One Mesmerizing Image, Six Cells, and a Space: The Story of Reactive Perivascular Astroglia, Enlarged Perivascular Spaces, and Small Vessel Disease

1. Introduction

Transmission Electron Microscopy (TEM) remains a powerful tool to investigate cellular and tissue remodeling changes in disease and various treatment models [1]. Indeed, author has examined hundreds of cortical, frontal layer III capillary neurovascular unit(s) (NVU) in diet-induced obesity, insulin resistance, metabolic syndrome, genetically induced female, obese, diabetes models (db/db and BTBR ob/ob), streptozotocin-induced diabetes in male, and control preclinical mouse models. These capillary NVUs demonstrated that the glial protoplasmic perivascular astrocytes endfeet were tightly adherent to the mural cells (endothelial and pericytes) shared basement membrane. Thus, the finding of a NVU with a definite sharply demarcated space separating the NVU capillary mural cells and their basal lamina/basement membrane from the surrounding-encompassing protoplasmic perivascular astrocyte endfeet basal lamina within the neuropil was indeed remarkable and mesmerizing as in the following observed original image from the neuroinflammatory lipopolysaccharide (LPS)-treated model as compared to control models (Figure 1) [2].

Notably, LPS-induced neuroinflammation in CD-1 male mice also depicted multiple response to injury ultrastructural remodeling changes including BECs activation supported by plasma membrane ruffling, aberrant mitochondria, increased macro-micropinocytosis (transcytosis), and increased extracellular vesicle formation. Pericyte(s) (Pc) nuclear rounding with retraction of Pc foot processes-endfeet (Pcfp) with increased lysosome, vacuoles, and vesicles numbers. Additionally, there were increased reactive amoeboid microglia cell attractions to the NVU note shown in Figure 1B [2]

The cellular components of the capillary NVU consists of brain endothelial cell(s) (BEC), Pcs and artery/arteriole vascular smooth muscle cells, protoplasmic perivascular astrocytes and their endfeet (pvACef), neurons, and interrogating microglial cell(s) (iMGC) as depicted (Figure 1) [3,4,5,6,7]. The recently described perivascular unit (PVU) consists of benign perivascular spaces (PVS) and the pathologically dilated or enlarged perivascular spaces (EPVS) identified in neuroinflammatory LPS-treated mice models (Figure 1B) [3,8] The PVS are bound by the innermost boundary created by NUV shared basement membrane of the BECs and Pcs with the outermost boundary formed by the compacted protoplasmic perivascular endfeet basement membrane also known as the glia limitans [8,9]. Importantly. resident perivascular macrophages represent the sixth cell that are known to reside within PVS/EPVS (Figure 1B) [10,11,12].

Descending pial arteries and arterioles PVS are important in delivering the cerebrospinal fluid (CSF) to the brain interstitial spaces (ISS) and interstitial fluid (ISF) of the parenchyma while the postcapillary ascending venular PVS are responsible for the efflux of the interstitial fluid (ISF) and its contents including some residual CSF, metabolic waste, solutes, and excess fluids/water to the subarachnoid space (Figure 2) [3,13,14,15].

Indeed, the PVS of the postcapillary venules, venules, and veins are responsible for the efflux of ISF with its metabolic/phagocytic waste and excess water to the subarachnoid space and hence to the peripheral circulation in addition to providing a conduit for the glymphatic system (GS) in the brain [16,17,18]. PVS are currently thought to form and mature postnatally under the influence of perivascular astrocytes along with other cellular components of the NVU. Perivascular astrocytes play a crucial role in shaping and maintaining the integrity and function of not only the NVU but also PVS. However, the complete mechanisms regarding the formation of PVS still remain an area of ongoing understanding and research [17,18].

It is important to define the GS since this narrative review focuses primarily on the post-capillary venule PVS as the existing anatomical conduit for the GS. Some have simply described the GS as the brain’s waste clearance system. However, the GS may be considered to be comprised by a brain-wide network of perivascular spaces/channels/conduits and their adjoining vascular luminal shared BEC and Pc basement membranes in postcapillary venules; vascular smooth muscle cell basement membranes, interstitium, and adventitia in arteries and arterioles of the intramural periarterial drainage (IPAD) system and specifically the outermost delimiting pvACef compacted basement membranes or glia limitans of the parenchymal components, which function to facilitate the exchange and the eventual clearance of the brain’s metabolic waste solutes carried in the interstitial fluid [18,19]. Further, the GS facilitates the exchange of fluid and solutes between the CSF and interstitial compartments organized along perivascular pathways and supported by arterial pulsation, vasomotion, respiration, and facilitated by pvACef AQP4 water channels [16,17,18,19]. Thus, this definition allows for not only the glymphatic system efflux hypothesis but also the retrograde IPAD efflux and mixing hypotheses within these PVS to be included. Importantly, the GS is thought to be related to two structures: the PVS and its outermost barrier/boundary formed by pvACef with their aquaporin 4 (AQP4) water channels [20].

