A blood–brain barrier overview on structure, function, impairment, and biomarkers of integrity

A highly controlled microenvironment is required to promote the normal functioning of the central nervous system (CNS). The existence of a biological barrier at the blood to brain interface effectively separating the brain from the rest of the body was established after the finding of Paul Ehrlich when he noticed that a peripherally infused dye did not stain the brain tissue. His finding was further supported by later observation from his associate Goldmann as he applied the same dye to the cerebrospinal fluid. It did stain only the brain tissue without extravasating in the periphery [1]. These biological barriers are established by different cells at three key interfaces: the blood–brain barrier (BBB), blood–CSF barrier (BCB), and the arachnoid barrier [2] (see Fig. 1).

The BBB is formed by microvascular endothelial cells lining the cerebral capillaries penetrating the brain and spinal cord of most mammals and other organisms with a well-developed CNS [3]. It is considered the largest interface for blood–brain exchange as the combined surface area per average adult is assumed to fall between 12 and 18 m² based on an average microvessels surface area of 150 and 200 cm² per gram tissue [4]. The BBB is playing a critical role in protecting the brain parenchyma from blood-borne agents and providing a significant obstacle to the entry of drugs and other exogenous compounds into the central nervous system.

The second barrier is the blood–cerebrospinal fluid barrier (BCSFB), which is formed by the epithelial cells of the choroid plexus. The CSF secretion into the ventricular brain system is controlled by the choroid plexus epithelial cells [5]. In contrast, the interstitial fluid (ISF), which is consisting of the rest of the brain extracellular fluid, is derived at least partially by secretion across the capillary endothelium of the BBB [6‐9]. ISF communicates freely with CSF over several locations, and its contribution to CSF is estimated between 10 and 60% [2].

Underlying the dura mater, we find the avascular arachnoid epithelium, which is considered the third barrier. The dura mater covers the CNS and completes the seal between the extracellular fluids of the central nervous system and the rest of the body [10]. Its contribution to the exchange between blood and brain is insignificant due to its avascular nature and the relatively small surface area compared to other barriers [11].

In this review, we will focus more on BBB as it is the main barrier contributing to CNS protection and maintaining the brain homeostasis. Unlike other vascular endothelial cells lining peripheral blood vessels, brain microvascular endothelial cells present distinctive morphological, structural, and functional characteristics that set them apart from other vascular endothelia. These include the following: (1) the expression of tight junctions (TJs), sealing the paracellular pathways between adjacent endothelial cells, thus preventing the unregulated passage of polar (water-soluble) molecules between the blood and the brain; (2) the absence of fenestrations; (3) the lack of pinocytic activity and the expression of active transport mechanisms to regulate the passage of essential molecule (including nutrients and essential amino acids) while blocking the passage of potentially undesired substance (both endogenous and xenobiotics) [12]. The transport barrier comprehends various efflux transporters, such as P-glycoprotein (P-gp) [13], breast cancer resistance protein (BCRP) [14], organic anion transporting polypeptide (OATP) [15]. These efflux systems may also share overlapping substrate affinities (such as P-gp and BCRP—[16]) actively pump compounds (including xenobiotics) out of the endothelial cells back into the blood circulation [17], resulting in reduced CNS exposure. Lastly, the presence of drug-metabolizing enzymes within the brain endothelial cells, such as CYP 450 enzymes (CYP1B1 and CYP2U1, CYP-3AF), results in the formation of the metabolic barrier [18‐21]. CYP mRNAs have been found in several areas of the human brain which plays a role in neurodegenerative disease by metabolizing drugs and fatty acids [20]. For instance, CYP2U1 metabolizes fatty acids like arachidonic CYP1B1 has a role in the metabolism of endogenous compounds in the CNS [19]. CYP3A4 also showed to be functionally expressed at drug resistance of epileptic BBB and oxidizes a large group of xenobiotics, including antiepileptic drugs [22].

These integrated barrier systems, while protecting the brain from potentially harmful compounds, are also the major obstacle for delivering drugs into the CNS [23]. Overall, the BBB function as a dynamic interface regulating the brain homeostasis and protecting the CNS, which can respond to different physiological and pathological conditions [10]. The Induction and maintenance of the barrier function fall primarily on the interaction between the microvascular endothelium, the astrocytic foot processes (which invest close to 99% of the abluminal surface area of the brain capillary), and pericytes [24] (see also Fig. 2).

In addition to microvascular endothelial cells, the capillary basement membrane (BM), astrocytes, pericytes (PCs) embedded within the BM, microglial and neuronal cells complete the structure of what we now refer to as the neurovascular unit (NVU) [25]. Below we provide a brief description of these cells and their role in maintaining the integrity of the BBB.

