A physiologically relevant blood-cerebrospinal fluid barrier model to study the permeability of neurodegenerative biomarkers.

In the brain, aquaporin-1 (AQP-1), a water channel for high osmotic water permeability, is mainly expressed in the apical membrane of the ventricular choroid plexus and regulates formation of cerebrospinal fluid (CSF). Although the physiology of AQP-1 has been the subject of several publications, much less is known about the trans-acting factors involved in the control of AQP-1 gene expression. Here we report that TTF-1, a homeodomain-containing transcriptional regulator, is coexpressed with AQP-1 in the rat brain choroid plexus and enhances AQP-1 gene transcription by binding to conserved core TTF-1-binding motifs in the 5'-flanking region of the AQP-1 gene. Intracerebroventricular administration of an antisense TTF-1 oligodeoxynucleotide significantly decreased AQP-1 synthesis and reduced CSF formation. In addition, blockade of TTF-1 synthesis increased survival of the animals following acute water intoxication-induced brain edema. These results suggest that TTF-1 is physiologically involved in the transcriptional control of AQP-1, which is required for CSF formation.

Characterization of thyroid hormone transport in a human choroid plexus papilloma cell line (HIBCPP) as an in vitro blood-cerebrospinal fluid barrier model.

BackgroundIdiopathic normal pressure hydrocephalus (iNPH) is a potentially reversible neurological condition of unresolved etiology characterized by a clinical triad of symptoms; gait disturbances, urinary incontinence, and cognitive deterioration. In the present study, we aimed to elucidate the molecular coupling between inflammatory markers and development of iNPH and determine whether inflammation-induced hyperactivity of the choroidal Na+/K+/2Cl− cotransporter (NKCC1) that is involved in cerebrospinal fluid (CSF) secretion could contribute to the iNPH pathogenesis.MethodsLumbar CSF samples from 20 iNPH patients (10 with clinical improvement upon CSF shunting, 10 without clinical improvement) and 20 elderly control subjects were analyzed with the novel proximity extension assay technique for presence of 92 different inflammatory markers. RNA-sequencing was employed to delineate choroidal abundance of the receptors for the inflammatory markers found elevated in the CSF from iNPH patients. The ability of the elevated inflammatory markers to modulate choroidal NKCC1 activity was determined by addition of combinations of rat version of these in ex vivo experiments on rat choroid plexus.Results11 inflammatory markers were significantly elevated in the CSF from iNPH patients compared to elderly control subjects: CCL28, CCL23, CCL3, OPG, CXCL1, IL-18, IL-8, OSM, 4E-BP1, CXCL6, and Flt3L. One inflammatory marker, CDCP1, was significantly decreased in iNPH patients compared to control subjects. None of the inflammatory markers differed significantly when comparing iNPH patients with and without clinical improvement upon CSF shunting. All receptors for the elevated inflammatory markers were expressed in the rat and human choroid plexus, except CCR4 and CXCR1, which were absent from the rat choroid plexus. None of the elevated inflammatory markers found in the CSF from iNPH patients modulated the choroidal NKCC1 activity in ex vivo experiments on rat choroid plexus.ConclusionThe CSF from iNPH patients contains elevated levels of a subset of inflammatory markers. Although the corresponding inflammatory receptors are, in general, expressed in the choroid plexus of rats and humans, their activation did not modulate the NKCC1-mediated fraction of choroidal CSF secretion ex vivo. The molecular mechanisms underlying ventriculomegaly in iNPH, and the possible connection to inflammation, therefore remains to be elucidated.

Caffeine, a common ingredient in energy drinks, crosses the blood–brain barrier easily, but the kinetics of caffeine across the blood–cerebrospinal fluid barrier (BCSFB) has not been investigated. Therefore, 127 autopsy cases (Group A, 30 patients, stimulant-detected group; and Group B, 97 patients, no stimulant detected group) were examined. In addition, a BCSFB model was constructed using human vascular endothelial cells and human choroid plexus epithelial cells separated by a filter, and the kinetics of caffeine in the BCSFB and the effects of 4-aminopyridine (4-AP), a neuroexcitatory agent, were studied. Caffeine concentrations in right heart blood (Rs) and cerebrospinal fluid (CSF) were compared in the autopsy cases: caffeine concentrations were higher in Rs than CSF in Group A compared to Group B. In the BCSFB model, caffeine and 4-AP were added to the upper layer, and the concentration in the lower layer of choroid plexus epithelial cells was measured. The CSF caffeine concentration was suppressed, depending on the 4-AP concentration. Histomorphological examination suggested that choroid plexus epithelial cells were involved in inhibiting the efflux of caffeine to the CSF. Thus, the simultaneous presence of stimulants and caffeine inhibits caffeine transfer across the BCSFB.

