Cerebrospinal Fluid-brain Barrier Research Articles

3,3',5-Triiodothyroacetic Acid Transporters.

Introduction: Thyroid hormone transporters are essential for thyroid hormones to enter target cells. Monocarboxylate transporter (MCT) 8 is a key transporter and is expressed at the blood-brain barrier (BBB), in neural cells and many other tissues. Patients with MCT8 deficiency have severe neurodevelopmental delays because of cerebral hypothyroidism and chronic sequelae of peripheral thyrotoxicosis. The T3 analog 3,3',5-triiodothyroacetic acid (TRIAC) rescued neurodevelopmental features in animal models mimicking MCT8 deficiency and improved key metabolic features in patients with MCT8 deficiency. However, the identity of the transporter(s) that facilitate TRIAC transport are unknown. Here, we screened candidate transporters that are expressed at the human BBB and/or brain-cerebrospinal fluid barrier and known thyroid hormone transporters for TRIAC transport. Materials and Methods: Plasma membrane expression was determined by cell surface biotinylation assays. Intracellular accumulation of 1 nM TRIAC was assessed in COS-1 cells expressing candidate transporters in Dulbecco's phosphate-buffered saline (DPBS)/0.1% glucose or Dulbecco's modified Eagle's medium (DMEM) with or without 0.1% bovine serum albumin (BSA). Expression of Slc22a8 was determined by fluorescent in situ hybridization in brain sections from wild-type and Mct8/Oatp1c1 knockout mice at postnatal days 12, 21, and 120. Results: In total, 59 plasma membrane transporters were selected for screening of TRIAC accumulation (n = 40 based on expression at the human BBB and/or brain-cerebrospinal fluid barrier and having small organic molecules as substrates; n = 19 known thyroid hormone transporters). Screening of the selected transporter panel showed that 18 transporters facilitated significant intracellular accumulation of TRIAC in DPBS/0.1% glucose or DMEM in the absence of BSA. In the presence of BSA, substantial transport was noted for SLCO1B1 and SLC22A8 (in DPBS/0.1% glucose and DMEM) and SLC10A1, SLC22A6, and SLC22A24 (in DMEM). The zebrafish and mouse orthologs of these transporters similarly facilitated intracellular accumulation of TRIAC. Highest Slc22a8 mRNA expression was detected in mouse brain capillary endothelial cells and choroid plexus epithelial cells at early postnatal time points, but was reduced at P120. Conclusions: Human SLC10A1, SLCO1B1, SLC22A6, SLC22A8, and SLC22A24 as well as their mouse and zebrafish orthologs are efficient TRIAC transporters. These findings contribute to the understanding of TRIAC treatment in patients with MCT8 deficiency and animal models thereof.

History of research concerning the ependyma: a view from inside the human brain.

The history of research concerning ependymal cells is reviewed. Cilia were identified along the surface of the cerebral ventricles c1835. Numerous anatomical and histopathological studies in the late 1800's showed irregularities in the ependymal surface that were thought to be indicative of specific pathologies such as syphilis; this was subsequently disproven. The evolution of thoughts about functions of cilia, the possible role of ependyma in the brain-cerebrospinal fluid barrier, and the relationship of ependyma to the subventricular zone germinal cells is discussed. How advances in light and electron microscopy and cell culture contributed to our understanding of the ependyma is described. Discoveries of the supraependymal serotoninergic axon network and supraependymal macrophages are recounted. Finally, the consequences of loss of ependymal cells from different regions of the central nervous system are considered.