When PVS (Virchow-Robin spaces) are large enough to be identified with magnetic resonance imaging (MRI), they are termed MRI-visible enlarged perivascular space (MVEPVS) that measure one to three millimeters in diameter that are usually symmetrically located within the basal ganglia or the centrum semiovale regions of brain MRIs (Figure 3) [3,21].

Importantly, EPVS/MVEPVS are known to be biomarkers for cerebral small vessel disease (SVD) that associate with multiple (at least a dozen or more) clinical neurologic diseases as follows: Alzheimer’s disease, Parkinson’s disease, Hunignton’s disease, multiple sclerosis, encephalitis (infectious or traumatic), traumatic brain injuries, post-traumatic stress disorder (still remaining to be fully established), autism spectrum disorders (still remaining to be fully established), migraine headaches, LPS infusions related to neuroinflammatory models or clinically related LPS excess due to gut dysbiosis [2,3], hypertension, and neurovascular clinical diseases that include: stroke (ischemic or hemorrhagic, transient ischemic attacks/events (TIAs), vascular dementia (VaD), vascular contributions to cognitive impairment and dementia (VCID), cerebral amyloid angiopathy (CAA), cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), neuromyelitis optica and/or neuromyelitis optica spectrum disorder, and normal aging [3,22].

SVD consists of a variety of aberrant structural and functional changes involving small cortical perforating arterioles, precapillary arterioles, capillaries, and ascending postcapillaries venules and veins in the brain [3]. The two most common pathological changes in SVD include cerebral arteriolosclerosis (hyalinosis and lipohyalinosis) and CAA. These changes may be divided into multiple categories identifiable by non-invasive MRI, which include enlarged perivascular spaces, cerebral microbleeds, small recent cortical infarcts, lacunes, and white matter hyperintensities (Box 1) [3,15,21,22,24,25,26,27,28,29].

The development of EPVS is complex and involves multiple functional and cellular mechanisms that are highly interconnected. These mechanisms include but are not limited to the following: 1). Brain endothelial cell activation/dysfunction (BECact/dys) with increased 2). Vascular inflammation; 3). Oxidative stress; 4). Neuroinflammation with decreased BEC protective nitric oxide and increased permeability of fluids (including ions, water, proteins including hormones, and polypeptides) and cells including leukocytes into the PVS due to endothelial blood-brain barrier dysfunction and disruption (BBBdd); 5). Microvessel stiffening especially in the arterial system with a loss of pulsatility that is important to drive the glymphatic system flow of waste and water clearance; 6). Astrocyte and pvACef dysfunction and or loss with decreased or dysfunctional aquaporin 4 (AQP4) water channel activity due to translocation from the plasma membrane or damage; 7). Neuroinflammation; 8). Oxidative stress with increased reactive oxygen, nitrogen, sulfur species; 9). Leukocyte accumulation within the PVS/PVU/GS; 10). Phagocytic debris accumulation of proteins and protein aggregation (specifically amyloid beta and tau), which disrupt GS drainage that result in EPVS; 11). Brain atrophy [3,9,15,22,24].

There are multiple reasons as to why PVS are important and at least eight core reasons are presented (Box 2) [3].

Even though there may exist an ongoing debate by some regarding whether these PVS are true spaces or only potential spaces, the current anatomical concepts suggest that PVS are indeed compartments between the outermost basement membranes of pvACef and innermost NVU mural cell (BEC and Pc) basement membranes. Regardless, author feels that the importance of EPVS will continue to grow along with the evolving role of the GS because current papers regarding these two mechanisms are growing exponentially in the literature as illustrated by the increasing number of published papers regarding these two interrelated systems identified in the National Institutes of Health (NIH) search engine PubMed [30].

The primary aim of this narrative is to increase the holistic understanding of the cells involved in the development of EPVS and SVD, while exploring the interrelationships of neurovascular, neuroimmune, and neurodegenerative mechanisms.

2. Cellular Involvement of the Neurovascular and Perivascular Unit

2.1. Brain Endothelial Cell(s) (BEC)

BECs are the first barrier between the blood and the brain. They provide the semipermeable interface-blood-brain barrier (BBB) (transfer and diffusion of solutes and fluids), trophic (metabolic homeostasis), tonic (vascular hemodynamics), and trafficking (vascular permeability, coagulation, and leukocyte extravasation) functions to provide brain homeostasis [31,32]. Further, BECs have a paucity of pinocytic vesicles, an absence of fenestrae, and the unique expression of paracellular tight and adherens junctions as compared to systemic endothelial cells [31,32,33,34]. BECs are constantly exposed to numerous injurious stimuli and are known to respond to injury via an innate response to injury mechanism (Figure 4) [31,35].