The choroid plexus (CP) plays a crucial role in cerebrospinal fluid (CSF) production and brain homeostasis. However, non-invasive imaging techniques to assess its function remain limited. This study was conducted to develop a novel, contrast-agent-free MRI technique, termed relaxation-exchange magnetic resonance imaging (REXI), for evaluating CP-CSF water transport, a potential biomarker of CP function. REXI utilizes the inherent and large difference in magnetic resonance transverse relaxation times (T2s) between CP tissue (e.g., blood vessels and epithelial cells) and CSF. It uses a filter block to remove most CP tissue magnetization (shorter T2), a mixing block for CP-CSF water exchange with mixing time tm, and a detection block with multi-echo acquisition to determine the CP/CSF component fraction after exchange. The REXI pulse sequence was implemented on a 9.4 T preclinical MRI scanner. For validation of REXI's ability to measure exchange, we conducted preliminary tests on urea-water proton-exchange phantoms with various pH levels. We measured the steady-state water efflux rate from CP to CSF in rats and tested the sensitivity of REXI in detecting CP dysfunction induced by the carbonic anhydrase inhibitor acetazolamide. REXI pulse sequence successfully captured changes in the proton exchange rate (from short-T2 component to long-T2 component [i.e., ksl]) of urea-water phantoms at varying pH, demonstrating its sensitivity to exchange processes. In rat CP, REXI significantly suppressed the CP tissue signal, reducing the short-T2 fraction (fshort) from 0.44 to 0.23 (p < 0.0001), with significant recovery to 0.28 after a mixing time of 400 ms (p = 0.014). The changes in fshort at various mixing times can be accurately described by a two-site exchange model, yielding a steady-state water efflux rate from CP to CSF (i.e., kbc) of 0.49 s-1. A scan-rescan experiment demonstrated that REXI had excellent reproducibility in measuring kbc (intraclass correlation coefficient = 0.90). Notably, acetazolamide-induced CSF reduction resulted in a 66% decrease in kbc within rat CP. This proof-of-concept study demonstrates the feasibility of REXI for measuring trans-barrier water exchange in the CP, offering a promising biomarker for future assessments of CP function.

High salt intake causes an increase in Na+ concentration in the CSF of SHR and Dahl S rats, and increased CSF [Na+] contributes to sympothoexcitation and hypertension. However, the transporters in the choroid plexus (CP) mediating the imbalance of water and electrolytes in the CSF are still not identified. Current study demonstrated that high salt intake did not alter UT‐A expression, but there was a significant reduction in UT‐B expression in the CP of Dahl S rats. Reduced UT‐B expression was associated with increased [Na+] in the CSF and elevated MAP, as measured with radiotelemetry in Dahl S rats treated with high salt diet. We next examined the effect of selectively silencing UT‐B in the CP on CSF [Na+] and MAP in normotensive rats on normal diet versus high salt diet. ICV injection of lentiviral vector containing UT‐B shRNA (Lv‐UTB‐shRNA) significantly reduced UT‐B expression in the CP of rats as compared with rats received scramble Lv‐SCR‐shRNA ICV injection. High salt diet significantly increased MAP and HR in rats received ICV injection of Lv‐UTB‐shRNA. The elevated MAP was associated with increased [Na+] in the CSF. Chronic ICV infusion of Na+‐rich CSF resulted in significant elevations in MAP and HR. In conclusion, high salt diet significantly reduced UT‐B expression in the CP of Dahl S rats and selective silencing of UT‐B expression in the CP of normotensive rats markedly increases salt‐sensitivity in MAP and CSF [Na+]. These data indicate that reduced UT‐B expression in the CP may contribute to an imbalance of water and electrolytes in the CSF of Dahl S rats on high salt diet, thereby contributing to the development of salt‐sensitive hypertension.

