Int. J. Devl. Neuroscience, Vol. 1, No. 6, pp. 369-373, 1983.

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T R A N S F E R R I N IN F E T A L R A T B R A I N AND C E R E B R O S P I N A L FLUID H. NEW,* K. M. DZIEGIELEWSKA*and N. R. SAUNDERS*t *Department of Physiologyand Centre for Neuroscience, UniversityCollege London, Gower Street, London WC1E 6BT, U.K.

Abstract--Concentrations of transferrin in plasma, cerebrospinal fluid (csf) and brain of fetal and newborn rats have been estimated by radial immunodiffusion assay. Transferrin was detected in both csf and brain at the earliest age investigated (12 days gestation). The plasma concentration increased throughout the gestational period studied and in the postnatal period: the overall increase was about 10-fold. In contrast, although the csf transferrin concentration increased by 3 times between 12 and 22 days (term) there was a considerable decline on reaching adult age. Brain transferrin concentration was highest at 18 days gestation. These results are important background information for studies of the significance of transferrin in brain development. Key words: Transferrin, Fetal rat, Csf.

Cerebrospinal fluid in the fetus contains a high concentration of protein. Several studies have shown that a small number of plasma proteins including transferrin make important quantitative contributions to this high concentration (see ref. 8 for references). Transferrin is also one of several proteins that have been demonstrated to be present within neurons of the immature brain at certain stages of development (see ref. 11 for references). Although transferrin has previously been identified in fetal rat csf 2° and is known to be present in fetal mouse brain, 22 no quantitative values for transferrin in rat csf and brain are available. It seems likely that transferrin may be of particular importance in brain development since it has recently been shown by several laboratories to be an essential constituent of artificial media for the culture of neurons in vitro. 6'7 This study was undertaken to obtain information about the levels of transferrin in rat fetal brain, csf and plasma as a preliminary to investigating the possible role of transferrin in brain development. METHODS Samples of csf and plasma were obtained from fetuses of time-mated Sprague-Dawley rats anaesthetized with sodium pentobarbitone (6 mg/100 g body wt) given i.p. Two- and ten-day-old litters of postnatal rats were also used (anaesthetized with ether). Clear csf, free of fetal blood contamination was obtained by cisternal puncture as described previously. 12 Blood was collected from an umbilical artery or by heart puncture into heparinized syringes and then centrifuged. The csf and plasma samples were stored at - 18°C. Estimations of a-fetoprotein, albumin, total protein t2 and acute phase a2-macroglobulin t9 on the same samples have already been published. Transferrin concentrations were established by radial immunodiffusion assay.17 A transferrin standard was obtained from Cappel Laboratories; it gave a single precipitation arc in crossed immunoelectrophoresis t6 against either anti-whole fetal or adult rat serum. The same standard was injected with Freunds incomplete adjuvant serially into New Zealand white rabbits. 15The resulting antiserum gave a single peak when run in crossed immunoelectrophoresis against fetal or adult rat plasma. This antiserum was used for the radial immunodiffusion assays. In a separate series of rat fetuses (12, 18, 21, 22 days) the brains were dissected free of pia-arachnoid and choroid plexuses were removed. This was part of a study of protein synthesis by fetal rat tissues that will be reported separately, t4 Samples of forebrain and midbrain were either extracted immediately (see below) or incubated for 1 h in Krebs solution (NaCI, 131 mM; KC1, 6 mM; MgSO4, 1.2 mM, NaH2PO4, 1.2 mM; CaC12, 2.5 mM; NaHCO3, 23 mM; glucose g/1 equilibrated t Author to whom correspondence should be addressed.

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with 5% COx in 02) containing [3H]leucine (Amersham, T R K 510) at 37°C. Incubation was stopped by placing the tubes on ice. The incubation medium was removed rapidly and transferred to tubes containing Triton X100 to give a final concentration of 2%. Triton X100 was also added to the tissue together with 200 ~1 Trasylol. 5 The samples were then homogenized using an all glass microhomogenizer and left overnight at 4°C. After centrifugation the supernatant was collected and assayed for its transferrin content: the transferrin content of the incubation medium was also estimated.

