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Development of the blood-brain barrier: A historical point of view
Abstract
Although there has been considerable controversy since the observation by Ehrlich more than 100 years ago that the brain did not take up dyes from the vascular system, the concept of an endothelial blood-brain barrier (BBB) was confirmed by the unequivocal demonstration that the passage of molecules from blood to brain and vice versa was prevented by endothelial tight junctions (TJs). There are three major functions implicated in the term "BBB": protection of the brain from the blood milieu, selective transport, and metabolism or modification of blood- or brain-borne substances. The BBB phenotype develops under the influence of associated brain cells, especially astrocytic glia, and consists of complex TJs and a number of specific transport and enzyme systems that regulate molecular traffic across the endothelial cells. The development of the BBB is a complex process that leads to endothelial cells with unique permeability characteristics due to high electrical resistance and the expression of specific transporters and metabolic pathways. This review article summarizes the historical background underlying our current knowledge of the cellular and molecular mechanisms involved in the development and maintenance of the BBB.
... Since Paul Ehrlich 's discovery of the blood-brain barrier, the mammalian BBB has been intensely studied in humans, rodents, rabbits, pigs, and dogs [32]. Despite the vast number of publications on the mammalian BBB, the subjects under examination remain exclusively restricted to "model" mammals that serve to better understand human health, disease, and therapeutics, with little regard for the ecological relevance. ...
... With the advent of the electron microscope, researchers were able to visualize and confirm the ultrastructural features of the mammalian BBB during the mid-1950s into the late 1980s, primarily in mice and rats [32]. It was determined through these studies that the mammalian BBB consisted of specialized endothelial cells that line the cerebral capillaries. ...
... It was determined through these studies that the mammalian BBB consisted of specialized endothelial cells that line the cerebral capillaries. The endothelial cells express membrane proteins called tight junction proteins (occludin, claudins, cingulin, and membrane-associated guanylate kinase proteins) and adhesion molecules (cadherins, platelet endothelial cell adhesion molecule, and junctional adhesion molecules) that regulate the selective paracellular diffusion of molecules into and out of the CNS [32,35]. ...
The need to protect neural tissue from toxins or other substances is as old as neural tissue itself. Early recognition of this need has led to more than a century of investigation of the blood-brain barrier (BBB). Many aspects of this important neuroprotective barrier have now been well established, including its cellular architecture and barrier and transport functions. Unsurprisingly, most research has had a human orientation, using mammalian and other animal models to develop translational research findings. However, cell layers forming a barrier between vascular spaces and neural tissues are found broadly throughout the invertebrates as well as in all vertebrates. Unfortunately, previous scenarios for the evolution of the BBB typically adopt a classic, now discredited ‘scala naturae’ approach, which inaccurately describes a putative evolutionary progression of the mammalian BBB from simple invertebrates to mammals. In fact, BBB-like structures have evolved independently numerous times, complicating simplistic views of the evolution of the BBB as a linear process. Here, we review BBBs in their various forms in both invertebrates and vertebrates, with an emphasis on the function, evolution, and conditional relevance of popular animal models such as the fruit fly and the zebrafish to mammalian BBB research.
... Initialement, Ehrlich avait interprété cela comme une faible affinité du solvant pour le cerveau. Figure 1 : Schéma simplifié des différentes étapes conduisant à la découverte de la Barrière hémato-encéphalique modifié de (Zlokovic, 2008 (Ribatti et al., 2006) (figure 1). Cette expérience a ainsi démontré l'existence d'une barrière séparant le sang du cerveau, limitant ainsi les échanges entre les deux compartiments. ...
... En parallèle, des études menées avec de l'acide gallique ou du ferrocyanure de potassium, ont permis à d'autres scientifiques de formuler la même hypothèse de l'existence d'une « barrière » entre le compartiment sanguin et le compartiment cérébral (Ribatti et al., 2006). ...
... Sa composition fut détaillée par Reese et Karnovsky en 1967, grâce à la microscopie électronique. Ils ont pu détailler au niveau des capillaires cérébraux, la présence de cellules endothéliales reliées entre elle grâce à des jonctions serrées (Ribatti et al., 2006). (Zlokovic and Apuzzo, 1998 (Hawkins and Davis, 2005). ...
La barrière hémato-encéphalique (BHE) contrôle le passage des médicaments, en partie par la présence d’ATP Binding Cassette (ABC) transporteurs. Dans de nombreuses pathologies cérébrales, la BHE est altérée. Parmi elles, les hémorragies intracérébrales (HIC), qui sont un effet iatrogène des anticoagulants. Des analyses cliniques montrent que les patients sous Anticoagulants Oraux Directs (AODs) présentent moins d’HIC que les patients traités avec les anticoagulants de référence, les anti-vitamine K (AVK), sans que les mécanismes cellulaires soient élucidés. Une des différences entre les AODs et les AVK résident dans leur profil pharmacocinétique, effectivement, les AODs sont des substrats des ABC transporteurs contrairement aux AVKs. Au cours des HIC, la thrombine est activée et entraine une altération de la BHE par clivage et des récepteurs protease activated receptor (PAR).Les objectifs de ce travail de thèse ont été de mettre en place un modèle in vitro de BHE afin d’étudier les interactions des médicaments avec les ABC transporteurs. Ensuite, le modèle est utilisé pour étudier les interactions des AODs en condition pathologique.Le modèle développé est basé sur la lignée HBEC-5i, peu décrite dans la littérature. Les cellules ont été cultivées en monocouche sur insert avec milieu conditionné issu d’astrocytes humains. Le modèle permet l’étude de l’interaction de thérapeutiques avec des ABC transporteurs par des mesures d’efflux ratios. Le modèle a été validé par des études de transport de molécules pharmacologiques. Ensuite, nous avons comparé, sur notre modèle, les effets de l’exposition à la thrombine avec ou sans prétraitement d’anticoagulants (rivaroxaban, dabigatran, apixaban, warfarine et héparine). Les AODs limitent l’ouverture de la BHE induite par la thrombine contrairement aux autres anticoagulants. Nos résultats ont montré que l’altération de la BHE est médiée par le clivage du récepteur PAR-1 par la thrombine. Ce clivage n’est pas le même en fonction de la classe d’anticoagulants utilisée, les AODs minimisant ce clivage. L’ensemble de ce travail de thèse a permis de donner des premières explications cellulaires quant aux mécanismes d’ouverture de la BHE consécutifs aux HIC sous AODs.
... The term "Blood-Brain Barrier" was introduced by Lewandowsky (1900) (Marcinowski, 2020), following the observations by the German bacteriologist Paul Ehrlich that peripherally injected soluble dyes stained all organs except the brain and spinal cord, suggesting the existence of two distinct compartments (Ribatti et al., 2006). Years later, Lewandowsky demonstrated that intraventricular injection of some compounds led to neuronal death, whereas intravenous injection had no effects on the central nervous system (CNS), and used the term blood-brain barrier (BBB) to describe this phenomenon (Ribatti et al., 2006). ...
... The term "Blood-Brain Barrier" was introduced by Lewandowsky (1900) (Marcinowski, 2020), following the observations by the German bacteriologist Paul Ehrlich that peripherally injected soluble dyes stained all organs except the brain and spinal cord, suggesting the existence of two distinct compartments (Ribatti et al., 2006). Years later, Lewandowsky demonstrated that intraventricular injection of some compounds led to neuronal death, whereas intravenous injection had no effects on the central nervous system (CNS), and used the term blood-brain barrier (BBB) to describe this phenomenon (Ribatti et al., 2006). ...
Ethanol consumption during pregnancy or lactation permanently impairs the development of the central nervous system (CNS), resulting in the spectrum of fetal alcohol disorders (FASD). FASD is a general term that covers a set of deficits in the embryo caused by gestational alcohol exposure, with fetal alcohol syndrome (FAS) considered the most serious. The clinical features of FAS include facial abnormalities, short stature, low body weight, and evidence of structural and/or functional damage to the central nervous system (CNS). The prevalence of FAS carriers worldwide is about 15 for every 10,000 live births (about 119,000 children with APS born per year). Epidemiological data in the US show that the incidence of FAS exceeds other congenital syndromes such as Down syndrome and spina bifida. The deleterious effects of ethanol appear in different brain regions, varying according to the dose and period of neural development when the embryo was exposed, and include: 1) microcephaly; 2) abnormalities in cortical development, with a significant decrease in gyrification; 3) agenesis or hypoplasia of the corpus callosum; and 4) cognitive and behavioral deficits (such as impaired memory and learning, speech difficulties, and hyperactivity). Current evidence indicates that CNS blood vessels are particularly affected by teratogenic ethanol. The CNS vasculature is composed of specialized endothelial cells that establish intimate interactions with astrocytes, pericytes, and microglia, constituting the neurovascular unit of the blood-brain barrier (BBB). Together with the fact that BBB exert protective function, it can prevent the passage of substances and drugs to treat diseases that affect the CNS. Pathological changes in the BBB, such as drug abuse during pregnancy, congenital infections, or ageing processes can drastically alter the molecular structure and vascular stability, disrupting the BBB and aggravating certain neurodegenerative and neurological diseases. In this review, we address the effects of alcohol exposure on the formation of the BBB, specifically describing the cellular and molecular events induced by ethanol in the physiology of endothelial cells and glial cells, as well as their interaction during CNS development.
... Many active substances with neuroprotective activities remain to be evaluated for the PLGA NP approach to improve their brain delivery [101][102][103][104][105][106][107][108][109][110][111][112][113][114][115]. Some of them, such as trehalose, exhort numerous properties ranging from anti-inflammatory to anti-oxidant, which can potentially be applied for out of current applications and NDDs treatment. ...
... Combining these compounds with PLGA NPs may be a very interesting new tool for brain disease treatment, such as AD and PD thanks to their neuroprotective activity. Indeed, Young Woo Lee and coworkers demonstrated that Sal B improved memory impairment caused by Aβ [25][26][27][28][29][30][31][32][33][34][35] peptide-induced neuronal damage in AD [103] while Zeng et al., published its ability to protect cells against MPPinduced apoptosis for PD treatment [112]. TS IIA inhibited transcription and translation of iNOS, MMP-2, and NF-κBp65, and suppressed expression of NADPH oxidase and iNOS, respectively, involved in AD [113] and PD [114] development. ...
Cette thèse a porté sur le développement de nouveaux vecteurs thérapeutiques pour le traitement de la maladie de Parkinson. La maladie de Parkinson est une maladie neurodégénérative qui se caractérise notamment par la présence d’inclusions protéiques intracytoplasmiques appelées « corps de Lewy ». L'une des causes de la formation de ces « corps de Lewy », principalement composés d’α-synucléine, serait l’atteinte du système autophagie-lysosome, nécessaire à la dégradation des composants cellulaires. Le rétablissement des fonctions de l’autophagie constitue donc une cible thérapeutique intéressante pour lutter contre la maladie de Parkinson. Une approche interdisciplinaire a permis de travailler sur les diverses étapes de la recherche à visée thérapeutique, de la synthèse chimique de substances actives à leur nano-formulation, puis à l’évaluation biologique de ces nanovecteurs. Premièrement, les propriétés biologiques de restauration du pH lysosomal des nanoparticules de poly(acide lactique-co-glycolique) (PLGA) ont été étudiées. Cette première partie a permis de conclure que ces nanoparticules possédaient une activité neuroprotectrice, tout en améliorant la synucléinopathie et un rétablissement de la fonction lysosomale chez un modèle murin de la maladie de Parkinson. Deuxièmement, l’effet thérapeutique d’une substance active reconnue comme neuroprotectrice, le tréhalose, a été évalué in-vitro sur une lignée cellulaire (BE (2)-M17), notamment pour sa capacité d’activation de l’autophagie. Des dérivés nucléolipide-tréhalose (appelés GlycoNucléoLipides ou GNL) ont donc été développés et synthétisés avant leur encapsulation à l’intérieur de nanoparticules de PLGA. Ces GNL ont également montré leur capacité d’auto-assemblage pour former des nanoparticules solides lipidiques (appelées SLN) capables, comme leurs analogues de PLGA, de traverser les membranes cellulaires et d’induire une activation de l’autophagie.
... 9 Similar to studies by Stern and Gautier, several investigations were carried out by dye injections to understand the circulations into the human brain. 10 The next important milestone on BBB was the identification of cellular structure that was responsible for its barrier properties. After the advent of electron microscopy, visualization of BBB became feasible and attempts were made to understand the cellular architecture of BBB ( Figure 1). ...
... Grafting experiments performed in vitro threw light on the involvement of astrocyte endfeet and several other cell types in the barrier function. 10 Early in vitro studies by Arthur and team 12 revealed the existence of tight junction facilitated by the coculturing of endothelial cells with astrocytes. Simultaneous search on other cell types involved in BBB was also conducted by several researchers in the early 19th century. ...
... This model has low clinical severity and enables activity studies in adult animals, with a fully developed blood-brain-barrier (BBB). Previous research has indeed established that mouse BBB is fully matured at 12-24 days of age (39). More severe, and short-lived mouse models of SMA (40) require injections directly after birth and are therefore not good proxies for delivery beyond a mature BBB. ...
Oligonucleotide therapeutics are an established class of drugs for the treatment of genetic disorders. Their clinical development is challenging, however, as they typically distribute poorly to extra-hepatic tissues after systemic injection. Here we tested the heteroduplex oligonucleotide (HDO) platform for systemic delivery of SMN2 splice-switching oligonucleotides of 2’- O- methoxyethyl phosphorothioate or phosphorodiamidate morpholino oligomer chemistries. We first showed that splice-switching HDO cargoes correct SMN2 splicing in cells derived from spinal muscular atrophy (SMA) patients, and validated extra-hepatic activity in spinal cord and muscle in a mouse model of SMA following systemic delivery. Our study raises prospects for delivery of nusinersen, the 2’- O- methoxylethyl phosphorothioate oligonucleotide therapy approved for SMA and currently delivered by intrathecal injection, by systemic injection exploiting the HDO chemistry platform. Our findings also suggest that oligonucleotide drugs lacking convincing in vivo efficacy in muscle tissue could be delivered effectively by the HDO technology.
