The role of shear stress in Blood-Brain Barrier endothelial physiology

<p>Abstract</p> <p>Background</p> <p>One of the most important and often neglected physiological stimuli contributing to the differentiation of vascular endothelial cells (ECs) into a blood-brain barrier (BBB) phenotype is shear stress (SS). With the use of a well established humanized dynamic <it>in vitro </it>BBB model and cDNA microarrays, we have profiled the effect of SS in the induction/suppression of ECs genes and related functions.</p> <p>Results</p> <p>Specifically, we found a significant upregulation of tight and adherens junctions proteins and genes. Trans-endothelial electrical resistance (TEER) and permeability measurements to know substances have shown that SS promoted the formation of a tight and highly selective BBB. SS also increased the RNA level of multidrug resistance transporters, ion channels, and several p450 enzymes. The RNA level of a number of specialized carrier-mediated transport systems (e.g., glucose, monocarboxylic acid, etc.) was also upregulated.</p> <p>RNA levels of modulatory enzymes of the glycolytic pathway (e.g., lactate dehydrogenase) were downregulated by SS while those involved in the Krebs cycle (e.g., lactate and other dehydrogenases) were upregulated. Measurements of glucose consumption versus lactate production showed that SS negatively modulated the glycolytic bioenergetic pathways of glucose metabolism in favor of the more efficient aerobic respiration. BBB ECs are responsive to inflammatory stimuli. Our data showed that SS increased the RNA levels of integrins and vascular adhesion molecules. SS also inhibited endothelial cell cycle via regulation of BTG family proteins encoding genes. This was paralleled by significant increase in the cytoskeletal protein content while that of membrane, cytosol, and nuclear sub-cellular fractions decreased. Furthermore, analysis of 2D gel electrophoresis (which allows identifying a large number of proteins per sample) of EC proteins extracted from membrane sub-cellular endothelial fractions showed that SS increased the expression levels of tight junction proteins. In addition, regulatory enzymes of the Krebb's cycle (aerobic glucose metabolism) were also upregulated. Furthermore, the expression pattern of key protein regulators of the cell cycle and parallel gene array data supported a cell proliferation inhibitory role for SS.</p> <p>Conclusions</p> <p>Genomic and proteomic analyses are currently used to examine BBB function in healthy and diseased brain and characterize this dynamic interface. In this study we showed that SS plays a key role in promoting the differentiation of vascular endothelial cells into a truly BBB phenotype. SS affected multiple aspect of the endothelial physiology spanning from tight junctions formation to cell division as well as the expression of multidrug resistance transporters. BBB dysfunction has been observed in many neurological diseases, but the causes are generally unknown. Our study provides essential insights to understand the role played by SS in the BBB formation and maintenance.</p

The Etiological Role of Blood-Brain Barrier Dysfunction in Seizure Disorders

A wind of change characterizes epilepsy research efforts. The traditional approach, based on a neurocentric view of seizure generation, promoted understanding of the neuronal mechanisms of seizures; this resulted in the development of potent anti-epileptic drugs (AEDs). The fact that a significant number of individuals with epilepsy still fail to respond to available AEDs restates the need for an alternative approach. Blood-brain barrier (BBB) dysfunction is an important etiological player in seizure disorders, and combination therapies utilizing an AED in conjunction with a “cerebrovascular” drug could be used to control seizures more effectively than AED therapy alone. The fact that the BBB plays an etiologic role in other neurological diseases will be discussed in the context of a more “holistic” approach to the patient with epilepsy, where comorbidity variables are also encompassed by drug therapy

Efficacy of Anti-Inflammatory Therapy in a Model of Acute Seizures and in a Population of Pediatric Drug Resistant Epileptics

Targeting pro-inflammatory events to reduce seizures is gaining momentum. Experimentally, antagonism of inflammatory processes and of blood-brain barrier (BBB) damage has been demonstrated to be beneficial in reducing status epilepticus (SE). Clinically, a role of inflammation in the pathophysiology of drug resistant epilepsies is suspected. However, the use anti-inflammatory drug such as glucocorticosteroids (GCs) is limited to selected pediatric epileptic syndromes and spasms. Lack of animal data may be one of the reasons for the limited use of GCs in epilepsy. We evaluated the effect of the CG dexamethasone in reducing the onset and the severity of pilocarpine SE in rats. We assessed BBB integrity by measuring serum S100β and Evans Blue brain extravasation. Electrophysiological monitoring and hematologic measurements (WBCs and IL-1β) were performed. We reviewed the effect of add on dexamethasone treatment on a population of pediatric patients affected by drug resistant epilepsy. We excluded subjects affected by West, Landau-Kleffner or Lennox-Gastaut syndromes and Rasmussen encephalitis, known to respond to GCs or adrenocorticotropic hormone (ACTH). The effect of two additional GCs, methylprednisolone and hydrocortisone, was also reviewed in this population. When dexamethasone treatment preceded exposure to the convulsive agent pilocarpine, the number of rats developing status epilepticus (SE) was reduced. When SE developed, the time-to-onset was significantly delayed compared to pilocarpine alone and mortality associated with pilocarpine-SE was abolished. Dexamethasone significantly protected the BBB from damage. The clinical study included pediatric drug resistant epileptic subjects receiving add on GC treatments. Decreased seizure frequency (≥50%) or interruption of status epilepticus was observed in the majority of the subjects, regardless of the underlying pathology. Our experimental results point to a seizure-reducing effect of dexamethasone. The mechanism encompasses improvement of BBB integrity. Our results also suggest that add on GCs could be of efficacy in controlling pediatric drug resistant seizures

Significance of ubiquitin carboxy-terminal hydrolase L1 elevations in athletes after sub-concussive head hits.

