Materials and Methods

1. EXPERIMANTAL PLAN

Healthy female S.D. rats weighing 180–230 g were procured from the Central Animal
House of the Panjab University, Chandigarh. Animals were acclimatized to laboratory
conditions prior to experimentation and were housed in polypropylene cages under a
hygienic bed of husk in a well-ventilated departmental animal house. The animals
were maintained on a standard laboratory pelleted feed (Ashirwaad Industries, Tirpari,
and Punjab) and water ad libitum throughout the period of experimentation. All the
procedures on rats were done in accordance with ethical guidelines for care and use of
laboratory animals, which were approved, by Institutional Animal Ethics Committee
(IAEC), Panjab University Chandigarh, India.

Experimental sessions were conducted by randomly dividing animals into 4 groups

and a six-week study was conducted to assess the various parameters.

Group 1: Sham control (vehicle group), Group 2: Aβ1-42 injected, Group 3: only
cranial X-irradiated (radiation control) and Group 4: Aβ1-42 injected animals
followed by cranial fractionated X-irradiation (2Gy X 5days).

2. CHEMICALS

Aβ1-42was purchased from Sigma (St. Louis. MO). Other chemicals were purchased
from Sigma (St. Louis, USA), Merck, Sisco Research Laboratories Pvt Ltd (Mumbai,
India) and Hi-Media Chemicals. The Allengers Medical Systems Ltd., USA, provided
the X-ray machine for the experiments to be conducted.

3. PREPARATION OF Aβ1-42 OLIGOMERS

Synthetic Aβ1-42 peptide was dissolved in sterile bidistilled water at a concentration of

200µmol/L and was aggregated by invitro incubation at 37°C for 24 hours to obtain a
solution for Aβ1-42 oligomers. The preparation was centrifuged at 14000 X g for 10
minutes and the supernatant containing Aβ oligomers was transferred to clean tubes
and stored at 4°C (Chunli et al., 2017).

4. SURGICAL PROCEDURE

The rats were anesthetized intraperitonally with sodium thiopentone (45 mg/kg i.p.)
and placed in a stereotaxic instrument. The scalp was incised and drilled at

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 Materials and Methods

stereotaxically defined intracerebroventricular (icv) points (1.2 mm behind the

bregma, 1.5 mm meta, 4 mm deep) according to the rat brain atlas by Paxinos and
Watson. The sham-operated rats (Group 1) received only the vehicle solution
(bidistilled water). Anaesthetized animals of Group 2 and Group 4 received single
injection of 5 µL synthetic Aβ1-42 peptide (1 µg/ µL in bidistilled water) bilaterally
with a 10 µL Hamilton microsyringe and the needle kept in place for a period of 2
minutes following injection. The animals of the group 4 were further subjected to 10
Gy X-irradiation (fractionated dose, 2 Gy X 5 days) 4 weeks post Aβ1-42 icv infusion
of the peptide.

Finally, the animals categorized as the radiation control group (Group 3) were
subjected to same dose of cranial fractionated X-irradiation (2 Gy X 5 days) only.

Figure 15: Stereotactic surgery for icv administration of Aβ1-42.

5. X- RAY MACHINE CALIBRATION

X-ray machine from Allengers, India was calibrated using FBX (Ferrous sulphate-
benzoic acid-xylenol orange) dosimeter as described by Gupta et al (1978). FBX is an
aqueous chemical dosimeter developed for measuring low-level radiation doses. Such
chemical dosimeters are based on the principle of interaction of radiation sensitive
chemicals with ionizing radiation and finally measuring the optical density of the end
product. Free radicals formed by radiolysis of aqueous medium oxidizes ferrous ion
of the solution to ferric ions that result in formation of complex (1:1) with xylenol
orange. Benzoic acid present in FBX increases the oxidation of ferrous ion to ferric

39
 Materials and Methods

ion up to four-fold thus amplifying the concentration of detectable ferric xylenol

orange complex. The optical density of the complex was spectrophotometrically
recorded at 548 nm.

Reagents

1) Benzoic acid – 5 mM
2) Ferrous ammonium sulphate- 0.2 mM
3) Xylenol orange - 0.2 mM
4) H2SO4-1 N

Procedure

Briefly, FBX dosimeteric solution was formed by dissolving benzoic acid in distilled
water in water bath. After cooling the solution to room temperature, 0.1 N sulfuric
acid, xylenol orange and ferrous ammonium sulphate were added. The solution was
kept in dark and was exposed to X-rays. FBX solution in a plastic petridish was
exposed to X-rays at fixed current of 20 mA and varying voltages from 40 kV to 110
kV. Thereafter, optical density OD was measured after 30 minutes of exposure at 548
nm wavelength. Measured optical density of different exposures were plotted against
different voltages and then compared with optical density of FBX sample exposed to
known different doses from LINAC (Department of Radiotherapy, Post Graduate
Institute of Medical Education and Research PGIMER), Chandigarh. From the graph
80 kV was selected which delivered dose of 70 rads in single fraction at constant
operating current of 20 mAs. The following table represents the dose response of
FBX at fixed operating current of 20 mAs and varying voltages (kV).

Table 1: Dose response of FBX against increasing voltages

VOLTAGE (kV) OD

40 0.021
60 0.053
80 0.067
100 0.079
110 0.062

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 Materials and Methods

0.08

0.07

0.06

0.05
OD

0.04

0.03

0.02

0.01

0
0 20 40 60 80 100
Dose in rads

Figure 16: Graph depicting dose in rads against OD of FBX exposed using LINAC.

6. NEUROBEHAVIORAL PARAMETERS

i. Motor function tests

a. Locomotor activity test

The locomotor activity was measured using a computerized actophotometer apparatus

(INCORP, India) by the method of Frussa-Filho et al (1997). Actophotometer is a
square arena apparatus that operates on photoelectric cells connected in circuit with a
counter. An array of infrared emitter pairs measures the animal activity and displays
the digital data on the front panel as the count of ambulatory movements. The
interruption of beam of light falling on the photocell following the animal movement
was recorded as account; higher the count, higher is the total locomotor activity of the
animal. Acclimatization of rats were allowed for a period of 2 minutes, following this
the activity of individual rat was measured for a total duration of 5 minutes and the
total ambulatory photobeam counts were recorded.

b. Muscular strength

Motor coordination in animals was evaluated using rota-rod measuring 3 cm in

diameter and rotating at a constant 20 rpm. Initial training sessions of 300 seconds
approximately 30 minutes apart were provided to the rats to maintain a steady posture
on the rotating rod following which a baseline trial of 120 seconds was conducted.
The animals were allowed a maximum of 120 seconds to complete the task. The

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 Materials and Methods

apparatus automatically records the time spent by each animal on the apparatus. The
animals that spent the maximum time on rotarod (without falling) were given a
maximum score of 120 seconds. The data was presented as the total time spent on the
rotating rod.

ii. Cognitive function tests

a. Active avoidance test

The paradigm assesses the cognitive behavior of rats and tests their ability to avoid
any aversive stimulus by performing a specific behavioral action. On the basis of the
number of times the animal escaped the test, the cognitive capability of the rats were
assessed. The apparatus consisted of two equal sized compartments (510 x 250 x 240
mm) separated by a partition. The one side of the partition has a conditioned stimulus
(light) and the other side is a dark chamber fitted with electrifiable rods on floor. In a
set of 10 test trials series, the animals were placed in lit chamber following which
after a time interval of 10 seconds the buzzer was switched on. After another 10
seconds, an electric shock was provided at 60 V and for 5 seconds to the animals. If
the animal jumps to the other side of the compartment as soon as the buzzer is set on,
it means the animal has avoided the test. But in case, if the animal jumps to the other
compartment after the electric shock or does not jump is termed as escapism. To
qualify, the animal has to avoid the test atleast 8 times. Animals in this specific test
learn the association between conditioned and unconditioned stimulus by shuttling
between the two compartments.