Focused Ultrasound-Mediated Blood–Brain Barrier Opening Best Promotes Neuroimmunomodulation through Brain Macrophage Redistribution

Neuroimmunomodulation is a promising form of drug-free treatment for neurological diseases ranging from Alzheimer’s disease to depression. The evidence supporting the efficacy of focused ultrasound (FUS) neuroimmunomodulation is encouraging; however, the method has yet to be standardized, and its mechanism remains poorly understood. Methods of FUS neuroimmunomodulation can be categorized into three paradigms based on the parameters used. In the first paradigm, focused ultrasound blood–brain barrier opening (FUS-BBBO) combines FUS with microbubbles (MB) to transiently and safely induce BBB opening. In the second paradigm, focused ultrasound neuromodulation (FUS-N) harnesses the acoustic effects of FUS alone (without MB). In the third paradigm, focused ultrasound with microbubbles without BBBO (FUS + MB) combines MB with FUS below the BBBO pressure threshold—harnessing the mechanical effects of FUS without opening the barrier. Due to the recent evidence of brain macrophage modulation in response to FUS-BBBO, we provide the first direct comparison of brain macrophage modulation between all three paradigms both in the presence and absence of Alzheimer’s disease (AD) pathology. Flow cytometry and single-cell sequencing are employed to identify FUS-BBBO as the FUS paradigm, which maximizes brain macrophage modulation, including an increase in the population of neuroprotective, disease-associated microglia and direct correlation between treatment cavitation dose and brain macrophage phagocytosis. Next, we combine spatial and single-cell transcriptomics with immunohistochemical validation to provide the first characterization of brain macrophage distribution in response to FUS-BBBO. Given their relevance within neurodegeneration and perturbation response, we emphasize the analysis of three brain macrophage populations—disease- and interferon-associated microglia and central-nervous-system-associated macrophages. We find and validate the redistribution of each population with an overall trend toward increased interaction with the brain–cerebrospinal fluid barrier (BCSFB) after FUS-BBBO, an effect that is found to be more pronounced in the presence of disease pathology. This study addresses the prior lack of FUS neuroimmunomodulation paradigm optimization and mechanism characterization, identifying that FUS-BBBO best modulates brain macrophage response via complex redistribution.

Ependyma in Neurodegenerative Diseases, Radiation-Induced Brain Injury and as a Therapeutic Target for Neurotrophic Factors.

The neuron loss caused by the progressive damage to the nervous system is proposed to be the main pathogenesis of neurodegenerative diseases. Ependyma is a layer of ciliated ependymal cells that participates in the formation of the brain-cerebrospinal fluid barrier (BCB). It functions to promotes the circulation of cerebrospinal fluid (CSF) and the material exchange between CSF and brain interstitial fluid. Radiation-induced brain injury (RIBI) shows obvious impairments of the blood-brain barrier (BBB). In the neuroinflammatory processes after acute brain injury, a large amount of complement proteins and infiltrated immune cells are circulated in the CSF to resist brain damage and promote substance exchange through the BCB. However, as the protective barrier lining the brain ventricles, the ependyma is extremely vulnerable to cytotoxic and cytolytic immune responses. When the ependyma is damaged, the integrity of BCB is destroyed, and the CSF flow and material exchange is affected, leading to brain microenvironment imbalance, which plays a vital role in the pathogenesis of neurodegenerative diseases. Epidermal growth factor (EGF) and other neurotrophic factors promote the differentiation and maturation of ependymal cells to maintain the integrity of the ependyma and the activity of ependymal cilia, and may have therapeutic potential in restoring the homeostasis of the brain microenvironment after RIBI or during the pathogenesis of neurodegenerative diseases.

Ependymal cells and neurodegenerative disease: outcomes of compromised ependymal barrier function.

Within the central nervous system, ependymal cells form critical components of the blood-cerebrospinal fluid barrier and the cerebrospinal fluid-brain barrier. These barriers provide biochemical, immunological and physical protection against the entry of molecules and foreign substances into the cerebrospinal fluid while also regulating cerebrospinal fluid dynamics, such as the composition, flow and removal of waste from the cerebrospinal fluid. Previous research has demonstrated that several neurodegenerative diseases, such as Alzheimer's disease and multiple sclerosis, display irregularities in ependymal cell function, morphology, gene expression and metabolism. Despite playing key roles in maintaining overall brain health, ependymal barriers are largely overlooked and understudied in the context of disease, thus limiting the development of novel diagnostic and treatment options. Therefore, this review explores the anatomical properties, functions and structures that define ependymal cells in the healthy brain, as well as the ways in which ependymal cell dysregulation manifests across several neurodegenerative diseases. Specifically, we will address potential mechanisms, causes and consequences of ependymal cell dysfunction and describe how compromising the integrity of ependymal barriers may initiate, contribute to, or drive widespread neurodegeneration in the brain.

