P0734 - Erythropoietin Mitigates Inflammatory and Oxidative Stress Responses to Oxygen-Glucose Deprivation (OGD) in a Ferret Organotypic Brain Slice Model

Background: The ferret brain more closely models the human brain than does the rodent, making it an excellent mid-sized animal in which to study hypoxia-ischemia (HI), a significant contributor to neurological injury in neonates. Erythropoietin (Epo) has antioxidant and anti-inflammatory properties in HI rodent studies, but it is not known if these properties are generalizable to other animal models.

Objective: To assess the downstream effects of Epo after oxygen-glucose deprivation (OGD) in a ferret organotypic brain slice model, which retains much of the cellular and structural architecture of the intact brain. We hypothesized that Epo would decrease cytotoxicity, inflammation and oxidative stress in OGD-exposed brain slices.

Design/

Methods: Term-equivalent ferrets were sacrificed at postnatal (P) day 21-23, and 300µM whole hemisphere slices were obtained using a vibratome. After 24h rest, slices were exposed to non-treated (NT) control conditions or 2h of OGD with or without Epo (1 U/mL). After 24h, relative cytotoxicity of brain slices was determined using an LDH assay, cell death was quantified via confocal microscopy of propidium iodide (PI) stained cells, cellular glutathione (GSH) levels were measured, and qRT-PCR evaluated target genes responsive to oxidative stress (CHAC, PTGES2, SLC7A11, GCLM, HMOX1) and inflammation (IL-1beta, IL-6, IL-8, IL-10, TNF-alpha).

Results: OGD resulted in increased PI uptake and significantly higher cell death (34.0%) compared to NT controls (7.3%; p=0.014); Addition of Epo after OGD decreased cell death to 23.3% (Figure 1).There was a trend towards increased LDH levels in the supernatant of slices exposed to OGD compared to controls (p=0.052). Slices exposed to Epo after OGD showed no difference in LDH levels compared to NT (p=0.23, n=18 per group; Figure 1). GSH was significantly reduced in slices exposed to OGD (p<0.002) with recovery in OGD slices treated with Epo (n=minimum 10 per group; Figure 2). OGD increased markers of oxidative stress, which were partly or fully-normalized with Epo treatment (n=18; Figure 3). OGD also resulted in amplification of markers of inflammation. Epo treatment significantly reduced IL-8, IL-10, IL-1B expression (n=18; p<0.05; Figure 2).

Conclusion: The ferret whole hemisphere OGD slice culture model provides a higher-throughput platform in which to investigate injury in living tissue and quantify oxidative stress and inflammation. Results from this pilot study in the ferret support the premise that acutely decreasing inflammation and oxidative stress contribute to the neuroprotective properties of Epo.