... The BBB was first proposed by Edwin Goldman in 1913, but the definite structure was only known to scientists when the scanning electron microscope appeared in 1937 (8). Specifically, BBB is a highly selective semipermeable barrier between blood plasma and neural cells, which can selectively prevent certain substances in blood circulation from entering the brain (5). ...
Glioma is the most common primary intracranial tumor in adults with poor prognosis. Current clinical treatment for glioma includes surgical resection along with chemoradiotherapy. However, the therapeutic efficacy is still unsatisfactory. The invasive nature of the glioma makes it impossible to completely resect it. The presence of blood-brain barrier (BBB) blocks chemotherapeutic drugs access to brain parenchyma for glioma treatment. Besides, tumor heterogeneity and hypoxic tumor microenvironment remarkably limit the efficacy of radiotherapy. With rapid advances of nanotechnology, the emergence of a new treatment approach, namely, reactive oxygen species (ROS)-based nanotherapy, provides an effective approach for eliminating glioma via generating large amounts of ROS in glioma cells. In addition, the emerging nanotechnology also provides BBB-crossing strategies, which allows effective ROS-based nanotherapy of glioma. In this review, we summarized ROS-based nanomedicine and their application in glioma treatment, including photodynamic therapy (PDT), photothermal therapy (PTT), chemodynamic therapy (CDT), sonodynamic therapy (SDT), radiation therapy, etc. Moreover, the current challenges and future prospects of ROS-based nanomedicine are also elucidated with the intention to accelerate its clinical translation.
... The BBB is the major site of blood oxygen-oxygen in the brain [82,83]. It has permeability for hydrophilic and small polar molecules while excluding larger hydrophilic molecules, providing protection to the CNS from toxins and pathogens [84][85][86]. ...
Simple Summary
Gliomas are the most common primary brain tumors. These cancers are universally fatal with limited treatment options. Glioma cells co-opt non-cancerous cells present in normal brain tissue. This manipulation results in a complex network of cell interactions. This interplay is further complicated by variations depending on specific mutations in glioma cells. In order to identify future treatments for gliomas, a better understanding of these interactions is needed. To address this, we review the literature to highlight these interactions and how they relate to different glioma mutations.
Abstract
Gliomas are the most common primary brain malignancy and are universally fatal. Despite significant breakthrough in understanding tumor biology, treatment breakthroughs have been limited. There is a growing appreciation that major limitations on effective treatment are related to the unique and highly complex glioma tumor microenvironment (TME). The TME consists of multiple different cell types, broadly categorized into tumoral, immune and non-tumoral, non-immune cells. Each group provides significant influence on the others, generating a pro-tumor dynamic with significant immunosuppression. In addition, glioma cells are highly heterogenous with various molecular distinctions on the cellular level. These variations, in turn, lead to their own unique influence on the TME. To develop future treatments, an understanding of this complex TME interplay is needed. To this end, we describe the TME in adult gliomas through interactions between its various components and through various glioma molecular phenotypes.
... Macromolecules (e.g. very high concentration of trypanblue) penetrate into the brain a few weeks after birth in mice (Ford, 1973;Risau et al., 1986;Ribatti et al., 2006;Rebecca et al., 2006). Alkaline phosphatase, one of ectoenzymes on the capillary endothelium in the brain (Calhau et al., 2002) and necessary for full BBB function, begin to appear 12-24 days after birth in mice and a few weeks in rats (Vorbrodt et al., 1986;Clark et al., 1993;Al-Sarraf et al., 1997;Rebecca et al., 2006). ...
... With the advent of electron microscopy in the 1950s, scientists were able to ascertain the ultrastructural features of the BBB (Ribatti et al. 2006). However, its pivotal role in maintaining homeostasis of the CNS and regulation of metabolism and circadian rhythm would not be elucidated until many years later (Abbott et al. 2009). ...
... The first concept of a compartmentalised CNS in scientific research was evident in the work of Paul Ehrlich in 1885 (Ribatti et al., 2006). Ehrlich injected a vital dye into the bloodstream of animals and examined the distribution throughout the organs. ...
... In particular, ontogenetic analyses at different developmental time points could clarify if the crosstalk between peripheral serotonin and brain wiring starts to occur during development. Indeed, the blood-brain barrier represents a clear boundary at adulthood, but during the first stages of life it is still immature and different molecules, including serotonin, can cross it [52]. Therefore, the reduction in serotonin amounts reaching the brain during the first period of life could interfere with the correct formation of neuronal circuits and inhibit their correct functioning [53]. ...
Serotonin is synthetized through the action of tryptophan hydroxylase (TPH) enzymes. While the TPH2 isoform is responsible for the production of serotonin in the brain, TPH1 is expressed in peripheral organs. Interestingly, despite its peripheral localization, alterations of the gene coding for TPH1 have been related to stress sensitivity and an increased susceptibility for psychiatric pathologies. On these bases, we took advantage of newly generated TPH1−/− rats, and we evaluated the impact of the lack of peripheral serotonin on the behavior and expression of brain plasticity-related genes under basal conditions and in response to stress. At a behavioral level, TPH1−/− rats displayed reduced anxiety-like behavior. Moreover, we found that neuronal activation, quantified by the expression of Bdnf and the immediate early gene Arc and transcription of glucocorticoid responsive genes after 1 h of acute restraint stress, was blunted in TPH1−/− rats in comparison to TPH1+/+ animals. Overall, we provided evidence for the influence of peripheral serotonin levels in modulating brain functions under basal and dynamic situations.
... The electron microscopic studies by Reese and Karnovsky [48] and by Brightman et al. [49] have shown that the mammalian blood-brain barrier (BBB) location is at the brain microvascular endothelial cells (BECs) [50]. Until recently, it was impossible to selectively identify or observe the individual cells of the BBB due to the lack of a specific marker for the functionally and morphologically unique microvessels [47,[51][52][53][54][55]. ...
Clinical and laboratory studies have shown that environmental exposure to cadmium produces damage to several organs, including bones, lungs, and kidneys. The involvement of cadmium in central nervous system (CNS) disorders has also been widely reported, but the precise pathophysiological mechanism is not yet fully understood. Children who were exposed to cadmium during pregnancy are known to suffer from developmental delays, learning difficulties, attention deficit hyperactivity disorder (ADHD), and other cognitive and neurobehavioral deficits. Results from numerous studies suggest that dysfunction of the blood-brain barrier (BBB) structures is an important step in the neurotoxicity of cadmium. A rat-specific BBB marker protein, the endothelial barrier antigen (EBA), has been previously isolated and classified by Sternberger and others. The mouse IgG1 clone, anti-endothelial barrier antigen (anti-EBA), detects a protein triplet (23.5kDa, 25 kDa, and 30kDa) localized to the luminal surface of central and peripheral nervous system (CNS and PNS) vascular endothelial cells with selective permeability barrier functions. This marker has been widely used for characterizing BBB alterations under demyelinating, inflammatory, and other CNS pathologies. Many studies have been published using the rat model system for studying the neurotoxic effect of acute and chronic exposure to cadmium. We applied the indirect immunofluorescent techniques using the anti-EBA antibody in conjunction with the Olympus cellSens computerized image analysis to detect and quantify the surface areas of BBB-competent microvessel profiles in paraformaldehyde-fixed, paraffin-embedded brains of term-delivered young rats after intraperitoneal injection of a single dose of cadmium chloride. We detected a statistically significant reduction in EBA-positive microvessel surface areas in the forebrain (t = 5.86, df = 1789, p-value < 0.001) and cerebellum (t=73.40, df=1337, p < 0.001) of cadmium-treated rats compared to the normal controls. Thus, this study supports the hypothesis that the EBA is a sensitive and measurable indicator for quantitative assessment of the impact of cadmium exposure in the developing rat brain.
... Biedl & Kraus (12) and Lewandowsky (13) first postulated the true barrier function of CNS vessels by 1900 after observing that intravenous (i.v.) injection of cholic acids or sodium ferrocyanide had no neurological effect, whereas intracerebroventricular injection produced symptoms. The anatomy of the BBB began to become understood in the 1950s through electron microscopy, which revealed a basement membrane and astrocytic endfeet on the abluminal surface of brain endothelial cells (14). Today we know that BBB function is an emergent property of several cellular and subcellular components collectively termed the neurovascular unit (NVU). ...
Brain disease remains a significant health, social, and economic burden with a high failure rate of translation of therapeutics to the clinic. Nanotherapeutics have represented a promising area of technology investment to improve drug bioavailability and delivery to the brain, with several successes for nanotherapeutic use for central nervous system disease that are currently in the clinic. However, renewed and continued research on the treatment of neurological disorders is critically needed. We explore the challenges of drug delivery to the brain and the ways in which nanotherapeutics can overcome these challenges. We provide a summary and overview of general design principles that can be applied to nanotherapeutics for uptake and penetration in the brain. We next highlight remaining questions that limit the translational potential of nanotherapeutics for application in the clinic. Lastly, we provide recommendations for ongoing preclinical research to improve the overall success of nanotherapeutics against neurological disease.
Expected final online publication date for the Annual Review of Chemical and Biomolecular Engineering, Volume 13 is October 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... The properties of the cerebral vasculature remained unknown due to the limited resolution of the light microscope, then discovery of electron microscopy allowed to define the ultrastructural features of blood vessels throughout the body. Early ultrastructural investigation (1955)(1956)(1957)(1958)(1959) questioned the selective role of ECs in the barrier function as well as its surrounding cells (Ribatti et al., 2006). Structure and function of the blood-brain barrier As already mentioned the CNS cells require a stable microenvironment, which is guaranteed by a cellular and molecular complex system involving brain microvessels and their close environment referred to as the BBB (Fig.3) (Abbott et al., 2010;Herndon et al., 2017). ...
Adult male mice exposure to low doses of ubiquitous endocrine disruptors of phthalate family, DEHP, induces alteration of neural function and of behaviors due to down-regulation of androgen receptors (AR). The link between reduced androgen sensitivity, neurovascular unit (NVU) dysfunction and neuroinflammation has been demonstrated. Whether phthalates exposure affects the integrity and function of the NVU remained to be explored. Exposure to low doses of DEHP alone or in an environmental phthalate mixture, increased blood-brain barrier (BBB) permeability, affecting the accessory tight junction protein Zona Occludens-1 and caveolae protein Cav-1 in capillaries of the hypothalamus mPOA and the hippocampal CA1 and CA3 areas, key cerebral areas involved in the sexual and cognitive behaviors in male, respectively. This was associated with an inflammatory profile including an astrocyte activation and iNOS expression in the mPOA, and a microglial activation in the mPOA and the hippocampal CA1 and CA3 areas. The protein levels of the inflammatory molecule, COX-2 were increased in activated microglial cells in the mPOA. None of the major effects induced by DEHP alone or in a mixture was detected in the hippocampal dendate gyrus. DEHP decreased AR protein levels in cerebral capillaries of the mPOA. Such exposure reduced the protein levels of basement membrane components and receptors involved in cell-matrix interactions while MMP-2 and MMP-9 activity was enhanced. These data point out a vulnerability of the NVU to exposure to DEHP alone or in a phthalate mixture, with similarities and differences in the induced effects between the studied androgen-sensitive brain regions.
... Compared with endothelial cells in the peripheral tissue, the continuous endothelial monolayer within the BBB lacks fenestrations and has the capability to strictly regulate the efflux and influx of ions, toxins, blood cells, nutrition, and pathogens by its unique permeability properties. The adjacent ECs are linked by the junction complex predominately comprising TJs and adherens junctions at the ultrastructural level, which contribute to limiting the diffusion of most hydrophilic molecules from plasma to the CNS through the paracellular pathway and the subsequent creation of high transendothelial electrical resistance (1500-2000 omega•cm 2 ) of the BBB [20,[191][192][193]. In addition, the maintenance of normal BBB physiological function is inseparable from the unique transport systems, including influx and efflux transporters, limited transcytosis rate, and low level of leukocyte adhesion molecules [17,190]. ...
Cerebral microbleeds (CMBs) are a disorder of cerebral microvessels that are characterized as small (<10 mm), hypointense, round or ovoid lesions seen on T2*-weighted gradient echo MRI. There is a high prevalence of CMBs in community-dwelling healthy older people. An increasing number of studies have demonstrated the significance of CMBs in stroke, dementia, Parkinson's disease, gait disturbances and late-life depression. Blood-brain barrier (BBB) dysfunction is considered to be the event that initializes CMBs development. However, the pathogenesis of CMBs has not yet been clearly elucidated. In this review, we introduce the pathogenesis of CMBs, hypertensive vasculopathy and cerebral amyloid angiopathy, and review recent research that has advanced our understanding of the mechanisms underlying BBB dysfunction and CMBs presence. CMBs-associated risk factors can exacerbate BBB breakdown through the vulnerability of BBB anatomical and functional changes. Finally, we discuss potential pharmacological approaches to target the BBB as therapy for CMBs.
... Although BBB has been primarily believed to be discovered by Paul Ehrlich's research, Liddelow evidenced that this idea was first observed by Ridley (1653-1708), where he noticed the difference in the permeability of beeswax and mercury in brain tissues from other tissues, and he mentioned this in the book Anatomy of the Brain, which was published in 1695 [6][7][8]. Following that, Ehrlich [9], Bield and Kraus [10], Lewandowsky [11], and Edwin Goldmann [12,13] performed groundbreaking research on the permeability of various materials from blood to brain tissues or the other way around, resulting in the discovery of a unique barrier structure in microvessels of the brain [14][15][16]. ...
The blood-brain barrier (BBB) is a semipermeable and extremely selective system in the central nervous system of most vertebrates, that separates blood from the brain’s extracellular fluid. It plays a vital role in regulating the transport of necessary materials for brain function, furthermore, protecting it from foreign substances in the blood that could damage it. In this review, we searched in Google Scholar, Pubmed, Web of Science, and Saudi Digital Library for the various cells and components that support the development and function of this barrier, as well as the different pathways to transport the various molecules between blood and the brain. We also discussed the aspects that lead to BBB dysfunction and its neuropathological consequences, with the identification of some of the most important biomarkers that might be used as a biomarker to predict the BBB disturbances. This comprehensive overview of BBB will pave the way for future studies to focus on developing more specific targeting systems in material delivery as a future approach that assists in combinatorial therapy or nanotherapy to destroy or modify this barrier in pathological conditions such as brain tumors and brain stem cell carcinomas.
