Brain Edema: From Molecular Mechanisms to Clinical Practice

2012;18:3645–3648.

Section I

General Introduction

Outline

Chapter 1 Physiology of Cerebral Blood Vessels

Chapter 2 Blood–Brain Interfaces Organization in Relation to Inorganic Ion Transport, CSF Secretion, and Circulation

Chapter 1

Physiology of Cerebral Blood Vessels

Ute Lindauer,    RWTH Aachen University, Aachen, Germany

Abstract

The vasculature in the brain is an actively regulated organ which, on the one hand, is influenced by global changes of systemic parameters (systemic blood pressure, blood gases, and blood pH) and, on the other hand, is capable of locally directing the blood to the regions of demand with high spatial and temporal resolution. Together with pericytes and astrocytes, as part of the neurovascular unit, cerebral vascular endothelium forms the tight blood-brain barrier and has an important immuno-regulatory function. Within this book chapter the main principles of the vascular architecture and the cellular components at the neurovascular unit forming the blood-brain barrier will be described before summarizing the main mechanisms of global blood flow regulation to changes in systemic parameters as well as giving an overview on the local regulation according to the cellular demand during neuronal activation.

Keywords

Vasculature architecture; neurovascular unit; cerebral vascular endothelium; neuronal activation; blood flow regulation; astrocyte; pericyte; vascular smooth muscle cell

Outline

Introduction 4

Cerebrovascular Architecture and Principle of Blood Perfusion 4

Cellular Components of Cerebral Vasculature and the Neurovascular Unit 5

Cerebrovascular Endothelial Cells 6

Cerebrovascular Smooth Muscle Cells 10

Pericytes 12

Astrocytes 14

Perivascular Neurons—Extrinsic and Intrinsic 15

Autoregulation and Myogenic Tone 16

Further Mechanisms of Global Blood Flow Regulation: Reaction to pCO2/pH and pO2 Changes 17

Mechanisms of Local Blood Flow Regulation—Neurovascular Coupling 17

Summary 18

References 20

Introduction

Approximately 20% of the body’s oxygen and 25% of the body’s glucose supply are consumed by the brain. Thus, in the brain a disproportionately high percentage of oxygen and glucose is consumed compared to its small fraction of approximately 2% of total body weight. Only very low amounts of glucose are stored as glycogen in astrocytes and most of the glucose is metabolized via oxidative phosphorylation, making the uninterrupted delivery of substrates for energy consumption an important prerequisite for proper brain function. The supply of the main energy substrates oxygen and glucose to the brain depends on the vasculature, and the brain receives approximately 20% of the cardiac output.¹,² The vasculature in the brain is not a stiff line system comparable to the plumbing system in a residential building. Rather, it is an actively regulated organ which, on the one hand, is influenced by global changes of systemic parameters (systemic blood pressure, blood gases, and blood pH) and, on the other hand, is capable of locally directing the blood to the regions of demand with high spatial and temporal resolution.³ Together with pericytes and astrocytes, as part of the neurovascular unit, cerebral vascular endothelium forms the tight blood-brain barrier and has an important immuno-regulatory function.⁴,⁵

Cerebrovascular Architecture and Principle of Blood Perfusion

Four main vessels are feeding the brain, the internal carotid arteries originating from the common carotid arteries, and the vertebral arteries. They form the Circle of Willis at the base of the brain, from which the main cerebral arteries are advancing and branching at the surface of the brain.⁶ Here they form the pial arteries, which send penetrating arterioles into the brain parenchyma with further ramification in precapillary arterioles and the capillary bed. Penetrating arterioles are responsible for approximately 40% of total cerebral vascular resistance;⁷ however, recent findings of large-scale microscopy data analysis combined with hemodynamic simulations suggest that the capillary bed is the largest contributor to vascular resistance.⁸ The venous blood is collected in small postcapillary venules followed by larger venules which reach the brain surface. The main draining systems are the deep and superficial cerebral veins which merge in dural venous sinuses and later on form the jugular vein, finally draining the blood into the superior vena cava.

