The brain and retina, which constitute the central nervous system (CNS), are highly complex tissues, requiring high levels of energy for function and tight control for health. To achieve this, they contain a specialized vasculature that controls parenchymal homeostasis, transport of metabolites, and confers, in part, so-called immune privilege (Forrester et al., 2018; Taylor and Ng, 2018). Most importantly, the bi-directional movement of molecules across the blood-tissue barriers is strictly controlled to maintain CNS health and brain function. For many decades the focus of this regulatory capacity lay at the endothelial cell (EC), the predominant cell of the blood-brain barrier (BBB), but more recently the wider neurovascular unit (NVU) has been recognized as providing functionality. The NVU is a complex multi-hetero-cellular structure of EC, neurons, glia, smooth muscle cells (SMCs), pericytes, and extracellular matrix (ECM). Together, these components regulate blood flow and metabolism, thus allowing the controlled exchange of nutrients and metabolic waste products (Hawkins and Davis, 2005; Lok et al., 2007). To meet the high metabolic demand of the CNS, particularly in response to an intensification of physical or mental activity, increased neuronal activity leads to subsequent changes in cerebral blood flow (functional hyperemia), in a process called neurovascular coupling. While neurons can directly regulate this system (McConnell et al., 2017), glial cells are often in direct physical contact with the vasculature and neurons, thus are critically positioned to interface between these cellular components where they may contribute to the relay of information and act as a modulator of such crosstalk. Additionally, NVU components are crucial for brain protection and homeostasis as ECs form the BBB, and glia physically ensheathing the blood vessels are seen as a secondary barrier (Kutuzov et al., 2018). With this close physical interaction between ECs and glia, nutrients required for CNS function are delivered from the blood vessels to neurons mainly via glia cells (Hurley et al., 2015), while waste compounds are passed via glial cell to microglia or back into the bloodstream (Marina et al., 2018). Dysfunction of the NVU is characterized by dysregulation of neurovascular coupling, neuron death, gliosis, microglia activation, mural cell transmigration, and BBB breakdown (Zlokovic, 2005; Willis, 2011). Dysfunction of the BBB is associated with increased vascular leakage, transcellular transport, immune cell infiltration, and reduction of intercellular junctions (Figure 1). Accordingly, glial cells and other NVU cells work closely together to maintain CNS function and maintenance.

In this review, we discuss glia cell types and their role in the NVU, by examining glia specializations to support neurons, the vasculature, and neuro-vascular interactions in the NVU. Lastly, we will highlight how rapidly improving techniques and tools will help us answer pressing outstanding questions in the field. While this review is not meant to be an exhaustive list of the many types of glia within the CNS, we aim to highlight their importance for the development, function, and dysfunction of the NVU in the brain and retina.

The complex interaction between NVU cells requires each cellular component to operate in a complex and coordinated manner to ensure homeostatic control of the BBB and blood retina barrier (BRB). Each component exhibits specialized features that are critical to the overall maintenance of NVU function (Figure 1). Briefly, ECs form a single-layer lining of tubular blood vessels, which are specialized depending on the vascular bed in which they are situated (Chico and Kugler, 2021). At the BBB/BRB, ECs exhibit reduced pinocytosis/transcytosis (O’Brown et al., 2019; Villaseñor et al., 2019), increased expression of tight junction molecules, such as claudins, occludin, or zonula occludens 1 (ZO-1; Figure 2A; Fanning et al., 1999; Nitta et al., 2003), and exclude free transport of substances over 400kDa (Pardridge, 2001). Mural cells, which constitute pericytes and vascular smooth muscle cells (vSMCs), are positioned in the basement membrane shared with ECs, maintain vascular stability, provide structural support for blood vessels, govern vasodilation/constriction (Tong et al., 2021), as well as contribute to NVU function by BBB maintenance (Armulik et al., 2010; Bell et al., 2010; Henshall et al., 2015). Neurons in the NVU transduce signals and control local cerebral blood flow directly, such as via nitric oxide (NO), as well as indirectly via glia cells, such as via arachnoid acid or potassium (K⁺) (Attwell et al., 2010). Additionally, neuronal activity itself shapes vascular and BBB formation via levels of neurotransmitter release (Lacoste et al., 2014; Whiteus et al., 2014). Glial cells physically ensheath blood vessels with their endfeet, creating the semi-permeable glia limitans (Kutuzov et al., 2018). Importantly, glia physically connect vessels to neurons (Zonta et al., 2003), modulate neurotransmission, and impact neurogenesis (Argente-Arizón et al., 2017; Falk and Götz, 2017). Microglia, macrophages, and perivascular macrophages (PVM) play roles in phagocytosis and the CNS inflammatory response, ensuring CNS maintenance and health (Guillemin and Brew, 2004; Serrats et al., 2010). The vascular basement membrane, which encompasses blood vessels, acts as a passage for fluid transport (Morris et al., 2016), while the perivascular basal laminae and ECM molecules support the glio-vascular interface (Hoddevik et al., 2020). Together, these components form a spatially and functionally integrated NVU with bidirectional communication, namely neuro-vascular-coupling and vascular-neuro-coupling. However, the precise mechanisms of the diverse roles of glial cells in NVU form, maintenance and function remain unclear. Answering these fundamental questions will be of particular importance as the NVUs’ main role is considered neurovascular coupling, but indeed various other aspects such as integration of signaling, metabolism, and homeostasis occur across NVU components and thus must also be considered.