Indeed, the central theme of brain injury involves gene activation and transcription of various factors in a response to injury wound healing mechanisms, which include hemostasis, inflammation, proliferation growth (re-growth), hypertrophy, differentiation, development, remodeling, and limited neural regeneration. If the injury is brief there will be regeneration; however, if the injurious stimuli are chronic there will be aberrant remodeling of cells and tissue to result in dysfunction and disease instead of regeneration and functional restoration (Figure 5) [35].

For example, in the case of LPS-treated models the BEC are injured and respond accordingly via TLR4 and NF-kB signaling to result in BECact/dys and BBBdd to result in increased permeability, neuroinflammation, and the formation of EPVS (Figure 6) [2,35,36,37,38].

Thus, injurious species to BECs of the NVU result in BECact/dys and BBBdd with increased permeability of neurotoxins and leukocytes into the adjacent PVU to allow multiple cellular crosstalk with resident and reactive antigen presenting perivascular macrophage(s) (rPVMΦ) and pvACef. This multicellular crosstalk within the PVS of the PVU results in increased oxidative stress, activation of matrix metalloproteinases (MMPs) to digest the outermost protective barrier of the pvACef glia limitans in the two-step process of neuroinflammation described by Owens et al [10]. Further, this allows BECact/dys-BBBdd to propagate neuroinflammation along with reactive atMGCs to increase cnsCC that may set in motion a self-perpetuating vicious cycle to result in EPVS and impaired waste and water clearance via the GS [3,8,9,10,11,12,14,17,22,29,30,39].

Importantly, the BEC glycocalyx plays a crucial role in protecting the capillary NVU and its BBB. The glycocalyx provides a surface coating, which consists of a complex interwoven polysaccharide mesh-like network of membrane-bound proteoglycans, glycoproteins, and glycosaminoglycans covering the apical luminal layer of the brain endothelial cells [40ecGCx]. It acts as the first barrier to circulating blood in the tripartite BBB consisting of (1) BEC glycocalyx; (2) BECs with their basement membranes; (3) an extravascular compartment of pericytes, the extracellular matrix, and perivascular astrocytes and their basement membranes [40,41]. Perturbations of this glycocalyx barrier (multiple injurious species including LPS) allow for increased permeability in the postcapillary venule that will be permissive to fluids, solutes, and proinflammatory peripherally derived leukocytes into the PVS which result in EPVS as well as increased neuroinflammation and neurodegeneration. LPS administration is known to cause endothelial glycocalyx injury (Figure 7) [42].

2.2. Capillary Neurovascular Unit Pericyte(s) (Pc)

Pcs along with BECs are vascular mural cells embedded in the basement membrane of blood microvessels and provide support to the capillary NVU. They contribute to the formation of the BBB tight and adherens junctions as well as to the shared basement membrane of BECs. Also, Pcs are uniquely positioned within the NVU to interact with BECs and pvACef to integrate, coordinate, and process signals from their neighboring cells to generate diverse functional responses that are critical for CNS functions in health and disease (Figure 1) [43,53]. These responses and interactions include the regulation of the blood-brain barrier permeability, angiogenesis, clearance of toxic metabolites, capillary hemodynamic responses (NVU coupling with vasorelaxation and constriction), neuroinflammation, and mesenchymal stem cell activity (Figure 8) [43,44,45,46,47,48,49,50,51,52,53,54].

Notably, some have elevated the roles of the BECs to be termed the ‘gatekeepers’ and/or the ‘sentinel’ cells, while the Pcs have been given the role of being the ‘guardian angel’ because of their critical support and protection they provide to BECs and NVUs. Further, these mural cells (BECs and Pcs) are co-dependent in that the BECs supply nitric oxide and platelet-derived growth factor beta (PDGFβ) for the Pc, while the Pc provides the BECs with VEGF for angiogenesis and also the receptor for PDGFβ [43,52].

2.3. Perivascular Astrocyte Endfeet (pvACef)

Astrocytes-astroglia are the most abundant glial cells and may be considered the master communicating, connecting, and creating cells within the brain (Figure 9) [55].

