动物实验教程(英文版).doc

PHARMACOLOGY EXPERIMENTINSTITUTE OF PHARMACOLOGYExperiment 1 The basic knowledge and technique of pharmacological experiment1. the purpose or aim for us to have this classlearn 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 experimentthere are many laboratory animals. In different experiment, we need to choose the appropriate animals. Next we will talk about the animals and their character1）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 animalin 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 animalsDuring 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 glove3）guinea pig: is easy to deal with. You can grab it anyway.4）rabbit: 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 methodsometime 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 anesthesiainhalation anesthesia: ether injection anesthesia: pentobarbital sodium/ urethane ip/iv6. drug administration1）how to use an injector/ or syringe2）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, stop and try again. The maximal content is 1ml per mouse.ip: 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 catheterizationI 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 Parametersof Phenol Sulfonephthalein(PSP)Aim To determine the pharmacokinetic parameters of PSP after single quick intravenous injection of PSP in a rabbitPrinciple 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-ketChange the Formula into logarithm: lgCt=lgCo-ket/2.303“lgCt”and “t”are linear correlationlgCo=a -ke/203.3=bCo= lg-1a (mol/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) =Vdk (L/min)Material1. Animal: rabbit2.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.Method1. Mark the tubes: 05; (1) blood (2)plasma2. 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 table3. 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: WaterNSheparin, inject 0.2ml heparin to the arterial catheter5. Administration: 6mg/ml PSP0.2ml/kg. Correctly and quicklyRecord the time as soon as finished administration6. 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 10min8. 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+233.72ODExperiment 3 Determination of ED50 of pentobarbital sodiumPurpose : 1. to determine thehypnotic ED 50 of intraperitonial injectiong of Pentobarbital sodium on mice2. grasp how to divide group randomlyPrinciple:1. the meaning of ED50(1) To graded reponse, ED50 refers to the dose of a drug required to produce 50% of that drugs 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 (Suns modified Kobers method)The conditions that needs to be satisfied when using this method to calculate ED50(1) 48 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 progressionthe formula: ED50= lg-1 Xm-i(P-0.5) + i/4(1-Pm-Pn)P: response rate. Pm: maximal response rate. Pn: minimal response rateXm: Log dose of the Pm group. i: log ratio of two adjacent doseMaterials . 1. animals :50 mice2. drugs: five dose of pentobarbital solution( 2.45,1.96,1.57,1.25,1.00 mg/ml)Methods1. weigh the animals and staining2. animal grouping : randomly 10 mice per group3. drug administration: ip 0.2ml/kg4. observe the number of sleeping mice within 15min of drug administrationResults:Using the following table to caculate the ED50Group nDrug concentration(mg/ml)dose (mg/kg)Log doseNumber of sleeping mouseP1102.45491.692101.96391.593101.57311.494101.25251.405101.00201.30Experiment 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-). HAH+ +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 mice4. drugs: strychnine solution (pH =8.0 or 1.0); picric acid solution.Methods5. weigh the animals and stain them6. animal grouping randomly: 12 mice per group7. drug administration: ig 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 administrationResults:groupnnumber of convulsive mouseThe time each mouse appearing convulsionstrychnine solution (pH =8.0)12strychnine solution (pH =1.0)12Experiment 5 Effects of Ach, atropine and BaCl2（barium chloride） on the isolated ileum of guinea-pigAIM: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 vitroPrinciples1. M-R: distribution and effect when M-R is activatedTo 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: Tyrodes 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 tyrodes solutionMATERIALS1. drugs: Ach, atropine, BaCl2 2. animal:guinea pig3. instruments: biolap98, tonotransducer，a series of eqiupments for experiment in vitroMethods1. set the temperature at 32 degree2. 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 lineset two parameters:（1）amplification mutiple: 100 （2）scan speed: 25s/div3. measure 20ml of tyrodes solution and pour it into the bathing tube4. 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 tyrodes 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 tyrodes 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. 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 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 a