (Peppiatt, et al., 2006; Attwell, et al., 2010) to make fine-tuned regulatory adjustments in response to stress stimuli (Fig. 2). The results of cell culture experiments indicate that adult brain microvascular pericytes have neural stem cell capability, expressing neuronal and glial cell markers during differentiation (Dore-Duffy, et al., 2006 Dore-Duffy, 2008). Such pericytes are primarily nestin/ neural glial antigen 2 (NG2)-positive and can even differentiate into cells of neural lineage (Dore-Duffy, et al., 2006). However, in general conditions, pericytes often differentiate into smooth muscle cells, fibroblasts (Silver, et al., 2001) or other cells.

2.1.

Pericytes as contractile cells

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The contraction and relaxation of capillaries affect nutrient delivery to the tissue, even when there is a constant total blood flow to a region (Jespersen & Østergaard, 2012). The role of pericytes in the regulation of capillary blood flow in response to synaptic transmission and the release of vasoactive mediators has long been speculated about and has had experimentally supported evidence (Bell, et al., 2010; Peppiatt, et al., 2006; Neuhaus, et al., 2017). Microvascular networks that have lost pericytes have frequently been described as being exceedingly tortuous and prone to tears and diffuse and/or focal dilations (microaneurysms) (Armulik, et al., 2010; Li, et al., 2011; Lindahl, et al., 1997; Bjarnegård, et al., 2004; Hellström, et al., 2001).

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Figure 2. General schematic of the interaction of intercellular signals between the components of the neurovascular unit. Individual proteins are labeled using the following standard abbreviations: gap junction protein - connexin-43 (CX43), calcium-sensing receptor (CaSR), interferon (IFN); Pericyte: tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), α-smooth muscle actin (α-SMA), nuclear-related factor 2 (Nrf2), cell-surface glycoprotein (CD146), neural glial antigen 2 (NG2), platelet-derived growth factor B (PDGF-B), transforming growth factor- β (TGF-β); Endothelial cells(ECs): notch homolog proteins (Notch), endothelial-expressed Jagged 1 (JAG1) protein, mediators of Smad, angiopoietin 1 (Ang1); Macrophages: intercellular adhesion molecule 1(ICAM1), macrophage differentiation antigen (Mac-1), leukocyte function-associated antigen-1 (LFA-1); Glia: stromal cell-derived factor 1 (SDF1). The targets of individual proteins are shown by arrows.

The color of the arrows associated with associated with the color of the cells they are indicating and their shapes coded respective proteins.

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2.1.1. The features of pericyte contractility The ability of pericytes to contract has been demonstrated in culture by the addition of vasoactive substances such as acetylcholine, bradykinin, histamine and serotonin (Murphy & Wagner, 1994; Speyer, et al., 1999; Wu, et al., 2003). In addition to their contractile properties, pericytes serve as receptors for vasoactive molecules, including catecholamines, endothelin-1, vasopressin, and angiotensin II (Díaz-Flores, et al., 2009; Hamilton, et al., 2010).

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The main mechanism by which pericytes contract is considered to be the expression of α-smooth muscle actin (α-SMA) (Kamouchi, et al., 2004), which is activated via Ca2+-dependent pathways (Kamouchi, et al., 2004; Ina, et al., 2011); however, discussions regarding the mechanism of pericyte contraction still continue (Hill, et al., 2015). Pericyte dilation occurs due to the activation of prostaglandin receptors (Kudryavtseva, et al., 2015), although NO production is also needed to suppress the synthesis of vasoconstrictive agents (Attwell, et al., 2010). Similar to vascular smooth muscle cells, pericytes strongly express an intermediate filament called vimentin, which is also indicative of their mechanical stress endurance, but they lack muscle-specific regulators such as desmin (Bandopadhyay, et al., 2001). However, lumping pericytes in the same class as vascular smooth muscle cells is incorrect because they differ greatly in their morphology and function.