Oxygen diffusion into the parenchyma occurs not only within the capillary bed but also to a significant amount at the level of penetrating arterioles.⁹ The oxygen content of the blood is lowest in high-branching-order capillary segments and postcapillary venules (pO2 ~ 30–35 mmHg) and slightly increases while passing the venules. It has been shown that under resting conditions, most of the oxygen is released to the tissue at the level of precapillary arterioles and low-branching-order capillaries, whereas high-branching-order capillaries only show a very low oxygen saturation gradient along their course toward the postcapillary venules. This phenomenon may be explained by a reuptake of oxygen from the tissue back into high-branching-order capillaries⁹ and/or into venules.¹⁰ Glucose is transported into the tissue mainly within the capillary bed by specific transporters.² Compared to other vascular systems, the capillaries in the brain are not fenestrated but the endothelial cells are connected by tight junctions, forming the blood-brain barrier together with adjacent pericytes and astrocytic foot processes (for more detail, see Section Blood-Brain Barrier Function). Capillaries, by definition, lack smooth muscle cells (SMCs). However, at distinct locations they are in close contact with pericytes which may take over the role of smooth muscle cells for adjusting blood flow to neuronal activity or metabolic needs at the very regional level.¹¹–¹³

Microvascular oxygen delivery strongly depends on the erythrocyte flux within the vessels. In contrast to the vasculature of skeletal muscles, where total closing and reopening of capillaries occurs (morphological capillary recruitment), in the brain all capillaries are perfused at least by blood plasma all the time.¹⁴,¹⁵ Perfusion by red blood cells is characterized by a significant heterogeneity under resting conditions, meaning that many capillaries are not maximally perfused. Within the capillary network, rapid fluctuations, spatial heterogeneity of velocity, and density of erythrocytes can be observed. During global as well as local stimulation of cerebral blood flow (CBF), the mean blood cell flux and velocity increases, capillary perfusion with red blood cells becomes more homogenous, and the number of poorly cell-perfused capillaries decreases.¹⁴,¹⁶,¹⁷ Hence, within the brain microcirculation a dynamic adjustment of red blood cell perfusion in continuously plasma-perfused capillaries takes place.

Cellular Components of Cerebral Vasculature and the Neurovascular Unit

The blood vessels of the cerebral macrocirculation (pial arteries and arterioles, larger branches of the penetrating arterioles, and larger venules and veins) are separated from the parenchyma by the Virchow–Robin space and are thus not in direct contact to changes of the milieu within the brain tissue. Within the microcirculation (smaller branches of penetrating arterioles, precapillary arterioles, capillaries, and postcapillary venules), the vessels come in close contact with parenchymal cells, forming the well-known entity of the neurovascular unit, a structure where neuronal-activity dependent parenchymal changes also have impact on mural cells of the vessels.¹⁸,¹⁹

The innermost cell barrier toward the blood is formed by endothelial cells. Separated from the endothelial cells by a basement membrane, the middle layer is composed of contractile cells. At the level of arteries and arterioles, diameter changes are induced by contraction or relaxation of smooth muscle cells which form a continuous layer within the vessel wall. Further down the vascular tree at the level of capillaries, smooth muscle cells disappear and are replaced by pericytes which are spatially isolated cells with contractile properties.²⁰,²¹ Larger arterioles and arteries within the pial vasculature are surrounded by the adventitial layer bearing a dense network of perivascular nerve endings of extracerebral origin, whereas intraparenchymal vessels are in contact with intrinsic nerves originating from subcortical areas or with local interneurons.¹⁹ In the following a more detailed description of the different cell types of the neurovascular unit will be provided.

