Age-associated physiological and pathological changes at the blood–brain barrier: A review

Structure of the blood–brain barrier (BBB)

Homeostasis of the extracellular microenvironment in the neural tissue of the brain as well as its protection against neurotoxic compounds and variations in the composition of the blood are important for normal function of the neurons. It is warranted by a structure formed between blood and brain, which is therefore called the BBB. ¹ Clear evidence for the existence of this permeability barrier in the brain emerged in 1909 with the demonstration by Edwin Goldman (a South African-German) that a dye injected into the blood stream of a rat stained the whole body – except for the brain and spinal cord. The opposite was also true: injection of the dye into the cerebral ventricles stained the brain and spinal cord but not the rest of the body.^2,3 This occurs because most organs of the body are perfused by capillaries lined with endothelial cells (ECs) that have small pores (fenestrations) to allow for the rapid movement of small molecules into the organ interstitial fluid from the circulation. However, the capillary endothelium of the brain and spinal cord lack these pores because the ECs of brain capillary are connected to each other by continuous tight junctions (TJs), produced by the interaction of several transmembrane proteins that project into and seal the paracellular pathway.^3–5 The interaction of these junctional proteins effectively blocks the free diffusion of polar solutes from blood along these potential paracellular pathways and so denies access to brain interstitial fluid. Thus, the BBB significantly impedes entry from blood to brain of virtually all molecules, except those that are small and lipophilic or those that enter the brain through an active transport mechanism, particularly with essential nutrients, precursors and cofactors. The other difference between the peripheral and central capillaries is that the brain capillaries are surrounded by pericytes, basal membrane and astrocyte end feet, which form a covering layer, that are missing at the peripheral microvessels. It is well established that in contrast to leaky vessels in peripheral organs, ⁶ the BBB restricts entry of polar molecules into the brain,^7–9 but can be transported into the brain via specific transporters expressed in the brain endothelium under physiological or pathological conditions.^10,11

There are five basic mechanisms by which solute molecules move across membranes. First is simple diffusion, which proceeds from high to low concentrations (e.g. sucrose and ethanol). Second is facilitated diffusion (glucose via GLUT-1), a form of carrier-mediated endocytosis, in which solute molecules bind to specific membrane protein carriers, also from high to low concentration. Third is simple diffusion through an aqueous channel, formed within the membrane. Fourth is active transport through a protein carrier (e.g. P-gp and BCRP) with a specific binding site that undergoes a change in affinity. Active transport requires ATP hydrolysis and conducts movement against the concentration gradient. The fifth is movement between cells is referred to as paracellular diffusion. The BBB has a number of highly selective mechanisms for transport of nutrients into the brain.

Several BBB peptide transport mechanisms (i.e. receptor-mediated, absorptive-mediated, carrier-mediated and non-specific passive diffusion), as well as non-transport processes (i.e. endocytosis without transcytosis, absorption and metabolism) are known. ¹²

The main cellular elements of BBB are (a) the brain capillary ECs, and these cells are associated with (b) pericytes that are responsible for the feeding of the ECs, (c) end feet of the astrocytes covering the capillaries, and (d) the interacting microglial cells and (e) neurons (Figure 1). The collective name of these cell types as a collaborative system in the brain is neurovascular unit (NVU). Beside the cellular elements, BBB comprises extracellular matrix and basal lamina as the parts of the protective system. All the cell types have special structures to ensure the regulation of the penetration of xenobiotics to the brain tissue. OPEN IN VIEWER

In the first part of this review, we will discuss the barrier function of these cellular elements in interaction with each other and supporting mechanisms. In the second part, the alterations of the protective molecular mechanisms during the process of maturation and aging will be summarized. In the final part, the age-related neurodegenerative disorders and the role of BBB disruption in their pathologies will be presented.

Endothelial barrier

Compared with peripheral vasculature, BBB ECs are characterized by increased mitochondrial content, exhibit minimal pinocytotic activity, and lack fenestrations.^14–17. Increased mitochondrial content is essential for these cells to maintain various active transport mechanisms such as those utilized to transport ions, nutrients and waste products into and out of brain parenchyma, thus contributing to precise regulation of the CNS microenvironment and ensuring proper neuronal function. The high concentration of mitochondria in cerebrovascular ECs might account for the sensitivity of the BBB to oxidant stressors. Furthermore, the physiology and pathophysiology of ECs are closely linked to the functioning of their mitochondria, and mitochondrial dysfunction is another important mediator of disease pathology in the brain. ¹⁸

