Ontogeny of the blood-brain barrier

Exp. Eye Res. (1977) Suppl., 523-550 Ontogeny of the Blood-Brain Barrier NORMANR. SAU~OERS

Department of Physiology, University CollegeLondon, London, U.K. l. Introduction The term blood-brain barrier was originally used to explain the experimental observation that acid dyes and certain other materials, when introduced into the blood, penetrated freely into many tissues but not into the brain. The first studies were carried out by Ehrlich (1885) and as pointed out by Davson (1976) his interpretation was that there was a failure of nervous tissue to take up the acid dyes. Other early workers (Biedl and Kraus, 1898; Lewandowsky, 1900) seem to have been responsible for the idea that the lack of penetration of certain substances into the brain was due to a barrier between the blood and the brain. That the restriction on dye penetration was due to a barrier to protein (to which many dyes bind in plasma) rather than to dye itself was shown by Tschirgi (1950). In the meantime the term blood-brain barrier has come into widespread use (some would say misuse) to cover a range of different mechanisms which control the internal environment of the brain. Stability of the internal environment of the brain is undoubtedly important for the normal functioning of the central nervous system. As Hugh Davson has put it, our sensory experience might be limited to a series of flashes and bangs were it not for the fact that the electrolyte composition of brain extracellular fluid is strictly controlled compared with that of the rest of the body. The various adult blood-brahl barrier mechanisms have been extensively studied and have been comprehensively reviewed by Davson (1967, 1972, 1976); see also Oldendorf (1977). Much less information is available about the development of these mechanisms although it has been thought for a long time that they are immature in the foetus and newborn. This paper will start with a brief review of earlier work which has been interpreted as suggesting that the foetal and newborn blood-brain barrier, at least to protein, is immature. Three aspects of the development of the blood-brain barrier have recently received some attention and will be the main subjects for this paper. (1) Morphology, particularly of intercellular junctions in developing brain. (2) Penetration of non-electrolytes from blood into brain and CSF in the foetus. (3) Protein composition of foetal CSF and penetration of protein from blood into brain and CSF in the foetus. Other aspects of the problem of blood-brain barrier development which will be touched upon more briefly are the extracellular space of developing brain and the secretion of the CSF in the foetus and newborn. The view that the foetal blood-brain barrier is immature was based on three main pieces of evidence : (1) In certain species some substances penetrate more freely into the brain of the foetus and new born than in the adult. (2) The concentration of protein in the newborn human infant, especially if prematurely born, is higher than in the adult. (3) The occurrence of kernicterus in the new born (brain damage due to deposition of bilirubin in the basal ganglia) was attributed by some to immaturity of the blood-brain barrier because the condition occurs only in the newborn period. 523

524

N. R. SAUNDERS

As will be discussed below the occurrence of kernicterus is due to other factors than an in,mature blood-brain barrier and the higher concentration of protein in newborn CSF may also have another explanation than immaturity or "leakiness" of the bloodbrain barrier. Early studies of penetration of materials into the developing brain were limited in scope but the results are fairly clear cut. However misquotation and counter misquotation have resulted in some confusion as will be considered in the next section. There is evidence from Flexner (1938) that some of the electrolyte gradients between CSF and plasma are established very early in foetal life. The implication of these observations, namely that the presence of such a gradient involves the development of both the mechanism which sets it up and a barrier or diffusion restriction which prevents re-equilibration, has largely been ignored until recently. In the past few years there have been several studies in various species which confirm and extend Flexner's results. These experiments were reviewed by Saunders and Bradbury (1973); as little new information on this aspect of the problem has been published since then it will not be dealt with in any detail in this review. There have been few studies of penetration of metabolically active materials from blood into brain and CSF. The difficulties of distinguishing between differences in metabolic usage and possible barrier effects have been discussed by Dobbing (1961, 1969) and the penetration of metabolic substrates such as a2p or amino acids into brain and CSF will not be considered here. 2. Early Studies of Blood-Brain Barrier Development

The fiIst experiments on the development of the blood-brain barrier followed naturally in the methodological footsteps of work on the barrier in adult animals. Behnsen (1927) using trypan blue observed that dye penetration occurred in the same restricted areas as in adult animals (e.g. choroid plexus, pineal and pituitary) but the staining was more intense and more extensive. Much of the brain was unstained as in adults. In the newborn of several species (cat, dog, rat and mouse) Stern and Rapoport (1927), Stern and Peyrot (1927) did not find any greater degree of penetration of trypan blue or congo red than in adults. Stern and Peyrot (1927) however, found that sodium ferrocyanide adnfinistered intraperitoneally or subcutaneously penetrated into both CSF and brain of newborn rats, rabbits and cats but not of foetal (term) newborn guinea-pigs, a finding which they correlated with the stage of development of the central nervous system. The work of Behnsen and of Stern has frequently been misquoted as suggesting that the blood-brain barrier in newborn animals is freely permeable to dyes. All the available evidence suggests that there is a considerable restriction of trypan blue penetration into the brains of foetal and newborn animals of a wide range of species. In addition to the above work, the problem has been studied in newborn rats by Millen and Hess (1958) who report similar findings to those of Behnsen, and in human (GrSntoft, 1954) and rat (Grazer and Clemente, 1957) foetuses in which no significant penetration of the dye into brain was observed. One source of confusion seems to have been that several authors have considered a single barrier to trypan blue (which binds to plasma protein) and to sodium ferrocyanide which is of much smaller molecular weight (304) than of protein bound trypan blue. Stern and co-workers clearly distinguished between the penetration of "colloid" (tr)3oan blue) and "crystalloid" (sodium ferrocyanide) and observed penetration only of the latter in the newborn of some species, as described above. There do not appear to be any

ONTOGENY OF THE BLOOD-BRAIN BARRIER

525

serious discrepancies between these studies particularly if differences in the stage of development of different species at birth are taken into account. The finding of Stern and Peyrot (1927) that a small molecular weight lipid insoluble compound (i.e. sodiunl ferrocyanide) penetrated more freely into CSF and brain of immature animals fits well with later findings (Ferguson and Woodbury, 1969; Evans, Reynolds, Reynolds, Saunders and Segal, 1974) which will be discussed below. In view of the recent observation that certain specific plasma proteins may be transported into brain and CSF of immature foetuses (Dziegielewska, Evans, Malinowska, Mellg~rd, Reynolds and Saunders, 1976) it is perhaps surprising that the dye try-pan blue, which binds to plasma protein, did not appear to penetrate into the brains of the youngest foetuses studied by GrSntoft and by Grazer and Clelnente However GrSntoft (1954) used a protein-free dye solution for perfusion in all his experiments as did GIaser and Clemente in their experiments on the least mature foetuses. Possibly in the absence of plasma protein, the dye did not penetrate. Also as pointed out by several authors, detection of dye in brain or CSF by microscopical means, is not a very reliable way of assessing penetration across the blood-brain and blood CSF barriers. Another possibility which could explain the observations of Behnsen (1927) and Millen and Hess (1958) that the restricted areas of dye penetration were more extensive in the developing brain is that the penetration was occurring only in vascularized areas of the brain and that the dye was indeed penetrating attached to some protein such as ~-fetoprotein by the specific transport mechanism which will be discussed later. The concentration of protein in human CSF in the newborn period is undoubtedly higher than in the adult; however, as will be considered in more detail in a later section, this does not necessarily mean that the adult blood-brain barrier to protein is immature in the new born. The suggestion that the newborn blood brain barrier to bilirubin may be immature (Spatz, 1934; Bakay, 1953; Lee, 1971) seems to be Eased on a misunderstanding of the nature of the bilirubin which may accumulate in the newborn period and give rise to jaundice followed by kernieterus if the degree of jaundice is severe. The bilirubin which accumulates in the newborn period is unconjugated and lipid soluble ; it would therefore cross membranes, including the blood-brain barrier, freely. That it does not usually do so is because it is bound to plasma protein. Should the binding capacity of plasma protein for unconjugated bilirubin be exceeded because of an excessive load of bilirubin, or because of a reduction in available binding sites caused by the presence of competing drugs or a fall in pH, then free lipid soluble bilirubin will enter the brain and kernicterus may result (see also Maisels, 1972; Saunders and Bradbury, 1973). The effects of adverse physiological conditions upon developing blood-brain barrier mechanisms will be considered later but in relation to kernieterus (see Lucey, Hibbard, Behrman, De Gallardo and Windle, 1964) their effect seems to be by affecting bilirubin binding to albumin or its affinity for central nervous system cells rather than on the blood-brain barrier itself. 3. CSF Protein During Development

