Blood-brain Barrier In Health Research Articles

Microglia at the blood brain barrier in health and disease.

The blood brain barrier (BBB) plays a crucial role in maintaining brain homeostasis by selectively preventing the entry of substances from the peripheral blood into the central nervous system (CNS). Comprised of endothelial cells, pericytes, and astrocytes, this highly regulated barrier encompasses the majority of the brain's vasculature. In addition to its protective function, the BBB also engages in significant crosstalk with perivascular macrophages (MΦ) and microglia, the resident MΦ of the brain. These interactions play a pivotal role in modulating the activation state of cells comprising the BBB, as well as MΦs and microglia, themselves. Alterations in systemic metabolic and inflammatory states can promote endothelial cell dysfunction, reducing the integrity of the BBB and potentially allowing peripheral blood factors to leak into the CNS compartment. This may mediate activation of perivascular MΦs, microglia, and astrocytes, and initiate further immune responses within the brain parenchyma, suggesting neuroinflammation can be triggered by signaling from the periphery, without primary injury or disease originating within the CNS. The intricate interplay between the periphery and the CNS through the BBB highlights the importance of understanding the role of microglia in mediating responses to systemic challenges. Despite recent advancements, our understanding of the interactions between microglia and the BBB is still in its early stages, leaving a significant gap in knowledge. However, emerging research is shedding light on the involvement of microglia at the BBB in various conditions, including systemic infections, diabetes, and ischemic stroke. This review aims to provide a comprehensive overview of the current research investigating the intricate relationship between microglia and the BBB in health and disease. By exploring these connections, we hope to advance our understanding of the role of brain immune responses to systemic challenges and their impact on CNS health and pathology. Uncovering these interactions may hold promise for the development of novel therapeutic strategies for neurological conditions that involve immune and vascular mechanisms.

Role of Intermediate Filaments in Blood-Brain Barrier in Health and Disease.

The blood–brain barrier (BBB) is a highly selective cellular monolayer unique to the microvasculature of the central nervous system (CNS), and it mediates the communication of the CNS with the rest of the body by regulating the passage of molecules into the CNS microenvironment. Limitation of passage of substances through the BBB is mainly due to tight junctions (TJ) and adherens junctions (AJ) between brain microvascular endothelial cells. The importance of actin filaments and microtubules in establishing and maintaining TJs and AJs has been indicated; however, recent studies have shown that intermediate filaments are also important in the formation and function of cell–cell junctions. The most common intermediate filament protein in endothelial cells is vimentin. Vimentin plays a role in blood–brain barrier permeability in both cell–cell and cell–matrix interactions by affecting the actin and microtubule reorganization and by binding directly to VE-cadherin or integrin proteins. The BBB permeability increases due to the formation of stress fibers and the disruption of VE–cadherin interactions between two neighboring cells in various diseases, disrupting the fiber network of intermediate filament vimentin in different ways. Intermediate filaments may be long ignored key targets in regulation of BBB permeability in health and disease.

Structure and Function of the Blood-Brain Barrier (BBB).

The blood-brain barrier (BBB) protects the vertebrate central nervous system from harmful blood-borne, endogenous and exogenous substances to ensure proper neuronal function. The BBB describes a function that is established by endothelial cells of CNS vessels in conjunction with pericytes, astrocytes, neurons and microglia, together forming the neurovascular unit (NVU). Endothelial barrier function is crucially induced and maintained by the Wnt/β-catenin pathway and requires intact NVU for proper functionality. The BBB and the NVU are characterized by a specialized assortment of molecular specializations, providing the basis for tightening, transport and immune response functionality.The present chapter introduces state-of-the-art knowledge of BBB structure and function and highlights current research topics, aiming to understanding in more depth the cellular and molecular interactions at the NVU, determining functionality of the BBB in health and disease, and providing novel potential targets for therapeutic BBB modulation. Moreover, we highlight recent advances in understanding BBB and NVU heterogeneity within the CNS as well as their contribution to CNS physiology, such as neurovascular coupling, and pathophysiology, is discussed. Finally, we give an outlook onto new avenues of BBB research.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells.

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 ± 80 Ω cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 ± 0.13 10-6 cm sec-1 (mean ± SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an <em>In Vitro</em> Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 ± 80 Ω cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 ± 0.13 10-6 cm sec-1 (mean ± SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Cryopreservation of Brain Endothelial Cells Derived from Human Induced Pluripotent Stem Cells Is Enhanced by Rho-Associated Coiled Coil-Containing Kinase Inhibition.

