**2. The BBB and the neurovascular unit**

*Connectivity and Functional Specialization in the Brain*

mechanism known as neurovascular coupling [5].

contributing to neurological effects.

disease (PD) and multiple sclerosis (MS).

the most metabolically active organs in the body, under physiological conditions, the human brain receives 20% of the total basal cardiac output and uses 20% of the body's oxygen and glucose [4]. Energy substrates are consumed by the brain from the blood via transport across the BBB, as the brain lacks a metabolic reservoir to store macromolecules for use when needed. In the mammalian brain, cerebral arteries, arterioles, and capillaries supply CNS with blood in response to neuronal stimuli by increasing the rate of cerebral blood flow (CBF), nutrients and oxygen delivery, a

The neurovascular coupling requires an integrated multicellular response to provide the perfusion needs for neuronal metabolism [5], different cell types are involved in this action, neurons and astrocytes generate mediators that trigger cellular responses in endothelium cells, pericytes, and smooth muscle cells (SMC), which contribute to vascular response in the BBB permeability. Functionally, these interactions are included in the concept of the neurovascular unit (NVU), which comprises various central and peripheral cell types that contribute to BBB structure and function (**Figure 1**) [6, 7]. However, in pathophysiological states, BBB breakdown and dysfunction leads to leakages of harmful blood components into the cerebral parenchyma, cellular infiltration, and aberrant transport and clearance of molecules [8], which is associated with CBF reductions and dysregulation [9],

Here, we first examine the cellular components that underlie the establishment of the BBB in NVU. Then, we focus on the cellular components of BBB and transport physiology. Complementary and in a translational way, examine how BBB breakdown and dysfunction related to acute vascular CNS disorders such as ischemic and hemorrhagic stroke, and BBB breakdown and dysfunction relate to neurological deficits and other pathologies in Alzheimer's disease (AD), Parkinson's

*(A) The multicellular structure of the neurovascular unit (NVU). The BBB is formed by endothelial cells at the level of the cerebral bed (arterial and venous). These endothelial cells interact with perivascular elements, such as the basal lamina (BMs), smooth muscle cells (SMCs) and astrocytic end-feet processes, perivascular neurons and pericytes to form a functional BBB. (B) The core anatomic elements of the NVU. Created with* 

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**Figure 1.**

*BioRender.com.*

The NVU is a relatively recent neuroscience concept, representing the structural and functional multicellular relationship between the brain and blood vessels [5]. The cellular components are the endothelial cells (EC), pericytes, perivascular astrocytes, microglia, the basement membrane (BM), and neuron (**Figure 1**) [10]. The NVU components share intimate and complex associations, and these associations have led to their classification as a single functioning unit. The NVU is responsible for the maintenance of a highly selective BBB and cerebral homeostasis, as well as the control of CBF [11]. Each NVU component seems to play a specific and active role, maintaining the dynamic linkages reciprocally under physiological conditions.

**Endothelial cells** are considered the BBB's anatomic basis since they form and tightly seal the wall of all cerebral vessels, thereby building a physical barrier between the blood and the brain parenchyma (**Figure 1**). Two different types of endothelial junctions exist: adherens junctions (AJ) and tight junctions (TJ) [12]. Adherens junctions comprise vascular endothelial (VE) cadherin and neural (N-) cadherin, both acting via homophilic interactions [13]. While VE-cadherin is vital for sealing adjacent endothelial cells, N-cadherin mediates their association with pericytes [13]. TJ contains transmembrane proteins such as claudins, occludins, and junction adhesion molecules, as well as the zona occludens cytoplasmic proteins (ZO). These proteins act collectively to close off interconnecting endothelial cells [14], restricting the paracellular diffusion of hydrophilic substances, even ions; this is a unique feature of the BBB endothelium [11] in the other hand, the neurovascular endothelial cells, in contrast to peripheral endothelial cells, is the low expression of adhesion molecules (e.g. member of the immunoglobulin superfamily VCAM-1), in this sense, immune cells never cross unstimulated BBB in the healthy CNS [15]. Interactions of endothelial cells with other NVU members mediate a decrease in transcytotic activity, downregulation of leukocyte adhesion molecules, and regulation of interendothelial junction stability during development and adulthood [14].

