Chapter 3

MATERIALS AND METHODS

s. No. Topics Page No.

3,1. Animals 81

3.2. induction o f Diabetes 81

3.3. Drugs 82

3.4. Chemicals and Reagents 84

3.5. Diagnostic Kits 84

3.6. Equipments and Instruments 85

3.7. Other materials 8 6

3.8 Methodology / Experimental procedure 87

3.9. Biochemical Estimations 87

3.9.1. Glucose Estimation 87

3.9.2. Insulin Estimation 89

3.10. Behavioral Estimations 93

3.10.1. Depression 93

3.10.2. Memory 94

3.10.3 Anxiety 94

3.11 in vitro study 95

3.12. STATISTICS 95

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3.1 AMiilALS

Wistar Albino rats of either sex weighing 150-250 g were obtained from the

Central Animal House, Jamia Harndard, New Delhi. They were housed in

polypropylene cages under controlled temperature (27°± 2°C), humidity (55 ±

5%) and in a natural light/dark cycle. They were fed with standard laboratory

pellets (Amrut Laboratory, rat and mice feed, Navmaharashrat Chakan Oil Mills

Ltd, Pune). Food and water were provided ad libitum. The experimental work was

conducted as per the guidelines of “Committee for the purpose of control and

Supervision of Experiments on Animals" (CPCSEA), Ministry of Social Justice

and Empowerment, Government of India, New Delhi.

3.2 INDUCTION OF DIABETES

Insulin dependent DM (!DDM| model:

Wistar albino rats (6 - 8 weeks old) weighing 150-250 g were fasted for 24 hr

before inducing diabetes with STZ. After light ether anesthesia, animals were

injected with STZ (55 mg/kg) intraperitoneally, STZ was dissolved in saline

solution just before each injection.

The volume injected was fixed at 0.5 ml /100 g body weight. After the injection,

all animals were housed and allowed access to food and water ad libitum. Serum

glucose and insulin levels were measured after 7 day of STZ injection and

animals exhibiting overt glucosuria were considered as STZ-diabetic resembling

type-1 diabetes in humans.

Non-Insulin dependent DM (NIDDM) model:

Diabetes was induced in newborn pups by a single injection of STZ (100j,ig/g

body weight), intraperitoneally. in 0.9 % saline and 0.05 M citrate buffer (pH-4.5)

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in equal volumes, on the first day of life (Portha et al., 1974). 12 weeks after the

injection of STZ, animals exhibiting fasting glucose levels >14mg/dl were

considered neonatal -STZ (n-STZ) - diabetic resembling type II diabetes in

humans.

NOTE - In our preliminary experiments, we observed high mortality (approx-

70%), hence this model was not selected again for further studies.

3.3 PROGS

1. Streptozotocin

Streptozotocin (STZ, mixed anomers) was procured from Sigma Chemicals,

USA. it was dissolved in 0.05 M citrate buffer (pH-4.5) and 0.9% normal saline in

equal volumes.

0.1 M Citrate buffer (pH 4.5)

Stock solutions

(A) 0.1 M solution of citric acid: 21.0 g of citric acid was dissolved in sufficient

quantity of distilled water and volume made up to 1 0 0 0 ml.

(B) 0.1 M solution of sodium citrate; 29.0 g of sodium citrate was dissolved in

sufficient quantity of water and volume made up to 1 0 0 0 ml.

For pH 4,5 28 ml of A + 22 ml of B -> diluted to 100 ml

2. Chromium Picolinate

Chromium picolinate (CrP) was a generous gift sample from Dr. R.K.Goyal,

which was originally procured from M/S Softcaps Pvt Ltd, Chennai. INDIA. It was

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82

dissolved in drinking water and the diabetic and normal rats received the same

for 4 weeks.

Two doses of CrP were used (Dose- 8 )ag/ml in drinking water, 4 ,ug/ml in drinking

water).

3. Glirriepiride

Glimepiride hydrochloride was procured from Ranbaxy India Ltd and was made

into suspension with double distilled water by adding 1% sodium Carboxy methyl

cellulose (CMC) as suspending agent.

Dose- 10mg/kg, p.o.

10 mg of Gli was suspended in 5 ml of distilled water using sodium CMC and

was administered 0.5 ml/100g body weight of rats.

