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of Physiol(gy (1991), 439, pp. 215-237 With 7 figures Printed in Great Britaini

SPECIES-SPECIFIC TRANSFER OF PLASMA ALBUMIN FROM BLOOD INTO DIFFERENT CEREBROSPINAL FLUID COMPARTMENTS IN THE FETAL SHEEP BY K. M. DZIEGIELEWSKA, M. D. HABGOOD, K. M0LLGARD*, M. STAGAARD* AND N. R. SAUNDERSt From Clinical Neurological Sciences, The Wessex Area Neurosciences Group, U`niversity of Southampton, Southampton General Hospital, Southampton SO9 4XY and the *Department of Medical Anatomy A, The Panum Institute, University of Copenhagen, Denmark

(Received 13 November 1990) SUMMARY

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), 61 + 10; cisterna magna (CM), 13-7+08. 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, 1P0 + 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 t To whom reprint requests should be sent at the University of Southampton. N1S 8926

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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. INTRODUCTION

The mechanisms that determine and control the exchange of materials between the blood and the cerebrospinal fluid (CSF) are usually referred to by the general term 'blood-CSF barrier'. Lipid-insoluble substances over a wide range of molecular sizes (from sucrose, molecular radius 0-51 nm, to large molecular weight proteins such as immunoglobulin G (IgG) 5.3 nm) penetrate from blood into CSF to a greater degree in the developing than in the mature brain (Dziegielewska, Evans, Malinowska, M0llgard, Reynolds, Reynolds & Saunders, 1979; Dziegielewska, Evans, Malinowska, M0llgard, Reynolds & Saunders, 1980 b). Whether this is because of developmental immaturity or developmental specialization remains a matter of some discussion (see Dziegielewska & Saunders, 1991; Saunders, 1991). The maturity of the barrier (blood-CSF or blood-brain) is often expressed as CSF-toplasma or brain-to-plasma ratios for a given molecule. In the adult brain the CSF/plasma ratio for many lipid-insoluble molecules is proportional to the molecular radius and concentration in plasma and the transfer seems to be largely due to diffusion (Felgenhauer, 1974). In the developing brain, it was observed that not only is the concentration of protein in CSF very high (up to 200 times that in the adult in some species; see Dziegielewska & Saunders, 1988) but the penetration of some proteins from plasma to CSF is not size dependent. Thus in 60-day-old fetal sheep (Dziegielewska et al. 1980 b) and in neonatal rats (Habgood, 1990), albumin CSF/plasma ratios are similar to those of inulin, but the molecular radius of albumin is some three times larger than inulin. Also the CSF/plasma ratios for individual proteins are not the same in spite of the fact that their molecular sizes are similar (Dziegielewska, Evans, Fossan, Lorscheider, Malinowska, M0llgard, Reynolds, Saunders & Wilkinson, 1980a; Dziegielewska et al. 1980b). It has been noted in earlier experiments on developing sheep brain that different species of the same protein, e.g. albumin, can be transferred across the choroid plexus to a different level (Dziegielewska et al. 1980b). These observations imply that there may be a specific protein transfer mechanism present in the immature brain that disappears later in brain development. The concentrations of proteins in different regions of the CSF space within the brain are not the same; these differences are apparent in the adult (see Davson, Welch & Segal, 1987) but the actual concentrations of protein are so low that they are often not much remarked upon. Because of the generally high concentration of protein in developing CSF, the gradient between different parts of the CSF system

BLOOD-CSF TRANSFER OF ALBUMINS IN FETAL SHEEP 217 is much more striking (e.g. Cavanagh, Cornelis, Dziegielewska, Evans, Lorscheider, M0llgard, Reynolds & Saunders, 1983) although proportionally similar to that in the adult. The only published studies of the penetration of lipid-insoluble markers into CSF in the developing brain appear to be those involving CSF from the cisterna magna (e.g. Ferguson & Woodbury, 1969; Dziegielewska et al. 1979). In the present study, we have investigated in fetal sheep the exchange of lipid-insoluble molecules ranging in molecular size from sucrose (0-51 nm radius) to albumin (3 5 nm radius) between blood and CSF at three different sites: lateral ventricle, cisterna magna and dorsal cortical subarachnoid space. In the choice of experimental design we set out to investigate whether (1) the gradient in protein concentration between different parts of the CSF system is due to non-specific differences in permeability of the choroid plexus at those sites or (2) it is due to a specific protein transfer system that may function differently at different sites. We have used the small molecular markers sucrose and inulin to assess the passive level of permeability of the system, and the plasma protein albumin as a marker for the specific element of the transfer mechanism. We have used albumins from different animal species, and isotopically labelled albumins. Endogenous sheep albumin was used as the internal marker. The results showed differences in the steady-state level of albumin at different CSF sites which could largely if not entirely be accounted for by a difference in specific transfer of albumins at these sites. In addition, there were clear differences in the permeability of different species of albumin. The molecular basis for such differences is considered in the Discussion. METHODS

Preparation of ewes and fetuses Details of the methods used have been published previously (Dziegielewska et al. 1979). Clun Forest ewes of known gestational age were anaesthetized with i.v. thiopentone (15-25 ml, 5 g (100 ml H2O)-1 according to the size of the ewe) followed by i.v. chloralose (1 g (100 ml isotonic NaCl solution)-'; 180 ml initial dose followed by 50-80 ml h-1). One horn of the uterus was delivered via a paramedian incision in the lower abdomen and placed on a heated side table. The exposed uterine horn was covered with plastic film ('Clingfilm') and overlaid with heat insulating material. Fine nylon (Portex No. PP202) cannulae were inserted into an artery and vein of a single placental cotyledon (Meschia, Makowski & Battaglia, 1969). The arterial blood gas and pH state of the anaesthetized ewe was monitored and controlled by variation of the composition of the inspired air and occasionally by positive pressure ventilation. Sheep fetuses in the age range 57-61 days gestation (n = 28) were used. The fetuses were left in utero until the end of the experiment.

