Nausea is the subjective experience of discomfort from an unpleasant wavelike sensation in the upper abdomen which may be accompanied with an urge to vomit; it can be induced by specific compounds and this response may be mediated by the vagus nerve, a direct neural connection in the brain-gut axis. Current research understands that gut-innervating vagal neurons can detect stomach stretch, hormones, irritants, and nutrients and send this sensory information from the gut to the area postrema (AP) and nucleus of the solitary tract (NTS), modifying behaviors such as nausea and feeding. To determine the nausea-related activation profiles through compound exposure, this study randomly screened compound libraries via in vitro assays by harvesting rat vagal nodose ganglia for cell culture and ratiometric calcium imaging to identify possible nausea signaling through the proxy of neuronal activity. Changes in intracellular calcium levels correlated to neuronal firing. This was repeated for identified compounds, capreomycin sulfate, zinc pyrithione, and caffeine, to validate dose response. Mice were gavaged in vivo with identified compounds and perfused for brain harvest; brains were sectioned then stained with c-Fos immunohistochemistry for imaging via confocal microscopy to detect c-Fos signal in the AP and/or NTS regions of the brainstem. By understanding how such identified compounds affect the brainstem regions related to nausea, we can expand our understanding of the mechanisms of nausea to develop better therapeutics.

Glioblastoma (GBM) is a highly aggressive brain cancer that is resistant to immunotherapy, largely due to poor infiltration of immune cells into the tumor microenvironment (TME). We are evaluating a novel mRNA lipid nanoparticle (LNP) vaccine for its immunogenicity, using a Pmel-1 T-cell receptor (TCR) transgenic mouse model. In this model, mosaic analysis by dual recombinase-mediated cassette exchange (MADR) glioma cells expressing gp100 antigen were implanted into the brains of C57BL/6 (Thy 1.2+) mice, followed by adoptive transfer of antigen-specific Pmel-1 T cells (Thy1.1+), and treatment with the mRNA-LNP vaccine or a placebo. To spatially analyze antigen-specific T-cell (Thy1.1+) behavior, multiplex immunofluorescence (mIF) was optimized using the Akoya Opal 6-plex Manual Detection Kit. Within the TME, preliminary data shows that vaccinated mice (EXP) had a robust expansion of T cells compared to controls (CTRL). There was a greater density of CD8+ killer T cells (815 vs 1.11 cells/mm²), CD4+ helper T cells (29 vs 3 cells/mm2), and antigen-specific T cells (110 vs 0 cells/mm2) in EXP mice. Additionally, aggregates of immune cells were observed around perivascular regions (CD31+) within the EXP TME, suggesting tertiary lymphoid structure (TLS)-like formation. These findings indicate that the mRNA vaccine successfully drives T cell infiltration into gp100-expressing GBM tumors, and provides a framework to immunophenotype T cell responses in Pmel-1 TCR tumor models.

Cardiovascular disease is the leading cause of death worldwide, and myocardial infarction (MI), or heart attack, often results in long-term damage due to poor vascular repair. Pericytes are cells that surround and support blood vessels, helping maintain vascular stability and tissue healing. Therefore, pericytes are proposed as a therapeutic target for improving cardiac repair, but the mechanisms regulating pericyte behavior are poorly understood. This project focused on protein p53, a cell cycle regulator, and its potential role in pericyte identity and function. We optimized an immunohistochemistry (IHC) protocol to reliably detect p53, and then evaluated the effects of pericyte ablation on cardiac structure and function using a mouse model that tracks pericytes with a tdTomato fluorescent reporter (Cspg4-Ai9) following diphtheria toxin (DTA) expression. Echocardiographic and ventricular fibrosis analysis revealed no significant differences in cardiac morphology or function between pericyte-ablated (DTA+) and control (WT) mice over time. These findings suggest pericyte loss alone may not lead to overt cardiac dysfunction under baseline conditions but may still subtly influence cardiac remodeling. Continuing studies using Fluorescence-Activated Cell Sorting, echocardiography, and IHC will investigate if pericyte-specific deletion of p53, which conversely preserves pericytes, affects vascular stability and cardiac repair following MI, with the goal of identifying new therapeutic strategies for cardiovascular disease.

As of 2023, type II diabetes impacts an estimated 33 to 35 million Americans. This potent metabolic disease is marked by high blood glucose levels and insulin resistance as a consequence of disrupted glucose homeostasis. Glucose metabolism at the single-cell level can be visualized using the HYlight biosensor, as noted by Koberstein et al. in a 2022 study. HYlight fluoresces when selectively binding with high affinity to fructose-1,6-bisphosphate (FBP), a product of the second rate limiting step in glycolysis. Thus HYlight can be utilized to visualize the energy state of the cell and glycolytic flux. While the Koberstein et al. study utilized pancreatic beta cells as a model, Teslaa Lab plans to utilize this HYlight system on muscle cells. Muscles act as a large reservoir of glycolytic intermediates, potentially playing a key role in glucose homeostasis. To establish a muscle cell line with stable expression of HYlight, we cloned the functional areas of the HYlight plasmid into a backbone with an antibiotic resistance gene for selection in mammalian cells. We are currently verifying whether HYlight responds reliably to perturbations expected to change FBP levels. Future experimentation involves utilizing this system for a small molecule screen for compounds that influence muscle glycolytic flux. The identified small molecules may be used for future study or treatment of Type II diabetes.

Sympathetic neural reinnervation of the heart post-myocardial infarction (MI) is a significant cause of arrhythmias. The role of extracellular matrix (ECM) remodeling processes which regulate reinnervation in large animal models remains incompletely understood. Chondroitin sulfate proteoglycans (CSPGs) have been shown to inhibit sympathetic axonal outgrowth in fibrotic scars. This study uses immunohistochemistry to illustrate the spatial relationship between CSPG deposition and noradrenergic nerve fibers. Sections of viable, border zone, and scar myocardium from pigs that had an MI were stained for CSPGs, dopamine β-hydroxylase (DBH) to identify sympathetic neurons that release norepinephrine, and phalloidin to delineate tissue structure. Qualitative analysis showed lesser DBH-positive fiber density where there was increased CSPG signal. This supports previous data demonstrating that CSPGs restrict sympathetic reinnervation post-cardiac ischemia-reperfusion injury in mice. Ongoing work will quantify the percent of area positive for CSPG signal, as well as the distribution of DBH-positive fibers using neurite tracing to quantify the extent of reinnervation. This study established a staining procedure to examine the interaction of ECM elements and neurons in a translationally significant model. Ultimately, the results of this study may provide valuable information regarding future therapies directed toward reducing arrhythmia risk associated with ECM remodeling.