Development of cerebrospinal fluid and the blood-cerebrospinal fluid barrier in rabbits.

Blood plasma and cerebrospinal fluid (CSF) samples were collected from adult female rabbits (New Zealand White), newborn, and embryos at 18, 20, 24, and 28 days of gestation. Samples were analyzed for total protein using the Folin phenol reagent. During development, mean total protein of blood plasma rose sharply from 12.45 to 12.51 mg/ml at 18 to 20 days to 37.56 mg/ml at 28 days. Levels further increased to 54.06 mg/ml in the newborn and to 66.18 mg/ml in the adult. The protein concentration of cerebrospinal fluid was constant at 5.20 to 5.29 mg/ml between 18 and 20 days of gestation, but steadily decreased to 3.53 mg/ml at 28 days. By birth, the CSF protein concentration was further reduced to 2.08 mg/ml, and this level differed only slightly (P < 0.05) from CSF protein values determined for adults. These data indicate that the blood-cerebrospinal fluid barrier to proteins begins to function by 18 to 20 days of gestation, and the protein concentration of cerebrospinal fluid approaches the normal adult value soon after birth.     

Glutathione metabolism at the blood-cerebrospinal fluid barrier

Glutathione metabolism and transport in the choroid plexus were probed by determining the effects of administration to rats of several compounds (buthionine sulfoximine, L-2-oxothiazolidine-4-carboxylate, L-(alpha 5,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazole acetic acid, gamma-glutamyl alanine, and glutathione monoethyl ester) on the levels of glutathione and cysteine in the cerebrospinal fluid. The findings indicate that glutathione is actively metabolized in the choroid plexus by pathways similar to those in kidney and other tissues. The level of glutathione in the cerebrospinal fluid can be decreased or increased by giving compounds that do not, under similar conditions, appreciably alter total brain levels of glutathione. Glutathione monoethyl ester is effectively transported into the cerebrospinal fluid.     

Structural basics for development of blood-cerebrospinal fluid barrier in man

Studies of development of hematoliquorian barrier in man represent significant difficulties, as it is not possible to employ the experimental-physiological approaches. In these conditions, the morphological analysis based on application of modern immunocytochemistry methods acquires the key role in fundamental physiological studies of onthogenesis of barrier central neurology systems. The current article presents an analytical review of publications and results of own authors research of structural organization of the hematoliquorian barrier in man during the prenatal ontogenesis.     

Strain-dependent disruption of blood-cerebrospinal fluid barrier by Streptoccocus suis in vitro

Streptococcus suis capsular type 2 is an important agent of diseases including meningitis among pigs worldwide, and is also a zoonotic agent. The barrier function of the choroid plexus epithelium that constitutes the structural basis for the blood-cerebrospinal fluid (CSF) barrier has not been elucidated yet in bacterial meningitis. We investigated the influence of various S. suis isolates on the barrier function of cultured porcine choroid plexus epithelial cells with respect to the transepithelial resistance and paracellular [(3)H]-mannitol flux. Preferentially apical application of S. suis isolates significantly decreased transepithelial resistance and significantly increased paracellular [(3)H]-mannitol flux in a time-, dose- and strain-dependent manner. Viable S. suis isolates caused cytotoxicity determined by lactate dehydrogenase assay and electron microscopy, whereas S. suis sonicates and UV-inactivated S. suis did not cause cytotoxicity. The observed effects on porcine choroid plexus epithelial cells barrier function could not exclusively be ascribed to known virulence factors of S. suis such as suilysin. In conclusion, S. suis isolates induce loss of blood-cerebrospinal fluid barrier function in an in vitro model. Thus, S. suis may facilitate trafficking of bacteria and leucocytes across the blood-cerebrospinal fluid barrier. The underlying mechanisms for the barrier breakdown have yet to be determined.     

