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Properties of the local internal environment of the adult brain are tightly controlled providing a stable milieu essential for its normal function. The mechanisms involved in this complex control are structural, molecular and physiological (influx and efflux transporters) frequently referred to as the 'blood-brain barrier'. These mechanisms include regulation of ion levels in brain interstitial fluid essential for normal neuronal function, supply of nutrients, removal of metabolic products, and prevention of entry or elimination of toxic agents. A key feature is cerebrospinal fluid secretion and turnover. This is much less during development, allowing greater accumulation of permeating molecules. The overall effect of these mechanisms is to tightly control the exchange of molecules into and out of the brain. This review presents experimental evidence currently available on the status of these mechanisms in developing brain. It has been frequently stated for over nearly a century that the blood-brain barrier is not present or at least is functionally deficient in the embryo, fetus and newborn. We suggest the alternative hypothesis that the barrier mechanisms in developing brain are likely to be appropriately matched to each stage of its development. The contributions of different barrier mechanisms, such as changes in constituents of cerebrospinal fluid in relation to specific features of brain development, for example neurogenesis, are only beginning to be studied. The evidence on this previously neglected aspect of brain barrier function is outlined. We also suggest future directions this field could follow with special emphasis on potential applications in a clinical setting., (© 2018 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

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.

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.

1. The blood-cerebrospinal fluid (CSF) exchange of a wide range of passively transported lipid insoluble compounds (0.43-5.4 nm molecular radius) has been investigated in rats at different stages of postnatal development (2 days old to adult). A novel 'litter-based' model for investigating blood-CSF barrier exchange in immature animals is described. 2. At each age investigated there was a clear inverse correlation between molecular radius and blood-CSF barrier exchange, in addition to an overall decrease in blood-CSF barrier exchange with increasing age. 3. The decrease in blood-CSF barrier exchange with age was not consistent with a reduction in pore diameters, nor does it appear to be due to an increase in the CSF sink effect with age. It seems likely to be due to a relative decrease in the number of a population of large diameter pores.

1. The transfer of albumin between the blood and the cerebrospinal fluid (CSF) has been investigated in neonatal (3 days old) and juvenile (20 days old) rats. At both stages of postnatal development, all of the albumin present in the CSF can be accounted for by transfer from the blood. Thus it is unlikely that in situ synthesis of albumin contributes to the naturally high levels of albumin in CSF in the developing brain. 2. The high concentration of albumin in CSF of the neonatal rat brain cannot be accounted for solely by diffusion from the blood. In the 3-day-old rat, only about one quarter of the albumin in CSF enters by diffusion from the blood, whilst the remainder appears to be transported into the CSF by a specific mechanism which can discriminate between different species of albumin. The specific transport component of albumin transfer between the blood and the CSF appears to be developmentally regulated and is not apparent in 20-day-old rats. 3. Chemical modification of albumin resulting in either an increase or a decrease in electrophoretic mobility (at pH 7.4), significantly reduces blood-CSF transfer of albumin in 3-day-old rats, but has little effect in the 20-day-old rat. Thus overall molecular charge does not appear to be an important feature of the species-specific blood-CSF albumin transport mechanism in neonatal rats.

