**3.1 BBB junctional molecules**

*Connectivity and Functional Specialization in the Brain*

capillaries [22, 23]. Astrocytes are also critical cellular support of BBB integrity. Recent molecular studies have shown several molecules released by astrocytes that enhance and maintain barrier tightness, such as cholesterol and phospholipid transporter molecule apolipoprotein E [24, 25]. Release of apolipoprotein E from astrocytes, for example, regulates endothelial TJs by signaling through the low-density lipoprotein receptor related protein 1 (LRP1) on both pericytes and endothelial cells of CNS microvessels [25]. Astrocytes have been identified as essential mediators of BBB formation and function because of purified astrocytes' ability to induce barrier properties in non-CNS blood vessels [26]. Based on these observations, it has been proposed that astrocytes are necessary for the

**Microglia** derive from hematopoietic precursors that migrate from the yolk sac into the CNS parenchyma, acting as the brain's main line of defense past the BBB and play a vital role in innate immune responses in the vascular bed and cerebral parenchyma (**Figure 1**) [27], little is known about how microglial-endothelial communications may shape and regulate the homeostatic BBB. However, studies have demonstrated that microglia are associated with endothelial's nascent vessels in the developing brain, and promote the fusion of cells in the stages following vascular endothelial growth factor-mediated induction [28]. Recent studies have shown the activation of microglia in CNS disorders like AD and multiple sclerosis, which are associated with BBB breakdown and neuroinflammation. In these conditions, microglial activation may be both a cause and consequence of BBB dysfunction [20]. Microglia can exist in one of two active states: in the activated pathway, microglia release proinflammatory cytokines like interleukin-1b and tumor necrosis factor-a. Whereas in alternative pathways, microglia are involved in tissue repair, phagocytosing neurons and foreign material, releasing chemokines and vascular endothelial growth factor [29]. On the other hand, brain endothelial cells can also secrete molecules that cause microglial activation [30]. In summary, a complex interplay between systemic and CNS derived immune cells exists at the BBB. **Basement Membrane:** The vascular tube is surrounded by two basement membranes (BMs), the inner vascular BM, and the outer parenchymal BM (**Figure 1**). The vascular BM is an extracellular matrix secreted by the ECs and pericytes, whereas the parenchymal BM is primarily secreted by astrocytic processes that extend towards the vasculature [31]. These BMs consist of different molecules, including type IV collagen, laminin, heparin sulfate proteoglycans, and other glycoproteins [32]. They provide an anchor for many signaling processes in the vasculature and also constitute an additional barrier for molecules and cells to cross before accessing the neural tissue. Disruption of these BMs by matrix metalloproteinases is an integral part of BBB dysfunction and posterior leukocyte infiltration, which can be observed in many different neurological disorders [32]. **Neurons and interneurons.** Neurons can detect small variations in their supply

of nutrients and oxygen and transform these signals into electrical and chemical messages to adjacent interneurons or astrocytes. In response to these signals, necessary adjustment mechanisms are initiated. Due to this phenomenon, neurons are considered NVU's pacemaker [15]. Neurons need to be able to signal to cerebral vessels when their energy demands change. Positive and negative feedback mechanisms exist to regulate cerebral blood flow, accompanied by adjustments of substrate delivery across the BBB, a process known as neurovascular coupling [33]. In this sense, one relevant mechanism for neurovascular coupling is direct innervation of astrocytic processes or the endothelial tube by, amongst others, serotonergic, noradrenergic, cholinergic, and GABAergic neurons [4]. Mechanisms of neurovascular coupling, particularly those that can explain direct molecular effects on BBB integrity, are yet to be established. Future knowledge will be of great interest since

formation of impermeable TJs in the developing vessels of the BBB.

**28**

The BBB is a diffusion barrier essential for the normal function of the CNS. The NVU endothelial cells differ from endothelial cells in the rest of the vascular system by their absence of fenestrations, and for having more extensive junctional molecules, mainly TJ, and sparse pinocytic vesicular transport [34]. These junctional molecules limit the paracellular flux of hydrophilic molecules across the BBB. In contrast, small lipophilic substances (O2 or CO2) can diffuse freely across plasma membranes along their concentration gradient [34]. Nutrients such as glucose and amino acids enter the brain via transporters, whereas receptor-mediated endocytosis mediates larger molecules' uptake, including insulin, leptin, and iron transferrin [35], it is believed that all the components of the BBB are essential for the normal function, stability, and permeability of the BBB.

