(548d) Elucidating Neuron-Glia Interactions in a Whole-Hemisphere Brain Slice Model of Neonatal Hypoxia Ischemia

Introduction: An oxygen-depriving event during birth can cause hypoxic-ischemic encephalopathy (HIE), a devastating brain injury that occurs in anywhere between 1.5 and 2.5 per 1000 live births. While some infants recover with minimal long-term deficits, more severe cases of HIE result in death or chronic disabilities, such as epilepsy or cerebral palsy. Moderate and severe cases of HIE are treated by non-curative therapeutic hypothermia, which is only effective in reducing morbidity by 40%. For mild cases of HIE, there is currently no approved therapeutic intervention, but infants with even mild HIE have worse long-term outcomes relative to those without. Better understanding the pathophysiology of HIE is crucial to mitigating the severity of poor outcomes. Of particular interest is the interplay between neuronal and glial cells, as their communication is essential in regulating neuroinflammation. Here, we demonstrate the use of organotypic brain slice cultures to probe cellular response to oxygen-glucose deprivation (OGD), an ex vivo model for hypoxia and ischemia. Organotypic slice models have advantages over primary cell culture, as multicellular interactions are retained and bias in cell type is eliminated. Additionally, tissue slices retain 3D architecture and the slices mature in vitro in a similar manner to native conditions, allowing slices to be used long term in culture without sacrificing physiological relevance. A single animal can yield numerous healthy organotypic slices, which reduces the required number of animals for in vivo animal testing. Relative to in vivo models, the extracellular environment can be better controlled, which allows for high throughput screening of various therapeutics or to study the biological response to an injury model.

Methods: Postnatal day (P) 10 or 17 Sprague-Dawley rats were sacrificed and the brain was promptly extracted. Hemispheres were separated and cut coronally into 300-micron slices. Slices were separated in ice cold dissection media (HBSS, 5 g/L D-glucose) and plated onto 0.4 µm semi-porous membranes (3 slices per membrane) in a 6 well plate with fresh media (50% HBSS, 25% MEM, 25% horse serum, 2 mM GlutaMAX, 1% penicillin streptomycin) and cultured for 1 week. Propidium iodide, lactate dehydrogenase assay, and alamar blue assay are used to assess viability over time. Gene gun particles were prepared by adding 25 mg gold (0.6-micron size), 50 µl of 0.05M spermidine, 20 µg tdTomato plasmid, and 50 µL of 1M CaCl₂, incubated, and the pellet washed with 100% ethanol. Bullets were formed using Tefzel tubing and then dried with nitrogen, cut with the Helios® Tubing Cutter and stored at 4 °C with a desiccation pellet until use. Slices are bombarded using a Helios® Gene Gun. A helium pulse at 160 psig propels tdTomato-coated, gold particles at the tissue to transfect individual cells on the tissue. A subset of slices was subjected to OGD by removing from glucose-treated media and depriving slices of oxygen by placing in a purged sealed chamber with nitrogen for 1h. Following insult, slices were cultured in normal culture media for either 6h, 24h, 48h, or 96h then fixed in 10% formalin for 1h. Slices were then stained with Iba-1 and PSD95 overnight for microglia and dendritic spines, respectively. Images of dendrites of transfected neurons (3 per slice) were acquired in three brain regions (thalamus, cortex, hippocampus) by confocal microscopy using a 60x objective and z-steps of 0.5 μm. Spine length, spine volume, spine count, microglia count, and microglia morphology was measured from confocal images by ImageJ. Additionally, we measured PSD95 intensity within microglia cells to determine the extent to which microglia are engulfing dendritic spines. Lactate dehydrogenase assay, propidium iodide staining, and alamar blue assay were used to assess viability at relevant end points.

Results: Whole-hemisphere brain slices can be cultured successfully for over two weeks. Propidium iodide staining showed region-specific viability throughout culture time, with the cortex being most effected early into culture. However, viability in all brain regions recovered after 4 days. AlamarBlue assay demonstrated that metabolic activity of slices stabilizes around 4 days. Slices from donor age P10 outperformed P17 in viability, with 80% and 50% viability, respectively. There was an increase in lactate dehydrogenase release and propidium iodidide staining in slices that underwent OGD. Microglia count increased and morphology took on an amoeboid shape, with loss of microglial branching, consistent with response to neuroinflammation; however, the extent of response varied regionally and temporally.

Conclusions: Organotypic brain slices allows for study of neuron-glia interactions with physiologically relevant cytoarchitecture and precise control of the extracellular environment. In the future, it will be possible to treat slices with potential therapeutics and monitor the cellular response. By utilizing biolistic transfection, we also have the ability to image the neuron-glia interactions within live cells, enabling long-term imaging of single cell(s). To our knowledge, this is the first work to quantify regionally-dependent neuronal-glia interactions using whole-hemisphere organotypic brain slices.