1. The blood-cerebrospinal fluid (CSF) transfer of endogenous sheep albumin and several exogenous species of albumin has been investigated in different CSF compartments of the immature fetal sheep brain, at an early stage of development (60 days gestation, term is 150 days) when the CSF concentration of total protein is high. 2. There were marked differences in the steady-state CSF/plasma ratios for all species of albumin (including endogenous sheep albumin) between different CSF compartments. Ratios measured in the cisterna magna were significantly higher than those in the dorsal subarachnoid space, which in turn were higher than those in the lateral ventricles. The ratios for endogenous sheep albumin were (%; mean +/- S.E.M.): lateral ventricle (LV), 4.0 +/- 0.03; dorsal subarachnoid (DSA), 6.1 +/- 1.0; cisterna magna (CM), 13.7 +/- 0.8. 3. Three hours after I.V. injection, the CSF/plasma ratios for bovine albumin (LV, 2.0 +/- 0.2; DSA, 2.4 +/- 0.1; CM, 7.2 +/- 0.7%) were significantly lower than the ratio for endogenous sheep albumin in all three compartments. The ratios for human albumin (LV, 0.7 +/- 0.2; DSA, 1.0 +/- 0.2; CM, 3.9 +/- 0.4%) were significantly lower than those for bovine albumin. 4. In all three CSF compartments, the endogenous sheep albumin ratios were higher than would be expected on the basis of transfer by passive mechanisms. Conversely, steady-state CSF/plasma ratios for [3H]sucrose and [14C]inulin were consistent with passive transfer, and there were no differences between the ratios for these markers measured in each of the three CSF regions. 5. Goat albumin and [35S]sheep albumin ratios were not significantly different, 5 h after injection, from the endogenous sheep albumin levels in each of the three CSF compartments. 6. It is concluded that in the 60-day-old fetal sheep, all of the endogenous albumin in CSF is derived from the plasma by a specific transfer mechanism that can distinguish between different species of the same protein. There is also some evidence of a small passive component of blood-CSF albumin transfer. 7. Immunocytochemical evidence suggests that the route of transfer from blood to CSF is transcellular, through the choroid plexus epithelial cells. 8. Regional variations in albumin ratios are probably due to differences in specific transfer into each CSF compartment. This is reflected in a differential immunocytochemical staining for albumin in choroid plexus epithelial cells from different regions of the brain. 9. The results are discussed in terms of differences in albumin amino acid sequences, structural homologies, and transfer by a specific transcellular mechanism.

1. The concentration of total protein in c.s.f. and plasma has been measured in fetal sheep of different gestational ages and in the adult. In c.s.f. it was highest (approximately 840 mg/100 ml.) in the youngest fetuses (35 days) and declined steeply by 60 days (260 mg/100 ml.). It decreased less markedly in the last half of gestation to reach about 50 mg/100 ml. at 125 days which is twice the adult value. Protein concentration in plasma was lowest in the youngest fetuses and did not rise much until the second half of gestation during which time it doubled. There was a further rather larger increase between the late fetal (125 days) stage and the adult.2. Albumin, fetuin, alpha-fetoprotein (AFP), transferrin and lipoprotein were identified in c.s.f. and plasma.3. The concentrations of albumin, AFP and fetuin in c.s.f. and plasma at different gestational ages were measured using immunodiffusion and radioimmuno-assays.4. Albumin was the major protein in plasma at all ages studied (35-128 days gestation and adult). Its concentration increased throughout gestation whereas that of fetuin was similar at all fetal ages and that of AFP declined markedly during the second half of gestation. In the adult, fetuin was only about 0.1% of the total protein in plasma and AFP was not detectable.5. In 35-40 day fetuses albumin, AFP and fetuin accounted for 90% of the total protein concentration in plasma and for about 70-80% of the total protein concentration of c.s.f. As gestation progressed the concentration of all three proteins in c.s.f. declined. But the concentration of AFP and fetuin fell more rapidly and to a greater extent than that of albumin; neither AFP nor fetuin could be detected in adult c.s.f.6. The c.s.f.: Plasma ratio was above 50% for AFP and above 60% for fetuin at 35 days compared with about 25% for albumin at the same fetal age. The c.s.f.: plasma ratios for all three proteins declined with increasing fetal age and were not significantly different from each other by about mid gestation.

1. The penetration of human and sheep plasma proteins from blood into c.s.f. of sheep fetuses (57-86 days gestation) has been studied. The proteins were injected intravenously via cotyledonary vessels. After different time periods the c.s.f. concentrations of marker proteins were estimated by radioactive counting of iodinated human or sheep proteins or by immunoassay of human proteins.2. Several proteins of similar molecular size penetrated into c.s.f. to a different extent in 60 day fetuses. The steady-state c.s.f.: plasma ratios were about 15% for human AFP, 10% for human transferrin and sheep albumin, 7% for human alpha(1)-antitrypsin, and 5% for human albumin. In older fetuses the penetration of protein from blood into c.s.f. was much reduced and no evidence for differential penetration of different proteins was found.3. The penetration of human AFP, transferrin and sheep albumin from blood into c.s.f. was greater than can be accounted for by passive diffusion.4. The results of this paper are discussed in relation to those of the preceding paper on the identification and quantification of proteins in fetal c.s.f. and plasma. The hypothesis is put forward that the choroid plexus of the immature (60 days and less) sheep fetus contains a mechanism for the transcellular transfer of plasma proteins which may be selective in nature and of importance for some aspects of brain development.

