Nanometer-sized luminescent semiconductor quantum dots (QDs) have been utilized as imaging and therapeutic agents in a variety of disease settings, including diseases of the central nervous system. QDs have several advantages over traditional fluorescent probes including their small size (5–10 nm), tunable excitation and emission spectra, tailorable surface functionality, efficient photoluminescence, and robust photostability, which are ideal characteristics for in vivo imaging. Although QDs are promising imaging agents in brain-related applications, no systematic evaluation of QD behavior in brain-relevant conditions has yet been done. Therefore, we sought to investigate QD colloidal stability, cellular uptake, and toxicity in vitro, ex vivo, and in vivo in the brain environment. We found that QD behavior is highly dependent on surface functionality and that treatment of cultured organotypic whole hemisphere (OWH) slices with QDs results in dose-dependent toxicity and metallothionein increase, but no subsequent mRNA expression level changes in inflammatory cytokines or other oxidative stress. QDs coated with poly(ethylene glycol) (PEG) were protected from aggregation in neurophysiologically relevant fluids and in tissue, allowing for greater penetration. Importantly, QD behavior differed in cultured slices as compared to monolayer cell cultures, and behavior in cultured slices aligned more closely with that seen in vivo. Irrespective of surface chemistry and brain-relevant platform, non-aggregated QDs were primarily internalized by microglia in a region-dependent manner both in slices and in vivo upon systemic administration. This knowledge will help guide further engineering of candidate QD-based imaging probes for neurological application.

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.

The gyrencephalic ferret brain is an excellent model in which to study hypoxia-ischemia (HI), a significant contributor to neurological injury in neonates. Vitamin E, an essential fat-soluble antioxidant, reduces oxidative stress and inflammation in both animal models and neonates. The aim of this study was to assess the effects of Vitamin E after oxygen glucose deprivation (OGD) in an organotypic ferret brain slice model of neonatal HI. We hypothesized that Vitamin E would decrease cytotoxicity, inflammation, and oxidative stress in OGD-exposed brain slices. Term-equivalent ferrets were sacrificed at postnatal (P) day 21-23 and 300µM whole hemisphere brain slices were obtained. During a 24h rest period, slices were cultured in either non-treated control conditions or with Erastin, a promotor of oxidative stress. Slices were then exposed to 2h of OGD followed by Vitamin E (25-100 IU/kg), Erastin (10µM) or Ferrostatin (1µM), an inhibitor of ferroptosis. Relative cytotoxicity was determined using an LDH assay, cell death was quantified via nuclear propidium iodide (PI) staining, oxidative stress was quantified via cellular GSH (glutathione) levels and target genes responsive to oxidative stress and inflammation were evaluated by qRT-PCR. OGD increased cytotoxicity, which was significantly reduced by treatment with Vitamin E. Vitamin E also preserved GSH after OGD and decreased amplification of certain markers of oxidative stress (CHAC1, SLC7A11) and inflammation (TNF-alpha, IL-8). Vitamin E remained protective after pretreatment with Erastin and was more protective than Ferrostatin, presumably due to its added anti-inflammatory properties. Results from the ferret whole hemisphere OGD model support the premise that Vitamin E neuroprotection is mediated by restoring GSH and acutely decreasing inflammation and oxidative stress after neonatal HI brain injury.

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