MINI PROJECT: ANTI-INFLAMMATORY TEST

PHARMACOLOGY & TOXICOLOGY UNIT.

Group Members↝ Anis Ayuni bt. Ismail (170607)↝ Nur Aqilah bt. Fauzi (170646)↝ Nurfarah Aqilah bt. Ahmad Nizam (170657)↝ Nurhayati bt. Mohd Yusoff (170921)

IntroductionANTI-INFLAMMATORY•Inflammation is the body's attempt at self-protection to remove harmful stimuli, including damaged cells, irritants, or pathogens - and begin the healing process. •The inflammatory response is usually quantified by increase in paw size (oedema) and is modulated by inhibitors of specific molecules within the inflammatory cascade.•In this experiment, we want to determine the anti-inflammation activity of Ficus Deltoidea in relieving the inflammation, so carrageenan-induced paw oedema experiment was conducted.•There are three groups:

1. Treatment group: Ficus Deltoidea

2. Positive group: Indomethacin

3. Negative group: Saline water

• Our group is a control or negative group.

• We gives the rat saline water by force feed.

• It is a procedure that differentiate the three group in this experiment.

• Every groups injected 0.1ml of 1% carrageenan and saline water respectively whether in left or right hind paw onto subcutaneous of plantar surface to induce oedema.

• The injection of Carrageenan and saline water will caused the inflammation the rat’s paws.

• The increase in percentage of paw volume was calculated based on the volume difference between the normal and abnormal paws (with or without carrageenan injection).

Introduction

Methods

1% carrageenan, 100 mg/kg plant extract, 10 mg/kg indomethacin in saline were prepared.

A line was marked above the ankle joint of the hind

limbs.

The solutions were administered to each

respective rats.

Both hind paws were then dipped

into small container of plethysmometer

to measure base volume.

After 30 minutes, subcutaneous injection

of 0.1 ml of 1% carrageenan was

performed onto plantar surface of right hind paw

of all rats.

Another subcutaneous injection of 0.1 ml

saline was performed onto plantar surface of

left hind paws.

Volume of both hind paws in all group was measured

on the next 30 minutes with 30 minutes interval

for 5 hours.

Hind paw swelling was calculated using the given

formula.

A. Carrageenan Induced Oedema Using Plethysmometer.

B. Carrageenan Induced Hyperalgesia Using Plantar Test.

After measuring base volume, the rats were

placed inside the container on Plantar Test.

Thermocouple was placed under the heels of

both hind paws, and withdrawal latencies

were recorded as base line.

Withdrawal latencies of both hind paws after

subcutaneous injection was performed were also

recorded.

The readings were recorded every 30

minutes interval for 5 hours.

Graphs for both experiment were plotted.

* Anti-hyperalgesia was detected when the right paw prolong the withdrawal time compared to the baseline values.

Plethysmometer.

During Plantar Test.

Results

RESULTSA)

Time (minutes) Right Paw Volume (Carrageenan)

Left paw Volume(Saline)

-30 1.29 1.23

0 1.85 1.63

30 1.9 1.53

60 2.25 1.76

90 2.50 2.28

120 2.34 1.90

150 3.06 2.02

180 3.14 2.32

210 2.42 1.54

240 2.02 1.69

Formula

% swelling = Vr – Vr0 - Vl – Vl0 X 100

Vr0 Vl0

Vr = Right Paw Volume

Vr0 = Right Paw initial Volume

Vl = Left Paw Volume

Vl0 = Left paw initial Volume

0 50 100 150 200 250 3000

10

20

30

40

50

60

70

80

Graph of percentage of Hind Paw swelling Against Time%

Sw

ellin

g

ResultsB)

Time (minutes) Right Paw ( Carrageenan) Left Paw ( Saline)

-30 14.9 11.9

0 17.5 15.1

30 15.6 7.4

60 26.7 10.9

90 13.9 11.9

120 14.9 14.6

150 14.1 8.9

180 9.9 7.3

210 9.9 6.4

240 8.9 7.8

Graph of Withdrawal Latencies vs Time (min)

-50 0 50 100 150 200 250 3000

5

10

15

20

25

30

Right Paw (Carrageenan)

Left Paw (Saline)Wit

hdra

wal

Lat

anci

es

Time (min)

Data Analysis

% S

wel

ling

Time (Minutes)

Data Analyses

• The paw swelling ratio is the percent increase of paw volume. The time-dependent curve

has shows that the injected carrageenan had cause the paw to swell.

• The graph show that the swelling have two phase in the given time. The different

phases(two different peaks) indicates cascade of biochemical events propagates to

matures the inflammatory response.

Graph of Withdrawal Latencies vs Time (min)

Time(min)

With

draw

al L

atan

cies

Data Analyses

• The graph shows the withdrawal latencies of rat’s hind paw.

• The inflamed paws by carrageenan have greater initial resting paw

temperatures than the paws receiving injections of saline. Also,

stimulation of the inflamed paws results in longer paw withdrawal

latencies than stimulation of saline-treated paws, and this longer latency

• There is some error in our result in which actually the theory that we found is the

inflamed paw should result in shorter paw withdrawal latencies.

Discussion

• The inflammatory effect was evaluated by carrageenan induced hind paw oedema. Carrageenan-induced paw oedema has been commonly used as an experimental animal model for acute inflammation and is believed to be biphasic.

• The oedema was induced by injection of 1% solution of carrageenan into rat’s plantar region. Different group of animal were administered with different solutions which are plant extract (100mg/kg) for treatment group, 1ml saline for negative control negative control group and 10mg/kg indomethacin for positive control group.

• Rat’ paw volume were measured by Plethysmometer. By using plantar test that measures response to infrared heat stimulus after the subcutaneous injection were performed, anti hyperalgesia can be detected.

• The graph show the value increased and decreased and have two phase.

• The early phase of the carrageenan model is mainly mediated by histamine, serotonin and increased synthesis of prostaglandins in the damaged tissue surroundings. While the late phase involved prostaglandin release mediated by the tissue macrophages.

• Apparatus used to measure the oedema:

• Plethysmometer

• Can measure the small volumes changes that occur.

• The water displacement produced by the immersion of the animal paw in the measuring tube is reflected into the second tube, inducing a change in the conductance between the two platinum electrodes.

• Plantar test

• Enables to discern the oedema to thermal stimulation caused by inflammatory in the unrestrained rat. 

Conclusion

• The volume of paw that have two phases indicates the cascade of the

different biochemical compound in body to respond to the inflammation.

• The inflamed paw has higher threshold temperature.

• Our part of experiment show the unstable result maybe influenced by

surrounding.

• Why must inject carrageenan and saline water?

• It is because carrageenan will cause the reaction of inflammation(oedema) by stimulating the release of specific antibody in the blood, while the saline water will not stimulate the reaction of inflammation but the inflammation still occur because of the tissue damage when we injecting the solution. Actually, saline water act as a blank because we can measure the inflammation(oedema) caused by cell injury and we can find the inflammation(oedema) caused by carrageenan compound

Thank You.