动物造模,兔先天性横膈疝气管阻塞模型 z-bio 2021-01-15 376

　　Method: Congenital diaphragmatic hernia (CDH) is a birth defect that occurs every 2000-4000 live births. Failure of normal diaphragm development leads to diaphragm defect, causing intra-abdominal contents to herniate into the chest cavity. CDH causes pulmonary dysplasia and pulmonary hypertension, leading to morbidity and 20% mortality in this population. Although medical advances are effective for CDH infants after birth, the mortality rate remains unchanged. Therefore, it is necessary to treat the fetus to stimulate the lung growth of CDH fetuses, reduce lung underdevelopment, and reduce the risk of respiratory damage. The concept of fetal tracheal obstruction (TO) comes from the observation of lung hyperplasia at birth in fetuses with laryngeal atresia. In the early 1990s, TO was first applied to the lamb CDH model in the context of CDH. In this model, we noticed significant lung growth and reversal of lung hypoplasia. Currently, there are three types of CDH animal models-genetics, pharmacology and surgery. The genetic model of CDH mainly exists in rodent models with knockout genes. This model is probably the least popular because most cases of CDH in humans are not related to genetic defects. Therefore, the genetic model of CDH is least suitable for human conditions. The pharmacological model involves the use of a teratogenic herbicide called nitrophene in pregnant rodents, which can cause CDH and lung dysplasia in their offspring. This model was first explored by Iritani in the 1980s. Normally, 100 mg of nitrate is dissolved in 1 ml of olive oil and is given on the 9th day of pregnancy to induce CDH in rodents. Nitrofen administration can cause 70-80% of fetuses to have right or left diaphragm defect, and 100% of fetuses have lung dysplasia. The advantages of nitrophene-induced CDH rodent models include low cost and convenient use. However, although nitrophene induces a human-like diaphragm defect in the early stages of lung development, since there is no known teratogenic-related CDH in humans, the use of teratogenic agents to induce such defects is worthy of attention . The surgical model of CDH helps to explore the treatment methods and innovative therapies of CDH. This model is mainly used for sheep and rabbits. In the 1960s, Delorimier used fetal lambs to establish the first CDH surgical model. The diaphragm defect is caused by surgery at 72-75 days of gestation, which corresponds to the tubule stage of lung development, which is 145-149 days of gestation. In the early 1990s, Fauza et al. established a CDH surgical model on fetal rabbits. Using rabbits instead of sheep has many advantages: lower cost, shorter gestation period, 32 days, and lung physiology is similar to humans. In the rabbit model, the diaphragm defect was created on the 23rd day of pregnancy and also during the pseudoglandular stage of lung development. Compared with other models, lung physiology is more similar to that of humans, because in rabbits, alveolarization begins before birth and completes after birth, while in sheep, alveolarization is almost completed at birth. In rodents, Alveolarification begins after birth. The feasibility of using rabbits as a surgical model and their similar lung physiology make it the most ideal and promising animal model for CDH research.

　　 Perioperative care: When the pregnancy time of New Zealand white rabbits is 13 days, each month, a group of 5 rabbits enter the facility, raised in a single cage, eating and drinking freely. Each animal underwent two operations, one with CDH on day 23 of gestation, and the second operation, with TO operation on day 28 of gestation. Before surgery, each pregnant rabbit received 0.12 mg/kg buprenorphine SR SQ (3 mg/ml) for pain relief, penicillin G 3000000 IU intramuscular injection to prevent infection, and Depo provera 4.5 mg intramuscular injection to reduce the risk of spontaneous abortion. Induce anesthesia with 5 mg/kg ketamine (100 mg/ml), 0.15 mg/kg dexmedetomidine (1 mg/ml), 0.01 mg/kg chamothrin SQ (0.2 mg/ml), The anesthesia was maintained with 5% isoflurane through a face mask. During the operation, sodium chloride was used for rehydration and eye gel was applied to the eyes. During the operation, a water circulation pad was used to maintain normal body temperature. Fix the limbs. Monitor and maintain vital signs within the normal range, including heart rate 180–250, respiratory rate 30–60, and temperature 38–40°C. Close monitoring of its weight, intake/exhaustion and surgical incision after surgery. Give meloxicam 0.2 mg/kg SQ (5 mg/ml) every 24 hours for 2 consecutive days for postoperative analgesia.