Monitoring The ewes' arterial blood pressure, heart rate and end-tidal CO2 concentrations were measured continuously as described previously (Bradbury, Crowder, Desai, Reynolds, Reynolds & Saunders, 1972). Arterial blood pH, Pco and PO were measured periodically using a Radiometer Blood Gas Apparatus (BMS3, Radiometer, Denmark). The arterial pH and blood gases were controlled by altering the composition of the inspired gases and, when necessary, by positive pressure ventilation. Fetal arterial blood pressure and heart rate were measured continuously. The initial fetal values were (means + S.E.M.): arterial blood pressure, 36-8 + 2-7 mmHg; heart rate, 213 ± 6 beats min-'. These values are similar to those reported previously (Dziegielewska et al. 1979) and did not change significantly during the course of the experiments. The initial fetal arterial blood pH and gas values were: pH, 7*44 + 0-03; P02, 35.9 + 3-5 mmHg; Pco2, 43 0 + 2- 1 mmHg. By the end of the experiments mean values were: pH, 7-41 + 0-06; Po,, 38 2 + 4-7 mmHg; Pco, 49-9 + 4-8 mmHg.

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Injected materials and sampling Various marker solutions (bovine serurn albumin (BSA, Fraction V. Pentex, ICN), goat serum albumin (GSA, Nordic, Netherlands), human serum albumin (HSA, A-8763, Sigma) 1251-labelled sheep serum albumin (SSA), or 355-labelled plasma proteins synthesized in vitro by dispersed liver cells (see below)) diluted in isotonic sodium chloride solution were administered intravascularly by slow infusion over the first 10-30 min of the experiment. The amounts injected were 20-25 mg of total protein as estimated both by dry weight and concentration of protein in the diluted solution (see below). In some experiments [14C]sucrose and [3H]inulin were also infused into the fetus. Arterial blood was sampled intermittently during the course of the experiment. At the end of each experiment a final blood sample was taken, the fetus was exteriorized and samples of CSF obtained as follows: (i) 10-15,l from the subarachnoid space over the dorsal surface of the cerebral hemispheres (4-5 mm lateral to the mid-line and 5 mm anterior to the lambdoid suture); (ii) 10-15 ul from the lateral ventricle at the same site as the subarachnoid CSF was sampled but at a depth of 5-6 mm from the skull surface; and (iii) 20-30 ,ul from the cisterna magna (except in the experiments using [35S]methionine-labelled proteins, when 50-60 ,l CSF were taken). Similar samples were obtained from six adult ewes. CSF and blood samples were centrifuged and the plasma separated. CSF samples were checked for blood contamination and only used if visibly clear; tests with blood added in serially increasing amounts to isotonic saline showed that less than 0 01 % blood contamination could be detected by eye. CSF and plasma samples were stored at -20 °C until measured.

Estimation of protein concentrations Total protein concentrations in CSF and plasma were estimated using the Bradford (1976) method and SSA as a standard. Concentrations of individual proteins were measured by radial immunodiffusion assays (Mancini, Carbonara & Heremans. 1965). Standard BSA, GSA, SSA and HSA were prepared and antisera used were rabbit anti-sheep albumin (Dziegielewska et al. 1980a), rabbit anti-human albumin (Dakopatts, Denmark), and sheep anti-bovine and anti-goat albumin (Binding Site Ltd). All antibodies were checked for cross-reactivity with fetal sheep serum and anti-human albumin was pre-absorbed with sheep albumin prior to its use in immunodiffusion assays. There was no cross-reactivity of sheep albumin with antibodies raised in sheep (anti-BSA and anti-GSA). The results were expressed as the ratio of CSF protein concentration/plasma protein concentration x 100, using a time-weighted mean value for the plasma protein concentration; this allows for any decline in marker protein concentration during the course of the experiment (generally around 10% decline over 3-5 h). The haematocrit of fetal blood samples was measured and the distribution volume of the injected proteins calculated.