Glia were originally described as scaffolds providing structural support and maintaining biophysical integrity (Virchow, 1856), making their role in supporting the structure and biophysical integrity of the CNS their most widely described function (Losada-Perez, 2018). However, glia are now being increasingly appreciated for their many other functional and regulatory roles, such as neurotransmission, BBB function, and controlling immunity (Table 1). To fulfill the variety of specialized functions required, glia cells are highly specialized according to each CNS region with respect to proteomic signatures, electrophysiology, Ca²⁺ signaling, morphology, and proximities to synapses (Tsai et al., 2012; Molofsky et al., 2014; Ben Haim and Rowitch, 2017; Chai et al., 2017). Moreover, even within morphological groups, such as astrocytes, there is heterogeneity of cell types and their pathological responses (John Lin et al., 2017; Hasel et al., 2021) that may also reflect regional differences in the structure and function of the NVU.

The most abundant and widespread glial cell type in the brain are astrocytes. Astrocytes are fivefold more numerous than neurons (Sofroniew and Vinters, 2010), with individual astrocytes contacting up to two million neuron synapses with elaborate morphologies (Oberheim et al., 2009). This high spatial correspondence between astrocytes and neurons is accompanied by astrocytes regulating neuron health by controlling neurotransmitters, such as glutamate or adenosine, as well as maintaining hydromineral brain homeostasis, such as Ca²⁺, Cl^–, or water (Simard and Nedergaard, 2004; Keaney and Campbell, 2015; Price et al., 2018). In addition to contacting neurons, astrocytes also contact blood vessels, affecting local blood flow by regulating blood vessel diameters by vasoconstriction (e.g., by arachnoid acid) and vasodilation (e.g., by prostaglandins) (Kimelberg, 2010). Astrocytes also physically and functionally contribute to the BBB and its permeability for factors such as molecular traffic of glucose or proteins (Abbott et al., 2006). Thus, astrocytes are central to the function of a healthy functioning NVU, and overall CNS function.

In the retina, the principal glial cell type are Müller glia (MG), which contact blood vessels and neurons, fulfilling similar functions as astrocytes in the brain (Newman and Reichenbach, 1996; Subirada et al., 2018). The structure, morphology, and species-specific differences of retinal MGs are well described (Cajal, 1995) and MG exhibit at least five apico-basal domains that stretch from the apical stem around photoreceptors to the basal endfoot in the inner limiting membrane (Wang et al., 2017). However, MG are not just highly organized apico-basally, but also laterally to interact with other cells; this intercalation between cells is in a so-called tiled fashion, thereby contacting almost all cells within the retina (MacDonald et al., 2017; Wang et al., 2017). The retina is protected by two separate components of the BRB. The outer (oBRB) consists of retinal pigment epithelium and the inner (iBRB) is located at the level of the retinal capillaries. The latter is established by MGs and pericytes, with dysfunction being implicated in several retinal diseases such as diabetic retinopathy (DR; Cunha-Vaz et al., 2011; Frey and Antonetti, 2011; Díaz-Coránguez et al., 2017; Park et al., 2017). In the mammalian retina, there are also astrocytes that contact blood vessels and are pivotal as a structural growth template during angiogenesis, mainly via vascular endothelial growth factor (VEGF) and Hypoxia-inducible factor (HIF) pathways, following ganglion cell templates (O’Sullivan et al., 2017; Paisley and Kay, 2021). This suggests that MG and astrocytes may work together in the mammalian retina to influence the development of the NVU as well as to regulate its function. In humans, the retinal astrocytes are limited to the inner vascular plexus, while MG contact both plexi and are likely to induce the BRB functionality/maturity in the deep plexus, raising the likelihood for differential coupling between astrocytes and MG in the healthy and potentially diseased retina (Tout et al., 1993; Fruttiger, 2007; Ashraf et al., 2020). However, MG are the only glia found in the fovea, which is free of astrocytes, microglia, and vascular EC, suggesting that in some regions of the retina MG are sufficient to solely meet the functional needs required for high acuity vision (Reichenbach and Bringmann, 2020).

