Localization of Metallothioneins-I & -II and -III in the Brain of Aged

Localization of Metallothioneins-I & -II and -III in the Brain of Aged Dog

Seiji KOJIMA, Akinori SHIMADA*, Takehito MORITA, Yoshiaki YAMANO1) and Takashi UMEMURA2) Departments of Veterinary Pathology and 1)Metabolic Biochemistry, Tottori University, Minami 4-101, Tottori-shi, Tottori 680-0945, and 2)Laboratory of Comparative Pathology, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo-shi, Hokkaido 060Ð0818, Japan

(Received 2 November 1998/Accepted 7 December 1998)

ABSTRACT. Localization of metallothionein (MT) -I & -II and MT-III and its significance in the brain aging in dogs were examined using immunohistological and molecular pathological techniques. MT-I & -II immunohistochemistry showed positive staining in the hypertrophic astrocytes throughout the aged dog brains; these MT-I & -II immunoreactive astrocytes were predominant in the cerebral cortex and around the blood vessels in the brain. These findings dominated in the brain regions with severe age-related morphological changes. In situ hybridization using MT-I mRNA riboprobes also demonstrated signals for MT-I mRNA in these hypertrophic astrocytes. Immunohistochemistry using a guinea pig antiserum against a synthetic polypeptide of canine MT-III demonstrated positive MT-III immunoreactivity predominantly in neurons in the Zn-rich regions such as hippocampus and parahippocampus. The findings were supported by in situ hybridization using MT-III mRNA riboprobes. Both MT-III immunoreactivity and signals for MT-III mRNA were demonstrated in neurons in the brain regardless of the intensity of the age-related changes. These results suggest, first, MT-I & -II may be induced in relation to the progress of the age-related morphological changes in the brain, playing an important role in the protection of the brain tissue from the toxic insults responsible for the brain aging, and second, MT-III may play a role in maintenance of Zn-related essential functions of the brain.—KEY WORDS: aging, brain, canine, in situ hybridization, metallothionein. J. Vet. Med. Sci. 61(4): 343Ð349, 1999

Metallothioneins (MTs) are a family of low molecular the recent discovery of the brain-specific MT-III, which weight (6,800 Da), cysteine-rich, metal-binding proteins that inhibits neuron survival in vitro, the implications of MTs in are found ubiquitously in a wide variety of species [9]. the brain pathophysiology are strengthened [29]. MT-III MTs are believed to play important roles in the homeostasis mRNA may or may not be down regulated in patients with of essential metals including zinc and copper, in the Alzheimer’s disease (AD) [5, 29]. There is also a detoxification of heavy metals, and in the scavenging of controversy as to the localization of MT-III in the brain. free radicals [4, 8]. Mammalian MT family consists of four MT-III immunoreactivity was localized predominantly in similar but distinct isoforms, designated MT-I, -II, -III and astrocytes of the human and rat brains [1, 30, 31]. On the -IV [16, 19]. MT-I and MT-II are believed to be essentially other hand, MT-III mRNA was demonstrated exclusively in the same because both proteins show high homology (93.4% neurons of mouse brain [12]. Thus, precise localization as identity) in amino acid sequence and share expression well as physiological roles of MTs in the brain are yet to be pattern; both MT-I and MT-II genes are expressed in various determined. organs including liver, kidney and brain, while MT-III is We have previously reported the age-related expressed only in the brain [16]. The brain specific MT-III morphological changes in the brain of dogs [22, 23]. In an contains 7 additional amino acids as compared to 61 amino attempt to better understand the pathophysiological roles of acids for MT-I & -II isoforms [16]. In comparison with MTs, we studied the localization of MT-I & -II and MT-III MT-I & -II, the genes of which are readily induced by heavy in both protein and mRNA levels in the brain of aged dogs. metals, MT-III do not show quick expression by these metals both in vivo and in vitro, suggesting MT-III is different MATERIALS AND METHODS from the other MT isotypes [5]. The expression of MT-IV is restricted to cornified and stratified squamous epithelium Animals: Ten aged dogs, five males and five females, of skin, tongue, and upper alimentary tract [19]. ranging in age from 12 to 18 years were used in the present Immunohistochemical localization of MT-I & -II in the study (Table 1). Three young beagle dogs ranging in age brain has been reported in humans [3, 27, 28], monkeys from 6 to 9 months were used as controls. The animals [26], mice [12, 14] and rats [14, 32]; MT-I & -II were were killed by euthanasia or died of a variety of disorders shown in choroid plexus epithelial cells, ependymal cells, including heart failure, tumor and renal failure. No signs of pia mater, and astrocytes in the hypothalamus, hippocampus, neurological disorders were observed in these dogs. cerebral cortex and white matter throughout the brain. With Histopathology: Brains were taken immediately after death and fixed in 10% phosphate-buffered formalin for three days. Brain samples at the level of frontal lobe, *CORRESPONDENCE TO: SHIMADA, A., Department of Veterinary Pathology, Tottori University, Minami 4-101, Tottori 680Ð occipital lobe, corpus striatum and diencephalon were 0945, Japan. trimmed and embedded in paraffin wax. Serial sections cut 344 S. KOJIMA ET AL.