Cortical grey matter protoplasmic astrocyte(s) (AC) have many important homeostatic functions in the brain including neurovascular coupling to match cerebral blood flow to neuronal activity. Additionally, ACs are a major supplier of energy to neurons (via glycogen storage and glycolysis to produce glucose and lactate) and are a rich source of antioxidants such as glutathione and superoxide dismutase. They also provide growth factors such as brain-derived growth factor, transforming growth factor beta and glial-derived growth factor [56,57,58,59]. ACs also define many aspects of synapse formation, plasticity, provide protective functions, synaptic maintenance and elimination [60]. Further, ACs are responsible for controlling the fluid volume of the brain including the interstitial fluid, interstitial fluid spaces, and PVS via the highly polarized plasma membrane water channel AQP4. This bidirectional water transport function of AQP4 in the plasma membrane of pvACef that faces the brain spaces including PVS is essential for the homeostasis of water balance in the brain [56]. Unfortunately, when these pvACef become reactive they lose their polarity of the bidirectional water channel resulting in dysfunction and fluid retention and expansion of spaces [61]. Notably findings in rodent models may not always translate well to human studies since human ACs are more complex, larger in size, and extend their cytoplasmic processes much farther when compared to rodent model brains [62].

Protoplasmic pvACef are known to tightly adhere to the basement membranes of the NVU shared by the BECs and Pcs of the capillary NVU as in Figure 1A [55,63,64]. However, LPS-treated models demonstrated that the pvACef became detached and separated from the NVU basement membranes [2], which would impair NVU coupling, similar to the findings associated with obesity, insulin resistance, and hyperglycemia as in the diabetic db/db female models [35]. pvACef are communicating cells via gap junction connexins Cx40, Cx43 and create a syncytium, connecting cells as in Figure 8, coupling cells as in NVU coupling and PVU coupling, and continuing and creating cells, such that they become continuous at the true capillary as it transitions to the postcapillary venule with normal PVUs, and pvACef that are continuous and creative in the development of the PVU, PVS/EPVS (Figure 9) [30,55].

It is important to note that the initial barrier of the PVU/PVS is the BEC BBB and the 2nd and final barrier of the PVS is the glia limitans or compacted basement membrane of the outermost pvACef. This places the pvACef as the critical barrier to prevent the extravasation of proinflammatory leukocytes into the parenchyma of the brain that results in the damaging role of neuroinflammation and its relation to neurodegeneration and impaired cognition [10Owens, 55HaypvACs]. Additionally, the outermost pvACef and their AQP4 water channels are crucial for the proper function of GS waste and water removal system [9,30,55,56]. Indeed, the PVU provides a crossroad and intersection holding area for the dynamic interactions of the vascular, immune and neuronal mechanisms to respond to injury that initially occurs at the BECs of the NVU in addition to the important glymphatic system of waste and excess water removal [8,9,27,65,66]

2.4. Perisynaptic Astrocyte Endfeet (psACef)

Perisynaptic astrocyte endfeet (psACef) provide synaptic plasticity, connectivity, and are important in information processing. They are responsible for forming the tripartite synapse including the presynaptic and postsynaptic terminals along with the cradling psACef, which are essential for synaptic maturation, isolative protection, synaptogenesis, and maintenance of synapses. Further, these functions provide for the synaptic fundamental mechanisms of not only connectivity and plasticity but also informational processing in the brain [35,55,56,58,60]

psACef may become detached similar to their detachment from the capillary NVU as a result of brain injuries that associate with and result from inflammation, oxidative stress with activation of regional proteases (MMP-2, 9) that allow for damage and dysfunction of the anchoring dystroglycans to allow for the psACef to detach and separate from the pre- and postsynaptic terminals of the tripartite synapse [35,55,56,58,60]. Detached and separated psACef will create a perisynaptic space (PSS) that may be referred to as the enlarged perisynaptic spaces (EPSS) similar to EPVS Additionally, these reactive detached and separated psACef could result in the translocation of the bidirectional water channel AQP4 to be translocated and dysfunctional with the possibility of excessive fluid contributing to even further psACef separation from the pre- and postsynaptic terminals (Figure 10) [35,55,60,67,68].

psACef support tripartite formation and function of synapses (synaptic transmission), neurotransmitter recycling, BBB, and the support of oligodendrocytes, which produce myelin [66Mz]. Indeed, if injury mechanisms (injurious species as in Figure 1) to either the NVU or the dendritic synapses are chronic there would be loss of NVU coupling with resulting regional hypoxia and/or hypometabolism with ensuing neuronal dysfunction and neurodegeneration with impaired cognition, especially in the very young or very old vulnerable individuals to result in multiple diseases such as autism spectrum disorder and LOAD respectively. Similarly, synaptic psACef detachment and separation could interfere with glutamate uptake at the involved synapses and the glutamate–glutamine cycle and thus, could contribute to glutamate excitotoxicity to further increase regional injury to perpetuate a chronic cycle of synaptic and synaptic plasticity - dysfunction and/or loss (synaptopathy) [55,69,70,71].

