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Early and transient increase in oxidative stress in the cerebral cortex of senescence-accelerated mouse
nrtdmhsofagaing Mechanisms of Ageing and Development
ELSEVIER
105
anddevelopment
86 (1996) 105-114
Early and transient increase in oxidative stress in the cerebral cortex of senescence-accelerated mouse Eiji Sato, Nanae Oda, Naoko Ozaki, Shin-ichi Hashimoto, Kurokawa*, Sadahiko Ishibashi
Tomonori
Department of Physiological Chemistry, Hiroshima University School of Medicine, Minami-lcu. Hiroshima 734, Japan
Received 1 August 1995; accepted 10 October 1995
Abstract Age-related changes in oxidative stress in the cerebral cortex of SAMP8, a substrain of senescence-accelerated mouse (SAM), were investigated in comparison with those in SAMRl, which were used as a control. The lipid peroxide and protein carbonyl contents were transiently increased in SAMP8 from 4- to &weeks of age. The increases in lipid peroxide were seen only in the cerebral cortex and not in other regions of the cerebrum. Furthermore, the net generation of reactive oxygen species in cerebral cells was also increased in SAMP8. In addition, the activity of glutamine synthetase, which is known as an enzyme highly sensitive to reactive oxygen, was decreased in the cerebral cortex of SAMP8 from 4- to 8-weeks of age. These results suggest that oxidative stress may be induced in the cerebral cortex of SAMP8 from 4- to 8-weeks of age, preceding the appearance of distinctive deficits in the brain of SAMPS. Keywords:
Senescence-accelerated
mouse (SAM); SAMP8; SAMRl; Reactive oxygen; Oxida-
tive stress
* Corresponding
author. Tel.: + 81 82 2575306; fax: + 81 82 2575309.
0047-6374/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved SSDI 0047-6374(95)01681-3
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1. Introduction Senescence-accelerated mouse (SAM), an experimental murine model of accelerated aging, was established by Takeda et al. [l]. SAM consists of two strains; accelerated senescence-prone mouse (SAMP), and accelerated senescence-resistant mouse (SAMR). Following a normal process of development, the SAMP shows an accelerated change with many signs of aging, such as a short life span, changes in general behavior, loss of skin glossiness, increased skin coarseness, hair loss, periophthalmic lesions, and increased lordokyphosis of the spine [l]. At present, SAMP consists of nine substrains (SAMPl, P2, P3, P6, P7, P8, P9, PlO, and Pl I), and each substrain has relatively specific pathologic phenotypes, in addition to having characteristics common to each of the SAMP strains (i.e. accelerated senescence). For example, senile amyloidosis in SAMPl [2,3], degenerative joint disease in SAMP3 [4], senile osteoporosis in SAMP6 [5], and cataract in SAMP9 [6] were demonstrated. In addition, SAMPS shows some distinctive defects in the brain, such as age-related deterioration of ability in learning and memory [7-g], and has been used as a useful animal model for research into senile memory impairment. While it has been shown that some pathomorphological changes, such as spongiform degeneration [lo], PAS-positive granular structures [l 11, and /?A4 protein-like immuno-reactive granular structures [12], were increased in SAMP8, the mechanisms responsible for these degenerative processes have remained unclear. In contrast to these SAMP, SAMR consists of three substrains (SAMRl, R4, and R5), which have essentially the same aging characteristics as SAMR2, an another control strain which was used in our previous studies. Oxygen free radicals have been implicated in the process of several degenerative diseases, as well as in the physiological decline associated with aging [15]. These highly reactive molecules produced within aerobic cells in the course of normal metabolic events, oxidize cellular components such as lipid, protein, and DNA, which in turn result in undesirable changes in cellular functions. In particular, oxidative stress in the brain is of interest in this regard because the brain consumes a lot of oxygen and contains relatively high concentrations of easily peroxidizable fatty acids. In fact, in some stressful conditions, such as various types of traumas and ischemia/reperfusion injury, excessive production of oxygen metabolites is induced, and it could constitute an important cause of neuronal degeneration. It has also been shown that administration of radical scavengers to aged animals restored loss of temporal and spatial memory, as well as increasing protein carbonyl content in the brain to the levels of younger ones [16]. These observations suggest that the oxidative modification of cellular components may play a critical role in the decline in the cellular functions associated with its degenerative process. Thus, we are interested in the involvement of oxidative stress in the early deficits observed in the brain of SAMP8. In this study, we report that oxidative stress may be induced in the cerebral cortex of SAMPS from 4- to 8-weeks of age, preceding the appearance of distinctive defects in the brain of SAMP8.