The Junction complex in the BBB comprises TJ, AJ, and Gap junctions (GJ). The TJ ultrastructurally appear as apparent fusion sites, involving the outer plasma membrane of adjacent endothelial cells [36]. The number of TJ strands, as well as the frequency of their ramifications, varies and consists of three integral membrane proteins: claudin, occludin, and junction adhesion molecules, as well as several other cytoplasmic accessory proteins, including members of the family zonula occludens (ZO-1, ZO-2, ZO-3) and cingulin (**Figure 2**). Cytoplasmic proteins link membrane proteins to actin, for maintaining the structural and functional integrity of the endothelium [36]. The Claudins were identified as the principal component of TJ and are localized exclusively at TJ strands. Claudins bind to other claudins on adjacent endothelial cells to form the primary seal of the TJ [37]. Closest to the

#### **Figure 2.**

*Basic molecular organization of BBB junctional molecules and transport. The endothelial cells confer unique properties on the BBB. They are the principal line of cerebral vasculature and have numerous junctional molecules such as tight junctions, adherens junction, gap junctions and accessory proteins that limit the passive paracellular diffusion of all but the smallest of solutes and ions. On the other hand, carriers, receptors and active efflux protein mediated transport allow substances such as peptides, amino acids, and glucose to selectively cross the BBB and release toxic substances and drugs into the blood preventing them from entering the brain. Created with BioRender.com.*

apical membrane, the claudin 1, 3, 5, 12, and occludins limit paracellular diffusion of solutes and ions across the BBB [38]. Loss of claudins is associated with permeability and BBB breakdown in neurodegenerative disorders and acute CNS diseases [39]. TJ proteins connect to actin and vinculin-based cytoskeletal filaments via scaffolding proteins of the membrane, associated with ZO 1, 2, and 3 [40]. Previous studies have shown that ZO-1 deficiency leads to BBB breakdown in many neurodegenerative and acute CNS disorders [41]. Occludin, another integral protein localized at the TJ, form the TJ's paracellular barrier when conjoined with neighboring cells' claudins [42]; the cytoplasmic domain of occludin directly associates with ZO proteins. The expression of occludin has also been documented in human adult brains, but not in average human newborn and fetal brain, suggesting their role as regulatory proteins that can alter paracellular permeability of the BBB [35]. The third type of TJ-associated membrane protein, junctional adhesion molecules (JAM), structurally consists of a single transmembrane domain and an extracellular portion with two immunoglobulin-like loops joined by disulfide bonds [43]. Three JAM-related proteins, JAM-1, JAM-2, and JAM-3 are expressed in human BBB and previous studies have shown their participation in cell-to-cell adhesion and monocyte transmigration through BBB [44].

The AJs are established between neighboring cells by homophilic interactions between the transmembrane proteins, vascular endothelial cadherin (VE-cadherin), and epithelial cadherin (E-cadherin) in CNS [13]. Nearby to the basolateral membrane, AJ proteins, VE-cadherin, and platelet endothelial cell adhesion molecule (PECAM-1) form homophilic endothelial-to-endothelial contacts limit paracellular diffusion of solutes [13]. GJ are other junctional molecules, whose connexin-37 (CX37), CX40, and CX43 form hemichannels between endothelial cells [45]. These membrane proteins enable direct cytoplasmic exchange of ions and low molecular weight metabolites between adjacent cells; these channels of communications are essential for propagating electrical signals and coordination of cell signaling by transfer of second messengers [46]. Furthermore, brain endothelial GP also support tight junction integrity.

