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Nutrition and Aging

Soy Isoflavones Improve Spatial Delayed Matching-to-Place Performance and Reduce Cholinergic Neuron Loss in Elderly Male Rats¹

Yoon-Bok Lee^*,, Hyong Joo Lee^,2, Moo Ho Won^**, In Koo Hwang^**, Tae-Cheon Kang^**, Jae-Yong Lee^**, Sang-Yoon Nam, Kang-Sung Kim, Eugene Kim^*, Sang-Hee Cheon^* and Heon-Soo Sohn^*

^* Central Research Institute, Dr. Chung’s Food Company Limited, Choongchungbuk-Do, 361-782, Korea; Department of Food Science and Technology, Seoul National University, Gwanak-gu, 151-742, Korea; ^** College of Medicine, Hallym University, Kangwon-Do, 200-702, Korea; College of Veterinary Medicine, Chungbuk National University, Choongchungbuk-Do, 361-763, Korea; and Department of Food and Nutrition, Yong-in University, Gyunggi-Do, 449-714, Korea

²To whom correspondence should be addressed. E-mail: leehyjo{at}snu.ac.kr.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
To investigate the protective activity of soy isoflavones on neurons, the effects of isoflavones on cholinergic enzyme activity, immunoreactivities of cholinergic enzyme, and delayed matching-to-place (DMP) performance were measured in normal elderly rats. Male Sprague-Dawley rats (n = 48; 10 mo old) were assigned to 3 groups: CD (control diet), ISO 0.3 (0.3 g/kg soy isoflavones diet), and ISO 1.2 (1.2 g/kg soy isoflavones diet). After 16 wk of consuming these diets, choline acetyltransferase (ChAT) activity in the ISO 0.3 group was greater in cortex and basal forebrain (BF; P < 0.05) than in controls. In BF, ChAT activity was also significantly greater in the ISO 1.2 group than in control rats. Acetylcholine esterase (AChE) activity in the ISO 0.3 group was significantly inhibited in cortex, BF, and hippocampus and in the ISO 1.2 group in cortex and hippocampus. Choline acetyltransferase immunoreactivity (ChAT-IR) in the ISO 1.2 group was significantly greater than in controls in the medial septum area. ChAT-IR in the ISO 0.3 and ISO 1.2 groups was significantly higher than in the CD group in the hippocampus CA1 area. Spatial DMP performance by the ISO 0.3 group showed significantly shorter swimming time than by the CD group. These findings show that soy isoflavones can influence the brain cholinergic system and reduce age-related neuron loss and cognition decline in male rats.

KEY WORDS: • soy isoflavones • cholinergic enzyme • aging • memory • spatial DMP performance

Phytoestrogens are defined as plant-derived nonsteroidal substances with a diphenolic structure that are structurally and functionally similar to 17ß-estradiol or that produce estrogenic effects (1). The phenolic ring is a key structural element of most compounds that bind to estrogen receptors (ER)³ (2). Specifically, isoflavones of phytoestrogens are strikingly similar in chemical structure to mammalian estrogens (3), and have been studied for their potential beneficial effects. Estrogen functions by binding to its receptor, including the "classic" ER- (4) and the second receptor, ER-ß (5). The tissue distribution and relative ligand binding affinities of ER-ß and ER- differ, and this finding may help to explain the selective action of estrogens in different tissues. It is fascinating that ER-ß is found in brain, bone, bladder, and vascular epithelia, tissues that are responsive to classical hormone replacement therapy. Furthermore, ER-ß is found in the neocortex, hippocampus, nuclei, and basal forebrain (BF), structures that are related to memory and learning (3).

During aging, neurons selectively degenerate in the hippocampus, entorhinal cortex, and temporal lobe areas related to recognition (6); cholinergic parameters decline continuously, and the deterioration of the brain cholinergic system leads to reduced recognition ability (7). Estrogen has neuroprotective effects (8) and affects the cholinergic neurotransmitter system (9). Estrogen-treated ovariectomized rats have increased choline acetyltransferase (ChAT) activity within the hippocampus and frontal cortex, and a high affinity for choline uptake (10). Woolley et al. (11) showed that estrogen replacement therapy (ERT) may regulate the density of dendritic spines and synapses of hippocampal pyramidal neurons. In addition, ERT has significant effects on cholinergic neurons in the medial septum (MS) and nucleus basalis magnocellularis (12). These neurons are the primary source of cholinergic innervation to the hippocampus and cortex (13). Although recently reported studies showed beneficial effects of soy protein and isoflavones on cognitive function and memory (14,15), very little is known about the potential effect of these compounds on the brain.