b. Passive avoidance test

The short-term memory analysis of animals were performed in a closed box with both
dark zone fitted with an electric grid and light chambers (Klenerova et al., 2003).
Both the chambers have similar dimensions (26 x 26 cm) separated through a sliding
door (8 x 8 cm). The animal was placed in the illuminated chamber for 50 seconds,
which it tends to avoid, and entered the dark compartment. The time taken to enter the
dark chamber from the illuminated open compartment is termed as acquisition trial
time. The sliding door was closed after the animal entered the dark compartment and
an electric stimulus of 60 V for 5 seconds was given to the animal. The procedure was
repeated after 24 hours (Day 2) and the time taken to enter the dark zone was noted
and termed as retention trial time for which the animal was placed in the open lighted

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 Materials and Methods

chamber for 300 seconds. The test basically is a fear-aggravated test that makes the
animals behave contrary to their innate tendencies for preference of dark areas and
avoidance of bright ones.

c. Morris water maze

Morris water maze navigation task is a spatial learning task for rodents assessed by
repeated trials and reference memory (Morris, 1984). The apparatus consisted of a
black circular water tank (diameter 200 cm, height 60 cm) with water filled to a height
of 30 cm above the base with water maintained at a temperature 23±1C. The tank
was divided into four quadrants with a platform submerged below the water surface in
4th quadrant at a fixed position throughout the experimentation. A seven day-trial
session was conducted on each rat for spatial acquisition with a maximum allowed
time of 120 seconds to reach the escape platform. If the animal failed to do so it was
manually guided to the platform and allowed to stay there for 30 seconds. On the final
day, the time taken by the rats to reach the hidden platform was recorded based on the
same protocol as described above.

d. Elevated plus maze

The elevated plus maze test was performed to evaluate cognition and anxiety-like
behavior in animals (Walf & Frye, 2007). The test exploits the innate rodent tendency
to prefer dark enclosed spaces to illuminated open areas. The apparatus consisted of
two open arms (50 x 10 cms) and two closed ones that are enclosed by side and end
walls (50 x 40 x 10 cms) (Lister, 1987). The arms were extended from a common
central platform (10 x 10 cm) and elevated to a height of 50 cms from the floor. The
trial sessions were conducted with animals being placed at the central platform with
head facing the open arm and were allowed to explore the maze for a maximum of
180 seconds. The time spent by the animals in the open and closed arms and their
number of entries in each arm is the key determinants of the anxiety levels of the
animals. An entry was confirmed when all the four paws of the animals were placed
on the arm (Pellow, 1985).

The spatial long-term cognition ability of rodents was also assessed by elevated plus
maze task (Itoh et al., 1991). Transfer latency indicates the time taken by animals to
move from open arm to enclosed arm and is a vital index to determine memory

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 Materials and Methods

processes. The animals were placed individually at the end of either of the open arms
and their transfer latency on Day 1 was noted down. The rodents were allowed to
explore and become acquainted with the maze for an additional 30 seconds on first
day. Further, the test was continued after every 24 hours for 4 days for which the
transfer latency time was recorded. A long latency period on the maze signified poor
cognition and retention tendency of the rodents.

7. BIOCHEMICAL INDICES

I. PROTEIN ESTIMATION:

Protein estimation in brain samples was estimated by the method of Lowry et al.,
(1951). The method is based on the reaction between the aromatic amino acids
(tryptophan and tyrosine) with folin phenol reagent. The alkaline copper tartarate
reacts with the proteins present in the sample to form cupric-amino acid complex.
Further, the formed complex and the aromatic amino acids reduce the
phosphotungstinic acid and phosphomolybdic acid that imparts blue color to the
solution, which was measured spectrophotometrically at 680 nm.

Reagents:

1. Lowry reagent: 1 part CuSO4: 1 part Na K tartarate: 100 Na2CO3

2. 2% (w/v) sodium carbonate in 0.1N NaOH
3. 1% (w/v) copper sulphate
4. 2% (w/v) Na K Tartarate
5. 1mg/ml Bovine serum albumin (BSA)
6. 1N Folin reagent diluted in 1:1 distilled water (freshly prepared)

Procedure:

Briefly, 20 µL of sample was diluted with distilled water to make a total volume of
700 µL. Blank and BSA standards were run in parallel tubes and their final volume
too was made to 700 µL. Lowry reagent (3 mL) was added and the mixture was
thoroughly mixed and allowed to stand for 10 minutes at room temperature. To this,
1N Folin Ciocalteau’s reagent was added followed by immediate vigorous shaking.
The contents were incubated at room temperature in dark for about 30 minutes. The
absorbance of blue coloured complex was measured at 680 nm. A standard curve was
plotted with BSA standards and protein content of samples were estimated from the
graph and expressed as mg protein/ mL.

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 Materials and Methods

II. NEUROCHEMICALPARAMETERS AND NEUROTRANSMITTERS

i. Acetylcholinesterase

Acetycholinesterase (AChE) was assayed in tissue homogenates by the method of

Ellman et al. (1961). The estimation was proceeded using acetylthiocholine iodide (an
ester of thiocholine and acetic acid) as a substrate for the reaction. AChE in the tissue
samples hydrolyzes acetylthiocholine into thiocholine and acetate. Thiocholine forms
a mercaptan, which reacts with an oxidizing agent DTNB (5,5-dithio-bis2-
nitrobenzoate) to form 5-thio-2-nitrobenzoate (yellow product), which has a
maximum absorption at 412 nm. The absorption intensity of DTNB adduct is used is
used to measure the amount of thiocholine formed which is proportional to the AChE
activity.

Reagents:

1. Sodium phosphate buffer (0.1M, pH 8.0)

2. Elman reagent (10 mM DTNB) in sodium phosphate buffer
3. Acetylthiocholine iodide 14.9 mM in double distilled water

Procedure:

Briefly, an appropriate amount of sample (100 μL) added to a cuvette containing 2.5
mL phosphate buffer and 0.2 mL Ellman’s reagent. The reaction was initiated by
adding 0.2 mL of substrate and the increase in absorbance was read at 412 nm for 2
minutes. The enzymatic activity was expressed as nanomoles of substrate hydrolyzed/
min/mg protein using molar extinction coefficient of DTNB (13600 M-1 cm-1).

ii. Monoamineoxidase

Estimation of the monoamine oxidase activity was done by the method of McEwen
and Cohen (1963). The oxidative deamination of benzylamine results in production of
benzaldehyde that allows convenient spectrophotometric assay of the enzyme.

