动物造模 z-bio 2021-10-09 378

　　All opioids (narcotic analgesics such as morphine) are addictive, including drug resistance and physical addiction. Drug resistance means that with repeated administration, the analgesic effect is weakened, and the amount of the drug needs to be increased to obtain the initial analgesic effect. When the painkillers are suddenly stopped, people and animals will salivate, vomit, dilate pupils, difficulty breathing, intense central agitation and various expressions of unbearable pain. These abnormal physical and behavioral dysfunctions, called "withdrawal symptoms" or physical dependence, indicate addiction. Currently, there are three common experimental methods that can be used to assess physical dependence on opioids in the world.

　　1 Classification dependent on experiment

　　1.1 Spontaneous withdrawal test (Spontaneous withdrawal test)

　　Continuous and gradual administration of experimental animals (small animals, rats, monkeys), stop the administration, and stabilize for a certain period of time. The time before taking the medicine. After that, the administration was stopped abruptly, and the animals' withdrawal symptoms were observed and recorded. .1.2 Precipitation withdrawal test

　　In a relatively short period of time, a large number of test drugs were used on animals, and then opioid antagonists were used to promote the development of withdrawal symptoms. reaction. If the animal has morphine-like withdrawal symptoms, it means that the study drug is of the same type as morphine. It promotes experimental withdrawal symptoms, severe and typical symptoms, and has a rapid onset in a short period of time, making it easy to observe and compare.

　　1.3 Alternative Test

　　After taking a certain amount of standard drugs (such as morphine) for a period of time, animals will become physically dependent. After the physical dependence increases, the standard drugs will be stopped, and the study drugs will be used to observe whether the animals show withdrawal symptoms. After replacing the study drug, if there are no withdrawal symptoms, it indicates that the physical dependence of the two drugs is similar. This type of testing is also called cross-physical dependency testing.

　　Animals, whether it is natural withdrawal or forced withdrawal, will show a series of signs or symptoms of varying degrees, namely withdrawal syndrome. However, not all withdrawal symptoms or symptoms appear in experimental animals. Therefore, it is necessary to consider several indicators in order to comprehensively evaluate each symptom, such as time, frequency, and severity, in different animals and different experiments.

　　1.4 Grading of withdrawal symptoms

　　Monkey is an internationally recognized experimental animal model used to assess physical dependence on opioids. The grading of withdrawal symptoms is based on a systematic study of morphine and heroin given to rhesus monkeys by Seevers in the 1930s after long-term continuous administration. It includes a variety of withdrawal symptoms and symptoms of autonomic function, physiological function and behavioral changes.

　　1. Grading of withdrawal symptoms () (1) Mild symptoms include fear, yawning, tearing, tremor, flushing, sweating, crying, lack of morale, loss of appetite, and loose stools. (2) Moderate animals exhibit intention tremor, loss of appetite, erect hair, muscle cramps, abdominal retention, diarrhea, general malaise and lying position.

　　(3) Extreme agitation, lying with eyes closed, abnormal posture, vomiting, pale complexion and obvious muscle cramps exacerbate the severe symptoms.

　　(4) Very severe fatigue (including facial expressions, difficulty breathing, severe dehydration), weight loss of more than 10% or even death.

　　2. Scoring criteria (level differences and symptom points)

　　(1) The slight grade difference is 5 points, each symptom is 3 points, the same symptoms are repeated within 1 day, and a 1-point countdown is performed.

　　(2) The moderate difference is 10 points, and each symptom is 4 points. If the same symptom recurs within a day, the time will be reduced by 1 point.

　　(3) The serious score difference is 17 points, and each symptom is 5 points. If the same symptom recurs within a day, the time will be reduced by 1 point.

　　3, scoring standards

　　The

　　score is a comprehensive judgment based on symptoms and grades. The score for each symptom should be different. The number of symptoms of the same grade must be different, but the score should not exceed the difference. Grade value.