Table 1. Clinical findings of the dogs

Dog No. Age Sex Breed Clinical findings (years)

1 8 Male Mongrel Renal failure 2 8 Female Mongrel Mammary gland tumor 3 11 Female Mongrel Intervertebral disc hernia 4 12 Male Yorkshire Terrier Dermatitis 5 12 Male Pomeranian Mammary gland tumor 6 15 Male Mongrel Renal failure 7 15 Female Mongrel Mastocytoma 8 16 Male Mongrel Renal failure 9 16 Female Mongrel Filariasis 10 18 Female Yorkshire Terrier Renal failure at 5 µm were stained with hematoxylin and eosin (HE) and mammalian species of MT [7]. Using the antibody, we periodic acid methenamine silver (PAM). have reported a part of findings of MT- Generation and characterization of the polyclonal anti immunohistochemistry in the brain of dogs under canine MT-III antibody: We selected amino acids sequence physiological and pathological conditions [21, 24, 25]. (51Ð63: Cys-Lys-Gly-Gly-Glu-Gly-Thr-Glu-Ala-Glu-Ala- Monoclonal antibody to glial fibrillary acidic protein Glu-Lys) of the canine MT-III protein for generation of the (GFAP) (Dako) was used to identify hypertrophic astrocytes polyclonal antibody; the residue contained six amino acid [17]. insertion, which is unique to the canine MT-III [10]. The After blocking endogeneous peroxidase activity with 3% polypeptide specific to canine MT-III was synthesized and H2O2, sections were treated with 10% normal bovine serum, conjugated at COOH-terminus SH group to keyhole limpet incubated with primary antibodies overnight at 4°C and then hemocyanine (Bio-Synthesis Inc., Texas, U.S.A.). After sequentially incubated with biotinylated goat anti mouse IgG preimmune serum was collected, Hartley guinea pigs were (Dako) (1: 400) for the monoclonal antibodies and with given subcutaneous injection of 500 µg of keyhole limpet biotinylated goat anti guinea pig IgG (Funakoshi) (1: 200) hemocyanine-peptide/50% Freund’s complete adjuvant. The for the polyclonal antibody for 30 min at room temperature, animals received three to four additional boosts at and in peroxidase-conjugated streptavidin (Dako) for 30 approximately 2 week intervals, consisting of 500 µg of the min. Primary antibodies were diluted with PBS (1: 200 for same adjuvant. Sera were collected after the immunizations. MT-III; 1:400 for MT-I & -II; 1:400 for GFAP). After the The specificity of the polyclonal anti canine MT-III antibody first and second incubations, the sections were given three 5 was determined by western blotting of total protein extracts min washes in phosphate-buffered saline (PBS) and then from frozen tissues including the liver, kidney and brain, developed with 3,3-diaminobenzidine tetrahydrochloride and respectively [13]. Briefly, the proteins were electrophoresed H2O2. The sections were counterstained with methyl green. on 15% sodium dodecyl sulfate polyacrylamide gel under The specificity of the MT-III polyclonal antibody was tested reducing conditions, and electroblotted to clear blot P by preabsorption techniques in which the antibody was membrane (ATTO, Tokyo, Japan). The membrane was preabsorbed with an excess amount (100 mM) of the initial blocked with 5% skim milk in Tris-buffer and incubated antigen. GFAP, MT-I & -II and MT-III immunopositive with anti canine MT-III antibody (1:200). The membrane cells were determined by light microscopy at × 200 was then, reacted with biotinylated antisera against guinea magnification. For each slide, 5 randomly selected fields pig immunoglobulin (1:200, Funakoshi, Tokyo, Japan). The were examined. The mean value per one field was bound immunoglobulin was visualized with 3,3- calculated from the total number of immunopositive cells in diaminobenzidine. The specificity of the MT-III polyclonal each slide. Their incidence was graded from (-) to (+++) : antibody was tested by the preabsorption techniques in －: 0/field, ±: 1-10/field, +: 11-20/field, ++: 21Ð30/field which the antibody was preabsorbed with an excess amount and +++ ≧ 31/field. (100 mM) of the initial antigen (MT-III specific synthetic In situ hybridization: Riboprobes were prepared from polypeptide, Cys-Lys-Gly-Gly-Glu-Gly-Thr-Glu-Ala-Glu- MT-I and -III cDNA constructs in pGEM-T Easy vector Ala-Glu-Lys). (Promega, Madison, U.S.A.). The MT-I construct contains Immunohistochemistry: The primary antibodies used in 109 base pair (bp) of the COOH-terminus non coding region this study were MT-III polyclonal antibody against canine (184 bp through 293 bp) of the canine MT-I cDNA [10]. MT-III and monoclonal antibody against horse MT-I & -II The MT-III construct contains 110 bp of the COOH- (E9; Dako, Glostrup, Denmark). The monoclonal MT-I & - terminus non coding region (291 bp through 401 bp) of II antibody is specifically reactive with a conserved epitope canine MT-III cDNA [11]. Homology between the MT-I of first five amino acid residues of the N-terminal portion and -III probe is 25%. Digoxigenin-labeled riboprobes were of the molecule; the epitope is common to several synthesized by in vitro transcription from the linearized METALLOTHIONEIN EXPRESSION IN THE AGED DOG BRAIN 345 cDNA templates using the appropriate RNA polymerase (SP6/T7) according to the manufacture’s protocol (Boehringer Mannheim, Mannheim, Germany). These riboprobes were used for in situ hybridization. Coronal paraffin sections (5 µm) cut at a level of diencephalon were used. Hybridization method used was reported before [11]. Briefly, after hybridization, the parafilm was dislodged by washing with 5 × SSC (1 × SSC = 0.15 M NaCl, 0.015 M Sodium citrate) and with 50% formamide, 2 × SSC for 60 min at 50°C, followed by two 10-min-washings at 50°C with 0.1 × SDS, 0.1 × SSC, 50% formamide. The slides were then incubated with alkaline-phosphatase-conjugated Fig. 1. Western blotting analysis of the brain, liver anti-digoxigenin antibody (Boehringer Mannheim). and kidney from a 6-month-old dog. Lane1-3: Following washing in Tris buffer nitroblue tetrazolium, proteins isolated from liver, kidney and brain, development solution was applied in accordance with the respectively. The polyclonal canine MT-III antibody stained a single band of approximately 6 manufacturer’s protocol and the slides were placed in a dark, kDa exclusively in the lane 3. Lane 4: protein humid chamber. After color reaction, the slides were rinsed isolated from brain. Antibody preabsorbed with with 10 mM Tris-HCl (pH 8.0), 1 mM EDTA and mounted an excess amount of the initial antigen stained no with counterstaining by methyl green. band.

