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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Placement of Aneurysm Clips
- Place a rat 30 G aneurysm clip on each uterine artery. Ensure cessation of blood flow, including proximal and distal pulses, and darkening of the uterine vessels, including individual placentas. Cover the exposed horns and entire surgical field with gauze and irrigate with sterile saline. Irrigate the field approximately every 10 minutes to keep it moist.
- After 60 min, remove the gauze and irrigate the field. Ensure that the uterine horns and vessels are adequately moistened for successful clip removal. Gently remove each aneurysm clip using forceps. Take care not to cause trauma to the vessel, and maintain tissue integrity during removal.
- Thoroughly irrigate the uterine horns and field, taking care to remove any stray gauze threads from the amniotic sacs.
2. Injection of Lipopolysaccharide into Amniotic Sacs
- At the base of each individual amniotic sac, just anterior to the placental plate, inject 100 µl of lipopolysaccharide (LPS) (4 µg/sac) with diluted Evan’s blue dye into the amniotic fluid. Use blunt forceps to stabilize and rotate each amniotic sac into an optimal position for injection. Dilute Evan’s blue dye is a contrast agent that is helpful in confirming proper syringe placement and injection.
NOTE: Use only an ultra-fine 0.3 ml insulin syringe with an attached 8 mm 31 G needle for the intra-amniotic injections. Using larger gauge needles will result in chronic amniotic fluid loss, fetal death, and reabsorption of the pregnancy. Small amounts of amniotic fluid leakage upon removal of the syringe can be mitigated by direct pressure to the amniotic sac. Some rat fetuses may tolerate a degree of oligohydramnios. However, acute amniotic fluid loss from a puncture with large gauge needles or accidental puncture resulting in chronic fluid leakage results in fetal loss and, in severe cases, loss of neighboring pregnancies. - Irrigate the uterine horns 3x with sterile saline.
3. Closing the Laparotomy
- Using forceps, carefully return the uterine horns to the peritoneal cavity. Ensuring adequate space between the amniotic sacs and the midline incision, re-approximate the musculofascial layer edges using a running 3-0 silk suture. Be aware of the placement of the amniotic sacs while closing the muscle incision. Be careful to not to suture into or through a sac.
- Re-approximate the skin layer, using a running 3-0 silk suture, closing the skin layer.
NOTE: The laparotomy should be closed in two layers of continuous sutures to allow for skin and muscular expansion with increasing gestation. Continuous sutures allow for evenly distributed wound tension. Interrupted sutures are less desired as multiple knots are irritating and can be easily chewed by the rat upon recovering from anesthesia. Surgical staples are not desired. Tails of the surgical knots should be cut very short (<3 mm). - Inject 1 ml of 0.125% bupivacaine subcutaneously around wound edges using a 26 G needle. Administer one dose of 0.1 mg/kg buprenorphine subcutaneously at the nape of the neck.
- Turn off isoflurane and towel dry the rat as necessary. Place the rat in a clean home cage and monitor its recovery from anesthesia. Ensure the rat does not become hypothermic.