Efflux transporters in rat placenta and developing brain: transcriptomic and functional response to paracetamol.

Adenosine triphosphate binding cassette (ABC) transporters transfer lipid-soluble molecules across cellular interfaces either directly or after enzymatic metabolism. RNAseq analysis identified transcripts for ABC transporters and enzymes in rat E19, P5 and adult brain and choroid plexus and E19 placenta. Their functional capacity to efflux small molecules was studied by quantitative analysis of paracetamol (acetaminophen) and its metabolites using liquid scintillation counting, autoradiography and ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). Animals were treated acutely (30 min) and chronically (5 days, twice daily) with paracetamol (15 mg/kg) to investigate ability of brain and placenta barriers to regulate ABC transport functionality during extended treatment. Results indicated that transcripts of many efflux-associated ABC transporters were higher in adult brain and choroid plexus than at earlier ages. Chronic treatment upregulated certain transcripts only in adult brain and altered concentrations of paracetamol metabolites in circulation of pregnant dams. Combination of changes to metabolites and transport system transcripts may explain observed changes in paracetamol entry into adult and fetal brains. Analysis of lower paracetamol dosing (3.75 mg/kg) indicated dose-dependent changes in paracetamol metabolism. Transcripts of ABC transporters and enzymes at key barriers responsible for molecular transport into the developing brain showed alterations in paracetamol pharmacokinetics in pregnancy following different treatment regimens.

Stimulation of defecation in spinal cord-injured rats by a centrally acting ghrelin receptor agonist.

STUDY DESIGN: Animal proof of principle study. OBJECTIVES: To determine whether capromorelin, a compound that causes defecation by stimulating ghrelin receptors within the lumbosacral defecation centers, is effective after spinal cord injury (SCI), and whether SCI significantly alters sensitivity to the compound. SETTING: University of Melbourne and Austin Hospital, Melbourne, Australia. METHODS: Rats were subjected to spinal cord contusion injury or were sham-operated. At 6 weeks after surgery, effects of capromorelin on blood pressure, heart rate and propulsive contractions of the colorectum were investigated. RESULTS: Capromorelin caused robust propulsive activity in the colorectum soon after its application. The compound was similarly effective in naïve, sham-operated and spinal cord-injured rats. Blood pressure increases caused by capromorelin were not exaggerated after SCI, and there was no evidence of phasic blood pressure increases when the colon was contracted by the compound. CONCLUSION: Capromorelin is a therapeutic compound that could potentially be used to relieve constipation by triggering defecation in spinal cord-injured patients.

Effects of neonatal systemic inflammation on blood-brain barrier permeability and behaviour in juvenile and adult rats.

Several neurological disorders have been linked to inflammatory insults suffered during development. We investigated the effects of neonatal systemic inflammation, induced by LPS injections, on blood-brain barrier permeability, endothelial tight junctions and behaviour of juvenile (P20) and adult rats. LPS-treatment resulted in altered cellular localisation of claudin-5 and changes in ultrastructural morphology of a few cerebral blood vessels. Barrier permeability to sucrose was significantly increased in LPS treated animals when adult but not at P20 or earlier. Behavioural tests showed that LPS treated animals at P20 exhibited altered behaviour using prepulse inhibition (PPI) analysis, whereas adults demonstrated altered behaviour in the dark/light test. These data indicate that an inflammatory insult during brain development can change blood-brain barrier permeability and behaviour in later life. It also suggests that the impact of inflammation can occur in several phases (short- and long-term) and that each phase might lead to different behavioural modifications.

Effect of minocycline on inflammation-induced damage to the blood-brain barrier and white matter during development.

