The Neurovascular Unit and Responses to Ischemia

Key Points

The neurovascular unit is a structural and functional concept that seeks to integrate microvessel events with those of the neurons, glia, and brain matrix and serves as a framework for fundamental and new findings (e.g., the glymphatic CSF transport network).
Within each compartment, networks of cells, matrix, and membranes interact and are moderated by supportive cells.
Coordinated changes in brain vascular and microvascular caliber and in blood flow provide an example of neurovascular coupling, although the precise signaling mechanisms are under study.
Disease processes (e.g., ischemic stroke, innate immunity, and inflammatory disorders) negatively impact the functional integrity of the neurovascular unit and may lead to territorial dysfunction.
Knowledge of cell–cell and compartment–compartment interactions is incomplete (e.g., precise roles of pericytes in microvessels and microglia and oligodendroglia in the neuropil). A full understanding of the signaling interactions between and among elements of the neurovascular unit may have predictive value.

Introduction

The central nervous system (CNS) consists of regions of microvessels and neural tissue that interact to maintain homeostasis and neuronal function, prevent the entry of substances from the vascular compartment that are toxic to neurons, promote organized neuronal and vascular responses to peripheral stimuli, and limit injury to cerebral regions. It was previously thought of as regions of neurons with cells supporting their nutrition, viability, and function; however, studies of the responses of cerebral tissue to ischemic injury reveal more complex cooperative relationships.

Cells of the CNS belong to at least six interacting networks: (i) neuronal networks that relay electrical information; (ii) syncytia of astrocytes that serve neurons and are part of microvessels; (iii) the endothelium that covers the luminal surface of microvessel and large vessel interiors; (iv) the extracellular matrix (ECM) that comprises the basal lamina of the vascular tree; (v) pericytes, positioned between the endothelium and astrocyte components of the microvasculature, that form a network within the vascular ECM; and, (vi) a glymphatic cerebrospinal fluid (CSF) transport network.

Control and modulation of regional and local cerebral blood flow (CBF) under conditions of normoxia depend upon neurovascular coupling. These rely upon neuron ⇒ microvessel communication. The proximity of microvessel endothelial cells to the circumferential astrocyte end-feet of capillaries, separated by the thin basal lamina ECM, ^, and the support of neurons by astrocytes suggest that actions could be directed from microvessels to the neurons they supply. ^, These rely upon microvessel ⇒ neuron communication. Hence, neuron ⇔ microvessel interactions can be described within the context of a neurovascular unit that consists of microvessels (endothelial cells, basal lamina ECM, astrocyte end-feet, and pericytes), astrocytes, and neurons and their axons, in addition to the supporting cells of the neuropil. This unit provides a structural framework for considering bidirectional actions of neurons and their supplying microvessels via intervening astrocytes and the potential modulation of these actions by other cells (e.g., microglia, oligodendroglia). It also provides a functional framework for considering the elements of the unit in their responses to changes in flow, ischemia, inflammatory stimuli, and other processes. These structural and functional relationships are built during brain tissue development as the vasculature matures. Growing insight into these cell and structural relationships sharpens research focus and explains the outcomes and limitations of fundamental and clinical research trials.

Architecture of the Central Nervous System and the Neurovascular Unit

Although the neuronal network subserves continuous input and output of the cerebral tissue, its functional complexity is supported by an organized vascular supply that contains the individual endothelial, basal lamina ECM, astrocyte, and pericyte networks. There is also heterogeneity within these networks, as displayed by endothelial cell function. Complexity within the vasculature is marked by local and regional endothelial cell specialization and variation in basal lamina ECM composition along the vascular axes. ^, It is likely that other networks have a similar local specialization.

Structural Relationships: Anatomy of the Cerebral Vasculature

Under normoxic conditions, the cerebral vasculature uniquely protects the neuropil from changes in plasma ion concentrations, as well as exposure to plasma substances toxic to neurons and other cells. It preserves neuronal cell function through shifting blood flow to regions of activation on demand, maintains an intravascular antithrombotic and antiinflammatory milieu, and supplies essential nutrients. This protection may be disrupted under conditions of focal or global ischemia, or other injury.