MMPs contribute to TNF-alpha-induced alteration of the blood-cerebrospinal fluid barrier in vitro

The epithelial cells of the choroid plexus separate the central nervous system from the blood forming the blood-cerebrospinal fluid (CSF) barrier. The choroid plexus is the main source of CSF, whose composition is markedly changed during pathological disorders, for example regarding matrix metalloproteases (MMPs) and tissue inhibitors of matrix metalloproteases (TIMPs). In the present study, we analyzed the impact of the proinflammatory cytokine tumor necrosis factor- (TNF-) on the blood-CSF barrier using an in vitro model based on porcine choroid plexus epithelial cells (PCPEC). TNF- evoked distinct inflammatory processes as shown by mRNA upregulation of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1. The cytokine caused a drastic decrease in transepithelial electrical resistance within several hours representing an enhanced permeability of PCPEC monolayers. In addition, the distribution of tight junction proteins was altered. Moreover, MMP activity in PCPEC supernatants was significantly increased by TNF-, presumably due to a diminished expression of TIMP-3 that was similarly observed. MMP-2, -3, and -9 as well as TIMP-1 and -2 were also analyzed and found to be differentially regulated by the cytokine. The TNF--induced breakdown of the blood-CSF barrier could partially be blocked by the MMP inhibitor GM-6001. Our results show a contribution of MMPs to the inflammatory breakdown of the blood-CSF barrier in vitro. Thus TNF- may mediate the binding of leukocytes to cellular adhesion molecules and the transmigration across the blood-CSF barrier. choroid plexus; matrix metalloproteases; tight junction; transepithelial electrical resistance; porcine choroid plexus epithelial cells; tumor necrosis factor-     

Intact and carboxyterminal PTH do not cross the blood-cerebrospinal fluid barrier

In order to examine whether parathyroid hormone (PTH) enters the cerebrospinal fluid (CSF), the blood levels of the hormone were acutely elevated either by infusion of parathyroid extract or by stimulation of the parathyroid glands by hypocalcemia. Despite marked elevations in the blood levels of the hormone, PTH could not be detected in the CSF. The data indicate the intact PTH or its carboxyterminal fragment do not cross the blood-CSF interface of the blood-brain barrier. The results, therefore, suggest that the action of PTH on brain must be mediated by an effect on the blood-brain interface of the blood-brain barrier.     

Development of blood-cerebrospinal fluid barrier to horseradish peroxidase in the avian choroidal epithelium

Summary Four neurons in the brain of the migratory locust were immunohistologically identified with an anti-met-enkephalin antiserum. The perikarya of two of these cells are located in the center of each of the two groups of lateral protocerebral neurosecretory cells. The fibres coming from these perikarya terminate in numerous immunoreactive ramifications visible at the periphery of both tractus I to the corpora cardiaca, through which pass the neurosecretory products of the pars intercerebralis. The other two cell bodies are located at the bases of the two optic lobes; their fibres enter the posterior part of the protocerebrum and ramify around the root of the nervus corporis cardiaci II, another area through which neurosecretory products pass. The topographic distribution of these met-enkephalin arborizations suggests that these four neurons may act as neuromodulators of the acitivity of the major neurosecretory cells in the brain of this insect.     

Impact of manganese on and transfer across blood-brain and blood-cerebrospinal fluid barrier in vitro

Manganese occupational and dietary overexposure has been shown to result in specific clinical central nervous system syndromes, which are similar to those observed in Parkinson disease. To date, modes of neurotoxic action of Mn are still to be elucidated but are thought to be strongly related to Mn accumulation in brain and oxidative stress. However, the pathway and the exact process of Mn uptake in the brain are yet not fully understood. Here, two well characterized primary porcine in vitro models of the blood-brain and the blood-cerebrospinal fluid (CSF) barrier were applied to assess the transfer of Mn in the brain while monitoring its effect on the barrier properties. Thus, for the first time effects of MnCl(2) on the integrity of these two barriers as well as Mn transfer across the respective barriers are compared in one study. The data reveal a stronger Mn sensitivity of the in vitro blood-CSF barrier compared with the blood-brain barrier. Very interestingly, the negative effects of Mn on the structural and functional properties of the highly Mn-sensitive blood-CSF barrier were partly reversible after incubation with calcium. In summary, both the observed stronger Mn sensitivity of the in vitro blood-CSF barrier and the observed site-directed, most probably active, Mn transport toward the brain facing compartment, reveal that, in contrast to the general assumption in literature, after oral Mn intake the blood-CSF barrier might be the major route for Mn into the brain.     

