This article describes a surgical technique for establishing a fetal tracheal occlusion model in pregnant mice. The method allows for the study of lung development in response to tracheal occlusion.
Place an anesthetized pregnant mouse of desired gestation age in the supine position. Sterilize the abdominal area. Perform laparotomy—a surgical incision through the abdominal wall. Dissect the abdominal muscles to expose the abdominal cavity.
Identify the uterine horns—long tubular structures that house multiple fetuses—allowing them to develop simultaneously. Gently exteriorize the uterine horns from the abdominal cavity, positioning them in a transverse orientation. Further, visualize the neck of the candidate mouse embryo.
Gently insert a surgical needle with the suture of appropriate thickness transversely through the uterine horn region, away from the placenta. Advance the needle further to the anterior part of the neck where the trachea is located. Direct the needle to exit the neck opposite the uterine horn. Carefully tighten the suture to occlude the trachea, ensuring uterine wall integrity. Return the uterine horns into the abdominal cavity.
Inject warm saline into the peritoneal cavity to facilitate pregnancy progression. Close the surgical incision and allow the mouse to recover. The established mouse fetal tracheal occlusion model can be used to study the impact of occluded trachea on lung development.
Clean the abdominal surface with alcohol and povidone-iodine, and maintain sterile conditions throughout the operation. Perform a vertical incision for the laparotomy of pregnant dams, and cut all layers separately. Identify the uterine horns on each side and determine the candidate fetuses for the surgery.
Operate on two fetuses in each uterine horn if there is an even number of fetuses on each side, and on one fetus in each uterine horn if there is an odd number. Using 2x magnification glasses for visualization, position the uterine horn in a transverse fashion. Position the pups facing upward between two fingers using the eyes and the tail as a guide.
Apply gentle pressure to the pup's head to allow extension of the head, and visualization of the neck. Perform tracheal occlusion, or TO, using an atraumatic needle and a 6-0 polypropylene suture. Keep the placenta on the side and far from the entrance and exit points of the needle.
Insert the needle transversely through the side of the uterus, away from the placenta, through the one-third anterior part of the neck. Then, move the needle gently to the midline of the neck. Direct it to the anterior part, then, exit the neck between the trachea and opposite the carotid sheath and uterus.
Knot the suture, taking care to maintain the integrity of the membranes and uterine wall. Replace the uterine horn in the abdomen and inject 2 milliliters of warm, sterile saline into the peritoneal cavity before closure.
Place a running 5-0 polyglactin suture to close the abdominal wall, and close the skin with a non-running silk suture. Apply 0.1 mg/kg of buprenorphine intraperitoneally for analgesia and allow the dam to recover in a warm incubator. Observe that the operated animal can feed itself and keep it alone in its individual cage.
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