1. Introduction
The human brain has 644 kilometers of blood vessels that provide oxygen, energy, metabolites, and nutrients to brain cells while also removing carbon dioxide as well as other metabolic wastes from the circulatory system [1]. The brain requires 20% of the body’s glucose and oxygen, while accounting for just 2% of total body mass, and can quickly increase blood supply and oxygen transfer to its active areas, a mechanism that is known as neurovascular coupling [1, 2]. This control is aided by barrier layers at the main interfaces between blood and neural tissue called blood-brain barrier (BBB) [3] (Figure 1).
... The tight junctions in the endothelial cells are a major reason for the limitation on the entry of molecules and ions into the brain from blood vessels. Reese and Karnovsky in 1967 described these tight junctions between the cells in the BBB vessels as continuous and only having a small number of vesicles [34,35]. This is contrary to non-cerebral vessels where vesicles are more frequent and abundant. ...
Neurosurgery as one of the most technologically demanding medical fields rapidly adapts the newest developments from multiple scientific disciplines for treating brain tumors. Despite half a century of clinical trials, survival for brain primary tumors such as glioblastoma (GBM), the most common primary brain cancer, or rare ones including primary central nervous system lymphoma (PCNSL), is dismal. Cancer therapy and research have currently shifted toward targeted approaches, and personalized therapies. The orchestration of novel and effective blood–brain barrier (BBB) drug delivery approaches, targeting of cancer cells and regulating tumor microenvironment including the immune system are the key themes of this review. As the global pandemic due to SARS-CoV-2 virus continues, neurosurgery and neuro-oncology must wrestle with the issues related to treatment-related immune dysfunction. The selection of chemotherapeutic treatments, even rare cases of hypersensitivity reactions (HSRs) that occur among immunocompromised people, and number of vaccinations they have to get are emerging as a new chapter for modern Nano neurosurgery.
... The BBB consists of endothelial cells of the capillary wall, astrocyte end-feet surrounding the capillary, and pericytes embedded in the capillary basement membrane [11]. The BBB protects the brain from the blood milieu, facilitates selective transport, and modifies blood-or brain-borne substances [12]. The BBB restricts the passage of pathogens, the diffusion of solutes in the blood, and the passage of peripheral immune factors [13][14]. ...
SARS‐CoV‐2 has infected hundreds of millions of people with over four million dead, resulting in one of the worst global pandemics in recent history. Neurological symptoms associated with COVID‐19 include anosmia, ageusia, headaches, confusion, delirium and strokes. These may manifest due to viral entry into the central nervous system (CNS) through the blood‐brain barrier (BBB) by means of ill‐defined mechanisms. Here we summarize the abilities of SARS‐CoV‐2 and other neurotropic RNA viruses, including Zika virus and Nipah virus, to cross the BBB into the CNS, highlight the role of magnetic resonance imaging (MRI) in assessing presence and severity of brain structural changes in COVID‐19 patients. We present new insight into key mutations in SARS‐CoV‐2 variants B.1.1.7 (P681H) and B.1.617.2 (P681R), which may impact on neuropilin‐1 (NRP1) binding and CNS invasion. We postulate that SARS‐CoV‐2 may infect both peripheral cells capable of crossing the BBB and brain endothelial cells to traverse the BBB and spread into the brain. COVID‐19 patients can be followed up with MRI modalities to better understand the long‐term effects of COVID‐19 on the brain. Coronaviruses and pandemic‐potential RNA viruses can reach the brain using various mechanisms and thereby induce neurological symptoms. Structural analysis of SARS‐CoV‐2 neuronal entry co‐receptor NRP1 interacting with the Spike protein revealed key mutations among existing variants of concern, which could affect NRP1 binding, and SARS‐CoV‐2 spread into the brain. Utilization of MRI modalities would be crucial for tracking viral‐mediated structural changes to the brain.
... The BBB was discovered in 1904 by Paul Ehrlich, a German physician, who conducted an initial experiment by injecting water-soluble dye into the bloodstream of mice (Desai 2007). The dye perfused through all organs in the mice, causing them to stain, excluding the brain (Ribatti 2006). This study was the first experimental evidence indicating there was an existing barrier between the body and brain. ...
The Blood-Brain Barrier (BBB) is a highly selective filter responsible for allowing certain gases such as oxygen and lipid-soluble molecules to pass (Anand 2014). Its selectiveness makes it challenging for many therapeutics to combat Alzheimer’s and Parkinson’s disease with external drug therapies. Large-molecule drug therapies never pass the BBB while small-molecule drugs pass only about 5% of the time (Pardridge 2005). In Alzheimer’s disease, tight junctions between endothelial cells degrade, causing an unregulated accumulation of amyloid-β (Aβ) protein (Ramanathan 2015). Consequently, this leads to the formation of neurofibrillary tangles that cut off the nutrient supply to the brain cells and kill neurons (Ramanathan 2015). In Parkinson’s disease, astrocyte mutations cause a build-up of α-synuclein (αSyn) which affects the neuroinflammatory response and causes dysfunction in dopaminergic neurons (Booth 2017; Meade 2019). New drug therapies for Alzheimer’s and Parkinson’s continue to undergo trials; some such as FPS-ZM1 and tilavonemab for Alzheimer’s and Ravicti for Parkinson’s have shown promising results. In addition, similarities in dysfunction for both diseases and some types of cancer have sparked possibilities in retargeting cancer drugs to improve Alzheimer's and Parkinson’s pathologies. This review will summarize current therapeutic advancements for Alzheimer’s and Parkinson’s disease and their possible future contributions.
... Einschränkend muss angesichts der Tatsache, dass mütterliches 5-HT während der Schwangerschaft über die Plazenta in den fetalen Organismus übertreten kann(Bonnin et al., 2011), und die Muttertiere als Tph2+/-Mäuse nur partiell 5-HT-defizient sind, angenommen werden, dass Tph2 -/-Jungtiere während der Schwangerschaft den Wirkungen von 5-HT dennoch in gewissem Ausmaß ausgesetzt sind. Darüber hinaus konnte gezeigt werden, dass die Blut-Hirn-Schranke vor P12 unreif ist, was es für von Tph1 peripher synthetisiertes 5-HT möglich machen könnte, bis zu diesem Zeitpunkt zu einem gewissen Grad die Blut-Hirn-Schranke zu passieren(Ribatti et al., 2006). Somit kann nicht ausgeschlossen werden, dass homozygot Tph2-defiziente Mäuse während der Entwicklung sowie in der frühen postnatalen Phase nicht doch in gewissem Umfang serotonergen Wirkungen ausgesetzt sind.In der ersten Kohorte zeigte sich in testnaiven MS-exponierten Mäusen interessanterweise eine geringere neuronale Aktivierung im La als in testnaiven Tieren der Kontrollgruppe. ...
Angsterkrankungen gehören zu den am weitesten verbreiteten psychischen Erkrankungen und stellen eine beträchtliche soziale und wirtschaftliche Herausforderung für unsere Gesellschaft dar. Aversive frühe Erfahrungen sind ein bekannter Risikofaktor für die Entwicklung verschiedener psychischer Erkrankungen, insbesondere Angststörungen. Während der frühen Entwicklung findet die Programmierung der Hypothalamus-Hypophysen-Nebennierenrinden- (HHN)-Achse, die die Ausschüttung des Stresshormons Cortisol in Menschen bzw. Corticosteron in Mäusen steuert, statt. Wenn Individuen in dieser kritischen Phase Stress ausgesetzt sind, wird die regelrechte Ausbildung der HHN-Achse gestört, was zu dysregulierten Verhaltensantworten auf Stressreize im späteren Leben führen kann. Das Serotonin (5-HT)-System als eines der ausgedehntesten Neurotransmittersysteme ist an der Vermittlung der Effekte von früher Stressexposition auf angstähnliche Verhaltensweisen beteiligt. Das Ziel dieser Studie ist es, die Interaktion zwischen genetischer Prädisposition und negativen Einflüssen in frühen Entwicklungsstadien auf die Ausbildung von Angstverhalten im Erwachsenenalter näher zu beleuchten. In dieser Studie wurden Tryptophanhydroxylase 2 (Tph2)-defiziente weibliche Mäuse als Modell für ein lebenslanges konstitutives 5-HT Synthesedefizit im zentralen Nervensystem verwendet. Nachkommen dieser Mauslinie wurden im frühen Lebensalter Maternaler Separation (MS), d.h. einem mütterlichen Trennungsparadigma, unterzogen und im Erwachsenenalter im „Open field“ (OF) oder in der „Dark-light box“ (DLB) getestet. Im Anschluss an die Verhaltensexperimente wurde die neuronale Aktivierung immunhistochemisch durch Darstellung des frühzeitig auftretenden Genprodukts c-Fos bestimmt. In der DLB zeigten homozygot Tph2-defiziente Mäuse eine verringerte motorische Aktivität im hellen Kompartiment, und dieser Effekt konnte durch MS normalisiert werden. Zusätzlich verstärkte MS bei diesem Genotyp das Auftreten von fluchtartigen Sprüngen. Im OF hat MS fluchtartige Verhaltensweisen in homo- und heterozygoten Tph2-defizienten Mäusen befördert. Beide Verhaltenstests führten zu spezifischen neuronalen Aktivierungsmustern, die mithilfe von c-Fos- Immunhistochemie ausgewertet wurden. Die Durchführung des DLB-Tests führte in Abhängigkeit vom Vorhandensein von Tph2 zur Aktivierung des paraventrikulären Kerns des Hypothalamus (PVN) und der basolateralen Amygdala (BL), wohingegen die Exposition gegenüber dem OF-Test zu einer Aktivierung der lateralen Amygdala (La) in Tieren, die einem mütterlichen Trennungsparadigma unterzogen wurden, sowie einer Aktivierung des ventrolateralen (VLPAG) und dorsolateralen (DLPAG) periaquäduktalen Höhlengraus in Abhängigkeit von Tph2 und MS führte. Zusammenfassend weisen die Ergebnisse dieser Studie darauf hin, dass MS aktive Verhaltensantworten auf aversive Reize in Abhängigkeit vom Vorhandensein von 5-HT im Gehirn fördert. Diese Effekte könnten durch die spezifische Aktivierung von mit Angstverhalten in Zusammenhang stehenden Gehirnregionen während der Verhaltensexperimente vermittelt werden.
... Serotonin is synthesized from the essential amino-acid L-tryptophan. In the blood stream, L-tryptophan is linked to serum-albumin but a free proportion that decreases with age and physiological status freely crosses the BBB (10% at postnatal day 12 when BBB is thought fully functional in rat [48]). In 5-HT-producing cells, tryptophan is then transported, accumulated and hydroxylated by the tryptophan hydroxylase (Tph) enzymes. ...
Post-Traumatic Stress Disorder (PTSD) is characterized by substantial physiological and/or psychological distress following exposure to trauma. Intrusive fear memories often lead to persistent avoidance of stimuli associated with the trauma, detachment from others, irritability and sleep disturbances. Different key structures in the brain are involved with fear conditioning, fear extinction and coping. The limbic system, namely, the amygdala complex in close relationship with the hippocampal hub and the prefrontal cortex play central roles in the integration and in coping with fear memories. Serotonin acting both as a neurotransmitter and as a neurohormone participates in regulating the normal and pathological activity of these anatomic structures. We review the literature analyzing how the different actors of the serotoninergic system (5-HT receptors, transporters and anabolic and catabolic pathways) may be involved in regulating the sensitivity to highly stressful events and hopefully coping with them.
... Primarily we think that OP and/or OC has ability to penetrate blood brain barrier(BBB).This penetration occur in time when BBB is still immature during lactation period and may be interfere with dopamine and /or another catacholamins and even other CNS neurotransmitters. It's reported that in developing mice BBB maturation is complete around the third postnatal week (13). Developing of elimination system of OP and/or OC beside gradual maturation of BBB may be were the main causes that reduced the observed clinical symptoms within last days of treatment. ...
The present study was aimed to evaluate neurotoxic effects of oseltamivir phosphate in lactating pups of orally dosed mice mothers during lactation. Twelve recently parturited female albino mice were divided equally into three groups, one control and two treated groups, each group consists of 4 dosed dams and 8 chosen pups .The nursing dams of T1 and T2 dosed daily orally with 1mg/kg and 5mg/kg,oseltamivir phosphate respectively representing the therapeutic dose and 5 fold dose of drug while control group dosed with distilled water. Lactating mice pups of all groups examined for the following parameters: First parameter was body weight changes and gain: In which T1group showed significant increase in mice pups body weight gain after 14 day of treatment in comparison with control group and T2. Second parameter was clinical symptoms observation /daily, all treatment groups that showed neurotoxic symptoms appeared from 1st dose and extended along the next few days of treatment to be gradually disappeared and completely lost within the last days of treatment in dose dependent manner.These neurotoxic symptoms were weakness, convulsions ,lay on back or side, extended body, incoordination ,extended limbs and limbs stiffness. Third parameter was gross and histopathological studies which demonstrate that the brain was the most affected organ beside extensive lesions in liver, kidney, stomach and small intestine of treated groups in dose dependent manner.
In conclusion of this study revealed that Oseltamivir phosphate produce neurotoxic effect in mice pups through indirect administration by nursing mothers dosing during lactation period and the level of toxicity was in dose dependent manner.
... L'intégrité et la perméabilité de la BHE sont aussi assurées par (i) les péricytes qui entourent les cellules endothéliales, (ii) la lame basale qui encercle les péricytes et les cellules endothéliales, ainsi que (iii) par les pieds astrocytaires qui couvrent environ 90% de la surface des cellules endothéliales ( Figure 13) (Löscher and Potschka, 2005). Le passage d'une substance à travers la BHE se restreint, notamment à cause de la présence des jonctions serrées, à quatre modes : la diffusion passive, la diffusion facilitée, le transport actif et la transcytose (Ribatti Domenico et al., 2006). La plupart des médicaments utilise la transcytose pour traverser la BHE, bien que cette voie soit le transport minoritaire de la BHE. ...