#### **3.2 BBB transport systems**

The major BBB transporters, receptors, and channels found in endothelial cells and pericytes have been validated by transcriptomic studies and protein analysis (**Figure 2**) [34]. Except for gases and small lipophilic molecules that freely diffuse across the endothelium, brain endothelial transport systems regulate molecular exchanges between blood and brain. The BBB's highly selective nature and the high metabolic demand of the brain demand other routes of entry for various nutrients to feed and nurture the brain [34]. Metabolic supply is achieved via several transporters expressed on the surface of CNS endothelial cells that drive the active transport of specific solutes and metabolites into the brain [47]. On the other hand, given the close proximity and highly interactive signaling between vascular pericytes and endothelial cells, it is relevant to describe in this chapter the BBB pericyte transporter.

**Endothelial carrier** enables solutes such as carbohydrates, amino acids (AA), monocarboxylic acids, hormones, fatty acids, nucleotides, inorganic ions, amines, choline, and vitamins to cross the BBB via substrate-specific transporters (**Figure 2**). In terms of carbohydrate transporters, GLUT1 (glucose transporter 1) is a uniporter that transports glucose. GLUT1 can transport glucose (and other hexoses) from either side of the luminal and abluminal endothelial membrane extracellularly or intracellularly [48]. Since glucose is lower in the brain interstitial fluid (ISF) than plasma, GLUT1 favors blood-to-brain transport of circulating glucose. GLUT1 is

**31**

K+

tions of Na<sup>+</sup>

and 2Cl−

*Blood-Brain Barrier Dysfunction in the Detrimental Brain Function*

expressed in endothelial cells, but not in neurons. Their importance is best illustrated by the fact that transcript encoding GLUT1 is one of the most abundant transcripts in brain endothelium. Their dysfunction and lack cause barrier breakdown and can prevent clearance of amyloid plaques, suggesting a contributing role in Alzheimer's

Regarding the transport of amino acids, all essential AA are transported into the brain across the BBB via endothelial AA transporter 1 and 2 (LAT1/2), that transport bidirectionally neutral AA such as tryptophan and tyrosine [50], and the cationic AA transporter 1 and 3 (CAT1/3) that transport cationic AA such as lysine and arginine [51]. Also, on the abluminal membrane transporters for excitatory AA (EAAT1/2/3) transport glutamate and aspartate out of the brain, limiting their excitotoxic effects on neurons [52]. Transporters of neutral and excitatory AA, such as glycine, taurine, and GABA are enriched abluminally and with highaffinity transport from brain to endothelium in a sodium dependent manner, and then, these AA are transported across the luminal membrane of the BBB into the blood via low-affinity transporters into the circulation [53]. Finally, essential fatty acids are essential for brain development and postnatal neural functions. The Brain endothelium expresses luminal transporters for fatty acids, including fatty acid transport protein 1 and 4 (FATP- 1/4) and the MFSD2A (Major Facilitator Superfamily Domain containing 2a) [54]. In the brain, MFSD2a is exclusively expressed in brain endothelium and is required for right BBB development and functional integrity. Finally, for Lactate released from skeletal muscles during exercise, and ketone bodies derived from liver from metabolism of fatty acids, the transport is facilitated by monocarboxylate transporter-1 (MCT1). Once inside the brain parenchyma, they are used as alternative energy metabolites by the brain, supply the brain with key substrates for DNA and RNA synthesis [54]. Nucleotides and nitrogenous base, e.g., cytosine, guanine, adenine, thymine and uracil, are all transported across the BBB via sodium-independent concentrative nucleoside transporter-2 (CNT2) and the sodium-independent equilibrative nucleoside

**Endothelial receptor** is the most important transporter because proteins and large macromolecules (e.g., fibrinogen, immunoglobulins, thrombin, plasminogen, and growth factors) cannot cross the BBB. However, some proteins and peptides use receptor transport to cross the BBB and enter the brain (**Figure 2**). Transferrin receptor (TfR) [56], insulin receptor (IR) [57], and leptin receptor (LEP-R) [58] mediate blood-to-brain transport of transferrin (iron-protein carrier), insulin, and leptin across the BBB, respectively. This characteristic has promoted its use for CNS drug delivery, including therapeutic antibodies [59]. Receptors LRP1 and LRP2 are expressed in the BBB's brain endothelium, with LRP1 binding Alzheimer's soluble

