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Pulmonary failure in SARS-CoV2-infected hospitalized COVID-19 patients is one such prominent example. Intervention strategies require characterization of the host-pathogen interaction by accurately assessing all of the above-mentioned disease parameters. To study infection in intact mammals, mice are often used as essential genetic models. Due to humane concerns, there is a constant unmet demand to develop studies that reduce the number of mice utilized while generating objective data. Here, we describe an integrated method of evaluating lung inflammation in mice infected with <jats:italic>Pseudomonas aeruginosa<\/jats:italic> or murine gammaherpesvirus (MHV)-68. This method conserves animal resources while permitting evaluation of disease mechanisms in both infection settings. Lungs from a single euthanized mouse were used for two purposes-biological assays to determine inflammation and infection load, as well as histology to evaluate tissue architecture. For this concurrent assessment of multiple parameters from a single euthanized mouse, we limit <jats:italic>in-situ<\/jats:italic> formalin fixation to the right lung of the cadaver. The unfixed left lung is collected immediately and divided into several segments for biological assays including determination of pathogen titer, assessment of infection-driven cytokine levels and appearance of cell death markers. <jats:italic>In situ<\/jats:italic> fixed right lung was then processed for histological determination of tissue injury and confirmation of infection-driven cell death patterns. This method reduces overall animal use and minimizes inter-animal variability that results from sacrificing different animals for different types of assays. The technique can be applied to any lung disease study in mice or other mammals.<\/jats:p>","DOI":"10.1371\/journal.pone.0246270","type":"journal-article","created":{"date-parts":[[2021,7,8]],"date-time":"2021-07-08T17:35:19Z","timestamp":1625765719000},"page":"e0246270","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":2,"title":["Integrated evaluation of lung disease in single animals"],"prefix":"10.1371","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6568-4747","authenticated-orcid":true,"given":"Pratyusha","family":"Mandal","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"John D.","family":"Lyons","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Eileen M.","family":"Burd","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michael","family":"Koval","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8362-9490","authenticated-orcid":true,"given":"Edward S.","family":"Mocarski","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Craig M.","family":"Coopersmith","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2021,7,8]]},"reference":[{"issue":"10","key":"pone.0246270.ref001","article-title":"The 3Rs and Humane Experimental Technique: Implementing Change.","volume":"9","author":"RC Hubrecht","year":"2019","journal-title":"Animals (Basel)."},{"issue":"1","key":"pone.0246270.ref002","doi-asserted-by":"crossref","first-page":"42","DOI":"10.1016\/j.immuni.2018.06.011","article-title":"Caspase-8 Collaborates with Caspase-11 to Drive Tissue Damage and Execution of Endotoxic Shock","volume":"49","author":"P Mandal","year":"2018","journal-title":"Immunity"},{"key":"pone.0246270.ref003","article-title":"Chronic Inflammation. 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Kloth","year":"2018","journal-title":"Toxins (Basel)."},{"issue":"12","key":"pone.0246270.ref025","doi-asserted-by":"crossref","first-page":"e1008192","DOI":"10.1371\/journal.ppat.1008192","article-title":"A role of hypoxia-inducible factor 1 alpha in Murine Gammaherpesvirus 68 (MHV68) lytic replication and reactivation from latency.","volume":"15","author":"DM Lopez-Rodriguez","year":"2019","journal-title":"PLoS Pathog."},{"issue":"10","key":"pone.0246270.ref026","doi-asserted-by":"crossref","first-page":"2389","DOI":"10.1128\/AAC.43.10.2389","article-title":"Effects of antibiotic therapy on Pseudomonas aeruginosa-induced lung injury in a rat model","volume":"43","author":"EJ Ernst","year":"1999","journal-title":"Antimicrob Agents Chemother"},{"issue":"92","key":"pone.0246270.ref027","first-page":"e52044","article-title":"Pseudomonas aeruginosa induced lung injury model","author":"V Suresh Kumar","year":"2014","journal-title":"J Vis 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