A high fraction of inspired oxygen does not mitigate atelectasis-induced lung tissue hypoxia or injury in experimental acute respiratory distress syndrome

A high fraction of inspired oxygen does not mitigate atelectasis-induced lung tissue hypoxia or injury in experimental acute respiratory distress syndrome | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A high fraction of inspired oxygen does not mitigate atelectasis-induced lung tissue hypoxia or injury in experimental acute respiratory distress syndrome Kentaro Tojo, Takuya Yazawa This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4449408/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Although alveolar hyperoxia exacerbates lung injury, clinical studies have failed to demonstrate the beneficial effects of lowering the fraction of inspired oxygen (F I O 2 ) in patients with acute respiratory distress syndrome (ARDS). Atelectasis, which is commonly observed in ARDS, not only leads to hypoxemia but also contributes to lung injury through hypoxia-induced alveolar tissue inflammation. Therefore, it is possible that excessively low F I O 2 may enhance hypoxia-induced inflammation in atelectasis, and raising F I O 2 to an appropriate level may be a reasonable strategy for its mitigation. In this study, we investigated the effects of different F I O 2 levels on alveolar tissue hypoxia and injury in a mechanically ventilated rat model of experimental ARDS with atelectasis. Methods Rats were intratracheally injected with lipopolysaccharide (LPS) to establish an ARDS model. They were allocated to the low, moderate, and high F I O 2 groups with F I O 2 of 21, 60, and 100%, respectively, a day after LPS injection. All groups were mechanically ventilated with an 8 mL/kg tidal volume and zero end-expiratory pressure to induce dorsal atelectatic regions. Arterial blood gas analysis was performed every 2 h. After six hours of mechanical ventilation, the rats were euthanized, and blood, bronchoalveolar lavage fluid, and lung tissues were collected and analyzed. Another set of animals was used for pimonidazole staining of the lung tissues to detect the hypoxic region. Results Lung mechanics, ratios of partial pressure of arterial oxygen (P a O 2 ) to F I O 2 , and partial pressure of arterial carbon dioxide were not significantly different among the three groups, although PaO2 changed with F I O 2 . The dorsal lung tissues were positively stained with pimonidazole regardless of F I O 2 , and the HIF-1α concentrations were not significantly different among the three groups, indicating that raising F I O 2 could not rescue alveolar tissue hypoxia. Moreover, changes in F I O 2 did not significantly affect lung injury or inflammation. In contrast, hypoxemia observed in the low F I O 2 group caused injury to organs other than the lungs. Conclusions Raising F I O 2 levels did not attenuate tissue hypoxia, inflammation, or injury in the atelectatic lung region in experimental ARDS. Our results indicate that raising F I O 2 levels to attenuate atelectasis-induced lung injury cannot be rationalized. Oxygen Acute Respiratory Distress Syndrome Atelectasis Alveolar Hypoxia Hypoxia-induced Inflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Acute respiratory distress syndrome (ARDS) is characterized by profound pulmonary edema and alveolar collapse 1 . Moreover, positive pressure ventilation suppressing spontaneous breathing to rescue patients from respiratory failure further augments dorsal alveolar collapse and atelectasis formation 2 . It has been reported that atelectasis itself enhances lung injury 3–6 , and alveolar hypoxia is the primary mechanism underlying atelectasis-induced lung inflammation and tissue injury 4 . Hypoxia enhances chemokine secretion from alveolar epithelial cells via NF-kb activation 4 and neutrophilic inflammation 7 . Mitigating alveolar hypoxia is expected to protect lung tissue from ARDS with atelectasis. The following two approaches may attenuate alveolar hypoxia: ( 1 ) the open lung approach involving alveolar recruitment and positive end-expiratory pressure (PEEP) application, and ( 2 ) increasing the inspired oxygen concentration. We previously demonstrated that the open lung approach has a protective effect against experimental ARDS by decreasing the atelectatic lung area 5 . Furthermore, it has been reported that mechanical ventilation with increased inspired oxygen levels can mitigate atelectasis-induced lung injury in animals without ARDS. 8 . However, the effects of raising fraction of inspired oxygen (F I O 2 ) on atelectatic lung tissue oxygen tension and injury in subjects with ARDS have not yet been evaluated. Alveolar hyperoxia has been demonstrated to exacerbate lung tissue injury 9,10 . However, randomized control trials have not shown the advantageous effects of decreasing the F I O 2 in critically ill patients including those with acute respiratory distress syndrome (ARDS) 11–14 . One possible explanation for this is that excessively low F I O 2 levels may also be detrimental for patients with atelectasis by enhancing hypoxia-induced inflammation. Consequently, raising F I O 2 to an appropriate level may mitigate atelectasis-induced lung tissue hypoxia and injury, while preventing hyperoxia-induced lung injury. Clarifying the biological effects of F I O 2 in ARDS with atelectasis will facilitate better future clinical studies. In this study, we evaluated the effects of three different F I O 2 levels on lung tissue hypoxia, inflammation, and injury in a mechanically ventilated rat model of experimental ARDS with atelectasis. We also evaluated the effects of F I O 2 on injuries to remote organs other than the lungs. Methods Animal experiments Eight-to-nine-week-old male Sprague-Dawley rats were used for the animal experiments. They were housed under a 12-h light/dark cycle with food and water available ad libitum. On the first day, lipopolysaccharides (LPS) were intratracheally administered to the rats, as previously described 5 . The trachea was exposed through a small incision in front of the neck under general anesthesia with intraperitoneal ketamine and xylazine administration. Thereafter, 300 µL of LPS solution in PBS (5 mg/mL) was intratracheally administered with air. Oxygen was administered (0.5 L/min) after intratracheal instillation until recovery from anesthesia on a warming board. The rats were anesthetized with intraperitoneal ketamine and xylazine 24 h after LPS instillation. Thereafter, an intravenous catheter was inserted through the left femoral vein, and an arterial catheter was placed through the right carotid artery. General anesthesia was maintained with propofol infusion (5 mg/h) via an intravenous line. Subsequently, the rats were tracheostomized, and mechanical ventilation was initiated. Mechanical ventilation was established, and pancuronium bromide was administered to prevent conflict with the ventilator. The initial settings for mechanical ventilation were as follows: F I O 2 , 0.21; tidal volume (TV), 8 mL/kg; frequency, 80/min; and PEEP, 4 cmH2O. Thereafter, the rats were randomized into three groups according to the F I O 2 : low F I O 2 Group with O 2 concentration of 30%, moderate F I O 2 Group with O2 concentration of 60%, and high F I O 2 Group with O 2 concentration of 100% (Fig. 1 ). The mechanical ventilation settings, except for the FIO2, were the same for all groups: TV, 8 mL/kg; frequency, 80/min; and zero end-expiratory pressure (ZEEP). Arterial blood gas analysis was performed before and every 2 h after group allocation. After six hours of mechanical ventilation, bronchoalveolar lavage fluid was collected by lavaging the right lung with two separate 4-ml aliquots of PBS containing 0.6 mM EDTA. The right lung was harvested, frozen, and stored for subsequent RNA and protein extraction. The left lung was fixed by intratracheal instillation of 4% paraformaldehyde in phosphate-buffered