SUBJECTS & METHODS

Neuroprotective effects of resveratrol against prenatal stress in rats

6.1. Animals and housing conditions
In-house bred male and female albino Wistar rats (90-120 days old) of weight 200-
230gm were selected for the study. The rats were maintained in 12 hours light and 12 hours
dark cycle and also in temperature and humidity controlled environment. All rats were fed
with standard rat food (contains, 21.96% crude oil, 3.10% crude fiber, 7.37% ash and 1.38%
sand silica) and water ad libitum, except during stressing procedure. The feed was supplied
by “Amrut laboratory animal feed, Amrut rat and mice pellet” manufactured by Pranav Agro
Industries Ltd, Maharashtra, India. Polypropylene cage (30 X 22 X 14 cm) with paddy husk
as bedding materials was used for housing the rats. The bedding material was changed every
alternate day. Breeding and maintenance of the animals were done as per the guidelines of
Government of India for use of Laboratory animals (Government of India notifies the rules
for breeding and conducting animal experiments, proposed in the gazette of India Dec 15,
1998: which was reproduced in Ind. Journal of Pharmacol 1999; 31:92-5). Institutional
Animal Ethical Committee (I.A.E.C) approval was obtained before the conduct of the study
(IAEC/KMC/11/06/2010) and care was taken to handle the rats in humane manner.
6.2. Timed pregnancy in rats
Three female rats were allowed to mate with one fertile sexually active male rat for 4
hours per day (separate male rats for each group). At the end of 4 hours, female rats were
separated and vaginal smears taken to detect the presence of sperm for the confirmation of
pregnancy and the rats were designated as day-0 of pregnancy for further counting the days.
The pregnant rats were housed individually in separate cages with proper label indicating the
day of conception and randomly allocated into six groups of six each. One male and one
female pups from each mother were considered for the study (n=12; six male and six female
pups). All the mothers delivered at term (22-24 th days of gestation). The offspring were raised
by their biological mothers until weaning (21 days after birth). On Day 21, after all offspring
weaned, male and female pups were separated and housed three in each cage respectively
until the completion of the study The number of offspring considered for this study is in
accordance with Holson and Pearce (1992).
SUBJECTS & METHODS
Neuroprotective effects of resveratrol against prenatal stress in rats
Figure M1: (A) Squamous cells in vaginal smear (B) Squamous cells and spermatozoa in clumps
6.3. Stressing procedure (Restraint stress)
The pregnant rats were stressed (restraint stress) using a wire mesh restrainer
(Mdhyastha et al., 2008) for three times daily for 45 minutes. To prevent habituation of
animals to the daily procedure, restraint periods were shifted randomly within certain time
periods (08:00 AM–11:00 AM, 12:00 AM–3:00 PM, and 4:00 PM–7:00 PM). The wire mesh
restrainer will have a wooden base and stainless steel wire mesh restrainer hinged to the base.
A pad lock and latch will help to secure the rat in the restrainer. The restrainer with
dimension 11 cm (L) x 6cm (B) x 6 cm (H) was used for rats with gestation day 1 to 10.
Restrainer of 11cm (L) x 8 cm (B) x 8 cm (H) was used for rats with gestation day 11 to till
delivery. This type of restrainer will only restrict the animal movement without any pain,
discomfort or suffocation.
Figure M2: Pregnant rat in wire mesh restrainer
6.4. Experimental study design
Neonatal parameters were investigated for each pup from the 1 st day of the birth till
weaning. After weaning, one male pup and one female pup from each mother (n=18) were
subjected to histological study of hippocampus (doublecortin and cresyl violet staining) on
SUBJECTS & METHODS
Neuroprotective effects of resveratrol against prenatal stress in rats
21st and 40th postnatal day as described below. Whereas another one male pup and one female
pup from each mother (n=12) were subjected to various behavioural tests on 21 st to 39th
postnatal day and biochemical estimations were performed on 40 th postnatal day as
summarized in the graphical representation as well as table below.
Graphical representation of study design:
(one ♂ & one ♀ pup from each mother)
6.5. Dosing procedure
The dose of resveratrol considered in the present study is according to the earlier
study by Kumar et al. (2007) and it was suspended with 0.5% carboxy methyl cellulose
(CMC). At dose of 10mg/kg body weight once daily was administered orally throughout
pregnancy.
Mother (n=18)
Histological studies
of hippocampus
(n=12; 6 ♂ & 6 ♀
pups)
Behavioural studies
followed by
biochemical
estimations in brain
homogenate (n=12;
6 ♂ & 6 ♀ pups)
HPA axis activity
(n=12; 6 ♂ & 6 ♀
pups)
Hippocampal
BDNF level
estimation (n=12; 6
♂ & 6 ♀ pups)
SUBJECTS & METHODS
Neuroprotective effects of resveratrol against prenatal stress in rats
6.6. Animal groupings
Pups belonging to pregnant rat received,
Group 1 (Control). vehicle (0.5% CMC, 10ml/kg body weight) throughout pregnancy.
Group 2 (RES). resveratrol alone throughout pregnancy.
Group 3 (EGS, Early Gestationl Stress). restrain stress from gestation day 1 to 10.
