(252a) Organotypic Whole Hemisphere Brain Slice Models to Study the Effects of Age and Oxygen-Glucose-Deprivation on the Extracellular Properties of Cortical and Striatal Tissue

Introduction: Chronic diseases are the leading cause of death and disability, affecting 6 in 10 adults in the United States. Developing novel therapeutics is necessary to combat chronic disease and reduce this burden, but the process to take a drug from discovery to marketplace is slow and costly. Failure to translate outcomes in animal models to success in human clinical trials is a major contributor to the time and cost associated with bringing drugs to market and is in part due to the inability of animal models to recapitulate the physiology of the disease in question. 2D in vitro cell culture models are simple and inexpensive, but lack contact between neighboring cells and native tissue architecture. 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. 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 any stimulus, including toxins and injury/disease models. The brain extracellular environment is involved in many critical processes associated with neurodevelopment, neural function, and repair following injury. Organization of the extracellular matrix and properties of the extracellular space vary throughout development and across different brain regions, motivating the need for platforms that provide access to multiple brain regions at different stages of development. Herein, we demonstrate the utility of organotypic whole hemisphere brain slices and establish them as a platform that can help satisfy this need. Whole hemisphere brain slices were then leveraged to better characterize how hypoxia impacts different regions of brain tissue.

Methods: Whole hemisphere brain slices taken from postnatal day 10 and postnatal day 17 rats were cultured up to two weeks. At set days in vitro (DIV), slices were either 1) screened for viability and metabolic activity using a lactate dehydrogenase assay (LDH), propidium iodide stain (PI), and Alamar Blue assay (AB), 2) fixed for antibody staining and imaging to study cellular response to culturing, or 3) used in particle tracking experiments to study changes in the extracellular . LDH and AB are colorimetric assays, the former quantifying toxicity via release of lactate dehydrogenase via damaged cell walls and the latter quantifying metabolic activity via conversion of a fluorophore (blue to red shift) by live cells. PI staining (only penetrates damaged cell membranes) allows quantification of­ viability with regional detail when imaged with a confocal microscope. Slices were fixed and antibody markers for neurons (Neun) and glial cells (Iba1, S100B) were applied prior to imaging by confocal microscopy. Cell distribution can be determined on a regional basis. Multiple particle tracking (MPT) is the tracking of fluorescent polystyrene nanoparticles in live tissue sections, using diffusion and other extracted parameters to better understand the microenvironment around particles. Oxygen glucose deprivation (OGD) is a model for hypoxia achieved by limiting oxygen (via nitrogen chamber) and glucose exposure (glucose-free media) to brain slices. Following 2h of OGD exposure, slices are rested for 24h or 72h until assays or imaging is performed at endpoints.

Results: Whole hemisphere brain slices taken from postnatal day 10 and postnatal day 17 rats retained viable, metabolically active cells through 14 days in vitro. Tissue extraction followed by oxygen-glucose-deprivation, used to model a hypoxic event ex vivo, resulted in reduced slice metabolic activity and elevated cytotoxicity, regardless of slice age. Multiple particle tracking in oxygen-glucose-deprived brain slices revealed that hypoxia-ischemia impacts the extracellular environment of brain tissue differently depending on brain age and brain region. In most instances, the extracellular space was most difficult to navigate immediately following insult, then gradually provided less hindrance to extracellular nanoparticle diffusion as time progressed. This response was not universal across all brain regions and . Slices from postnatal day 10 rats were highly viable for at least two weeks, while slices from postnatal day 17 rats had poor viability. In both ages, viability was lowered after slicing, but only recovered in the postnatal day 10 slices. There is a strong microglial response (change in morphology to proinflammatory state) initially after slicing and in response to OGD. OGD resulted in cell death in both neuronal and glial populations. Postnatal day 17 slices had higher cell death response to OGD and did not recover to the same extent as postnatal day 10 slices, which had a viability level return to baselines after 72h.

Conclusions: In addition to in vivo work being highly costly, which makes age-based studies resource intensive, experimental control/access is limited in these models. As a result, quantifying spatiotemporal response to brain disease is more difficult to achieve. We culture whole hemisphere brain slices taken from postnatal day 10 and postnatal day 17 rats to demonstrate our experimental approach. As onset of some neurological diseases is age-related, the ability to culture slices from older animals is advantageous to developing accurate ex vivo models. Our work demonstrates that brain slices can be used to probe extracellular and cellular responses to OGD with the ability to resolve the response by donor rat age, time after insult, and brain region. Importantly, this workflow is not limited to the OGD model. We anticipate that the combination of multiple particle tracking and antibody staining can be applied to other disease models, such as for Alzheimer’s, to quantify extracellular and cellular effects of disease in tandem on a time, region, and age basis.