动物造模,DOCA盐性高血压动物模型 z-bio 2021-04-06 509

　　1. Overview of DOCA salt hypertension

　　Human hypertension is divided into two major categories: primary hypertension and secondary hypertension according to its occurrence mechanism. In secondary hypertension, primary aldosteronism occupies an important position. Primary aldosteronism (primary aldosteronism, referred to as primary aldosteronism) is due to the adrenal cortex (mostly adenomas, a few hyperplasia), excessive secretion of aldosterone, resulting in water and sodium retention, increased blood volume, renin- The angiotensin system is inhibited, and a group of clinical syndromes with hypertension, hypokalemia, myasthenia, and nocturia as the main clinical manifestations appear, belonging to mineralocorticoid hypertrophy independent of renin-angiotensin. DOCA salt hypertension is a kind of deoxycorticosterone acetate that causes hypertension. The animal model of this type of hypertension is the basis of experimental research on dietary nutrients. It is similar to the primary aldosteronism in human hypertension, and the use of aldosterone antagonists can treat the disease of DHR (hypertensive rats caused by deoxycorticosterone acetate). This animal model can be used to study human hyperaldosteronism disease.

　　2. The mechanism of DOCA salt hypertension blood pressure increase

　　The pathogenesis of DOCA salt hypertension has nothing to do with RAS, because DHR has low renin levels and AT1 receptor antagonists do not lower its blood pressure. The factors that cause DHR are:

　　(1) The role of salt Salt is a very important factor in the formation of DHR. DOCA alone does not increase blood pressure. After salt is added, extracellular fluid and blood volume increase first, renal function and structure change, renal arterioles, renal and Hypertrophy of the heart and arterial walls, and then a significant increase in blood pressure.

　　(2) The role of DOCA Because large doses of DOCA (precursor of aldosterone) are used in the model making process, it has a similar physiological effect as aldosterone, which can cause sodium retention, potassium excretion, expansion of extracellular fluid, and increased blood volume. , The concentration of sodium ions in the blood vessel wall and blood circulation increases, and the blood vessel's response to norepinephrine is strengthened, causing high blood pressure. Due to the expansion of body fluid volume, feedback inhibition of the circulating renin-angiotensin system results in low plasma renin activity. Therefore, it is a kind of low. Renin-induced hypertension model.

　　(3) The role of vasopressin (AVP) CNS plays an important role in the pathogenesis of DHR. Sodium retention affects vascular function. DHR will increase the pressure of exogenous NE, E and AngⅡ before the blood pressure rises significantly. The reaction increased. While rats are given DOCA, chronic intraventricular infusion of NaCl solution can cause blood pressure to rise, and the vasopressin in DHR plasma increases several times, especially malignant hypertension. Vasopressin antiserum or injection of vasopressin analogs that can block the boosting effect of vasopressin can lower the blood pressure of DHR. There is a kind of rat (Brattboro rat) whose blood pressure does not rise after DOCA-salt is administered, but when vasopressin is injected, the blood pressure rises steadily. It shows that vasopressin plays an important role in the occurrence of DHR, but some people hold different views. The role of vasopressin needs to be further studied.

　　3. Establishment of an animal model of deoxycorticosterone acetate-salt induced hypertensive rats (DHR)

　　This model is a secondary hypertension model. Deoxycorticosterone acetate is made strictly in accordance with dietary control conditions

　　An animal model of hypertension caused by

　　. The preparation of this kind of animal model of hypertension is the basis of experimental research on dietary nutrients. And similar to primary aldosteronism in human hypertension, aldosterone antagonists can prevent the formation of DHR.

　　(1) The establishment of the SelyeDHR model As early as 1943, Selye reported that Addison patients had increased blood pressure after drinking excessive sodium chloride. Later, he gave young rats a subcutaneous injection of synthetic deoxycorticosterone acetate (DOCA), and Drinking 1% saline can cause a significant increase in blood pressure in rats. After that, in order to form DOCA salt-induced hypertension rats (DHR), the following methods are often used: 4 to 6-week-old rats, both male and female, under anesthesia with ether or ketamine, remove one kidney and bury it under the skin on the back of the neck For capsules containing DOCA, the dose of DOCA is 100 mg/kg. After the operation, the rats were given 1% NaCl solution. Stable hypertension can be formed after 4 to 6 weeks. SBP is generally above 180mmHg, and it can still develop into malignant hypertension. DOCA alone can only cause a temporary increase in extracellular fluid and blood pressure. After 4 weeks, blood pressure returns to normal levels. Only the combined application of DOCA and high salt can cause a stable increase in blood pressure. The increase in blood pressure is more pronounced after removal of one kidney.