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2.1.2. Neurovascular relationships The control of vasomotor effects as a result of a neurovascular relationship remains controversial (Maolood & Meister, 2009; Norsted, et al., 2008). Most researchers are aware of the fact that neuronal activity leads to noradrenaline release from the locus coeruleus axon terminals, two-thirds of which end near capillaries (Cohen, et al., 1997). This noradrenaline release activates outward membrane currents in pericytes and first dilates capillaries up to the fourth branching order followed by the arterioles (Hall, et al., 2014). Because the capillaries are closer to neurons and detect neuronal activity earlier than arterioles, they may pass a hyperpolarizing vasodilatory signal back to arterioles via gap junctions between adjacent pericytes or ECs (Peppiatt, et al., 2006; Puro, 2007).

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The longitudinal processes of pericytes located on the basal lamina are able to change the stiffness of the vessel wall (Attwell, et al., 2016) and the vascular diameter via contraction or relaxation. This promotes the passage of red blood cells through small capillaries. Most contractile pericytes are located near capillary bifurcations (Wu, et al., 2003). Vasodilation of the capillary itself contributes significantly to an increased blood flow. Furthermore, pericytes can block neural impulses designed for the contraction of large vessels (Hill, et al., 2015), leaving the microvascular diameters unchanged.

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In experiments examining the blood flow and dynamic changes in the capillary diameter (Hill, et al., 2015; Fernández-Klett, et al., 2010), it was concluded that dilations are produced by the relaxation of smooth muscle cells rather than pericytes. Therefore, all contractile pericytes were redefined as smooth muscle cells. However, the notion that the dilation of capillaries is achieved by the relaxation of contractile pericytes (Peppiatt, et al., 2006; Hall, et al., 2014) still persists. Nevertheless, after strong capillary constriction, blood flow is limited and capillary constriction is evoked by pericyte processes, followed by the death of a portion of the pericytes (Hall, et al., 2014). In addition, pericyte death is expected to result in the loss of a BBB (Bell, et al., 2010; Armulik, et al., 2010; Daneman, et al., 2010). Together, these pericyte malfunctions promote neuronal dysfunction or even death (Attwell, et al., 2016).

The data from models of brain pathology suggest that new pericytes are recruited from the bone marrow (Krueger & Bechmann, 2010). During brain pericyte repair, approximately 40% of the cells migrate from their microvascular location into the adjacent neuropil (Dore-Duffy, et al., 2000). The remaining pericyte population is restored via cell division in the brain tissue.

2.2.

Pericytes as regulators of endothelial cell activity

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On average, for every one brain pericyte there are up to three ECs. In the retina, this ratio is 1:1 (SáPereira et al.,2012). Therefore, given the vessel length, the CNS is one of the tissues that is most saturated by pericytes in the human body. Their processes cover 30-90% of the abluminal surface of the microvessel wall (Dalkara, et al., 2011; Mathiisen, et al., 2010). The differences in coverage are linked to EC activity. Greater pericyte coverage promotes a reduction in the area with single EC reactivity (Díaz-Flores, et al., 2009). Furthermore, pericytic coverage is not dependent upon age, although aged pericytes have a larger presence of lysosomes and inclusions (Peters & Sethares, 2012). The thickness of the outer basal lamina of the cerebral capillaries also increases significantly with age, but this does not contribute to the underlying cause of cognitive decline (Peters & Sethares, 2012). Pericyte deficiency correlates with endothelial hyperplasia, an abnormal EC shape and ultrastructure, the altered cellular distribution of certain junctional proteins, and morphological signs of increased transendothelial permeability, and it may interfere with nutrient and electrolyte transport (Hellström, et al., 2001).

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2.2.1. Paracrine signaling of pericytes and the effect on angiogenesis Pericytes are able to affect tight junctions and vesicle trafficking in ECs by paracrine signals via gap junctions between endothelial cells that extend through a discontinuous basal lamina (Daneman, et al., 2010; Dziewulska & Lewandowska, 2012; Wevers & de Vries, 2015; Cuevas, et al., 1984), direct physical contact, adhesion plaques and peg-and-socket contacts that allow the intercellular exchange of ions and by small molecules (Rucker, et al., 2000; Winkler, et al., 2014). In particular, junction proteins underlie the actin cytoskeletal networks between pericytes and the endothelium (Díaz-Flores, et al., 2009). Among them, the most studied are N-cadherin, (Gerhardt, et al., 2000; Li, et al., 2011; Winkler, et al., 2011) and the connexin-43 (CX43) hemichannels (Bobbie, et al., 2010; Chew, et al., 2010; Kovács, et al., 2012). Both of these junction proteins play an important role in angiogenesis and pericyte differentiation.

