Acta physiol. scand. 1976. 96. 399-406 From the Institute of Medical Physiology, Dept. A University of Copenhagen, Denmark
Transfer of 1125-Albumin from Blood into Brain and Cerebrospinal Fluid in Newborn and Juvenile Rats BY
OLEAMTORP Received 13 August 1975
Abstract AMTORP,0. Transfer of 1126-albumin from blood into brain and cerebrospinalfluid in newborn and juvenile rats. Acta physiol. scand. 1976. 96. 399406. Human fetuses, rabbit fetuses and newborn rats have a relatively high concentration of protein in cerebrospinal fluid (csf) as compared with the adult. The present study was undertaken in an attempt to evaluate whether this high protein concentration in newborn rats is caused by a high permeability of the blood-braincsf barrier to protein or by a low production rate of csf. The results suggest that the high concentration of protein in csf in newborn rats is due to a low rate of production of csf rather than to an increased transfer of protein across the blood-brain-csf barrier.
Key words: blood-brain barrier, macromolecules, ontogenesis of the blood-brain barrier.
Since Ehrlich in 1885 reported that aniline dyes when injected into the blood stained all tissues except the central nervous system, the concept of a blood-brain barrier has been established. Subsequent chemical and radioactive estimations by many workers demonstrated restraints in the rate of entry of many low molecular weight substances into brain and csf from blood (Davson 1967). The morphological basis for this barrier is probably the tight junctions between cerebral endothelial cells and between the epithelial cells in the choroid plexus (Reese and Karnovsky 1967, Brightman and Reese 1969). Previous data have indicated that the transfer of various hydrophilic solutes into the brain is less restricted in the immature brain as compared with the mature brain (Davson 1967, Ferguson and Woodbury 1969, Evans et al. 1974). Klosowskii (1963) reported the findings of Purtn that in human fetuses 8 weeks of gestational age the protein concentration in csf was some twenty times higher than that of the adult. In a recent study (Amtorp and Ssrensen 1974) we have found that newborn rats have a high concentration of protein in csf. The high concentration of protein in csf might reflect either a high rate of entry of protein into the brain as compared with the adult or a low rate of production of csf. 399
400
OLE AMTORP
The present study was undertaken in an attempt to evaluate which of the two explanations might be the correct. This was done by measuring the transport of IlZ6-albuminfrom blood into brain interstitial fluid and csf in newborn and juvenile rats.
Methods Newborn and juvenile Sprague-Dawley rats were used. The uptake of 11z6-albumin in brain and csf in rats, newborn, 5 days old and 30 days old was studied following a single intraperitoneal injection of the tracer (0.1-0.2pCi). At either 2, 4, 6, 16 or 24 h after injection of the tracer the animalswereanesthetized with pentobarbital (60 mg/kg) and samples of csf and blood were collected with a glass capillary introduced into the cisterna magna and by heart puncture. The rats were decapitated and exsanguinated to minimize the vascular blood content of the brain. Afterwards the brain was removed and weighed samples were counted in a y-spectrometer (Packard Instruments). Human I1as-albumin was obtained from Studsvik, Sweden. The relative concentration of free I l z 6 in the batches was determined following separation of the albumin and the free by paper electrophoresis, and subsequent counting of cut-out pieces of the paper. Approximately 15" free Itz6 was present in the different batches. Since the injectate contained about I % free 1Iz6 it must be considered to which extent the results have Although the amount of free been influenced by the uptake of free in the injectate was about 1 OU the concentration of free in the blood will decrease rapidly after injection due to uptake of the trace by the thyroid gland. The view that the results refer to uptake of 11z6-albuminby the amounts of free brain and not to the uptake of free 1lZ6 is further supported by the fact that iodide is actively transported out from the central nervous system across the choroid plexus even in immature animals (Robinson cf af. 1968). Samples of csf which were visibly contaminated with blood were discarded. This would ensure a blood contamination of less than 0.1 %.The small amount of residual blood in the brain was considered to be of no importance in this series of experiments. The results were expressed as (cpm/g brain/cpm/g blood).
