EXPERIMENT 4.
7
Object To study the analgesic effect of morphine in mice using tail-flick method. .
Principle Analgesia is defined as a state of reduced awareness to pain, and analgesics
are substances which decrease pain sensation (painkillers) by increasing threshold to
painful stimuli. The commonly used analgesics are aspirin, paracetamol (non-narcotic
type) and morphine (narcotic type).
Painful reaction in experimental animals can be produced by applying noxious
(unpleasant) stimuli such as (i) thermal (radiant heat as a source of pain), (ii) chemical
(irritants such as acetic acid and bradykinin) and (iii) physical pressure (tail
compression). In the laboratory, commonly used procedures are tail-flick (tail-
withdrawal from the radiant heat) method using analgesiometer, hot plate Gumping
from the hot plate at 55°C) method and acetic acid-induced writhing.
Students are advised to study the pharmacology of analgesics before
performing this experiment.
Requirements
Animal Mice (20-25 g)
Drugs Morphine sulphate (Dose 5 mg/kg s.c., prepare stock solution containing 0.5
mg/ml and inject I ml/IOO g of body weight of mouse).
Equipment Analgesiometer (Techno)
Procedure
1. Weigh and number the mice.
2. Take basal reaction time-: to radiant heat by placing the tip (last 1-2 cm) of the tail
on the radiant heat source. The tail-withdrawal from the heat (flicking response) is
taken as the end point. Normally a mouse withdraws its tail within 3-5 sec. A cut off
period of 10-12 sec is . observed to prevent damage to the tail. Any animal failing to
withdraw its tail in 3-5 sec is rejected from the study. Take at least 3-5 basal reaction
times for 'each mouse at a gap of 5 min to confirm normal behaviour of the animal.
3. Inject morphine and note the reaction time at 5, IS, 30 and 60 min after the drug. As
the reaction time reaches 10 sec it is considered maximum analgesia and the tail is
removed from the source of heat to avoid tissue damage.
4. Calculate percentage increase in reaction time (index of analgesia) at each time
interval.
Morphine given in dose of 5mg/kg, s.c.
k * A cut off time of 10 sec. is taken as maximum analgesic response to avoid
amage to the tail due to heat.
EXPERIMENT 4.8
Object To study the ;. effect of morphine in mice using hot plate method..
Principle In this method heat is used as a source of pain. Animals are individually
placed on a hot plate maintained at constant temperature (55°C) and the reaction of
animals, such as paw licking or jump response is taken as the end point. Analgesics
increase the reaction-time. The method was first described by Eddy and Leimbach
(1953) (Fig. 4.6).
Requirements
Animals Mice (20-25 g) .
Drugs Morphine sulphate (Dose 5 mg/kg, s.c., prepare a stock solution containing 0.5
mg/ml and inject 1 mlll00 g of body weight of mouse.)
Equipmen!. Eddy's hot plate (Techno)
Procedure
1. Weigh and number the mice.
2. Take the basal reaction-time by observing hind paw licking or
jump response (whichever appears first) in animals when placed on the hot plate
maintained at constant temperature (55°C). Normally animals show such response in
6-8 sec. A cut off period of 15 sec is observed to avoid damage to the paws.
3. Inject morphine to animals and note the reaction time of animals on the hot plate at
15, 30, 60 and 120 min after the drug administration. As the reaction time increases
with morphine, 15 sec is taken as maximum analgesia and the animals are removed
from the hot plate to avoid injury to the paws.
4. Calculate percent increase in reaction-time (as index of analgesia) at each time
interval.
Observations
Sr. No. Tail-flick method Basal reaction time (see) Reaction time (see) after
Morphine administration
15 min 30 min
Fig. 4.6 Eddy's Hot Plate (Techno) Observations-Hot-plate method
Basal reaction time (see) Reaction-tif!.l'Y(sec) aj!.g.-.
morphin_d'!!.!!!istratio_ I
Paw-licking Jump response\_
Jump
Paw-licking
response
l. 4
2. 6
3. 8
4. 5
5. 6
Morphine given in a dose of 5 mglkg, s.c.
. A cut off-time of 15 sec is taken as maximum analgesic response to avoid. injury to
the paws.
EXPERIMENT 4.9
Object To study the analgesic effect of morphine against acetic
acid-induced writhing in nilce.
Principle Painful reactions in animals may be produced by chemicals also.
Intraperitoneal injection of phenylquinone_ bradykinin or acetic acid produces pain
reaction which is characterised as a writhing response. Constriction of abdomen,
turning of trunk (twist) and extension of hind legs are taken as reaction to chemically
induced pain. Analgesics, both narcotic and non-narcotic type, inhibit writhing
response.
Requirements
Animals Mice (25-30 g)
Drugs Morphine sulphate (Dose 5 mg/kg, s.c.; preparethe stock
solution as described in previous experiments), acetic acid 1 % v/v (Inject 1 mVIOO g of
body weight of the animal).
Procedure
1. Weigh and number the animals.
2. Divide the animals into two groups, each consisting of 5 animals. Administer
appropriate volume of acetic acid solution to the first group (which serves as control),
place them individually under glass jar for observation.
3. Note the onset on wriths. Reconj! the number of abdominal contractions, trunk twist
response and extension of hind limbs as well as the number of animals showing such
response during a period of 10 min.
4. To the second group of animals inject morphine. Fifteen minutes later, administer
acetic acid solution to these animals. Note the onset and severity of writhing response
as done in step 3.
5. Calculate the mean thing scores in control and morphine treated groups. Note the
inhibition of pain response by morphine.
Observations
Acetic acid-induced writhing
Treatment Number of writhing (10 min) Responders n/n.
S.
No.
1. 2. 3. 4. 5. Body wt. (g) Control (acetic acid) 1. 2. 3. 4. 5. Mean
Morphine + Acetic acid
Mean
