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RESULTS
Neuroprotective effects of resveratrol against prenatal stress in rats Page 70

7.1. Neonatal parameters
7.1.1. Gestational length, litter size, mortality and early physical developments
Sexually dimorphic effect was not observed in any of the parameters, hence mean
values for both male and female were collapsed together (male and female data of each
parameter are given in Table 6, 8, 10, 12 & 14). Neither the Restraint stress nor the
resveratrol treatment had any effect on gestational length, litter size, and early physical
developments (day of ear pinna detachment, day of eruption of upper & lower incisors and
day of eye opening). (Table 4)
RESULTS
Table 4: Neonatal parameters following prenatal exposure to stress, resveratrol and their
combination
CON
RES
EGS
LGS
EGS+RES
LGS+RES
P value
ANOVA
significance
Gestational
length
22.83±0.16
22.83±0.16
23.33±0.21
22.50±0.22
22.33±0.21
22.83±0.30
p=0.077
F=2.231
Litter size
6.83±0.60
6.50±0.34
7.33±0.55
7.16±0.47
6.66±0.33
7.33±0.33
p=0.689
F=0.614
Day of ear
pinna
detachment
3.33±0.21
4.00±0.00
3.83±0.16
3.83±0.16
3.83±0.16
3.83±0.16
p=0.1073,
F=2.000
Day of
eruption of
incisors
12.66±0.33
12.83±0.16
12.66±0.33
13.5±0.22
13.16±0.30
12.33±0.21
p=0.0675
F=2.342
Day of eye
opening
16.0±0.25
16.5±0.22
16.0±0.25
16.66±0.40
16.33±0.21
16.0±0.25
p=0.711
F=0.584
Data are expressed as mean±SEM (n=24). Animal groups: CON: control, RES: pups received prenatal resveratrol
during entire gestation period, EGS: pups exposed to
prenatal stress during gestation day1 to 10, LGS: pups exposed to stress during gestation day 11 till delivery,
EGS+RES:pups received prenatal stress during gestation day 1
to 10 along with resveratrol entire gestation period and LGS+RES: pups received prenatal stress during gestation day
11 to till delivery along with resveratrol entire gestation
period. (One way ANOVA, Bonferroni’s test).
RESULTS
7.1.2. Birth weight and weight gain
A significant correlation between prenatal environment (stress, resveratrol and their
combinations) and the birth weight/weight gain was observed. ANOVA multiple comparison
tests revealed that birth weight, weight on day 7 and day 14 was significantly lower in the
offspring exposed to late gestational stress (LGS) compared to control group offspring (birth
weight & weight on day 7; CON Vs LGS, p<0.001 & weight on day 14: CON Vs LGS,
p<0.01). Offspring received prenatal stress during late gestation along with resveratrol
treatment (LGS+RES) showed a significant increase in birth weight and weight on day 7
compared to the offspring exposed the late gestational stress (birth weight & weight on day 7;
LGS Vs LGS+RES, p<0.05). However the weight gain calculated on day 14 and day 21
remained unaffected. (Figure R1)
0
10
20
30
40
CON
EGS
LGS
RES
EGS+RES
*** LGS+RES
a$
***
c
#$
**
Birth weight Day 7 Day 14 Day 21
Weight in g
Figure R1: Birth weight (in g) and weight gained at different neonatal period following prenatal exposure to
stress, resveratrol and their combination.
ANOVA significance (Bonferroni’s test, each bar represents mean±SEM, n=12)
Birth weight, P<0.01 & F=5.089; Day-7, P<0.0001 & F=15.30; Day-14, P<0.01 & F=3.835; Day-21, P<0.189
& F=1.624
CON Vs EGS & LGS, *** p<0.001, ** p<0.01,
RES Vs LGS, c p<0.001, a p<0.05
EGS Vs EGS+RES, # p<0.05
LGS Vs LGS+RES, $ p<0.05
RESULTS
7.2. HPA axis activity
7.2.1. Serum cortisol level
A significant increase of serum cortisol level was observed in the offspring exposed to
prenatal stress (both early and late) compared control group offspring (CON Vs EGS, p<0.01
& CON Vs LGS, p<0.001). Offspring received prenatal stress during late gestation along with
resveratrol treatment (LGS+RES) showed a significant decrease in serum cortisol level
compared to offspring received prenatal stress during late gestation (LGS Vs LGS+RES,
p<0.05). However offspring received early gestational stress along with resveratrol treatment
(EGS+RES) failed to normalize the elevated serum cortisol level in the offspring exposed to
early prenatal stress (EGS). (Figure R2)
CON
RES
EGS
LGS
EGS+RES
LGS+RES
0
1
2
3
4
***
**
b
**
$
μg/dl
Figure R2: Serum Cortisol level (μg/dl) in rats.
P<0.0001 & F=9.590
CON Vs EGS & EGS+RES, ** p<0.01; CON Vs LGS, *** p<0.001
RES Vs LGS, b p<0.01
RESULTS
7.2.2. Adrenal gland weight and ascorbic acid (Vit C) level
Gestational stress, resveratrol treatment and their combination did not have any
significant effect on adrenal gland weight in 40 th day rats (p=0.942, F=0.242). The mean
adrenal gland weight (in mg, mean±SEM, n=12 per group) for group-1 was 8.57±0.30, for
group-2, 8.21±0.39, for group-3, 8.55±0.35, for group-4, 8.42±0.39, for group-5, 8.15±0.38
and for group-6, 8.21±0.39.
Adrenal ascorbic acid level was significantly decreased in the offspring exposed to
prenatal stress (both early and late) compared control group offspring (CON Vs EGS,
p<0.001 & CON Vs LGS, p<0.01). Pre-treatment with resveratrol during pregnancy able to
alleviate the decreased adrenal ascorbic acid level in prenatally stressed offspring but not
significantly (p=0.0599). (Figure R3)
CON
RES
EGS
LGS
EGS+RES
LGS+RES
0
200
400
600
800
***
a **
μg/mg Protein
Figure R3: Adrenal ascorbic acid (Vit-C) level (μg/mg Protein) in rats.
P<0.0001 & F=6.710
CON Vs EGS, *** p<0.001; CON Vs LGS, ** p<0.01
RES Vs EGS, a p<0.05
RESULTS
Summary of prenatal stress induced hyperactive HPA axis and its normalization by
resveratrol treatment:
Offspring exposed to prenatal stress during early and late gestation resulted in an
increase level of serum cortisol and a decrease level adrenal ascorbic acid. This indicating
prenatal stress induced hyperactive HPA axis. Resveratrol treatment along with prenatal
stress during late gestation could able to normalize the prenatal stress induced increased
cortisol level and adrenal ascorbic acid level in the offspring. However resveratrol treatment
failed to reverse the hyperactive HPA axis activity in early gestational stressed offspring.
