Imaging of Gut Bacterial Macroscopic Changes in Simulated Microgravity-Exposed Rats via <i>In Vivo</i> Metabolic Labeling

The impact of the microgravity environment on gut bacteria has been widely recognized to induce notable gastrointestinal pathology during extended spaceflight. However, most current studies for gut microbiome homeostasis profiling are based on the 16S rRNA gene sequencing of fecal samples; this technology faces challenges in analyzing gut bacterial alterations <i>in situ</i>, dynamically, and with high spatiotemporal resolution. Herein, we present the utilization of bioorthogonal metabolic labeling for noninvasive imaging of gut bacterial macroscopic changes in simulated microgravity (SMG) rats. After being subsequently labeled with the metabolic reporters d-Ala-N₃ and ICG-DBCO through click chemistry, it was shown that SMG can trigger obvious perturbation of gut bacteria, evidenced by the significant increase in the total bacterial content and spatial distribution variations. Such a difference was accompanied by the occurrence of intestinal inflammation and tissue damage. Compared with 16S rRNA genome analysis focusing on composition and diversity, the metabolic labeling strategy provides unprecedented insights into the macroscopic changes of the gut bacterial content and distribution under SMG. Our study will be helpful for investigating the biological implication of SMG-induced imbalance in gut bacteria, potentially promoting the deep investigation of the complex gastrointestinal pathology in space biomedicine.