Choroid plexus epithelial cells (CPECs) secrete most of the cerebrospinal fluid (CSF) and regulate its ionic composition through poorly understood mechanisms. CPECs express Na+/K+-ATPase and Na+–K+–2Cl− cotransporter 1 (NKCC1) on their apical membrane (CSF facing), deviating from typical basolateral membrane location in Cl−-secreting epithelia (Quinton et al. 1973; Piechotta et al. 2002; Praetorius & Damkier, 2017; Gregoriades et al. 2019). Given this peculiar polarity, only shared with retinal pigment epithelial cells (Miller & Edelman, 1990; Gundersen et al. 1991; Joseph & Miller, 1991; Lobato-Alvarez et al. 2016), the direction of basal net ion fluxes mediated by apical NKCC1 and associated water fluxes in CPECs is controversial, and the cotransporter function unclear. There are two opposing viewpoints: one proposes that under basal conditions apical NKCC1 works in the net efflux mode, cotransporting ions and water, directly contributing to sustained CSF production (Steffensen et al. 2018). In contrast, data from our lab support the hypothesis that under basal conditions apical NKCC1 of CPECs is indeed continuously active, but it works in the net inward flux mode, transporting ions and associated water towards the cell interior, maintaining intracellular Cl− concentration ([Cl−]i), and cell water volume (CWV) needed for CSF secretion (Gregoriades et al. 2019). According to this viewpoint, apical NKCC1 has an absorptive function, contributing indirectly to CSF secretion, and probably working as the sensor and regulator of CSF K+ suspected by Husted & Reed (1976). NKCC1 transport is electroneutral, with a coupling stoichiometry of 1Na+:1K+:2Cl−. Thus, the direction and magnitude of net ion cotransport is determined by the vectorial sum of the chemical potential gradients of the three cotransported ions: ΔμNa+ + ΔμK+ + 2ΔμCl−. Like other membrane transporters, NKCC1 is reversible; it can work in the net inward- or net outward-flux mode, or near equilibrium. The net free energy driving NKCC1-mediated ion transport (ΔμNKCC1) is defined by the equation in Fig. 1. From this equation, it is evident that variations in the chemical gradients of any of the transported ions can alter the direction of transport, but this is particularly critical for the extracellular K+ concentration ([K+]o) and the intracellular Na+ concentration ([Na+]i). Thus, modest changes in [K+]o or [Na+]i have a major impact on the sign and magnitude of ΔμNKCC1 (Fig. 1). The hypothesis that NKCC1 works in the net efflux mode, directly contributing to CSF secretion, largely originated from two sets of observations. First, estimates of [Na+]i in CPECs using flame photometry measurements of whole choroid plexus (CP) tissue pieces from rodents yield an unusually high value (30–50 mM), compared to other epithelial cells (Johanson & Murphy, 1990; Murphy & Johanson, 1990; Keep et al. 1994; Steffensen et al. 2018). This high [Na+]i makes NKCC1-mediated net ion efflux thermodynamically feasible (Fig. 1A). Second, intracerebroventricular bumetanide at concentrations expected to block apical NKCC1 produced partial (50%) inhibition of basal CSF secretion in canines (Javaheri & Wagner, 1993) and mice (Steffensen et al. 2018), and 50% inhibition of unidirectional blood–CSF flux of radiolabelled Cl− (Johnson et al. 1987). Third, wide-field imaging on ex vivo CP with the fluorescent Na+ indicator SBFI showed an increase in the dye emission signal during tissue exposure to bumetanide (10 μM), which was attributed to an increase in [Na+]i of CPECs, resulting from NKCC1 inhibition. The rationale is that if NKCC1 transports Na+ outwardly, its inhibition with bumetanide should lead to an increase in [Na+]i (Steffensen et al. 2018). The hypothesis that apical NKCC1 works in the net inward flux mode, having an absorptive function (Gregoriades et al. 2019), originated from measurements of in vitro CP tissue showing that 86Rb+ uptake, as surrogate of K+, depended on the presence of external Na+ and Cl−, and was blocked by bumetanide (Bairamian et al. 1991). Further, bumetanide decreased intracellular water of in vitro CP. Later work in single CPECs from rat suggested that NKCC1 functions as a K+-reabsorption mechanism from CSF to blood (Wu et al. 1998). In addition, Inhibition of NKCC1 produced shrinkage of single CPECs. In these experiments, relative cell volume (CV) changes were estimated from measurement of cross-sectional areas obtained by video-enhanced differential interference contrast (DIC) microscopy. Bumetanide (100 μm) caused a 9% decrease in basal CV, whereas increasing [K+]o from 3 to 25 mm produced a 33% increase in CV that was blocked by bumetanide or external Na+ removal. These observations were consistent with the in vitro CP tissue measurements of Johanson's lab (Bairamian et al. 1991), but they were challenged because of the high concentration of bumetanide, which could have effects in other transporters expressed in CPECs (Brown et al. 2009; Hughes et al. 2010). However, high sensitivity (1%) live-cell-imaging microscopy (LCIM) measurement of changes in CWV in acutely dissociated single CPECs loaded with the fluorescent dye calcein definitely demonstrated that bumetanide (10 μm) produced a reversible decrease (16%) in basal CWV in CPECs from NKCC1+/+ mouse. In contrast, CPECs from NKCC1−/− did not respond to the same bumetanide concentration (Gregoriades et al. 2019), indicating that the bumetanide-induced cell shrinkage was specifically mediated by NKCC1. Methodological differences between labs are the most likely explanation for the divergent views concerning the apical NKCC1 net flux direction under basal conditions. In our lab, we measured CV changes following genetic or pharmacological inactivation of apical NKCC1, in single CPECs, both in situ and in vitro, using three different methods: transmission electron microscopy, DIC-LCIM, and real-time changes in CWV in single CPECs loaded with the fluorescent dye calcein. The three methods gave the same result, namely CPEC shrinkage following inactivation of NKCC1. This is the CV response predicted if apical NKCC1 is continuously active under basal conditions, inwardly transporting ions and associated water, thereby keeping basal CWV constant. We proposed that the sustained cell shrinkage observed upon genetic or pharmacological inactivation of NKCC1 results from the suppression of solute and water influx, and the presence of unbalanced net efflux pathways that continue working until the cells adopt a new shrunken steady state (Gregoriades et al. 2019). The identity of these net efflux pathways remains elusive. A critical issue in the debate about the directionality of NKCC1-mediated transport is the [Na+]i of CPECs. We measured [Na+]i in individually calibrated single CPECs, using the fluorescent dye ANG-2 (Gregoriades et al. 2019). We found that [Na+]i of NKCC1+/+ is 9.2 ± 0.6 mM, a value similar to that reported for amphibian CPECs using ion-sensitive microelectrodes (Saito & Wright, 1987), but significantly lower than that estimated from flame photometry of whole CP tissue, based on unproven assumptions about CP water and blood content (Delpire & Gagnon, 2019). Thus, the [Na+]i measured with ANG-2 and ion-sensitive microelectrodes favours NKCC1 working in the inward flux mode (Fig. 1). Interestingly, there were no differences in [Na+]i between CPECs from NKCC1−/− and NKCC1+/+, indicating that the mechanisms determining [Na+]i are not affected by NKCC1 deletion. Another observation supporting the net inward flux hypothesis is the basal [Cl−]i measured with the fluorescent indicator MQAE. Basal [Cl−]i of NKCC1+/+ CPECs was approximately 60 mm, whereas in CPECs from