Traumatic brain injury involves a primary mechanical injury that is followed by a secondary<br/>inflammatory cascade. The inflammatory cascade in the CNS releases cytokines which are<br/>associated with leukocytosis and a systemic immune response. Acute changes to peripheral<br/>immune cell populations post-TBI include a 4.5-fold increase of neutrophils 3 hours post-injury,<br/>and 2.7-fold or higher increase of monocytes 24 hours post-injury. Flow Cytometry is a<br/>technique that integrates fluidics, optics, and electronics to characterize cells based on their light<br/>scatter and antigen expression via monoclonal antibodies conjugated to fluorochromes. Flow<br/>cytometry is a valuable tool in cell characterization however the standard technique for data<br/>analysis, manual gating, is associated with inefficiency, subjectivity, and irreproducibility.<br/>Unsupervised analysis that uses algorithms packaged as plug-ins for flow cytometry analysis<br/>software has been discussed as a solution to the limits of manual gating and as an alternative<br/>method of data visualization and exploration. This investigation evaluated the use of tSNE<br/>(dimensionality reduction algorithm) and FlowSOM (population clustering algorithm)<br/>unsupervised flow cytometry analysis of immune cell population changes in female mice that<br/>have been exposed to a LPS-induced systemic inflammatory challenge, results were compared to<br/>those of manual gating. Flow cytometry data was obtained from blood samples taken prior to and<br/>24 hours after LPS injection. Unsupervised analysis was able to identify populations of<br/>neutrophils and pro-inflammatory/anti-inflammatory monocytes, it also identified several more<br/>populations however further inquiry with a more specific fluorescent panel would be required to<br/>establish the specificity and validity of these populations. Unsupervised analysis with tSNE and<br/>FlowSOM demonstrated the efficient and intuitive nature of the technique, however it also<br/>illustrated the importance of the investigator in preparing data and modulating plug-in settings.

Damage to the Central Nervous System (CNS), such as traumatic brain injury (TBI) can often lead to a systemic inflammatory response since inflammatory mediators can be carried through the cardiovascular system. Past studies indicate that this inflammatory response that started at the CNS can increase the risk of heart disease. This growing interest in the heart-brain axis led our lab to explore if there is any impact of TBI on cardiac function and remodeling. TBI has been shown to have short-term effects on the heart, but few studies evaluate the long-term impact of TBI on the heart. To analyze any long-term impacts, we extracted hearts from rats 6 months post TBI, or sham that had been treated with vehicle or lipopolysaccharide (LPS) injections. LPS was administered to assess how inflammation could impact protein expression in the heart. Reactive oxygen species (ROS) targets such as NOX2, NOX4, SOD1, SOD2, catalase, and osteopontin were measured as potential indicators of cardiac remodeling. Rats that received vehicle TBI and LPS TBI resulted in no statistically significant differences (p>0.05) when evaluated as fold-change over the vehicle. This trend was consistent when normalizing to LPS sham. Since there were no changes in ROS targets, the hypothesis that there is long-term cardiac remodeling in the heart post-TBI was rejected. Further investigation is warranted since the present design of this study may not be ideal for evaluating long-term impact as histology samples were not obtained nor cardiac function assessments.