Archives of Gerontology and Geriatrics
25 (1997) 321 – 331

Age-related changes in barrier function in mouse
brain
II. Accumulation of serum albumin in the
olfactory bulb of SAM mice increased with aging
Masaki Ueno a,*, Ichiro Akiguchi b, Masanori Hosokawa c,
Masahiko Shinnou a, Haruhiko Sakamoto a, Manabu Takemura b,
Keiichi Higuchi c
a

Second Department of Pathology, Kagawa Medical Uni6ersity, 1750 -1 Ikenobe, Miki-cho, Kita-gun,
Kagawa 761 -07, Japan
b
Department of Neurology, Faculty of Medicine, Kyoto Uni6ersity, Kyoto 606, Japan
c
Department of Senescence Biology, Chest Disease Research Institute, Kyoto Uni6ersity,
Kyoto 606, Japan
Received 6 February 1997; accepted 5 June 1997

Abstract
Brain transfer rate (BTR) and brain to plasma concentration ratio (BPCR) of radiolabelled human serum albumin injected intravenously were calculated to evaluate the brain
transfer of serum albumin in the cerebral cortex, the right hemi-brain and the olfactory bulb
of senescence accelerated prone mice (SAMP8) and senescence accelerated resistant mice
(SAMR1). BTR and BPCR in SAMP8 and SAMR1 were signiﬁcantly higher in the olfactory
bulb than in the cerebral cortex or the right hemi-brain. Age-related signiﬁcant changes in
BTR and BPCR were observed in the olfactory bulb of SAMP8 and SAMR1. These ﬁndings
suggest that the barrier function to serum albumin in the olfactory bulb of SAMP8 and
SAMR1 is not so tight as it is in the cerebral cortex and becomes weaker with age and that
the age-related changes are manifested at a younger age in SAMP8 than in SAMR1. © 1997
Elsevier Science Ireland Ltd.

* Corresponding author. Tel.: + 81 878 912115; fax: +81 878 912116.
0167-4943/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved.
PII S 0 1 6 7 - 4 9 4 3 ( 9 7 ) 0 0 0 2 2 - 8

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M. Ueno et al. / Arch. Gerontol. Geriatr. 25 (1997) 321–331

Keywords: Aging; Blood-brain barrier; Olfactory bulb; Senescence accelerated mice; Serum
albumin

1. Introduction
In the central nervous system, the blood-brain barrier (BBB) in the cerebral
microvessels prevents intravascular macromolecules from entering the cerebral
parenchyma (Brightman and Reese, 1969; Davson, 1976). At the level of the
olfactory bulb, however, the pathway between the nose and the brain has been
shown to be bidirectional to molecules delivered intranasally and intraventricularly (Baker and Spencer, 1986; Balin et al., 1986). It is also known that
cerebral interstitial ﬂuid drains via subarachnoid spaces of the olfactory bulb
into deep cervical lymph node (Bradbury et al., 1981) and that radiolabelled
human serum albumin injected intravenously can be transported into the olfactory bulb more easily than into other BBB-regions of DDD mice (Ueno et al.,
1991).
Senescence-accelerated mouse (SAM), a murine model of accelerated senescence, was established by Takeda and his colleagues (Takeda et al., 1981). The
SAM model is a valid one for research on effects of aging (Takeda et al., 1991,
1994). Some papers on age-related changes in the BBB have already been reported (Rapoport et al., 1979; Rudick and Buell, 1983; Mooradian, 1988). We
have also evaluated age-related changes in the barrier function in several brain
regions of SAMP8 mice, which showed age-related deﬁcits in learning and memory (Miyamoto et al., 1986; Yagi et al., 1988, 1989), and reported that the
barrier function to exogenous radiolabelled serum albumin signiﬁcantly changed
with aging in special areas (the hippocampus, the pons and the cerebellum) but
not the frontal cortex nor the parietal cortex (Ueno et al., 1993). Recently the
barrier function to endogenous serum albumin in the olfactory bulb of SAMP8
mice was ultrastructurally examined by using a immunocytochemical procedure
and it was reported that the density of immunolabelling of the subendothelial
space and the surrounding neuropil of microvessels in the olfactory bulb of aged
SAMP8 was signiﬁcantly higher than that observed in control mice (Ueno et al.,
1996). These ﬁndings prompted us to examine whether the high labelling density
for endogenous albumin in the subendothelial space of microvessels in the olfactory bulb of aged SAMP8 resulted from the increased permeability of the microvessels to serum albumin and to quantitatively evaluate the transfer of
exogenous serum albumin from the blood stream into the olfactory bulb of
SAMP8 mice and the differences in the transfer of serum albumin between
SAMP8 and SAMR1.

