Barbeta et al. Critical Care (2023) 27:239 Critical Care https://doi.org/10.1186/s13054-023-04512-8 PERSPECTIVE Open Access A long‑lasting porcine model of ARDS caused by pneumonia and ventilator‑induced lung injury Enric Barbeta1,2,3,4†, Marta Arrieta3,4†, Ana Motos2,3,4*, Joaquim Bobi3,4,5,12, Hua Yang3,4,6, Minlan Yang3,4,7, Giacomo Tanzella3,9, Pierluigi Di Ginnatale3,8, Stefano Nogas3,9, Carmen Rosa Vargas3,4, Roberto Cabrera2,3, Denise Battaglini3,4,9, Andrea Meli3,11, Kasra Kiarostami3,4, Nil Vázquez3,4, Laia Fernández‑Barat2,3,4, Montserrat Rigol3,4,12, Ricard Mellado‑Artigas1,2,3, Gerard Frigola14, Marta Camprubí‑Rimblas2,14, Pau Ferrer3,15, Daniel Martinez3,13, Antonio Artigas2,14, Carlos Ferrando1,2,3,4, Miquel Ferrer2,3,4,10 and Antoni Torres2,3,4,10* Abstract Background Animal models of acute respiratory distress syndrome (ARDS) do not completely resemble human ARDS, struggling translational research. We aimed to characterize a porcine model of ARDS induced by pneumonia— the most common risk factor in humans—and analyze the additional effect of ventilator-induced lung injury (VILI). Methods Bronchoscopy-guided instillation of a multidrug-resistant Pseudomonas aeruginosa strain was performed in ten healthy pigs. In six animals (pneumonia-with-VILI group), pulmonary damage was further increased by VILI applied 3 h before instillation and until ARDS was diagnosed by ­PaO2/FiO2 < 150 mmHg. Four animals (pneumonia-without- VILI group) were protectively ventilated 3 h before inoculum and thereafter. Gas exchange, respiratory mechanics, hemodynamics, microbiological studies and inflammatory markers were analyzed during the 96-h experiment. During necropsy, lobar samples were also analyzed. Results All animals from pneumonia-with-VILI group reached Berlin criteria for ARDS diagnosis until the end of experiment. The mean duration under ARDS diagnosis was 46.8 ± 7.7 h; the lowest ­PaO2/FiO2 was 83 ± 5.45 mmHg. The group of pigs that were not subjected to VILI did not meet ARDS criteria, even when presenting with bilateral pneumonia. Animals developing ARDS presented hemodynamic instability as well as severe hypercapnia despite high-minute ventilation. Unlike the pneumonia-without-VILI group, the ARDS animals presented lower static compli‑ ance (p = 0.011) and increased pulmonary permeability (p = 0.013). The highest burden of P. aeruginosa was found at pneumonia diagnosis in all animals, as well as a high inflammatory response shown by a release of interleukin (IL)-6 and IL-8. At histological examination, only animals comprising the pneumonia-with-VILI group presented signs con‑ sistent with diffuse alveolar damage. Conclusions In conclusion, we established an accurate pulmonary sepsis-induced ARDS model. † Enric Barbeta and Marta Arrieta contributed equally to this work. *Correspondence: Ana Motos amotos@clinic.cat Antoni Torres atorres@clinic.cat Full list of author information is available at the end of the article © The Author(s) 2023. 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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Barbeta et al. Critical Care (2023) 27:239 Page 2 of 14 Keywords ARDS, Ventilator-induced lung injury, Porcine model, Pneumonia, Double hit, Injurious mechanical ventilation Background and ventilator-induced lung injury (VILI). To evaluate Acute respiratory distress syndrome (ARDS) is a syn- pneumonia as a cause of ARDS within the presence and drome characterized by capillary endothelial injury and absence of VILI, we studied two animal groups: one that diffuse alveolar damage secondary of cytokines release comprised pigs with bilateral pneumonia with a high after a direct risk factor exposure (i.e., infection, aspira- pulmonary strain and the other (control) with bilateral tion, major trauma) or an indirect risk factors (i.e., sepsis, pneumonia and no significant pulmonary strain. After blood transfusion, major surgery) [1]. the Berlin criteria for ARDS were met, we monitored The latest definition (i.e., Berlin definition) [1] describes gas exchange, pulmonary mechanics, pulmonary and an acute clinical–radiological syndrome characterized by systemic hemodynamics, and pulmonary and systemic acute respiratory failure and bilateral pulmonary opaci- inflammatory markers. We also studied the microbiology ties not fully caused by heart failure, fluid overload or and histology of the injured lungs. other pulmonary pathologies such as lung tumors or ate- lectasis. As a result, patients with ARDS require oxygen Methods therapy and mechanical ventilation, which is deliverable This is an experimental animal study that aimed to char- either invasively or non-invasively. acterize the respiratory, hemodynamic, inflammatory, Animal experimentation has greatly contributed to microbiological and histological features of ARDS caused generating knowledge about ARDS. It was first done in by a bilateral Pseudomonas aeruginosa pneumonia and animals with the application of mechanical ventilation with/without the application of injurious ventilation (a and high tidal volume to promote lung damage before high or low pulmonary strain). being shown in several randomized clinical trials [2]. The The study was carried out in 10 Large White Landrace benefits of prone position were initially described in ani- pigs (40–48 kg), subjected to 96 h of mechanical ventilation mal