Besides the principal glial cells in the brain and retina (i.e., astrocytes and MG), other glial cells exist to fulfill crucial functions in the CNS. During development, progenitors, so-called radial glia, will divide to generate neurons and glia (Rakic, 2003), while also making contact with the vasculature where they contribute to the regulation of CNS angiogenesis. In certain CNS regions, radial glia persist into adulthood as stem cells, contributing to BBB maintenance via retinoic acid signaling (Sharif et al., 2018). Microglia are the primary immune cells in the CNS, surveying their environment and responding to insult, fulfilling roles in phagocytosis and inflammation where they express both pro-inflammatory (e.g., IL-1β) and anti-inflammatory (e.g., IL-10) molecules, with subsequent upregulation of factors such as Glial Fibrillary Acidic Protein (GFAP). Additionally, microglia-to-astrocyte crosstalk in response to glutamate plays a role in neuro-immune-interactions (Nimmerjahn et al., 2005; Macht, 2016), and microglia are required for normal neurogenesis, mostly by nerve growth factor (NGF) and tumor necrosis factor (TNF; Matejuk and Ransohoff, 2020). Another type of glia, called ependymal cells, line the brain ventricles, producing cerebrospinal fluid, and subpopulations acting as progenitors for astrocytes and oligodendrocytes (Bigio, 2010; Furube et al., 2020). Thus, there are several types of glia found within each CNS tissue, yet several pressing questions remain to understand the importance of glia in the NVU. These include what role do glial cells play in driving NVU formation during development, how are glial cells directed to contact and support multi-cellular units within the NVU, and what are the consequences of dysfunction of these glial components on NVU function.

The fact that glia are a structurally integral part of the NVU physically linking the vasculature and neurons, emphasizes their functionality in BBB/BRB formation and CNS development.

Glia cells provide structural support to neuronal tissues for anisotropic mechanical tension (Nagashima et al., 2017), and loss of MG in the retina results in tearing of the tissue due to the loss of their biophysical support (MacDonald et al., 2015). Another biophysical role of glia is that they can swell, which subsequently affects the NVU by spatial changes. The impact of glia volume changes on neurons is facilitated and relayed by the fact that glia ensheath pre- and post-synaptic terminals of neurons to form the “tripartite synapse” (Araque et al., 1999; Santello et al., 2012; Hillen et al., 2018). Due to this physical proximity, changes in glia cell size can modulate the extracellular space and subsequently neuron excitability (Florence et al., 2012; Vecino et al., 2016). This is achieved by the synergistic activity of Aquaporin 4 (Aqp4), a channel protein, which is needed for water transport and is enriched in astrocytic endfeet (Gleiser et al., 2016), as well as the transient receptor potential cation channel TRPV4 for Ca²⁺ influx (Jo et al., 2015). However, even though Aqp4 and TRPV4 are considered main factors, glia swelling is a complex process and, depending on the context, other factors were shown to play a role in glia volume changes. These include K⁺ ion transport via connexin 43, Kir 4.1, or Na⁺/K⁺-ATPase, and ion flux via Na⁺-K⁺-Cl^– co-transporter (NKCC1), or glutamate movement via specialized transporters (Lafrenaye and Simard, 2019). These factors can also change in disease or upon injury, as exemplified by sulfonylurea receptor 1 – transient receptor potential melastatin 4 (SUR1-TRPM4) which is upregulated in CNS injury (Mehta et al., 2015), but the impact of such changes on NVU function remain poorly defined.