(Table 5 & 6)
Table 5: Summary of HPA axis activity in rats in different groups
HPA axis activity
Serum cortisol
(μg/dl)
Adrenal ascorbic
acid
(μg/mg Protein)
Adrenal
weight
(gm)
CON (n=12) 1.29±0.22 666.1±30.0 8.57±0.30
RES (n=12) 1.49±0.17 599.2±16.1 8.21±0.39
EGS (n=12) 2.35±0.17***b 480.04±16.8*** b 8.55±0.35
LGS (n=12) 2.66±0.18** 515.4±31.4** 4.2±0.39
EGS+RES (n=12) 2.25±0.10** 566.6±17.8 8.15±0.38
LGS+RES (n=12) 1.88±0.07$ 568.5±28.9 8.21±0.39
F value 9.590 6.710 0.242
ANOVA
significance
(p value)
<0.0001 <0.0001 0.942
ANOVA significance (Bonferroni’s test) for serum cortisol
P<0.0001 & F=9.590
CON Vs EGS & EGS+RES, ** p<0.01; CON Vs LGS, *** p<0.001
RES Vs LGS, b p<0.01
ANOVA significance (Bonferroni’s test) for adrenal ascorbic acid
P<0.0001 & F=6.710
CON Vs EGS, *** p<0.001; CON Vs LGS, ** p<0.01
RES Vs EGS, a p<0.05
ANOVA significance (Bonferroni’s test) for adrenal gland weight
p=0.942 & F=0.242, No significant difference between the groups
RESULTS
Table 6: Summary of HPA axis activity in male and female rats
HPA axis activity
Serum cortisol
(μg/dl)
Adrenal ascorbic
acid
(μg/mg Protein)
Adrenal weight
(gm)
CON
Male
(n=6)
1.08±
0.06
p, 0.474
t, 0.874
F, 2.284
633.56±
38.15
p, 0.381
t, 0.914
F, 1.517
8.83±
0.45
p, 0.417
t, 0.846
Female F,1.278
(n=6)
1.50±
0.47
688.93±
46.99
8.31±
0.40
RES
Male
(n=6)
1.35±
0.27
p, 0.536
t, 0.739
F, 1.210
588.80±
25.49
p, 0.544
t, 0.627
F, 1.440
8.65±
0.65
p, 0.289
t, 1.118
Female F, 2.532
(n=6)
1.63±
0.25
609.63±
21.25
7.78±
0.41
EGS
Male
(n=6)
2.62±
0.17
p, 0.079
t, 3.329
F, 1.138
494.73±
25.13
p, 0.401
t, 0.859
F, 1.178
8.86±
0.53
p, 0.395
t, 0.887
Female F, 1.231
(n=6)
2.09±
0.08
465.35±
23.15
8.23±
0.47
LGS
Male
(n=6)
2.37±
0.17
p, 0.246
t, 1.621
F, 3.038
531.11±
42.12
p, 0.642
t, 0.479
F, 1.395
8.98±
0.65
p, 1.172
t, 1.468
Female F, 2.988
(n=6)
2.94±
0.30
499.86±
49.76
7.86±
0.38
EGS+RES
Male
(n=6)
2.10±
0.09
p, 0.163
t, 2.159
F, 1.000
564.08±
27.21
p, 0.892
t, 0.139
F, 1.129
8.23±
0.57
p, 0.856
t, 0.186
Female F, 1.051
(n=6)
2.39±
0.09
569.28±
29.61
8.08±
0.56
LGS+RES
Male
(n=6)
1.82±
0.15
p, 0.523
t, 0.767
F, 3.610
610.20±
50.24
p, 0.159
t, 1.52
F, 5.268
7.91±
0.56
p, 0.470
t, 0.749
Female F, 1.027
(n=6)
1.95±
0.05
526.88±
21.89
8.51±
0.57
Note: Unpaired, two tailed Student’s t-test. Each data represents mean±SEM
RESULTS
7.3. Neuronal assay of hippocampus
7.3.1. Quantification of DCX +ve neurons (marker of neurogenesis) in 21 and 40 days
old rat brain
The DCX +ve neuron is a marker of neurogenesis. Both early and late gestational
stress showed a significant decrease in DCX positive neurons in subglanular zone
(SGZ)/granular cell layer (GCL) of hilus of DG in both 21 st and 40th postnatal rat brain (CON
Vs EGS, p<0.01 & CON Vs LGS, p<0.001). Offspring received prenatal stress during late
gestation (LGS) showed a significant decrease in DCX positive neurons in DG compared to
the offspring received early gestational stress (EGS) in both 21 st and 40th postnatal rat brain
(EGS Vs LGS, p<0.001). Offspring exposed to prenatal stress (both early and late) along with
resveratrol (EGS+RES and LGS+RES) showed a significant increase in DCX positive
neurons in SGZ/GCL of hilus of DG when compared to prenatally stressed offspring (EGS
Vs EGS+RES, p<0.05 & LGS Vs LGS+RES, p<0.001) in both 21st and 40th postnatal rat
brain. No significant change in the number of DCX positive neurons in DG of offspring
exposed to prenatal resveratrol treatment alone (RES) was observed. (Figure R4 to R7)
CON
RES
EGS
LGS
EGS+RES
LGS+RES
0
20
40
60
80
**
C
***
C
###
# $$$
DCX +ve neurons in
SGZ/GCL in hilus of DG
Figure R4: Effect of prenatal stress and resveratrol treatment on neurogenesis (21 days old rat brain).
ANOVA significance (Bonferroni’s test, each bar represents mean±SEM, n=66 slides per group)
P<0.0001 & F=28.635
CON Vs EGS, ** p<0.01; CON Vs LGS, *** p<0.001
RES Vs EGS & LGS, c p<0.001
EGS Vs LGS, ### p<0.001; EGS Vs EGS+RES, # p<0.05
LGS Vs LGS+RES, $$$ p<0.001
RESULTS
CON
RES
EGS
LGS
EGS+RES
LGS+RES
0
10
20
30
40
50
**
b
***
C
$$$
##
###
DCX +ve neurons in
SGZ/GCL in hilus of DG
Figure R5: Effect of prenatal stress and resveratrol treatment on neurogenesis (40 days old rat brain).
ANOVA significance (Bonferroni’s test, each bar represents mean±SEM, n=66 slides per group)
P<0.0001 & F=65.401
CON Vs EGS, ** p<0.01; CON Vs LGS, *** p<0.001
RES Vs EGS, b p<0.01; RES Vs LGS, c p<0.001
EGS Vs LGS, ### p<0.001; EGS Vs EGS+RES, # # p<0.01
RESULTS
Figure R6: Photomicrographs of doublecortin positive (DCX +ve) neurons in hilus of DG in 21 days old rat brain
under 20X; CON: control, RES: pups received prenatal
resveratrol during entire gestation period, EGS: pups received prenatal stress during day 1 to 10, LGS: pups received
stress during day 11 till delivery, EGS+RES: pups
received prenatal stress during day 1 to 10 and resveratrol during entire gestation period and LGS+RES: pups received
prenatal stress during day 11 to till delivery and
resveratrol during entire gestation period. The arrows denote the DCX +ve neurons. The DCX +ve neurons were
counted in subglanular zone (SGZ)/granular cell layer
(GCL) of hilus of dentate gyrus using occulomicrometer (250μm length per horn).
CON RES EGS
LGS EGS+
RES
LGS+
RES
RESULTS
Figure R7: Photomicrographs of doublecortin positive (DCX +ve) neurons in hilus of DG in 40 days old rat brain
under 20X; CON: control, RES: pups received prenatal
resveratrol during entire gestation period, EGS: pups received prenatal stress during day 1 to 10, LGS: pups received
stress during day 11 till delivery, EGS+RES: pups
received prenatal stress during day 1 to 10 and resveratrol during entire gestation period and LGS+RES: pups received
prenatal stress during day 11 to till delivery and
resveratrol during entire gestation period. The arrows denote the DCX +ve neurons. The DCX +ve neurons were
counted in subglanular zone (SGZ)/granular cell layer
(GCL) of hilus of dentate gyrus using occulomicrometer (250μm length per horn).
CON RES EGS
LGS EGS+
RES
LGS+
RES
RESULTS
7.3.2. Cresyl violet staining
Prenatal stress produced a regional specific effect on hippocampal neurons. The CA1
and CA2 region of the hippocampus did not show any significant structural or numerical
changes in response to prenatal stress. The number of viable neurons in DG in prenatally
stressed offspring were significantly lower compared to control group offspring (CON Vs
EGS & LGS, p<0.001). Only late gestational stress (LGS) showed a significant loss (p<0.01)
of hippocampal CA3 neurons compared to control group. Similar results were obtained when
prenatally resveratrol treated offspring (RES) compared with prenatally stressed offspring
(EGS & LGS). Offspring who received prenatal stress along with resveratrol (EGS+RES and
LGS+RES) showed a significant increase in number of viable neurons in DG compared to
prenatally stressed offspring (EGS Vs EGS+RES, p<0.05 & LGS Vs LGS+RES, p<0.05).
Resveratrol treatment during late gestational period failed to exert protective effect on CA 3
hippocampal neurons. (Figure R8 to R13)
RESULTS
0
10
20
30
40
50
*** ***
$#
bb
** *
DG CA3 CA2 CA4
CON
RES
EGS
LGS
EGS+RES
LGS+RES
b
Neuronal number
Figure R8: Prenatal stress, resveratrol and their combination induced changes in the neuronal numbers in
250μm length area in hippocampal Cornu Ammonis (CA 3, CA2, CA1) and Dentate Gyrus (DG) in rat offspring.
Values are expressed as Mean±SEM (n=66 slides per group).
ANOVA significance (Bonferroni’s test) for normal DG neurons
P<0.0001, F=8.445
CON Vs EGS & LGS, ***p<0.001
RES Vs EGS & LGS, bp<0.01
EGS Vs EGS+RES, $p <0.05
LGS Vs LGS+RES, #p <0.05
ANOVA significance (Bonferroni’s test) for CA3 neurons
P=0.006, F=4.46
CON Vs LGS, ** p<0.01, and CON Vs LGS+RES, *p <0.05
RES Vs LGS, bp <0.01
P=0.2274, F= 1.389, No significant difference between the groups
P=0.1801, F=1.528, No significant difference between the groups
RESULTS
Figure R9: Photomicrographs of DG under 20X; CON: control, RES: pups received prenatal resveratrol during
entire gestation period, EGS: pups received prenatal
stress during day 1 to 10, LGS: pups received stress during day 11 till delivery, EGS+RES: pups received prenatal
stress during day 1 to 10 and resveratrol during entire
gestation period and LGS+RES: pups received prenatal stress during day 11 to till delivery and resveratrol during
entire gestation period. The arrow in CON group,
denotes the viable neuron with intact membrane, while in LGS group, the arrow denotes the pyknotic neuron with
distorted membrane. The numbers of viable neurons
were counted using occulomicrometer (250μm length)
CON RES EGS
LGS EGS+
RES
LGS+
RES
RESULTS
Figure R10: Photomicrographs of CA3 region of hippocampus under 20X; CON: control, RES: pups received
prenatal resveratrol during entire gestation period, EGS:
pups received prenatal stress during day 1 to 10, LGS: pups received stress during day 11 till delivery, EGS+RES:
pups received prenatal stress during day 1 to 10 and
resveratrol during entire gestation period and LGS+RES: pups received prenatal stress during day 11 to till delivery
and resveratrol during entire gestation period. The
arrow in CON group, denotes the viable neuron with intact membrane, while in LGS group, the arrow denotes the
pyknotic neuron with distorted membrane. The
numbers of viable neurons were counted using occulomicrometer (250μm length)
CON RES EGS
LGS EGS+
RES
LGS+
RES
RESULTS
prenatal resveratrol during entire gestation period,
EGS: pups received prenatal stress during day 1 to 10, LGS: pups received stress during day 11 till delivery,
EGS+RES: pups received prenatal stress during day 1 to 10
and resveratrol during entire gestation period and LGS+RES: pups received prenatal stress during day 11 to till
delivery and resveratrol during entire gestation period.