Nanoparticle Drug Delivery System for Glioma and Its Efficacy Improvement Strategies: A Comprehensive Review.

Gliomas are the most common tumor of the central nervous system. However, the presence of the brain barrier blocks the effective delivery of drugs and leads to the treatment failure of various drugs. The development of a nanoparticle drug delivery system (NDDS) can solve this problem. In this review, we summarized the brain barrier (including blood–brain barrier (BBB), blood–brain tumor barriers (BBTB), brain–cerebrospinal fluid barrier (BCB), and nose-to-brain barrier), NDDS of glioma (such as passive targeting systems, active targeting systems, and environmental responsive targeting systems), and NDDS efficacy improvement strategies and deficiencies. The research prospect of drug-targeted delivery systems for glioma is also discussed.

Selective localization of IgG from cerebrospinal fluid to brain parenchyma

BackgroundEncounter of autoantibodies with specific antigens can lead to hypersensitivity reactions and pathology. In multiple sclerosis and neuromyelitis optica spectrum disease (NMOSD), immunoglobulin-G (IgG) deposition has been observed in pathological lesions in the central nervous system. The paradigmatic autoantibodies in NMOSD are specific for the water channel aquaporin-4, localized to astrocytic end-feet at the blood-brain barrier and ependymal cells at the cerebrospinal fluid-brain barrier. We have previously observed that IgG antibodies from NMO patients (NMO-IgG) access brain parenchyma from the cerebrospinal fluid and induce subpial and periventricular NMO-like lesions and blood-brain barrier breakdown, in a complement-dependent manner.ObjectiveTo investigate how IgG trafficking from cerebrospinal fluid to brain parenchyma can be influenced by injury.MethodsIgG from healthy donors was intrathecally injected into the cerebrospinal fluid via cisterna magna at 1, 2, 4, or 7 days after a distal stereotactic sterile needle insertion to the striatum.ResultsAntibody deposition, detected by staining for human IgG, peaked 1 day after the intrathecal injection and was selectively seen close to the needle insertion. When NMO-IgG was intrathecally injected, we observed complement-dependent NMO-like pathology (loss of aquaporin-4 and glial fibrillary acidic protein) proximal to the insertion site, with similar kinetics. A fluorescent tracer did not show the same distribution indicating IgG-selective localization.ConclusionThese findings suggest that IgG from cerebrospinal fluid localize selectively in brain parenchyma at the site of injury and pathogenic NMO-IgG induce astrocyte pathology at the same location.

Parkinson’s disease

Adeno-associated virus (AAV) vectors are derived from the non-pathogenic parvovirus. The genome of AAV vector does not integrate into the host chromosome and persists as an episome in the nucleus of host cells. Although their small packaging capacity (about 3 kb) can limit the use of AAV vectors, most therapeutic genes fall within this size range. Use of multiple vectors and simultaneous expression of the respective genes into the target cell can overcome this drawback. AAV vectors transduce neurons efficiently and can express therapeutic genes or miRNA for prolonged periods without causing any toxic effects. To date, AAV vectors have been used in more than 183 clinical trials conducted for various diseases such as retinal degeneration, hemophilia, and Parkinson disease, and have shown no adverse effects. The expression of dopamine-synthesizing enzymes persisted 15 years after gene transfer in a primate model of Parkinson’s disease. In clinical studies, intraputaminal delivery of AAV vectors that contain aromatic l-amino acid decarboxylase (AADC) gene show the beneficial effects in patients with either advanced Parkinson’s disease or AADC deficiency. Some AAV vectors can cross the blood or cerebrospinal fluid-brain barrier and transduce neurons of the central nervous system after systemic injection. Use of these AAV vectors in proof-of-principle investigations of animal models for various metabolic and neurologic diseases such as lysosomal storage disease, Alzheimer’s disease, spinocerebellar ataxia, and motor neuron disease has yielded favorable results. Various advantages of AAV vector-based gene therapy compared to ordinary drugs necessitate the development of more manufacturing systems that can optimize large-scale production of GMP-grade AAV vectors.