Group 4 (LGS, Late Gestationl Stress). restrain stress from gestation day 11 to till
delivery.
Group 5 (EGS+RES). restrain stress from gestation day 1 to 10 along with resveratrol
throughout pregnancy.
Group 6 (LGS+RES). restrain stress from gestation day 11 to till delivery along with
resveratrol throughout pregnancy.
6.7. Chemicals
Resveratrol (Cat. no. 70675, Cayman chemicals, USA) was obtained from Pro Lab
marketing, New Delhi, India. Brain derived neurotrophic factor (BDNF) levels were
quantified using a commercially available ELISA kit (CUSABIO, Cat. no. CSB-E04504r).
Polyclonal antidoublecortin antibody (Doublecortin: C-18: Cat. No. sc-8066) as primary
antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, A, U.S.A.).
Biotinylated antigoat IgG (Cat. no. BA-9500), Avidin Biotin Complex (ABC Kit: Cat. no.
PK-4005), Diaminobezedine (DAB Kit: Cat. no. SK-4100) and Normal Horse Serum (NHS:
Cat no.S-2000) were obtained from Vector Laboratories, Inc. (Vector Laboratories, USA).
Cresyl violet stain (Cat. no. C5042) and Poly-L-Lysine was purchased from Sigma Chemical
Co. (St Louis, MO, U.S.A.). Other chemicals and reagents are HPLC or analytical grade
(Sigma, St. Louis, Mo, U.S.A.) procured from Sri Durga Laboratories, Mangalore, India.
SUBJECTS & METHODS
Neuroprotective effects of resveratrol against prenatal stress in rats
6.8. Table 3: Study parameters
Purpose Day on study performed Study parameters
Safety of resveratrol
Till postnatal day 21
Gestational length
Litter size
Mortality rate
Day of ear pinna detachment
Day of eye opening
Day of eruption of teeth
Birth weight & weight gain
HPA axis activity
40th postnatal day
Serum cortisol estimation
Adrenal gland weight
Adrenal ascorbic acid
estimation
Hippocampal neurogenesis 21st & 40th postnatal day Doublecortin
immunostaining
Neuronal assay 21st postnatal day Cresyl violet staining
Hippocampal neurotrophic
factor expression
40th postnatal day Brain derived neurotrophic
factor (BDNF) estimation by
ELISA method
Motor functions 22nd & 25th postnatal day Open field test
Rota rod test
Cognitive functions 30th & 35th postnatal day Passive avoidance test
Water maze test
Brain oxidant/antioxidant
system
40th postnatal day
Estimation of lipid
peroxidation
Estimation of advanced
oxidative products of protein
Estimation of reduced
glutathione
Estimation of total
antioxidants
Assay of glutathione
reductase
Assay of superoxide
dismutase
Assay of Na+, K+ -ATPase
Note: brain oxidant and antioxidant systems were estimated in whole brain homogenate and hippocampal BDNF
level was estimated in hippocampal homegenate
SUBJECTS & METHODS
Neuroprotective effects of resveratrol against prenatal stress in rats
6.8.1. Neonatal study parameters
The safety of resveratrol during pregnancy was evaluated by screening mortality rate,
gestational length and early physical developments.
6.8.1.1. Gestational length and Mortality
Number of still born pups and total number of pups born to each control or stressed
rat were counted at birth to calculate the mortality at birth. During postnatal period (till 21
days) number of death was also counted to get the postnatal mortality rate.
6.8.1.2. Litter size
Total number of pups born to each mother was counted to get the litter size.
6.8.1.3. Birth weight and weight gain
Weight of pups born from stressed mother were taken at birth and compared with
similar weight of pups born to control rats. Weight of pups was measured on 1 st, 7th, 14th and
21st day.
6.8.1.4. Pinna detachment
During early postnatal period, pups were observed daily to see the development of
external ear (pinna of external ear). The day on which external ear was 1.0 mm (length) x 0.5
mm (breadth) defined as day of pinna detachment, and that day was noted for each pup.
Figure M3: Rat pups born to stressed mothers. (A) at birth;
(B) 7 days old; (C) 14 days old; (D) 21 days old
SUBJECTS & METHODS
Neuroprotective effects of resveratrol against prenatal stress in rats
Figure M4: Rat pups showing development of external ear pinna detachment
6.8.1.5. Eruption of upper and lower incisors
Eruption of teeth is a good developmental index. Pups were observed daily during
early postnatal period to witness the eruption of upper and lower incisor teeth. The day on
which teeth were 0.5 mm was considered as day of teeth eruption.
6.8.1.6. Eye opening
A visible opening (2 mm length) of palpebral fissure defined as eyes are opened. The
day on which eyes are opened was noted for each pup.
Figure M6: Rat pup showing opening of palpebral fissure
Figure M5: Rat pup showing eruption of lower incisiors
SUBJECTS & METHODS
Neuroprotective effects of resveratrol against prenatal stress in rats
6.8.2. Assessment of HPA axis activity
6.8.2.1. Quantification of cortisol concentrations in blood serum
Principle of the assay: Competitive Principle - This principle is applied to analyte of low
molecular weight such as cortisol. The sequences of the reactions involved are given below,
 In the first step, sample and a specific anti-cortisol antibody labelled with a ruthenium
complex were combined in the assay cup.