The numbers of viable neurons were counted using occulomicrometer (250μm length).
CON RES EGS
LGS EGS+
RES
LGS+
RES
RESULTS
prenatal resveratrol during entire gestation period,
EGS: pups received prenatal stress during day 1 to 10, LGS: pups received stress during day 11 till delivery,
EGS+RES: pups received prenatal stress during day 1 to 10
and resveratrol during entire gestation period and LGS+RES: pups received prenatal stress during day 11 to till
delivery and resveratrol during entire gestation period.
The numbers of viable neurons were counted using occulomicrometer (250μm length).
CON RES EGS
LGS EGS+
RES
LGS+
RES
RESULTS
Figure R13: Photomicrographs of hippocampal neurons under 40X. The arrows in the left figure
denote the normal viable neurons with intact
cell membrane however, arrows in the right figure denote pyknotic neurons with distorted cell
membrane
RESULTS
7.4. BDNF level in hippocampus
Prenatal stress during early (EGS) and late (LGS) gestation resulted in a significant
decrease in the hippocampal BDNF level in 40th postnatal day offspring compared to control
group offspring (CON Vs EGS, p<0.05 & CON Vs LGS, p<0.001). Offspring received
parental stress during late gestation (LGS) showed a significant decrease in the hippocampal
BDNF level compared to the offspring received early gestational stress (EGS Vs LGS,
p<0.05). Offspring exposed to prenatal stress (early and late) along with resveratrol
(EGS+RES and LGS+RES) showed a significant increase in the level of BDNF in
hippocamus when compared to prenatally stressed offspring (EGS Vs EGS+RES, p<0.01 and
LGS Vs LGS+RES, p<0.001). No significant change in hippocampal BDNF level was
observed in offspring exposed to prenatal resveratrol treatment alone (RES). (Figure R14)
CON
RES
EGS
LGS
EGS+RES
LGS+RES
0
100
200
300
400
*
a
***
c
#
##
$$$
pg/mg brain tissue
Figure R14: Effect of prenatal stress and resveratrol treatment on hippocampal BDNF level (pg/mg brain
tissue)
P<0.0001 & F=16.239
CON Vs EGS, * p<0.05; CON Vs LGS, *** p<0.001
RES Vs EGS, a p<0.05; RES Vs LGS, c p<0.001
EGS Vs LGS, # p<0.05; EGS Vs EGS+RES, ## p<0.01
RESULTS
Summary of prenatal stress induced decrease neurogenesis, survival of neurons,
hippocampal BDNF level and its and its prevention by resveratrol treatment:
Neurogenesis: Prenatal stress during early and late gestation resulted in a decrease in DCX
positive neurons in subglanular zone (SGZ)/glanular cell layer (GCL) of hilus of DG in both
21st and 40th postnatal day rat brain. Offspring received parental stress during late gestation
showed a more decrease in DCX positive neurons in DG compared to the offspring received
early gestational stress. Administration of resveratrol during pregnancy has reversed the
prenatal stress induced decrease postnatal neurogenesis in DG. (Table 7 & 8)
Survival of neurons: Prenatal stress (early and late) has produced regional specific effect on
hippocampal neurons. The prenatally stressed offspring (EGS & LGS) showed decrease in
the number of neurons in the dentate gyrus and CA 3 regions of the hippocampus. The CA1
and CA2 regions of the hippocampus did not show any numerical changes in response to
prenatal stress. Administration of resveratrol during pregnancy has reversed the prenatal
stress induced neuronal damage of dentate gyrus but not the CA 3 hippocampal neurons.
(Table 7 & 8)
BDNF level in hippocampus: Prenatal stress during early and late gestation resulted in a
decrease in the level of BDNF in 40th postnatal day rat hippocampus. Late gestational stress
caused a more decrease in BDNF level compared to early gestational stress. Administration
of resveratrol during pregnancy has reversed the prenatal stress induced decrease in BDNF
level in hippocampus. (Table 7 & 8)
RESULTS
Table 7: Summary of prenatal stress, resveratrol and their combination induced changes in
hippocampal neurogenesis (doublecortin
immunostaining), survival of neurons (cresyl violet staining) and hippocampal BDNF level
in different group rats
Cresyl violet staining Doublecortin (DCX) immunostaining in
DG
BDNF
No. of DG
neurons
No. of CA3
neurons
No. of CA2
neurons
No. of CA1
neurons
No. of DCX +ve
neurons (21st day)
No. of DCX +ve
neurons (40th day)
Pg/mg brain tissue
CON (n=12) 40.95±0. 41 15.72±0.26 21.6±0.34 21.42±0.24 57.35±1.43 40.23±0.37 343.29±15.79
RES (n=12) 40.13±0.37 15.5±0.26 21.28±0.24 20.92±0.24 59.15±1.49 39.92±0.75 350.93±14.28
EGS (n=12) 38.22±0.36*** b 14.81±0.24 20.71±0.32 21.48±0.27 51.29±0.95**c 35.34±0.78**b 284.35±14.22*a
LGS (n=12) 38.16±0.41***b 14.50±0.23**b 21.5±0.30 21.8±0.24 40.43±0.03*** c ### 27.75±0.72*** b ###
224.23±9.12***c#
EGS+RES (n=12) 40.00±0.36# 14.78±0.18 21.06±0.19 21.21±0.19 57.17±1.49# 38.57±0.37## 254.30±7.74##
LGS+RES (n=12) 39.80±0.37$ 14.60±0.18* 21.07±0.22 21.51±0.23 55.78±1.34$$$ 39.78± 0.40$$$ 328.60±12.67$$
F value 8.445 4.46 1.389 1.528 28.635 65.401 16.239
ANOVA significance
(p value)
<0.0001 0.006 0.2274 0.1801 <0.0001 <0.0001 <0.0001
ANOVA significance (Bonferroni’s test, each data represents mean±SEM)
CON Vs EGS, *** p<0.001, ** p<0.01,* p<0.05; CON Vs LGS+RES, * p<0.05
RES Vs EGS & LGS, c p<0.001, b p<0.01, a p<0.05
EGS Vs LGS, ### p<0.001, # p<0.05; EGS Vs EGS+RES, ## p<0.01, # p<0.01
LGS Vs LGS+RES, $$$ p<0.001, $$ p<0.01, $ p<0.05
RESULTS
Table 8: Summary of prenatal stress, resveratrol and their combination induced changes in
hippocampal neurogenesis (doublecortin
immunostaining), survival of neurons (cresyl violet staining) and hippocampal BDNF level
in male and female rats
Cresyl violet staining Doublecortin (DCX)
immunostaining in DG
BDNF
No. of DG
neurons
No. of CA3
neurons
No. of CA2 neurons No. of CA1 neurons No. of DCX +ve
neurons (21st day)
No. of DCX +ve
neurons (40th day)
Pg/mg brain tissue
CON
Male
(n=6)
41.03±
0.68
p, 0.85
t, 0.18
F, 2.02
15.27±
0.45
p, 0.13
t, 0.15
F, 1.66
21.90±
0.57
p, 0.38
t, 0.87
F, 2.17
21.15±
0.32
p, 0.27
t, 1.11
F, 1.27
55.00±
1.82
p, 0.09
t, 1.67
F, 1.39
