220. Utilizing in vitro Models of Neonatal Intraventricular Hemorrhage to Screen Anti-inflammatory and White Matter-protective Compounds

Authors: Brandon Andrew Miller, MD; Chirayukumar Pandya, PhD; Danielle Goulding; R Vogel; Aaron Silverstein; Josh Morganti (Lexington, KY)

Introduction:

Neonatal intraventricular hemorrhage (IVH) is a consequence of preterm birth.  We have found that in vitro studies mimic certain aspects of in vivo neonatal IVH models including macrophage activation and white matter oxidative stress.  We have used these in vitro models to test therapeutics that may be clinically useful for treatment of neonatal IVH. 

Methods:

We utilized a rat model of neonatal intraventricular hemorrhage, stereotactically injecting 10ul of 150 mg/ml hemoglobin into the lateral ventricle of post-natal day five rat pups.  A meso-scale detection ELISA was used to quantify inflammatory cytokines and Western blot was used to measure oxidative stress in the ipsilateral and contralateral hemisphere post injury.  Immunohistochemistry was used to assess macrophage activation and oxidative stress.  Five to six animals were utilized in each experimental group.  For in vitro experiments, mixed cortical cultures were obtained from post-natal day two rats and allowed to expand. Microglia and oligodendrocytes were isolated and exposed to different concentrations of hemoglobin.  Outcomes after in vitro exposure to hemoglobin were assessed via meso-scale ELISA, immunohistochemistry, genetic analysis, and free radical activated dyes.

Results:

Both in vivo and in vitro model systems demonstrated inflammation and oxidative stress in response to hemoglobin.  The inflammatory profile in vitro mirrors the cytokine profile in vivo, both via genetic analysis of CCL2 expression and analysis of inflammatory cytokine proteins.  Immunohistochemistry shows that inflammation and oxidative stress are specifically elevated within white matter in vivo and that in vitro hemoglobin directly elevates oxidative stress in oligodendrocyte progenitor cells.  We tested the FDA-approved therapeutics azithromycin and phenelzine which showed the potential to reduce hemoglobin-induced inflammation and oxidative stress, respectively. 

Conclusion:

in vitro modeling recapitulates many features of neonatal IVH.  This reductionistic strategy can be used to screen drugs that may have therapeutic efficacy.