【Animal Experiment】-Establishment of Morphine Dependence Rat Model
动物实验,大鼠模型
z-bo
2022-02-03
845
1. The purpose of the experiment
(1) Familiar with the method of establishing morphine-dependent rat model, and copy the morphine-dependent rat model.
(2) Observe and score the withdrawal symptoms of morphine-dependent rats to determine whether a morphine-dependent rat model has been successfully established.
2. Experimental materials and methods
(1) Experimental materials
1. Required equipment
(1) Glass specimen cylinder with lid (height 45 cm or more, diameter 30 cm): observe the withdrawal symptoms of rats.
(2) Weigh the mouse on a standard electronic balance.
(3) Squirrel cages and related facilities (including drinking water, feed and bedding)
2. Reagents needed
(1) Morphine hydrochloride injection: 1 ml specification is 10 mg x 10 tablets/box, batch number is H21022436, stored at room temperature.
(2) Morphine hydrochloride (injectable as a poison), "School Poison Management Regulations", including pre-approved use plan, purchase, daily management and use time of double lock dual safety management and strict compliance with regulations
(3) 0.9% sodium chloride injection: 500ml/bottle, batch number H20056626, stored at room temperature.
(4) Naloxone Hydrochloride Injection: The specification is 1ml x 5 tablets/box, 0.4mg, and the batch number is H20055758. Store at room temperature.
3. There are no special reagent preparation requirements for the preparation of reagents.
(II) Experiment-specific methods and procedures
1. Experimental animals: 40 healthy SD rats, weighing (260±20) g, were provided by the Experimental Animal Center of the School of Experimental Animal Science, Kunming Medical University, half of the males and half of them served. According to the principle of random grouping, they were divided into blank control group and morphine dependent group. In addition, the morphine-dependent component is divided into four subgroups: a 1-week morphine-dependent group, a 2-week morphine-dependent group, a 4-week morphine-dependent group, and an 8-week morphine-dependent group. In other words, the experimental rats were divided into 5 groups, 8 in each group, and they were placed in separate cages, where they could eat and drink freely. Observe and record their activities, food intake, water consumption, stools, etc. 2. Experimental methods Rats in the morphine-dependent group were injected subcutaneously with morphine hydrochloride for three consecutive days (8:00, 12:00, 16:00) every day for 5 consecutive days. The dose of each injection is 5 mg/kg on day 1, 10 mg/kg on day 2, 20 mg/kg on day 3, 40 mg/kg on day 4, and day 5 50 mg/kg. Yes. Before each injection, it should be thoroughly disinfected, and the animal should be weighed once a day with a disposable syringe. The blank control group was injected with the same dose of saline. Four groups of morphine-dependent rats were randomly selected on the 6th day of each group. Two rats were injected subcutaneously with naloxone hydrochloride 5 mg/kg 2 hours before the morphine injection to promote the experiment of inducing withdrawal symptoms, and they were placed in a glass specimen jar to observe the withdrawal symptoms. Then, given a score, the empty control group is given the same treatment and a given score.
After confirming morphine addiction by ine, perform 1 week of morphine addiction group, 2 weeks of morphine addiction group, 4 weeks of morphine addiction group and 8 weeks of morphine addiction group every day, subcutaneous injection of 30 mg/kg into the back once a day twice a week Morphine hydrochloride. Four, four and eight weeks were established in the four different periods of the morphine-dependent rat model.
Countless symptoms include drooping, salivation and diarrhea. During the observation process, ptosis was evaluated every 2 minutes, and other uncountable symptoms were scored according to the severity. The asymptomatic score is 0 points, mild is 1 point, and severe is 2 points. The total withdrawal score is obtained by summing the symptoms. All rats shouted 2 points during the contact. The sum of all symptom scores is the total withdrawal score.
(3) Precautions for each step of the experimental operation (1) On the 5th day of the first phase of the morphine injection experiment in rats, especially on the 3rd day (the injection dose is 10 mg/increase from kg). Maximum 20mg/kg), and on the 4th day (increase the injection dose from 20mg/kg to 40mg/kg). Due to the rapid change in dosage, some animals may die due to drug intolerance. Careful observation is needed to reduce animal mortality.
(2) Pay attention to aseptic operation during injection. Before injection into the rat's dorsal skin, the hair should be shortened to expose the skin, the skin should be sterilized with 75% medical alcohol, and a 1 ml disposable syringe should be used for injection.
(3) Every time you use a glass bottle to observe withdrawal symptoms, you need to wipe it carefully with 75% medical alcohol and observe another animal. The purpose is to clean and remove peculiar smell, and avoid interference with the previous experimental rats in the subsequent experimental rats.
3. Experimental results
Observe and record the withdrawal symptoms of the two groups of rats. Rats in the morphine-dependent group had obvious withdrawal symptoms, which occurred significantly higher than the control group. After the t test, there was a significant difference between the two groups (P\u003c0.05 or P\u003c0.01), which was based on the rat model that proved the successful establishment of morphine. Countless withdrawal symptoms were observed (trembling like a dog, stretching, fur cleaning, swallowing, standing, jumping, tooth tremor) and countless symptoms (sagging, saliva). Secretions, diarrhea, etc.) Maldonade, etc. Scores are recorded according to methods and scoring standards, the number of countable symptoms during the observation period is recorded, and the scores are scored according to the exit scoring standards.
Analysis of the four results
Increased daily, subcutaneous injection of morphine hydrochloride injection and naloxone hydrochloride injection induced withdrawal symptoms, and scored the withdrawal symptoms. The control group and the compliance group successfully established a morphine-dependent rat model. The advantages of this method are simple operation, low animal mortality, intuitive observation of withdrawal symptoms and strong operability of the scoring standard. It is an excellent method to establish a morphine-dependent rat model..
5. Experience and experience
(1) This experiment is based on observing countless withdrawal symptoms and countless withdrawal symptoms in experimental rats, and comparing the scoring results. Successfully established.
(2) There are two issues that require special attention during the experiment. One is the large change in the injection dose on the 3rd and 4th day of the above experiment, which requires close attention and observation to reduce animal mortality. Please note that the withdrawal symptoms are scored by trained personnel who are not part of the study group. Avoid the interference of subjective factors.
