New tools for studying microglia in the mouse and human CNS.
Proceedings of the National Academy of Sciences of the United States of America
2016; 113 (12): E1738-46
SnapShot: Astrocytes in Health and Disease.
2015; 162 (5): 1170-1170 e1
The specific function of microglia, the tissue resident macrophages of the brain and spinal cord, has been difficult to ascertain because of a lack of tools to distinguish microglia from other immune cells, thereby limiting specific immunostaining, purification, and manipulation. Because of their unique developmental origins and predicted functions, the distinction of microglia from other myeloid cells is critically important for understanding brain development and disease; better tools would greatly facilitate studies of microglia function in the developing, adult, and injured CNS. Here, we identify transmembrane protein 119 (Tmem119), a cell-surface protein of unknown function, as a highly expressed microglia-specific marker in both mouse and human. We developed monoclonal antibodies to its intracellular and extracellular domains that enable the immunostaining of microglia in histological sections in healthy and diseased brains, as well as isolation of pure nonactivated microglia by FACS. Using our antibodies, we provide, to our knowledge, the first RNAseq profiles of highly pure mouse microglia during development and after an immune challenge. We used these to demonstrate that mouse microglia mature by the second postnatal week and to predict novel microglial functions. Together, we anticipate these resources will be valuable for the future study and understanding of microglia in health and disease.
View details for DOI 10.1073/pnas.1525528113
View details for PubMedID 26884166
Development of the choroid plexus and blood-CSF barrier.
Frontiers in neuroscience
2015; 9: 32-?
Astrocytes are central nervous system (CNS) glial cells with many important functions for normal development and neural functioning. They help control extracellular ion and neurotransmitter concentrations; provide neurotrophic support; are implicated in synapse formation, function, and pruning; and help maintain the blood-brain barrier. Following injury and in disease, they undergo rapid and chronic alterations in function that can either promote or hinder recovery, depending on the disease.
View details for DOI 10.1016/j.cell.2015.08.029
View details for PubMedID 26317476
Influx mechanisms in the embryonic and adult rat choroid plexus: a transcriptome study.
Frontiers in neuroscience
2015; 9: 123-?
Well-known as one of the main sources of cerebrospinal fluid (CSF), the choroid plexuses have been, and still remain, a relatively understudied tissue in neuroscience. The choroid plexus and CSF (along with the blood-brain barrier proper) are recognized to provide a robust protective effort for the brain: a physical barrier to impede entrance of toxic metabolites to the brain; a "biochemical" barrier that facilitates removal of moieties that circumvent this physical barrier; and buoyant physical protection by CSF itself. In addition, the choroid plexus-CSF system has been shown to be integral for normal brain development, central nervous system (CNS) homeostasis, and repair after disease and trauma. It has been suggested to provide a stem-cell like repository for neuronal and astrocyte glial cell progenitors. By far, the most widely recognized choroid plexus role is as the site of the blood-CSF barrier, controller of the internal CNS microenvironment. Mechanisms involved combine structural diffusion restraint from tight junctions between plexus epithelial cells (physical barrier) and specific exchange mechanisms across the interface (enzymatic barrier). The current hypothesis states that early in development this interface is functional and more specific than in the adult, with differences historically termed as "immaturity" actually correctly reflecting developmental specialization. The advanced knowledge of the choroid plexus-CSF system proves itself imperative to understand a range of neurological diseases, from those caused by plexus or CSF drainage dysfunction (e.g., hydrocephalus) to more complicated late-stage diseases (e.g., Alzheimer's) and failure of CNS regeneration. This review will focus on choroid plexus development, outlining how early specializations may be exploited clinically.
View details for DOI 10.3389/fnins.2015.00032
View details for PubMedID 25784848
The inner CSF-brain barrier: developmentally controlled access to the brain via intercellular junctions.
Frontiers in neuroscience
2015; 9: 16-?