saline (PBS at 20 cmH 2 O pressure) and embedded in paraffin for histopathological examination. Pimonidazole Staining Another set of animals was used for pimonidazole staining of lung tissues to detect the hypoxic region. The experimental protocols were the same as those described above for the experimental group allocation. Four hours after group allocation, 18 mg of pimonidazole hydrochloride (HypoxyprobeTM-1; Hypoxyprobe, MA, USA) was injected via an intravenous femoral catheter. After 2 h, the rats were euthanized, and the lungs were fixed with paraformaldehyde and embedded in paraffin, as described above. The paraffin-embedded lung sections were immunohistochemically stained using an anti-pimonidazole mouse IgG1 monoclonal antibody and an avidin-biotin complex (ABC) kit (Vector Laboratories, CA, USA) according to the manufacturer’s instructions. Analysis of bronchoalveolar lavage fluids A portion of the collected bronchoalveolar lavage fluid (BALF) was stained with Samson’s reagent solution, and the white blood cells were counted. The remaining BALF was centrifuged at 2,000 × g, and the supernatants were collected. The total protein concentration in BALF supernatants was quantified using bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, MA, USA). The cytokine and myeloperoxidase concentrations were measured using commercially available ELISA kits as follows: TNF-α: DY510 (R&D Systems, MN, USA); IL-1β: DY501 (R&D Systems); IL-6: DY506 (R&D Systems); IL-10: DY522 (R&D Systems); CXCL-1: DY515 (R&D Systems); CCL2: DY3144-05 (R&D Systems); RAGE: DY1616 (R&D Systems); ICAM-1: DY583 (R&D Systems). Protein analysis of lung tissues The proteins were extracted from the entire right lung tissue homogenized in Radio-Immunoprecipitation Assay buffer containing a protease inhibitor cocktail. The concentration of HIF-1α was quantified by ELISA (DYC1935-2, R&D Systems), and the value was normalized to the total protein concentration determined by the BCA assay. We measured the HIF-1α concentrations in the lung tissues from the animals in the present study and those from the ARDS rat model ventilated with an open lung approach. The latter tissue samples had been collected in our previous study. Reverse transcription-quantitative PCR analysis RNA was extracted from lung tissue homogenates using a spin column (FastGene™ RNA Premium Kit, Nippon Genetics, Tokyo, Japan). The extracted RNA was reverse-transcribed using a reverse transcription kit (RevertraAce, Toyobo, Tokyo, Japan). Quantitative PCR was performed using TB Green® Premix Ex Taq™ II (Takara Bio, Shiga, Japan) with specific primers (Thermo Fisher Scientific) (Table 1 ) under the following conditions 30 s at 95°C and 40 cycles for 5 s at 95°C and 30 s at 60°C (CFX96 real time system, Bio-Rad Laboratories, CA, USA)). The expressions of target genes relative to the expression of beta-actin were calculated. Table 1 Primers for qPCR Gene Primer Sequence TNF-α Forward Reverse 5′-CCACCACGCTCTTCTGTCTAC-3′ 5′-GCTTGGTGGTTTGCTACGAC-3′ IL-1β Forward Reverse 5′-TCTCACAGCAGCATCTCGAC-3′ 5′-CATCATCCCACGAGTCACAG-3′ IL-6 Forward Reverse 5′-GGAACAGCTATGAAGTTTCTCTCC-3′ 5′-GGGTGGTATCCTCTGTGAAGTC-3′ IL-10 Forward Reverse 5′-GACAATAACTGCACCCACTTCC-3′ 5′-CAACCCAAGTAACCCTTAAAGTCC-3′ CXCL-1 Forward Reverse 5′-GCACCCAAACCGAAGTCATA-3′ 5′-GCCATCGGTGCAATCTATCT-3′ CCL-2 Forward Reverse 5′-GCTTCTGGGCCTGTTGTTC-3′ 5′-CTGCTGCTGGTGATTCTCTTGT-3′ ACTB Forward Reverse 5′-TGACGTTGACATCCGTAAAGAC-3′ 5′-AGAGCCACCAATCCACACA-3′ Histological analysis Paraffin-embedded left lung sections were stained with hematoxylin and eosin. Histopathological scores were evaluated by a pathologist in a blinded manner following previously described methods 5 . Analysis of the plasma concentrations of GOT, GPT, creatinine, and cystatin C The plasma concentrations of GOT, GPT, creatinine and cystatin C were determined using commercially available kits following the manufacturer’s instructions : GOT and GPT: Transaminase CII Test Wako (FUJIFILM Wako Pure Chemical, Osaka, Japan); Creatinine: LabAssay™ Creatinine (FUJIFILM Wako Pure Chemical); cystatin C (MSCTC0, R&D systems). Statistical Analysis Data are presented as means ± standard deviation (SD). GraphPad Prism 10 (GraphPad Software, CA, USA) was used for all the statistical analyses. Statistical significance was set at p < 0.05. Longitudinal physiological parameters, arterial blood gas analysis data, and lactate values were analyzed using two-way repeated-measures analysis of variance and post-hoc Tukey’s multiple comparison test. The concentrations of lung tissue HIF-1α, inflammatory mediators, and liver and kidney injury markers were analyzed using one-way repeated-measures analysis of variance and post-hoc Tukey’s multiple comparison test. Results Physiological parameters and arterial blood gas analysis The physiological parameters and results of the arterial blood gas analysis are shown in Fig. 2 . The driving pressures increased after the discontinuation of PEEP application and were not significantly different among the three groups (Fig. 2 A). The Partial pressure of arterial oxygen (PaO 2 ) changed with the F I O 2 (Fig. 2 B), while the PaO 2 / F I O 2 (P/F) ratios (Fig. 2 C) and PaCO 2 concentrations (Fig. 2 D) of the three groups were not significantly different. Collectively, the lung mechanics and respiratory function were not significantly affected by F I O 2 . In the low F I O 2 group, the mean arterial pressure gradually decreased (Fig. 2 E), and the lactate concentrations were significantly elevated (Fig. 2 F), possibly due to severe hypoxemia. Increased FIO2 did not attenuate atelectasis-induced lung tissue hypoxia We evaluated the effects of F I O 2 on atelectasis-induced alveolar tissue hypoxia. Pimonidazole staining, which indicated hypoxic tissues, showed that dorsal atelectatic alveolar tissues became hypoxic, irrespective of F I O 2 (Fig. 3 A). The HIF-1α concentrations in the lung tissues in all the experimental groups with atelectasis were significantly higher than those in the lung tissue ventilated with the open-lung approach (Fig. 3 B). However, F I O 2 had non-significant effects on the HIF-1α concentration in the lung tissues with atelectasis (Fig. 3 B). These results demonstrate that elevated F I O 2 does not attenuate hypoxia in atelectatic lung tissues. Increased F I O 2 did not attenuate alveolar inflammation or tissue injury in mechanically-ventilated ARDS lungs with atelectasis Next, we evaluated the effects of F I O 2 on alveolar inflammatory responses and tissue injury in mechanically ventilated animals with ARDS and atelectasis. The mRNA expression of TNF-α, IL-1β, IL-6, IL-10, CXCL-1, and CCL-2 in lung tissue was not significantly different among the groups (Fig. 4 A). The protein concentrations of these cytokines and chemokines in the BALF were also not significantly different (Fig. 4 B). Moreover, there were no significant differences in the BALF leukocyte counts or myeloperoxidase concentrations among the three groups (Fig. 4 C, D). The concentrations of alveolar tissue injury markers, total protein, ICAM-1, and sRAGE in the BALF were not significantly different among the three groups (Fig. 5 A-C). The lung histological scores of both the dorsal and ventral alveolar regions were not significantly different among the three groups (Fig. 5 D, E). Collectively, F I O 2 did not affect the inflammatory responses or alveolar tissue injury in the animals with ARDS with accompanying atelectasis. Severe hypoxemia induced damage in organs other than lungs Finally, we evaluated the effects of F I O 2 on organs other than the lungs. The GOT and GPT concentrations were significantly increased in the low F I O 2 group (Fig. 6 A, B), possibly due to hypoxemia and hypotension. The creatinine concentrations also tended to increase in the low F I O 2 group (Fig. 6 C), and the cystatin C levels were significantly higher in the low F I O 2 group than in