PHARMACOLOGY EXPERIMENT
INSTITUTE OF PHARMACOLOGY
Experiment 1 The basic knowledge and technique of pharmacological
experiment
1. the purpose or aim for us to have this class
learn basic or fundamental laboratory technique and skill. And verify the theory we have learned in
pharmacology.
2. the laboratory animals that we often used in our experiment
there are many laboratory animals. In different experiment, we need to choose the appropriate
animals. Next we will talk about the animals and their character
1 ） frog or toad:
they are often used in physiological studies, we rarely use it in our
pharmacological studies.
2）mouse or mice: mouse is the most frequently used animal in our experiment. The main reason
they are frequently used is they are very cheap, about 2-3 yuan per mouse. The second reason is
they have high reproduction rate and easy to raise. Mice are often used in the screening test of
drug or When we study the effect of antineoplastic drugs, analgesics , antitussive drugs etc, or
determine ED50（median effective dose） or LD50（median lethal dose） of a drug.
3）rat: it is very like mouse in shape but is much bigger than mouse. It is very fierce, they have
very sharp teeth and bad temper. If you are not careful, you will be bite by them. So if you want to
grab them, you need to put on thick gloves. Rats are not used often in our experiment but are
widely used in research study. For example, rats are often used in chronic toxicity test or long-term
toxicity test in the preclinical study of a drug.
4）guinea pig:
they are very beautiful and lovely animals. They are very timid or docile （tender）
and never attack people. In the street, you can see some people sell guinea pig. Someone raise it in
their home as a pet. It has a shortcoming: it can not synthesize Vitamin C by itself. So it needs to
get Vit C from food. You need to feed it with some cabbage, carrot etc .
Guinea pig is very sensitive to a kind of substance Histamine (HA). As a result of that, it was often
used in the hypersensitive test( allergic reaction) or the screening of anti-asthmatic drug.
5）rabbit: we are very familiar with rabbit, it is often used too but a little expensive. Its body
temperature is very stable. So it is the assigned animal in the pyrogen test during the quality
control of drug.（pharmacopeia） In our experiment, we often use it to observe the effect of some
drug on the blood pressure.
6）cat: cat is also bad tempered and irritable and not often used in our experiment. Its blood
pressure is very stable. So it is the assigned animal in the hypotensive substance test during the
drug quality control.
7）dog: dog belongs to the large animal in our experiment and is not often used either. A dog can
cost several hundred yuan. During the research, dog was often chosen to study some drug works on
cardiovascular system. For example, when we do some research on anti-arrhythmic drugs,
anti-anginal or anti-myocardial ischemia drugs.
8）others : including pig, goat, monkey. They are rarely used.
3. the staining of the animal
in our experiment, we often use more than one animal, so we need to make some marks to
discriminate each animal. Because they all look alike, we need to stain on the different place of
their skin to identify them. Such as neck, head, tail , back, leg and so on.
The most common dye we use is picric acid ( trinitrophenol) solution, it has a yellow color. This
color can last for a long time on the skin of the animal and can not easily fade.
4. the fixing of the animals
During our experiment, we need to fix the animal for drug administration and for other operations.
To different animal, the method of fixing is also different. Next, I will show you how to grasp and
fix the animal safely.
1）mouse: there are two ways we fix the mouse. The first is double-hand method: look at me. I will
show you how to get the mouse with two hands, grab the tail of the mouse with your right hand, and
put it on a rough (coarse) surface, not a smooth surface. Use the left hand to grab the skin around
the neck and back.
The second way is single-hand method. Only use your left hand to fix the animal.
2）rat:
the same as mouse. Need to be more careful and a thick glove
3）guinea pig:
4）rabbit:
is easy to deal with. You can grab it anyway.
the right way to fix the rabbit is : grab the skin in the back with the right hand, and
carry its buttock with the left hand. This way the rabbit will feel very comfortable and not struggle.
Then you can put the rabbit into the fixing box.
The wrong way: grab its two ears, that will hurt
the rabbit, and it will not cooperate。
5. anesthesia method
sometime when we do some operation or collect blood, we need to anesthetize the animal. The
method include two:
1）local anesthesia: we usually need two drugs: procaine and lidocaine . the way of injection is
subcutaneous injection.
