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. 2020 Dec 10;10:21629. doi: 10.1038/s41598-020-78410-2

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Experimental procedures. (a) The procedure for measuring blood BHB concentration is shown. Rats were injected with equal volumes of BHB (250 mg/kg) or PBS. Tail vein blood was collected from the same rats, at the following time points: pre-injection, 10, 20, 30, 45, and 60 min after injection with BHB. (b) Schematic representation of the SPS procedure and behavioral tests conducted. The three behavioral experiments performed were as follows: (1) non-SPS (control) vs SPS as a PTSD model (n = 12/group), (2) SPS-induced PTSD without BHB treatment vs SPS-induced PTSD with BHB treatment (n = 15/group), (3) controls without BHB vs controls with BHB (n = 9/group). On day 1, rats were exposed to SPS. From days 2–13, rats were injected with BHB, subcutaneously, twice per day (except for the experiment (1): non-SPS vs SPS). Habituation to the EPM was done on day 8 and the OFT was conducted on day 10, and the EPM on day 14. On behavioral test days, BHB or PBS, was injected following testing. (c) Schematic representation of the SPS procedure and blood collection. Three groups were allocated as follows: A non-SPS without BHB administration group (n = 18), a SPS without BHB administration group (n = 9), and a SPS with BHB administration group (n = 9). On day 1, rats were exposed to SPS. From days 2–14, rats were injected with BHB or PBS, twice per day. On day 15, the rats were euthanized, and blood was collected. BHB: beta-hydroxybutyrate, SPS: single prolonged stress, Veh: Vehicle (PBS), EPM: elevated plus maze test. OFT: open field test.