the high F I O 2 group (Fig. 6 D). Discussion In the present study, we demonstrated that increasing F I O 2 levels did not rescue tissue hypoxia in the atelectatic lung regions of a mechanically ventilated animal model of ARDS. Moreover, F I O 2 2 did not affect atelectasis-induced inflammation or tissue injury. These findings suggest that raising F I O 2 levels to attenuate hypoxia-induced lung inflammation and injury cannot be rationalized. However, organs other than the lungs are more vulnerable to hypoxemia, and caution should be exercised to maintain oxygen tension above the limit inducing organ damage. This study is the first to directly demonstrate the effects of F I O 2 on oxygen tension in atelectatic lung tissues. Our results indicated that these effects were negligible. A previous study demonstrated that high F I O 2 levels rescued right-sided heart failure in animals with atelectasis 8 , which reflects pulmonary hypertension due to lung tissue hypoxia. However, the study did not evaluate oxygen tension in the atelectatic lung tissue, and it is possible that elevated oxygen partial pressure in the ventilated lung region, rather than in the atelectatic lung region, may lower pulmonary vascular resistance. Based on the concentrations of HIF-1α and pimonidazole staining of lung tissues, the oxygen diffusion to atelectatic lung regions seems very limited and cannot rescue atelectatic lung tissue hypoxia. On the other hand, HIF-1α stabilization in lung tissue was suppressed by the open lung approach with PEEP and recruitment maneuver. Our results suggest that the open-lung approach is the only efficient method for rescuing lung tissue from hypoxia. Alveolar tissue hypoxia causes inflammation. We previously demonstrated that alveolar hypoxia during atelectasis increases CXLC-1 expression in alveolar epithelial cells through NF-kB activation 4 . Other previous studies have also demonstrated alveolar macrophage 15–17 and neutrophil 7,18,19 activation under hypoxic conditions. Moreover, our previous study showed that improving lung aeration through an open-lung approach can reduce lung inflammation and injury 5 , possibly by attenuating lung tissue hypoxia. However, the present study demonstrated that changes in F I O 2 levels did not significantly affect alveolar tissue damage or inflammatory responses in experimental animals with ARDS and atelectasis. As mentioned above, this may be attributed to the inability of the inspiratory gas to diffuse into the atelectatic lung tissue, not significantly affecting inflammation or tissue injury. In other words, lowering F I O 2 levels in patients with ARDS with atelectasis does not seem to aggravate lung tissue inflammation or injury. Hyperoxia also causes lung injury through the generation of reactive oxygen species 9,10,20 . Based on these observations, several randomized controlled trials have evaluated the efficacy of conservative oxygen therapy in critically ill patients 11–14 . However, all the studies have not demonstrated the benefits of conservative oxygen therapy. Consistent with these clinical studies, our study did not demonstrate the harmful effects of high F I O 2 concentrations, although the duration of exposure was relatively short. In the discussion regarding oxygen toxicity, attention should be paid to the fact that several experimental animal studies have evaluated oxygen toxicity in small rodents, which are more vulnerable to hyperoxia than large mammals 10 . Further clinical investigations are necessary to evaluate whether conservative oxygen therapy truly has beneficial effects. We observed that hypoxemia in the low F I O 2 group led to hypotension, lactic acidosis, and liver and kidney injury. A recent clinical study evaluating the effects of FIO2 on ARDS demonstrated that conservative oxygen therapy was associated with mesenteric ischemia in patients with ARDS 12 Moreover, targeting lower arterial partial oxygen pressure may impair cognitive function and adversely affect long-term outcomes in survivors of ARDS 21,22 . Oxygen toxicity has mainly been demonstrated in the lungs. The toxicity of oxygen to other organs is not clear, except in special settings such as ischemia-reperfusion injury. Oxygen tension was highest in the lungs and decreased thereafter to the end organs. Caution should be exercised to prevent hypoxemia in organs other than the lungs. This study had some limitations. First, the duration of mechanical ventilation was shorter than that used in the clinical setting. However, analysis of mRNA, which changes promptly after the stimulus, also showed no significant changes, suggesting that the effects of F I O 2 against atelectasis-induced lung injury are minimal. Second, we used LPS, not bacteria, to induce experimental ARDS. Therefore, the effects of F I O 2 on pathogen load and immunological responses are unclear. Several studies have demonstrated that atelectasis causes immunosuppression in the lungs. Therefore, F I O 2 may affect immunological responses in infection-induced ARDS accompanied by atelectasis. Conclusions In conclusion, increasing F I O 2 levels did not attenuate tissue hypoxia, inflammation, or injury in the atelectatic lung region in LPS-induced ARDS. Our results indicate that increasing the F I O 2 concentration to attenuate atelectasis-induced lung injury cannot be rationalized. However, attention should be paid to preventing severe hypoxemia because it is harmful to organs other than the lungs. Abbreviations ARDS, acute respiratory distress syndrome; BALF, broncho-alveolar lavage fluid; CT, computed tomography; F I O 2 , fraction of inspired oxygen; LPS, lipopolysaccharide; P a CO 2 , partial pressure of arterial carbondioxide; P a O 2 , partial pressure of arterial oxygen; PEEP, Positive end-expiratory pressure; P/F ratio, P a O 2 / F I O2 ratio; TV, tidal volume; ZEEP, zero end-expiratory pressure; Declarations Ethics approval All experimental animal protocols were reviewed and approved by the Animal Research Committee of Yokohama City University, Japan. Consent to participate Not applicable Consent for publication Not applicable Availability of data and materials The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests Funding Supported, in part, by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (17K17062, 22K09146) . Authors' contributions K. Tojo conceived and designed the study, performed experiments, and wrote the manuscript. T. Yazawa performed histological examinations and revised the manuscript. Acknowledgements We thank Ms. Akiko Adachi and Ms. Yuki Yuba for their technical assistance. References Puybasset L, Cluzel P, Gusman P, Grenier P, Preteux F, Rouby J-J. Regional distribution of gas and tissue in acute respiratory distress syndrome. I. Consequences for lung morphology. Intensiv Care Med 2000; 26 : 857–869. Albert RK. The Role of Ventilation-induced Surfactant Dysfunction and Atelectasis in Causing Acute Respiratory Distress Syndrome. Am J Respir Crit Care Med 2012; 185 : 702–708. Retamal J, Bergamini BC, Carvalho AR, Bozza FA, Borzone G, Borges JB et al. 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The Adult Respiratory Distress Syndrome Cognitive Outcomes Study. Am J Respir Crit Care Med 2012; 185 : 1307–1315. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4449408","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":309663676,"identity":"0874a965-ebfd-430c-b95e-541d7db208dc","order_by":0,"name":"Kentaro Tojo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYBACCSA2YKgAMXiQxXlwqAdpYQNpOUOqFgbGNgwteIDk/OYDBR/nHZaTbO89+OlGDYO8wQHmhx8YZO7g1CLNxpZgOHPbYWNpnnPJ0jnHGAw3HGAzBlr5DKcWOTYeA2PebYcT50nkGEjnsP1PMDjAYAb0y2ECWuaAtRj/zvnHANTC/g2vFmmwlobDibMlcsykc9tAWnjw2yLZlpZgOONYurFkzxkz69w+BsOZh3mKJRLw+EXi8OFjBh9qrOUkjvcY3875xiDPd7x944ePPbhDDAjYDBgYmpH4zECc2HMAnxbmBwwMdeiCP/BqGQWjYBSMgpEFANAPTWj6GqSeAAAAAElFTkSuQmCC","orcid":"","institution":"Yokohama City University School of Medicine and Graduate School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Kentaro","middleName":"","lastName":"Tojo","suffix":""},{"id":309663677,"identity":"d933024a-60eb-4568-93bf-a9189fad03ed","order_by":1,"name":"Takuya Yazawa","email":"","orcid":"","institution":"Dokkyo Medical University School of Medicine and Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Takuya","middleName":"","lastName":"Yazawa","suffix":""}],"badges":[],"createdAt":"2024-05-20 13:27:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4449408/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4449408/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57942134,"identity":"f7881763-32a0-42b4-acbc-66d7f7fcda9d","added_by":"auto","created_at":"2024-06-07 19:00:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":99506,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of experimental design.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4449408/v1/0ddfee42c05fc26305107566.png"},{"id":57942171,"identity":"4ee5d226-2947-4821-88cd-727c71404208","added_by":"auto","created_at":"2024-06-07 19:00:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":489890,"visible":true,"origin":"","legend":"\u003cp\u003ePhysiological parameters and arterial blood gas analysis. A, Dynamic driving pressure. B, Partial pressure of arterial oxygen. C, P\u003csub\u003ea\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/ F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e ratio. D, Partial pressure of arterial carbon dioxide. E, Mean arterial blood pressure. F, Blood lactate concentration. †\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e Moderate F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group, ‡\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e High F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group. Data represent the means ± SEM.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4449408/v1/7cacb6dc94b926ed568abe0f.png"},{"id":57942170,"identity":"c4b1c7b4-3c19-4599-ae0a-cd69ec5ca9a8","added_by":"auto","created_at":"2024-06-07 19:00:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2822263,"visible":true,"origin":"","legend":"\u003cp\u003eA, Lung tissue pimonidazole staining and B, HIF-1α protein levels in lung the lung tissue. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05\u003cem\u003e, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001\u003c/em\u003e. Data represent the means ± SEM.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4449408/v1/8eb186389a087384554a94d9.png"},{"id":57942135,"identity":"925825f4-3212-4e98-baf4-d4eb38b51db1","added_by":"auto","created_at":"2024-06-07 19:00:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":569458,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of inflammatory mediators and leukocytes in bronchoalveolar lavage fluids (BALF). A, mRNA expressions of cytokines and chemokines in the lung tissues. B, Protein concentrations of cytokines and chemokines in the BALF. C, White blood cell counts and D, myeloperoxidases (MPO) in the BALF. *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05\u003cem\u003e, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001\u003c/em\u003e. Data represent the means ± SEM.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4449408/v1/86292e40927063f5287fe6da.png"},{"id":57942174,"identity":"554331fd-4871-4953-9d2c-1bfe9b02c575","added_by":"auto","created_at":"2024-06-07 19:00:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3019269,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of tissue injury markers in bronchoalveolar lavage fluids (BALF) and histology. A, total protein concentration, B, soluble RAGE, and C, ICAM-1 in the BALF. D, Representative images of lung sections stained with hematoxylin and eosin. E, Histological scores assessed in a blinded manner. *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05\u003cem\u003e, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001\u003c/em\u003e. Data represent the means ± SEM.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4449408/v1/78975d07ae89de5d6194c5b9.png"},{"id":57942177,"identity":"61853679-9478-4e54-82ea-00c0f8813ddb","added_by":"auto","created_at":"2024-06-07 19:00:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":179809,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of liver and kidney injury markers in plasma. A, GOT, B, GPT, C, Creatinine, and D, Cystatin C in the plasma. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05\u003cem\u003e, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001\u003c/em\u003e. Data represent the means ± SEM.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-4449408/v1/1a7483f94b2921dd8caa4529.png"},{"id":57943937,"identity":"f45bc9e1-b715-46d1-a64f-acc3dd21c5ef","added_by":"auto","created_at":"2024-06-07 19:08:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9164894,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4449408/v1/d5d41f5f-2524-42de-81ee-7bc2f2a79bf5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A high fraction of inspired oxygen does not mitigate atelectasis-induced lung tissue hypoxia or injury in experimental acute respiratory distress syndrome","fulltext":[{"header":"Background","content":"\u003cp\u003eAcute respiratory distress syndrome (ARDS) is characterized by profound pulmonary edema and alveolar collapse\u003csup\u003e1\u003c/sup\u003e. Moreover, positive pressure ventilation suppressing spontaneous breathing to rescue patients from respiratory failure further augments dorsal alveolar collapse and atelectasis formation\u003csup\u003e2\u003c/sup\u003e. It has been reported that atelectasis itself enhances lung injury\u003csup\u003e3\u0026ndash;6\u003c/sup\u003e, and alveolar hypoxia is the primary mechanism underlying atelectasis-induced lung inflammation and tissue injury\u003csup\u003e4\u003c/sup\u003e. Hypoxia enhances chemokine secretion from alveolar epithelial cells via NF-kb activation\u003csup\u003e4\u003c/sup\u003e and neutrophilic inflammation\u003csup\u003e7\u003c/sup\u003e. Mitigating alveolar hypoxia is expected to protect lung tissue from ARDS with atelectasis.\u003c/p\u003e \u003cp\u003eThe following two approaches may attenuate alveolar hypoxia: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) the open lung approach involving alveolar recruitment and positive end-expiratory pressure (PEEP) application, and (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) increasing the inspired oxygen concentration. We previously demonstrated that the open lung approach has a protective effect against experimental ARDS by decreasing the atelectatic lung area\u003csup\u003e5\u003c/sup\u003e. Furthermore, it has been reported that mechanical ventilation with increased inspired oxygen levels can mitigate atelectasis-induced lung injury in animals without ARDS.\u003csup\u003e8\u003c/sup\u003e. However, the effects of raising fraction of inspired oxygen (F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) on atelectatic lung tissue oxygen tension and injury in subjects with ARDS have not yet been evaluated.\u003c/p\u003e \u003cp\u003eAlveolar hyperoxia has been demonstrated to exacerbate lung tissue injury\u003csup\u003e9,10\u003c/sup\u003e. However, randomized control trials have not shown the advantageous effects of decreasing the F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in critically ill patients including those with acute respiratory distress syndrome (ARDS)\u003csup\u003e11\u0026ndash;14\u003c/sup\u003e. One possible explanation for this is that excessively low F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels may also be detrimental for patients with atelectasis by enhancing hypoxia-induced inflammation. Consequently, raising F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to an appropriate level may mitigate atelectasis-induced lung tissue hypoxia and injury, while preventing hyperoxia-induced lung injury. Clarifying the biological effects of F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in ARDS with atelectasis will facilitate better future clinical studies.