That the concentration of protein in CSF is higher in the human newborn, especially if preterm, than in the adult has been suggested by many authors (see Table I). These observations are one of the main reasons for the rather frequent statement that the blood-brain burlier in the foetus and newborn is immature. For obvious reasons, in many cases CSF has been taken only from ill infants and it is therefore not always

526

N.R.

SAUNDERS TABLE I

Total protein concentration (mg/lO0 ml) in CSF offun term and pre-term infants estimated during thefirst few days of life

Authors

~,{ean (my/100 ml)

s.~:.

Range

n

Condition

---194 -240 -90 -180

14 11 34 135 35 51

ncrmal healthy normal healthy normal cerebral axotia

-138 -259 -292 -1600 -144 --

19 49 9 70 59 9

Full term

Spiegel-Adolf et al. (1954) Widell (1958) :Nasralla et al. (1958) Naidoo (1968) Piliero and Lending (1959) W a t s o n (1964)

103 80.9 115 63 70 77

9.9 6.2 5.8 1.6 6.0 5-3

46 32 25 26

Pre term

Otila (1948)* Nasralla et al. (1958)t Gyllensw~rd a n d MalmstrSm (1962)~ Bauer et al. (1965)§ Bartolozzi et al. (1967)[] Cole et al. (1974)¶ I

* Birth weights t Birth weights J; Birth weights § Birth weights [[ Birth weights ~I Birth weights

100 167 176 187 62 120

5.5 6.4 26 28 4.3 10

50 81 57 30 12

hcalthy normal healthy various normal normal

920-2150 g, from Table 19, p. 91. < 4 . 5 lbs, from Table 2, p. 1404. < 2 0 0 0 g, from Table 5, p. 60. 800-2620 g, from Fig. 1, p. 1018. < 2 5 0 0 g, from Fig. 2, p. 299. 1080-1710 g, from Table 1, p. 724.

clear if the values are indeed normal. However, there are a few reports (Table I) in which samples were taken from these normal children. There is considerable variation between results, even from the normal infants, but it seems clear that at full term, CSF protein concentration (60-100 rag/100 ml) is somewhat above the adult value (15-65 rag/100 ml, Schultze and Heremans, 1966). In preterm infants the concentration is rather higher (Table I). There are very few reports of CSF protein concentration in the hmnan foetus. According to Klosovskii (1963) the level at 16 weeks gestation is about 20 times that in the adult. Adinolfi, Beck, Haddad and Seller (1976) have reported values of up to 770 rag/100 ml at 20 weeks gestation. The level of total protein in foetal CSF is undoubtedly much higher than in the newborn or adult; this is confirmed by the animal studies mentioned below. However, a problem with the human foetal studies is that the foetuses will have been asphyxiated for an unknown length of time. Since hypercapnia has been shown to increase the penetration of protein into CSF (e.g. Hochwald, Mathan and Brown, 1973) it is possible that this nfight affect both the total protein concentration in CSF and also the relative amounts of individual proteins in the CSF. Klosovskii (1963) reported very high levels of CSF protein in foetal cats, although he did not give any absolute values and made no comment upon the physiological state of the foetuses. Bito and Myers (1970) reported that during the last third of gestation in the rhesus monkey cisternal CSF protein concentration was about six times that in the adult. Amtorp and Sorensen (1974) found that the concentration of

O N T O G E N Y OF T H E B L O O D - B R A I N B A R R I E R

527

protein in CSF of newborn rats was about 260 mg/100 ml although again little information was given about the physiological state of the animals ; foetal rabbits of 23 days gestation (term 31 days) were said to have high levels of CSF protein but no absolute values were given. Birge, Rose, Haywood and Doolin (1974) carried out a detailed study of CSF protein concentration in the chick embryo. They showed that it reached a peak of about 530 rag/100 ml at embryonic day 11. At University College London we have recently obtained similar information from sheep foetuses of different gestational ages delivered by caesarean section. Figure 1 shows clearly the high concentration of foetal CSF protein early in development. Some of the CSF samples were obtained from foetuses whose blood gas values were shown to be normal for their gestational age (cf. Evans et al., 1974). The CSF protein concentration did not seem to be affected even if the samples were not obtained until a few (2 5)rain after bleeding out the foetus. Samples of CSF obtained from pig foetuses gave total protein values which were similar to those of'sheep foetuses at the equivalent gestational age.

600

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{ 500

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Age (deys)

FIc. l. Changes in the CSF total protein concentration during development in the sheep. Pooled data fronl several breeds of sheep. The peak at 55 days is probably spurious since there appear to be some differences in CSF protein conceutration between breeds early in gestation. Bars are ±S.E.

Q~talitative ide~hification of foetal CSF 29roteins A large amount of work has been done on identification of proteins in adult CSF (e.g. Schultze and Heremans, 1966; Bock, 1973). Comparatively little has been done on newborn CSF and practically nothing is known about foetal CSF proteins, either human or animal. Recently Adinolfi et al. (1976) have reported the presence of high concentrations of ~-fetoprotein (a foetal specific protein) and albumin in the CSF of immature human foetuses obtained following therapeutic abortion. Adinolfi et al. also obtained estimates of transferrin, IgG and fl-2-mieroglobulin on the same samples. Amtorp and Sorensen (1974) estimated the concentration of albumin and of ~globulin in CSF of foetal and newborn rabbits. In studies of sheep foetal CSF at

528

N. R. S A U N D E R S

FIG. 2. Two dimensional immunoelectrophoresis of 60-day foetal sheep plasma and CSF (a) and adult sheep plasma and CSF (b). A, albumin; T, transferrin; F, fetuin.