Reagents:

1. Phosphate buffer solution: 0.2 M, pH 7.2

2. Benzylamine : 10 mM in PBS
3. 60% perchloric acid
4. Cyclohexane

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 Materials and Methods

Procedure:

The assay tube consisted of 100 µL brain sample to which 0.3ml benzylamine was
added; the total was made to 3 mL with PBS. The tubes were incubated with
metabolic shaker bath at 37•C for 3 hrs. The reaction was stopped with the addition of
0.3 mL 60% perchloric acid following which the extraction of the product
(benzaldehyde) was done by the addition of 3 mL cyclohexane. The contents in the
tube were emulsified using glass-stirring rod and the tubes were kept at room
temperature for 15 minutes. The tubes were again emulsified with rod and centrifuged
at 2000 x g for 30 minutes. The absorbance of the cyclohexane extract was then
measured at 242 nm. Control tubes were also run in parallel, except that the substrate
benzylamine was added at the end of the incubation. The results were expressed as
nanomoles of benzaldehyde/min/mg protein.

iii. Dopamine

The concentration of biogenic amine- dopamine was estimated by the method of Cox
and Perhach, 1973.

Reagents:

1. Standards: 100 µg/mL of dopamine hydrochloride in 0.1 N HCl, the solution

was serially diluted to give concentrations upto 2 µg/mL.
2. Acidified butanol: 50 mL of n-butanol washed successively with 5 mL 1N
NaOH, 5 mL 1N HCl, 4 x 5 mL of distilled water in a separating funnel. The
solution was further saturated with NaCl followed by sequential addition of 50
mL salt saturated butanol, 0.0425 mL conc HCl, 0.05 g potassium
metabisulphite, , 5 mg EDTA (disodium salt) and was thoroughly shaken at
each step.
3. EDTA reagent: 0.1 M EDTA in 1.0 M sodium acetate, pH 7 adjusted with
NaOH.
4. Alumina: 20 g alumina boiled in 100 mL 1.0N HCl for 30 minutes, washed
with 20 x 10 mL of distilled water and was left to dry in oven at 100C for
approximately 4 hours.
5. Iodine agent: 0.1 N iodine solution prepared in absolute ethanol
6. Alkaline sulphite: 5 g sodium sulphite in 1mL distilled water, add 4.5 mL of 5
N NaOH mixed with 0.5 mL sodium sulphite solution.

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 Materials and Methods

Procedure:

The cerebral cortex was homogenized in acidified butanol, the homogenates were
centrifuged for 5 minutes at 800 x g.

The extracted neurotransmitters in 2.5 mL of the supernatant fluid were then

transferred to a 13 mL glass stoppered centrifuge tube containing 2.5 mL distilled
water and 5 mL heptane. Post this layering, the tubes were shaken for 5 minutes and
centrifuged again at 800 x g for 5 minutes. 2.5 mL of the aqueous phase obtained was
further mixed in a test tube containing 200mg alumina, and1ml sodium acetate. The
tubes were again centrifuged for 5 minutes at 800 x g. The aqueous phase was
aspirated from alumina. For dopamine estimation the alumina extract was treated with
0.1 N 2 mL acetic acid and were centrifuged at 800 x g for 5 minutes. To the 1 mL
aqueous phase thus obtained 0.1 mL EDTA was added followed by addition of 0.1
mL iodine reagent, mixed thoroughly for 2 minutes and finally 0.2 mL alkaline
sulphite was added. Further, 0.5 N acetic acid was added to this reaction mixture,
followed by heating at 100•C for 5 minutes. The tubes were cooled, reheated for
another 5 minutes at 100C and the fluorescence was read at 320 nm and 370 nm
respectively.

Internal standards were prepared by adding known amounts of each standard to the
homogenate pool and running in parallel with the tissue samples. The pool
homogenates without the standards served as blank for the standards.

The results were expressed as nanogram per milligram of wet tissue weight.

iv. Serotonin

Estimation of serotonin, a biogenic amine was performed by the method of Church

(2005) by HPLC technique with an electrochemical detector. On the day of
experimentation, the freshly dissected brain samples were homogenized in 0.1M
perchloric acid (60 µL -10 M perchloric acid/100 mL). The homogenates were
centrifuged at 12000 x g for 5 minutes, the supernatant thus obtained was further
filtered through 0.25 micron nylon filters before injecting in HPLC injection pump.
Our experimentation was performed on Waters system consisting of an injector valve
(20 µL sample), isocratic pump capable of tolerating high pressures, C18 reverse

47
 Materials and Methods

phase column and EC detector. The mobile phase consisted of 10mM sodium citrate,
2 mM heptane sulphonic acid, 25 µM EDTA, 25 mM sodium hypophosphate buffer at
a flow rate of 0.80 mL/min and 0.8 V under isocratic elution were used for the
estimation. Further, pH was set at 4.5 with orthophosphoric acid, the buffer and
acetonitrile were taken in the ratio of 87:13 and the temperature of the column was
maintained at 30•C. Empower software recorded and analyzed the obtained
experimental data.

III. ANTIOXIDANT DEFENSE INDICES

Animals were sacrificed by decapitation after anesthetization with diethyl ether. The
brains were immediately removed and rinsed with ice-cold isotonic saline. Tissue
homogenates (20% w/v) were prepared in ice cold 10 mM PBS (pH 7.4).
Homogenates were centrifuged at 1000 g for 10 minutes at 4ºC. Pellets were
discarded and the supernatants were used to determine the reduced glutathione and
lipid peroxidation levels. The supernatants were further re-centrifuged at 10,000 g for
30 minutes at 4ºC to obtain Post Mitochondrial Fraction (PMF) used for the rest of the
biochemical estimations.

i. Superoxide dismutase

The assay was performed according to the method of Kono, 1978. The assay of SOD
is based on the inhibition of the formation of NADH-phenazine methosulphate-
nitroblue tetrazolium formazon. The reduction of nitroblue tetrazolium (NBT) to blue
formazon mediated by hydroxylamine hydrochloride was determined under aerobic
conditions. Addition of Superoxide dismutase inhibits the reduction of NBT by
superoxide anions generated by photo-oxidation of hydroxylamine hydrochloride and
the extent of inhibition was taken as measure of enzyme activity.

Reagents:

1. Solution A: 50 mM Na2CO3 in 0. 1mM EDTA (pH 10.8)

2. Solution B: 90 µM Nitro Blue Tetrazolium (NBT) in solution A
3. Solution C: 60 µL Triton X
4. Solution D: 20 mM hydroxylamine HCl

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 Materials and Methods

Procedure:

The reaction mixture contained 1.3 mL of 50 mM sodium carbonate solution in 0.1

mM EDTA, 0.5 mL of 96 μM NBT and 0.1 mL of 0.6% Triton X-100. The reaction
was initiated by the addition of 0.1 mL of 20 mM Hydroxylamine hydrochloride (pH
5.0) and appropriate amount of tissue homogenate and the rate of NBT reduction was
measured by a spectrophotometer. The activity of enzyme was expressed as
international unit/min/mg protein, where one unit of enzyme is the amount of enzyme
inhibiting the rate of reaction by 50%.

ii. Catalase
Catalase was spectrophotometrically determined by using the method described by
Luck (1971). The decomposition of hydrogen peroxide (H2O2) by catalase is
monitored spectrophotometrically by following the decrease in absorbance at 240 nm.
H2O2 2H2O + O2

Reagents:

1. 0.05M PBS, pH 7.0

2. H2O2 12mM

Procedure:

Briefly, 3 mL reaction mixture in a sample cuvette contained 50 mM potassium

phosphate buffer (pH 7.0), H2O2 at a concentration where it shows an optical
absorbance of 0.5, and the brain homogenate. Every sample was analyzed
simultaneously with appropriate blanks without H2O2. The decrease in absorbance on
decomposition of H2O2 was measured at 240 nm on a double beam
spectrophotometer.The amount of H2O2 decomposed was calculated on the basis of
molar extinction coefficient of H2O2 (0.0394 M-1 cm-1) and the results were expressed
as μmoles of H2O2 decomposed /min/mg protein).

iii. Lipid Peroxidation

The quantitative measurement of lipid peroxidation was done according to the method
of Wills (1966). The reactive oxygen species formed renders the lipids in cell
membranes vulnerable to peroxidation. Peroxidation of lipids induces the formation

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 Materials and Methods

of malondialdehyde (MDA), which on reaction with thiobarbituric acid (TBA) formed

a pink colour complex that showed maximum absorption at 532 nm.