experimentation as well [3]. Other concepts related in the prone position (SERVO-I, Maquet, NJ, USA). The to ventilator-induced lung injury such as pulmonary experiment was divided into two time frames: (1) induc- strain, stress and mechanical power were demonstrated tion and (2) model development. During induction, pigs in animals before humans [4, 5]. Paradoxically, though, were challenged with a multidrug-resistant (MDR) strain none of these insightful experiments were performed in of P. aeruginosa and, based on the study group, mechani- animal models of ARDS. They were, however, done in cally ventilated with protective or injurious settings. Model animals without any lung injury. development began when ARDS diagnosis was established; Reproducing ARDS in animals is challenging. The most it concluded once the animal was put down. common cause of ARDS in humans is pneumonia [6], As previously described, we prepared the animal and and its definition is based on a constellation of clinical maintained deep anesthesia throughout the experiment characteristics (acute respiratory failure) and radiologi- [11]. Figure 1 displays main assessments throughout the cal signs (bilateral pulmonary edema) [1]. However, the experiment detailed in Additional file 1. definition of ARDS in animal models is rarely supported by a disorder of oxygenation or thoracic imaging. Indeed, most pulmonary insults used in an experimental setting Bilateral pneumonia with VILI differ substantially from human ARDS physiopathology This group comprised six animals. Briefly, after animal (i.e., intravenous oleic acid or endotoxin administration, preparation, we performed 3 h of VILI with a ventila- surfactant depletion with saline lavage, etc.) [7, 8]. There- tor setting that ensured harmful pulmonary dynamic fore, animal models usually present features inconsistent strain (> 1.5) [12]. Following this, we instilled 15 mL of with human ARDS like short duration [7, 9] and a signifi- ­107 colony-forming unit (CFU)/mL of MDR P. aerugi- cant rate of reversibility [10]. All these drawbacks ham- nosa, susceptible to both meropenem and levofloxacin, per research in animal experimentation, as it relates to into each lobe via bronchoscopy (see Additional file 1: therapies such as new ventilation and extracorporeal life Table S1). Injurious mechanical ventilation resumed support strategies and targeted treatments. with the same previous setting, albeit with a positive In this study, we aimed to characterize a double-hit end-expiratory pressure (PEEP) of 5 c­ mH2O. We diag- porcine model of ARDS induced by the most com- nosed ARDS when arterial partial pressure of oxygen mon risk factors identified in humans [6]: pneumonia ­(PaO2)/fraction of inspired oxygen (­FiO2) < 150 mmHg Barbeta et al. Critical Care (2023) 27:239 Page 3 of 14 Fig. 1 Flowchart of the study. Study assessments are displayed in black dots, while protective and injurious ventilation phases are shown for both study groups. Major check includes: Arterial and mixed venous blood gases, hemodynamics, urine output and bispectral index. Abbreviations: ARDS: acute respiratory distress syndrome; P.V.: protective ventilation. ARDS diagnosis time point in the pneumonia-without-VILI group corresponds to 30 h from bacterial inoculum, which is the median time from inoculum to ARDS diagnosis in the pneumonia-with-VILI group. This equivalence needed to be done for comparison reasons because the pneumonia-without-VILI group never met the Berlin criteria for ARDS diagnosis was achieved. Once criteria for ARDS were met, ven- Statistical analysis tilator mode was changed to volume control (VC) and The results were reported as the mean and standard pigs were ventilated according to a lung protective error of the mean. Categorical variables were presented strategy. Tidal volume was set to aim for normocapnia as umbers and percentage. For continuous variables, with a DP that did not exceed 15 ­cmH2O [13]. we compared groups and over time using the repeated measure two-way ANOVA test. Bonferroni’s post hoc multiple comparison was used to control the experiment- Bilateral pneumonia without VILI wise error rate. Comparison between groups at a fixed The control arm included four animals which pre- time point was assessed using the Mann–Whitney non- sented bilateral pneumonia and received treatment parametric test. Either the Chi-square test or Fisher’s with protective ventilator parameters (no significant exact test was used to compare categorical variables. A strain). Like the previous group, 3 h of ventilation was two-sided p value < 0.05 was considered statistically sig- applied before the bacterial challenge, with a setting nificant. All statistical analyses were performed using the that assured a non-harmful pulmonary strain (< 1.5) GraphPad Prism 8 (version 8.0.1 (244), San Diego, CA, [12]. After that, we instilled 15 mL of ­107 CFU/mL of USA). MDR P. aeruginosa into each pulmonary lobe. We con- tinued applying protective ventilation until the end of Results the experiment. Apart from a DP < 15 c­ mH2O [13], we Ten animals (42.9 ± 1.13 kg) were divided into two maintained the thresholds for the most important VILI groups: six animals comprised the pneumonia-with-VILI parameters under a safe range per literature [5, 12, 13] group and four animals the pneumonia-without-VILI (Table 1). group. In the