Modeling Posthemorrhagic Hydrocephalus of Prematurity in Rats

Our laboratory seeks to characterize cellular and molecular pathophysiologic mechanisms of different perinatal brain injuries, with a specific interest being in posthemorrhagic hydrocephalus of prematurity, or PHHP. Our goal is to uncover novel, translatable therapies for these challenging diseases. This protocol helps us better understand the damage done to the developing brain after intraventricular hemorrhage and hydrocephalus.

With this model, we are uncovering potential strategies to reverse that brain damage and develop new and less invasive treatments. This protocol recapitulates essential clinical features of PHHP in rats by combining in utero chorioamnionitis and postnatal IVH using lysed red blood cells. The protocol yields rats with sustained macrocephaly, ventriculomegaly, increased intracranial pressure, and functional disability into adulthood, facilitating translational PHHP studies with a full spectrum of outcome measures.

To begin, obtain blood from a male and a female Sprague-Dawley rat pup at postnatal day one from a litter that experienced in utero chorioamnionitis on embryonic day 18. Vortex the tube well to mix the contents. Using small surgical scissors, chop and mince any blood clots in the tube.

Centrifuge the blood suspension at 500g for 10 minutes at four degrees Celsius. Remove the supernatant. Resuspend the pellet in 0.2 milliliters of sterile saline, and vortex well to ensure proper mixing.

Chop and mince any residual blood clots in the suspension post-vortex using small surgical scissors. After the final centrifugation, add 0.25 milliliters of sterile saline to the pellet and vortex the tube well to mix the contents. Place the suspension on dry ice for five minutes.

Now transfer the suspension into an incubator set at 37.5 degrees Celsius for five minutes until completely thawed, and vortex the suspension well after thawing. Repeat the freeze and thaw cycles three times in total. After the final thaw, vortex the tube and perform a quick spin in the centrifuge.

Position a small platform on wet ice and place a dry laboratory wipe on the platform to protect the pup's skin during the procedure. After anesthetizing the pup by hypothermia, position an external surgical lamp set to its brightest settings. Transilluminate the skull to visualize the lateral ventricles through the skull.

Use a 0.3-milliliter, eight-millimeter long, 31-gauge insulin syringe with an ultra-fine percutaneous needle to inject 20 microliters of lysed red blood cells into the right lateral ventricle. Rats with posthemorrhagic hydrocephalus of prematurity, or PHHP, exhibited enlarged, domed craniums and macrocephaly, as shown by an increased head circumference surrogate measure observable as juveniles. Rats with PHHP had increased intra-aural distance and increased intracranial pressure on postnatal day 21 compared to sham controls.

Ventriculomegaly and increased ventricular volume in rats with PHHP were visible through MRI, confirmed by comparison with sham controls.