2）general anesthesia: include inhalation anesthesia and injection anesthesia
inhalation anesthesia: ether
injection anesthesia:
pentobarbital sodium/ urethane
ip/iv
6. drug administration
1）how to use an injector/ or syringe
2）the way of drug administration include: ig（administration by gavage）, im (intramuscular
injection), iv( intravenous injection), sc( subcutaneous injection), ip (intraperitoneal injection)
abdominal cavity (腹腔)
3）Mouse:
ig: Use an special injector. The tip of it is blunted for not hurting the esophagus. The injector was
across the palate to the esophagus. Be careful not insert the injector to trachea （air-tube）, if the
mouse became cyanosis,
ip:
stop and try again. The maximal content is 1ml per mouse.
there are three points that you need to pay attention: position/ angel / draw back.. first the
position of injection is in the abdomen, not too high, not too low, if too high, liver may be hurt, if
too low, bladder may be hurt. Second, the angle should be about 45 degree. If too small, the drug
may be injected into subcutaneous layer. If too large(perpendicular), may hurt the organ in the
abdomen. Third, after the syringe needle has been in the abdomen, before injection, you should
draw back the stylet to see if can draw out something, if not , you can go on. If draw out blood or
urine, that shows you have fail.
4）rabbit:
ig: is a little difficult. To finish this process need at least two person. I will give you a model. One
person sit on the chair, and keep the rabbit. The other one give the drugs. We need to use mouth-gag
to open its mouth, and then use the catheter , insert the cathether into the stomach of the rabbit. Be
careful not insert the catheter into the trachea （air-tube）. You can test it using a glass of water. Put
the other end of the catheter in the water, if you can see bubble, that means you made a great
mistake. Extract the catheter and try again.
iv : is often used in the following experiment, you need to grasp it skillfully. There is a vein on the
border of the ear of the rabbit, this is the most suitable place for the iv injection. You should begin
your injection from the tip of the ear and proceed gradually.
7. Operation (rabbit)—artery catheterization
I want to introduce an operation that you also need to grasp. Because next time we will use it. It is
CCA（common carotid artery） intubation or catheterization of CCA. The animal we used is
rabbit.
First anesthetize the rabbit with 20% urethane（5-6ml/kg）, then fix it on the operating table. And
open the skin in the neck, then separate the CCA. Ligate at the proximal end of the heart with a
thread, ligate the other end of the CCA with an artery clamp. And incise a V shape orifice on the
artery with a ophthalmology operation scissors. And insert the artery catheter into the CCA, and
then fix it with a thread. Remove the clamp when needed.
Experiment 2 Determination of the Pharmacokinetic Parameters
of Phenol Sulfonephthalein(PSP)
[Aim] To determine the pharmacokinetic parameters of PSP after single quick intravenous injection
of PSP in a rabbit
[Principle] 1) Another name of PSP is phenol red.
In alkaline (basic) Solution it turns red. As we
all know that it can be used to indicate the pH of the solution.( pH indicator)
2) PSP is eliminated from kidney in the unchanged form in urine and belongs to one compartment
model, First-order elimination kinetics.
One compartment model: Drug distributed rapidly to the organs of tissues with high blood flow.
First-order elimination kinetics: Blood concentration of drug is reduced in equal rate; the
eliminated rate is direct ratio with blood concentration of a drug.
Formula: Ct=C0.e-ket
Change the Formula into logarithm: lgCt=lgCo-ke·t/2.303
“lgCt”and “t”are linear correlation
lgCo=a
-ke/203.3=b
Co= lg-1a (μmol/l) ke = －2.303b (min-1)
t1/2 (half-life time) is the time required to Change the amount of drug in the body by one-half during
elimination. t1/2 depends on drug physicochemical Character and body conditions.
t1/2=0.693/ke (min)
Vd (apparent volume of distribution) is used for measuring distribution range, relates the amount of
drug in the body to the concentration of dug in the blood.
Vd=Xo/Co (L)
CL (clearance) =Vd×k (L/min)
[Material]
1. Animal: rabbit
2.Drugs and agents:
6mg/ml PSP solution (MW: 354.36); 20% urethane (5-6ml/kg); Normal Saline, 500u/ml heparin
solution; diluent solution (9mg/ml NaCl 29ml+1mol/L NaOH 1ml)
3. Apparatus and instruments: operating instruments (13); Liquid handling equipment; Tubes;
colorimeters; etc.
[Method]
1. Mark the tubes: 0—5; (1) —blood
2.
(2)—plasma
Anesthetize rabbit. (1) weigh the rabbit (2) iv 20% urethane 5-6ml/kg. After giving one-half
of the anesthetic drugs, please take out the rabbit from the fixing box, and investigate the reflection
of the rabbit at the same time and continue giving drugs more slowly.