former group, two pigs were put down before the end of the experiment due to severe respira- tory failure and severe respiratory acidosis. The mortality Pneumonia diagnosis rate reached 33.3%. All animals in the pneumonia-with- Pneumonia was clinically diagnosed in both groups out-VILI group completed the study. based on a decline in ­PaO2 and one of the following signs of infection: body temperature > 38.5 °C, a white blood count > 14·109/L and purulent secretions [14]. At pneu- Induction period in pigs with pneumonia according monia diagnosis, we started antimicrobial therapy with to the presence or absence of VILI 25 mg/kg of meropenem every 8 h and 10 mg/kg of levo- Table 1 and Fig. 2 display parameters related to mechani- floxacin every 24 h, as dual therapy recommended by cal ventilation, gas exchange and respiratory mechan- international guidelines [15]. ics observed during the induction period. During this Barbeta et al. Critical Care Table 1 Induction and model development periods (2023) 27:239 Pneumonia with VILI Pneumonia without VILI Injurious ventilation P. Injurious Injurious ventilation ARDS (to the Protective P. Protective Pneumonia ARDS aeruginosa ventilation (pneumonia end of the ventilation aeruginosa ventilation (to the inoculum (inoculum to diagnosis to ARDS experiment) inoculum (inoculum to end of the pneumonia diagnosis) pneumonia experiment) diagnosis) diagnosis) Duration (h) 3.1 ± 0.1 0.9 ± 0.1 12.0 ± 0.7 16.0 ± 2.3 46.8 ± 7.7 3.1 ± 0.2 NA 7.2 ± 1.1 82.4 ± 1.2 NA FiO2 (%) 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 41.3 ± 0.0 NA 40.6 ± 0.6 44.4 ± 1.1 NA TV (mL/kg) 33.3 ± 2.4 10.5 ± 0.0 27.5 ± 1.6 25.1 ± 0.9 5.7 ± 0.2 8.1 ± 0 NA 7.8 ± 0.2 7.2 ± 0.1 NA RR (breaths/min) 12.3 ± 0.0 20.0 ± 0.0 11.3 ± 0.9 10 ± 0.0 45.8 ± 1.0 15.4 ± 0.4 NA 16.8 ± 0.1 23.7 ± 1.1 NA PEEP ­(cmH2O) 0.0 ± 0.0 3.0 ± 0.0 4.9 ± 0.0 4.9 ± 0.1 9.5 ± 0.3 4.0 ± 0.0 NA 4.3 ± 0.0 4.8 ± 0.2 NA DP (mmHg) 27.7 ± 0.2 12.5 ± 0.0 30.1 ± 0.3 29.9 ± 0.2 17.7 ± 0.5 9.5 ± 0.5 NA 12.3 ± 0.0 13.5 ± 0.4 NA MP NA 10.5 ± 1.6 34.7 ± 0.8 28.35 ± 1.2 26.9 ± 1.7 5.23 ± 0.7 NA 6.65 ± 1.0 10.1 ± 0.3 NA Ventilatory mode PC VC PC PC VC VC NA VC VC NA After the inoculum, a short period of protective ventilation was performed to ease bacteria engraftment. Note that this was not necessary in the other group because protective ventilation was already ongoing. Data are reported as mean ± standard error ARDS acute respiratory distress syndrome, DP driving pressure, FiO2 fraction of inspired oxygen, MP mechanical power, P. aeruginosa, Pseudomonas aeruginosa, PC pressure control, PEEP positive end-expiratory pressure, RR respiratory rate, TV tidal volume, VC volume control Page 4 of 14 Barbeta et al. Critical Care (2023) 27:239 Page 5 of 14 period, we performed a 15-mL P. aeruginosa challenge of (14.54 ± 1.30 and 21.77 ± 0.32 ­cmH2O/L) (p = 0.011). 7.77 ± 0.11 log CFU/mL in each pulmonary lobe in both Furthermore, DP and mechanical power showed signifi- groups. cant differences between groups (p = 0.011 and p = 0.001, After the bacterial challenge, we continued injurious respectively). Mean values were 17.65 ± 0.50 ­cmH2O and ventilation in the VILI group for 28.04 ± 3.03 h until the 26.87 ± 1.72 J/min in the group of pneumonia with VILI Berlin criteria were met, with the mean P ­ aO2/FiO2 being and 13.46 ± 0.34 ­cmH2O and 10.09 ± 0.27 J/min in the 129.62 ± 6.1 mmHg at ARDS diagnosis. Bilateral opaci- group of pneumonia without VILI. After the ARDS diag- ties were identified by X-ray in all six pigs. In contrast, nosis time point, ­PaCO2 in animals with VILI was signifi- the pneumonia-without-VILI group never met the Ber- cantly higher than in those without VILI (69.28 ± 10.95 lin criteria for ARDS. P­ aO2/FiO2 did not decrease below and 38.85 ± 1.076 mmHg, p = 0.027). This also was 300 mmHg at any time point (mean ­ PaO2/FiO2 was observed in exhaled minute volume (11.71 ± 0.32 and 389.90 ± 3.97 mmHg) (p < 0.0002) (Fig. 2). 7.02 ± 0.10 L/min, p < 0.001). Furthermore, mean EVLW No significant differences were found in the hemody- was 840.2 ± 61.73 mL/kg in the pneumonia-with-VILI namic parameters between groups during the induction group and 385.6 ± 6.90 mL/kg in the pneumonia-with- period, as shown in Fig. 3. out-VILI group (p = 0.013), as shown in Fig. 2. Ventilator Pneumonia was clinically and microbiology established setting adjustments are displayed in Additional file 1: Fig. at 11.99 ± 0.67 and 7.25 ± 1.12 h post-inoculum in the S1. pneumonia-with-and-without-VILI groups, respectively (p = 0.01). Additional file 1: Table S2 shows the achieved criteria of pneumonia diagnosis. No differences in P. aer- Model development: systemic and pulmonary uginosa burden were observed between study groups hemodynamics at pneumonia diagnosis, neither in tracheal secretions Mean pulmonary pressure increased in pigs with ARDS: (p = 0.46) nor in BAL samples (p = 0.57) (Fig. 4). Mean values were 26.23 ± 0.73 mmHg compared with 22.79 ± 0.28 mmHg in in the pneumonia-without-VILI group (p = 0.056). Mean arterial pressure in pigs with Model development: respiratory function ARDS remained at 72.48 ± 1.23 mmHg. In the other Animals comprising the pneumonia-with-VILI group group, it was 84.79 ± 5.21 mmHg (p = 0.003). Pigs from maintained a ­PaO2/FiO2 below 300 mmHg until the end the pneumonia-with-VILI group presented a higher of experiment (model