39.90±
0.55
p, 0.38
t, 0.87
F, 1.23
331.50±
18.1
p, 0.51
t, 0.71
Female F, 2.36
(n=6)
40.87±
0.48
16.39±
0.58
21.30±
0.39
21.69±
0.36
59.71±
2.15
40.56±
0.50
355.10±
27.90
RES
Male
(n=6)
40.03±
0.55
p, 0.77
t, 0.28
F, 1.12
15.21±
0.43
p, 0.20
t, 1.08
F,2.06
21.69±
0.32
p, 0.09
t, 1.70
F, 1.20
21.00±
0.35
p, 0.75
t, 0.31
F, 1.13
59.09±
2.07
p, 0.96
t, 0.04
F, 1.10
38.90±
0.91
p, 0.17
t, 1.35
F, 1.65
335.70±
21.10
p, 0.34
t, 1.08
Female F, 1.29
(n=6)
40.24±
0.50
15.78±
0.30
20.87±
0.35
20.84±
0.33
59.21±
2.18
40.93±
1.18
366.10±
18.50
EGS
Male
(n=6)
38.39±
0.47
p, 0.64
t, 0.45
F,1.33
14.81±
0.39
p, 1.00
t, 0.00
F, 1.61
20.15±
0.53
p, 0.08
t, 1.75
F, 2.25
21.00±
0.37
p, 0.07
t, 1.78
F, 1.11
52.09±
1.40
p, 0.41
t, 0.88
F, 1.16
35.18±
0.99
p, 0.67
t, 0.41
F, 1.32
296.90±
17.10
p, 0.43
t, 0.86
Female F, 1.91
(n=6)
38.06±
0.55
14.81±
0.30
21.27±
0.35
21.96±
0.39
50.50±
1.30
35.79±
1.23
271.70±
23.50
LGS
Male
(n=6)
38.24±
0.66
p, 0.85
t, 0.18
F, 1.17
14.39±
0.36
p, 0.65
t, 0.44
F, 1.42
21.39±
0.43
p, 0.72
t, 0.34
F, 1.06
21.78±
0.31
p, 0.95
t, 0.05
F, 1.74
39.75±
1.41
p, 0.46
t, 0.73
F, 1.32
26.96±
1.02
p, 0.28
t, 1.07
F, 1.01
217.50±
8.10
p, 0.52
t, 0.96
Female F, 4.56
(n=6)
38.06±
0.50
14.60±
0.30
21.66±
0.42
21.81±
0.40
41.12±
1.22
28.53±
1.02
230.90±
17.40
EGS+RES
Male
(n=6)
38.72±
0.54
p, 0.78
t, 0.28
F, 1.15
13.54±
0.25
p, 0.07
t, 1.78
F, 1.16
21.27±
0.24
p, 0.26
t, 1.11
F, 1.36
21.54±
0.29
p, 0.07
t, 1.78
F, 1.76
59.63±
2.06
p, 0.08
t, 1.73
F, 1.04
38.09±
0.52
p, 1.94
t, 1.31
F,1.08
359.60±
13.70
p, 0.55
t, 0.64
Female F, 2.24
(n=6)
38.48±
0.67
14.21±
0.27
20.84±
0.27
20.87±
0.22
54.54±
2.08
39.09±
0.51
348.90±
9.10
LGS+RES
Male
(n=6)
38.09±
0.39
p, 0.05
t, 1.27
F, 1.36
13.87±
0.20
p, 0.92
t, 0.09
F,1.43
21.63±
0.29
p, 0.06
t, 1.64
F, 1.07
22.45±
0.25
p, 0.06
t, 1.92
F, 1.81
57.18±
1.61
p, 0.28
t, 1.07
F, 1.73
40.93±
0.52
p, 0.54
t, 1.036
F, 1.11
351.90±
6.10
p, 0.12
t, 1.92
Female F, 5.86
(n=6)
39.02±
0.60
13.84±
0.24
20.51±
0.30
21.57±
0.31
54.29±
2.19
39.62±
0.55
311.80±
19.80
RESULTS
7.5. Behavioural test
7.5.1. Open field test
Prenatally stressed offspring (EGS and LGS) made significantly fewer (p<0.001) central
squares entries compared to control and prenatally alone resveratrol treated offspring (Figure
R15A). There was also a significant decrease (p<0.001) in time spent in central squares and
increase (p<0.001) in time spent in peripheral squares in prenatally stressed offspring
compared to control and prenatally alone resveratrol treated offspring (Figure R15B). Early
or late gestational stress along with resveratrol treatment, increased central squares entries
(EGS Vs EGS+RES, p<0.001 & LGS Vs LGS+RES, p<0.05). Similarly, resveratrol
treatment with early or late gestational stress significantly increase in time spent in central
squares (EGS Vs EGS+RES, p<0.001 & LGS Vs LGS+RES, p<0.001) and decrease in time
spent in peripheral squares (EGS Vs EGS+RES, p<0.001 & LGS Vs LGS+RES, p<0.001)
compared to prenatally stressed offspring. No alteration in the number of rearings (p=0.715,
F= 0.579) and groomings (p=0.0508, F=2.345) was detected in any of the group.
RESULTS
0
20
40
60
80
CON
RES
EGS
LGS
EGS+RES
LGS+RES
***
c
*
c ### $
|
Central squares Peripheral squares
(A)
Number of squares moved
0
100
200
300
400
CON
RES
EGS
LGS
EGS+RES
LGS+RES
***
c
***
c
## $$
***
c
***
c
## $$
|
Cental squares Peripheral squares
(B)
Time spent in squares (sec)
Figure R15: Effect of prenatal stress and resveratrol treatment on number of central and peripheral squares
moved (A) and the time spent in central and peripheral squares (B) in the open field exploration test in 21-dayold
rats.
ANOVA significance (Bonferroni’s test, each bar represents mean±SEM, n=12 per group)
Number of central squares moved: P<0.0001 & F=11.640
Number of central squares moved: P<0.919 & F=0.474
Time spent in central and peripheral squares: P<0.0001 & F=11.695
CON Vs EGS & LGS, *** p<0.001, ** p<0.01, * p<0.05
EGS Vs EGS+RES, ### p<0.001, ## p<0.01
LGS Vs LGS+RES, $$ p<0.01, $ p<0.05
RESULTS
7.5.2. Rota-rod test
In all the groups except LGS-group rats had no effects on the motor deficit. Offspring
received prenatal stress during late gestation (LGS) showed a shorter latency to fall off the
revolving bar relative to control (p<0.05), indicating a decline in motor co-ordination. (Figure
R16)
CON
RES
EGS
LGS
EGS+RES
LGS+RES
0
20
40
60
80
100
*
Latency of falling down
from rota-rod bar (sec)
Figure R16: effects of prenatal stress, resveratrol and their combinations on motor performance of rats in the
Rota-rod test.
P=0.0124& F=5.026
CON Vs LGS, *p<0.05
RESULTS
7.5.3. Passive avoidance test
There was no significant difference between the different study groups (prenatal
stress, resveratrol and their combination) on the initial latency to enter the dark compartment,
time spent in dark and bright compartments during the passive exploration test. Therefore any
differences seen subsequently are a reflection of differences in the retrieval and are not
related to the initial baseline activities.
(A) Latency to enter the dark compartment
During passive avoidance retention test, the offspring exposed to prenatal stress
during early (EGS) and late (LGS) gestation took shorter time to enter the dark compartment
compared to control group offspring (CON Vs EGS, p<0.001, CON Vs LGS, p<0.01).
Shorter latency indicates poor retrieval of learned behaviour. Prenatal resveratrol treatment
reversed the cognitive impairment (took longer time to enter in to the dark compartment) of
the offspring exposed to late gestational stress (LGS Vs LGS+RES, p<0.001) but not in early
gestational stress (LGS Vs LGS+RES, p>0.05) (Figure R17A)
CON
RES
EGS
LGS
EGS+RES
LGS+RES
0
50
100
150
***
c **
c
###
(A) $$
Latency to enter the
dark compartment (sec)
Figure R17(A): Effect of prenatal stress, resveratrol and their combinations on latency to enter the dark
compartment in the passive avoidance apparatus during the retention test.
