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However, the underlying biological mechanisms remain unclear. This study set out to investigate dynamic changes in urinary proteome due to MH in rats with cerebral I\/R injury and explore the neuroprotective mechanisms of MH. A Pulsinelli\u2019s four-vessel occlusion (4-VO) rat model was used to mimic global cerebral I\/R injury. Liquid chromatography-tandem mass spectrometry (LC\u2013MS\/MS) was employed to profile the urinary proteome of rats with\/without MH (32\u00a0\u00b0C) treatment after I\/R injury. Representative differentially expressed proteins (DEPs) associated with MH were validated by western blotting in hippocampus.\u00a0A total of 597 urinary proteins were identified, among which 119 demonstrated significant changes associated with MH. Gene Ontology (GO) annotation of the DEPs revealed that MH significantly enriched in endopeptidase activity, inflammatory response, aging, response to oxidative stress and reactive oxygen species, blood coagulation, and cell adhesion. Notably, changes in 12 DEPs were significantly reversed by MH treatment. Among them, 8 differential urinary proteins were previously reported to be closely associated with brain disease, including NP, FZD1, B2M, EPCR, ATRN, MB, CA1and VPS4A. Two representative proteins (FZD1, B2M) were further validated by western blotting in the hippocampus and the results were shown to be consistent with urinary proteomic analysis. Overall, this study strengthens the idea that urinary proteome can sensitively reflect pathophysiological changes in the brain, and appears to be the first study to explore the neuroprotective effects of MH by urinary proteomic analysis. FZD1 and B2M may be involved in the most fundamental molecular biological mechanisms of MH neuroprotection.<\/jats:p>\n                <jats:p><jats:bold>Graphical Abstract<\/jats:bold><\/jats:p>\n                <jats:p>In a 4-VO rat model, 119 urinary proteins demonstrated significant changes associated with MH. MH is enriched in endopeptidase activity, inflammatory response, oxidative stress, etc, and significantly reversed changes in 12 DEPs. FZD1 and B2M are thought to be involved in the most fundamental molecular biological mechanisms of MH neuroprotection.<\/jats:p>","DOI":"10.1007\/s10571-024-01483-4","type":"journal-article","created":{"date-parts":[[2024,6,5]],"date-time":"2024-06-05T07:01:41Z","timestamp":1717570901000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Urine Proteomic Signatures of Mild Hypothermia Treatment in Cerebral Ischemia\u2013Reperfusion Injury in Rats"],"prefix":"10.1007","volume":"44","author":[{"given":"Dandan","family":"Zhang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dapeng","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xueting","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanyan","family":"Sui","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fuguo","family":"Ma","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuting","family":"Dai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mingshan","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Weiwei","family":"Qin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,6,5]]},"reference":[{"issue":"6","key":"1483_CR1","doi-asserted-by":"publisher","first-page":"345","DOI":"10.1016\/j.gpb.2015.08.005","volume":"13","author":"M An","year":"2015","unstructured":"An M, Gao Y (2015) Urinary biomarkers of brain diseases. 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The animal experiments were reviewed and approved by Qingdao Municipal Hospital Medical Ethics Committee (2021Y39).","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethical Approval"}},{"value":"We declared that this study was carried out in compliance with the ARRIVE guidelines.","order":5,"name":"Ethics","group":{"name":"EthicsHeading","label":"Statement on ARRIVE Guidelines"}}],"article-number":"49"}}