　　 Creation of CDH: A total of 75 fetuses developed CDH at the 23rd day of pregnancy. As mentioned above, pregnant rabbits are pre-administered. The abdomen fur was cut off, the abdomen was prepared and covered with 2% chlorhexidine, and the uterus was exposed by laparotomy at the lower midline. Count the number of fetuses per uterine horn. Two fetuses are selected from the most ovarian end of each uterine horn, and there are 4 fetuses in each uterine horn. Gently palpate to determine the position of the fetus. Use a 15# knife to perform a 1 cm hysterectomy on the anti-media side of the uterus. Use 6-0 Prolene@ stitching money to stitch. The left upper limb of the fetus was identified, exposed, and head retracted. At the mark between the thoracic blood vessels, a left thoracotomy was performed with a 25 gauge needle and a hemostatic forceps. The lungs contract, exposing the fetal diaphragm. Grasp the diaphragm with hemostatic forceps and cut a piece with fine scissors to cause a defect in the diaphragm. The chest wall was sutured with 6-0 Prolene@. The fetus is placed back into the uterus and the uterine incision is sutured. The incision of laparotomy was sutured with 3-0 Vicryl®. In the last group of 5 pregnant rabbits, 10 fake CDH fetuses were produced. Follow the surgical protocol as described earlier. After CDH is produced, two more fetuses are selected at the very end of the ovary. False CDH is produced by performing a left thoracotomy without producing a diaphragm defect. We recommend that each pregnant rabbit undergo up to 4 fetal operations to improve the survival rate of the fetus. We found that increasing the number of fake fetuses and increasing the number of fetuses to 6 will only increase the operation time and pressure of pregnant rabbits and fetuses, and lead to higher mortality.

　　 Tracheal obstruction: A total of 17 fetuses underwent TO at 28 days of pregnancy. As mentioned above, pregnant rabbits are pre-administered. Under induction anesthesia, the abdomen is sterilized and prepared and covered in a sterile manner. Open the lower laparotomy incision and count the number of fetuses. Viability is observed. Half of the likely surviving CDH fetuses are selected as TO. Do a hysterectomy to expose the fetal head. Immediately cover the mouth and nose with a non-woven sponge soaked in saline to ensure that no fetus is breathing. Make a horizontal incision on the upper edge of the thyroid. Cut the trachea directly. After separation, the trachea was double-ligated with 4-0 Vicryl@ suture. The fetal skin is sutured with 6-0 Prolene@. The uterus is sutured continuously with 6-0 Prolene@. The fascia of pregnant rabbits is sutured with 3-0 Vicryl@, and the skin is sutured with 4-0 Monoryl@. The postoperative treatment is as described above.

　　 Euthanasia and organ recovery: fetal body weight and lung tissue collection were performed on the 31st day of pregnancy. Each pregnant rabbit was sedated with 10-50 mg/kg ketamine (100 mg/ml) and 2.5-10 mg/kg xylazine (100 mg/ml), and 100 mg/kg pentobarbital sodium IV was given through the ear vein (50 mg/ml) Euthanasia. A midline laparotomy was performed and the heart of the pregnant rabbit was palpated to confirm that there was no heart activity. All fetuses have been removed. Immediately cover the mouth and nose with a non-woven sponge soaked in saline, and put each fetus to death to ensure that no fetus is breathing. All fetuses are weighed. Perform sternotomy and midline laparotomy to assess whether there is a diaphragm defect in the diaphragm. At the same time, an incision is made at the CDH+ of the fetus to assess whether the trachea is ligated. Choose a control unoperated fetus from each female rabbit containing other surgical specimens, and select it according to its weight (ie the average number of litters). Measure the total lung weight and right lung weight of each fetus, and calculate the left lung weight by subtraction. The upper lung lobes were collected and stored in 4% paraformaldehyde, and the lower lung lobes were frozen in liquid nitrogen and stored at -80°C.

　　 Method verification: This surgical technique to create CDH caused lung hypoplasia, which was reversed by tracheal obstruction. Recently, our study on the effect of tracheal obstruction in rabbit CDH model on Wnt signal transduction confirmed this point.