Damage to white matter in some premature infants exposed to intrauterine infections is thought to involve disruption of the blood-brain barrier. We have examined the effect of minocycline, an agent reported to reduce brain damage resulting from inflammation, on inflammation-induced disruption of the blood-brain barrier and damage to white matter. Post-natal marsupial opossums (Monodelphis domestica) were studied as most brain development in this species occurs after birth. Single intraperitoneal lipopolysaccharide (LPS) injection (0.2 mg/kg) with or without minocycline (45 mg/kg) at post-natal day (P)35 caused short-lasting barrier breakdown to plasma proteins but not to (14)C-sucrose. By P44, blood-brain barrier integrity was intact but a reduced volume of white matter was present. At P44 after prolonged inflammation (5 x 0.2 mg/kg LPS at 48 h intervals), proteins from blood were observed within brain white matter and permeability to (14)C-sucrose in the hindbrain increased by 31%. The volume of the external capsule and the proportion of myelin were 70 and 57%, respectively, of those in control animals. Minocycline administered during prolonged inflammation restored blood-brain barrier integrity but not LPS-induced damage to white matter. These data suggest that long-term changes in blood-brain barrier permeability occur only after a prolonged period of inflammation during development; however, damage to white matter can result from even a short-lasting breakdown of the barrier. Manipulation of the inflammatory response may have implications for prevention of some developmentally induced neurological conditions.

Changes in blood-brain barrier permeability to large and small molecules following traumatic brain injury in mice.

The entry of therapeutic compounds into the brain and spinal cord is normally restricted by barrier mechanisms in cerebral blood vessels (blood-brain barrier) and choroid plexuses (blood-CSF barrier). In the injured brain, ruptured cerebral blood vessels circumvent these barrier mechanisms by allowing blood contents to escape directly into the brain parenchyma. This process may contribute to the secondary damage that follows the initial primary injury. However, this localized compromise of barrier function in the injured brain may also provide a 'window of opportunity' through which drugs that do not normally cross the blood-brain barriers are able to do so. This paper describes a systematic study of barrier permeability in a mouse model of traumatic brain injury using both small and large inert molecules that can be visualized or quantified. The results show that soon after trauma, both large and small molecules are able to enter the brain in and around the injury site. Barrier restriction to large (protein-sized) molecules is restored by 4-5 h after injury. In contrast, smaller molecules (286-10,000 Da) are still able to enter the brain as long as 4 days postinjury. Thus the period of potential secondary damage from barrier disruption and the period during which therapeutic compounds have direct access to the injured brain may be longer than previously thought.

Blood-CSF barrier function in the rat embryo.

Blood-cerebrospinal fluid (CSF) barrier function and expansion of the ventricular system were investigated in embryonic rats (E12-18). Permeability markers (sucrose and inulin) were injected intraperitoneally and concentrations measured in plasma and CSF at two sites (lateral and 4th ventricles) after 1 h. Total protein concentrations were also measured. CSF/plasma concentration ratios for endogenous protein were stable at approximately 20% at E14-18 and subsequently declined. In contrast, ratios for sucrose (100%) and inulin (40%) were highest at the earliest ages studied (E13-14) and then decreased substantially. Between E13 and E16 the volume of the lateral ventricles increased over three-fold. Decreasing CSF/plasma concentration ratios for small, passively diffusing molecules during embryonic development may not reflect changes in permeability. Instead, increasing volume of distribution appears to be important in this decline. The intracellular presence of a small marker (3000 Da biotin-dextranamine) in plexus epithelial cells following intraperitoneal injection indicates a transcellular route of transfer. Ultrastructural evidence confirmed that choroid plexus tight junctions are impermeable to small molecules at least as early as E15, indicating the blood-CSF barrier is morphologically and functionally mature early in embryonic development. Comparison of two albumins (human and bovine) showed that transfer of human albumin (surrogate for endogenous protein) was 4-5 times greater than bovine, indicating selective blood-to-CSF transfer. The number of plexus epithelial cells immunopositive for endogenous plasma protein increased in parallel with increases in total protein content of the expanding ventricular system. Results suggest that different transcellular mechanisms for protein and small molecule transfer are operating across the embryonic blood-CSF interface.

Aquaporin-1 in the choroid plexuses of developing mammalian brain.