Tight and adherens junction (AJs) constitutes the junction complex of the BBB. The TJs are present at the sites of fusion involving the outer surface of the plasma membrane of adjacent endothelial cells or the same cell (see Fig. 2). Adherens junctions are composed of a cadherin–catenin complex and its associated proteins. The TJs consists of three integral membrane proteins, namely, claudin, occludin, and junction adhesion molecules (JAMS), and several cytoplasmic accessory proteins including Zonula occludens-1, -2, -3 (ZO-1, ZO-2, ZO-3), cingulin, and others (see Fig. 2). The cytoplasmic proteins link membrane proteins to actin, which is the primary cytoskeleton protein for the maintenance of structural and functional integrity of the endothelium.

After early detection of the cytoplasmic accessory proteins, intramembrane junctional proteins were found to be directly contributing to the restricted interendothelial permeability. Occludin, a protein with four transmembrane domains, was the first discovered protein, which seemed to play a less important role in permeability restriction as occludin-deficient mouse mutant is viable and has intact biological barriers. The discovery of claudins, another four transmembrane domains protein with no homology to occludin, was of fundamental importance for TJs research. Also, JAMs and the endothelial selective adhesion molecule (ESAM), which are members of the immunoglobulin superfamily, are considered TJs components, which probably fulfill regulatory functions of the barrier [59].

Tight junction formation appears to be a very early feature of BBB development, and a barrier to the free movement of proteins and macromolecules is formed at the primary stages of brain development [2].

Cadherin is the main component of these junctional membrane proteins, which joins the actin cytoskeleton through intermediary proteins, catenins, to form adhesive contacts between cells. AJs assemble via homophilic interactions between the extracellular domains of calcium-dependent cadherin on the surface of adjacent cells. The submembrane proteins β- or γ-catenin connects the cytoplasmic domains of cadherins to the actin cytoskeleton via α-catenin (see Fig. 2). AJs components, including cadherin, alpha-actinin, and vinculin (α-catenin analog), have been demonstrated in intact microvessels of the BBB in rats. TJs and AJs components are known to interact, particularly ZO-1 and catenins, and influence TJs assembly [79].

The intracellular scaffold proteins ZO-1, ZO-2, and ZO-3, which link the junctional molecules claudin and occludin via cingulin to intracellular actin and the cytoskeleton appear to play a crucial role in the efficiency of the TJs [52, 59, 88]. Studies show that changes in calcium concentration both intracellular and extracellular can have a great impact on tight junction assembly and efficiency as a barrier and can alter the electrical resistance across the cell layer [10, 89]. As mentioned early, soluble factors released by many of the cell types associated with brain microvessels (including microglia and astrocytes) as well as nerve terminals adjacent to the endothelial extracellular matrix/basal lamina (such as vasoactive agents and cytokines) can modify tight junction assembly and barrier permeability [10, 90].

Mammalian endothelial cells are known to undergo dynamic changes under different stimuli. Cellular, molecular, and physical stimuli are the essential players that help ECs to acquire specialized functions. For most cell types, chemical signaling seems to play a pivotal role in cell physiology. Physical stimulation, such as shear stress, is one of the most important but underestimated physiological stimuli, which contributes to vascular ECs differentiation and maturation besides other cellular and molecular signaling. Akin to responses to inflammatory cytokines, shear stress has been shown to cause dramatic changes in ECs morphology [91], gene expression [92], and function [93]. Shear stress showed to induce the production of reactive oxygen species (ROS) and nitric oxide (NO). NO is known to cause vasodilation, autocrine signaling, and also increase the production of free radicals. A study showed that ECs might scavenge free radicals by increasing levels of GAPDH and other intracellular enzymes [94]. Another study showed that NO might have a protective role in BBB during reperfusion after the transient loss of flow in a condition mimicking ischemic stroke [95].

Shear stress was shown to induce a significant upregulation of tight and adherens junction proteins and genes in human brain microvascular endothelial cells (HBMECs). The study reported a 5.91- and 2.13-fold increase in claudin-5 and cadherin-5 gene expression, respectively. HBMECs showed higher maturation and differentiation under shear stress, as indicated by the increase in the trans-endothelial electrical resistance (TEER) from 100 to 700 Ω cm². Also, SS induced the endothelial expression of different drug transporters and metabolic properties that allow the BBB to protect the CNS from harmful substances [96]. In contrast, a study on iPSCs-HBMECs d didn't show any significant changes in tight junctions, TEER, or cell morphology. These discrepancies highlight the importance of further studies to optimize culture models based on non-primary BBB ECs to better reflect the physiological responses of the BBB in vivo [97].

Akin to cytokines effect, shear stress has been shown to play a critical role in maintaining blood vessels’ homeostasis and a protective role. A study in HBMECs showed that shear stress was a much more potent stimulus for thrombomodulin (TM) release, yielding media TM levels of 1000 pg/10⁵ cells when compared to 175 and 210 pg/10⁵ cells for, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), respectively. Thrombomodulin is an integral membrane receptor constitutively expressed on the luminal surface of vascular ECs and an essential determinant of blood vessel homeostasis. The study also showed that shear-conditioned media was able to completely block thrombin-induced permeabilization of HBMECs, which confirm the protective role of shear stress [98]. Overall, BBB studies must consider the effect of shear stress as it is playing an essential role in BBB maturation and homeostasis. In this respect, our group has summarized the advances in in-vitro BBB models [99, 100].