　　The three secondary symptoms are equal to a higher level of symptom score, for example, three mild symptom scores (5 + 3×3) = a moderate symptom score (10 + 4).

　　3 points for moderate symptoms (10 + 4×3) = 1 point for severe symptoms (17 + 5). If the animal is exhausted or dead, no other symptoms are visible, so the given score corresponds to the sum of mild, moderate and severe symptoms.

　　Below, we will separately describe commonly used animal experimental models.

　　2 Spontaneous withdrawal test

　　Spontaneous withdrawal test is a chronic test in which the study drug is usually given in increased or fixed doses. The administration method can be injection (subcutaneous, intramuscular, intraperitoneal and intravenous), tube feeding, transplantation of drug tablets, and can be administered by mixing and drinking.

　　2.1 Spontaneous withdrawal test in mice (mice)

　　"Mice are mainly administered by mixing and drinking water. Male and female mice weighing 20-22 g were divided into a blank control group, a positive drug control group and a test drug group. For positive drugs, morphine hydrochloride or codeine powder can be mixed with powder (morphine hydrochloride: 1.0 mg/1 g powder, codeine phosphate: 2.0 mg/1 g powder) and administered for 7 days. On the 8th day, we pulled out the mice and started feeding them a drug-free diet. The mice were weighed one day before refueling, and weighed every 4 hours after refueling until they returned to normal. Calculate the weight loss percentage of mice at each time point after withdrawal, and perform t test between each group.

　　2.2 Spontaneous withdrawal test in rats (rats)

　　(1) Healthy rats weighing 200-220 g were injected subcutaneously. There are 10 animals in each group, half are males and half are males. The experimental animals were randomly divided into a negative control group, a positive drug control group and a test drug group, and the positive drug was morphine. The morphine-dependent rat model was formed by a dose-escalation method, and the duration was 1 month. The method of administration is subcutaneous injection of morphine (sc) 3 times a day. For the first two weeks, the model dose is 5 days, 10 mg/kg body weight, for 4 days, 15, 20 mg/kg body weight, for 3 days, and the injection dose is 0.1 ml/100 g body weight. The doses in the 3rd and 4th weeks were 30 mg/(kg body weight? D). The rats were weighed the day before the morphine was stopped, and changes in body weight were observed on the 29th day after the morphine was stopped. The body weight was measured 3 times a day for 4 consecutive days. Calculate the average weight change rate of each group of rats, and perform statistical tests to assess whether the study drug is physiologically dependent. (2) Healthy rats ingest mixed drug food, weighing 200-220g, each group of 10 animals, half male and half male. The experimental animals were randomly divided into a negative control group, a positive drug control group and a test drug group. The positive drug can be morphine hydrochloride or codeine phosphate. The test drug morphine or codeine is mixed into the powder. Usually, each gram of powder contains 1.0 mg of morphine hydrochloride or 2.0 mg of codeine phosphate. Weigh the consumed powder and replace it once a day. The rats were fed the mixed powder for 1 week to form a dependence model. On day 8, the rats were replaced with morphine or codeine-free powder, and the rats spontaneously showed withdrawal symptoms. The positive control continued to receive powder containing morphine or codeine. The rats were weighed every 4 hours for 3 days after stopping the drug. Calculate the average weight change rate of each group of rats, and perform the t test between each group.

　　After withdrawal from morphine, the weight of the rats dropped sharply. This is most obvious in 24-36 hours and is the best indicator of the body's dependence on morphine. The body weight of the rats in the test drug group was compared with the body weight of the rats in the positive and negative control groups to determine the possibility of physical dependence. Weight loss is relatively objective, quantitative, and accurate, and is widely used as an indicator of physical dependence.