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2. Materials and methods 2. I. Reagents 2’,7’-dichlorofluorescin diacetate (DCFH-DA) was purchased from Eastman Kodak (New York, USA). 2’,7’-dichlorofluorescein (DCF) was from Nacalai Tesque Inc. (Kyoto, Japan). Monochlorobimane (mBC1) was from Molecular Probes Inc. (Eugene, Oregon, USA). All other agents used were of reagent grade from standard commercial sources. 2.2. Animals SAMP8 and SAMRl, as control strains, were generously donated by Professor Toshio Takeda (Kyoto University, Japan). They were bred under conventional conditions with food and water ad libitum. Male and female mice of 2, 4-8, 20-28, and 40-56 weeks of age were used in this study. 2.3. Tissue preparations After decapitation of the mouse, the cerebral cortex, hippocampus and residual region of the cerebrum (remainder) were rapidly dissociated and placed on a chilled surface. The superficial blood vessels were removed and the surface of the brain was washed with ice-cold Hanks’ solution buffered with Pipes (pH 7.3, 137 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO,, 1.3 mM CaCl,, 0.4 mM Na,HPO,, 0.4 mM KH,PO,, 5.5 mM D-glucose, and 8 mM Pipes). The dissociated cerebral cells were prepared as previously reported [13] with a minor modification in which all solutions included 5.5 mM D-glucose. Cerebral cortex extracts were prepared as follows. The dissociated cerebral cortex was homogenized in 10 mM Hepes buffer (pH 7.4) containing 137 mM NaCl, 4.6 mM KCl, 1.1 mM KH,PO,, 1.I mM EDTA, 0.6 mM MgSO,, leupeptin (0.5 pg/ml), pepstatin (0.7 pg/ml), aprotinin (0.5 pg/ml), and phenylmethylsulfonyl fluoride (40 pg/ml). Insoluble debris was pelleted at 100 000 x g for 1 h. The clear supernatant was recovered and used for the protein carbonyl and glutamine synthetase assays. Homogenates for the measurement of lipid peroxide content were prepared from the hippocampus and remainder, as well as the cerebral cortex, by using a Potter-Elvehjem homogenizer in 150 mM KC1 and 50 mM Tris-HCl (pH 7.4). The protein content of the samples was determined by the method of Lowry et al. [17]. 2.4. Assays The lipid peroxide (thiobarbituric acid reactive substance (TBARS)) content of the homogenates of the cerebral cortex, hippocampus and remainder were determined by the method of Buege and Aust [18] using 1,1,3,3_tetraethoxypropane as a standard. The protein carbonyl content was determined spectrophotometrically by the 2,4_dinitrophenylhydrazine (DNPH)-labeling procedure [19]. Results are ex-
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pressed as nmols of DNPH incorporated/mg protein, calculated on the basis of an extinction coefficient of 22.0 mM - ’ cm - l for aliphatic hydrazones. Glutamine synthetase activity was determined by a y-glutamyl transfer assay, as previously reported [20]. One unit of glutamine synthetase activity is defined as the amount of activity catalyzing the formation of 1 pmol of product (y-glutamyl hydroxamate) per min under specified assay conditions. The values were corrected for nonspecific glutaminase activity by comparing the activities in the presence and absence of ADP and arsenate. The net cellular generation of the reactive oxygen species in the dissociated cells was measured using the fluorescent probe 2’,7’-dichlorofluorescin diacetate (DCFH-DA) [21], using 2’,7’-dichlorofluorescein (DCF) as a standard. In brief, the dissociated cells were incubated at 37°C with DCFH-DA (2.5 pug/ml) in the presence, or absence, of 1 mM diethyl maleate for O-90 min, and then fluorescence at 485 nm, in response to excitation at 530 nm, was measured. Cellular glutathione content was determined using monochlorobimane (mBC1) [22]. In brief, after treatment of the dissociated cells with 1 mM diethyl maleate for O-60 min, mBCl(40 PM) was added and the tills were further incubated for 15 min, and then fluorescence at 460 nm, in response to excitation at 395 nm, was measured. 2.5. Statistical analysis Student’s unpaired t-test and, for multiple group comparisons, a one-way analysis of variance (ANOVA) test were employed to determine statistical significance.
3. Results Age-related changes in the lipid peroxide and protein carbonyl content in the brain of SAM mice were examined in the cerebral homogenate and extract, respectively. In both strains, the lipid peroxide content increased at 40-56 weeks of age, and that of protein carbonyl exhibited a biphasic change with age; it increased up to 20-28 weeks of age, and then decreased (Fig. lA,B). Interestingly, the content of both increased at 4-8 weeks of age in SAMP8, as compared with those in SAMRI. No significant differences were observed between these two strains in other aged mice. Next, age-related changes in the activity of glutamine synthetase, which is known as an enzyme highly sensitive to reactive oxygen, were examined in the cerebral extracts. As shown in Fig. lC, the activity was decreased in SAMP8 at 4-8 weeks of age. No significant differences were observed between these two strains in other aged mice. To characterize further the increase in oxidative stress in the brain of 4-8-week-old SAMP8, the content of lipid peroxide in the hippocampus and residual region of the cerebrum (remainder) were examined in addition to the cerebral cortex.The significant increase in lipid peroxides was seen only in the cerebral cortex and not in other regions of the cerebrum (Fig. 1D). These results indicate that a possible increase in oxidative stress exists in the cerebral cortex of SAMP8 at an earlier stage.
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Next, the net generation of reactive oxygen species was determined using the fluorescent probe 2’,7’-dichlorofluorescin diacetate (DCFH-DA). DCFH-DA, a non-polar and non-fluorescent compound that readily diffuses into cells, is hydrolyzed by intracellular esterases to the non-fluorescent derivative 2’,7’-dichlorofluorescin (DCFH). In the presence of intracellular hydrogen peroxide, DCFH is oxidized to the highly fluorescent compound 2’,7’-dichlorofluorescein (DCF) [21].