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323

2. Materials and methods

2.1. Animals
SAMP8 mice aged 3, 7 and 13 months (n = 36) (median survival time (MST):
12.1 months) and 3, 13 and 22-month-old SAMR1 mice (n= 36) (MST: 18.9
months) were maintained under conventional conditions and fed a commercial diet
(CE-2, Nihon CLEA) and tap water ad libitum. All mice were housed in cages, the
room temperature was 24 92°C and a 12 h light/dark cycle with lights on at 07:00
h was maintained. Throughout the experiments, all the mice were alert, up to
decapitation.

2.2. Experimental procedures
With a double isotope technique reported previously (Ueno et al., 1991, 1993)
using 125I-human serum albumin (HSA) as a test tracer and 131I-HSA as a plasma
marker (Leibowitz and Kennedy, 1972; Koh and Paterson, 1987; Poduslo et al.,
1994), we evaluated the brain accumulation of HSA. Iodination of HSA by carrier
free Na125-I (IMS-30, Amersham) or carrier free Na131-I (IBS-3, Amersham) was
performed by the ICI method (McFarlane, 1958). Unbound iodine was removed by
an anion exchange resin column (column size 0.5 ml) and overnight dialysis against
0.01 M phosphate buffered saline (PBS) pH 7.2 at 4°C. Speciﬁc activities ranged
from 0.74 to 1.26 × 102 (KBq/vg protein) for 125I-HSA and 0.56–1.30× 102 (KBq/
vg protein) for 131I-HSA, respectively. The ratio of trichloroacetic acid (TCA)
precipitable to total counts in the injection solution, including iodinated HSA,
always exceeded 99%. Protein concentrations were determined by the method of
Bradford with bovine serum albumin as the standard (Bradford, 1976). Mice were
slowly injected via the tail vein with 0.5 vg of 125I-HSA per gram of body weight
(BW), the total volume of which was below 200 vl. These animals were bled from
the tail vein at different time intervals (see below) and decapitated at different
times. Before decapitation at 3 and 24 h postinjection (p.i.), 50 vl of blood from the
tail vein was drawn into capillary tubes 5 min and 0.5, 1 and 2 h p.i. and 5 min and
1, 6 and 12 h p.i., respectively. These same mice, 5 min prior to decapitation, were
slowly injected with 0.15 – 0.25 vg of 131I-HSA (plasma marker)/BW (g), the total
volume of which was below 150 vl and were then given an overdose of pentobarbital sodium i.p. A sample of 500 vl of blood was taken from the heart just before
decapitation. The brain was removed and the dura and subarachnoidal vessels
carefully excised and discarded. The brain surface was washed clean with PBS,
hemisectioned at the midline and quickly dissected into several regions in a room
kept at 4°C. Radioactivities in the cerebral cortex, the olfactory bulb and the right
hemi-brain were measured for 5 min using a gamma counter (Aloka Auto Well
Gamma System, ARC-300). The 125I-activity was corrected for counts due to 131I
‘spill-over’ into the 125I channel.
Parenchymal brain radioactivity (BR) was calculated by:

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BR = (125Bra − 125WB × 131Bra/131WB)
where 125Bra and 125WB were the radioactivities of 125I-HSA in the brain and the
whole blood per unit volume, respectively and 131Bra and 131WB were the radioactivities of 131I-HSA in the brain and the whole blood per unit volume, respectively.
Brain transfer rate (BTR) was deﬁned as:
BTR = BR/(Wbr ×mean PR)
where Wbr was brain weight and mean PR was mean plasma radioactivity of
I-HSA per unit volume and was calculated from the integral of plasma radioactivity of 125I-HSA per unit volume divided by circulating time of test tracer (Ueno
et al., 1993). BTR was calculated in the mice, which were decapitated at 3 h p.i., to
evaluate the transfer of 125I-HSA in the brain. Brain to plasma concentration ratio
(BPCR) was deﬁned as:
125

BPCR =BR/(Wbr ×PRdec)
where PRdec was plasma radioactivity of 125I-HSA per unit volume at decapitation
(Ueno et al., 1991, 1993). BPCR was calculated in the mice, which were decapitated
at 24 h p.i. to evaluate the accumulation of 125I-HSA in the brain.

2.3. Plasma clearance
In order to evaluate the plasma clearance of 125I-HSA injected intravenously in
SAMP8 and SAMR1 mice of different ages, time course of plasma concentrations
of 125I-HSA was examined and then a plasma concentration curve of 125I-HSA was
ﬁtted to the data points. The curve was obligated to be ﬁtted to two exponential
curves by a computer (Ueno et al., 1991, 1993). Two kinds of the exponential
decline correspond to initial fall and late decay. The initial fall and late decay in
plasma clearance of 125I-HSA were evaluated by calculating the half life (Cohen et
al., 1956).