The brain endothelium is highly reactive because it serves as both a source of, and a target for, inflammatory proteins and reactive oxygen species (ROS). Neuroinflammation and oxidative stress lead to BBB breakdown, which are implicated in the pathogenesis of central nervous system (CNS) disease. Cell polarity of ECs is ascribed to differing functional expression of transporter proteins and metabolic enzymes that are differentially expressed on the luminal (or apical) and abluminal (or basolateral) membranes, which further contribute to the high selectivity of the BBB.^19–21 Of the many transporters expressed at the BBB endothelium, several have been implicated in influx and/or efflux of drugs into the CNS. Decreased expression and/or defective function of transporters, leads to increased BBB permeability, toxic adverse effects of drugs and can be an early event in the pathology of several disorders. Factors that can disrupt the BBB are varied, ranging from secreted elements to immune cells and pathogens. Compromised BBB integrity manifests mainly as increased barrier permeability. In addition to direct effects on ECs, other members of the NVU can be affected, that is pericytes, astrocytes and basement membrane (BM), which in turn aggravate impairment of BBB functions. Consequences vary from dysregulated molecular and ionic flux across the damaged BBB to the initiation of a central inflammatory response. Despite manifold causes, characteristics and consequences, BBB breakdown generally culminates in neuronal dysfunction, neuroinflammation and neurodegeneration. Downstream pathological outcomes and potential for recovery are diverse ²² (Figure 2). OPEN IN VIEWER

Astrocytes

Astrocytes are the most abundant cell type in the brain and display a fibroblast-like morphology within grey matter^15,31; however, this morphology can be influenced by their CNS location and associations with other cell types that are in close proximity. ³² Astrocyte end-feet cover over 99% of cerebral capillaries, ³³ leading to critical cell–cell interactions that directly modulate and regulate BBB characteristics. ³² Several studies have demonstrated that astrocytes play a vital role in maintenance, and perhaps induction, of BBB characteristics.

Astrocytes are fundamental for homeostasis, defence and regeneration of the CNS. Loss of astroglial function and astroglial reactivity contributes to the aging of the brain and to neurodegenerative diseases. Changes in astroglia in aging and neurodegeneration are highly heterogeneous and region-specific. ³⁴

Several inducing factors secreted by astrocytes have been identified, including TGF-β, GDNF and BFGF, which are involved in induction and regulation of the BBB phenotype. Additionally, astrocytes can regulate brain microvascular permeability via Ca²⁺ signalling involving astrocyte-endothelial gap junctions and purinergic transmission.^15,26,30,32 Astrocytes play a critical role in preventing excitotoxicity induced by acute elevations of glutamate in the brain. This is mediated via expression of astrocyte glutamate transporters excitatory amino acid transporters (EAAT1 and EAAT2) that are responsible for glutamate uptake into the astrocyte cell, thus reducing glutamate levels in the parenchyma. ³⁵

Lipoproteins in plasma transport lipids between tissues, however, only high-density lipoproteins (HDL) appear to traverse the BBB; thus, lipoproteins found in the brain must be produced within the CNS. Apolipoproteins E (ApoE) and ApoJ are the most abundant apolipoproteins in the brain, which are mostly synthesized by astrocytes. ³⁶ ApoE has an important role in the pathology and BBB injury during the development of Alzheimer's disease (see more details later in this article).

There is also evidence that astrocytes progressively accumulate in the normal aging brain, increasing in both number and size. These astrocyte changes in normal brain aging may, in the event of an injury, contribute to the exacerbated injury response and poorer outcomes observed in older traumatic brain injury survivors. ³⁷ Astrocyte morphology changes and GFAP expression increases progressively during aging in humans and rodents.^38,39

Pericytes

Pericytes are BBB associated cells. During aging, pericytes show ultrastructural changes such as vesicular and lipofuscin-like inclusions^1,40–43; an increased size of mitochondria ⁴⁴ and a foamy transformation. ⁴⁵ Ueno et al. ⁴⁶ reported on membranous inclusions within the basal lamina or the ECM of the microvessels and suggested the degeneration of pericytes. de Jong et al. ⁴⁷ in the aging rat and Stewart et al. ⁴⁸ in the aging human brain showed the degeneration and loss of pericytes. Burns et al.^49,50 described the reduction of the cross-sectional area of pericytes in aging monkey and rat brain. However, Peters et al. ⁴² found no change in the number of pericytes in monkey, and Heinsen and Heinsen ⁵¹ and Peinado et al. ⁴¹ showed an increase in the number of pericytes in the rat aging brain. The obvious discrepancies in the literature could be explained by considering the degeneration and the proliferation of pericytes as distinct age-related mechanisms. Accordingly, Scheibel and Fried ⁵² could show that pericytes in aging brain begin to lose their contact to the EC and begin to migrate. This behaviour seems to be a response of the pericytes to a yet unidentified age-related effect. The PDGF released by ECs is known to bind to the PDGF receptor expressed by pericytes and to attract pericytes to ECs.^53,54 If this ligand-receptor system is disturbed, the functional integrity of the vessel is impaired.^53,55 With age, this system could deteriorate as well, but data are not available as yet.