Besides this physical link, glia also functionally link NVU components, exemplified by their impact on EC junctions, transporters, and pathways (Figure 1; Hayashi et al., 1997; McAllister et al., 2001; Haseloff et al., 2005). This functional link is in part achieved by factors such as glial cell line-derived neurotrophic factor (GDNF), VEGF, fibroblast growth factor 2 (FGF2), or angiopoietin-1 (Figures 2B,C; Igarashi et al., 1999; Lee et al., 2003; Lécuyer et al., 2016; Blanco-Suarez et al., 2018; Jha et al., 2018). But glia cells are also key to supporting neurotransmission by removal of neurotransmitters to terminate synaptic transmission and reestablish neuronal excitability, thus avoiding toxic overstimulation, called excitotoxicity (Turner and Adamson, 2011). Glia also directly regulate neuronal activity within the synapse (Pannasch and Rouach, 2013; Sibille et al., 2014) and synchronize/modulate synaptic inputs (Fellin et al., 2004; Fellin, 2009) on the level of signaling via gliotransmitters, such as gamma-aminobutyric acid (GABA), glutamate, or cytokines (Kim et al., 2020). The major neurotransmitters are the excitatory glutamate and the inhibitory GABA, working together to regulate CNS function. Following the removal of neurotransmitters from the synaptic cleft, glia cells transfer these neurotransmitters back to neurons in a process called the glutamate-glutamine cycle that requires ammonia (NH₃) and ammonium ion (NH₄⁺) derived from NVU blood vessels (Bak et al., 2006; Limón et al., 2021), thus reestablishing functional neuron neurotransmitter pools. This maintenance of synaptic potentials comes at a very high metabolic cost with the energy for this provided by astrocytes and blood vessels (Vergara et al., 2019). While it was previously assumed that both glutamate and GABAergic neurons are under astrocytic control, a recent in vitro study challenges this, suggesting that GABAergic neurons establish functional synaptic transmission without glia (Turko et al., 2019). Another neuromodulator released from neurons or glia (Butt, 2011), impacting neuronal function, is adenosine derived from adenosine triphosphate (ATP) breakdown. Adenosine stimulates receptors that regulate the release of GABA, glutamate, acetylcholine, noradrenaline, 5-HT, and dopamine (Sperlágh and Vizi, 2011).

Thus, glia are specialized morphologically, biophysically, and molecularly to support and regulate the NVU. However, the multitude of glial functions within the NVU makes it challenging to discern which mechanisms are necessary and sufficient for NVU form and function. To understand the role of glia cells, further studies are needed where glia cells are disrupted (i.e., lacking, inhibited, or overactive). Such studies will allow us to disentangle NVU interactions, establish the exact role of glia cells within it and the pathophysiological consequences.

As glia directly contact and ensheath blood vessels, they directly influence EC structure and function rather than passively co-exist. Indeed, in the last decade, it has become clear that glial cells play an active role in facilitating vascular angiogenesis via expression of factors, such as VEGF or transforming growth factor 1β (TGF-1β) in radial glia cells (Virgintino et al., 2003; Welser et al., 2010; Biswas et al., 2017; Siqueira et al., 2018). Moreover, radial glia were shown to stabilize murine nascent cortex vessels via inhibition of Wnt signaling and proliferation in EC in a contact- and age-dependent manner, a process which is potentially mediated by MMP-2 (Ma et al., 2013). Glia cells, specifically CNS-specific macrophages (microglia), were also shown to play pivotal roles in the fusion of blood vessels, called anastomosis. This is achieved by Notch1-expressing macrophages that link path-seeking dll4-expressing vascular tip cells (Outtz et al., 2011) as well as by acting as physical chaperones which express TIE2 and NRP1, regulating anastomosis downstream of VEGF-mediated endothelial tip cell induction (Fantin et al., 2010). After vessel- and NVU-formation, glia are also pivotal for BBB maturity, via factors such as retinoic acid supplied by radial glial cells, which increases BBB stability and the expression of BBB-specific genes such as p-glycoprotein (P-gp), occludin, and Glut-1 (Mizee et al., 2013) [see details for BBB transport systems and junctions (Zhao et al., 2015)]. Similarly, astrocytic Src suppressed C kinase substrate (SSeCKS) reduces VEGF and increases EC tight junctions (Lee et al., 2003), or astrocytic angiotensin-converting enzyme-1 (ACE-1), which produces angiotensin II that facilitates BBB maturation and junction protein stabilization (Lavoie and Sigmund, 2003; Wosik et al., 2007). ECs and glia interact bidirectionally in NVU development. Firstly, radial glia support EC maturation toward decreased proliferation, reduced tip cell marker DLL4, and reduced vascular permeability, thus supporting BBB maturation. Subsequently, ECs increase GFAP-positivity in radial glia in a VEGF-A dependent manner, leading to astrocyte differentiation and NVU formation (da Silva et al., 2019). In addition to these molecular impacts, the migration patterns of blood vessels, astrocytes, and neurons are closely associated with each other like scaffolds, with astrocytes providing VEGF for EC migration and, vice versa, ECs in turn provide oxygen for astrocyte differentiation (Bozoyan et al., 2012; Duan et al., 2017; O’Sullivan et al., 2017). Critically, in pathology, lactate-stimulated MG express G-protein–coupled receptor 81 (GPR81), which triggers neovascularization via pathways such as Wnt or Norrin (Madaan et al., 2019).

