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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. 

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.