Isotopic methods SSA (Fraction V, Miles Laboratories) was iodinated with 125I (IMS.30, Amersham International) using the method of Greenwood & Hunter (1963). Free iodide was removed by overnight dialysis at 4 °C against 0 9 % sodium chloride. The sample was checked for aggregation by electrophoresis in 12 % polyacrylamide gel followed by autoradiography of the gel. Over 80 % of all radioactivity was associated with the monomeric form of albumin as judged by densitometric scanning. Radioactivity was counted in an LKB gamma counter at an efficiency of 64 +4-9 %. Results were expressed as the ratio of CSF activity/time-weighted plasma activity x 100 (in d.p.m. I&h'; where d.p.m. is disintegrations per minute). In some experiments 6,6-[3H]sucrose (TRA.332, Amersham International; 40-80 #Ci) and ['4C]inulin carboxylic acid (CFA.399, Amersham International; 5-20 ,uCi) were injected slowly over 20-30 min with one of the protein solutions in a total volume of 1-5-2-5 ml; aliquots of 0-1-0 2 ml were injected at hourly intervals to maintain an approximately constant plasma concentration (Evans, Reynolds. Reynolds, Saunders & Segal, 1974; Habgood, 1990). In individual experiments the plasma concentration of labelled sucrose or inulin varied by about + 20%. However, it has previously been shown that even following a single I.v.injection of labelled marker, the use of time-weighted mean plasma estimates of isotope concentration gives similar results to estimates obtained from steady-state plasma experiments (cf. Evans et al. 1974; Dziegielewska et al. 1979). Activities of 3H and 14C were counted in an LKB liquid scintillation counter after dispersion of brain, CSF and plasma samples at 45 °C in 0 3 ml LKB 'Optisolve'

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solution before the addition of 4 5 ml LKB RIA liquid scintillant cocktail. Counting efficiency was 63 + 15% for 3H and 916+00-3% for '4C. Blood contamination of brain samples was estimated using 113mIn as previously described (Dziegielewska et al. 1979).

Calculations After correcting for background and blood contamination the results were expressed as disintegrations per minute (d.p.m.) per microlitre (or milligram) of sample. The individual plasma levels were multiplied by the sample collection time, summed and divided by the total experiment time to give a time-weighted average. Brain/plasma and CSF/plasma ratios were calculated as sample/plasma x 100 (in d.p.m. ul-'). Distribution spaces were calculated as injection/plasma (d.p.m. ,tl-1). No allowance was made for differences in water content of CSF and plasma; in fetal sheep this is much less than in the adult because fetal CSF contains a higher concentration, and plasma a lower concentration, of protein than in the adult (see Table 2). In vitro labelling of sheep plasma proteins Liver samples were obtained from fetal sheep of various gestational ages (40-95 days) and placed immediately in ice-cold Eagle's minimal essential medium (Imperial Laboratories) supplemented with 2 g 1'- sodium bicarbonate and 2 mM-L-glutamine. Livers were then gently dissociated either by cutting with a sterile scalpel blade into very fine pieces (for older livers) or simply teasing with sterile syringe needles. Tissue was then centrifuged at 2000 r.p.m. for 5 min and the pellet resuspended in 5-20 ml of the same medium containing a total of 1 mCi of [35S]methionine (Amersham International). The volume of the incubation medium depended on the amount of the tissue and was estimated not to exceed 10 % w/v. The cultures were incubated at 37 'C in 5 % CO2 in air (CO2 incubator, Heraeus, Germany) for 90 min. The viability of the hepatocytes was checked by staining with Trypan Blue. At the end of 90 min, the cultures were transferred to a centrifuge tube and spun for 10 min. Supernatants were collected and checked for total protein, total radioactivity, albumin concentration and radioactivity associated with albumin. In most cases it was necessary to concentrate the supernatants in order to obtain enough counts for subsequent experiments. The concentration was done by forced dialysis. The radioactivity associated with albumin was determined by running crossed immunoelectrophoretic plates of the sample against anti-sheep albumin (Dziegielewska et al. 1980 a) followed by careful cutting out of the albumin peak precipitated in the gels (see below). Approximately 1 x 106 d.p.m. were injected into the experimental 60-day-old fetuses as described earlier.

Estimation of [35J]methionine-labelled sheep proteins CSF and plasma samples from the experiments using in vitro-labelled proteins were processed as follows. First, total protein and albumin concentrations were estimated as described above. Then total radioactivity was counted on the '4C channel in an LKB liquid scintillation counter. CSF (50-60,1) was freeze-dried and reconstituted in 5,ul of distilled water prior to application to crossed electrophoresis gels. Plasma (4 ,ul) was run in parallel. The crossed electrophoretic plates were run as described previously (Dziegielewska et al. 1980a). In the second dimension the intermediate gel contained 2-5 ml anti-sheep albumin (Dziegielewska et al. 1980a), and the top gel contained 2 ml anti-sheep plasma (Dakopatts, Denmark) plus 0 5 ml anti-calf fetuin (Dakopatts, Denmark). At the end of the electrophoretic separation the plates were washed in 0 9 % sodium chloride and visible peaks of precipitation corresponding to individual proteins were cut out and transferred into weighed scintillation vials. The weight of each gel was determined. Four control pieces of gel (from precipitate-free parts of the plate) were also included from each separation and used to estimate the background. Gels were dispersed in LKB 'Optisolve' solution and scintillant added (LKB RIA scintillant cocktail). Samples were counted on the 14C channel at 90-92% efficiency. Results were expressed as d.p.m. (utl original fluid)-' after correcting for the background (d.p.m. (mg of gel)-').