We investigated the effects of soy isoflavones in elderly rats using behavioral tests and cholinergic enzyme analysis, and analyzing the immunoreactivities of cholinergic enzyme and receptors using quantitative immunohistochemical techniques.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Animals and diets. Male Sprague-Dawley rats (3–4 mo old) were purchased from Bio-Genomics. They were acclimated in an environmentally controlled animal laboratory and given free access to a commercial standard diet (EP 322–6) and distilled water. They were kept at a constant temperature (22 ± 1°C) and relative humidity (55 ± 5%), with a 12-h light:dark cycle. All rats were treated in accordance with the NIH guidelines (16).

When the rats were 10 mo old, they were assigned to 3 groups (n = 16) based on body weight: CD (control diet), ISO 0.3 (0.3 g/kg soy isoflavones diet), and ISO 1.2 (1.2 g/kg soy isoflavones diet). The isoenergetic and isonitrogenous diets were based on the AIN76A formulation (17) and were fed to the rats for 16 wk. Distilled water was always available to the rats. During the experiment period, the body weight was measured once each week, and the food intake was measured every day. Behavioral tests were performed at 15 wk, and rats were killed for the collection of brains at 16 wk.

    Extraction of soybean isoflavones. For extraction of isoflavones, soybean hypocotyls were extracted with 10 volumes of 80% aqueous ethanol with stirring for 2 h at 70°C. The methanol extract was condensed with a rotary evaporator at 50°C. Finally, the soybean isoflavones extract was obtained by freeze-drying the concentrated methanol extract. Once isolated, the soybean isoflavone extract was stored at –80°C until analysis and formulation of the experimental diets. The HPLC analysis of 3 isoflavones and their 9 derivatives was performed using a diode-array detector (HP1100 system, Agilent) and an eclipse XDB C-18 column (Agilent). UV detection was at 260 nm, and the injection volume was 5 µL. The mobile phases were 0.1% (v:v) acetic acid in H₂O (solvent A) and 0.1% (v:v) acetic acid in acetonitrile (solvent B). A flow rate of 1.2 mL/min under an initial condition of 93:7 (A:B) was held for 25 min, brought to 15% B over 25 min, to 20% B over 5 min, and to 25% B over 15 min, all with a linear gradient. Genistein, daidzein, and glycitein were purchased from Sigma Chemical, and malonyl-, acetyl-, and glycoside forms were purchased from Fujico for use as standards in HPLC analyses.

    Behavioral test: delayed matching-to-place water maze. The learning and memory performance of elderly rats was tested using the delayed matching-to-place (DMP) water maze test, which is a modified version of the Morris water maze test (18). A circular pool (135 cm in diameter and 55 cm deep) was filled with water to a depth of 40 cm, and powdered milk was mixed with the water to produce an opaque medium. The pool was then divided into 4 quadrants, and a platform was placed in 1 quadrant 2.5 cm below the water surface. The location of platform was changed daily. Daily sessions consisted of 5 trials separated by a 90-min interval. Rats were trained until they reached the platform on 3 consecutive days. After mastering the DMP water maze rule, rats were tested for the next 3 consecutive days in the same way as during training. The means of the swimming time were used to evaluate the performance of rats, and the values are the means per trial from the last 3 d of testing. Rat positions in the pool were automatically tracked, and swimming time was automatically calculated by a commercial video/computer system (Videotrack 512 System, version 2.67, View Point) above the center of the pool.

    Cholinergic enzyme activities. After behavioral testing, the rats were decapitated and cerebral cortex, hippocampus, and BF were collected. ChAT activity was determined spectrophotometrically using the method of Chao and Wolfgram (19). The tissue was homogenized (10 g/L) in ice-cold 0.05 mol/L sodium phosphate buffer (pH 7.0). In each test tube, 20 µL of 0.05 mol/L phosphate buffer, 1 mol/L choline chloride, 3 mmol/L sodium chloride, 1.1 mmol/L EDTA, 6.5 mmol/L dithioerythritol, 0.76 mmol/L neostigmine bromide, and distilled water were added along with 40 µL of 6.2 mmol/L acetyl CoA and 0.1 mol/L creatine. The mixture was preincubated at 37°C for 5 min and 200 µL of tissue homogenate was added. For the blank, 200 µL of boiled homogenate was added. The mixture was incubated at 37°C for 20 min and boiled at 100°C for 2 min; 800 µL of 2.5 mmol/L sodium arsenite was then added. The test tubes were centrifuged at 12,000 x g for 5 min, and 1 mL of suspension was collected. 4-Dithiopyrimidine (10 µL, 1 mmol/L) was added, and the tubes were allowed to stand for 15 min before being read spectrophotometrically at 342 nm.

Acetylcholine esterase (AChE) activity was determined by the method of Ellman et al. (20). In a test tube, 2.6 mL of 0.05 mol/L sodium phosphate buffer (pH 7.0), 0.4 mL of homogenate, and 100 µL of dithiobisnitrobenzoic acid were added. The test tube was mixed and 20 µL of the substrate of acetylcholine iodide was added. The initial absorbance and the absorbance after 4 min were read at 412 nm.