**Endothelial active efflux and ion transport.** ATP-binding cassette (ABC) transporters utilize ATP as an energy source and are expressed at the luminal side of the BBB endothelium. They function to prevent brain accumulation of drugs, xenobiotics and macromolecules via active efflux from endothelium to blood [**Figure 2**]. Some examples are ABCB1 (also known as P-glycoprotein, P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated proteins (MRP). The BBB also has a significant role in regulating ions' concentration in the

K+

ological equilibrium of the resting membrane and action potentials [61].

into the brain and potassium efflux from the brain, maintaining high concentra-

Luminal Na-K- Cl (chloride) cotransporter (NKCC) mediates entry of Na<sup>+</sup>

from blood-to-endothelium. The bicarbonate (HCO3)

ATPase, is a key regulator of sodium influx

in the brain, critical for the electrophysi-

−

,

Cl exchanger

Ab fragments and mediating its brain-to-blood clearance [60].

*DOI: http://dx.doi.org/10.5772/intechopen.94572*

disease progression [49].

transporter-1 and 2 (ENT1/2) [55].

CNS. The luminal sodium pump, Na<sup>+</sup>

and low concentrations of K<sup>+</sup>

#### *Blood-Brain Barrier Dysfunction in the Detrimental Brain Function DOI: http://dx.doi.org/10.5772/intechopen.94572*

*Connectivity and Functional Specialization in the Brain*

cyte transmigration through BBB [44].

also support tight junction integrity.

**3.2 BBB transport systems**

apical membrane, the claudin 1, 3, 5, 12, and occludins limit paracellular diffusion of solutes and ions across the BBB [38]. Loss of claudins is associated with permeability and BBB breakdown in neurodegenerative disorders and acute CNS diseases [39]. TJ proteins connect to actin and vinculin-based cytoskeletal filaments via scaffolding proteins of the membrane, associated with ZO 1, 2, and 3 [40]. Previous studies have shown that ZO-1 deficiency leads to BBB breakdown in many neurodegenerative and acute CNS disorders [41]. Occludin, another integral protein localized at the TJ, form the TJ's paracellular barrier when conjoined with neighboring cells' claudins [42]; the cytoplasmic domain of occludin directly associates with ZO proteins. The expression of occludin has also been documented in human adult brains, but not in average human newborn and fetal brain, suggesting their role as regulatory proteins that can alter paracellular permeability of the BBB [35]. The third type of TJ-associated membrane protein, junctional adhesion molecules (JAM), structurally consists of a single transmembrane domain and an extracellular portion with two immunoglobulin-like loops joined by disulfide bonds [43]. Three JAM-related proteins, JAM-1, JAM-2, and JAM-3 are expressed in human BBB and previous studies have shown their participation in cell-to-cell adhesion and mono-

The AJs are established between neighboring cells by homophilic interactions between the transmembrane proteins, vascular endothelial cadherin (VE-cadherin), and epithelial cadherin (E-cadherin) in CNS [13]. Nearby to the basolateral membrane, AJ proteins, VE-cadherin, and platelet endothelial cell adhesion molecule (PECAM-1) form homophilic endothelial-to-endothelial contacts limit paracellular diffusion of solutes [13]. GJ are other junctional molecules, whose connexin-37 (CX37), CX40, and CX43 form hemichannels between endothelial cells [45]. These membrane proteins enable direct cytoplasmic exchange of ions and low molecular weight metabolites between adjacent cells; these channels of communications are essential for propagating electrical signals and coordination of cell signaling by transfer of second messengers [46]. Furthermore, brain endothelial GP

The major BBB transporters, receptors, and channels found in endothelial cells and pericytes have been validated by transcriptomic studies and protein analysis (**Figure 2**) [34]. Except for gases and small lipophilic molecules that freely diffuse across the endothelium, brain endothelial transport systems regulate molecular exchanges between blood and brain. The BBB's highly selective nature and the high metabolic demand of the brain demand other routes of entry for various nutrients to feed and nurture the brain [34]. Metabolic supply is achieved via several transporters expressed on the surface of CNS endothelial cells that drive the active transport of specific solutes and metabolites into the brain [47]. On the other hand, given the close proximity and highly interactive signaling between vascular pericytes and endothelial cells, it is relevant to describe in this chapter the BBB pericyte