 After the first incubation, cortisol-specific biotinylated antibody and streptavidin-coated
paramagnetic microparticles were added. The still free binding sites of the labelled
antibody became occupied with the formation of an antigen-hapten complex. The entire
complex was bound to the micro particle via interaction of biotin and streptavidin.
 After the second incubation, the reaction mixture containing the immune complexes was
transported into the measuring cell. The immune complexes were magnetically entrapped
on the working electrode, but unbound reagent and sample are washed away by a system
buffer.
 In the Electrochemiluminescence reaction, the conjugate is a ruthenium based derivative
and the chemiluminescent reaction is electrically stimulated to produce light. The amount
of light produced was indirectly proportionately to the amount of antigen in the sample.
Sample collection: Blood samples were taken between 8.00 and 10.00 AM to avoid circadian
variations of serum cortisol concentrations. The 40 th postnatal day rats were anesthetized
individually in a glass jar containing saturated ether vapour and intracardiac blood was
collected.
Assay procedure: The sample was analyzed using Elecsys 2010 autoanalyzer and modular
analytics E170 established at the Clinical Biochemistry Laboratory, Kasturba Medical
College, Mangalore, India.
Calculation of results: Calculation of the concentration of the cortisol was carried out by
means of a 2-point calibration curve that was established using standards of known cortisol
concentration. The concentration of cortisol was expressed in ng/ml serum.
SUBJECTS & METHODS
Neuroprotective effects of resveratrol against prenatal stress in rats
6.8.2.2. Adrenal gland weight
The abdominal cavity was opened and the adrenal glands were recovered, carefully
freed from adjacent tissues under a stereo dissecting microscope, and weighed individually
by using high precision single pan electronic weighing balance (Adventure TM).
6.8.2.3. Estimation of adrenal ascorbic acid
Tissue processing: On 40th postnatal day, aadrenal gland were removed and 10%(w/v) tissue
homogenate were prepared in ice-cold 0.1M saline phosphate buffer (pH 7.4), centrifuged for
15 minutes 10,000 X g at 4°C and the supernatant was for estimation of adrenal ascorbic
acid.
Adrenal ascorbic acid level was measured as described previously by Roe & Kuether (1949)
and Lyle et al., 2009.
Principle of the assay: Ascorbic acid is first dehydrogenated by thiourea. The
dehydroascorbic acid is then reacted with 2, 4 dinitrophenyl hydrazine (DNPH) to form
osazone and dissolved in sulphuric acid to give an orange-red color complex which was
measured at 540nm.
Assay procedure: In the adrenal gland homogenate, 6% Tricaboxylic acid (TCA) was added
and centrifuged at 3000g for15 minutes. The supernatant was coupled with 2,4,Ndinitrophenyl
hydrazine (DNPH) in presence of thiourea as a mild reducing agent. 2ml cold
conc. H2SO4 (12 M) was added drop by drop, which converts DNPH into a orange-red
compound, which was assayed spectrophotometrically with a Systronic-117 UV-Visible
spectrophotometer at 520 nm. The value was expressed in μg/mg protein. Protein was
measured using the Lowery et al., (1951) method.
SUBJECTS & METHODS
Neuroprotective effects of resveratrol against prenatal stress in rats
6.8.3. Histological study of hippocampus
6.8.3.1. Immunohistochemical Assay for Doublecortin (DCX)
Perfusion: On 21st and 40th postnatal day six animals from each group were deeply
anesthetized with ether and secured on a dissection board, and its chest cavity was opened to
expose the heart. About 75-100 ml of 0.9% saline was perfused through the left ventricle, at
the rate of 1ml/min followed by perfusion of cold 4% paraformaldehyde in phosphate buffer
(PH =7.4). The animals will be decapitated and the brains were removed and kept in 4%
paraformaldehyde in phosphate buffer for 48hr (post fixation) at 4˚C. Paraffin blocks were
made in an embedding bath. Coronal sections of 5μm thickness were cut in the dorsal
hippocampus using rotary microtome (Jung Biocut 2035, Leica,Germany). Twenty five from
each animal were mounted serially on air dried poly-L-lysine coated slides.
Preparation of paraffin blocks: The animal was decapitated and the brain was removed and
kept in 10% formalin for 48h (post fixation). Paraffin blocks were made as described below:
Dehydration : a. 70% alcohol (2 hours)
b. 90% alcohol (2 hours)
c. 100% alcohol (3 changes for 2 hours)
Clearing : xylene (2 hours)
Embedding : a. four changes of paraffin wax for ½ hour each
b. embedding in fresh filtered paraffin wax
Coronal sections of 5μm thickness were cut in the dorsal hippocampus using a rotary
microtome (Jung Biocutt 2035, Leica, Germany). Twenty five sections from each animal
were mounted serially on on the slides which were already smeared with Poly-L-Lysine.