Calculations Assuming that the transfer of 11z3-albuminfrom blood into brain follows a simple exponential function, i.c. the amount transferred is proportional to the difference between plasma concentration and brain concentration, then the following equation describes the brain concentration of 1lZ6-albumin C,(t)
=
C,(I -e-kt)
(1)
where C,(t) is the concentration of 112K-albuminin brain, C, the concentration of 11z6-albuminat steady state and k is the rate constant of the transfer process. The values of C,(t) are obtained experimentally by determining the radio-activity in brain tissue. Cm is determined graphically from a plot of C,(t) vs time. k is then obtained by logarithmic transformation of eq. (I), i.e. In ((2,- C,(t))= In (C,- kst. As seen in Fig. 4 the results plotted in this way fitted closely to a straight line and k can simply be obtained by measuring tt. k 0.6931t). ~
Results The concentration of 11z6-albuminin blood varied less than 10% between 2 and 24 h after intraperitoneal injection of the tracer. The concentration of 11z3-albuminin the brain was always less than 10% of that in the blood. Whole brainlblood ratios
Fig. 1 shows the concentration of 11z6-albuminin brain relative to blood at various times after injection of Itz6-albumin in newborn rats, 5-day-old rats and 30-day-old rats. As can
401
ALBUMIN TRANSFER INTO BRAIN
I
c m l brain x 100 c p i l g blood
7 6 -
Fig. 1. The concentration of Ileaalbumin in brain in newborn rats, 5-day-old rats, and 30-day-old rats expressed relative to the concentration in whole blood. All curves are fitted by eye. The bars indicate+ - S.E.
I
,
,
2
4
6
I
16 Time (hrs)
I
24
be seen there was a decrease in the relative concentration of 11z6-albuminin whole brain with increasing age.
Csflblood ratios Fig. 2 shows the concentration of 11z6-albuminin csf relative to blood at various times after injection of the tracer. As can be seen there was a decrease in the relative concentration of 1126-albuminin csf with increasing age.
Csflbrain interstitialfluid ratios Since Ilz6-albumin is located extracellularly its concentration in brain interstitial fluid (CIsF)may be calculated from the concentration in whole brain. Cbr*lIl
""=%
ISF in brain tissue
x
100
Since the calculations were made without corrections for the amount of IlZ6-albuminin the residual blood in brain tissue the calculated CIsF overestimates the real concentration in cpmlg cst x 100 c p m l g blood newborn
Fig. 2. The concentration of P6albumin in csf in newborn rats, 5-day-old rats, and 30-day-old rats expressed relative to the concentration in whole blood. All curves are fitted by eye. The bars indicatekS.E.
5 days old
30 days old 2
4
6
16 lime Ihrs)
24
402
OLE AMTORP
c p m l g x 100 cpmlg blood u 16 -
s
12'-
7
6
'I
nrwborn 8
8 /cst
5911YUlM 8
5
7
0
the interstitial fluid somewhat but the calculated values may be used to indicate the different patterns in rats of different ages. CI,, in newborn and juvenile rats was calculated using the values for extracellular space reported by Ferguson and Woodbury (1969): 45 % in newborn rats, 40% in rats 5 days of age and 15% in rats 30 days of age. As can be seen in Fig. 3 the relation between the concentration of I1"-albumin in csf relative to blood and the concentration of IIPs-albumin in brain interstitial fluid relative to blood was different in the various groups of rats which have been studied. In newborn rats the relative concentration of 11z6-albuminin csf and in brain interstitial fluid approaches unity with time. At 5 days of age this pattern changes as the concentration in csf relative to that in brain interstitial fluid decreases. This trend is even more conspicuous in rats 30 days
403
ALBUMIN TRANSFER INTO BRAIN
t
cpm l g brain I 100 cpmlg blood
--
Newborn
Fig. 4. Semilogarithmic diagram of the timeuptake curves into brain of 1126-albuminin newborn rats, 5-day-old rats and 30-day-old rats. t i are indicated on the fitted straight lines. The curves are fitted by eye. (For further explanations: see text.)
e
I
16
I
u
Time I h l
of age. Fig. 4 shows a semilogarithmic diagram of the curves of uptake into brain of ILesalbumin in newborn rats 5-day-old rats and 30-day-old rats. t# in hours as found from the fitted straight lines was 4.8, 5.7 and 3.8 respectively. The calculated k was 0.002 min-’ in newborn rats, 0.002 min-’ in 5-day-old rats and 0.003 min-l in 30-day-old rats.
Discussion A substance in the blood passes into csf across the blood-csf barrier by way of the choroid plexus or across the blood-brain barrier and further by diffusion from the cerebral capillaries by way of the brain interstitial fluid. Csf acts as a “sink” for substances entering the brain interstitial fluid from brain capillaries across the blood brain barrier because of its bulk flow through the ventricles and subarachnoid cavities and its unselective exit through the arachnoid villi.