Reagents:

1. 10% (w/v) TCA

2. 0.67% (w/v) TBA
3. 0.1M Tris-HCl buffer (pH 7.4)

Procedure:

Brain homogenates were mixed with ice cold 10% TCA and were centrifuged at 800 g
for 10 minutes. To the supernatant, 0.67% TBA was added and the pink colour
developed by boiling at 100ºC was read at 532 nm using UV Visible
spectrophotometer. Since Malondialdehyde is a degradation product of peroxidized
lipids, the development of pink colour with the absorption characteristics (maximum
at 532 nm) as TBA-MDA chromophore has been taken as an index of lipid
peroxidation. The results were expressed as nanomoles of malondialdehyde per
milligram of protein. The amount of MDA formed was calculated on the basis of
Molar Extinction coefficient of MDA-TBA chromophore (1.56X 105 M-1 cm-1).

iv. Reactive oxygen species

Reactive oxygen species (ROS) was estimated using the modified method of Driver et
al., 2000. The esterases get activated in the presence of ROS in the samples that in
turn cleave the acetate group of non-fluorescent dye, dichlorofluoresceindiacetate
(DCFH-DA) to form a fluorescent probe dichlorofluorescein (DCF), the intensity of
which can be measured at 530 nm.

Reagents:

1. Locke’s buffer (pH 7.4): 154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3, 2.0
mM CaCl2, 10mM D-glucose, 5 mM HEPES
2. DCFH-DA: 1mM in 5 mM DMSO (10 μM working solution was prepared by
diluting in PBS).

Procedure:

Brain homogenates were made in ice-cold Locke’s buffer (5 mg tissue/ml). Reaction

was initiated by addition of appropriate amount of homogenate to 10 μM DCFH-DA

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 Materials and Methods

and the reaction mixture was incubated for 30 minutes in dark at room temperature to
allow incorporation of the probe into membrane bound vesicles. The amount of DCF
fluorescence produced was measured at 488 nm (excitation wavelength) and 521 nm
(emission wavelength) using fluorescence spectrophotometer. The fluorescence
intensity unit of DCF/mg protein denotes the amount of ROS in different treatment
groups.

v. Glutathione system

a. Reduced Glutathione

Reduced Glutathione is a non-protein sulphydryl compound estimated by the method

of Ellman (1959) in which DTNB [5,5’-dithiobis-(2-nitrobenzoic acid)] , a disulphide
compound is readily reduced by sulphydryl compounds (SH) to release
nitromercaptobenzoic acid anion that yields an intense yellow colour to be read at 412
nm.

Reagents:

1. TCA 25%
2. PBS (0.2 M, pH 8.0)
3. DTNB 0.6 mM

Procedure:

Briefly, 0.1mL of TCA (25%) was added to tissue homogenate (500µl) and
centrifuged at 2000 g for 15 minutes. The supernatant obtained was further diluted
with 900 μL 0.2M PBS (pH 8.0) and mixed with freshly prepared 1mL -10 mM 5,50-
dithiobis (2-nitrobenzoic acid) made in 0.2 M phosphate buffer (pH 8.0). Optical
density of the yellow colored complex formed by the reaction of GSH and DTNB
(Ellman’s reaction) was measured on a double beam spectrophotomter at 412 nm
against a reference that contained 25% TCA instead of the sample.

Molar absorption coefficient of GSH 13600 M-1 cm -1

and the GSH content was
expressed as μmoles/mg protein.

b. Total and Oxidized glutathione content

The total glutathione content as measured by the method of Zahler and Cleland
(1968). The method is based on the principle of reduction with dithioerythritol and
determination of the resulting monothiols with DTNB in presence of arsenite.

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 Materials and Methods

Reagents:

1. Dithioerthritol : 0.003 M
2. TrisHCl buffer: 1.0 M (pH 8.1)
3. DTNB: 0.003 M in sodium acetate
4. Sodium acetate: 0.05 M (pH 5.0) in distilled water
5. Sodium arsenite: 0.005 M

Procedure:

The disulfide in the sample was mixed with dithioerythritol and the reduction is
allowed to proceed for 20 minutes. After reduction, tris buffer and sodium arsenite
were mixed followed by the addition of DTNB and the absorbance is recorded for 3
minutes. The TG contents were expressed in terms of µmoles of GSH/mg protein.
Oxidized glutathione was quantitated by subtracting the value of glutathione reduced
from total glutathione.

c. Glutathione reductase

The Glutathione Reductase (GR) is a flavoprotein that catalyzes the NADPH-

dependent reduction of Glutathione disulphide (GSSG) to reduced Glutathione
(GSH). The utilization of NADPH is directly related to the activity of GR. The assay
was performed by the method of Carlberg and Mannervik (1985) wherein GR
catalyzes the following reaction. The decrease in absorbance following NADPH
oxidation was spectrophotometrically read at 340 nm.

GSSG+ NADPH+ H+ 2GSH + NADP+

Reagents:

1. PBS: 0.2 M (pH 7.0)

2. EDTA: 60 mM in PBS
3. BSA: 2% in PBS
4. NADPH: 2 mM in PBS (freshly prepared light sensitive)
5. GSSG: 60 mM in PBS

The reaction was initiated by the addition of sample in the cuvette containing buffer,
distilled water, EDTA, BSA, NADPH and GSSG and the decrease in absorbance was

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 Materials and Methods

followed for 2 minutes at 340 nm. A unit of GR activity is defined as the amount of
enzyme that catalyzes the oxidation of 1 µM NADPH/min/mg protein.

6.22X10-3 mM-1 cm -1 is taken as the molar absorption coefficient of NADPH.

d. Glutathione-S-transferase

GST activity was determined by using the method of Habig et al (1974). GST
catalyzes the formation of glutathione conjugates of CDNB (substrate) that absorbs
maximum at 340 nm. The principle of estimation is based on the increase in
absorption of light due to GSH-CDNB conjugate in the region of 330-350 nm that
reflects the GST activity.

Reagents:

1. PBS (0.2 M, pH 6.5)

2. CDNB (2 mg/10 mL in absolute alcohol)
3. GSH 1mM (3 mg/10 mL buffer, light sensitive)

Procedure:

The assay involved the use of CDNB (1-chloro-2,4-dinitrobenzene,1,2-dichloro-4-

nitrobenzene) as substrate for GST. The tissue homogenate was added to the reaction
mixture in the cuvette consisting of PBS, CDNB and GSH and the absorbance was
recorded at 340 nm for 3 minutes. The activity was expressed as μmol CDNB
conjugate/min/mg protein by using extinction coefficient of 9.6 mM-1 cm -1.

8. RADIOISOTOPIC STUDIES

i. Radiotracer Uptake studies

99m
The distribution of the TcO4-and 99m
TcDTPA in different regions of brain was
evaluated by the administration of 2.96-4.44 MBq of the radiopharmaceutical
intravenously in heart of anaesthesized female rats. The bio-distribution was
ascertained by sacrificing the animals and carefully dissecting out the different
regions of brain at desired time intervals.

