Blood-Brain Barrier

The BBB is a highly specialized structure of the neurovascular system composed of a microvascular endothelium, as well as proximal astrocytes, basement membrane, and pericytes. It maintains separation between the components of the circulating blood and those of the central nervous system (CNS) and controls homeostatic balance between these systems. Under physiologic conditions, the BBB ensures constant supply of nutrients (oxygen, glucose, and other substances) for brain cells and guides the inflammatory cells to respond to the changes of the local environment. To meet the energetic demands of the brain, glial cells work with neurons to regulate the function and dilation of blood vessels in response to metabolic requirements. The concerted efforts of all of these cell types together are conceptualized as the neurovascular unit (NVU), which couples brain activity to vascular supply, allowing the import of metabolic materials, as well as providing for the export of waste, via the BBB.

Anatomically and functionally, endothelial cells (ECs) situate at the interface between the blood and the brain. Cerebral ECs are not fenestrated but are interconnected by tight junctions forming a continuous BBB [10]. It has become apparent that ECs require contacts with various CNS cell types to establish BBB characteristics [11]. They compose the vessel walls and generate a physical barrier impermeable to most large or charged molecules through abundant tight junctions. From this position, they perform essential biological functions, including barrier, transport of micronutrients and macronutrients, receptor-mediated signaling, leukocyte trafficking, and osmoregulation. They express a wide variety of transporters and receptors, engaging in transporter- and vesicle-based transfer of molecules between the brain and the blood. Specialized receptors on the membrane of ECs initiate intracellular signaling cascades in response to agonists that activate specific receptors or changes in shear stress at the cell surface produced by changes in the rate of blood flow. The best described transporter is the glucose transporter isoform 1 that becomes highly expressed upon BBB formation. Gap junctions permit cross talk between adjacent ECs allowing the transmission of intracellular responses. Once initiated, these cascades trigger the release of potent vasodilator substances such as nitric oxide (NO) and prostacyclin and vasoconstrictors, such as endothelin and endothelium-derived constrictor factor [12, 13].

Astrocytes are the most numerous cell type in the CNS, and their specialized end-feet cover nearly the entire surface of CNS microvessels. Astrocytes are star-shaped cells with many processes emanating from the cell body and surround most portions of the microvessels and capillaries and as part of the NVU interact with ECs through the end-feet of their processes [14, 15]. Astrocytes in the brain perform an array of homeostatic functions. Their processes encircle the synapses, where they control the extracellular pH, ion balance, and neurotransmitter concentrations necessary for optimal neuronal function. These activities make them ideally suited for sensing the demands of the associated neurons, which they translate to the other cells of the BBB via their end-feet. Thus, they act as a link between synaptic activity and the cerebrovascular cells by translating information between neurons and cerebral microvessels. Astrocytes and ECs also influence each other’s structure and function. For example, interaction of astrocytes with ECs can greatly enhance EC tight junctions and reduce gap junctional area, resulting in decreased permeability of the EC layer. Therefore, astrocyte-microvascular EC interactions are essential for a functional NVU [15]. Moreover, during intense activity, astrocytes signal the energy demands of neurons to the vascular cells mediating cerebral blood flow (CBF) which then can signal an increased demand in blood flow [16]. Astrocytes have also been shown to regulate CBF responses by influencing contractile properties of small penetrating intracerebral arteries [17].

Pericytes are mural cells covering the abluminal surface of microvessels. Pericytes are flat, undifferentiated, tissue cells with contractile potential that develop around capillary walls, contributing to stability of microvessels and covering a major part of the abluminal endothelial surface. In the neurovascular unit, pericytes are embedded in a thin layer of basement membrane, which separates them from ECs and end-feet of astrocytes. While most of the pericyte bodies and processes do not attach to ECs because of the basement membrane, interdigitations of pericyte and EC membranes can directly connect in the area lacking basement membrane, forming “peg-and-socket” connections [18, 19]. Together with the ensheathment of brain capillaries by astrocytic end-feet, the close contact of ECs with pericytes via the peg-and-socket junctions within a common basal lamina is crucial for establishing and maintaining the BBB [20]. The CNS vasculature has significantly higher pericyte coverage compared with peripheral vessels, and pericytes have an important role in regulating capillary diameter, CBF, and extracellular matrix protein secretion. Perivascular pericytes release a large number of growth factors and angiogenic molecules, which regulate microvascular permeability, remodeling, and angiogenesis.

