Vikram Puvenna

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

BackgroundOne 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 in vitro BBB model and cDNA microarrays, we have profiled the effect of SS in the induction/suppression of ECs genes and related functions.ResultsSpecifically, 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.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 Krebbs 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.ConclusionsGenomic 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.

Consequences of repeated blood-brain barrier disruption in football players

The acknowledgement of risks for traumatic brain injury in American football players has prompted studies for sideline concussion diagnosis and testing for neurological deficits. While concussions are recognized etiological factors for a spectrum of neurological sequelae, the consequences of sub-concussive events are unclear. We tested the hypothesis that blood-brain barrier disruption (BBBD) and the accompanying surge of the astrocytic protein S100B in blood may cause an immune response associated with production of auto-antibodies. We also wished to determine whether these events result in disrupted white matter on diffusion tensor imaging (DT) scans. Players from three college football teams were enrolled (total of 67 volunteers). None of the players experienced a concussion. Blood samples were collected before and after games (n = 57); the number of head hits in all players was monitored by movie review and post-game interviews. S100B serum levels and auto-antibodies against S100B were measured and correlated by direct and reverse immunoassays (n = 15 players; 5 games). A subset of players underwent DTI scans pre- and post-season and after a 6-month interval (n = 10). Cognitive and functional assessments were also performed. After a game, transient BBB damage measured by serum S100B was detected only in players experiencing the greatest number of sub-concussive head hits. Elevated levels of auto-antibodies against S100B were elevated only after repeated sub-concussive events characterized by BBBD. Serum levels of S100B auto-antibodies also predicted persistence of MRI-DTI abnormalities which in turn correlated with cognitive changes. Even in the absence of concussion, football players may experience repeated BBBD and serum surges of the potential auto-antigen S100B. The correlation of serum S100B, auto-antibodies and DTI changes support a link between repeated BBBD and future risk for cognitive changes.

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.

Pathophysiological impact of cigarette smoke exposure on the cerebrovascular system with a focus on the blood-brain barrier: Expanding the awareness of smoking toxicity in an underappreciated area

Recent evidence has indicated that active and passive cigarette smoking are associated, in a dose-dependent manner, with dysfunction of normal endothelial physiology. Tobacco smoke (TS) may predispose individuals to atherogenic and thrombotic problems, significantly increasing the risk for ischemic manifestations such as acute coronary syndrome and stroke. Despite the strong evidence for an association between smoking and vascular impairment, the impact of TS exposure on the blood-brain barrier (BBB) has only been marginally addressed. This is a major problem given that the BBB is crucial in the maintenance of brain homeostasis. Recent data have also shown that chronic smokers have a higher incidence of small vessel ischemic disease (SVID), a pathological condition characterized by leaky brain microvessels and loss of BBB integrity. In the brain TS increases the risk of silent cerebral infarction (SCI) and stroke owing to the pro-coagulant and atherogenic effects of smoking. In this article we provide a detailed review and analysis of current knowledge of the pathophysiology of tobacco smoke toxicity at the cerebrovascular levels. We also discuss the potential toxicity of recently marketed “potential-reduced exposure products”.

Cellular localization and functional significance of CYP3A4 in the human epileptic brain

Purpose:  Compelling evidence supports the presence of P450 enzymes (CYPs) in the central nervous system (CNS). However, little information is available on the localization and function of CYPs in the drug‐resistant epileptic brain. We have evaluated the pattern of expression of the specific enzyme CYP3A4 and studied its co‐localization with MDR1. We also determined whether an association exists between CYP3A4 expression and cell survival.

Modulation of peripheral cytotoxic cells and ictogenesis in a model of seizures

Purpose:  A link between seizure susceptibility, blood–brain barrier (BBB) failure, and the activation of peripheral white blood cells has been recently proposed. However, the molecular players involved in this cascade of events are unknown. We tested the hypothesis that immunosupression by splenectomy or lack of perforin, a downstream factor of natural killer (NK) and cytotoxic T cells, could reduce seizure onset.

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.

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 phosphorylated tau unique to chronic traumatic encephalopathy? Phosphorylated tau in epileptic brain and chronic traumatic encephalopathy.

Repetitive traumatic brain injury (rTBI) is one of the major risk factors for the abnormal deposition of phosphorylated tau (PT) in the brain and chronic traumatic encephalopathy (CTE). CTE and temporal lobe epilepsy (TLE) affect the limbic system, but no comparative studies on PT distribution in TLE and CTE are available. It is also unclear whether PT pathology results from repeated head hits (rTBI). These gaps prevent a thorough understanding of the pathogenesis and clinical significance of PT, limiting our ability to develop preventative and therapeutic interventions. We quantified PT in TLE and CTE to unveil whether a history of rTBI is a prerequisite for PT accumulation in the brain. Six postmortem CTE (mean 73.3 years) and age matched control samples were compared to 19 surgically resected TLE brain specimens (4 months-58 years; mean 27.6 years). No history of TBI was present in TLE or control; all CTE patients had a history of rTBI. TLE and CTE brain displayed increased levels of PT as revealed by immunohistochemistry. No age-dependent changes were noted, as PT was present as early as 4 months after birth. In TLE and CTE, cortical neurons, perivascular regions around penetrating pial vessels and meninges were immunopositive for PT; white matter tracts also displayed robust expression of extracellular PT organized in bundles parallel to venules. Microscopically, there were extensive tau-immunoreactive neuronal, astrocytic and degenerating neurites throughout the brain. In CTE perivascular tangles were most prominent. Overall, significant differences in staining intensities were found between CTE and control (P<0.01) but not between CTE and TLE (P=0.08). pS199 tau analysis showed that CTE had the most high molecular weight tangle-associated tau, whereas epileptic brain contained low molecular weight tau. Tau deposition may not be specific to rTBI since TLE recapitulated most of the pathological features of CTE.

Expression and functional relevance of UGT1A4 in a cohort of human drug-resistant epileptic brains

Brain drug bioavailability is regulated by the blood–brain barrier (BBB). It was recently suggested that cytochrome P450 (CYP) enzymes could act in concert with multidrug transporter proteins to regulate drug penetration and distribution into the diseased brain. The possibility that phase II metabolic enzymes could be expressed in the epileptic brain has been not evaluated. Phase II enzymes are involved in the metabolism of common antiepileptic drugs (AEDs).