RESULTS Table 2. Summary of GFAP, MT-I & -II and MT-III immunohistochemistry Western blotting analysis: Western blotting of the brain, liver and kidney using anti canine MT-III antibody revealed Dog No. GFAP MT-I & II MT-III a single band exclusively in the lane of brain at Control 1 ±±++ approximately 6 kDa that corresponded in size to the Control 2 Ð ± +++ reported molecular size of MTs [16, 19] (Fig. 1). No band Control 3 ±±+++ was shown in the lane of the liver and kidney, indicating 1 ± ++ ±± the antibody obtained is specific to MT-III. The absorption 2 ++ 3+ + ++ of the polyclonal anti canine MT-III antibody with initial 4 + ++ ++ antigen abolished the positive band. 5++++++ Histological and Immunohistochemical findings: Gross 6++++++ and microscopic examinations of the brain of the aged dogs 7 +++ ++ ++ revealed no signs of neurological disorders other than typical 8 ++ +++ +++ aged changes, such as thickening of the meninges, mild 9 +++ +++ ++ 10 +++ +++ ++ dilation of the ventricles, lipofuscin deposits in nerve cell bodies, neuronal loss, diffuse gliosis, occasional dystrophic Ð: 0/field, ±: 1Ð10/field, +: 11Ð20/filed, ++: 21Ð30/field, axons and senile plaques. +++: ≧31/field. Results of the immunohistochemical study on GFAP, MT- I & -II and MT-III were summarized in Table 2. An increase MT-III immunoreactivity was shown exclusively in nerve in GFAP positive astrocytes in the aged dogs, as compared cell bodies in all dogs examined. The intensity of the to controls, was the most consistent histological feature immunoreactivity differed from case to case and from region observed in the GFAP immunohistochemistry. These GFAP to region in the brain. However, MT-III immunoreactive immunoreactive astrocytes were seen in the cerebral cortical neurons were consistently demonstrated in the hippocampus layer from III to V, subpial area, white matter and CA1 and CA2 regions; the intensity was stronger in the periventricular area in the diencephalon (Fig. 2A). In control CA1 than CA2 (data not shown). Consistent dogs, there were a few GFAP immunoreactive astrocytes immunoreactivity was also shown in large neurons of (Fig. 2B). MT-I & -II immunoreactivity was shown in both parahippocampus and thalamic nuclei including nucleus nucleus and cytoplasm of astrocytes, which distributed in anterior thalami, nucleus reticularis thalami and nucleus the thalamus, white matter, hippocampus, periventricular ventralis anterior in all dogs examined; neurons in the area and cerebral cortical layer from III to V; the finding nucleus ventralis anterior showed strongest was remarkable around the blood vessels of each regions immunoreactivity (Fig. 4). There was no MT-III (Fig. 3A). The intensity of the MT-I & -II immunostaining immunolabelling in the other cell types including astrocyte, was stronger in the brain regions with severe age-related oligodendrocyte and microglia. The absorption of the anti morphological changes. A small number of MT-I & -II canine MT-III antibody with the initial antigen abolished all immunoreactive astrocytes were observed in the brain of staining. There was no staining observed when the anti control dogs (Fig. 3B). canine MT-III antibody was omitted from the procedure. 346 S. KOJIMA ET AL.

Fig. 2. Cerebral cortices from a 16-year-old dog (A) and from a 6-month-old dog (B). Note the intense GFAP immunoreactivity in the process of hypertrophic astrocytes in A. GFAP immunostain. Bars=30 µm.

Fig. 3. Cerebral cortices from a 16-year-old dog (A) and a 6-month-old dog (B). Increased number of MT-I & -II immunoreactive astrocytes are observed in A. Note a few astrocytes (arrows) showing weak MT-I & -II immunoreactivity in B. Bars=30 µm. Inset: Signals for MT-I mRNA are shown in an astrocyte (arrow). Note no signals in neurons. × 120. In situ hybridization (MT-I mRNA).