The impact of sub-concussive head hits (sub-CHIs) has been recently investigated in American football players, a population at risk for varying degrees of post-traumatic sequelae. Results show how sub-CHIs in athletes translate in serum as the appearance of reporters of blood-brain barrier disruption (BBBD), how the number and severity of sub-CHIs correlate with elevations of putative markers of brain injury is unknown. Serum brain injury markers such as UCH-L1 depend on BBBD. We investigated the effects of sub-CHIs in collegiate football players on markers of BBBD, markers of cerebrospinal fluid leakage (serum beta 2-transferrin) and markers of brain damage. Emergency room patients admitted for a clinically-diagnosed mild traumatic brain injury (mTBI) were used as positive controls. Healthy volunteers were used as negative controls. Specifically this study was designed to determine the use of UCH-L1 as an aid in the diagnosis of sub-concussive head injury in athletes. The extent and intensity of head impacts and serum values of S100B, UCH-L1, and beta-2 transferrin were measured pre- and post-game from 15 college football players who did not experience a concussion after a game. S100B was elevated in players experiencing the most sub-CHIs; UCH-L1 levels were also elevated but did not correlate with S100B or sub-CHIs. Beta-2 transferrin levels remained unchanged. No correlation between UCH-L1 levels and mTBI were measured in patients. Low levels of S100B were able to rule out mTBI and high S100B levels correlated with TBI severity. UCH-L1 did not display any interpretable change in football players or in individuals with mild TBI. The significance of UCH-L1 changes in sub-concussions or mTBI needs to be further elucidated

Is peripheral immunity regulated by blood-brain barrier permeability changes?

S100B is a reporter of blood-brain barrier (BBB) integrity which appears in blood when the BBB is breached. Circulating S100B derives from either extracranial sources or release into circulation by normal fluctuations in BBB integrity or pathologic BBB disruption (BBBD). Elevated S100B matches the clinical presence of indices of BBBD (gadolinium enhancement or albumin coefficient). After repeated sub-concussive episodes, serum S100B triggers an antigen-driven production of anti-S100B autoantibodies. We tested the hypothesis that the presence of S100B in extracranial tissue is due to peripheral cellular uptake of serum S100B by antigen presenting cells, which may induce the production of auto antibodies against S100B. To test this hypothesis, we used animal models of seizures, enrolled patients undergoing repeated BBBD, and collected serum samples from epileptic patients. We employed a broad array of techniques, including immunohistochemistry, RNA analysis, tracer injection and serum analysis. mRNA for S100B was segregated to barrier organs (testis, kidney and brain) but S100B protein was detected in immunocompetent cells in spleen, thymus and lymph nodes, in resident immune cells (Langerhans, satellite cells in heart muscle, etc.) and BBB endothelium. Uptake of labeled S100B by rat spleen CD4+ or CD8+ and CD86+ dendritic cells was exacerbated by pilocarpine-induced status epilepticus which is accompanied by BBBD. Clinical seizures were preceded by a surge of serum S100B. In patients undergoing repeated therapeutic BBBD, an autoimmune response against S100B was measured. In addition to its role in the central nervous system and its diagnostic value as a BBBD reporter, S100B may integrate blood-brain barrier disruption to the control of systemic immunity by a mechanism involving the activation of immune cells. We propose a scenario where extravasated S100B may trigger a pathologic autoimmune reaction linking systemic and CNS immune responses

Lack of significant correlation between serum UCH-L1 levels and a diagnosis of mild TBI.

<p><b>A</b>) S100B levels are significantly elevated in patients with a diagnosis of mild TBI compared to healthy controls (<i>p</i><0.01) and, <b>B</b>) S100B levels correlate with post-traumatic findings on head CT (<i>p</i><0.01). <b>C</b>) <b>and D</b>) UCH-L1 levels remain unchanged in patients with a diagnosis of mTBI compared to controls as well as in those patients with positive findings on head CT. Significance is determined by ANOVA and denoted as (*) for p<0.05.</p

S100B serum surges correlate with the extent and number of head hits, while UCH-L1 does not appear to correlate with sub-concussive head hits.

<p>Mean percent change values of S100B and UCH-L1 levels (see equation (1)) were plotted against Head Hit Index (HHI) scores (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096296#s2" target="_blank">Methods</a>). A statistically significant difference (by Wilcoxon Mann Whitney) was found between S100B surges at HHI of 0 and HHI of 1 and 2. UCH-L1 did not correlate with any of the HHI used for this study, and the levels of UCH-L1 measured after the game and normalized for their pre-game value as in (1) did not discriminate between a HHI of 0 (special team or played without any head hit) and any HHI (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096296#pone.0096296-Marchi2" target="_blank">[10]</a> for details on HHI). Statistical differences were analyzed by the Wilcoxon Mann Whitney test, both in the comparison between S100B and UCH-L1 at a particular HHI and in the comparison of the individual marker at a HHI 0 vs. HHI 1, 2, 3, and 4,6. To correct for type 1 error in multiple comparisons the Dunnett’s correction was used. Significance is shown as (*) for p<0.05; and <i>n.s.</i> for not significant.</p

Markers’ change following a game confirms lack of correlation between S100B and UCH-L1.

<p><b>A–B</b>) refers to normalized serum surges of S100B and UCH-L1 after game 1 and game 2. Note lack of correlation between these markers (<i>p</i> = 0.19; R² = 0.37; game 1, <i>p</i> = 0.11; R² =  −0.45; game 2). The <i>red line</i> is the linear regression fit while the outer lines show confidence intervals of 95%. We measured the normalized percent change in each game in an attempt to correct for the possibility of different measuring sensitivities for subconcussive head hits for the two markers measured by ELISA. Significance is determined by ANOVA and denoted as (*) for p<0.05.</p