RESULTS

Csf and plasma The concentrations of transferrin in csf and plasma between fetal day 12 and postnatal day 10 and in the adult are shown in Table 1. The csf concentration at 12 days gestation was 19.5 + 2 . 2 mg/100 ml (13.5% of total protein in csf, estimated from the total protein data of Dziegielewska et al.) 12 It increased significantly by 17 days gestation to 40.3 -+ 4.7 mg/100 ml ( 14.5% total protein). The concentration of transferrin in csf remained at about this level from 17 days until the early postnatal period. A significant decline was detected by 10 days of age when it was 19 _+ 1.7 rag/100 ml. The adult value was substantially less at about 2 mg/100 ml. Table 1. Concentrationsof transferrin (mg/100ml) in plasma and csf of fetal and neonatal rats. Age in days from conception or from day of birth. Mean _+S.E.M. Csffpl= csf concentration:plasma concentration x 100; the ratio was calculated from means because paired samples of csf and plasma were not available at all ages. The right-hand columns show transferrin concentrations in csf and plasma expressed as percentage of total protein concentration in these fluids (values from 12) Csf:PI Plasmatransferrin Csftransferrin (usingmean Age (days) (rag/100ml) (mg/100ml) at each age) 12-13 15 17 19 20-21 22 Newborn +2 +10 Adult

--130 _+30 (2) 384 +22.1(10) 359.3_+ 9 (14) 542 _+57 (5) 639 _+39 (15) 636 -+43 (18) 1093 -+52 (22) 1288 -+84 (8)

19.5_ + 2.2(4) 21.8_+ 2.5(6) 40.3_+ 4.7(6) 47.5_+ 2.0(14) 39.6_+ 2.3(16) 60.6_+17.5(6) 36.3_+ 1.4(13) 37.9-+ 4.8(10) 19.0-+ 1.7(12) 2.4 (2)

--31.0 12.4 11.0 10.4 5.7 6.0 1.7 0.1

Transferrin concentrationas % of total protein Csf

Plasma

13.5 12.7 14.5 16.7 14.7 17.9 11.7 16.8 18.4 9.8

--8.2 19.4 18.4 211.3 22.2 20.2 27.4 ---

In plasma the transferrin concentration was 130 + 30 mg/100 ml (8% total plasma protein) at 17 days gestation, which was the earliest age at which satisfactory blood samples could be obtained. The concentration increased about 3-fold by 19 days and showed a further significant increase by 22 days gestation. There was a nearly 2-fold increase between 2 and 10 days postnatal and a further increase in the adult rats. Between 19 days gestation and 2 days postnatal transferrin constituted about 20% of the total plasma protein concentration. The relation between csf and plasma concentration is also shown (as csf: plasma × 100) in Table 1. There was a steady decline in csf: plasma ratio throughout the development period studied, from 31% at 17 days gestation to 1.7% at 10 days postnatal. As the csf transferrin concentration was approximately stable during the period 17 days gestation to 2 days postnatal, this decline in csf:plasma ratio was clearly due to the marked increase in plasma transferrin concentration that occurred during this period.

Tissue transferrin concentrations Most of the values for brain content of transferrin were obtained from experiments in which brain samples were incubated for 1 h and then extracted. In a control experiment using paired brain

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samples from 21 day fetuses, there was no significant difference in the transferrin content of samples extracted immediately compared with the transferrin content of samples extracted after 1 h of incubation (adding the contents of the incubation medium and tissue residue). The concentrations of transferrin in forebrain and midbrain (mg/100 g wet tissue weight) between 12 days gestation and term (22 days) are shown in Table 2. At 12 days gestation the concentrations of transferrin in liver and brain were not significantly different. By 18 days the concentration in both tissues had increased to the highest concentration estimated (forebrain, 72---23 mg/ 100 g; liver, 85 --- 23 mg/100 g) but there was still no significant difference between brain and liver. Subsequently, the liver showed no significant change up to 22 days gestation, but the level in the brain had decreased significantly to about 15% of the peak level by this age. Table 2. Transferrin content (mg/100g wet wt) of fetal rat brain and liver. Age in days from conception, n--number of samples; mean -+S.E.M. Age (days)

n

Forebrain

Midbrain

Liver

12 18 21 22

6 6 16 10

25.9-+15.7 16.2-+ 2.9 15.9-+ 6.8 72.0 -+23.0 55.0 +-45.0 85.0 -+23,0 16.1-+ 3.3 25.2-+11.6 59.2--- 7,6 11.3-+ 3.0 11.2 + - 4.0 85.0-+20,6