development period). The mean vasopressor dependency index (VDI); however, it was duration of an ARDS diagnosis in animals that survived not significant (0.63 ± 0.16 vs 0.03 ± 0.02, p = 0.158). After was 58.7 ± 1.3 h. For those who were killed due to clini- an ARDS diagnosis, systemic vascular resistances were cal instability, it was 29.9 h and 16.3 h each. During this lower in the pneumonia-with-VILI group than in the period (model development), the mean ­PaO2/FiO2 was pneumonia-without-VILI group (907.3 ± 44.76 dynes/s/ 187.80 ± 28.46 mmHg, with the lowest measure recorded cm−5 1432.71 ± 124.41 dynes/s/cm−5; p = 0.02). Figure 3 at 83.00 ± 5.45 mmHg. Figure 2 displays the evolution shows other hemodynamic parameters. Laboratory find- of respiratory system mechanics during model devel- ings and fluid balance are reported in Additional file 1: opment. Respiratory system compliance was lower in Table S3 and Fig. S2. animals with VILI compared with those without VILI (See figure on next page.) Fig. 2 Respiratory function. Pink lines and dots represent animals from the pneumonia-with-VILI group, and the green ones represent those animals from the pneumonia-without-VILI group. A Evolution of airway pressures in the pneumonia-with-VILI group. B Evolution of airway pressures in the pneumonia-without-VILI group. C Evolution of P ­ aO2/FiO2 throughout the study with periods of moderate and severe respiratory failure in the pneumonia-with-VILI group. Note that in the group of pigs without VILI, it does not decrease below 300 mmHg. D, E ­PaCO2 and exhaled minute volume were increased in the group of pneumonia with VILI after ARDS diagnosis and were stable in animals that were ventilated protectively. Note the high variability in ­PaCO2 at 36 h of ARDS diagnosis is due to the death of two animals. F Higher compliance was observed in the pneumonia-with-VILI model during induction period, whereas after ARDS diagnosis, it was lower. G A significantly higher mechanical power was observed between groups and at every time point. H Extravascular lung water increased in both groups throughout the study time points, showing evidence of an increase in lung permeability. However, in the pneumonia-with-VILI group, it was significantly higher. Abbreviations: ARDS: acute respiratory distress syndrome; DP: driving pressure; PEEP: positive end-expiratory pressure; Ppeak: peak pressure; VILI: ventilator-induced lung injury. Data are reported as mean ± standard error of mean (SEM). # ARDS diagnosis time point in the pneumonia-without-VILI group corresponds to 30 h from bacterial inoculum, which is the median time from inoculum to ARDS diagnosis in the pneumonia-with-VILI group. This equivalence needed to be done for comparison reasons because the pneumonia-without-VILI group never met the Berlin criteria for ARDS diagnosis. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 Barbeta et al. Critical Care (2023) 27:239 Page 6 of 14 Fig. 2 (See legend on previous page.) Barbeta et al. Critical Care (2023) 27:239 Page 7 of 14 Imaging, necropsy and histology Systemic and local inflammatory markers We performed X-ray images of two different pro- As shown in Additional file 1: Fig. S5, systemic IL-6 and jections at baseline and the end of the experiment. IL-8 had significant time-dependent changes (p < 0.0001 While normal lung appearance was found at baseline, and p = 0.024). At pneumonia diagnosis, plasma bilateral opacities were observed at the end of the IL-6 was higher in the pneumonia-with-VILI group experiment in both groups. X-ray images were also (579.94 ± 138.79 vs. 356.79 ± 233.66 pg/mL, p = 0.038). performed at ARDS diagnosis in animals with VILI, However, after the ARDS diagnosis (model development revealing bilateral opacities too. At necropsy, macro- period), these differences disappeared. IL-6 and IL-8 also scopic findings were more severe in those with VILI presented an increase in BAL, albeit without significant than in animals presenting pneumonia without VILI, differences between groups. as shown in Additional file 1: Fig. S3. Figure 5 displays the main lung findings. Macroscop- Microbiology assessments ically, all pigs presented with findings consistent with Figure 5 displays the main microbiological results. bilateral pneumonia. However, animals that devel- Throughout the model development period, animals oped ARDS also had widespread hepatization, severe from the pneumonia-with-VILI group presented a lower edematous appearance, pleural effusion and adher- burden of P. aeruginosa in tracheal aspirates (4.24 ± 0.68 ence. Importantly, in the pneumonia-with-VILI group, log CFU/mL in the pneumonia-with-VILI group and we found higher vascular permeability, as revealed by 6.01 ± 0.32 log CFU/mL in the pneumonia-without-VILI both a higher lung/body weight ratio (2.71 ± 0.26 in group, p = 0.027). However, it was not different for lung the pneumonia-with-VILI group vs. 1.61 ± 0.13% in the tissue or BAL. At the end of the experiment, tracheal pneumonia-without-VILI group, p = 0.009) and lung aspirate cultures from both groups tested > 3 log CFU/ wet-to-dry ratio (7.56 ± 0.73 in the pneumonia-with- mL. In lung tissue, mean values were below 3 log CFU/ VILI group vs. 4.03 ± 1.03% in the pneumonia-with- mL in both groups. out-VILI group, p = 0.029). In the pneumonia-with-VILI group, all except one Discussion (83%) presented hyaline membrane formation con- We developed a model of pulmonary sepsis-induced sistent with diffuse alveolar damage (DAD). How- ARDS in pigs that had long-lasting features consistent ever, DAD and hyaline membranes were absent in all with human ARDS. Our model was characterized by the the animals in which pneumonia was induced without following aspects: (I) development of severe oxygena- VILI. Furthermore, when compared with the pneumo- tion disorders; (II) development of hypercapnia despite nia-without-VILI group, animals that developed ARDS the application of high-minute ventilation (reflecting an also presented a rise in intra-alveolar (p = 0.033) and increase in dead-space ventilation); (III) decrease in res- interstitial neutrophil infiltration (p = 0.005), as well piratory system static compliance; (IV) increase in pul- as increased septal edema (p = 0.038). Alveolar edema, monary permeability; (V) presence of cardiovascular intra-alveolar fibrin and hemorrhage, proliferation of dysfunction (sepsis-induced vasoplegia); (VI) pulmonary alveolar type II cells and epithelial denudation were and systemic inflammation; and (VII) signs of diffuse reported in both groups without differences. In Addi- alveolar damage. tional file 1: Fig. S4, the same features are displayed by The model presented fulfills recent recommendations dependent and non-dependent areas. for animal experimentation in the field of acute lung (See figure on next page.) Fig. 3 Hemodynamics. Pink lines and dots represent animals from the pneumonia-with-VILI group, and the green ones represent those animals from pneumonia-without-VILI group. A The evolution of VDI showed the need to use norepinephrine to maintain mean arterial pressure in the group of pigs with VILI and in those pigs ventilated protectively. B Mean pulmonary pressure increased at pneumonia diagnosis and remained stable in the pneumonia-with-VILI group, whereas it decreased in the group without VILI. C, D CVP and PAOP did not present significant differences between groups. E, F A higher cardiac output and heart rate were found in animals with VILI in comparison with those ventilated protectively. G Animals developing ARDS presented higher maximal left ventricular pressure rise (LV dPmax) as a marker of systolic function. H Systemic vascular resistance decreased in animals with pneumonia and VILI in comparison with the group of pigs without VILI. Abbreviations: ARDS: acute respiratory distress syndrome; CVP: central venous pressure; LV dPmax: maximal left ventricle pressure rise; MAP: mean arterial pressure; PAOP: pulmonary arterial occlusion pressure; VDI: vasopressor dependency index. Data are reported as mean ± standard error of mean (SEM). # ARDS diagnosis time point in the pneumonia-without-VILI group corresponds to 30 h from inoculum, which is the median time from inoculum to ARDS diagnosis in the pneumonia-with-VILI group. This equivalence needed to be done for comparison reasons because the pneumonia-without-VILI group never met the Berlin criteria for ARDS diagnosis. *p < 0.05 Barbeta et al. Critical Care (2023) 27:239 Page 8 of 14 Fig. 3 (See legend on previous page.) Barbeta et al. Critical Care (2023) 27:239 Page 9 of 14 injury [16]. That considered, we demonstrated histologi- disorders in oxygenation are maintained up to approxi- cal evidence of tissue injury; alteration of the alveolar– mately 60 h. In the surfactant depletion model, some capillary barrier; presence of systemic and pulmonary authors reported an increase in BAL cytokines, but the inflammatory response; and severe respiratory dysfunc- role of inflammation is unclear [9]. We demonstrated, tion. Furthermore, other relevant characteristics specific nonetheless, an increase in both systemic and pulmonary to the model should be underscored: First and foremost, inflammation and signs of sepsis-associated cardiovascu- the pulmonary insult used in the induction period is the lar dysfunction. main cause of ARDS in humans (bacterial pneumonia). Similar flaws are identified in the oleic acid (OA) Moreover, the second hit used in the model (VILI) might model. This one mimic ARDS caused by fat embolism, an be unavoidable in human ARDS, as ventilation with high exceptional etiology of syndrome [10]. In the OA model, strain and stress is needed to maintain gas exchange in the pathophysiology of pulmonary injury is unknown and the most severe patients [6]. Second, the large duration of unlikely similar to the most common causes of ARDS the experiment allows for tracking changes in respiratory such as pneumonia, the risk factor used in our model physiology. Third, large animals allow for advanced mon- [6]. OA administration raises pulmonary arterial pres- itoring (i.e., electrical impedance tomography, and analy- sure and intrapulmonary shunt [19–21] and produces a sis of chest wall and pulmonary respiratory mechanics, marked increase in EVLW [20]. We also showed a rise pulmonary and systemic hemodynamics, etc.) as well as in the mean pulmonary arterial pressure and EVLW. We evaluation of complex therapies such as extracorporeal tracked changes in ­PaO2/FiO2 as surrogate of pulmonary membrane oxygenation and mechanical ventilation. shunt, identifying periods with a value below 100 mmHg (i.e., severe ARDS). In the OA model, an inflammatory Comparison of animal models and their characteristics