**Endothelial carrier** enables solutes such as carbohydrates, amino acids (AA), monocarboxylic acids, hormones, fatty acids, nucleotides, inorganic ions, amines, choline, and vitamins to cross the BBB via substrate-specific transporters (**Figure 2**). In terms of carbohydrate transporters, GLUT1 (glucose transporter 1) is a uniporter that transports glucose. GLUT1 can transport glucose (and other hexoses) from either side of the luminal and abluminal endothelial membrane extracellularly or intracellularly [48]. Since glucose is lower in the brain interstitial fluid (ISF) than plasma, GLUT1 favors blood-to-brain transport of circulating glucose. GLUT1 is

**30**

transporter.

expressed in endothelial cells, but not in neurons. Their importance is best illustrated by the fact that transcript encoding GLUT1 is one of the most abundant transcripts in brain endothelium. Their dysfunction and lack cause barrier breakdown and can prevent clearance of amyloid plaques, suggesting a contributing role in Alzheimer's disease progression [49].

Regarding the transport of amino acids, all essential AA are transported into the brain across the BBB via endothelial AA transporter 1 and 2 (LAT1/2), that transport bidirectionally neutral AA such as tryptophan and tyrosine [50], and the cationic AA transporter 1 and 3 (CAT1/3) that transport cationic AA such as lysine and arginine [51]. Also, on the abluminal membrane transporters for excitatory AA (EAAT1/2/3) transport glutamate and aspartate out of the brain, limiting their excitotoxic effects on neurons [52]. Transporters of neutral and excitatory AA, such as glycine, taurine, and GABA are enriched abluminally and with highaffinity transport from brain to endothelium in a sodium dependent manner, and then, these AA are transported across the luminal membrane of the BBB into the blood via low-affinity transporters into the circulation [53]. Finally, essential fatty acids are essential for brain development and postnatal neural functions. The Brain endothelium expresses luminal transporters for fatty acids, including fatty acid transport protein 1 and 4 (FATP- 1/4) and the MFSD2A (Major Facilitator Superfamily Domain containing 2a) [54]. In the brain, MFSD2a is exclusively expressed in brain endothelium and is required for right BBB development and functional integrity. Finally, for Lactate released from skeletal muscles during exercise, and ketone bodies derived from liver from metabolism of fatty acids, the transport is facilitated by monocarboxylate transporter-1 (MCT1). Once inside the brain parenchyma, they are used as alternative energy metabolites by the brain, supply the brain with key substrates for DNA and RNA synthesis [54]. Nucleotides and nitrogenous base, e.g., cytosine, guanine, adenine, thymine and uracil, are all transported across the BBB via sodium-independent concentrative nucleoside transporter-2 (CNT2) and the sodium-independent equilibrative nucleoside transporter-1 and 2 (ENT1/2) [55].

**Endothelial receptor** is the most important transporter because proteins and large macromolecules (e.g., fibrinogen, immunoglobulins, thrombin, plasminogen, and growth factors) cannot cross the BBB. However, some proteins and peptides use receptor transport to cross the BBB and enter the brain (**Figure 2**). Transferrin receptor (TfR) [56], insulin receptor (IR) [57], and leptin receptor (LEP-R) [58] mediate blood-to-brain transport of transferrin (iron-protein carrier), insulin, and leptin across the BBB, respectively. This characteristic has promoted its use for CNS drug delivery, including therapeutic antibodies [59]. Receptors LRP1 and LRP2 are expressed in the BBB's brain endothelium, with LRP1 binding Alzheimer's soluble Ab fragments and mediating its brain-to-blood clearance [60].

**Endothelial active efflux and ion transport.** ATP-binding cassette (ABC) transporters utilize ATP as an energy source and are expressed at the luminal side of the BBB endothelium. They function to prevent brain accumulation of drugs, xenobiotics and macromolecules via active efflux from endothelium to blood [**Figure 2**]. Some examples are ABCB1 (also known as P-glycoprotein, P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated proteins (MRP). The BBB also has a significant role in regulating ions' concentration in the CNS. The luminal sodium pump, Na<sup>+</sup> K+ ATPase, is a key regulator of sodium influx into the brain and potassium efflux from the brain, maintaining high concentrations of Na<sup>+</sup> and low concentrations of K<sup>+</sup> in the brain, critical for the electrophysiological equilibrium of the resting membrane and action potentials [61].