Besides these roles in angiogenesis, glia are also pivotal in regulating blood flow via regulating NVU synaptic activity as well as by releasing factors such as calcium, NO, arachidonic acid, and prostaglandins (Gordon et al., 2007; Attwell et al., 2010; Biesecker et al., 2016; Magaki et al., 2018). It was also shown that angiotensinogen-to-angiotensin II cleavage occurs in glia, with angiotensin I receptor (AT1-R) causing vasoconstriction (Kawamura et al., 2004), while AT2-R causing vasodilation (Fletcher et al., 2010). Moreover, glia contribute to vasodilation indirectly by interaction with other NVU components such as pericytes, which then, in turn, impacts the vasodilatory state (Nortley and Attwell, 2017). Here, one important molecule is calcium, with calcium signaling not only being coordinated between glia cells (Muñoz et al., 2015), but also ECs (Thakore et al., 2021). It remains to be understood to which extent glia-EC signaling is indirectly (via neurons or pericytes) or directly coupled. Lastly, neuron-derived NO regulates glia-mediated vasodilation via prostanoids and epoxyeicosatrienoic acids (Someya et al., 2019). Together, glial cells therefore not only play a role in angiogenesis, anastomosis, EC maturation, and blood flow regulation, with glia dysfunction potentially leading to BBB breakdown, pathological vascularization, dysregulation of vasoregulation and failure to deliver sufficient oxygenation.

As the CNS has high metabolic demands and lacks a carbohydrate storage system, the NVU is critical to serving the retina and brain metabolic needs as glucose has to be continuously supplied via the blood to meet the constant CNS energy demand. This is particularly crucial as neurons rely on oxidative metabolism, making them sensitive to changes in levels of oxygen and potentially ischemia (Turner and Adamson, 2011); on the other hand, astrocytes rely on glycolytic metabolism, and glucose can be stored in them in the form of glycogen, rendering them central players in NVU and CNS metabolism (Öz et al., 2007). Crucially, pyruvate carboxylase, an enzyme that is key to synthesizing the neurotransmitter glutamate from glucose via the anaplerotic pathway, is almost exclusively expressed in astrocytes, positioning them as essential producers of the neurotransmitters GABA and glutamate (Schousboe et al., 2019). Recently it was shown that retinal MG also conduct anaplerosis (Singh et al., 2020), suggesting that MG support retinal NVU metabolism similar to astrocytes in the brain.

While the majority of vascular-to-neuron glucose metabolism occurs directly via glia in the NVU, a minor proportion of the glucose flux happens directly between blood vessels and neurons (Maoz et al., 2018). At the end of glycolysis, lactate and pyruvate are produced, but instead of being merely “end-products” they are utilized further to generate energy. Indeed, lactate is taken up by neurons and is metabolized in preference to glucose when both are available (Bouzier-Sore et al., 2003; Bouzier-Sore et al., 2006). Once transported into the neuron, lactate is converted to pyruvate and used for ATP production. Thus, glia not only provide compounds for neurons but also complement them in their metabolic requirements. Besides glucose metabolism, astrocytes are also crucial for fatty acid oxidation (FAO) to generate ATP, and catabolic ketogenesis to generate ketone bodies for neuronal metabolism; with ketone bodies or lactate being used by neurons as an energy substrate to produce ATP (Bélanger and Magistretti, 2009; Souza et al., 2019). Importantly, neurons produce toxic fatty acids that are endocytosed by NVU astrocytes for cytoprotection and CNS health (Ioannou et al., 2019). Metabolically, astrocytes also play a critical role in L-serine de novo synthesis, which is converted to D-serine in neurons, acting as a co-agonist of N-methyl-D-aspartate (NMDA) receptors (Yamasaki et al., 2001). Together, these data demonstrate that glial cells play a key role in maintaining the homeostatic status of the metabolism of neurotransmitters, glucose, FAO, L-serine, as well as NO, essential for maintaining normal function of the NVU. Any disturbance of this fine-tuned balance, therefore, such as would occur in diseases as diverse as stroke and diabetes, will influence NVU function resulting in further failure to supply adequate essential substrates for normal neuronal function.