　　1. Preparation method Deoxycorticosterone acetate-salt induced hypertensive rats (DHR) model can be made in two ways.

　　For a long time, people have used the subcutaneous injection method created by Selye in 1943 to form a DOCA-salt hypertensive rat model, which is a classic method of modeling. The production method is as follows:

　　(1) Both male and female SD rats can be used, and the weight is 150-～200g.

　　(2) After intraperitoneal injection of ether or 3% sodium pentobarbital (30mg/kg) anesthetized, fixation, cut off the hair of the abdominal surgical field, and disinfect the skin with 0.05% chlorhexidine alcohol. At 1.5cm below the xiphoid process, incise the skin and muscle layer along the midline of the abdomen, extend the index finger of your right hand into the abdominal cavity and touch the left kidney, and gently squeeze the kidney from the outside of the abdomen with your left finger. Use cotton soaked with physiological saline. Wrap it, and then fix it with your left index finger and thumb. Use the right hand to carefully separate the renal pedicle fascia with unhooked straight ophthalmic forceps, separate the renal artery along the deep vein, and ligate the renal artery near the aorta. Follow this method to ligate the deep vein and ureter, and then use a scalpel to remove the left kidney and adrenal gland (the left adrenal gland is cut with a small knife on the capsule, and the adrenal gland is gently pressed with elbow unhooked ophthalmic forceps to remove the medulla and the large adrenal gland. Part of the cortex is removed). Finally, the abdominal muscle and skin incision were sutured and disinfected with iodine.

　　(3) Rats after the operation were injected subcutaneously with 10,000 to 20,000 U of penicillin sodium salt (5×10,000 UPG/d).

　　(4) After the operation, 1% saline was given and DOCA oil 10mg/W was injected subcutaneously for four weeks. The injection should be strictly disinfected and the injection site should be changed frequently to prevent infection and ensure the effective absorption of DOCA.

　　(5) The blood pressure and plasma deoxycorticosterone content of rats increased significantly 2 weeks after the operation, and the plasma deoxycorticosterone dropped to normal at 7 to 14 weeks after the operation, but the blood pressure remained at a high level.

　　2. The usage and dosage of DOCA oil

　　(1) Once a week method: Once a week, DOCA 50mg/kg each time.

　　(2) Twice a week method: Twice a week, 5mg/head. As for the treatment time of penicillin sodium (5×10000UPG/d), some people recommend anti-infection for 1 week.

　　(3) Surgical method: using a back incision, the method is: anesthetize with ether or 3% sodium pentobarbital (30mg/kg) by intraperitoneal injection, the rat is fixed in the prone position, and the height of the lower abdomen is about 2~3cm Cut off the hair of the surgical field, disinfect the skin with 0.05% chlorhexidine alcohol, cut the skin along the midline of the spine from the 10th thoracic vertebra to the 3rd lumbar vertebra, 1.5-2cm below the left quarter rib and 1cm from the spine Separate the muscles with small vascular forceps, use two fingers to squeeze out the kidney from the lower part of the abdomen, carefully peel off the kidney from the surrounding tissues, remove the kidney after separation, and then suture the muscle and skin wounds separately.

　　The surgical instruments do not need to be autoclaved, as long as they are immersed in 75% alcohol for 30 minutes, rinsed with boiled saline before use, and still immersed in alcohol after use.

　　Because this method requires repeated subcutaneous injections of DOCA oil or suspension into rats, the time for hypertension to form is longer, which increases the chance of infection of rats and the labor intensity of the test personnel.