Morphological techniques At the end of most experiments, following collection of CSF samples, brains were removed from the skull and fixed overnight by immersion in Bouin's fixative for immunocytochemistry. The tissue was washed for 1 0-20 min in tap water, dehydrated in graded alcohols, cleared in chloroform or xylene and embedded in paraffin wax (melting point 56 °C). Serial sections of the entire brains

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were cut in sagittal or frontal planes, deparaffinized and rehydrated. Sections were stained with Haematoxylin and Eosin or Toluidine Blue for routine histological examination. Adjacent sections were stained immunocytochemically, using rabbit polyclonal primary antibodies against sheep (Dziegielewska et al. 1980a) and human (Dakopatts, Denmark; Nordic, Netherlands) albumins or goat anti-human albumin (Nordic, Netherlands). The antibodies were pre-absorbed with human and sheep albumin respectively. Their cross-reactivities were checked both in immunodiffusion assays (Dziegielewska et al. 1980a) and on sections from fetal sheep brain and 10- to 12-week-old fetal human brain obtained in connection with legal abortions. In terms of choroid plexus development these stages are equivalent (Jacobsen, M0llg'ard, Reynolds & Saunders, 1983). Sections for immunocytochemistry were washed in Tris-buffered saline (TBS: 0-05 M-Tris, pH 7-4; 0 15 M-NaCl) with 0-01 % Nonidet P-40, and then pre-incubated in TBS with 0 5 % ovalbumin, 03% gelatin and 0-05 % Tween-20 as blocking reagents (M0llgard & Balslev, 1989). Overnight incubation with the primary antibody, diluted in pre-incubation buffer, was carried out at 4 'C. The primary goat antibody was detected using rabbit anti-goat IgG (Dakopatts, Denmark) and goat peroxidase-anti-peroxidase (PAP; Dakopatts, Denmark) and the rabbit antibodies were detected using a modified PAP or alkaline phosphatase-anti-alkaline phosphatase (APAAP) method (M. Stagaard, R. S. Nowakowski, 0. B. F. Terkelsen & K. M0llgard, unpublished observations). The peroxidase reaction was developed in diaminobenzidine (Sigma, Denmark) whereas the alkaline phosphatase was developed in bromo-chloroindolephosphate and Nitroblue Tetrazolium. The sections were cleared and cover-slipped with DPX mountant (BDH) or cover-slipped immediately with Aquamount. RESULTS

Haematocrit and distribution volumes of injected sucrose, inulin and proteins [3H]sucrose and [14C]inulin distribution spaces (as a percentage of body weight) were (%; mean+ S.E.M.) 31-1 + 2-5 and 29-3 + 3-1 respectively. Injected protein distribution spaces were: BSA, 15-9+0± 9 ml (range 13-3-19-2); and HSA, 22-7 + 2-1 ml (range 14-2-32-7). Haematocrits from arterial blood samples from some 60-day-old fetuses were estimated. The initial value was 24-8+1±2 % (n= 8) with a tendency to increase during the course of the experiment (30 1+ 3 1 %, n= 5). Thus if most of the injected protein remained within the vascular compartment, the total circulating blood volume in 60-day-old fetuses can be estimated from these results as at least 30 ml. Penetration of [3H]sucrose and ['4C]inulin into differentCSF compartments and brain regions In five experiments in fetuses with a mean age of 59 4+0 7 days (range 57-61), radiolabelled sucrose and inulin were injected on a schedule designed to maintain an approximately steady plasma level over 3 h (see Methods). Blood and CSF samples from the sites described in Methods were removed and the brains were then divided into hindbrain, midbrain and diencephalon, and forebrain (separated into anterior and posterior cerebral hemispheres). Radioactivity levels were determined and the results expressed as ratios (tissue or CSF activity/plasma activity x 100) as described in Methods. These results are shown in Table 1. With the exception of the choroid plexus, in all brain regions and CSF compartments studied the [3H]sucrose ratios were consistently higher than the [14C]inulin ratios. The CSF/plasma ratios for [3H]sucrose were similar in all three CSF compartments as were the ratios for [14C]inulin. This is in marked contrast to the results for albumin permeability (see next section). There was no significant difference between the ratios for [3H]sucrose and[14C]inulin in the choroid plexus, indicating that both molecules have equivalent access to the same (extracellular) tissue space within the choroid plexus. [3H]Sucrose

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TABLE 1. Distribution of small molecular markers in central nervous system n Sucrose Inulin Whole brain 5 10 0+007 5-1 +0 4 Cerebral hemispheres 5 75 +0 7 3-1 +0-3 Midbrain 5 11-7 + 1-4 4-9+0-6 Hindbrain 5 5-4+ 11 10-5+1-4 Choroid plexus 5 37-7 +43 352 + 2-4 Ventricular CSF 4 12-7+2-3 70+03 4 Subarachnoid CSF 6-2 +2-0 14-5+ 2-0 Cisternal CSF 4 15-7 + 1 3 7-6+2 1 Distribution of [3H]sucrose and ['4C]inulin in brain, choroid plexuses and CSF of 60-day-old fetal sheep following 3 h i.v. exposure to approximately constant plasma levels of [3H]sucrose and [14C]inulin. Results are expressed as ratio of tissue or CSF/plasma x 100, in d.p.m. ml-'. Mean + s. E. M.; n = number of fetuses. TABLE 2. Protein concentrations in CSF and plasma Protein concentration (mg (100 ml)-')

CSF compartment Sheep albumin 60 days gestation Cortical subarachnoid 63+ 10 (16) Lateral ventricle 38 + 2 (18) Cisterna magna 136+8 (19) Plasma 1008+41 (20) Adult Cortical subarachnoid 10-4 + 2-1 (6) Lateral ventricle 8-4+ 11 (7) Cisterna magna 9-8 + 2-3 (6)