NKCC1−/− it was close to electrochemical equilibrium, indicating that NKCC1 mediates uphill Cl− transport, and maintains [Cl−]i above equilibrium. In contrast to the methods used in our lab, Steffensen and colleagues (Steffensen et al. 2018) did not measure single CPEC volume changes, but relative 2D changes in size of ex vivo pieces of whole CP loaded with calcein; however, this fluorescent dye was not used as a CWV indicator, but to stain the CP pieces. Images of square 'regions of interest' encroaching CP tissue and background were converted to black and white, respectively. Relative changes in CP tissue size caused by large osmotic challenges were inferred from changes in the black to white ratio. These measurements represent underestimates of 3D volume changes of the CP pieces, but even if those changes in whole tissue volume could be measured, the signals are difficult to interpret for various reasons. The most important is that CP tissue is a highly vascularized structure composed not only of CPECs, but also of connective tissue and immune cells, endothelial and vascular smooth muscle cells, and blood cells, mainly erythrocytes (Ghersi-Egea et al. 2018). The relative cellular composition of rat CP determined by differential counts show that only 25–40% of the total number of cells are epithelial (Quay, 1966). In the IV ventricle CP, where functional imaging experiments are typically done, only 37.7% of cells are epithelial; 48.6% are intravascular erythrocytes and 13.7% are stromal cells such as fibroblasts, smooth muscle and endothelial cells. Another concern in the interpretationation of measurements in whole CP tissue is the lack of consideration of the impact of unstirred layers and other diffusion barriers. Thus, inferences about NKCC1 dynamics in single CPECs from changes in volume of whole CP tissue are difficult to interpret, and conclusions are unwarranted. The SBFI measurements of Steffensen's group, asserting that inhibition of NKCC1 with bumetanide leads to an increase in [Na+]i, are also difficult to interpret for the following reasons. First, lack of proof that the recorded SBFI signals resulted from actual changes in intracellular Na+ (e.g. by transient exposure to ouabain). Second, SBFI was not calibrated. Third, bumetanide interferes with SBFI signals, and an apparent increase in the ratio 340/380 can be explained by bumetanide autofluorescence, and not by an increase in [Na+]i. When excited at 340–345 nm, bumetanide fluorescence increases in a concentration-dependent manner, thereby overlapping with the SBFI excitation at 340 nm (Zhang et al. 1993; Robertson & Foskett, 1995; Rose & Ransom, 1996). When bumetanide is excited at 340 nm, at pH 7.4, fluorescence emission peaks between 400 and 450 nm (Fiori et al. 2003), the emission wavelength at which Steffensen's group recorded the signals. Another methodological difference, which was already mentioned, is that one group used whole CP (Steffensen et al. 2018) whereas the other used freshly isolated CPECs (Gregoriades et al. 2019). The advantages of the acutely isolated single-cell method include the following: (1) their optical properties, which make them ideal for real-time single-cell imaging microscopy measurements of ion concentrations and fluxes, using fluorescent probes combined with DIC optics; (2) the direction and magnitude of net fluxes through electroneutral transporters, like NKCC1, can be assessed by measuring changes in CWV, combined with pharmacological and molecular approaches (KO models, pharmacological inhibitors, external ions requirements); (3) minimization of fluid exchange artefacts resulting from diffusion barriers and unstirred layers; (4) cell viability can be readily assessed with calcein; and (5) preservation of epithelial cell structural and functional polarity (Reuss, 2001). The main disadvantage of acutely isolated epithelial cells is the loss of epithelial structure by detaching the cells from each other, and from the basal lamina, thereby eliminating paracellular pathways. A potential limitation of the studies of Steffensen et al. and Gregoriades et al. is that the experiments were performed in Hepes-buffered solutions. However, solutions were equilibrated with air and thus most likely contained some bicarbonate (HCO3−) resulting from the spontaneous hydration of CO2. This is important because two Na+ entry pathways in CPECs are HCO3−-dependent (Praetorius & Damkier, 2017): NCBE (SLC4A10) and NBCn1 (SLC4A7). It could be argued that decreased Na+ entry through these pathways reduces [Na+]i. However, inhibition of the Na+/K+-ATPase with ouabain resulted in an increase in [Na+]i in single CPECs and thus Na+ entry pathways were present in these cells (Fig. 7A in Gregoriades et al. 2019). Mechanisms of intracellular Na+ regulation have not been studied in CPECs but predictably, the ultimate determinant of [Na+]i will be the Na+/K+-ATPase that counteracts the Na+ 'leaks'. Interestingly, early work in hippocampal neurons and astrocytes, which express Na+-coupled HCO3− transporters, demonstrated that basal [Na+]i measured with SBFI was not significantly different in Hepes-buffered compared with CO2/HCO3−-buffered saline (Rose & Ransom, 1996, 1997). The overwhelming evidence from both in situ and in vitro models shows that under basal conditions NKCC1 normally works in the net influx mode, maintaining both [Cl−]i and intracellular water volume of CPECs needed for CSF secretion. This explains the counterintuitive observation that inhibition of NKCC1 by intracerebroventricular bumetanide reduces CSF secretion by 50% (Javaheri & Wagner, 1993; Karimy et al. 2017; Steffensen et al. 2018). NKCC1 most likely works as a K+ absorption mechanism, transporting K+ from CSF to blood and contributing, together with the Na+/K+-ATPase, to maintenance of low (2.9 mm) and constant CSF [K+]. Several unanswered questions remain in this model, one of which is the identity of the basolateral exit pathways for K+. Readers are invited to give their views on this and the accompanying CrossTalk articles in this issue by submitting a brief (250 word) comment. Comments may be submitted up to 6 weeks after publication of the article, at which point the discussion will close and the CrossTalk authors will be invited to submit a 'LastWord'. Please email your comment, including a title and a declaration of interest, to jphysiol@physoc.org. Comments will be moderated and accepted comments will be published online only as 'supporting information' to the original debate articles once discussion has closed. Francisco J. Alvarez-Leefmans MD, PhD, is Full Professor at the Department of Pharmacology and Toxicology, Boonshoft School of Medicine, Wright State University, Dayton, OH, USA. He received his PhD in physiology from University College London, where he continued his postdoctoral training with Professors Sir Bernard Katz and Ricardo Miledi. In 1997, he was awarded the Guggenheim Fellowship in Natural Sciences (Neuroscience) for his studies on chloride transport mechanisms in primary sensory neurons, where he first described and functionally characterized the Na+–K+−2Cl− cotransporter (NKCC1) in vertebrate nervous system. He developed the 'calcein' method to measure changes in cell water volume in single cells by live-cell imaging microscopy. He is interested in the molecular and cellular mechanisms used by choroid plexus epithelial cells to sense and regulate the cerebrospinal fluid potassium concentration, a fundamental problem of broad physiological and clinical significance for brain function. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. None. Sole author. Dayton Children's Hospital Foundation WSU grant 670180.