The blood-brain barrier (BBB) maintains brain homeostasis but also presents a major obstacle to brain drug delivery. Brain microvascular endothelial cells (BMECs) form the principal barrier and therefore represent the major cellular component of in vitro BBB models. Such models are often used for mechanistic studies of the BBB in health and disease and for drug screening. Recently, human induced pluripotent stem cells (iPSCs) have emerged as a new source for generating BMEC-like cells for use in in vitro human BBB studies. However, the inability to cryopreserve iPSC-BMECs has impeded implementation of this model by requiring a fresh differentiation to generate cells for each experiment. Cryopreservation of differentiated iPSC-BMECs would have a number of distinct advantages, including enabling production of larger scale lots, decreasing lead time to generate purified iPSC-BMEC cultures, and facilitating use of iPSC-BMECs in large-scale screening. In this study, we demonstrate that iPSC-BMECs can be successfully cryopreserved at multiple differentiation stages. Cryopreserved iPSC-BMECs retain high viability, express standard endothelial and BBB markers, and reach a high transendothelial electrical resistance (TEER) of ∼3000 Ω·cm2, equivalent to nonfrozen controls. Rho-associated coiled coil-containing kinase (ROCK) inhibitor Y-27632 substantially increased survival and attachment of cryopreserved iPSC-BMECs, as well as stabilized TEER above 800 Ω·cm2 out to 7 days post-thaw. Overall, cryopreservation will ease handling and storage of high-quality iPSC-BMECs, reducing a key barrier to greater implementation of these cells in modeling the human BBB.

Regulation of brain endothelial barrier function by microRNAs in health and neuroinflammation.

Brain endothelial cells constitute the major cellular element of the highly specialized blood-brain barrier (BBB) and thereby contribute to CNS homeostasis by restricting entry of circulating leukocytes and blood-borne molecules into the CNS. Therefore, compromised function of brain endothelial cells has serious consequences for BBB integrity. This has been associated with early events in the pathogenesis of several disorders that affect the CNS, such as multiple sclerosis, HIV-associated neurologic disorder, and stroke. Recent studies demonstrate that brain endothelial microRNAs play critical roles in the regulation of BBB function under normal and neuroinflammatory conditions. This review will focus on emerging evidence that indicates that brain endothelial microRNAs regulate barrier function and orchestrate various phases of the neuroinflammatory response, including endothelial activation in response to cytokines as well as restoration of inflamed endothelium into a quiescent state. In particular, we discuss novel microRNA regulatory mechanisms and their contribution to cellular interactions at the neurovascular unit that influence the overall function of the BBB in health and during neuroinflammation.-Lopez-Ramirez, M. A., Reijerkerk, A., de Vries, H. E., Romero, I. A. Regulation of brain endothelial barrier function by microRNAs in health and neuroinflammation.

Glial regulation of the blood-brain barrier in health and disease.

The brain is the organ with the highest metabolic demand in the body. Therefore, it needs specialized vasculature to provide it with the necessary oxygen and nutrients, while protecting it against pathogens and toxins. The blood-brain barrier (BBB) is very tightly regulated by specialized endothelial cells, two basement membranes, and astrocytic endfeet. The proximity of astrocytes to the vessel makes them perfect candidates to influence the function of the BBB. Moreover, other glial cells are also known to contribute to either BBB quiescence or breakdown. In this review, we summarize the knowledge on glial regulation of the BBB during development, in homeostatic conditions in the adult, and during neuroinflammatory responses.

Exploring the effects of cell seeding density on the differentiation of human pluripotent stem cells to brain microvascular endothelial cells.