404
OLE AMTORP
The concentration of a substance in csf and in brain is determined by its volume of distribution, by the differences between its rate of entrance into brain and csf from plasma across the blood-brain-csf barrier and its rate of exit from csf by bulk flow through the arachnoid villi, the latter being determined by the rate of production of csf. Decreasing extracellular space, barrier formation between blood and brain and between blood and csf and a larger bulk flow of csf due to an increased production all contribute to decreasing the concentration of a particular substance in brain and csf and to lowering the brain/plasma and csf/plasma ratios during the ontogenetic development. Three factors have to be taken into account when considering the conclusion from recent studies that immature animals have a relatively high concentration of protein in csf as compared with mature animals: Immature animals have a larger brain extracellular space as compared with the adult. In immature animals the transfer of a solute between blood and brain and between blood and csf might be less restricted as compared with mature animals. Further, in immature animals the bulk flow of csf through the arachnoid villi probably is sluggish whilst it progressively increases as the choroid plexus matures. Fig. 1 and Fig. 2 show the uptake data for 1126-albuminin brain and csf obtained simultaneously in newborn rats, 5-day-old rats and 30-day-old rats. It is evident from the figures that the concentration of IIPs-albumin in brain and csf was higher in newborn rats than in juvenile rats and that the concentration decreases with increasing age. On the basis of the brain/csf ratio for inulin, Ferguson and Woodbury (1969) calculated the extracellular space in pre- and postnatal rats. They found that the extracellular space decreases until 16 days of age but in older animals the extracellular space as calculated on the basis of brain/csf ratio apparently increases again. This is an unexpected finding and in disagreement with the finding of an inulin space of 13.5% in adult rats (Woodward et a/. 1967). Ferguson and Woodbury (1969) apparently also disregard the unexpected increase in the extracellular space and rather accept that the extracellular space is continuously decreasing from 50% in 4 days prenatal rats until it reaches a final value of 13.50/o.The larger extracellular space in the brain of the newborn rats may contribute to a larger accumulation of 1125-albuminin the present experiments. In the present study there was found no difference in the rate of transfer of 1125-albumin from blood into brain tissue in newborn rats as compared with 5-day-old rats and 30-dayold rats. However, the permeability of the cerebral capillaries can be larger in newborn rats when considered the results of Bar and Wolff (1973) on the vascularization of the rat cerebral cortex. They found that the postnatal development of capillary length and number of branchings continuously increased from birth until 30 days of age. The permeability of cerebral capillaries can for this reason be greater than the rate constant of the transfer process for 112s-albuminfrom blood into brain tissue suggests in newborn rats and 5-day-old rats. From a morphological point of view there is no evidence for maturation of the barrier mechanisms for large molecules between blood and brain and between blood and csf with increasing age. The barrier mechanisms show a considerable maturity of function very early in the development. Olsson et al. (1968) could not detect intravascularly administered fluorescein albumin outside the vessels in the brain of neither prenatal, newborn or postnatal rats. This is in agreement with the morphological evidence that the endothelial cells in capillaries
ALBUMIN TRANSFER INTO BRAIN
405
in prenatal rat cerebral cortex are connected by tight junctions (Carley and Maxwell 1970). Thus it is possible that an increased permeability of cerebral capillaries in newborn rats reflects a greater transcapillary pinocytotic transport of macromolecules in immature animals compared with mature animals. In the choroid plexus in adult rats Becker et al. (1967) found a rapid uptake of the protein tracer horseradish peroxidase from the vascular compartment. The injected protein passed out of the fenestrated capillaries and was rapidly distributed throughout the extracellular space. The tracer then entered the choroidal epithelial cells via pinocytotic vesicles and migrated as far as to the ventricular surface. The tracer also moved between the epithelial cells of the choroid plexus until stopped by tight junctions near the ventricular surface. In developing rat choroid plexus Morecki et al. (1969) could identify horseradish peroxidase when administered intravenously in the vascular compartment and in the extracellular space, but not in the choroid epithelium prior to 4th to 6th days postpartum. The conclusion that newborn rats produce little csf is based on two pieces of evidence. Fig. 3 shows that the relative concentration of ILaS-albuminin csf approaches the calculated relative concentration of 11e6-albuminin brain interstitial fluid. During development this pattern changes. The concentration of 1126-albuminin csf relative to that in brain interstitial fluid decreases and the concentration of 1126-albuminin csf remains below that of brain interstitial fluid indicating an increased “sink action” of csf due to an increased production rate during development. If the high extra-cellular space as suggested by Ferguson and Woodbury (1969) in newborn rats largely overestimates the real space, the validity of the present conclusion that newborn rats produce little csf can be spurious. The concentration of albumin in a smaller extracellular space will exceed the concentration of the isotope in the csf. If the estimates of a large extracellular space is fairly correct the accumulation of P6albumin in the csf space can hardly be explained other than by a decreased exit through the arachnoid villi. In a previous study (Amtorp and Snrrensen 1974) it was found that administration of Acetazolamide did not increase the concentration of protein in cisternal csf in newborn rats whereas it did so in older animals, another suggestion that the newborn rats produced little if any csf. This conclusion is further supported by the fact that csf production in 5-day-old rats is only 40% of that in 30-day-old rats (Bass and Lundborg 1973). By exclusion then it seems that the relatively high protein concentration in csf in immature rats could be explained by a smaller bulk flow of csf through the arachnoid villi due to a lower production rate of csf as compared with mature rats. This work was supported in part by the Warwar-Larsen Foundation, Denmark.