Luminal Na-K- Cl (chloride) cotransporter (NKCC) mediates entry of Na<sup>+</sup> , K+ and 2Cl− from blood-to-endothelium. The bicarbonate (HCO3) − Cl exchanger mediates the entry of intracellular Cl<sup>−</sup> and the extracellular release of HCO3 − , regulating intracellular endothelial pH levels [62]. The Na+ -Ca2+ (sodium-calcium) exchanger cotransporter mediates Ca2+ efflux from endothelium into brain ISF, which maintains low intracellular Ca2+ levels in the microvascular endothelium [34]. Abluminal transient receptor potential (TRP) channels, also known as non- selective Ca2+ conducting cation channels, are expressed in both arterial endothelium and brain microvascular endothelial cell lines. TRP channels regulate Ca2 influx into brain endothelium, which in turn promotes the release of soluble factors such as NO, prostaglandins, and endothelial-derived hyperpolarizing factor initiating endothelium-dependent vasodilation [63]. BBB dysfunction also generates a leak of molecules across it, enabling considerable vascular fluid movement across the microvascular endothelium and the development of vasogenic edema [64]. Increased expression and activity of Na-K-Cl cotransporter (NKCC), sodiumhydrogen antiporter 1 and 2 (NHE1 and NHE2), and TRP channels promote the influx of Na<sup>+</sup> , and Cl− , generating a subsequent gradient osmotical that force the water movement across the BBB.

**Pericyte transporters.** Recent studies suggest that pericytes also express several transporters, receptors, and ion channels (**Figure 2**), such as carbohydrate transporters like the insulin-regulated glucose transporter GLUT4 and GLUT10 [65] and AA transporters, including the high-affinity excitatory AA transporter EAAT2, sodium-dependent neutral AA transporter SLC6A17, sodium and chloride dependent transporter SLC6A20 for small AA including glycine and proline, GABA transporter-1 and 2 (GAT1; GAT2), and the cationic AA transporter CAT2 [34, 65]. These transporters contribute to the removal of excitatory AA from the brain to prevent excitotoxicity, similar to endothelial transporters.

As endothelial cells, pericytes express lipoprotein receptor LRP1, mediating cellular uptake followed by its intracellular degradation and clearance [66] Pericytes regulate cerebrovascular integrity in an APOE-dependent way, inhibiting the proinflammatory CypA-MMP-9 pathway which prevents the degradation of BBB's TJ and basement membrane proteins [67]. These findings support that pericytes play an active role in regulating CBF and permeability of the BBB.

## **4. BBB dysfunction**

BBB's integrity is essential for the normal functioning of the nervous system. It comes as no surprise then that its disruption initiates and perpetuates several neurological pathophysiological events. Although the nature and extent of such changes vary from every condition, one key commonality is the breakdown of BBB and the detrimental functioning of the NVU [4]. The BBB prevents neurotoxic plasma components, blood cells, and pathogens from entering the brain (integrity of BBB). At the same time, the BBB regulates transport of molecules into and out of the central nervous system (CNS) (permeability of BBB). In cerebrovascular diseases, BBB breakdown and dysfunction leads to leakages of components into the CNS, contributing to neurological deficits [68].

The cells of the NVU are extremely sensitive to a number of different substances, including pro-inflammatory cytokines (e.g. IL-1, IL-6, TNF-α, interferon-γ), lipid mediators, oxidative compounds (free radical), vasogenic agents (e.g. glutamate, serotonin, histamine) and other endogenous stimuli (e.g. extracellular K+ and intracellular Ca2+). Many of these substances are released under pathophysiological conditions and changes of their levels in BBB is a critical event in the development and progression of CNS dysfunction [69]. In some cases, increased BBB permeability is a consequence of the pathology, such as with ischemic stroke