Backgroundpara-Tyramine (p-TA) is a biogenic amine which is involved in multiple neuronal signal transductions. Since the concentration of p-TA in dog cerebrospinal fluid (CSF) has been reported to be greater than that in plasma, it is proposed that clearance of cerebral p-TA is important for normal function. The purpose of this study was to examine the role of the blood–brain barrier and blood-cerebrospinal fluid barrier (BCSFB) in p-TA clearance from the brain.MethodsIn vivo [3H]p-TA elimination from rat cerebral cortex and from CSF was examined after intracerebral and intracerebroventricular administration, respectively. To evaluate BCSFB-mediated p-TA transport, [3H]p-TA uptake by isolated rat choroid plexus and conditionally immortalized rat choroid plexus epithelial cells, TR-CSFB3 cells, was performed.ResultsThe half-life of [3H]p-TA elimination from rat CSF was found to be 2.9 min, which is 62-fold faster than that from rat cerebral cortex. In addition, this [3H]p-TA elimination from the CSF was significantly inhibited by co-injection of excess unlabeled p-TA. Thus, carrier-mediated p-TA transport process(es) are assumed to take part in p-TA elimination from the CSF. Since it is known that transporters at the BCSFB participate in compound elimination from the CSF, [3H]p-TA transport in ex vivo and in vitro models of rat BCSFB was examined. The [3H]p-TA uptake by isolated rat choroid plexus and TR-CSFB3 cells was time-dependent and was inhibited by unlabeled p-TA, indicating carrier-mediated p-TA transport at the BCSFB. The p-TA uptake by isolated choroid plexus and TR-CSFB3 cells was not reduced in the absence of extracellular Na+ and Cl−, and in the presence of substrates of typical organic cation transporters. However, this p-TA uptake was significantly inhibited by cationic drugs such as propranolol, imipramine, amantadine, verapamil, and pyrilamine. Moreover, p-TA uptake by TR-CSFB3 cells took place in an oppositely-directed H+ gradient manner. Therefore, this suggested that p-TA transport at the BCSFB involves cationic drug-sensitive transport systems which are distinct from typical plasma membrane organic cation transporters.ConclusionOur study indicates that p-TA elimination from the CSF is greater than that from the cerebral cortex. Moreover, it is suggested that cationic drug-sensitive transport systems in the BCSFB participate in this p-TA elimination from the CSF.

