Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

The overall goal of this surgical procedure is to provide a reproducible model of brain injury associated with extremely premature birth, via the induction of in utero, transient systemic hypoxia, ischemia, and intra amniotic inflammation to recapitulate, placental insufficiency and chorioamnioitis in rats. This method can help answer key questions about the pathogenesis of perinatal brain injury that arise from intrauterine processes facilitating the development of directed therapeutic strategies. The main advantage of this technique is that it induces the systemic injury that mimics intrauterine insults from both infection and hypoxia, ischemia producing functional neurological deficits in the mature rat.

This model is particularly suited to dissecting the mechanisms of injury related to inflammation. As the hypoxia, ischemia and infectious stimuli can be tested individually or as a combined injury, demonstrating the procedure will be Lindsey Chan, a technician from our laboratory. Before beginning the procedure, use small animal clippers to remove all of the hair from the lower abdominal region of an embryonic day.

18 pregnant rat in a rectangular pattern, taking care not to nick the nipples or to generate razor rash. Next, using sterile cotton swabs, scrub the skin with three alternating applications of povidone iodine, and 70%ethanol. When the skin is dry, confirm the appropriate depth of anesthesia by toe, pinch and drape the animal.

Then use a scalpel to make a three centimeter midline incision in the animal's abdomen, followed by blunt dissection of the skin layer from the abdominal fascia with scissors. Using forceps and surgical scissors. Elevate the abdominal fascia layer and make an incision through the avascular linear elbow.

When the peritoneal cavity is exposed, place surgical gauze on the exterior of the incision and moisten the gauze with sterile saline. Then using blunt forceps and external pressure on the abdomen, gently remove the uterine horns from the peritoneal cavity and arrange the horns on the moistened gauze. Taking care to avoid entanglement and contact with the intestines, use only the muscular tissue between the individual amniotic sacs to arrange the fetuses.

Then using blunt dissection, expose and isolate the four uterine arteries. Taking care not to damage the maternal vessels 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 em 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.

Then thoroughly irrigate the horns and field. One last time, taking care to remove any stray threads of gauze from the amniotic sacs. 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 ultra fine 0.3 milliliter 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 ultra fine 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 three oh silk suture to re approximate the musculo fascial layer edges without suturing into or through a sack, followed by closure of the skin with another running 3.0 silk suture. Next, use a 26 gauge needle to subcutaneously.

Inject one milliliter of 0.125%bupivacaine around the wound edges. Towel dry the rat as necessary, inject 0.1 milligram per kilogram of buprenorphine at the nape of the neck. Place the animal in a clean home cage with monitoring until fully recovered.

Following transient systemic hypoxia ischemia and LPS administration at embryonic day 18, as just demonstrated het and eosin staining reveals significant histopathological abnormalities in both the placenta and in the brain. Indeed, placenta examined on embryonic days 19 and 21 are grossly emits with micro hemorrhage and necrosis throughout the decidua and labyrinth. Significant inflammatory infiltrate and increased vascularity are also observed.

Further brains examined on postnatal day two reveal ventricular magaly, as well as white matter and subplate neuron loss compared to shams transient systemic hypoxia, ischemia and LPS also significantly decreased the expression of potassium chloride cotransporter two protein, A chloride cotransporter central to the development of GABAergic inhibition in the cortex at postnatal day 15, which is consistent with the impaired developmental upregulation observed during the critical postnatal period of circuit maturation. After watching this video, you should have a solid understanding of how to perform this surgery in pregnant rats and how to induce prenatal transient hypoxia ischemia via uterine artery occlusion with in-utero inflammation via intra amniotic injection of lipopolysaccharide solution. Once mastered, this technique can be completed in 90 minutes if it is performed properly.