Glycerol-Induced Interventricular Hemorrhage in Premature Rabbit Pups

Timed pregnant New Zealand white rabbits (Oryctolagus cuniculus) were purchased from Charles River Laboratories Inc. (Wilmington, MA, USA). The premature pups were delivered by cesarean section (Supplementary Fig. S1A) at E29 gestational age (term gestation = 32 days). Newborn premature pups were maintained and fed according to methods published in our previous publications.^38,39,43

We induced hemorrhage and confirmed presence and absence using sonography (Supplementary Fig. S1B-S1F). The grades of IVH were defined based on the ventricular volume of echogenic area measured (length, breadth, and depth in coronal and sagittal views) as described.^38,39 Hemorrhage was classified as: (1) no gross IVH, (2) moderate (clot 30-100 mm³), gross hemorrhage into lateral ventricles with some ventricular enlargement (2 separate lateral ventricles discerned); or (3) severe (clot 100-250 mm³) IVH with considerable ventricular enlargement (fusion of ventricles into a common chamber) and/or intraparenchymal hemorrhage. After head ultrasound grading, the pups are assigned to 3 groups: (1) Control pups with no IVH, (2) Glycerol-IVH saline injected pups, and (3) Glycerol-IVH USSC injected. We use one premature rabbit pup in each experimental group (no IVH healthy control, IVH saline injected control, and IVH each treatment) for each experimental endpoint from the same litter. The New York Medical College Institutional Animal Care and Use Committee (IACUC) approved all experimental interventions, endpoint sample collections, and laboratory assays.