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The Journal of Neuroscience, October 1, 1998, 18(19):8047-8055
Long-Term Dietary Strawberry, Spinach, or Vitamin E
Supplementation Retards the Onset of Age-Related Neuronal
Signal-Transduction and Cognitive Behavioral Deficits
J. A.
Joseph1,
B.
Shukitt-Hale1,
N. A.
Denisova1,
R. L.
Prior1,
G.
Cao1,
A.
Martin1,
G.
Taglialatela2, and
P. C.
Bickford3
1 United States Department of Agriculture Human
Nutrition Research Center on Aging at Tufts, Boston, Massachusetts
02111, 2 Department of Chemistry and Genetics, University
of Texas, Galveston, Texas 77555, and 3 Department of
Veteran's Affairs Medical Center, Denver, Colorado 80262
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ABSTRACT |
Recent research has indicated that increased vulnerability to
oxidative stress may be the major factor involved in CNS functional declines in aging and age-related neurodegenerative diseases, and that
antioxidants, e.g., vitamin E, may ameliorate or prevent these
declines. Present studies examined whether long-term feeding of Fischer
344 rats, beginning when the rats were 6 months of age and continuing
for 8 months, with diets supplemented with a fruit or vegetable extract
identified as being high in antioxidant activity, could prevent the
age-related induction of receptor-mediated signal transduction deficits
that might have a behavioral component. Thus, the following parameters
were examined: (1) oxotremorine-enhanced striatal dopamine release
(OX-K+-ERDA), (2) cerebellar  receptor
augmentation of GABA responding, (3) striatal synaptosomal
45Ca2+ clearance, (4)
carbachol-stimulated GTPase activity, and (5) Morris water maze
performance. The rats were given control diets or those supplemented
with strawberry extracts (SE), 9.5 gm/kg dried aqueous extract (DAE),
spinach (SPN 6.4 gm/kg DAE), or vitamin E (500 IU/kg). Results
indicated that SPN-fed rats demonstrated the greatest retardation of
age-effects on all parameters except GTPase activity, on which SE had
the greatest effect, whereas SE and vitamin E showed significant but
equal protection against these age-induced deficits on the other
parameters. For example, OX-K+-ERDA enhancement was
four times greater in the SPN group than in controls. Thus,
phytochemicals present in antioxidant-rich foods such as spinach may be
beneficial in retarding functional age-related CNS and cognitive
behavioral deficits and, perhaps, may have some benefit in
neurodegenerative disease.
Key words:
antioxidants; aging; diet; dopamine; GABA; norepinephrine; striatum; cerebellum; cognitive behavior
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INTRODUCTION |
It is well known that there are
numerous declines in central neuronal functioning that can occur in
aging in the absence of neurodegenerative disease. These alterations
may be manifested as a loss of neurotransmitter receptor sensitivity
such as: (1) muscarinic (Amenta et al., 1989 ; Araujo et al., 1990;
Joseph et al., 1990; Sherman and Friedman, 1990; Vannucchi, 1991; Viana et al., 1992; Yufu et al., 1994; Egashira et al., 1996), (2) adrenergic (Burnett et al., 1990; Gelbmann and Muller, 1990; Gould and Bickford, 1997), (3) dopaminergic (Joseph et al., 1978; Roth and Joseph, 1994;
Gould et al., 1996; Volkow et al., 1996; Araki et al., 1997; Zhang et al., 1997; Levine and Cepeda, 1998), and (4) opioid (Dondi et
al., 1992; Kornhuber et al., 1996; Nagahara et al., 1996).
Although a great deal of research has been devoted toward the
delineation of the most critical factors that may account for this
functional neuronal loss in aging and enhanced loss in age-related neurodegenerative diseases [Alzheimer's (AD) and Parkinson's (PD) diseases], their specification has been elusive. However, recent studies have suggested that one of the most important may be
age-related decrements in the ability to mitigate long-term oxidative
stress (OS) effects. For example, OS may be a primary etiological
factor in both AD (Finch and Cohen, 1997) and PD (Jenner, 1996), and there are increases in OS vulnerability as a function of age (Joseph et
al., 1996). Evidence also indicates that there are reductions in
endogenous antioxidants in aging (e.g., glutathione, Ohkuwa et al.,
1997; and glutamine synthetase, Carney et al., 1994), with
increases in lipid peroxidation (Migheli et al., 1994; Yu, 1994).