In addition to the above metabolic coupling, neuron-to-vascular coupling is achieved by NO, a gaseous neurotransmitter acting as a vasodilator that is needed for neurovascular coupling and regulating the vasodilatory vascular response, called functional hyperemia (Hoiland et al., 2020). In hyperemia, glia cells are essential in relaying either vasodilation or vasoconstriction depending on the available NO concentration (Metea and Newman, 2007). Further to physiological NO, glia-mediated NO and gliosis-related production of reactive oxygen (ROS) or nitrogen species (RNS), play a role in nitro-oxidative stress such as in neuroinflammation and disease such as epileptogenesis. Indeed, Sharma et al. suggest that epilepsy is preceded by a cascade of reactive gliosis, ROS/RNS, inflammatory cytokines, and neurodegeneration (Sharma et al., 2019). This neuro-vascular metabolic coupling of glucose, fatty acid, L-serine, and NO via glia is dependent on glia directly contacting ECs within the NVU. The required spatial connections between endfeet and ECs are partially achieved via connexins, which form intercellular gap junction channels and hemichannels that are expressed in ECs and astrocyte endfeet, as well as are associated with BBB maturation (Zhao et al., 2018). Also, Pannexins (Panx) that resemble connexin-based hemichannels, are thought to play a role in vasculo-neuro coupling although there is still a debate on their function in CNS development and health (Giaume et al., 2020).

Beyond their roles in physiological conditions, glia and microglia contribute to neuroinflammation in response to injury, stroke, or other neurological diseases (Cekanaviciute and Buckwalter, 2016). They act as primary initiators of the inflammation cascade by increased reactivity and the secretion of factors such as chemokines (Sofroniew and Vinters, 2010; Karve et al., 2016). One ubiquitous biomarker of so-called glial activation is increased expression of GFAP (Diaz-Arrastia et al., 2013). Despite the extensive list of definitions for different glia cell states, the field agrees that inactive and reactive glia cells display changes in molecular profiles, including alterations in the cytoskeleton, metabolism, chaperones, secreted proteins, signaling proteins, and transporters. These molecular changes are accompanied by morphological transformation in cellular phenotype, such as hypertrophy (Escartin et al., 2021), which may impact the ability of glial cells to provide multiple support functions for each NVU component(s) and hence its uncoupling.

Besides the physiological functions of glial cells in inflammation, their roles in pathological settings are of wider interest. For example, increasing evidence also shows glia as a link between vascular and neurological contributions in cognitive impairment, Alzheimer’s Disease (AD), and seizures (Burda and Sofroniew, 2014; Edison et al., 2018; Price et al., 2018; Diaz Verdugo et al., 2019). Neuropathologically, AD is characterized by intracellular neurofibrillary tangles and brain parenchymal amyloid β-peptide (Aβ) deposits. The latter form neuritic plaques and cerebral amyloid angiopathy, leading to angiopathy and NVU dysregulation (Soto-Rojas et al., 2021). Astrocytes have been shown to degrade amyloid-beta in an apolipoprotein E (APOE)-dependent manner, a process that could be impaired in AD (Koistinaho et al., 2004), leading to plaque formation. Neurodegenerative disorders are complex conditions with multiple underlying causes and the role of glia has not been fully elucidated (Gleichman and Carmichael, 2020). Nevertheless, several astrocyte/glia risk factor genes, such as APOE, particularly the E4 isoform (Pihlstrøm et al., 2018) or Clusterin (CLU) and FERM Domain Containing Kindlin 2 (FERMT2) (Verheijen and Sleegers, 2018), have been identified for AD. Also, it is generally acknowledged that AD involves inflammatory responses, initiated, or mediated via microglia and astrocytes that lead to BBB breakdown (Akiyama et al., 2000; Nagele et al., 2004). Throughout AD disease progression, different aspects were found to affect the NVU. In pre-senile AD, increased proliferation (Ki-67), gliosis (GFAP), and vascular changes, but not neurogenesis were shown (Boekhoorn et al., 2006). In late-onset AD, vascular dysregulation and BBB breakdown are considered as the earliest biomarker (Iturria-Medina et al., 2016; Sweeney et al., 2018), with vascular dysregulation preceding changes in amyloid beta deposition, metabolism, function, structure, and memory. However, future work is needed to link genetic and mechanistic causes of AD and the spatiotemporal impacts on individual NVU components and NVU function.