The transcriptome of embryonic and adult rat lateral ventricular choroid plexus, using a combination of RNA-Sequencing and microarray data, was analyzed by functional groups of influx transporters, particularly solute carrier (SLC) transporters. RNA-Seq was performed at embryonic day (E) 15 and adult with additional data obtained at intermediate ages from microarray analysis. The largest represented functional group in the embryo was amino acid transporters (twelve) with expression levels 2-98 times greater than in the adult. In contrast, in the adult only six amino acid transporters were up-regulated compared to the embryo and at more modest enrichment levels (<5-fold enrichment above E15). In E15 plexus five glucose transporters, in particular Glut-1, and only one monocarboxylate transporter were enriched compared to the adult, whereas only two glucose transporters but six monocarboxylate transporters in the adult plexus were expressed at higher levels than in embryos. These results are compared with earlier published physiological studies of amino acid and monocarboxylate transport in developing rodents. This comparison shows correlation of high expression of some transporters in the developing brain with higher amino acid transport activity reported previously. Data for divalent metal transporters are also considered. Immunohistochemistry of several transporters (e.g., Slc16a10, a thyroid hormone transporter) gene products was carried out to confirm translational activity and to define cellular distribution of the proteins. Overall the results show that there is substantial expression of numerous influx transporters in the embryonic choroid plexus, many at higher levels than in the adult. This, together with immunohistochemical evidence and data from published physiological transport studies suggests that the choroid plexus in embryonic brain plays a major role in supplying the developing brain with essential nutrients.
View details for DOI 10.3389/fnins.2015.00123
View details for PubMedID 25972776
Cellular Specificity of the Blood-CSF Barrier for Albumin Transfer across the Choroid Plexus Epithelium
2014; 9 (9)
An RNA-Sequencing Transcriptome and Splicing Database of Glia, Neurons, and Vascular Cells of the Cerebral Cortex
JOURNAL OF NEUROSCIENCE
2014; 34 (36): 11929-11947
In the adult the interface between the cerebrospinal fluid and the brain is lined by the ependymal cells, which are joined by gap junctions. These intercellular connections do not provide a diffusional restrain between the two compartments. However, during development this interface, initially consisting of neuroepithelial cells and later radial glial cells, is characterized by "strap" junctions, which limit the exchange of different sized molecules between cerebrospinal fluid and the brain parenchyma. Here we provide a systematic study of permeability properties of this inner cerebrospinal fluid-brain barrier during mouse development from embryonic day, E17 until adult. Results show that at fetal stages exchange across this barrier is restricted to the smallest molecules (286Da) and the diffusional restraint is progressively removed as the brain develops. By postnatal day P20, molecules the size of plasma proteins (70 kDa) diffuse freely. Transcriptomic analysis of junctional proteins present in the cerebrospinal fluid-brain interface showed expression of adherens junctional proteins, actins, cadherins and catenins changing in a development manner consistent with the observed changes in the permeability studies. Gap junction proteins were only identified in the adult as was claudin-11. Immunohistochemistry was used to localize at the cellular level some of the adherens junctional proteins of genes identified from transcriptomic analysis. N-cadherin, β - and α-catenin immunoreactivity was detected outlining the inner CSF-brain interface from E16; most of these markers were not present in the adult ependyma. Claudin-5 was present in the apical-most part of radial glial cells and in endothelial cells in embryos, but only in endothelial cells including plexus endothelial cells in adults. Claudin-11 was only immunopositive in the adult, consistent with results obtained from transcriptomic analysis. These results provide information about physiological, molecular and morphological-related permeability changes occurring at the inner cerebrospinal fluid-brain barrier during brain development.
View details for DOI 10.3389/fnins.2015.00016
View details for PubMedID 25729345
Age-Dependent Transcriptome and Proteome Following Transection of Neonatal Spinal Cord of Monodelphis domestica (South American Grey Short-Tailed Opossum)
2014; 9 (6)
The major cell classes of the brain differ in their developmental processes, metabolism, signaling, and function. To better understand the functions and interactions of the cell types that comprise these classes, we acutely purified representative populations of neurons, astrocytes, oligodendrocyte precursor cells, newly formed oligodendrocytes, myelinating oligodendrocytes, microglia, endothelial cells, and pericytes from mouse cerebral cortex. We generated a transcriptome database for these eight cell types by RNA sequencing and used a sensitive algorithm to detect alternative splicing events in each cell type. Bioinformatic analyses identified thousands of new cell type-enriched genes and splicing isoforms that will provide novel markers for cell identification, tools for genetic manipulation, and insights into the biology of the brain. For example, our data provide clues as to how neurons and astrocytes differ in their ability to dynamically regulate glycolytic flux and lactate generation attributable to unique splicing of PKM2, the gene encoding the glycolytic enzyme pyruvate kinase. This dataset will provide a powerful new resource for understanding the development and function of the brain. To ensure the widespread distribution of these datasets, we have created a user-friendly website (http://web.stanford.edu/group/barres_lab/brain_rnaseq.html) that provides a platform for analyzing and comparing transciption and alternative splicing profiles for various cell classes in the brain.