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New blood vessels are blood vessel renewal occurs formed through the sprouting of EC and are stabilized by the formation of the perivascular matrix, which is associated with pericyte signaling when it is torn (see Fig. 2). Among the pericyte signaling factors are platelet-derived growth factor B (PDGF-B); transforming growth factor-β (TGF-β), which is secreted by both of the cells; sphingosine1 phosphate and other angiopoietin signaling molecules (Gaengel, et al., 2009; Onogi, et al., 2017). PDGF-B and its derivatives are essential for the recruitment of brain pericytes during EC tube formation (Gaengel, et al., 2009; Lindahl, et al., 1997; Tallquist, et al., 2003). Neurogenic locus notch homolog protein 3 (Notch 3) signaling is critical for the proangiogenic abilities of pericytes, pericyte–endothelium interactions, endothelial-dependent mural cell differentiation, and the regulation of microcirculation in the brain (Hofmann & Iruela-Arispe, 2007; Liu, et al., 2009). The expression of signals focused on Notch 3 depends on signaling during neurogenesis or neural differentiation and is initiated by the endothelial-expressed Jagged 1 (JAG1) protein. The gamma-secretase inhibitor (DAPT) blocks Notch 3 signaling upregulation (Liu, et al., 2009). Another signal that inhibits pericyte recruitment during angiogenesis is a growth arrest-specific protein 6 (Gas6) signal that acts on the luteolin pathway (Li, et al., 2017).

Pericytes as macrophages

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Smad proteins play an extremely important role in cerebrovascular integrity, especially in the beginning of new vessel development (Lan, et al., 2007; Van Geest, et al., 2010), and promote the modification of cells (differentiation or cancer development) (Costello, et al., 2009). Smad proteins are activated by transmembrane serine-threonine receptor kinases in response to TGF-β signaling (Lan, et al., 2007; Li, et al., 2011). TGF-β is a multifunctional cytokine that is secreted by different cells of the NVU (Gaengel, et al., 2009; Lebrin, et al., 2005; Dudvarski Stankovic, et al., 2016) and exerts a number of effects on vascular development (Gaengel, et al., 2009; Li, et al., 2011; Sieczkiewicz & Herman, 2003; Lebrin, et al., 2005; Walshe, et al., 2009). This signaling allows the pericytes to control the assembly of the vascular basal lamina matrix during angiogenesis (Stratman, et al., 2009) via expression of several matrix metalloproteinases (Candelario-Jalil, et al., 2009; Saharinen, et al., 2008) and directly contributes to the synthesis of essential extracellular matrix proteins (Díaz-Flores, et al., 2009; Stratman, et al., 2009; Van Geest, et al., 2010), such as laminin, nidogen or fibronectin. In addition, pericytes have the capacity to enhance the extracellular matrix and alter both the degree of angiogenesis (Winkler, et al., 2011) and the population of neuronal-glial precursor cells (Braun, et al., 2007; Dudvarski Stankovic, et al., 2016). Differentiated pericytes bear the receptors for vascular endothelial growth factor (VEGF), which may act in a paracrine manner as a survival and stabilizing factor for ECs in microvessels (Darland, et al., 2003; Ozerdem & Stallcup, 2004). Pericyte deficiency has direct effects on EC number, increases the VEGF levels, promotes vascular permeability, and further abrogates the microvessel architecture (Hellström, et al., 2001).

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Pericytes constitutively express numerous histocompatibility complex antigens, allowing them to be classified as antigen-presenting or immune-regulating cells (Rustenhoven, et al., 2017; Domev, et al., 2014). Brain pericytes are capable of acting in a similar manner to brain phagocytes (van Deurs, 1976). Additionally, these pericytes are capable of immune regulation effects due to their stem cell properties (Pombero, et al., 2016). They may convert to tissue macrophages when they enter the brain parenchyma (Thomas, 1999; Proebstl, et al., 2012) and perform antibody-dependent phagocytosis (Balabanov, et al., 1996). Furthermore, some brain pericytes are derived from the monocyte lineage (Krueger & Bechmann, 2010). Nevertheless, not all brain pericytes have immune properties. Therefore, a separate subtype of phagocyte-like pericytes has been proposed (Krueger & Bechmann, 2010).

