Characterization of a mGluR5 Knockout Rat Model with Hallmarks of Fragile X Syndrome.

The number of reported cases of neurodevelopmental disorders has increased significantly in the last few decades, but the etiology of these diseases remains poorly understood. There is evidence of a fundamental link between genetic abnormalities and symptoms of autism spectrum disorders (ASDs), and the most common monogenetic inheritable form of ASDs is Fragile X Syndrome (FXS). Previous studies indicate that FXS is linked to glutamate signaling regulation by the G-protein-coupled metabotropic glutamate receptor 5 (mGluR5), which has been shown to have a regulatory role in neuroinflammation. We characterized the effect of knocking out mGluR5 in an organism known to have complex cognitive functions-the rat. The heterozygous phenotype is the most clinically relevant; therefore, we performed analysis in heterozygous pups. We showed developmental abnormalities in heterozygous mGluR5 knockout rats, as well as a significant increase in chemokine (C-X-C motif) ligand 1 (CXCL) expression, a hallmark indicator of early onset inflammation. We quantified an increase in microglial density in the knockout pups and quantified morphological phenotypes representative of greater reactivity in the male vs. female and postnatal day 28 heterozygous pups compared to postnatal day 14 heterozygous pups. In response to injury, reactive microglia release matrix metalloproteases, contribute to extracellular matrix (ECM) breakdown, and are responsible for eradicating cellular and molecular debris. In our study, the changes in microglial density and reactivity correlated with abnormalities in the mRNA expression levels of ECM proteins and with the density of perineuronal nets. We saw atypical neuropsychiatric behavior in open field and elevated plus tests in heterozygous pups compared to wild-type litter and age-matched controls. These results demonstrate the pathological potential of the mGluR5 knockout in rats and further support the presence of neuroinflammatory roots in ASDs.

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

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.

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

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 human infants. 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 300 µM whole-hemisphere brain slices were obtained. During a 24-h rest period, slices were cultured in either nontreated control conditions or with erastin, a promotor of oxidative stress. Slices were then exposed to 2 h of OGD followed by vitamin E (25-100 IU/kg), erastin (10 µM), or ferrostatin (1 µM), an inhibitor of ferroptosis. Relative cytotoxicity was determined using a lactate dehydrogenase assay, cell death was quantified via nuclear propidium iodide staining, oxidative stress was quantified via cellular glutathione (GSH) 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.

Quantum Dot Cellular Uptake and Toxicity in the Developing Brain: Implications for Use as Imaging Probes.

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.