A ferret brain slice model of oxygen–glucose deprivation captures regional responses to perinatal injury and treatment associated with specific microglial phenotypes

Abstract

Organotypic brain slice models are an ideal technological platform to investigate therapeutic options for hypoxic‐ischemic (HI) brain injury, a leading cause of morbidity and mortality in neonates. The brain exhibits regional differences in the response to HI injury in vivo. This can be modeled using organotypic brain slices, which maintain three‐dimensional regional structures and reflect the regional differences in injury response. Here, we developed an organotypic whole hemisphere (OWH) slice culture model of HI injury using the gyrencephalic ferret brain at a developmental stage equivalent to a full‐term human infant in order to better probe region‐specific cellular responses to injury. Each slice encompassed the cortex, corpus callosum, subcortical white matter, hippocampus, basal ganglia, and thalamus. Regional responses to treatment with either erythropoietin (Epo) or the ketone body acetoacetate (AcAc) were highly heterogenous. While both treatments suppressed global injury responses and oxidative stress, significant neuroprotection was only seen in a subset of regions, with others displaying no response or potential exacerbation of injury. Similar regional heterogeneity was seen in the morphology and response of microglia to injury and treatment, which mirrored those seen after injury in vivo. Within each region, machine‐learning‐based classification of microglia morphological shifts in response to injury predicted the neuroprotective response to each therapy, with different morphologies associated with different treatment responses. This suggests that the ferret OWH slice culture model provides a platform for examining regional responses to injury in the gyrencephalic brain, as well as for screening combinations of therapeutics to provide global neuroprotection after injury.

Brain Tissue-Derived Extracellular Vesicle Mediated Therapy in the Neonatal Ischemic Brain

“…To study EV therapeutic activity and its impact on cellular behavior, we performed oxygen glucose deprivation (OGD) in organotypic whole hemisphere (OWH) slices from the P10 rat brain to model HI in term-equivalent neonates [ 32 , 33 ]. OWH brain slices uniquely capture the 3D cytoarchitecture and regional complexity of the brain [ 33 , 34 ]. OWH slices allow for the simultaneous assessment of different brain regions, such as the cortex, hippocampus, thalamus, and others, which are not present collectively in standard organoid, cortical, or hippocampal culture models [ 35 ].…”

“…BEV solution was then diluted with sterile 1X PBS to achieve experimental dosages (5 μg, 12.5 μg, 25 μg, 50 μg) suspended in a total volume of 100 μL per slice. The BEV solution was topically applied onto the tissue slices at various application timepoints (−24, 0, 4, and 24 h post-OGD conditioning), aligned with previous therapeutic efficacy studies performed in OWH slices [ 33 , 34 ]. After various BEV exposure timepoints of interest (4, 24, and 48 h), slices were stained with propidium iodide (PI) to quantify cell viability and fixed with 4% formalin.…”

Organotypic whole hemisphere brain slice models to study the effects of donor age and oxygen-glucose-deprivation on the extracellular properties of cortical and striatal tissue

“…We leverage PI, a fluorescence-based imaging approach for quantifying cell damage [ 35 , 36 ], to characterize how both the cortical and striatal regions of P10 and P17 OWH slices respond to the transition ex vivo. The cortex and striatum play critical roles in normal brain function [ 37 – 40 ] and are susceptible to hypoxic-ischemic events like cerebral ischemia [ 41 – 43 ], which we model using OGD.…”

Vitamin E Decreases Cytotoxicity and Mitigates Inflammatory and Oxidative Stress Responses in a Ferret Organotypic Brain Slice Model of Neonatal Hypoxia-Ischemia

“…The primary goal of the studies presented here was to examine regional and sex-based differences in cell death as a result of OGD and vitamin E treatment. However, we have previously shown that regional differences in treatment response are linked to local microglial responses to injury [40]. This may be a plausible reason for why the hippocampus, one of the brain regions most densely populated with microglia [41], showed a significant decrease in cytotoxicity after vitamin E exposure post-injury.…”