4. Sertraline

Sertraline hydrochloride salt was procured from Ranbaxy India Ltd. and was

made into suspension with double distilled water by adding 1% sodium CMC as

suspending agent.

Dose- 30 mg/kg, p.o.

30 mg of Ser was suspended in 5 ml of distilled water using sodium CMC and

was administered 0.5 ml/IOOg body weight of rats for 3 weeks.

5. Reboxetine

Reboxetine methanesulphonate was procured from Zydus Neurosciences

(Cadila Healthcare Ltd), Ahmedabad, India and was made into suspension with

double distilled water by adding 1% sodium CMC as.suspending agent.

Dose - 2.5 mg/kg, i.p.

2,5 rtig of reboxetine was suspended in 5 ml of distilled water using sodium CMC

and was administered 0.5 ml/100g body weight of rats for 3 weeks.

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83

Chapter 3 M aterials & M ethods

S. Pinaciclil

Pinacidil hydrochloride was procured from Leo Pharmaceuticals, Denmark. The

solution was made by dissolving the required quantity in double distilled water.

Dose - 1 mg/kg, p.o.

1 mg of pinacidil was dissolved in 5 ml of distilled water and was administered

0.5 ml/100g body weight of rats for 3 weeks.

3.4 CHEMICALS AND REAGENTS

Anesthetic diethyl ether

Calcium chloride

Distilled water

Glucose

Magnesium chloride

Potassium chloride

Potassium hydroxide

Sodium Bicarbonate

Sodium Carbonate

Sodium Chloride

Sodium dihydrogen phosphate

Sodium hydroxide

STZ

CDH, Mumbai

CDH, Mumbai

Lab Distillation Assembly

CDH, Mumbai

E.Merck, Mumbai

E.Merck, Mumbai

CDH, Mumbai

CDH, Mumbai

CDH, Mumbai

E.Merck, Mumbai

E.Merck. Mumbai

CDH, Mumbai

Sigma Chemicals, USA

3.5 DIAGNOSTIC KITS

1. Glucose Kit

2. Insulin Kit (ELISA)

Span Diagnostics Ltd, Surat.

Diagnostic System Lab, Inc.

Webster, TX, USA.

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Chupier 3 M a te ria ls & M ethods

3 / 0 EQXJIPMENTS AND INSTRUWIENTS

1 . Photoelectric colorimeter

2. Spectrophotometer (double Beam)

3. UV Spectrophotometer

4. Fluorescence Spectrophotometer

5. Centrifuge Remi- T8A

6. Micropipette (10|.il - 100(.il)

7. Oven

8. BOD incubator

9. Electronic digital single pan balance

(100^g-100g)

10. pH Meter

11. Chemical Balance

12. Refrigerator

13. Magnetic Stirrer

14. Distillation Unit

15. Microplate Reader

16. Orbital Microplate Shaker

17. Water Bath

18. Organ Bath

19. Sherrington’s Rotating Drum

Systronic 101, Delhi

Hitachi, Japan

Lambda Bio 20 VI-20

UV/ Vis Perkin Elmer

Hitachi- 65.0-105

Remi Scientific Industry,

Mumbai

J. Mitra Brothers & Co.

Ltd, Delhi

Wirswow Instruments,

Delhi

Wirswow Instruments,

Delhi

Dhona Instruments, Delhi

Control Dynamics,

Manbalore

Roy Balances Work,

Varanasi

Kelvinator India Ltd.

Metrex, India

Bio-Tek Instruments Inc,

USA

New Lab, Mumbai

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3 ;/ QTiJER ii/VTERIALS

1. Rat feeding needle

2. Disposable syringes and needles

3. Hand gloves

4. Sterilized cotton

5. Capillary tubes

6. Wax trays

7. Surgical Instruments

8. Glass wares and other glass apparatus

AUTOMATED MICRO PLATE READER

It is a single channel assay reader system for in-vitro diagnostic and research

use, designed to automatically perform endpoint analysis. It can measure the

optical density of solutions in 24, 48 or 96 well micro plates. The data files

created can be printed in report form or can be stored as data files for a variety of

enzyme linked immunosorbent assay (ELISA) based applications. It features

superior optical specifications; with an extended dynamic range of up to 3.000

absorbance units in some read modes. The wavelength range is from 400nm to

750nm. Kinetic analysis can be performed using computer control.