BBB dysfunction is reported in many CNS pathological conditions including multiple sclerosis [161]; hypoxic and ischemic insult [162]; Parkinson’s and Alzheimer’s disease [163]; epilepsy [164]; brain tumors [165]; glaucoma [166], and lysosomal storage diseases [167]. The observed barrier dysfunction can range from mild and transient changes in BBB permeability, resulting from tight junction opening, to chronic barrier breakdown, and changes in transport systems and enzymes can also occur. This process can also be associated with the degradation of the basement membrane [168]. Microglial activation and infiltration of different plasma components and immune cells into the brain parenchyma result in disturbance of CNS homeostasis and variable damage to the surrounding brain. In most cases, it is not possible to determine whether barrier compromise is causal in disease onset or a result of neurological disease progression. Still, barrier disturbance can often be seen to contribute to and exacerbate developing pathology [169].

As discussed earlier, under normal conditions, the BBB is relatively impermeable. In pathologic conditions, several vasoactive agents, cytokines, and chemical mediators are released that increase BBB permeability. Several in vitro and in vivo studies showed the opening effect on BBB of several mediators include glutamate, aspartate, taurine, ATP, endothelin-1, NO, TNF-α, and macrophage-inflammatory protein 2 (MIP2) which are produced by astrocytes [132, 170, 171]. Other humoral agents reported to increase BBB permeability are bradykinin, 5HT, histamine, thrombin, UTP, UMP, substance P, quinolinic acid, platelet-activating factor, and free radicals [132, 172, 173]. They are variable in the source as some of these agents are released by endothelium and have an autocrine effect on endothelium itself. For example, endothelin (ET-1) acts on Endothelin A (ETA) receptors in ECs. In physiologic conditions, chemical mediators released by nerve terminals of neurons associated with blood vessels such as histamine, substance P, and glutamate, influence and regulate BBB permeability. Table 1 summarizes different pathological conditions that are affecting BBB integrity.

The raised interest in exploring BBB—after many studies showed its critical role in a wide range of neurological disorders—mandate the use of accurate and representative markers to demonstrate the integrity of the barriers between the blood, the brain, and the CSF [216]. The drug used should be metabolically inert, non-toxic at the applied doses, not bound to other molecules such as proteins in plasma or tissues, be available in a range of molecular sizes, can be visualized in the range from the naked eye to the electron microscopical level, and be reliably quantifiable.

To date, there is no single available marker that fulfills all the criteria mentioned above. Dextran, available in a wide range of molecular sizes labeled with either biotin or a fluorescent tag, has been extensively used in permeability studies, but it is tedious to quantify. Radiolabeled markers, which are also available in a wide range of molecular sizes, can be easily quantified. Still, they cannot be visualized with enough resolution and have many other limitations, which will be mentioned below. Recently ¹³C sucrose has been introduced as a small molecular weight marker, which can be precisely quantified but also cannot be visualized.

For the quantification of subtle BBB impairment, markers with small molecular weight, such as sucrose should be used, because even a minor degree of barrier damage is expected to have a noticeable effect on their permeability which cannot be quantified with larger molecular weight markers [217]. In general, physicochemical properties, such as size and polarity should be considered for measuring the BBB permeability. When the BBB is compromised and loses its integrity, the chance of large molecular weight markers such as dextran to enter the brain increases, and this could be considered as an option to evaluate the integrity and permeability of the BBB however, for accurately examining small changes in BBB permeability that occur in many neurological conditions, small molecular weight markers (with MW of less than 400 Da) have superiority. Thus, a combination of different markers is currently the most reliable approach to sufficiently assess barrier integrity in the developing or pathological brain. We will try to briefly explore the most used markers and elaborate on their advantages and disadvantages, hoping that will help researchers pick the best marker which can fit their application. A summary of different markers used for BBB permeability studies is represented in Table 2.

The BBB is a fundamental component of the CNS. Its functional and structural integrity is vital in maintaining the homeostasis of the brain microenvironment. Deterioration in BBB function may play a significant role in the pathogenesis of disease since the BBB dynamically responds to many events associated with flow disturbances, free radical release, and cytokine generation. Furthermore, many neurological disorders and lesions are associated with increased BBB permeability such as neoplasia, hypertension, dementia, epilepsy, infection, multiple sclerosis, and trauma. Any disorder which affects BBB function will cause secondary effects on cerebral blood flow and vascular tone, further influencing transport across the BBB. In this review, we covered several critical aspects of the BBB encompassing the role and functions of its cellular components as well as the contribution of physical stimuli such as shear stress. We also examined several neurological disorders related to the impairment of the BBB. Finally, we provided a comprehensive list of methods currently available to assess the viability of the BBB in vivo and in vitro and discuss the pros and cons of each method.