Microglia are the resident macrophages of the brain and play critical roles in innate and adaptive immune responses of the CNS. Once thought to be relatively quiescent in their “resting” state, their ramified processes actually constantly survey the brain parenchyma. Upon stimulation, activated microglia engage in a variety of pro- as well as anti-inflammatory morphologies including phagocytic phenotypes. The pro-inflammatory activated state of microglia typically affects the permeability of the BBB, increasing its permeability via production of reactive oxygen species and inflammatory cytokines such as TNF-α.

Basement membranes in the NVU also significantly contribute to BBB integrity through several mechanisms. The predominant constituents of the cerebrovascular basement membranes include collagen IV, laminin, perlecan, nidogen, and fibronectin, which are extracellular matrix proteins produced by each cell type in the NVU [21, 22]. There are two types of basement membranes in the NVU: (1) an endothelial basement membrane composed of extracellular matrix produced by ECs and pericytes and (2) a parenchymal basement membrane formed by those from astrocytes [23, 24]. Basement membranes function as a physical barrier surrounding the abluminal surface of endothelial cells and anchor the cells in place at the BBB. In addition, they also contribute to BBB regulation, where the extracellular matrix mediates diverse signaling in endothelial cells and pericytes [24].

Neurovascular Unit and Neurovascular Coupling

In brain capillaries, ECs form the tube structure with barrier integrity, in which the abluminal surface is covered by basement membranes composed of extracellular matrix. The endothelial tubes are surrounded by pericytes, astrocyte end-feet, and neurons, comprising the NVU. The cells of the NVU work in concert to maintain homeostasis of the cerebral microenvironment, regulate CBF, control exchange between the BBB and blood, contribute to immune surveillance in the brain, and provide support to brain cells. While physical barrier structures in ECs predominantly control BBB integrity, molecular barrier systems through endothelial transporters can mediate the influx and efflux of specific molecules at the BBB [25–27].

Cerebrovascular health is essential to maintain adequate brain perfusion and preserve normal brain function. This is accomplished through coupling alterations in local CBF (e.g., by increasing vascular dilation in response to elevated metabolic demand) to neuronal activity, a process termed neurovascular coupling. The brain’s structural and functional integrity relies on an uninterrupted and well-regulated blood supply, and consequently brain dysfunction and ultimately death often involve interruption of CBF [28]. Mechanisms that ensure adequate neuronal blood supply and CBF are essential to meet the brain’s metabolic demand and support its overall health [16]. Adjustments of CBF must be highly regulated to maintain proper cellular homeostasis and function [29]. This is accomplished through neurovascular coupling orchestrated by an intercellular signaling network [30]. The NVU is functionally integrated to regulate CBF responses to neuronal stimulation which ensures a rapid increase in CBF and oxygen delivery to activated brain regions [1, 31].

Cerebrovascular Dysfunction in AD

AD is a neurodegenerative disorder often associated with neurovascular dysfunction, cognitive decline, and accumulation in brain of amyloid beta (Aβ) peptide and tau-related lesions in neurons. With age, increasing prevalence of coincident AD and cerebrovascular disease (CVD) has been well-recognized. CVD and vascular risk factors are associated with AD and contribute to neuropathological changes such as selective brain atrophy and accumulation of Aβ in AD [40]. Intracerebral Aβ is removed from the brain through vascular mechanisms involving the lipoprotein receptor protein 1 (LRP1) and P-glycoprotein [41, 42]. A study of the association of vascular pathology and cerebrovascular disease with neuropathologically confirmed neurodegenerative disease revealed that AD has a significantly higher prevalence of cerebrovascular disease and vascular pathology than other related neurodegenerative diseases [43]. Furthermore, AD is more likely to occur in patients presenting cerebral infarctions, as around 70% of patients diagnosed with AD have evidence of coexistent cerebrovascular disease.

Cerebrovascular abnormalities are a common phenomenon in AD. Multiple epidemiological studies have shown a remarkable overlap among risk factors for cerebrovascular disorder and sporadic, late-onset AD. AD is associated with marked alterations in cerebrovascular structure and function of the NVU in both large intracranial vessels and microvessels [44]. Microvascular pathophysiological alterations have a causal role both in the development of AD and related cognitive decline. BBB transport systems are significantly altered in AD patients compared to controls [45]. Amyloid β (Aβ) is a major contributor to BBB dysfunction in AD, and Aβ deposits in the vasculature enhance BBB permeability in the AD brain. Cerebral amyloid angiopathy (CAA), which is characterized by deposition of amyloid fibrils in the walls of small- to medium-sized blood vessels, promotes the degeneration of smooth muscle cells and pericytes, leading to compromised BBB or simply breakdown of BBB [26]. Reduced CBF occurs early in the development of AD, most significantly in areas where tau pathology is associated with AD. These perfusion deficits develop in pre-symptomatic stages before brain atrophy [46, 47]. CBF reductions correlate with dementia, cortical atrophy, and vascular disease in the white matter. AD patients exhibit significant impairment of neurovascular coupling responses. In AD or in CAA, accumulation of Aβ leads to the damage of the vessel wall, increasing the chance of lobar hemorrhages [44]. As neuronal activity continuously determines neurovascular and neurometabolic coupling [48], functional deterioration of the neurons is a pathophysiological characteristic in brain aging and several age-related neuropathological conditions like AD (see below for PD) [1, 49].