Total protein 249+ 38 (13) 131 + 15 (15) 277+ 18 (16) 1670+ 140 (17)

Albumin (%) 30-6+5-6 (10)

400±+ 6-1 (10) 49-2+4-7 (13) 52-9+4-9 (9)

48-5 + 6-5 25-1 + 5-5 22-0+7-8 47-4+8-4 28-2 + 5-2 23-9+4 0 Plasma* 4089+ 234 (7) 6912 + 304 59-5+ 3-9 Concentrations of sheep albumin and total protein in CSF from different compartments and in plasma of 60-day-old (56-61) fetal sheep and adult ewes. Means+S.E.M.; values in parentheses indicate n. The right-hand column shows the proportion of albumin as a percentage of total protein in each compartment at both ages. * Values from Dziegielewska (1982).

and ['4C]inulin were significantly lower in the cerebral hemispheres than in the midand hindbrain regions. This may reflect differences in vascularity (see Discussion). Albumin and total protein in plasma and different CSF compartments in 60-day-old fetal sheep The concentrations of total protein measured with the Bradford (1976) method and of sheep albumin measured by radial immunodiffusion assay (Mancini et al. 1965) in plasma and CSF from the three different compartments studied are shown in Table 2. The total protein concentration was highest in CSF from the cisterna magna (277 + 18 mg (100 ml)-1) and lowest in CSF from the lateral ventricles (131 + 15 mg (100 ml)-'). The cortical subarachnoid CSF total protein concentration was not significantly different from that of the cisterna magna. The albumin concentration was highest in cisternal CSF (136+8 mg (100 ml)-') and lowest in ventricular CSF (38± 2 mg (100 ml)-'). The concentration of albumin in cortical subarachnoid CSF (63±10 mg (100 ml)-') was intermediate and significantly different from that in both

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proteins

Cisterna magna Lateral ventricular Dorsal subarachnoid 157+1-3% 14-5+2-0% 12-7+2-3% [llH]sucrose r = 0-838 r = 0-873 r = 0-722 P = 0-011* P = 0-005*t I' = 0(043 7-6+2-1% 4 6-2+2-0% 70+03% ["4C]inulin r = 0-863 r = 0(921 r = 0770 P = 0*001*t P = 0-006*t P = 0-025* 13 7+0-8% 18 40+0003% 61+1-0% Sheep albumin r = -0-026 r = -0-160 r = -0-114 P = 0-908 P = 0-487 P = 0-067 14 7+0-8% 4 2+0-3% 3 49+04% Goat albumin r = 0-325 r = 0-817 r = 0-912 P = 0 530 P = 0-067 P = 0-012* 7-2+0 7% 2-0+0 2% 5 2-4+0-1 % Bovine albumin r = 0-843 r = 0-829 r = 0 903 P = 0 073 P = 0 083 P = 0-076 3 9+04% 1 0+02% 0-7+0 2 % Human albumin 8 r = 0-858 r = 0-640 r = 0 953 P = 0 006*t P < 0001*t p = 0 008*t 3-9+0-8% I'll-labelled SSA 3 1-2+0-5%Yo 1-7+02% CSF/plasma ratios ( x 100) (means+ S.E.M.) and least squares linear regression correlations between CSF and plasma levels of [3Hisucrose, ["4C]inulin and various species of albumin (sheep, goat, bovine, human) in different CSF regions of the 60-day-old fetal sheep brain. Significance levels of *P < 005 and of tP < 001 are indicated. These results show significant correlations between plasma and CSF concentrations for [3H]sucrose, ['4C]inulin, HSA and dorsal subarachnoid GSA, but not for the other albumins. n 4

the lateral ventricle and the cisterna magna. The proportion that albumin contributed to the total protein concentration varied from about 25% for cortical subarachnoid CSF and 49% for cisternal CSF to 60% for plasma (Table 2). The

BLOOD-CSF TRANSFER OF ALBUTMINS ILNr FETAL SHEEP

223

variation in CSF albumin concentration, particularly in the cisterna magna, was greater and more clearly related to fetal age (Fig. 1). This variation made it important to compare the levels of exogenous albumin with that of endogenous sheep albumin in the same fetus (see below). There was some variation in the plasma albumin concentration (1048+48 mg (100 ml)-', range 700-1420) which was generally but not always related to the age of the fetus. For comparison with the adult, plasma and CSF samples were obtained from the same three sites in some of the ewes. The total protein and albumin concentrations of these samples and the proportion that albumin contributed to total protein are shown in Table 2 (see Discussion for comment).