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The brain has a tightly controlled immune surveillance system (Galea, et al., 2007; Louveau, et al., 2015) in which pericytes are assigned a regulatory role. Microglia are the first immune cells that are activated upon cerebral dysfunction (Schilling, et al., 2003). During microglial activation, TGF-β signals targeted for tissue macrophages and pericytes are released (Dudvarski Stankovic, et al., 2016). Tissue macrophages transmigrate through the microvascular walls to activate mast cells. During this process, they pass through ECs, pericytes and the basal lamina. Then, white blood cell interact directly with pericytes by moving along the pericyte processes to the gaps between adjacent pericytes with the support of the expression of ICAM-1-, Mac-1-, and LFA-1-dependent signals (Proebstl, et al., 2012; Daneman, et al., 2010). Furthermore, quantitative real-time analysis has shown that brain pericytes can increase the expression of typical inflammatory marker proteins after stimulation with tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), interferon-gamma (IFN-γ), or lipopolysaccharides (LPS) (Pieper, et al., 2014). In addition, pericytes suppress vascular permeability and immune cell activity during vascularization by secreting intercellular adhesion molecule 1 (Icam1), activated leukocyte adhesion molecule (Alcam) and lectin galactose binding soluble 3 (Lgals3) (Daneman, et al., 2010).

3. Roles of pericytes in neurodegenerative diseases

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The preceding discussion that the pericytes can be distinguished not only by their position along the vascular tree and their location in relation to the tunica media of the vessel (Zimmermann, 1923) but also by their functional properties. On the one hand, it is evident that there is a sub-group of pericytes that constantly regulate the brain microcirculation in order to preserve homeostasis by small contractions and by affecting ECs. On the other hand, there is a subtype of pericytes that provide a rapid response that is dependent on specific signals and are capable of performing standard functions or additionally show immune or even stem cell properties. However, additional quantitative and qualitative studies are needed to devise invent suitable nomenclature for the different pericyte subtypes (Attwell, et al., 2016).

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In animal models of pericyte-deficient mutants, transendothelial fluid flow was found to be increased during the degeneration of brain pericytes (Armulik, et al., 2010; Bell, et al., 2010). As a result of reduced tight junction protein expression, paracellular transport was also increased (Bell, et al., 2010), which included the deposition of several potentially vasculotoxic and neurotoxic blood-derived macromolecules (Winkler, et al., 2011) that result in BBB breakdown.

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Pericyte loss leads to brain vascular damage in two general ways: (1) transformation of the brain microcirculation and (2) BBB breakdown (Bell, et al., 2010). The first pathway is associated with diminished brain capillary perfusion via occlusion (Craggs, et al., 2015; Dziewulska & Lewandowska, 2012), and the second pathway is associated with the infringement of accumulated serum proteins and several vasculotoxins (Armulik, et al., 2010; Montagne, et al., 2015).

Reorganization in the brain microcirculation

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Pericyte constriction depends on intracellular calcium flux (Peppiatt, et al., 2006; Hamilton, et al., 2010; Chow, et al., 2007). Excessive calcium accumulation in pericytes causes capillary constriction resulting in blood flow obstruction. However, mutations in Notch 3 lead to the loss of vascular smooth muscle cells and pericytes (Hofmann & Iruela-Arispe, 2007), which also leads to narrowing and/or occlusion of the capillary lumen. In both cases, degenerative changes in endothelial-pericytic connectivity, especially within peg-and-socket junctions, have been observed (Dziewulska & Lewandowska, 2012; Glinskii, et al., 2013). As a result, the brain microvascular network is destabilized and becomes remodeled. Furthermore, pericyte-induced ischemia may negatively affect brain regeneration by limiting substrate and drug delivery to tissue and by promoting metabolic stress (Yemisci, et al., 2009). Moreover, pericyte dissociation promotes a deficiency in the contribution of matrix metalloproteinases (Trivedi, et al., 2016), resulting in a sharp decline in angiogenesis and vascular stability, especially during the early phase of regeneration.

