Impairment of decentralization leads to virus of endometrial disorders such as in fatality, a recurrent miscarriage. This method provides a good and reliable animal model for the study of endometrial decentralization. This artificial decentralization model with Ovariectomy can avoid the influence of angelina over hormones which can often highly reproducible results with minor within group differences.
To begin sterilize the surgical instruments with a high temperature and high pressure sterilizer one day before the operation. Apply the protective eye ointment to the eyeballs to prevent dryness during the anesthesia. Start the operation when mice are completely anesthetized.
Place the mice in the prone position on the operating surface and shave the hair on the back after wetting them with soapy water. Disinfect the skin with 70%ethanol. Find the operation incision site at 0.5 centimeters on the upper edge of the thigh root of both hind limbs parallel to the spine.
Take the incision site as the center and disinfect the incision area with iodophor, three times. Make a longitudinal incision of about 0.5 to one centimeter using scissors. Cut the fascia and passively separate the muscles with tweezers.
Find a piece of the white fat pad in the incision field close to the lower pole of the kidney through the thin muscle layer. Clamp the fat mass with hemostatic clips and pull the ovary wrapped by the fat pad out of the incision. Clamp the junction of the ovary and fallopian tubes with curved tweezers and remove the ovary along the ovarian pedicle with scissors.
Stop bleeding using an electrocoagulation pen or a needle of a one milliliter syringe heated on the alcohol lamp. Rinse the surgical incision with normal saline to prevent tissue adhesion. Use a 4-0 suture to sow the muscle and skin respectively and disinfect the surgical incision with iodophor again.
Place the mice in the prone position in the cage and resuscitate them in a 25 degree Celsius incubator. Equipped with a light source and ventilation system for about two hours. Pay attention to the mice after the operation.
Put them back in the original feeding place alone until they recover completely and give them enough water and food. Subcutaneously inject 100 nanograms of estrogen in 50 microliters of sesame oil into each mouse for three days followed by two days of rest. Inject one milligram of progesterone and 10 nanograms of estrogen in 50 microliters of sesame oil into each mouse for another three days.
Operate the mice to induce the artificial residualization model six hours after the third estrogen progesterone combined injection. Find the uterus horn at the lower end of the fallopian tubes and inject 20 microliters of sesame oil slowly along the uterine horn. Push the tissue back, suture the incision and allow the mouse to recover.
Fix three to five milliliters of the uterus tissue with 10%formalin embedded in paraffin and section them. Stain the sections with hematoxylin and eosin to observe the morphological changes. Embed another three to five millimeters of the uterus tissue in optimal cutting temperature compound freeze in liquid nitrogen and store in a minus 80 degree Celsius freezer.
Frozen section the tissue in 10 micrometers and detect alkaline phosphatase activity in the uterine tissue by the Azo coupling method. The general morphology of the artificial decentralized uterus of mice induced by oil is closer to that of the uterus in pregnancy. The uterine body becomes thick and the uterine cavity becomes smaller than the non induced side.
The volume and weight of the induced uterine horn are significantly higher than that of the non induced side. Hematoxylin and eosin staining of the uterine tissue showed that the glands disappear and the end endometrial stromal cells on the induced torn differentiated into large round cytoplasmic and multinucleated decidual cells with unclear cell boundaries. The non induced side sections show the endometrial tissue morphology under a normal physiological state.
The result of the Azo coupling method showed that the alkaline phosphatase content of the oil induced side was significantly higher than that of the non induced side. The QPCR results showed that the mRNA expression of PRL-8-A-2, ALPL and IGFBP-1 in the induced horn was significantly higher than that in the non induced one, suggesting that oil can induce artificial decentralization in mice. Homo supplementation is a lengthy process that requires special attention contamination between homos which is the key to success.
On the basis of this model uterine horn injection of Adenovirus can be performed which can study the relevant mechanism of specific molecules regulating residualization.