(3) fix the rabbit on the operating table
3. Operation: artery catheterization
First you need to anesthetize the rabbit with 20% urethane, then fix it on the operating table. And
open the skin in the neck, then separate the CCA. Ligate the CCA at the far end of the heart with a
thread, ligate with an artery clamp at the proximal end of the heart. And incise a V shape orifice on
the artery with a ophthalmology operation scissors. And insert the artery catheter into the CCA,
and then fix it with a thread. Remove the clamp when needed.
Points that should be paid attention to
(1) The injector and arterial catheter must be washed by heparin solution to protect the blood from
coagulation.
4. Getting 1.5ml blood before administration of PSP, then put the blood into the 0-tube containing
heparin, and mix it gently immediately. Pay attention to wash the injector: Water—NS—heparin,
inject 0.2ml heparin to the arterial catheter
5. Administration: 6mg/ml PSP—0.2ml/kg. Correctly and quickly
Record the time as soon as finished administration
6. Getting blood sample respectively at 2min, 5min, 10min, 15min, 25min after administration.
If you can not get blood at the planned time, please record the actual time.
7. Centrifugation
centrifuge at 1500rpm for 10min
8. Collect 0.2ml supernatant fluid (plasma) into the new corresponding tube, then add 3ml
diluent solution to each tube, the solution would display light or shade red (pink).
9. Colorimetric analysis
The plasmic PSP concentration was determined to OD value at 560nm （ namometer ） .
C=1.604+233.72OD
Experiment 3 Determination of ED50 of pentobarbital sodium
Purpose :
1. to determine thehypnotic ED 50 of intraperitonial injectiong of Pentobarbital sodium on mice
2. grasp how to divide group randomly
Principle:
1. the meaning of ED50
(1) To graded reponse, ED50 refers to the dose of a drug required to produce 50% of that
drug’s maximal effect.
(2) To quantal response, it means the dose at which 50% of individuals exhibit the special
quantal effect.
ED50 provide a convnient way of comparing the potency of drug in experiment.
In our present experiment, we want to know the dose of pentobarbital sodium (ED50) that
cause half of the mice to sleep.
2. Pentobarbital sodium the effect of it is dose-related.
The dose-effect relationship are very typical. In small dose, it will cause sedation, when the
dose increase, it will cause hypnosis. In high dose, it can cause anesthesia. In higher dose, it will
depress the CNS ans cause respiratory depression, even cause death of the animal.
3. positive index: The dissaperance of righting reflex (shows the mouse has sleeped)
4. The formula for the caculation of ED50 (Sun’s modified Kober’s method)
The conditions that needs to be satisfied when using this method to calculate ED50
(1) 4~8 different groups , usually more than 5 group
(2) the number of the animals are eaual in each group. Usually more than 10 every group
(3) the ratio of two adjacent dose is equal. The five doses are geometric progression
the formula: ED50= lg-1[ Xm-i(∑P-0.5) + i/4(1-Pm-Pn)]
P: response rate.
Pm: maximal response rate.
Pn: minimal response rate
Xm: Log dose of the Pm group.
i: log ratio of two adjacent dose
Materials .
1. animals :50 mice
2. drugs: five dose of pentobarbital solution( 2.45,1.96,1.57,1.25,1.00 mg/ml)
Methods
1. weigh the animals and staining
2. animal grouping : randomly 10 mice per group
3. drug administration: ip 0.2ml/kg
4. observe the number of sleeping mice within 15min of drug administration
Results:
Using the following table to caculate the ED50
Group
n
1
2
3
4
5
10
10
10
10
10
Drug concentration(mg/ml)
2.45
1.96
1.57
1.25
1.00
dose
(mg/kg)
Log dose
49
39
31
25
20
1.69
1.59
1.49
1.40
1.30
Number
of
sleeping mouse
P
Experiment 4 Influence of pH on the absorptive rate of strychnine
Purpose:
3. To investigate the different effects (quick or slow) produced by strychnine in different pH
solution per oral (ig) to mice.
4. To know about the influence of pH on the absorptive rate of strychnine.
Principle:
5. Most drugs are either weak acids or weak bases. Acidic drugs (HA) release a (hydrogen ion) H+,
and causing a charged anion (A-). HA
H+ +A-
Weak bases (BH+) can also release a H+, however the protonated form of basic drugs is usually
charged and loss of a proton, produces the uncharged base (B).
B + H+
BH+.
2. A drug passes through membranes more readily if it uncharged. AABB increase absorption;
ABBA accelerate excretion.
pKa-pH=lg（BH+ / B）
3. Strychnine is a weak basic CNS stimulant. It can excite spine selectively and cause convulsion
(legs or limbs are rigid), even die.