ONTOGENY OF THE BLOOD-BRAIN BARRIER

529

University College London, the Laurell (1965) two-dimensional immunoelectrophoretic technique has been used to compare foetal CSF proteins with those of plasma from the same foetuses. These studies show clearly that many of the CSF proteins are identical with those of the plasma [Fig. 2(a)]. So far only albumin, transferrin and fetuin (a foetal specific protein which is similar to ~-fetoprotein) have been identified definitely. But there appear to be more types of protein in foetal CSF than in the adult [Figs 2(a), (b)]. 4. Morphology of the Developing Blood-Brain and Blood-CSF Barriers

It is clear from the studies of Reese and Karnovsky (1967), Brightman and Reese (1969) and others that in adult animals the principal barrier to protein penetration from blood into brain and CSF is constituted by the tight junctions between endothelial cells in cerebral vessels and between the epithelial cells in the choroid plexus. Several authors have assumed that if the blood-brain barrier to protein is immature in the foetus or newborn then the tight junctions in developing brain would be poorly formed or incomplete. Cerebral endothelial tight iunctions have been seen in chick embryos as early as 6 days (Delorme, 1972) and even 4 days (Roy, Hirano, Kochen and Zimmerman, 1974). However, Delorme, Gayet and Grignon (1970) suggested that many of the junctions are incompletely formed at 6-8 days and only become properly tight by around 10-12 days, at which time they also reported a decline in penetration of horseradish peroxidase from blood into brain. Birge et al. (1974) made a similar correlation at 10-12 days between the formation of tight junctions in chick choroid plexus epithelial cells (the blood-CSF barrier to protein) and the maxinmm concentration of protein in CSF during embryonic development. Both Delorme et al. and Birge et al. suggested that these findings are evidence that there is immaturity of the blood-brain barrier to protein before 10 days in the chick embryo and that it takes the form of incomplete tight junctions. It is clear from their papers that at least some cerebral endothelial and ehoroid plexus epithelial tight junctions develop before 10 days in the chick embryo. Caley and Maxwell (1970) reported junctional complexes between endothelial cells of the cerebral cortex of newborn rats. Similarly early development of tight j unctions in the choroid plexus is suggested by the work of Tennyson (1975). Definition of a particular junction as "tight" or "gap" is difficult if only thin-section electronmicroscopy is used since serial sectioning is needed to be sure that a junction which has a "gap" appearance in one section may not in fact be "tight" in another. Also even if a junction has a "tight" appearance it may not form a complete intercellular barrier. Another problem is that development in chick embryos is on such a compressed timescale that important changes in development may appear to be correlated simply because their time separation is only a few hours whereas the material tends to be examined on a day-by-day basis. An important observation by Ddorme et al. (1970) in their horseradish peroxidase (HRP) experiments was the occurrence of pinoeytotic vesicles in cerebral endothelial cells containing HRP reaction product and which could be seen in some eases to penetrate into the cerebral extracellular space. Delorme et al. dismiss this finding as unimportant for the penetration of HRP into developing brain on the grounds that the vesicles were not very frequent. Such a conclusion cannot be drawn on the basis of the qualitative evidence presented. Recent studies in sheep, pig and human foetnses (Bohr and Mollg~rd, 1974; Nollggrd and Saunders, 1975) confirm that cerebral endothelial and choroid plexus

FIG. 3. (a) Freeze-fracture replica of a choroid plexus epithelial cell from a 45-day gestation foetal sheep. The fracture has exposed a large area of tight junction towards the apex (CSF surface) of the cell. There are at least four strands running roughly parallel to the apical cell surface. Bar indicates 0.5 tzm. (b) Freeze-fracture replica of a choroid plexus epithelial cell from a 125-day gestation foetal sheep. Note the similar number of strands compared with 45 days (a) and that the junctional depth is at least as great at 45 days as at 125 days. Bar indicates 0.5/~m (Dr K. Mollgb~rd, unpublished).

ONTOGENY

OF T H E B L O O D - B R A I N

BARRIER

531

epithelial tight junctions develop very early indeed. These junctions have been demonstrated both in thin section EM material and by freeze fracture. The latter technique has the advantage that a much greater part of a single junction can be examined than in thin sections; as pointed out above, the appearance of a tight junction in a single thin section does not necessarily mean that the junction forms the belt-like structure around the circumference of adjacent cells that is the typical appearance of the fully developed junction. Tight junctions are easier to define in freeze-fracture material because their appearance is characteristically very different from that of gap junctions. The completeness of a junction can also be more satisfactorily assessed, although not absolutely so. An advantage of the sheep over the chick is that tile gestational period of the sheep is much longer (147 days compared with 21 days). Thus developmental events are on a longer time scale. It is clear from a compari,son of the development of tight jnnctions in foetal sheep brain and of the levels of foetal CSF protein (Dziegielewska et al., 1976) that there is substantial tight junction formation well before the high concentration of CSF protein begins to decline. In the earliest foetuses examined so far (35 days) there was extensive tight junction formation in the choroid plexus. An example of the freeze fracture appearance of a choroid plexus tight junction at 45 days gestation is shown in Fig. 3. The junctional strands, which correspond to the points of membrane contact seen in thin section EM, are of moderate complexity. In all junctions studied so far between 35 and 125 days gestation there were at least two or three strands (Mollg~rd, Malinowska and Saunders, 1976). At an early stage of brain development there are very few vessels in the cortical plate. Tight junctions have not yet been identified in endothelial cells of the sheep foetus early in gestation, although they have been demonstrated in early hmnan foetuses (Mollg~rd and Saunders, 1975). From Fig. 1 it can be seen that the CSF protein concentration in sheep foetuses does not begin to decline until around 55 days. Furthermore there is no obvious difference between tight junction appearance in the 60-day brain and choroid plexus (e.g. Fig. 4) compared with 125 days (Fig. 5) nor does there appear to be any change in the complexity of tight junction strands (Fig. 3), yet between these times the foetal CSF protein concentration has declined from about 350 rag/100 ml to around 50 rag/100 ml. Thus in the sheep at least (and similar information has been obtained from the pig foetus) immaturity of tight junctions does not seem to account for the high level of CSF protein; other mechanisms have therefore been considered. It could be that a lot of the protein in foetal CSF comes from the brain side of the barrier rather than from the blood. This seems to be excluded front being quantitively important by the finding, which will be discussed later, that many of the CSF proteins are imnmnoelectrophoreticallyidentical with those in plasma and that certain plasma proteins penetrate rapidly from blood into CSF and brain. The route of penetration is probably transcellular. The direct evidence for this proposition is not yet substantial but experiments arc in progress to investigate the hypothesis. It is supported by the observation of Mollggrd and Saunders (1975) that the dye Alcian blue, which binds to plasma proteins, penetrates into foetal sheep brain and CSF at 60 days but not at 125 days. The dye is electrondense when treated with osmimn tetroxide In EM thin sections it is present not in intercellular junctions (which are tight at this stage) but within a tubular system of smooth endoplasmic reticulum (Figs 6 and 7). It is not yet clear whether this tubular system extends right across both endothelial and choroid plexus epithelial cells; there may be an intermediate step of vesicular transport between the cell surface and the tubular system. However, the tubular

FIG. 4. Thin section of choroid plexus epithelium from a 60-day sheep foetus. A well-formed tight junction is seen at the apical end of the lateral cell margin. Within the cell are elements of a tubular system of smooth endoplasmie retieulum. Bar indicates 0.5 ~m (Dr K. Mollg&rd: unpublished). FIG. 5. Thin section of choroid plexus epithelium from a 125-day sheep foetus. A well formed tight junction is seen at the apical end of the lateral cell margins. Note that in these thin sections the tight junctions in Figs 4 and 5 appear to be of similar depth and complexity. Bar indicates 0.5 ~m (Dr K. Mollghrd, unpublished).

