200±20g Wistar non-pregnant female mice, 5 days postpartum and lactation Wistar nest mice, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. Shanghai Branch (SPF grade, license number: SCXK(Shanghai) 2017-0011). Metoclopramide(specification:10mg:1ml,N.05190803,manufacturer: Shanghai Hefeng Pharmaceutical Co.,Ltd.).Oxytocin Injection(Specification:1ml:10IU,National Medicine Standard H31020862, Manufacturer: Shanghai Shanghai First Biochemical Pharmaceutical Co., Ltd.),Chloral Hydrate (250G, N.A600288-0250, Manufacturer: Shenggong Bioengineering(Shanghai)Co.,Ltd.,complete Freund's adjuvant (50ml,N.P2036,manufacturer:Shanghai Biyuntian Biotechnology Co.,Ltd.), incomplete Freund's adjuvant(50ml,N.P2031,manufacturer: Shanghai Biyuntian Biological Technology Co.,Ltd.). Rat PRL Elisa kit (manufacturer: Shanghai Hengyuan Biotechnology Co., Ltd., N.20H09L11)
Refer to the collection method described in the literature, the specific operation method is as follows: before the experiment, all the experimental items are placed in an ultra-clean table for ultraviolet disinfection for 20 minutes; during the experiment, the lactating female rat are pressed 2-3ml/kg with 10% chloral hydrate minal cavity for anesthesia. After the rat was unconscious, the rat abdomen was quickly depilated with depilatory cream. After the hair was cleaned, the exposed skin of the chest and abdomen was disinfected with an alcohol cotton ball. Inject 10IU/mL oxytocin into the rat's abdominal cavity at 2ml/kg according to the rat's body weight. Wear sterile gloves to take milk. Gently squeeze around the nipple to make a small amount of milk appear on the nipple. Use the right hand to transfer 200ul. The device sucks the milk away at any time, and discharges the milk into a 1.5ml cryotube that has been sterilized in advance. After the milk is collected, the mother mouse is disinfected again, and the milk is frozen at -20°C for later use.
On the night before use, the mother's milk was mixed with complete Freund's adjuvant or incomplete Freund's adjuvant in a ratio of 1:1, and placed on a shaker at 4 ℃ overnight to prepare a milky white oil-in-bag milk mixture, and rat mammary gland injection was performed on the second day.
Wistar female rats were randomly divided into 4 groups, group A: normal control group, 2 female rats; group B: single-use gastric compound group, 2 female rats, each female mouse at a dose of 30 mg/kg at 9:00 on the day, 0.6ml metoclopramide was injected subcutaneously on the back for 2 weeks; group C: 6 female rats, on the basis of group B, at the same time each female mouse was injected with 0.2ml oil through the third and fourth pairs of mammary glands respectively Milk suspension (milk + complete Freund's adjuvant), 0.2ml of oil-in-oil suspension (milk + incomplete Freund's adjuvant) was injected again after 1 week; group D: 8 female rats, on the basis of group C Above, after 2 weeks, the mammary glands were again injected with 0.2ml of emulsion-in-oil suspension (milk + incomplete Freund’s adjuvant).
Injection Method and Route
Rats were admitted to the animal room SPF grade feeding environment to adapt to 5 days, at 9:00 every day, the Metoclopramidere admitted to the animal room SPF grade feeding environment to adapt to 5 days, at 9:00 every day, the stomach was subcutaneously injected on the back of rats for 2 weeks; the prepared mixed rats were injected into the subcutaneous fat pad through the 3rd and 4th pair of mammary glands of dams every other week.
Specimen Collection and Processing
Before the start of the experiment, blood was collected from the orbit of each rat in a quiet condition to retain serum for future use. After 2 weeks of metoclopramide administration, orbital blood samples were taken again, and rat serum was subjected to Elisa to detect changes in prolactin levels. Two rats were randomly selected at 1 and 2 weeks after injection in groups C and D. Blood samples were collected from the abdominal aorta after anesthesia, and the second, third, and fourth pairs of breast tissues were taken, washed with normal saline and fixed in paraformaldehyde for 24 hours before paraffin tissue embedding and HE staining for observation. Three weeks after the injection of D group, all the remaining rats were sacrificed and sampled, and the operation was the same as above.
Observation of general conditions
After the first injection of the 3rd and 4th pair of mammary glands of rats, the size of mammary masses was measured once a week until sacrifice, and the skin changes, redness, swelling, and ulceration of the rats were recorded.
The diagnosis of non-puerperal mastitis mainly depends on the pathological diagnosis of the diseased tissue, ductal dilatation, secretion, granuloma formation, giant cell reaction, plasma cell infiltration, lymphocyte infiltration, eosinophil infiltration, histiocyte infiltration and other conditions in the breast lesion tissue. It is mainly based on Ackerman Pathological Diagnosis (the 10th edition) as the diagnostic criteria for non-puerperal mastitis.
GraphPadPrism7.0 software was used for statistical analysis. The mean value of measurement data was expressed as `x±s，t Test was performed for comparative analysis. P<0.05 was considered statistically significant.