Unmasking the Role of Uptake Transporters for Digoxin Uptake Across the Barriers of the Central Nervous System in Rat.

The role of uptake transporter (organic anion–transporting polypeptide [Oatp]) in the disposition of a P-glycoprotein (P-gp) substrate (digoxin) at the barriers of central nervous system, namely, the blood-brain barrier (BBB), blood-spinal cord barrier (BSCB), and brain-cerebrospinal fluid barrier (BCSFB), was studied using rat as a preclinical species. In vivo chemical inhibition of P-gp and Oatp was achieved using elacridar and rifampicin, respectively. Our findings show that (1) digoxin had a low brain-to-plasma concentration ratio (B/P) (0.07) in rat; (2) in the presence of elacridar, the B/P of digoxin increased by about 12-fold; (3) rifampicin administration alone did not change the digoxin B/P significantly when compared with digoxin B/P alone; (4) rifampicin administration along with elacridar resulted only in 6-fold increase in the B/P of digoxin; (5) similar fold changes and trends were seen with the spinal cord-to-plasma concentration ratio of digoxin, indicating the similarity between BBB and the BSCB; and (6) unlike BBB and BSCB, the presence of rifampicin further increased the cerebrospinal fluid-to-plasma concentration ratio (CSF/P) for digoxin, suggesting a differential orientation of the uptake transporters at the BCSFB (CSF to blood) compared with the BBB (blood to brain). The observations for digoxin uptake, at least at the BBB and the BSCB, advocate the importance of uptake transporters (Oatps). However, the activity of such uptake transporters became evident only after inhibition of the efflux transporter (P-gp).

The inner CSF-brain barrier: developmentally controlled access to the brain via intercellular junctions.

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.

Systemic factors mediate reversible age-associated brain dysfunction.

Brain function declines in aging mammals. Recent work has identified dysregulation of key blood-borne factors whose altered expression during aging diminishes brain function in mice. Increased C-C motif chemokine 11 (CCL11) expression with aging is detrimental to brain function. On the other hand, plasma levels of the trophic factor growth/differentiation factor 11 (GDF11) decrease with aging. Restoration of youthful levels of GDF11 by injection partially restores brain function and neurogenesis by improving endothelial cell function and vasculature. Moreover, GDF11 has a rejuvenative effect on cardiac and skeletal muscle. Decreased type II interferon (IFN-II) and increased type I interferon (IFN-I) signaling during aging at the choroid plexus (CP), which constitutes the brain-cerebrospinal fluid barrier (B-CSF-B), negatively effects brain function. Blood from young mice contains factors that restore IFN-II levels. IFN-II is required for maintenance of the CP, and low IFN-II levels are associated with decreased cognitive abilities. IFN-I levels appear to drive increased CCL11 expression through the CSF. Blood from young animals does not restore IFN-I levels. However, injecting anti-interferon-α/β receptor (IFNAR) antibodies into the CSF inhibits downstream IFN-I gene and protein expression and decreases expression of CCL11, partially restoring neurogenesis and cognitive function. These results suggest that IFN-I plays a critical role in increasing CCL11 during aging of the brain. An emerging theme is that aging-associated loss of function in mammals may involve a set of defined, potentially reversible changes in many tissues and organs, including the brain, permitting development of potential rejuvenative therapies.

Ependymal damage in a Plasmodium yoelii yoelii lethal murine malaria model.