\u003c/p\u003e \u003cp\u003eIn this study, we evaluated the effects of three different F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels on lung tissue hypoxia, inflammation, and injury in a mechanically ventilated rat model of experimental ARDS with atelectasis. We also evaluated the effects of F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e on injuries to remote organs other than the lungs.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimal experiments\u003c/h2\u003e \u003cp\u003eEight-to-nine-week-old male Sprague-Dawley rats were used for the animal experiments. They were housed under a 12-h light/dark cycle with food and water available ad libitum.\u003c/p\u003e \u003cp\u003eOn the first day, lipopolysaccharides (LPS) were intratracheally administered to the rats, as previously described\u003csup\u003e5\u003c/sup\u003e. The trachea was exposed through a small incision in front of the neck under general anesthesia with intraperitoneal ketamine and xylazine administration. Thereafter, 300 \u0026micro;L of LPS solution in PBS (5 mg/mL) was intratracheally administered with air. Oxygen was administered (0.5 L/min) after intratracheal instillation until recovery from anesthesia on a warming board.\u003c/p\u003e \u003cp\u003eThe rats were anesthetized with intraperitoneal ketamine and xylazine 24 h after LPS instillation. Thereafter, an intravenous catheter was inserted through the left femoral vein, and an arterial catheter was placed through the right carotid artery. General anesthesia was maintained with propofol infusion (5 mg/h) via an intravenous line. Subsequently, the rats were tracheostomized, and mechanical ventilation was initiated. Mechanical ventilation was established, and pancuronium bromide was administered to prevent conflict with the ventilator. The initial settings for mechanical ventilation were as follows: F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 0.21; tidal volume (TV), 8 mL/kg; frequency, 80/min; and PEEP, 4 cmH2O.\u003c/p\u003e \u003cp\u003eThereafter, the rats were randomized into three groups according to the F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e: low F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e Group with O\u003csub\u003e2\u003c/sub\u003e concentration of 30%, moderate F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e Group with O2 concentration of 60%, and high F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e Group with O\u003csub\u003e2\u003c/sub\u003e concentration of 100% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The mechanical ventilation settings, except for the FIO2, were the same for all groups: TV, 8 mL/kg; frequency, 80/min; and zero end-expiratory pressure (ZEEP).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eArterial blood gas analysis was performed before and every 2 h after group allocation. After six hours of mechanical ventilation, bronchoalveolar lavage fluid was collected by lavaging the right lung with two separate 4-ml aliquots of PBS containing 0.6 mM EDTA. The right lung was harvested, frozen, and stored for subsequent RNA and protein extraction. The left lung was fixed by intratracheal instillation of 4% paraformaldehyde in phosphate-buffered saline (PBS at 20 cmH\u003csub\u003e2\u003c/sub\u003eO pressure) and embedded in paraffin for histopathological examination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePimonidazole Staining\u003c/h2\u003e \u003cp\u003eAnother set of animals was used for pimonidazole staining of lung tissues to detect the hypoxic region. The experimental protocols were the same as those described above for the experimental group allocation. Four hours after group allocation, 18 mg of pimonidazole hydrochloride (HypoxyprobeTM-1; Hypoxyprobe, MA, USA) was injected via an intravenous femoral catheter. After 2 h, the rats were euthanized, and the lungs were fixed with paraformaldehyde and embedded in paraffin, as described above. The paraffin-embedded lung sections were immunohistochemically stained using an anti-pimonidazole mouse IgG1 monoclonal antibody and an avidin-biotin complex (ABC) kit (Vector Laboratories, CA, USA) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of bronchoalveolar lavage fluids\u003c/h2\u003e \u003cp\u003eA portion of the collected bronchoalveolar lavage fluid (BALF) was stained with Samson\u0026rsquo;s reagent solution, and the white blood cells were counted. The remaining BALF was centrifuged at 2,000 \u0026times; g, and the supernatants were collected. The total protein concentration in BALF supernatants was quantified using bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, MA, USA). The cytokine and myeloperoxidase concentrations were measured using commercially available ELISA kits as follows: TNF-α: DY510 (R\u0026amp;D Systems, MN, USA); IL-1β: DY501 (R\u0026amp;D Systems); IL-6: DY506 (R\u0026amp;D Systems); IL-10: DY522 (R\u0026amp;D Systems); CXCL-1: DY515 (R\u0026amp;D Systems); CCL2: DY3144-05 (R\u0026amp;D Systems); RAGE: DY1616 (R\u0026amp;D Systems); ICAM-1: DY583 (R\u0026amp;D Systems).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eProtein analysis of lung tissues\u003c/h2\u003e \u003cp\u003eThe proteins were extracted from the entire right lung tissue homogenized in Radio-Immunoprecipitation Assay buffer containing a protease inhibitor cocktail. The concentration of HIF-1α was quantified by ELISA (DYC1935-2, R\u0026amp;D Systems), and the value was normalized to the total protein concentration determined by the BCA assay. We measured the HIF-1α concentrations in the lung tissues from the animals in the present study and those from the ARDS rat model ventilated with an open lung approach. The latter tissue samples had been collected in our previous study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eReverse transcription-quantitative PCR analysis\u003c/h2\u003e \u003cp\u003eRNA was extracted from lung tissue homogenates using a spin column (FastGene\u0026trade; RNA Premium Kit, Nippon Genetics, Tokyo, Japan). The extracted RNA was reverse-transcribed using a reverse transcription kit (RevertraAce, Toyobo, Tokyo, Japan). Quantitative PCR was performed using TB Green\u0026reg; Premix Ex Taq\u0026trade; II (Takara Bio, Shiga, Japan) with specific primers (Thermo Fisher Scientific) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) under the following conditions 30 s at 95\u0026deg;C and 40 cycles for 5 s at 95\u0026deg;C and 30 s at 60\u0026deg;C (CFX96 real time system, Bio-Rad Laboratories, CA, USA)). The expressions of target genes relative to the expression of beta-actin were calculated.