$j * g
~~~~
i
0.5
0
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20I
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age (weeks)
age (weeks)
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‘j= z
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age (weeks) Fig. 1. Age-related changes in lipid peroxide content (A), protein carbonyl content (B), in the activities of glutamine synthetase (C) in the cerebral cortex, and in the differences of lipid peroxide content among cerebral regions in 4-8-weeks-old mice (D). (A) and (D), and (B) and (C) were determined in the brain homogenates and extracts, respectively, as described in Materials and methods. (A)-(C): Differences between the two strains were observed only at 4-8 weeks of age: 0, SAMPI: and 0, SAMRI. (D): An increase in lipid peroxides in SAMPI was observed only in the cerebral cortex. Data are expressed as a mean k SE. of S- 13 mice. “P < 0.01 vs. those of corresponding SAMRl values. b,cP < 0.05 vs. those of 2- and 20-28-week-old SAMPB and SAMRl, respectively. d,eP <: 0.01 vs. those of 2- and 40-56-weekold SAMP8 and SAMRl, respectively. ‘Pi 0.01 vs. those of 2- and 4-8-week-old SAMPI. gP <. 0.01 vs. that of 2-week-old SAMRI.
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0
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40 time
60 (min)
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Fig. 2. Net generation of reactive oxygen species (A) and changes in cellular glutathione content (B) with diethyl maleate treatment. Dissociated cerebral cells prepared from 4-&week-old SAMP8 (00) and SAMRl (A A) were incubated with DCFH-DA (A) or mBC1 (B) in the presence (0 a) or absence (.A) of 1 mM diethyl maleate, and then fluorescence intensity was measured as described in Materials and methods. Data are expressed as a mean _+S.E. of 4- 13 mice.“P < 0.05 vs. those of corresponding SAMRl values.
The DCF content of cerebral cells increased during the incubation for both strains, and was significantly higher in SAMP8 than in SAMRl at 90 min (Fig. 2A). The difference between these strains was more significant when the cells were treated with 1 mM diethyl maleate to deplete cellular glutathione. Since the elevated accumulation of DCF might result from the difference in cellular glutathione content between these two strains, the glutathione content of cerebral cells was measured using the fluorescent probe monochlorobimane (mBC1). While the fluorescence intensity was markedly decreased during the treatment with diethyl maleate in both strains, no difference was observed between these two strains (Fig. 2B). The fluorescent intensity after incubation for 60 min in the absence of diethyl maleate was slightly decreased and there was no difference between the two strains. These results suggest that the increased net generation of reactive oxygen species, without a particular decrease in cellular glutathione, may possibly contribute to the oxidative stress in 4-8-week-old SAMP8, which leads to the increase in lipid peroxide and protein carbonyl levels.
4. Discussion It has been reported that some defects in the brain, such as deterioration of ability in learning and memory, begins to progress as early as 2 months after birth in SAMP8 [7]. It is not clear, however, what precedes this appearance. In this study, we found that the lipid peroxide and protein carbonyl content increased, and the activity of glutamine synthetase decreased, in the cerebral cortex of SAMP8 at 4-8
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weeks of age. Glutamine synthetase is known as being an enzyme highly sensitive to reactive oxygen, and a loss of enzyme activity, associated with an increase in protein carbonyl content, has been reported in human brain [23], cultured fibroblasts [24], and rat liver [25], as well as in ischemia/reperfusion-injured gerbil brain [26]. In addition, the net generation of reactive oxygen species was actually increased in SAMP8. These results suggest that oxidative stress may increase in the cerebral cortex of SAMP8 at an earlier stage. Because the increase was observed transiently, prior to such defects in the brain, and was not found in the control senescence-resistant strain, it is plausible that some relationships may exist between oxidative stress at an earlier age and degenerative processes in the brain observed later in SAMPS. However, it remains an interesting problem as to how the oxidative stress at 4-8 weeks of age, with its complete recovery to the level of control mice after the age of 20-28 weeks, has etiologic importance for the following chronic neurological symptoms in these mice. In normal aging, oxidative stress does not exhibit such transient characteristics. It may be assumed that the transient nature observed in oxidative stress is a specific characteristic of accelerated aging in the brain of SAMP8. We did not observe the age-related differences in the lipid peroxide and protein carbonyl content and in the activities of glutamine synthetase of 4-&-week-old SAMP8, indicating that oxidative