Hemoglobin

P7 mice received unilateral intraventricular hemoglobin injection using a modification of a previously described rat model of neonatal IVH (Goulding et al., 2020 (link)). Prior to IVH induction, pups were sedated throughout the procedure with isoflurane (5% for induction and 2-3% for maintenance). Pups were secured in a warmed stereotaxic frame using nonrupture ear bars (Stoelting, Wood Dale, IL). The scalp was prepped with 10% povidone-iodine, and a midline skin incision was made to expose bregma. Using a stereotaxic injector (Stoelting, Wood Dale, IL) equipped with a Hamilton syringe (model 701 RN, 30G, point style 4, removable needle), the right lateral ventricle was accessed at coordinates 1 mm lateral, 3 mm posterior, and 2 mm deep from bregma. Injections of 150 mg/ml of hemoglobin (MP Biomedicals, Irvine, CA) prepared in phosphate-buffered saline (PBS) or PBS alone (control group) were delivered at a rate of 6.67 μl per minute, until a total of 10 μl was injected. The syringe was left in place for an additional 1 minute to reduce retrograde flow upon removal. Incisions were closed with Vetbond tissue adhesive (3M Corp, St. Paul, MN), and animals were returned to their dams. All mice were then deeply sedated with isoflurane, cervically dislocated, and decapitated at 4 hours post-surgery to harvest brain tissue. No animals died unintentionally during this procedure.