Cerebrovascular Endothelial Cells

The endothelial cells form the innermost cell layer of the barrier separating the blood from the parenchyma. On the luminal (apical) side, the cells are coated by the glycocalyx, a negatively charged dense network of membrane bound molecules (proteoglycans, glycoproteins, and glycolipids), and supplemented by absorbed plasma proteins and glycosaminoglycanes. This coating has a thickness of 0.4–0.5 µm,²² and is known as the endothelial surface layer. It has important physiological roles in regulating cerebral blood flow by significantly contributing to hemodynamic resistance²³ and by mechanotransduction of blood flow-induced forces (shear stress).²⁴ It impacts the permeability of solutes and water,²⁵–²⁷ as well as the adhesion and emigration of leukocytes²⁸ and coagulation²⁹,³⁰ (for more detailed information, see Refs. 31,32).

Blood-Brain Barrier Function

The endothelial cells of cerebral vessels are tightly connected by tight junctions and adherence junctions, continuously blocking permeability via the paracellular route and by this morphologically building the blood-brain barrier. As summarized by three very profound reviews (see Refs. 33–35), the main transmembrane components of the tight junctions are claudins, junctional adhesion molecules, and occludin, which are anchored within the cytoplasm by zonula occludens proteins and by this connected to the actin cytoskeleton. Adherence junctions are composed of the cytoplasmic components ß-catenin and afadin and the transmembrane proteins nectins and cadherins. The tightness of the blood-brain barrier is not accomplished by the endothelial cells alone. At their abluminal side the endothelial cells are covered by the basal lamina (basal membrane), with extracellular matrix proteins being involved in proper tight junction protein expression and by this in barrier function.³⁶ The dense coverage of capillary endothelial cells with astrocytic end-feet³⁷ plays an important role in building up transendothelial resistance. In addition, pericytes are essential for blood-brain barrier function, and it has been shown that pericyte deficiency in the brain leads to increased endothelial transcytosis and increased permeability to immunoglobulins and other plasma proteins.³⁸,³⁹ Both astrocytes and pericytes interact with endothelial cells, and by growth factor signaling and transcriptional regulation of tight and adherence junction proteins, the tightness of the blood-brain barrier is dynamically regulated.³⁵ Beside tight junctions, endothelial cells are connected with each other by gap junctions formed by connexins, responsible for the direct passage of ions and small molecules between adjacent cells.⁴⁰ Gap junction proteins are very often colocalized with tight junction proteins and a dynamic interaction of connexins with other junction proteins has been described.⁴¹,⁴² A cross-talk of the regulation of these two intercellular junctions has been suggested, with gap junction proteins probably also playing a role in blood barrier function.

Vasoactive Factors Released by Endothelial Cells: Endothelium-Dependent Cerebrovascular Reactivity

For several vasoactive humoral or parenchymal stimuli an intact cerebrovascular endothelium is a precondition to induce diameter changes of cerebral vessel. Under resting conditions as well as upon receptor or ion channel activation under physiological or pathophysiological conditions the endothelium is capable of releasing vasoactive factors acting on the underlying smooth muscle cells inducing vasodilation or vasoconstriction.

Probably the most important and best known molecule released by endothelial cells is the vasodilator nitric oxide (NO), identified as the molecule behind the endothelium-derived relaxing factor, released by endothelial cells upon acetylcholine (ACh) receptor activation and acting via cGMP production in vascular smooth muscle cells.⁴³–⁴⁶ Along with ACh, other molecules like bradykinin, substance P, or arginine vasopressin (only in selected vascular beds)⁴⁷–⁴⁹ also induce NO-dependent vasodilation. Beside this receptor activation-dependent augmentation of NO production, NO is also continuously produced and perivascularly released by endothelial cells and perivascular neurons (see below) by the constitutively expressed NO synthases (NOS) (isoforms I and III), inducing a basal vasodilative tonus of the vessels. Endothelial-derived NO also provides important antiinflammatory properties preventing coagulation and leukocyte aggregation and adhesion to the vessel wall, for example, by reducing endothelial expression of adhesion molecules and pro-inflammatory cytokines⁵⁰–⁵² (for review, see Refs. 53,54).