1. The development of the blood-brain and blood-c.s.f barriers to lipid insoluble substances of different molecular radii has been studied in fetal sheep, early (60 days) and late (125 days) in gestation, using labelled erythritol (C14), sucrose (3H or 14C), inulin (3H or 14C) and albumin (125I), or albumin and IgG detected by immunoassay. 2. Morphological studies of fetal brain and choroid plexus at the same gestational stages were carried out using thin section electron microscopy and the freeze fracture techniques. 3. Penetration of markers into c.s.f. was substantially greater at 60 days than at 125 days, but at both ages the steady-state level achieved appeared to be related to molecular size. 4. A simple model describing penetration from blood into c.s.f. at 60 days is proposed. It involves the assumption that c.s.f. and brain extracellular fluid are effectively separate compartments; morphological and permeability data which supports this assumption is presented. The data for c.s.f. at 60 days are consistent with the suggestion that the markers penetrate into c.s.f. by diffusion and are not restricted by small pores in the interface between blood and c.s.f. 5. The reduction in penetration which occurred by 125 days for all markers except erythritol appears to be accounted for by an increase in the sink effect and a decrease in the effective surface area for exchange between blood and c.s.f. 6. Intercellular tight junctions of both cerebral endothelial cells and choroid plexus epithelial cells were well formed at 60 days gestation. There was no change in junctional characteristics previously thought to correlate with transepithelial permeability (tight junction depth and strand number) between the two ages studied, although there were marked changes in permeability. 7. Evidence is advanced in support of the hypothesis that in the fetus much of the penetration of lipid insoluble non-polar substances across the blood-c.s.f. barrier and perhaps across the blood-brain barrier occurs via a transcellular route consisting of a system of tubulo-cisternal endoplasmic reticulum. Penetration via the choroid plexus appears to be the dominant route for penetration from blood into c.s.f. in the 60 day fetus.

1. Axonal transport of phospholipids labelled by lumbosacral spinal cord injection of [3H]choline has been studied in normal and injured sciatic nerves of the rat. 2. The appearance of labelled material in progressively increasing amounts in the sciatic nerve following spinal cord injection was consistent with a maximum velocity of axonal transport of about 20 mm/hr. There was also evidence of substantial amounts of labelled phospholipids being transported at much slower velocities. 3. In sciatic nerves injured by crushing there was an accumulation of labelled phospholipid immediately proximal to the crush. The accumulation was progressive with time. There was also an increase of labelled phospholipid in all the more proximal segments of the crushed nerves; this reached a maximum of about twice that in uncrushed nerves at 10 hr. after spinal cord injection. 4. The labelled phospholipid was shown to be about 80-90% phosphatidylcholine both in uncrushed and crushed nerves. 5. The nature of the mechanism of this very rapid response of neurones to peripheral injury did not appear to be due to loss of 'information' from the periphery or action potentials initiated at the site of injury. The phenomenon has been further investigated by injection of drugs into the injured or control nerves. KCl injected at (but not proximal to) the site of injury was effective in blocking the injury response providing it was injected between a few minutes before or up to 30 min after the time of injury. Injection of either tetrodotoxin or local anaesthetic was as effective as injury in increasing the amount of labelled phospholipid transport. 6. These results suggest that the occurrence of an injury in a distant process of a neuron can be signalled retrogradely to the cell body by a mechanism involving a signal velocity of at least 140 mm/hr.