    Immunohistochemistry. After behavioral testing, the rats remaining and not used for the cholinergic enzyme activity assay were anesthetized with pentobarbital sodium, and perfused via the ascending aorta with 200 mL of 4% paraformaldehyde in phosphate buffer. The brains were removed, postfixed in the same fixative for 4 h, and rinsed in phosphate buffer containing 300 g/L sucrose at 4°C for 2 d. Thereafter the tissues were frozen and sectioned with a cryostat at 30 µm and consecutive sections were collected in 6-well plates containing PBS. These free-floating sections were then incubated with 5% normal goat serum for 30 min at room temperature. Some of the sections were then incubated in the mouse ChAT antibody (diluted 1:500, Chemicon) in PBS containing 0.3% Triton X-100 and 2% normal goat serum overnight at room temperature. After being washed 3 times for 10 min with PBS, sections were incubated sequentially in horse anti-mouse IgG or goat anti-rabbit IgG (Vector) and streptavidin (Vector), and diluted 1:200 in the same solution as the primary antiserum. Between the incubations, the tissues were washed with PBS 3 times for 10 min each. The sections were visualized with DAB (3,3'-diaminobezidine) in 0.1 mol/L Tris buffer and mounted on gelatin-coated slides. The immunoreactions were then observed under a light microscope (Axioscope, Carl Zeiss).

    Quantitative analysis. Sections (5 sections per rat) of the hippocampus and BF areas were viewed through a microscope connected via CCD camera to a PC monitor. At a magnification of 25–50, the region was outlined on the monitor and the area measured. Images of immunoreactivities in the hippocampus and BF areas for each rat were captured with an Applescanner. The brightness and contrast of each image file were calibrated by Adobe Photoshop version 2.4.1. and analyzed using NIH image 1.59 software. Values of background staining were obtained and subtracted from the immunoreactive intensities.

    Statistical analysis. The effect of diet treatment was assessed by ANOVA with SPSS software. Values are presented as means ± SD. Differences among groups were tested by Duncan’s multiple-range test or by Dunnett’s test and were considered significant at P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Isoflavone concentrations of the diets. We obtained a yield of 5018.2 mg isoflavones/100 g isoflavone extracts (Table 1). The isoflavone concentrations of the ISO 0.3 and ISO 1.2 diets were 0.321 and 1.284 g/kg diet, respectively. Glycoside and malonyl forms were dominant in the extract. The daily isoflavone intake in the ISO 0.3 group was 8.47 mg of isoflavone extract (0.425 mg of isoflavones), and in the ISO 1.2 group, 29.74 mg of isoflavone extract (1.49 mg of isoflavones).

    Cholinergic enzyme activities. In cortex, ChAT activity was significantly greater in the ISO 0.3 group than in the CD group (Fig. 1). In BF, ChAT activity in the ISO 0.3 and ISO 1.2 groups was significantly higher than that in the CD group.

View larger version (32K): [in this window] [in a new window]   FIGURE 1 ChAT enzyme activities in cortex, basal forebrain, and hippocampus of elderly male rats fed the CD and the diets containing 0.3 g/kg soy isoflavones (ISO 0.3) and 1.2 g/kg soy isoflavones (ISO 1.2) for 16 wk. Values are means ± SD, n = 8. Means for a brain section without a common letter differ, P < 0.05.

View larger version (23K): [in this window] [in a new window]   FIGURE 2 AChE enzyme activities in cortex, basal forebrain, and hippocampus of elderly male rats fed the CD and the diets containing 0.3 g/kg soy isoflavones (ISO 0.3) and 1.2 g/kg soy isoflavones (ISO 1.2) for 16 wk. Values are means SD, n = 8. Means for a brain section without a common letter differ, P < 0.05.

View larger version (92K): [in this window] [in a new window]   FIGURE 3 ChAT immunoreactivity of medial septal region in elderly male rats fed the CD and the diets containing 0.3 g/kg soy isoflavones (ISO 0.3) and 1.2 g/kg soy isoflavones (ISO 1.2) for 16 wk. Bar = 50 µm.

View larger version (69K): [in this window] [in a new window]   FIGURE 4 ChAT immunoreactivity of hippocampus CA1 region in elderly male rats fed the CD and the diets containing 0.3 g/kg soy isoflavones (ISO 0.3) and 1.2 g/kg soy isoflavones (ISO 1.2) for 16 wk. Bar = 50 µm.

