Collection and measurement of circadian minimum stress hormones in nocturnal mice to create baseline concentrations.

1. Research Question: Will measurement of early morning stress hormone concentrations as baselines improve results of previous mouse experiments?2. Hypothesis (prediction): Measurement of early morning stress hormone concentrations as baselines will improve results of previous mouse experiments.

3. Method to Test the Hypothesis: Enzyme Linked Immuno Sorbent Assays (ELISA) will be used to collect data to test the prediction.In previous experiments raw ELISA data from both Corticosterone and Cortisone blood serum samples had large variances after several hours of anesthesia and surgical manipulations.  Those raw data results were interpreted as just barely significant or just barely non-significant.  Confidence intervals hovered near or below 90% instead of 95% which was expected.  Today, many journals require that studies report confidence intervals as well as statistical significance and those confidence intervals need to be improved here.  Therefore, this 2023 proposal will collect baseline data and address those statistical issues by transforming the raw concentration data into a percent change from baseline data.  A baseline concentration for both stress hormones will be created by collecting blood serum from stress-free (anesthesia only) mice.  This will take place at a point in their nocturnal circadian rhythm when their stress hormone levels are at the lowest, which is just before 7am and lights on in their room.  Thirty mice for each of the two hormones will be needed to produce these two baseline concentrations.  It is expected that the improvement from raw concentration data to percent change from baseline data will improve significance and confidence intervals and expedite publishing those experiments. The reason our previous studies could not use a Pre-test / Post-test design to produce within-subjects controls was because the mice were euthanized by heart puncture during blood collection.  Heart puncture was preferred because tail vein blood samples and retro-orbital blood collection would not have provided enough blood for ELISA. Thus, no within-subjects stress hormone baseline concentrations could have been produced for each individual mouse making the experimental design Post-test only.  Therefore, an after the fact (ex post facto) establishment of stress hormone baselines based on circadian rhythm low points for both hormones is now required.