In situ hybridization: Canine MT-I mRNA was brain, especially in those in the cerebral cortex (Fig. 3A). demonstrated predominantly in astrocytes throughout the Canine MT-III mRNA was shown exclusively in the nerve METALLOTHIONEIN EXPRESSION IN THE AGED DOG BRAIN 347

Fig. 4. Thalamus from a 15-year-old dog (A) and from a 6-month-old dog (B), showing MT-III positive finding exclusively in nerve cell bodies. MT-III immunostain. Bars=30 µm. cell bodies in the hippocampus from CA1 to CA4 regions and in large neurons of parahippocampus (Fig. 5). Intense hybridization signals were also demonstrated in the thalamic nuclei; neurons in the nucleus ventralis anterior showed strongest signals. A lower level of hybridization signal was also observed in neurons in the cerebral cortical layer from II to V. The findings of in situ hybridization corresponded to those of immunohistochemistry. No signal was demonstrated when MT-I and MT-III sense riboprobes were used as negative controls (data not shown).

DISCUSSION

Aged dog brain shows a variety of age-related morphological changes including atrophy, signs of neuronal loss, axonal dystrophy, β-amyloidosis and astrocytic gliosis: these changes are very similar to those observed in the brain of aged humans [22, 23]. In the present study, GFAP- positive astrocytic gliosis was demonstrated in all the aged dog brain. The change is believed to be a reaction to the loss of neurons and dendrites [2, 20]. Astrocytes are shown to produce trophic factors, playing important roles in the regulation of the microenvironment in the brain [6, 18]. The possibility exists that astrocytic hypertrophy represents a primary effect of aging rather than a mere secondary Fig. 5. Hippocampus from an 18-year-old dog, showing predominant signals for MT-III mRNA in neurons. response to degenerative events in the brain [15]. Bar=30 µm. In situ hybridization (MT-III mRNA). MT-I & -II immunoreactivity was shown in astrocytes, ependymal cells and choroid plexus epithelial cells in the brain of dogs. The findings agreed with those reported in and mice [12, 14]. Intense MT-I & -II immunoreactivity the brain of humans [3, 27, 28], monkeys [26], rats [14, 32] was observed in astrocytes in the periventricular area and 348 S. KOJIMA ET AL. those around the blood vessels, indicating that MT-I & -II 11. Kojima, S., Kodan, A., Kobayashi, K., Morita, T., Shimada, may play a role as a barrier against the toxic substances A., Yamano, Y. and Umemura, T. 1998. Molecular cloning including metals. MT-I & -II positive astrocytes were also and expression of the canine metallothionein-III gene. Can. predominant in the brain regions with severe age-related J. Vet. Res. 62: 148Ð151. changes, such as neuronal loss and axonal dystrophy. 12. Masters, B. A., Quaife, C. J., Erickson, J. C., Kelly, E. J., Froelick, G. J., Zambrowicz, B. P., Brinster, R. L. and Therefore, MT-I & -II may play an important role in the Palmiter, R. D. 1994. Metallothionein III is expressed in neu- protection of the brain tissue from the toxic insults rons that sequester zinc in synaptic vesicles. J. Neurosci. 14: responsible for the brain aging. 5844Ð5857. As to the role of MT-III, maintenance of Zn-related 13. Mizzen, C. A., Cartel, N. J., Yu, W. H., Fraser, P. E. and essential functions of the brain has been reported based on McLachalan, D. R. 1996. Sensitive detection of the study of the spatio-temporal pattern of MT-III gene metallothioneins-1, -2 and -3 in tissue homogenates by im- expression in the brain of mice [12]. Predominant and munoblotting: A method for enhanced membrane transfer and consistent expression of MT-III was also shown in neurons retention. J. Biochem. Biophys. Methods 32: 77Ð83. in the Zn-rich regions such as hippocampus and 14. Nishimura, N., Nishimura, H., Ghaffar, A. and Tohyama, C. parahippocampus in the brain of dogs. Both MT-III 1992. Localization of metallothionein in the brain of rat and mouse. J. Histochem. Cytochem. 