The choroid plexus (CP) epithelium is a unique structure in the brain that forms an interface between the peripheral blood and the cerebrospinal fluid (CSF), which is mostly produced by the CP itself. Because the CP transcriptome is regulated by the sex hormone background, the present study compared gene/protein expression profiles in the CP and CSF from male and female rats aiming to better understand sex-related differences in CP functions and brain physiology. We used data previously obtained by cDNA microarrays to compare the CP transcriptome between male and female rats, and complemented these data with the proteomic analysis of the CSF of castrated and sham-operated males and females. Microarray analysis showed that 17128 and 17002 genes are expressed in the male and female CP, which allowed the functional annotation of 141 and 134 pathways, respectively. Among the most expressed genes, canonical pathways associated with mitochondrial dysfunctions and oxidative phosphorylation were the most prominent, whereas the most relevant molecular and cellular functions annotated were protein synthesis, cellular growth and proliferation, cell death and survival, molecular transport, and protein trafficking. No significant differences were found between males and females regarding these pathways. Seminal functions of the CP differentially regulated between sexes were circadian rhythm signalling, as well as several canonical pathways related to stem cell differentiation, metabolism and the barrier function of the CP. The proteomic analysis identified five down-regulated proteins in the CSF samples from male rats compared to females and seven proteins exhibiting marked variation in the CSF of gonadectomised males compared to sham animals, whereas no differences were found between sham and ovariectomised females. These data clearly show sex-related differences in CP gene expression and CSF protein composition that may impact upon neurological diseases.