response is shown with an increase in some inflamma- Given the clinically relevant insults used (pneumonia and tory markers in lung such as IL-6 and IL-1 beta [22–24]. VILI) as well as the physiological characteristics exhib- At histological examination, however, only mild signs of ited during the 96-h experiment period, we believe that lung injury are found with no hyaline membrane forma- the model presented has higher accuracy in reproducing tion [20, 23, 25]. In our model, though, we demonstrated human ARDS compared with others [7, 8, 16, 17]. DAD in 83% of histological specimens of pigs with In line with our study, the repeated lavage model pre- ARDS. In the OA model, the severity of lung injury is sents remarkable gas exchange disorders and diminished somewhat unpredictable because animals present differ- compliance due to surfactant depletion. Specifically, our ent responses per OA doses and sudden, intense hemo- ARDS model shows a mean P ­ aO2/FiO2 which, through- dynamic instability during infusion [23, 25]. Like the out the model development, ranges in moderate sever- repeated lavage model, the oleic acid-induced lung injury ity with periods of severe instability. Moreover, notably is also reversible within several hours, contrasting with diminished and progressively deteriorating compliance is our 60-h ARDS period [21, 23]. found throughout the model development period as well. Systemic administration of bacterial lipopolysaccha- Surfactant depletion is not the main mechanism of res- rides (LPS) was one of the earliest approaches used to piratory failure in adult ARDS; hence, this model fails to study sepsis-induced ARDS. LPS administration mark- reproduce other clinical features of ARDS. For example, edly decreases ­PaO2 and systemic arterial pressure. It also in contrast from our model, only modest epithelial injury provides important information about host inflammatory signs are present; permeability changes, scarce; and responses in BAL and serum [7, 26]. For that reason, it clinical features described, reversible, lasting less than is a good choice for pathophysiological studies of infec- 24 h [9, 10, 18]. Our model, however, presents a histol- tion and ARDS [7, 26]. Our model follows this approach; ogy that demonstrates that severe epithelial damage and however, we instill viable bacteria—rather than bacterial (See figure on next page.) Fig. 4 Lung findings. Pink lines and dots represent animals from the pneumonia-with-VILI group, and the green ones represent those animals from the pneumonia-without-VILI group. A Macroscopic findings: on the left, non-dependent images of an animal from the pneumonia-with-VILI group and on the right, from the pneumonia-without-VILI group. B Significantly higher EVLW in the pneumonia-with-VILI group at the end of the study (p = 0.02). C Lung weight-to-body weight ratio was significantly higher in animals with pneumonia and VILI (p = 0.009). D The wet lung weight-to-dry lung weight ratio was significantly higher in the pneumonia-with-VILI group (p = 0.029). E Percentage of lobes affected with histological features in both study groups. F Histological score evaluated in both groups (0–3). G I: Lung tissue microscopic findings in pneumonia-without-VILI animals presenting inflammatory neutrophilic infiltrate ( +). G II and III: Lung tissue microscopic findings in pneumonia-with-VILI animals showed moderate neutrophil infiltration of the alveolar space and septae, severe edema and hemorrhage, as well as neutrophil infiltration ( +), severe thickening of interlobular septae (#) and evident presence of hyaline membranes (black arrows) surrounding alveoli. Abbreviations: ARDS: acute respiratory distress syndrome; ATII: alveolar epithelial type II cells; VILI: ventilator-induced lung injury. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 Barbeta et al. Critical Care (2023) 27:239 Page 10 of 14 Fig. 4 (See legend on previous page.) Barbeta et al. Critical Care (2023) 27:239 Page 11 of 14 Fig. 5 Microbiology assessments. Pink lines, dots and boxes represent animals from the pneumonia-with-VILI group, and the green ones represent those animals from the pneumonia-without-VILI group. A After pneumonia diagnosis and antibiotic start, Pseudomonas aeruginosa concentration in tracheal secretions decreased (p = 0.007), more evident in animals with VILI. B With respect to BAL fluids, the burden decreased throughout the study (p = 0.001). C In lung tissue, mean values resulted below 3 log CFU/mL after having received 8.2 ± 0.9, 3 ± 0.4 and 7 ± 0.7 doses of meropenem, levofloxacin and ceftriaxone, respectively. Abbreviations: ARDS: acute respiratory distress syndrome; CFU: colony-forming units; VILI: ventilator-induced lung injury. Pulmonary lobes: RUL, right upper lobe; RML, right medium lobe; RLL-D, right lower lobe-dependent; RLL-ND, right lower lobe-non-dependent; LUL, left upper lobe; LLL-D, left lower lobe-dependent; LLL-ND, left lower lobe-non-dependent. Data are reported as mean ± standard error. # ARDS diagnosis time point in the pneumonia-without-VILI group corresponds to 30 h from inoculum, which is the median time from inoculum to ARDS diagnosis in the pneumonia-with-VILI group. This equivalence needed to be done for comparison reasons because the pneumonia-without-VILI group never met the Berlin criteria for ARDS diagnosis. *p < 0.05 toxins—into