P<0.0001& F=21.882
CON Vs EGS & LGS, *** p<0.001, ** p<0.01
EGS Vs LGS+RES, ### p<0.001
LGS Vs LGS+RES, $$ p<0.01
RESULTS
(B) Time spent in bright compartment
On assessing total time spent in bright compartment of the passive avoidance
apparatus during the retention test, it was found that prenatally stressed offspring (both early
and late) spent significantly less time in bright compartment compared to control group
offspring (CON Vs EGS & LGS, p<0.05) and offspring received prenatal resveratrol
treatment alone (RES Vs EGS & LGS, p<0.001). Offspring exposed to prenatal stress during
late gestation along with resveratrol treatment spent significantly more time in bright
compartment (LGS Vs LGS+RES, p<0.001). (Figure R17B)
CON
RES
EGS
LGS
EGS+RES
LGS+RES
0
100
200
300
*
cc
###
$$$
*
(B)
Time spent in the bright
compartment (sec)
Figure 17(B): Effect of prenatal stress, resveratrol and their combinations on time spent in the bright
P<0.0001& F=15.975
CON Vs EGS & LGS, * p<0.05
EGE Vs LGS+RES, ### p<0.001
RESULTS
(C)Time spent in dark compartment
On assessing total time spent in dark compartment of the passive avoidance apparatus
during the retention test, it was found that prenatally stressed offspring (both early and late)
spent significantly more time in dark compartment compared to control group offspring
(CON Vs EGS & LGS, p<0.05) and offspring received prenatal resveratrol treatment alone
(RES Vs EGS & LGS, p<0.001). Offspring exposed to prenatal stress during late gestation
along with resveratrol treatment spent significantly less time in dark compartment (LGS Vs
LGS+RES, p<0.001). (Figure R17C)
CON
RES
EGS
LGS
EGS+RES
LGS+RES
0
50
100
150
200
*
cc
*
$$$
###
(C)
Time spent in the dark
compartment (sec)
Figure R17(C): Effect of prenatal stress, resveratrol and their combinations on time spent in the dark
P<0.0001& F=15.975
CON Vs EGS & LGS, * p<0.05
EGS Vs LGS+RES, ### p<0.001
RESULTS
7.5.4. Morris water maze test
Latency to escape platform during learning sessions
The spatial memory of offspring was examined with the Morris water maze test. In
the first session, rats in all groups went on swimming around water tank and failed to reach
the escape platform. Second session onwards, rats in all groups (except prenatally stressed
rats) learnt to reach the escape platform quickly and escape from there and their escape
latency period decreased progressively from session to session. Prenatal restraint stress
during early or late gestation, significantly increased (p<0.001) the escape latency compared
with control. These results confirmed that prenatal stress can induce memory impairment in
the offspring. No significant variation in escape latency during training sessions was
observed in the offspring exposed to prenatal resveratrol treatment alone (RES). Offspring
exposed to prenatal stress (both early and late) along with resveratrol treatment showed
significant decrease in the escape latency compared to prenatally stressed offspring (EGS Vs
EGS+RES, p<0.001; LGS Vs LGS+RES, p<0.001). (Figure R18)
123456789
0
20
40
60
80
100 CON
RES
EGS
LGS
EGS+RES
LGS+RES
***
C ###
$$$
Learning session
Escape latency (sec)
Figure R18: Latency to escape on to the platform during learning sessions in water maze test (sec) by rats in
different groups.
Two way ANOVA significance (Bonferroni’s test, each bar represents mean±SEM, n=12)
P<0.0001
CON Vs EGS & LGS, *** p<0.001
EGS Vs EGS+RES, ### p<0.001
RESULTS
(A) Latency to enter target quadrant
Prenatally stressed offspring took longer time to reach the target quadrant during
water maze retention test 24hrs after last learning session (probe test) compared to control
rats compared to control group offspring (CON Vs EGS & LGS, p<0.001). Similar results
were obtained with the offspring received prenatal resveratrol treatment alone when
compared with prenatal stressed offspring (RES Vs EGS & LGS, p<0.001). Offspring who
received prenatal stress during early and late gestation along with resveratrol treatment took
significantly less time to reach the target quadrant compared to prenatally stressed offspring
(EGS Vs EGS+RES, p<0.001 & LGS Vs LGS+RES, p<0.001). (Figure R18A)
CON
RES
EGS
LGS
EGS+RES
LGS+RES
0
2
4
6
8
***
***
c
$$$
c
###
(A)
Latency to enter
the target quadrant(sec)
Figure 18(A): Latency to enter the target quadrant by rats in different groups during water maze retention test.
P<0.0001 & F=24.620
RESULTS
(B) Time spent in target quadrant
Prenatally stressed offspring spent significantly (p<0.001) less time in the target
quadrant compared to control group offspring (CON Vs EGS & LGS, p<0.001). Similar
results were obtained with the offspring received prenatal resveratrol treatment alone when
compared with prenatal stressed offspring (RES Vs EGS & LGS, p<0.001). Offspring who
received prenatal stress during early and late gestation along with resveratrol treatment spent
significantly more time in the target quadrant compared to prenatally stressed offspring (EGS
Vs EGS+RES, p<0.01 & LGS Vs LGS+RES, p<0.001). (Figure R18B)
CON
RES
EGS
LGS
EGS+RES
LGS+RES
0
10
20
30
***
***
c
c
## $$$
(B)
Time spent in
target quadrant (sec)
Figure R18(B): Time spent in the target quadrant by rats in different groups during water maze retention test.
P<0.0001 & F=31.121
EGS Vs EGS+RES, ## p<0.01
RESULTS
Summary of prenatal stress induced motor and cognitive impairment and its amelioration by
Motor functions
Open field exploratory test: The rats exposed to prenatal stress during late gestation (LGS)
spent less time and moved less number of peripheral squares. The number of grooming and
rearing was unaltered in all the groups, indicating anxiogenic behaviour. Administration of
resveratrol reversed these effects. (Table 9 & 10)
Rota rod test: Offspring received prenatal stress during late gestation (LGS) showed a shorter
latency to fall off the revolving bar, indicating a decline in reflex development, which was
reversed by resveratrol treatment. (Table 9 & 10)
Cognitive functions
Passive avoidance test: The latency in entering the dark compartment (where they received
shock earlier) is more in control rats. However prenatally stressed (both early and late) rats
showed a decrease in latency (delayed entry in to the dark compartment) in spite of shock
received earlier in the dark compartment. Prenatal resveratrol treatment reversed the
cognitive impairment (took longer time to enter in to the dark compartment) of the offspring
received late gestational stress but not in early gestational stress. (Table 11 & 12)
Water maze test: The prenatally stressed offspring (early and late) took longer time to reach
the target quadrant and spent less time in the target quadrant during water maze retention test
(probe test) 24 hrs after last learning session, indicating prenatal stress induced-cognitive
dysfunction. Offspring who received prenatal stress (both early and late) along with
resveratrol treatment took less time to reach the target quadrant and spent longer time in
target quadrant compared to prenatally stressed offspring, suggests maternal administration of
resveratrol effectively prevented prenatal stress induced-cognitive dysfunction. (Table 11 &
12)
RESULTS
Table 9: Summary of open field and rota-rod test in rats in different groups
Open field test Rota-rod test
No. of central
squares moved
No. of
peripheral
squares moved
Time spent in
central squares
(seconds)
Time spent in
peripheral squares
(seconds)
Latency of falling
from rota rod bar
(seconds)
CON (n=12) 12.41±1.00 58.83±6.09 24.00±1.56 276.00±1.56 CON (n=12) 47.91±4.59
RES (n=12) 15.83±1.61 69.41±4.13 24.91±2.66 275.25±2.66 RES (n=12) 45.50±2.92
EGS (n=12) 4.33±1.19***c 58.75±4.34 6.66±1.76***c 293.44±1.72***c EGS (n=12) 43.41±5.56*
