Choroid Plexus Preparations to Study Transport

and Permeability It has been particularly challenging to develop CP preparations/ models that closely simulate the barrier properties in the intact organism. The in vitro counterpart should, as much as possible, mimic the natural in vivo system. How can the true physiological significance of in vitro experimental data be properly gauged? For a size range of water-soluble test agents, the relative values of the permeability or influx coefficients determined in vitro should correlate closely with in vivo assessments. Also, the steady-state concentration ratios, CSF/plasma or CP/CSF, should be comparable when relating data from an artificial environment to the organism.

Each CP preparation has advantages as well as drawbacks (1, 29, 98). In vivo experimentation provides a natural context but has complex variables, whereas the transwell approach with a cultured monolayer offers the capability of adjusting apical vs. basolateral medium composition (99). It is sound to corroborate in vitro data, i.e., cell culture with possible altered transporter expression (100) and modified tissue structure, with BCSFB information procured from more intact systems (in situ CP (101), in vivo CP (102), and isolated CP (34)). It is desirable to have a CP preparation with tight junction integrity, normal transepithelial electrical resistance, cellular energy reserve, and adequate CSF-forming ability. Reliable CP transport models can be built on data obtained from a battery of in vitro and intact preparations.

7. Permeation of Water, Nonelectrolytes, and Proteins Across BCSFB

7.1. Water

For molecules distributing passively, the rapidity of movement across CP is inversely proportional to the molecular weight of the migrating species. Diffusion through membranes of BCSFB is promoted by lipid solubility, as indexed by the oil/water partition coefficient of a given molecule. Water-soluble molecules, as discussed here, traverse most efficiently along diffusion pathways with the least steric hindrance. Thus, the smallest molecules, such as water, penetrate the BCSFB rapidly by moving through small pores or channels. As the largest molecules, proteins encounter considerable impediment to diffusion and therefore have the highest reflection coefficients and lowest penetration rates. The discussion below treats the permeations of water (quantitatively the most preponderant molecule in formation of CSF, which is 99% water), urea (an endogenous prototype nonelectrolyte moderately sieved at the BCSFB), and proteins (which are mainly excluded from CSF, but undergo slow leakage across CP in reverse rank order to size). The BCSFB is presented here as a screen or sieve that decreases the diffusion of hydrophilic molecules in proportion to their size.

The BCSFB is uniquely equipped to transfer a lot of fluid, hence it displays fast water permeation. Brisk CP blood flow, substantial Na+,K+-ATPase, and carbonic anhydrase activities, along with a rapid metabolic rate that reflects extensive transport and syntheses, all support voluminous CSF turnover. Fluid throughput across CP, when normalized for mass of tissue, is equivalent to glomerular filtration rate in the kidneys. Human CP secretes about 400,000 mL/day. Efficient transfer of water across the BCSFB is assured by Aquaporin-1 (AQP-1), a channel conducive to the osmotic flow of water. Rapid and extensive permeation from blood into the CP-CSF system is demonstrated by kinetic analysis of uptake curves for tritiated-water, which penetrates the entire water compartment (extracellular plus intracellular) within minutes (31). This is consistent with water moving transcellularly through epithelial cells of CP.

AQP-1, present in both the red blood cell and CP epithelium, facilitates water movement across the plasma membrane. There is nearly universal agreement that AQP-1 is on the CSF-facing apical membrane, and that water efflux (Fig. 4, step h) is passively conducted through AQP-1 in response to elevated osmotic pressure set up by solute extrusion (Fig. 4, step h) into the ventricles (Fig. 3). Less conclusive is how water is taken up at the basolat-eral membrane (Fig. 4, step f) from the plasma ultrafiltrate. Basolateral staining for AQP-1 in CP was reported in a study of early development (103). Also, AQP-3, the glyceroaquaporin in the CP basolateral membrane (81), deserves further study in regard to water loading of the choroid epithelial cells from the blood side.