References AMTORP, 0.and s. c. S~RENSEN, The ontogenetic development of concentration differences for protein and ions between plasma and cerebrospinal fluid in rabbits and rats. J . Physiol. (Lond.) 1974. 243. 387-400. BAR, T. and J. R. WOLFP,On the vascularization of the rat’s cerebral cortex. Bibl. anar. (Basel) 1973. If. 515-519. BASS, N. H. and P. LUNDBORG, Postnatal development of bulk flow in the cerebrospinal fluid system of
406
OLE AMTORP
the albino rat: Clearance of carboxyl-(14C) inulin after intrathecal infusion. Brain Res. 1973.52. 323332.
BECKER,N. H., A. B. NOVIKOFF and H. M. ZIMMERMAN, Fine structure observations of the uptake of intravenously injected peroxidase by the rat choroid plexus. J. Histochem. Cytochem. 1967. 15. 160-165. BRIGHTMAN, H.W. and T. S. REESE,Junctions between intimately apposed cell membranes in the vertebrate brain. J. cell. Biol. 1969. 40. 648-677. CARLEY, W. D. and D. S. MAXWELL, Development of the blood vessels and extracellulai spaces during postnatal maturation of rat cerebral cortex. J. Comp. Neurol. 1970. 138. 3 1-48. DAVSON,H., The normal protein concentration. In: Physiology of the Cerebrospinal Fluid 1970 pp. 272273. London. Little, Brown & Company. EHRLICH,P., Das Sauersroff-Bediirfnis des Organismus. Eine farbenanalytische Studie. Berlin 1885. EVANS,C. A. N., J. M. REYNOLDS, M. L. REYNOLDS, N. R. SAUNDERS and M. B. SEGAL,The development of a blood-brain barrier mechanism in foetal sheep. J. Physiol (Lond.) 1974. 238. 371-386. FERGUSON, R. K. and WOODBURY, Penetration of 14C inulin and 14C sucrose into brain, cerebrospinal fluid and skeletal muscle in developing rats. Exp. Brain Res. 1969. 7. 181-194. KLOSOWSKII, B. N., The development of the brain and its disturbance by harmful factors. 1963 p. 8 . Oxford. Pergamon. and N. H.BECKER, Transport of peroxidase by the developing rat choroid MORECKI, R.. H. M. ZIMMERMAN plexus. Acra neuropath. (Berl.) 1969. 14. 14-18. OLSSON,Y.,I. KLATZO,P. SOURANDER and 0. STEINWALL, Blood-brain barrier to albumin in embryonic, newborn and adult rats. Acta neuropath. (Bed.) 1968. 10. 117-122. REESE,T. S. and M. J. KARNOWSKY, Fine structural localization of a blood-brain barrier to exogenous peroxydase. J. cell. Biol. 1967. 34. 207-217. ROBINSON, R. J., R. W. P. CUTLER,A. V.LORENZO and C. F. BARLOW, Development of transport mechanisms for sulphate and iodide in immature choroid plexus. J. Neurochem. 1968. 15. 455-458. WOODWARD, D. L., D. J. REEDand D. M. WOODBURY, The extracellular space of rat cerebral cortex. Amer. J . Physiol. 1961. 212. 367-370.