Antiretroviral drugs and the central nervous system.

The host–pathogen interaction during meningitis can be investigated with blood-cerebrospinal-fluid-barrier (BCSFB) cell culture models. They are commonly handled under atmospheric oxygen conditions (19–21% O2), although the physiological oxygen conditions are significantly lower in cerebrospinal fluid (CSF) (7–8% O2). We aimed to characterize oxygen levels in a Streptococcus (S.) suis-infected BCSFB model with transmigrating neutrophils. A BCSFB model with human choroid plexus epithelial cells growing on transwell-filters was used. The upper “blood”-compartment was infected and blood-derived neutrophils were added. S. suis and neutrophils transmigrated through the BCSFB into the “CSF”-compartment. Here, oxygen and pH values were determined with the non-invasive SensorDish® reader. Slight orbital shaking improved the luminescence-based measurement technique for detecting free oxygen. In the non-infected BCSFB model, an oxygen value of 7% O2 was determined. However, with S. suis and transmigrating neutrophils, the oxygen value significantly decreased to 2% O2. The pH level decreased slightly in all groups. In conclusion, we characterized oxygen levels in the BCSFB model and demonstrated the oxygen consumption by cells and bacteria. Oxygen values in the non-infected BCSFB model are comparable to in vivo values determined in pigs in the CSF. Infection and transmigrating neutrophils decrease the oxygen value to lower values.

Intrathecal methotrexate (MTX) has been associated with severe neurotoxicity. Because carrier-associated removal of MTX from the cerebrospinal fluid (CSF) into blood remains undefined, we determined the expression and function of MTX transporters in rat choroid plexus (CP). MTX neurotoxicity usually manifests as seizures requiring therapy with antiepileptic drugs (AEDs) such as phenobarbital (PB). Because we have demonstrated that PB reduces activity of MTX influx carrier reduced folate carrier (Rfc1) in liver, we investigated the influence of the AEDs PB, carbamazepine (CBZ), or gabapentin on Rfc1-mediated MTX transport in CP. Reverse transcriptase-polymerase chain reaction and Western blot analysis showed similar expression of the MTX influx carrier Rfc1 and organic anion transporter 3 or efflux transporter multidrug resistance-associated protein 1 (Mrp1) and breast cancer resistance protein (Bcrp) in rat CP tissue and choroidal epithelial Z310 cells. Confocal microscopy revealed subcellular localization of Rfc1 and Bcrp at the apical and of Mrp1 at the basolateral CP membrane. Uptake, efflux, and inhibition studies indicated MTX transport activity of Rfc1, Mrp1, and Bcrp. PB and CBZ but not gabapentin significantly inhibited Rfc1-mediated uptake of MTX in CP cells. Studies on the regulatory mechanism showed that PB significantly inhibited Rfc1 translation but did not alter carrier gene expression. Altogether, removal of intrathecal MTX across the blood-CSF barrier may be achieved through Rfc1-mediated uptake from the CSF followed by MTX extrusion into blood, particularly via Mrp1. Antiepileptic treatment with PB or CBZ causes post-transcriptional down-regulation of Rfc1 activity in CP. This mechanism may result in enhanced MTX toxicity in patients with cancer who are receiving intrathecal MTX chemotherapy by reduced CSF clearance of the drug.