the lungs, thus fulfilling the complete clini- endotoxin preparations [8]. In contrast, the duration of cal picture of severe pneumonia. During the model devel- our model is enough to perform multiple analyses, given opment, a high burden of P. aeruginosa was found in BAL its reproducible nature as well. and tracheal aspirates. While we did not find differences The smoke/burn is a reproducible model that resem- in BAL samples, we did identify a lower bacterial burden bles the clinical time course of ARDS during the first in tracheal aspirates from animals with ARDS in com- 24 h. It can be used in studies up to 96 h [28]. However, parison with those who did not develop it. The interpre- smoke inhalation as etiology of ARDS is not frequent, tation of this finding is not clear. We speculate that the and the pathophysiology of lung injury is different from plasma leak into the lungs of pigs with ARDS prompted sepsis [6]. Although some studies achieve severe respira- tracheal aspirations more frequently and promoted bac- tory failure [28], this does not last longer 24 h [29, 30]. terial clearance. Histological alterations observed in the Inflammatory markers as IL-6 are found elevated in BAL LPS model are mainly neutrophil parenchymal infiltra- and plasma [28, 31]. Histology observations demonstrate tion and mild edema, features that resemble those seen a presence of inflammatory infiltrate, mild hemorrhage in pneumonia. However, in this model, hyaline mem- and edema, albeit no hyaline membranes [28, 31, 32]. branes are lacking [7, 27], suggesting milder epithelial Injurious ventilation has been used as a unique or damage and lower pulmonary permeability compared double-hit model to reproduce ARDS in animals [8, 32]. with our model. Other limitations of this model are short In rodent animal models, VILI causes an impairment in duration, significant differences in endotoxin response respiratory mechanics [33, 34], tissue injury [33, 35] and between species and variability in biological activity of pulmonary permeability [34], but oxygenation disorders Barbeta et al. Critical Care (2023) 27:239 Page 12 of 14 are mild and do not usually meet ARDS criteria [36–39]. ease progression to ARDS in pneumonia and perhaps These findings in small animals are difficult to translate raise respiratory-attributable death. in terms of humans. Contrarily, in larger animal mod- els, VILI provokes severe respiratory failure. Unlike our Limitations model, though, it does not last longer than 24 h [38–42]. Some limitations are present in this study. First, as a In these models, inflammation caused by VILI was also result of severe respiratory failure, mortality reported evaluated by several methods including cytokine analysis in this model was high (33.3%). Second, we only stud- in BAL fluid, plasma and lung tissue, and either immu- ied ten non-randomized pigs and just those treated with nohistochemistry or immunofluorescence [8, 33-44]. high pulmonary strain fulfilled the diagnostic criteria While some studies confirmed that VILI induces a pul- for ARDS. Nevertheless, the dispersion of data was low, monary inflammatory response [33–39], others did not and our findings were consistent. Due to logistical con- [44]. These differences might be explained by either the straints, animals were not randomized. Third, critically intensity of VILI observed between studies or the pri- ill patients usually present with significant comorbidi- mary pulmonary insult performed before aggressive ven- ties that determine prognosis. However, animals used in tilation was started [45]. Interpretations of our findings the model were female, young and healthy. Moreover, should consider that we used a DP of up to 30 ­cmH2O to the sequence of events in the clinical setting differs, as ensure a clinically significant pulmonary strain and that patients initially experience an insult that results in res- such ventilation was performed in lungs affected by a pri- piratory insufficiency, followed by the need for mechani- mary inflammatory insult (pneumonia). Interestingly, as cal ventilation. In our model, healthy animals underwent observed in our model, other authors reported the pres- injurious ventilation, were then subjected to bacterial ence of hyaline membranes and diffuse alveolar damage challenge and finally continued to receive injurious ven- in animals ventilated in an injurious fashion [40]. tilation. Fourth, P. aeruginosa is a cause of pneumonia in patients with specific risk factors such as airway dis- The effect of ventilator‑induced lung injury in pneumonia eases (i.e., chronic obstructive pulmonary disease) and In our model, the group with low pulmonary strain—no immunosuppression. Fifth, there was not a control group VILI—presented oxygen disorders that were mild and did of pigs without pneumonia that received treatment with not meet the Berlin criteria for ARDS. On the other hand, aggressive ventilation. pigs comprising the pneumonia-with-VILI group had sustained life-threatening gas exchange disorders, as well Conclusion as other remarkable disturbances. In the current model, In conclusion, we established an accurate pulmonary however, we did not find clinically relevant extrapulmo- sepsis-induced ARDS model. To carry out this model, it nary organ dysfunctions other than hemodynamic failure is necessary to induce bilateral