3.2.

Blood-brain barrier breakdown

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All pericytes play varying pathophysiological roles in BBB breakdown during age-dependent learning and memory impairment disorders associated with neuronal degenerative changes (Bell, et al., 2010). Disorders such as Alzheimer’s disease (AD) (Montagne, et al., 2015; Kisler, et al., 2017), Parkinson's disease (PD) (Padel, et al., 2016), multiple sclerosis (Ortiz, et al., 2014; Geraldes, et al., 2017) and CADASIL (cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy) (Dziewulska & Lewandowska, 2012) are most often implicated. The development of BBB dysfunction during all of these disorders has many similarities. Furthermore, this dysfunction may be affected by the presence of neuroinflammatory agents.

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3.2.1. Pathogenesis of BBB dysfunction A simpler way to understand this mechanism is to examine the pathogenesis of BBB dysfunction in AD. During the initial stages of AD, hippocampal pericytes begin to degrade, and reduce PDGF-B production (Montagne, et al., 2015; Halliday, et al., 2016). Interestingly, the ε4 allele of the apolipoprotein E gene is believed to be responsible for these detrimental changes (Tai, et al., 2016). Violation of endothelial/pericyte interactions via the related molecules PDGFR-β, α-SMA and aminopeptidase N (CD13) promotes the reduction in vascular endothelial cadherin (or CD144) in endothelial adherens junctions across the BBB (Gertz, et al., 2016; Bell & Zlokovic, 2009). This decreases the capillary diameter and causes the development of endothelial hyperplasia and an abnormal endothelial shape and ultrastructure (Hellström, et al., 1999). As a consequence, amyloid deposits occur (Dalkara, et al., 2011). Furthermore, the accumulation of undesirable substances such as amyloid in the brain tissue is closely associated with an increase in gliosis and a loss of astrocytic end-feet contacts (Duncombe, et al., 2017; Iadecola, 2017), thereby promoting profound neurovascular unit dysfunction.

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With the development of this pathological process, amylin cell inclusions collect in the cytoplasm of pericytes (Winkler, et al., 2014; Schultz, et al., 2017). This pancreatic β-cell-derived peptide causes nuclear changes associated with cell autophagy and reduced expression of neuron-glial antigen (Molteni & Rossetti, 2017). Under such conditions, not only amyloid deposits but also leakage and deposition of several potentially toxic and metabolic-associated macromolecules can occur. Among these macromolecules are fibrin (Paul, et al., 2007), thrombin (Chen, et al., 2010; Mhatre, et al., 2004), plasmin (Chen & Strickland, 1997), hemoglobin-derived hemosiderin and others that cause an accumulation of iron and reactive oxygen species (Bell, et al., 2012; Halliday, et al., 2016; Zhong, et al., 2008). In addition, pericyte functional failure entails the violation of adhesion molecule synthesis and promotes T-cell entry into the brain (Verbeek, et al., 1995; Wevers & de Vries, 2015), which occurs during neurodegenerative diseases such as multiple sclerosis.

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The nitrative/oxidative stress induces by ischemia/reperfusion of affected microcirculatory areas affects nutrient delivery to the tissue (Jespersen & Østergaard, 2012; Dalkara & AlarconMartinez, 2015) and plays a critical role in neuronal activity and survival (Hall, et al., 2014; Yemisci, et al., 2009; Dalkara & Alarcon-Martinez, 2015). A G-protein-coupled calcium-sensing receptor (CaSR) acts as a modulator of systemic calcium homeostasis (Noh, et al., 2015). CaSR is mostly located in reactive astrocytes and some neurons. Its expression regulates the altered extracellular ionic environment within the ischemic and border zones (Noh, et al., 2015) associated with particular endothelial cells and pericytes. Furthermore, in extreme cases of ischemia, a profound and irreversible contraction of pericytes, clearly preceding their death (Neuhaus, et al., 2017), leads to capillary obstruction. This capillary obstruction promotes angiogenesis and neovascularization via the multipotential stem cell activity of other brain pericytes (Eyden, 2005; Truettner, et al., 2013; Yao, et al., 2016). Notably, PDGF-B treatment has been shown to cause vascular stabilization and neurorestorative effects in the striatum in mice models of PD (Padel, et al., 2016). Additionally, other important risk factors further the progression of the disease and can affect the components of the brain vessels including the pericytes (Ghori, et al., 2017; Geraldes, et al., 2017; Patel & Shah, 2017). 3.2.2. The features of viral infections Viral infections often support the destruction of tight junction proteins to dysregulate the BBB during neuroinflammation (Kazakos, et al., 2017; Castro, et al., 2016). Pericyte exposure to viruses or cytokines results in the decreased secretion of basement membrane components and factors such as angiopoietin-1 or TGF-β (Persidsky, et al., 2016). This is why cerebellar astrocytes promote cellular responses of the BBB during neurotropic viral infection via active interferon production (Daniels, et