Given these considerations, we believed that it might be possible to
counter dietarily the decreases in antioxidant protection that occur in
aging by increasing the intake of fruits and vegetables identified as
being high in antioxidant activity (Yamori and Horie, 1994; Cao et al.,
1995, 1996; Meydani et al., 1995; Taylor and Nowell, 1997; Wang et al.,
1996). Such consumption has already been found to reduce cancer
incidence (Doll, 1990; Willett, 1994a,b) and ischemic heart disease
(Hughes, 1995; Mayne, 1996).
In the brain, the consumption of the flavonoid glycosides of ginkgo
biloba decreased memory impairment (Rai et al., 1991), difficulties in
concentration (Kleijnen and Knipshild, 1992a,b), Ca+2-induced increases in neuronal oxidative
metabolism (Oyama et al., 1993, 1994), and AD progression (Kanowski et
al., 1996). Thus, present research was directed toward determining if
the early appearance of decrements in receptor sensitivity (at 15 months of age in Fischer 344 rats), loss of calcium homeostasis (Landfield and Eldridge, 1994), and cognitive performance could be
prevented by 8 months (6-15 months) of feeding of a control diet or
diets containing vitamin E or extracts of strawberries or spinach.
Strawberries and spinach have been identified previously (Cao et al.,
1995, 1996; Wang et al., 1996) by the oxygen radical absorbance
capacity (ORAC) assay as being high in antioxidant activity.
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MATERIALS AND METHODS |
Animals
The subjects consisted of 80 male Fischer 344 rats (Harlan
Sprague Dawley, Indianapolis, IN). The rats were individually housed in
stainless steel mesh suspended cages, provided food and water ad
libitum, and maintained on a 12 hr light/dark cycle. All animals were observed daily for clinical signs of disease.
After a 12 d acclimatization period to the facility, the
6-month-old rats were weight-matched, given 2 weeks on the control (modified AIN-93) diet (Table 1), and
randomly assigned to one of four groups: control diet, or the control
diet supplemented with 500 IU vitamin E acetate, 0.95% (w/v)
strawberry extract, or 0.64% spinach extract (Table 1). They were fed
these diets for 8 months before experimental testing. The amounts of
strawberry or spinach extracts added into the control diets were based
on an equivalent ORAC activity so that each diet provided equivalent antioxidant activity (1.36 mmol Trolox equivalent per kilogram of
diet). Monthly weights and food intakes (over a 48 hr period) were
recorded. These animals were used in compliance with all applicable
laws and regulations as well as principles expressed in the National
Institutes of Health, United States Public Health Service Guide for the
Care and Use of Laboratory Animals. This study was approved by the
Animal Care and Use Committee of our center.
Diet preparation
We added 400 gm of sample to water in the ratio of 2:1 for
strawberries and spinach, then homogenized it in a blender for 2 min.
The recovered homogenate was centrifuged at 13,000 × g for 15 min at 4°C. The supernatant was then recovered and combined in
freezer bags, 500 ml/bag. The extract was frozen and then crushed and
placed in the freeze drier until dry, which usually required ~7 d.
The freeze-dried extracts were shipped to Research Diets Inc. (New
Brunswick, NJ) where they were combined with a control diet (Table 1).
The amount of corn starch in the control diet was adjusted accordingly
when vitamin E acetate and strawberry or spinach extracts were
added.
Procedures
Dopamine release. Dopamine (DA) release was conducted
as previously described (Joseph et al., 1988a,b; 1990). Briefly,
cross-cut (300 µm, McIlwain tissue chopper) striatal slices were
obtained from the animals maintained on the various diets. The slices
were placed in small glass vials containing modified
Krebs'solution-Ringer's solution basal release medium (BRM) that had
been bubbled for 30 min with 95% O2 and 5%
CO2 and which contained (in mM)
NaHCO3 21, glucose 3.4, NaH2PO4
1.3, EGTA 1, MgCl2 0.93, NaCl 127, and KCl 2.5 (low KCl),
pH 7.4. They were then placed in the perfusion chambers in which they
were maintained at 37°C and perfused with the BRM for 30 min. After
this equilibration period, the medium was then switched to one
containing (in mM) KCl 30, CaCl2 · 2 H2O 1.26 (in place of EGTA), and NaCl 57 and 0 or 500 µM oxotremorine, and the enhancement of
K+-evoked striatal dopamine release
(K+-ERDA) was assessed. DA release was then
quantitated by HPLC coupled to electrochemical detection. Data were
expressed as picomoles per milligram of protein as determined by the
Lowry procedure (Lowry et al., 1951).
