CHAPTER 4

lATERIALS

CONTENTS

Page

Chapter 4.1 Animals 57

Chapter 4.2 Test Drugs

1. Kushta Tila Kalan (KTK) 58

2. Swanta Bhasma (SB) 58

3. Auranofin (AN) 59

Chapter 4.3 Other Drugs 60

Chapter 4.4 Chemicals and Reagents 62

Chapter 4.5 Equipment

1. Traction Test 64

2. Photoactometer 64

3. Tail Clip 64

4. Eddy’s Hot Plate 65

5. Analgesiometer 65

6. Pododolorimeter/Aggressometer 65

7. Electroconvulsiometer 66

8. Cook’s Pole Climbing Response Apparatus 66

9. Rota Rod Apparatus 66

10.Passive Avoidance Response Apparatus 67

11.Videopath Analyzer 67

12-Anxiometer 69

13.Elevated Plus Maze 71

14.Behavioural Despair Apparatus 72

15.Jumping Box 72

16.CataIepsy Blocks 72

17.Activity Wheel 73

IS.Brain Block Holder 73

19.Spectrophotometer 73

20.Hlgh Performance Liquid Chromatography 75

(HPLC) Apparatus

21.Miscellaneous Apparatus 75

56

Chapter 4.1

ANIMALS

The investigations were carried on adult animals of either sex: Wistar strain Albino rats

(100-200 g) and Swiss Albino mice (20-30 g). The animals were kept in a group of 8-10

housed in polypropylene cages kept in air-conditioned rooms (25-28°C) and maintained

on standard pellet diet (Amrut Laboratory rat and mice feed, Navmaharashtra Chakan Oil

Mills Ltd., Pune) and water ad libitum. For experiments on growth and body weight

young rats pups (50-60 g) were used. Approximately, equal number of male and female

animals were taken in each group except for experiments related to antiaggressive activity

and immunomodulating studies (mice) where only male animals were used. The

experiments were performed in a noise-free atmosphere at ambient temperatures ranging

between 18-30'^C.

57

Chapter 4.2

TEST DRUGS

1. Kushta Tila Kalan (KTK)

The drug was procured from M/s Hamdard (Wakf) Laboratories, Delhi, India. Pure gold

leaves (30 g) are thoroughly triturated with mercury (60 g) to a butter-like smooth paste.

The latter is triturated with 50 g of powdered sulphur for 2 days and made into Ig tablets.

The tablets are sealed in earthen pots and subjected to ashing using cow dung cakes as

fuel. The scrapped material is pulverized and triturated̂ with 50 g of sulphur and 62.5 ml

of juice of Aloe vera (GHEEKANWAR) and subjected to ashing in similar manner. The

process is repeated several times using alternately sulphur and juice oi^A.vera and

distillate o f rose flowers. The final product is a very fine pink powder (Kabeeruddin and

Wahid 1922; Said 1969).

2. Swarna Bhasma (SB)

The drug was procured from M/s Ayurveda Ras Shala, Pune, India. It is prepared,

following the method of Ayurvedic texts as described by Chopra et al (1982). The gold

leaves are subjected to a process o f purification, which involves burning it in fire and

steeping the red hot metal successively in oil, cow's urine, whey and extract o f Dolichos

uniflorus (KULATHA KALAI). In reducing gold, one part of purified metal and two

parts o f mercury are rubbed with an acid and made into one ball. This ball is then encased

in earthen containers with powdered sulphur and subjected to ashing using cow dung

58

cakes as fuel. The bhasma is scrapped and subjected to repeated ashing in a similar

manner for 14 times before therapeutic use. Thorough trituration at each stage of the

fourteen putas is necessary to yield a very fine powder and this ensures proper quality of

bhasma (Said 1969; Chopra et al 1982).

3. Auranofin (AN)«

The drug, procured from M/s Alidac Genetics and Pharmaceuticals, Ahmedabad (Goldar

tablets), is known to contain 29% of gold (Tripathi 1995).

59

Chapter 4.3

OTHER DRUGS

1. Acetyl salicylic acid (Disprin, Reckitt and Colman, Calcutta): Aqueous solution (10

mg/ml).