2.5. Resident Perivascular Macrophages (PVMΦ)

NVUs and PVUs/PVS provide a crossroad and an opportunity for multicellular crosstalk that eventually allow for the breech of the outermost glia limitans of the reactive pvACef and reactive PVMΦs (rPVMΦs) with subsequent neuroinflammation via its antigen presenting cell (APC) capabilities (Figure 11) [8,10,30,55,72].

Importantly, PVMΦs are only found in the PVS that lie immediately adjacent to the NVUs, which allow them the ability to have intimate contacts with the mural cells (BECs and Pcs) as well as the outermost pvACef boundary cells of the PVS in addition to their direct exposure and interactions with the CSF and ISF within PVS/EPVS [8,10,72]. Additionally, rPVMΦs are capable of interacting with the leukocytes that are delivered to the PVS once the BBB has been compromised by multiple injurious species due to BECact/dys and BBBdd as previously presented (Figure 5; section 2.1.) [8,10,72,73,74]. Also, PVMΦ are yolk sac-derived innate immune cells that are important for the development of neuroinflammation [73,74,75,76]. Clinically, previous publications have shown that PVMΦs are involved in the pathology of AD [77], PD [78], stress [79], obesity [80s], stroke [81], hypertension [82], and metainflammation associated with insulin resistance, MetS, and diabetes [72]

PVMΦs are considered as a specialized subset of border-associated macrophages (BAMs), which also include border-associated macrophages as also identified in the meninges and the choroid plexus (epiplexis and Kolmer cells) [8,10,83,84]. Indeed, the brain’s border interfaces are important hubs for not only the uptake and efflux of solutes, ions, and cells but also provide the necessary and essential hubs for neuroimmune regulation (PVS/EPVS, meninges, choroid plexuses, and circumventricular organs).

3. Perivascular Spaces (PVS) of the Perivascular Unit (PVU), EPVS, and Interstitial Fluid (ISF)

According to the ancient Chinese Taoist/Daoist philosophy a space is not an absence or a void but acts as a dynamic and essential element of existence, harmony, and homeostasis [85]. To this end, PVS provide not only a crossroad and a specific space for multicellular crosstalk but also provide for the natural flow of fluids (pial arterial CSF influx and postcapillary venular ISF efflux) including water as well as metabolic and phagocytic waste of the GS within this enclosure. When PVS are healthy they are seldom observed by MRH; however, in multiple neurologic pathologies these PVS undergo aberrant structural remodeling to become EPVS/ MVEPVS. Importantly, PVS function as an efflux system or GS for waste and excess water removal in postcapillary venules [2,8,9,17,30].

PVMΦs normally reside within the confines of PVS/EPVS (section 2.4.) [10]. These PVMΦs constantly surveille the PVU spaces and are capable of not only being an antigen presenting cell but also capable of undergoing reactive changes to stressful stimuli as occurs with BECact/dys and BBBdd. They typically stain positive for CD206 and CD163 that are seldom stained in border-associated microglia cells. These PVMΦs allow the PVS their ability to act as a unit to function as a neuromodulator within the brain to prime the neuroimmune system.

The development of EPVS is complex and involves multiple functional and cellular mechanisms that are highly interconnected. These mechanisms include but are not limited to the following: 1). BECact/dys due to multiple injurious species within precapillary arterioles, capillary NVU’s, postcapillary venules, and the subsequent development of increased 2). Vascular inflammation; 3). Oxidative stress; 4). Neuroinflammation with decreased BEC protective nitric oxide (BEC dysfunction) and increased permeability of fluids (including ions, water, proteins including hormones, and polypeptides) and cells including leukocytes into the PVS due to endothelial blood-brain barrier dysfunction and disruption (BBBdd); 5). Microvessel stiffening due to increased pulsatile pressure resulting in microvessel remodeling with further stiffening in the arterial system resulting in impaired pulsatile flow that is important to drive the glymphatic system flow to promote waste and water clearance from the perivascular spaces. 6). Glial pvACef dysfunction and or loss with decreased or dysfunctional AQP4 activity; 7). Neuroinflammation; 8). Oxidative stress with increased reactive oxygen (ROS), nitrogen, sulfur species (RONSS); 9). Leukocyte accumulation within the PVS/PVU/GS; 10). Phagocytic debris accumulation of proteins, misfolded proteins, and protein aggregation (specifically amyloid beta and tau), which disrupt GS waste and excess water removal resulting in stalled flow that further dilates the PVS to remodel into EPVS proximal to regions of stalled flow. Thus, the EPVS structural crossroad and holding space allow for interactions between the neurovascular, neuroimmune, and neuronal mechanisms that relate to clinically relevant neurodevelopmental and neurodegenerative disorders [3,8,9,10,15,23,24,27,30,35,39,55]. In addition to the previous 10 mechanisms, one overarching theme seemed to reoccur in the development and progression of EPVS. This theme involved the response to injury that occurred at the capillary NVU BEC initial injury and the subsequent response to injury aberrant remodeling. The overarching finding of the NVU BECs to develop BECact/dys with subsequent BBBdd with increased permeability was noted to be the primary instigator in the mechanistic evolution of EPVS and SVD [3,8,9,10,30,31,35,40]. The other notable finding was that obstruction to the PVS via excessive metabolic waste, phagocytic debris, phagocytized misfolded and soluble proteins such as amyloid beta and tau, neuroinflammatory cells, or dysfunction of the abluminal perivascular astrocyte endfeet resulting in the dysfunction or loss of their polarized aquaporin-4 water channels might allow for the remodeling obstructive or stalled flow resulting in the proximal dilation of the PVS within the PVU to become EPVS [8,9,10,15,30,86,87,88,89].