Les tumeurs gliales sont les tumeurs cérébrales les plus fréquemment retrouvées chez l’enfant. Les gliomes infiltrant du tronc cérébral (DIPG) sont la forme la plus agressive des gliomes pédiatriques. Les épendymomes (EPN) restent actuellement difficilement curables et à l’origine de fréquentes rechutes. Le manque de matériel biologique et l'absence de modèles in vitro et in vivo pertinents ont longtemps entravé le développement de nouvelles thérapeutiques dans ces deux cancers. Des études récentes ont montré que le DIPG est caractérisé par une mutation unique située sur la queue régulatrice de l’histone H3 dans l’un des deux gènes HIST1H3B/C ou H3F3A. En revanche, aucune cible moléculaire n’a pu être identifiée par séquençage du génome dans les EPN. Récemment, il a été montré que le profil de méthylation des EPN permet de diviser les EPN de la fosse postérieure (EPN-PF) et les EPN supratentoriels (EPN-ST) en neuf sous-groupes différents.Dans un premier temps, nous avons développé des xénogreffes par stéréotaxie dans la souris Nude (i) de manière directe en greffant directement les cellules tumorales afin d’obtenir des modèles PDOX (Patient Derived Orthotopic Xenograft), et (ii) de manière indirecte en cultivant in vitro les cellules tumorales avant injection in vivo afin d’obtenir des modèles de CDOX (Cell Derived Orthotopic Xenograft). Ainsi, nous avons obtenu 15 PDOX et 8 CDOX bioluminescentes de DIPG différents à partir de biopsies de patient au diagnostic ; ainsi que 3 PDOX d’EPN-PF et 1 PDOX d’EPN-ST à partir de résections tumorales de patient au diagnostic ou à la rechute. Une analyse approfondie des tumeurs de DIPG obtenues montre que les xénogreffes conservent le phénotype de la tumeur du patient, en particulier les principales caractéristiques du DIPG, tout en reflétant l’hétérogénéité interindividuelle observée chez les patients. L’histologie des xénogreffes d’EPN relève leur pertinence vis-à-vis de la maladie. A partir de ces PDOX, nous avons pu générer 3 PDOX bioluminescentes sans sélection clonale in vitro au préalable. Puis, nous avons évalué le mébendazole dans deux modèles CDOX de DIPG. La toxicité du médicament utilisé à forte dose de manière chronique n’a pas permis de mettre en évidence un effet thérapeutique décisif. Enfin, nous avons adapté un système d’ouverture de la barrière hématoencéphalique (BHE) à l’aide d’ultrasons non focalisés couplés à des microbulles dans nos modèles in vivo deDIPG. Malgré l’ouverture de la BHE, il n’a pas été possible de potentialiser l’effet thérapeutique du panobinostat dans une CDOX de DIPG, le passage du médicament n’étant pas augmenté dans le tissu cérébral. Nous sommes actuellement en cours d’évaluation d’un nouveau médicament candidat, l’irinotécan.
... About 100 years ago, after the intravenous injection of a proper dye, it was observed that most of the organs other than the brain were dyed. It was understood that this situation is due to a specific structure of vessels between the brain and blood, which is called the blood-brain barrier (BBB) [1]. The BBB is also one of the most complicated barrier to pass for the therapeutic drugs, and because of the structure of the BBB, only a few small molecules with appropriate lipophilicity, molecular weight, and charge can penetrate through the BBB and pass in the central nervous system (CNS). ...
The blood-brain barrier is one of the most complicated barrier to pass for therapeutic drugs. Because of the structure of the blood-brain barrier, only a few small molecules with appropriate lipophilicity, molecular weight, and charge can penetrate through the blood-brain barrier and pass in the central nervous system. Because of this unique property, blood-brain barrier is still a major problem for the treatment of central nervous system diseases. In the last decades, many strategies to overcome this barrier have been investigated. Compared to other drug delivery strategies, due to the reduced side effects and no requirement for surgical operations, brain targeted nanoparticle is one the most promising and popular strategy used do deliver drugs to the brain. Many in vitro and in vivo preclinical studies have been conducted to determine optimum brain targeted nanoparticles. These studies were reported that characteristics of nanoparticles such as particle size, zeta potential, and targeting ligand are critical to achieving the goals. In this review, first of all, the structure of the blood-brain barrier and possible causes of blood-brain barrier disruption were summarized. Later, previous strategies of brain targeted drug delivery and characteristic prosperities for optimized brain-targeted nanoparticles were evaluated. Moreover, different strategies, such as focus ultrasound, which can increase the effectiveness of nanoparticular system applications, are mentioned.
... Tight junctions between the cuboidal epithelial cells create a third CNS barrier, the blood-CSF barrier (18) (Figure 2). The BBB concept was introduced in 1885, when Paul Ehrlich observed that certain aniline dyes injected into the blood stained all the tissues of the body except those of the CNS (19). CNS selective impermeability derives from its structure of endothelial cells connected by tight junctions, with densely populated pericytes embedded in an ensheathing basement membrane, and astrocytic end feet wrapped around these individual vessels (20). ...
Neuroimmunology, albeit a relatively established discipline, has recently sparked numerous exciting findings on microglia, the resident macrophages of the central nervous system (CNS). This review addresses meningeal immunity, a less-studied aspect of neuroimmune interactions. The meninges, a triple layer of membranes—the pia mater, arachnoid mater, and dura mater—surround the CNS, encompassing the cerebrospinal fluid produced by the choroid plexus epithelium. Unlike the adjacent brain parenchyma, the meninges contain a wide repertoire of immune cells. These constitute meningeal immunity, which is primarily concerned with immune surveillance of the CNS, and—according to recent evidence—also participates in postinjury CNS recovery, chronic neurodegenerative conditions, and even higher brain function. Meningeal immunity has recently come under the spotlight owing to the characterization of meningeal lymphatic vessels draining the CNS. Here, we review the current state of our understanding of meningeal immunity and its effects on healthy and diseased brains.
... Paul Ehrlich and his student Edwin Goldmann demonstrated the impermeability of the CNS to numerous dyes administered by peripheral injection. 1 To date, three dynamic and homeostatic interfaces are known to collectively contribute to the regulation of the movement of molecules, ions and cells between the CNS and the peripheral blood, the degree of which is subjected to the substance itself and the present pathophysiological condition(s) 2 , in a physical, transport, immunological and metabolic manner 3 : 1) the blood-brain barrier (BBB) separating the brain interstitial fluid from the blood, 2) the blood-cerebrospinal fluid (CSF) barrier (BCSFB) separating the choroid plexus, the secretion site of the CSF, and from the blood and 3) the arachnoid epithelium separating the subarachnoid CSF from the blood. 3,4 The BBB was a concept first proposed by Lina Stern in the 1921 and it was a widely held view that the high density of astrocytes around the epithelium was the main contributor to the barrier function. ...
An explosion of research using numerous in vitro and in vivo techniques highlighted the significant roles of P-glycoprotein (P-gp), an ATP-binding cassette (ABC) efflux transporter, in the blood-brain barrier (BBB) in physiology, pathology, pharmacotherapy and drug delivery. A relatively new in vitro technique exploiting fluorescent substrates, nonfluorescent inhibitors, confocal microscopy and quantitative image analysis enabled
transporter research in isolated mammalian and fish brain capillaries, leading to numerous novel discoveries. Two fluorescent P-gp substrates, Rhodamine 123 (Rho123) and EverFluor FL verapamil HCl (EFV) commonly used in P-gp research have not been investigated using
this particular technique, whereas verapamil HCl (Ver) has been previously employed as a non-fluorescent P-gp inhibitor. This thesis endeavoured to 1) establish this technique in the laboratory using Rho123 and Ver as probes, before evaluating the in vitro P-gp efflux transport of 2) Rho123 and 3) EFV using Ver as the P-gp inhibitor with this technique. Brain capillaries were isolated from 6-week old Slc:Wistar/ST rats by brain dissection, homogenisation and serial centrifugations and filtrations. The capillaries were incubated in the fluorescent substrate (1 μM Rho123 or 1 μM EFV) ± the inhibitor (100 μM Ver) for 30 min (Rho123) or 60 min (EFV), followed by confocal imaging. After the images have been transformed to greyscale on Adobe Photoshop, the fluorescence intensities were measured on
ImageJ. The average net fluorescence intensities for the two treatment groups, i.e. control and +inhibitor, were compared using two-tailed student t-test, where the null hypothesis was rejected if p < 0.05. The established procedure was successful in isolating rat brain capillaries
of retained morphology, high quantity, purity and viability which were suitable for the transport study. Several identified procedural issues, i.e. not freshly prepared chemicals and dissimilar incubation periods for the capillaries in their respective chemical(s), were improved accordingly. The importance of maintained and calibrated confocal microscopy
was also highlighted. In the case of Rho123, the luminal fluorescence intensities in the control and +inhibitor capillaries were 23.89 ± 2.39 and 18.32 ± 1.45, respectively (p > 0.05). It was proposed that further procedural optimisation would be required to accommodate the sensitivity and promiscuity of Rho123, which could possibly be surmounted by the use of a more specific P-gp inhibitor, e.g. cyclosporine A. For EFV, luminal fluorescence intensities in the control and +inhibitor capillaries were 112.56 ± 4.94 and 58.85 ± 4.52, respectively (p < 0.01). This demonstrated the in vitro efflux transport of EFV by P-gp in the BBB of the isolated rat brain capillaries, which is in accordance with other in vitro and in vivo studies in the literature. Notably, this finding also indirectly ascertained the localisation of P-gp on the luminal membrane of the BECs, where it is involved in xenobiotic efflux from BECs to peripheral blood. Overall, this thesis demonstrated and endorsed the practicality of investigating in vitro P-gp efflux transport of fluorescent substrates in the BBB using isolated rat brain capillaries. Notably, research in correlating the findings generated using this technique with those from other in vitro and in vivo studies is warranted to endorse its translationability.
In a myriad of developmental and degenerative brain diseases, characteristic pathological biomarkers are often associated with cerebral blood flow and vasculature. However, the relationship between vascular dysfunction and markers of brain disease is not well-defined. Additionally, it is difficult to deliver effective therapeutics to the brain due to the highly regulated blood-brain barrier (BBB) at the microvasculature interface of the brain. This Review first covers the need for modeling the BBB and the challenges of modeling the BBB. In vitro models of the BBB enable the study of the relationship between vascular dysfunction, BBB function, and disease progression and can serve as a platform to screen therapeutics. In particular, microfluidic-based in vitro BBB models are useful for studying brain vasculature as they support cell culture within the presence of continuous perfusion, which mirrors the in vivo flow and associated stress conditions in the brain. Early microfluidic models of the BBB created the most simplistic models possible that still displayed some functional aspects of the in vivo BBB. Therefore, this Review also discusses the emerging unique ways in which microfluidics in tandem with recent advancements in cell culture, biomaterials, and in vitro modeling can be used to develop more complex and physiologically relevant models of the BBB. Finally, we discuss the current and future state-of-the-art application of microfluidic BBB models for drug development and disease modeling, and the ongoing areas of needed innovation in this field.
For the brain to operate normally and to develop with structural integrity in addition to neuronal function, blood-brain barrier present in brain capillaries serves as a vital barrier mechanism. In addition to the transport barrier created by membranes, transporters, and vesicular processes, the structure and function of the BBB are summarised. The physical barrier is created by endothelial tight junctions. The permeability and transport of molecules between extracellular fluid and plasma are constrained by the presence of tight junctions between neighbouring endothelial cells. Each solute must pass through both membranes in the luminal and abluminal divisions. The functions of the neurovascular unit are described, with special emphasis on the pericytes, microglia, and astrocyte endfeet. The luminal membrane contains five separate facilitative transport mechanisms, each of which is exclusive to a few substrates. Nevertheless, the import of big-branched and aromatic neutral amino acids is facilitated by two key carriers (System L and y+) in the plasma membrane. It is asymmetrically present in both membranes. The sodium pump Na+/K+-ATPase is highly expressed in the abluminal membrane, where many Na+ dependent transport mechanisms move amino acids against its concentration gradient. The trojan horse strategy, which uses molecular tools to bind the medication and its formulations, is also preferred in drug delivery. The BBB's cellular structure, the transport systems unique to each substrate, and the necessity to identify transporters with changes that assist the transfer of various medications have all been changed in the current work. Nevertheless, to rule out the BBB passage for the new class of neuroactive medications, the mixing of traditional pharmacology and nanotechnology needs to be focused on outcomes that show promise.
Neurodegenerative diseases (NDs) such as dementia, Alzheimer’s disease, Parkinson’s disease, frontotemporal dementia, and amyotrophic lateral sclerosis are some of the most prevalent disorders currently afflicting healthcare systems. Many of these diseases share similar pathological hallmarks, including elevated oxidative stress, mitochondrial dysfunction, protein misfolding, excitotoxicity, and neuroinflammation, all of which contribute to the deterioration of the nervous system’s structure and function. The development of diagnostic and therapeutic materials in the monitoring and treatment of these diseases remains challenging. One of the biggest challenges facing therapeutic and diagnostic materials is the blood–brain barrier (BBB). The BBB is a multifunctional membrane possessing a plethora of biochemical, cellular, and immunological features that ensure brain homeostasis by preventing the entry and accumulation of unwanted compounds. With regards to neurodegenerative diseases, the recent application of tailored nanomaterials (nanocarriers and nanoparticles) has led to advances in diagnostics and therapeutics. In this review, we provide an overview of commonly used nanoparticles and their applications in NDs, which may offer new therapeutic strategies for the prevention and treatment of neurodegenerative diseases.
