Inflammation Mediated Brain Damage and Cytokine Expression In A Maternally Derived Murine Model For Preterm Hypoxic Ischemic Encephalopathy

Tyler C Hillman^1,2Braeden Jacobson¹Kiara Piaggio Hurtado De Mendoza³Marlene Lopez²Nicholas Iwakoshi²Christopher G Wilson^1,2,4*

• ¹School of Medicine, Loma Linda University, Loma Linda, United States
• ²Lawrence D. Longo Center for Perinatal Biology, School of Medicine, Loma Linda University, Loma Linda, California, United States
• ³Whittier College, Whittier, California, United States
• ⁴Department of General Pediatrics, Loma Linda University Children's Hospital, Loma Linda, California, United States

Preterm hypoxic-ischemic encephalopathy (pHIE) is a complex brain injury that contributes to chronic neural inflammation and neurological disorders. The signs and symptoms of in utero pHIE can often be overlooked, untreated or lumped into more generic conditions such as encephalopathy of prematurity (EOP). Clinical interventions like hypothermia and erythropoietin do not improve pHIE. We characterized a murine model for pHIE, which includes hypoxia and maternal factors as a cost-effective alternative to large animal models of HIE. We injected pregnant mouse dams with LPS to stimulate an inflammatory response on embryonic days 15-16 (E15-E16), and whole cage hypoxia exposures occurred from postnatal days 3 to 9. To quantify the development of inflammation in the pHIE model, we used immunohistochemistry to stain for Caspase-9 in the cortex (20 µm per slice) and then counted Caspase-9 positive cells using unbiased stereology. We stained brain tissue with MAP2 to quantify neuronal intermediate filament expression and staining using a machine-learning based image analysis approach. We quantified cytokines (IL-1ꞵ, IL-6, IL-10, IL-18 and TNF-ɑ) using RT-qPCR and (IL-18) ELISA to characterize differential expression in all treatment groups. The pHIE animals were compared with controls (LPS-Normoxia, Saline-Hypoxia, Saline-Normoxia, and Naïve) and with a model of only hypoxia (10% O2) exposure in mouse pups.The pHIE pups showed significantly higher expression of Caspase-9 throughout the cortex compared to Naïve pup brains (p < 0.05). MAP2 expression was significantly decreased (p < 0.05) between 1.5 -6.0 mm of the brain compared to Saline-Hypoxia and Naïve animals. Both IL-1ꞵ and IL-10 expression in LPS-Hypoxia animals was significantly higher (p < 0.05) than in Saline-Hypoxia and Naive animals. TNF-ɑ expression was not significantly different between LPS-Hypoxia and Saline-Hypoxia animals. However, both showed significantly different transcription, compared to Naive animals. The model we describe here shows cortical damage similar to that seen in human HIE.