Electrophysiology. Rats from the various diet groups were
anesthetized with urethane (0.75-1.25 gm/kg), intubated, and allowed to breath spontaneously. Corneal reflex and toe pinch was used to
monitor anesthetic level to establish equal planes of anesthesia. A
heating pad was used to maintain body temperature at 37°C. Animals were placed in a stereotaxic frame, and the skin and muscle over the
posterior vermis was removed. The cistern was drained, and the skull
and dura over the vermis were removed. A solution of 2% agar in saline
covered the brain. Recordings were made in lobules VI and VII of
cerebellar vermis from Purkinje cells as identified by anatomical
location and the characteristic complex spiking of Purkinje cells.
Neuronal signals were amplified and filtered ( 3 dB at 0.3 and 5 kHz)
and displayed on a storage oscilloscope. Action potentials were
isolated using a window discriminator, and the output was displayed
using a strip chart recorder. Single units had to have a
signal-to-noise ratio of at least 2:1. Multibarrel glass micropipettes were used for single-cell recording and local drug application via
microiontophoresis (resistance of the recording electrodes 1.5-3.3
 ). In the multibarrel glass micropipettes, two barrels were
filled with 3 M NaCl, and the other two barrels were filled with GABA (0.25 M, pH 4.0-4.5) and with the -adrenergic
agonist, isoproterenol (ISO) (0.25 M, pH 4.0-4.5),
respectively. A constant current source provided ejection and retaining
currents for the drug barrels and passed an equal current of opposite
polarity through the balance barrel to neutralize the tip potential.
Uniform pulses of drug were applied at regular intervals.
GABA was locally applied by microiontophoresis to produce a 10-30%
inhibition of spontaneous firing rates. Isoproterenol was then applied
concurrently until either a change in the response to GABA was observed
or a change in baseline spontaneous rate was observed. Four
applications of GABA were given before ISO was coadministered.
After ISO was turned off, GABA was given until it could be determined
if the pre-ISO level of GABAergic inhibition would return. Only cells
in which the post-ISO level of GABAergic inhibition matched the pre-ISO
level of GABAergic inhibition were analyzed. Drug-induced responses
were quantified by computer. The rate meter data were digitized, and
the percent inhibitions of firing rate resulting from drug applications
were calculated.
45Ca recovery. Striatal synaptosomes were
isolated from the individual Fischer 344 rats as described previously
(Yeh et al., 1993). The final pellets of Ficoll-purified synaptosomes
were washed twice and resuspended in the basal medium (B-cond, in
mM: 136 NaCl; 5 KCl; 1.2 CaCl2; 1.3 MgCl2; 10 glucose; and 20 Tris, pH 7.65) at the
protein concentration 1-1.3 mg/ml. Aliquots of synaptosomes (50 µl)
were preincubated for 5 min at 37°C with constant shaking. OS in
synaptosomes was induced by adding H2O2 (final
concentration 300 µM) for 15 min. 45Ca uptake
studies were performed as described (Leslie et al., 1980). The
45Ca uptake was started by transferring oxidized and
control synaptosomes to basal medium or depolarizing medium (D-cond, in
mM: 60 KCl; 1.2 CaCl2; 1.3 MgCl2; 10 glucose; and 20 Tris, pH 7.65). Both media
had previously been supplemented with 45CaCl2
(2 µCi). The reaction was stopped after 2 min by rapid filtration of
samples through Whatman GF/B (Maidstone, UK) filters, followed by
washing three times with ice-cold stop medium (in mM: 136 NaCl; 5 KCl; 3 EGTA; 1.3 MgCl2; 10 glucose; and 20 Tris, pH 7.65) by using vacuum filtration (BRANDEL, model
ML-48). The radioactivity retained in the filters was measured
by a liquid scintillation counter (WALLAC 1409; WALLAC Oy, Turku,
Finland) programmed for automatic quenching correction. Results were
expressed as nanomoles of Ca2+ per milligram of
protein. Percent of increase in 45Ca uptake (Increase) and
recovery at 30 sec after depolarization (Recovery) were calculated as
follows:
Cognitive testing. The working memory version of the
Morris water maze (MWM), with a 10 min intertrial interval, was
performed to test spatial learning and memory (Morris, 1984; Brandeis
et al., 1989). Performance on the maze, including the working memory paradigm, has been shown to deteriorate with aging (Gage et al., 1984;
Rapp et al., 1987; Gallagher and Pelleymounter, 1988; Brandeis et al.,
1989; Van der Staay and de Jonge, 1993; Ingram et al., 1994) because of
a specific deficit in the ability of aged rats to use spatial
information (Rapp et al., 1987).
