The role of the blood-brain barrier during neurological disease and infection.

A healthy brain is protected by the blood-brain barrier (BBB), which is formed by the endothelial cells that line brain capillaries. The BBB plays an extremely important role in supporting normal neuronal function by maintaining the homeostasis of the brain microenvironment and restricting pathogen and toxin entry to the brain. Dysfunction of this highly complex and regulated structure can be life threatening. BBB dysfunction is implicated in many neurological diseases such as stroke, Alzheimer's disease, multiple sclerosis, and brain infections. Among other mechanisms, inflammation and/or flow disturbances are major causes of BBB dysfunction in neurological infections and diseases. In particular, in ischaemic stroke, both inflammation and flow disturbances contribute to BBB disruption, leading to devastating consequences. While a transient or minor disruption to the barrier function could be tolerated, chronic or a total breach of the barrier can result in irreversible brain damage. It is worth noting that timing and extent of BBB disruption play an important role in the process of any repair of brain damage and treatment strategies. This review evaluates and summarises some of the latest research on the role of the BBB during neurological disease and infection with a focus on the effects of inflammation and flow disturbances on the BBB. The BBB's crucial role in protecting the brain is also the bottleneck in central nervous system drug development. Therefore, innovative strategies to carry therapeutics across the BBB and novel models to screen drugs, and to study the complex, overlapping mechanisms of BBB disruption are urgently needed.

Diagnostic biomarker kinetics: how brain-derived biomarkers distribute through the human body, and how this affects their diagnostic significance: the case of S100B.

Blood biomarkers of neurological diseases are often employed to rule out or confirm the presence of significant intracranial or cerebrovascular pathology or for the differential diagnosis of conditions with similar presentations (e.g., hemorrhagic vs. embolic stroke). More widespread utilization of biomarkers related to brain health is hampered by our incomplete understanding of the kinetic properties, release patterns, and excretion of molecules derived from the brain. This is, in particular, true for S100B, an astrocyte-derived protein released across the blood-brain barrier (BBB). We developed an open-source pharmacokinetic computer model that allows investigations of biomarker's movement across the body, the sources of biomarker's release, and its elimination. This model was derived from a general in silico model of drug pharmacokinetics adapted for protein biomarkers. We improved the model's predictive value by adding realistic blood flow values, organ levels of S100B, lymphatic and glymphatic circulation, and glomerular filtration for excretion in urine. Three key variables control biomarker levels in blood or saliva: blood-brain barrier permeability, the S100B partition into peripheral organs, and the cellular levels of S100B in astrocytes. A small contribution to steady-state levels of glymphatic drainage was also observed; this mechanism also contributed to the uptake of organs of circulating S100B. This open-source model can also mimic the kinetic behavior of other markers, such as GFAP or NF-L. Our results show that S100B, after uptake by various organs from the systemic circulation, can be released back into systemic fluids at levels that do not significantly affect the clinical significance of venous blood or salivary levels after an episode of BBB disruption.

GFAP and S100B: What You Always Wanted to Know and Never Dared to Ask.

Traumatic brain injury (TBI) is a major global health issue, with outcomes spanning from intracranial bleeding, debilitating sequelae, and invalidity with consequences for individuals, families, and healthcare systems. Early diagnosis of TBI by testing peripheral fluids such as blood or saliva has been the focus of many research efforts, leading to FDA approval for a bench-top assay for blood GFAP and UCH-L1 and a plasma point-of-care test for GFAP. The biomarker S100B has been included in clinical guidelines for mTBI (mTBI) in Europe. Despite these successes, several unresolved issues have been recognized, including the robustness of prior data, the presence of biomarkers in tissues beyond the central nervous system, and the time course of biomarkers in peripheral body fluids. In this review article, we present some of these issues and provide a viewpoint derived from an analysis of existing literature. We focus on two astrocytic proteins, S100B and GFAP, the most commonly employed biomarkers used in mTBI. We also offer recommendations that may translate into a broader acceptance of these clinical tools.

Hypoxemia increases blood-brain barrier permeability during extreme apnea in humans.