2. Amphetamine D. (Sigma Laboratories, USA): Aqueous Solution (2mg/ml).

3. Apomorphine hydrochloride (Sigma Laboratories, USA): Aqueous solution (0.25

mg/ml).

4. Buspirone hydrochloride (Dadiha Pharmaceuticals, Merind, and Tamil Nadu):

Aqueous solution (500 fig/ml).

5. Chlorpromazine (CDH, Delhi); Aqueous solutions made from pure salt of the drug

(250-500 ^ig/ml).

6 . Diazepam LP. (Calmpose, Ranbaxy, Dewas): Injection, 2 ml ampoules (5 mg/ml).

7. Ginseng (Korean Ginseng) Courtesy: Professor Hyung Suk Kim of Kyung-Hee

University-Seoul, Korea; available in teabags containing 3 g water-soluble powder.

Aqueous solution (35 mg/ml).

8. Haloperidol (Sigma Laboratories, USA) was dissolved in 10% lactic acid. This was

then diluted to give concentrations of 25 and 50 |^g/ml.

8. Imipramine (Dapsonil, S,G. Pharma, Mumbai) Tablets containing 25 mg imipramine

hydrochloride

9. Levodopa (Wallace Pharmaceuticals Ltd, Goa): Aqueous solution (15 mg/ml).

60

10. Morphine (Govt. Opium and Alkaloid Works, Ghazipur); water-soluble salt was used

to give the desired concentration (100-500 |ag/ml).

11. Naloxone (Sigma Laboratories, USA): Aqueous solution (100 ng/ml).

12. Pentobarbitone Sodium: Freely soluble sodium salt of pentobarbitone (Loba

chemicals, Mumbai). Aqueous solutions (4-6 mg/ml).

13. Pentylene tetrazol (Sigma Laboratories, USA): Aqueous solution (10 mg/ml).

14. Phenytoin (Sigma Laboratories, USA): Aqueous solution (2.5 mg/ml).

15. Reserpine hydrochloride (Sigma Laboratories, USA): Aqueous solution (0.5 mg/ml).

16. Strychnine chloride (CDH, Delhi): Aqueous solution (400 |ig/ml).

17. Yeast extract powder (CDH, Mumbai): was suspended in distilled water (15%) and

activated by incubating at 37°c for 48 h.

61

Chapter 4.4

CHEMICALS AND REA GENTS

Source

Sigma Laboratories, USA

Analytical grade reagents and chemicals were used. The drugs and chemicals were

dissolved in double distilled water; those which were not soluble were suspended in

distilled water using 1% gum acacia.

Chemicals/Reagents

Acetylthiocholine iodide, Bovine serum albumin (BSA),

Cholesterol standard, l-chloro-2,4 dinitrobenzene (CDNB),

3,4 dihydroxyphenyl acetic acid, dinitrobenzene--(DTNB),

Dopamine, Epinephrine, Fecal calf serum, Glucose-6-

phosphate, 5 hydroxyindole acetic acid. Hank’s Balanced salt

Solution (HBSS), Homovanillic acid, Horseradish peroxide,

Nicotinamide adenine dinucleotide phosphate (NADP),

Oxidised and reduced glutathione, Phenylmethylsulphonyl

fluoride (PMFS), Phosphate Buffer saline (PBS), Reduced

nicotinamide adenine dinucleotide phosphate (NADPH),

RPMI - 1640, Serotonin, Wright - Giemsa Stain.

Benzylamine hydrochloride. Citric acid, Disodium EDTA, Hi Media Laboratories

62

Disodium hydrogen orthophosphate, Eosin, Ethylene diamine New Delhi

tetracetate (EDTA), Folin’s reagent. Glycerol, Hydrogen

Peroxide, Monobasic sodium phosphate, Sodium dihydrogen

ortho phosphate. Sucrose, Sulphosalicylic acid, Thiobarbituric

acid (TBA), Tris-HCl, Trisodium citrate.

Acetonitrile, Ascorbic acid, Chloroform, HPLC water, Merck India Ltd., Mumbai

Hydrochloric acid, Isoproterenol, Methanol, Nitric acid,

Potassium Ferrocynide, Potassium iodide, Sulphuric acid.