Passing time has seen numerous developments in the computational approaches and applications. All this has shown a specialized positive impact in the field of Biomedicine and lead to encouragement of interdisciplinary fields like medical informatics, bio-informatics, nano-technology, nano-informatics, computational biology, system biology, etc. Present work embraces the analytics of the advancements and unearthing across the most deadly diseases in the world. Major emphasis is on the improvements in the drug delivery methods to ensure site-specificity and effectiveness of the potential drugs. It is the most important aspect of the present targeted therapies is the drug delivery vehicles. Paper revolves around the characteristics of an ideal drug delivery system. Is it Efficiency? Biocompatibility? Or just Nonimmunogenicity? Yes, here drug-carriers are on the spotlight. Additionally we also focused the deciding factors like drug circulation time, its ADMET aspects and chemical descriptors that are the indispensable part of a drug-carrier system. In the end publication survey results provides a suitable podium to present work. The possibilities of novel developments offer the clear cut proof of increasing popularity of biological lipid vesicles and nano-scale drug delivery systems. Citations involve current market and clinical status of such systems in our present day RandD and pharma-market.
Background of the blood brain barrier (BBB) in brain capillaries constitutes an essential barrier mechanism for normal functioning and development of the brain of structural integrity besides neuronal function. The structure and function of the BBB is summarized besides the physical barrier formed by the endothelial tight junctions, and the transport barrier resulting from membrane transporters and vesicular mechanisms. The presence of tight junctions between adjacent endothelial cells restricts permeability and movement of molecules between extracellular fluid and plasma. It is divided into luminal and abluminal where each solute must cross both membranes. Methodology was followed by including all information regarding transporters available and its role in brain by systematic search of various electronic databases, including Science Direct, Scopus, Google Scholar, Research Gate and PubMed. The keywords Neuroimaging, Alzheimer’s, transporters, endothelial membrane, blood brain barrier, Cognition were used for the study. No time limitation was set for including the studies. Results showed the roles of neurovascular unit are outlined, especially the astrocyte endfeet, pericytes and microglia. Five different systems of facilitative transport are found in luminal membrane and specific for few substrates. Nonetheless, two major facilitative carriers (System L and y+) are located in both membranes asymmetrically. In contrast, several Na+ dependent transport systems transport amino acids against its concentration gradient present in abluminal membrane, where the sodium pump Na+/K+-ATPase is highly expressed. Trojan horse mechanism is also favored in drug delivery by employing molecular tools to bind drug and its formulations. In Conclusion, the blending of the classical pharmacology with nanotechnology needs to be focused with promising results to rule out the BBB passage for the new generation of neuroactive drugs.
Endothelial cells form the innermost layer of all blood vessels and are the only vascular component that remains throughout all vascular segments. The cerebral vasculature has several unique properties not found in the peripheral circulation; this requires that the cerebral endothelium be considered as a unique entity. Cerebral endothelial cells perform several functions vital for brain health. The cerebral vasculature is responsible for protecting the brain from external threats carried in the blood. The endothelial cells are central to this requirement as they form the basis of the blood-brain barrier. The endothelium also regulates fibrinolysis, thrombosis, platelet activation, vascular permeability, metabolism, catabolism, inflammation, and white cell trafficking. Endothelial cells regulate the changes in vascular structure caused by angiogenesis and artery remodeling. Further, the endothelium contributes to vascular tone, allowing proper perfusion of the brain which has high energy demands and no energy stores. In this article, we discuss the basic anatomy and physiology of the cerebral endothelium. Where appropriate, we discuss the detrimental effects of high blood pressure on the cerebral endothelium and the contribution of cerebrovascular disease endothelial dysfunction and dementia. © 2022 American Physiological Society. Compr Physiol 12:3449-3508, 2022.
Blood-brain barrier (BBB), a unique membrane barrier formed by closely stitched brain capillary endothelial cells (BCEC) with tight cellular junctions, separates brain from the circulating blood to protect it from bloodborne pathogens. BBB greatly limits the entry of chemotherapeutics to brain, and in consequence, it is a major obstacle for treating brain tumor. Advances in designing efficient nano-drug carriers are opening new avenues for overcoming this uphill systemic challenge. This book chapter describes current understanding of nanocarriers-mediated noninvasive drug targeting to brain tumor. Design principles behind the construction of the most promising recently designed receptor and transporter selective nano-drug carriers for combating brain tumors have been highlighted.
At present, therapeutics for the diseases of central nervous system (CNS) are complicated by the presence of blood‐brain barrier (BBB). BBB maintains intracranial homeostasis and facilitates brain‐body communications, but hinders the effectiveness of drug delivery system based on nanoparticles (NPs). Great efforts have been devoted to the elevation of NPs‐based brain delivery over the past decades. In addition to the chemical modifications, biomimetic technologies such as cell membranes or neurotropic viruses camouflage, immune cells or extracellular vesicles (EVs) loading are promising candidates to make NPs ideal vehicles. This review summarizes the characteristics and transport mechanisms of BBB, and the recent advances in biomimetic technologies, administration methods that enhance the BBB penetration and targeting capabilities of NPs.
The endocannabinoid system (ECS) has been found at the blood-brain barrier (BBB), as Cannabinoid receptors were characterized in human brain microvascular endothelial cells and astrocytes. In several in vitro and in vivo studies, cannabinoids decreased BBB permeability and enhanced membrane integrity, which may be achieved through endothelial tight junctions and other mechanisms. These permeability regulation effects of cannabinoids suggested that the ECS may protect the brain by enhancing barrier integrity. Related questions about cannabinoid-drug interaction and drug distribution across the BBB are also raised. Specifically, can cannabinoids significantly reduce drug bioavailability to the brain? More in-depth and systematic investigations are needed to characterize and quantify these effects of cannabinoids on brain microvasculature physiopathology. Therefore, this review summarizes literatures from different disciplines to promote more research on assessing the therapeutic benefits and risks of using cannabinoids to protect BBB from dysfunctions or breakdown, and to avoid consequent brain damages due to inflammation, neurodegenerations, hemorrhage, ischemia, or other causes.
Brain metastases account for the majority of malignant brain tumors and as newer cancer treatments improve patient survival, the reported incidence of brain metastases is increasing. Lung cancer is the most frequent origin of metastases to the brain, and this diagnosis is associated with significant morbidity as well as decreased quality of life and a worse prognosis. The treatment for brain metastases in NSCLC has historically been local therapy, either surgery or radiation, as many chemotherapies have limited efficacy in the brain. These strategies are highly effective but can be a source of morbidity themselves. Newer systemic therapies, including targeted small molecule drugs and immunotherapy, have shown promise in treating NSCLC associated CNS disease either alone or in combination with local therapies. Increasing evidence for this strategy is accumulating as more clinical trials allow the inclusion of patients with untreated asymptomatic brain metastases. This chapter summarizes the current use of systemic therapy in the treatment of brain metastases in NSCLC.
Alzheimer's disease (AD) drug discovery is the alarming need for the generation-alpha, given the world population is getting older and the cost of healthcare increasing day by day. The intricacy is to correctly judge the targeted drug delivery methods parallel to the evaluation of new-age drugs. In the current era intranasal (IN) delivery is overly being discussed as a repurposing topical treatment tool. This bona fide route has been experimentally explored for trauma care and emergency medication services but not extensively to treat neurodegenerative diseases, especially AD. Until date, despite the progress in nanotechnology-based targeted drug delivery, the only two formulae tested in clinical trials for AD therapeutics through IN delivery are insulin and rivastigmine. Keeping in mind the advantages of IN drug delivery, including enhanced bioavailability, slow drug decomposition, avoidance of the first-pass metabolism in the liver, the chance of AD drug regimens' expansion has been discussed in this present review. Here we present a glimpse of the roadmap towards the AD-IN therapeutics with an update of current researches along with the scope of further modifications.
Over the last few years, organic bioelectronics has experienced an exponential growth with applications encompassing platforms for tissue engineering, drug delivery systems, implantable, and wearable sensors. Although reducing the physical and mechanical mismatch with the human tissues allows to increase the coupling efficiency, several challenges are still open in terms of matching biological curvature, size, and interface stiffness. In this context, the replacement of bulky with more flexible and conformable devices is required, implying the transition from inorganic conventional electronics to organic electronics. Indeed, the advent of organic materials in bioelectronics, due to the indisputable benefits related to biocompatibility, flexibility, and electrical properties, has granted superior coupling properties with human tissues increasing the performances of both sensing and stimulation platforms. In this review the ease of functionalization and patterning of conductive polymers (CPs) will be analyzed as a strategy that enables the fabrication of platforms with high structural flexibility ranging from the macro to the micro/nano‐scales, leading to the increase of devices sensitivity. Drawing from the concept of biomimicry, the human body tissues interfaces will be explored through an ideal journey starting from organic platforms for epidermal sensing and stimulation. Then, devices capable of establishing a dynamic coupling with the heart will be reviewed and finally, following the circulatory system and crossing the blood‐brain barrier, the brain will be reached and novel sensing and computing implants advances that pave the way to the possibility to emulate as well as to interact with the neural functions will be analyzed.
Background
Glioma is the most aggressive and lethal tumor of the central nervous system. Owing to the cellular heterogeneity, the invasiveness, and blood-brain barrier (BBB), current therapeutic approaches, such as chemotherapy and radiotherapy, are poorly to obtain great anti-tumor efficacy. However, peptides, a novel type of therapeutic agent, displayed excellent ability in the tumor, which becomes a new molecule for glioma treatment.
Method
We review the current knowledge on peptides for the treatment of glioma through a PubMed-based literature search.
Results
In the treatment of glioma, peptides can be used as (i) decoration on the surface of the delivery system, facilitating the distribution and accumulation of the anti-tumor drug in the target site;(ii) anti-tumor active molecules, inhibiting the growth of glioma and reducing solid tumor volume; (iii) immune-stimulating factor, and activating immune cells in the tumor microenvironment or recruiting immune cells to the tumor for breaking out the immunosuppression by glioma cells.
Conclusion
The application of peptides has revolutionized the treatment of glioma, which is based on targeting, penetrating, anti-tumor activities, and immunostimulatory. Moreover, better outcomes have been discovered in combining different kinds of peptides rather than a single one. Until now, more and more preclinical studies have been developed with multifarious peptides, which show promising results in vitro or vivo with the model of glioma.
Glioma is the most common type of Central Nervous System (CNS) neoplasia and it arises from glial cells. As glial cells are formed by different types of cells, glioma can be classified according to the cells that originate it or the malignancy grade. Glioblastoma multiforme is the most common and aggressive glioma. The high lethality of this tumor is related to the difficulty in performing surgical removal, chemotherapy, and radiotherapy in the CNS. To improve glioma treatment, a wide range of chemotherapeutics have been encapsulated in nanosystems to increase their ability to overcome the blood-brain barrier (BBB) and specifically reach the tumoral cells, reducing side effects and improving drug concentration in the tumor microenvironment. Several studies have investigated nanosystems covered with targeting ligands (e.g., proteins, peptides, aptamers, folate, and glucose) to increase the ability of drugs to cross the BBB and enhance their specificity to glioma through specific recognition by receptors on BBB and glioma cells. This review addresses the main targeting ligands used in nanosystems to overcome the BBB and promote the active targeting of drugs for glioma. Furthermore, the advantages of using these molecules in glioma treatment are discussed.
Among all central nervous diseases, malignant glioma is a crucial part that deserves more attention since high fatality and disability rate. There are several therapeutic strategies applied to the treatment of malignant glioma, especially certain chemotherapy-related treatments. However, the existence of the blood-brain barrier (BBB) seriously hinders the strategy's progress, so how to escape from the barriers is a fascinating question. Herein, we comprehensively discussed the details of malignant glioma and the BBB's functional morphology and summarized several routes bypassing the BBB. Additionally, since possessing excellent properties for drug delivery, we provided an insight into various promising natural polymeric materials and highlighted their applications in the treatment of malignant glioma.
Les nanoémulsions huile dans eau (H/E) sont utilisées depuis plus de 50 ans en clinique humaine comme source de lipides en nutrition parentérale. Si cette dernière décennie a vu émerger la mise à profit de cette forme comme véhicule de substances actives lipophiles, l’utilisation des nanoémulsions comme vecteur d'agents thérapeutique ou diagnostique reste encore sous-exploitée. L’objectif de cette thèse a été le développement d'une plateforme de nanoémulsions comme vecteur alternatif aux nanosystèmes classiquement utilisés. Deux applications ont été visées : le diagnostic de la plaque vulnérable d'athérosclérose et le traitement de la maladie de Parkinson. Les nanoémulsions ont été fonctionnalisées avec des anticorps humanisés dirigés contre l’athérome et chargées avec des particules magnétiques pour servir d’agent de contraste moléculaire pour l’imagerie par résonance magnétique (IRM) et pour une nouvelle technique : l’imagerie par particules magnétique (IPM). L'efficacité du nanosystème pour le ciblage de la plaque a été démontré sur des souris athéromateuses. L’inclusion de chromophores lipophiles originaux et ultrabrillants ainsi que la possibilité d'incorporer des substances actives ont permis d’ouvrir la voie vers le développement de formulations multimodales et théranostiques. Les nanoémulsions thérapeutiques contre Parkinson ont été développées pour rétablir le pH lysosomal des neurones dopaminergiques par l'encapsulation d'un polymère (PLGA). Ce défaut d’acidification favorise la mort cellulaire par l’accumulation de déchets dans les neurones. La formulation a été optimisée pour le passage intracérébral par voie intraveineuse ou intranasale. Les résultats montrent un passage cérébral in vivo par voie intraveineuse avec une confirmation in vitro de la régénération du pH. Les perspectives de ce travail sont la poursuite de la plateforme et l'ouverture vers de nouvelles applications comme l'hyperthermie magnétique dans les cancers.
The administration of drugs to the CNS has become a difficult task because several barriers in the brain, i.e. the blood-brain barrier (BBB) and a blood-cerebrospinal fluid barrier must be overcome. Several methods to assist in overcoming the BBB have been implemented in recent years, such as osmotic obstruction with the BBB and chemical amplification of prodrugs. Brain targeting creates new doors for scientists to move forward for better investigations so that people with brain diseases can be treated. The current demand is to establish drug delivery techniques to allow the molecules of the drug to efficiently cross the BBB and this study discusses these strategies that efficiently improve the delivery of brain-targeted drugs. Therefore, drugs powered by nanoparticles are being developed via advanced non-invasive methods to control neurological disorders. This study will explore and examine the anatomy and physiology of the BBB, the concept of Magic Bullet, the pathways for drug transport, drug permeability assessment strategies for BBB, and endogenous cells possible interventions as nanoparticles supply cells.