Passage of delta sleep-inducing peptide (DSIP) across the blood-cerebrospinal fluid barrier

Unidirectional flux of 125I-labeled DSIP at the blood-tissue interface of the blood-cerebrospinal fluid (CSF) barrier was studied in the perfused in situ choroid plexuses of the lateral ventricles of the sheep. Arterio-venous loss of 125I-radioactivity suggested a low-to-moderate permeability of the choroid epithelium to the intact peptide from the blood side. A saturable mechanism with Michaelis-Menten type kinetics with high affinity and very low capacity (approximate values: Kt = 5.0 +/- 0.4 nM; Vmax = 272 +/- 10 fmol.min-1) was demonstrated at the blood-tissue interface of the choroid plexus. The clearance of DSIP from the ventricles during ventriculo-cisternal perfusion in the rabbit indicated no significant flux of the intact peptide out of the CSF. The results suggest that DSIP crosses the blood-CSF barrier, while the system lacks the specific mechanisms for removal from the CSF found with most, if not all, amino acids and several peptides.     

Polar bacterial invasion and translocation of Streptococcus suis across the blood-cerebrospinal fluid barrier in vitro

Previous experimental studies in a standard Transwell culture system have shown Streptococcus suis ability to compromise barrier function of porcine choroid plexus epithelial cells (PCPEC). The development of an 'inverted' Transwell filter system of PCPEC enables us now for the first time to investigate bacterial invasion and translocation from the physiologically relevant basolateral (blood) to the apical (cerobrospinal fluid) side. Most importantly, we observed specific invasion and translocation of S. suis across the PCPEC exclusively from the basolateral side. During this process, bacterial viability and the presence of a capsule as well as cytoskeletal regulation of PCPEC seemed to play an important role. No loss of barrier function was observed. Bacterial translocation could be significantly inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002, but not by its inactive analogue   Ly303511 or dexamethasone. Apotome imaging as well as electron microscopy revealed intracellular bacteria often in cell vacuoles. Thus, possibly regulated by the presence of a capsule, S. suis induces signals that depend on the lipid kinase phosphatidylinositol 3-kinase pathway, which paves the way for cellular uptake during the bacterial transcellular translocation process. Taken together, our data underline the relevance of the blood–cerebrospinal fluid barrier as a gate for bacterial entry into the central nervous system.     

Alpha-2-macroglobulin, albumin, and chymotrypsin inhibitory capacity in cerebrospinal fluid as indices of blood-cerebrospinal fluid barrier

Concentration of alpha-2-macroglobulin, albumin, and chymotrypsin inhibitory capacity representing mainly alpha-1-proteinase inhibitor were estimated in cerebrospinal fluid in disorders of the central nervous system. While chymotrypsin inhibitory capacity was elevated in all cases with derangement of the blood-cerebrospinal fluid barrier, in 30% of the cases alpha-2-macroglobulin levels were in the normal range. The difference can be attributed to the much larger size of the latter. Better correlation between albumin concentration and chymotrypsin inhibitory capacity (r = 0.84) than between albumin and alpha-2-macroglobulin (r = 0.62) supports the view that the rate of entry of proteins from blood into cerebrospinal fluid is inversely related to their size.     

The blood-cerebrospinal fluid barrier is a major pathway of cerebral creatinine clearance: involvement of transporter-mediated process

There is still incomplete evidence for the cerebral clearance of creatinine (CTN) which is an endogenous convulsant and accumulates in the brain and CSF of patients with renal failure. The purpose of this study was to clarify the transporter-mediated CTN efflux transport from the brain/CSF. In vivo data demonstrated that CTN after intracerebral administration was not significantly eliminated from the brain across the blood-brain barrier. In contrast, the elimination clearance of CTN from the CSF was 60-fold greater than that of inulin, reflecting CSF bulk flow. Even in renal failure model rats, the increasing ratio of the CTN concentration in the CSF was lower than that in the plasma, suggesting a significant role for the CSF-to-blood efflux process. The inhibitory effects of inhibitors and antisense oligonucleotides on CTN uptake by isolated choroid plexus indicated the involvement of rat organic cation transporter 3 (rOCT3) and creatine transporter (CRT) in CTN transport. rOCT3- and CRT-mediated low-affinity CTN transport with K(m) values of 47.7 and 52.0 mM, respectively. Our findings suggest that CTN is eliminated from the CSF across the blood-CSF barrier as a major pathway of cerebral CTN clearance and transporter-mediated processes are involved in the CTN transport in the choroid plexus.     