Montagne and coworkers showed that pericyte injury and possibly early degeneration correlates with increased BBB permeability within the hippocampus, ⁵⁶ a region known to be affected by pericyte loss and BBB breakdown on post-mortem tissue analysis in Alzheimers' disease. ⁵⁷

Microglia

Microglia, the primary immune cells of the brain, are ubiquitously distributed in the CNS and are activated in response to systemic inflammation, trauma and several CNS pathophysiologies.^15,58–60 This cell type is not the basic element of the BBB, but it has an impact on the BBB function and integrity. Microglia present with a ramified morphology that is characterized by a small soma and fine cellular processes during their “resting state”. Microglial activation in response to pathophysiological stressors can trigger changes cell morphology, which include reduced complexity of cellular processes and transition from a ramified morphology to an amoeboid appearance. ⁵⁹ Activated microglia produces high levels of neurotoxic and proinflammatory mediators such as nitric oxide, peroxide, TNF-α and proteases, all of which result in cell injury and neuronal death, ³⁰ As immune cells microglia scavenge apoptotic cells, tissue debris after trauma, or microbes. ³⁰ They can also act as scavengers of extracellular molecules such as amyloid-β.^59,60 Activation of microglia is associated with altered TJ protein expression and increased BBB permeability. ⁶¹

Briefly the major physiological protective effector functions of microglia: (a) Proliferation, (b) morphological transformation, (c) motility and migration, (d) intercellular communication, (e) phagocytosis and (f) proteostasis. With aging, microglia shift their morphology and may display diminished capacity for normal functions related to migration, clearance and the ability to shift from a proinflammatory to an anti-inflammatory state to regulate injury and repair. ⁶²

Neurons

There is considerable evidence for direct innervation of both brain microvessel ECs and associated astrocyte processes via distinct connections with noradrenergic,^15,62,63 serotonergic, ⁶⁴ cholinergic^65,66 and GABAergic ⁶⁷ neurons. Therefore, neurons are in connection with the BBB and have influence on the behaviour of capillary endothelial barrier. For example, studies have shown that loss of direct noradrenergic input from the locus coeruleus results in increased BBB susceptibility to effects of acute hypertension, resulting in significantly increased permeability to 125-I labelled albumin. ⁶⁸

With aging, cell senescence can contribute to BBB compromise. The compromised BBB allows an influx of inflammatory cytokines to enter the brain. These cytokines lead to neuronal and glial damage. Several factors contribute to the deterioration of synaptic plasticity with age and one of these factors appears to be a heightened level of activation of microglia, which may reflect impairment in the homeostatic ability of these cells with age, or an increase in responsiveness to modulatory molecules. The age-related decrease in tissue perfusion, together with the increase in BBB permeability may alter the microenvironment in the brain; this, combined with the proposed age-related compromised homeostatic capability of microglia, may be a significant factor in maintaining the neuroinflammatory changes, which have been described in the aged brain and which exert a negative impact on synaptic function. These combined changes may contribute to the deficit in long-term potentiation (LTP) in perforant path-granule cell synapses of aged animals. ⁶⁹ Ultimately, the functional neuronal changes within the brain can cause age-related disease.

Changes in the BBB with physiological aging

How to define physiological aging? It is evident even from casual observation of physical activities, such as walking, that elderly people exhibit a deterioration of physiological processes. Moreover, the inability of athletes to continue peak performance when they reach their 30s or 40s indicates that deterioration begins at a relatively young age and progresses in severity from that point on. Indeed, many studies have confirmed that most physiological processes deteriorate progressively after about 30 years of age, some functions more severely affected than others. Diseases that do not occur until, or increase in frequency at, advanced ages are called age-associated diseases. Coronary heart disease, stroke, many types of cancer, osteoporosis, Alzheimer's disease, Parkinson's disease (PD) and other neurodegenerative disorders are examples of such diseases commonly found in elderly people. Indeed, age-associated disease underlies much of the physiological deterioration of old age. However, many studies focus on subjects who are free of discernible disease in what they refer to as the study of ‘normal aging or physiological aging’ (Edward J Masoro, Medicine Encyclopedia, Aging Healthy). Concerning aging of the brain and BBB, physiological aging can be defined as a deterioration of the functions without cognitive decline and dementia.