For these experiments, the maze consisted of a circular black
fiberglass pool (134 cm in diameter × 50 cm in height), filled to
a depth of 30 cm with water maintained at 23°C. The pool was divided
into four equal-size quadrants. The circular escape platform (10 cm in
diameter) was colored black and, therefore, hidden from sight. The
platform was submerged 2 cm below the surface of the water in the
center of one of the quadrants; its location was changed to a different
quadrant for each session of testing. The maze was placed in a room
with the lights dimmed, and there were numerous extramaze cues on the
walls.
MWM testing was performed daily for 4 consecutive d, with a morning and
an afternoon session, two trials each session, with a 10 min intertrial
interval between the two trials. At the beginning of each trial, the
rat was gently immersed in the water at one of four randomized start
locations (located 90° apart on the perimeter of the pool). Each rat
was allowed 120 sec to escape onto the platform; if the rat failed to
escape within this time, it was guided to the platform. Once the rat
reached the platform, it remained there for 15 sec (trial 1, reference
memory or acquisition trial). The rat was returned to its home cage
between trials (10 min). Trial 2 (the working memory or retrieval
trial) used the same platform location and start position as trial 1. Performance (latency to find platform in seconds, distance swam in
centimeters, and swim speed in centimeters per second) on each trial
was videotaped and analyzed with image tracking software (HVS Image,
Hampton, England).
Analyses of oxidative stress. The effects of oxidative
stress on the production of reactive oxygen species (ROS) in the brain tissue obtained from the various diet groups were assessed using 2',7'-dichlorofluorescein diacetate (DCFH-DA; Molecular Probes, Eugene,
OR) analysis (Ueda et al., 1997). It has been shown that DCFH-DA is
nonpolar, nonionic, crosses cell membranes, and is enzymatically
hydrolyzed by intracellular esterases to nonfluorescent DCFH-DA. In the
presence of ROS, DCFH-DA is rapidly oxidized to highly fluorescent
2',7'-dichlorofluorescein (DCF) (Lebel et al., 1992). For these
experiments, striatum and cerebellum were quickly removed from the
brain. Pellets of membranes (synaptosomes, myelin, and mitochondria)
were obtained as described previously (Denisova et al., 1998). Each
pellet was then resuspended in incubation media (IM: in mM:
136 NaCl; 5 KCl; 1.2 CaCl2; 1.3 MgCl2; 10 glucose; and 20 Tris, pH 7.65) at the
protein concentration 1-1.3 mg/ml. Aliquots (100 µg of protein) were
preincubated for 5 min at 37°C with constant shaking. DCF was added
to each sample (final concentration 50 µM) for 30 min.
Samples were washed, resuspended in IM, and placed on a 96-well plate.
Fluorescence was monitored for 15 min on CytoFluor multi-well plate
reader (PerSeptive Biosystem, Framingham, MA). The dye was excited at
485 nm, and emission was filtered using 530 nm filter (slit 20 and 25 for excitation and emission, respectively). The results were expressed
as DCF fluorescence.
GTPase activity Striatal membranes were prepared, and low
KM GTPase analysis was performed according
to the method of Cassel and Selinger (1976) as modified by Joseph et
al. (1998). Briefly, membranes were prepared by homogenizing the
striatal tissue in 10 ml of Tris buffer, 50 mM, pH 7.4, EDTA, 10 mM, and phenylmethylsulfonyl fluoride, 0.1 mM in a Tekmar Company (Cincinnati, OH) Tissuemizer (setting 5, 5 sec). Membranes were then centrifuged at 20,000 × g for 10 min, and the pellet was resuspended and washed at
the same speed and time and resuspended in 1 ml Tris-EDTA. Membranes (10 ug membrane protein) were then incubated in a reaction mixture containing (in mM): 100 NaCl; 20 Tris-HCl, pH 7.4,; 5 MgCl2; 1 ATP; 2 AppNHp; 10 phosphocreatine; 2 dithiothreitol; 0.1 EDTA; 0.1 EGTA; 60 U/ml creatine phosphokinase; 0.3 µM [ -32P]GTP (NEN, 30 Ci/mmol) for 10 min at 37°C, and carbachol (0, 10 5 103 M). After stopping the reaction
with 900 µl of ice-cold 5% activated charcoal in 20 mM
phosphoric acid, an aliquot was taken, and radioactivity was determined
by liquid scintillation counting. Low KM GTPase activity was calculated by subtracting the activity measured in the
presence of 100 µM unlabeled GTP from total activity.