1. The blood-brain, blood-CSF and CSF-brain barriers to protein have been studied in post-natal tammar wallabies (newborn to 70 days) using morphological and physiological techniques. 2. Endogenous and exogenous plasma proteins, and in some experiments horseradish peroxidase, were used as indicators of barrier integrity or permeability. 3. Immunocytochemical studies of endogenous tammar proteins showed that these (e.g. albumin) were present in the lumen of vessels in the brain, in cerebrospinal fluid (CSF) and within some cells in the choroid plexus and brain. No staining of the brain extracellular space was obtained; in particular there was no perivascular staining. Possible artifacts that could account for this lack of staining are discussed. 4. Ultrastructural studies showed the presence of well-formed tight junctions between cerebral endothelial cells and between choroid plexus epithelial cells, even as early as the day of birth. A membrane specialization between adjacent neuropendymal cells that had the same ultrastructural appearance as the 'strap junction' previously described in human and sheep fetuses was observed. These junctions may act as a barrier (CSF-brain barrier) to the passage of protein from CSF into brain in these immature animals, as has previously been described in eutherian fetuses. 5. In experiments in which exogenous plasma proteins or horseradish peroxidase were injected intravenously, care was taken to limit both the volume and protein load injected. These proteins penetrated into CSF. The naturally occurring steady-state CSF/plasma ratio for several proteins was approached by several of the injected (human) proteins within a few hours of I.V. injection, suggesting that much of the protein in CSF, at least when sampled from the hindbrain, originates from plasma in this species. No penetration across cerebral vessels was observed. Uptake of some proteins (e.g. albumin), occurred into neuroependymal cells at some ages. 6. These results suggest that the very immature brain of the newborn tammar is protected from protein present in the circulating plasma even at an embryonic stage of development by a combination of a well-formed blood-brain barrier to protein in the cerebral vessels and a CSF-brain barrier to protein at the level of the neuroependyma. The adult-type blood-CSF barrier to protein (tight junctions between adjacent choroid plexus epithelial cells) is present but appears to be bypassed in the immature brain, probably by a transcellular route across the choroid plexus.

1. Plasma proteins have been demonstrated to be present in early fetal sheep brain in amounts which cannot be accounted for by blood contamination. 2. The distribution of alpha-fetoprotein, albumin, fetuin, alpha 1-antitrypsin and transferrin has been studied by immunoassay of extracts from brain homogenates and by immunoperoxidase histochemistry of fetal brains between 37 and 125 days gestation (term is 150 days). 3. At 35 days gestation fetuin and albumin were quantitatively the most important proteins in fetal brain, both as estimated by extraction and by immunohistochemistry. Both of these proteins, and also alpha-fetoprotein and alpha 1-antitrypsin, declined considerably in amount by 60 days gestation. After 60 days the concentrations of albumin and alpha-fetoprotein were not significantly different from that due to blood contamination and only occasional cells could be demonstrated by immunohistochemistry. Fetuin and alpha 1-antitrypsin were present in reduced but significant amounts at least up to 125 days gestation. 4. The immunohistochemical results showed that considerable numbers of immature neurones were stained for some plasma proteins. Fetuin positive cells predominated both in terms of the larger number of cells which stained at 35-40 days gestation and in the persistence of positive cells up to 125 days gestation. Numerous cells in the neuroependymal layer and in several layers of the developing cortical plate were positive, especially early in gestation. Only a few transferrin or alpha 1-antitrypsin positive cells were observed. 5. Permeability of the blood-brain barrier to sheep or human serum albumin (labelled with 125I) was tested in 60 day fetal sheep by intravenous injection and estimation of brain radioactivity at 3 or 6 hr with allowance for blood contamination. Only a very small (but significant) penetration of protein was detected. Unlike penetration of protein into c.s.f. at the same age, it did not reach its natural steady state in brain. 6. It is concluded that the blood-brain barrier to protein is well developed in the immature fetal sheep but that developing neurones probably acquire certain plasma proteins directly from the c.s.f. when they are differentiating in the neuroependyma. The subsequent distribution of plasma protein positive cells in different brain regions is suggested to be due to the migration of developing neurones for the neuroependyma although the possibility of local synthesis of plasma proteins has not been excluded.