Transfer of 1125-Albumin from Blood into Brain and Cerebrospinal Fluid in Newborn and Juvenile Rats BY
OLEAMTORP Received 13 August 1975
Abstract AMTORP,0. Transfer of 1126-albumin from blood into brain and cerebrospinalfluid in newborn and juvenile rats. Acta physiol. scand. 1976. 96. 399406. Human fetuses, rabbit fetuses and newborn rats have a relatively high concentration of protein in cerebrospinal fluid (csf) as compared with the adult. The present study was undertaken in an attempt to evaluate whether this high protein concentration in newborn rats is caused by a high permeability of the blood-braincsf barrier to protein or by a low production rate of csf. The results suggest that the high concentration of protein in csf in newborn rats is due to a low rate of production of csf rather than to an increased transfer of protein across the blood-brain-csf barrier.
Key words: blood-brain barrier, macromolecules, ontogenesis of the blood-brain barrier.
Since Ehrlich in 1885 reported that aniline dyes when injected into the blood stained all tissues except the central nervous system, the concept of a blood-brain barrier has been established. Subsequent chemical and radioactive estimations by many workers demonstrated restraints in the rate of entry of many low molecular weight substances into brain and csf from blood (Davson 1967). The morphological basis for this barrier is probably the tight junctions between cerebral endothelial cells and between the epithelial cells in the choroid plexus (Reese and Karnovsky 1967, Brightman and Reese 1969). Previous data have indicated that the transfer of various hydrophilic solutes into the brain is less restricted in the immature brain as compared with the mature brain (Davson 1967, Ferguson and Woodbury 1969, Evans et al. 1974). Klosowskii (1963) reported the findings of Purtn that in human fetuses 8 weeks of gestational age the protein concentration in csf was some twenty times higher than that of the adult. In a recent study (Amtorp and Ssrensen 1974) we have found that newborn rats have a high concentration of protein in csf. The high concentration of protein in csf might reflect either a high rate of entry of protein into the brain as compared with the adult or a low rate of production of csf. 399
400
OLE AMTORP
The present study was undertaken in an attempt to evaluate which of the two explanations might be the correct. This was done by measuring the transport of IlZ6-albuminfrom blood into brain interstitial fluid and csf in newborn and juvenile rats.
Methods Newborn and juvenile Sprague-Dawley rats were used. The uptake of 11z6-albumin in brain and csf in rats, newborn, 5 days old and 30 days old was studied following a single intraperitoneal injection of the tracer (0.1-0.2pCi). At either 2, 4, 6, 16 or 24 h after injection of the tracer the animalswereanesthetized with pentobarbital (60 mg/kg) and samples of csf and blood were collected with a glass capillary introduced into the cisterna magna and by heart puncture. The rats were decapitated and exsanguinated to minimize the vascular blood content of the brain. Afterwards the brain was removed and weighed samples were counted in a y-spectrometer (Packard Instruments). Human I1as-albumin was obtained from Studsvik, Sweden. The relative concentration of free I l z 6 in the batches was determined following separation of the albumin and the free by paper electrophoresis, and subsequent counting of cut-out pieces of the paper. Approximately 15" free Itz6 was present in the different batches. Since the injectate contained about I % free 1Iz6 it must be considered to which extent the results have Although the amount of free been influenced by the uptake of free in the injectate was about 1 OU the concentration of free in the blood will decrease rapidly after injection due to uptake of the trace by the thyroid gland. The view that the results refer to uptake of 11z6-albuminby the amounts of free brain and not to the uptake of free 1lZ6 is further supported by the fact that iodide is actively transported out from the central nervous system across the choroid plexus even in immature animals (Robinson cf af. 1968). Samples of csf which were visibly contaminated with blood were discarded. This would ensure a blood contamination of less than 0.1 %.The small amount of residual blood in the brain was considered to be of no importance in this series of experiments. The results were expressed as (cpm/g brain/cpm/g blood).