al., 2017), resulting in the profound production of IL-1, IL-6, prostaglandin E2 (PGE2) and C-C motif ligand 5 (CCL5) by microvascular pericytes (Chang, et al., 2017; Persidsky, et al., 2016). IL-6 and CCL5, in turn, play an active role in the disruption of the endothelial barrier integrity, and leukocyte chemotaxis is regulated in favor of brain infiltration by leukocytes (Kazakos, et al., 2017; Nyúl-Tóth, et al., 2017). Notably, damage to individual brain structures is depends on the virus and infected cell type (Hlavatý, et al., 2017). Such differential effects may be a consequence of differences in the stability of NVU components and the pericytes within the individual brain structures in response to the particular virus.

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4. Pericytes in brain cancer

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The role of pericytes during the development of brain cancer is variable and deserves a separate review. Nevertheless, we believe that discussing it generally in this context is necessary. Tumor blood vessels have multiple abnormalities in the organization of their pericytes (Morikawa, et al., 2002). One abnormality is a defective integration of the pericytes into the development of tumor microvessels (Yotsumoto, et al., 2015). Because they are affected by tumor necrosis factor-alpha (TNF-α), the brain capillary pericytes increase ICAM-1 protein expression (Pieper & Galla, 2014) which has antagonistic effects on the tight junctions between the cells forming the BBB.

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An insufficiency in pericyte-endothelial cell interactions via the NG2 proteoglycan results in the loss of the ability of pericytes to decrease the formation of endothelial cell junctions and decrease the assembly of the vascular basal lamina (Maolood & Meister, 2009; Yotsumoto, et al., 2015), leading to less efficient tumor blood flow and increased intratumoral hypoxia and angiogenesis. The cell–cell ephrinB2/EphB4 signals between endothelial and perivascular cells in tumors are more effective than those in healthy tissue and promote the recruitment of pericytes during tumor growth and angiogenesis (Noren, et al., 2004; Ghori, et al., 2017). Moreover, perivascular soft tissue across all tumors contains cells that are immunoreactive for RGS5 (a regulator of G-protein signaling 5) and αSMA (Shen, et al., 2016). Another signal of the intercellular interaction between vasculature wall components, pericytes in particular, in brain tumors is CD90 (Inoue, et al., 2016), which is a marker typical of angiogenesis in malignant tumors that can be involved in angiogenesis through the recruitment and functional regulation of tumor-associated macrophages. Furthermore, the pericytes express a special GT198 and can proliferate into tumor cells (Zhang, et al., 2017) under the condition of NOTCH inhibition (Huang, et al., 2017). The resulting fragility promotes tumor-associated hemorrhage (Abramsson, et al., 2003), edema and necrosis (Behling, et al., 2016) as a consequence of increased perfusion and reduced diffusion.

5. Detection and quantification of pericytes

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Studies of brain vascular pericytes that are based on specific imaging techniques with adequate and representative sampling and exact data quantification are extremely important for understanding their theoretical and practical application. However, researchers face some challenges in this field. 5.1. Detection of pericytes The first complication is the absence of a single marker capable of histologically identifying all pericytes. Different researchers have their own approaches for visualizing pericytes using antibodies (Nikolajsen, et al., 2016; Domev, et al., 2014; Schultz, et al., 2017, etc.). For this reason, a combination of vessel morphology markers that indicate contractile, cytoskeleton, and surface proteins are required for the identification of brain vessel pericytes. Transcardial perfusion with phosphate

buffer and a mild zinc fixative (Nikolajsen, et al., 2016) is commonly used to better visualize pericytes. A list of the most commonly used antibodies for the detection of pericyte function at different research levels is presented in Table 1. Among the less commonly markers used to detect pericytes are the following: CD13 (aminopeptidase N), membrane alanyl (Bandopadhyay, et al., 2001; Armulik, et al., 2011), CD 105 (Crisan, et al., 2008), CD 146 (Bardin, et al., 2001; Tigges, et al., 2012; Domev, et al., 2014), vascular cell adhesion molecule 1 (VCAM-1) (Dalkara, et al., 2011).