Drug delivery is basically the process of transferring a drug entity or formulation to the desired target following administration via various routes in the human body. In this chapter we will be focusing on nose to brain drug delivery. Nose to brain drug delivery, as the title indicates, is delivering a drug or targeting the drug to brain via the nasal route. This technique is noninvasive and relies on the highly permeable nasal mucosa, which allows rapid drug absorption. Drug enters the brain from the olfactory region bypassing the blood-brain barrier. The blood-brain barrier has tight junctions that allows only a specific amount and a specific size of drug particles to enter or pass through to provide a desired therapeutic effect in brain. Nanoparticles of size below 100 nm may only pass through blood-brain barrier and have been used in the form of various formulations for the treatment of different neurological diseases. In recent years, nanoformulations are widely being used for the treatment of CNS diseases. Epilepsy is a chronic neurodegenerative disease with recurrent seizure episodes affecting nervous system and it is a life-threatening disease that requires immediate treatment. Current conventional therapy has limited bioavailability via oral, intravenous, and rectal administration. To overcome these limitations, nose to brain delivery using nanoformulations has shown promising results for the treatment of this disease.
Knowledge about the transport of active compounds across the blood-brain barrier is of essential importance for drug development. Systemically applied drugs for the central nervous system (CNS) must be able to cross the blood-brain barrier in order to reach their target sites, whereas drugs that are supposed to act in the periphery should not permeate the blood-brain barrier so that they do not trigger any adverse central adverse effects. A number of approaches have been pursued, and manifold in silico, in vitro, and in vivo animal models were developed in order to be able to make a better prediction for humans about the possible penetration of active substances into the CNS. In this particular case, however, in vitro models play a special role, since the data basis for in silico models is usually in need of improvement, and the predictive power of in vivo animal models has to be checked for possible species differences. The blood-brain barrier is a dynamic, highly selective barrier formed by brain capillary endothelial cells. One of its main tasks is the maintenance of homeostasis in the CNS. The function of the barrier is regulated by cells of the microenvironment and the shear stress mediated by the blood flow, which makes the model development most complex. In general, one could follow the credo “as easy as possible, as complex as necessary” for the usage of in vitro BBB models for drug development. In addition to the description of the classical cell culture models (transwell, hollow fiber) and guidance how to apply them, the latest developments (spheroids, microfluidic models) will be introduced in this chapter, as it is attempted to get more in vivo-like and to be applicable for high-throughput usage with these models. Moreover, details about the development of models based on stem cells derived from different sources with a special focus on human induced pluripotent stem cells are presented.
The intravital deposition of silver in the chorioid plexuses, area postrema, intercolumnar tubercle, neurohypophysis, and pineal body of rats, given 1.5 gm. of silver nitrate per liter of drinking water for periods of up to one year, has been investigated by electron microscopy. Unlike other parts of the central nervous system, these regions store large amounts of silver. In all of these structures, silver is deposited in the form of dense granules in the basement membrane upon which the capillary endothelium rests, in and upon the connective tissue cells and fibers constituting a loose pericapillary sheath, and in an outer membrane separating this sheath from the parenchymatous cells. Parts of the central nervous system which do not store silver, for example the spinal cord, cerebellar cortex, cerebral cortex, and reticular formation, lack a connective tissue investment of the capillaries. In these locations, the glial processes or end-feet are closely applied to the walls of the capillaries. Only a narrow space, filled by an amorphous, moderately electron-dense substance, separates the plasma membranes of the endothelial cells and glial processes. The significance of these observations is discussed with respect to the questions of the Virchow-Robin perivascular spaces, the interstitial ground-substance of the brain, and the location of the hematoencephalic barrier.
The development of the human blood-brain and blood-CSF barriers
The commonly held belief that the fetal blood-brain and blood-CSF barriers are immature is reviewed. Results obtained from carefully conducted experiments with horseradish peroxidase and optimal freeze-fracturing suggest that the chick, rat and monkey brain barrier systems to proteins are tight from the earliest stages of development. Previous studies are reviewed in the light of new information on retrograde axonal transport, circumventricular organs, the proper use of horseradish peroxidase, freeze-fracturing, immunocytochemistry and plasma protein gene expression in the developing human brain. Original data on the development of human brain barrier systems are included. Tight junctions between cerebral endothelial and choroid plexus epithelial cells form the morphological basis for these systems. CSF in the fetus contains a remarkably high concentration of protein in contrast to adult CSF which is characterized by a very low protein concentration. This has previously been interpreted as due to immaturity of barriers in the fetal brain. Tight junctions between cerebral endothelial cells and between choroid plexus epithelial cells have been investigated in human embryos and fetuses by freeze fracture and thin section electron microscopy. As soon as the choroid plexus and the brain capillaries differentiated they exhibited well formed tight junctions. These junctions were very complex at early stages of development. A new barrier consisting of ‘strap junctions’ was found in the developing germinal matrix. The very high concentration of protein in early human fetal CSF cannot be accounted for by a lack of tight junctions
The commonly held belief that the fetal blood-brain and blood-CSF barriers are immature is reviewed. Results obtained from carefully conducted experiments with horseradish peroxidase and optimal freeze-fracturing suggest that the chick, rat and monkey brain barrier systems to proteins are tight from the earliest stages of development. Previous studies are reviewed in the light of new information on retrograde axonal transport, circumventricular organs, the proper use of horseradish peroxidase, freeze-fracturing, immunocytochemistry and plasma protein gene expression in the developing human brain. Original data on the development of human brain barrier systems are included. Tight junctions between cerebral endothelial and choroid plexus epithelial cells form the morphological basis for these systems. CSF in the fetus contains a remarkably high concentration of protein in contrast to adult CSF which is characterized by a very low protein concentration. This has previously been interpreted as due to immaturity of barriers in the fetal brain. Tight junctions between cerebral endothelial cells and between choroid plexus epithelial cells have been investigated in human embryos and fetuses by freeze fracture and thin section electron microscopy. As soon as the choroid plexus and the brain capillaries differentiated they exhibited well formed tight junctions. These junctions were very complex at early stages of development. A new barrier consisting of 'strap junctions' was found in the developing germinal matrix. The very high concentration of protein in early human fetal CSF cannot be accounted for by a lack of tight junctions in the developing brain barrier systems. Some transfer of proteins from blood to CSF, possibly via an intracellular route, has been demonstrated in immature experimental animals, but it seems that an important contribution to CSF proteins in the fetus may be synthesis by the developing brain and choroid plexuses with subsequent release into the CSF.
Certain junctions between ependymal cells, between astrocytes, and between some electrically coupled neurons have heretofore been regarded as tight, pentalaminar occlusions of the intercellular cleft. These junctions are now redefined in terms of their configuration after treatment of brain tissue in uranyl acetate before dehydration. Instead of a median dense lamina, they are bisected by a median gap 20-30 A wide which is continuous with the rest of the interspace. The patency of these "gap junctions" is further demonstrated by the penetration of horseradish peroxidase or lanthanum into the median gap, the latter tracer delineating there a polygonal substructure. However, either tracer can circumvent gap junctions because they are plaque-shaped rather than complete, circumferential belts. Tight junctions, which retain a pentalaminar appearance after uranyl acetate block treatment, are restricted primarily to the endothelium of parenchymal capillaries and the epithelium of the choroid plexus. They form rows of extensive, overlapping occlusions of the interspace and are neither circumvented nor penetrated by peroxidase and lanthanum. These junctions are morphologically distinguishable from the "labile" pentalaminar appositions which appear or disappear according to the preparative method and which do not interfere with the intercellular movement of tracers. Therefore, the interspaces of the brain are generally patent, allowing intercellular movement of colloidal materials. Endothelial and epithelial tight junctions occlude the interspaces between blood and parenchyma or cerebral ventricles, thereby constituting a structural basis for the blood-brain and blood-cerebrospinal fluid barriers.
From 10 minutes to 3(1/2) hours after the intraventricular injection into rats of 15 to 100 mg of ferritin, an appreciable fraction of the protein, visualized electron microscopically, traverses the ependymal epithelium by diffusing along the dense intercellular substance of the luminal open junction and thence, by circumventing discrete intercellular fusions which partition rather than seal the interspace. These partitions shunt additional protein into the cell, where ferritin is transported within pinocytotic vesicles to the lateral and basal plasma-lemma and, presumably, back into the interspace again. The basal interspace is irregularly distended by pools of moderately dense "filler" within which ferritin accumulates. The larger fraction of protein enters the ependyma by pinocytosis and is eventually segregated within membrane-enclosed organelles such as vacuoles, multivesicular bodies, and dense bodies, where the molecules may assume a crystalline packing. As a result of the accumulation of ferritin within these inclusions and within filler substance, only a small amount of protein remains to enter the underlying parenchyma. Presentation of ferritin to prefixed cells leads to a random dispersion of free cytoplasmic ferritin. This artifactual distribution in both prefixed and postfixed cells is concurrent with disruption of cell membranes.
Horseradish peroxidase was administered to mice by intravenous injection, and its distribution in cerebral cortex studied with a recently available technique for localizing peroxidase with the electron microscope. Brains were fixed by either immersion or vascular perfusion 10-60 min after administration of various doses of peroxidase. Exogenous peroxidase was localized in the lumina of blood vessels and in some micropinocytotic vesicles within endothelial cells; none was found beyond the vascular endothelium. Micropinocytotic vesicles were few in number and did not appear to transport peroxidase while tight junctions between endothelial cells were probably responsible for preventing its intercellular passage. Our findings therefore localize, at a fine structural level, a "barrier" to the passage of peroxidase at the endothelium of vessels in the cerebral cortex. The significance of these findings is discussed, particularly with reference to a recent study in which similar techniques were applied to capillaries in heart and skeletal muscle.
The transendothelial passage of horseradish peroxidase, injected intravenously into mice, was studied at the ultrastructural level in capillaries of cardiac and skeletal muscle. Peroxidase appeared to permeate endothelial intercellular clefts and cell junctions. Abnormal peroxidase-induced vascular leakage was excluded. Neutral lanthanum tracer gave similar results. The endothelial cell junctions were considered to be maculae occludentes, with gaps of about 40 A in width between the maculae, rather than zonulae occludentes. Some observations in favor of concurrent vesicular transport of peroxidase were also made. It is concluded that the endothelial cell junctions are most likely to be the morphological equivalent of the small pore system proposed by physiologists for the passage of small, lipid-insoluble molecules across the endothelium.
In order to establish criteria for the identification of the neural and glial cells of the central nervous system, sections of the brains and spinal cords of mice, rabbits, guinea pigs, and rats; and portions of tumors of the human brain have been examined by electron microscopy.
Identification of neurons is made possible by the characteristic cytoplasmic picture, in which there is a distinct granular and less constant membranous ergastoplasmic pattern. In no other cell of the central nervous system is such a distinct granular component present in the ergastoplasm. The shape of the neuron in electron microscopic preparations is similar to that seen by light microscopy with several dendrites containing a similar cytoplasm arising from the perikaryon. Synapses are relatively common on the surface of the neuron and its dendrites.
Microglial cells are relatively small and dense with few processes, and are arranged as perineuronal and perivascular satellites for the most part. Occasionally phagocytized material is present in their cytoplasm. The oligodendroglial cells are identifiable by their position as perineuronal satellites and in the white matter as cells arranged in rows. They have a uniformly round to ovoid nucleus with a pale cytoplasm, which has a sparse, finely granular component and a few small mitochondria. The processes are few and relatively straight when cut in longitudinal section. The predominant cellular type in an oligodendroglioma was similar, with a pale cytoplasm. The astrocytes are variable in appearance. Their nuclei are moderately large, irregularly ovoid, and the cytoplasm adjacent to the nucleus is finely granular and scant. In the protoplasmic astrocytes the cytoplasm has a complicated infolded arrangement with reduplication of the plasma membrane, numerous processes extending radially from the cell and rebranching. To a certain extent this same folded plasma membrane was noted in the fibrous astrocytes. However, their more distant processes were narrowed, relatively straight, and filled with numerous dense fibrils. The processes of the astrocyte often surrounded axons, and other cellular processes, and surrounded some vessels, while attaching to a part of the wall of another vessel. Proliferating cells in experimentally produced gliosis and in astrocytic neoplasms were similar in structure.
The ependymal cells and the epithelium of the choroid plexus have a specialized surface with microvillous projections of the cytoplasm covered by the plasma membrane. Cilia in varying numbers are present in both epithelia.
It was attempted to preserve the water distribution in central nervous tissue by rapid freezing followed by substitution fixation at low temperature. The vermis of the cerebellum of white mice was frozen by bringing it into contact with a polished silver mirror maintained at a temperature of about -207 degrees C. The tissue was subjected to substitution fixation in acetone containing 2 per cent OsO(4) at -85 degrees C for 2 days, and then prepared for electron microscopy by embedding in Maraglas, sectioning, and staining with lead citrate or uranyl acetate and lead. Cerebellum frozen within 30 seconds of circulatory arrest was compared with cerebellum frozen after 8 minutes' asphyxiation. From impedance measurements under these conditions, it could be expected that in the former tissue the electrolyte and water distribution is similar to that in the normal, oxygenated cerebellum, whereas in the asphyxiated tissue a transport of water and electrolytes into the intracellular compartment has taken place. Electron micrographs of tissue frozen shortly after circulatory arrest revealed the presence of an appreciable extracellular space between the axons of granular layer cells. Between glia, dendrites, and presynaptic endings the usual narrow clefts and even tight junctions were found. Also the synaptic cleft was of the usual width (250 to 300 A). In asphyxiated tissue, the extracellular space between the axons is either completely obliterated (tight junctions) or reduced to narrow clefts between apposing cell surfaces.
