The persistence of high uptake of serum albumin in the olfactory bulbs of mice throughout their adult lives

Arch. Gerontol. Geriatr., 13 (1991) 201-210

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© 1991 Elsevier Science Publishers B.V. All rights reserved 0167-4943/91/$03.50 AGG 00410

The persistence of high uptake of serum albumin in the olfactory bulbs of mice throughout their adult lives Masaki Ueno a, Ichiro Akiguchi a, Hironobu Naiki c, Yasuhisa Fujibayashi b, Hidenao Fukuyama a, Jun Kimura a, Masakuni Kameyama d and Toshio Takeda c Departments of a Neurology and h Nuclear Medicine, Faculty of Medicine, and c Department of Senescence Biology, Chest Disease Research Institute, Kyoto University, Sakyo-ku, Kyoto 606, Japan, and ~ Department of Neurology, Sumitomo Hospital, Osaka 530, Japan (Received 12 February 1991; revised version received 15 May 1991; accepted 17 May 1991)

Summary Brain to plasma concentration ratios of i.v. administered human serum albumin (HSA) in the olfactory bulb, frontal cortex and cerebellum were evaluated in DDD mice of different ages. We measured the brain uptake of serum albumin excluding intravascular content by using a double isotope technique and examined the time course of the brain uptake to evaluate the brain uptake at different time intervals. In young adult mice, the value was significantly higher in the olfactory bulb than in other brain regions 3-24 h after 125I-HSA injection. It was about 2.3 times higher in the olfactory bulb than in the cerebellum (P < 0.01). The high concentration ratios in the olfactory bulb were observed in all 4-22-month-old mice. Moreover, the ratio in the olfactory bulb 24 h after 125I-HSA injection was higher in 22-month-old mice than in younger animals. The high uptake of serum albumin in the olfactory bulb suggests that intravascular macromolecules can be transported into the olfactory bulb more easily than in other brain regions with tight endothelium, and the persistence of high uptake during adult life may be associated with age-related morphological changes in the olfactory bulb. Mouse olfactory bulb; Blood-brain barrier; Aging; Human serum albumin; Intravenous injection

Introduction In the central nervous system (CNS), tight junctions and scarce pinocytosis or fenestrations in the cerebral endothelium compose the blood-brain barrier (BBB)

Correspondence to: Masaki Ueno, M.D., Department of Neurology, Faculty of Medicine, Kyoto University, Sakryo-ku, Kyoto 606, Japan, Tel.: 075-751-3766.

202 and protect the brain parenchyma from circulating substances (Davson et al., 1987; Brightman, 1989). However, at the level of the olfactory bulb, 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 (Bradbury et al., 1981). In this way, the olfactory bulb is an important intracerebral region which is in contact with extracerebral regions. Rhinencephalo-limbic structures are affected in patients with Alzheimer's disease (Esiri and Wilcock, 1984; Pearson et al., 1985; Roberts, 1986) and in those with herpes encephalitis (Johnson, 1964). It has been reported that the rat olfactory bulb shows dramatic morphological changes during adult life (Hinds and McNelly, 1977). The aim of this study is to determine whether there are any differences in the brain uptake by the olfactory bulb and by other brain regions with tight endothelium of macromolecules injected intravenously and whether there are any age-related differences in brain uptake (Mooradian, 1988). Serum albumin, the most abundant circulating protein, which is frequently used to examine BBB integrity (Amtorp, 1976; Johansson and Martinsson, 1980; Koh and Paterson, 1987) and age-related changes in the vulnerability of the brain to environmental stimuli (Sanker et al., 1983) was used as the marker in this study.

Material and Methods

Animals DDD mice aged 3-5, 11, 15 and 22 months (n = 52) were maintained under conventional conditions and fed a commercial diet (CE-2, Nihon CLEA) and tap water ad libitum (Staats, 1985; Kawamata et al., 1990). The mean life span of this colony in our animal quarters is 18.1 months. The major pathologies in their later lives are infectious diseases, such as pneumonia, pulmonary abscess and liver abscess.