Activity was expressed in picomoles of [-32P]
hydrolyzed per milligram of protein, per minute. Values were expressed
as mean ± SEM of the differences between basal and
carbachol-stimulated low KM GTPase
activity ( G) in picomoles per milligram of protein per minute.
Proteins were determined by the Lowry et al. (1951) method.
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RESULTS |
Weights and food intakes
The rats gained weight from 6 to 15 months
[F(11,36) = 215.44; p < 0.001] from an average of 356.3 ± 0.4 precontrol diet to 473.4 ± 3.4 at age 15 months. However, there were no differences in weight between the diet groups over time (p > 0.05) or at age 15 months (p > 0.05). There
were also no differences in food intakes between the diet groups over
the course of the study (p > 0.05).
DA release
As can be seen from Figure 1, all
striatal slices obtained from the animals in the various diet groups
(eight animals per group) showed significantly greater oxo-enhanced
K+-ERDA than that seen in those obtained from
animals maintained on the control diet [F(3,28) = 10.6, p < 0.0001; Fisher's least significant
difference test: control vs strawberry, p < 0.03; control vs spinach, p < 0.0001; and control vs
vitamin E, p < 0.014]. Additional post
hoc comparisons indicated that oxo-enhanced K+ ERDA in the spinach-fed group was greater than
the strawberry-fed (p < 0.002) or vitamin E
(p < 0.006) groups.
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Figure 1.
Oxotremorine enhancement of dopamine release from
striatal slices obtained and prepared from animals maintained on the
control or the various antioxidant diets. For this figure,
a differs from the strawberry, spinach, and high vitamin
E groups (p < 0.034; p < 0.0001; and p < 0.014, respectively).
c differs from the strawberry
(p < 0.002) and high vitamin E
(p < 0.006) groups, whereas
bs do not differ from each other
(p > 0.05).
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Electrophysiology
Electrophysiological recordings from cerebellar Purkinje neurons
indicated that the ability of isoproterenol to modulate GABA inhibitions decreased in the rats maintained on the control diet. When
the effect of ISO was tested in aged control rats, as had been seen
previously (Gould and Bickford, 1997), only 32% of the neurons tested
demonstrated an ISO augmentation of the GABAergic responses. Aged rats
that had been on the vitamin E, strawberry, or spinach diet had
significantly more cells that responded to ISO
(p < 0.05 Fisher's exact test). Examples of
ratemeter records for the control and strawberry-supplemented rats are
shown in Figure 2, whereas the mean
responses for all groups are shown in Figure
3.
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Figure 2.
Examples of ratemeter records from extracellular
recordings of Purkinje neurons from an aged rat maintained for 8 months
(from 6-15 months of age) on a control
(A-C) or strawberry
(D-F) diet. In A
and D, the baseline response to GABA is shown. The dose
of GABA was adjusted to produce a 15-30% inhibition of the Purkinje
cell firing rate. In B and E,
isoproterenol was coapplied with GABA. In the control, aged rat,
isoproterenol had no effect at lower doses (data not shown) and, at a
high dose the GABA inhibition was reduced, and the baseline firing rate
was diminished. In contrast, in the aged rat that was maintained on the
strawberry diet, isoproterenol increased the GABA inhibition to 98%.
This is similar to what has been previously observed in young rats.
C and F show recovery back to
preisoproterenol levels of GABA inhibition. Bars above
the ratemeter indicate the drug application times. The
horizontal calibration bar indicates time in seconds,
whereas the vertical calibration bar indicates the
firing rate of the Purkinje neuron in action potentials per
second.
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Figure 3.
Mean isoproterenol facilitation of GABAergic
inhibition of cerebellar Purkinje cells in animals maintained on
control or various antioxidant diets for 8 months (6-15 months of
age). For this figure, as differ from the control group
at p < 0.05. No antioxidant groups differed from
each other.