For the purpose of studying the hematoencephalic barrier as it is concerned with silver circulating in the blood stream, silver nitrate was vitally administered to rats in their drinking water over periods of 6 to 8 months. The cerebrum, cerebellum, medulla, area postrema, and choroid plexus were prepared for light and electron microscopy. Silver deposition was found in the perivascular spaces in the choroid plexus, area postrema, in the medulla surrounding the area postrema, and in minute quantities in the cerebrum, cerebellum, and most of the medulla. Two levels of the hematoencephalic barrier were apparently demonstrated in our investigations. The endothelial linings of the vessels in the cerebrum, cerebellum, and medulla constitute the first threshold of the hematoencephalic barrier (specifically here, blood-brain barrier). The cell membranes adjacent to the perivascular spaces form the second threshold, as follows:—the neuroglial cell membranes in the cerebrum, cerebellum, and medulla (blood-brain barrier); the membranes of the neuroglial cells in the area postrema (blood-brain barrier); and the membranes of the epithelial cells of the choroid plexus (blood-cerebrospinal fluid barrier). This study deals with silver deposition and does not infer that the penetration of ionic silver, if present in the blood stream, would necessarily be limited to the regions described.
Bleb-like structures were observed to cover the epithelial cell surfaces in the choroid plexus. They may be cellular projections increasing the cell surface area or they may be secretory droplets.
The vascular system of the central nervous system is derived from capillary endothelial cells, which have invaded the early embryonic neuroectoderm. This process is called angiogenesis and is probably regulated by brain-derived factors. Vascular endothelial cell growth factor (VEGF) is an angiogenic growth factor whose expression correlated with embryonic brain angiogenesis, i.e. expression is high in the embryonic brain when angiogenesis occurs and low in the adult brain when angiogenesis is shut off under normal physiological conditions. VEGF receptors 1 and 2 (flt-1 and flk-1) as well as another pair of receptors (tie-1 and tie-2) are receptor tyrosine kinases specifically expressed in endothelial cells. Expression of these receptors is high during brain angiogenesis but low in adult blood-brain barrier endothelium. Signal transduction by these or other receptors involved in endothelial cell growth and differentiatian may be mediated by lyn, a nonreceptor tyrosine kinase expressed in brain endothelium. Induction and maintenance of blood-brain barrier endothelial cell characteristics (complex tight junctions, low number of vesicles, specialized transport systems) are regulated by the local brain environment; e.g. astrocytes. Tight junctions between brain endothelial cells are the structural basis for the paracellular impermeability and high electrical resistance of blood-brain barrier endothelium. Association of tight junction particles with the P-face along with the intercellular adhesion forces rather than the number or branching frequency of tight junction strands correlated with BBB development and function suggesting that the cytoplasmic anchoring of the tight junctions plays an important role.
1. The study of the blood–brain barrier and its various realms offers a myriad of opportunities for scientific exploration. This review focuses on two of these areas in particular: the induction of the blood–brain barrier and the molecular mechanisms underlying this developmental process.
2. The creation of the blood–brain barrier is considered a specific step in the differentiation of cerebral capillary endothelial cells, resulting in a number of biochemical and functional alterations. Although the specific endothelial properties which maintain the homeostasis in the central nervous system necessary for neuronal function have been well described, the inductive mechanisms which trigger blood–brain barrier establishment in capillary endothelial cells are unknown.
3. The timetable of blood–brain barrier formation is still a matter of debate, caused largely by the use of varying experimental systems and by the general difficulty of quantitatively measuring the degree of blood–brain barrier “tightness.” However, there is a general consensus that a gradual formation of the blood–brain barrier starts shortly after intraneural neovascularization and that the neural microenvironment (neurons and/or astrocytes) plays a key role in inducing blood–brain barrier function in capillary endothelial cells. This view stems from numerous in vitro experiments using mostly cocultures of capillary endothelial cells and astrocytes and assays for easily measurable blood–brain barrier markers. In vivo, there are great difficulties in proving the inductive influence of the neuronal environment. Also dealt with in this article are brain tumors, the least understood in vivo systems, and the induction or noninduction of barrier function in the newly established tumor vascularization.
4. Finally, this review tries to elucidate the question concerning the nature of the inductive signal eliciting blood–brain barrier formation in the cerebral microvasculature.
Autonomic preganglionic, sensory, and lower motoneuron perikarya within the central nervous system, as well as cell bodies with axons projecting to the circumventricular organs, are retrogradely labeled with horseradish peroxidase (HRP) delivered to their axon terminals by cerebral and extracerebral blood. Subsequent to vascular injection of HRP into mice, blood‐borne peroxidase passes across permeable vessels in muscle, ganglia, and in all circumventricular organs except for the subcommissural organ in which no leak could be discerned. Brain parenchyma adjacent to each of the permeable circumventricular organs quickly becomes inundated with the protein. By four to six hours post‐injection, this extracellular HRP reaction product has disappeared, and by eight hours perikarya of specific hypothalamic nuclei contain HRP‐positive granules indicative of the intra‐axonal retrograde transport of the protein. Hypothalamic neurons so labeled are presumed to send axons to such circumventricular organs as the median eminence or neurohypophysis and include neurons of the magnocellular neurosecretory supraoptic and paraventricular nuclei, the accessory magnocellular nuclei, the parvicellular arcuate nucleus, and a band of periventricular cells extending rostrally into the medial preoptic area. Labeled somata are also adjacent to the organum vasculosum of the lamina terminalis and in the vertical limb of the nucleus of the diagonal band of Broca. No similarly labeled cell bodies were identified near the subfornical organ.
At 12 hours post‐injection, HRP labeling of specific brain stem and spinal cord somata with axons efferent from the central nervous system indicates that protein from the peripheral blood can be incorporated by neurons of different functional categories for retrograde transport. Thus, blood‐borne peroxidase, imbibed presumably from myoneural clefts by motoneuron axon terminals, is transported to perikarya in cranial motor nerve nuclei III, IV, V, VI, VII, XII, the ambiguus nucleus, and in the ventral horn of the spinal cord. Sensory endings afferent to muscle spindles also take up HRP for retrograde transport, as manifested by the labeling of cell bodies in the mesencephalic nucleus of V. Autonomic preganglionic terminals take up HRP for transport back to their cell bodies in the intermediolateral sympathetic cell column in the spinal cord and to parasympathetic cell groups such as the brain stem dorsal motor vagus and salivatory nuclei.
Three cell groups in the brain stem that presumably have their efferent projections intrinsic to the central nervous system contain peroxidase‐labeled perikarya. These cell groups include a portion of the nucleus of the solitary tract rostral to the area postrema and the noradrenergic A1 and A5 nuclei of Dahlström and Fuxe ('64). The area postrema is thought to receive axon collaterals from the nucleus of the solitary tract (Morest, '60). Of the circumventricular organs, only the median eminence is believed to have a prominent noradrenergic innervation originating from somewhere in the brainstem. The peroxidase‐labeled A1 and A5 neurons may represent the origin of this innervation.
Vascular infusion of peroxidase results in retrograde neuronal labeling of neurosecretory, motor, sensory, and autonomic systems. The inference is made that other substances, such as toxins and neurovirulent viruses, can also enter these neuronal systems, as does peroxidase, from cerebral and extracerebral blood.
One to 20 mg of horseradish peroxidase (HRP) was injected into the allantoic vein of chick embryos between the 7th to 21st day of incubation and its penetration from blood into the interstitium of cerebellum and spinal cord (upper thoracic) was examined by light and electron microscopy in order to study development of the blood-brain barrier. Until the 12th day of incubation (stage 38) the reaction products, indicating the presence of peroxidase, were distributed in every region of the intercellular spaces in both cerebellum and spinal cord, whereas in 13th and 14th day embryos (stage 39 and 40) they were observed only in the medullary regions. In the embryos after 15th day (stage 41) they filled only the intravascular lumen and some micropinocytotic vesicles in the endothelium.
These results show that the blood-brain barrier to HRP develops roughly synchronously in various parts of the central nervous system and evolves independently of neurogenesis, the course of which differs considerably in the cerebellum and spinal cord.
1. Ion permeability of the blood-brain barrier was studied by in situ measurement of transendothelial electrical resistance in anaesthetized rats aged between 17 days gestation and 33 days after birth, and by electron microscopic examination of lanthanum permeability in fetal and neonatal rats aged up to 10 days old. 2. The blood-brain barrier in 17- to 20-day fetuses had a resistance of 310 omega cm2 but was impermeable to lanthanum, and therefore had properties intermediate between leaky and tight epithelia. 3. From 21 days gestation, the resistance was 1128 omega cm2, indicating a tight blood-brain barrier and low ion permeability. There was little further change in barrier resistance after birth, and in 28- to 33-day rats, when the brain barrier systems are mature in other ways, vessels had a mean resistance of 1462 omega cm2. 4. In the tight blood-brain barrier, arterial vessels had a significantly higher resistance than venous vessels, 1490 and 918 omega cm2 respectively. In vessels less than 50 microns diameter and within the normal 60 min experimental period, there was no significant variation in vessel resistance. 5. Hyperosmotic shock caused a rapid decay in resistance (maximal within 5 min), and after disruption of the blood-brain barrier, vessel resistance was 100-300 omega cm2 in both arterial and venous vessels, and the effect was reversible. After the application of metabolic poisons (NaCN plus iodoacetate) and low temperature there was a similarly low electrical resistance. 6. It is concluded that the increase in electrical resistance at birth indicates a decrease in paracellular ion permeability at the blood-brain barrier and is required for effective brain interstitial fluid ion regulation.
The blood-brain barrier is a specific property of differentiated brain endothelium. To study the differentiation of blood vessels in the brain, we have correlated the expression of a number of proteins in brain endothelial cells with the development of the blood-brain barrier in mouse, quail, and chick embryos. Using histochemical methods, alkaline phosphatase activity was found to be present in all species and appeared around embryonic Days 17 (mouse), 14 (quail), and 12 (chick). Butyrylcholinesterase activity was found in the mouse and quail but not the chick brain vasculature, and appeared around Days 17 (mouse) and 15 (quail). gamma-Glutamyltranspeptidase activity was demonstrated histochemically in mouse but not in chick and quail brain capillaries, beginning at Day 15. Transferrin receptor was localized on brain endothelium in all species by immunofluorescence methods using monoclonal antibodies. It appeared at Days 15 and 11 in mouse and chick embryonic brain, respectively. The staining of all markers in embryonic brain was compared with adult brain endothelium and the leptomeningeal blood vessels. The expression of these proteins was correlated with the development of the blood-brain barrier by studying the permeability of brain endothelium for the protein horseradish peroxidase during mouse embryogenesis. Vessels in the telencephalon were found to become impermeable around Day 16 of development. Taken together the results of previous investigations and those presented here, we conclude that a number of proteins are sequentially expressed in brain endothelial cells correlating in time with the formation of the blood-brain barrier in different species.
The final development of specializations by brain capillary endothelial cells, which characterize them as distinct from non-central nervous system (CNS) endothelium, is thought to be controlled by astrocyte-derived factors produced locally within the CNS. One specialization, the complex intercellular tight junction, which is unique to these cells and a major component of the blood-brain barrier, is controlled by an astrocyte-derived factor(s) and a "competent' extracellular matrix (Arthur et al., 1987). In order to test whether these factors can also trigger development of brain endothelium-like tight junctions in non-CNS microvessel endothelial cells, passaged bovine aorta and pulmonary artery endothelial cells were cultured in either 50% astrocyte-conditioned medium and 50% alpha-MEM, or in alpha-MEM alone (control). Only endothelial cells maintained in conditioned medium exhibited ultrastructural features indicative of synthesis and plasma membrane-insertion of junction components (Shivers et al., 1985). No assembled tight junctions were seen in these cells. Endothelial cells plated onto coverslips coated with ECM (Cedarlane Labs., Hornby, Ont.) and maintained in astrocyte-conditioned medium, displayed large, complex tight junctions and extraordinarily large gap junctions. Cells plated onto plastic or fibronectin-coated substrates possessed no tight or gap junctions. Results of this study show that CNS astrocytes produce a soluble factor(s) that promotes synthesis and insertion of tight junction components in non-CNS endothelial cells. Moreover, an intact, endothelial-derived extracellular matrix is required for assembly of tight junctions to complete development of this brain capillary-like specialization. This study confirms the notions that: a) the final fine-tuning of cell differentiation is under local control, and b) that endothelial cells in general do not express their final destination-specific differentiated features until those features are induced by local environment-produced conditions.
The structural basis for the decline in non-specific permeability (tightening) of the blood-brain barrier (bbb) during development was investigated in fetal, newborn and weanling mice cerebral hemispheres. Permeability was assessed by measuring the peroxidase activity in the brain 4 h after i.p. injection of horseradish peroxidase--a commonly used vascular tracer that does not cross the intact bbb. Peroxidase activity in brain declined by about two-thirds between birth and weaning, despite increases in both circulating levels and vessel density. Morphometric analysis of the cerebral vessels showed that vesicular density was low (less than 4/micron 2 cytoplasm) at all ages examined , and therefore, is unlikely to be related to permeability changes. Interendothelial junctions changed in that the proportion of the junction composed of zonulae occludens increased, while junctional clefts decreased, and expanded junctional clefts virtually disappeared. We conclude, therefore, that junctional changes underlie the developmental tightening of the bbb. The observation that junctional changes also showed a strong inverse correlation with vessel density suggests that junctional leakiness, and therefore high non-specific permeability, may be a consequence of vessel proliferation in the developing brain.
Fourth passage rat brain capillary endothelial cell cultures, which no longer possess the tight junctions characteristic of this highly specialized component of the blood-brain barrier, were used to study induction of zonulae occludentes in vitro. These cells, when grown in 50% rat brain astrocyte-conditioned medium and 50% alpha-MEM on an endothelial cell matrix-coated substrate (Cedarlane Labs, Hornby, Ont.), possessed numerous, elaborately complex, tight junctions which were identical to those displayed in vivo by intact brain capillary endothelium. Endothelial cells grown in 50% astrocyte-conditioned medium and 50% alpha-MEM on bare plastic or fibronectin-coated substrate, possessed no tight junctions. Results of this study clearly demonstrate the local control of tight junction biogenesis in brain capillary endothelial cells depends on: (1) an astrocyte-produced factor(s), and (2) a 'competent' (cell-produced) extracellular matrix.
