Specific Aim 2. Confirm that prolonged exposure to hypergravity leads to a breakdown in communication between distinct physiological systems, causing decrements in function. The same mice used in Aim 1 will be used here. Our model focuses on brainstem control of cardiopulmonary function, driven primarily by sympathetic/vagal innervation. The brainstem, spinal cord, heart and lungs will be collected. Analysis will focus specifically on the nucleus tractus solitarii (NTS), the first-order integrative site for vagal afferents projecting from the gut, heart, and lungs to the central nervous system. The NTS coordinates efferent output to targets that are critical for the control of breathing, heart/vasculature, and gut. In vivo and in vitro electrophysiology will be used to quantify changes in sympathetic tone and confirm that synaptic activity within the NTS is altered by changes in microbiome. Immunohistochemistry and RNAseq will be used to assess changes in inflammation and stress-related message, allowing us to determine the role cytokines and downstream effectors play in modulating NTS neuronal activity correlated with cardiorespiratory control.
Specific Aim 3. Determine that centrifugation-induced changes in health status signaling are linked to changes in microbiome. Based on our previous findings, we hypothesize that ingestion of a prebiotic supplement blend [galactooligosaccharides (GOS)+polydextrose (PDX)+lactoferrin + milk fat globule membrane protein (MFGMP)] will both prevent gut microbial dysbiosis produced by centrifugation and increase selective stress protective gut bacteria (i.e., lactobacillus and bifidobacteria). Prebiotic-enriched chow or calorically matched chow will be available ad libitum throughout the experiment, starting 1 week prior to centrifugation. Mice will be placed on the 8 ft diameter centrifuge at Ames Research Center and exposed to 2 G for 4 weeks. Parameters described in Aims 1 & 2 will be assessed as described.
Year 1: Experiment Overview Male and Female, C57BL/6 mice (10/group) were ordered from Jackson Laboratories and shipped to the Ames Research Center Animal Care Facility (ACF). After a 1-week acclimation period, the mice were moved to the 8 ft. diameter centrifuge facility for an additional 1-week acclimation period. During this time, we collected baseline ECG measurements and fecal samples. Thereafter, the mice were placed on the centrifuge and exposed to 2 G for 4 weeks. Controls were kept in the same room in similar lighting, temperature and noise conditions. The centrifuge was stopped briefly twice a week to change bedding, food, hydrogel packs and to perform health checks. Two weeks into the run, we collected fecal pellets from all mice and measured ECG activity on a subset of mice. After centrifugation, we again took ECG measurements on all mice prior to tissue collection. Adrenals, blood and spleen were collected to assess changes in sympathetic and inflammatory activity via flow cytometry, multiplexed cytokine arrays and ELISAs. Fecal pellets and intestines were collected to evaluate changes in the adherent commensal bacteria of the gut microbiome. Assays include 16S rRNA sequencing, taxonomic analyses, and selective bacterial culture.
Year 2: Experiment Overview For part two of the study, female C57BL/6 mice (10/group) will be used. All experiment details will remain the same as in year one except for the addition of a vivarium control group and the prebiotic diet (specific aim 3). The vivarium control group will be housed at the Animal Care Facility for the entire experiment duration and shall serve as the unexposed condition. Additionally, all mice will be provided either a prebiotic or control diet to evaluate the responses from changes in the gravitational environment.
This experiment is currently in progress. Results will be available at the conclusion of the study.
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