BackgroundBrain microvascular-like endothelial cells (BMECs) derived from human pluripotent stem cells (hPSCs) have significant promise as tools for drug screening and studying the structure and function of the BBB in health and disease. The density of hPSCs is a key factor in regulating cell fate and yield during differentiation. Prior reports of hPSC differentiation to BMECs have seeded hPSCs in aggregates, leading to non-uniform cell densities that may result in differentiation heterogeneity. Here we report a singularized-cell seeding approach compatible with hPSC-derived BMEC differentiation protocols and evaluate the effects of initial hPSC seeding density on the subsequent differentiation, yield, and blood–brain barrier (BBB) phenotype.MethodsA range of densities of hPSCs was seeded and differentiated, with the resultant endothelial cell yield quantified via VE-cadherin flow cytometry. Barrier phenotype of purified hPSC-derived BMECs was measured via transendothelial electrical resistance (TEER), and purification protocols were subsequently optimized to maximize TEER. Expression of characteristic vascular markers, tight junction proteins, and transporters was confirmed by immunocytochemistry and quantified by flow cytometry. P-glycoprotein and MRP-family transporter activity was assessed by intracellular accumulation assay.ResultsThe initial hPSC seeding density of approximately 30,000 cells/cm2 served to maximize the yield of VE-cadherin+ BMECs per input hPSC. BMECs displayed the highest TEER (>2,000 Ω × cm2) within this same range of initial seeding densities, although optimization of the BMEC purification method could minimize the seeding density dependence for some lines. Localization and expression levels of tight junction proteins as well as efflux transporter activity were largely independent of hPSC seeding density. Finally, the utility of the singularized-cell seeding approach was demonstrated by scaling the differentiation and purification process down from 6-well to 96-well culture without impacting BBB phenotype.ConclusionsGiven the yield and barrier dependence on initial seeding density, the singularized-cell seeding approach reported here should enhance the reproducibility and scalability of hPSC-derived BBB models, particularly for the application to new pluripotent stem cell lines.Electronic supplementary materialThe online version of this article (doi:10.1186/s12987-015-0007-9) contains supplementary material, which is available to authorized users.

Primary porcine brain microvessel endothelial cell isolation and culture.

Cell culture models of the blood-brain barrier (BBB) are useful tools to study the functionality of the BBB in health and disease. Several good in vitro BBB models are available from different species. However, most brain endothelial cells lose some of their in vivo BBB phenotype in culture. Porcine brain endothelial cells (PBECs) tend to retain most of their in vivo BBB characteristics and usually give higher transendothelial electrical resistance (TEER, representing functional well-developed tight junctions) compared to brain endothelial cells from other species. The protocol described in this unit gives detailed instructions for isolation and culture of PBECs from fresh porcine brains. This porcine BBB model generates high TEER without the need for co-culture with astrocytes. However, astrocyte-derived factors can be introduced to the system through the use of astrocyte-conditioned medium or co-culture with astrocytes, which may be necessary for further enhancing the BBB phenotype for certain complex studies.

Breaking and building the wall: the biology of the blood-brain barrier in health and disease

The blood-brain barrier (BBB) is a complex feature of brain endothelial cells that restricts the passage of blood-borne molecules into the brain parenchyma, while ensuring the delivery of essential nutrients and selected biomolecules. Brain vasculature is anatomically distinct from that of other organs and comprises in addition to endothelial cells, pericytes and astrocytes, which collectively form the neurovascular unit (NVU). This review focuses on the regulation of BBB properties by the NVU and the periphery. A brief overview of cellular components of the NVU, and BBB characteristics will be provided, with more emphasis placed on the molecular mechanisms involved in the development of brain vasculature and human genetic diseases primarily affected by dysfunction of components of the NVU. In addition, the regulation of brain vasculature by peripheral factors such as diet and systemic disease is discussed.

Carbon-11 Labeled Tracers for In Vivo Imaging of P-Glycoprotein Function: Kinetics, Advantages and Disadvantages

P-glycoprotein (P-gp) is a drug efflux transporter with broad substrate specificity localized in the blood-brain barrier and in several peripheral organs. In order to understand the role of P-gp in physiological and patho-physiological conditions, several carbon-11 labelled P-gp tracers have been developed and validated. This review provides an overview of the spectrum of radiopharmaceuticals that is available for this purpose. A short overview of the physiology of the blood-brain barrier in health and disease is also provided. Tracer kinetic modelling for quantitative analysis of P-gp function and expression is highlighted, and the advantages and disadvantages of the various tracers are discussed.