**33**

*Blood-Brain Barrier Dysfunction in the Detrimental Brain Function*

and traumatic brain injury, increased of intrinsic cellular proinflammatory, oxidative stress and dysregulation of vasogenic mediators, whereas in other cases BBB opening may be another condition in which cerebrovascular abnormalities have been noted, such as neurodegenerative disease [70]. As a result, there is a direct association between integrity impairment and high permeability of these substances in the brain. Some of the steps that follow include alteration or breakdown

In ischemic stroke, there is a sudden cessation of blood supply to the brain tissue, which translates into reduced oxygen and glucose delivery, both essential for ATP production. Depletion of ATP levels can lead to impaired functioning of Na/K-ATPase and Ca+2ATPase activity, generating ion- gradient failure and abnormal intracellular ion accumulation. By contrast, endothelial transporters' activity, such as Na/H ion-exchanger and Na-K-Cl cotransporter are stimulated. This second-

parenchyma, which results in characteristic cytotoxic edema secondary to ischemia [71]. The stimulation of this transporter's activity also triggers endothelial cell Na+ accumulation, generating swelling that contributes to BBB breakdown [72]. The Na+ cellular uptake depolarizes the cell's membrane, opening voltage-gated ion channels and promoting Ca2+ further cell uptake. These changes, in turn, prompt the release of excitatory neurotransmitters, which can be toxic [71]. BBB's breakdown in stroke occurs in a biphasic subacute fashion [70]. In the initial hit, activated metalloproteinases MMP-2 attack tight junction proteins. This activation is mediated by membrane-type MMP (MMP-14) and the fur gene expression, regulated by hypoxia-induced factor 1a (HIF-1a) [73]. Decreased expression and disorganization of tight junction constituent proteins, claudins, are the first signs of BBB damage, with further dysfunction of influx and efflux BBB transporters' expression. These

changes are limit the hypoxic area and revert after the acute insult [70].

rebral hemorrhage mechanisms are shown in **Figure 3**.

After 24 and 48 hours post-reperfusion, a non-reversible second phase takes place. Proinflammatory local cytokines activate inducible and freely available metalloproteinases MMP-3 and MMP-9, whose destructive activity characterizes this phase [74]. The most abundant cytokines present in focal cerebral ischemic areas are TNF-alfa and IL-1b [75] and have also been observed to decrease the expression of occludin and ZO-1 [76]. Cyclooxygenase-2 also plays a role in this second and more harmful opening of the BBB. Although this inflammation is local and mainly initiated by the activation of glia and pericytes, the BBB's damage and opening allow monocytes and neutrophils' entrance, perpetuating and amplifying the local inflammatory response [74]. The breakage of BBB in ischemic stroke is also the precursor of further complications such as the hemorrhagic transformation of the infarcted parenchyma [77]. A schematic view of ischemic stroke and intrace-

BBB can also be disrupted by the action of reactive oxygen species (ROS) and ensuing oxidative stress. Superoxide anion (O2−) is a known mediator of cellular damage after ischemic stroke. Under oxidative stress conditions such as stroke, superoxide dismutase's (SOD) metabolic capacity of controlling the biological activity of O2− gets surpassed. When combined with nitric oxide (NO), O2− forms peroxynitrite, a cytotoxic and proinflammatory molecule that can initiate and amplify BBB's injury by its ability to nitrosylate tyrosine and inducing endothelial damage [78]. Oxidative stress plays a critical role in ischemia/reperfusion (I/R)

, and water across to the barrier and into the brain

*DOI: http://dx.doi.org/10.5772/intechopen.94572*

of the physical, transport, and immune barriers.

**4.1 Alteration of BBB by cerebrovascular injury**

, Cl−

*4.1.1 Ischemic stroke*

arily induces increased Na+

and traumatic brain injury, increased of intrinsic cellular proinflammatory, oxidative stress and dysregulation of vasogenic mediators, whereas in other cases BBB opening may be another condition in which cerebrovascular abnormalities have been noted, such as neurodegenerative disease [70]. As a result, there is a direct association between integrity impairment and high permeability of these substances in the brain. Some of the steps that follow include alteration or breakdown of the physical, transport, and immune barriers.