Vascular dysfunction also plays a central role in the development of AD. Typical amyloid plaques and neurofibrillary tangles (NFTs) may result from hypoperfusion due to inadequate blood supply. Ischemic conditions may trigger Aβ accumulation and facilitate amyloidogenic cleavage leading to increased levels of toxic Aβ. Indeed, dramatically and consistently increasing accumulation of Aβ has been observed after cerebral ischemia [50]. In vitro, Aβ has been demonstrated to be toxic to both cerebral and peripheral endothelium [51, 52]. The toxic effects of Aβ on cerebral blood vessels may also induce cerebral hypoperfusion and increase vulnerability to ischemic damage. Since amyloid plaques appear to promote ischemia and vice versa, it is plausible that there is a synergistic relationship between the amyloidogenic and vascular features of AD pathology. Cerebral hypoperfusion is an early feature of AD, which occurs several years before the onset of clinical symptoms. The perfusion of precuneus is firstly affected, followed by cingulate gyrus and the lateral part of the parietal lobe, about 10 years before the development of dementia, and then the frontal and temporal lobes.

A strong relationship between age-associated CVD and AD exists; however, it is complicated by the high level of risk factors and overlap that increases with age. As such, the boundary between physiological and pathological aging is not categorical. For example, midlife hypertension and AD are strongly correlated, especially in those not undergoing treatment with antihypertensive drugs [53]. Furthermore, increased blood pressure (BP) in midlife not only leads to pathological changes in blood vessels but also brain atrophy and an increase in senile Aβ plaques and NFTs in the neocortex and hippocampus [54]. Moreover, long-lasting increases in BP may worsen the risk of AD by inducing small vessel disease (SVD), structural white matter alterations, and cerebral hypoperfusion through impairment of normal vascular regulation or atherosclerosis.

Cerebrovascular Dysfunction in PD

PD is the second most common neurodegenerative disorder in the elderly population. It is clinically characterized by parkinsonism (resting tremor, bradykinesia, rigidity, and postural instability), and histopathological changes include progressive loss of neurons in multiple regions, specifically the substantia nigra pars compacta, accompanied by the presence of aggregated deposits known as Lewy bodies and Lewy neurites. The relationship between CVD, vascular risk factors, and PD is variable in clinical, radiological, and pathological studies. Furthermore, large-scale cohort studies have demonstrated that PD is associated with higher risk of CVD [55, 56]. Cerebrovascular pathologies and vascular risk factors are associated with an increased prevalence of PD and cognitive impairment in the elderly [57, 58]. In a clinicopathological study, prevalence of concomitant PD and cerebrovascular disease, mainly SVD, was 26–40% [59, 60]. A meta-analysis demonstrated that PD is associated with CVD, and patients with PD were at a higher risk of CVD later in their life [61].

The loss of dopaminergic neurons is a hallmark of PD. Dopaminergic neurons are equipped with abundant mitochondria and are therefore easily exposed to high levels of ROS [62]. Mitochondrial DNA is easily damaged by ROS, which results in mitochondrial malfunction. In aged cells, mitophagy is disturbed [63]. Meanwhile, low energy support results in reduced mitochondrial repair [64]. All of these changes can contribute to dopaminergic neuronal loss. In physiological conditions, dopaminergic neurons are well equipped to deal with oxidative stress. Superoxide dismutase (SOD) and glutathione peroxidase (GPX) nonspecifically decompose ROS/NOS. However, when these protective mechanisms are compromised during aging, excess ROS will be produced. The subsequent oxidative stress will then facilitate the aggregation of α-synuclein, eventually leading to formation of Lewy bodies [65].

Neuronal degeneration and bioenergetic derailment in PD are accompanied by cerebrovascular dysfunction and alteration in cerebral metabolism. Decreases in CBF values are observed in basal ganglia, hippocampus, prefrontal cortex, and parietal white matter in PD patients when compared to healthy subjects [66]. In PD patients, CBF reductions in parietal regions correlated with cognitive dysfunction, suggesting a link between cognitive deficits and perfusion [67]. Impaired autoregulation of brain perfusion, independent of dopaminergic treatment, has been demonstrated in PD patients subjected to a drop in BP compared with controls [68].