Voluntary asphyxia imposed by static apnea challenges blood-brain barrier (BBB) integrity in humans through transient extremes of hypertension, hypoxemia and hypercapnia. In the present study, ten ultra-elite breath-hold divers performed two maximal dry apneas preceded by normoxic normoventilation (NX: severe hypoxemia and hypercapnia) and hyperoxic hyperventilation (HX: absence of hypoxemia with exacerbating hypercapnia) with measurements obtained before and immediately after apnea. Transcerebral exchange of NVU proteins (ELISA, Single Molecule Array) were calculated as the product of global cerebral blood flow (gCBF, duplex ultrasound) and radial arterial to internal jugular venous concentration gradients. Apnea duration increased from 5 m 6 s in NX to 15 m 59 s in HX (P = <0.001) resulting in marked elevations in gCBF and venous S100B, glial fibrillary acidic protein, ubiquitin carboxy-terminal hydrolase-L1 and total tau (all P < 0.05 vs. baseline). This culminated in net cerebral output reflecting mildly increased BBB permeability and increased neuronal-gliovascular reactivity that was more pronounced in NX due to more severe systemic and intracranial hypertension (P < 0.05 vs. HX). These findings identify the hemodynamic stress to which the apneic brain is exposed, highlighting the critical contribution of hypoxemia and not just hypercapnia to BBB disruption.

Inflammation in pediatric epilepsies: Update on clinical features and treatment options.

The role of inflammation is increasingly recognized in triggering or sustaining epileptic activity. In the last decades, increasing research has provided definite evidence to support the link between immunity, inflammatory process, and epilepsy. Neuro- and systemic inflammation play a pivotal role in driving epileptogenesis through different pathogenetic mechanisms: the activation of innate immunity in glia, neurons, and microvasculature, the brain mediated by blood-brain barrier (BBB) impairment, and the imbalance of pro- and anti-inflammatory molecules produced by both arms of immunity. More recently, research has focused on the adverse effects of maternal or early-life immune activation and cytokine imbalance on fetal neurodevelopment and postnatal epilepsy. A complex crosstalk between the immune and nervous system, and a crucial interplay of genetic, epigenetic, and environmental factors may influence structures and functions of the developing brain. A better understanding of the inflammatory process in promoting epilepsy implies that targeting specific pathways may be effective in seizure control. Multiple targets have been identified so far, and several antiseizure interventions are obtained by inhibiting inflammatory signaling or protecting/restoring BBB. All this evidence has changed the field of epilepsy research and neuropharmacology. Further developments and new treatments will rapidly emerge to improve seizure management in inflammation-related epilepsies. This article is part of the Special Issue "Severe Infantile Epilepsies".

Brain dysfunction in COVID-19 and CAR-T therapy: cytokine storm-associated encephalopathy.

OBJECTIVE: Many neurological manifestations are associated with COVID-19, including a distinct form of encephalopathy related to cytokine storm, the acute systemic inflammatory syndrome present in a subgroup of COVID-19 patients. Cytokine storm is also associated with immune effector cell-associated neurotoxicity syndrome (ICANS), a complication of chimeric antigen receptor T-cell (CAR-T) therapy, a highly effective treatment for refractory hematological malignancies. We investigated whether COVID-19-related encephalopathy, ICANS, and other encephalopathies associated with cytokine storm, share clinical and investigative findings. METHODS: Narrative literature review. RESULTS: Comparisons between COVID-19-related encephalopathy and ICANS revealed several overlapping features. Clinically, these included dysexecutive syndrome, language disturbances, akinetic mutism and delirium. EEG showed a prevalence of frontal abnormalities. Brain MRI was often unrevealing. CSF elevated cytokine levels have been reported. A direct correlation between cytokine storm intensity and severity of neurological manifestations has been shown for both conditions. Clinical recovery occurred spontaneously or following immunotherapies in most of the patients. Similar clinical and investigative features were also reported in other encephalopathies associated with cytokine storm, such as hemophagocytic lymphohistiocytosis, sepsis, and febrile infection-associated encephalopathies. INTERPRETATION: COVID-19-related encephalopathy and ICANS are characterized by a predominant electro-clinical frontal lobe dysfunction and share several features with other encephalopathies associated with cytokine storm, which may represent the common denominator of a clinical spectrum of neurological disorders. Therefore, we propose a unifying definition of cytokine storm-associated encephalopathy (CySE), and its diagnostic criteria.