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Table 1. Commonly used antibodies for pericyte detection. To this date, no marker exclusively specific for pericytes has been published and widely acknowledged. Identification of pericytes requires a combination of immunohistochemical markers and a careful microscopic assessment of the position and morphology of each cell. Wherever known, the Pros (advantages) and Cons (disadvantages) of each method are presented together with the references demonstrating the practical use and reactivity in mammalian species.

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RGS5

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Desmin

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αSMA

Mouse / Rat α-Smooth muscle actin (Dore-Duffy, Pros: indicates the contractile phenotype et al., of pericytes 2006;NyúlCons: positive also in adjacent vascular Tóth, et al., smooth muscle cells 2016) Muscle-specific intermediate filament (Ghori, et al., Pros: is absent in pericytes, may be used 2017) as an exclusion criterion The pericyte-specific gene (Mitchell, et Pros: suitable for studies on angiogenesis al., 2008) as it is involved in the regulation of G proteins during angiogenesis

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Proteins affecting endothelial cell activity Protein of the extracellular matrix and (Yao, et al., component of the basal lamina 2016) laminin Pros: clearly outlining the pericytes Cons: usable in capillaries with continuous basal lamina only. Collagen found primarily in the basal (Nikolajsen, lamina et al., 2016; Pros: clearly surrounding the position of Duncombe, et Type IV pericytes al., 2017) collagen Cons: not applicable in newly sprouting or tumor capillaries without fully developed basal lamina Platelet endothelial cell adhesion (Nikolajsen, molecule et al., 2016; CD31 Pros: enhancing the contrast between Ghori, et al., or pericytes and adjacent endothelial cells 2017) PECAM-1 Cons: careful morphological assessment needed

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(Nyúl-Tóth, et (Shen, et al., al., 2016; 2016) Thanabalasun daram, et al., 2011) (Thanabalasu (Lyle, et al., ndaram, et al., 2016) 2011) (Shen, et al., 2016)

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Contractile agents of pericytes

(Lyle, et al., 2016; Schultz, et al., 2017)

Nestin

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CD13 or aminopeptidase N

Beta-type platelet-derived growth factor (Nyúl-Tóth, et (Nyúl-Tóth, et (Glinskii, et Pros: seems to be exclusively expressed al., 2016; al., 2016) al., 2013) in pericytes in the adult brain Onogi, et al., Cons: not reliable in early ontogenesis 2017) and growing individuals Neuroectodermal stem cell marker (Thanabalasu Pros: useful for analysisof pericyte ndaram, et al., differentiation in vitro 2011) Cons: positive in many other undifferentiated cell types including endothelium A member of the type II integral (Gertz, et al., (Lyle, et al., membrane metalloproteases 2016; He, et 2016) Pros: useful in studies on blood-brainal., 2016) barrier Cons: positive also in some nerve synapses and in a variety of tumor cells

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CD 140 or PDGF-B

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Immune-associated markers of pericytes Neural glial antigen 2, (Dore-Duffy, et (Nyúl-Tóth, et (Schultz, et Pros: positive in pericytes of nascent al., 2006; al., 2016) al., 2017) NG 2 capillaries Urrutia, et al., Cons: expressed also by oligodendrocyte 2016; He, et al., precursor cells 2016) Intracellular adhesion molecule-1 (Pieper & CD 54 or Pros: expressed in activated pericytes Galla, 2014) ICAM-1 response to cytokines Cons: positive also on endothelial cells (Zhang, et al., 2017)

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Cancer-associated pericyte markers A steroid hormone receptor Pros: positive in pericytes in tumors GT198 Cons: unreliable in adjacent nontumorous regions