Calculations Assuming that the transfer of 11z3-albuminfrom blood into brain follows a simple exponential function, i.c. the amount transferred is proportional to the difference between plasma concentration and brain concentration, then the following equation describes the brain concentration of 1lZ6-albumin C,(t)
=
C,(I -e-kt)
(1)
where C,(t) is the concentration of 112K-albuminin brain, C, the concentration of 11z6-albuminat steady state and k is the rate constant of the transfer process. The values of C,(t) are obtained experimentally by determining the radio-activity in brain tissue. Cm is determined graphically from a plot of C,(t) vs time. k is then obtained by logarithmic transformation of eq. (I), i.e. In ((2,- C,(t))= In (C,- kst. As seen in Fig. 4 the results plotted in this way fitted closely to a straight line and k can simply be obtained by measuring tt. k 0.6931t). ~
Results The concentration of 11z6-albuminin blood varied less than 10% between 2 and 24 h after intraperitoneal injection of the tracer. The concentration of 11z3-albuminin the brain was always less than 10% of that in the blood. Whole brainlblood ratios
Fig. 1 shows the concentration of 11z6-albuminin brain relative to blood at various times after injection of Itz6-albumin in newborn rats, 5-day-old rats and 30-day-old rats. As can
401
ALBUMIN TRANSFER INTO BRAIN
I
c m l brain x 100 c p i l g blood
7 6 -
Fig. 1. The concentration of Ileaalbumin in brain in newborn rats, 5-day-old rats, and 30-day-old rats expressed relative to the concentration in whole blood. All curves are fitted by eye. The bars indicate+ - S.E.
I
,
,
2
4
6
I
16 Time (hrs)
I
24
be seen there was a decrease in the relative concentration of 11z6-albuminin whole brain with increasing age.
Csflblood ratios Fig. 2 shows the concentration of 11z6-albuminin csf relative to blood at various times after injection of the tracer. As can be seen there was a decrease in the relative concentration of 1126-albuminin csf with increasing age.
Csflbrain interstitialfluid ratios Since Ilz6-albumin is located extracellularly its concentration in brain interstitial fluid (CIsF)may be calculated from the concentration in whole brain. Cbr*lIl
""=%
ISF in brain tissue
x
100
Since the calculations were made without corrections for the amount of IlZ6-albuminin the residual blood in brain tissue the calculated CIsF overestimates the real concentration in cpmlg cst x 100 c p m l g blood newborn
Fig. 2. The concentration of P6albumin in csf in newborn rats, 5-day-old rats, and 30-day-old rats expressed relative to the concentration in whole blood. All curves are fitted by eye. The bars indicatekS.E.
5 days old
30 days old 2
4
6
16 lime Ihrs)
24
402
OLE AMTORP
c p m l g x 100 cpmlg blood u 16 -
s
12'-
7
6
'I
nrwborn 8
8 /cst
5911YUlM 8
5
7
0
the interstitial fluid somewhat but the calculated values may be used to indicate the different patterns in rats of different ages. CI,, in newborn and juvenile rats was calculated using the values for extracellular space reported by Ferguson and Woodbury (1969): 45 % in newborn rats, 40% in rats 5 days of age and 15% in rats 30 days of age. As can be seen in Fig. 3 the relation between the concentration of I1"-albumin in csf relative to blood and the concentration of IIPs-albumin in brain interstitial fluid relative to blood was different in the various groups of rats which have been studied. In newborn rats the relative concentration of 11z6-albuminin csf and in brain interstitial fluid approaches unity with time. At 5 days of age this pattern changes as the concentration in csf relative to that in brain interstitial fluid decreases. This trend is even more conspicuous in rats 30 days
403
ALBUMIN TRANSFER INTO BRAIN
t
cpm l g brain I 100 cpmlg blood
--
Newborn
Fig. 4. Semilogarithmic diagram of the timeuptake curves into brain of 1126-albuminin newborn rats, 5-day-old rats and 30-day-old rats. t i are indicated on the fitted straight lines. The curves are fitted by eye. (For further explanations: see text.)
e
I
16
I
u
Time I h l
of age. Fig. 4 shows a semilogarithmic diagram of the curves of uptake into brain of ILesalbumin in newborn rats 5-day-old rats and 30-day-old rats. t# in hours as found from the fitted straight lines was 4.8, 5.7 and 3.8 respectively. The calculated k was 0.002 min-’ in newborn rats, 0.002 min-’ in 5-day-old rats and 0.003 min-l in 30-day-old rats.
Discussion A substance in the blood passes into csf across the blood-csf barrier by way of the choroid plexus or across the blood-brain barrier and further by diffusion from the cerebral capillaries by way of the brain interstitial fluid. Csf acts as a “sink” for substances entering the brain interstitial fluid from brain capillaries across the blood brain barrier because of its bulk flow through the ventricles and subarachnoid cavities and its unselective exit through the arachnoid villi.