View larger version (24K): [in this window] [in a new window]   FIGURE 5 Quantitative data of ChAT immunoreactivities in basal forebrain medial septum (ChAT-MS) and hippocampus CA1 (ChAT-CA1) of elderly male rats fed the CD and the diets containing 0.3 g/kg soy isoflavones (ISO 0.3) and 1.2 g/kg soy isoflavones (ISO 1.2) for 16 wk. Values are means ± SD, n = 8. Means for a brain section without a common letter differ, P < 0.05.

View larger version (14K): [in this window] [in a new window]   FIGURE 6 Spatial DMP performance in elderly male rats fed the CD and the diets containing 0.3 g/kg soy isoflavones (ISO 0.3) and 1.2 g/kg soy isoflavones (ISO 1.2) for 16 wk. Values are means ± SD, n = 16. Means for a trial without a common letter differ, P < 0.05.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

    Cholinergic enzymes in hippocampus, cortex, and basal forebrains. In this study, soy isoflavones increased ChAT activity in cortex (ISO 0.3), and BF (ISO 0.3, ISO 1.2), and inhibited AChE activity in cortex (ISO 0.3, ISO 1.2), BF (ISO 0.3), and hippocampus (ISO 0.3, ISO 1.2). In particular, there was a dramatic increase in ChAT activity in the BF region, and a dramatic decrease in AChE activity in the cortex region. These findings demonstrate that soy isoflavones can have different effects on the cholinergic enzyme activities in the cortex, BF, and hippocampus, i.e., soy isoflavones upregulated the ChAT activity in BF and downregulated the AChE activity in cortex and hippocampus. Moreover, the data showed that low-concentration soy isoflavones (ISO 0.3) downregulated AChE activity in all regions of the cortex, hippocampus, and BF.

    Cholinergic MS cells and cholinergic hippocampus CA1 cells. The present study demonstrated that soy isoflavones influence the cell density of cholinergic neurons in MS and hippocampus CA1, as well as the cholinergic enzyme activity in the BF, hippocampus, and cortex. We investigated ChAT-IR in MS and hippocampus CA1 because cholinergic neurons project from the septum to the hippocampus and appear to play a key role in modulating the activity of the hippocampal pyramidal cells (23).

Soy isoflavones increased ChAT-IR in MS and hippocampus CA1. Cholinergic MS neurons have abundant efferent connections to the hippocampal CA1, CA2, and CA3 regions, and the dentate gyrus, the so-called septohippocampal pathway (24). The soy isoflavone–induced ChAT-IR increase in the hippocampus may reflect cell populations in the MS. Thus, it is tempting to hypothesize that the transsynaptic effects of soy isoflavones influence the synapse plasticity of pyramidal cell spines in the CA1 subfield of hippocampus via MS by the septohippocampal pathway. Although the hippocampus also receives moderate ChAT immunoreactive projections from the diagonal band and nbM/SI in rats (25), the present findings imply that the soy isoflavone–induced increase in cholinergic fiber density in the hippocampus may be influenced by the cholinergic MS magnocellular increase (Fig. 5).

    Spatial memory in elderly male rats. Soy isoflavones had a positive influence on cognitive ability in elderly male rats, and the consumption of a low level of isoflavones [10.89 mg/(kg body wt · d)] resulted in shorter swimming times than a high level of isoflavones [38.53 mg/(kg body wt · d)] (Fig. 6).

The effect of phytoestrogens was reported to differ depending on the dose and the sex. In this study, both low and high levels of isoflavones increased cholinergic enzyme activity, but in the DMP water maze task, the swimming times were shorter in the group fed a low level of isoflavones than in controls but not in the group fed a higher level. A high-phytoestrogen diet was found to improve memory in female rats, but to impair it in male rats (26). It was reported that a relatively low-phytoestrogen intake might improve spatial learning and memory in male rats (27), which is supported by the suggestion that the basal forebrain cholinergic system differs between male and female rats and in male rats, the basal forebrain does not have the same response to estradiol as that of females (28).

The present study suggests that soy isoflavones can ameliorate deficits in memory tasks resulting from the loss of cholinergic input to the hippocampus or cholinergic degeneration in elderly male rats.

	FOOTNOTES

 
¹ Supported by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (no. 00-PJI-PG4-PT04–0003).

³ Abbreviations used: AChE, acetylcholine esterase; BF, basal forebrain; CD, control diet; ChAT, choline acetyltransferase; ChAT-IR, choline acetyltransferase immunoreactivity; DMP, delayed matching-to-place; ER, estrogen receptor; ERT, estrogen replacement therapy; ISO, soy isoflavone diet; MS, medial septum.

Manuscript received 3 November 2003. Initial review completed 28 December 2003. Revision accepted 25 March 2004.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Lee, Y.-B. Articles by Sohn, H.-S. Search for Related Content PubMed PubMed Citation Articles by Lee, Y.-B. Articles by Sohn, H.-S.