　　2.3 Study on spontaneous withdrawal of monkeys (monkeys)

　　(1) Before entering the clinic, the use of new painkillers is because the dependence of painkillers on monkeys is similar to that of humans, and the withdrawal symptoms are more obvious and easier to observe, so this is a necessary experiment. Guangxi Zhuang Autonomous Region (Rhesus subspecies) is a common animal, weighing 4-6 kg, and each group has 3 or more animals, male and female.

　　(2) Experimental method

　　1) The Siever method has two basic methods. One is the rapid gain method, in which the initial dose of morphine is 10 mg/kg body weight and 10 mg/kg body weight per day. .. It reached a maximum of 115 mg/kg body weight in about 6 weeks, and the monkeys showed severe withdrawal symptoms after stopping the drug. The second is the dose of morphine, the total dose and a rapid increase after 6 days. -Similarly, more than 12 months, the above-mentioned withdrawal symptoms will still occur even after the drug is stopped.

　　2) Yanagta method is an improved Seevers method. Morphine was injected subcutaneously every 6 hours at a dose of 3.0 mg/kg body weight and administered continuously for at least 60 days to enable monkeys to form an experimental model of morphine dependence.

　　3) Comprehensive method of subcutaneous injection of morphine 3 times a day, maintaining 3, 6, and 10 mg/kg body weight from the first week to the third week, and 15 mg/kg body weight from the fourth week. Until 90 days. (3) Experimental procedure The monkeys were randomly divided into a negative control group, a positive drug control group and a test drug group. Study drugs are usually divided into three dose groups. The low dose generally adopts the clinical dose. For the high-dose group, select the corresponding dose according to the characteristics of the drug. Drugs that are less likely to be addictive should choose a dose close to the toxic reaction. Maximum tolerated dose; the dose of the middle dose group is between the high dose and the low dose. Study drugs can be administered at a constant dose or in increased doses. The animals in each group were given continuous administration for up to 90 days, and the drug was discontinued. After stopping the drug, the patient was observed continuously for 7 days, 3 times a day. Record and weigh the various behaviors of the monkeys. We calculated the rate of weight change based on the degree of withdrawal symptoms.

　　3 Precipitationwithdrawaltest

　　An important issue of Precipitationwithdrawaltest is the choice of opioid receptor antagonists. The antagonist itself must be very specific, have no other pharmacological effects, and do not interfere with the evaluation of withdrawal symptoms. Antagonists are usually given parenterally to make the withdrawal reaction the fastest, so they are also the most likely to cause the withdrawal reaction.

　　3.1 Mouse prompt withdrawal test (mice)

　　Mouse prompt withdrawal test is a commonly used screening model to assess physical dependence on opioid analgesics.

　　1. Mouse erect test (erectthetailtestinice)

　　(1) The experimental method is to divide 20-40 mice of 14-16 g into two groups, and subcutaneously inject morphine (15 mg/kg body weight) to the recipient. Try medicine. Fifteen minutes after administration, animals in the morphine group showed a vertical tail, continued to walk, and tended to recover only 2-4 hours later. After each group of animals continued to use the drug, the vertical tail phenomenon of the morphine group was more obvious. After taking the medicine for 14-28 days, the dose was increased to 44 mg/kg body weight. Then, allyl morphine was injected subcutaneously at a body weight of 16 mg/kg, and the morphine and the test drug were injected separately. The erection on the tail of the morphine animal disappeared and gathered together. This is very similar to the normal saline control group. Observe the study drug group in parallel with the morphine group to observe whether the animal's external symptoms are resisted by allylmorphine, and to determine whether it is a morphine-type drug. For example, tetrahydropalmatine has been proven not to belong to the morphine type.