40: 309Ð315. immunoreactivity and signals for MT-III mRNA were 15. O’callaghan, J. P. and Miller, D. B. 1991. The concentration demonstrated in neurons in the brain of dogs regardless of of glial fibrillary acidic protein increase with age in the mouse the intensity of the age-related changes. Further study is and rat brain. Neurobiol. Aging 12: 171Ð174. required for the elucidation of detailed functions of MT-III 16. Palmiter, R. D., Findley, S. D., Whitmoer, T. E. and Durnam, in the brain. D. M. 1992. MT-III, a brain-specific member of the metallothionein gene family. Proc. Natl. Acad. Sci. U.S.A. ACKNOWLEDGMENTS. We thank Dr. T. Sanekata, 89: 6333Ð6337. Department of Veterinary Microbiology, Tottori University, 17. Petito, C. K., Morgello, S., Felix, J. C. and Lesser, M. L. for his usefull suggestion in the generation of the polyclonal 1990. The two patterns of reactive astrocytosis in postischemic antibody. This study was supported by a Grant-in-Aid for rat brain. J. Cereb. Blood Flow Metab. 10: 850Ð859. 18. Politis, M. J. and Miller, J. E. 1985. The roles of reactive General Scientific Research (08660382) from the Ministry gliosis and mitosis on trophic factor production in trauma- of Education, Science and Culture, Japan. tized nervous system tissue. Brain Res. 346: 186Ð189. 19. Quaife, C. J., Jay, S. D., Glenda, C. E., Froelick, J., Kelly, E. REFERENCES J., Zambrowicz, B. P. and Palmiter, R. D. 1994. Induction of a new metallothionein isoform (MT-IV) occurs during differ- 1. Anezaki, A., Ishiguro, H., Hozumi, I., Inuzuka, T., Hiraiwa, entiation of stratified squamous epithelia. Biochemistry 33: M., Kobayashi, H., Yuguchi, T., Wanaka, A., Uda, Y., 7250Ð7259. Miyatake, T., Yamada, K., Tohyama, M. and Tsuji, S. 1995. 20. Scheibel, M. E. and Scheibel, A. B. 1975. Structural changes Expression of growth inhibitory factor (GIF) in normal and in the aging brain. pp. 11Ð37. In: Clinical, Morphologic, and injured rat brains. Neurochem. Int. 27: 89Ð94. Neurochemical Aspects in the Aging Central Nervous Sys- 2. Beach, T. G., Walker, R. and McGeer, E. G. 1989. Patterns tem, vol. 1 (Brody, H., Harman, D. and Ordy, J. M. eds.), of gliosis in Alzheimer’s disease and aging cerebrum. Glia 2: Raven Press, New York. 420Ð436. 21. Shimada, A., Irie, M., Kojima, S., Yamano, Y., Kobayashi, 3. Blaauwgeers, H. G. T., Sillevis, P. A. E., Dejong, J. M. B. V. K. and Umemura, T. 1996. Immunohistochemical localiza- and Troost, D. 1993. Distribution of metallothionein in the tion of metallothionein in the olfactory pathway of dogs. J. human central nervous system. Glia 8: 62Ð70. Vet. Med. Sci. 58: 983Ð988. 4. Dunn, M. A., Blalock, T, L. and Cousins, R. J. 1987. 22. Shimada, A., Kuwamura, M., Awakura, T., Umemura, T. and Metallothionein. Proc. Soc. Exp. Biol. Med. 185: 107Ð119. Itakura, C. 1992. An immunohistochemical and ultrastruc- 5. Erickson, J. C., Hollopeter, G., Thomas, S. A., Froelick, G. J. tural study on age-related astrocytic gliosis in the central and Palmiter, R. D. 1997. Disruption of the metallothionein- nervous system of dogs. J. Vet. Med. Sci. 54: 29Ð36. III gene in mice: Analysis of brain zinc, behavior, and neuron 23. Shimada, A., Kuwamura, M., Awakura, T., Umemura, T., vulnerability to metals, aging, and seizures. J. Neruosci. 17: Takeda, K., Ohama, E. and Itakura, C. 1992. Topographic 1271Ð1281. relationship between senile plaques and cerebrovascular amy- 6. Frei, K., Bodmer, S., Schwerdel, C. and Fontana, A. 1985. loidosis in the brain of aged dogs. J. Vet. Med. Sci. 54: Astrocytes of the brain synthesize interleukin 3-like factors. 137Ð144. J. Immunol. 135: 4044Ð4047. 24. Shimada, A., Yamamura, Y., Uemura, T., Kojima, S. and 7. Jasani, B. and Elmes, M. E. 1991. Immunohistochemical de- Umemura, T. 1998. Localization of metallothionein in a vari- tection of metallothionein. Methods Enzymol. 205: 95Ð107. ety of brain lesions in dogs. J. Vet. Med. Sci. 60: 351Ð358. 8. Karin, M. 1985. Metallothionein: Proteins in search of func- 25. Shimada, A., Yanagida, M. and Umemura, T. 1997. An im- tion. Cell 41: 9Ð10. munohistochemical study on the tissue specific localization 9. Kille, P., Hemmings, A. and Lunney, E. A. 1994. Memories of metallothionein in dogs. J. Comp. Pathol. 116: 1Ð11. of metallothionein. Biochem. Biophys. Acta 1205: 151Ð161. 26. Suzuki, K., Nakajima, K., Kawaharada, U., Hara, F., Uehara, 10. Kobayashi, K., Shimada, A., Yamano, Y. and Umemura, T. K., Otaki, N., Kimura, M. and Tamura, Y. 1992. Localization 1997. Molecular cloning of a canine metallothionein cDNA. of metallothionein in the brain of Macaca fascicularis. Acta J. Vet. Med. Sci. 59: 819Ð823. Histochem. Cytochem. 25: 609Ð616. METALLOTHIONEIN EXPRESSION IN THE AGED DOG BRAIN 349

