Cerebral ischemia is a major cause of death in both neonates and adults, and currently has no cure. Nanotechnology represents one promising area of therapeutic development for cerebral ischemia due to the ability of nanoparticles to overcome biological barriers in the brain. ex vivo injury models have emerged as a highthroughput alternative that can recapitulate disease processes and enable nanoscale probing of the brain microenvironment. In this study, we used oxygen-glucose deprivation (OGD) to model ischemic injury and studied nanoparticle interaction with microglia, resident immune cells in the brain that are of increasing interest for therapeutic delivery. By measuring cell death and glutathione production, we evaluated the effect of OGD exposure time and treatment with azithromycin (AZ) on slice health. We found a robust injury response with 0.5 hr of OGD exposure and effective treatment after immediate application of AZ. We observed an OGD-induced shift in microglial morphology toward increased heterogeneity and circularity, and a decrease in microglial number, which was reversed after treatment. OGD enhanced diffusion of polystyrene-poly(ethylene glycol) (PS-PEG) nanoparticles, improving transport and ability to reach target cells. While microglial uptake of dendrimers or quantum dots (QDs) was not enhanced after injury, internalization of PS-PEG was significantly increased. For PS-PEG, AZ treatment restored microglial uptake to normal control levels. Our results suggest that different nanoparticle platforms should be carefully screened before application and upon doing so; disease-mediated changes in the brain microenvironment can be leveraged by nanoscale drug delivery devices for enhanced cell interaction.
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.
Hypoxic-Ischemic Encephalopathy (HIE) in the brain is the leading cause of morbidity and mortality in neonates and can lead to irreparable tissue damage and cognition. Thus, investigating key mediators of the HI response to identify points of therapeutic intervention has significant clinical potential. Brain repair after HI requires highly coordinated injury responses mediated by cell-derived extracellular vesicles (EVs). Studies show that stem cell-derived EVs attenuate the injury response in ischemic models by releasing neuroprotective, neurogenic, and anti-inflammatory factors. In contrast to 2D cell cultures, we successfully isolated and characterized EVs from whole brain rat tissue (BEV) to study the therapeutic potential of endogenous EVs. We showed that BEVs decrease cytotoxicity in an ex vivo oxygen glucose deprivation (OGD) brain slice model of HI in a dose- and time-dependent manner. The minimum therapeutic dosage was determined to be 25 μg BEVs with a therapeutic application time window of 4–24 h post-injury. At this therapeutic dosage, BEV treatment increased anti-inflammatory cytokine expression. The morphology of microglia was also observed to shift from an amoeboid, inflammatory phenotype to a restorative, anti-inflammatory phenotype between 24–48 h of BEV exposure after OGD injury, indicating a shift in phenotype following BEV treatment. These results demonstrate the use of OWH brain slices to facilitate understanding of BEV activity and therapeutic potential in complex brain pathologies for treating neurological injury in neonates.
BackgroundWe performed a systematic review that identified at least 9,000 scientific papers on PubMed that include immunofluorescent images of cells from the central nervous system (CNS). These CNS papers contain tens of thousands of immunofluorescent neural images supporting the findings of over 50,000 associated researchers. While many existing reviews discuss different aspects of immunofluorescent microscopy, such as image acquisition and staining protocols, few papers discuss immunofluorescent imaging from an image-processing perspective. We analyzed the literature to determine the image processing methods that were commonly published alongside the associated CNS cell, microscopy technique, and animal model, and highlight gaps in image processing documentation and reporting in the CNS research field.MethodsWe completed a comprehensive search of PubMed publications using Medical Subject Headings (MeSH) terms and other general search terms for CNS cells and common fluorescent microscopy techniques. Publications were found on PubMed using a combination of column description terms and row description terms. We manually tagged the comma-separated values file (CSV) metadata of each publication with the following categories: animal or cell model, quantified features, threshold techniques, segmentation techniques, and image processing software.ResultsOf the almost 9,000 immunofluorescent imaging papers identified in our search, only 856 explicitly include image processing information. Moreover, hundreds of the 856 papers are missing thresholding, segmentation, and morphological feature details necessary for explainable, unbiased, and reproducible results. In our assessment of the literature, we visualized current image processing practices, compiled the image processing options from the top twelve software programs, and designed a road map to enhance image processing. We determined that thresholding and segmentation methods were often left out of publications and underreported or underutilized for quantifying CNS cell research.DiscussionLess than 10% of papers with immunofluorescent images include image processing in their methods. A few authors are implementing advanced methods in image analysis to quantify over 40 different CNS cell features, which can provide quantitative insights in CNS cell features that will advance CNS research. However, our review puts forward that image analysis methods will remain limited in rigor and reproducibility without more rigorous and detailed reporting of image processing methods.ConclusionImage processing is a critical part of CNS research that must be improved to increase scientific insight, explainability, reproducibility, and rigor.
Translation of nanotherapeutics from preclinical research to clinical application is difficult due to the complex and dynamic interaction space between the nanotherapeutic and the brain environment. To improve translation, increased insight into nanoformulation-brain interactions in preclinical research is necessary. We developed a nanoformulation-brain database and wrote queries to connect the complex physical, chemical, and biological features of neurotherapeutics based on experimental data. We queried the database to select nanoformulations based on specific physical characteristics that enable effective penetration within the brain, including size, polydispersity index, and zeta potential. Additionally, we demonstrate the ability to query the database to return select nanoformulation characteristics, including nanoformulation methodology or methodological variables such as surfactant, polymer, drug loading, and sonication times. Finally, we show the capacity of our database to produce correlations relating nanoparticle formulation parameters to biological outcomes, including nanotherapeutic impact on cell viability in cultured brain slices.
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