This article describes a surgical procedure in a pregnant rat model to induce hypoxic-ischemic conditions in fetuses. The method involves occluding uterine arteries and injecting lipopolysaccharide (LPS) to study the resulting neuroinflammation and brain damage.
Take an anesthetized pregnant rat with exteriorized uterine horns containing fetuses positioned on moist gauze.
Place aneurysm clips on the exposed uterine arteries to restrict blood flow, evidenced by darkening uterine vessels.
This restriction induces hypoxic-ischemic conditions by depriving the fetuses of oxygen and nutrients, leading to central nervous system damage.
Regularly hydrate the uterine horns with sterile saline.
After the occlusion period, rinse the uterine horns and remove the aneurysm clips to restore blood flow.
Inject a lipopolysaccharide or LPS and dye mixture into the amniotic fluid of each amniotic sac, using the dye to confirm the injection visually.
Rinse the uterine horns, return them to the peritoneal cavity, and close the incision.
In the amniotic fluid, LPS binds to immune cell receptors, triggering the release of pro-inflammatory cytokines.
These cytokines reach the fetal brain, activate microglial cells, and cause neuroinflammation, resulting in brain damage and fetal encephalopathy.
When the arteries have been isolated, place a 30 gauge rat aneurysm clip on each vessel.
Before applying the clips, it's important to ensure that all the surrounding tissue is completely clear. When placing the clips, secure them with the blunt forceps and then place them in parallel. Observing the cessation of pulse is critical to ensure complete hypoxia ischemia.
When all the clips have been placed, confirm the cessation of the proximal and distal pulses and the darkening of the uterine vessels, including in the individual placentas. Then cover the entire surgical field with gauze, and irrigate the tissue with sterile saline every 10 minutes for an hour. After 60 minutes, remove the gauze and irrigate the field again. When the uterine horns and vessels are adequately moistened, use forceps to gently remove each aneurysm clip, taking care not to cause trauma to the vessel and to maintain the tissue integrity.
To treat the fetuses with LPS, use blunt forceps to stabilize and rotate each amniotic sac so that the base of each individual amniotic sac is visible. Then, load one ultrafine 0.3 milliliters insulin syringe with an attached eight millimeter 31-gauge needle with LPS solution and inject 100 microliters of the solution just anterior to each placental plate. As the needle is removed, apply direct pressure to the amniotic sac to stop any fluid leakage.
Insertion of an ultrafine 31 gauge needle at the base of the amniotic sac is critical for proper LPS delivery without allowing any amniotic fluid to escape.
After irrigating the uterine horns three times with sterile saline, use forceps to return the horns to the peritoneal cavity. Then, use a running 3/0 silk suture to re approximate the musculo fascial layer edges without suturing into or through a sac, followed by closure of the skin with another running 3/0 silk suture.
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