Materials .
3. animals: 24 mice
4. drugs: strychnine solution (pH =8.0 or 1.0); picric acid solution.
Methods
5. weigh the animals and stain them
6. animal grouping randomly:
7. drug administration:
ig
12 mice per group
0.3ml/10g, record the time after administration.
8. observe the effects of every mice and the time each mice appearing convulsion within 15min
after drug administration
Results:
group
n
strychnine solution (pH =8.0)
12
strychnine solution (pH =1.0)
12
number of convulsive mouse
The time each mouse appearing
convulsion
Experiment 5 Effects of Ach, atropine and BaCl2（barium chloride） on the
isolated ileum of guinea-pig
AIM:
1. to observe the effect of acetylcholine, atropine and BaCl2 on smooth muscle of ileum and
analyze the mechanism of action of atropine.
2. to grasp how to do experiment in vitro
Principles
1. M-R: distribution and effect when M-R is activated
To the ileum, the effect when M-R was stimualted is contraction of smooth muscle and increase
of tension of ileum.
Ach is an agonist of M-R, while atropine is an antagonist of M-R, so it can antagonize the
effect of Ach.
2. three conditions that an isolated organ to be alive in vitro:
（1） temperature: 32 ℃
to decrease its autonomic contraction
（2） gas: air is ok to ileum, no need oxygen
（3） nutrient solution: Tyrode’s solution (include glucose, Nacl, Kcl, Mgcl2 Cacl2,
NaH2PO4 etc)
to different organs, the specific conditions are a little different, today the condition that we need
to provide for the ileum is 32 ℃, air and tyrode’s solution
MATERIALS
1. drugs: Ach, atropine, BaCl2
2. animal:guinea pig
3. instruments: biolap98, tonotransducer，a series of eqiupments for experiment in vitro
Methods
1. set the temperature at 32 degree
2. setup the software named“biolap98”
enter “biolap98”---- choose number 1 channel----press “input signal”- “channel 1” -“tension”----press “start”.
Press a button to let the curve lower 5 divisions from the middle line
set two parameters:（1）amplification mutiple: 100 （2）scan speed: 25s/div
3. measure 20ml of tyrode’s solution and pour it into the bathing tube
4. prepare the samples: 2cm ileum
get a guinea pig, and beat its head, when it dies, open its abodemen and get its ileum out, wash it
with tyrode’s solution, and cut it into several part, the lengh of each section is about 2cm.
Be careful when prepare the samples. Do not hurt the ileum with too much force .
5. hang the sample diagonally . suspend the sample quickly and incubated it with tyrode’s solution
and air. The air bubble 2-4 every second
Sample: one end on L type hook, the other end on thread hook. Fix the L type hook on iron
stand, fix the thread on tonotransducer.
6. adjust the load:
Let the thread not too loose, not too tense.
adjust the height of L type hook and let the curve ascending two divisions and
fix the L type hook.
7. add the drugs to the bathing tube : 0.2ml each time.
The sequence is : Ach---- Atropine-----Ach------ BaCl2-----Atropine
First add Ach, when the effects of Ach reach its peak, add Atropine, when the curve returned to
stable, add Ach for another time. Add BaCl2 , when its action reach peak, add Atropine .
8. stop the experiment and save the results.
RESULTS
Disscusion: analyze the results.
.
Experiment 6 Anticoagulant Effects of Drugs in Vitro
AIM:
To compare the characteristic of different anticoagulant drugs and analysize their mechanism.
Principles:
Heparin enhance the effect of antithrombin III to inactivate the coagulant factor 2,
9，10，11，12，
so it can cause anticoagulant effect in vivo and in vitro.
Dicoumarin inhibit the modification or synthesis of the coagulant factor 2, 7, 9，10 in liver.
Sodium citrate solution always is used in storing blood, which produces anticoagulant effect by
combining calcium.
MATERIALS
drugs: Heparin，Dicoumarin，Sodium citrate solution，normal saline, potassium oxalate solution,
calcium chloride solution
animal: rabbit
Methods
3. Mark the tubes 0, 1, 2, 3.
Add drugs: add 0.25ml drugs into every tube respectively.
0----NS; 1---- Dicoumarin; 2----Heparin; 3----Sodium citrate solution
2. Anesthetize rabbit.
3. Operation: artery catheterization.
4. Get 5ml blood by injector washed by potassium oxalate solution and put 0.9ml blood into every
tube, then add 3mg/ml calcium chloride solution 0.1 ml at once, and after mixing gently, put the
tubes into 37centigrade water bath, record the time.