99m
TcECD, a neutral lipophilic complex with high cerebral retention is used as a tracer
for cerebral perfusion studies. 37 MBq of the radiopharmaceutical was injected

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 Materials and Methods

intravenously following which the animals were sacrificed and the different regions of
brain was carefully removed, isolated and weighed to measure the radioactive
localization of the pharmaceutical. The counts were taken using an automated well
scintillation counter and expressed as % injected dose per gram of tissue weight.

ii. Radiorespirometric determination of 14C-D-glucose turnover

14
One of the most sensitive analytical procedures, C-D- glucose radiorespirometric
technique employs 14C–labeled substrates and measures the activity of evolved 14CO2
signifying differential 14C glucose uptake and metabolism in normal and pathological
conditions. 14C, a low beta energy emitter is measured in liquid scintillation counter.
14
The double-vial geometry for radiorespirometric determination of the evolved CO2
is an efficient technique for measuring the respiration of 14C-labeled substrates.

Reagents:

1. Kreb’s Ringer Buffer (100 mL): 0.6 g NaCl,

0.0075 g KCl,
0.01 g NaHCO3,
0.01 g CaCl2
100 mL-distilled water

2. Bray’s Scintillation Fluid: 30 g Napthalene,

200 mg 1,4-bis(5-phenyloxazol-2-yl)benzene
[POPOP]
2 g 2,5 Diphenyloxazole (PPO)
10 mL Ethylene glycol
10 mL Glacial acetic acid
50 mL Methanol
Final volume adjusted to 500 mL with 1,4 Dioxan

3. Methanolic NaoH 8:2

4. KOH: 20%

Procedure:

A small vial (outer diameter 1.5 cm, inner diameter 4.5 cm) was placed inside the
standard borosilicate glass scintillation vial forming two compartments. The inner vial

54
 Materials and Methods

14
contained C labeled substrate buffer, the brain tissue (50 mg cerebral cortex). The
outer compartment was lined on the inside with a filtered wick (Whatman filter paper
no.1, 2.5cm x 6.5cm) previously soaked with a mixture of scintillation cocktail
(Bray’s scintillation fluid), methanolic NaOH (8:2) and allowed to dry under vacuum.
In general, counting efficiency increased with greater homogeneity and stability in the
mixture used to treat wicks. Thus, mixtures containing methanolic NaOH showed
higher efficiency than those containing aqueous NaOH.

The dried strips were then rolled and placed in the inner side of the outer scintillation
vial. 0.37 MBq radiolabelled glucose was added to 25 mL Kreb’s ringer buffer. Brain
tissue was then added to the inner vial containing trace amounts of radiolabeled
glucose (0.0148 MBq Kreb’s ringer buffer). The set-up was allowed to incubate for
14
45 minutes at 37C. CO2 produced through respiration in the sample compartment
was trapped on the wick and was measured directly and continuously in a liquid
scintillation counter under 14C energy peak. The results were expressed as % of 14CO2
of trapped as Na214CO3/ min/g tissue.

iii. In vitro uptake of 14C glucose

14
C-D-glucose uptake was assessed by the tissue accumulation method of Crane and
Mandelstam (1960). Glucose metabolism and its disruption is a vital indicator of
diseased state that imparts altered uptake and turnover of the radiolabeled glucose.

Reagents:

1. Kreb’s ringer buffer: 0.9%NaCl

1.15% KCl
1.22% CaCl2
3.82% MgSO4.H2O
1.3% NaHCO3 (adjusted to 100 mL distilled water)

2. Bray’s Scintillation Fluid: 30 g Napthalene,

200 mg 1,4-bis(5-phenyloxazol-2-yl)benzene [POPOP]
2 g 2,5 Diphenyloxazole (PPO)
10 mL Ethylene glycol
10 mL Glacial acetic acid
50 mL Methanol
Final volume adjusted to 500 mL with 1,4 Dioxan

55
 Materials and Methods

The cerebral cortex was sliced into thin cortical sections of 0.5mm thickness. Briefly,
5 mM of unlabeled D-glucose and 8 µCi -radiolabeled glucose were added to 100 mL
stock Kreb’s ringer buffer. Brain slices were incubated for 10 minutes at 37°C in
stoppered flasks containing 5.0 ml Kreb’s ringer bicarbonate buffer (pH 7.4), with
unlabeled glucose and trace amounts of radiolabeled glucose (0.4 µCi/5 ml in flask).
The tissue slices were then taken out; filter dried and dissolved overnight in
scintillation vials containing 20% KOH. Further, Bray’s scintillation cocktail was then
added to the vials and the radioactivity was measured by using a liquid scintillation
counter. Prior to counting, the liquid scintillation counter was set for 14C energy, and
background counts of empty vials were recorded. The results were expressed in
µmoles of glucose/min/g tissue.

9. MEMBRANE FLUIDITY STUDIES

The plasma membranes were isolated form the cerebral cortex regions of brain
following the Lin & Way (1982) protocol. Briefly, the cerebral cortices from freshly
dissected rats were homogenized in 10 volumes of 0.32 mmol/L sucrose solution
containing1 mmol/L EDTA (pH 7.5) and 5 mmol/L HEPES. The homogenates were
centrifuged at 2000 x g for 10 minutes and the resultant supernatants were further
recentrifuged at 17000 x g for 30 minutes to obtain crude plasma membrane fractions.
All the procedures were carried out at 4C.

Membrane fluidity analysis was performed by using a non-fluorecent probe 1,6-

diphenyl-1,3,5-hexatriene (DPH); following the method of Swapna et al (2006). DPH
is a hydrophobic molecule that is non-fluorescent in aqueous solutions and fluoresces
after localization in membrane hydrophobic core and its mobility measurement forms
the basis to investigate the membrane fluidity parameter. The probe has been reported
get incorporated in membranes of cellular organelles and determine the flexibility
(rotational diffusion) of fatty acyl chains of the lipid bilayer. The rearrangement in
membrane lipids is known to influence the fluorescence anisotropy parameter by
changing the rotational and lateral diffusion of DPH that gets excited by plane
polarized light at 365 nm.

56
 Materials and Methods

Reagents:

1. DPH 1 mM in Tetrahydrofuran (light sensitive)

2. TrisHCl buffer 1M

Procedure:

A stock solution of 1 mM probe in THF was prepared and stored in dark bottles being
light sensitive. 10 µL DPH solution was added with rapid stirring into 1000 volumes
of Tris-HCl buffer (10 mL) at room temperature. The suspension was stirred till no
THF odour was detected and the suspension showed negligible fluorescence. Plasma
membrane samples (0.1 mL)) were incubated in 3 mL) of the above-prepared
suspension containing 3 µM of DPH for 2-4hours at 37°C. Thereafter, the
fluorescence anisotropy (r) fluorescence polarization (P) was estimated with exciting
wavelength of 365 nm and emission wavelength of 430 nm using a Perkin Elmer
Luminescence spectrophotometer LS55 (Beaconsfield). Fluidity, order parameter and
anisotropy parameters were then calculated by using the following expressions:

Fluidity = 1/Polarization
Anisotropy = [(r0/r)-1]-1 where r0 value for DPH =0.362
Order parameter S2 = [(4/3r- 0.1)]/ r0

10. CALCIUM HOMEOSTASIS ASSESSMENT PARAMETERS

I. Total Calcium levels:

Estimation of trace elemental concentrations of calcium in the brain tissues (cerebral

cortices) of the different treatment groups was carried out by the method of atomic
absorption spectroscopy (AAS). The spectrophotometer measures the concentration of
gas-phase atoms but since the analyte used in the biochemical estimations are usually
solids or liquids, the sample is vaporized in a furnace (either flame or graphite). The
atoms absorb light in either visible or UV range and get excited to higher energy
levels thereby determining the analyte concentration.