Assay definitions (consisting of protocols, templates and formulas) and the data

they produce, are managed by an onboard processor, via a 2X24 LCD screen

and membrane switch. It is designed to serve as a stand-alone system, or as a

part of a larger laboratory data network, sending, receiving and nnanipulating

assay data as needed.

Package Contents

The contents of the Automated Micro plate Reader package includes:

> Micro plate Reader

> Power Cord

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86

Filter wheel with 4 standard filters: 405nm, 450 nm, 490nm, 630nm and one

blank filter.

3.8 METHODOLOGY / EXPERIMENTAL PROCEDURE

3.8.1 Collection of blood samples; Blood was withdrawn from the tail vein of rat

in the sterilized test tubes, under light ether anesthesia. All rats were

sacrificed under anesthesia by carotid bleeding.

3.8.2 Separation of serunn: About 5 ml of blood was collected in a sterile

centrifuge tube and left undisturbed at 37“C, till the formation of ciot for 1

h. The clot was dislodged using a sterile loop and refrigerated at 2-8°C for

3-4 h. During this period, serum exuded and the clot retracted, The serum

was aspirated using a sterile pipette after centrifugation at 3000 rpm for 15

min. Serum collected was analyzed for biochemical parameters

immediately or within 24 h after storing at 0-4°C.

3,9 BIOCHEMICAL ESTiMATiONS:

3.9.1 Blood Glucose

Method: Glucose Oxidase Peroxidase (GOD/POD) Method (Braham and

Trinder, 1972)

Principle: Glucose is oxidized by Glucose Oxidase to Gluconic acid and

Hydrogen Peroxide. In a subsequent peroxidase reaction, the oxygen liberated is

accepted by the chromogen system to give a red colored quinoneamine

compound. The red color so developed is measured at SOSnm and is directly

proportional to glucose concentration.

Sample: Serum 0.02 ml

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87

Glucose Oxidase D“ Glucose + O2 + H2 O ------------------------> Gluconic acid + H2 O2

Glucose peroxidase H2 O + 4 - amino antipyrine----------------- ---------- Quinoneirnine + H2 O

(chromogen oxygen acceptor ( chrome red colored complex)system)

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Reagents (Supplied in the kit)

Reagent 1: Glucose enzyme kit

Reagent 2; Glucose Diluent

Reagent 3: Glucose Standard, 100 mg%

Preparation of working glucose reagent

The contents of one vial of glucose reagent (Reagent 1) were quantitatively

transferred to the black bottle (provided in the kit) and reconstituted with 50 ml

glucose diluent (Reagent 2). It was mixed slowly to dissolve completely.

Storage and Stability

Reagent 1 and 3 were stable at 2-8°C and Reagent 2 was stable at room

temperature, till the expiry date mentioned on the individual label.

Working glucose reagent was stable at 2-8°C up to 6 months.

Procedure

Preparation of the Blank, Standard and Test samples were carried as follows;

The solutions in the tubes were mixed weli and incubated for 10 minutes at 37°C.

The optical density at 505 nm was measured against purified water. The final

color was stable for one hour.

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Chapter 3 M aterials & M ethods

Blank (B) Standard (S) Test (T)

Serum - - 0.02mi

Glucose Standard, - 0,02ml -

100mg% 1.5ml 1.5ml 1.5ml

Working Glucose Reagent

Purified Water

1.5ml 1.5ml 1.5ml

Calculations

Serum Glucose in mg/100ml- O .D T es t-O .D , Blank

O.D Std. -O .D . Blank

3.9.2 Serum Insulin Estimation

X 100

ELISA was used to estimate insulin quantitatively, for this purpose, DSL-10-1600

ACTIVE ™ ELISA kit was used.

Principle

The DSL-10-1600 ACTIVE ™ insulin ELISA is an enzymatically amplified 'one

step' type immunoassay. In the assay, Standards, Controls and unknown serum

samples are incubated with anti-insulin antibody in micro titration wells that have

been coated with another anti- insulin antibody. After incubation and washing, the

wells are incubated with the substrate tetramethybenzidine (TMB). An acidic

stopping solution is then added and the degree of enzymatic turn over of the

substrate is determined by dual wavelength absorbance measurement at 450

and 620 nm. The absorbance measured is directly proportional to the

concentration of insulin presenL A set of insulin standards is used to plot a

standard curve of absorbance versus insulin concentrations from which the

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insulin concentrations in the unknowns can be calculated (Nakagawa et al.,

1973; Hwang et ah, 1985).