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimers for qPCR\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSequence\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTNF-α\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;-CCACCACGCTCTTCTGTCTAC-3\u0026prime;\u003c/p\u003e \u003cp\u003e5\u0026prime;-GCTTGGTGGTTTGCTACGAC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eIL-1β\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;-TCTCACAGCAGCATCTCGAC-3\u0026prime;\u003c/p\u003e \u003cp\u003e5\u0026prime;-CATCATCCCACGAGTCACAG-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eIL-6\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;-GGAACAGCTATGAAGTTTCTCTCC-3\u0026prime;\u003c/p\u003e \u003cp\u003e5\u0026prime;-GGGTGGTATCCTCTGTGAAGTC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eIL-10\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;-GACAATAACTGCACCCACTTCC-3\u0026prime;\u003c/p\u003e \u003cp\u003e5\u0026prime;-CAACCCAAGTAACCCTTAAAGTCC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCXCL-1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;-GCACCCAAACCGAAGTCATA-3\u0026prime;\u003c/p\u003e \u003cp\u003e5\u0026prime;-GCCATCGGTGCAATCTATCT-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCCL-2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;-GCTTCTGGGCCTGTTGTTC-3\u0026prime;\u003c/p\u003e \u003cp\u003e5\u0026prime;-CTGCTGCTGGTGATTCTCTTGT-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eACTB\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;-TGACGTTGACATCCGTAAAGAC-3\u0026prime;\u003c/p\u003e \u003cp\u003e5\u0026prime;-AGAGCCACCAATCCACACA-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHistological analysis\u003c/h2\u003e \u003cp\u003eParaffin-embedded left lung sections were stained with hematoxylin and eosin. Histopathological scores were evaluated by a pathologist in a blinded manner following previously described methods\u003csup\u003e5\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of the plasma concentrations of GOT, GPT, creatinine, and cystatin C\u003c/h2\u003e \u003cp\u003eThe plasma concentrations of GOT, GPT, creatinine and cystatin C were determined using commercially available kits following the manufacturer\u0026rsquo;s instructions : GOT and GPT: Transaminase CII Test Wako (FUJIFILM Wako Pure Chemical, Osaka, Japan); Creatinine: LabAssay\u0026trade; Creatinine (FUJIFILM Wako Pure Chemical); cystatin C (MSCTC0, R\u0026amp;D systems).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). GraphPad Prism 10 (GraphPad Software, CA, USA) was used for all the statistical analyses. Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Longitudinal physiological parameters, arterial blood gas analysis data, and lactate values were analyzed using two-way repeated-measures analysis of variance and post-hoc Tukey\u0026rsquo;s multiple comparison test. The concentrations of lung tissue HIF-1α, inflammatory mediators, and liver and kidney injury markers were analyzed using one-way repeated-measures analysis of variance and post-hoc Tukey\u0026rsquo;s multiple comparison test.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePhysiological parameters and arterial blood gas analysis\u003c/h2\u003e \u003cp\u003eThe physiological parameters and results of the arterial blood gas analysis are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The driving pressures increased after the discontinuation of PEEP application and were not significantly different among the three groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The Partial pressure of arterial oxygen (PaO\u003csub\u003e2\u003c/sub\u003e) changed with the F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), while the PaO\u003csub\u003e2\u003c/sub\u003e/ F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (P/F) ratios (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) and PaCO\u003csub\u003e2\u003c/sub\u003e concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) of the three groups were not significantly different. Collectively, the lung mechanics and respiratory function were not significantly affected by F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. In the low F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group, the mean arterial pressure gradually decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), and the lactate concentrations were significantly elevated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), possibly due to severe hypoxemia.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIncreased FIO2 did not attenuate atelectasis-induced lung tissue hypoxia\u003c/h2\u003e \u003cp\u003eWe evaluated the effects of F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e on atelectasis-induced alveolar tissue hypoxia. Pimonidazole staining, which indicated hypoxic tissues, showed that dorsal atelectatic alveolar tissues became hypoxic, irrespective of F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The HIF-1α concentrations in the lung tissues in all the experimental groups with atelectasis were significantly higher than those in the lung tissue ventilated with the open-lung approach (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). However, F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e had non-significant effects on the HIF-1α concentration in the lung tissues with atelectasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). These results demonstrate that elevated F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e does not attenuate hypoxia in atelectatic lung tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIncreased F\u003c/b\u003e \u003csub\u003e \u003cb\u003eI\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eO\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003edid not attenuate alveolar inflammation or tissue injury in mechanically-ventilated ARDS lungs with atelectasis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNext, we evaluated the effects of F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e on alveolar inflammatory responses and tissue injury in mechanically ventilated animals with ARDS and atelectasis. The mRNA expression of TNF-α, IL-1β, IL-6, IL-10, CXCL-1, and CCL-2 in lung tissue was not significantly different among the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The protein concentrations of these cytokines and chemokines in the BALF were also not significantly different (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Moreover, there were no significant differences in the BALF leukocyte counts or myeloperoxidase concentrations among the three groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe concentrations of alveolar tissue injury markers, total protein, ICAM-1, and sRAGE in the BALF were not significantly different among the three groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C). The lung histological scores of both the dorsal and ventral alveolar regions were not significantly different among the three groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, E). Collectively, F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e did not affect the inflammatory responses or alveolar tissue injury in the animals with ARDS with accompanying atelectasis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSevere hypoxemia induced damage in organs other than lungs\u003c/h2\u003e \u003cp\u003eFinally, we evaluated the effects of F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e on organs other than the lungs. The GOT and GPT concentrations were significantly increased in the low F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B), possibly due to hypoxemia and hypotension. The creatinine concentrations also tended to increase in the low F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), and the cystatin C levels were significantly higher in the low F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group than in the high F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we demonstrated that increasing F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels did not rescue tissue hypoxia in the atelectatic lung regions of a mechanically ventilated animal model of ARDS. Moreover, F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e2 did not affect atelectasis-induced inflammation or tissue injury. These findings suggest that raising F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels to attenuate hypoxia-induced lung inflammation and injury cannot be rationalized. However, organs other than the lungs are more vulnerable to hypoxemia, and caution should be exercised to maintain oxygen tension above the limit inducing organ damage.