The cerebromicrovasculature of immature mice ranging in age from 1-24 days after birth was studied by electron microscopy. A micromanipulator apparatus enabled us to facilitate direction of a needle cannula and injection into leg veins or hearts with horseradish peroxidase (HRP) or ferritin (anionic and cationic) tracers and subsequent perfusion fixatives. Numerous HRP-filled vesiculocanalicular transport structures appearing in endothelial cells (ECs) of brain microblood vessels (MBVs) were observed in time periods ranging from 1-14 days. In addition to HRP transport across the ECs by tubulovesicular profiles, some of these structures appeared to become connected to multivesicular bodies. Between 14 and 24 days after birth, limited HRP was transported across the ECs to the basement membrane in only a few short segments of subpial arterioles. The decoration pattern with cationized ferritin (CF) on the luminal surface of the ECs depends upon whether the surface was exposed to the ligand before or after fixation. Quenching of aldehyde groups in fixed brain tissue has critical importance for the decoration pattern of CF on the luminal plasmalemmal surface. The absence of CF labeling on the delimiting membranes of plasmalemmal vesicles and tubular structures suggests that these structures represent differentiated microdomains engaged in macromolecular transport in the ECs of the developing mouse brain.
The relationship between alkaline phosphatase (AP) activity and maturation of blood-brain barrier (BBB) in mouse brain was studied ultracytochemically. The permeability of micro-blood vessels (MBVs) to intravenously injected horseradish peroxidase was considered to be a criterion of BBB maturation, which occurs between the 12th and 24th day of mouse life. This process coincides with the appearance of cytochemically detectable AP activity in luminal plasma membrane (PM) of the endothelial cell (EC) of capillaries or in both luminal and abluminal PM in arterioles. No reaction appears in ECs of venules. These observations indicate that the development of BBB function is accompanied by the formation of enzymatic barrier in MBVs endothelia. AP constitutes one of the components of this barrier. Comparative study indicates that in various MBVs of non-BBB type, except in skeletal muscle, no AP activity is detected in similar incubation conditions.
The distribution of anionic sites detected in vitro with cationized ferritin and lectin-binding sites on the endothelial cell (EC) surface of brain micro-blood vessels was studied by electron microscopy. Gold-labeled lectins and glycoproteins and Lowicryl K4M-embedded brain samples obtained from mouse embryos (19th day), and from 1-, 5-, 12-, 24- and 48-day-old and adult mice were used. It was shown that the functional maturation of the blood-brain barrier (BBB) occurring in the mouse after birth between the 12th and 24th day of life is accompanied by a disappearance of vesicular transport in capillaries and by the formation of a uniform, thin, negatively charged layer on the surface of the EC. Concomitantly the binding of lectins specific for beta-D-galactosyl (RCA) and sialyl (LFA and WGA) residues become progressively more intense and uniform on both luminal and abluminal fronts of the EC. The concentration of HPA-binding sites on the abluminal side of the EC and in the basement membrane increases. Similarly the binding of Con A becomes more intense on abluminal than on luminal front of the EC. These observations suggest that extensive remodeling of anionic sites and surface glycoprotein layer and also the elaboration of ECs polarity occur during BBB maturation.
The formation of a blood-brain barrier to horseradish peroxidase was microscopically and ultrastructurally investigated in the tectum opticum of the chick during development of the intraneural blood vessel network from the 6th incubation day to hatching, and in adult specimens. Extravasation of the circulating marker, apparently unimpeded during early stages of vasculogenesis, starts to diminish from the 14th incubation day (i.d.) and is prevented after the 18th i.d. The tracer seems to get out of the vessel lumina through the sites of reciprocal contact between adjacent endothelial cells, and the differentiation of tight junctions there hinders the passage of peroxidase particles. The formation of numerous endothelial vacuoles during early vasculogenesis and the setting of the blood-brain barrier are discussed in connection with the mechanisms of transendothelial transport, and respectively, the processes of moulding of the growing endothelia.
The entry of radioactively labeled protein from blood into cerebrospinal fluid (CSF) and into cerebral tissue is much slower and less in amount, than that of tritiated water and certain ions. The barrier that impedes the entry of labeled protein into the CSF has been interpreted as being responsible also for the exclusion of intravascularly injected acid dyes which, because of their negative electric charge, bind to serum proteins. The second portion of this paper is concerned with this direction of transfer and offers a brief, preliminary description of the movement of peroxidase from ventricular CSF not only toward the vessels of the choroid plexus but also toward the vessels of the cerebral parenchyma. Some observations on the passage of the protein ferritin in this direction are also included. Movement of peroxidase from choroidal blood to epithelium and also the movement of peroxidase and ferritin from ventricle into choroidal epithelium and cerebral parenchyma are given in detail in the abstract. As a conclusion, the chapter states that the anatomical barriers to the movement of peroxidase from blood to ventricular CSF consist of: (a) the structures (which are probably tight junctions) between the apices of the choroidal epithelial cells and, perhaps, (b) their ventricular surface. This surface has been regarded as the barrier to the movement of fluorescent proflavine dyes out of the choroid plexus, though the only part of the epithelial cells enclosing demonstrable dye was the nuclear membrane rather than any portion of the cell membrane itself.
Activity of the non-specific alcaline phosphatase appears in brain capillaries in the course of intrauterine development, whereas that of the pseudocholinesterase (butyrylcholinesterase) appears only after birth. This latter enzyme is located in a perinuclear localization at the beginning and occupies later the entire cytoplasm of the endothelial cell. The definitive enzymo-histochemical structure of the capillary is achieved on the 21st day of postnatal development. By means of electron histochemistry, the reaction product of the butyrylcholinesterase reaction appears to be located in and around pinocytotic vesicles and in the perinuclear cysternae of endothelial cells. The results obtained are in harmony with the idea that butyrylcholinesterase is somehow involved in the function of the blood-brain barrier.
The electron lucent channels, about 200 Å‐wide, between the plasma‐lemmas of adjacent cell processes within the brain are true pericellular spaces along which ferritin molecules can move. The micelles, visualized electronmicroscopically, move through the spaces until they encounter an interglial fusion or its myelin counterpart. The interspace between glial processes is abruptly sealed by these inter‐membranous fusions or occasionally distended by a dense filler capable of trapping ferritin. The cerebral interspace is thus highly variable in width and content.
Molecules initially enter the interspace by passing across the basement membrane of the glial border fronting the subarachnoid space whence they are pinocytosed by the underlying glial processes and, at the subarachnoid border of the anterior medullary velum, by also passing directly between adjacent ependymal extensions. Once having entered the parenchymal interspace, micelles can be pinocytosed by neuronal somata and processes and, to a greater degree, by glial fibers. Pinocytosis by the thicker rather than the attenuated portions of glial fibers suggests that the plasmalemmas on the opposite sides of a process must be separated by a critical distance before pinocytotic indentations can be formed. The thinner portions thus offer greater barriers to the movement of micelles than do thicker regions.
The presence of naturally occurring plasma proteins in the cell bodies of some neurons whose axons project outside the central nervous system was sought using the peroxidase-antiperoxidase (PAP) immunocytochemical technique. Rabbit antisera to whole rat serum and rat albumin were used as the primary antisera. The finding of immunoperoxidase reaction product within the perikarya is indicative of the presence of plasma proteins. It is suggested that the endocytosis and retrograde transport of exogenous protein tracers by neurons is paralleled by the neuronal incorporation of naturally occurring plasma proteins.
The neuronal microenvironment in the vertebrate brain is isolated from plasma by a series of selective membranes, including the blood-brain barrier, the choroid plexus, and the meningeal barrier. This review deals with the structure and function of these selective membranes in the different vertebrate classes. Present knowledge indicates that all vertebrates have brain barrier membranes and, further, that functional characteristics of these membranes are basically similar in all the vertebrate classes. The blood-brain barrier (or capillary-glial complex) and the meningeal barrier have many of the properties of a tight epithelium, including the presence of tight junctions and specific transport mechanisms. The choroidal epithelium is a typical secretory epithelium. The functional significance of the specialized membranes located at the blood-brain interface is considered, and we suggest that the phylogenetic development of a blood-brain barrier provided neurons of the vertebrate brain with a unique extracellular milieu optimal both for synaptic communication and for nonsynaptic communication via the entire extracellular space.
Infusion of hyperosmolar solutions into the internal carotid artery causes opening of blood-brain barrier to macromolecules. Ultrastructural tracer studies indicate that extravasation of macromolecules takes place primarily in segments of large penetrating cortical blood vessels. The purpose of the present study was to examine fracture faces of cerebral endothelium in normal and hyperosmolar mannitol-treated rat brains in an attempt to elucidate: (a) the organization of endothelial cell junctions in various segments of the cerebral vascular bed and (b) the structural basis of blood-brain barrier opening in hyperosmotic conditions. We found that in control rat brains: (a) capillary endothelium is provided with complex networks of continuous multistranded tight junctions; (b) continuous capillary-type tight junctions extend, although in a simpler beltlike fashion, into the endothelium of postcapillary venules; (c) the endothelium of collecting veins possess widely discontinuous single- or double-stranded tight junctions associated with gap junctions; (d) arteries have endothelial tight junctions containing focal discontinuities associated with gap junctions. In hyperosmolar mannitol-treated rat brains, there appeared focal distensions of compartments but no definitive structural discontinuities in capillary-type tight junctions. Our data suggest that the blood-brain barrier consists of: (a) an extended tight region (comprising both capillaries and postcapillary venules) and (b) focal, potentially leaky regions (restricted to collecting veins and possibly arteries). Our studies furnished no direct evidence for the structural basis of blood-brain barrier opening in hyperosmolar mannitol-treated rat brains.
Populations of isolated brain capillaries have been proposed as useful models for in vitro studies of the blood-brain barrier. Preliminary investigations of barrier properties using such preparations of brain microvessels have suggested that the tight interendothelial junctions (zonulae occludentes) are intact and retain the impermeability to the protein tracer horseradish peroxidase, exhibited by them in vivo. The endothelial junctions of isolated capillaries are therefore assumed to be functionally "tight' in vitro. In order to determine the precise structural organization of these occluding junctions, including an estimate of their tightness (complexity), and to demonstrate a method for simple but precise assessment of junctional integrity, pellets of isolated rat brain capillaries were freeze-fractured and then replicated with platinum and carbon. The freeze-fracture images of interendothelial zonulae occludentes revealed complex arrays of intramembrane ridges and grooves characteristic of tight junctions. Longitudinal fractures of the cellular lining of capillaries exposed vast expanses of interendothelial plasma membrane interfaces and the junctional complexes situated between the cells. From such arrays, the elaborate and complex architecture of the zonulae occludentes could be readily appreciated. Situated on the PF fracture faces are 6-8 parallel ridges which display a high degree of anastomosing between adjacent strands. The EF fracture face contains grooves complementary to the PF face ridges. The zonulae occludentes of these capillary endothelial cells are similar in complexity to those reported in the literature for reptilian brain capillaries and therefore can be presumed "very tight'. This study demonstrates that freeze-fracture of pellets of brain capillaries alleviates sampling problems inherent in whole tissue preparations and, in addition, demonstrates the usefulness of freeze-fracture as a tool to monitor junction structure during in vitro investigation of the blood-brain barrier.
The viscoelastic behavior of single resting vascular smooth muscle cells from bovine coronary artery was studied. No maintained passive force could be recorded, even when the cells were stretched to two to four times their initial length; this finding suggests that the smooth muscle cells do not contribute to the parallel elastic component in arterial smooth muscle tissue. The force during stretch of resting arterial cells was proportional to the rate of stretch (which varied between 20 and 60% of the initial length per second). This linear viscous resistance was also found for toad stomach cells when similar stretches were applied. The stress-relaxation curves of the arterial cells could be fitted to the sum of two exponential components (with half-lives of 13.1 and 0.5 s, respectively). As a result of the above findings, a model consisting of two viscoelastic elements in parallel was proposed for a single resting arterial smooth muscle cell. The viscous resistance to stretch of resting cells in a Ca2+-containing solution was not significantly (P greater than 0.01) different from that in a Ca2+-free solution. The same result was obtained for bovine coronary arterial rings. It is concluded that an adequate model for resting arterial smooth muscle should include an intracellular viscous element.
A newly developed technique for determination of the electrical resistance of the capillary wall was applied to microvessels at the surface of the frog brain. Current was injected into a capillary or venule via a microelectrode and the ensuing intravascular potential profile away from the current source was determined with a second microelectrode placed at various positions along the capillary. The membrane resistance was calculated according to the theory for leaky cables used in determinations of axon membrane resistance. The average resistance was 1870 omega . cm2. Since the surface vessels of the frog brain are devoid of glial investment but otherwise similar to brain parenchymal vessels, the results prove that the endothelium is the site of the blood-brain barrier. The electrical resistance is similar to that of a 'tight' epithelium.
The penetration of horseradish peroxidase (HRP) from blood into ventricle via the avian choroid plexus was examined by electron microscopy in order to study the development of the blood-cerebrospinal fluid barrier (BCSFB). 1–20 mg HRP was injected into the allantoic vein of chick embryos between the 7th and 21st day of incubation. Until the 8th day (stage 34) the reaction products of injected HRP were observed in the interepithelial clefts at both luminal (ventricular) and abluminal sides. At the 9th day (stage 35), their penetration was blocked at most apical junctional complexes of the choroidal epithelia. At the 10th day (stage 36) and at every subsequent stage, HRP molecules were completely impeded at the apical tight junctions.
These results show that the BCSFB to HRP in the avian choroidal epithelium is completely established by the 10th day, when molecules of HRP are still able to permeate between the capillary endothelia in the cerebellum and the spinal cord (Wakai and Hirokawa 1978).
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Sulla colorazione vitale del sistema nervoso centrale negli animali neonati
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