　　Take another 40 mice and divide them into two groups. After subcutaneous injection of the study drug and morphine, another experiment was performed. For example, the study drug is tetrahydropalmatine (80 mg/kg body weight), the dose is gradually increased to 200 mg/kg body weight on day 21, and morphine increases from 35 mg/kg body weight to 53 mg/kg body weight... . From the 22nd day after the injection of 53 mg/kg body weight of morphine to the tetrahydropalmatine group, the animals showed excitement and a vertical tail, showing a morphine-like effect. After injecting 200 mg/kg body weight of tetrahydropalmatine into the morphine group, the animals showed severe central pressure, their limbs relaxed, and the animals were placed in a resting cage. The nature of the effect is similar to tetrahydropalmatine. (2) Model characteristics: The tail response of mice parallels the addiction of narcotic analgesics. Addictive drugs such as heroin and morphine have a large tail erection index, while drugs with less addictive properties such as codeine have a small tail erection index. Antagonists such as morphine do not make the tail erect and can antagonize the erect tail response of morphine. This is an easy way to quickly identify drugs.

　　2. Mouse jumping experiment (jumptestinmice)

　　(1) The experimental method uses 20-25g male mice, 10 mice in each group. Morphine was injected subcutaneously every 12 hours at a dose of 100 mg/kg body weight for a total of 14 times. The addiction is 6 hours after the last injection of morphine. Subcutaneous injection of naloxone at a dose of 5 mg/kg body weight can cause poisoning. The mice were immediately placed on a table with a height of 35 cm and a diameter of 30 cm. The number of animals jumping within 30 minutes after the naloxone injection was observed. Then observe and record every 1.5, 3, or 5 hours. Withdrawal symptoms are most severe within 30 minutes after naloxone injection. In addition, it was observed that the weight of the mice decreased significantly, the rectal temperature decreased (90-180 minutes), and spontaneous activity and resistance increased. (2) Model characteristics This method is based on the fact that naloxone can induce mice to jump to assess their dependence on analgesics. Naloxone-induced mouse beating is a sensitive indicator for studying morphine withdrawal, but this is not a unique phenomenon that depends on morphine and other painkillers, and can be inhibited by other drugs. Due to the short duration of action of naloxone, withdrawal symptoms should be observed within 30 minutes after naloxone injection. It is also possible to determine half of the effective dose of naloxone (ED50) that triggers the jump, and the ED50 value can be used to determine the physical dependence of the study drug. This method can be used to screen the physical dependence of analgesics. Its advantages are economy, simplicity and speed, clear indicators and high sensitivity. 3.2 Rats Rats were treated with opioid receptor antagonists after becoming morphine dependent. Within a few minutes, the animal showed tears, salivation, diarrhea, clenched teeth, violent restlessness, and abnormal posture. Withdrawal symptoms and weight loss gradually. About 2 hours after the administration of the antagonist, the withdrawal symptoms basically disappeared, and the weight loss no longer continued. (1) Experimental method Healthy rats, weighing 200-220g, each group of 10 animals, half of the male and half of the female. They were randomly divided into a negative control group, a positive drug control group and a test drug group. The morphine dependence model is formed by a dose escalation method, usually using a one-week or two-cycle test model. Morphine was injected subcutaneously at 0.1 ml/kg body weight 3 times a day, and the doses were 5 and 10 mg/kg body weight for 4 days and 15, 20 mg/kg body weight for 3 days. At 8:00 am on the 15th day, morphine was subcutaneously injected 40 minutes after the last opioid receptor antagonist to promote withdrawal. The usual dose of naloxone is 4 mg/kg body weight. Immediately put the rat in a transparent plexiglass container, observe and record the rat’s withdrawal symptoms within 1 hour, within 30 minutes and 60 minutes

　　Weigh the rats and record the withdrawal symptoms. You can also take morphine multiple times within 2 days, then inject naloxone, and then use a rapid addiction method to encourage withdrawal.