\u003c/p\u003e \u003cp\u003eThis study is the first to directly demonstrate the effects of F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e on oxygen tension in atelectatic lung tissues. Our results indicated that these effects were negligible. A previous study demonstrated that high F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels rescued right-sided heart failure in animals with atelectasis\u003csup\u003e8\u003c/sup\u003e, which reflects pulmonary hypertension due to lung tissue hypoxia. However, the study did not evaluate oxygen tension in the atelectatic lung tissue, and it is possible that elevated oxygen partial pressure in the ventilated lung region, rather than in the atelectatic lung region, may lower pulmonary vascular resistance. Based on the concentrations of HIF-1α and pimonidazole staining of lung tissues, the oxygen diffusion to atelectatic lung regions seems very limited and cannot rescue atelectatic lung tissue hypoxia. On the other hand, HIF-1α stabilization in lung tissue was suppressed by the open lung approach with PEEP and recruitment maneuver. Our results suggest that the open-lung approach is the only efficient method for rescuing lung tissue from hypoxia.\u003c/p\u003e \u003cp\u003eAlveolar tissue hypoxia causes inflammation. We previously demonstrated that alveolar hypoxia during atelectasis increases CXLC-1 expression in alveolar epithelial cells through NF-kB activation\u003csup\u003e4\u003c/sup\u003e. Other previous studies have also demonstrated alveolar macrophage\u003csup\u003e15\u0026ndash;17\u003c/sup\u003e and neutrophil\u003csup\u003e7,18,19\u003c/sup\u003e activation under hypoxic conditions. Moreover, our previous study showed that improving lung aeration through an open-lung approach can reduce lung inflammation and injury\u003csup\u003e5\u003c/sup\u003e, possibly by attenuating lung tissue hypoxia. However, the present study demonstrated that changes in F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels did not significantly affect alveolar tissue damage or inflammatory responses in experimental animals with ARDS and atelectasis. As mentioned above, this may be attributed to the inability of the inspiratory gas to diffuse into the atelectatic lung tissue, not significantly affecting inflammation or tissue injury. In other words, lowering F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels in patients with ARDS with atelectasis does not seem to aggravate lung tissue inflammation or injury.\u003c/p\u003e \u003cp\u003eHyperoxia also causes lung injury through the generation of reactive oxygen species\u003csup\u003e9,10,20\u003c/sup\u003e. Based on these observations, several randomized controlled trials have evaluated the efficacy of conservative oxygen therapy in critically ill patients\u003csup\u003e11\u0026ndash;14\u003c/sup\u003e. However, all the studies have not demonstrated the benefits of conservative oxygen therapy. Consistent with these clinical studies, our study did not demonstrate the harmful effects of high F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentrations, although the duration of exposure was relatively short. In the discussion regarding oxygen toxicity, attention should be paid to the fact that several experimental animal studies have evaluated oxygen toxicity in small rodents, which are more vulnerable to hyperoxia than large mammals\u003csup\u003e10\u003c/sup\u003e. Further clinical investigations are necessary to evaluate whether conservative oxygen therapy truly has beneficial effects.\u003c/p\u003e \u003cp\u003eWe observed that hypoxemia in the low F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group led to hypotension, lactic acidosis, and liver and kidney injury. A recent clinical study evaluating the effects of FIO2 on ARDS demonstrated that conservative oxygen therapy was associated with mesenteric ischemia in patients with ARDS\u003csup\u003e12\u003c/sup\u003e Moreover, targeting lower arterial partial oxygen pressure may impair cognitive function and adversely affect long-term outcomes in survivors of ARDS\u003csup\u003e21,22\u003c/sup\u003e. Oxygen toxicity has mainly been demonstrated in the lungs. The toxicity of oxygen to other organs is not clear, except in special settings such as ischemia-reperfusion injury. Oxygen tension was highest in the lungs and decreased thereafter to the end organs. Caution should be exercised to prevent hypoxemia in organs other than the lungs.\u003c/p\u003e \u003cp\u003eThis study had some limitations. First, the duration of mechanical ventilation was shorter than that used in the clinical setting. However, analysis of mRNA, which changes promptly after the stimulus, also showed no significant changes, suggesting that the effects of F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e against atelectasis-induced lung injury are minimal. Second, we used LPS, not bacteria, to induce experimental ARDS. Therefore, the effects of F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e on pathogen load and immunological responses are unclear. Several studies have demonstrated that atelectasis causes immunosuppression in the lungs. Therefore, F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e may affect immunological responses in infection-induced ARDS accompanied by atelectasis.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, increasing F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels did not attenuate tissue hypoxia, inflammation, or injury in the atelectatic lung region in LPS-induced ARDS. Our results indicate that increasing the F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration to attenuate atelectasis-induced lung injury cannot be rationalized. However, attention should be paid to preventing severe hypoxemia because it is harmful to organs other than the lungs.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eARDS, acute respiratory distress syndrome; BALF, broncho-alveolar lavage fluid; CT, computed tomography; F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, fraction of inspired oxygen; LPS, lipopolysaccharide; P\u003csub\u003ea\u003c/sub\u003eCO\u003csub\u003e2\u003c/sub\u003e, partial pressure of arterial carbondioxide; P\u003csub\u003ea\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, partial pressure of arterial oxygen; PEEP, Positive end-expiratory pressure; P/F ratio, P\u003csub\u003ea\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/ F\u003csub\u003eI\u003c/sub\u003eO2 ratio; TV, tidal volume; ZEEP, zero end-expiratory pressure;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental\u0026nbsp;animal protocols were reviewed and approved by the\u0026nbsp;Animal Research Committee of Yokohama City University, Japan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupported, in part, by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (17K17062, \u0026nbsp;\u0026nbsp;22K09146)\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK. Tojo conceived and designed the study, performed experiments, and wrote the manuscript. T. Yazawa performed histological examinations and revised the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Ms. Akiko Adachi and Ms. Yuki Yuba for their technical assistance.\u003c/p\u003e\n\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePuybasset L, Cluzel P, Gusman P, Grenier P, Preteux F, Rouby J-J. Regional distribution of gas and tissue in acute respiratory distress syndrome. I. Consequences for lung morphology. \u003cem\u003eIntensiv Care Med\u003c/em\u003e 2000; \u003cstrong\u003e26\u003c/strong\u003e: 857\u0026ndash;869.\u003c/li\u003e\n\u003cli\u003eAlbert RK. 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Hyperoxia-induced lung injury in acute respiratory distress syndrome: what is its relative impact? \u003cem\u003eAm J Physiol-Lung Cell Mol Physiol\u003c/em\u003e 2023; \u003cstrong\u003e325\u003c/strong\u003e: L9\u0026ndash;L16.\u003c/li\u003e\n\u003cli\u003eGroup I-RI and the A and NZICSCT, Mackle D, Bellomo R, Bailey M, Beasley R, Deane A \u003cem\u003eet al.\u003c/em\u003e Conservative Oxygen Therapy during Mechanical Ventilation in the ICU. \u003cem\u003eN Engl J Med\u003c/em\u003e 2019; \u003cstrong\u003e382\u003c/strong\u003e: 989\u0026ndash;998.\u003c/li\u003e\n\u003cli\u003eBarrot L, Asfar P, Mauny F, Winiszewski H, Montini F, Badie J \u003cem\u003eet al.