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45Ca recovery
When differences in 45Ca recovery were examined in the
striatal synaptosomes obtained from the controls and various groups, the results indicated that there were significant differences as a
function of diet and H2O2 treatment
[F(3,26) = 7.79; p < 0.001],
with all of the diet groups showing greater 45Ca recovery,
i.e., a greater ability to extrude or sequester calcium after
depolarization than the control group. As shown in Figure 4, 45Ca recovery was
significantly decreased in the H2O2-exposed
diet control group as compared with nonexposed controls
(p < 0.001, post hoc t
tests). These tests also revealed that no deficits were seen in
45Ca recovery after exposure to
H2O2 in any of the diet-fed groups (All
p values > 0.05 in comparisons between
non-H2O2-exposed and H2O2-treated groups for the spinach, vitamin E,
and strawberry groups, Fig. 4). Additionally, the spinach-fed,
H2O2-exposed group showed greater recovery than
either the vitamin E- or
strawberry-H2O2-treated groups
(p < 0.01 for both comparisons).
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Figure 4.
Calcium recovery in synaptosomes obtained from
animals in the various diet groups and exposed to 0 or 300 µM H2O2 (15 min) and depolarized
with 60 mM KCl. For this figure, a differs
from untreated (no H2O2) control
(p < 0.001), b differs from
H2O2-treated control
(p < 0.02; p < 0.001;
and p < 0.05, respectively), and c
differs from vitamin E (p < 0.01) or
strawberry-fed (p < 0.01)
H2O2-treated groups.
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Cognitive testing
For the MWM, latency to find the platform, distance swam, and
speed were calculated separately for trials 1 and 2. Subsequently, to
analyze the effect of the diets on cognitive performance, ANOVAs were
run only on data for days 3 and 4, because no difference was expected
between the diet groups before this time, and because the rats were
still learning the task (as seen by a significant effect of time). For
trial 1, ANOVA showed a significant effect of diet group for latency
[F(3,71) = 3.39; p < 0.05]
and distance [F(3,71) = 5.18; p < 0.01] (Fig. 5). The group fed the
spinach diet had a shorter latency to find the platform in the
reference memory trial of the MWM compared with the control group
(p < 0.05) (Fig. 5A). Additionally,
both the spinach and vitamin E groups showed a shorter distance to the
platform on trial 1 compared with the control group
(p < 0.05) (Fig. 5B). These
differences were not caused by swim speed, because there was no
significant effect of diet group on this measure. There were also no
differences between any of the diet groups on working memory (trial 2)
performance.
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Figure 5.
Morris water maze performance in the various diet
groups. Performance was assessed over 4 d (2 sessions per day, 2 trials per session). Results are given as latencies
(A) and distances (B) to
find the hidden platform from the first and second trials for each
session on days 3 and 4. For this figure, as differ from
the control group at p < 0.05 (trial
1).
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Oxidative stress
As can be seen in Figure
6A, there were
significant differences in DCF fluorescence among the various groups
[F(3,20) = 87.61; p < 0.0001]
in the striatum. In this regard, the striata obtained from the
strawberry-supplemented group did not exhibit any increased level of OS
protection relative to the control group (p > 0.05 control vs strawberry group). Only the spinach and vitamin E
groups showed greater native OS protection than controls
(p < 0.0001 for both spinach vs control and
vitamin E vs control), whereas the vitamin E group showed less native
OS protection than the spinach group (p < 0.007).
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Figure 6.
Interaction of diets and oxidative stress in
striatum (A) and cerebellum
(B). Oxidative stress in brain tissue was
evaluated by using DCFH-DA, as described in Material and Methods. Data
for the formation of reactive oxygen species were obtained from 6-13
individual animals per group, performed with 6-10 replicates, and
expressed as mean ± SEM. For this figure, means not sharing a
common letter are significantly different (p < 0.01).
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In the cerebellum (Fig. 6B), there were also
significant differences in DCF fluorescence among the various groups
[F(3,42) = 156.62; p < 0.0001], indicating increased native protection against oxidative
stress in these groups. All of the supplemented groups differed from
control (p < 0.0001), whereas the animals maintained on the high vitamin E-supplemented diet showed the highest
protection (p < 0.0001 as compared with the
strawberry- and spinach-supplemented groups). The cerebellar tissue
obtained from animals maintained on the strawberry-supplemented diet
also showed less fluorescence (more antioxidant protection) than the spinach-supplemented group (p < 0.0001).
GTPase activity
The results with respect to age-induced decrements in
carbachol-stimulated GTPase activity differed from those of the other parameters in that the strawberry and the vitamin E supplementation prevented the decrements, whereas spinach did not [control vs spinach,
p > 0.05; control vs strawberry, p < 0.0001; control vs vitamin E, p < 0.05;
F(3,20) = 12.6; p < 0.0001 overall]. The results expressed as between basal and
carbachol-stimulated GTPase activity are shown in Figure
7.