Experimental procedures With intravenous administration technique using 125I-HSA as a test tracer and 131I-HSA as a plasma marker (Leibowitz and Kennedy, 1972; Ohno et al., 1978; Lee et al., 1986; Koh and Paterson, 1987; Smith, 1989), we evaluated the brain to plasma concentration ratio of human serum albumin (HSA) following i.v. administration. Iodination of HSA by carrier free Na125I (IMS-30, Amersham Corp.) or carrier free Nal31I (IBS-3, Amersham Corp.) was performed by the IC1 method (McFarlane, 1958). Unbound iodine was removed by an anion exchange resin column (column size, 0.5 ml) and overnight dialysis against phosphate buffered saline (PBS), pH 7.2 at 4°C. Specific activities were 0.78-1.26× 102 KBq//zg protein for 125I-HSA and 0.67-1.18 × 10 2 KBq//zg protein for 131I_HSA. The ratio of trichloroacetic acid precipitable to total counts always exceeded 99%. Protein concentrations were determined by the method of Bradford with bovine serum albumin as the standard (Bradford, 1976).

203 TABLE I Regional blood volume Regions

4M (n = 20)

11 M (n = 8)

15 M (n = 8)

22 M (n = 8)

2.93 _+0.21 1.16 -+ 0.07 1.45 _+0.06

2.69 _+0.16 1.36 _+0.07 1.49 _+0.04

3.21 -+0.30 1.42 _+0.05 1.51 _+0.05

( × 10 - 2 / ~ l / m g ) Olfactory bulb Frontal cortex Cerebellum

2.10 _+0.12 1.17 + 0.04 1.41 _+0.04

Regional blood volume = 131Br/131WB, where 131Br is the measured activity of 1311-HSA in the brain at the time of decapitation per unit wet weight and 131WB is the activity of t3~I-HSA in the whole blood per unit volume at the time of decapitation ( c p m / m g b r a i n ) / ( c p m / p H whole blood).

Mice were injected via the tail vein with 0.5 p~g protein of 125I-HSA per gram of body weight and were decapitated 3, 6, 12 or 24 h later. Before decapitation, 50 ~I of blood from the tail vein were drawn into capillary tubes 5 min and 0.5, 1 and 2 h post-injection; 5 min and 1, 2 and 3 h post-injection; 5 min and 1, 4 and 8 h post-injection; 5 min and 1, 6 and 12 h post-injection, respectively. Five minutes prior to decapitation, the mice were injected with 0.15-0.25 ~g protein of ~3~I-HSA per gram of body weight and were then given i.p. an overdose of pentobarbital sodium. The 13aI-HSA space 5 min after the i.v. injection was used as an estimate of regional blood volume. A sample of 500/.d of blood was taken from the heart just before decapitation. The brain was removed, and the dura and subarachnoidal vessels were carefully excised and discarded. The brain surface was washed clean with phosphate buffered saline, hemisectioned at the midline and quickly dissected into several regions in a room kept at 4 ° C. The radioactivity of each region was measured by a gamma counter (Aloka Auto Well Gamma System, ARC-300) for 5 min. The 125I-activity was corrected for counts due to 131I 'spill-over' into the 125I channel. Age-related changes in the brain to plasma concentration ratios were evaluated in mice of different ages decapitated 3 and 24 h after 125I-HSA injection. Parenchymal brain radioactivity was determined as measured brain radioactivity minus intravascular radioactivity which was calculated from the radioactivity in whole blood per unit volume and regional blood volume (see Table I). Brain to plasma concentration ratios were expressed as ratios of parenchymal brain radioactivity per unit wet weight to plasma radioactivity per unit volume.

TCA precipitation The ratio of non-protein-bound 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 non-protein-bound 12sI radioactivity in the brain and

204

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Fig. 1. Plasma concentrations of 125I-HSA following intravenous injection. The curve is a computer fit to the data points. Each value shown is the mean + S.E.M. of 5 values.

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Fig. 2. Time course of brain to plasma concentration ratios in three regions in 3 - 5 month-old mice. Each value shown is the m e a n + S . E . M , of 5 values. Means of values in the frontal cortex, the cerebellum and the olfactory bulb are shown by closed circles, closed triangles and closed squares, respectively. A ( P < 0.01): significantly different from values in other regions.

205

the plasma were determined by precipitation of the protein-bound 125I with 10% trichloroacetic acid.

Data analysis Comparison among different brain regions was made by a one-way analysis of variance (ANOVA). All values in the paper are expressed as means + S.E.M. of n experiments.