\u003c/em\u003e Liberal or Conservative Oxygen Therapy for Acute Respiratory Distress Syndrome. \u003cem\u003eN Engl J Med\u003c/em\u003e 2020; \u003cstrong\u003e382\u003c/strong\u003e: 999\u0026ndash;1008.\u003c/li\u003e\n\u003cli\u003eSchj\u0026oslash;rring OL, Klitgaard TL, Perner A, Wetterslev J, Lange T, Siegemund M \u003cem\u003eet al.\u003c/em\u003e Lower or Higher Oxygenation Targets for Acute Hypoxemic Respiratory Failure. \u003cem\u003eN Engl J Med\u003c/em\u003e 2021; \u003cstrong\u003e384\u003c/strong\u003e: 1301\u0026ndash;1311.\u003c/li\u003e\n\u003cli\u003eWal LI van der, Grim CCA, Prado MR del, Westerloo DJ van, Boerma EC, Jong HGR \u003cem\u003eet al.\u003c/em\u003e Conservative versus Liberal Oxygenation Targets in Intensive Care Unit Patients (ICONIC): A Randomized Clinical Trial. \u003cem\u003eAm J Respir Crit Care Med\u003c/em\u003e 2023; \u003cstrong\u003e208\u003c/strong\u003e: 770\u0026ndash;779.\u003c/li\u003e\n\u003cli\u003eLeeper-Woodford SK, Detmer K. Acute hypoxia increases alveolar macrophage tumor necrosis factor activity and alters NF-\u0026kappa;B expression. \u003cem\u003eAm J Physiol-Lung Cell Mol Physiol\u003c/em\u003e 1999; \u003cstrong\u003e276\u003c/strong\u003e: L909\u0026ndash;L916.\u003c/li\u003e\n\u003cli\u003eChao J, Wood JG, Blanco VG, Gonzalez NC. The Systemic Inflammation of Alveolar Hypoxia Is Initiated by Alveolar Macrophage\u0026ndash;Borne Mediator(s). \u003cem\u003eAm J Respir Cell Mol Biol\u003c/em\u003e 2009; \u003cstrong\u003e41\u003c/strong\u003e: 573\u0026ndash;582.\u003c/li\u003e\n\u003cli\u003eChao J, Donham P, Rooijen N van, Wood JG, Gonzalez NC. Monocyte Chemoattractant Protein\u0026ndash;1 Released from Alveolar Macrophages Mediates the Systemic Inflammation of Acute Alveolar Hypoxia. \u003cem\u003eAm J Respir Cell Mol Biol\u003c/em\u003e 2011; \u003cstrong\u003e45\u003c/strong\u003e: 53\u0026ndash;61.\u003c/li\u003e\n\u003cli\u003eWatts ER, Howden AJM, Morrison T, Sadiku P, Hukelmann JL, Kriegsheim A von \u003cem\u003eet al.\u003c/em\u003e Hypoxia drives murine neutrophil protein scavenging to maintain central carbon metabolism. \u003cem\u003eJ Clin Investig\u003c/em\u003e 2021; \u003cstrong\u003e131\u003c/strong\u003e. doi:10.1172/jci134073.\u003c/li\u003e\n\u003cli\u003eLodge KM, Vassallo A, Liu B, Long M, Tong Z, Newby PR \u003cem\u003eet al.\u003c/em\u003e Hypoxia Increases the Potential for Neutrophil-mediated Endothelial Damage in Chronic Obstructive Pulmonary Disease. \u003cem\u003eAm J Respir Crit Care Med\u003c/em\u003e 2022; \u003cstrong\u003e205\u003c/strong\u003e: 903\u0026ndash;916.\u003c/li\u003e\n\u003cli\u003eMinkove S, Dhamapurkar R, Cui X, Li Y, Sun J, Cooper D \u003cem\u003eet al.\u003c/em\u003e Effect of low-to-moderate hyperoxia on lung injury in preclinical animal models: a systematic review and meta-analysis. \u003cem\u003eIntensiv Care Med Exp\u003c/em\u003e 2023; \u003cstrong\u003e11\u003c/strong\u003e: 22.\u003c/li\u003e\n\u003cli\u003eHOPKINS RO, WEAVER LK, POPE D, Jr. JFO, BIGLER ED, LARSON-LOHR V. Neuropsychological Sequelae and Impaired Health Status in Survivors of Severe Acute Respiratory Distress Syndrome. \u003cem\u003eAm J Respir Crit Care Med\u003c/em\u003e 1999; \u003cstrong\u003e160\u003c/strong\u003e: 50\u0026ndash;56.\u003c/li\u003e\n\u003cli\u003eMikkelsen ME, Christie JD, Lanken PN, Biester RC, Thompson BT, Bellamy SL \u003cem\u003eet al.\u003c/em\u003e The Adult Respiratory Distress Syndrome Cognitive Outcomes Study. \u003cem\u003eAm J Respir Crit Care Med\u003c/em\u003e 2012; \u003cstrong\u003e185\u003c/strong\u003e: 1307\u0026ndash;1315.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Oxygen, Acute Respiratory Distress Syndrome, Atelectasis, Alveolar Hypoxia, Hypoxia-induced Inflammation","lastPublishedDoi":"10.21203/rs.3.rs-4449408/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4449408/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAlthough alveolar hyperoxia exacerbates lung injury, clinical studies have failed to demonstrate the beneficial effects of lowering the fraction of inspired oxygen (F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) in patients with acute respiratory distress syndrome (ARDS). Atelectasis, which is commonly observed in ARDS, not only leads to hypoxemia but also contributes to lung injury through hypoxia-induced alveolar tissue inflammation. Therefore, it is possible that excessively low F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e may enhance hypoxia-induced inflammation in atelectasis, and raising F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to an appropriate level may be a reasonable strategy for its mitigation. In this study, we investigated the effects of different F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels on alveolar tissue hypoxia and injury in a mechanically ventilated rat model of experimental ARDS with atelectasis.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eRats were intratracheally injected with lipopolysaccharide (LPS) to establish an ARDS model. They were allocated to the low, moderate, and high F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e groups with F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e of 21, 60, and 100%, respectively, a day after LPS injection. All groups were mechanically ventilated with an 8 mL/kg tidal volume and zero end-expiratory pressure to induce dorsal atelectatic regions. Arterial blood gas analysis was performed every 2 h. After six hours of mechanical ventilation, the rats were euthanized, and blood, bronchoalveolar lavage fluid, and lung tissues were collected and analyzed. Another set of animals was used for pimonidazole staining of the lung tissues to detect the hypoxic region.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eLung mechanics, ratios of partial pressure of arterial oxygen (P\u003csub\u003ea\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) to F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and partial pressure of arterial carbon dioxide were not significantly different among the three groups, although PaO2 changed with F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. The dorsal lung tissues were positively stained with pimonidazole regardless of F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and the HIF-1α concentrations were not significantly different among the three groups, indicating that raising F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e could not rescue alveolar tissue hypoxia. Moreover, changes in F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e did not significantly affect lung injury or inflammation. In contrast, hypoxemia observed in the low F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group caused injury to organs other than the lungs.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eRaising F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels did not attenuate tissue hypoxia, inflammation, or injury in the atelectatic lung region in experimental ARDS. Our results indicate that raising F\u003csub\u003eI\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels to attenuate atelectasis-induced lung injury cannot be rationalized.\u003c/p\u003e","manuscriptTitle":"A high fraction of inspired oxygen does not mitigate atelectasis-induced lung tissue hypoxia or injury in experimental acute respiratory distress syndrome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-07 18:59:52","doi":"10.21203/rs.3.rs-4449408/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0638a152-308d-4a75-8b69-25badc09b50c","owner":[],"postedDate":"June 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-10T04:26:22+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-07 18:59:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4449408","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4449408","identity":"rs-4449408","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}