Xylene

Acetic acid (Glacial), Alcohol, Ammonium / Potassium alum, CDH, New Delhi

Charcoal powder, Copper sulpahte. Ferric chlorideTG^ti^

violet, Gum acacia, Hematoxylin crystal. Magnesium

chloride, Mercuric chloride. Mercuric oxide red,

Orthophosphoric acid. Paraffin, Perchloric acid. Petroleum

jelly, Phloxine, Potasium chloride, Silver sulphate. Sodium

chloride. Sodium octanesulfonic acid, Sodium potassium

taratrate. Sodium sulphate. Trichloroacetic acid (TCA)

Double Sided Tape St. Paul, USA

63

Chapter 4.5

EQUIPMENT

1. Traction Test

The set up is essentially similar to that described by Turner (1965). A metallic wire

(length: 250 mm, diameter: 0.8 mm) stretched horizontally by means of two stands, one

on each end, was used to evaluate the spontaneous motor activity (SMA) by suspending

the mice by their fore paws to it.

2. Pbotoactometer

The apparatus consist of a chamber with 6 photocells and cris*crossing lights and on

electromagnetic counter. When a rat introduced into chamber^whenever it intercepts a

light beam, the counter is activated. This apparatus measures the spontaneous motor

activity of animal over / prolonged periods without the presence of the experimenter. .

3. Tail Clip

Rubber-protected artery forceps were used as tail clips to give mechanical noxious

stimuli.

64

4. Eddy’s Hot Plate

The apparatus consists of a square perspex chamber (30 x 30 x 20 cm) with a hot plate

floor. It is provided with a temperature regulator, pilot bulb and an indicator scale. The

required temperature (55 ± 2°C in this case) can be set by adjusting the regulator. A push ̂

button device confirms the adjusted temperature. When the heating process is over and

the adjusted temperature is attained, it is shown by automatic switching off the heating

indicator.

5. Analgesiometer

The apparatus (Techno electronics, Lucknow) gives of a pain stimulus by application of

radiant heat (Nichrome wire). It is surrounded, by a chrome-plated water jacket.

Circulation of the water through this jacket maintains a constant temperature round the

stimulus area. The stimulus intensity can be adjuste^to-lngh or low levels. Current is

indicated on the meter. It should be adjusted between 3-4 amp^and it should never

exceed 6 amps. The apparatus has a groove to place the tall of the rat over It'to give the

stimulus.

6. Pododolorimeter / Aggressometer

An Aggessometer (Techno Electronics, Lucknow) was used as a pododolorimeter. It

consists of two perspex chambers (any of these could be used for the present study) with

a wire grid floor to give foot shock of desired voltage (0 to 150 volts, in steps of 10

volts). Besides voUage regulator the panel has pilot bulb and stimulus indicator lamps.

65

7. Electro Convulsiometer

The apparatus (Techno Electronics, Lucknow) has three controls for different intensities

of current stimulus: A= 0.25 to 0.5 mA, B= 0.5 to 3 mA and the third control provides

multiples between 1-12. The time for stimulus can be adjusted (0.1 to 1 second). The ear

electrodes are connected by means of leads to stimulus out put terminals provided on the

panel. Other controls on the panels are off-on switch, pilot bulbs indicating power supply

and stimulus and switch for starting the stimulus and resetting the apparatus.

8. Cook’s Pole Climbing Response Apparatus

This apparatus first described by Cook and Widely (1957) was used both for the study of

conditioned avoidance response (CAR) and learning experiments. The apparatus used

(Techno Electronics, Lucknow) has an experimental chamber with a wire grid floor fitted

in a sound proof enclosure. Instrument circuit provides two types of stimuli (buzzer and

6 volts D.C. shock). The stimuli may be presented singly or together for a fixed duration

(approx. 45 sec.) and can be terminated at any time.]^ A wooden pole fixed into the

detachable lid provides the only escape, as the walls of the cuaniuer are slippery.

9. Rota Rod Apparatus

Omni Rotor (Omnitech Electronics Inc., Columbus USA) was used. It consists of a

rotating rod (75 mm diameter). The ro ̂ is divided by circular partitions into four

compartments permuting test on four rats at a time. There are controls for adjustment of

maximum speed and time to reach this speed. A digital display shows the speed (RPM) at

which the apparatus has been set. The apparatus is provided with automatic counters

66

recording time in units of 0.1 sec with pilot lights and controls for start and termination of

a session. When the rats falls off the rotating shaft, the counter stops regarding the staying

time of the rat.