Immunostaining procedure: The sections were stained with doublecortin immunostain (Rao
and Shetty, 2004) as follows,
a. The sections were deparaffinized with xylene for 10 minutes (twice)
b. The slides were kept in 100% ethanol (absolute alcohol) for 5 minutes (2 times)
c. The sections were rehydrated with descending grades 90%, 70% and 50% of alcohol
(5 minutes each)
d. Immersed in distilled water for one dip
e. Warmed in 0.01 M citrate buffer (pH 6) for 30 minutes in water bath at 60˚C
f. Washed in distilled water
g. Then placed in Phosphate Buffer (PB) at pH 7.4 for 5 minutes
SUBJECTS & METHODS
Neuroprotective effects of resveratrol against prenatal stress in rats
h. The slides were removed from PB bath, only good sections were retained
(unnecessary sections were removed) and the slides were cleaned with the tissue
paper & circle was put around the section
i. Incubated for 30 minutes with 3-5 % H 2O2 (Peroxidase block) contains methanol and
PBS, for blocking nonspecific background
j. Sections were washed in PBS for three times 5 minute each
k. Blocking with Normal Horse Serum (NHS)
 5% NHS prepared in PBS contains Triton X-100/Tween 20
 Sections were covered with NHS, incubated for 30mins in room temperature
l. Application of primary antibody (Doublecortin)
 Doublecortin was diluted in PBS contained NHS (1:200)
 The sections were covered with diluted Doublecortin
 Incubated for 3-4 hours in room temperature
 Adequate moisture was maintained to prevent drying of tissue
m. Sections were washed in PBS for three times 5 minute each
n. Application of secondary antibody (Biotinylated Antigoat IgG)
 Biotinylated Antigoat IgG was diluted in PBS (1:200)
 The excess PBS was blotted from the section
 The sections were covered with diluted Biotinylated Antigoat IgG
 Incubated for 1hr in room temperature
o. Sections were washed in PBS for three times 5 minute each
p. Application of ABC (Avidin Biotin Complex) reagent
 Preparation of ABC reagent:
 ABC reagent was prepared by mixing reagent-A and reagent-B in PBS (1:50)
 mixed well and allowed to stand for 30min before use
 The excess PBS was blotted from the section
 The sections were covered with prepared ABC reagent
 Incubated for 1hr
q. Sections were washed in PBS for three times 5 minute each
r. Using diaminobezedine (DAB) staining
 The reagent was prepared just before use
 To 5ml of distilled water, 1 drops of buffer stock solution was added and
mixed well
SUBJECTS & METHODS
Neuroprotective effects of resveratrol against prenatal stress in rats
 2 drops of DAB stock solution was added and mixed well
 1 drops of H202 solution was added and mixed well
 The tissue sections were incubated with the substrate at room temperature
until suitable stain developed
s. Sections were washed in PBS for three times 5 minute each
t. The sections were dehydrated with ascending grades 50%, 70%, 90% and 100% of
alcohol (2minutes each)
u. Washed in distilled water and the slides were kept in xylene and mounted with cover
slip
calibration of micrometers: Stage micrometer is a ruler that mounted on a microscope slide
that does have units [millimetres (mm) and micrometers (μm)]. A typical stage micrometer
scale is 1mm (1000 μm) long and divided into 100 divisions, so that each division equal to
10μm. An ocular micrometer consists of 100 divisions. During calibration, the stage
micrometer is lined up with the ocular micrometer and the numbers of divisions on the ocular
micrometer per millimeter or micrometer on the stage micrometer are counted. The number
of divisions will change as the magnification changes.
At the magnifications of 40X: It was observed that sixteen ocular micrometer divisions
(OMD) overlapped five stage micrometer divisions (SMD), where one stage micrometer
division is equal to 10μm.
16 OMD = 5 SMD
1 OMD = 0.3125 SMD
1 OMD = 0.312 X 10 = 3.12μm (1 SMD = 10μm)
80 OMD = 80 X 3.12 = 250μm
In this study 250μm hippocmpal length (80 ocular micrometer divisions) was
measured for neuronal count under 40 X magnification.
SUBJECTS & METHODS
Neuroprotective effects of resveratrol against prenatal stress in rats
Quantitative evaluation of doublecortin positive (+ve) neurons: All sections were observed
using light microscopy at 40X magnification. The numbers of newly born neurons, i.e.
doublecortin +ve neurons were counted in subglanular zone (SGZ)/granular cell layer (GCL)
of hilus of dentate gyrus using occulomicrometer (250μm length per horn).
Figure M7: Photomicrographs of DCX +ve neurons in SGZ/GCL in holus of DG under 40X
The arrows denote the DCX +ve neurons.
6.8.3.2. Cresyl violet staining
Perfusion: On the 21st postnatal day, six male and six female offspring were used for each
group (n=12). The perfusion was performed as described above in doublecortin
immunostaining procedure.
Preparation of paraffin blocks: The animal was decapitated and the brain was removed and
kept in 10% formalin for 48h (post fixation). Paraffin blocks were made as described above
in doublecortin immunostaining procedure.