Albumnin permeability in different CSE compartments The natural steady-state CSF/plasma ratios for sheep albumin at 60 days gestation in each of the three compartments studied are shown in Table 3. The CSF/plasma ratios for total protein in each compartment are also shown. The highest ratio for sheep albumin was in cisternal CSF (13-7+0-8%) and the lowest ratio was in lateral ventricular CSF (40+003 %). Most of the albumin permeability experiments were run for 3 h because previous studies in this preparation had shown that several exogenous proteins including human albumin approached a steady-state level in cisternal CSF by 3-6 h after i.v. injection of the protein solution (Dziegielewska et al. 1980b). HSA and 1251-labelled SSA achieved mean cisternal CSF/plasma ratios of 39%, compared with 7-2 % for BSA, 147 % for GSA and 137 % for the natural steady state for sheep albumin. Similarly for dorsal cortical subarachnoid and lateral ventricular CSF there were clear and statistically significant differences between HSA and BSA, GSA or SSA. The actual ratios for each albumin were lowest in lateral ventricular CSF, highest in cisternal CSF and intermediate in dorsal cortical subarachnoid CSF. Because of the variations in estimates of plasma and CISF endogenous albumin concentration mentioned above, the results were normalized for each fetus, using the sheep albumin concentration as 100 % and expressing the exogenous albumin concentration as a corresponding percentage of the sheep albumin value from the same fetus. The results are shown in Fig. 2. On this basis human albumin reached 41 % of the natural sheep albumin steady-state value in cisternal CSF. 29 % of that in subarachnoid CSF and 26 % of that in ventricular CSF. The comparable values for BSA in each space were all higher: 52, 39 and 52 % respectively (Fig. 2). Bovine albumin was used in these experiments because of its closer immunological and structural relationship to sheep albumin, in the hope that it might act as a marker for sheep albumin. However, as shown by the results in Fig. 2, BSA only reached about 50 % of the SSA ratio at 3 h. Because of its closer immunological relationship to sheep albumin, goat albumin (GSA) was also used. This was possible because of the availability of anti-goat albumin raised in sheep, which did not crossreact with endogenous SSA. GSA reached a cisternal CSF/plasma ratio of 14 7 % at 3 h (Table 3), which was not significantly different from the endogenous SSA ratio. In addition we studied the permeability of radiolabelled sheep albumin. Jodination of sheep albumin proved unsatisfactory (see Table 3 and Methods) therefore it was decided to radiolabel sheep proteins in vitro by incubating fetal liver cells with [35S]methionine (see Methods). The radioactive proteins were infused slowly i.v.

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Fig. 2. CSF/plasma ratios for several different species of albumin (including 35S- and 1251_ labelled sheep albumin) 3 h after i.v. injection, normalized to the naturally occurring sheep albumin ratio. CSF samples were collected from the cisterna magna (open bars) and from within the lateral ventricles (filled bars). Error bars shown are standard errors of the mean. Note that the absolute concentrations of albumins in lateral ventricular CSF are only about 20-30 % of those in cisternal CSF (cf. Table 3). TABLE 4. Penetration of proteins from blood into CSF Cisternal CSF/plasma x 100 (d.p.m. (100 ml)-')

Total protein

Albumin

BSA 35S-labelled SSA 35S-labelled SSA Total 13-0 (n = 2) 14-8+2-1 17-0+2-2 13-6+19 16-2+3-4 Penetration of radioactively labelled proteins from blood into cisternal CSF 3-5 h after i.v. injection into 60-day-old fetuses (n = 4). Total protein in CSF and plasma was estimated by the Bradford (1976) method and albumins by radial immunodiffusion assay (Mancini et al. 1965). Radioactivity of total protein and albumin was estimated as described in Methods. Values given are means+s.E.M.

taking half an hour or more, because of the large volume that had to be injected in order to achieve measurable levels of radioactivity in the CSF of the fetuses. By comparing the CSF/plasma ratios for total protein and total radiolabelled protein, and the ratios for the naturally occurring and radiolabelled individual proteins (separated by immunoprecipitation, see Methods) it was possible to see whether sheep albumin and other in vitro-labelled sheep proteins reached their natural steady states. BSA was included in the injected protein solution for comparison in two of these experiments. The results for cisternal CSF are shown in Table 4. Fig. 3. Consecutive sections from the fore- and midbrain stained for SSA (A) and for HSA (B). Note the strong reaction in meninges (M), the choroid plexus of third ventricle (3V) and the large blood vessels in the subarachnoid space and of fasciculus retroflexus (FR) following staining for endogenous SSA. The visual cortex (VC) is completely unstained. The adjacent section stained for HSA (B) exhibits a marked reactivity in blood vessels and third ventricular choroid plexus, but fibre bundles are unstained. A and B are of same magnification. Bars indicate 500 ,um. This and all following figures were obtained from fetuses injected with human serum albumin and immunostained for either sheep serum albumin (SSA) or human serum albumin (HSA).

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The results show that 35S-labelled SSA approached the natural steady state of unlabelled endogenous SSA by 3-5 hours after i.v. injection. This was also the ease for the 'total proteins' labelled radioactively. In these longer experiments BkSA reached 76% (n = 2) of the SSA steady-state concentration ratio for cisternal CSF in the same samples, which is closer than in the 3 h experiments (ef. Tables 3 and 4). Samples of ventricular CSF were obtained in some of the longer experiments. For total protein, the 35S-labelled protein reached a CSF/plasma ratio of 6-0 %, compared with 6-5 % for the endogenous total protein (n = 2). For albumin 35S-labelled SSA reached 8-7%, for bovine albumin, 6-4% and for endogenous SSA, 6-3% (n = 2). Thus in ventricular CSF both total protein and marker albumins approached their natural steady states even more closely than in cisternal CSF.