2.4. TCA precipitation
The ratio of protein-unbound 125I counts to total counts in the brain was
evaluated in mice decapitated 24 h after 125I-HSA injection without 131I-HSA
injection. The intravascular contents were removed by perfusion via the left
ventricle with 15 ml PBS. The protein-unbound 125I radioactivity in the brain and
the plasma were determined by precipitation of the protein-bound 125I with 10%
trichloroacetic acid. The ratio of protein-unbound 125I radioactivity to total radioactivity in the brain and the plasma ranged from 9 to 13% and from 1 to 2%,
respectively.

M. Ueno et al. / Arch. Gerontol. Geriatr. 25 (1997) 321–331
Table 1
Half life of plasma concentration of
Mice

325

125

I-HSA in SAMP8 and SAMR1 mice

Half life (h)
Initial
0.679 0.06
0.7590.13
0.789 0.11
0.659 0.09
0.759 0.07
0.619 0.08

SAMP8-3M
SAMP8-7M
SAMP8-13M
SAMR1-3M
SAMR1-13M
SAMR1-22M

Late
12.2 90.5
11.1 90.4
11.9 9 0 9
12.9 9 0.5
13.3 9 0.6
13.2 90.8

Plasma concentration values were simulated to two exponential curves, corresponding to initial fall and
late decay, then two kinds of half life were calculated. The number of samples in each group is six.

2.5. Data analysis
Values in this paper are expressed as means9 S.E.M. Statistical signiﬁcance was
evaluated by analysis of variance followed by the Tukey method.

3. Results
Time-dependent plasma concentrations of 125I-HSA following i.v. injection of the
tracer were well ﬁtted to two exponential curves both in SAMP8 and SAMR1, as
reported previously (Ueno et al., 1993). Table 1 shows the half lives of intravascular
125
I-HSA in SAMP8 and SAMR1 of different ages. There were no signiﬁcant
differences in the initial rapid fall and late slow decay in plasma clearance of
125
I-HSA among mice of different ages both in SAMP8 and SAMR1 mice. Table 2
Table 2
Blood volume
Region of
the brain

SAMP8 (months-old)

SAMR1 (months-old)

3
CX
HB
OB

7

13

3

13

22

1.219 0 04
1.299 0.04
2.739 0.28a

1.229 0.03
1.309 0.02
2.609 0.20a

1.41 9 0.05
1.38 9 0.04
3.28 9 0.32a

1.16 9 0.05
1.279 0.03
2.71 9 0.23a

1.23 9 0.03
1.23 9 0.03
2.91 9 0.27a

1.24 9 0.09
1.30 9 0.07
3.21 9 0.28a

The blood volume is deﬁned as 131Bra/(Wbr×131WB), where 131Bra is the measured radioactivity of
131
I-HSA in the brain, 131WB is the radioactivity of 131I-HSA in the whole blood per unit volume and
Wbr is the regional brain weight. Values are expressed as ×10−2 vl/mg. The number of samples in each
group is 12.
CX, cortex; HB, hemi-brain; OB; olfactory bulb.
a
PB0.01, signiﬁcantly different from the value in the cortex of the same group.

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Fig. 1. Brain transfer rate (BTR) of 125I-HSA in 3-, 7- and 13-month-old SAMP8 (a) and 3-, 13 and
22-month-old SAMR1 (b). The number of animals in each group is six. a PB 0.01: Signiﬁcantly different
from the value in the cortex in the mice of the same age. b PB 0.05: Signiﬁcantly different from the value
for younger mice in each region.

M. Ueno et al. / Arch. Gerontol. Geriatr. 25 (1997) 321–331

327

Fig. 2. Brain to plasma concentration ratio (BPCR) of 125I-HSA in 3-, 7- and 13-month-old SAMP8 (a)
and 3-, 13- and 22-month-old SAMR1 (b). The number of animals in each group is six. a PB 0.01:
Signiﬁcantly different from the value in the cortex in the mice of the same age. b P0.05: Signiﬁcantly
different from the value for younger mice in each region.

shows the blood volume in SAM mice, deﬁned as 131Bra/(Wbr×131WB). The
blood volume in the olfactory bulb was 2.1–2.6 times higher than in the cerebral
cortex (SAMP8: F(2, 66) =101.28 (PB0.01), SAMR1: F(2, 66)= 145.68 (PB
0.01)), as noted by other workers (Ohno et al., 1978; Rapoport et al., 1979). There
were no signiﬁcant age-related changes (SAMP8: F(2, 33)=3.21 (P\ 0.05),
SAMR1: F(2, 33) =0.98 (P \0.05)).
Fig. 1(a) and (b) show BTR in SAMP8 and SAMR1 mice, respectively. BTR in
the olfactory bulbs of SAMP8 mice were signiﬁcantly higher than those in the
cerebral cortex or the right hemi-brain (F(2, 30)= 120.20, PB 0.01). The value was
about 3.3 times higher in the olfactory bulb than in the cerebral cortex. Age-related
changes in BTR were observed in the olfactory bulb of SAMP8 (F(2, 15)= 6.50,
PB 0.01). BTR in the olfactory bulbs of SAMR1 mice were signiﬁcantly higher
than in the cerebral cortex or the right hemi-brain (F(2, 30)= 120.94, PB0.01). It
was about 3.9 times higher in the olfactory bulb than in the cerebral cortex.
Age-related change was observed in the olfactory bulb of SAMR1 (F(2, 15)= 3.91,
PB 0.05).