View details for DOI 10.1523/JNEUROSCI.1860-14.2014
View details for Web of Science ID 000341765400008
View details for PubMedID 25186741
Cellular specificity of the blood-CSF barrier for albumin transfer across the choroid plexus epithelium.
2014; 9 (9)
This study describes a combined transcriptome and proteome analysis of Monodelphis domestica response to spinal cord injury at two different postnatal ages. Previously we showed that complete transection at postnatal day 7 (P7) is followed by profuse axon growth across the lesion with near-normal locomotion and swimming when adult. In contrast, at P28 there is no axon growth across the lesion, the animals exhibit weight-bearing locomotion, but cannot use hind limbs when swimming. Here we examined changes in gene and protein expression in the segment of spinal cord rostral to the lesion at 24 h after transection at P7 and at P28. Following injury at P7 only forty genes changed (all increased expression); most were immune/inflammatory genes. Following injury at P28 many more genes changed their expression and the magnitude of change for some genes was strikingly greater. Again many were associated with the immune/inflammation response. In functional groups known to be inhibitory to regeneration in adult cords the expression changes were generally muted, in some cases opposite to that required to account for neurite inhibition. For example myelin basic protein expression was reduced following injury at P28 both at the gene and protein levels. Only four genes from families with extracellular matrix functions thought to influence neurite outgrowth in adult injured cords showed substantial changes in expression following injury at P28: Olfactomedin 4 (Olfm4, 480 fold compared to controls), matrix metallopeptidase (Mmp1, 104 fold), papilin (Papln, 152 fold) and integrin α4 (Itga4, 57 fold). These data provide a resource for investigation of a priori hypotheses in future studies of mechanisms of spinal cord regeneration in immature animals compared to lack of regeneration at more mature stages.
View details for DOI 10.1371/journal.pone.0099080
View details for Web of Science ID 000340947700059
View details for PubMedID 24914927
Mechanisms That Determine the Internal Environment of the Developing Brain: A Transcriptomic, Functional and Ultrastructural Approach
2013; 8 (7)
To maintain the precise internal milieu of the mammalian central nervous system, well-controlled transfer of molecules from periphery into brain is required. Recently the soluble and cell-surface albumin-binding glycoprotein SPARC (secreted protein acidic and rich in cysteine) has been implicated in albumin transport into developing brain, however the exact mechanism remains unknown. We postulate that SPARC is a docking site for albumin, mediating its uptake and transfer by choroid plexus epithelial cells from blood into cerebrospinal fluid (CSF). We used in vivo physiological measurements of transfer of endogenous (mouse) and exogenous (human) albumins, in situ Proximity Ligation Assay (in situ PLA), and qRT-PCR experiments to examine the cellular mechanism mediating protein transfer across the blood-CSF interface. We report that at all developmental stages mouse albumin and SPARC gave positive signals with in situ PLAs in plasma, CSF and within individual plexus cells suggesting a possible molecular interaction. In contrast, in situ PLA experiments in brain sections from mice injected with human albumin showed positive signals for human albumin in the vascular compartment that were only rarely identifiable within choroid plexus cells and only at older ages. Concentrations of both endogenous mouse albumin and exogenous (intraperitoneally injected) human albumin were estimated in plasma and CSF and expressed as CSF/plasma concentration ratios. Human albumin was not transferred through the mouse blood-CSF barrier to the same extent as endogenous mouse albumin, confirming results from in situ PLA. During postnatal development Sparc gene expression was higher in early postnatal ages than in the adult and changed in response to altered levels of albumin in blood plasma in a differential and developmentally regulated manner. Here we propose a possible cellular route and mechanism by which albumin is transferred from blood into CSF across a sub-population of specialised choroid plexus epithelial cells.
View details for DOI 10.1371/journal.pone.0106592
View details for PubMedID 25211495
Transporters of the blood-brain and blood-CSF interfaces in development and in the adult.