404
OLE AMTORP
The concentration of a substance in csf and in brain is determined by its volume of distribution, by the differences between its rate of entrance into brain and csf from plasma across the blood-brain-csf barrier and its rate of exit from csf by bulk flow through the arachnoid villi, the latter being determined by the rate of production of csf. Decreasing extracellular space, barrier formation between blood and brain and between blood and csf and a larger bulk flow of csf due to an increased production all contribute to decreasing the concentration of a particular substance in brain and csf and to lowering the brain/plasma and csf/plasma ratios during the ontogenetic development. Three factors have to be taken into account when considering the conclusion from recent studies that immature animals have a relatively high concentration of protein in csf as compared with mature animals: Immature animals have a larger brain extracellular space as compared with the adult. In immature animals the transfer of a solute between blood and brain and between blood and csf might be less restricted as compared with mature animals. Further, in immature animals the bulk flow of csf through the arachnoid villi probably is sluggish whilst it progressively increases as the choroid plexus matures. Fig. 1 and Fig. 2 show the uptake data for 1126-albuminin brain and csf obtained simultaneously in newborn rats, 5-day-old rats and 30-day-old rats. It is evident from the figures that the concentration of IIPs-albumin in brain and csf was higher in newborn rats than in juvenile rats and that the concentration decreases with increasing age. On the basis of the brain/csf ratio for inulin, Ferguson and Woodbury (1969) calculated the extracellular space in pre- and postnatal rats. They found that the extracellular space decreases until 16 days of age but in older animals the extracellular space as calculated on the basis of brain/csf ratio apparently increases again. This is an unexpected finding and in disagreement with the finding of an inulin space of 13.5% in adult rats (Woodward et a/. 1967). Ferguson and Woodbury (1969) apparently also disregard the unexpected increase in the extracellular space and rather accept that the extracellular space is continuously decreasing from 50% in 4 days prenatal rats until it reaches a final value of 13.50/o.The larger extracellular space in the brain of the newborn rats may contribute to a larger accumulation of 1125-albuminin the present experiments. In the present study there was found no difference in the rate of transfer of 1125-albumin from blood into brain tissue in newborn rats as compared with 5-day-old rats and 30-dayold rats. However, the permeability of the cerebral capillaries can be larger in newborn rats when considered the results of Bar and Wolff (1973) on the vascularization of the rat cerebral cortex. They found that the postnatal development of capillary length and number of branchings continuously increased from birth until 30 days of age. The permeability of cerebral capillaries can for this reason be greater than the rate constant of the transfer process for 112s-albuminfrom blood into brain tissue suggests in newborn rats and 5-day-old rats. From a morphological point of view there is no evidence for maturation of the barrier mechanisms for large molecules between blood and brain and between blood and csf with increasing age. The barrier mechanisms show a considerable maturity of function very early in the development. Olsson et al. (1968) could not detect intravascularly administered fluorescein albumin outside the vessels in the brain of neither prenatal, newborn or postnatal rats. This is in agreement with the morphological evidence that the endothelial cells in capillaries
ALBUMIN TRANSFER INTO BRAIN
405
in prenatal rat cerebral cortex are connected by tight junctions (Carley and Maxwell 1970). Thus it is possible that an increased permeability of cerebral capillaries in newborn rats reflects a greater transcapillary pinocytotic transport of macromolecules in immature animals compared with mature animals. In the choroid plexus in adult rats Becker et al. (1967) found a rapid uptake of the protein tracer horseradish peroxidase from the vascular compartment. The injected protein passed out of the fenestrated capillaries and was rapidly distributed throughout the extracellular space. The tracer then entered the choroidal epithelial cells via pinocytotic vesicles and migrated as far as to the ventricular surface. The tracer also moved between the epithelial cells of the choroid plexus until stopped by tight junctions near the ventricular surface. In developing rat choroid plexus Morecki et al. (1969) could identify horseradish peroxidase when administered intravenously in the vascular compartment and in the extracellular space, but not in the choroid epithelium prior to 4th to 6th days postpartum. The conclusion that newborn rats produce little csf is based on two pieces of evidence. Fig. 3 shows that the relative concentration of ILaS-albuminin csf approaches the calculated relative concentration of 11e6-albuminin brain interstitial fluid. During development this pattern changes. The concentration of 1126-albuminin csf relative to that in brain interstitial fluid decreases and the concentration of 1126-albuminin csf remains below that of brain interstitial fluid indicating an increased “sink action” of csf due to an increased production rate during development. If the high extra-cellular space as suggested by Ferguson and Woodbury (1969) in newborn rats largely overestimates the real space, the validity of the present conclusion that newborn rats produce little csf can be spurious. The concentration of albumin in a smaller extracellular space will exceed the concentration of the isotope in the csf. If the estimates of a large extracellular space is fairly correct the accumulation of P6albumin in the csf space can hardly be explained other than by a decreased exit through the arachnoid villi. In a previous study (Amtorp and Snrrensen 1974) it was found that administration of Acetazolamide did not increase the concentration of protein in cisternal csf in newborn rats whereas it did so in older animals, another suggestion that the newborn rats produced little if any csf. This conclusion is further supported by the fact that csf production in 5-day-old rats is only 40% of that in 30-day-old rats (Bass and Lundborg 1973). By exclusion then it seems that the relatively high protein concentration in csf in immature rats could be explained by a smaller bulk flow of csf through the arachnoid villi due to a lower production rate of csf as compared with mature rats. This work was supported in part by the Warwar-Larsen Foundation, Denmark.