In addition to NO, arachidonic acid metabolites are important endothelial mediators of vasodilation. Besides the enzyme responsible for NO production, endothelial cells also constitutively express cyclooxygenase 1 (COX-1, also called prostaglandin H synthase⁵⁵,⁵⁶), converting arachidonic acid, liberated from membrane phospholipids by phospholipases, to prostacyclin (prostaglandin I2), inducing relaxation of the underlying vascular smooth muscle cells by activation of adenylate-cyclase and production of cAMP.⁵⁷,⁵⁸ As with NO synthase within endothelial cells, COX-1 is continuously activated by blood flow-induced shear stress or can be further activated by receptor-dependent activation.⁵⁹ Under pathophysiological conditions, arachidonic acid can also be metabolized by the COX-pathway to prostaglandin PGH2/thromboxaneA2 (TXA2) with vasoconstricting properties.⁶⁰

Another pathway of arachidonic acid metabolism represents the formation of epoxyeicosatrienoic acids (EETs) by cytochrome P450 epoxygenase located within endothelial cells. According to a recent study EETs relax smooth muscle cells by activation of a novel Ca²+ signaling complex in cerebral vascular smooth muscle cells, consisting of the calcium-permeable member of the transient receptor potential vanilloid cation channels TRPV4, of ryanodine receptors, and of large-conductance Ca²+-activated potassium channels (BKCa) located on smooth muscle cells.⁶¹,⁶² However, suppression of EETs production by NO within endothelial cells has been suggested.⁶³

A third important endothelium-dependent vasodilating mechanism is endothelium-derived hyperpolarization (EDH), also significantly contributing to resting diameters of cerebral vessels and thus to resting CBF. Following a long search for identifying a factor mediating the hyperpolarization of smooth muscle cells, the current knowledge argues for a mechanism rather than a real mediator molecule. Activation of small- and intermediate-conductance Ca²+-activated potassium channels (SKCa, KCa2.3, and IKCa, KCa3.1) within the membrane of endothelial cells (e.g., by stimulation of endothelial P2 purinoceptors⁶⁴) induces endothelial cell hyperpolarization. This hyperpolarization propagates towards the vascular smooth muscle probably through myo-endothelial gap junctions, inducing relaxation by closure of voltage gated Ca²+ channels at the smooth muscle cells (see Section Cerebrovascular Smooth Muscle Cells).⁵⁹,⁶⁵–⁶⁸ In addition to their role for EDH, IKCa channels may also play an important role in endothelial barrier dysfunction and edema formation in brain pathologies.⁶⁹

Two other gaseous mediators or modulators of vascular tone, carbon monoxide (CO), and hydrogen sulfide (H2S), may also be produced by endothelial cells via the constitutively expressed enzymes heme oxygenase-2⁷⁰,⁷¹ and cystathionine-γ-lyase/cystathionine β-synthase/3-mercaptopyruvate sulfurtransferase,⁷² respectively. Acute elevations of CO in newborn pigs dilate cerebral vessels by directly acting on BKCa channels on smooth muscle cells by elevating the channel-apparent Ca²+ sensitivity⁷³ and by elevating BKCa channel activity by increasing the coupling of Ca²+ sparks to BKCa channels.⁷⁴,⁷⁵ In addition, a permissive role of endothelial NO has been reported for CO-induced dilation in piglet pial arterioles.⁷⁶ In contrast to the acute effects of CO in piglets, prolonged CO production in the adult rat cerebral microcirculation has been shown to induce vasoconstriction, apparently by inhibiting NOS.⁷⁷ Species and age related differences in the anatomical localization of the NO- and CO-gas-producing enzymes and their reception systems may be responsible for these discrepant observations. For H2S it has been convincingly shown within the mesenteric circulation and the aorta that H2S is a major EDHF, causing vascular endothelial and smooth muscle cell hyperpolarization and relaxation by activating ATP-sensitive (KATP) as well as IKCa and SKCa potassium channels through cysteine S-sulfhydration.⁷⁸,⁷⁹ Within the cerebral circulation H2S has been shown to dilate cerebral arterioles mainly by activating KATP channels on smooth muscle cells,⁸⁰ which may become particularly important during NO deficiency.⁸¹

