Mechanisms of Endothelial Injury and Blood–Brain Barrier Dysfunction in Stroke

Introduction

The blood–brain barrier (BBB) is an essential physical and biochemical barrier that separates the CNS from the systemic circulation. It is formed by a monolayer of capillary endothelial cells that interact with each other as well as with other components of the neurovascular unit (i.e., astrocytes, microglia, neurons, pericytes, extracellular matrix) to precisely maintain CNS homeostasis. In addition, the BBB restricts brain permeation of xenobiotics in an effort to reduce the probability of CNS toxicity. During ischemic stroke, brain microvascular endothelial cells are triggered by pathophysiological stimuli [i.e., reactive oxygen species (ROS), inflammatory mediators], leading to disruption of tight junction (TJ) protein complexes and subsequent barrier opening. This process is clinically significant because BBB dysfunction is well known to contribute to deleterious events following stroke, such as intracerebral hemorrhage and vasogenic edema. Understanding cellular responses in the setting of stroke provides an opportunity to develop novel pharmacological approaches that promote BBB repair by modulating endothelial injury.

Disruption of the Blood–Brain Barrier in Ischemic Stroke

Considerable research has demonstrated that BBB dysfunction in stroke is biphasic in nature. To this end, acute breakdown of the BBB is demarcated by an immediate enhancement in solute permeability observed at approximately 4–6 h after an ischemic insult followed by a second barrier opening that occurs 2–3 days after stroke . The onset of BBB dysfunction directly corresponds to the severity of ischemia . Acute BBB breakdown involves four distinct processes: (1) endothelial swelling, (2) endothelial membrane disruption, (3) disruption of TJ protein complexes between adjacent endothelial cells, and (4) total vascular dysfunction . Perturbation of extracellular matrix (i.e., type IV collagen, heparan sulfate proteoglycan, laminin, fibronectin, perlecan) is prominently involved in this acute BBB dysfunction. Activation of proteinases, including matrix metalloproteinases (MMPs), is a critical component of early breakdown of extracellular matrix and subsequent BBB dysfunction in stroke . Involvement of MMPs in BBB dysfunction has been demonstrated in in vivo experimental stroke models. Furthermore, elevation of MMP9 levels has been observed in patients diagnosed with acute ischemic stroke . MMPs degrade the extracellular matrix that comprises the basal lamina and directly compromises the BBB by degradation of TJ constituent proteins (i.e., claudin-5, occludin). In addition, MMP-mediated opening of the BBB in ischemic stroke is associated with activation of endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS), leading to an enhancement in nitric oxide (NO) signaling.

Experimental models of focal cerebral ischemia have provided essential information on solute leak across the BBB in the context of stroke. Using the transient middle cerebral artery occlusion (MCAO) rodent model, increased leak of sucrose, a vascular marker that does not typically cross the BBB, was demonstrated in the ischemic hemisphere but not in the contralateral hemisphere . BBB disruption following an ischemic insult is significant and can allow blood-to-brain leak of large molecules, such as Evans blue-albumin . Evans blue dye, when unconjugated to plasma proteins, is a relatively small molecule with a molecular weight of 960.8 Da. In vivo, Evans blue dye irreversibly binds to serum albumin causing formation of a very large, solute–protein complex (i.e., in excess of 60,000 Da) that can only traverse the BBB under significant pathological stress as is known to occur following focal ischemia. Of particular note, Evan’s blue-albumin leak following experimental ischemic stroke was directly correlated with redistribution of critical TJ proteins occludin, claudin-5, and zonula occludens (ZO)-1 .

Reorganization of TJ protein complexes and associated leak across the BBB enables vascular fluid (i.e., water) to readily move across the microvascular endothelium, which leads to the development of vasogenic edema. MCAO studies have shown that enhanced blood-to-brain movement of sodium exacerbates water movement across the BBB. This change in the sodium gradient across the microvascular endothelium is facilitated by increased functional expression of the Na–K–Cl cotransporter as well as Na–H exchangers NHE1 and/or NHE2 . Disruption of sodium gradients across the BBB during ischemic stroke can also involve upregulation of sodium-dependent glucose transporters, such as sodium-glucose cotransporter (SGLT). Specifically, pharmacological inhibition of SGLT in MCAO rats significantly reduced infarct and edema ratios, which implies that this transporter may be a critical determinant of stroke outcome .