　　(4) Silica gel infiltration method: In 1973, HS Ormsbee and CF Ryan first used a DOCA-infiltrated silicone tube (50mm long and about 6mm in diameter) to be embedded in the rat subcutaneously to form DOCA-salt hypertension, which overcomes the shortcomings of the subcutaneous injection method. In recent years, some foreign researchers have begun to adopt this new method. Due to the complexity of the production process of DOCA silicone cable, it is difficult to popularize this method.

　　(2) Establishment of improved DHR model In 1993, domestic scholars Wang Qing and others took the lead in using and improving the technology in China. The detailed preparation method is as follows:

　　1. The production method of DOCA silicone tube uses a human body silicone tube with an outer diameter of 4mm and an inner diameter of 2.5mm. The length is 25mm. The wall of the tube is drilled with 10-14μm micro-holes. The tube is filled with 100mg DOCA powder, and then a thin tube with a length of 1.5mm and a diameter of 2.5mm is used. The silicone tube seals the openings at both ends of the tube. Before use, soak the DOCA silicone tube in a penicillin saline solution (1×100000UPG/ml) for 30 minutes.

　　2. Preparation method of DOCA-salt hypertensive rat model Male SD rats weighing (140±9) g (provided by the Experimental Animal Center of the First Military Medical University) were anesthetized intraperitoneally with 3% pentobarbital sodium 30 mg/kg. After anesthesia, fixation, remove abdominal hair, and disinfect.

　　3. Remove the left kidney and adrenal gland (the method is the same as before).

　　4. DOCA subcutaneous embedding tube A DOCA silicone tube was placed subcutaneously in the right lower abdomen of the rat, and finally the abdominal muscle and skin incision were sutured and disinfected with iodine.

　　5. After the operation, 1% saline was fed, and all rats were injected intramuscularly with penicillin sodium (5×10000UPG/d) for 3 consecutive days.

　　Wang Qing et al. believe that compared with the subcutaneous injection method, the blood pressure of the DOCA subcutaneous tube increases linearly in the first 8 weeks after the operation, but the former has a significantly faster rate of pressure increase than the latter. Increasing the number of holes on the DOCA silicone tube cannot further increase Boost rate (10 holes are enough). In short, compared with the DOCA subcutaneous injection method, the DOCA subcutaneous implantation method has the following advantages: ①The formation time of hypertension is shorter and the formation rate is high (88%), the pressure rise rate is faster (3 weeks), and the pressure rise is larger. ②The method is simple, reliable, reproducible, and easy to popularize; ③It reduces the chance of animal infection and reduces the labor intensity of experimenters.

　　Four, application value

　　This model is a secondary hypertension model, similar to the primary hyperaldosteronism in human hypertension. Therefore, it can be used for the study of hypertension caused by primary aldosterone. In addition, the literature uses DOCA salt-induced hypertensive rats to study the kidneys, such as the protective effect of bosentan on the kidneys of hypertensive rats; or use this model to observe the protective effect of antihypertensive drugs on vascular function, such as Enala Prilpril has no effect on DOCA-salt-induced hypertensive rats, and Cilazapril can stimulate the expression of nitric oxide synthase to improve myocardial remodeling in hypertensive rats induced by deoxycorticosterone acetate. At the same time, it is also widely used in the research of traditional Chinese medicine and traditional Chinese medicine, such as the expression of matrix metalloproteinase-2 in the heart of DHR and the influence of Shexiang Baoxin Pills.

　　rats (MHS, Milan), Lyon hypertensive rats (LH, Lyon), etc. Among them, SHR is one of the most widely used model animals.

　　SHR was bred from Wistar rats by Okamoto, Tokyo in 1963, and its coat is white. This mouse has a high incidence of hypertension. Hypertension has formed at 16 weeks of age, with systolic blood pressure> 160mmHg, no obvious primary kidney or adrenal damage, and a high incidence of cardiovascular disease. This strain has high blood pressure and stable inheritance. The increase in blood pressure occurs under natural conditions and can form typical complications of hypertension. The etiology of SHR hypertension is multi-factorial. It is similar to the mechanism of human primary hypertension. The increase in blood pressure is related to the increase in peripheral vascular resistance and responds to antihypertensive drugs. Therefore, it is An ideal animal model for the study of the mechanism of human essential hypertension and the screening of hypertension drugs.