Results

Fig. 1 shows a plasma concentration curve of ~25I-HSA following i.v. injection. Table I shows the regional blood volume defined as the 131I_HSA space 5 min after i.v. injection. The volume in the olfactory bulb was approximately two-fold higher than in other areas, as noted by other workers (Ohno et al., 1978; Rapoport et al., 1979). Fig. 2 shows the time course of the brain to plasma concentration ratios in 3-5-month-old mice. The ratio was significantly higher in the olfactory bulb than A

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Fig. 3. Age-related changes in the brain to plasma concentration ratios of Ie~I-HSA 3 h after injection. Each value shown is the mean +_S.E.M. of 5 values. Means of values in the frontal cortex, the cerebellum and the olfactory bulb are shown by closed circles, closed triangles and closed squares, respectively. A ( P < 0.01): significantly different from values in other regions.

206 A,B

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Fig. 4. Age-related changes in the brain to plasma concentration ratios of 12SI-HSA 24 h after injection. Each value shown is the mean _+S.E.M. of 4 values. Means of values in the frontal cortex, the cerebellum and the olfactory bulb are shown by closed circles, closed triangles and closed squares, respectively. A ( P < 0.01): significantly different from values in other regions. B ( P < 0.05): significantly different from values in younger mice in each region.

in other areas of the brain 3-24 h after 125I-HSA injection. It was about 2.3 times higher in the olfactory bulb than in the cerebellum (F(2,32) = 63.01, P < 0.01). Figs. 3 and 4 show age-related changes in the brain to plasma concentration ratio 3 and 24 h after 125I-HSA injection, respectively. We found the persistence of high concentration ratios in the olfactory bulbs of mice in the age range from 4 to 22 months 3 and 24 h after i.v. injection (3 h experiment: F(2,24) = 62.14, P < 0.01; 24 h experiment: F(2,32)= 88.96, P < 0.01). The brain to plasma concentration ratios in three different regions all tended to rise with age. The brain to plasma concentration ratio in the olfactory bulb 24 h after 125I-HSA injection was significantly higher in 22-month-old mice than in 11 or 15-month-old mice (F(3,16) = 3.28, P < 0.05). The ratio of non-protein-bound 125I radioactivity to total radioactivity in the brain and plasma ranged from 9.3% to 13.0% (n = 5) and from 1% to 2% (n = 4), respectively.

207

Discussion In this study, we evaluated the brain uptake of HSA injected intravenously by using a double isotope technique. We found a similar time course of the brain uptake of HSA in the mouse as reported earlier in the rat (Amtorp, 1976). Moreover, we found a significantly higher concentration ratio of 125I-HSA in the olfactory bulb than in other brain regions with tight endothelium and the persistence of high uptake of serum albumin during adult life. However, the brain uptake of 125I-HSA in the olfactory bulb was smaller than that in BBB-free regions reported so far (Ohno et al., 1978; Ermisch et al., 1984). Ermisch et al. (1984) and Ohno et al. (1978) reported that the accumulation of smaller molecule markers in the olfactory bulb tended to be somewhat higher in regions with tight endothelium. The higher brain to plasma concentration ratio of 125I-HSA in the olfactory bulb than in other brain regions is probably due to various factors, such as an increase in vesicular transport which carries serum albumin transendothelially (Westergaard and Brightman, 1973; Broadwell et al., 1988), patent intercellular clefts in the arachnoid (Balin et al., 1986), high blood volume, redistribution (Bradbury et al., 1981) or some unknown factors. The increase in vesicular transport is likely, because the olfactory bulb is relatively widely covered with pia mater and therefore would be expected to be widely supplied by pial and parenchymal arterioles the endothelium of which contains many vesicles and pits unlike capillary endothelium (Westergaard et al., 1973; Roggendorf et al., 1976). Balin et al. (1986) showed that a peroxidase reaction product injected intravenously was observed on the ventricular and pial surfaces of the mouse brain, within the Virchow-Robin spaces of large vessels penetrating the pial surfaces. The precise mechanism of higher uptake of HSA in the olfactory bulb must be clarified. We obtained direct evidence for the ready penetration into and accumulation in the olfactory bulb of macromolecules administered intravenously and the persistence of the high uptake of intravascular substances throughout the adult lives of the animals. Some human serum proteins are known to inhibit ligand binding at neurotransmitter receptors in the human brain (Andorn et al., 1986; Pappolla and Andorn, 1987). Therefore these results may be associated with age-related neuronal changes in the olfactory bulb (Hinds and McNelly, 1977).

Acknowledgements Gratitude is extended to Dr. T. Kita, Kyoto University for kindly providing IC1 solution, Dr. A. Cary for critical comments and S. Iwai and K. Kogishi for technical assistance. This study was supported by a grant from the Ministry of Education, Culture and Science of Japan, and a grant from the Mihara Foundation.

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