stress may continue for at least 4 weeks at the younger stage in these mice. Because 4 weeks is a very big period in the life of a mouse to be considered as one age point (i.e. it is about one tenth of the life span of SAMP8), it could be assumed that the continued stress, even if it is relatively transient and not to a marked degree, results in severe changes in the cellular functions, which could potentially be triggering effects for the subsequent chronic neurological symptoms. To confirm this hypothesis, further study, such as’administration of radical scavengers to the mice, is required to show that oxidative stress at an earlier age is actually aggravating the neurological manifestation in this mouse model . The mechanism responsible for the appearance of the oxidative stress observed in this study has remains unclear. Oxidative damage to a biological system results from a disturbed balance of the deteriorating potential and the antioxidant capacity of the tissue. The involvement of antioxidant capacity in oxidative stress was shown in the brain. In Parkinson’s disease subjects, a reduction in the level of catalase, glutathione peroxidase [27,28] and glutathione [29], and an increase in superoxide dismutase [30], were observed in the substantia nigra. Sohal et al. [31] reported that mitochondrial 0, and H,O, production increased, and catalase and glutathione peroxidase activities decreased, in the brain of house mouse (MUS musculus) as compared with those in white-footed mouse (Perornyscus Zeucopus) at 3.5 months of age; the latter has twice the life span of the former. In our preliminary experiments, superoxide dismutase activity in SAMPS at younger ages was not different from that in control mice. The capacity of other antioxidants in the brain is under investigation. On the other hand, deteriorating potential, including metabolic changes in SAMP8, may be involved in increased oxidative stress. In previous papers, we reported an increase in glucose metabolism, as well as in membrane glucose
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transporters, in the brain of SAMP8 of 4-8 weeks of age [13,14], as observed for oxidative stress in this study. In addition, the changes in these parameters were seen only in the cerebral cortex. These results indicate that some relationship might exist between oxidative stress and increased glucose metabolism in SAMPS. Since an increase in glucose metabolism may produce some undesirable changes in oxidative metabolism, such an increase may be involved in oxidative stress in SAMP8. On the other hand, it could be that the increase in glucose utilization in the brain is a compensating reaction to cope with the oxidative stress. In normal aging, however, a parallel increase in oxidative stress and glucose metabolism is not observed in the brain. In Alzheimer’s disease subjects, although glucose metabolism was decreased on positron emission tomography (PET) scans, that measured in brain slices was increased [32]. In this regard, it might be assumed that an increase in glucose metabolism observed in vitro does not reflect the actual metabolic state in vivo. Further study must be done to evaluate the significance of such parallel changes observed in SAMPS. In conclusion, we demonstrated in this study that oxidative stress (increased lipid peroxide and protein carbonyl content, increased net generation of reactive oxygen species, and decreased activity of glutamine synthetase) exists in the cerebral cortex of 4-g-week-old SAMP8. At present, it remains unclear whether or not such increases in oxidative stress are primarily responsible for the deterioration in learning and memory in SAMPI;. However, our present observations do appear to provide some clues towards understanding the mechanism responsible for initiating the process of accelerated aging in the brain of SAMP8.
Acknowledgements The authors are grateful to professor Toshio Takeda (Kyoto University, Japan) for kindly supplying SAM.
References [l] T. Takeda, M. Hosokawa, S. Takeshita, M. Irino, K. Higuchi, T. Matsushita, Y. Tomita, K. Yasuhira, H. Hamamoto, K. Shim& M. Ishii and T. Yamamuro, A new murine model of accelerated senescence. Mech. Ageing Dev., 17 (1981) 183-194. [2] K. Higuchi, T. Yonezu, K. Kogishi, A. Matsumura, S. Takeshita, K. Higuchi, A. Kohno, M. Matsushita, M. Hosokawa and T. Takeda, Purification and characterization of a senile amyloid-related antigenic substance (apoSASSAM) from mouse serum. ApoSASSAM is an apoA-II apolipoprotein of mouse high density lipoproteins. J. Biol. Chem., 261 (1986) 12 834-12840. [3] A. Matsumura, K. Higuchi, K. Shimizu, M. Hosokawa, K. Hashimoto, K. Yasuhira and T. Takeda, A novel amyloid fibril protein isolated from senescence-accelerated mice. Lab. Znuest., 47 (1982) 270-275. [4] W. H. Chen, M. Hosokawa, T. Tsuboyama, T. Ono, T. Iizuka and T. Takeda, Age-related changes in the temporomandibular joint of the senescence accelerated mouse. SAM-P/3 as a new murine model of degenerative joint disease. Am. J. Parhol., 135 (1989) 379-385.