Immunocytochemical distribution of endogenous (sheep) albumin and exogenous (humtan) albumin The distribution of sheep albumin in control fetuses was compared with the distribution of human albumin in the experimental group that was injected intravascularly with human serum albumin (e.g. Fig. 3). The antiserum to sheep albumin was pre-absorbed so as not to cross-react with human albumin and the antiserum to human albumin was pre-absorbed so as not to cross-react with sheep albumin. We were unable to detect either sheep or human albumin in the perivascular or extracellular spaces; i.e. there was no sign of a 'leaky' blood-brain barrier to endogenous albumin. WAe did not attempt to quantify the number of albumin-positive cells at different choroid plexus sites because we were mainly concerned with determining whether the route of entry of HSA corresponded to the distribution of endogenous SSA. Some quantitative information is available from an earlier paper (Jacobsen, M0llgard, Reynolds & Saunders, 1983). Even a cursory examination of Figs 3-6 shows that there are clear differences in the proportion of albumin-positive cells in different choroid plexuses that correspond to the differences in albumin permeability in different CSF compartments, as is described in detail below.

Sheep albumin At 60 davs gestation a strong immunoreaction for endogenous albumin was present in CSF, the stroma of the choroid plexuses, plasma and meninges (Fig. 3A). Large fibre bundles particularly in the brain stem also showed some immunoreactivity (Fig. 3A). In contrast very little intracellular staining was observed, in particular in the forebrain, where only scattered positively stained neuroepithelial cells were seen. Stained neuroepithelial cells were more abundant in the wall of the third ventricle (Fig. 5A), where some of the stained cells extended from the ventricular surfaces into the subventricular zone. In the brain stem, positive staining was observed in a proportion of the well-developed neurones of the motor columns (Fig. 6B). In the cerebellum, some neurones of the dentate and deep roof nuclei, some young neurones in the external granular layer and a few Purkinje cells exhibited a positive staining reaction. A strong reactivity was found within epithelial cells of the circumventricular organs, e.g. the area postrema and the median eminence, in which a blood-brain barrier to protein does not exist. A high proportion of the epithelial cells of the

BLOOD-CSF TRANSFER OF ALBUMINS IN FETAL SHEEP

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rostral-most part of the third ventricular choroid plexus (Fig. 5A) showed a strong reactivity whereas only a few cells were positive in the fourth ventricular choroid plexus (Fig. 6A), and even fewer in the choroid plexus of the lateral ventricle (Fig. 4A). In the lateral ventricular plexuses the stained cells were found particularly at

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the root of the plexus. The positive staining for albumin was distributed throughout the cytoplasm from the basolateral to the apical cell membrane (e.g. Fig. 6A).

Human albumin In parallel to the findings of endogenous sheep albumin, positive staining f6r human albumin was present in CSF and the choroid plexus stroma (Fig. 3B). Some of the neuroepithelial cells lining all four cerebral ventricles were positive for human 8-2

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albumin; these are cells which are in direct contact with the positively stained CSF (e.g. Fig. 5B). In the brain stem neither fibre bundles nor neurones of the motor columns were stained (Figs 3B and 6C). The cerebellum was also unstained. In the circumventricular organs, e.g. the median eminence, a positive reaction was observed intracellularly and in the neuropil. In addition to the positive stroma of the

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choroid plexus, a proportion of the epithelial cells were positive in each of the plexuses (lateral and third ventricles, see Figs 4B and 5B; fourth ventricle not illustrated for HSA). Cells were clearly either positive or negative and were often present as a single positive cell within a group of negative cells. A higher proportion of cells was positive for human albumin and staining was generally stronger in the third ventricular choroid plexus than in the lateral and fourth ventricular plexuses. DISCUSSION

The injected volumes of fluid containing different albumins were generally less than 10 % of the estimated total circulating blood volume in these 60-day-old fetuses. There was no evidence of disruption of blood vessels in the histological sections examined for the distribution of injected and endogenous albumin. This is an important point, since many previous studies claiming evidence of barrier immaturity in the fetus and newborn appear to have used very large volumes and/or high concentrations of protein (see Saunders, 1991 for review).

Blood-brain and blood-CSF barriers to radiolabelled sucrose and inulin in different CSF compartments and brain regions The CSF/plasma ratios for [3H]sucrose and ["4C]inulin obtained for cisternal CSF were very similar to the 3 h values reported by Dziegielewska et al. (1979) who showed that a steady state for these markers in both CSF and brain was approached by 4-5 h after the start of an i.v. injection at 60 days gestation. The 3 h ratios for sucrose and inulin were about 70-80 % of the steady-state values for both sucrose and inulin in CSF or brain (cf. Dziegielewska et al. 1979). The penetration of these markers into other CSF compartments has not been previously studied. The results show that the level of penetration into all three compartments is similar although the larger [14C]inulin molecule gave values that were only about 50 % of the [3H]sucrose ratios. In brain tissue there was a similar proportional difference in the penetration of labelled inulin and sucrose. The ratios achieved for whole brain at 3 h were similar to those obtained for the same time by Dziegielewska et al. (1979), i.e. about 70-80 % of the steady-state level (cf. Dziegielewska et al. 1979). However, in the present experiments there was a clear difference in the brain/plasma ratios in different brain regions (see Table 1) which has not been reported previously. The [3H]sucrose ratio was nearly 50 % higher in the hind- and midbrain when compared with the cerebral hemispheres and for ["4C]inulin there was 65 % more in the mid- or hindbrain than in the cerebral hemispheres. This greater penetration is probably due to the greater vascularity of the more developed hind- and midbrain. At 60 days gestation Dziegielewska et al. (1979) estimated that the capillary density of the thalamus was more than twice that of the cerebral hemispheres. A proportional increase in permeability would not necessarily be expected since the vessels in the more mature parts of the brain are probably individually less permeable to small molecular weight compounds than those in the less mature parts of the brain. The similarity of the CSF/plasma ratios for sucrose and for inulin in the different CSF spaces contrasts with the regional differences for each of these markers in brain tissue (Table 1).