The normal brain develops within a well-controlled stable internal "milieu" protected by specialised mechanisms referred to collectively as blood-brain barriers. A fundamental feature of this environment is the control of water flow in and out of the developing brain. Because of limited vascularisation of the immature brain, choroid plexuses, via the cerebrospinal fluid, have been proposed as the main route of fluid exchange between the blood and brain interfaces. We describe the temporal expression and appearance of aquaporin-1 (AQP1) which is important for water transfer across adult choroid plexuses. AQP1 expression was studied in rat embryos using real time reverse transcription/polymerase chain reaction. mRNA for AQP1 was present in rat brain at embryonic day 12 (E12) one day before the protein was detectable in the fourth ventricular choroid plexus (the first plexus to appear); its relative levels increased at E13-E14 when more AQP1-immunoreactive cells appeared in all plexuses. The presence of AQP1 was determined immunocytochemically in five different mammalian species (rat, mouse, human, sheep and opossum) in all four choroid plexuses from their earliest appearance. In all five species studied, the appearance of AQP1 immunoreactivity followed the same developmental sequence: the fourth, lateral and, finally, third ventricular choroid plexus. The stage of choroid plexus development when AQP1 was first detected in all five species and in all four choroid plexuses corresponded to the transition between Stages I and II. The cellular localisation of AQP1 in all choroid plexuses, as soon as it was detectable, had the characteristic apical membrane distribution previously described in the adult; a basolateral membrane localisation was also observed.

Breakdown of the blood-brain barrier to proteins in white matter of the developing brain following systemic inflammation.

Compromised blood-brain barrier permeability resulting from systemic inflammation has been implicated as a possible cause of brain damage in fetuses and newborns and may underlie white matter damage later in life. Rats at postnatal day (P) 0, P8 and P20 and opossums (Monodelphis domestica) at P15, P20, P35, P50 and P60 and adults of both species were injected intraperitoneally with 0.2-10 mg/kg body weight of 055:B5 lipopolysaccharide. An acute-phase response occurred in all animals. A change in the permeability of the blood-brain barrier to plasma proteins during a restricted period of postnatal development in both species was determined immunocytochemically by the presence of proteins surrounding cerebral blood vessels and in brain parenchyma. Blood vessels in white matter, but not grey matter, became transiently permeable to proteins between 10 and 24 h after lipopolysaccharide injection in P0 and P8 rats and P35-P60 opossums. Brains of Monodelphis younger than P35, rats older than P20 and adults of both species were not affected. Permeability of the blood-cerebrospinal fluid (CSF) barrier to proteins was not affected by systemic inflammation for at least 48 h after intraperitoneal injection of lipopolysaccharide. These results show that there is a restricted period in brain development when the blood-brain barrier, but not the blood-CSF barrier, to proteins is susceptible to systemic inflammation; this does not appear to be attributable to barrier "immaturity" but to its stage of development and only occurs in white matter.

Permeability and route of entry for lipid-insoluble molecules across brain barriers in developing Monodelphis domestica.

1. We have studied the permeability of blood-brain barriers to small molecules such as [(14)C]sucrose, [(3)H]inulin, [(14)C]L-glucose and [(3)H]glycerol from early stages of development (postnatal day 6, P6) in South American opossums (Monodelphis domestica), using a litter-based method for estimating steady-state cerebrospinal fluid (CSF)/plasma and brain/plasma ratios of markers that were injected I.P. 2. Steady-state ratios for L-glucose, sucrose and inulin all showed progressive decreases during development. The rate of uptake of L-glucose into the brain and CSF, in short time course experiments (7-24 min) when age-related differences in CSF production can be considered negligible also decreased during development. These results indicate that there is a significant decrease in the permeability of brain barriers to small lipid-insoluble molecules during brain development. 3. The steady-state blood/CSF ratio for 3000 Da lysine-fixable biotin-dextran following I.P. injection was shown to be consistent with diffusion from blood to CSF. It was therefore used to visualise the route of penetration for small lipid-insoluble molecules across brain barriers at P0-30. The proportion of biotin-dextran-positive cells in the choroid plexuses declined in parallel with the age-related decline in permeability to the small-molecular-weight markers; the paracellular (tight junction) pathway for biotin-dextran appeared to be blocked, but biotin-dextran was easily detectable in the CSF. A transcellular route from blood to CSF was suggested by the finding that some choroid plexus epithelial cells contained biotin-dextran. 4. Biotin-dextran was also taken up by cerebral endothelial cells in the youngest brains studied (P0), but in contrast to the CSF, could not be detected in the brain extracellular space (i.e. a significant blood-brain barrier to small-sized lipid-insoluble compounds was already present). However, in immature brains (P0-13) biotin-dextran was taken up by some cells in the brain. These cells generally had contact with the CSF, suggesting that it is likely to have been the source of their biotin-dextran. Since the quantitative permeability data suggest that biotin-dextran behaves similarly to the radiolabelled markers used in this study, it is suggested that these markers in the more immature brains were also present intracellularly. Thus, brain/plasma ratios may be a misleading indicator of blood-brain barrier permeability in very immature animals. 5. The immunocytochemical staining for biotin-dextran in the CSF, in contrast to the lack of staining in the brain extracellular space, together with the quantitative permeability data showing that the radiolabelled markers penetrated more rapidly and to a much higher steady-state level in CSF than in the brain, suggests that lipid-insoluble molecules such as sucrose and inulin reach the immature brain predominantly via the CSF rather than directly across the very few blood vessels that are present at that time.

