Hutchinson, M. R. | bioRxiv Science

bioRxiv2023

Time course of lipopolysaccharide- and capsaicin-induced cytokine release in an ex vivo mouse model of vaginal inflammation

ProblemThe neuroimmune interface has been characterised in few areas of the body. The objective of this study was to investigate the neuroimmune interface in the mouse vagina through a novel ex vivo model, to determine whether LPS could directly activate and produce TRPV1-mediated neuronal activation. Method of StudyConcentrations of IL-1{beta} and IL-6 release into the supernatant at different times post ex vivo stimulation with LPS, capsaicin, or a combination of the two were assessed using enzyme-linked immunosorbent assay. ResultsThere were no differences in the supernatant concentration of IL-6 with different stimulation type nor stimulation time. Supernatant concentrations of IL-1{beta} were greater at the 20 hour time point than the 4 hour time point, and were greater for stimulations involving LPS. ConclusionThere is a clear pattern of pro-inflammatory neuroimmune responses following ex vivo stimulation of mouse vaginal tissues with capsaicin and LPS, evident as an increased IL-1{beta} output. This output is greatest at 20 hours post-stimulation, indicating this neuroimmune response is time-dependent.

neuroscience↗

bioRxiv2020

BrainPhys neuronal medium optimized for imaging and optogenetics in vitro

The capabilities of imaging technologies, fluorescent sensors, and optogenetics tools for cell biology have improved exponentially in the last ten years. At the same time, advances in cellular reprogramming and organoid engineering have quickly expanded the use of human neuronal models in vitro. Altogether this creates an increasing need for tissue culture conditions better adapted to live-cell imaging. Here, we identified multiple caveats of traditional media when used for live imaging and functional assays on neuronal cultures (e.g., phototoxicity, suboptimal fluorescence signals, and unphysiological neuronal activity). To overcome these issues, we developed a new neuromedium, "BrainPhys Imaging", in which we adjusted fluorescent and phototoxic compounds. The new medium is based on the formulation of the original BrainPhys medium, which we designed to better support the neuronal activity of human neurons in vitro 1. We tested the new imaging-optimized formulation on human neurons cultured in monolayers or organoids, and rat primary neurons. BrainPhys Imaging enhanced fluorescence signals and reduced phototoxicity throughout the entire light spectrum. Importantly, consistent with standard BrainPhys, we showed that the new imaging medium optimally supports the electrical and synaptic activity of midbrain and human cortical neurons in culture. We also benchmarked the capacity of the new medium for functional calcium imaging and optogenetic control of human neurons. Altogether, our study shows that the new BrainPhys Imaging improves the quality of a wide range of fluorescence imaging applications with live neurons in vitro while supporting cell viability and neuronal functions.

neuroscience↗