The recognition of PVS/EPVS has also brought forth an increased interest in the brain’s interstitial space(s) (ISS) and how they empty into the PVS of the postcapillary venules glymphatic pathway (GS). Even though the ISS diameter is only nanometers in size (e.g. approximately 20–50 nanometers in mouse brains), its total volume is known to account for up to 20% of the total brain volume and is crucial for interstitial fluid (ISF) solute and fluid transport, signal transmission, and communication amongst neuronal parenchyma within the neuropil (Figure 12) [90].

4. Glia and PVS/EPVS Ultrastructural Remodeling in LPS Neuroinflammatory, Glucotoxicity, and Hyperhomocysteinemia (HHCY) Models

In LPS-treated neuroinflammatory models, MGCs have been demonstrated to be attracted to the NVU along with the formation of EPVS [3]. Additionally, MGCs appeared to be invasive to the capillary NUV that was associated with the detachment and separation of pvACef in obese diabetic db/db female models at 20-weeks of age (Figure 13) [91].

Indeed, reactive astrocytes (macroglia) and microglia play an important role in the remodeling of various animal models. Remodeling changes range from attracted MGCs of the NVU in female LPS-treated models [2], to invasive and destructive effects of MGCs that envelope the NVU with subsequent detachment and separation of pvACef in the female obese, insulin resistant, and diabetic db/db models in Figure 13 [91], to the damaged BEC glycocalyx and formation of postcapillary venular enlarged perivascular spaces as identified in the female obese diabetic BTBR.Cg-Lepob/WiscJ ob/ob mouse (BTBR ob/ob) models, glia are actively involved in brain ultrastructural remodeling changes (Figure 14) [92].

In diabetes, elevated brain glucose (neural glucotoxicity) not only undergoes autoxidation to generate ROS but also is capable of forming complex non-enzymatic reactions between reducing sugars and the amino groups of proteins advanced glycation end-products (AGEs), which reacts with the BEC receptor for AGE (RAGE) to generate even larger amounts of ROS along with oxidative damage to proteins, lipids and nucleotides. This glucotoxicity also promotes increased mitochondrial ROS (mtROS) [91]. Importantly, diabetes is known to be associated with increased SVD and EPVS [93,94,95,96,97]. Notably, homocysteine (Hcy) is another injurious species in addition to LPS and glucotoxicity to the BECs of the NVU as in figure. Homocysteine (Hcy) is a physiologic sulfur/sulfhydryl-containing amino acid that is liberated upon protein degradation and breakdown that may lead to a pathological condition called hyperhomocysteinemia (HHCY) especially if there is a nutrient deficiency or impaired uptake of vitamin B cofactors (B6, B9, and B12) that are important for running the folate-methionine cycles of folate one carbon metabolism (FOCM) (Figure 15) [98,99].

When Hcy becomes elevated as in HHCY, it undergoes autoxidation (similar to glucose autoxidation) via the following set of reactions [98,99]: Two homocysteines (2Hcy-SH) plus oxygen yeilds → homocystine: oxidized disulfide, plus two hydrogens. plus two electrons (HcyS-SHcy +2 H+ and 2e-) to generate ROS such as superoxide, hydroxyl radical, and/or hydrgen peroxide. Additionally, formation of mixed disulfides may contribute to the additional formation of ROS such as: Hcy-SH + R-SH ---- O₂ -------→ Hcy-S-SR + H2O→ reactive oxygen species or Hcy-SH + R1-S-S-R2 ← → R1-S-S-Hcy + R2-SH → reactive oxygen species where R = any organic compound in the plasma with a thiol group (-SH) accessible to react with Hcy, such as proteins, cysteine, glutathionine, gamma-glutamylcysteine, or cystinylglycine. Further, HHCY may undergo complicated rearrangements to form Hcy thiolactone (a cyclic thioester), which is chemically reactive and acylates free amino groups such as the side-chain lysine groups of proteins (termed N-homocysteinylation), forms an amide bond with the lysine residue and introduces a free thiol group, which can lead to protein damage and is implicated not only cardiovascular disease but also in brain microvessel dysfunction and disease. Further, the process of N-homocysteinylation promotes further ROS/oxidative stress in addition to the homocysteinylated proteins that may become damaged or dysfunctional to result in a loss of their biological activity [98,99,100,101,102].