Malaria continues to be a major global health problem, and over 40% of the world's population is at risk. Severe or complicated malaria is defined by clinical or laboratory evidence of vital organ dysfunction, including dysfunction of the central nervous system (CNS). The pathogenesis of complicated malaria has not been completely elucidated; however, the development of the multiorgan affection seems to play an important role in the disruption of the blood brain barrier (BBB) that protects the CNS against chemical insults. Historically, the BBB has received more attention in the pathogenesis of malaria than have the cerebrospinal fluid-brain barrier (CSFBB) and ependymal cells. This perspective may be misguided because, in the context of disease or toxicity, the CSFBB is more vulnerable to many foreign invaders than are the capillaries. Given the lack on studies of the damage to the CSFBB and ependymal epithelium in experimental murine malaria, the present study evaluated morphological changes in the ependymal cells of CD-1 male mice infected with lethal Plasmodium yoelii yoelii (Pyy) via histopathology and scanning electron microscopy (SEM). Samples were taken two, four and six days post-infection (PI). No lesions were observed upon the initial infection. By the fourth day PI, fourth ventricle ependymal samples exhibited disruptions and roughened epithelia. More severe injuries were observed at six days PI and included thickened cilia and deep separations between the ependymal intercellular spaces. In some of the analyzed areas, the absence of microvilli and cell layer detachment were observed, and some areas exhibited blebbing surfaces. The ependymal cell lesions observed in the CD1 male mice infected with lethal Pyy seemed to facilitate the paracellular permeability of the CSFBB and consequently promote the access of inflammatory mediators and toxic molecules through the barrier, which resulted in damage to the brain tissue. Understanding the mechanism of ependymal disruption during lethal murine malaria could help to elucidate the local and systemic factors that are involved in the pathogenesis of the disease and may provide essential clues for the prevention and treatment of complicated human malaria.

Superficial siderosis associated with aceruloplasminemia. Case report

A 63-year-old woman with a past history of right subdural hematoma (SDH) at the age of 61years was referred to our hospital under a suspicion of aceruloplasminemia (ACP). A neurological examination revealed very mild cognitive impairment and cerebellar ataxia. Blood chemistry data showed deficient ceruloplasmin (Cp), decreased copper, and increased ferritin. A nonsense mutation (c.2630G>A, p.Trp858Ter) was detected in the Cp gene. Brain magnetic resonance imaging (MRI) showed marked hypointensity at the surface of the cerebrum, cerebellum, and brainstem bilaterally, in addition to the bilateral basal ganglia, thalamus, and dentate nucleus, suggesting the coexistence of ACP and superficial siderosis (SS). The characteristics of SS in ACP have not been examined neuroradiologically or neuropathologically in great detail, while SDH and its curative surgery are known to cause SS. The distribution of the hypointensity areas on MRI was expanded bilaterally to the subtentorial areas of this patient, which was much more widespread than observed in typical SS after SDH. We speculate that the underlying ACP may expand the SS induced by SDH. Cp would accelerate iron export from the brain via the blood–cerebrospinal fluid (CSF) barrier, or CSF–brain barrier when excessive iron is loaded into the subarachnoid space.

Localization of the Na +-coupled neutral amino acid transporter 2 in the cerebral cortex