Molecular aspects of medicine
2013; 34 (2-3): 742-752
We provide comprehensive identification of embryonic (E15) and adult rat lateral ventricular choroid plexus transcriptome, with focus on junction-associated proteins, ionic influx transporters and channels. Additionally, these data are related to new structural and previously published permeability studies. Results reveal that most genes associated with intercellular junctions are expressed at similar levels at both ages. In total, 32 molecules known to be associated with brain barrier interfaces were identified. Nine claudins showed unaltered expression, while two claudins (6 and 8) were expressed at higher levels in the embryo. Expression levels for most cytoplasmic/regulatory adaptors (10 of 12) were similar at the two ages. A few junctional genes displayed lower expression in embryos, including 5 claudins, occludin and one junctional adhesion molecule. Three gap junction genes were enriched in the embryo. The functional effectiveness of these junctions was assessed using blood-delivered water-soluble tracers at both the light and electron microscopic level: embryo and adult junctions halted movement of both 286Da and 3kDa molecules into the cerebrospinal fluid (CSF). The molecular identities of many ion channel and transporter genes previously reported as important for CSF formation and secretion in the adult were demonstrated in the embryonic choroid plexus (and validated with immunohistochemistry of protein products), but with some major age-related differences in expression. In addition, a large number of previously unidentified ion channel and transporter genes were identified for the first time in plexus epithelium. These results, in addition to data obtained from electron microscopical and physiological permeability experiments in immature brains, indicate that exchange between blood and CSF is mainly transcellular, as well-formed tight junctions restrict movement of small water-soluble molecules from early in development. These data strongly indicate the brain develops within a well-protected internal environment and the exchange between the blood, brain and CSF is transcellular and not through incomplete barriers.
View details for DOI 10.1371/journal.pone.0065629
View details for Web of Science ID 000321341000007
View details for PubMedID 23843944
Immune responses at brain barriers and implications for brain development and neurological function in later life.
Frontiers in integrative neuroscience
2013; 7: 61-?
The protective barriers of the brain provide a complex series of physical and chemical obstacles to movement of macromolecules from the periphery into the central nervous system. Studies on these barriers have been focused on two main research areas: (i) anatomical and physiological descriptions of their properties, including during development where functioning barriers are likely to be important for normal neuronal growth; and (ii), investigations of these barriers during disease and attempts at overcoming their defenses in order to deliver drugs to the central nervous system. Both fields are now advanced by the application of molecular gene expression studies of cerebral endothelia (blood vasculature, site of the blood-brain barrier) and choroid plexus epithelia (site of the blood-cerebrospinal fluid barrier) from developing and adult brains, particularly with respect to solute-linked carriers and other transporters. These new techniques provide a wealth of information on the changing nature of transporters at barrier interfaces during normal development and following disease. This review outlines published findings from transcriptome and qPCR studies of expression of genes coding for transporters in these barriers, with a focus on developing brain. The findings clearly support earlier published physiological data describing specific transport mechanisms across barrier interfaces both in the adult and in particular in the developing brain.
View details for DOI 10.1016/j.mam.2012.11.006
View details for PubMedID 23506907
Developmental changes in the transcriptome of the rat choroid plexus in relation to neuroprotection.
Fluids and barriers of the CNS
2013; 10 (1): 25-?
For a long time the brain has been considered an immune-privileged site due to a muted inflammatory response and the presence of protective brain barriers. It is now recognized that neuroinflammation may play an important role in almost all neurological disorders and that the brain barriers may be contributing through either normal immune signaling or disruption of their basic physiological mechanisms. The distinction between normal function and dysfunction at the barriers is difficult to dissect, partly due to a lack of understanding of normal barrier function and partly because of physiological changes that occur as part of normal development and ageing. Brain barriers consist of a number of interacting structural and physiological elements including tight junctions between adjacent barrier cells and an array of influx and efflux transporters. Despite these protective mechanisms, the capacity for immune-surveillance of the brain is maintained, and there is evidence of inflammatory signaling at the brain barriers that may be an important part of the body's response to damage or infection. This signaling system appears to change both with normal ageing, and during disease. Changes may affect diapedesis of immune cells and active molecular transfer, or cause rearrangement of the tight junctions and an increase in passive permeability across barrier interfaces. Here we review the many elements that contribute to brain barrier functions and how they respond to inflammation, particularly during development and aging. The implications of inflammation-induced barrier dysfunction for brain development and subsequent neurological function are also discussed.