　　(2) Score of withdrawal symptoms in rat impulse test

　　1) Behavior

　　Abnormal posture: 2 points

　　Vigorous stirring, touch: 1 minute; approach: 2 minutes

　　Intermittent teeth: 0.5 points; continuity: 1 point 2) Autonomic nervous system symptoms

　　Flow 4 points

　　Diarrhea and loose stool: 4 points; Irregularity: 8 points

　　Saliva secretion is mild: 1 point; obvious: 2 points

　　3.3 The monkeys that established the model are the same as the natural withdrawal experiment. For subcutaneous administration, the starting dose is 3 mg/kg body weight every 6 hours, and the dose can be gradually increased and lasted for 15 or 30 days. Two hours after the last morphine injection, naloxone 0.1 mg/kg body weight or allylmorphine 1.0 mg/kg body weight was subcutaneously injected according to the monkey withdrawal symptom classification criteria, and the animals’ withdrawal symptoms were observed and recorded within 2 hours. Do it. Find the average withdrawal score and weight loss rate for each group of monkeys. Generally speaking, the severity of withdrawal symptoms is related to the duration of morphine administration. From the time of administration of test drugs and standard drugs to monkeys to the dose of opioid antagonist needed to promote withdrawal symptoms, the nature and potential of the test drug to cause physical dependence can be determined. The new drug will be tested according to the above-mentioned morphine monkey impulsive withdrawal test procedure to provide information on the physical dependence of the drug. Combined with the results of other tests, you can predict potential physical dependence.

　　4Substitutiontest (substitutiontest)

　　Substitution test is to study the ability of drugs to suppress opioid withdrawal symptoms. By comparing the ability of drugs with different dependencies to inhibit withdrawal, it provides an experimental basis for the physical dependence of the test drug and morphine. 4.1 Mice replacement test (mice) Randomly divide healthy mice into several groups, and use the above spontaneous withdrawal method to establish a morphine dependence model. After stopping morphine, mice in different groups were given morphine, L-methadone, pethidine (meperidine), dihydromorphine and other analgesics and research drugs. Compared with the above narcotic analgesics, the The study drug is withdrawn and has the ability to suppress the response. It is determined. The physical dependence of the test drug is similar to that of morphine. 4.2 Rat substitution test (rat) The various techniques described above can be used to establish experimental models of morphine-dependent rats. After morphine withdrawal, use research drugs instead of morphine (mouse experimental methods can also be used, using morphine, L-methadone, pethidine, dihydromorphine and other analgesics and research drugs), and the quantitative observation of withdrawal research is based on the design Withdrawal scoring standard, the degree to which the tested drug inhibits withdrawal.

　　4.3 Monkey substitution test (monkey)

　　The

　　monkey substitution test is the most common method to assess whether a study drug has a morphine-like effect. Morphine was injected subcutaneously into monkeys twice a day. The body weight was 9 mg/kg on the first day and 53 mg/kg on the 14th day. The body weight was gradually increased and the dose was maintained. 14 to 21 days after administration (total dose is about 497-631 mg/kg body weight), monkeys will be poisoned by morphine. Before injecting morphine every day, I often feel irritable. As the experimenter approaches, the forelimbs will automatically stretch so that the experimenter can administer the drug, and then move freely after the administration. The drug was stopped 16 hours after the last injection of morphine, and allyl morphine was injected subcutaneously at 2.8-7.9 mg/kg body weight for a poisoning study. A few minutes after the administration, the monkey showed withdrawal symptoms, initially showing tremor and nausea, and then lay on the floor and rolled and screamed. This is most noticeable in 10 to 20 minutes. At this time, inject the test drug (equivalent to morphine) subcutaneously to observe whether it can replace morphine to solve the withdrawal symptoms. All alternative methods show that painkillers are also addictive and belong to the same type of narcotic effect as morphine. Otherwise, it does not belong to this type. For example, in the replacement experiment, tetrahydropalmatine attenuated morphine withdrawal symptoms, and continued to produce withdrawal symptoms were not observed, resulting in severe breathing difficulties and death in the monkeys. If the replacement is ineffective, morphine will be injected. Within 3-5 minutes, the withdrawal symptoms will be significantly reduced or completely eliminated, and they will return to their original state and move freely.