**Pericytes** are mural cells enwrapping capillary blood vessels on their abluminal side. Structurally, pericytes extend processes from their cell body, covering several endothelial cells (**Figure 1**). In contrast to peripheral tissues, the brain has the highest pericyte to endothelial cell ratio [16]. Pericytes are embedded within the basement membrane (BM) of capillary endothelial cells and are thus centrally positioned between endothelial cells, astrocytes, and neurons [3]. In total, pericytes cover a large cerebral vascular area which can reach up to 40% of the neurovascular surface [17]. One of the main functions of pericytes is the control of the vasoreactivity and cerebral blood flow in response to neuronal activity [18]. As a recently explored example, glutamate induces prostaglandin E2 and nitric oxide release, which leads to actively relaxing pericytes to dilate capillaries [19]. Vascular permeability increases with decreasing pericyte coverage, which is partly due to the regulation of endothelial transcytosis. Moreover, other parts of the NVU are also influenced by pericytes, including neurons, immune cells, and the basement membrane [20].

**Astrocytes** are the most abundant cell type in the brain with a variety of functions. Beyond BBB regulation, they participate in synapse formation, uptake and recycling of neurotransmitters and ions, regulation of extracellular potassium levels, nutrition of neurons, and control of inflammatory responses within the CNS [21]. Astrocytes provide a cellular link between the neuronal circuitry and blood vessels. This neurovascular coupling enables astrocytes to relay signals that regulate blood flow in response to neuronal activity; this includes regulating the contraction/dilation of vascular SMC surrounding arterioles and

capillaries [22, 23]. Astrocytes are also critical cellular support of BBB integrity. Recent molecular studies have shown several molecules released by astrocytes that enhance and maintain barrier tightness, such as cholesterol and phospholipid transporter molecule apolipoprotein E [24, 25]. Release of apolipoprotein E from astrocytes, for example, regulates endothelial TJs by signaling through the low-density lipoprotein receptor related protein 1 (LRP1) on both pericytes and endothelial cells of CNS microvessels [25]. Astrocytes have been identified as essential mediators of BBB formation and function because of purified astrocytes' ability to induce barrier properties in non-CNS blood vessels [26]. Based on these observations, it has been proposed that astrocytes are necessary for the formation of impermeable TJs in the developing vessels of the BBB.

**Microglia** derive from hematopoietic precursors that migrate from the yolk sac into the CNS parenchyma, acting as the brain's main line of defense past the BBB and play a vital role in innate immune responses in the vascular bed and cerebral parenchyma (**Figure 1**) [27], little is known about how microglial-endothelial communications may shape and regulate the homeostatic BBB. However, studies have demonstrated that microglia are associated with endothelial's nascent vessels in the developing brain, and promote the fusion of cells in the stages following vascular endothelial growth factor-mediated induction [28]. Recent studies have shown the activation of microglia in CNS disorders like AD and multiple sclerosis, which are associated with BBB breakdown and neuroinflammation. In these conditions, microglial activation may be both a cause and consequence of BBB dysfunction [20]. Microglia can exist in one of two active states: in the activated pathway, microglia release proinflammatory cytokines like interleukin-1b and tumor necrosis factor-a. Whereas in alternative pathways, microglia are involved in tissue repair, phagocytosing neurons and foreign material, releasing chemokines and vascular endothelial growth factor [29]. On the other hand, brain endothelial cells can also secrete molecules that cause microglial activation [30]. In summary, a complex interplay between systemic and CNS derived immune cells exists at the BBB.