Endothelial cells are also capable of releasing vasoconstricting molecules. Beside the arachidonic acid metabolites PGH2/TXA2 (see above), one of the most potent endothelium-derived constrictors is endothelin-1, which is produced as pre-pro-ET-1, cleaved to big ET-1 by an endopeptidase and then converted to ET-1 by endothelin-converting enzyme.⁸² Vasoconstriction is induced by activation of endothelin-A and endothelin-B receptors located on vascular smooth muscle cells. Beside this strong predominant vasoconstrictive action, endothelin-1 at very low concentrations can also induce endothelium-dependent and NO-mediated vasodilation by activation of endothelin-B receptors located at endothelial cell membranes.⁸³,⁸⁴

Mechanotransduction of Shear Stress

Endothelial cells are exposed to mechanical frictional forces by the circulating blood. This shear stress induces vasodilation and is thus also called flow-induced vasodilation. Membrane molecules and cellular microdomains (e.g., the glycocalyx, adhesion proteins, G proteins of G protein-coupled receptors, and the cytoskeleton) contribute to sensing of shear stress.⁸⁵ Ca²+ influx via activation of transient receptor potential (TRP) channels²⁴ leads to eNOS activation and NO production and thus vasodilation. In addition, a autocrine/paracrine action of endogenous ACh may be involved, where ACh is produced and released by endothelial cells following increased shear stress, again resulting in enhanced NO production.⁸⁶

Cerebrovascular Smooth Muscle Cells

Vascular smooth muscle cells are the main effectors responsible for the regulation of global blood flow at the level of larger pial arteries as well as for the spatially defined regulation at the level of penetrating and precapillary arterioles. Besides the intrinsic tone of the arterial circulation induced by intraluminal pressure and sheer stress at the endothelial cell layer, the balance between vasoconstrictive and vasodilative molecules released by vascular endothelial or brain parenchymal cells and acting on smooth muscle cells determines the diameters and thus the blood flow within the vasculature. Dependent on the activity of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP), the balance of phosphorylation and de-phosphorylation of myosin light chain determines the contractility of the smooth muscle cell.⁸⁷

Principle Mechanisms of SMC Constriction or Relaxation

Constriction or relaxation of SMCs occurs mainly by two mechanisms: (1) via changes of the intracellular Ca²+ concentration due to exchange from the extracellular compartment or due to release and reuptake from and to internal stores, determining the activity of the MLCK,⁸⁸ and (2) via alteration of the Ca²+ sensitivity of the contractile apparatus without changes in Ca²+ concentration (so-called Ca²+ sensitization), determined by RhoA/Rho-associated protein kinase (ROCK)-dependent phosphorylation status of MLCP with ROCK promoting contraction by phosphorylating and thus inhibiting MLCP.⁸⁹,⁹⁰ Beside induction of Ca²+-sensitization, it has recently been shown that the RhoA/ROCK pathway can also induce membrane depolarization via activation of transient receptor potential cation channels (subfamily M member 4, TRPM4) and thus opening of voltage-dependent Ca²+ channels (VDCC).⁹¹

Intracellular Ca²+ increases via flux through receptor operated ion channels (ROCs) or by activation of VDCC following depolarization of the smooth muscle cell membrane. ROCs are nonselective cation channels, mainly members of the transient receptor potential canonical (TRPC) channels family. They simultaneously induce the entry of Na+ and Ca²+ and thus triggering cell membrane depolarization. Whether the increased Ca²+ flux through the channels directly activate the contractile process is not known so far.⁹²

Membrane potential changes are responsible for the degree of Ca²+ influx through VDCC. In cerebral vasculature, L-type (CaV1.2) and T-type (CaV3.1 and CaV3.2) voltage-dependent Ca²+ channels are expressed in smooth muscle cells.⁹³