HHCY is known to be associated with multiple neurological diseases and/or disorders including cognitive impairment [103Elias], cardiovascular disease [104,105,106,107,108,109,110,111,112,113], stroke (ischemic) [102,114], vascular dementia (VaD) [102,115], LOAD [99,102,116], Parkinson’s disease [102,117], schizophrenia, major depressive, and bipolar disorders [102,118], epilepsy and antiepileptic therapies [102,119], and autism spectrum disorder(s) (ASD) [120,121]. Further, it is interesting to note that LPS treatment and HHCY may co-occur and interact synergistically regarding neuroinflammation to result in the interactive vicious cycle between inflammation and oxidative stress [122]

5. Conclusions and Future Directions

Ruoli Chen (guest editor) of this special series in the Neuroglia Journal [https://www.mdpi.com/journal/neuroglia/special_issues/3750Q69655] has put forth the concept that neuroglia (astrocytes and microglia) are at a crossroad from their unifying role of maintaining support and homeostasis in the brain to that of exploring how neuroglia might contribute to disease onset, progression, and potential recovery in multiple neurological diseases and disorders. Indeed, the neuroglia cells are at a definite crossroad along with BECs, Pcs, neurons along with their synapses in addition to neurovascular and perivascular units (NVU and PVU) as discussed in this narrative review.

The PVS within the PVU provide a holding space or a crossroad intersection for multiple cell-cell communications that are bounded by the innermost shared basement membranes of mural cells (BECs and Pcs) and the outmost abluminal basement membranes (glia limitans) of the pvACef. The initial or 1st barrier of PVU consists of the BBB, while the outermost highly protective pvACef basement membrane-glia limitans provide the 2nd barrier. This 2nd barrier created by the pvACef glia limitans prevents excessive fluids, neurotoxins, and proinflammatory cells from entering the interstitial spaces of the brain as in figures 1B,9, 14C, D (Figure 16) [3,8,9,10,30,55].

There are multiple factors responsible for the continuous flow of the admixture of CSF, ISF, and metabolic waste within the PVS including: the continuous production of CSF via the choroid plexuses within the ventricles [14,123], the adjacent pia arterial pulsations [124,125], respiratory motion [126], pvACef-AQP4 water channel proteins [125], and all important sleep-wake cycle (since most glymphatic flow occurs during sleep [14,17,127,128]. Additionally, the psACef also provide a protective barrier to support and protect the synaptic transmission of the tripartite synapses [35,55,60,67,68] as well as the transfer of information throughout multiple informational networks including the default mode network (thought and reflection), central executive network (attention and working memory), and the salience network (important in processing internal and external stimuli) in the brain [129,130,131].

There are certain limitations to any narrative review as they reflect the interests of the author that assist with the knowledge of the topic at hand. Also, this narrative relies heavily upon the use of rodent models to explain and increase the understanding of ultrastructural remodeling changes, which may not always directly translate to human individuals, especially since astrocyte morphology in human individuals are much more complex than in rodent models [62]. Additionally, images were derived primarily from the frontal grey matter cortex and represent primarily layer III and the transition to only the immediate subcortical regions of the brain. Future directions include the need for greater MRI resolution to better identify and understand the venular side of the capillary NVU, which include EPVS and impaired glymphatic waste clearance from the ISF contents of the brain such as the use of 7Tesla instead of 1-3Tesla currently in use [3Why, 9A closer look,]. Also, the venular system with its PVU and PVS are essential for waste removal including the effective removal of neurotoxins including soluble oligomeric amyloid beta, tau, other neurotoxic misfolded proteins, and metabolic waste to slow or prevent neurovascular, neuroinflammatory, and neurodegenerative diseases discussed in this narrative review via the GS that is dependent on the normal functioning of the postcapillary venular PVS as it is known to be the structural conduit for waste removal within the PVU. Additionally, EPVS can now be identified and quantitated via algorithms that have been created for artificial intelligence and deep machine learning, which reduce time and effort, and increase specificity in contrast to earlier visual quantifications to better understand the relationships between EPVS, SVD and impaired cognitive abilities in large, combined cohorts [132].

