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

Abstract

Background: 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. We demonstrate the utility of organotypic whole hemisphere brain slices as a platform to probe regional and developmental changes in the brain extracellular environment. We also leverage whole hemisphere brain slices to characterize the impact of cerebral ischemia on different regions of brain tissue.

Results: Whole hemisphere brain slices taken from postnatal (P) day 10 and P17 rats retained viable, metabolically active cells through 14 days in vitro (DIV). Oxygen-glucose-deprivation (OGD), used to model a cerebral ischemic event in vivo, resulted in reduced slice metabolic activity and elevated cell death, regardless of slice age. Slices from P10 and P17 brains showed an oligodendrocyte and microglia-driven proliferative response after OGD exposure, higher than the proliferative response seen in DIV-matched normal control slices. Multiple particle tracking in oxygen-glucose-deprived brain slices revealed that oxygen-glucose-deprivation 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. However, changes in diffusion were not universal across all brain regions and ages.

Conclusions: We demonstrate whole hemisphere brain slices from P10 and P17 rats can be cultured up to two weeks in vitro. These brain slices provide a viable platform for studying both normal physiological processes and injury associated mechanisms with control over brain age and region. Ex vivo OGD impacted cortical and striatal brain tissue differently, aligning with preexisting data generated in in vivo models. These data motivate the need to account for both brain region and age when investigating mechanisms of injury and designing potential therapies for cerebral ischemia.

Keywords: Brain microenvironment; Brain slices; Extracellular; Nanoparticle diffusion; Organotypic; Particle tracking.

Fig. 1

Schematic of OWH slice culture methodology and experimental workflow. A Left: Coronal slices containing cortex (CTX) and striatum (STR) are plated onto PTFE membrane inserts. Right: a single OWH brain slice after explantation. Although not imaged in this study, hippocampal (HIP) and corpus callosum (CC) regions are also labeled for reference. Scale bars = 15 cm and 5 cm. B Experimental workflow. Slices from postnatal (P) day 10 or P17 aged rat donors are cultured up to 14 days, performing OGD on 4 DIV with endpoints at 5 days in vitro (DIV) (24 h post-OGD), 7 DIV (72 h post-OGD), and 10 DIV

Fig. 2

Metabolic activity and LDH release profiles for P10 and P17 brain slices for 14 DIV following explantation. Metabolic activity values are normalized to the metabolic activity at the acute timepoint. LDH release (%) values are normalized to the LDH release of acute slices immediately treated with TX-100. A-B Metabolic activity of (A) P10 and (B) P17 slices. Error bars represent the mean ± SD. C-D % LDH release profile of (C) P10 and (D) P17 slices. Error bars represent median ± IQR (C) and mean ± SD (D). n = 6–42 OWH slices. * denotes significant differences (p < 0.05). For metabolic activity data (A&B) and LDH release data in P10 brain slices (C), comparisons were made between the 4DIV timepoint and all other time points. For LDH release data in P17 brain slices (D), comparisons were made between the 11DIV timepoint and all other time points. In all instances (A-D), comparisons were also made between the 14DIV timepoint and the 14DIV, TX-100 treated group

Fig. 3

Propidium iodide imaging in normal control P10 and P17 slices through 14DIV. Representative 40x images of either (A) P10 or (B) P17 brain slices taken at acute, 1DIV, 4DIV, 7DIV, 11DIV, and 14DIV timepoints. Images are split into two rows for each age. Top row: cortex; bottom row: striatum. Error bars: 100 μm. C-H Quantification of propidium iodide imaging in normal control P10 and P17 slices through 14DIV. Profiles for P10 slices are given for (C) all data combined, (D) the cortex, and (E) the striatum. P17 profiles are given for (F) all data combined, (G) the cortex, and (H) the striatum. Error bars represent the median ± IQR