The elemental estimation was performed following the wet acid digestion of brain
tissues by the method of Zumkley et al(1979). 100mg tissue was dissolved in an acid
mixture of perchloric acid and HNO3 (1:3) followed by digestion in sand bath for 44
hours till formation of white ash/ residue. The residue was dissolved in 2 mL 10 mM

57
 Materials and Methods

HNO3 and was placed in the sample holder of the instrument. The nebulizer sucks the
samples and dissociates into atomic form in the flame cell. The flame is constituted of
a mixture of compressed air and acetylene. When light passes the sample, atoms
absorb this light giving the corresponding absorbance, which is directly proportional
to the number of metal atoms present in the sample.

II. Intracellular free Ca2+:

Preparation of purified synaptosomes:

Synaptosomes are osmotically sensitive pinched off nerve endings that contain
numerous clear synaptic vesicles. When isolated from mammalian brain, they retain a
piece of attached postsynaptic membrane facing the active zone. Synaptosomes are
commonly used to study synaptic transmission in invitro conditions as they contain
relevant molecular machinery necessary for uptake, storage and release of
neurotransmitters (catecholamines, acetylcholine peptides etc) in a calcium-dependent
manner. Synaptosomes were prepared on a discontinuous sucrose gradient according
to the procedure of Gray & Whittaker (1962). Briefly, the cerebral homogenate (10%)
was prepared in 0.32 M sucrose in 20 mM TrisHCl (pH 7.4) and was centrifuged at
1000 x g for 10 minutes. The supernatant obtained was further re-centrifuged at 12500
x g for 20 minutes to obtain crude mitochondrial pellets that was resuspended in 0.32
M sucrose in 20 mM TrisHCl. The suspension was layered over a step density
gradient of 0.8 M, 1.0 M, 1.2 M sucrose layers. The suspension was ultracentrifuged
at 80000 X g for 2 hours and the interface over 1.0 M sucrose was recovered that
constituted the synaptosomal fraction. The obtained fraction was resuspended in
physiological buffer that constitute synaptosomal preparations.

Intrasynaptosomal free Ca2+

Intracellular free Ca2+ was determined in synaptosomes with Fura 2-AM

(acetoxymethyl ester), a cell permeant dye used as calcium indicator according to the
method of Adamson et al (1989).

Intracellular esterases cleave intracellular Fura 2-AM into the active Fura 2. The
spectral characteristics of Fura 2-AM are the same as those of unbound Fura 2, but
only Fura 2 is sensitive to calcium ion. Addition of [Ca2+]i increases the fluorescence
of Fura 2 when excited at 330-350 nm and decreases the fluorescence from excitation

58
 Materials and Methods

at 380 nm. Monitoring the decreasing signal generated by 380 nm excitation will
indicate the extent of cleavage of the Fura 2-AM and subsequent binding of [Ca2+]i
ion.

Reagents:

1. 10 mM Fura 2-AM made in DMSO

2. 5 mM EGTA
3. 20% SDS

Procedure:

Isolated synaptosomes (in physiological buffer) were loaded with 1 mM Fura-2/AM

at 37°C for 1 hour (protected from light). The excess Fura 2-AM was removed by
centrifugation. The fluorescence (F) at 340 to 380 nm excitation and 510 nm emission
was measured and [Ca2+]i was calculated according to the formula:

[Ca2+]i=[(F-Fmin)/(Fmax-F)] x Kd

The Kd for Fura free acid is 225 nM. Maximal fluorescence (Fmax) was measured
after lysis of synaptosomes with SDS (2 mL) and minimal fluorescence (Fmin) was
measured in the presence of 5 mM EGTA (1 mL).

II. Preparation of synaptosomal plasma membranes

Synaptosomal plasma membranes were prepared from the region of cerebral cortices
of freshly dissected rats according to the method of Jones and Matus (1974). Briefly,
the crude mitochondrialfraction was suspended in hypotonic buffer (5 mM TrisHCl;
pH 8.0) at 0C for 30 minutes. followed by homogenization. The lysate was made
34% (w/w) with sucrose by the addition of an appropriatevolume of 48% (w/w)
sucrose. An upper phase of 28.5% (w/w) sucrose was layered over the sample phase
and a small volume of 10% (w/w) sucrose was overlaid the upper phase. The density
gradient was centrifuged at 60,000 X g for 110 minutes. The synaptic plasma
membranes were recovered from the interface between 34% (w/w) and 28.5% (w/w)
sucrose. The upper white flocculent layer contained myelin, the middle gray band
contained the synaptosomal plasma membrane and the lowest brown pellet contained
mitochondria. The membranes were suspended in 40 mM TrisHCl (pH 7.4).

59
 Materials and Methods

a) Na+ K+ ATPase

The enzyme activity was estimated by the method of Wallech & Kamat (1966). The
enzyme activity was determined by measuring the amount of inorganic phosphate (Pi)
liberated from ATP during the incubation of synaptosomal fraction. Ouabin was used
as Na+ K+ ATPase inhibitor for measurement of inorganic phosphate already present
in the cells. Inorganic phosphate forms blue coloured complex on reacting with 1,2,4
amino napthiolsulphonic acid (ANSA) measured spectrophotometrically at 595 nm.

Reagents:

1. TrisHCl buffer: 30 mM (pH 7.2)

2. Ouabin: 10 mM (in warm water)

3. 1.4 mM ATP solution: 0.072 g in 10 mL tris buffer

4. 10% TCA

5. Standards: 5 mg KH2PO4 in 100 mL DDW (0.007 g/ 50 mL)

Procedure:

For the estimation, three sets of test tubes were employed per sample; first one was
for test, second as control and the third one for ouabin insensitive enzymes. To the
test (tube1), 0.1 mL of sample was added followed by 0.4 mL of buffer and 0.1 mL of
substrate (ATP) in a sequential manner to which finally 0.4 mL DDW was added.

To the second set, 0.1 mL DDW was added instead of sample and in addition to the
contents of tube one, 1 mL TCA was added immediately. To the third set, 0.4 mL of
buffer was added followed by 0.1 mL ouabin and 0.1 mL of ATP followed by
addition of 0.3 mL DDW. Tubes were then incubated for 15 minutes at 37°C in water
bath. 10% TCA was then added to set one and three to arrest the reaction. The
contents were centrifuged at 3000 rpm for 30 minutes and the inorganic phosphorous
released was estimated spectrophotometrically by method of Fiske & Subarrow
(1925). Na+ K+-ATPase activity was calculated from the difference between amounts
of inorganic phosphate found after incubation in the absence and presence of 10 mM
ouabin.

60
 Materials and Methods

b) Ca2+ ATPase

Ca2+ATPase was assayed by the method of Hjerten & Pan (1983) in the synaptic
plasma membrane. The inorganic phosphorous liberated was estimated by Fiske and
Subbarrow (1925) method.