We studied the distribution and cellular localization of Na +-coupled neutral amino acid transporter 2, a member of the system A family of amino acid transporters, in the rat and human cerebral cortex using immunocytochemical methods. Na +-coupled neutral amino acid transporter 2-positive neurons were pyramidal and non-pyramidal, and Na +-coupled neutral amino acid transporter 2/GABA double-labeling studies revealed that Na +-coupled neutral amino acid transporter 2 was highly expressed by GABAergic neurons. Double-labeling studies with the synaptophysin indicated that rare axon terminals express Na +-coupled neutral amino acid transporter 2. Na +-coupled neutral amino acid transporter 2-immunoreactivity was also found in astrocytes, leptomeninges, ependymal cells and choroid plexus. Electron microscopy showed robust Na +-coupled neutral amino acid transporter 2-immunoreactivity in the somato-dendritic compartment of neurons and in glial processes, but, as in the case of double-labeling studies, failed to reveal Na +-coupled neutral amino acid transporter 2-immunoreactivity in terminals. To rule out the possibility that the absence of Na +-coupled neutral amino acid transporter 1-[Melone M, Quagliano F, Barbaresi P, Varoqui H, Erickson JD, Conti F (2004) Localization of the glutamine transporter SNAT1 in rat cerebral cortex and neighboring structures, with a note on its localization in human cortex. Cereb Cortex 14:562–574] and Na +-coupled neutral amino acid transporter 2-positive terminals was due to insufficient antigen detection, we evaluated Na +-coupled neutral amino acid transporter 1/synaptophysin and Na +-coupled neutral amino acid transporter 2/synaptophysin coexpression using non-standard immunocytochemical procedures and found that Na +-coupled neutral amino acid transporter 1 and Na +-coupled neutral amino acid transporter 2+ terminals were rare in all conditions. These findings indicate that Na +-coupled neutral amino acid transporter 1 and Na +-coupled neutral amino acid transporter 2 are virtually absent in cortical terminals, and suggest that they do not contribute significantly to replenishing the Glu and GABA transmitter pools through the glutamate–glutamine cycle. The strong expression of Na +-coupled neutral amino acid transporter 2 in the somato-dendritic compartment and in non-neuronal elements that are integral parts of the blood–brain and brain–cerebrospinal fluid barrier suggests that Na +-coupled neutral amino acid transporter 2 plays a role in regulating the levels of Gln and other amino acids in the metabolic compartment of cortical neurons.

Prolonged intrathecal release of soluble Fas following severe traumatic brain injury in humans

The mechanisms underlying cell death following traumatic brain injury (TBI) are not fully understood. Apoptosis is believed to be one mechanism contributing to a marked and prolonged neuronal cell loss following TBI. Recent data suggest a role for Fas (APO-1, CD95), a type I transmembrane receptor glycoprotein of the nerve growth factor/tumor necrosis factor superfamily, and its ligand (Fas ligand, FasL) in apoptotic events in the central nervous system. A truncated form of the Fas receptor, soluble Fas (sFas) may indicate activation of the Fas/FasL system and act as a negative feedback mechanism, thereby inhibiting Fas mediated apoptosis. Soluble Fas was measured in cerebrospinal fluid (CSF) and serum of 10 patients with severe TBI (GCS≤8) for up to 15 days post-trauma. No sFas was detected in CSF samples from patients without neurological pathologies. Conversely, after TBI 118 out of 120 CSF samples showed elevated sFas concentrations ranging from 56 to 4327 mU/ml. Paired serum samples showed above normal (8.5 U/ml) sFas concentrations in 5 of 10 patients. Serum levels of sFas were always higher than CSF levels. However, there was no correlation between concentrations measured in CSF and in serum ( r 2=0.078, p=0.02), suggesting that the concentrations in the two compartments are independently regulated. Also, no correlation was found between sFas in CSF and blood brain barrier (BBB) dysfunction as assessed by the albumin CSF/serum quotient ( Q A), and concentrations of the cytotoxic cytokine tumor necrosis factor-alpha in CSF, respectively. Furthermore, there was no correlation with two markers of immune activation (soluble interleukin-2 receptor and neopterin) in CSF. Maximal CSF levels of sFas correlated significantly ( r 2=0.8191, p<0.001) with the early peaks of neuron-specific enolase in CSF (a marker for neuronal cell destruction), indicating that activation of the Fas mediated pathway of apoptosis may be in part the direct result of the initial trauma. However, the prolonged elevation of sFas in CSF may be caused by the ongoing inflammatory response to trauma and delayed apoptotic cell death.