ONTOGEN¥

OF THE BLOOD-BRAIN

BARRIER

533

Fins 6 and 7. These electronmicrographs were obtained from the choroid plexus of another 60-day sheep foetus which received Alcian blue i.v. about 10 min prior to fixation. A particulate precipitate (P) is observed inside some parts of the endoplasmic reticulum. In :Fig. 6 precipitate can also be seen on the apical cell membrane (curved arrows). The tubular system makes very close contact with the apical cell membrane, the outer surface of which is exposed to the CSF (open arrows, Fig. 6). An electron dense structure (small arrow, Fig. 6) seems to separate the CSF and the tubular system. In :Fig. 7 a similar precipitate-containing tubular system can be seen (arrows). The system appears to make very close contact with the lateral cell membrane (LM) and the lateral intercellular space which is separated from the CSF (L) by a junctional complcx (JC) ( :~:50 000). Reproduced, with permission, from Mollg£rd and Saunders (1975). J. Neurocytol. 4~ 453-68.

534

N. R. SAUNDERS

system has been observed to make direct contact with the CSF surface of choroid plexus epithelial cells (Fig. 6). It seems that Delorme et al. may have been premature in dismissing the importance of transcellular transfer as a mechanism of horseradish l)eroxidase penetration in chick embryo brain. 5. Development of the Blood-Brain Barrier to Non-electrolytes

One of the striking demonstrations of a blood-brain barrier effect in adult animals is the small degree of penetration of extracellular markers such as sucrose or inulin from blood into brain and CSF compared with other tissues such as nmscle (Davson, 1967). Although some authors attributed this difference to a lack of extracellular space in adult brain this is clearly not the ease since the brain space obtained with for example sucrose, is much larger if the marker is introduced via the CSF by ventrieulo-cisternal perfusion (Oldendorf and Davson, 1967). In adult rat)bits Oldendorf and Davson found that the brain:plasma ratio of sucrose was 2.7% after 3 hr of intravenous sucrose, but reached 5.8% after a similar period of ventriculoeisternal perfusion with fluid containing sucrose. However, since a small amount of sucrose did penetrate into the brain from the blood, following i.v. injection, it might be expected that in very long experiments a higher brain :plasma ratio would be obtained. However, this did not occur and Oldendorf and Davson suggested that this was due to a sink effect of CSF draining away the penetrating marker back into the blood via the arachnoid granulations. This is a concept which was proposed by Davson (1963) and which Davson and Welch (e.g. 1971) have developed in later papers. In order to investigate this barrier mechanism in the foetus it is necessary to obtain estimates of the rate and degree of penetration of non-electrolytes into brain and CSF from blood ; it is also necessary to have information about the size of the extracellular space in developing brain and of the production of CSF by the developing ehoroid plexus. Woodbury and his colleagues (Vernadakis and Woodbury, 1965; Ferguson and Woodbury, 1969) studied the problem in foetal and neonatal rats. They found that both sucrose and inulin penetrated into brain and CSF at a greater rate and to a greater extent in immature rats compared with more mature ones (e.g. Fig. 8). There are clearly considerable difficulties involved in carrying out experiments in such small animals, and there is necessarily some uncertainty about their physiological state. Also the intraperitoneal injection into the mother of labelled sucrose and inulin meant that the markers could only reach the foetal brain and CSF by a rather complex route including the maternal peritonemn and the placenta. No information was obtained about the blood-level of markers during the course of the experiments so that brain : plasma and CSF:plasma ratios could only have been estimated on the basis of terminal samples. This may give an incorrect estimate of the ratios since the blood levels are likely to have been changing rather than remaining steady during an experiment. The brain spaces are also likely to be overestimates because they did not include a correction for isotope in any blood which remained in the brain samples. No information was given about the nature of the labelled material counted in the samples. Particularly because of the intraperitoneal route of injection there is a possibility that some of the markers may have reached the gut and been metabolized to other materials which could have been taken up by the brain and CSF of the foetal or neonatal rats. These observations were extended by the recently published study of Amtorp (1976) on the penetration of [*25I]albumin from blood into brain and CSF

ONTOGEhTY OF T H E B L O O D - B R A I N B A R R I E R [ Cl4"l inulin

[CI4"l inulin Ioo 80

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Time (hr) Fro. 8. The simultaneous uptake of [l~C]inulin into (a) cerebrospinal fluid (CSF) and (b) cerebral cortex (called brain in subsequent figures) of rats during maturation, as a function of the time after injection of the inulin. (A) --4 days, 1.15 g; ( ~ ) newborn, 4.6 g; (D) 3 days, 6.20 g; (O) 9 days, 12.7 g; (×) 16 days, 29.9 g; ( ~ ) 26 days, 60.0 g; (O) adult, 250 g. Redrawn, with permission, from Ferguson and Woodbury (1969). Expl. Brain Res. 7, 181-94.

of developing rats from the newborn period to 30 days. The same technical objections apply to the experiments of Amtorp as have been mentioned for those of Ferguson and Woodbury. The results of Ferguson and Woodbury and of Amtorp are replotted in Figs 9 and 10 to show the dependence upon molecular size of both the initial rate IOO

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Time (hr) FIG. 9. The simultaneous penetration of [tdCJsucrose ( 0 ) and [14C]inulin (©) into CSF and brain of 3-day-old rats as a function of time after intraperi~oneal injection, replotted from Ferguson and Woodbury (1969); and the penetration of [125I]albumin (A) into brain and CSF of 5-day-old rats as a function of time after intraperitoneal injection, replotted from Amtorp (1976).

of penetration and the steady state level achieved. It is also clear that in the older animals the degree of penetration of all three markers declines in a similar manner. In spite of the above criticisms these results in tiny fragile rats represent a considerable technical achievement and appear to be consistent with those of later experiments in which there was some information abo~t the state of the foetuses and

536

N.R. SAUNDERS 50

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Time (hr)

Fro. 10. The penetration of [laC]sucrose and ['4C]inulin into CSF and brain of 14-day-old rats as a fnnction of time after injection, replotted from Ferguson and Woodbury (1969); and the penetration of [12~I]albumin into brain and CSF of 30-day-old rats as a function of time after injection, replottcd from Amtorp (1976).

the nature of the markers estimated in brain, CSF and plasma. Olsson, Klatzo, Sourander and Steinwall (1968) used intravenous fluorescent labelled albumin detected microscopically in rats from foetal to adult ages. They did not observe any greater penetration of albumin into brain even in 15-day embryos than in the adult. It may he that this microscopical technique was insufficiently sensitive to detect very small amounts of protein which may have penetrated into the brain. The small level (about 6~o) reported by Amtorp for newborn rats is likely to have been partly due to blood contamination of the brain specimens. In foetal sheep brains the albumin level was only about 0.5~o after correction for blood contamination (see below and Fig. 17). Ferguson and Woodbury (1969) and Amtorp (1976) discussed their results in terms of possible changes in extracellular space volume, CSF secretion rate ("sink effect") and barrier formation during brain development. They tended to emphasize the possible importance of a larger extracellular space and reduced sink effect rather than a reduction in permeability of the system during development. However, this interpretation is not entirely supported by their data. Extracellular space was estimated from the brain:CSF ratios of sucrose and inulin at different ages (Fig. 11). Standard 80

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FIG. 11. Relation between the brain to fluid (CSF or plasma water) ratio × 100 (space in %) of [I~C]inulin and [14C]sucrose and the age in days before or after birth of rats. Redrawn, with permission, from Ferguson and Woodbury (1969). Expl. Brain Res. 7, 181-94.