In addition to neuronal injury, glial cells play a pivotal part in PD development. Dopaminergic neurons are equipped with unmyelinated axons, and thus astrocytes make the most intimate contact with them [69]. Intact astrocytes provide protection and support to dopaminergic neurons through production of antioxidants such as glutathione and by removing toxic molecules including glutamate and α-synuclein [70]. Meanwhile, astrocytic neurotrophic factors protect neurons from damage [71]. However, chronic inflammation in the aged CNS changes the phenotype of astrocytes that become more pro-inflammatory. The senile astrocytes fail to provide protection and support; instead, they produce cytokines, chemokines, and ROS, which harm the integrity of the NVU [69].

Cerebrovascular Dysfunction in ALS

ALS is characterized by progressive loss of motor neurons in the anterior horn of the spinal cord and brain, resulting in progressive weakness, muscle atrophy, and respiratory failure. Although the pathogenesis of ALS remains largely unknown, neuropathologic features and gene mutations associated with ALS have shed important light on the etiology of the disease. Mutations in superoxide dismutase-1 (SOD1 ) are the most common form of inherited ALS, accounting for almost 25% of familial cases. Mutations in SOD1 and overproduction of ROS/RNS and dramatic gliosis characterized by abnormalities of astrocytes, widespread astrocytosis, and activated microglial cells are evident in ALS [72].

Impairments of the BBB, blood-spinal cord barrier, or blood-cerebrospinal fluid barrier (BCSFB), aggravating motor neuron damage, are also possible pathogenic mechanism in ALS. These barriers control the exchanges of various substances between the blood and brain/spinal cord and maintain proper homeostasis of the CNS. The first evidence of altered BCSFB affected permeability in ALS was provided in 1980s, with the finding of abnormally high levels of IgG, albumin, and complement component C3a in the CSF of ALS patients [73, 74]. At the same time, IgG deposits and C3 and C4 were found in the spinal cord and motor cortex of patients with ALS, suggesting BBB/BSCB disruption [75]. Compelling evidence of changes in all NVU components, including the BBB/BSCB, in both patients and animal models identifies ALS as a neurovascular disease. Qualitative analyses of spinal cord tissue from ALS patients evidenced markedly decreased perivascular tight junction and basement membrane, as well as astrocyte end-feet dissociated from the endothelium. These results confirmed lost endothelial integrity as one characteristic of BSCB disruption that might contribute to disease progression [76]. It has been suggested that entry of harmful blood-borne substances into areas of motor neuron degeneration may have implications for the pathogenesis of ALS [77]. Circulating ECs are considered markers for endothelial damage [78] and are unexpectedly reduced in peripheral blood of ALS patients with moderate or severe disease [79], providing further support for vascular damage in ALS.

Oxidative stress also plays a crucial role in ALS [80]. Oxidative stress is a major component of the BBB impairment. In physiological conditions, ROS are generated largely from mitochondrial activity in neural cells. Specific endogenous antioxidants such as superoxide dismutase and glutathione peroxidase are able to scavenge ROS. In pathological conditions, however, injury leads to excitotoxicity, activation of inflammatory pathways, mitochondrial dysfunctions, leukocyte recruitment, and microglia activation, all of which increase levels of free radicals [81].

Vasculoprotective Approaches

Nutritional factors are essential to neuroprotection. Development or progression of cognitive impairment and dementia can be slowed by consumption of certain foods and vitamin supplements [82]. Regular consumption of fish is related to lower risk of AD [56] and slower rate of cognitive decline [83]. Fish offers protection by countering inflammation and enhancing vascular tone and countering atherosclerosis. The B vitamins, especially folate, B6, and B12 are implicated as likely to sustain or improve cognitive function in older age [84]. Chronic accumulation of reactive oxygen species in older brains may exhaust antioxidant capacity and trigger neurodegenerative processes as characterized in AD. Dietary supplementation with fruit or vegetable extracts high in antioxidants helps to decrease the enhanced vulnerability to oxidative stress and improve neuronal communication via increases in neuronal signaling and animal behavior [85].

Changes in lifestyle factors will decrease the risk of developing dementia in later life [86]. Regular exercise can reduce rate of age-related cognitive decline and decreased risk of incident dementia or AD [87]. Increased physical activity in midlife has been found to be associated with less neocortical atrophy in the elderly [88]. Vasculoprotective factors such as nutritional factors, lifestyle factors, and physical activity will not only reduce the risk of onset of degenerative diseases but also slow down the progression of these disorders.