Confocal microscopy and image analysis were used to compare driving forces, specificity, and regulation of transport of the fluorescent organic anion, Texas Red (sulforhodamine 101 free acid; TR), in lateral choroid plexus (CP) isolated from rat and an evolutionarily ancient vertebrate, dogfish shark (Squalus acanthias). CP from both species exhibited concentrative, specific, and metabolism-dependent TR transport from bath to subepithelial/vascular space; at steady state, TR accumulation in vascular/subepithelial space was substantially higher than in epithelial cells. In rat CP, steady-state TR accumulation in subepithelial/vascular spaces was reduced by Na(+)-replacement, but was not affected by a 10-fold increase in buffer K(+). In shark CP, Na(+)-replacement did not alter TR accumulation in either tissue compartment; subepithelial/vascular space levels of TR were reduced in high-K(+) medium. In both species, steady-state TR accumulation was not affected by p-aminohippurate or leukotriene C4, suggesting that neither organic anion transporters (SLC22A family) nor multidrug resistance-associated proteins (ABCC family) contributed. In rat CP, digoxin was without effect, indicating that organic anion transporting polypeptide isoform 2 was not involved. Several organic anions reduced cellular and subepithelial/vascular space TR accumulation in both tissues, including estrone sulfate, taurocholate, and the Mrp1 inhibitor MK571. In rat CP, TR accumulation in subepithelial/vascular spaces increased with PKA activation (forskolin), but was not affected by PKC activation (phorbol ester). In shark, neither PKA nor PKC activation specifically affected TR transport. Thus, rat and dogfish shark CP transport TR but do so using different basic mechanisms that respond to different regulatory signals.

BackgroundFemale sex is a known risk factor of brain disorders with raised intracranial pressure (ICP) and sex hormones have been suggested to alter cerebrospinal fluid (CSF) dynamics, thus impairing ICP regulation in CSF disorders such as idiopathic intracranial hypertension (IIH). The choroid plexus (CP) is the tissue producing CSF and it has been hypothesized that altered hormonal composition could affect the activity of transporters involved in CSF secretion, thus affecting ICP. Therefore, we aimed to investigate if expression of various transporters involved in CSF secretion at CP were different between males and females and between females in different estrous cycle states. Steroid levels in serum was also investigated.MethodsFemale and male rats were used to determine sex-differences in the genes encoding for the transporters Aqp1 and 4, NKCC1, NBCe2, NCBE; carbonic anhydrase enzymes II and III (CA), subunits of the Na+/K+-ATPase including Atp1a1, Atp1b1 and Fxyd1 at CP. The estrous cycle stage metestrus (MET) and estrous (ES) were determined before euthanasia. Serum and CP were collected and subjected to RT-qPCR analysis and western blots. Serum was used to measure steroid levels using liquid chromatography tandem mass spectrometry (LC–MS/MS).ResultsSignificant differences in gene expression and steroid levels between males and ES females were found, while no differences were found between male and MET females. During ES, expression of Aqp1 was lower (p < 0.01) and NKCC1 was higher in females compared to males. CAII was lower while CAIII was higher in ES females (p < 0.0001). Gene expression of Atp1a1 was lower in ES compared to male (p = 0.0008). Several of these choroidal genes were also significantly different in MET compared to females in ES. Differences in gene expression during the estrus cycle were correlated to serum level of steroid hormones. Protein expression of AQP1 (p = 0.008) and CAII (p = 0.035) was reduced in ES females compared to males.ConclusionsThis study demonstrates for the first time that expression at CP is sex-dependent and markedly affected by the estrous cycle in female rats. Further, expression was related to hormone levels in serum. This opens a completely new avenue for steroid regulation of the expression of CSF transporters and the close link to the understanding of CSF disorders such as IIH.

The cerebrospinal fluid (CSF) provides mechanical protection for the brain and serves as a brain dispersion route for nutrients, hormones, and metabolic waste. The CSF secretion rate is elevated in the dark phase in both humans and rats, which could support the CSF flow along the paravascular spaces that may be implicated in waste clearance. The similar diurnal CSF dynamics pattern observed in the day-active human and the nocturnal rat suggests a circadian regulation of this physiological variable, rather than sleep itself. To obtain a catalog of potential molecular drivers that could provide the day–night-associated modulation of the CSF secretion rate, we determined the diurnal fluctuation in the rat choroid plexus transcriptomic profile with RNA-seq and in the CSF metabolomics with ultraperformance liquid chromatography combined with mass spectrometry. We detected significant fluctuation of 19 CSF metabolites and differential expression of 2,778 choroid plexus genes between the light and the dark phase, the latter of which encompassed circadian rhythm–related genes and several choroid plexus transport mechanisms. The fluctuating components were organized with joint pathway analysis, of which several pathways demonstrated diurnal regulation. Our results illustrate substantial transcriptional and metabolic light–dark phase–mediated changes taking place in the rat choroid plexus and its encircling CSF. The combined data provide directions toward future identification of the molecular pathways governing the fluctuation of this physiological process and could potentially be harnessed to modulate the CSF dynamics in pathology.