SUBJECTS & METHODS
Neuroprotective effects of resveratrol against prenatal stress in rats
Staining: One hundred milligrams of cresyl violet was dissolved in 100 ml of distilled water
(0.1%). To this 0.5 ml of 10% acetic acid was added to give a pH of 3.5-3.8. The stain was
filtered before use. The sections were stained with cresyl violet stain (Madhystha et al., 2002)
as follows:
a. Xylene (2 changes each of 5 minute)
b. Descending grades of alcohol (100%, 90%,70% and 50%) for each 2 minute
c. Distilled water (15 minute)
d. 0.1% cresyl violet stain (30 minute at 60˚C)
e. Cool to room temperature
f. Ascending grades of alcohol (50%, 70%, 90% and 100%) for 1-2 minute each
g. Xylene (2 minute)
h. Mount with DPX
Scoring: In each hippocampal section, cornua amonis (CA 1, CA2, CA3; 250μm length) of
hippocampus and dentate gyrus (250μm length) were selected (using an oculomicometer) and
the numbers of viable neurons were counted. The slides were screened using a light
microscope at 40X magnification (Magnus, Olympus Pvt. Ltd. New Delhi, India). Ten
sections from each rat were considered. Cells which were darkly stained, shrunken and
fragmented nuclei were excluded from the count. Slides from different groups of rats were
decoded to avoid manual bias while counting the cells. The cell counts were expressed as the
number of cells per unit length of the cell field (cells/250μm length).
Figure M8: Coronal section of the rat brain showing different parts of the hippocampus under 4X
(cresyl violet stain). CA-cornua amonis, DG-denate gyrus
SUBJECTS & METHODS
Neuroprotective effects of resveratrol against prenatal stress in rats
6.8.4. ELISA analysis of brain derived neurotrophic factor (BDNF) level in hippocampus
Principle of the assay: The microtiter plate provided in this kit has been pre-coated with an
antibody specific to BDNF. Standards or samples are then added to the appropriate microtiter
plate wells with a biotin-conjugated antibody preparation specific for BDNF and Avidin
conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated.
Then a TMB (3,3',5,5' tetramethyl-benzidine) substrate solution is added to each well. Only
those wells that contain BDNF, biotin-conjugated antibody and enzyme-conjugated Avidin
will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of
a sulphuric acid solution and the color change is measured spectrophotometrically at a
wavelength of 450 nm ± 2 nm. The concentration of BDNF in the samples is then determined
by comparing the O.D. of the samples to the standard curve.
Tissue preparation: Hundred milligram hippocampal tissues were rinsed with 1X phosphate
buffered saline (PBS), homogenized in 1 ml of 1X PBS and stored overnight at -20˚C. After
two freeze-thaw cycles were performed to break the cell membranes, the homogenates were
centrifuged for 5 minutes at 5000 X g, 2-8˚C. The supernatants were used for ELISA
analysis.
Assay procedure: BDNF levels were quantified using am commercially available ELISA kit
(CUSABIO, catalogue number. CSB-E04504r) according to the manufacturer’s protocol.
Calculation of results: The level of BDNF protein in each sample was determined using the
standard curve. The professional software “curve expert 1.3” was used to make the standard
curve. Standard curve was constructed by plotting the mean absorbance for each standard on
the X-axis against the concentration on the Y-axis and a best fit curve was drawn through the
points on the graph. Values were expressed as pg BDNF/100 mg tissue.
SUBJECTS & METHODS
Neuroprotective effects of resveratrol against prenatal stress in rats
6.8.5. Behavioural tests
6.8.5.1. Open field exploration test
This is one of the mostly widely used methods to access the motor, exploratory
activities and emotional reactivity of neonatal rats at 21 day (Bures et al., 1983; Madhyastha
et al., 2002). The open field apparatus (Techno, Lucknow, India) consists of a rectangular
box (100x100x40 cms); the floor area marked into 25 squares (5x5 cms). A uniform
illumination was provided with 60 watts bulb fixed 60 cm above the centre of field. In this
novel environment, rat was placed in one corner of the chamber. The fraction of total
exploratory time spent in peripheral area (close to wall) and in the central area will be
measured for individual rat. Total time for exploration in each session will be 5 min, and each
rat will have 3 sessions with 30 minute interval. In addition to this rearing (elevated hind limb
& pelvis with elevation of fore limb) and grooming (use of head, tongue and fore limb for the
process of cleaning various part of the body) behaviour were quantified.
6.8.5.2. Rota rod test
The rota rod test is an established method for evaluating muscle tone and strength
(Bures et al., 1983; Bairy et al. 2007). This apparatus (Techno, Lucknow, India) consists of
rotating wooden rods of different diameter for rats of different age (3.5 cm for post weaning
rats) powered by a motor capable of rotating the rod up to maximum 15 rpm. Circular
cardboard discs divided the rod in to six compartments each 15.25 cm length. During training
session, any rat that fell from the rod was immediately placed back on the rod. Repeated falls
excluded an animal from continuing the further trials. Each rat was made to hold the rotating
rod, till it failed to hold and falls down. Total time it could hold was noted. Each rat was
given 4 trials with an inter trial interval of 15 min.