Findings from several studies point to CP apical expression of AQP-1 as being intimately related to CSF formation rate (Table 2). Relatively low levels of AQP-1 are found in early fetal development (104), before CSF formation has reached a peak level (103), and then again in late stages of life (105) when CP secretion of CSF is dwindling (12). AQP-1 null mice have lowered CSF formation and pressure (106). Acute hyponatremia, when water enters too rapidly into the CNS, causes up-regulation of AQP-1 channels in CP (107); down-regulation of BCSFB AQP-1 in hyponatremia might alleviate ICP elevation by reducing osmotic flow into the ventricles. Basic research is creating new opportunities for controlling CSF flow (Table 2), either by regulating transcription factors (108) that modulate AQP-1 expression, applying fluid-homeostatic peptides to alter AQP-1 conductance (109), or synthesizing new drugs, e.g., bumetanide-related agents, that inhibit AQP-1 water channels (110).

Does Starling's law of filtration apply to water distribution across AQP-1 and AQP-4? Clearly, AQP-1 is osmosensitive. In response to osmolar gradients between plasma and CSF, there is substantial transfer of fluid across the BCSFB (107, 111). But what about elevations in ICP that reduce the cerebral perfusion pressure? Under these conditions of changing hydrostatic pressure gradients between CSF and blood, there may be greater clearance of fluid from the CSF across aquaporins in CP. Still, experimental evidence is needed to verify this point. The differential expression of AQP-1 and AQP-4, respectively, at the BCSFB and BBB prompt the pursuit of new agents that can target water channels at each barrier interface separately. Such pharmacologic selectivity would be a step forward in attaining fluid homeostasis by more finely controlling water movements among various CNS compartments.

7.2. Urea Transport and partitioning data for urea, which is smaller than sucrose and mannitol, are useful for evaluating changes in barrier permeability to nonelectrolytes. Steady-state concentrations of

Table 2

Aquaporin 1 (AQP-1), choroid plexus, and fluid turnover across the BCSFB

Experiment or state Observation Comments Species

Table 2

Aquaporin 1 (AQP-1), choroid plexus, and fluid turnover across the BCSFB

Fetal choroid plexus development of AQP-1 channels

Exclusive apical staining from 14th gestational week

By 18th week, cytoplasm as well as CP apical membrane stained

Human ( 14^0 week of gestation) (104)

Effects of natural aging on AQP-1 expression

Reduced AQP-1 and Na\K+-ATPase in CP of old (20 mo) vs. young animals

The decreased CSF formation in old rats (12) is consistent with less AQP-1 and Na+ pumps

Sprague-Dawley rats (105)

Peptidergic inhibition of AQP-1 function

Atrial natriuretic peptide (ANP) reduced basal to apical fluid transport

ANP altered the conductance of CP AQP-1 channels (cGMP gated)

Rat choroid plexus primary culture (109)

Effects of acute hyponatremia on AQP-1 channels

Hyponatremia increased CP AQP-1 expression by 28%

AQP-1-mediated excessive water flux across BCSFB may elevate the ICP

Rats (injected with hypotonic dextrose and dDAVP for 2 h) (107)

Deletion of AQP-1 channels in null mice

Deleted AQP-1 in CP led to a 25% decline in CSF formation rate

Secondary to reduced CSF turnover, the ICP fell by 55%

Wild-type control and AQP-1 null mice (106)

Thyroid hormone transcription factor-1 (TTF -1) regulates AQP-1 expression

TTF-1 is coexpressed with AQP-1 in CP and enhances AQP-1 gene transcription

Antisense to TTF-1 given i.c.v. lowered AQP-1 synthesis and CSF formation rate

Rat (108)

Inhibition of AQP-1 by a derivative of the bumetanide loop diuretic

The AQP-1 inhibitor, AqB013

(bumetanide analog), reduced H,0 permeability in oocytes, with an IC50 of 20 |im

Bumetanide inhibits CSF production by effects on CP apical membrane (139); AqB013 might be useful in brain H,0 homeostasis

Oocytes (110)

urea in adult human and animal fluids reveal a CSF level of urea only 0.7-0.8 of that in plasma (112). Since there is not active transport of urea by carrier from CSF to blood (113), this indicates that the CSF/plasma ratio, R , of <1 is due to normal

blood-to-CSF molecular sieving of urea by adult CP. Urea therefore permeates BCSFB more slowly than water due to CP restriction on diffusion, even when factoring for the effect of molecular size on unrestricted diffusion. In states in which the barrier is more effectively permeable to nonelectrolytes, e.g., in pig fetal life, Rurea approaches unity. Such equilibrium distribution, i.e., the Rurea of 1.0 in the CP-CSF system of fetal pigs (114) and neonatal rats (102), points to early-life lack of BCSFB tightness/secretion (67, 115) and therefore attenuated molecular sieving. It is expected that many water-soluble drugs would be similarly sieved by the CP epithelial membranes in transit to the ventricles (116), thereby resulting in lower agent levels in CSF.