Is Salivary S100B a Biomarker of Traumatic Brain Injury? A Pilot Study.

Traumatic brain injury (TBI) results in short and long-term disability neurodegeneration. Mild traumatic brain injury (mTBI) represents up to 85% of head injuries; diagnosis and early management is based on computed tomography (CT) or in-hospital observation, which are time- and cost- intensive. CT involves exposure to potentially harmful ionizing radiation and >90% of the scans are negative. Blood-brain barrier (BBB) damage is suspected pathological event post-TBI contributing to long-term sequelae and a reliable and rapid point-of-care test to screen those who can safely forego acute head CT would be of great help in evaluating patients with an acute mTBI. In this pilot study, 15 adult patients with suspected TBI (mean age = 47 years, range 18-79) and 15 control subjects (mean age = 33 years, range 23-53) were enrolled. We found that the average salivary S100B level was 3.9 fold higher than blood S100B, regardless of the presence of pathology. [S100B]saliva positively correlated with [S100B]serum (Pearson' coefficient = 0.79; p < 0.01). Salivary S100B levels were as effective in differentiating TBI patients from control subjects as serum levels (Control vs. TBI: p < 0.01; Serum ROCAUC = 0.94 and Saliva ROCAUC = 0.75). I These initial results suggest that measuring salivary S100B could represent an alternative to serum S100B in the diagnosis of TBI. Larger and confirmatory trials are needed to define salivary biomarker kinetics in relation to TBI severity and the possible roles of gender, ethnicity and age in influencing salivary S100B levels.

Peripheral Blood and Salivary Biomarkers of Blood-Brain Barrier Permeability and Neuronal Damage: Clinical and Applied Concepts.

Within the neurovascular unit (NVU), the blood-brain barrier (BBB) operates as a key cerebrovascular interface, dynamically insulating the brain parenchyma from peripheral blood and compartments. Increased BBB permeability is clinically relevant for at least two reasons: it actively participates to the etiology of central nervous system (CNS) diseases, and it enables the diagnosis of neurological disorders based on the detection of CNS molecules in peripheral body fluids. In pathological conditions, a suite of glial, neuronal, and pericyte biomarkers can exit the brain reaching the peripheral blood and, after a process of filtration, may also appear in saliva or urine according to varying temporal trajectories. Here, we specifically examine the evidence in favor of or against the use of protein biomarkers of NVU damage and BBB permeability in traumatic head injury, including sport (sub)concussive impacts, seizure disorders, and neurodegenerative processes such as Alzheimer's disease. We further extend this analysis by focusing on the correlates of human extreme physiology applied to the NVU and its biomarkers. To this end, we report NVU changes after prolonged exercise, freediving, and gravitational stress, focusing on the presence of peripheral biomarkers in these conditions. The development of a biomarker toolkit will enable minimally invasive routines for the assessment of brain health in a broad spectrum of clinical, emergency, and sport settings.

In vitro Models of the Blood-Brain Barrier: Tools in Translational Medicine.

Medical progress has historically depended on scientific discoveries. Until recently, science was driven by technological advancements that, once translated to the clinic, fostered new treatments and interventions. More recently, technology-driven medical progress has often outpaced laboratory research. For example, intravascular devices, pacemakers for the heart and brain, spinal cord stimulators, and surgical robots are used routinely to treat a variety of diseases. The rapid expansion of science into ever more advanced molecular and genetic mechanisms of disease has often distanced laboratory-based research from day-to-day clinical realities that remain based on evidence and outcomes. A recognized reason for this hiatus is the lack of laboratory tools that recapitulate the clinical reality faced by physicians and surgeons. To overcome this, the NIH and FDA have in the recent past joined forces to support the development of a "human-on-a-chip" that will allow research scientists to perform experiments on a realistic replica when testing the effectiveness of novel experimental therapies. The development of a "human-on-a-chip" rests on the capacity to grow in vitro various organs-on-a-chip, connected with appropriate vascular supplies and nerves, and our ability to measure and perform experiments on these virtually invisible organs. One of the tissue structures to be scaled down on a chip is the human blood-brain barrier. This review gives a historical perspective on in vitro models of the BBB and summarizes the most recent 3D models that attempt to fill the gap between research modeling and patient care. We also present a summary of how these in vitro models of the BBB can be applied to study human brain diseases and their treatments. We have chosen NeuroAIDS, COVID-19, multiple sclerosis, and Alzheimer's disease as examples of in vitro model application to neurological disorders. Major insight pertaining to these illnesses as a consequence of more profound understanding of the BBB can reveal new avenues for the development of diagnostics, more efficient therapies, and definitive clarity of disease etiology and pathological progression.