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Figure 7.
Differences (expressed as G from baseline) in
carbachol-stimulated low KM GTPase activity
from striatal slices obtained from the various diet groups (see
Materials and Methods). For this figure, b differs from
the control, spinach, and vitamin E groups
(p < 0.0001; p < 0.0001; and p < 0.003, respectively), whereas
c differs from the control and spinach groups
(p < 0.05).
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DISCUSSION |
As indicated in the introductory remarks, there is an abundance of
literature to suggest that diets high in fruits and vegetables are
important in preventing or moderating such major disorders as cancer
and heart disease. Even extracts of single foods such as garlic (Pinto
et al., 1997) and tomato (lycopene; Sharoni et al., 1997) can have some
antitumor properties. In the brain, evidence was also cited that
indicated that it might be possible to reduce the deleterious effects
of aging and AD through dietary supplementation with ginkgo biloba or
vitamin E. In addition, recent studies have indicated that garlic
extract was effective in preventing brain atrophy (Moriguchi et al.,
1997) as well as learning and memory impairments (Nishiyama et al.,
1997) in the senescence-accelerated mouse.
Moreover, preliminary findings from our laboratories have suggested
that fruit and vegetable extracts high in both flavonoid levels (e.g.,
spinach and strawberries) as well as total antioxidant activity, as
assessed via the ORAC assay (Cao et al., 1995, 1996; Wang et al.,
1996), also antagonize the deleterious "age-like" neuronal effects
of 48 hr of exposure to 100% normobaric O2. Rats fed diets
containing these extracts for 6 wks before exposure showed no loss in
striatal muscarinic or cerebellar GABAergic receptor sensitivity
(Chadman et al., 1997). These oxygen-induced decreases in neuronal
function have been investigated in numerous experiments and have also
been shown to be sensitive to aging and have been associated with
behavioral deficits.
However, there is a paucity of research concerned with the positive
effects of fruit and/or vegetable supplementation in retarding age-related neuronal and behavioral dysfunctions using age-valid tests.
The results of these experiments have provided such evidence and have
suggested, for the first time, that dietary supplementation with foods
identified as being high in antioxidant activity (via ORAC) can retard
the effects of aging on four rather diverse indices of neuronal and
behavioral functions that are sensitive to both oxidative stress and
aging.
Thus, each of these diets was effective in retarding the age-associated
deficits in muscarinic receptor sensitivity, as assessed via
oxotremorine enhancement of striatal DA release; isoproteronal facilitation of GABAergic inhibition of cerebellar Purkinje neurons; calcium regulation; and Morris water maze performance. Spinach supplementation consistently produced the greatest retardation of the
aging effects in calcium regulation, oxotremorine-enhanced K+-ERDA, and the onset of cognitive deficits; all
three diets were similarly effective in preventing the loss of
NE sensitivity in the Purkinje cells. In contrast to the other
parameters, when decrements in carbachol-stimulated GTPase activity
were assessed, the results indicated that the vitamin E and
strawberry-supplemented diets showed the greatest efficacy in
preventing their onset.
We are presently attempting to delineate the sites of actions of the
phytochemicals present in these foods and particular classes that are
the most effective in preventing these age-related deficits. One
important class may be the flavonoids. Flavonoids are recognized as one
group of phytochemicals which include, among others: allium compounds
(diallyl sulfide and allyl methyl trisulfide) and carotenoids
( -carotene, -carotene, lutein, and lycopene). Because flavonoids
are present to a considerable degree in vegetables and fruits, they
make up an important part of the human diet (Kuhnau, 1976). Their daily
intake has been estimated to be as high as 1 gm per day, with the
primary dietary source being vegetables. However, although some
flavonoids may have higher antioxidant activity than others (Cao et
al., 1997), it may be that ultimately it is the "Gestalt" of the
myriad of interactions among various classes of phytochemicals present
in foods with high ORAC activity that may confer this potent
antioxidant protection. In other words, "the whole is more than the
sum of its parts".
One point that can be made in this regard is that preliminary analyses
of the regional differences in vitamin C among cortical, hippocampal,
cerebellar, and striatal tissues obtained from the diet groups in this
study indicate that there are no differences as a function of diet.
These findings indicate that the prevention of the age changes among
the various diets are probably not the result of vitamin C contained in
the strawberry or spinach extracts. Moreover, in a preliminary analysis
of the brain levels of vitamin E among the various diets, only the high
vitamin E diet increased regional brain levels of vitamin E relative to
controls. However, the striata obtained from the animals maintained on
the high vitamin E diet showed the lowest levels of vitamin E of any
brain region examined, whereas the hippocampus showed the highest.