10. Passive Avoidance Response Apparatus

Continuous avoidance response apparatus (Techno Electronics, Lucknow) with an

inverted petridish (shock-free zone) was used. The apparatus can give shock stimulus (0-

200 volts) with adjustable delay, timings (5-60 sec). The animal can avoid the shock by

pressing the lever before expiry of the time delay. The set time aeiay starts again from the

moment the lever is pressed. The counters record stimulus, delivered and stimulus

avoided. It measures ability of the test animal to anticipate the time duration to press a

lever to avoid shock. As we used mice (instead of rats) the animals were unable to press

the lever and the only place to avoid the shock was the petridish.

11, Videopath Analyzer

Videopath analyzer (Coulboum Instruments, USA) consistsVf an open field chamber (50

X 50 X 35 cm), a video camera fixed over the chamber by an adjustable rod, an activity

monitor, a programmer and a printer. (Figure 4.1) The animal is placed over a black paper

(to provide contrast) in the open field chamber and the camera is activated. The animal}

can now be viewed on the monitor screen and its position can be adjusted using

horizontal/vertical controls. Proper brightness and contrast levels can also be adjusted.

The desired number of sessions, duration and session intervals can be programmed on the

activity programmer. The apparatus is provided with indicator lights to show the progress

67

M

V

a:

(*

■V,V'

f t . '.— ".% nr'tt:;

■a(

p f rA

Figure 4.1; VIDEOPATIl ANALYZER FOR MONITORING THE NEl'ROBEHAVIOURAL -\CTI\ ITY OF RATS

of the session. The data is fed to the printer which gives print outs for the following

parameters according to the program

a) Data interval

b) Wall Hugging (sec)

c) Comer (sec)

d) Quadrant 1 (sec)

e) Quadrant 2 (sec)

0 Quadrant 3 (sec)

g) Quadrant 4 (sec)

h) Locomotion (sec)

i) Rest (sec)

j) Rearing (sec)

k) Stereo events

1) Rotation (CW)

m) Rotation (CCW)

fi) Distance (cm)

Session totals for all the parameters are also printed. Values for average speed (cm/m) are

also available. The apparatus permits observations and recording of behavioral

parameters with the observer siting at a distance without disturbing the animal.

12. Anxiometer

The apparatus (Columbus Instruments, Ohio, USA) consists of three parts: (Figure 4.2)

69

Figure \1: COLllM lll'S A N X IO IT E R (Aiiiiiiiil showing lickiiig of spou

(a) A perspex rat chamber (47 x 26 x 20 cm). This chamber is provided with a metal grid

floor and a metal lid. A plastic bottle with a cork and metallic spout is placed on the top

o f the chamber with the spout protruding through the lid. The grid floor and the spout are

connected to (b) A shock generating- device. This is provided with a dial indicator for

subject current ( 0 - 5 milli amperes) and controls for on/off switch, meter range and shock

intensity. It has a power indicator lamp and leads for initiating shock as also for shock

out put. (c) Anxiometer - It has digital records for nimiber of licks and a number of

shocks and controls for monitoring with or without shock, starting and stopping the test

session and resetting the instrument. The indicator bulbs for power, testing, licks and

shocks are also provided on the panel. When the raUbcates the spout and licks the water

from the bottle, it gets a mild electric shock (0.5 mA, 1 sec). The number o f drinking

episodes and the shocks delivered during a desired period o f observation (Usually 3 min)

are recorded. The apparatus thus provides reward (drinking water, which may be

sweetened) and punishment (shock) simultaneously creating a conflict situation. The

apparatus was first devised by Vogel and coworkers (1971) and is named after him.

13. Elevated Plus Maze

This apparatus, first devised by Pellow and coworkers (1985), was fabricated by the

Maintenance Department of Jamia Hamdard. It consists of a plus-shaped maze elevated

45 cm above ground level with two open (10 x 50 cm) and two closed (10 x 50 x 40 cm)

arms. There is a central square area (10 x 10 cm) at the junction of open and closed arms

where the animal is placed at the start of the experiment. The apparatus is designed to

71

measure anxiety-related behavior in rats with closed arms representing security and open

arms in security and fear of heights.