View details for DOI 10.3389/fnint.2013.00061
View details for PubMedID 23986663
Molecular Characterisation of Transport Mechanisms at the Developing Mouse Blood-CSF Interface: A Transcriptome Approach
2012; 7 (3)
The choroid plexuses are the interface between the blood and the cerebrospinal fluid (CSF) contained within the ventricular spaces of the central nervous system. The tight junctions linking adjacent cells of the choroidal epithelium create a physical barrier to paracellular movement of molecules. Multispecific efflux transporters as well as drug-metabolizing and antioxidant enzymes functioning in these cells contribute to a metabolic barrier. These barrier properties reflect a neuroprotective function of the choroid plexus. The choroid plexuses develop early during embryogenesis and provide pivotal control of the internal environment throughout development when the brain is especially vulnerable to toxic insults. Perinatal injuries like hypoxia and trauma, and exposure to drugs or toxic xenobiotics can have serious consequences on neurogenesis and long-term development. The present study describes the developmental expression pattern of genes involved in the neuroprotective functions of the blood-CSF barrier.The transcriptome of rat lateral ventricular choroid plexuses isolated from fifteen-day-old embryos, nineteen-day old fetuses, two-day old pups, and adults was analyzed by a combination of Affymetrix microarrays, Illumina RNA-Sequencing, and quantitative RT-PCR.Genes coding for proteins involved in junction formation are expressed early during development. Overall perinatal expression levels of genes involved in drug metabolism and antioxidant mechanisms are similar to, or higher than levels measured in adults. A similar developmental pattern was observed for multispecific efflux transporter genes of the Abc and Slc superfamilies. Expression of all these genes was more variable in choroid plexus from fifteen-day-old embryos. A large panel of transcription factors involved in the xenobiotic- or cell stress-mediated induction of detoxifying enzymes and transporters is also expressed throughout development.This transcriptomic analysis suggests relatively well-established neuroprotective mechanisms at the blood-CSF barrier throughout development of the rat. The expression of many transcription factors early in development raises the possibility of additional protection for the vulnerable developing brain, should the fetus or newborn be exposed to drugs or other xenobiotics.
View details for DOI 10.1186/2045-8118-10-25
View details for PubMedID 23915922
Barrier mechanisms in the developing brain.
Frontiers in pharmacology
2012; 3: 46-?
Exchange mechanisms across the blood-cerebrospinal fluid (CSF) barrier in the choroid plexuses within the cerebral ventricles control access of molecules to the central nervous system, especially in early development when the brain is poorly vascularised. However, little is known about their molecular or developmental characteristics. We examined the transcriptome of lateral ventricular choroid plexus in embryonic day 15 (E15) and adult mice. Numerous genes identified in the adult were expressed at similar levels at E15, indicating substantial plexus maturity early in development. Some genes coding for key functions (intercellular/tight junctions, influx/efflux transporters) changed expression during development and their expression patterns are discussed in the context of available physiological/permeability results in the developing brain. Three genes: Secreted protein acidic and rich in cysteine (Sparc), Glycophorin A (Gypa) and C (Gypc), were identified as those whose gene products are candidates to target plasma proteins to choroid plexus cells. These were investigated using quantitative- and single-cell-PCR on plexus epithelial cells that were albumin- or total plasma protein-immunopositive. Results showed a significant degree of concordance between plasma protein/albumin immunoreactivity and expression of the putative transporters. Immunohistochemistry identified SPARC and GYPA in choroid plexus epithelial cells in the embryo with a subcellular distribution that was consistent with transport of albumin from blood to cerebrospinal fluid. In adult plexus this pattern of immunostaining was absent. We propose a model of the cellular mechanism in which SPARC and GYPA, together with identified vesicle-associated membrane proteins (VAMPs) may act as receptors/transporters in developmentally regulated transfer of plasma proteins at the blood-CSF interface.
View details for DOI 10.1371/journal.pone.0033554
View details for Web of Science ID 000303857100033
View details for PubMedID 22457777
SPARC/osteonectin, an endogenous mechanism for targeting albumin to the blood-cerebrospinal fluid interface during brain development
EUROPEAN JOURNAL OF NEUROSCIENCE
2011; 34 (7): 1062-1073
The adult brain functions within a well-controlled stable environment, the properties of which are determined by cellular exchange mechanisms superimposed on the diffusion restraint provided by tight junctions at interfaces between blood, brain and cerebrospinal fluid (CSF). These interfaces are referred to as "the" blood-brain barrier. It is widely believed that in embryos and newborns, this barrier is immature or "leaky," rendering the developing brain more vulnerable to drugs or toxins entering the fetal circulation from the mother. New evidence shows that many adult mechanisms, including functionally effective tight junctions are present in embryonic brain and some transporters are more active during development than in the adult. Additionally, some mechanisms present in embryos are not present in adults, e.g., specific transport of plasma proteins across the blood-CSF barrier and embryo-specific intercellular junctions between neuroependymal cells lining the ventricles. However developing cerebral vessels appear to be more fragile than in the adult. Together these properties may render developing brains more vulnerable to drugs, toxins, and pathological conditions, contributing to cerebral damage and later neurological disorders. In addition, after birth loss of protection by efflux transporters in placenta may also render the neonatal brain more vulnerable than in the fetus.