O N T O G E N Y OF T H E B L O O D - B R A I N B A R R I E R

537

errors for the ratios were not given but it seems unlikely that there was any significant difference in the sucrose ratios between 3 and 16 days postnatal and between 0 and 9 days for the inulin ratios (although the ratios for both molecules may have been slightly higher in the foetal rats studied). The ratios for adult rats were very large indeed. Ferguson and Woodbury suggested that this may have been due to development of an ependymal barrier as the animals matured. An alternative possibility is that the large brain: CSF ratios occurred because of the contribution of isotope in any blood contained in the brain samples. This would be more important in older animals because of the much greater degree of vaseularization of the brain. There was, however, a considerable change in both the initial slope for penetration into brain and CSF and in the steady state level achieved at the different ages (Fig. 8). Ferguson and Woodbury also cite evidence (Brizzee and Jacobs, 1959) for a larger extracellular space in the newborn rat brain, but it seems doubtful if such a conclusion can be drawn from the original data of these authors since it describes only changes in neuronal cell population in relation to brain size and does not mention brain extracellular space directly. In contrast Caley and Maxwell (1970) estimated from morphological studies that the cerebral cortical extracellular space actually increased between the newborn (12%), 5 days postnatal (20~o) in the rat and then declined to 1% at 21 days. Bondareff and Pysh (1968) estimated a value of 40.5% for rat cortical extracellular space at 10 days postnatal which declined to 26% at 21 days and was 22% in the adult. Measurements of extracellular space obtained from morphological data are probably not very reliable because of the very variable shrinkage involved in fixation, but Caley and Maxwell's results certainly cannot explain the decline in nonelectrolyte penetration described by Ferguson and Woodbury and in albmnin described by Amtorp. More recent papers (Johanson and Woodbury, 1974; Woodbury, Johanson and Bronsted, 1974) have given information about the sink effect in developing rat brain. In 3-day-old rats the secretion rate was 0.2 ~l/min compared with 0.8 t~l/min at 24 days and 2.0 t~l/min in the adult. These values are likely to have been overestimates of the true secretion rate since in the same experiments inulin was found to penetrate into brain tissue (presmnably across the ependyma and into the brain extracellular space). As this happened to a greater extent in the younger brains the estimates of CSF secretion rate for these brains are likely to have been overestimated disproportionately compared with the older brains. This much lower rate of secretion in immature rat brain is interpreted by Woodbury and his co-workers as evidence for a reduced sink effect in the immature brain. However, even accepting the above rates of secretion, the absolute rate of CSF secretion is not a very good guide to the sink effect since the secretion rate should be compared with the size of the ventricular system and brain for which it acts as a sink. The precise geometrical relationships between choroid plexus, cerebral ventricles, extracellular space and blood vessels are extremely complex and for most species unknown. Also they change markedly during development. Therefore it is very difficult to devise a satisfactory basis for comparison of the sink effect at different stages of brain development. The relationship between the size of the choroid plexus and its rate of production of CSF is not necessarily a reliable index of the sink effect at any particular stage of development. It does of course indicate how close the choroid plexus at any stage of development approaches the secretory capacity of the adult ehoroid plexus. In fact if the CSF secretion rate is expressed per unit weight of choroid plexus the difference between immature rats and the adult is much less than the difference when the absolute rates of secretion are

538

N.R.

SAUNDERS

compared. Thus between 8 days postnatal and the adult the rate of secretion per gram of choroid plexus doubled, whereas the absolute rate of secretion increased by nearly five times (Johanson and Woodbury, 1974). Unfortunately no values were given for brain weight or CSF volume at the different ages but similar studies in sheep foetuses for which this data was available (see below) suggested that the sink effect may be very effective even in immature brain. Ferguson and Woodbury (1969) also examined the ratios of some electrolytes between CSF and plasma in rats of different ages and concluded that CSF is an ultrafiltrate of plasma at 4-6 days before birth. However, at this stage of development of the cerebral ventricular system in the rat, there is no direct communication between the IVth ventricle and subarachnoid space (Cammermeyer, 1971). Thus it is not clear how the composition of fluid obtained bypuncture of the cisterna magna would compare with fluid obtained from within the ventricular system. The values of CSF and plasma electrolytes obtained at later ages suggest that the choroid plexus was capable of secretion at those ages, as was confirmed by the later studies of Johanson and Woodbury (1974). Amtorp (1976) accepted the evidence of Ferguson and Woodbury (1969) for a larger extracellular space in developing rat brain and considered that it contributed to the greater accumulation of [l~SI]albumin which he found in brain and CSF of immature rats. He also considered that the sink effect in these immature rats was likely to be less than in older rats. The evidence quoted (Bass and Lundborg, 1973), although indicating a lower rate of secretion of CSF in 5-day-old compared with 10- and 30-day-old rats, did not take adequate account of the age differences in brain and ventricular size discussed above. Also at 5 days, but not at later ages, there was evidence that the inulin nlarker used for estimation of CSF clearance penetrated to a significant extent across the ependyma into brain, thus rendering the dilution method unreliable. Woodbury and co-workers did consider that part of the greater penetration of nonelectrolytes into immature rat brain was due to increased permeability of the brain blood vessels early in development. In fact the initial rate of entry of sucrose or inulin would not be expected to be much affected by a change in sink effect (Davson and Welch, 1971) so the observation that the initial slopes were different at different ages (Fig. 8) suggests that the barrier to sucrose and inulin in the rat is indeed substantially more permeable in the immature state than later in development and that this is the main reason for the greater penetration of non-electrolytes in the immature brain, rather than any differences in extracellular space or CSF secretion rate although these may make some contribution. This conclusion was suggested by Bradbury (1975) who estimated permeability-surface area products from their data ; it is supported by later studies in the sheep (Evans et al., 1974, 1976; Dziegielewska et al., 1977), as will now be described. It has already been pointed out that the sheep foetus has a number of advantages for studies of physiological function during development. At any particular stage of development the sheep foetus is large compared with the foetuses of more usual laboratory animals. Thus their physiological state can be more easily assessed and controlled. Also blood levels of isotopic and other markers can be estimated and controlled during an experiment. The nmlticotyledenary nature of the placenta means that vessels of only a single cotyledon can be cannulated rather than the more gross interference required in species where the placenta is only a single plate. Also surgical interference is easier without precipitating the placental sepsoration which occurs in many species when the uterus is opened.