LGS (n=12) 6.85±0.80*c 58.83±6.09 12.41±1.16***c 287.58±1.16***c LGS (n=12) 33.58±3.98
EGS+RES
(n=12)
13.33±1.35### 57.33±5.74 17.00±1.55## 283.00±1.55## EGS+RES (n=12) 40.16±3.98
LGS+RES
(n=12)
11.33±1.47$ 66.08±4.11 21.25±1.37$$ 278.75±1.37$$ LGS+RES (n=12) 44.00±2.82
F value 11.640 0.919 16.695 16.695 F value 5.026
ANOVA
significance
(p value)
<0.0001 0.474 <0.0001 <0.0001 ANOVA significance
(p value)
0.0124
CON Vs EGS & LGS, *** p<0.001, * p<0.05
EGS Vs EGS+RES, ### p<0.001, ##p<0.01
LGS Vs LGS+RES, $$ p<0.01, $ p<0.05
RESULTS
Table 10: Summary of open field and rota-rod test in males and female rats
Open field test Rota-rod test
No. of central squares
moved
No. of peripheral squares
moved
Time spent in central
squares (seconds)
Time spent in peripheral
squares (seconds)
Latency of falling from
rota rod bar (seconds)
CON
Male
(n=6)
12.83±
1.19
p, 2.054
t, 0.399
F, 2.054
59.66±
4.97
p, 0.898
t, 0.130
F, 5.605
24.83±
2.94
p, 0.617
t, 0.515
F, 4.930
275.16±
2.94
p, 0.617
t, 0.515
F, 4.937
40.66±
4.37
p, 0.118
t, 1.710
Female F, 2.764
(n=6)
12.00±
1.71
58.00±
11.77
23.16±
1.32
276.83±
1.32
55.16±
7.26
RES
Male
(n=6)
14.50±
2.21
p, 0.434
t, 0.813
F, 1.185
66.16±
4.80
p, 0.457
t, 0.772
F, 2.069
22.83±
3.97
p, 0.388
t, 0.901
F, 1.078
277.33±
2.78
p, 0.450
t, 0.785
F, 2.618
45.16±
4.82
p, 0.915
t, 0.108
Female F, 1.622
(n=6)
17.16±
2.41
72.66±
6.91
28.00±
4.13
273.16±
4.51
45.83±
3.79
EGS
Male
(n=6)
4.50±
1.80
p, 0.896
t, 0.133
F, 1.073
59.50±
6.40
p, 0.872
t, 0.164
F, 1.028
5.83±
1.64
p, 0.655
t, 0.455
F, 3.977
293.33±
1.45
p, 0.892
t, 0.138
F, 5.160
36.00±
3.39
p, 0.195
t, 1.389
Female F, 8.857
(n=6)
4.16±
1.74
58.00±
6.48
7.50±
3.27
292.83±
3.30
50.83±
10.12
LGS
Male
(n=6)
7.66±
0.91
p, 0.188
t, 1.410
F, 1.796
66.16±
4.80
p, 0.475
t, 0.772
F, 2.069
18.50±
1.08
p, 0.100
t, 1.808
F, 2.798
289.50±
1.08
p, 0.101
t, 1.808
F, 2.798
35.33±
7.03
p, 0.440
t, 0.802
Female F, 3.072
(n=6)
5.50±
1.23
72.66±
6.91
14.33±
1.82
285.66±
1.82
41.83±
4.01
EGS+RES
Male
(n=6)
14.83±
1.01
p, 0.287
t, 1.124
F, 5.930
58.66±
5.77
p, 0.829
t, 0.221
F, 3.342
17.16±
2.86
p, 0.920
t, 0.102
F, 3.485
282.83±
2.86
p, 0.920
t, 0.102
F, 3.485
41.00±
3.54
p, 0.800
t, 0.259
Female F, 2.280
(n=6)
11.83±
2.46
56.00±
10.55
16.83±
1.53
281.16±
1.53
39.33±
5.34
LGS+RES
Male
(n=6)
13.50±
2.36
p, 0.150
t, 1.557
F, 2.584
67.66±
7.73
p, 0.719
t, 0.369
F, 4.330
22.16±
1.49
p, 0.529
t, 0.650
F, 2.564
279.16±
1.85
p, 0.327
t, 1.029
F, 1.127
40.33±
3.69
p, 0.209
t, 1.343
Female F, 1.186
(n=6)
9.16±
1.47
64.50±
3.69
20.30±
2.39
281.83±
1.77
47.66±
4.02
RESULTS
Table 11: Summary of passive avoidance and water maze test performance in rats in
different groups
Passive avoidance test Water maze test
Latency to enter
dark compartment
(seconds)
Time spent in bright
compartment
(seconds)
Time spent in dark
compartment
(seconds)
Latency to enter
target quadrant
(seconds)
Time spent in target
quadrant
(seconds)
CON (n=12) 111.00±5.44 188.50±5.69 111.50±5.69 CON (n=12) 3.33±0.18 24.41±1.17
RES (n=12) 116.50±10.47 210.16±7.82 89.83±7.81 RES (n=12) 3.83±0.16 27.00±0.66
EGS (n=12) 44.00±5.73***c 153.50±7.11*c 146.50±7.11*c EGS (n=12) 6.19±0.29***c 16.83±0.53**c
LGS (n=12) 54.41±4.79**c 156.91±4.89*c 143.08±4.89*c LGS (n=12) 6.83±0.36***c 15.08±0.55**c
EGS+RES (n=12) 46.22±2.52### 169.58±12.19### 130.41±12.19### EGS+RES (n=12) 5.16±0.27### 21.08±0.72##
LGS+RES (n=12) 52.87±4.63$$ 242.00±11.21$$$ 58.00±1.21$$$ LGS+RES (n=12) 4.75±0.27$$$ 20.66±0.96$$$
F value 21.882 15.975 15.975 F value 24.620 31.121
ANOVA
significance
(p value)
<0.0001 <0.0001 <0.0001 ANOVA significance
(p value)
<0.0001 <0.0001
CON Vs EGS & LGS, *** p<0.001, ** p<0.01, * p<0.05
EGS Vs EGS+RES, ### p<0.001, ##p<0.01
LGS Vs LGS+RES, $$$ p<0.001, $$ p<0.01
RESULTS
Table 12: Summary of passive avoidance and water maze test performance in males and
female rats
Passive avoidance test Water maze test
Latency to enter dark
compartment (seconds)
Time spent in bright
Time spent in dark
Latency to enter target
quadrant (seconds)
Time spent in target
quadrant (seconds)
CON
Male (n=6) 103.33±
5.37
p, 0.169
t, 1.482
F, 2.713
179.50±
8.35
p, 0.117
t, 1.713
F, 1.720
120.52±
8.35
p, 0.117
t, 1.713
F, 1.720
3.16±
0.30
p, 0.401
t, 0.877
F, 1.889
25.50±
1.31
p, 0.379
t, 0.918
Female F, 2.239
(n=6)
118.66±
8.84
197.50±
6.37
102.50±
6.37
3.50±
0.22
23.33±
1.96
RES
Male (n=6) 99.83±
10.96
p, 0.114
t, 1.729
F, 2.091
207.66±
10.14
p, 0.765
t, 0.306
F, 1.595
92.33±
10.14
p, 0.765
t, 0.306
F, 1.595
3.66±
0.21
p, 0.340
t, 1.000
F, 1.500
26.83±
1.07
p, 0.814
t, 0.240
Female F, 1.526
(n=6)
133.16±
15.85
212.66±
12.81
87.33±
12.81
4.00±
0.25
27.16±
0.87
EGS
Male (n=6) 33.83±
6.92
p, 0.073
t, 2.000
F, 1.157
148.50±
11.94
p, 0.508
t, 0.685
F, 2.037
151.50±
11.94
p, 0.508
t, 0.685
F, 2.037
6.33±
0.21
p, 0.599
t, 0.542
F, 7.500
15.83±
0.47
p, 0.055
t, 2.162
Female F, 2.756
(n=6)
54.16±
7.44
158.50±
8.36
141.50±
8.36
6.00±
0.57
17.83±
0.79
LGS
Male (n=6) 51.66±
7.80
p, 0.590
t, 0.555
F, 1.637
161.16±
7.73
p, 0.411
t, 0.857
F, 1.516
138.83±
7.73
p, 0.411
t, 0.857
F, 1.516
7.16±
0.47
p, 0.387
t, 0.903
F, 1.390
14.83±
0.67
p, 0.674
t, 0.432
Female F, 1.708
(n=6)
57.16±
6.09
152.66±
7.73
147.33±
6.19
6.50±
0.56
15.33±
0.91
EGS+RES
Male (n=6) 85.33±
9.69
p, 0.362
t, 0.549
F, 2.447
162.00±
14.24
p, 0.559
t, 0.603
F, 2.113
138.00±
14.24
p, 0.559
t, 0.603
F, 2.113
5.33±
0.42
p, 0.563
t, 0.597
F, 1.333
22.16±
0.60
p, 0.140
t, 1.603
Female F, 4.062
(n=6)
68.16±
15.16
177.16±
20.18
122.84±
20.18
5.00±
0.36
20.00±
1.21
LGS+RES
Male (n=6) 134.16±
17.05
p, 0.141
t, 1.595
F, 2.264
222.50±
14.69
p, 0.080
t, 1.947
F, 1.162
77.50±
14.69
p, 0.080
t, 1.947
F, 1.162
5.00±
0.36
p, 0.395
t, 0.888
F, 1.370
20.66±
1.17
p, 1.000
t, 0.000
Female F, 2.968
(n=6)
85.00±
25.66
261.50±
13.62
38.50±
13.62
4.50±
0.42
20.66±
1.64
RESULTS
7.6. Brain oxidants and anti-oxidants
7.6.1. Lipid peroxidation in brain
The level of the thiobarbituric acid reactive substances (TBARS) is a marker of lipid
peroxidation. Offspring exposed to prenatal stress (both early and late) showed a significant
increase in TBARS level in the brain compared to control (CON Vs EGS & LGS, p<0.001).
Offspring exposed to prenatal stress (both early and late) along with resveratrol (EGS+RES
and LGS+RES) showed a significant decrease in TBARS level in the brain compared to
prenatally stressed offspring (EGS Vs EGS+RES, p<0.05 & LGS Vs LGS+RES, p<0.001).
No significant change in the level of TBARS was observed in the brain of offspring exposed
to prenatal resveratrol treatment alone (RES). (Figure R19)
CON
RES
EGS
LGS
EGS+RES
LGS+RES
0.0
0.5
1.0
1.5
2.0
2.5
***
***
c
#
$$$
c
μmoles/gm protein
Figure R19: Effects of prenatal stress and resveratrol administration on the TBARS (μmoles/gm protein) level
in the brain.
P<0.0001 & F=24.12
EGS Vs EGS+RES, # p<0.05
RESULTS
7.6.2. Advanced oxidative products of protein (AOPPs) level in the brain
AOPPs level significantly increased in the brain of prenatally stressed offspring (EGS
and LGS) compared to control group offspring (CON Vs EGS & LGS, p<0.001). Offspring
exposed to late gestational stress (LGS) showed significant increase (p<0.001) in AOPPs
level in the brain compared to offspring exposed to early gestational stress (EGS). Offspring
exposed to prenatal stress (both early and late) along with resveratrol showed a significant
decrease in AOPPs level compared to prenatally stressed offspring (EGS Vs EGS+RES,
p<0.001 & LGS Vs LGS+RES, p<0.001). No significant variation in AOPPs level was
observed in the offspring exposed prenatal resveratrol treatment alone (RES). (Figure R20)
CON
RES
EGS
LGS
EGS+RES
LGS+RES
0
5
10
15
20
***
***
C
###
###
$$$
C
μmoles/lit
Figure R20: Effects of prenatal stress and resveratrol administration on advanced oxidative products of protein
(μmoles/lit) level in the brain.