　　(1) genetic background

　　1. Originated from the Tokyo outbred Wistar rat, using a male rat with spontaneous hypertension (blood pressure value of 145～175mmHg) and a female rat with elevated blood pressure (blood pressure value of 130～140mmHg) Mate and reproduce, and then continue to mate with brothers and sisters to obtain an animal model of spontaneous hypertension. SHR is a mutant animal, which is bred through genetic orientation.

　　2. Different strains of SHR spontaneously hypertensive rats have been bred in various countries, such as SHR bred by Professor Okamoto in Japan; GHR bred by Smirk and others in New Zealand; MHS successfully bred by Bianchi and others in Milan; bred by Dahl and others Successful Brookhaven strains of hypertension-sensitive rats HSR.

　　3. Coat color albino.

　　(2) Strain characteristics and uses The animals in this strain tend to have cerebrovascular injury and stroke. After 10 weeks of age, the arterial systolic blood pressure is 200-350mmHg for male rats and 180-200mmHg for female rats. The incidence of cardiovascular disease is high, but there is no organ damage in the kidneys and adrenal glands. Genetic analysis shows that this situation is controlled by 3 to 4 genes, one of which may be the main one. The incidence of male SHR rats is as high as 70% to 80%, and the highest blood pressure can reach 230mmHg; the incidence of female SHR rats is low, and the highest blood pressure is only 170mmHg. Thus, relatively speaking, the incidence of male SHR rats is high and stable. The symptoms of SHR rats are susceptible to startle, unstable standing, and abnormal irritability during startling. SHR male rats have an average blood pressure of 135mmHg at about 7 weeks of age, and their blood pressure values increase gradually with age. After 4 months of age, blood pressure can reach or exceed 200mmHg.

　　(3) Feeding and management of SHR SHR has higher requirements for environment and nutrition.

　　1. Environmental control adopts SPF barrier environment for breeding, free ingestion of 60Co irradiated sterilized pellets, the temperature of the breeding room is (22±3.5) ℃, the relative humidity is 45% to 55%, and the light is controlled manually (day 12 hours, night 12 hours), The ammonia concentration is less than 20ppm, the feed and litter are autoclaved, and the drinking water is cold boiled water. The litter is changed every day in summer and the nest is changed three times a week in winter. The floor and cage are wiped clean with disinfectant every day.

　　2. Feeding In order to meet the nutritional needs of breeding mice and suckling mice, from one week before the breeding mice to weaning of the suckling mice, the breeding mice and suckling mice need to be fed a cake made of milk powder and eggs in addition to the full-price pellet feed. .

　　(4) Growth and development of SHR

　　1. In the early stage of the development of the suckling mouse, the whole body of the newborn suckling mouse is naked and hairless, with some fine whiskers only around the mouth; the 4-day-old suckling mouse can see sparse fine hairs on its head, back, limbs and other parts under the light, and the 6-day-old suckling mouse can start crawling ; 9-day-old suckling rat's coat becomes thicker and denser

　　At the age of 10 days, the crown of the lower incisor teeth broke through the gingival membrane and began to expose; at the age of 11 days, the upper teeth began to be exposed; at the age of 15 days, the baby rat's abdomen grew hairy, and the whole body coat began to grow: 17-18 days old, the baby rat opened Eyes; weaned at about 30 days of age.

　　2. After birth, weight gain pups are weighed every one week, and the weight status of each period. The 4th to 12th week is an important period for its rapid weight gain. This period should provide adequate nutrition. During the entire growth and development process, SHR's The growth rate is slower than that of Wistar and SD rats in the closed group, which reflects the difference in growth and development between the inbred mutant line and the closed group.

　　3. Breeding uses the full-sibling method of inbred lines for breeding. Generally, SHR has a large number of first and second litters, with an average of 9 litters, and the third litter begins to decline, with an average of 7 litters, and the fourth litter. The average number is 5. After the fourth birth, the breeding rats generally lose their fertility and should be eliminated in time. It is worth noting that during breeding, the male rats are placed in the female cage for ~ weeks, and then the male rats are separated. The pregnancy rate of female rats can reach 100%. Otherwise, if the male and female breeding rats are not separated in time, the suckling rats may be trampled to death, causing weaning. The rate is reduced.