Fig. 4

MPT in P10 and P17 NC brain slices through 14DIV. D_b,eff distributions at acute, 1DIV, 4DIV, 7DIV, 11DIV, and 14DIV. Data is provided for the (A) cortex of P10 slices, (B) striatum of P10 slices, (C) cortex of P17 slices, and (D) striatum of P17 slices. For D_b,eff distributions, error bars represent the median value and IQR. E Median D_{b, eff} values are provided for each age and brain region at each timepoint

Fig. 5

Metabolic activity, LDH release, and cell proliferation profiles following OGD. A Metabolic activity of P10 slices after 1 h, 2 h, or 3 h OGD. Error bars represent median and IQR. B LDH release (%) profiles for P10 slices after 1 h, 2 h, or 3 h OGD. Error bars represent mean ± SD. C Metabolic activity profiles for P17 slices after 1 h, 2 h, or 3 h OGD. Error bars represent median and IQR. * denotes significant differences (p < 0.05) between all OGD groups (1 h, 2 h, and 3 h) and the NC. D LDH release (%) profiles for P17 slices after 1 h, 2 h, or 3 h OGD. Error bars represent mean ± SD. * denotes significant differences (p < 0.05) between all OGD groups (1 h, 2 h, and 3 h) and the NC. ☨ denotes significant differences (p < 0.05) between the 1 h and 3 h OGD groups and the NC. For (A) and (C), n = 6–12 slices per condition. For (B) and (D), n = 6 slices for all OGD conditions, and n = 12–42 slices for NC conditions. Each data point represents a single slice. E Percentage of proliferating cells in P10 slices throughout culture and after 2 h OGD. 24 h time points refer to time after OGD, equivalent to 5DIV; 72 h time points refer to 72 h after OGD, equivalent to 7DIV. Data from a NC slice at 144 h (10DIV) is provided for comparison. F Percentage of proliferating cells in the cortex and striatum following 2 h OGD and compared to NC. Each data point represents a single image from 3 total slices per group

Fig. 6

Profiles of % of cells damaged following 1 h, 2 h, or 3 h OGD quantified via PI staining, live-dead cell counting, and HIF1α intensity. Data from each age are contained in their own row. Each dot represents a single ROI, where n = 10–12 ROIs with 5–6 ROIs per slice. P10 (A) cortex and (B) striatum data are presented in the top row. P17 (C) cortex and (D) striatum data are given in the bottom row. Error bars represent the median ± IQR. E Representative images of HIF1α staining 24 h after 2 h OGD at 4DIV from P10 slices (scale bar = 50 μm). F Mean intensity of HIF1α staining 24 h and 72 h after 2 h OGD. Each point represents a single image. G Mean intensity of HIF1α staining 24 h and 72 h after 2 h OGD in the cortex and striatum. Error bars represent the median ± IQR for (E, F, G)

Fig. 7

Cellular response immediately following 2 h OGD. Representative images are shown for (A) P10 and (B) P17 slices for PI (red) and NeuN (green) co-staining. The left column has a 40x magnification image; higher magnification images from the white dashed line box display a merged image, an image of PI alone, and an image of NeuN alone. Scale bars for the 40x magnification images: 100 μm. Scale bars for the zoomed in images: 25 μm. C Representative images of Iba + microglia (green) and Olig2+ oligodendrocytes (green) co-stained with EdU (yellow) marker for proliferation in the striatum 24 h after OGD or at equivalent culture time for NC. In all images, cell nuclei are stained with DAPI (blue). Scale bars = 50 μm

Fig. 8

D_b,eff distributions for 40 nm PS-PEG nanoparticles navigating the extracellular space of P10 and P17 brain slices following 1 h, 2 h, or 3 h OGD. D_b,eff distributions were generated from MPT experiments performed at an acute, 24 h-, and 72-96 h post-OGD timepoint. Each row represents a brain age. The top row consists of data generated from the (A) cortex and (B) striatum of P10 OWH brain slices. The bottom row contains D_b,eff distributions from the (C) cortex and (D) striatum of P17 OWH brain slices. In all instances, the red, green, and blue markers represent 1 h, 2 h, and 3 h OGD groups, respectively. Error bars represent the median D_b,eff ± IQR