Matthew R. Rusling James DeMar Nabarun Chakraborty Allison V. Hoke ^* Stacy A. Miller ^* John G. Rosenberger ^* Andrew Batuure Donna M. Wilder ^* V Sujith Sajja Joseph B. Long ^* Rasha Hammamieh Aarti Gautam ^*

• Walter Reed Army Institute of Research, Silver Spring, United States

Sequelae from traumatic brain injuries (TBI) and post-traumatic stress disorder (PTSD), are major career-limiting factors for combat soldiers. Overlap between TBI and PTSD symptoms alongside other common comorbidities complicate the diagnosis and treatment. Systems level and high throughput approaches are key in understanding the underlying biomolecular mechanisms and differentiating these conditions. The present study identifies dietary factors & proposes mechanisms behind psychological stress and TBI, using established pre-clinical animal models and a multi-omics approach. Here we used microbiome characterizations of rats exposed to simulations of blast-induced TBI and underwater trauma (UWT)-induced psychological stress. We further studied the effect of dietary omega-6 versus omega-3 polyunsaturated fatty acid (n-6, n-3 PUFA) enrichment on the insult responses. Use of excess n-6 PUFA was chosen due to its high prevalence in the Western diet and pro-inflammatory nature. Prior to TBI or UWT, animals were maintained for six weeks and continued thereafter on either a standard diet or two customized chows imbalanced and diminished in omega-3 content. Corresponding shams were done for all groups. Fecal bacterial microbiome populations were assessed using 16S rRNA gene sequencing. Physiologic outcome modelling identified that dietary status affected post-TBI Lactate Dehydrogenase (LDH) & Triglyceride (TG) levels with n-3 PUFA having a large attenuating influence. The UWT model showed similar trends, where diet significantly altered LDH, terminal corticosterone (14 days post-exposure), and a fear behavior susceptibility. Fecal microbiome alpha diversity was significantly reduced by high levels of n-3 PUFA. Likewise, beta diversity of the microbiome was significantly affected by both diet & time but not exposure to TBI or UWT. Compositionally, temporal effects on the microbiome were more likely to be observed with the diets. The most affected features fell within the Proteobacteria phyla, in which n-3 PUFA enrichment significantly reduced Alphaproteobacteria in the TBI model, and increased Gammaproteobacteria in the UWT group. All these observations can influence vulnerability or resilience of the Warfighter to blast induced TBI and acute psychological stress. The microbiome mechanisms facilitate and provide a knowledge-driven unbiased panel of signatures to discriminate between the two insults and is an essential tool for designing precise care management.