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[S] M. Matsushita, T. Tsuboyama, R. Kasai, H. Okumura, T. Yamamuro, K. Higuchi, K. Higuchi, A. Kohno, T. Yonezu, A. Utani, M. Umezawa and T. Takeda, Age-related changes in bone mass in the senescence-accelerated mouse (SAM). SAM-R/3 and SAM-P/6 as new murine models for senile osteoporosis. Am. J. Pathol., 125 (1986) 276-283. [6] M. Hosokawa, Y. Ashida. T. Matsushita, K. Takahashi and T. Takeda, Persistent hyaloid vascular system in age-related cataract in a SAM strain of mouse. Exp. Eye Res.. 57 (1993) 427-434. [7] A. Ohta, T. Hirano, H. Yagi, S. Tanaka, M. Hosokawa and T. Takeda, Behavioral characteristics of the SAM-P/8 strain in Sidman active avoidance task. Brain Res.. 498 (1989) 195-198. [8] M. Miyamoto, Y. Kiyota, N. Yamazaki, A. Nagaoka, T. Matsuo, Y. Nagawa and T. Takeda. Age-related changes in learning and memory in the senescence-accelerated mouse (SAM). Phvsiol. Behau., 38 (1986) 399-406. [9] H. Yagi, S. Katoh, I. Akiguchi and T. Takeda, Age-related deterioration
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of ability of acquisition in memory and learning in senescence accelerated mouse: SAM-P/8 as an animal model of disturbances in recent memory. Brain Res., 474 (1988) 86-93. T. Takeda, M. Hosokawa and K. Higuchi, Senescence-accelerated mouse (SAM): a novel rnurine model of accelerated senescence. J. Am. Geriatr. Sot., 39 (1991) 911-919. H. Akiyama, M. Kameyama, I. Akiguchi, H. Sugiyama, T. Kawamata, H. Fukuyama, H. Klmura, M. Matsushita and T. Takeda, Periodic acid-Schiff (PAS)-positive, granular structures increase in the brain of senescence accelerated mouse (SAM). Acta Neuropathol. Bet%. 72 (1986) l24- 129. M. Takemura, S. Nakamura, I. Akiguchi, M. Ueno, N. Oka, S. Ishikawa. A. Shimada, J. Kimura and T. Takeda, Beta/A4 proteinlike immunoreactive granular structures in the brain of senescenceaccelerated mouse. Am. J. Pathol.. 142 (1993) l887- 1897. E. Sato, A. Inoue, T. Kurokawa and S. Ishibashi, Early changes in glucose metabolism in the cerebrum of senescence accelerated mouse: involvement of glucose transporter. Brain RcT., 637
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and S. Ishibashi, Early changes in glucose metabolism in the cerebrum of senescence accelerated mouse: coupling with glucose transporter. In T. Takeda (ed.), The SAM Model of Senescence, Elsevier, Amsterdam, 1994, pp. 3133316. [15] D. Harman, The aging process. Proc. Natl. Acad. Sci. USA, 78 (1981) 712447128. [ 161 J.M. Carney, P.E. Starke-Reed, C.N. Oliver, R.W. Landum, M.S. Cheng, J.F. Wu and R.A. Floyd. Reversal of age-related increase in brain protein oxidation, decrease in enzyme activity, and loss in temporal and spatial memory by chronic administration of the spin-trapping compound N-tertbutyl-a-phenylnitrone. Proc. Natl. Acad. Sci. USA, 88 (1991) 3633-3636. [17] O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurment with the folin phenol reagent. J. Biol. Chem., 193 (1951) 265-275. 1181J.A. Buege and S.D. Aust, Microsomal lipid peroxidation. Methods Enzymol., 52 (1978) 302-310. [19] R.L. Levine, D. Garland, C.N. Oliver, A. Amici, I. Climent, A. G. Lenz, B.W. Ahn, S. Shahiel and E.R. Stadtman, Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol., 186 (1990) 464478. [20] R.E. Miller, R. Hackenberg and H. Gershman, Regulation of glutamine synthetase in cultured 3T3-Ll cells by insulin, hydrocortisone, and dibutyryl cyclic AMP. Proc. Natl. Acad. Sci. USA. 75 (1978) 1418-1422. [2l] D.A. Bass, J.W. Parce, L.R. Dechatelet, P. Szejda, M.C. Seeds and M. Thomas, Flow cytometric
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8277838.
ELSEVIER
105
anddevelopment
86 (1996) 105-114
Early and transient increase in oxidative stress in the cerebral cortex of senescence-accelerated mouse Eiji Sato, Nanae Oda, Naoko Ozaki, Shin-ichi Hashimoto, Kurokawa*, Sadahiko Ishibashi
Tomonori
Department of Physiological Chemistry, Hiroshima University School of Medicine, Minami-lcu. Hiroshima 734, Japan
Received 1 August 1995; accepted 10 October 1995
Abstract Age-related changes in oxidative stress in the cerebral cortex of SAMP8, a substrain of senescence-accelerated mouse (SAM), were investigated in comparison with those in SAMRl, which were used as a control. The lipid peroxide and protein carbonyl contents were transiently increased in SAMP8 from 4- to &weeks of age. The increases in lipid peroxide were seen only in the cerebral cortex and not in other regions of the cerebrum. Furthermore, the net generation of reactive oxygen species in cerebral cells was also increased in SAMP8. In addition, the activity of glutamine synthetase, which is known as an enzyme highly sensitive to reactive oxygen, was decreased in the cerebral cortex of SAMP8 from 4- to 8-weeks of age. These results suggest that oxidative stress may be induced in the cerebral cortex of SAMP8 from 4- to 8-weeks of age, preceding the appearance of distinctive deficits in the brain of SAMPS. Keywords:
Senescence-accelerated
mouse (SAM); SAMP8; SAMRl; Reactive oxygen; Oxida-
tive stress
* Corresponding
author. Tel.: + 81 82 2575306; fax: + 81 82 2575309.