Potassium Channels on VSMCs

In addition to the activity of ROCs, membrane potential changes are mainly induced by changes of the conductance of potassium channels within the cell membrane. Upon activation of potassium channels, cell membrane hyperpolarization occurs due to potassium outward flow, inducing closure of VDCCs followed by a decrease in intracellular Ca²+ concentration and vascular relaxation (for detailed review on potassium channels of VSMCs, see Ref. 53). The main types of classical potassium channels within smooth muscle cells are ATP-sensitive potassium channels (KATP), large-conductance Ca²+-activated potassium channels (BKCa), voltage-dependent potassium channels (KV), and inward rectifier potassium channels (KIR). Whereas KIR channels are mainly activated by low concentrations of potassium (up to 20 mM) and KV channels by membrane depolarization, KATP and KCa channels can be activated by agonists following receptor activation as well as second messengers (cGMP and cAMP). KATP channels are also sensitive to hypoxia and energy failure induced ATP reduction. BKCa channels are also activated by local Ca²+-release events (Ca²+ sparks) through ryanodine-sensitive channels in the sarco/endoplasmic reticulum⁹⁴ probably gated by Ca²+ released through L-type as well as T-type VDCCs residing near ryanodine receptor,⁹⁵ resulting in spontaneous transient outward currents (STOCs).⁹⁶–⁹⁸ Following membrane depolarization and rise in cytosolic Ca²+ concentration, KV and BKCa-mediated K+ conductances drive a negative feedback mechanisms to prevent excessive myogenic constriction.⁹⁶,⁹⁸

In addition to the classical K+ channels with a single-pore region mentioned above, the expression of nontypical, two-pore domain K+ (K2P) channels has recently been confirmed in cerebrovascular SMCs, which can be directly activated by unmetabolized arachidonic acid inducing K+ outward flux, membrane hyperpolarization, and vasodilation.⁹⁹ The overall significance of these channels is not known so far and requires further investigation.

Intracellular Ca²+ concentration can also be changed by activation of recently identified acid sensing ion channels expressed within the SMC membrane of cerebral arteries.¹⁰⁰ It has been shown that they are involved in regulation of smooth muscle cell migration,¹⁰¹ and they may play a role in pressure-induced constriction and basal myogenic tone.¹⁰²

Role of Internal Ca²+ Stores

Beside Ca²+ entry from extracellular space through ion channels located within the cell membrane, Ca²+ can also be released from internal stores via inositol 1,4,5-trisphosphate (IP3) receptor channels located in the membrane of the sarco/endoplasmic reticulum. Activation of smooth muscle cell membrane bound receptors followed by phospholipase C activation leads to hydrolysis of phosphatidylinosol 4,5-bisphosphate (PIP2) to IP3. IP3 receptors located on the endoplasmic reticulum respond to this by releasing Ca²+ and thus inducing contraction.¹⁰³,¹⁰⁴ (Re-)loading of the sarco/endoplasmic reticulum with Ca²+ occurs via activity of the Ca²+ pump sarco/endoplasmic reticulum Ca²+-ATPase (SERCA).¹⁰⁵ Depletion of sarco/endoplasmic reticulum Ca²+ stores, on the other hand, activates store operated Ca²+ channels increasing cytosolic Ca²+ concentration specifically within noncontractile compartments without vasoconstriction.¹⁰⁶

Mechanisms of NO-Induced Vasodilation

As mentioned above, NO released by endothelial cells and perivascular neurons provides a basic vasodilative tonus within the vasculature in the brain. NO stimulates soluble guanylate cyclase within SMCs converting GTP to cGMP followed by activation of cGMP-dependent protein kinase and target protein phosphorylation, resulting in vasodilation by various mechanisms (BKCa channel activation, inhibition of Ca²+ release from endoplasmic reticulum, and inhibition of Rho kinase/ROCK pathway by phosphorylation of Rho).¹⁰⁷ In addition, cGMP-independent effects exist: Besides inducing nitrosylation of target proteins (e.g., causing activation of potassium channels), NO is known to inhibit cytochrome P450 (CYP450, CYP) ω-hydroxylase activity (CYP 4A and 4F families) that builds 20-hydroxyeicosatetraenoic acid (20-HETE) from arachidonic acid in cerebral arteries.¹⁰⁸ When perivascular NO is reduced or absent, 20-HETE accumulates and potently constricts VSMCs by inhibiting BKCa channels; by activating L-type VDCCs; and by stimulating protein kinase C, Rho kinase, and the mitogen-activated protein kinase pathway (for detailed review, see Ref. 109). In addition, NO has been shown to suppress the Ca²+ currents of T-type and L-type VDCCs and by this reducing Ca²+ concentration in SMCs.¹¹⁰,¹¹¹ Furthermore, NO may also induce vasodilation by increasing the uptake of Ca²+ by SERCA.¹¹²

