P4 rodents were anesthetized (isoflurane 2–3% induction and 1.5% maintenance) and fixed in a stereotaxic frame. A 2.5 mm midline incision was made and a 0.3 cc syringe with a 30-gauge needle was inserted into the right lateral ventricle (LV) at the following coordinates from bregma: 1.4 mm lateral, 0.5 mm anterior, and 2.0 mm deep. A small volume (20 μL) of artificial CSF (aCSF) (Tocris Bioscience, Bristol, UK) or hemoglobin constituted in aCSF at a 150 mg/mL concentration was injected at a rate of 8000 nL/min using a micro-infusion pump (World Precision Instruments, Sarasota, FL) to create the aCSF control and IVH-PHH conditions respectively. The needle was left in for 5 min post injection to prevent backflow. The incision was closed with 6–0 Ethilon suture (Ethicon Inc, Raritan, NJ). Rodents recovered from anesthesia and were returned to their cage with the dam for 72 h before CSF tracer injection at P7. Rats injected with hemoglobin developed ventriculomegaly by the time of tracer injection at P7 [15 (link)–17 (link)].
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