Development of the choroid plexus.

Mammalian choroid plexuses develop at four sites in the roof of the neural tube shortly after its closure, in the order IVth, lateral, and IIIrd ventricles. Bone morphogenetic proteins and tropomyosin are involved in early specification of these sites and in early plexus growth. Four stages of lateral ventricular plexus development have been defined, based on human and sheep fetuses; these depend mainly on the appearance of epithelial cells and presence or absence of glycogen. Other plexuses and other species are probably similar, although marsupials may lack glycogen. Choroid plexuses form one of the blood-brain barrier interfaces that control the brain's internal environment. The mechanisms involved combine a structural diffusion restraint (tight junctions between the plexus epithelial cells) and specific exchange mechanisms. In this review, it is argued that barrier mechanisms in the developing brain are different in important respects from those in the adult brain, but these differences do not necessarily reflect immaturity of the system. Absence of a barrier mechanism or presence of one not found in the adult may be a specialisation that is appropriate for that stage of brain development. Emphasis is placed on determining which mechanisms are present in the immature brain and relating them to brain development. One mechanism unique to the developing brain transfers specific proteins from blood to cerebrospinal fluid (CSF), via tubulocisternal endoplasmic reticulum in plexus epithelial cells. This results in a high concentration of proteins in early CSF. These proteins do not penetrate into brain extracellular space because of "strap" junctions between adjacent neuroependymal cells, which disappear later in development, when the protein concentration in CSF is much lower. Functions of the proteins in early CSF are discussed in terms of generation of a "colloid" osmotic pressure that expands the ventricular system as the brain grows; the proteins may also act as specific carriers and growth factors in their own right. The pathway for low molecular weight compounds, which is much more permeable in the developing choroid plexuses, appears also to be a transcellular one, rather than paracellular via tight junctions. There is thus good evidence to support a novel view of the state of development and functional significance of barrier mechanisms in the immature brain. It grows in an environment that is different from that of the rest of the fetus/neonate and that is also different in some respects from that of the adult. But these differences reflect developmental specialisation rather than immaturity.

Fetuin in human bone marrow: detection in foetal tissue and patients with mastocytosis.

Fetuin, a foetal protein of unknown function, has been shown to be expressed in both the immune and nervous systems, especially during development. Here, we show for the first time, that fetuin is abundantly present in many cells of the foetal human bone marrow, but is restricted to cells of the monocytic lineage in the adult. Fetuin's immunoreactivity increased considerably in adult human bone marrow in some pathological conditions, particularly in mastocytosis and was also increased in bone marrows in some cases of acute leukaemias, especially in acute myeloid leukaemia. This increase in the presence of fetuin in neoplastic bone marrows is not reflected by an increased level of circulating fetuin. This last observation contradicts earlier suggestions that fetuin is specifically reduced in cancer patients. A consistent increase in fetuin immunoreactivity in bone marrow of most cases of mastocytosis, as demonstrated in this paper, could become a useful tool in the diagnosis of this disease.

Fetuin in the developing neocortex of the rat: distribution and origin.