The general strategy to protect cortical and deep brain structures from obstruction of CBF involves the interconnection of territories of brain-supplying vessels via pial collateral anastomoses over the cerebral hemispheres and the circle of Willis. Pial and cortical penetrating arteries consist of (i) an endothelial cell layer, (ii) the basal lamina, (iii) layers of smooth muscle cells encased in ECM (myointima), and (iv) an adventitia derived from the leptomeninges. In the cortex and striatum, an extension of the subarachnoid space forms the Virchow-Robins (V-R) space, which surrounds penetrating arterioles until it “disappears” into the glia limitans . The abluminal boundary of the glia limitans is formed by the astrocyte end-feet. With further arborization of the microvasculature, the glia limitans at the capillary level fuses with the thin basal lamina. The ECM composition varies depending upon the vessel of interest. ^, Astrocytes serve both microvessels and neurons in this setting ( Figs. 7.1–7.3 ) and are considered the vascular portion of the neurovascular unit . The post-capillary venule network bears close ultrastructural resemblance to the capillaries, except for the presence of a limited myointimal layer. It is the site of polymorphonuclear (PMN) leukocyte-platelet interactions, leukocyte adhesion, and transmigration.

Fig. 7.1, Characteristics of cerebral microvessels under normoxic conditions. Upper left panel, Depiction of a neurovascular unit displaying relationships between neurons and their supply capillaries. Upper right panel, Capillary from rat inferior colliculus. Note endothelial cell, basal lamina matrix, and surrounding astrocyte end-feet. 4.5–5.0 μm diameter. Lower-left panel, Post-capillary venule from rat cortex. ∼10 μm diameter. Lower right panel, Key to the lower-left panel, indicating the endothelium (endo), basal lamina matrix (arrowheads), astrocyte, and astrocyte end-feet (A), and pericytes (∗).

Fig. 7.2, Vascular astrocyte end-feet surround all parts of the vasculature in central nervous system. Spatial organization of astrocytes and neurons in the neurovascular unit . Astrocytes extend two types of processes: (1) vascular end-feet that surround the vessel wall, and (2) fine peri-synaptic processes that enwrap synapses. In this figure, astrocytes are expressing enhanced green-fluorescent protein (eGFP; yellow ), whereas neurons are labeled with antibody to microtubule-associated protein 2 (MAP-2; blue ). A small vessel is outlined by eGFP-positive astrocytic end-feet.

Fig. 7.3, A schematic drawing of the vascular tree (pial artery→penetrating artery→arteriole→capillary). The cross-section is at the level of the capillary. Astrocyte end-feet, that are connected by gap junctions and express aquaporin-4 (AQP4), surround the entire vasculature in central nervous system. The perivascular space is a donut-shaped tunnel system formed by vascular astrocytic end-feet (outer wall) and the vessel (inner wall). The perivascular space around penetrating arteries is also called the Virchow-Robins space. The term “basal lamina” refers to the matrix between the endothelium and the astrocyte end-feet in cerebral capillaries and bounds the perivascular space in microvessels. The glymphatic system utilizes the perivascular space for rapid influx (arteries) of cerebrospinal fluid that flows out along veins.

Also, recent work suggests that the paravascular space plays an important physiologic function as a highway for the influx of cerebrospinal fluid that subsequently mixes with interstitial fluid as part of the so-called glymphatic clearance system. The glymphatic fluid system intersects with the V–R space and affords clearance of CSF and solutes from the CNS.

The neuropil consists of neurons of a variety of subtypes, astrocytes, oligodendroglia, microglia, mast cells, and ECM containing chondroitin sulfate proteoglycans (CSPG) (e.g., CAT-301pg), neurexin, and other proteoglycans. The relative content of these cells and components and their interrelationships likely depend upon the region of the CNS under consideration.

A unique feature of the cerebral capillary is the permeability barrier, a strategy that prevents contact of the neuropil with the blood and its plasma constituents while allowing O ₂ delivery. The permeability barrier consists of (i) the inter-endothelial cell tight junctions and limited pinocytosis, (ii) the basal lamina ECM, ^, (iii) pericytes, and (iv) adhesion of the astrocyte end-feet to the intact subtending basal lamina. Developmental interrelationships between the endothelium and astrocytes highlight the close functional association of endothelial cells and astrocytes in cerebral capillaries. Astrocytes and endothelial cells interact to form the intervening basal lamina barrier and the inter-endothelial tight junctions as part of the capillary permeability barrier. Elegant xenograft experiments have shown that endothelial cells and astrocyte end-feet are required for the appearance and maintenance of the low permeability barrier phenotype. Cerebral microvessel endothelial cells display regional functional specialization along the microvascular axis, ^, which appears to depend on the interaction of its cellular components. ^, It has been postulated that soluble factors generated by astrocytes maintain the endothelial characteristics of the blood-brain barrier, including the tight junctions, transendothelial resistance, and the polarity of glucose-amino acid transport. ^,