In the retina, understanding the NVU and glia contribution is of particular interest as NVU dysfunction can precede neural dysfunction in patient retinas, such as in patients with Type 1 Diabetes who exhibit DR (Lasta et al., 2013). In DR and other retinal diseases, inflammation and accompanying side effects are critical contributors to disease progression and vision loss. Crucially, it has been shown that MG provide VEGF, which contributes to the upregulation of inflammatory markers, such as ICAM1 and TNFα (Le, 2017). These pro-inflammatory compounds induce pathological vascular leakage and retinal neovascularization, making anti-VEGF therapies an important therapeutic strategy in DR. In DR, it was shown that the MGs that express VEGF display a distinct morphology (Pierce et al., 1995), suggesting that they enter a reactive phenotype with altered function before expressing VEGF, which in turn leads to changes in NVU and vascular function. Besides morphological changes and increased VEGF expression, MG also show altered expression of trophic factors in DR, such as NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), ciliary neurotrophic factor (CNTF), or glial cell line-derived neurotrophic factor (GDNF), as well as inflammatory factors such as interleukin 1β (IL-1β), IL-6, IL-8, and TNF-α. Together, these factors contribute to disease progression and neuron survival (Boss et al., 2017). Indeed, in diabetes, it is likely that such changes result in early NVU dysfunction and subsequent failure of neurovascular coupling and hence an insufficient supply of nutrients, which then cause further damage to the NVU. Another retinal disease with clear implications of MG dysfunction is macular telangiectasia 2 which causes loss of central vision due to vascular defects (MacTel 2) (Powner et al., 2010a,b). Critically, it was shown that loss of MG matches the area of macular pigment depletion in MacTel 2 patients (Powner et al., 2010b). Moreover, the loss of these MG impacts on the NVU as a secondary hallmark feature of MacTel 2 is the formation of dysfunctional telangiectatic vessels. One potentially crucial aspect when thinking about the regional restriction of MG loss and vascular defects is that macular MG rely more on serine biosynthesis than peripheral MG (Zhang T. et al., 2019), with serine synthesis disruption resulting in mitochondria dysfunction and oxidative stress and ultimately retinal pathology (Zhang et al., 2018). This retinal pathology is also associated with swelling-induced MG volume changes (Lafrenaye and Simard, 2019) and penetration of astrocytes into deeper layers (Coffey et al., 2007). Thus, rather than having a passive role in pathogenesis, the activation state of glia, together with changes in their morphology and molecular expression, is directly associated with disease progression of diseases such as DR and MacTel 2.

Finally, it is important to note that retinal demand for oxygen is even greater than that of the brain. This is especially so during dark adaptation when metabolic activity and oxygen demand is high. The configuration of the vascular supply to the retina means that during high demand the functional reserve of oxygen is minimal, even when neurovascular coupling maximizes functional hyperemia. This makes the retina extremely vulnerable to hypoxic damage and so even small alterations to the function of any component of the NVU, including MG, is likely to result in compromised retinal function.

It is increasingly clear that glial cells are critical for NVU development, function, maintenance, and dysfunction in disease. However, uncovering the precise contribution of glial cells to the NVU has been challenging to date, particularly when trying to understand and integrate their dual contribution to neurons and the vasculature. With the advancement of imaging, computational and genetic tools, it will be possible to use multidimensional approaches (morphology, function, genetics, interactions, and dynamics) to clarify the exact role(s) of glial cells in the NVU. Examining the role of glia in the “neuro-glial-vascular unit” with such a holistic approach will enhance the understanding, diagnosis, and treatment of aging and disease in the nervous system.

EK, JG, and RM contributed to the conception and design of the study. EK wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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