Elevated expression of glucose transporter‐1 in hypothalamic ependymal cells not involved in the formation of the brain–cerebrospinal fluid barrier

Elevated expression of glucose transporter‐1 in hypothalamic ependymal cells not involved in the formation of the brain–cerebrospinal fluid barrier

Elevated expression of glucose transporter-1 in hypothalamic ependymal cells not involved in the formation of the brain-cerebrospinal fluid barrier

Glucose transporters play an essential role in the acquisition of glucose by the brain. Elevated expression of glucose transporter-1 has been detected in endothelial cells of the blood-brain barrier and in choroid plexus cells of the blood-cerebrospinal fluid barrier. On the other hand, there is a paucity of information on the expression of glucose transporters in the ependymal cells that line the walls of the cerebral ventricles. The tanycytes are specialized ependymal cells localized in circumventricular organs such as the median eminence that can be segregated into at least three types, alpha, beta1 and beta2. The beta2 tanycytes form tight junctions and participate in the formation of the cerebrospinal fluid-median eminence barrier. Using immunocytochemistry and in situ hybridization, we analyzed the expression of hexose transporters in rat and mouse hypothalamic tanycytes. In both species, immunocytochemical analysis revealed elevated expression of glucose transporter-1 in alpha and beta1 tanycytes. Intense anti-glucose transporter-1 staining was observed in cell processes located throughout the arcuate nucleus, in the end-feet reaching the lateral sulcus of the infundibular region, and in cell processes contacting the hypothalamic capillaries. On the other hand, there was very low expression of glucose transporter-1 in beta2 tanycytes involved in barrier function. In contrast with the results of the cytochemical analysis, in situ hybridization revealed that tanycytes alpha, beta1, and beta2 express similar levels of glucose transporter-1 mRNA. Further analysis using anti-glial fibrillary acidic protein antibodies to identify areas rich in astrocytes revealed that astrocytes were absent from areas containing alpha and beta1 tanycytes, but were abundant in regions containing the barrier-forming beta2 tanycytes. Overall, our data reveal a lack of correlation between participation in barrier function and expression of glucose transporter-1 in hypothalamic tanycytes. Given the virtual absence of astrocytes in areas rich in alpha and beta1 tanycytes, we speculate whether the tanycytes might have astrocyte-like functions and participate in the metabolic coupling between glia and neurons in the hypothalamic area.

Intercellular barriers to and transcellular transfer of albumin in the fetal sheep brain.

The nature of the barriers that keep proteins out of the developing brain has been studied in tissues obtained from fetal sheep in experiments conducted under controlled physiological conditions. In anaesthetised pregnant ewes, 60 day gestation fetuses (term is 150 days) were exposed to human albumin injected intravenously for periods up to 6 h. The immunocytochemical distribution of exogenous human albumin was compared with that of endogenous sheep albumin at both the light and electron-microscopical level. Immunogold labelling of ultracryosections suggests that a tubulocisternal endoplasmic reticulum system in immature choroid-plexus epithelial cells is the route by which albumin crosses from blood to cerebrospinal fluid (CSF) in the developing brain. The integrity of the blood-brain barrier, the blood-cerebrospinal fluid barrier and the cerebrospinal fluid-brain barrier to protein, was confirmed. In addition, at the outer surface of the developing brain there also appears to be a restriction on the passage of albumin from CSF into the brain. These observations support earlier proposals that the immature brain develops within an internal environment from which proteins in plasma and CSF are largely excluded.

Magnetic resonance imaging and histopathological study of brain lesions in rabbits given intravenous verotoxin 2.