**Basement Membrane:** The vascular tube is surrounded by two basement membranes (BMs), the inner vascular BM, and the outer parenchymal BM (**Figure 1**). The vascular BM is an extracellular matrix secreted by the ECs and pericytes, whereas the parenchymal BM is primarily secreted by astrocytic processes that extend towards the vasculature [31]. These BMs consist of different molecules, including type IV collagen, laminin, heparin sulfate proteoglycans, and other glycoproteins [32]. They provide an anchor for many signaling processes in the vasculature and also constitute an additional barrier for molecules and cells to cross before accessing the neural tissue. Disruption of these BMs by matrix metalloproteinases is an integral part of BBB dysfunction and posterior leukocyte infiltration, which can be observed in many different neurological disorders [32].

**Neurons and interneurons.** Neurons can detect small variations in their supply of nutrients and oxygen and transform these signals into electrical and chemical messages to adjacent interneurons or astrocytes. In response to these signals, necessary adjustment mechanisms are initiated. Due to this phenomenon, neurons are considered NVU's pacemaker [15]. Neurons need to be able to signal to cerebral vessels when their energy demands change. Positive and negative feedback mechanisms exist to regulate cerebral blood flow, accompanied by adjustments of substrate delivery across the BBB, a process known as neurovascular coupling [33]. In this sense, one relevant mechanism for neurovascular coupling is direct innervation of astrocytic processes or the endothelial tube by, amongst others, serotonergic, noradrenergic, cholinergic, and GABAergic neurons [4]. Mechanisms of neurovascular coupling, particularly those that can explain direct molecular effects on BBB integrity, are yet to be established. Future knowledge will be of great interest since

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**Figure 2.**

*brain. Created with BioRender.com.*

*Blood-Brain Barrier Dysfunction in the Detrimental Brain Function*

new therapeutic tools could help modulate intercellular communication in diseases

The BBB is a diffusion barrier essential for the normal function of the CNS. The NVU endothelial cells differ from endothelial cells in the rest of the vascular system by their absence of fenestrations, and for having more extensive junctional molecules, mainly TJ, and sparse pinocytic vesicular transport [34]. These junctional molecules limit the paracellular flux of hydrophilic molecules across the BBB. In contrast, small lipophilic substances (O2 or CO2) can diffuse freely across plasma membranes along their concentration gradient [34]. Nutrients such as glucose and amino acids enter the brain via transporters, whereas receptor-mediated endocytosis mediates larger molecules' uptake, including insulin, leptin, and iron transferrin [35], it is believed that all the components of the BBB are essential for the normal

The Junction complex in the BBB comprises TJ, AJ, and Gap junctions (GJ). The TJ ultrastructurally appear as apparent fusion sites, involving the outer plasma membrane of adjacent endothelial cells [36]. The number of TJ strands, as well as the frequency of their ramifications, varies and consists of three integral membrane proteins: claudin, occludin, and junction adhesion molecules, as well as several other cytoplasmic accessory proteins, including members of the family zonula occludens (ZO-1, ZO-2, ZO-3) and cingulin (**Figure 2**). Cytoplasmic proteins link membrane proteins to actin, for maintaining the structural and functional integrity of the endothelium [36]. The Claudins were identified as the principal component of TJ and are localized exclusively at TJ strands. Claudins bind to other claudins on adjacent endothelial cells to form the primary seal of the TJ [37]. Closest to the

*Basic molecular organization of BBB junctional molecules and transport. The endothelial cells confer unique properties on the BBB. They are the principal line of cerebral vasculature and have numerous junctional molecules such as tight junctions, adherens junction, gap junctions and accessory proteins that limit the passive paracellular diffusion of all but the smallest of solutes and ions. On the other hand, carriers, receptors and active efflux protein mediated transport allow substances such as peptides, amino acids, and glucose to selectively cross the BBB and release toxic substances and drugs into the blood preventing them from entering the* 

**3. BBB physiology: building blocks and transport routes in BBB**

*DOI: http://dx.doi.org/10.5772/intechopen.94572*

function, stability, and permeability of the BBB.

linked to vascular dysfunction.

**3.1 BBB junctional molecules**

new therapeutic tools could help modulate intercellular communication in diseases linked to vascular dysfunction.