DISCUSSION Transferrin concentrations in plasma during fetal and postnatal development have been estimated in several species: rat, 24 cattle, 1 human, 2 sheep 13 and pig. 8 However, the patterns of change and the absolute level in the fetal period compared with the adult concentration appear to be different in different species. In the rat, Yeoh & Morgan 24 reported an increase in fetal transferrin concentration from about 280 mg/100 ml at 19 days gestation up to about 450 mg/100 ml at term. These are similar to the values obtained for the same ages in this study (see Table 1). However, in the postnatal period Yeoh & Morgan found a decline to about 300 mg/100 ml followed by an increase at 15 days postnatal and a further increase in the adult; whereas in our study there was little change in the early postnatal period followed by a considerable rise at 10 days and a further increase in the adult to 1288 --- 84 mg/100 ml, a value which is two and a half times that found by Yeoh & Morgan. It is possible that the difference in postnatal and adult transferrin concentrations in the two studies was due to differences in breed of rats or in their diet. Pantelouris & Hale 2° have reported a large increase in transferrin between the fetal and adult stages in the mouse. Transferrin appears to make a significant quantitative contribution to the high concentration of total protein found in the fetal csf of all species so far investigated. 2,8,13 In the rat the contribution of transferrin to total protein concentration in csf during development varied between 12 and 18% without any particular age related trend being apparent (from comparison of data in Table 1 with csf total protein concentration of Dziegielewska et al.) ;12 in the adult the contribution of transferrin to total protein in csf was about 10%. The pattern of change in transferrin concentration in fetal rat csf at different ages is similar to that previously described for albumin 12 and et2-macroglobulin. 19The concentrations of all 3 proteins increased progressively after 15 days gestation to reach a peak on the day of birth. AFP reached its peak concentration in csf at 17-19 days ~5 and had declined to less than half the peak by the newborn period. All 4 proteins show a considerable decline in their concentrations between 2 and 10 days postnatal. The differences in changes of csf concentration with fetal age do not seem to be related to changes in plasma concentration of these proteins. Although there is a general tendency for the csf: plasma ratio to fall with increasing fetal age the pattern of fall was different for each protein (cf. Table 1, Panrucker et al. 19 and Table 2). For example, the transferrin csf:plasma ratio decreased from 10.4 to 5.7% between 22 days and the newborn whereas that of albumin did not change; ~2etamacroglobulin increased significantly and a-fetoprotein decreased, but only slightly, during the same period. Such changes do not appear consistent with passive diffusion of proteins from plasma to