14. Behavioural Despair Apparatus

The apparatus is essentially similar to the one used by Porsolt et al (1978). In this study a

•perspex beaker (height: 25 cm, diameter: 18 cm, capacity: 5 L) was used. When a rat is

forced to swim in a restricted space from which there is no escape, it eventually ceases

further attempts and becomes immobile apart from the slight passive movements for

survival or to keep its head above the water level." Periods o f irnmobility reflect the state

and degree of despair in the rat.

15. Jumping Box

Jumping Box (Techno Lucknow) consists of two-chamber compartments divided by a

transparent perspex hurdfe. Floors of any of the chambers can be charged by appropriate

grill selector switch. Stimulus can be measured by voltmeter (0-50 volts). The desiredI

shock intensity can be adjusted. A buzzer is also provided. Crossing over to the shock-

free chamber on warning buzzer alone (conditioned avoidance) or with shock

(unconditioned avoidance) are noted. The apparatus was used for the study of learned

helplessness.

16. Catalepsy Blocks

Simple equipment described byJDandiya and Baneijee (1986) was used. It comprised of

72

two wooden blocks (height: 3cm and 9cm) for measurement of stages III and IV of

catalepsy.

17. Activity Wheel

The apparatus (Hicon Eminence, Delhi) is suitable for the study o f forced motor activity

with or without temperature stress. It consists of a perspex chamber (32 x 20 x 14 cm)

containing a wheel shaped cage (diameter: 30 cm) which freely moves on its axis. When

a rat is placed inside this wheel cage with water in the chamber, it tries to climb on the

wheel to escape from water. The animal is unable to climb out completely due to rotation

of the water wheel. As a result, the animal continues to turn the wheel.'The movements

(both forward and backward) of the wheel are record^d^^;digital counter.

18. Brain Block Holder

It consist o f perspex block ^ 5 x 29 x 24 mm) with a central oval depression (25 x 15

mm) and 12 cuts to the depth of 10 mm each to enable 11 sections of 1.5 mm thickness.

(Figure 4.3)

1 Spectrophotometer

Spectrophotometer DU-64 (Beckman, USA) was used to analyze the samples for various

biochemical parameters in the visible range at 610 nm. The apparatus has provisions to

select the desired range (visible or ultraviolet) in which the estimations are to be.done and

operated at the required wavelength. It is calibrated with the blank sample by means

cuvette. Absorbance of standard and test samples can be taken in a similar manner.

73

H.

f v r

Wit; "4hLi>.,

l ln i i r o I j : |A ) Rill l in i i i i h k ' k lidklei', ( l i ) Rut lii'iiiii l id ldcr willi r:il hriiiii O m l i i i l |i(isiliiiii) iiiiil ( ( ' ) Riil hriiin ) lock lidliler w ill) n\ hriiiii mid hliiile p i i s s in j l l in ) i i« l i S c f l io i i 1; Rli(iiiik'iu'e|)l iiil(ii i | ii) scpiiriilin}; l)v (riii isvcrso

sec t ion into tw o m i i s c c r e b d l i i i i ) mid ii irdiilla oliliiiijiiitii, k t i i i i i h O p t ic ( lii i isin:i wliicli deli i i i i ts tlic m iler idr la i i o f liypollialaiiii i!; a n d p asses lliniii;;li an ter io r con i i i i i ss i ire and sep era t i i iu c cr e l in i in iiilii t » o n lo t l ia lan i i is , s t r ia tu m , n iid l)ra in , lii i locani m s and |c c o r t c \ .

20. High Performance Liquid Chromatography (HPLC) Apparatus

Merck Hitachi HPLC apparatus with two units L7100 pump, an amperometric L3500. A

detector and ODS / Lichro CART 250-45 nm was used. The apparatus was equipped with/

a PC with HSM 7000 (HPLC system manager) software and Hewlett Packard Inkjet

Printer.

21, Miscellaneous Apparatus

Haemocytometer, Haemoglobinometer, Microscope, Chemical Balances, Animal

Balances, Water baths, Homogenisers, Centrifuge machines, Distillation'^apparatuses,

Micropipettes Thermometers, Glassware, Plasticwar^Surgical instruments, Syringes,

Feeding needles etc.

75