NEUROBID—an EU-funded project to study the developing brain barriers

Brain diseases are one of the most prevalent groups of diseases in Europe with estimated annual costs amounting to €386 billion. Data collected by the WHO suggest that brain diseases are responsible for 35% of Europe's total disease burden. In the treatment of neurological disease, the blood brain barrier (BBB) still represents an obstacle for the delivery of drugs to the brain and thus a major challenge for the development of therapeutic regimens. Understanding the molecular basis and functioning of the BBB in health and disease, including transport mechanisms across the BBB, therefore holds significant potential for future strategies to prevent and ameliorate neurological disease.Recent research indicates that some neurological disorders have a developmental etiologic component. The major goal of the NEUROBID project is thus to understand the molecular mechanisms and function of the BBB in health and disease both in the developing brain and the adult central nervous system. With an interdisciplinary consortium from the fields of developmental neurobiology and BBB research, NEUROBID aims to (i) understand the involvement of normal and disturbed BBB function in normal and abnormal brain development and (ii) to develop novel strategies for drug delivery to the brain. Unique transport mechanisms across the BBB will be used to target potential therapeutic macromolecular and cellular agents specifically to the brain barriers and transport them into the brain. The main target disorders of NEUROBID are non-inherited neurodevelopmental disorders arising from perinatal adverse exposure, such as cerebral palsy, and classic adult neurological disorders such as multiple sclerosis and stroke.In the long term, NEUROBID hopes to pave the way for new treatment strategies and thus reduce the economic and social burden of neurological disease.

Blood-brain barrier in health and disease

Blood-brain barrier in health and disease

An examination of the blood-brain barrier in health and disease

The brain is protected from bloodborne toxins by the walls of the brain capillaries. The capillaries make up the primary part of the so-called blood-brain barrier (BBB). This is an exclusive barrier that precludes a large number of substances from entering the brain. This is because of its specific structural and biochemical properties that arise from interactions of a number of cell types. This review introduces the concept of the BBB to the practitioner. It examines the elements that are presently understood to be necessary for its formation. Finally, the influence of the BBB on disease is examined. This will enable the practitioner to have a comprehensive understanding of the effect the presence or absence of the BBB has on central nervous system health.

Selective binding of peripheral blood lymphocytes to the walls of cerebral vessels in frozen sections of human brain

In order to identify the factor that control the binding of blood leucocytes to cerebral blood vessels we have modified and applied the frozen section assay of Stamper and Woodruff to the study of human brain. Cryostat sections of brain tissue obtained at post mortem were overlaid with blood lymphocytes and experimental conditions were defined which permitted optimum binding of the cells to transected blood vessel walls. The maximal binding of lymphocytes to cerebral vessels occurred when 6 × 10 6 lymphocytes were overlaid onto brain sections for 30 min at 7°C with gentle agitation. Only a small proportion (0.01%) of the added lymphocytes bound to exposed cerebral vessels. However, lymphocytes were far more adherent than monocytes and polymorphonuclear cells (7-fold and 11-fold respectively: p < 0.001) and activation of lymphocytes with IL-2 enhanced their binding to blood vessel walls (mean 130% increase; p < 0.03). Further analysis revealed that CD4-positive T lymphocytes were the predominant cell population binding to the blood vessels. Antibody blocking studies showed that lymphocyte binding to cerebral blood vessels was inhibited by pretreating the lymphocytes with anti-CD11a, anti-CD18 or anti-CD49d ( p ≤ 0.02) and immunohistochemical studies revealed the presence of the counter-receptors ICAM-1 (CD54) and VCAM-1 (CD106) for these adhesion molecules in addition to the presence of E-selectin (CD62E) and P-selectin (CD62P) on the cerebral blood vessels. The establishment of a technique in situ which measures selective binding of CD4-positive peripheral lymphocytes to sections of cerebral blood vessels will assist in the molecular characterization of factors that control the interaction of leucocytes with the blood-brain barrier in health and disease.

Expression of adhesion molecules and histocompatibility antigens at the blood-brain barrier.

The migration of inflammatory cells through the blood-brain barrier in health and disease involves complex interactions between hematogenous cells, endothelial cells, the basement membrane and the perivascular glia limitans. Recent evidence is presented, suggesting that part of these interactions involve antigen independent mechanisms, mediated by cellular adhesion molecules. The accessibility of endothelial adhesion molecules in the intact blood-brain barrier is lower compared to vessels in other organs. This may account for the low traffic of hematogenous cells through the normal blood-brain barrier. However, in inflammatory conditions the expression of endothelial adhesion molecules is upregulated, which may lead to recruitment of inflammatory cells into the lesions. Antigen specific activation of T-cells at the blood-brain barrier apparently takes place mainly at perivascular monocytes and microglia cells. In severe inflammatory lesions, however, astrocytes may be additionally involved in antigen presentation.

The Blood-Brain Barrier in Health and Disease

The Blood-Brain Barrier in Health and Disease

Blood-brain transfer of glucose and other molecules measured by rapid indicator dilution.

Blood-brain transfer of glucose and other molecules measured by rapid indicator dilution.