Establishment of a Rat Model for Intrauterine Adhesions via Dual Injury: Curettage and Infection

Intrauterine adhesions, IUAs, the most common pathological consequence of endometrial injury severely impair reproductive function. Tumor and infection are widely recognized as key risk factor for IUA development. We have successfully established a reproducible rat model of IUAs by integrating two critical pathogenic mechanisms, curettage and infection.

House female SD rats that are 8 to 10 weeks old, sexually mature, and unpaired in groups under SPF conditions. Take daily vaginal discharge smear from rats to determine their as estrous cycle stage. Slides are stained with hematoxylin.

Perform surgery during the estrous period. Before surgery, dissolve lipopolysaccharide, LPS, powder in PBS at a concentration of six milligram per liter. Immerse sterile 4/0 surgical cutting sutures in the LPS solution for 24 hours.

Then, store them in a four degrees centigrade refrigerator to prepare LPS-impregnated sutures. Prior to anesthesia induction, administer buprenorphine subcutaneously as pre-operative analgesia. Then, anesthetize the rat by isoflurane inhalation.

Assess anesthetic depth via toe pinch reflex. Proceed only if no withdrawal response is observed. Shave a four centimeter plus six centimeter area on the lower abdominal wall of the rat with a hair clipper.

Disinfect the area with three alternating scrubs of 1%povidone-iodine and 70%ethanol and cover it with a sterile disposable surgical drape. Make a skin incision in the midline of the lower abdomen above the pubic symphysis. Expose the abdominal muscle.

Carefully make a small incision into the peritoneal cavity and extend the incision. Take care to avoid inadvertently damaging the abdominal organs. Rotate and expose the two uterine horns situated above the pubic symphysis behind the bladder.

Make a three millimeter incision in the right uterine horns five millimeter above the bifurcation while avoiding blood vessels. Insert a scraping spoon into the right horn through the uterine incision. Scrape the uterine cavity until it feels uneven and the inner wall become rough.

Scratch approximately 30 times in each direction, front, back, left, and right. When scratching around the direction of the uterine horn, apply force evenly to prevent tearing or perforation the uterine wall. Insert a straightened surgical needle with LPS-impregnated suture through the incision of the right uterine horn to the end of the cavity.

Then, withdraw the needle, leaving the LPS suture inside the entire right uterine cavity with three to four centimeters of suture length exposed above the bifurcation. Leave the LPS suture in the uterine cavity for 48 hours. Close the right horn incision intermittently with 7/0 absorbable sutures.

Treat the right uterine horn in the same manner. Thoroughly wash the peritoneal cavity with sterile normal saline to remove any remaining endometrial debris. Close the peritoneal cavity with continuous 4/0 absorbable sutures.

Inject 0.5%cefazolin sodium solution into the abdominal wall muscle to prevent surgical site infection. Close the skin with continuous 4/0 absorbable sutures. Leave approximately two centimeter of the LPS suture protruding outside the skin.

To prevent the rats from biting the remaining sutures, fix them to the skin with 4/0 absorbable suture one centimeter from the abdominal incision. Trim excess suture material. Perform no uterine injury procedures when the uterine horns are exposed following laparotomy.

Wash the peritoneal cavity with sterile normal saline. Close the peritoneal cavity and the skin with 4/0 absorbable sutures. Two days after surgery, use forceps to gentle eject the LPS sutures from the uterine cavity of rats in the dual injury group.

Collect and examine uteri 7 and 14 days after the initial surgery in both groups, corresponding to approximately two and four regular estrous cycles respectively. Reopen the abdominal cavity after anesthetize the rat with isoflurane. Remove both uterine horns and evaluate macro characteristics.

Observe for swelling, congestion, luminal narrowing, surface roughness, or loss of elasticity. Check bilateral symmetry of the horns and inspect uterine cavities for occlusions, stenosis, or effusion. Euthanize rats via overdose of isoflurane inhalation, approved by the Animal Ethics Committee, after uterine horn collection as this a non-survival surgery.

Gross morphological observation of uterine horns. At seven days after surgery, the bilateral uterine horns in the sham operation group remain symmetrical, sharp, and elastic with no evidence of localized stiffness or fluid accumulation. By contrast, the bilateral horns in the dual injury group exhibit mild rigidity, uneven uterine wall thickness, focal stiffness, and ruminal stenosis.

At 14 days after surgery, the bilateral uterine horns maintained morphology comparable to presurgical baseline in the sham operation group, with intact symmetry and texture. In the dual injury group, however, both horns display contracture, pale coloration, reduce elasticity, and irregular wall thickness. Localized stiffness and luminal narrowing were exacerbated.

Histological change at seven days post-surgery. In the sham operation group, under HE and Masson trichrome staining, the endometrial epithelium, glandular architecture, and uterine lumen morphology closely resembled normal uterine structures. Endometrial surface exhibit a wave contour and the glands are round to elliptical in shape.

The stroma demonstrates no evidence of edema, congestion, or hemorrhage, and collagen fibers remain sparsely distributed with loose organization. In the dual injury group, under HE staining, the uterine cavity exhibits narrowing, partial occlusion, or filling with necrotic debris and leukocytes. The single layer columnar epithelium is thin or absent, replaced by flat or row-columnar epithelial cells.

Endometrial glands are reduced in number or absent. Under Masson trichrome staining, the number of endometrial gland decreases with the relative reduction in the epithelial area. Conversely, the blue-stained fibrous in the stroma increases in quantity, exhibit disorganized arrangement, and appears darker in staining intensity.

Histological change at 14 days post-surgery. In the sham operation group under HE and Masson trichrome staining, the endometrium of rats shows no significant changes with morphology consistent with that observed at seven days post-surgery. In the dual injury group, under HE staining, glandular and epithelial regeneration is nearly absent.

Instead, extensive collagen depositioned and interstitial fibrosis developed, leading to intrauterine adhesions and near-complete or complete cavity occlusion. Under Masson trichrome staining, normal columnar epithelia and glands are replaced by fibrous tissue, and intrauterine adhesions developed which are manifested by a large number of atrophic glands and surrounded by a mass of collagen fibers. The interstitial fibrous tissue increases significantly, with collagen appearing denser and darker compared to the seven day post-surgery stage.

At 14 days post-surgery, the area ratio of endometrial fibrosis in the dual injury group is significantly higher than that in the sham operation group. Our findings demonstrate that the dual injury approach establish a stable, reproducible IUA model in rats with procedural feasibility and consistent pathological features. This model provides a practical approach for investigating IUA pathogenesis and testing therapeutic strategies.