pneumonia and increase (sepsis-induced vasoplegia). The latter was more promi- pulmonary damage with VILI. The clinically relevant nent in the pneumonia-with-VILI group. Of note, while pulmonary insults and features exhibited, long duration pigs with ARDS exhibited a high inflammatory response, and capacity for invasive procedures and continuous this was not clearly influenced by the presence of VILI monitoring to be performed constitute the most impor- during model development. Importantly, high mortality tant elements of this model. For those reasons stated, we (33.3%) found only in the group of pigs ventilated with believe that this model is appropriate for studying novel the high pulmonary strain was driven by respiratory aci- lung protective strategies and respiratory rescue thera- dosis and refractory respiratory failure. pies for very severe ARDS. The findings described between groups merit clini- cal interpretation. Refractory respiratory failure—either Abbreviations manifested as severe hypoxemia or respiratory acido- ARDS Acute respiratory distress syndrome sis—represents a non-negligible cause of death and mor- BAL Bronchoalveolar lavage CFU Colony-forming unit bidity in ARDS [46, 47]. Given the extreme oxygenation CVP Central venous pressure and ventilation disorders found in the group of pigs with DP Driving pressure VILI, maintaining protective ventilation was challenging EVLW Extravascular lung water FiO2 Fraction of inspired oxygen after the ARDS diagnosis (model development period). ICU Intensive care unit This is revealed by an increase in mechanical power LV dPmax Maximal left ventricular pressure rise throughout all the experiment which, consequently, MAP Mean arterial pressure MDR Multidrug resistance might have dramatically diminished the chances of pul- OA Oleic acid monary recovery in the group with VILI. Our results, PaO2 Partial pressure of oxygen in arterial blood therefore, suggest that a high pulmonary strain might PAOP Pulmonary artery occluded pressure Barbeta et al. Critical Care (2023) 27:239 Page 13 of 14 PaCO2 Partial pressure of carbon dioxide in arterial blood Institute, Hospital Clinic of Barcelona, Villarroel st. 170, 08036 Barcelona, Spain. 11 PC Pressure control Department of Anesthesia and Intensive Care, Fondazione IRCCS Ca’Granda PEEP Positive End-expiratory pressure Ospedale Maggiore Policlinico, Milan, Italy. 12 Cardiology Department, Institute RR Respiratory rate Clinic Cardiovascular (ICCV), Hospital Clinic, Barcelona, Spain. 13 Department VC Volume control of Pathology, Hospital Clinic, Barcelona, Spain. 14 Critical Care Center, Parc VDI Vasopressor dependency index Taulí Hospital Universitari, Institut d’Investigació i Innovació Parc Taulí (I3PT), VILI Ventilatory-induced lung injury Universitat Autònoma de Barcelona, Sabadell, Spain. 15 Universitat Pompeu ZEEP Zero end-expiratory pressure Fabra, Barcelona, Spain. Received: 20 February 2023 Accepted: 30 May 2023 Supplementary Information The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s13054-​023-​04512-8. References Additional file 1. Additional methods, tables and figures. 1. Ranieri VM, Rubenfeld GD, Thompson BT, Ferguson ND, Caldwell E, Fan E, Camporota L, Slutsky AS. Acute respiratory distress syndrome: the Berlin Acknowledgements definition. JAMA. 2012;307(23):2526–33. We would like to thank Cristina Miralles who included and processed all of the 2. Kolobow T, Moretti MP, Fumagalli R, Mascheroni D, Prato P, Chen V, Joris histological samples. We would also like to thank Anthony Armenta for review‑ M. Severe impairment in lung function induced by high peak airway ing the English language and syntax used throughout the manuscript. pressure during mechanical ventilation. An experimental study. Am Rev Respir Dis. 1987;135(2):312–5. Author contributions 3. Broccard A, Shapiro RS, Schmitz LL, Adams AB, Nahum A, Marini JJ. Prone AM, EB, JB and AT designed the study; EB, MA, JB, HY, MY, GT, PDG, SN, AM, CRV, positioning attenuates and redistributes ventilator-induced lung injury in RC, DB, AM, KK, NV, MR, MCR and PF performed the experiments; GF, DM and dogs. Crit Care Med. 2000;28(2):295–303. MA analyzed and interpreted data; LFB, RMA, AA, CF and MF critically revised 4. Protti A, Andreis DT, Monti M, Santini A, Sparacino CC, Langer T, Votta E, the design of the study and the manuscript. All authors read and approved Gatti S, Lombardi L, Leopardi O, Masson S, Cressoni M, Gattinoni L. Lung the final manuscript. stress and strain during mechanical ventilation: any difference between statics and dynamics? Crit Care Med. 2013;41(4):1046–55. Funding 5. Cressoni M, Gotti M, Chiurazzi C, Massari D, Algieri I, Amini M, Cammaroto MF received a grant from the Fondo de Investigación Sanitaria (PI18/00974), A, Brioni M, Montaruli C, Nikolla K, Guanziroli M, Dondossola D, Gatti Instituto de Salud Carlos III, co-funded by the European Union, and a grant S, Valerio V, Vergani GL, Pugni P, Cadringher P, Gagliano N, Gattinoni L. from SEPAR 2018. EB has received financial support from the Instituto de Mechanical power and development of ventilator-induced lung injury. Salud Carlos III (Rio Hortega 2019: CM19/00133), co-funded by European Anesthesiology. 2016;124(5):1100–8. Social Fund (ESF)/ “Investing in your future”, and the "Beca Becario" by SOCAP. 6. 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