6.8.5.3. Passive avoidance test
To test the memory retention, 30 days old rat pups in all the groups were subjected to
passive avoidance test earlier described by Bures et al., 1983 and Cherian et al., 2009. The
test determines the ability of a rat to remember a foot shock delivered 24 hr prior to the
memory retention test.
The apparatus used for this purpose was a digital passive avoidance apparatus
(Techno, Lucknow, India). Passive avoidance apparatus consists of a wooden box with two
compartments: (a) larger, bright compartment and (b) smaller, dark compartment equipped
SUBJECTS & METHODS
Neuroprotective effects of resveratrol against prenatal stress in rats
with grid floor, which is attached to a shock source. The connection between the two
compartments could be closed with a sliding door.
The experiment included three parts, i) an exploration test, ii) an aversive stimulation
and learning phase (passive avoidance acquisition), and iii) retention test
i) Exploration test: On the first day of test, rat was placed in the centre of the illuminated
large compartment facing away from the entrance to the dark small compartment, for
exploration. The door between the two compartments remained open at this time. The rat was
allowed to explore both compartments for 5 minutes. This is followed by three test trials of 5
min each. At the end of the trial, the rat was placed in the home cage, where it remained
during an inter-interval of 5 minutes. In each trial, fraction of time spent in each compartment
was noted.
ii) Aversive stimulation and learning phase (passive avoidance acquisition): At the end of 3rd
test trial, as soon as the animal stepped into dark compartment, a foot shock was delivered
through the grid floor (50 Hz, 1.5 mA, for 1 second). The rat was held additional 10sec, to
allow the animal to form an association between the properties of the chamber and foot
shock. The rat was then returned to its home cage.
iii) Retention test: The memory retention test was done 24 hr after foot shock. The rat was
placed in the bright compartment and the time taken (the step-through latency) for it to enter
the dark compartment for the first time was recorded using a stop watch. A maximum of 300
sec were given for the rat to explore. Fraction of time spent in dark and bright compartment
for each rat was noted. Normal rats avoid entering the dark chamber, where they received
shock on previous day, suppressing their normal behavior of exploring the dark compartment.
Decreased latency to enter the dark compartment will suggest poor memory retention.
6.8.5.4. Water maze test
This is one of the mostly widely used methods to access the spatial learning, place
learning, cognitive maps and memory in rats. To test the spatial memory, rats were subjected
to Morris water maze test (Morris, 1984; Bairy et al. 2007) from 34th to 39th postnatal day.
The water maze apparatus consists of a circular water tank of 1.83 meters in diameter,
divided into 4 quadrants. There will be a 4’’x 4’’ size escape platform submerged in one of
the quadrant, the target quadrant. The top surface of the platform was hidden approximately
1cm below the surface of the water. The pool is filled with water at a temperature of 18-26˚C
SUBJECTS & METHODS
Neuroprotective effects of resveratrol against prenatal stress in rats
to a depth of about 40 cm. The rats were trained in the water maze in 11 sessions on 6
consecutive days, two sessions on each day except on first day where only one session was
given. Each session consists of 4 trials. In each trial, time taken to reach the hidden platform
was recorded. If the rat was unable to find the platform within two minutes, the training
session was terminated and a maximum score of two minutes was assigned. Twenty-four
hours after the last session, rats were subjected to memory retention (probe test). This session
was of 30 sec duration. Here time taken to reach the target quadrant and time spent in the
target quadrant were measured. Greater latency to reach the target quadrant and less time
spent in the target quadrant suggests memory impairment.
SUBJECTS & METHODS
Neuroprotective effects of resveratrol against prenatal stress in rats
6.8.6. Oxidant/antioxidant systems measurement in brain
Tissue processing: Biochemical tests were carried out 24 h after the last behavioral test (on
40th postnatal day). Six male and six female rats were sacrificed by decapitation, always
between 8.00 - 10.00 AM to avoid any possible rhythmic variations in the antioxidant
enzyme levels.
The whole brain was removed rapidly and rinsed with 0.1M/L saline phosphate buffer
(pH 7.4). Tissue was weighed and homogenized (1:10w/v) in 0.1M/L saline phosphate buffer.
The homogenate was centrifuged at 10,000 X g for 20 min at 4°C and aliquots of supernatant
were separated and used for following biochemical estimations except Na +, K+ ATPase
activity estimation.
While in case of Na+, K+ -ATPase activity estimation the whole brain was weighed
and homogenized (1:10w/v) in sucrose isotonic buffer (0.32 mM sucrose, 12.5 mM Tris and
1 mM EDTA), pH 7.4 and dilution 1:19. The homogenate was centrifuged at 10,000 X g for
20 min at 4°C and aliquots of supernatant were separated and used for assay of Na +, K+-
ATPase activity.
6.8.6.1. Assay for lipid peroxidation (LPO)
The lipid peroxidation product in brain homogenate were measured through the
estimation of thiobarbituric acid reactive substances (TBARS) levels by the method as
described by Buege and Aust, 1978 and Gayathri et al., 2000a.
Principle of the assay: This assay is based on the reaction of a chromogenic reagent, 2-
thiobarbituric acid, with malanoaldehyde (MDA) at 25°C. One molecule of MDA reacts with
2 molecules of 2-thiobarbituric acid via a Knoevenagel-type condensation to yield a
chromophore with absorbance maximum at 532 nm .