View details for DOI 10.3389/fphar.2012.00046
View details for PubMedID 22479246
Modification of protein transfer across blood/cerebrospinal fluid barrier in response to altered plasma protein composition during development
EUROPEAN JOURNAL OF NEUROSCIENCE
2011; 33 (3): 391-400
Specialized populations of choroid plexus epithelial cells have previously been shown to be responsible for the transfer of individual plasma proteins from blood to the cerebrospinal fluid (CSF), contributing to their characteristically high concentrations in CSF of the developing brain. The mechanism of this protein transfer remains elusive. Using a marsupial, Monodelphis domestica, we demonstrate that the albumin-binding protein SPARC (osteonectin/BM-40/culture-shock protein) is present in a subset of choroid plexus epithelial cells from its first appearance, throughout development, and into adulthood. The synthesis of SPARC by the lateral ventricular plexus was confirmed with real-time PCR. The expression level of SPARC was higher in plexuses of younger than older animals. Western blot analysis of the gene product confirmed the quantitative PCR results. The co-localization of SPARC and albumin shown by immunocytochemistry and its cellular location indicate that this glycoprotein may act as a recognition site for albumin. In addition, the numbers of SPARC-immunopositive cells and its expression were responsive to experimental changes of albumin concentration in the blood. It is suggested that SPARC may be one of the molecules that govern the uptake and delivery of proteins from blood to the CSF. The results also confirm that protein transfer across the blood-CSF barrier is developmentally and physiologically regulated.
View details for DOI 10.1111/j.1460-9568.2011.07821.x
View details for Web of Science ID 000296202200004
View details for PubMedID 21899600
Fluids and barriers of the CNS: a historical viewpoint.
Fluids and barriers of the CNS
2011; 8 (1): 2-?
A developmentally regulated protein-specific transfer mechanism across choroid plexus epithelial cells has previously been proposed to contribute to the characteristically high concentration of protein in cerebrospinal fluid (CSF) in the immature brain. Here we demonstrate that this mechanism is sensitive to protein variations in plasma resulting in changed numbers of transferring cells for individual proteins and altered transfer into the CSF. Pups of Monodelphis domestica at postnatal day (P)9, P65 and P110 were injected intraperitoneally with either adult Monodelphis plasma or exogenous bovine fetuin. Samples of CSF, blood and brain were collected from terminally anaesthetized animals 3-48 h later. The concentration of total protein was measured and levels of albumin, hemopexin, ?-fetoprotein and bovine fetuin were estimated by western blotting. Numbers of lateral ventricular choroid plexus cells positive for total and individual plasma proteins were counted in paraffin sections of brains stained with appropriate antibodies. Following intraperitoneal injections, the content of proteins in the CSF increased at all three ages, but the concentration increased only in the CSF of older animals. The total numbers of plexus cells positive for plasma protein did not change significantly, but cells positive for individual proteins did. Fetuin was detected in all protein-positive cells, but apparently displaced ?-fetoprotein and, to a lesser degree, hemopexin. The results indicate that protein transfer across the blood/CSF barrier appears to be regulated by a molecular recognition mechanism that is probably saturable but may not be as specific for individual proteins as previously suggested.
View details for DOI 10.1111/j.1460-9568.2010.07509.x
View details for Web of Science ID 000286769800002
View details for PubMedID 21138490
Assessing blood-cerebrospinal fluid barrier permeability in the rat embryo.
Methods in molecular biology (Clifton, N.J.)
2011; 686: 247-265
Tracing the exact origins of modern science can be a difficult but rewarding pursuit. It is possible for the astute reader to follow the background of any subject through the many important surviving texts from the classical and ancient world. While empirical investigations have been described by many since the time of Aristotle and scientific methods have been employed since the Middle Ages, the beginnings of modern science are generally accepted to have originated during the 'scientific revolution' of the 16th and 17th centuries in Europe. The scientific method is so fundamental to modern science that some philosophers consider earlier investigations as 'pre-science'. Notwithstanding this, the insight that can be gained from the study of the beginnings of a subject can prove important in the understanding of work more recently completed. As this journal undergoes an expansion in focus and nomenclature from cerebrospinal fluid (CSF) into all barriers of the central nervous system (CNS), this review traces the history of both the blood-CSF and blood-brain barriers from as early as it was possible to find references, to the time when modern concepts were established at the beginning of the 20th century.