Cerebrospinal fluid dynamics and intracranial pressure elevation in neurological diseases.

The fine balance between the secretion, composition, volume and turnover of cerebrospinal fluid (CSF) is strictly regulated. However, during certain neurological diseases, this balance can be disrupted. A significant disruption to the normal CSF circulation can be life threatening, leading to increased intracranial pressure (ICP), and is implicated in hydrocephalus, idiopathic intracranial hypertension, brain trauma, brain tumours and stroke. Yet, the exact cellular, molecular and physiological mechanisms that contribute to altered hydrodynamic pathways in these diseases are poorly defined or hotly debated. The traditional views and concepts of CSF secretion, flow and drainage have been challenged, also due to recent findings suggesting more complex mechanisms of brain fluid dynamics than previously proposed. This review evaluates and summarises current hypotheses of CSF dynamics and presents evidence for the role of impaired CSF dynamics in elevated ICP, alongside discussion of the proteins that are potentially involved in altered CSF physiology during neurological disease. Undoubtedly CSF secretion, absorption and drainage are important aspects of brain fluid homeostasis in maintaining a stable ICP. Traditionally, pharmacological interventions or CSF drainage have been used to reduce ICP elevation due to over production of CSF. However, these drugs are used only as a temporary solution due to their undesirable side effects. Emerging evidence suggests that pharmacological targeting of aquaporins, transient receptor potential vanilloid type 4 (TRPV4), and the Na+-K+-2Cl- cotransporter (NKCC1) merit further investigation as potential targets in neurological diseases involving impaired brain fluid dynamics and elevated ICP.

Breakdown of blood brain barrier as a mechanism of post-traumatic epilepsy.

Traumatic brain injury (TBI) accounts for approximately 16% of acute symptomatic seizures which usually occur in the first week after trauma. Children are at higher risk for post-traumatic seizures than adults. Post-traumatic seizures are a risk factor for delayed development of epilepsy. Delayed, chronic post-traumatic epilepsy is preceded by a silent period during which therapeutic interventions may arrest, revert or prevent epileptogenesis. A number of recent review articles summarize the most important features of post-traumatic seizures and epilepsy; this review will instead focus on the link between cerebrovascular permeability, epileptogenesis and ictal events after TBI. The possibility of acting on the blood-brain barrier (BBB) and the neurovascular unit to prevent, disrupt or treat post-traumatic epilepsy is also discussed. Finally, we describe the latest quest for biomarkers of epileptogenesis which may allow for a more targeted intervention.

How do we use in vitro models to understand epileptiform and ictal activity? A report of the TASK1-WG4 group of the ILAE/AES Joint Translational Task Force.

In vitro brain tissue preparations allow the convenient and affordable study of brain networks and have allowed us to garner molecular, cellular, and electrophysiologic insights into brain function with a detail not achievable in vivo. Preparations from both rodent and human postsurgical tissue have been utilized to generate in vitro electrical activity similar to electrographic activity seen in patients with epilepsy. A great deal of knowledge about how brain networks generate various forms of epileptiform activity has been gained, but due to the multiple in vitro models and manipulations used, there is a need for a standardization across studies. Here, we describe epileptiform patterns generated using in vitro brain preparations, focusing on issues and best practices pertaining to recording, reporting, and interpretation of the electrophysiologic patterns observed. We also discuss criteria for defining in vitro seizure-like patterns (i.e., ictal) and interictal discharges. Unifying terminologies and definitions are proposed. We suggest a set of best practices for reporting in vitro studies to favor both efficient across-lab comparisons and translation to in vivo models and human studies.