0047-6374/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved SSDI 0047-6374(95)01681-3
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1. Introduction Senescence-accelerated mouse (SAM), an experimental murine model of accelerated aging, was established by Takeda et al. [l]. SAM consists of two strains; accelerated senescence-prone mouse (SAMP), and accelerated senescence-resistant mouse (SAMR). Following a normal process of development, the SAMP shows an accelerated change with many signs of aging, such as a short life span, changes in general behavior, loss of skin glossiness, increased skin coarseness, hair loss, periophthalmic lesions, and increased lordokyphosis of the spine [l]. At present, SAMP consists of nine substrains (SAMPl, P2, P3, P6, P7, P8, P9, PlO, and Pl I), and each substrain has relatively specific pathologic phenotypes, in addition to having characteristics common to each of the SAMP strains (i.e. accelerated senescence). For example, senile amyloidosis in SAMPl [2,3], degenerative joint disease in SAMP3 [4], senile osteoporosis in SAMP6 [5], and cataract in SAMP9 [6] were demonstrated. In addition, SAMPS shows some distinctive defects in the brain, such as age-related deterioration of ability in learning and memory [7-g], and has been used as a useful animal model for research into senile memory impairment. While it has been shown that some pathomorphological changes, such as spongiform degeneration [lo], PAS-positive granular structures [l 11, and /?A4 protein-like immuno-reactive granular structures [12], were increased in SAMP8, the mechanisms responsible for these degenerative processes have remained unclear. In contrast to these SAMP, SAMR consists of three substrains (SAMRl, R4, and R5), which have essentially the same aging characteristics as SAMR2, an another control strain which was used in our previous studies. Oxygen free radicals have been implicated in the process of several degenerative diseases, as well as in the physiological decline associated with aging [15]. These highly reactive molecules produced within aerobic cells in the course of normal metabolic events, oxidize cellular components such as lipid, protein, and DNA, which in turn result in undesirable changes in cellular functions. In particular, oxidative stress in the brain is of interest in this regard because the brain consumes a lot of oxygen and contains relatively high concentrations of easily peroxidizable fatty acids. In fact, in some stressful conditions, such as various types of traumas and ischemia/reperfusion injury, excessive production of oxygen metabolites is induced, and it could constitute an important cause of neuronal degeneration. It has also been shown that administration of radical scavengers to aged animals restored loss of temporal and spatial memory, as well as increasing protein carbonyl content in the brain to the levels of younger ones [16]. These observations suggest that the oxidative modification of cellular components may play a critical role in the decline in the cellular functions associated with its degenerative process. Thus, we are interested in the involvement of oxidative stress in the early deficits observed in the brain of SAMP8. In this study, we report that oxidative stress may be induced in the cerebral cortex of SAMPS from 4- to 8-weeks of age, preceding the appearance of distinctive defects in the brain of SAMP8.
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2. Materials and methods 2. I. Reagents 2’,7’-dichlorofluorescin diacetate (DCFH-DA) was purchased from Eastman Kodak (New York, USA). 2’,7’-dichlorofluorescein (DCF) was from Nacalai Tesque Inc. (Kyoto, Japan). Monochlorobimane (mBC1) was from Molecular Probes Inc. (Eugene, Oregon, USA). All other agents used were of reagent grade from standard commercial sources. 2.2. Animals SAMP8 and SAMRl, as control strains, were generously donated by Professor Toshio Takeda (Kyoto University, Japan). They were bred under conventional conditions with food and water ad libitum. Male and female mice of 2, 4-8, 20-28, and 40-56 weeks of age were used in this study. 2.3. Tissue preparations After decapitation of the mouse, the cerebral cortex, hippocampus and residual region of the cerebrum (remainder) were rapidly dissociated and placed on a chilled surface. The superficial blood vessels were removed and the surface of the brain was washed with ice-cold Hanks’ solution buffered with Pipes (pH 7.3, 137 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO,, 1.3 mM CaCl,, 0.4 mM Na,HPO,, 0.4 mM KH,PO,, 5.5 mM D-glucose, and 8 mM Pipes). The dissociated cerebral cells were prepared as previously reported [13] with a minor modification in which all solutions included 5.5 mM D-glucose. Cerebral cortex extracts were prepared as follows. The dissociated cerebral cortex was homogenized in 10 mM Hepes buffer (pH 7.4) containing 137 mM NaCl, 4.6 mM KCl, 1.1 mM KH,PO,, 1.I mM EDTA, 0.6 mM MgSO,, leupeptin (0.5 pg/ml), pepstatin (0.7 pg/ml), aprotinin (0.5 pg/ml), and phenylmethylsulfonyl fluoride (40 pg/ml). Insoluble debris was pelleted at 100 000 x g for 1 h. The clear supernatant was recovered and used for the protein carbonyl and glutamine synthetase assays. Homogenates for the measurement of lipid peroxide content were prepared from the hippocampus and remainder, as well as the cerebral cortex, by using a Potter-Elvehjem homogenizer in 150 mM KC1 and 50 mM Tris-HCl (pH 7.4). The protein content of the samples was determined by the method of Lowry et al. [17]. 2.4. Assays The lipid peroxide (thiobarbituric acid reactive substance (TBARS)) content of the homogenates of the cerebral cortex, hippocampus and remainder were determined by the method of Buege and Aust [18] using 1,1,3,3_tetraethoxypropane as a standard. The protein carbonyl content was determined spectrophotometrically by the 2,4_dinitrophenylhydrazine (DNPH)-labeling procedure [19]. Results are ex-
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pressed as nmols of DNPH incorporated/mg protein, calculated on the basis of an extinction coefficient of 22.0 mM - ’ cm - l for aliphatic hydrazones. Glutamine synthetase activity was determined by a y-glutamyl transfer assay, as previously reported [20]. One unit of glutamine synthetase activity is defined as the amount of activity catalyzing the formation of 1 pmol of product (y-glutamyl hydroxamate) per min under specified assay conditions. The values were corrected for nonspecific glutaminase activity by comparing the activities in the presence and absence of ADP and arsenate. The net cellular generation of the reactive oxygen species in the dissociated cells was measured using the fluorescent probe 2’,7’-dichlorofluorescin diacetate (DCFH-DA) [21], using 2’,7’-dichlorofluorescein (DCF) as a standard. In brief, the dissociated cells were incubated at 37°C with DCFH-DA (2.5 pug/ml) in the presence, or absence, of 1 mM diethyl maleate for O-90 min, and then fluorescence at 485 nm, in response to excitation at 530 nm, was measured. Cellular glutathione content was determined using monochlorobimane (mBC1) [22]. In brief, after treatment of the dissociated cells with 1 mM diethyl maleate for O-60 min, mBCl(40 PM) was added and the tills were further incubated for 15 min, and then fluorescence at 460 nm, in response to excitation at 395 nm, was measured. 2.5. Statistical analysis Student’s unpaired t-test and, for multiple group comparisons, a one-way analysis of variance (ANOVA) test were employed to determine statistical significance.