In mature mammals, the CSF sink action or drain effect (102, 112) occurs when CP gears up to actively form CSF (11). With BCSFB tightening and greater fluid turnover across adult CP, hydrophilic solutes such as urea undergo more sieving, relative to water movement, in diffusion pathways as reflected by decreasing values (102) for Rurea. Urea, like water, diffuses transcellularly (102) via membrane channels or pores as well as paracellularly (117) across tight junctions. When CSF in adult animal investigations is sampled from the cisterna magna, which is a mixture of subarachnoid and ventricular fluid, R values of 0.7-0.8 are urea observed. However, a lower Rurea value of 0.6, indicating greater sieving, is obtained for ventricular fluid sampled upstream of the basal cisterns. This more accurately reflects CSF-inward molecular fluxes across the BCSFB, when the ventricular CSF sample is taken closer to the secreting CP tissues.

Transfer and influx coefficients, analogous to the permeability-surface area product, quantify the penetration of radiolabeled urea and other nonelectrolytes (67) from blood to CSF (31, 102, 113, 117). As in other transport investigations, the assumption is made that the radiolabeled test molecule penetrates in the same manner as the nonlabel. The influx coefficient Km in mL/g/min is calculated from the initial slope of the curve for test solute volume of distribution, Vd vs. time allowed for distribution. Permeability is proportional to Kin. To determine BCSFB permeability in rats, the Vd data for early uptake into CSF at 30 m are strongly associated with a Kn value across CP. Thus, there is anatomical, developmental (67), and pharmacologic evidence (118) that Km for the BCSFB can be obtained from the fast component of CSF uptake (119).

Kin for urea transfer across rat BCSFB decreases with postnatal age from 1 week to adulthood (67). This is likely due to CP tightening/maturation, as manifested by a substantial increase after 1 week in the Kin for Na+ actively transported into the ventricles (67, 102). Moreover, the K for CSF uptake of man-nitol, a nonelectrolyte larger than urea, also decreases over this same postnatal interval (Fig. 6). Slower penetrations of water-soluble urea and mannitol with advancing age are expected as the CP tightens up for more efficient CSF secretion into the ventricles. Barrier tightening should be reflected by an increase in the BCSFB K ratio of urea/mannitol. This is the case as the K

in ' in ratio triples, from 4.4 to 14.3, between 2 weeks and adulthood (Fig. 6). Another instance of an augmented Km ratio for urea/ mannitol occurs after acute hypertension when CP tight junctions are damaged, consequently allowing greater molecular leakage into CSF (117). Thus, by comparing permeation rates of two solutes, as with the Kin ratio above, and relating this to the ratio of their free diffusion coefficients, D, cm2/s (117), insight is gained on the degree of restriction offered to molecules diffusing through barriers (Fig. 6).