Assay procedure: One milliliter of supernatant was precipitated with 2.5 ml of ice cold
trichloro acetic acid (TCA). The samples were centrifuged at 3000g for 10min. To 2ml of this
supernatant, 0.67% of thiobarbituric acid (TBA) was added and kept in boiling water bath for
10min and cooled it. The pink chromogen developed was read immediately at 532 nm using a
Systronic-117 UV-Visible spectrophotometer. Thiobarbituric acid-reactive substances
(TBARS) concentration was calculated using molar extinction coefficient of chromophore
(1.56 x 105(mol/l) -1cm -1) and the values were expressed in μmoles/gm protein. Total protein
concentration of tissues was measured by the method of Lowry et al., 1951.
SUBJECTS & METHODS
Neuroprotective effects of resveratrol against prenatal stress in rats
6.8.6.2. Estimation of advanced oxidative products of protein (AOPPs)
Advanced oxidative products of protein was estimated according to the method
described by Witko-Sarsat et al., 1996.
Principle of the assay: The advanced oxidative products of protein assay is a bioassay tool
for the direct quantitative measurement of AOPPs in brain homogenate. The unknown
AOPP-containing brain homogenate are first mixed with an assay reaction initiator like
potassium iodide (KI) that begins a color development process. After a brief incubation, a
stop solution acetic acid is added and the samples can be read with a standard
spectrophotometer.
Assay procedure: Brain supernatant was diluted with normal saline (1:10). To that, 200 μl of
1.16 M KI was added and kept for 2 minute. Later 400μl of acetic acid was added. Similarly a
sample blank was prepared containing only normal saline and other reagents as above. The
absorbance was measured spectrophotometrically with a Systronic-117 UV-Visible
spectrophotometer at 340 nm. Concentration of AOPPs was calculated by using the extinction
coefficient of 26 mM-1xcm-1 and the values were expressed in μmoles/lit.
6.8.6.3. Assay of reduced glutathione (GSH)
Tissue GSH concentration was estimated according to the method described by
Ellman, 1959 and Jyothi et al., 2007
Principle of the assay: The general thiol reagent, 5-5'-dithiobis[2-nitrobenzoic acid] (DTNB,
Ellman’s Reagent) reacts with GSH to form the 412 nm chromophore, 5-thionitrobenzoic
acid (TNB) and GSH-TNB. The GSH-TNB is subsequently reduced by glutathione reductase
and b-nicotinamide adenine dinucleotide phosphate (NADPH), releasing a second TNB
molecule and recycling the GSH; thus amplifying the response. Any oxidized (GSSG)
initially present in the reaction mixture or formed from the mixed disulfide reaction is rapidly
reduced to GSH.
Assay procedure: One milliliter of supernatant was precipitated with 1 ml of metaphosphoric
acid and cold digested at 4ºC for 1 h. The samples were centrifuged at 1,200g for 15 min at
4ºC. To 1ml of this supernatant, 2.7ml of phosphate buffer and 0.2ml of 5, 5’ dithio-bis-2-
nitrobenzoic acid (DTNB) was added. The yellow color that developed was read immediately
at 412nm using a Systronic-117 UV-Visible spectrophotometer. The values were expressed in
SUBJECTS & METHODS
Neuroprotective effects of resveratrol against prenatal stress in rats
mg/gm protein. The total protein concentration of tissues was measured by the method of
Lowry et al., 1951.
6.8.6.4. Estimation of total antioxidants (TAO)
The total antioxidants level was estimated according to the method described by
Koracevic et al., 2001.
Principle of the assay: A standardized solution of Fe-EDTA complex reacts with hydrogen
peroxide by a Fenton - type reaction, leading to the formation of hydroxyl radicals. These
reactive oxygen species degrade benzoate, resulting in the release of thiobarbituric acidreactive
substances (TBARS). Antioxidants from the added sample (brain homogenate) cause
the suppression in the production of TBARS. The reaction can be measured
spectrophometrically and the rate of inhibition of colour development is proportional to the
concentration of anti-oxidative activity.
Assay procedure: Each sample had its own control in which Fe-EDTA mixture, H 2O2 and
sodium benzoate were added after 20% acetic acid. For each series of analysis a negative
control was prepared containing the same reagents as A, except that brain homogenate was
replaced with 0.1M sodium phosphate buffer, pH 7.4. Uric acid (1 Mm/L) was used as a
standard. The reaction mixture was incubated at 37ºC for 60 min. Then 20% acetic acid and
0.8% TBA were added and incubate for 10 min at 100ºC, then cooled in ice bath. The
absorbance was measured at 532 nm against deionised water using Systronic-117 UV-Visible
spectrophotometer. The total antioxidants level in whole brain was expressed as mmoles/L.
6.8.6.5. Assay of glutathione reductase (GSH-Rd)
The GSH-Rd activity was measured using the method originally described by Moron
et al, 1979 and Gayathri et al., 2000b.