ONTOGENY

OF THE

BLOOD-BRAIN

BARRIER

539

Results from experiments on blood-brain and blood-CSF permeability to nonelectrolytes in the sheep foetus are summarized below. The experimental techniques have been described in detail previously (Bradbury et al., 1972; Evans et al., 1974). Briefly, pregnant ewes of known gestational age (49 days to term, 147 days) were anaesthetized with thiopentone and chloralose. The uterus was delivered through an anterior abdominal incision and an artery and vein of a single cotyledon were cannulated (0.1-2.0 mm diameter nylon tubing depending upon size of vessels). This is relatively easy even in the smallest foetuses (at 50 days gestation body weight = 10 g) because the placental development is much in advance of that of the foetus (Barcroft, 1946). Intravenous injections of isotopically labelled markers were made using [3H]or [14C]sucrose, [nC]erythritol, [3H]- or [14C]innlin and [425I]- or [laqJalbumin. In the earlier studies (Evans et al., 1974) only sucrose was used and an approximately constant plasma level was maintained for intervals of 0-5-6 hr by intermittent injection (()ldendorf and Davson, 1967) or by continuous infusion. At the end of each experiment brain and cisternal CSF samples were taken. By using both 14C and aH labels in the same foetus but for different times two time points could be obtained in a single experiment. In later experiments a single initial injection of several different markers was given (e.g. [l~C]erythritol and Jail]inulin) and the blood levels estimated from intermittent samples. In most experiments blood contamination of brain and CSF samples was estimated by giving indium-ll3m mixed with ewe plasma 5-10 rain before the end of each experiment (Sisson, Oldendorf and Cassen, 1970). The indimnl13m binds to transferrin and in such a short time remains within the circulation. Radioactivity in CSF, brain and plasma was estimated either by liquid scintillation counting in a PPO/POPOP/Instagel mixture following dispersion of the samples in "Soluene" (Packard) or by direct counting in a Panax Gamma One-Sixty Counter, as appropriate. d/rain per g wet brain or CSF x 100 d/rain per g plasma

Rcsults were expressed as

49 days A

t2

.9

9~8

i6 4

/~7 / I

4

125days

2 00

I

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:5

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Fro. 12. The rate of penetration of labelled sucrose from blood into brain at different gestational age in the sheep. A steady level of plasma sucrose was maintained by i.v. infusion for the times indicated by t h e abscissa. Bars are ±s.E., small n u m b e r s are n u m b e r of experiments. Redrawn, with permission, from E v a n s et al. (1974). J. Pl~ysiol. (Lond.) 238, 371-86.

540

N. R. S A U N D E R S

Figure 12 shows the results of experiments at three different foetal ages for penetration of sucrose into brain. It is clear from this that both the initial rate of penetration and the steady state ratio were greater in younger foetuses. The main decline in sucrose penetration both into brain and into CSF occurred between the gestational ages 50 and 70 days as is shovel in Fig. 13. Here are plotted the brain:plasma and CSF:plasma ratios after 90 min intravenous sucrose. The extracellular space of brain at 50-60 days does not seem to be greater than at term. The chloride, sodium and potassium levels in brain at these ages were rather similar

20

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Gestational age

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Term

Fro. 13. Level of labelled sucrose in sheep brain and CSF after 90 min i.v. infusion of sucrose. Abscissa = age of foetus. Bars are :kS.E, small n u m b e r s are n u m b e r of experiments. Redrawn, with permission, from E v a n s et al. (1974). J. Physiol. (Load.) 238, 371-86.

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F:o. 14. CSF secretion rates, expressed as ~l/min/g brain during development in the -heep. Bars are i s . z . (Data, with permission, from E v a n s et al. (1974). J. Physiol. (Load.) 238, 371-86.

O N T O G E N Y OF T H E B L O O D - B R A I N B A R R I E R

541

(Bradbury et al., 1972). Also the brain sucrose space, estimated from brain : CSF ratios (Evans et al., 1974) appeared to be similar at these widely different gestational ages. Thus it seems unlikely that a larger extracellular space in brain at 50 60 days could have contributed much to the substantially higher steady state level of sucrose at this age compared with later in development. At 60 days gestation, estimates of CSF secretion rate (Evans et al., 1974) showed that the sink effect, estimated as the secretion rate/g brain, was at least as great if not greater than at later stages of development (Fig. 14). This was also so if the sink effect were expressed as percent turnover per rain (Table II, Evans ct al., 1974). The secretory capacity of the choroid TABLE II

Effect of hypercapnia (PaCO2 = 102±2 mmHg) on volume of distribution of [all]- or [14C]sucrose in brain and CSF after 90 rain i.v. infusion in foetal sheep and newborn lambs PeP 2 values are for foetal arterial blood. Mean ±s.E., n values in brackets Age (days)

Hypcrcapnia F()ctal 131-4-2 (14) Newborn 1 2 i 4 (8)

CSF/plasma (%)

Brai./plasma (%)

14.5±5.3 (5)

2.26±0.31 (14)

10.8i3.4

1.96-4-0.47

Control (P.co 2 40~:1 mmHg) Foetal 123±2 (5) 2.98-4-0.30 (3) Newborn 7±1 (4) 2.71±0.30 (3)

1.38±0.26 (5) 1.49i0"20

plexus, as judged by the secretion rate per gram of choroid plexus was indeed less at 60 days (0.16 ~l/min/g) than at 125 days (0.50/~l/min/g) but as pointed out above, this may not be a very good index of the sink effect, given the large differences in ventricular and brain size and in cerebral capillary density at different ages. A reduced sink effect compared with adult brain does not therefore seem to account for the greater penetration of sucrose in immature sheep foetuses. It seems likely that the permeability of the blood-brain and blood-CSF barriers to sucrose is substantially greater at 50-60 days than later in gestation. The problem has been investigated in more detail by using several molecules of different radius (erythritol, 0-32 nm, inulin 1.39 nm and albumin 3-5 nm) in addition to the sucrose, 0-51 nm, already described. The results of experiments to investigate the time course of penetration of these materials into CSF at 60 days gestation are summarized in Fig. 15. It is quite clear from these data that the initial rate of penetration depends upon molecular size. Similar results were obtained for penetration into brain, although of course the steady state levels were lower because the markers penetrated predominantly into extracellular fluid. In contrast, at 125 days, although the time course of penetration of erythritol was similar to that at 60 days, that for sucrose was much reduced as has already been shown in Figs 12 and 13. Penetration of inulin has also been found to be much reduced at 125 days reaching a csf/plasma ratio of only about 0.9% by 6 hr. Analysis of the steady state ratios achieved for

542

N. R. S A U N D E R S

inulin and sucrose, compared with their molecular radii, shows that at both 60 and 125 days these molecules are only nfinimally restricted in their penetration into csf and brain (Dziegielewska et al., 1977). The reduced penetration at 125 days is probably due to a decrease in surface area of contact between blood, CSF and brain, the anatomical basis for which is currently under investigation (see footnote on p. 243).

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Time{hr) FIO. 15. The uptake of labelled erythritol (Q), sucrose (O), inulin ( ~ ) and albumin (A) into CSF of 60-day sheep foetuses as a function of time after administration. Bars are ±S.E., small n u m b c r s are n u m b e r of experiments. (Dziegielewska, Evans, Malinowska, Mollghrd, Reynolds and Saunders, unpublished.