Reagents:

1. 125 mM TrisHCl buffer (pH=8)

2. 50 mM CaCl2
3. 10 mM ATP

Procedure:

The incubation mixture contained 0.1 mL of each of the reagents and mixed well. To
this mixture, 0.1 mL water was also added. After equilibrating the tubes at 37•C the
reaction was initiated by the addition of 0.1mL homogenate. The reaction mixture was
then incubated for 15 minutes at 37°C. After incubation, the reaction was arrested by
addition of 1.0 mL 10%TCA. The control tubes received enzymes after the addition
of 10%TCA. The tubes were centrifuged at 3000 x g for 10 minutes and the inorganic
phosphorous content was estimated by the Fiske & Subbarrow (1925) method. The
activities of Ca2+ ATPase were expressed as µmoles of Pi liberated/min/mg protein.

III. cAMP levels

Adenosine 3’5’-cyclic monophosphate (cAMP) is a ubiquitous second messenger

critical in a number of signal transduction pathways. The R&D Systems cAMP
immunoassay (kit) is based on direct competitive binding technique for sensitive and
quantitative determination of cAMP levels. A monoclonal antibody specific for
cAMP binds to goat anti-mouse antibody coated on to the microplate. Following a
wash to remove excess monoclonal antibody, cAMP present in the sample competes
with horseradish peroxidase (HRP)-labeled cAMP to bind the sites on the monoclonal
antibody on the plate. After incubation and washing to remove excess conjugate and
unbound sample, the amount of cAMP-HRP bound to the plate was determined by
reading HRP activity at 450 nm. The intensity of the color is inversely proportional to
the concentration of cAMP in samples.

61
 Materials and Methods

11. WESTERN TRANSFER ANALYSIS FOR PROTEIN EXPRESSION

Protein expression of various molecular markers associated with disruption of calcium

homeostasis- Protein kinase C (PKC), Protein kinase A (PKA), Inositol triphosphate
(IP3), Phospholipase C (PLC) and biomarkers associated with neurodegeneration-
amyloid precursor protein (APP), tau, glial fibrillary acidic protein (GFAP), alpha-
synuclein and ubiquitin were investigated using Western blot technique. Beta actin
was used as a loading control throughout the experimentation period.

I. Sample Preparation:

20% tissue homogenates were prepared in fresh ice-cold lysis Buffer A [10 mM Tris,
100 mM NaCl, 1% Triton X 100, 5 mM EDTA, 1 mM PMSF (phenylmethylsulfonyl
fluoride) and 2 mM DTT (dithiothreitol; pH 8.0)]. The extracts were separated by
centrifugation at 5000×g for 5 minutes (4°C) and the supernatant (cytoplasmic
fraction) was collected. Further, the pellets were resuspended in lysis buffer B [50
mM NaCl, 10 mM HEPES (pH 7.6), 0.1 mM EDTA, 25% glycerol and 0.5 mM
PMSF (17.6 mg/1 mL isopropanol] at 4°C and pelleted by recentrifugation at 5000×g
for 15 minutes at 4°C.

Resulting nuclear debris is incubated in the same lysis buffer B for 30 minutes on ice
following which it is recentrifuged at 10,000×g for 10 minutes at 4o C and the
resultant supernatant was collected and used as the nuclear extract.The protein
concentration of the tissue samples was determined by the method of Bradford, 1976.

II. Sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE):

SDS-PAGE is a common analytical method to separate proteins on the basis of the

electrophoretic mobility of proteins, dependent primarily on their molecular weights.

The protein samples (80 μg) were subjected to separation on 10% SDS-PAGE by the
method given by Laemili et al(1970) and was carried out by mini dual gel apparatus
(GENEI, India).

1. Buffer A: 1.5 M TrisHCl (pH 8.8)

2. Buffer B: 0.5 M TrisHCl (pH 6.8)

62
 Materials and Methods

3. Stock acrylamide solution (for 100 mL): 30g acrylamide, 0.8 g bis-acrylamide.
Final solution was made to 100 mL with double distilled H2O. The solution was
filtered through Whatman’s filter paper and stored in dark bottle at 4°C.

4. 10% Ammonium persulphate solution (APS, freshly prepared)

5. 10% Sodium dodecyl sulphate (SDS)

6. Plug gel composition: 1 mL acrylamide stock, 40 μl APS and 4 μl TEMED.

7. Separating gel composition:

a. Acrylamide stock: 1.7 mL

b. Autoclaved water:1.9 mL
c. Buffer A:1.3 mL
d. APS:40 μL
e. SDS:50 μL
f. TEMED:4.4 μL

8. Stack gel composition:

a. Acrylamide stock: 670 μL

b. Autoclaved water: 2.7 mL
c. Buffer B: 50 μL
d. APS: 40 μL
e. SDS: 40 μL
f. TEMED: 4 μL

9. Sample buffer (5 mL):

a. Tris Buffer B (0.5 M)

b. 2 % SDS: 0.1 g
c. 10% glycerol: 0.5 mL
d. DTT: 0.1 g
e. Bromophenol Blue (0.1%): 5 mg

10. Running buffer:

a. Tris Base: 3 g
b. Glycine: 14.4 g
c. 10% SDS: 1 mL [Final volume made 1000 mL with distilled water]

63
 Materials and Methods

11. Coomassie brilliant blue stain:

a. Methanol: 250 mL

b. Coommasie blue: 250 mg

c. Glacial acetic acid: 50 mL [Final volume made 500 mL with distilled

water]

12. Destaining solution:

a. Methanol: 62.5 mL

b. Glacial acetic acid: 17.5 mL [Final volume made to 250 mL with distilled water]

Procedure:

a. Gel Casting

The glass plates are carefully sealed on the three sides with spacers between them of
1.2 mm each, prior to the casting of gel. Next, the plug gel (500 μl) was quickly
poured to seal the bottom formed between the plates. The plug gel was allowed to set
for 5 minutes that was followed by pouring the separating gel and was immediately
overlaid with butanol and water (1:1) to prevent drying of the resolving gel. The set-
up was left undisturbed for the gel to polymerize. Further, the top of the separating
gel was rinsed with distilled water and dried using Whatman sheet. Finally, the
stacking gel was casted with the comb placed in between the sealed glass plates for
the well formation. The set-up was again allowed to stand for another 45 minutes at
room temperature to polymerize.

b. Sample preparation

80 µg of protein extract were mixed in equal ratio with sample buffer and heated in a
boiling water bath for 5 minutes. Samples were loaded in the respective wells and
electrophoresis was carried out at a constant current of 15 mA through stacking and
30 mA through separating (resolving) gel. Commassie blue stain was used to assess
protein expression in the gels before proceeding further. After electrophoresis, the gel
was then processed further for western transfer.

III. Western transfer

Procedure:

The immunoblotting is a sensitive procedure for the detection of proteins post-

electrophoresis. The separated proteins on the gel were transferred to a polyvinylidene

64
 Materials and Methods

fluoride (PVDF) membrane that was activated by immersing the membrane in

methanol for about 2 minutes followed by immersing it in chilled transfer buffer for
the process of equilibration. The gel, whatman filter paper (approximately of the gel
size) and padding foam were also soaked in transfer buffer for equilibration prior to
electro transfer.