Principle of the assay: This assay is based on the reduction of oxidized glutathione (GSSG)
to reduced glutathione (GSH) by NADPH in the presence of glutathione reductase. The
reaction is measured by the decrease in absorbance at 340nm using an extinction coefficient
of chromospheres (1.36x104 (mol/l)-1 cm-1). The activity of glutathione reductase is used as
an indicator for oxidative stress.
Assay procedure: The reaction mixture consisted of1.6 ml of 0.067 M potassium phosphate
buffer (pH 6.6), 0.12 ml of 0.06% NADPH, 0.12 ml 1.15% GSSG, 0.1ml of enzyme source
and water in a final volume of 2 ml. All mixtures and solutions were prepared at room
SUBJECTS & METHODS
Neuroprotective effects of resveratrol against prenatal stress in rats
temperature. Control cuvettes then received 180μL of deionized water while sample cuvettes
received 60μL of deionized water and 120 μL of GSSG solution. NADPH oxidation was
followed for 5 min and was recorded using a Systronic-117 spectrophotometer. The reduction
of GSSG to GSH was determined indirectly by the measurement of the consumption of
NADPH, as demonstrated by a decrease in absorbance at 340 nm as a function of time. The
enzyme activity was calculated using extinction coefficient of chromophore (1.36x10 4
(mol/l)-1 cm-1) and expressed as nmol NADPH oxidized/min/mg protein. Protein content was
determined by the method of Lowry et al., (1951) with bovine serum albumin as standard.
6.8.6.6. Assay of superoxide dismutatase activity (SOD)
Superoxide Dismutatase activity was determined by the method of Marklund et al.,
1974 and Gayathri et al., 2000b.
Principle of the assay: Superoxide Dismutase (SOD) catalyzes the dismutation of the
superoxide radical (O2
-) into hydrogen peroxide (H 2O2) and elemental oxygen (O2) and as
such provides an important defense against the toxicity of the superoxide radical. In the
assay, superoxide ions (O2
-), generated due to fluorescent illumination, converts nitroblue
tetrazolium (NBT) to NBT-diformazan, which absorbs light at 560 nm. SOD reduces the
superoxide ion concentration and thereby lowers the rate of NBT-diformazan formation. The
extent of reduction in the appearance of NBT-diformazan is a measure of SOD activity
present in the experimental sample.
Assay procedure: The reaction was perfomed in an mixture containing 5.6x10 -5 M nitroblue
tetrazolium (NBT), 1.17X10-6 M riboflavin, 1x10-2M methionine in 0.05M potassium
phosphate buffer, pH 7.8 with suitably diluted tissue homogenate in a total volume of 3ml.
Illumination of solution was carried out in an aluminium lined foil box fitted with an 15v
fluorescent lamp. The solution taken in a beaker was kept in the box and illuminated exactly
for 10 min. Control without the enzyme source was prepared. The absorbance was measured
spectrophotometrically with a Systronic-117 UV-Visible spectrophotometer at 560 nm. SOD
activity was expressed as specific activity of the enzyme in units per mg protein (U/mg
protein). Protein content was determined by the method of Lowry et al., 1951.
SUBJECTS & METHODS
Neuroprotective effects of resveratrol against prenatal stress in rats
6.8.6.7. Assay of Na+, K+-ATPase activity
Na+, K+-ATPase activity was determined by the method of Wyse et al., 2000.
Principle of the assay: Sodium potassium ATPase transporters Na+ out and K+ in against
concentration gradients at the lost an adenosine triphosphate (ATP) molecules, that gives
adenosine diphosphate (ADP) and inorganic phosphate (iP). The librated iP is estimated by
Fiske Subba Rao Methods
Assay procedure: Na+, K+ -ATPase activity was measured by adding 200 μL of brain
homogenate to the reaction mixture contained 1 M NaCl, 1 M KCl, 0.1 M MgCl 2, 0.2 M
EDTA and 0.5 M Tris-HCl buffer, pH 7.4, in a final volume of 500 μL. The reaction was
started by the addition of 100 μL of 30 mM ATP (disodium salt, vanadium free). Control was
assayed under the same conditions with the addition of 200 μL 10 mM ouabain. Reaction was
terminated by the addition of 1 ml 10% ice cold TCA after 60 min. Then the mixture was
centrifuged for 10 min at 1000 X g to remove the precipitate. Na +, K+ -ATPase activity was
calculated by the difference between the two assays. Released inorganic phosphate (Pi) was
measured by the method of Fiske-Subbarow (1925). Enzyme specific activity was expressed
as μmol iP released /mg of protein /hr. Protein content was determined by the method of
Lowry et al., 1951.
6.9. Statistical analysis
All statistical analysis were performed using GraphPad prisim 5 software (GraphPad
Software, Inc. USA). Data were presented as the Mean±SE. Statistical analysis for multiple
comparisons was performed by one-way analysis of variance (ANOVA) with Bonferroni’s
corrections. Data from the training sessions were analyzed separately by two-way ANOVA
taking the number of the trial as a repeated measure. Comparison of data between male and
female group was assessed by unpaired “t” test. Comparison of total number of DCX positive
neurons in 21st day and 40th day was also assessed by unpaired “t” test. P value < 0.05 was
considered as significant.