Concluding Remarks

The above mentioned mechanisms leading to VSMC contraction or relaxation and by this vasoconstriction or vasodilation are not uniformly active in all vascular beds within the cerebral vasculature. They may significantly differ in their importance and specificity between larger pial arteries and intraparenchymal arterioles. For a comprehensive overview of these differences with respect to the control of smooth muscle membrane potential see the recent review by Longden et al.¹¹³

Pericytes

Pericytes are contractile cells located within leaves of the basement membrane. They form bulging cell bodies with distinct circular and longitudinal processes and sheet the capillaries with variable density and morphology along the capillary bed. The coverage is most compact at the transition from precapillary arterioles to first-order capillaries, forming a mesh-like structure, whereas further on they become more delicate with thin circular and small longitudinal strands. Toward the postcapillary venules they regain their mesh-like structure, however, with lower degree of coverage compared to the first-order level.²⁰,²¹,¹¹⁴ Pericytes are in close contact with endothelial cells, connected by cap junctions and adherence junctions,¹¹⁵,¹¹⁶ and reside in close proximity to astrocytic end-feet and perivascular neuronal processes, and thus, together with VSMCs, being an integral part of the neurovascular unit.

Pericytes are characterized by expressing contractile and cytoskeletal proteins (amongst others: α-smooth muscle actin and myosin), and are commonly identified by their expression of the cell surface antigens transmembrane chondroitin sulfate proteoglycan NG2, platelet-derived growth factor receptor-β, and aminopeptidase A (for overview, see Ref. 117).

They play an important role in tightening of the blood-brain barrier by supporting and controlling endothelial cells expressing tight and adherence junction proteins and by inducing polarization of astrocytic end-feet.³⁸ In addition, they seem to be involved in the regulation of signaling for leukocyte adhesion and transmigration at endothelial cells and communication of inflammatory signals within the neurovascular unit.¹¹⁸,¹¹⁹ Furthermore, pericytes react to many vasodilative and vasoconstrictive molecules and mechanisms comparable to VSMCs.¹²⁰,¹²¹ Based on this, in recent years, profound experimental evidence has been described for a prominent role in regulation of cerebral blood flow at the level of capillaries,¹¹,¹³,¹²² with pericytes probably being responsible for the very early increase of capillary blood flow to neuronal activation, even before precapillary and penetrating arterioles dilate.¹² Discrepant results²¹ have opened a discussion on the precise definition of capillaries at the transition zone from precapillary arterioles to capillaries and by this of pericytes and VSMCs within the microvasculature.²⁰ It is worth to notice in this context that pericytes can constrict and by this can narrow the lumen of capillaries to an extent leading to the complete blockade of red blood cell passage under pathophysiological conditions.¹¹,¹²

Many signaling pathways and molecules (e.g., the platelet-derived growth factor (PDGF)-BB—PDGF receptor β pathway, the transforming growth factor-β (TGF-β)—TGF-β receptor-2 pathway, the notch pathway, the vascular endothelial growth factor (VEGF)-A-VEGF receptor-2 pathway, the arachidonic acid pathway, NO, and potassium) are involved in the above mentioned functions of pericytes mediating the communication to and receiving information from endothelial cells, astrocytes, and perivascular neurons and thus within the neurovascular unit. A detailed description of these pathways is beyond the scope of this chapter and is excellently provided in a recent review by Sweeney et al.,¹¹⁷ which can be recommended for further reading.