27. Suzuki, K., Nakajima, K., Kawaharada, U., Uehara, K., Hara, 30. Uchida, Y., Takio, K., Titani, K., Ihara, Y. and Tomonaga, F., Otaki, N., Kimura, M. and Tamura, Y. 1992. M. 1991. The growth inhibitory factor that is deficient in the Metallothionein in the human brain. Acta Histochem. Alzheimer’s disease brain is a 68 amino acid metallothionein- Cytochem. 25: 617Ð622. like protein. Neuron 7: 337Ð347. 28. Suzuki, K., Nakajima, K., Otaki, N., Kimura, M., Kawaharada, 31. Yamada, M., Hayashi, S., Hozumi, I., Inuzuka, T., Tsuji, S. U., Uehara, K., Hara, F., Nakazato, Y. and Takatama, M. and Takahashi, H. 1996. Subcellular localization of growth 1994. Localization of metallothionein in aged human brain. inhibitory factor in rat brain: Light and electron microscopic Pathol. Internat. 44: 20Ð26. immunohistochemical studies. Brain Res. 735: 257Ð264. 29. Tsuji, S., Kobayashi, H., Uchida, Y., Ihara, Y. and Miyatake 32. Young, J. K., Garvey, J. S. and Huang, P.C. 1991. Glial T. 1992. Molecular cloning of human growth inhibitory fac- immunoreactivity for metallothionein in the rat brain. Glia 4: tor cDNA and its down-regulation in Alzheimer’s disease. 602Ð610. EMBO J. 11: 4843Ð4850.