Dysfunction of BBB appears and it is associated with inflammation and loss of TJs without leukocyte recruitment at adult period and aging. Cell damage is characterized by nuclear DNA damage and can contribute to aging either indirectly by increasing apoptosis or cellular senescence or directly by increasing cell dysfunction.

The matured immune system is also entering into function to regulate the passage of cells and molecules from the periphery to the CNS through BBB. For example, ECs selectively regulate trans-endothelial migration of T-helper lymphocytes (Th1 and Th2) suppressing the initiation of CNS autoimmune. ⁷⁰ TJ barriers of ECs prevent the free entry of blood-derived substances; thereby maintain the extracellular environment of the brain. Astrocytes express TJ proteins and densely and tightly surround mature neurons to protect them from blood-derived neurotoxic substances. ⁷¹

The main changes at the BBB during physiological aging are presented in Table 1.

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Table 1.

Alterations of BBB constituents during physiological aging.

BBB elements	Properties	References
ECs	Capillary wall thickness: increased in humans decreased in rats decreased in monkeys Number of ECs: decreased in humans Number of mitochondria: decreased	Hunziker et al. 72 Hicks et al. 44 Burns et al. 49 Hunziker et al. 72 Burns et al.49,50 Grammas et al. 18
Tight junctions	Expression of tight junction proteins: decreased	Elahy et al., 71 Enciu et al. 73
Basal lamina	Thickness of basement membrane: Increased Concentration of collagen IV and argin: Increased Concentration of laminin: decreased	Candiello et al., 29 Ravens 74 Candiello et al. 29 Candiello et al. 29
Astrocytes	Astrocyte proliferation: Increased number and size GFAP expression: Increased	Rodriguez-Arellano et al., 34 Harris et al. 37 Chisholm and Sohrabji, 38 Middeldorp and Hol 39
Microglia	Changes to amoeboid morphology Production of neurotoxic proinflammatory mediators	Kettenmann et al. 59 Ronaldson and Davis 30
Pericytes	Number of pericytes: Degeneration and loss of pericytes Ultrastructural changes: vesicular and lipofuscin-like inclusions, increased size of mitochondria, foamy transformation	Stewart et al., 48 Burns et al.49,50 Rascher and Wolburg 1 Hicks et al. 44 Sturrock 45
Neurons	Deterioration of synaptic plasticity Deficit in long-term potentiation Impaired neurogenesis Increased apoptosis Neuronal damage due cytokine release	Blau et al. 69 Blau et al. 69 Lucke-Wold et al. 75 Lucke-Wold et al., 75 Cerbai et al. 76 Buschini et al. 77

Changes at the BBB associated with age-related neurodegenerative pathologies

An important component of age-related pathology is neurodegeneration. It can be defined as progressive loss of neuronal structure and function, finally leading to neuronal cell death. Most neurodegenerative diseases have their onset in mid-life and can be characterized by motor and/or cognitive symptoms that progressively worsen with age and may reduce life expectancy.

Discussion and outlook

In summary, this review highlights several important key points of the disruption of BBB during the process of aging. First, the main players of the barrier formation at the blood-brain interface are introduced. Second, the possible multifactorial nature of maturation of BBB and NVU during embryonic development and postnatal period is presented. Then the changes in phenotypes and functionality in physiological aging and the consequent neurological impairment are summarized. Third, the review focuses on the cellular and molecular events and associated cerebrovascular factors contributing pathologically to different neurodegenerative disorders. Although the timing of disease symptoms and progression is very important in e.g. Alzheimer's disease, often patients present with significant tau and Aβ load by the time the symptoms begin. Given the contributions of vascular impairments at certain points during the disease process, it is important to investigate the vascular pathology and BBB permeability conditions before the disease becomes too severe to reverse. Finally, it is clear that the majority of dementia types involve an underlying small-vessel disease or cerebrovascular/BBB dysfunction at some points during disease progression. However, the causality dilemma still exists in identifying the circular cause and consequences between vascular dysfunction and the clinical signs of different dementias and age associated neurodegenerative processes, which is an important exploratory topic of future research.

The recognition of the role of efflux and uptake transporters in the pathology of Alzheimer's and PDs and many other CNS diseases might offer the avenue for new therapeutic intervention strategies for the pharmacological and clinical drug research for the therapy of currently untreatable chronic neurodegenerative disorders.