The overall goal of this procedure is to surgically induce endometriosis in mice by autologous transfer of uterine tissue to the arterial cascade of the intestinal mesentary. This is accomplished by first ligating, removing and exposing the endometrium of the uterus. The second step of the procedure is to create three endometrioid implants from the exposed endometrium using a two millimeter biopsy punch.
The third step of the procedure is to suture the three endometrioid implants to arteries in the arterial cascade of the intestinal mesentery. The final step of the procedure is to close the abdominal wall and skin and allow the animal to recover the endometrioid lesions. Excised from mice resemble those from women with endometriosis as determined by histological analysis and gene and protein expression patterns.
Generally, individuals new to this method will struggle for three main reasons. First, the mouse tissues are just very small. Second, the intestinal mesentery is easily damaged, and third, sutures if placed too tightly, will result in bowel necrosis.
Begin by gathering all the necessary surgical equipment for a successful aseptic surgery, including solutions for both analgesia and anesthesia. After anesthetizing the animal using inhaled isoflurane, confirm full anesthesia by negative response to a toe pinch stimulus to prevent dryness during surgery, apply ophthalmic ointment to the eyes and then shave the surgery site using small clippers. Disinfect the surgery site with three alternating swipes of chlorhexidine scrub and 70%ethanol, and then drape the animal with a sterile field.
Make a small midline incision using either small scissors or a scalpel blade ending 0.5 to 1.0 centimeters rostral to the vaginal opening. Gently blunt, dissect the body wall from the abdominal wall in order to facilitate closure of the surgery site. After blunt dissecting, make a small midline incision in the abdominal wall.
Next, using small forceps, gently locate the left uterine horn. The uterus is dorsal to the intestine, which is what is typically visible upon first entering the incision site, gently pull up on the uterine horn and slide an open forceps underneath it to serve as a retractor. It is also helpful to note the appearance of the ovaries and uterus at this time.
To assist with estrus cycle dating. Slide two, six to eight centimeter pieces of five aught black braided silk suture underneath the stretched uterine horn. Securely ligate the horn at the utero tubule junction.
Just coddle to the fallopian tube and at the utero cervical junction. Using a square knot at each location, leave the ends of the suture uncut. Cut out the section of uterine horn between the two ligations and place the tissue in a glass Petri dish containing approximately 100 microliters of PBS containing penicillin and streptomycin.
Cut the ends of the silk suture Last. If suture comes loose or there is bleeding, find the stump and tie another knot or not using small scissors. Strip the fat off the uterine horn.
Open the uterine horn by inserting one blade of a small pair of scissors into the lumen. Gently slide the scissors down the uterine horn while holding the horn with forceps In the glass Petri dish, use a two millimeter biopsy punch to cut out three equal sized implants. Being sure to keep them from drying out.
Place sterile gauze immediately above the incision site and thoroughly wet with sterile PBS containing penicillin and streptomycin. It is important to maintain hydration throughout the following steps with small, smooth forceps. Gently find the cecum and move roly along the small intestine.
Pull out a small four to five centimeter section of intestine and arrange it like a fan on the pre wetted gauze so that the arterial cascade of the intestinal mesentery is clearly visible. Be sure to keep the bowel moist at all times with sterile saline. Place one implant on a needle, then use six aut black alon suture to gently secure the implant to an artery approximately 0.5 centimeters from the bowel.
Use only one. Throw over the forceps and do not tighten the suture very hard. As doing so may result in loss of blood flow and subsequent necrosis of the intestine.
Complete two knots with one, throw each and trim the suture within two millimeters of the implant, hydrate the intestine. Then moving in a rostral direction. Pull out the next three to four centimeters of intestine and gently replace the section that already contains an implant.
Skip one or two arteries from the previous implant site and suture the next implant repeat for the third implant. Finally, suture the abdominal wall and close the skin with wound clips. After closing the surgery site and administering analgesics, place the animal ventral side down in cage, partially atop a recirculating heated water pad until the animal is recovered.
To recover the endometrioid lesions, locate the black sutures in the intestinal mesentery. Measure the length and width of each endometrioid lesion using calipers. Dissect out the endometrioid lesions, being careful not to lance them.
Remove any non endometrioid tissue from the lesion. Wave the three fluid-filled endometrioid lesions prior to removing the suture. Gently remove the suture and lance two of the endometrioid lesions.
Weigh these again before proceeding onto tissue processing. Next excise and weigh the remaining uterine horn histological analysis of endometriosis in both women and in the mouse model shown here indicates that the endometrioid lesion contains endometrial glands and stroma endometriotic lesions in mice also contain hemosiderin laden macrophages, which are a common hallmark of endometriosis in women. Endometrioid lesions removed from mice three days post induction appear inflamed and hemorrhagic as shown on the left after two to four weeks of growth.
Endometriotic lesions in the mouse model are cyst like fluid-filled and surrounded by peritoneal adhesions compared to lesion weight at induction. Fluid-filled lesions were 306%and 862%larger and lance lesions were 51%and 172%larger at one and two months post induction respectively. Fluid-filled and lad endometrioid lesion.
Weights remain consistent at one month post induction over five different experiments Following this procedure. This model can be used to answer questions about the pathophysiology of endometriosis and to explore novel therapeutics for the treatment of pain and infertility associated with this disease.
