016 - Regional Transcriptomic Responses to Injury in an Extremely Preterm Ferret Organotypic Brain Slice Model

Publication Number: 16.6326

Olivia C. Brandon, University of Washington: Magnuson Health Sciences Center – RR 544A, Seattle, WA, United States; Henry S. Jacqmotte, University of Washington School of Medicine, Seattle, WA, United States; Zheyu (Ruby) Jin, University of Washington, Seattle, WA, United States; Kylie Corry, University of Washington School of Medicine, Seattle, WA, United States; Daniel Moralejo, University of Washington School of Medicine, Seattle, WA, United States; Robell Mateo M. Bassett, University of Washington School of Medicine, Missoula, MT, United States; Hawley Helmbrecht, Johns Hopkins University School of Medicine, Baltimore, MD, United States; Sarah E. Kolnik, University of Washington - Seattle Children's Hospital, Seattle, WA, United States; Sandra E. Juul, University of Washington, Seattle, WA, United States; Elizabeth Nance, University of Washington School of Medicine, Seattle, WA, United States; Thomas R. Wood, University of Washington School of Medicine, Seattle, WA, United States

Background: Over 50% of extremely preterm (EP, < 28 weeks’ gestation) newborns die or develop a significant disability. A key challenge in translating neuroprotective treatments for EP infants is varied regional responses of the brain to injury and therapy. The ferret brain, with its similarities to the human brain in gyrification and white matter development, serves as an ideal model to better understand these regional differences, which could improve translation of therapies to the clinic.

Objective: To examine regional transcriptomic responses to injury in the cortex, deep grey matter (including the hippocampus, thalamus, and basal ganglia), and white matter in an EP-equivalent ex vivo ferret brain slice model of oxygen glucose deprivation (OGD).

Design/Methods: 300μm whole-hemisphere postnatal day 14 live ferret brain slices were cultured for 72h in vitro and then randomized to control or OGD (2h of glucose-depleted media at 0% oxygen for 2 h). Slices were cultured for another 24h before being micro dissected. Regional digital transcriptomics were analyzed used a ferret-specific NanoString nCounter panel. A log2-fold change was calculated for transcripts comparing expression after OGD to control using a t-test. Gene ontology enrichment analyses were performed on differentially expressed transcripts using ShinyGO graphical gene-set enrichment tool.

Results: Of the 255 transcripts assessed, n=143 were differentially regulated in response to OGD in the cortex, n=125 in the white matter, and n=32 in the deep grey matter (Fig 1). N=24 transcripts were differentially regulated in response to OGD across all regions, while n=33 were only affected in the cortex, n=9 in the white matter, and n=2 in the deep grey matter (Fig 2). Transcripts related to neuron projection morphogenesis were the most upregulated in both the cortex and the white matter, while those related to regeneration and glial cell differentiation were significantly downregulated in the cortex and white matter, respectively (Fig 3A-B, D-E). In the deep grey matter, transcripts related to regulation of glucose transport were upregulated, while those related to regeneration were downregulated (Fig 3C, F).

Conclusion(s): In our EP-equivalent ferret brain slice model, distinct regional patterns of gene expression were observed in response to OGD. In addition to region-specific responses, neuron projection morphogenesis transcripts were the most upregulated in both the cortex and white matter, while regeneration-related transcripts were downregulated across all regions. These pathways may be potential targets for future therapeutic research in EP brain injury.

Figure 1. Regional transcriptomics in response to oxygen glucose deprivation (OGD).
Volcano plots of differentially expressed transcripts in the cortex (A), white matter (B), and deep grey matter (C) after OGD. Colored dots represent transcripts that were significantly differentially expressed after OGD (p < 0.05). Positive log2 fold-change (red) indicates significantly upregulated expression after OGD, and negative log2 fold-change (blue) indicates significantly downregulated expression. The tables describe the top 5 most upregulated (top rows) or downregulated (bottom rows) transcripts and their functions in the cortex (D), white matter (E), and deep grey matter (F).

Figure 2. Transcripts significantly differentially expressed after oxygen glucose deprivation (OGD) in only one region.
Gene ontology enrichment analyses were performed on differentially expressed transcripts in only one region with the ShinyGO graphical gene-set enrichment tool using the ferret genome (A-B). Only 2 transcripts were significantly altered in the deep grey matter relating to myelination, cell-cell adhesion, and anti-inflammatory response. Transcripts and their corresponding log2 fold change that are significantly upregulated (red) or downregulated (blue) after OGD (C).

Figure 3. Biological processes related to differentially regulated transcripts of interest.
Upregulated (top row) or downregulated (bottom row) transcripts after OGD in the cortex (left column; A, D), white matter (middle column; B, E), and deep grey matter (right column; C, F). Gene ontology enrichment analyses were performed on differentially expressed transcripts with the ShinyGO graphical gene-set enrichment tool using the ferret genome.