Biomarkers in traumatic brain injury (TBI): a review.

Biomarkers can be broadly defined as qualitative or quantitative measurements that convey information on the physiopathological state of a subject at a certain time point or disease state. Biomarkers can indicate health, pathology, or response to treatment, including unwanted side effects. When used as outcomes in clinical trials, biomarkers act as surrogates or substitutes for clinically meaningful endpoints. Biomarkers of disease can be diagnostic (the identification of the nature and cause of a condition) or prognostic (predicting the likelihood of a person's survival or outcome of a disease). In addition, genetic biomarkers can be used to quantify the risk of developing a certain disease. In the specific case of traumatic brain injury, surrogate blood biomarkers of imaging can improve the standard of care and reduce the costs of diagnosis. In addition, a prognostic role for biomarkers has been suggested in the case of post-traumatic epilepsy. Given the extensive literature on clinical biomarkers, we will focus herein on biomarkers which are present in peripheral body fluids such as saliva and blood. In particular, blood biomarkers, such as glial fibrillary acidic protein and salivary/blood S100B, will be discussed together with the use of nucleic acids (eg, DNA) collected from peripheral cells.

IFN-γ, IL-17A, or zonulin rapidly increase the permeability of the blood-brain and small intestinal epithelial barriers: Relevance for neuro-inflammatory diseases.

Breakdown of the blood-brain barrier (BBB) precedes lesion formation in the brains of multiple sclerosis (MS) patients. Since recent data implicate disruption of the small intestinal epithelial barrier (IEB) in the pathogenesis of MS, we hypothesized that the increased permeability of the BBB and IEB are mechanistically linked. Zonulin, a protein produced by small intestine epithelium, can rapidly increase small intestinal permeability. Zonulin blood levels are elevated in MS, but it is unknown whether zonulin can also disrupt the BBB. Increased production of IL-17A and IFN-γ has been implicated in the pathogenesis of MS, epilepsy, and stroke, and these cytokines impact BBB integrity after 24 h. We here report that primary human brain microvascular endothelial cells expressed the EGFR and PAR2 receptors necessary to respond to zonulin, and that zonulin increased BBB permeability to a 40 kDa dextran tracer within 1 h. Moreover, both IL-17A and IFN-γ also rapidly increased BBB and IEB permeability. By using confocal microscopy, we found that exposure of the IEB to zonulin, IFN-γ, or IL-17A in vitro rapidly modified the localization of the TJ proteins, ZO-1, claudin-5, and occludin. TJ disassembly was accompanied by marked depolymerization of the peri-junctional F-actin cytoskeleton. Our data indicate that IFN-γ, IL-17A, or zonulin can increase the permeability of the IEB and BBB rapidly in vitro, by modifying TJs and the underlying actin cytoskeleton. These observations may help clarify how the gut-brain axis mediates the pathogenesis of neuro-inflammatory diseases.

Modulation of glucocorticoid receptor in human epileptic endothelial cells impacts drug biotransformation in an in vitro blood-brain barrier model.