Unidirectional uptake of enkephalins at the blood-tissue interface of the blood-cerebrospinal fluid barrier: a saturable mechanism

The cellular uptake at the blood-tissue interface of the blood-cerebrospinal fluid (CSF) barrier to tyrosyl-3,5-[³H]enkephalin-[5-l-leucine] (abbreviated to Leu-enkephalin) and of its synthetic analogue d-alanine²-tyrosyl-3,5-[³H]enkephalin-[5-d-leucine] (abbreviated to d-Ala²-d-Leu⁵-enkephalin) was studied in the isolated perfused choroid plexuses from the lateral ventricles of the sheep, using the rapid (<30 s), single circulation, paired-tracer dilution technique, in which d-[¹⁴C]-mannitol serves as an extracellular marker. Cellular uptake of peptides was estimated by directly comparing venous dilution profiles of [³H] and [¹⁴C] radioactivities in the absence and presence of unlabelled peptide, the N-terminal amino acid (l-tyrosine), the typical l-transport system substrate, 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH) and the inhibitor of aminopeptidase activity, bacitracin. The cellular uptake of both enkephalins was strongly (65–76%) but not completely inhibited by the addition of 5 mM unlabelled peptide to the bolus; the self-inhibition was significantly higher for d-Ala²-d-Leu⁵-enkephalin than for Leu-enkephalin. The addition to the bolus of l-tyrosine (5 mM), BCH (10 mM) or bacitracin (2 mM) reduced the ³H-radioactivity uptake by the choroid plexus of both enkephalins by 20–40%, the degree of inhibition being greater for [³H]-Leu-enkephalin than for its analogue.It is concluded that during single passage of enkephalins through the choroid plexus circulation, unidirectional uptake at the blood-tissue interface of the blood-CSF barrier consists of two components; a saturable component, which represents uptake of the intact peptide by the choroid epithelium, and a non-saturable component, which reflects enzymatic degradation of peptide in the blood and/or at the barrier, with a liberation of the N-terminal tyrosyl residue. Higher penetration of the blood-CSF barrier by d-Ala²-d-Leu⁵-enkephalin can be attributed to its greater resistance to hydrolysis.     

Clinical and laboratory comparisons of tetracycline penetration through the blood-cerebrospinal fluid barrier in central nervous system diseases]

The data on tetracycline penetration from the blood into the serebrospinal fluid of patients with different diseases of the central nervous system are presented. Clinico-laboratory comparisons showed that the antibiotic penetration did not depend on the character and severity of the main disease of the brain (tumor, trauma, abscess). No dependence on the surgical intervention was either found. The index of tetracycline penetration from the blood into the liquor did not depend on the drug administration route, i.e. intramuscularly or orally. The presence of the post-operative meningitis increased permeability of the brain membranes for tetracycline.     

Polar invasion and translocation of Neisseria meningitidis and Streptococcus suis in a novel human model of the blood-cerebrospinal fluid barrier

Acute bacterial meningitis is a life-threatening disease in humans. Discussed as entry sites for pathogens into the brain are the blood-brain and the blood-cerebrospinal fluid barrier (BCSFB). Although human brain microvascular endothelial cells (HBMEC) constitute a well established human in vitro model for the blood-brain barrier, until now no reliable human system presenting the BCSFB has been developed. Here, we describe for the first time a functional human BCSFB model based on human choroid plexus papilloma cells (HIBCPP), which display typical hallmarks of a BCSFB as the expression of junctional proteins and formation of tight junctions, a high electrical resistance and minimal levels of macromolecular flux when grown on transwell filters. Importantly, when challenged with the zoonotic pathogen Streptococcus suis or the human pathogenic bacterium Neisseria meningitidis the HIBCPP show polar bacterial invasion only from the physiologically relevant basolateral side. Meningococcal invasion is attenuated by the presence of a capsule and translocated N. meningitidis form microcolonies on the apical side of HIBCPP opposite of sites of entry. As a functionally relevant human model of the BCSFB the HIBCPP offer a wide range of options for analysis of disease-related mechanisms at the choroid plexus epithelium, especially involving human pathogens.