REAGENTS

Anti-insulin coated Micro titration Strips: One strip holder contains 96

polystyrene micro titer wells with anti-insulin antibody immobilized to the inside

wall of each well.

Insulin Standards (Lyophilized)

One vial labeled A, containing Of.ilU/ml insulin, and five vials, labeled B-F

containing concentrations of approximately 3.0, 10.0, 30.0, 100.0 and 300.0

f.tlU/rnl insulin in human serum with a non- mercury preservative.

The standard A was reconstituted with 1.0 ml of deionized water. After

reconstitution, they were stored at 2-8 °C for two weeks. The DSL-10-1600

standards have been calibrated to the WHI 1®' IRP 66/304.

Insulin Controls (iyophiiized)

Two vials, levels 1 and 11 contains low and high concentrations of insulin in human

serum with a non-mercury preservative. Each vial was reconstituted with 0.5ml of

deionized water. After reconstitution, they were stored at 2-8 °C for two weeks.

Insulin Antibody Enzyme conjugate concentrate

One vial, containing 0.3 ml of a solution of insulin conjugated to horseradish

peroxidase in buffer with a non-mercury preservative. This was stored at 2-8°C.

Assay Buffer B

One bottle, 28 ml, containing a protein based buffer with a non-mercury

preservative. It was stored at 2-8°C until expiration date.

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90

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TMB Chrornogen Solution

One vial, 11 ml, containing a solution of TMB in citrate buffer with H2 O2 . It was

stored at 2-8°C.

Wash Concentrate

One bottle, 60 ml, containing buffered saline with a nonionic detergent, it was

diluted 25 fold with deionized water prior to use. It was stored at 2-8“C.

Stopping Solution

One vial 11 n'il, containing 0.2 M sulphuric acid, it was stored at 2-8°C.

All reagents and samples were allowed to reach room temperature and mixed

thoroughly, before use.

Procedural Notes

Serum specimens were frozen at ~ -20°C. All kit reagents and specimens were

brought to room temperature (~25°C) before use. They were also thoroughly

mixed before use.

Double distilled water was used since the enzyme is highly sensitive to microbial

contamination, sodium azide, hypochlorus acid and aromatic chlorohydrocarbons

often found in laboratory water supply.

Exposure of reagents to excessive heat or direct sunlight was avoided during

storage and incubation.

Test Procedures

Preparation o f Reagents

(i) Wash Solution

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It was prepared by diluting wash concentrate 25 folds with deionized water,

(ii) Antibody Enzyme Conjugate Solution

The antibody Enzyme Conjugate concentrate was diluted just prior to use in the

assay at the ratio of 1 part into 50 parts of the assay buffer, according to the

number of wells used.

(iii) Micro Titration Wells

The coated wells required for the assay were selected first. The remaining

unused wells were placed in the resealable pouch with a desiccant.

Assay Procedure

All reagents and specimens were brought to room temperature (~25°C) before

use. They were also thoroughly mixed before use. Standards, controls and

unknown were assayed in duplicate.

(i) The micro titration strips to be used were marked.

(ii) 25j.ll of the Standards A-E, Controls and unknowns were pipetted into

the appropriate wells.

(iii) The antibody enzyme conjugate solution was prepared and 100f.il was

added to each well using a semiautomatic dispenser.

(iv) The wells were incubated, shaking at a fast speed (500-700 rpm) on

an orbital micro plate shaker, at room temperature (-25 °C) for 1 hr,

(v) Each well was aspirated and washed 5 times with the wash solution

using an automated micro plate washer.

(vi) 100 ml of TMB chromogen solution was added to each well using a

semiautomatic dispenser.

(vii) The wells were incubated, shaking at a fast speed (500-700 rpm) on

an orbital micro plate shaker, at room temperature (-25 °C) for 10 min.

(viii) 1 0 0 )il of the stopping solution (0 . 2 M H2 SO4 ) was added to each well

using a semiautomatic dispenser.

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92

(ix) The absorbance of the solution in the wells were read within 30 min,

using a micro plate reader set to 450 nm with background wavelength

correction set at 620 nm.