OBJECTIVE: Nuclear receptors and cytochrome P450 (CYP) regulate hepatic metabolism of several drugs. Nuclear receptors are expressed at the neurovascular unit of patients with drug-resistant epilepsy. We studied whether glucocorticoid receptor (GR) silencing or inhibition in human epileptic brain endothelial cells (EPI-ECs) functionally impacts drug bioavailability across an in vitro model of the blood-brain barrier (BBB) by CYP-multidrug transporter (multidrug resistance protein 1, MDR1) mechanisms. METHODS: Surgically resected brain specimens from patients with drug-resistant epilepsy, primary EPI-ECs, and control human brain microvascular endothelial cells (HBMECs) were used. Expression of GR, pregnane X receptor, CYP3A4, and MDR1 was analyzed pre- and post-GR silencing in EPI-ECs. Endothelial cells were co-cultured with astrocytes and seeded in an in vitro flow-based BBB model (DIV-BBB). Alternatively, the GR inhibitor mifepristone was added to the EPI-EC DIV-BBB. Integrity of the BBB was monitored by measuring transendothelial electrical resistance. Cell viability was assessed by glucose-lactate levels. Permeability of [3 H]sucrose and [14 C]phenytoin was quantified. CYP function was determined by measuring resorufin formation and oxcarbazepine (OXC) metabolism. RESULTS: Silencing and inhibition of GR in EPI-ECs resulted in decreased pregnane X receptor, CYP3A4, and MDR1 expression. GR silencing or inhibition did not affect BBB properties in vitro, as transendothelial electrical resistance and Psucrose were unaltered, and glucose metabolism was maintained. GR EPI-EC silencing or inhibition led to (1) increased Pphenytoin BBB permeability as compared to control; (2) decreased CYP function, indirectly evaluated by resorufin formation; (3) improved OXC bioavailability with increased abluminal (brain-side) OXC levels as compared to control. SIGNIFICANCE: Our results suggest that modulating GR expression in EPI-ECs at the BBB modifies drug metabolism and penetration by a mechanism encompassing P450 and efflux transporters. The latter could be exploited for future drug design and to overcome pharmacoresistance.

The role and diagnostic significance of cellular barriers after concussive head trauma.

The onset of concussive head trauma often triggers an intricate sequence of physical consequences and pathophysiological responses. These sequelae can be acute (i.e., hematoma) or chronic (i.e., autoimmune response, neurodegeneration, etc.), and may follow traumas of any severity. A critical factor for prognostication of postconcussion outcome is the pathophysiological response of cellular barriers, which can be measured by several biomarkers of the acute and chronic postinjury phases. We present herein a review on the postconcussion mechanisms of the blood-brain barrier, as well as the diagnostic/prognostic approaches that utilize differential biomarker expression across this boundary. We discuss the role of the blood-saliva cellular barrier as a regulatory filter for brain-derived biomarkers in blood, and its implications for saliva-based diagnostic assays.

The role of brain barriers in fluid movement in the CNS: is there a 'glymphatic' system?

Brain fluids are rigidly regulated to provide stable environments for neuronal function, e.g., low K+, Ca2+, and protein to optimise signalling and minimise neurotoxicity. At the same time, neuronal and astroglial waste must be promptly removed. The interstitial fluid (ISF) of the brain tissue and the cerebrospinal fluid (CSF) bathing the CNS are integral to this homeostasis and the idea of a glia-lymph or 'glymphatic' system for waste clearance from brain has developed over the last 5 years. This links bulk (convective) flow of CSF into brain along the outside of penetrating arteries, glia-mediated convective transport of fluid and solutes through the brain extracellular space (ECS) involving the aquaporin-4 (AQP4) water channel, and finally delivery of fluid to venules for clearance along peri-venous spaces. However, recent evidence favours important amendments to the 'glymphatic' hypothesis, particularly concerning the role of glia and transfer of solutes within the ECS. This review discusses studies which question the role of AQP4 in ISF flow and the lack of evidence for its ability to transport solutes; summarizes attributes of brain ECS that strongly favour the diffusion of small and large molecules without ISF flow; discusses work on hydraulic conductivity and the nature of the extracellular matrix which may impede fluid movement; and reconsiders the roles of the perivascular space (PVS) in CSF-ISF exchange and drainage. We also consider the extent to which CSF-ISF exchange is possible and desirable, the impact of neuropathology on fluid drainage, and why using CSF as a proxy measure of brain components or drug delivery is problematic. We propose that new work and key historical studies both support the concept of a perivascular fluid system, whereby CSF enters the brain via PVS convective flow or dispersion along larger caliber arteries/arterioles, diffusion predominantly regulates CSF/ISF exchange at the level of the neurovascular unit associated with CNS microvessels, and, finally, a mixture of CSF/ISF/waste products is normally cleared along the PVS of venules/veins as well as other pathways; such a system may or may not constitute a true 'circulation', but, at the least, suggests a comprehensive re-evaluation of the previously proposed 'glymphatic' concepts in favour of a new system better taking into account basic cerebrovascular physiology and fluid transport considerations.