D Urea

Kin Urea Kjn Mannitol

25 20 15 10

D Mannitol 0 0

BCSFB

1 WK 2 WK Adult

D Mannitol 0 0

1 WK 2 WK Adult

Kin Urea

0.558

0.456

0.300

Kin Mannitol

0.122

0.103

0.021

1 WK 2 WK Adult

0.480

0.402

0.282

0.051

0.039

0.012

Fig. 6. Nonelectrolyte influx coefficient ratio analysis: Kn values for 14C-urea (31, 102) and 3H-mannitol (67) were obtained for fast-component CSF uptake across CP, the BCSFB, vs. cortical BBB uptake in Sprague-Dawley rats (n=6 for 1 week, 2 weeks, and adult > 5 weeks). Mean values for Kn (mL/g/m) are presented below the graph; slopes for the early linear part of uptake curve, l/d vs. time, were statistically compared and, for each region, the adult values were significantly different (P < 0.05) from those at both 1 and 2 weeks. The ratio of the free diffusion coefficients (D), urea/mannitol, in agar gel is 2, as indicated by horizontal broken line. In biological systems, e.g., capillaries or epithelial membranes, the larger solute will undergo proportionally more sieving due to greater restriction to diffusion, thus increasing the Kn (urea/ mannitol) ratio. Therefore, the extent to which Kn (urea/mannitol) exceeds D (urea/mannitol) presumably reflects the degree of restriction to diffusion across the barrier interfaces. With ongoing maturation, there was tightening of both the BCSFB and BBB, i.e., more molecular sieving after 2 week, as indicated by the sharply rising values in the ratio of Knurea/Knmannitol; after 2 week the CSF formation in rats also rapidly increases (67). Thus, in infant animals at 1-2 week with evidently less barrier hindrance to diffusion into CSF and cortex, the radiolabeled urea and mannitol were cleared from blood at rates closer to free unhindered diffusion.

Polar molecules with low-permeability properties have also been tested for penetrability across in situ and in vitro preparations of CP. A classical experiment (120) analyzed permeation rates of several nonelectrolytes of graded molecular weights, diffusing from isolated CP (bathed in CSF containing the test molecules) to venous blood draining the plexus preparation; this enabled calculation of BCSFB permeability coefficients, P, cm/s, for urea, mannitol, sucrose, and inulin (120). More recently, Strazielle et al. (98) used primary cultures of rat CP as a transwell monolayer system to quantify fluxes of extracellular markers that penetrate tight junctions vs. drugs that permeate CP epithelial cells. Their determination of P values for hydrophilic nonelectro-lyte penetration across the rat monolayer compares favorably with previous P values for in vitro rabbit CP (120) (Fig. 7). Comparable P values and impedance, found for in vitro vs. in vivo preparations, augurs well for using cultured CP preparations to pharmacologically assess the BCSFB. Because CP cell culture monolayer permeability varies, however, according to differential expression and localization of tight junction proteins (121), it is imperative in transport and permeability analyses to characterize each cultured CP preparation for its particular barrier properties.

7.3. Proteins CSF protein concentration in adult mammals is much lower than plasma, by 2-3 orders of magnitude. This substantial protein gradient, from blood to CSF, reflects the degree of the BCSFB

Fig. 7. A comparison of in vitro vs. in vivo CP permeability to water-soluble substances: Permeability coefficients, Pe, were determined for a series of graded molecular weights of hydrophilic nonelectrolytes. The in vitro coefficients were measured in a Transwell system, with means ± SEM for n=3 or 4 [98]. Pe values were similar in the in vivo rabbit CP epithelium [120] vs. an in vitro primary culture monolayer of rat CP cells [98]. The straight line is from linear regression analysis, r = 0.98. Thus, the four polar molecules assessed displayed similar permeativities in the in vitro and intact systems. Reproduced with permission [98].

Fig. 7. A comparison of in vitro vs. in vivo CP permeability to water-soluble substances: Permeability coefficients, Pe, were determined for a series of graded molecular weights of hydrophilic nonelectrolytes. The in vitro coefficients were measured in a Transwell system, with means ± SEM for n=3 or 4 [98]. Pe values were similar in the in vivo rabbit CP epithelium [120] vs. an in vitro primary culture monolayer of rat CP cells [98]. The straight line is from linear regression analysis, r = 0.98. Thus, the four polar molecules assessed displayed similar permeativities in the in vitro and intact systems. Reproduced with permission [98].

permeability to macromolecules. Even though the "leaky" tight junctions of the BCSFB (70), like the proximal tubule, are substantially more permeable than the BBB, still, plasma proteins penetrate the BCSFB very slowly compared to Na+, urea, and water. Even small proteins like microperoxidase (~2,000 Da) are blocked by CP tight junctions (122). This molecular sieving of plasma proteins and polypeptides by CP is important for homeostasis of CSF osmotic pressure and sink action (102), as well as for regulation of CSF-mediated immune and endocrine signaling (11). Protein trafficking across the epithelial BCSFB is finely controlled by mechanisms differing from the endothelial BBB.