View details for DOI 10.1186/2045-8118-8-2
View details for PubMedID 21349150
Efflux mechanisms at the developing brain barriers: ABC-transporters in the fetal and postnatal rat
2010; 197 (1): 51-59
The rat is a useful model for studies of embryonic blood-CSF function in that the embryos are large enough to collect sufficient fluid samples for analysis and exteriorized embryos can be kept viable for several hours in order to conduct longer term experiments. Both quantitative and qualitative methods that are similar to those used in adult studies can be used to assess blood-CSF function in the rat embryo; however, there are technical aspects of these studies that are more challenging. The choice of the methods to be used depends largely on the question being asked. This chapter describes in detail the precise steps that need to be taken to keep rat embryos in a good physiological state while conducting the experiments, how to administer markers into the embryonic circulation, and how to sample blood and/or CSF from embryos. How to evaluate the results obtained is outlined at the end of each method, together with notes on some limitations that are inherent in developmental studies.
View details for DOI 10.1007/978-1-60761-938-3_11
View details for PubMedID 21082375
Development of the lateral ventricular choroid plexus in a marsupial, Monodelphis domestica.
Cerebrospinal fluid research
2010; 7: 16-?
Proteins of the ATP-binding cassette (ABC) family, present at the blood-brain barrier interfaces, have been shown to reduce the entry of compounds from blood into the brain by active efflux. Their substrates are diverse including many drugs and toxins and therefore provide an important mechanism for brain neuroprotection. However, knowledge of their presence and function in the developing brain is very limited. We have used qPCR and immunocytochemistry to determine gene expression and localisation of four main barrier ABC-transporters (pgp/ABCB1, MRP1/ABCC1, MRP4/ABCC4 and BCRP/ABCG2) in the fetal and neonatal rat brain cerebral blood vessels (site of blood-brain barrier) and choroid plexus (site of blood-CSF barrier). The study shows that ABC-transporters localise to the brain barriers even at early fetal stages and although pgp expression was lower in the fetus, the other transporters were expressed at comparable levels in fetal and adult brains suggesting direct neuroprotection of the brain in addition to that provided by the placenta. BCRP was expressed at higher levels in developing choroid plexus and was only detected at fetal stages on the blood-facing side of epithelial cells indicating a particular role of this transporter for early brain efflux mechanisms.
View details for DOI 10.1016/j.toxlet.2010.04.025
View details for Web of Science ID 000279989500008
View details for PubMedID 20466047
Cellular transfer of macromolecules across the developing choroid plexus of Monodelphis domestica
EUROPEAN JOURNAL OF NEUROSCIENCE
2009; 29 (2): 253-266
Choroid plexus epithelial cells are the site of blood/cerebrospinal fluid (CSF) barrier and regulate molecular transfer between the two compartments. Their mitotic activity in the adult is low. During development, the pattern of growth and timing of acquisition of functional properties of plexus epithelium are not known.Numbers and size of choroid plexus epithelial cells and their nuclei were counted and measured in the lateral ventricular plexus from the first day of its appearance until adulthood. Newborn Monodelphis pups were injected with 5-bromo-2-deoxyuridine (BrdU) at postnatal day 3 (P3), P4 and P5. Additional animals were injected at P63, P64 and P65. BrdU-immunopositive nuclei were counted and their position mapped in the plexus structure at different ages after injections. Double-labelling immunocytochemistry with antibodies to plasma protein identified post-mitotic cells involved in protein transfer.Numbers of choroid plexus epithelial cells increased 10-fold between the time of birth and adulthood. In newborn pups each consecutive injection of BrdU labelled 20-40 of epithelial cells counted. After 3 injections, numbers of BrdU positive cells remained constant for at least 2 months. BrdU injections at an older age (P63, P64, P65) resulted in a smaller number of labelled plexus cells. Numbers of plexus cells immunopositive for both BrdU and plasma protein increased with age indicating that protein transferring properties are acquired post mitotically. Labelled nuclei were only detected on the dorsal arm of the plexus as it grows from the neuroependyma, moving along the structure in a 'conveyor belt' like fashion.The present study established that lateral ventricular choroid plexus epithelial cells are born on the dorsal side of the structure only. Cells born in the first few days after choroid plexus differentiation from the neuroependyma remain present even two months later. Protein-transferring properties are acquired post-mitotically and relatively early in plexus development.