Some authors have suggested (e.g. Wright, 1972) that the route of penetration of non-electrolytes across epithelia is via the tight junctions. Casley-Smith (1969) has observed deposits of sodium ferrocyanide in cerebral capillary endothelial tight junctions in EM thin sections following i.v. administration. He has suggested that cerebral endothelial tight junctions are permeable to ions and other small molecules. However, evidence outlined in the section on morphology suggested that there were no obvious changes in tight junction structure either in cerebral endothelial or choroid plexus epithelial cells between 60 (or even earlier) and 125 days gestation in the sheep. (see also Mollg~rd, Malinowska and Saunders, 1976). This indicates that the marked decrease in permeability between these ages is unlikely to have been due to an obvious change in tight junction structure, such as the closeness of apposition of adjacent membrane outer leaflets or junction strand number (cf. Claude and Goodenough, 1973). However, this does not exclude the possibility that some other change in tight junction structure (not necessarily detectable by electronmicroscopical observa.tion) may be involved in the decline in non-electrolyte and albumin penetration in the sheep foetus and developing rat. There are considerable technical difficulties in visualizing water soluble molecules such as inulin and sucrose in electronmicroscopical thin sections. This direct method of investigating the route by which non-electrolytes and albumin penetrate into brain and CSF has not so far been attempted. The finding of Mollg~rd and Saunders (1975) that the dye Alcian blue (Figs 6 and 7) penetrates into brain and CSF via a transcellular route in cerebral endothelial and choroid plexus epithelial cells suggests the possibility that passively penetrating markers such as the non-electrolytes and albumin used in the above experiments may also penetrate by a

ONTOGENY

OF THE BLOOD-BRAIN

BARRIER

543

transcellular route.* Although the dye Alcian blue is predominantly bound to plasma proteins when injected intravenously (Mollg~rd and Sorensen, 1974), it is not clear if the osmiophilic material resolved intracellularly (Figs 6 and 7) is dye or dye bound to protein; nor is it clear to which plasma proteins the Alcian blue was bound in these experiments. These are important points since evidence discussed in the next section indicates that certain specific proteins may penetrate into early foetal brain and CSF to a much greater extent than would be expected from their molecular weights. 5. Penetration of Protein from Plasma into CSF and Brain in Foetal Sheep

The evidence discussed in the section on morphology indicates that tight junctions in foetal brain are well-formed at a time when foetal CSF protein concentration is still high and that there is no obvious change in tight junction structure which could account for the subsequent marked decline in the concentrations of CSF protein in later foetal life. Thus the presence of a high concentration of CSF protein is not itself evidence for "immaturity" or "leakiness" of the blood-brain and blood-CSF barriers to protein. As was shown in the section on CSF proteins, during development many of the proteins in early foetal CSF are immunoelectrophoretically identical with those in plasma. In order to investigate the possibility that plasma proteins might penetrate

-.o

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o

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2

3

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Era. 16. The uptake of albumin (A), transferrin (0) and y-fetoprotein (O) into 60-day sheep CSF as a function of time after administration. Bars are :hS.E., small numbers are number of experiments (Dziegielewska, Evans, Malinowska, Mellghrd, Reynolds and Saunders, unpublished).

more easily into early foetal brain than later on (in spite of the presence of well formed tight junctions even in very immature brain), experiments have been carried out to estimate the time-course of penetration of several plasma proteins from blood into CSF and brain of 60 and 125-day foetuses (Dzieglielewska et al., 1976). The experiments were essentially similar to those described in the previous section on nonelectrolytes. Either human serum proteins (Behringwerke A G) detected by radial diffusion immunoassay using specific antisera (Dako) or isotopically labelled proteins (59Fe or 11stain- sheep transferrin and 125Ihuman serum albumin) were used. Following a single intravenous injection of protein, intermittent samples of plasma were taken * It has recently been observed (M~,llg~rd and Saunders, 1977) that the number of contacts between tubulocistemal endoplasmic reticulum and the CSF surface of the epithelial cell membrane in choroidplexus is much fewer at 125 days t h a n at 60 days.

544

N. R. S A U N D E R S

for measurement of the blood levels of the proteins during the course of the experiment. After 3 or 6 hr the experiments were terminated, the CSF sampled by eisternal puncture and the brain removed. Figure 15 shows the time course of penetration of albumin (reel. wt. 69 000), transferrin (reel. wt. 74 000) and ~-fetoprotein (mol. wt. 64 000) from blood into CSF in 60-day sheep foetttses. It is quite clear that the rate of penetration bore no relation to molecular weight. If these curves are compared with those for non-electrolytes (Fig. 16) it seems likely that although the albumin was probably penetrating passively at a rate and to an extent which would be expected flom its molecular size, both transferrin and ~-fetoprotein penetrated to a much greater extent than would be expected on this basis. Less information is available about the penetration of these proteins into brain, as not all the experimental samples have been analysed at the time of writing. The very low penetration of albumin even at 60 days gestation (detected isotopically) has already been mentioned. In Fig. 17 it is compared with the penetration of transferrin labelled with mmIn/51Fe. The shape of the time-course of penetration of the latter is difficult to interpret without further information but it seems likely that, initially,

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Albumin (125I/'3mi)

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Time(hr) FIo. 17. The uptake of [1251/131I]albumin (O) and [1131nIn/ 59 Fe]transferrin ( 0 ) into 60-day sheep brain as a function of time after administration. Bars are -4-s.]~. (Dziegielewska, Evans, Malinowska, Reynolds and Saunders, unpublished).

transferrin penetrated rapidly into brain as it did into CSF (Fig. 15), but at a later stage in these experiments there was a possibility that either the label (llamIn or 59Fe) or the transferrin itself penetrated into the brain cells. This should be resolved when the immunoassays for the human serum protein penetration into brain have been performed. The only experiments which have so far been carried out involving protein penetration in older foetuses (125 days gestation) used transferrin labelled with mmIn or 5°Fe. Six hours after intravenous injection the CSF:plasma ratio was 1.8±0.45~o (n = 4) and the brain :plasma ratio was 4.034-0.36% (n = 4). Thus the penetration of transferrin appeared to be markedly less in more mature foetuses. Since the level of fetnin in CSF at this late stage in gestation was much lower than at 60 days, the

O N T O G E N Y OF T H E B L O O D - B R A I N B A R R I E R

545

penetration of fetuin/~-fetoprotein from blood into brain and CSF would probably also be much less at 125 days. The evidence is not yet available for it to be clear when in gestation the rapid penetration of specific proteins, such as transferrin and ~-fetoprotein, first becomes markedly reduced. From the rapid decline in total protein in CSF (Fig. 1) one can speculate that it is probably around 65-70 days gestation. 6. Effect of Adverse Conditions on Barrier Permeability During Development

There have been several studies in adult animals of adverse conditions such as asphyxia or hypercapnia on blood-brain and blood-CSF barrier permeability to materials such as trypan blue and sodium ferrocyanide (Stern, Romei and Gertchikowa, 1934); albumin (Cutler and Barlow, 1966; Hochwald et al., 1973) and sucrose (Cameron, Davson and Segal, 1969) ; but very little is known about the effect of these or other adverse conditions on barrier permeability during the foetal or newborn periods. Stern and co-workers published brief reports of effects of conditions such as carbon monoxide poisoning, hypothermia and chronic alcohol poisoning (e.g. Stern and Lokchina, 1927) but otherwise little seems to have been done apart from studies on kernicterus in asphyxiated animals (e.g. Lucey et al., 1964, see above). Evans, et al. (1976) investigated the effect of severe hypercapnia (PaCO2 approx. 100 mmHg), hypoxia (PaO2 ~ 10-15 mmHg) or nonrespiratory acidosis (pH approx. 7.00) on labelled sucrose penetration into CSF and brain in foetal and newborn lambs. Some of their results are summarized in Tables II and III. From TABLE III

Effect of abnormal arterial Pc%, PO~ or pH on volume of distribution of [aH]- or [14C]sucrose in foetal sheep and newborn lamb brain after 90 min i.v. infusion of isotope. Gestational age range 113-142 days, newborn age range 1-10 days. Mean ±s.E., n values in brackets