Flux regulation for most proteins at the BCSFB does not begin at the choroidal capillary wall. In marked contrast to microvascular/interstitial functional relationships in the brain, where plasma protein diffusion is constrained by the cerebral capillary wall, many plasma proteins in CP readily escape the plexus capillaries and gain entrance to the interstitium (Fig. 4). Lacking a lymphatic drainage system, the CP disposes of unneeded protein by phagocytosis via fibroblasts and macrophages in the interstitium (46). In addition, other extravasated protein molecules are either endocytosed by epithelial cells/lyso-somes for breakdown or diffuse into the basolateral clefts up to the tight junctions (46). Zonulae occludentes at the apex of CP epithelial cells, together with the basolateral membrane, are the true anatomic substrates of the BCSFB and therefore greatly restrict diffusion passage of most proteins and polypeptides into CSF. CP tight junctions appear early in fetal development (123), indicating that protein access to CSF is carefully regulated at all life stages.

Due to very slow penetration rates of proteins from blood to CSF, it has not been feasible to determine influx coefficient, K ,

or permeability-surface area products for plasma-borne radiolabeled proteins, as has been the case for ions and small nonelec-trolytes (67). Rather, protein steady-state ratio data, CSF/ plasma, along with biochemical information about protein size, yield deductive information about macromolecular transport across BCSFB (124, 125). CSF sampling source is significant. The closer the CSF specimen is to CP, e.g., ventricular or nascent CSF, the more valid it is to attribute BCSFB modeling, using CSF/plasma ratios, to transport and permeability mechanisms in CP. Due to anatomical and functional complexity of CP, and the wide variety of protein species (ranging from a1-antitrypsin ~45,000 Da to b-lipoprotein ~2,239,000 Da) translocated across BCSFB, it is not surprising that multimechanistic heterogeneous transport models (125) provide better data fit than homogeneous ones.

There are several routes/mechanisms in CP of adults, for plasma proteins to reach CSF involving diffusion as well as nondiffusion processes. At least one CSF accession route involves diffusion through pore-like structures or channels (124). Evidence for diffusion stems from observations that the concentration of many plasma proteins in CSF is inversely proportional to their respective molecular weights (124). It is challenging with EM to unequivocally identify tight junction pores or discontinuities relating to protein permeation (126), but a tight junction discontinuity of even 0.08% of the total perimeter (124) would enable a pore-like route for a small leak of protein into CSF. A dual-mechanism model of protein permeation across the CP epithelium, incorporating both diffusion of smaller proteins across pores of uniform size and putative pinocytosis of larger proteins, was proposed (124). CSF protein levels have been correlated (125) with the CP tight junction "pores" of varying diameters as described previously (127). The latter findings were supported by 3-D tight junctional analyses (128).

Both in vitro (120) and in vivo (129) models of CP-CSF have furnished evidence for bulk flow or convective, nondiffusion components of transport through CP. Pinocytotic uptake of protein occurs at the basolateral membrane, but it is difficult to ultrastructurally demonstrate subsequent apical membrane exo-cytosis into CSF (46). Transcellular transfer of protein, especially early in development, likely occurs in CP by the tubulocisternal endoplasmic reticulum; the selectivity of this process for transport to fetal CSF has been demonstrated for different forms of albumin (123), suggesting specific endocytotic mechanisms that initiate the translocation. Transcellular protein transport at the BCSFB awaits further analyses in adult mammals, including the possible role of cytoplasmic tubular networks in conveying proteins across CP to CSF.

The steady-state CSF/serum albumin ratio, Qalbumin, has long been used as an endogenous index of BBB permeability status. Normally Qalbumin is about 0.005 (130). An elevated Qalbumin ratio reflects BBB breakdown. In a generic sense, "BBB" collectively denotes barriers in brain capillaries as well as elements of the BCSFB. Although changes in Qalbumin manifest altered brain capillary function, they may also indicate damage to CP as well. Indeed, a study of Qalbumin in aging patients revealed increased permeability of the BCSFB in older subjects (131).

For IgG (~160,000 Da), a much larger molecule than albumin (~68,000 Da), the steady-state CSF/plasma ratio (QIgG) is only 0.0027 (130). IgG penetration of CP, at least transcellularly (132), was not detectable by peroxidase or electron microscopy. Leakage of IgG from plasma to CSF is so slow that IgG is used as a reference protein in CSF to indicate central IgG synthesis, i.e., from analysis of elevated values for the ratio of QIgG/Qalbumin. CSF protein ratio analyses, then, provide valuable information about central protein metabolism as well as BCSFB permeability.

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