References AMTORP, 0.and s. c. S~RENSEN, The ontogenetic development of concentration differences for protein and ions between plasma and cerebrospinal fluid in rabbits and rats. J . Physiol. (Lond.) 1974. 243. 387-400. BAR, T. and J. R. WOLFP,On the vascularization of the rat’s cerebral cortex. Bibl. anar. (Basel) 1973. If. 515-519. BASS, N. H. and P. LUNDBORG, Postnatal development of bulk flow in the cerebrospinal fluid system of
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the albino rat: Clearance of carboxyl-(14C) inulin after intrathecal infusion. Brain Res. 1973.52. 323332.
BECKER,N. H., A. B. NOVIKOFF and H. M. ZIMMERMAN, Fine structure observations of the uptake of intravenously injected peroxidase by the rat choroid plexus. J. Histochem. Cytochem. 1967. 15. 160-165. BRIGHTMAN, H.W. and T. S. REESE,Junctions between intimately apposed cell membranes in the vertebrate brain. J. cell. Biol. 1969. 40. 648-677. CARLEY, W. D. and D. S. MAXWELL, Development of the blood vessels and extracellulai spaces during postnatal maturation of rat cerebral cortex. J. Comp. Neurol. 1970. 138. 3 1-48. DAVSON,H., The normal protein concentration. In: Physiology of the Cerebrospinal Fluid 1970 pp. 272273. London. Little, Brown & Company. EHRLICH,P., Das Sauersroff-Bediirfnis des Organismus. Eine farbenanalytische Studie. Berlin 1885. EVANS,C. A. N., J. M. REYNOLDS, M. L. REYNOLDS, N. R. SAUNDERS and M. B. SEGAL,The development of a blood-brain barrier mechanism in foetal sheep. J. Physiol (Lond.) 1974. 238. 371-386. FERGUSON, R. K. and WOODBURY, Penetration of 14C inulin and 14C sucrose into brain, cerebrospinal fluid and skeletal muscle in developing rats. Exp. Brain Res. 1969. 7. 181-194. KLOSOWSKII, B. N., The development of the brain and its disturbance by harmful factors. 1963 p. 8 . Oxford. Pergamon. and N. H.BECKER, Transport of peroxidase by the developing rat choroid MORECKI, R.. H. M. ZIMMERMAN plexus. Acra neuropath. (Berl.) 1969. 14. 14-18. OLSSON,Y.,I. KLATZO,P. SOURANDER and 0. STEINWALL, Blood-brain barrier to albumin in embryonic, newborn and adult rats. Acta neuropath. (Bed.) 1968. 10. 117-122. REESE,T. S. and M. J. KARNOWSKY, Fine structural localization of a blood-brain barrier to exogenous peroxydase. J. cell. Biol. 1967. 34. 207-217. ROBINSON, R. J., R. W. P. CUTLER,A. V.LORENZO and C. F. BARLOW, Development of transport mechanisms for sulphate and iodide in immature choroid plexus. J. Neurochem. 1968. 15. 455-458. WOODWARD, D. L., D. J. REEDand D. M. WOODBURY, The extracellular space of rat cerebral cortex. Amer. J . Physiol. 1961. 212. 367-370.