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Fig. 2(a) and (b) show BPCR in SAMP8 and SAMR1 mice, respectively. BPCR
in the olfactory bulbs of SAMP8 were signiﬁcantly higher than those in the cerebral
cortex or the right hemi-brain (F(2, 30)= 112.43, PB 0.01). The value was about
2.5 times higher in the olfactory bulb than in the cerebral cortex. Age-related
change in BPCR was observed in the olfactory bulb of SAMP8 (F(2, 15)=5.71,
P B 0.05). BPCR in the olfactory bulbs of SAMR1 were signiﬁcantly higher than
those in the cerebral cortex or the right hemi-brain (F(2, 30)= 39.11, PB 0.01). It
was about three times higher in the olfactory bulb than in the cerebral cortex.
Age-related change in BPCR was observed in the olfactory bulb of SAMR1
(F(2, 15) =5.88, P B 0.05). Age-related changes in BTR and BPCR were observed
both in the olfactory bulbs of SAMP8 and SAMR1 and were manifested at a
younger age in SAMP8 than in SAMR1.

4. Discussion
In this study, we quantitatively evaluated the transfer and the accumulation of
serum albumin from the blood stream into the olfactory bulb of SAM mice by

Fig. 2 (continued).

M. Ueno et al. / Arch. Gerontol. Geriatr. 25 (1997) 321–331

329

using a double isotope technique reported previously (Ueno et al., 1991, 1993)
and found that the accumulation of serum albumin was 2.5–3.9 times higher in
the olfactory bulb than in the cerebral cortex of SAM mice. Moreover age-related increases in the accumulation of serum albumin in the olfactory bulb were
observed both in SAMP8 and SAMR1. In addition, the age-related increases
were manifested at younger age in SAMP8 than in SAMR1. These results are
compatible with our previous ultrastructural ﬁnding that the high labelling density for endogenous albumin was observed in the subendothelial space and the
surrounding neuropil of microvessels in the olfactory bulb of aged SAMP8
(Ueno et al., 1996). The previous ultrastructural ﬁnding and these quantitative
results show that the barrier function to serum albumin in the olfactory bulb of
SAM mice is not as tight as it is in the cerebral cortex and becomes weaker with
age. As the greater accumulation of serum albumin could be observed in the
olfactory bulb than in the cerebral cortex even at 3 h after the injection of the
tracer, it is likely that the high labelling density for endogenous albumin in the
neuropil around the microvessels in the olfactory bulb of aged SAMP8 resulted
from the increased permeability to serum albumin in the microvessels in the
olfactory bulb but not from the invasion of serum albumin through the neuropil
from the neighboring areas. The BTR in the cerebral cortex in this study coincided well with the values in the frontal or parietal cortex in the paper reported
by Harik and McGunigal (Harik and McGunigal, 1984), who controlled the
concentration of 125I-HSA in the plasma under steady state conditions to evaluate the leakage of 125I-HSA across the BBB.
The rat olfactory bulb was reported to show dramatic morphological changes
during adult life (Hinds and McNelly, 1977). It is known that some human
serum proteins inhibit ligand binding at neurotransmitter receptors in the human
brain (Andorn et al., 1986; Pappola and Andorn, 1987) and that selective neuronal necrosis following cerebral ischemia is accompanied by the extravasation of
serum proteins in the damaged area (Schmidt-Kastner et al., 1990). Accordingly,
age-related increases in the accumulation of serum albumin in the olfactory bulb
may be associated with age-related morphological changes in the olfactory bulb.
On the contrary, such slightly permeable vasculature in the olfactory bulb in
adult mice may have some functional signiﬁcance for postnatal neurogenesis,
which occurs in the olfactory bulb in adult animals (Altman, 1969; Kaplan and
Hinds, 1977). The clear meaning of the greater accumulation of serum albumin
in the olfactory bulb than in the cerebral cortex remains to be elucidated.

Acknowledgements
Gratitude is extended to Dr T. Kita, Kyoto University, for kindly providing
the ICI solution and Dr T. Takeda, Kyoto University, for always encouraging
us. This work was supported by a grant-in-aid for scientiﬁc research and for
developmental scientiﬁc research from the Ministry of Education, Science and
Culture of Japan.

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