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 - 8 322 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. M. Ueno et al. / Arch. Gerontol. Geriatr. 25 (1997) 321–331 323 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: 324 M. Ueno et al. / Arch. Gerontol. Geriatr. 25 (1997) 321–331 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. 326 M. Ueno et al. / Arch. Gerontol. Geriatr. 25 (1997) 321–331 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). 328 M. Ueno et al. / Arch. Gerontol. Geriatr. 25 (1997) 321–331 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. 330 M. Ueno et al. / Arch. Gerontol. Geriatr. 25 (1997) 321–331 References Altman, J., 1969. Autoradiographic and histological studies of postnatal neurogenesis. IV. Cell proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb. J. Comp. Neurol. 137, 433 – 458. Andorn, A.C., Pappola, M.A., Fox, H., Klemens, F.K., Martello, P.A., 1986. Human serum cohn fraction 4 (h-globulin enriched) inhibits ligand binding at neurotransmitter receptors in human brain. Proc. Natl. Acad. Sci. USA 83, 4572– 4575. Baker, H., Spencer, R.F., 1986. Transneuronal transport of peroxidase-conjugated wheat germ agglutinin (WGA-HRP) from the olfactory epithelium to the brain of the adult rat. Exp. Brain Res. 63, 461–473. Balin, B.J., Broadwell, R.D., Salcman, M., El-Kalliny, M., 1986. Avenues for entry of peripherally administered protein to the central nervous system in mouse, rat and squirrel monkey. J. Comp. Neurol. 251, 260–280. Bradbury, N.W.B., Cserr, H.F., Westrop, R.J., 1981. Drainage of cerebral interstitial fluid into deep cervical lymph of the rabbit. Am. J. Physiol. 240, 329 – 336. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248 – 254. Brightman, M.W., Reese, T.S., 1969. Junctions between intimately apposed cell membranes in the vertebrate brain. J. Cell Biol. 40, 648 – 677. Cohen, S., Holloway, R.C., Matthews, C., McFarlane, A.S., 1956. Distribution and elimination of 131Iand 14C-labelled plasma proteins in the rabbit. Biochem. J. 62, 143 – 154. Davson, H., 1976. The blood-brain barrier. J. Physiol. 255, 1 – 28. Harik, S.I., McGunigal, T. Jr., 1984. The protective influence of the locus ceruleus on the blood-brain barrier. Ann. Neurol. 15, 568–574. Hinds, J.W., McNelly, N.A., 1977. Aging of the rat olfactory bulb: growth and atrophy of constituent layers and changes in size and number of mitral cells. J. Comp. Neurol. 171, 345 – 368. Kaplan, M.S., Hinds, J.M., 1977. Neurogenesis in the adult rat: Electron microscopic analysis of light radioautographs. Science 197, 1092–1094. Koh, C.S., Paterson, P.Y., 1987. Suppression of clinical signs of cell-transferred experimental allergic encephalomyelitis and altered cerebrovascular permeability in Lewis rats treated with plasminogen activator inhibitor. Cell. Immunol. 107, 52 – 63. Leibowitz, S., Kennedy, L., 1972. Cerebral vascular permeability and cellular infiltration in experimental allergic encephalomyelitis. Immunology 22, 859 – 869. McFarlane, A.S., 1958. Efficient trace-labelling of proteins with iodine. Nature 182, 53. Miyamoto, M., Kiyota, Y., Yamazaki, N., Nagaoka, A., Matsuo, T., Nagawa, Y., Takeda, T., 1986. Age-related changes in learning and memory in the senescence-accelerated mouse (SAM). Physiol. Behav. 38, 399–406. Mooradian, A.D., 1988. Effect of aging on the blood-brain barrier. Neurobiol. Aging 9, 31 – 39. Ohno, K., Pettigrew, K.D., Rapoport, S.I., 1978. Lower limits of cerebrovascular permeability to nonelectrolytes in the conscious rat. Am. J. Physiol. 235, 299 – 307. Pappola, M.A., Andorn, A.C., 1987. Serum protein leakage in aged human brain and inhibition of ligand binding at h2-adrenergic and cholinergic binding sites. Synapse 1, 82 – 89. Poduslo, J.F., Geoffry, L.C., Carole, T.B., 1994. Macromolecular permeability across the blood-nerve and blood-brain barriers. Proc. Natl. Acad. Sci. USA 91, 5705 – 5709. Rapoport, S.I., Ohno, K., Pettigrew, K.D., 1979. Blood-brain barrier permeability in senescent rats. J. Gerontol. 34, 162–169. Rudick, R.C., Buell, S.J., 1983. Integrity of blood-brain barrier to peroxidase in senescent mice. Neurobiol. Aging 4, 283–287. Schmidt-Kastner, R., Szymas, J., Hossmann, K.-A., 1990. Immunohistochemical study of glial reaction and serum-protein extravasation in relation to neuronal damage in rat hippocampus after ischemia. Neuroscience 38, 527–540. M. Ueno et al. / Arch. Gerontol. Geriatr. 25 (1997) 321–331 331 Takeda, T., Hosokawa, M., Takeshita, S., Irino, M., Higuchi, K., Matsushita, T., Tomita, Y., Yasuhira, K., Hanamoto, H., Shimizu, K., Ishii, M., Yamamuro, T., 1981. A new murine model of accelerated senescence. Mech. Ageing Dev. 17, 183194. Takeda, T., Hosokawa, M., Higuchi, K., 1991. Senescence-accelerated mouse (SAM): A novel murine model of accelerated senescence. J. Am. Geriatr. Soc. 39, 911919. Takeda, T., Hosokawa, M., Higuchi, K., Hosono, M., Akiguchi, I., Katoh, H., 1994. A novel murine model of aging, senescence-accelerated mouse (SAM). Arch. Gerontol. Geriatr. 19, 185 – 192. Ueno, M., Akiguchi, I., Naiki, H., Fujibayashi, Y., Fukuyama, H., Kimura, J., Kameyama, M., Takeda, T., 1991. The persistence of high uptake of serum albumin in the olfactory bulbs of mice throughout their adult lives. Arch. Gerontol. Geriatr. 13, 201 – 210. Ueno, M., Akiguchi, I., Yagi, H., Fujibayashi, Y., Kimura, J., Takeda, T., 1993. Age-related changes in barrier function in mouse brain. I. Accelerated age-related increase in brain transfer of serum albumin in accelerated senescence prone SAM-P/8 mice with deficits in learning and memory. Arch. Gerontol. Geriatr. 16, 233–248. Ueno, M., Dobrogowska, D.H., Vorbrodt, A.W., 1996. Immunocytochemical evaluation of the bloodbrain barrier to endogenous albumin in the olfactory bulb and pons of senescence-accelerated mice (SAM). Histochem. Cell Biol. 105, 203 – 212. Yagi, H., Katoh, S., Akiguchi, I., Takeda, T., 1988. Age-related deterioration of ability of acquisition in memory and learning in senescence accelerated mouse. Brain Res. 474, 86 – 93. Yagi, H., Irino, M., Matsushita, T., Katoh, H., Umezawa, M., Tsuboyama, T., Hosokawa, M., Akiguchi, I., Tokunaga, R., Takeda, T., 1989. Spontaneous spongy degeneration of the brain stem in SAM-P/8 mice, a newly developed memory-deficient strain. J. Neuropathol. Exp. Neurol. 48, 577–590. .