Immunocytochemical distribution of the fetal protein fetuin in the neocortex of developing rat brain and the presence of its mRNA, as detected by using reverse transcriptase-polymerase chain reaction analysis, was studied in fetuses at embryonic day 15 (E15) through E22, in neonates at postnatal day 0 (P0) through P20, and in adults. Quantitative estimates of fetuin in cerebrospinal fluid (CSF) and plasma were obtained over the same period. Exogenous (bovine) fetuin injected intraperitoneally into fetal and postnatal rats was used to study the uptake of fetuin into CSF and brain and its distribution compared with endogenous fetuin; bovine albumin was used as a control. Fetuin was identified immunocytochemically in the cortical plate and subplate cells of the developing neocortex. In the rat fetus, fetuin first was apparent at E17, mainly in cell processes, but a few subplate cells also were positive. By E18, there was strong staining in subplate neurons and in inner cells of the cortical plate. At E21, these inner cells of the cortical plate were beginning to differentiate into layer VI neurons, many of which were positive for fetuin. By P0-P1, more layer VI neurons and some layer V neurons had become positive for fetuin. Fetuin immunoreactivity generally was weaker at P1, and, by P2-P3, it had disappeared from all of the layers of the developing neocortex. Bovine fetuin (but not albumin), probably taken up through CSF over the neocortical dorsal surface, had a cytoplasmic distribution; endogenous rat fetuin was both cytoplasmic and membrane bound. Thus, much of this fetuin can be accounted for by uptake, although the presence of fetuin mRNA indicates that in situ synthesis may also contribute.

Determinants of passive drug entry into the central nervous system.

1. The blood-brain barriers restrict the passive diffusion of many drugs into the brain and constitute a significant obstacle in the pharmacological treatment of central nervous system diseases and disorders. The degree of restriction they impose is variable, with some lipid-insoluble drugs effectively excluded from the brain, while many lipid-soluble drugs do not appear to be subject to any restriction. 2. The ease with which any particular drug diffuses across the blood-brain barrier is determined largely by the number and strength of intermolecular forces "holding" it to surrounding water molecules. By quantifying the molecular features that contribute to these forces, it is possible to predict the in vivo blood-brain barrier permeability of a drug from its molecular structure. Dipolarity, polarizability, and hydrogen bonding ability are factors that appear to reduce permeability, whereas molecular volume (size) and molar refraction are associated with increased permeability. 3. Increasing the passive entry of "restricted" drugs into the central nervous system can be achieved by disrupting the blood-brain barrier (increased paracellular diffusion) or by modifying the structure of "restricted" drugs to temporarily or permanently increase their lipid solubility (increased transcellular permeability). 4. Competitive inhibition of outwardly directed active efflux mechanisms (P-glycoprotein and MRP, the multidrug resistance-related protein) can also significantly increase the accumulation of certain drugs within the central nervous system.

Investigation into the correlation between the structure of hydroxypyridinones and blood-brain barrier permeability.

Bidentate hydroxypyridinones are under active development as orally active iron chelators. With applications for the treatment of general body iron overload, for instance with thalassaemia, the distribution of the chelators should be limited to peripheral tissue and they should not enter the central nervous system. This study compares the predictive abilities of LogPoctanol and LogPcyclohexane and reports the existence of good correlations between blood-brain barrier (BBB) permeability and both values for N-alkylpyridinones. 1,omega-Hydroxyalkyl hydroxypyridinones penetrate the BBB much more slowly than the simple 1-alkyl derivatives. This observation is likely to have important toxicological implications.

Barrier mechanisms in the brain, II. Immature brain.

1. It is widely believed that 'the' blood-brain barrier is immature in foetuses and newborns. 2. Much evidence in support of this belief is based on experiments that were unphysiological and likely to have disrupted fragile blood vessels of the developing brain. Some confusion about barrier development arises from insufficient recognition that the term 'blood-brain barrier' describes a complex series of mechanisms controlling the internal environment of the brain. 3. We present evidence showing that the brain develops within an environment that, particularly with respect to protein, is different from that of the rest of the body and that possesses a number of unique features not present in the adult. 4. Barriers to protein at blood-brain and blood-cerebrospinal fluid (CSF) interfaces (tight junctions) are present from very early in development; immunocytochemical and permeability data show that proteins are largely excluded from extracellular space in developing brain. 5. Cerebrospinal fluid in developing brain contains high concentrations of proteins largely derived from plasma. This protein is transferred from blood by an intracellular mechanism across the epithelial cells of the immature choroid plexus. Only a small proportion of choroid plexus cells is involved. The route is an intracellular system of tubulo-endoplasmic reticulum continuously connected across the epithelial cells only early in brain development. 6. High concentrations of proteins in CSF in developing brain are largely excluded from the brain's extracellular space by barriers at the internal and external CSF-brain interfaces. These consist of membrane specializations between surfaces of cells forming these interfaces (neuroependyma on the inner surface; radial glial end feet on the outer surface). In contrast with tight junctions present at the blood-brain and blood-CSF barriers, at the CSF-brain barriers of the immature brain, other junctional types are involved: strap junctions in the neuroependyma and a mixture of junctions at the outer CSF-brain barrier (plate junctions, strap junctions and wafer junctions). These barriers are not present in the adult. 7. Permeability to small lipid-insoluble molecules is greater in developing brain; more specific mechanisms, such as those involved in transfer of ions and amino acids, develop sequentially as the brain grows.