3. Results Age-related changes in the lipid peroxide and protein carbonyl content in the brain of SAM mice were examined in the cerebral homogenate and extract, respectively. In both strains, the lipid peroxide content increased at 40-56 weeks of age, and that of protein carbonyl exhibited a biphasic change with age; it increased up to 20-28 weeks of age, and then decreased (Fig. lA,B). Interestingly, the content of both increased at 4-8 weeks of age in SAMP8, as compared with those in SAMRI. No significant differences were observed between these two strains in other aged mice. Next, age-related changes in the activity of glutamine synthetase, which is known as an enzyme highly sensitive to reactive oxygen, were examined in the cerebral extracts. As shown in Fig. lC, the activity was decreased in SAMP8 at 4-8 weeks of age. No significant differences were observed between these two strains in other aged mice. To characterize further the increase in oxidative stress in the brain of 4-8-week-old SAMP8, the content of lipid peroxide in the hippocampus and residual region of the cerebrum (remainder) were examined in addition to the cerebral cortex.The significant increase in lipid peroxides was seen only in the cerebral cortex and not in other regions of the cerebrum (Fig. 1D). These results indicate that a possible increase in oxidative stress exists in the cerebral cortex of SAMP8 at an earlier stage.
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Next, the net generation of reactive oxygen species was determined using the fluorescent probe 2’,7’-dichlorofluorescin diacetate (DCFH-DA). DCFH-DA, a non-polar and non-fluorescent compound that readily diffuses into cells, is hydrolyzed by intracellular esterases to the non-fluorescent derivative 2’,7’-dichlorofluorescin (DCFH). In the presence of intracellular hydrogen peroxide, DCFH is oxidized to the highly fluorescent compound 2’,7’-dichlorofluorescein (DCF) [21].
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age (weeks) Fig. 1. Age-related changes in lipid peroxide content (A), protein carbonyl content (B), in the activities of glutamine synthetase (C) in the cerebral cortex, and in the differences of lipid peroxide content among cerebral regions in 4-8-weeks-old mice (D). (A) and (D), and (B) and (C) were determined in the brain homogenates and extracts, respectively, as described in Materials and methods. (A)-(C): Differences between the two strains were observed only at 4-8 weeks of age: 0, SAMPI: and 0, SAMRI. (D): An increase in lipid peroxides in SAMPI was observed only in the cerebral cortex. Data are expressed as a mean k SE. of S- 13 mice. “P < 0.01 vs. those of corresponding SAMRl values. b,cP < 0.05 vs. those of 2- and 20-28-week-old SAMPB and SAMRl, respectively. d,eP <: 0.01 vs. those of 2- and 40-56-weekold SAMP8 and SAMRl, respectively. ‘Pi 0.01 vs. those of 2- and 4-8-week-old SAMPI. gP <. 0.01 vs. that of 2-week-old SAMRI.
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Fig. 2. Net generation of reactive oxygen species (A) and changes in cellular glutathione content (B) with diethyl maleate treatment. Dissociated cerebral cells prepared from 4-&week-old SAMP8 (00) and SAMRl (A A) were incubated with DCFH-DA (A) or mBC1 (B) in the presence (0 a) or absence (.A) of 1 mM diethyl maleate, and then fluorescence intensity was measured as described in Materials and methods. Data are expressed as a mean _+S.E. of 4- 13 mice.“P < 0.05 vs. those of corresponding SAMRl values.
The DCF content of cerebral cells increased during the incubation for both strains, and was significantly higher in SAMP8 than in SAMRl at 90 min (Fig. 2A). The difference between these strains was more significant when the cells were treated with 1 mM diethyl maleate to deplete cellular glutathione. Since the elevated accumulation of DCF might result from the difference in cellular glutathione content between these two strains, the glutathione content of cerebral cells was measured using the fluorescent probe monochlorobimane (mBC1). While the fluorescence intensity was markedly decreased during the treatment with diethyl maleate in both strains, no difference was observed between these two strains (Fig. 2B). The fluorescent intensity after incubation for 60 min in the absence of diethyl maleate was slightly decreased and there was no difference between the two strains. These results suggest that the increased net generation of reactive oxygen species, without a particular decrease in cellular glutathione, may possibly contribute to the oxidative stress in 4-8-week-old SAMP8, which leads to the increase in lipid peroxide and protein carbonyl levels.