Reagents:

1. Transfer buffer:

a. Tris buffer: 5.8 g

b. Glycine: 8.9 g
c. SDS: 3 g
d. Methanol: 200 mL
e. Distilled water: 800 mL

2. Phosphate buffer saline (PBS 0.01 M, pH 7.4)

a. Na2HPO4: 1.452 g
b. NaH2PO4: 0.132 g
c. NaCl: 9 g
d. Distilled water: 1000 mL

The equilibrated filter sheets, activated membrane, gel and foam pads were all put
together to form a sandwich, where in the electrophoresed gel and PVDF membrane
were covered carefully with layers of filter sheets and foam pads on either side. The
gel was kept at the cathode side and membrane at the anode side of the unit. The gel,
membrane and cushioning pads were all tightened together with the help of provided
screws and the process was carefully done to avoid any air bubbles in it. The entire
assembly was placed in chilled transfer buffer in transfer chamber. Electro transfer of
the proteins from the gel to the membrane was carried out at 200 mA for 2 hours at -
4°C.

After transfer, the membrane was washed with phosphate buffered saline (PBS) for 10
minutes and placed in blocking buffer (1 % BSA and 0.01 % tween 20 in PBS) for 1
hour with constant shaking. The membrane was then incubated with various protein
specific primary antibodies for 12 hours at 4°C. The membrane was sequentially
washed with PBS, 0.05 % tween 20 in PBS and PBS for 5 minutes each.

65
 Materials and Methods

Then the membrane was incubated with peroxidase labeled secondary antibody
{Sigma-Aldrich Inc. (USA); 1:2000} for 3 hours at 4o C. Thereafter the membrane
was washed again as described above. The blot was developed in dark, by adding
diaminobezidine (DAB 10 mg/15 mL PBS containing 15 µL of H2O2).Reaction was
terminated by rinsing the membrane with double distilled water and the membrane
was air-dried.

Densitometric analysis of bands

Bands obtained were densitometrically analysed using Image J software and the
density was expressed as gray values in the densitrometric units.

12. HISTOARCHITECTURAL STUDIES

Cerebral cortex sections of the different treatment groups were assessed for
histoarchitectural changes by keeping the tissues immersed in 10% formalin for about
24-48 hours and further processed for the different stains.

i. Haematoxylin and Eosin staining

The histopathological alterations were studied in the different treatment groups by

subjecting the tissue of interest to Haematoxylin and Eosin (H&E) staining. The
cortical sections from freshly dissected rats were washed with 0.9% NaCl (ice cold)
and immediately fixed with in 10% formalin for about 24 hours. Further, the tissues
were processed according to standard lab techniques (Pearse, 1968). Post fixation, the
tissues were dehydrated in ascending grades of alcohol followed by embedding in a
mixture of benzene and paraffin wax (1:1). The tissues were finally immersed in pure
molten wax for about 3 hours and were finally embedded (block making). Finally,
paraffin sections were cut with 5-7 microns thickness with the manually driven
microtome, which were mounted on glass slides.

The sections so obtained were dewaxed in xylene and rehydrated with descending
grades of alcohol from absolute alcohol to 30%, finally brought to distilled water and
stained in haematoxylin for about 20 seconds. The slides were further dipped in
ammonia water till the appearance of blue color and observed under light microscope
to ensure proper staining. In case of over staining, the slides were rinsed with acid
water. After ensuring optimum stain, the slides were treated further with ascending

66
 Materials and Methods

series of alcohol grades (30%, 50%, 70%) and stained with 1% alcoholic eosin for 30-
60 seconds, differentiated in 90% and absolute alcohol, cleared in xylene and finally
mounted in DPX. The tissue sections were then observed under the light microscope
(LEICA DM 3000).

ii. Vonkossa stain:

Vonkossa stained sections of the cerebral cortices indicated the calcium deposition in
the course of investigation in all the four groups. The stain principle is a precipitation
reaction in which the silver ions react with phosphate in the acidic medium.

Tissue sections were deparaffinized and brought to distilled water and rinsed
thoroughly. Further, the sections were immersed in 0.5-1% AgNO3 for 10-15 minutes
under sunlight. Next, the sections were rinsed with distilled water again and immersed
in 5% sodium thiosulphate for 30 seconds to remove unreacted silver. The slides were
again rinsed in distilled water and counterstained with 1% neutral red (30 seconds).
The sections were dehydrated through graded alcohol, cleared in xylene and
eventually mounted in DPX.

iii. Congo Red

Paraffin embedded sections from the cerebral cortex was used to detect Aβ oligomeric
aggregation stained by Congo red staining technique (Highman’s Congo red method,
1946).

Reagents:

1. Congo Red: 0.5 g in 50% ethanol (100 mL)

2. Potassium hydroxide: 0.2 g in 80% ethanol (100 mL)

Procedure:

Deparaffinize the tissue sections and then immersed in congo-red (1-5 minutes).
Further the slides were differentiated in KOH (1-3 minutes) and later rinsed in tap
water followed by stain in Haematoxylin for 2-3 minutes.The tissue sections were
again rinsed multiple times in distilled water, dehydrated in alcohol gradations and
finally mounted and visualized under light microscope.

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 Materials and Methods

iv. Thioflavin S

Thioflavin binds to beta-sheet rich structures that are a defining feature of amyloid
fibrils, however they are not particularly specific to amyloid. The sections were
deparaffinized, and subsequently hydrated in a series of downgraded alcohols and
finally brought to distilled water for a few seconds. The sections were now immersed
in 1% Thioflavin S for 10 minutes at room temperature. The stained slides should be
stored in cold, dark place since the dye is fluorescent and light sensitive. The slides
were further differentiated in 70% alcohol for 5 minutes, rinsed with distilled water
twice, mounted and analyzed under fluorescence microscope.

II. Transmission Electron microscopic studies

Electron microscopic studies were conducted to assess the ultrastructural alterations

introduced by the different treatments in the brain tissues of the four groups. Freshly
incised cortex tissues were fixed in 3% (v/v) glutaraldehyde made in 0.2 M sodium
cacodylate buffer (pH 7.2) for 10-12 hours at 4•C. The fixed sections were cut into
1mm cubes approximately. Further, the specimens were washed thrice with 0.1 M
cacodylate buffer and then these cubes were post fixed in 1% (w/v) osmium
tetraoxide (2 hours at 4•C) prepared in the same buffer. Next, the specimens were
washed thoroughly in the buffer to remove any extraneous traces of osmium
tetraoxide and dehydrated in in ascending gradations of acetone permitting 20 minutes
in each change. Specimens were then infiltrated with EPON and propylene oxide
mixture (1:10 for 2 hours at room temperature followed by embedding in resin. The
specimen blocks were made by polymerization of pure embedded resin at 60•C for 48-
72 hours.

This was followed by ultrasectioning of the specimen blocks using ultramicrotome.

Initially, semi thin section (1µm) was cut using sharp glass knives to locate the area of
interest in different treatment groups. Later, these semi-thin sections were stained
with 0.5% toluidine blue prepared in 1% borax solution. The ultrathin sections of
interference colors from golden to silver were cut and loaded on fine copper grids of
100-300 mesh size. The sections were double stained with uranyl acetate and lead
citrate and examined under Transmission Electron Microscope at PGIMER,
Chandigarh.

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 Materials and Methods

13. Statistical Analysis

The results are expressed as Mean± SD of six animals per group. The data was
analyzed using one-way analysis of variance (ANOVA), followed by Newman-Keuls
test for multiple inter-group comparisons (pair wise) comparisons by employing the
Statistical Package for social sciences (SPSS) 17 software (IBM Corporation, New
York, USA).

ANOVA is a parametric test based on the assumption that all samples are drawn from
normally distributed populations with the same variances. The correlation coefficients
were calculated by linear regression analysis and the values with p0.05 were
considered as statistically significant.

69