Barrier mechanisms in the brain, I. Adult brain.

1. The adult brain functions within a well-controlled (internal) environment that is separate from that of the internal environment of the rest of the body as a whole. 2. The underlying mechanism of control of the brain's internal environment lies in the presence of tight junctions between the cerebral endothelial cells at the blood-brain interface (blood-brain barrier) and between choroid plexus epithelial cells (blood-cerebrospinal fluid (CSF) barrier). 3. The effect of tight junctions at the blood-brain and blood-CSF barriers is to convert the properties of the individual endothelial and epithelial cells into properties of these interfaces as a whole. 4. Superimposed on the diffusion restriction provided by the tight junctions in the blood-brain and blood-CSF barriers is a series of transport mechanisms into and out of the brain and CSF that determine and control the internal environment of the brain with respect to a wide range of molecules, such as electrolytes, amino acids, glucose, vitamins and peptides. 5. The physical characteristics of drugs, together with their interaction with the properties of the barriers between blood, brain and CSF, determine the extent to which drugs penetrate into the brain. 6. Drugs can be targeted to the brain by making use of knowledge of this interaction between the physical properties of a drug (which can be modified by manipulation of the structure of the molecule in predictable ways) and the influx/efflux mechanisms present in the blood-CSF and blood-brain interfaces.

Permeability of the developing and mature blood-brain barriers to theophylline in rats.

1. In the present study, the uptake of theophylline and L-glucose into the adult and neonatal rat brain has been investigated. Steady state cerebrospinal fluid (CSF) and brain concentrations of theophylline were reached within 1 h following a single intraperitoneal (i.p.) injection, whereas steady state CSF and brain concentrations of L-glucose were not approached until after 5 h. 2. Steady state brain:plasma and CSF:plasma concentration ratios for theophylline and L-glucose in neonatal rats were significantly higher than ratios in adult rats. Erythrocyte:plasma ratios for theophylline in neonatal rats were also significantly higher than ratios in adult rats. Steady state ratios for theophylline were significantly higher than those for L-glucose in both neonatal and adult rats. 3. Respiratory acidosis (pH 6.9-7.0) did not affect steady state CSF:plasma or brain:plasma ratios for theophylline in neonatal or adult rats. In contrast, steady state CSF:plasma and brain:plasma ratios for L-glucose were increased by respiratory acidosis. 4. The lower steady state CSF:plasma, brain:plasma and erythrocyte:plasma ratios for theophylline in adult rats are likely to be due to a higher concentration of plasma proteins in adult blood compared with neonates, with a greater retention of protein-bound (non-exchangeable) theophylline in adult blood, and are unlikely to be due to p-glycoprotein-mediated efflux of theophylline at the adult blood-brain barrier.

Uptake of Ga-67 into rat cerebral hemisphere and cerebellum. Comparison with Fe-59.

Transferrin and transferrin receptors play an important role in the transport of iron into the brain. To determine whether gallium enters the brain by the same mechanism, uptakes of 67Ga and 59Fe have been compared under controlled conditions. Rates of gallium penetration into brain (K(in)) were four times slower than those for 59Fe. K(in) for 67Ga when infused with citrate were 0.88 +/- 0.24 and 0.94 +/- 0.39 x 10(-3) ml g-1h-1 for cerebral hemisphere and cerebellum, respectively. When infused as the transferrin complex, 67Ga uptake into the brain was not different from that when infused with citrate. The presence of the anti-transferrin receptor antibody OX-26 significantly reduced uptake of 59Fe by 60% and 64% into cerebral hemisphere and cerebellum, respectively. By contrast, pretreatment of rats with OX-26 enhanced the uptake of 67Ga into brain, particularly when infused with citrate; mean increases in uptake of 67Ga were 120% and 144% for cerebral hemisphere and cerebellum, respectively. Purified 67Ga-transferrin was also taken up into both brain regions examined in the presence of OX-26. These results indicate that the transport of non-transferrin bound gallium is an important mechanism for gallium uptake into brain.