Control (foetuses) Hypercapnia (foetuses) Post-hypercapnia* Hypoxia (foetuses) Non-respiratoryt acidosis

Po 2

Pc%

pH

Brain/plasma (%)

274-1

404-1

7.32±0.01

1.384-0.26

(5)

2.98±0.30

37L1

102±2

7.06±0.04

13±2

524-3

7.234-0.03

2.26±0.31 (14) 1-17±0.30 (5) 0.644-0.08 (3)

14.5 ±5.3 -3.20=[=0.64

404-2

7.074-0.01

1.434-0.21

(4)

CSF/plasma (%)

3.522=0.17

* Four foetuses and one newborn: 90 miu hypereapnia (Paco2 approx. 100 mmHg) followed by 90 min i.v. sucrose at blood gives values similar to those of Control groups. Poa within control range for foetuses and newborn ventilated lambs. t Two foetuses and two newborn: infused with lactic acid (IN, 0.1-0-9 ml/min). Po 2 within control range for foetuses and newborn ventilated lambs.

these it is clear that hypereapnia had a marked effect in increasing the penetration of sucrose from blood into CSF, an effect which was greater in the less mature animals. The effect on penetration into brain was extremely variable, possibly due to reduction of brain extracellular space by brain oedcma (see Evans et al., 1976). Nonrespiratory acidosis did not significantly affect sucrose penetration into brain or

546

N.R.

SAUNDERS

CSF. Hypoxia actually reduced the level of sucrose accumulating in brain, possibly because of reduced brain extraeellular space; this interpretation was supported by the finding of a reduced brain :CSF ratio of sucrose following ventrieulo-eisternal perfusion (Oldendorf and Davson, 1967) of the brain under hypoxie conditions compared with control conditions. Hypoxia did not seem to affect penetration into CSF. If hypereapnia also increases the penetration of plasma protein into CSF in the immature brain, as has been demonstrated for the adult (e.g. Hochwald et al., 1973), then this effect could account for the very high levels of CSF protein reported in some ill preterm infants with high P o e 2 values (e.g. Cole et al., 1974). 7. Concluding Discussion The problem of the maturity and permeability of the foetal blood brain and bloodCSF barriers to protein thus seems rather more complex than previously thought. It seems likely that the barrier mechanism to lipid insoluble materials that penetre,te passively, such as non-electrolytes and albumin, undergoes a substantial reduction in permeability between 50 and 70 days in the sheep foetus and in the newborn period in the rat. The early experiments using sodium ferroeyanide (Stern and Peyrot, 1927) may also have been investigating this same barrier. But since it remains unclear whether dyes such as trypan blue would have bound predominantly to a protein which penetrates passively such as albumin or to one such as ~-fetoprotein which appears to penetrate much more rapidly (presumably by another mechanism) the significance of the results from these dye studies remains unclear, more especially in view of the fact that microscopical observation for the presence or absence of dye is not a very reliable method. Clearly more plasma proteins need to be investigated in order to establish which types of protein penetrate rapidly into early foetal brain and which penetrate passively. The route by which the proteins penetrate is presumably intracellular since, as described above, the junctions between the endothelial and epithelial cells are tight. Whether it is via the smooth endoplasmic reticulum, as appears to be the case for Alcian blue dye, seems speculative at this stage. Indeed one important question is: since the Alcian blue experiments provide at present the only indication of a possible route for penetration from blood into brain and CSF in these immature foetuses, is this the route both for passive permeation by non-electrolytes and albumin as well as for the more rapid transfer of certain specific proteins (~-fetoprotein and transferrin)? If so what are the significant features of the tubular system which allow it to discriminate in terms either of molecular size or of protein specificity? The possibility that certain plasma proteins may be moved selectively and rather rapidly into the brain of an immature foetus is presumably of significance for brain development in its early stages. Transferrin is one of the first proteins to be synthesized in embryo (Schultze and Heremans, 1966, p. 528) and its significance in development may be much more than its known adult function of iron transport. ~-fetoprotein is a foetal specific protein which is synthesized in the yolk-sac, liver and possibly placenta early in gestation (Adinolfi, Adinolfi and Lessof, 1975). The function of :¢-fetoprotein is unknown. There is some evidence that it binds certain hormones, notably oestrogens (Uriel, Nechaud and Dupiers, 1972); thus it may have a transport function analogous to that of transferrin for iron. ~-Fetoprotein has been shown to have an inhibitory effect upon antibody synthesis and so could play an immuneregulatory role in foetal development (Adinolfi et al., 1975). It may also affect the growth of cells in tissue culture (Fisher, Puck and Sate, 1958). It may be that certain

ONTOGENY OF THE BLOOD-BRAIN BARRIER

547

proteins such as transferrin and ~-fetoprotein play an important part, either nutritional or growth controlling, in the early development of the brain; it may be significant that the increase in number of neuroblasts ceases around 60-65 days gestation in sheep (Astrom, 1967), i.e. about the time when the concentration of protein in foetal CSF is declining rapidly. However, the germinal layer supporting cells continue to divide, differentiate and migrate oat to the cerebral hemispheres, probably at least until 80-90 days gestation. The observation that certain plasma proteins may be transported into immature foetal brain may have another significance for brain development. Namely, that there may be pathological situations in which abnormal proteins are taken up and cause some maldevelopment of the brain. In conclusion, the period of 50-70 days gestation in the sheep foetus and late foetal, early newborn period in the rat seem to be critical for the development of a number of different blood-brain barrier mechanisms in these species. It is the time when CSF total protein concentration falls rapidly. Passive penetration of lipid insoluble molecules declines considerably. CSF/plasma gradients for several electrolytes in both species become apparent about these times, indicating the adequate functioning of some ion pumps and of a barrier sufficient to prevent re-equilibration. Finally, at least in the sheep, penetration of specific proteins (transferrin and ~.-fetoprotein) is probably switching off. ACKNOWLEDGMENTS I should like to thank all my colleagues at University College London and elsewhere for their considerable contributions to all aspects of the work carried out at University College London, which is described in this review: Mr Charles A. N. Evans, Mrs Margaret L. Reynolds, Miss Katarzyna M. Dziegielewska, Dr Danuta H. Malinowska, Mrs Teresa Feldman, Dr Michael W. B. Bradbury, Dr Geoffrey M. Durbin, Dr Kjeld Mollgfird, Dr John M. Reynolds, Dr Malcolm B. Segal. I should particularly like to thank Dr Danuta Malinowska for her skilful help in preparation of the manuscript and Dr Kjeld Mollg£rd for his preparation of and permission to reproduce Figs 3-7. I should also like to thank Mrs Anne Gorrod for deciphering nay execrable handwriting and typing the manuscript. The advice and support of Dr Hugh Davson, Professor Sir Andrew Huxley and Professor Leonard B. Strang were essential for the initiation and development of these projects. This research has been supported by grants from the Medical Research Council (U.K.) and by travel grants from the Wellcome Trust, whose help is gratefully acknowledged. l~EFERENCES Adinolfi, A., Adinolfi, M. and Lessor, M. H. (1975). Alpha-feto-protein during development and in disease. J. Med. Gen. 12, 138-51. Adinolfl, M., Beck, S. E., Haddad, S. A. and Seller, M. J. (1976). Permeability of the bloodcerebrospinal fluid barrier to plasma proteins during foetal and perinatal life. Nature, Lond.

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