　　5 other withdrawaltest

　　5.1 Bodyshakingtestinrats

　　(1) Experimental method Male rats weighing 300-350 g. After anesthesia, the catheter is placed in the neck of the jugular vein and connected to the outdoor injection system via a piston. The animals were kept in cages throughout the experiment. Morphine is dissolved in sterile saline and administered gradually for at least 5 days. On day 1, 1.5 mg/kg body weight of morphine was injected every hour as a 24-hour maintenance dose. Increase the dose every day, and inject 15 mg/kg body weight of morphine every 4 hours on the 5th day. Starting from day 6, a maintenance dose of morphine of 15 mg/kg body weight was injected every 6 hours. At this time, the rats had obvious tremor responses during each injection interval. Injection of morphine 15 mg/kg body weight can eliminate this reaction. This wobble response is a reliable indicator of withdrawal symptoms because it can be observed repeatedly in all rats. Six hours after the morphine injection, 30 minutes of observation can be used to count body shaking. In the control group, the number of shakers with or without saline injection was observed in the same manner. Another morphine control group (15 mg/kg body weight 30 minutes after intravenous injection) observed its effect.

　　(2) Model features Body swing is a feature of morphine withdrawal in rats. When a fallen dog lands and spins water, the head and body swing similarly. The jitter phenomenon is as follows. The swaying body is like a wet dog. When rats become morphine-dependent, the dopamine receptors in the brain become allergic, and the function of neurotransmitters outside the pyramidal tract becomes imbalanced, causing the body to tremble. Since this phenomenon has a certain relationship with morphine withdrawal symptoms, it has been used as one of the indicators to observe the morphine addiction test. It should be noted that the body tremor response is only a parallel indicator of morphine withdrawal symptoms, not a specific effect. Because DA receptor agonists and antagonists can affect the overactive DA receptors in the brain, false positives or false negatives that may appear in the experiment should be ruled out.

　　5.2 AttackTestInTats

　　(1) Experimental method: male rats, weighing 250-300 g. The dose of morphine was increased in accordance with regulations, and intraperitoneally injected 3 times a day, 15 mg/kg body weight per day on the first day, and 15 mg/kg body weight per day thereafter. On the 9th day, the body weight was 405 mg/kg body weight. At this time, each injection of morphine was 135 mg/kg body weight. After the maintenance dose for 3-5 days, the morphine injection was stopped. Two hours later, the rats showed withdrawal symptoms such as weight loss, body tremors, drooping eyelids and twisting. At this time, if the rats live together in the same cage, they will attack each other. Challenge experiments are usually performed 3 days after the last morphine injection. Place four mice in the same cage, observe for one hour, and record the number of times the battle has passed (ie, the front limbs are in contact in the air, the heads face each other, and the hind limbs stand during the battle). Attack and bite each other.

　　(2) Model feature: Positive reaction is also one of the observation indicators of addiction experiment. This experiment actually reflects the changes in brain dopaminergic nervous system function caused by morphine poisoning. The withdrawal symptoms that appear after the drug is stopped are symptoms of high dopaminergic activity. Agonists may enhance the seizure response, while antagonists may weaken the seizure effect. Therefore, in order to use aggressive testing as an indicator of withdrawal symptoms, it is also necessary to eliminate false positives or false negatives caused by drugs interfering with the dopamine function system. The time course of epilepsy symptoms does not exactly match other withdrawal symptoms. After morphine withdrawal, autonomic and physical symptoms appeared 24 hours after the last dose, and the intensity of symptoms decreased significantly at 72 hours. Aggression was not obvious 24 hours after the last dose, and was most pronounced at 72 hours. Even if the other withdrawal symptoms are reduced to the lowest level, the seizures are very strong, and even 30 days after the last medication, the seizures are also very strong. Therefore, observation should be made 72 hours after stopping the morphine injection.