Astrocytes

Astrocytes are ideally located within the neurovascular unit with having contact to neurons at the synapses as well as to the vasculature by astrocytic end-feet. At the level of larger penetrating arteries they form the glia limitans, with their end-feet layer bordering the Virchow–Robin space which separates the arteries from the parenchyma, contains interstitial fluid, and builds the so-called cerebral glymphatic system.³⁵,¹²³ Further down the vascular tree, the Virchow–Robin space becomes smaller and disappears, and astrocytes come in close contact to the SMCs at the vessel wall of arterioles and venules and to the endothelial cells and pericytes at the level of capillaries, respectively.

Astrocytes have an important role in supporting neuronal activity by uptake and recycling of neurotransmitters and, via gap junction coupling, in redistribution of K+ ions.¹²⁴,¹²⁵ In addition, they substantially participate in glucose metabolism, the main fuel of neurons.¹²⁶

For vascular function, the signaling from and to their end-feet is essential. Besides being involved in blood-brain barrier tightening, astrocytes are the key contributors to water exchange from and to the brain parenchyma passing through the blood-brain barrier. Aquaporin (AQP) channels permit passive bidirectional water flux along the osmotic diffusion gradient, with AQP4 being highly expressed in astrocytic perivascular end-feet.¹²⁷

Finally, astrocytes are important players in blood flow regulation.⁴,¹²⁸–¹³³ The key event of vasoactive signaling of astrocytes is the increase of Ca²+ within the cells. Activation of metabotropic glutamate receptors located at the membrane of the cell bodies induce phospholipase C activation, IP3 production and release of Ca²+ from endoplasmic reticulum stores resulting in increased Ca²+ concentration in somata and end-feet.¹³³,¹³⁴ Recently, the proposed key role of IP3-dependent Ca²+ signaling for vascular regulation has been questioned,¹³⁵ and besides IP3 receptor activation-dependent Ca²+ signaling, transmembrane Ca²+ fluxes may be involved in wave and microdomain Ca²+ fluctuations in astrocytic processes.¹³⁶ Other neurotransmitters like ATP released by neurons may also induce astrocytic Ca²+ elevation by purinergic receptor stimulation via IP3-dependent (purinergic metabotropic P2Y receptors¹³⁷) or IP3-independent pathways (purinergic ionotropic P2X receptors¹³⁸,¹³⁹). Several mechanisms have been proposed to induce vasodilation due to Ca²+ increases in astrocyte somata and end-feet processes. Activation of phospholipase A or phospholipase D¹³⁹ liberates arachidonic acid from membrane phospholipid pools. Metabolism of arachidonic acid by cytochrome P450 epoxygenase results in astrocytic EETs production,⁶³ which may dilate the vessel by activation of BKCa channels directly on SMCs¹⁴⁰ or on astrocytic end-feet membranes, followed by K+ release with subsequent activation of KIR channels on SMCs,¹⁴¹ both inducing membrane hyperpolarization and thus relaxation of the cells. The role of astrocytic EETs production in blood flow regulation is not fully proven so far, as vasodilation due to local Ca²+ uncaging in astrocytic end-feet was not prevented by CYP450 epoxygenase inhibition.¹³² Arachidonic acid can also be metabolized by COX forming prostaglandin E2. However, it is not resolved whether astrocytes uniformly express COX-1 or cyclooxygenase 2 (COX-2) and whether prostaglandin E2 (PGE2) dilates or rather constricts parenchymal arterioles (for review, see Ref. 113). In addition to arachidonic acid metabolism induced vasodilation, Ca²+ waves reaching the end-feet processes may also induce K+-dependent vascular dilation by activation of astrocytic BKCa channels followed by K+ release with subsequent activation of KIR channels on SMCs. In this context, it has recently been proposed that Ca²+-sensitive TRPV4 channels, which are expressed in the astrocytic end-feet membranes, may also be activated by Ca²+ waves, stimulating further Ca²+ influx and by this locally boosting the effect on BKCa channels and amplifying the