P<0.0001 & F=50.07
EGS Vs LGS & EGS+RES, ### p<0.001
RESULTS
7.6.3. Reduced glutathione (GSH) level in brain
Offspring received prenatal stress during early (EGS) and late gestation (LGS) showed
a significant decrease of GSH level in the brain compared to control group offspring (CON
Vs EGS & LGS, p<0.001). Offspring who received late gestational stress along with
resveratrol treatment (LGS+RES) showed a significant increase (p<0.01) in GSH level when
compared to prenatally stressed offspring (LGS). However offspring exposed to early
gestational stress along with resveratrol (EGS+RES) failed to increase (p>0.05) the GSH
level in the offspring exposed to early gestational stress alone (EGS). No significant change
in GSH level was observed in offspring whose mothers were treated with resveratrol alone
(RES). (Figure R21)
CON
RES
EGS
LGS
EGS+RES
LGS+RES
0
2
4
6
8
*** ***
***
***
cc
c
c
##
$$
mg/gm protein
Figure R21: Brain reduced Glutathione level (mg/gm protein) in rats.
P<0.0001 & F=67.95
CON Vs EGS, LGS, EGS+RES & LGS+RES, *** p<0.001
RES Vs EGS, LGS, EGS+RES & LGS+RES, c p<0.001
LGS Vs LGS+RES, $$ p<0.01
RESULTS
7.6.4. Total antioxidants in the brain
The total antioxidants level in the brain decreased significantly (p<0.001) in
prenatally stressed offspring (both early and late) compared to control group offspring.
Offspring exposed to prenatal stress (both early and late) along with resveratrol showed a
significant increase in total antioxidants level compared to prenatally stressed offspring (EGS
Vs EGS+RES, p<0.001 & LGS Vs LGS+RES, p<0.05). No significant variation in total
antioxidants level was observed in brain of offspring exposed to prenatal resveratrol
treatment alone (RES). (Figure R22)
CON
RES
EGS
LGS
EGS+RES
LGS+RES
0.0
0.5
1.0
1.5
2.0
***
***
$
C
C
###
mmoles/lit
Figure R22: Effects of prenatal stress and resveratrol administration on the total antioxidant level (mmoles/lit)
in the brain.
P<0.0001 & F=35.03
RESULTS
7.6.5. Glutathione reductase (GSSG-Rd) activity in brain
The activity of brain GSSG-Rd was significantly decreased in prenatally stressed
offspring (EGS and LGS) compared to control group offspring (CON Vs EGS & LGS,
p<0.001). Resveratrol treatment significantly elevated the GSSG-Rd activity in the brain of
offspring who received prenatal stress during late gestation but not in early gestation
compared to prenatally stressed offspring (EGS Vs EGS+RES, p>0.05 & LGS Vs LGS+RES,
p<0.001). No significant change in GSSG-Rd activity in the brain was observed offspring
received prenatal resveratrol treatment alone (RES). (Figure R23)
CON
RES
EGS
LGS
EGS+RES
LGS+RES
0
2
4
6
8
10
***
***
***
c
c
c
a
$$$
nmoles/NADPH/min/mg protein
Figure R23: Brain Glutathione reductase activity (nmol NADPH oxidized/min/mg protein) in rats.
P<0.0001 & F=28.831
CON Vs EGS, LGS & EGS+RES, *** p<0.001
RES Vs EGS, LGS, EGS+RES & LGS+RES, c p<0.001, a p<0.05
RESULTS
7.6.6. Superoxide Dismutase (SOD) activity in brain
The brain SOD activity was significantly decreased in the offspring exposed to
prenatal stress (both early and late) compared to control group offspring (CON Vs EGS,
p<0.01 & CON Vs LGS, p<0.001). But resveratrol treatment failed to restore the inhibited
SOD activity in prenatally stressed offspring (p>0.05). No significant change in SOD activity
was observed in offspring exposed to prenatal resveratrol treatment alone (RES). (Figure
R24)
CON
RES
EGS
LGS
EGS+RES
LGS+RES
0
1
2
3
4
**
*** a ** *
Units/mg protein
Figure R24: Brain Superoxide dismutase activity (U/mg protein) in rats.
P<0.0001 & F=6.283
CON Vs EGS & EGS+RES, ** p<0.01; CON Vs LGS, *** p<0.001; CON Vs LGS+RES, * p<0.001
RES Vs LGS, a p<0.05
RESULTS
7.6.7. Na+, K+ -ATPase activity in brain
Linearity of enzyme reaction was assessed by measuring the amount of inorganic
phosphate liberated at different time intervals (15, 30, 45, 60, 75, 90 min respectively), while
maintaining the concentrations of other ions (Na +, K+, Mg2+), ATP and brain homogenate
constant. Na+,K+ -ATPase activity was linear (R2 linear = 0.87). The brain Na+, K+ -ATPase
activity level was decreased significantly (p<0.05) only in the offspring who recieved
prenatal stress during late gestation (LGS) compared to control group offspring (CON).
Though the resveratrol treatment during late gestational period has enhanced the Na +,K+ -
ATPase activity, but it is statistically not significant (p=0.0577). No significant variation in
Na+,K+ -ATPase activity was observed in the offspring exposed to prenatal resveratrol
treatment alone (RES). (Figure R25)
CON
RES
EGS
LGS
EGS+RES
LGS+RES
0
2
4
6
8
**
b
μmol of phosphorus/mg of protein/h
Figure R25: Brain Na+,K+ -ATPase activity (μmol of phosphorus/mg of protein/h) in rats.
P=0.0006 & F=20.228
CON Vs LGS, ** p<0.01
RES Vs LGS, bp<0.01
RESULTS
Summary of prenatal stress induced neuronal oxidative stress and its amelioration by
The findings of this study confirm the deleterious effect of prenatal stress during
different gestational period (early as well as late) on the antioxidant systems in the brain of
offspring (Table 13 & 14)
In comparison to the control group, the prenatally stressed offspring showed a marked
increase in the level of thiobarbituric acid reactive substances (TBARS) and advanced
oxidative products of protein (AOPPs) in the brain, indicating intensified lipid peroxidation
(LPO) and oxidized protein products. The prenatally stressed offspring groups also exhibited
decreased activity of superoxide dismutase (SOD), glutathione reductase (GGSH-Rd) and
Na+, K+ -ATPase activities in the brain and also decreased in the levels of reduced
glutathione (GSH) and total antioxidants (TAO), validating the suppressed antioxidant
efficiency in combating the prenatal stress induced free radical damage.
As seen in the Table 13, the offspring received prenatal stress during early pregnancy
(EGS) showed the great percentage decrease in the activity of GGSH-Rd (–53.39%), SOD (–
25.69%) and level of GSH (-62.2%) and TAO (-57.96%) in the brain. There is also an
increased in LPO (+144.59%) and AOPP (+156.1%) in the brain. These results indicate that
prenatal stress during early gestational period causes oxidative damage in the offspring brain.
Similarly the offspring received prenatal stress during late pregnancy (LGS) showed
the greatest percentage decreased in the activity of GGSH-Rd (–45.74%), SOD (–29.16%),
Na+, K+ -ATPase (-42.58%) and level of GSH (-59.88%) and TAO (-40.76%) in the brain.