(x) The mean absorbance for each standard, control or unknown was

calculated. A graph was plotted between log of the mean absorbance

reading on the y-axis versus the log of concentration on the X-axis,

The straight line so obtained was the standard curve.

Now the mean absorbance reading of the controls was observed from the

standard curve. These concentrations were compared with the concentration

given in the bottles to check the authenticity of the method.

The mean absorbance readings of the samples were calculated and their

concentrations were observed from the standard curve.

Normal Range (insulin); 2.1 - 30.8 lU/ml

(24 jui lU/ml = 1 ng /ml)

3,JO BEHAVIORAL ESTIMATIONS

3J0.1 DEPRESSION

Modified FST: Modified FST as described by Cfyan and co-workers was used

(2002a; 2002b). Briefly, the rats were forced to swim individually in cylinder (40

cm height, 15 cm diameter) containing fresh water (temperature 22 ± 2°C) up to

a height of 30 cm for 15 minutes. This constituted the ‘pre-test’ swim. Twenty-

four hours later, each mouse was re-exposed to the swimming condition in a

similar environment in a 6 minutes 'test session’. The total duration of climbing,

swimming and immobility in the last 5 minutes of the 6 minutes test session was

recorded for each animal.

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93

3.102 MEMORY

Spontaneous Alternation behavior; This mode! was used to test Short-Term

memory with respect to spatial orientation and perception as described by

Ragozzino et at (1998). Briefly, rats were removed from the cages and placed

individually in a four-arm cross maze. The maze constructed of wood, painted

gray and contained a central platform (25 cm dia) from which radiated 4

symmetrical arms (55 cm long X 10 cm wide) with 12 cm wails. After being

placed in the central platform, rats were allowed to transverse the maze freely for

12 min. The number and sequence of entries were recorded; an alternation was

defined as entry into four different arms on an overlapping quintuple set. Five

consecutive arm choices within the total set of arm choices made up of a

quintuple set. A quintuple set consisting of arm choices B, A, C, B, D comprised

as ‘Alternation’ while the set with B, A, D, B, A did not (using this procedure,

possible alternation sequences were equal to the number of arm entries minus

four).

Percent alternation was calculated as follows:

Actual alternation

Alternation % = --------------------------X 100

Possible alternation *

* Number of arm entries - 4

3.10.3 ANXIETY

The elevated plus maze test developed by Pellow et al (1985) was employed to

unveil the effect of CrP on anxiety. The maze apparatus consists of 2 open (55 X

10 cm) and 2 closed arms (55 X 10 X 12 cm) having an open roof with the entire

plus maze elevated 50 cm from the floor. Each rat was placed individually at the

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94

center of the elevated plus maze with its head facing an open arm. During the 5

min test, the performance of the animal for the first entry, the number of entries

into the open \ closed arms and the time spent in each arm of the maze were

recorded. An arm entry was defined as the entry of all four feet into one arm.

Each animal was used only once and the test carried out during a fixed time of

the day. The arms and the center were thoroughly cleaned after each trial.

3.11 IN VITRO STUDY

Segments of terminal ileum (3-4 cm long) were removed from the rats. The

tissues were suspended in 40 ml inner organ baths containing Tyrode solution

{(mM); NaCI 137, KCI 2.7, CaClj 1,5, MgCI2 1, NaHCOa 12, NaHjPOU 0.4,

and glucose 5.5}, which was bubbled with air and maintained at 37±1°C.

Responses to the drugs were recorded on a drum using an isotonic frontal writing

lever, which exerted a 500 mg tension on the tissue that gave 8-10 fold

magnification at the responses. The tissue was allowed to equilibrate for 30 min.

During this time, the bathing solution was changed every 10 min.

In the first series of experiments, after 30 min stabilization, the smooth muscle

was contracted by 80 mM KCI and the responses were allowed to reach a

plateau. This was obtained within 10-15 min after the addition of KCI, The

responses of different drugs were then recorded in a cumulative manner.

Sufficient time was allowed before the next dose was added. On complete

relaxation, the tissue was washed every 5 min and the base line was restored,

3.12 STATISTICS

The data were expressed as Mean + SEM and results were analyzed by AN OVA

followed by Dunnett’s t test. P values < 0.05 were considered significant.

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95