Albumin transfer across the choroid plexus of South American opossum (Monodelphis domestica).

1. Blood-cerebrospinal fluid (CSF) transfer of various exogenous albumins has been investigated in developing Monodelphis domestica (South American grey short-tailed opossum) and compared with the steady-state CSF: plasma ratios for endogenous (Monodelphis) albumin. Ratios for Monodelphis albumin and human albumin were similar and were the highest at postnatal day 5 (P5) (48.2 +/- 4.4 and 40.6 +/- 4.5%, respectively). The ratio for bovine albumin was similar to the steady-state ratio for Monodelphis albumin at P7-8 but became consistently lower than the Monodelphis albumin ratio at all other ages until P32-36 when all albumins tested attained a similar low ratio. The CSF:plasma ratio of chemically modified (succinylated) bovine albumin was always significantly lower than that of other albumins, except at the oldest age examined (P32-36). 2. Immunocytochemistry showed that within the brain, albumin was confined to the lumen and endothelial cells of blood vessels. In the choroid plexus only a small proportion (0.2-1.7% of the total cell number) of epithelial cells was positive for albumin, both endogenous and exogenous, at all ages studied (except the 3rd ventricle where cells were only positive from P8). The CSF was strongly positive for all albumins. The peak proportion of positive cells and of albumin concentrations in CSF occurred at P8. These findings suggest that the primary route for penetration of albumin into CSF is directly across the choroid plexus rather than via the brain. 3. Double-labelling immunocytochemistry revealed that the same epithelial cells contained both endogenous (Monodelphis) and exogenous (human) albumin. In contrast, for succinylated albumin, at P7 only about 35% (lateral ventricle) and 50% (4th ventricle) of Monodelphis albumin-positive cells were also positive for succinylated albumin, but by P30 this proportion increased to 90% at both sites. 4. Thus the developing choroid plexus distinguishes between different albumins. Chemical modification of albumin (succinylation) disrupts this mechanism. It is proposed that in older animals (P32-36) all of the albumin in the CSF is derived from plasma by diffusion (as in adult animals). At earlier stages of development, a proportion of the albumin in CSF also appears to be transferred from the plasma by diffusion with an additional component transferred by a mechanism that can distinguish between different species of albumin. The main route of entry of albumin to CSF seems likely to be via the choroid plexus epithelial cells.

The nature of increased blood-cerebrospinal fluid barrier exchange during CO2 inhalation in newborn and adult rats.

Exposure of newborn (2-day-old) and adult rats to increasing levels of inspired carbon dioxide (5-15% CO2) resulted in increased steady-state cerebrospinal fluid/plasma ratios for a wide range of different-sized, lipid-insoluble permeability markers (molecular radius ranged from 0.43 nm for L-glucose to 5.3 nm for immunoglobulin G). In control animals breathing room air and animals exposed to an elevated level of inspired CO2, steady-state CSF/plasma ratios for all permeability markers were proportional to their free diffusion coefficient. Steady-state CSF/plasma ratios in newborn animals were significantly higher than in adult animals, and at all ages the ratios for animals exposed to CO2 were higher than the ratios in control animals. In contrast to the increased steady-state CSF/plasma ratios in animals exposed to elevated levels of inspired CO2, there was no significant difference in short-term (10 min after i.v. injection) CSF/plasma ratios for [14C]L-glucose between 10- to 20-day-old control rats and rats of similar age exposed to 10% inspired CO2. Steady-state experiments confirmed that CSF/plasma ratios for [14C]L-glucose in 20-day-old rats exposed to 10% inspired CO2 were raised significantly (twice those measured in control animals breathing room air). The lack of effect of raised CO2 on short-term CSF/plasma ratios indicates that the significant increases in steady-state CSF/plasma ratios, in animals exposed to elevated levels of inspired CO2, are not due to a general increase in the permeability of the blood-CSF or blood-brain barriers; they are likely to be accounted for by CO2-induced reductions in the rate of CSF secretion.