There is also an increased in LPO (+199%) and AOPP (+235.5%) in the brain. These results
indicate that prenatal stress during late gestational period also causes oxidative damage in the
offspring brain. (Table 13)
Resveratrol supplementation proved to be effective in restoring oxidative damage in
brain evidenced by diminishing elevated TBARS and AOPP levels in prenatally stressed
offspring. Resveratrol treatment elevated the diminished GSH and TAO levels and also
enhanced the inhibited activities of GSH-Rd, SOD and Na +, K+-ATPase activities in
prenatally stressed offspring brain. Resveratrol treatment failed to enhance the suppressed
SOD activity. The maximum ameliorative effect of resveratrol supplement was observed in
the offspring of late gestational stress than early gestational stress. The effectiveness of
resveratrol treatment during pregnancy on various neuronal oxidants and antioxidants
RESULTS
parameters were expressed in terms of % recovery against the prenatal stress during early and
late gestation in the offspring brain. (Table 13)
Offspring received prenatal stress during early gestation along with resveratrol
treatment (EGS+RES) showed ameliorative activity on the level of LPO (+48.59%), GSH
(-12.44%), TAO (-97.8%), AOPP (+57.45%) and on enzyme activities of GSH-Rd (-33.26%)
compared to prenatally stressed pups (EGS). (Table 12)
Similarly, the offspring which received prenatal stress during late gestation along with
resveratrol treatment (LGS+RES) showed maximum ameliorative activity on the level of
LPO (+84.38%), GSH (-27.63%), TAO (-50.0%), AOPP (+81.51%) and on enzyme activities
of GSH-Rd (-70.09%), SOD (-19.03%) and Na+, K+-ATPase (-9.59%) compared to
prenatally stressed offspring (LGS). (Table 12)
The % recovery was calculated using the formula:
% change from control by prenatal stress – (% change from the control by
prenatal stress + resveratrol supplement)
% Recovery = X 100
% change from control by prenatal stress
RESULTS
Table 12: Summary of brain oxidants and antioxidants level in rats in different groups
Brain oxidant/antioxidants
CON
(n=12)
RES
(n=12)
EGS
(n=12)
LGS
(n=12)
EGS+RES
(n=12)
LGS+RES
(n=12)
F value
ANOVA
significance
Lipid Peroxidation
(μmoles/gm protein)
0.74±0.04 0.62±0.04
1.81±0.1*** c
(+144.59%)
1.99±0.21*** c
(+199%)
1.29±0.09 * b #
(+74.32%)
0.97±0.09 $$$
(+31.08%)
24.12 P<0.0001
Advanced oxidative products
of protein (μmoles/L)
4.87±0.16 5.0±0.19 12.18±0.46*** c
(+150.1%)
16.34±0.42*** c ###
(+235.5%)
7.98±0.29*** c ###
(+63.86%)
6.99±0.22*** c $$$
(+43.53%)
5.007 P<0.0001
Reduced glutathione
(mg/gm protein)
6.83±0.25 6.16±0.33 2.60±0.07*** c
(-62.2%)
2.74±0.16*** c
(-59.88%)
3.11±0.19*** c
(-54.46%)
3.87±0.21*** c $$
(-43.33%)
67.95 P<0.0001
Total Antioxidants
(mmoles/L)
1.57±0.06 1.99±0.06 0.66±0.03*** c
(-57.96%)
0.93±0.07*** c
(-40.76%)
1.55±0.10 ###
(-1.27%)
1.25±0.05 * c $
(-20.38%)
35.03 P<0.0001
Glutathione Reductase (nmol
NADPH oxidized/min/
mg protein)
8.11±0.46 8.87±0.35 3.78±0.21*** c
(-53.39%)
4.40±0.21*** c
(-45.74%)
5.22±0.37*** c
(-35.63%)
7.00±0.56 a $$
(-13.68%)
28.83 P<0.0001
Superoxide dismutase (U/mg
protein)
2.88±0.17 2.63±0.14 2.14±0.13 **
(-25.69%)
2.04±0.07*** a
(-29.16%)
2.11±0.10 **
(-26.73%)
2.20±0.16 *
(-23.61%)
6.28 P<0.0001
Na+, K+ -ATPase activity
(μmol of phosphorus/mg of
protein/h)
5.73±0.04 5.98±0.66 5.73±0.59 3.29±0.33** b
(-42.58%)
3.78±0.58
(-34.03%)
5.18±0.37
(-9.59%)
20.22 P=0.0006
CON Vs EGS, *** p<0.001, ** p<0.01; CON Vs LGS, *** p<0.001, ** p<0.01; CON Vs EGS+RES, *** p<0.001,
** p<0.01, * p<0.05;
CON Vs LGS+RES, *** p<0.001, ** p<0.01: RES Vs EGS, c p<0.001; RES Vs LGS, c p<0.001, b p<0.01, a p<0.05;
RES Vs EGS+RES, c p<0.001, b p<0.01;
RES Vs LGS+RES, c p<0.001, a p<0.05: EGS Vs LGS, ### p<0.001: EGS Vs EGS+RES, ### p<0.001, # p<0.001: LGS
Vs LGS+RES, $$$ p<0.001, $$ p<0.01, $ p<0.05
Values in parenthesis are % change: ‘–’ sign indicates decrease, ‘+’ sign indicates increase over controls
RESULTS
Table 13: Summary of brain oxidants and antioxidants level in male and female rats
Brain oxidant/antioxidants
Lipid
Peroxidation
(μmoles/gm
protein)
Advanced
oxidative
products of
protein
(μmoles/L)
Reduced
glutathione
(mg/gm protein)
Total
Antioxidants
(mmoles/L)
Glutathione
Reductase
(nmol NADPH
oxidized/min/m
g protein)
Superoxide
dismutase
(U/mg protein)
Na+, K+ -ATPase
activity (μmol of
phosphorus/mg of
protein/h)
CON
Male
(n=6)
0.68±
0.03
p, 0.22
t, 1.29
F, 5.74
4.67±
0.25
p, 0.99
t, 0.01
F, 1.17
6.59±
0.45
p, 0.38
t, 0.91
F, 2.92
1.57±
0.09
p, 0.94
t, 0.06
F, 1.50
7.73±
0.75
p, 0.47
t, 0.75
F, 1.59
2.57±
0.10
p, 0.08
t, 1.93
F, 8.64
5.69±
0.71
p, 0.93
t, 0.08
Female F, 2.45
(n=6)
0.81±
0.08
4.87±
0.23
7.07±
0.26
1.58±
0.11
8.46±
0.60
3.19±
0.30
5.76±
0.45
RES
Male
(n=6)
0.61±
0.07
p, 0.74
t, 0.33
F, 2.31
4.91±
0.19
p, 0.65
t, 0.46
F, 3.14
6.00±
0.50
p, 0.64
t, 0.47
F, 1.12
1.79±
0.09
p, 0.49
t, 0.71
F, 1.37
8.50±
0.60
p, 0.30
t, 1.07
F, 2.98
2.51±
0.25
p, 0.44
t, 0.79
F, 2.45
5.87±
0.80
p, 0.87
t, 0.16
Female F, 2.04
(n=6)
0.64±
0.04
5.10±
0.35
6.30±
0.47
1.68±
0.08
9.21±
0.34
2.75±
0.16
6.10±
1.14
EGS
Male
(n=6)
1.64±
0.12
p, 0.10
t, 1.80
F, 1.35
12.40±
0.59
p, 0.66
t, 0.45
F, 1.63
2.65±
0.08
p, 0.50
t, 0.68
F, 2.30
0.70±
0.03
p, 0.30
t, 1.08
F, 5.21
3.93±
0.27
p, 0.49
t, 0.70
F, 1.51
2.29±
0.18
p, 0.28
t, 0.79
F, 2.45
4.28±
0.37
p, 0.73
t, 0.34
Female F, 1.82
(n=6)
1.98±
0.14
11.97±
0.57
2.54±
0.12
0.62±
0.66
3.63±
0.33
2.00±
0.19
4.06±
0.51
LGS
Male
(n=6)
2.02±
0.32
p, 0.97
t, 0.03
F, 1.11
16.35±
0.69
p, 0.98
t, 0.02
F, 1.53
2.72±
0.19
p, 0.91
t, 0.11
F, 2.28
0.84±
0.09
p, 0.24
t, 0.72
F, 5.46
4.64±
0.37
p, 0.26
t, 1.17
F, 3.52
1.92±
0.10
p, 0.28
t, 1.12
F, 1.10
3.84±
0.51
p, 0.10
t, 1.81
Female F, 2.90
(n=6)
1.98±
0.30
16.33±
0.56
2.76±
0.29
1.02±
0.11
4.15±
0.19
2.09±
0.09
2.73±
0.30
EGS+RES
Male
(n=6)
1.28±
0.18
p, 0.94
t, 0.70
F, 5.61
8.31±
0.33
p, 0.28
t, 1.12
F, 2.06
3.16±
0.37
p, 0.80
t, 0.25
F, 6.35
1.56±
0.22
p, 0.94
t, 0.07
F, 5.94
5.63±
0.68
p, 0.29
t, 1.11
F, 5.81
2.30±
0.13
p, 0.25
t, 1.21
F, 1.23
4.19±
0.76
p, 0.51
t, 0.67
Female F, 1.44
(n=6)
1.29±
0.07
7.65±
0.48
3.06±
0.14
1.54±
0.04
4.81±
0.28
1.92±
0.12
3.37±
0.92
LGS+RES
Male
(n=6)
0.99±
0.18
p, 0.80
t, 0.24
F, 4.65
6.66±
0.35
p, 0.14
t, 1.56
F, 2.58
3.98±
0.33
p, 0.61
t, 0.51
F, 5.32
1.28±
0.09
p, 0.54
t, 0.62
F, 2.78
7.84±
0.78
p, 0.14
t, 1.56
F, 0.86
2.17±
0.23
p, 0.06
t, 2.09
F, 1.27
5.21±
0.53
p, 0.93
t, 0.77
Female F, 1.18
(n=6)
0.94±
0.08
7.33±
0.24
3.76±
0.28
1.22±
0.05
6.16±
0.72
2.14±
0.25
5.15±
0.58
RESULTS
Neuroprotective effects of resveratrol against prenatal stress in rats Page 117__

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RESULTS

Neuroprotective effects of resveratrol against prenatal stress in rats Page 70

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