Regarding future directions, it will be necessary to better understand the development and progression of the recent findings of EPVS in neurodevelopmental disorders such as autism spectrum disorders (ASD). ASD may be broadly defined as a heterogeneous group of neurodevelopmental disorders that are characterized by impaired social interaction and/or communication that are associated with restrictive or repetitive patterns of behavior [133,134]. The recent findings of EPVS in the neurodevelopmental disorder ASD [135,136,137] and the recent increase of ASD have alarmed clinicians, neurologists, neuroscientists (specifically pediatric neuroscientists), the public, and recently governmental agencies [138]. Therefore, it is absolutely necessary to evaluate and better understand why there are increased EPVS in ASD. Not only is ASD associated with EPVS but also Hcy is elevated and known to associate with increased EPVS and is also elevated in ASD as well as being a biomarker for impaired FOCM [99,120,139]. Thus, deficient systemic and brain folate induced either by nutritional deficiency or by the development of autoantibodies to the folate receptor alpha that will impair brain FOCM and result in HHCY as in Figure 15, which acts as an injurious stimulus to BECs due to the excessive autoxidation of Hcy and oxidative stress be examined. Once injured the BECs will undergo both structural and functional changes with the development of BECact/dys and BBBdd with increasing neuroinflammation with the development of EPVS as in figures 9 and 16. These functional and structural remodeling changes to the BECs and EPVS will result in impaired glymphatic system clearance of neurotoxins and metabolic waste to produce an ongoing vicious self-perpetuating cycle of injury and response to injury remodeling. Additionally, HHCY will impair the necessary FOCM donation of methyl groups, which are critically necessary for supporting the rapid neural expansion with neurogenesis, synaptic plasticity. and neural network connectivity in infants and young children due to their positive effects on biosynthesis of nucleic acids, proteins, and lipids required for growth and epigenetic modifications important for regulating gene expression [140,141,142].

While this narrative review has focus primarily on LPS and glucose excess, it is important to note that nutritional deficiencies (deficient folate) or genetic abnormalities including the methylenetetrahydrofolate reductase (MTHFR) mutation gene polymorphism (677C>T) enzyme plays an important role in the folate cycle, and autoantibody formation against the folate receptor alpha also impair FOCM donation of one-carbon units to neurons with resultant neurodevelopmental disorders. Additionally, multiple nutrients are important for proper neurodevelopment [141,142]. Thes nutrients consist of lipids-polarlipids, minerals, vitamins (A, D, B6. B9(folate), B-12), proteins and amino acids, tryptophane, tyrosine, phenylalanine, branched chain amino acids, pro-prebiotics [141].

Treatment for lowering Hcy consists of folic acid or folate supplements (0.5-5 mg folic acid and 0.5mg of vitamin B-12 [143] and/or the newer folinic acid treatments, should there be autoantibodies to the folate receptor alpha [143]. When there are autoantibodies to folate receptor alpha this may be treated with the use of folinic acid (5-formyltetrahydrofolate) that is physiologically converted enzymatically to 5MTHF that can enter the CSF via the reduced folate carrier on the choroid epithelial cells at the choroid plexus [144]. Also, folinic acid is capable of displacement of the blocking autoantibodies to folate receptor alpha in addition to increased diffusion when the plasma level of folinic acid is increased [144]. Interestingly, Beversdorf et al. have recently proposed a precision medicine approach for heterogeneous conditions with autism spectrum disorders [145]. Indeed, Beversdorf Phase 2m proposal might consider the findings of EPVS on MRI as biomarkers for impaired (FOCM) such as elevations in Hcy, deficient nutrient intake of folate, autoantibodies for folate receptor alpha, oxidative stress/ROS due to autoxidation of Hcy, BBBact/dys, BBBdd, decreased GSH, and increased GSSG. Not only will these findings serve as biomarkers for ASD but also will serve to follow the response to various future treatment paradigms [145].

While there are multiple injurious species (neurotoxins) to the BECs of the NVU as in Figure 5, this narrative overview has focused primarily on LPS-induced neuroinflammation, hyperglycemia of diabetes with elevated glucose and the AGE/RAGE interaction to increase ROS, Hcy/HHCY due to impaired FOCM, pC/C due to metainflammation as a result of visceral obesity and gut dysbiosis, and ROS due to multiple causes that are all capable of inducing mesmerizing images. Regardless of the multiple neurotoxins, there seems to be an overarching mechanism of BECact/dys resulting in inflammation, oxidative stress, and decreased nitric oxide that also associates with BBBdd to increase permeability and leaky brains resulting in EPVS. Further clarification of the prognostic values in the development and progression of EPVS and SVD in neurodegenerative diseases such as LOAD in aging and neurodevelopmental and neuropsychiatric disorders such as ASD in youth need to be studied in greater depth in animal models and human individuals. Importantly, the astroglia cells (inclusive of pvACef and psACef) are playing an important and crucial role in the development of EPVS.