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Therefore, various methods can be used for pericyte detection. Nevertheless, determining the optimal method for routine use is not clear (Nyúl-Tóth, et al., 2016; Duncombe, et al., 2017; Nikolajsen, et al., 2016). For this reason, we systematically cut a wild-type mouse cerebellum into 18-µm-thick serial sections. Then, randomly selected sections from the middle part of cerebellum (Franklin & Paxinos, 2008) were stained with hematoxylin-eosin and the three most commonly used antibodies (laminin, CD31 and α-SMA). Although cerebellar pericytes were detected with each stain (Fig. 3), in our opinion, the pericyte bodies were best visualized when a laminin or α-SMA antibody agonist was used. 5.2. Quantitative histology of pericytes Another complication that researchers face is that the various methodical approaches most often used describe qualitative, not quantitative, changes. Thus, unbiased stereology design-based methods, which are meant to quantify objects in tissue sections to generate reliable structural data that describes biological features, are most suitable for quantitative analysis (Sterio, 1984; Nyengaard & Gundersen, 2006; Mouton, 2014). In the final sample, the structural quantities such as the total number of pericytes, their density and the number of pericytes per vessel length (Mayhew, 2005) can be used as the structural quantity for evaluating these parameters.

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Figure 3. Comparison of the staining for pericyte detection in the cortex of mouse cerebellum. Randomly selected sections from the middle part of mouse cerebellum were immunohistochemically labeled with laminin, CD31 and α-SMA antibodies that were visualized using horseradish peroxidase/diaminobenzidine (dark brown) and were counterstained using hematoxylin. Hematoxylineosin staining was used as a reference. A random microscopic field containing pericytes (arrow) is shown. Scale bar: 50 μm.

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Unfortunately, manual subjective image capture and analysis with the aid of computer software as currently performed is a time- and labor-intensive process that is affected by the subjective counting decisions. The “best practice” for quantifying morphometric parameters is using an automatic stereology system that achieves unbiased stereology counts of immunostained cells (Mouton, et al., 2017). This approach uses the same hardware, e.g., a microscope equipped with a motorized XYZ stage, a digital camera and a PC. We believe that in the short term, such approaches for automatic analysis of biological structures will become faster and achieve higher quality basic neuroscience research.

Concluding remarks A greater understanding of how the brain microvascular pericytes affect brain metabolism can allow us to develop diagnostic and therapeutic targets for neurodegenerative or mental disease. Unfortunately, the heterogeneity of the methods used and the insufficient amount of quantitative results in the published studies prevents us from clearly and quantitatively describing the pericyte changes that occur in neurodegenerative diseases and tumorigenesis. The use of different methods,

even those that are not optimal for a particular purpose, may lead to contradictory results in studies of the same disease. Despite the abundance of studies of pericyte changes, an enormous amount of information is still lacking. Knowledge regarding the effect of pericytes on limiting the membrane of perivascular glia is very limited as well. Furthermore, quantitative analysis of the pericytes of various brain regions in different viral infections, mental illnesses, chronic alcohol consumption, and neurodegenerative disorders would be of interest. The efficacy of pericyte signaling is also important for the performance of the endogenous regenerative mechanisms of brain tissue after injury and for brain plasticity.

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Therefore, further comprehensive studies of the brain pericytes that consider both the multiple functions of the BBB and the associated complexities of the development of illnesses are extremely important and crucial for clinical applications. Such studies should describe changes in pericytes in diseased tissue and, if possible, the course of their development during the progression of the disease. In addition, an unbiased quantification of pericytes using automatic stereology systems should be utilized.

Declaration of interest

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Competing interests: The authors declare that they have no competing interests.

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Author contributions: The first author performed the literature search and wrote the manuscript. The last two authors contributed equally to the generation of ideas, general editing, and commenting on the text. The final manuscript preparation was conducted by the first author. All authors have read and approved the final manuscript.

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Acknowledgements: We would like to thank Šlajerová M. for their valuable help during the histological processing of the tissue samples.

References

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Funding: This study was supported by the National Sustainability Program I (NPU I) Nr. LO1503 provided by the Ministry of Education Youth and Sports of the Czech Republic.

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