5. Observe: slope the tubes every 30 second gently. If the blood stops flowing, it is coagulant.
Please record the time.
6. If the blood in the tubes will never appear coagulant, add another high concentration 10mg/ml
3mg/ml calcium chloride solution 0.1 ml, go on observing just like before.
RESULTS
Disscusion: analysize the results.
Experiment 7 Effects of drugs on arterial blood pressure of anesthetic rabbit
AIM: to observe the effects of autonomic drugs on rabbit arterious blood pressure
Principles
1. heart: M-R and β1-R
vascular smooth muscle: α-R andM-R, β2-R
2. autonomic drugs include drugs acting on cholinoceptor and drugs acting on adrenoceptor. These
drugs exhibit their effcts through binding to receptors.
Materials:
1. animal: rabbit
2. drugs : Ach, neostigmine, atropine, NA, AD, Isop, phentolamine, propranolol
3. eqipmemts: BL-410, operating instruments, artery catheter, trachea catheter, pressure transducer
Methods
1. setup the software named“BL-410”
enter “BL-410”---- choose number 1 channel----press “input signal”- “channel 1” -- “tension”
----press “start”.
2. anesthetize the rabbit: 20% urathene 5~6ml/kg
3. operation:
（1） trachea catheterization
（2） CCA catheterization
（3） Fix the scalp needle ( for drug administration )
4. Drug administration: iv through the scalp needle
Group A
（1） Ach 0.1ml/kg
（2） Neostigmine 0.1ml/kg
（3） Ach 0.1ml/kg
（4） Atropine 0.4ml/kg
（5） Ach 0.1ml/kg
Group B
（1） AD 0.1ml/kg
（2） NA 0.1ml/kg
（3） Isop 0.1ml/kg
（4） Phetolamine (regitine)
0.2ml/kg slowly
（5） 5min later, repeat （1）（2）（3）
Group C
（1）AD 0.1ml/kg
（2）NA 0.1ml/kg
（3）Isop 0.1ml/kg
（4）propranolol
0.1ml/kg slowly
（5）5min later, repeat （1）（2）（3）
Results:
Print the figure and analyze the effects of the drugs
Experiment 8 Effects of Drugs on the isolated Guinea-pig Tracheal Ring
AIM:
To observe the effects of drugs on tracheal smooth muscle and select the asthmatic drugs.
Principles:
3. There are M-R, H1-R, β2-R in tracheal smooth muscle.
4. When M-R and H1-R are stimulated, the tracheal smooth muscle will be contraction and the muscular tension
will be increased.
When β2-R was stimulated, the tracheal smooth muscle will be relaxation and the muscular tension will be
decreased.
5. The conditions for an isolated tracheal ring to be alive in vitro:
（1） temperature: 37 ℃
（2） gas: oxygen
（3） nutrient solution: Krebs-Henseleit’s solution
MATERIALS
4. drugs: histamine, isoprenaline, Ach, aminophyline, propranolol
5. animal: guinea pig
6. instruments: bl-410E software, tonotransducer，a series of eqiupments for experiment in vitro
Methods
4. Set the temperature at 37 centigrade.
5. Start the software names“BL-410E”
enter “BL-410E”---- choose number 1 channel----press “input signal”-“channel 1” -- “tension”----press
“start”.
set parameters: amplification mutiple: 20
3. Measure 15 ml of Krebs-Henseleit’s solution and pour it into the bathing tube
4. Prepare the samples: 0.5cm tracheal, get a guinea pig, and beat its head, when it dies, get the tracheal out, and
cut it into several part, the lengh of each section is about 0.5 cm. Be gentle when prepare the samples. Do not
hurt the ileum with too much power.
5. Hang the sample parallel. Hang the sample quickly and incubated it with K-H’s solution and oxygen, Make
the air bubble continuously (2-3 every second).
Sample: one end on L type hook, the other end on thread hook. Fix the L type hook on iron stand, fix the
thread on tonotransducer. Let the thread not too loose, not too tense.
6. Adjust the load: adjust the height of L type hook and let the curve ascending 2g and fix the L type hook.
7. Add the drugs to the bathing tube: 0.15 ml each time. Firstly, stabilize 15 min
The sequence is:
Ach (quiescently scan) ---- reach its peak (need about 5min-10min, then scan 1cm distance.)----Isop (like
before)
clean 3 times, stabilize 10 min
HA-----Amino
Prop------ Isop-----Amino
8. Stop the experiment and save the results.
RESULTS
Disscusion: analyze the results.