Because the high vitamin E diet group showed significantly less
protection against loss of oxotremorine enhancement of DA, Morris water
maze performance, and striatal synaptosomal Ca2+
recovery relative to the spinach-supplemented group, these findings indicate that other phytochemicals (e.g., flavonoids) contained in the
diets of the supplemented groups may be more effective in protecting
against the deleterious effects of aging on these parameters.
However, given the findings with respect to the DCF analyses, it may be
that factors other than those having to do with protection against
oxidative stress may be involved. For example, in the cerebellum,
whereas the supplemented diets were effective in increasing the level
of OS protection (reduced DCF fluorescence), in all of the groups as
compared with control the spinach-supplemented group had the lowest
level of protection of any of the groups, although this supplementation
significantly retarded the onset of loss of NE inhibition as a function
of aging. In the striatum, the strawberry-supplemented group showed the
lowest level of OS protection (highest fluorescence) but had the best
protection against the loss of carbachol-stimulated GTPase activity of
any group, whereas vitamin E did not necessarily provide the greatest long-term protection in any of the parameters examined but showed the
greatest efficacy in reducing fluorescence in the DCF assessments in
both the striatum and cerebellum. Thus, it may be that there are other
effects of the phytochemicals contained in spinach and strawberries in
addition to antioxidant protection. One of these may be alterations in
membrane rigidity. It is known, for example, that flavonoids increase
membrane fluidity (Ramassamy et al., 1993; Stoll et al., 1996; Halder
and Bhaduri, 1998), and we have shown previously (Joseph et al., 1995)
that by incubating striatal slices in
S-adenosyl-L-methionine, a potent
membrane-fluidizing agent, we were able to reverse the age-related
deficits in oxotremorine-enhanced K+-ERDA. We are
currently investigating whether the flavonoids contained in the
strawberry- and spinach-supplemented diets, especially the
anthocyanins, can have similar effects in these assessments.
In addition, attempts are being undertaken to determine whether these
diets will also be effective in reversing the deleterious effects of
aging on the above parameters as well as motor behavior. However,
present findings, thus far, suggest that nutritional intervention with
fruits and vegetables may play an important role in preventing or
perhaps even reversing the effects of oxidative stress in aging on
brain function.
| |
FOOTNOTES |
Received April 14, 1998; revised July 13, 1998; accepted July 20, 1998.
This work was supported by the United States Department of Agriculture
(USDA) and United States Public Health Service Grants AG04418,
AG007728, and VA MRS. We thank Dr. Donna Bilinski for her valuable help
with this manuscript and Mr. Donald E. Smith of the animal care staff
(Department of Comparative Biology and Medicine) of the USDA Human
Nutrition Research Center at Tufts University for the feeding and
maintenance of the rats used in this study.
Correspondence should be addressed to Dr. J.A. Joseph, United States
Department of Agriculture Human Nutrition Research Center on Aging at
Tufts University, Room 919, 711 Washington Street, Boston, MA 02111.
| |
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M. C. Cartford, C. Gemma, and P. C. Bickford
Eighteen-Month-Old Fischer 344 Rats Fed a Spinach-Enriched Diet Show Improved Delay Classical Eyeblink Conditioning and Reduced Expression of Tumor Necrosis Factor alpha (TNFalpha ) and TNFbeta in the Cerebellum
J. Neurosci.,
July 15, 2002;
22(14):
5813 - 5816.
[Abstract]
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A. Martin, R. Prior, B. Shukitt-Hale, G. Cao, and J. A. Joseph
Effect of Fruits, Vegetables, or Vitamin E-Rich Diet on Vitamins E and C Distribution in Peripheral and Brain Tissues: Implications for Brain Function
J. Gerontol. A Biol. Sci. Med. Sci.,
March 1, 2000;
55(3):
144B - 151.
[Abstract]
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J. A. Joseph, B. Shukitt-Hale, N. A. Denisova, D. Bielinski, A. Martin, J. J. McEwen, and P. C. Bickford
Reversals of Age-Related Declines in Neuronal Signal Transduction, Cognitive, and Motor Behavioral Deficits with Blueberry, Spinach, or Strawberry Dietary Supplementation
J. Neurosci.,
September 15, 1999;
19(18):
8114 - 8121.
[Abstract]
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Top
Abstract
Introduction