When rabbits were given intravenously purified verotoxin 2 (VT2) at 5 microg/kg of body weight, they developed hemorrhagic diarrhea, flaccid paresis, an ataxic gait, an opisthotonic posture, and convulsions. To examine the effects of VT2 toxemia on the rabbit central nervous system, magnetic resonance imaging and ultrastructural studies were performed. At 24, 57, and 80 h after injection of VT2 into 12 rabbits, T2-weighted images of the central nervous system were obtained. The initial lesion was noted at 24 h in the hypothalamic areas of all experimental animals. At 57 h, the T2 value increased in the medulla of the cerebral hemisphere or the hippocampus, with a brain stem lesion in six rabbits (50%). The rabbits with the brain stem lesions, in which neurological signs were very severe, died within 6 days. Lesions in the cerebellar hemisphere and/or vermis were noted in four rabbits (33%) that survived more than 1 month. To better understand the pathogenesis of VT2 in these brain lesions, we examined the deterioration of the blood-brain barrier and cerebrospinal fluid-brain barrier by using horseradish peroxidase as a tracer. The tracer was detected by electron microscopy both in the subendothelial layer, including the basal lamina, and throughout the cytoplasm of the ependymal cell layer covering the ventricle after intravenous or intrathecal treatment with horseradish peroxidase. We also determined the localization of VT2 by immunoelectron microscopy and found that it was localized on edematous endothelial cells of capillaries, ependymal cells, and myelin sheaths. The present study suggests that VT2 was conveyed from the endothelial and ependymal cell layers and caused edematous changes in the rabbit brain.

Rapid distribution of intraventricularly administered sucrose into cerebrospinal fluid cisterns via subarachnoid velae in rat

The intracranial distribution of [ 14C]sucrose, an extracellular marker infused for 30 s into one lateral ventricle, was determined by autoradiography of frozen-dried brain sections. Within 3.5 min [ 14C]sucrose appeared in: (i) the third ventricle, including optic, infundibular and mammillary recesses; (ii) the aqueduct of Sylvius; (iii) the velum interpositum, a part of the subarachnoid space that runs along the roof of the third ventricle and contains many blood vessels; (iv) the mesencephalic and fourth ventricles; and (v) the superior medullary velum, a highly vascular extension of the subarachnoid space that terminates at the walls of the mesencephalic and fourth ventricles. Within 5 min, radioactivity was present in the interpeduncular, ambient and quadrigeminal cisterns, which encircle the midbrain. By 10 min, approximately 11% of the radioactivity had passed into the subarachnoid space via a previously undescribed flow pathway that included the velum interpositum and superior medullary velum. At many places along the ventricular system, [ 14C]sucrose appeared to move from cerebrospinal fluid into the adjacent tissue by simple diffusion, as reported previously [Blasberg R. G. et al. (1974) J. Pharmac. exp. Ther. 195, 73–83; Levin V. A. et al. (1970) Am. J. Physiol. 219, 1528–1533; Patlak C. and Fenstermacher J. D. (1975) Am. J. Physiol. 229, 877–884; Rosenberg G. A. and Kyner W. T. (1980) Brain Res. 193, 56–66; Rosenberg G. A. et al. (1986) Am. J. Physiol. 251, F485–F489]. Little sucrose was, however, taken up by: (i) circumventricular organs such as the subfornical organ; (ii) medullary and cerebellar tissue next to the lateral recesses; and (iii) the superior and inferior colliculi and cerebral peduncles. For the latter two groups of structures, entry from cerebrospinal fluid was apparently blocked by a thick, multilayered glia limitans. Although [ 14C]sucrose was virtually absent from the rest of the subarachnoid system after 1 h, it remained in the perivascular spaces and/or walls of pial arteries and arterioles for more than 3 h. Certain transport proteins, protease inhibitors, growth factors and other neurobiologically active materials are present in cerebrospinal fluid, and their distribution to the brain and its blood vessels may be important. The present work shows, in the rat, that the flow of cerebrospinal fluid and the disposition of its constituents is fairly complex and differs among regions. Flow was rapid throughout the ventricular system and into various subarachnoid velae and cisterns, but was surprisingly slow and slight over the cerebral and cerebellar cortices. The cerebrospinal fluid-to-tissue flux of material was relatively free at many interfaces, but was greatly restricted at others, the latter indicating that the old concept of a “cerebrospinal fluid–brain barrier” may hold at such places. Finally, radiolabeled sucrose was retained longer within the walls and perivascular spaces of pial arteries and arterioles than in other subarachnoid tissues; one function of the cerebrospinal fluid system or “third circulation” may thus be delivering factors and agents to these pial blood vessels.