View details for DOI 10.1186/1743-8454-7-16
View details for PubMedID 20920364
The blood-CSF barrier explained: When development is not immaturity
2008; 30 (3): 237-248
Choroid plexus epithelial cells secrete cerebrospinal fluid (CSF) and transfer molecules from blood into CSF. Tight junctions between choroidal epithelial cells are functionally effective from early in development: the route of transfer is suggested to be transcellular. Routes of transfer for endogenous and exogenous plasma proteins and dextrans were studied in Monodelphis domestica (opossum). Pups at postnatal (P) days 1-65 and young adults were injected with biotinylated dextrans (3-70 kDa) and/or foetal protein fetuin. CSF, plasma and brain samples were collected from terminally anaesthetized animals. Choroid plexus cells containing plasma proteins were detected immunocytochemically. Numbers of plasma protein-positive epithelial cells increased to adult levels by P28, but their percentage of plexus cells declined. Numbers of cells positive for biotinylated probes increased with age, while their percentage remained constant. Colocalization studies showed specificity for individual proteins in some epithelial cells. Biotinylated probes and endogenous proteins colocalized in about 10% of cells in younger animals, increasing towards 100% by adulthood. Injections of markers into the ventricles demonstrated that protein is transferred only from blood into CSF, whereas dextrans pass in both directions. These results indicate that protein and lipid-insoluble markers are transferred by separate mechanisms present in choroid plexuses from the earliest stage of brain development, and transfer of proteins from plasma across choroid plexus epithelial cells contributes to the high protein concentration in CSF in the immature brain.
View details for DOI 10.1111/j.1460-9568.2008.06571.x
View details for Web of Science ID 000262515600005
View details for PubMedID 19200232
Blood-CSF barrier function in the rat embryo
EUROPEAN JOURNAL OF NEUROSCIENCE
2006; 24 (1): 65-76
It is often suggested that during development the brain barriers are immature. This argument stems from teleological interpretations and experimental observations of the high protein concentrations in fetal cerebrospinal fluid (CSF) and decreases in apparent permeability of passive markers during development. We argue that the developmental blood-CSF barrier restricts the passage of lipid-insoluble molecules by the same mechanism as in the adult (tight junctions) rendering the paracellular pathway an unlikely route of entry. Instead, we suggest that both protein and passive markers are transferred across the epithelium through a transcellular route. We propose that changes in volume of distribution can largely explain the decrease in apparent permeability for passive markers and that developmentally regulated cellular transfer explains changes in CSF protein concentrations. The blood-CSF tight junctions are functionally mature from very early in development, and it appears that transfer from blood into embryonic brain occurs predominately via CSF rather than the vasculature.
View details for DOI 10.1002/bies.20718
View details for Web of Science ID 000253960600006
View details for PubMedID 18293362
Blood-cerebrospinal fluid (CSF) barrier function and expansion of the ventricular system were investigated in embryonic rats (E12-18). Permeability markers (sucrose and inulin) were injected intraperitoneally and concentrations measured in plasma and CSF at two sites (lateral and 4th ventricles) after 1 h. Total protein concentrations were also measured. CSF/plasma concentration ratios for endogenous protein were stable at approximately 20% at E14-18 and subsequently declined. In contrast, ratios for sucrose (100%) and inulin (40%) were highest at the earliest ages studied (E13-14) and then decreased substantially. Between E13 and E16 the volume of the lateral ventricles increased over three-fold. Decreasing CSF/plasma concentration ratios for small, passively diffusing molecules during embryonic development may not reflect changes in permeability. Instead, increasing volume of distribution appears to be important in this decline. The intracellular presence of a small marker (3000 Da biotin-dextranamine) in plexus epithelial cells following intraperitoneal injection indicates a transcellular route of transfer. Ultrastructural evidence confirmed that choroid plexus tight junctions are impermeable to small molecules at least as early as E15, indicating the blood-CSF barrier is morphologically and functionally mature early in embryonic development. Comparison of two albumins (human and bovine) showed that transfer of human albumin (surrogate for endogenous protein) was 4-5 times greater than bovine, indicating selective blood-to-CSF transfer. The number of plexus epithelial cells immunopositive for endogenous plasma protein increased in parallel with increases in total protein content of the expanding ventricular system. Results suggest that different transcellular mechanisms for protein and small molecule transfer are operating across the embryonic blood-CSF interface.
View details for DOI 10.1111/j.1460-9568.2006.04904.x
View details for Web of Science ID 000239004100009
View details for PubMedID 16800861