4. Discussion It has been reported that some defects in the brain, such as deterioration of ability in learning and memory, begins to progress as early as 2 months after birth in SAMP8 [7]. It is not clear, however, what precedes this appearance. In this study, we found that the lipid peroxide and protein carbonyl content increased, and the activity of glutamine synthetase decreased, in the cerebral cortex of SAMP8 at 4-8
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weeks of age. Glutamine synthetase is known as being an enzyme highly sensitive to reactive oxygen, and a loss of enzyme activity, associated with an increase in protein carbonyl content, has been reported in human brain [23], cultured fibroblasts [24], and rat liver [25], as well as in ischemia/reperfusion-injured gerbil brain [26]. In addition, the net generation of reactive oxygen species was actually increased in SAMP8. These results suggest that oxidative stress may increase in the cerebral cortex of SAMP8 at an earlier stage. Because the increase was observed transiently, prior to such defects in the brain, and was not found in the control senescence-resistant strain, it is plausible that some relationships may exist between oxidative stress at an earlier age and degenerative processes in the brain observed later in SAMPS. However, it remains an interesting problem as to how the oxidative stress at 4-8 weeks of age, with its complete recovery to the level of control mice after the age of 20-28 weeks, has etiologic importance for the following chronic neurological symptoms in these mice. In normal aging, oxidative stress does not exhibit such transient characteristics. It may be assumed that the transient nature observed in oxidative stress is a specific characteristic of accelerated aging in the brain of SAMP8. We did not observe the age-related differences in the lipid peroxide and protein carbonyl content and in the activities of glutamine synthetase of 4-&-week-old SAMP8, indicating that oxidative stress may continue for at least 4 weeks at the younger stage in these mice. Because 4 weeks is a very big period in the life of a mouse to be considered as one age point (i.e. it is about one tenth of the life span of SAMP8), it could be assumed that the continued stress, even if it is relatively transient and not to a marked degree, results in severe changes in the cellular functions, which could potentially be triggering effects for the subsequent chronic neurological symptoms. To confirm this hypothesis, further study, such as’administration of radical scavengers to the mice, is required to show that oxidative stress at an earlier age is actually aggravating the neurological manifestation in this mouse model . The mechanism responsible for the appearance of the oxidative stress observed in this study has remains unclear. Oxidative damage to a biological system results from a disturbed balance of the deteriorating potential and the antioxidant capacity of the tissue. The involvement of antioxidant capacity in oxidative stress was shown in the brain. In Parkinson’s disease subjects, a reduction in the level of catalase, glutathione peroxidase [27,28] and glutathione [29], and an increase in superoxide dismutase [30], were observed in the substantia nigra. Sohal et al. [31] reported that mitochondrial 0, and H,O, production increased, and catalase and glutathione peroxidase activities decreased, in the brain of house mouse (MUS musculus) as compared with those in white-footed mouse (Perornyscus Zeucopus) at 3.5 months of age; the latter has twice the life span of the former. In our preliminary experiments, superoxide dismutase activity in SAMPS at younger ages was not different from that in control mice. The capacity of other antioxidants in the brain is under investigation. On the other hand, deteriorating potential, including metabolic changes in SAMP8, may be involved in increased oxidative stress. In previous papers, we reported an increase in glucose metabolism, as well as in membrane glucose
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transporters, in the brain of SAMP8 of 4-8 weeks of age [13,14], as observed for oxidative stress in this study. In addition, the changes in these parameters were seen only in the cerebral cortex. These results indicate that some relationship might exist between oxidative stress and increased glucose metabolism in SAMPS. Since an increase in glucose metabolism may produce some undesirable changes in oxidative metabolism, such an increase may be involved in oxidative stress in SAMP8. On the other hand, it could be that the increase in glucose utilization in the brain is a compensating reaction to cope with the oxidative stress. In normal aging, however, a parallel increase in oxidative stress and glucose metabolism is not observed in the brain. In Alzheimer’s disease subjects, although glucose metabolism was decreased on positron emission tomography (PET) scans, that measured in brain slices was increased [32]. In this regard, it might be assumed that an increase in glucose metabolism observed in vitro does not reflect the actual metabolic state in vivo. Further study must be done to evaluate the significance of such parallel changes observed in SAMPS. In conclusion, we demonstrated in this study that oxidative stress (increased lipid peroxide and protein carbonyl content, increased net generation of reactive oxygen species, and decreased activity of glutamine synthetase) exists in the cerebral cortex of 4-g-week-old SAMP8. At present, it remains unclear whether or not such increases in oxidative stress are primarily responsible for the deterioration in learning and memory in SAMPI;. However, our present observations do appear to provide some clues towards understanding the mechanism responsible for initiating the process of accelerated aging in the brain of SAMP8.
Acknowledgements The authors are grateful to professor Toshio Takeda (Kyoto University, Japan) for kindly supplying SAM.
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