(131g) Organotypic Whole Hemisphere Brain Slice Model of Hypoxic Ischemic Brain Injury in Low-and Middle- Income Countries

Background: The incidence of hypoxic-ischemic encephalopathy (HIE) brain injury after perinatal (≥ 36 weeks gestation) oxygen deprivation is around 1-4 per 1,000 live births in the Western world and 2-3 times higher in Low- and Middle-Income Countries (LMIC) contributing to 8% of childhood deaths worldwide. The recent phase-III HELIX (hypothermia for moderate or severe neonatal encephalopathy in low- and middle-income countries) trial showed that HIE treatment used in high-resource areas is not beneficial in LMICs and may, in fact, result in increased deaths. This is largely due to a different pattern of HIE injury in LMIC populations characterized by an intermittent insult and growth-restriction indicating nutrient insufficiency. This shifts the injury from affecting the basal ganglia/thalamus region of the brain to a “watershed” or widespread white matter injury pattern that does not respond to the current standard of treatment. Additionally, dysfunction of the blood-brain barrier is often associated with disease biomarkers and white matter damage seen in many diseases including HIE. A few clinical and animal studies have shown that intermittent oxygen deprivation results in a similar injury pattern. However, to our knowledge, no in vitro models fully mimic this complex injury process and in vitro models are needed for accurate screening of therapeutics, including how pathological changes alter neurotherapeutic transport and uptake in different regions of the brain.

Methods: Here, we present a tunable organotypic whole hemisphere (OWH) brain slice model to investigate stimuli-responsiveness to replicate the injury pattern seen in HIE in LMICs. We prepare 300µm thick OWH slices isolated from postnatal day 10 (P10) rats as we’ve previously described. Slices are cultured in normal slice culture conditions for 4 days in vitro (DIV). To induce injury resulting from nutritional deficiency featured in LMIC HI, we cultured slices with media containing 2.5% serum and compared them to slices cultured in the standard 5% serum concentration. To induce the additional intermittent injury also featured in LMIC HI, at 4 DIV we alternated 30 minutes of oxygen-glucose deprivation (OGD) exposure with 10 minute periods of reoxygenation (OGDR). OGDR was applied with 1, 3, and 5 cycles to OWH slices that had previously been cultured in 2.5% and 5% serum. 24 hours after the end of the last OGDR, we evaluated global cell death via LDH assay and region-specific differences in the striatum, thalamus, and cortex via live-dead staining and confocal imaging.

Results: We showed lower cell viability corresponding to the slices cultured with lower serum and increased cell death with increased OGDR exposure. Regional differences in cell death in the thalamus and cortex were seen most distinctly in slices exposed to 1 cycle of OGDR and cultured in 5% serum. Cell death and pyknotic nuclei were seen in the striatum, thalamus, and cortex for all slices cultured in 2.5% serum with the highest cell death corresponding with the slices exposed to 5 cycles of OGDR. Current work in progress involves staining vessels with Zona-Occludens (ZO-1) and Occludin to evaluate differences in vessel coverage, vascular cell damage, and evaluate differences in injury in cells located in areas further from large vessels which would further indicate recapitulation of LMIC HI-like injury. This will also allow for analysis of the interplay between the tissue microenvironment and vasculature interfaces in response to nutrient-deficient OGDR.

Conclusions: These results show our ability to exhibit HI injury compounded by nutritional deficiency and intermittent insult in brain slice models. The lower serum concentration and repetitive OGDR exposure resulted in injury throughout the slice compared to cell death being more pronounced in the thalamus alone which reflects the different injury pattern seen in HI injury in LMIC populations. Future work will use the intermittent nutrient deprived OGDR model to screen combinatorial therapeutics, including nano-technology based therapeutics, with promise for translation and use in LMIC HIE cohorts. This can lead to the development of more effective treatments for LMIC HIE as well as serve as a paradigm for adapting existing ex vivo models to represent more diverse diseases.