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csf in the fetus a n d d o not s u p p o r t the f r e q u e n t a s s e r t i o n that the b l o o d - b r a i n b a r r i e r in the fetus a n d n e w b o r n is i m m a t u r e . 21 N e v e r t h e l e s s t h e r e is e v i d e n c e in b o t h n e w b o r n rats 4 a n d fetal s h e e p Za for t r a n s f e r o f p l a s m a p r o t e i n s f r o m b l o o d to csf. H o w e v e r , in the e a r l y stages of d e v e l o p m e n t in fetal s h e e p , p e n e t r a t i o n of l a b e l l e d p l a s m a p r o t e i n s f r o m b l o o d was insufficient to a c c o u n t for the n a t u r a l s t e a d y state o f p l a s m a p r o t e i n in fetal csf at 30-40 d a y s g e s t a t i o n . 9 It is p o s s i b l e t h a t transferrin a n d o t h e r p l a s m a p r o t e i n s in c h o r o i d plexus a n d b r a i n at s o m e stages of d e v e l o p m e n t m a y c o n t r i b u t e to the high c o n c e n t r a t i o n of p r o t e i n in fetal csf. T h e m a r k e d d e c l i n e in csf p r o t e i n conc e n t r a t i o n in the n e w b o r n p e r i o d is p r o b a b l y a s s o c i a t e d with the i n c r e a s i n g flow of csf t h r o u g h the v e n t r i c u l a r system which occurs at this age. 23 T h e d i f f e r e n c e s b e t w e e n i n d i v i d u a l p r o t e i n s in t h e i r p a t t e r n s of c o n c e n t r a t i o n c h a n g e s with age a r e likely to be d u e to d i f f e r e n c e s in t r a n s f e r from b l o o d t° a n d c o n t r i b u t i o n s from b r a i n or c h o r o i d plexus; Ali etal. ~ have r e c e n t l y p u b l i s h e d results of c u l t u r e of rat b r a i n cells that suggest that n e w b o r n rat b r a i n is a b l e to s y n t h e s i z e a n d s e c r e t e p l a s m a p r o t e i n s (c~-fetoprotein a n d a l b u m i n ) . T h e p r e s e n c e o f t r a n s f e r r i n in h o m o g e n a t e s o f fetal b r a i n s u p p o r t s p r e v i o u s i m m u n o h i s t o c h e m i c a l o b s e r v a t i o n s o f the p r e s e n c e of t r a n s f e r r i n within the d e v e l o p i n g b r a i n in s e v e r a l species (mouse22 s h e e p ) . ~ P a r t of the t r a n s f e r r i n e x t r a c t e d c o m e s f r o m u n a v o i d a b l e b l o o d c o n t a m i n a t i o n but this is likely to have b e e n < 1-2"/o of the b l o o d level. ~1 T h u s it can be e s t i m a t e d f r o m the d a t a of T a b l e 2 t h a t b l o o d c o n t a m i n a t i o n w o u l d only have c o n t r i b u t e d a small fraction of that e x t r a c t e d from 12 a n d 18 d a y brains. H o w e v e r , in b r a i n s f r o m fetuses a r o u n d t e r m , it c o u l d have c o n t r i b u t e d s u b s t a n t i a l l y to the e s t i m a t e s of e x t r a c t a b l e t r a n s f e r r i n . A s o u r c e of c o n t a m i n a t i o n by s o m e p l a s m a p r o t e i n s , at 12 d a y s g e s t a t i o n , was from m e s e n c h y m a l tissue p r e s e n t o v e r the surface of the brain. H o w e v e r , this stains only slightly for t r a n s f e r r i n by i m m u n o c y t o c h e m i s t r y ( C o r n e l i s , p e r s o n a l c o m m u n i c a t i o n ) . A t 12 days it p r o v e d difficult to r e m o v e this surface m e m b r a n e so t h e r e m a y have b e e n s o m e c o n t a m i n a t i o n of b r a i n by t r a n s f e r r i n f r o m m e s e n c h y m e ; this was not a p r o b l e m in o l d e r fetuses as t h e m e s e n c h y m e was m o r e clearly d i f f e r e n t i a t e d into p i a - a r a c h n o i d a n d c o u l d easily be removed. S t u d i e s o f the i m m u n o c y t o c h e m i c a l d i s t r i b u t i o n of t r a n s f e r r i n a n d o t h e r p l a s m a p r o t e i n s in the fetal rat b r a i n are in p r o g r e s s . In fetal s h e e p at 35 a n d 40 d a y s g e s t a t i o n w h e n the cortical p l a t e is at an e a r l y stage of f o r m a t i o n it has b e e n f o u n d ~4 that t r a n s f e r r i n is d i s t r i b u t e d on e i t h e r side of the c e l l u l a r layer of the p r i m i t i v e cortical p l a t e in r e g i o n s w h e r e s y n a p t o g e n e s i s is k n o w n to be occurring. ~s Such an a s s o c i a t i o n m a y be r e l a t e d to r e c e n t findings in tissue c u l t u r e studies that t r a n s f e r r i n is an essential i n g r e d i e n t of d e f i n e d m e d i a for n e u r o n cultures. 6'7 T h e f u n c t i o n a l role of t r a n s f e r r i n in n e u r o n d e v e l o p m e n t w h e t h e r in vitro o r in v i v o is not clear. It is not k n o w n if it is r e l a t e d to its iron b i n d i n g a n d iron t r a n s p o r t p r o p e r t i e s o r to s o m e o t h e r p r o p e r t y .

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