BLOOD-CSF TRANSFER OF ALBUMINS IN FETAL SHEEP

231

Total protein and albumin concentrations in different (1,SF compartments It is well established that in the adult the concentration of total protein is different, depending upon where the CSF is sampled from. Davson et al. (1987) cite a number of human studies which gave results in the ranges (mg (100 ml)-1): 15-26 in lateral ventricular CSF, 17-36 in cisternal CSF and 30-56 in lumbar CSF. Differences between different studies were probably due to the methods and standards used. In all studies reported in which samples were obtained from the same individual from different sites and protein concentrations were estimated by the same method, the consistent finding was that ventricular CSF had the lowest concentration and lumbar CSF the highest, with cisternal CSF being intermediate. The present measurements of total protein concentration in adult (ewe) CSF show that in this species the concentration of protein in the lateral ventricle (22 0 + 7 8 mg (100 ml)-') is lower than in the cisterna magna (28-2+5-2 mg (100 ml)-') and the cortical subarachnoid space (485+65 mg (100 ml)-'); the latter value was significantly different from those for the other two compartments. Estimations of protein concentrations in CSF from the subarachnoid space over the dorsal cerebral cortex do not appear to have been obtained previously. The value for the dorsal cortical subarachnoid space is similar to that reported for adult human lumbar CSF (see above). This high value for dorsal cortical subarachnoid CSF seems surprising since it is generally assumed to be in continuity with cisternal CSF. If it is so, then either non-protein fluid is withdrawn or protein is added during the passage of CSF from the cisterna magna to the subarachnoid villi where it returns to the venous side of the circulation (Davson et al. 1987). The results for total protein and albumin concentration in cisternal CSF and plasma and for albumin in lateral ventricular CSF of 60-day-old fetal sheep reported here (Table 2) are similar to those obtained previously (Dziegielewska et al. 1980a; Dziegielewska, 1982; Cavanagh et al. 1983). Results for total protein in lateral ventricular CSF and for total protein and albumin in dorsal cortical subarachnoid CSF during brain development have not been obtained previously, apart from a few 'relative' values given by Bito & Myers (1970) for cortical subarachnoid CSF total protein in fetal monkeys of 50-100 days gestational age. Unlike the adult, the concentration of total protein is not significantly higher in the dorsal cortical subarachnoid CSF than in cisternal CSF (Table 2). The concentration of albumin was nearly 50% lower in the cortical subarachnoid space as compared to the cisterna magna, in spite of the similar concentration of total protein. Crossed immunoelectrophoresis of total CSF protein from both compartments did not reveal any particular protein making up for the difference - rather there was a small relative general increase in concentration of all major proteins other than albumin in CSF from the cortical subarachnoid space. It does seem though that the relatively lower levels of protein in the subarachnoid space may be the result of specific removal of albumin from the CSF fluid between the cisterna magna and the subarachnoid space. Whether this is a specific mechanism designed to recirculate albumin (or substances carried by albumin) or a means of keeping the fetal CSF protein concentration at a stable level remains to be investigated. It would be interesting to speculate that in view of immunocytochemical evidence of the strongest staining for albumin in the

232

K. M. DZIEGIELEWSKA AND OTHERS

third ventricular choroid plexus cells, the CSF ventricular network might act as a 'macro-cell' actively transporting specific proteins in and out of the system. Blood-CSF penetration of albumin in different CSF compartments The CSF/plasma ratios for endogenous SSA and for all of the exogenous albumin species (BSA, GSA, HSA and 125I-labelled SSA) are significantly lower in subarachnoid CSF and within lateral ventricular CSF when compared with cisternal CSF. This is in marked contrast to the penetration of smaller inert markers which was similar in all three compartments (Table 1). This would suggest that the penetration of albumin and that of the inert markers (sucrose and inulin) may be by different mechanisms or at different sites.

Species differences in albumin penetration from blood to CSF These results confirm earlier findings (e.g. Dziegielewska et al. 1980 b) that, at least at 60 days gestation in sheep, the high concentration of proteins in CSF can be accounted for largely, if not entirely, by penetration of albumin and other proteins from blood (Table 4), probably across the choroid plexus (see below). The most striking finding of the present study was the marked species difference in penetration of albumin from blood into CSF. Thus GSA penetrated to the same extent as SSA, but BSA penetrated only to about 50% of the SSA level in 3 h. Human albumin penetrated least, as did sheep albumin that had been iodinated with 1251 (Table 3, Fig. 2). Table 3 also shows the correlation coefficients for the concentration of markers in CSF compared with plasma, for each of the three compartments studied. For [3H]sucrose, ['4C]inulin and human albumin the correlation coefficients were significant (P < 005) or highly significant (P