Two additional barriers include the intact basal lamina and receptor-associated adhesion of the endothelium and astrocyte end-feet to their ECM ligands. The intact basal lamina ECM protects the brain parenchyma from hemorrhage. ^, The basal lamina contains laminins, collagen type IV, fibronectin, and other components, including heparan sulfate proteoglycans (HSPGs; e.g., perlecan), entactin, nidogen, and other minor components. ^, Adhesion of both endothelial cells and astrocytes to the basal lamina requires the interaction of cells with their matrix ligands. ^, Haring et al. described integrin receptor expression patterns of integrin subunits in normal CNS microvessel subclasses. Their potential roles in microvessel and permeability barriers have been considered recently. Integrin α1β1 on endothelial cells appears in all cerebral microvessels, including cerebral capillaries. ^, ^, ^, Integrins α3β1 and α6β1 are also expressed by cerebral endothelial cells. Integrin α6β4 is expressed on astrocyte end-feet around select microvessels, whereas integrin α1β1 can be found on their fibers, although on select astrocytes and much less frequently. ^, αβ-dystroglycan, the sole member of a separate family of matrix adhesion receptors, is predominantly expressed by astrocyte end-feet. Middle cerebral artery occlusion (MCA:O) perturbs adhesion receptor–matrix relationships that coincide with the detachment of astrocyte end-feet from the basal lamina.

Little is known, yet, about the signaling functions of the adhesion receptors expressed in cerebral microvessels. Osada et al. and Izawa et al. have demonstrated that interference with integrin–matrix adhesion disrupts permeability and tight junction fidelity, ^, indicating that inter-endothelial cohesion and endothelial cell–basal lamina adhesion are connected.

Pericytes are embedded within the basal lamina matrix of all cerebral microvessels. These pluripotential cells become activated and can migrate in response to specific stimuli including focal ischemia and inflammation. Pericytes also have been shown to have properties found in select inflammatory cells. ^, A role for pericytes in the formation of the microvessel permeability barrier and its fidelity has been demonstrated. Pericytes are also involved in the development of the glymphatic system. The number of pericytes decreases during aging, along with microvessel density and glymphatic flow. Pericytes have also been postulated to influence capillary diameter and may contribute to local blood flow regulation under normoxia, although other groups have challenged these findings. ^,

Specialization of the microvasculature is implied by differences in endothelial function, basal lamina ECM composition, and adhesion receptor expression that may accord with regional specialization of the neurons. Currently, for microvessel–astrocyte–neuron partners, such specialization has not been explored. However, evidence for neuron–microvessel functional relations is known.

Functional Relationships

Functional features supporting the concept of the neurovascular unit have been revealed by (i) physiologic studies of regional CBF dynamics and (ii) interneuronal communication hinting at functional pairings between the microvasculature and the neuropil. These may appear to be unidirectional (e.g., neuron ⇒ microvessel); however, they are more likely multidirectional based on the established networks (see aforementioned section).

Neuron—Microvessel Communication

The structural relationships established during brain development allow neurons to effect changes in flow through the (micro)vasculature. Neurovascular coupling has been recognized since the time of Sherrington and refers to the inter-relationship between CBF and neuronal activity that balances energy demand with nutrient supply. The mechanism that achieves coupling between flow and neuronal activity does not appear to involve direct sensing of energy consumption per se , at least under physiologic conditions. It appears to depend upon the integrity of the neurovascular unit and cross-talk among neurons, astrocytes, arterioles (vascular smooth muscle), and microvessels via neurotransmitters, astrocyte signaling, and exocrine effectors. Effectors released from neuronal and glial compartments implicated in the vascular response include NO, adenosine, H ⁺ , K ⁺ , CO ₂ , and arachidonic acid metabolites. Astrocytes provide one link between neuronal activity and transmitter release (i.e., glutamate) and local CBF changes. In the formulation reviewed in the following section, astrocytes release vasoactive molecules that regulate diameter changes of supply vessels. More recently, it has been shown that hyperemia is initiated in capillaries. Possible mechanisms may involve pericytes, PO ₂ dips, or raised extracellular K ⁺ . ^, ^,

According to another formulation, axon terminals releasing neurotransmitters approximate vascular smooth muscle cells and may contribute to flow. Currently, our view of neurovascular coupling is incomplete. Alterations in local CBF may reflect (i) astrocyte–smooth muscle–endothelial interactions, (ii) neuron-vascular interactions, and/or (iii) other couplings.

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