Acute Respiratory distress syndrome (ARDS) is recognized as one of the most severe manifestations of organ dysfunction following ischemia-reperfusion injury (IRI) [1]. ARDS is defined as acute hypoxemic respiratory failure with bilateral pulmonary infiltrates on chest X-ray or computed tomography (CT) of a non-cardiac origin, and a Horowitz index (PaO₂/FiO₂; the ratio of partial pressure of oxygen in arterial blood to the fraction of inspiratory oxygen concentration) for lung function of less than 300 mmHg. The incidence of ARDS in the United States is estimated to be between 62.2 and 78.9 cases per 100,000, among which 75% is moderate or severe. The overall mortality was evaluated at 43% [2,3]. Although intensively investigated for years, the fundamental mechanisms contributing to ARDS have not been completely understood [3].

Acute limb ischemia (ALI) is defined as a sudden decrease in limb arterial perfusion that jeopardizes limb viability [4]. It is a common vascular emergency, with an incidence of 22–26 per 100,000 patients per year [5]. One of the consequences of revascularization is IRI, defined as worsening of cellular dysfunction and death following restoration of the blood flow to previously ischemic tissues. The mechanism behind the phenomenon is mostly related to dysregulation of metabolic pathways during ischemia, accumulation of metabolites and generation of reactive oxygen species (ROS) with reintroduction of oxygen during reperfusion [6]. This is particularly important in large-scale combat operations, such as the war in Ukraine, where, due to loss of air supremacy, the tourniquet time is significantly prolonged [7]. More notably though, this process is not limited to the affected area (for instance, the limb), but may lead to extensive systemic insult with multiple organs, including ARDS [8]. Alongside its high mortality, ARDS is very costly to manage, as these patients require prolonged intensive care hospitalization, and there are limited therapeutic modalities available to combat this condition.

This has necessitated the creation of a reliable, reproducible and cost-effective animal model for further investigations and translational research to develop new therapies for ARDS. Commonly described animal models of ARDS were established by surfactant washout, oleic acid (OA) intravenous injection or lipopolysaccharide (LPS) injection. These, however, differ from the actual pathomechanism of lung insult [9]. A physiologically comparable model was implemented by inducing pulmonary ischemia by clamping the pulmonary artery, the bronchial artery, and the bronchus in the affected lung, but this model requires a rather complex and invasive surgical approach [1].

In the current work we propose a swine model of ARDS secondary to ALI. In this setting, in a time-dependent manner, the corresponding reperfusion injury releases a systemic burst of pro-inflammatory mediators and ROS, precipitating non-cardiogenic pulmonary edema. This approach offers a practical, physiological, less invasive and more accessible alternative to traditional ARDS models, which are often complex and costly. Facilitating this model in the medical field could enhance the understanding of ARDS mechanisms and allow preclinical studies of novel therapies with better translatability.

Two female Yorkshire Swine were enrolled in the study. Instrumentation was successfully performed. Baseline angiogram was obtained depicting normal swine vasculature (Figure 1a). Baseline ABG measurements were obtained, with a lactate of 1.8–2.1 mmol/L and potassium of 4.2–4.5 mmol/L. Baseline FiO₂ was 59%, blood pressure 130/80 mmHg, heart rate 100 bpm, SpO₂ 99%, respiratory rate 16 and end-tidal CO₂ 30 mmHg. The baseline Horowitz index was calculated to be 503.9 and 510 mmHg, for each animal, respectively. Subsequently, EIA and MSA balloon occlusion was achieved, and confirmed with angiogram images (Figure 1b,c).

During the nine-hour occlusion phase, no significant events occurred, and the animals remained hemodynamically stable. Upon reperfusion, blood flow was restored (Figure 1d). The lactate was 1.9–2.0 mmol/L, the potassium 4.8–4.9 mmol/L and the Horowitz index 340–349.5 mmHg.

During the reperfusion-induced ARDS phase, oxygen saturation decreased to 92%, necessitating ventilator adjustments, including increasing FiO₂ to 100%, transitioning to pressure-controlled ventilation, raising PEEP while maintaining a plateau pressure below 30 cmH₂O, and implementing an inverse inspiration-to-expiration ratio greater than 1:1.

After six hours of reperfusion, lung imaging including ultrasound and CT was obtained depicting B-lines and dense consolidations, respectively (Figures 2 and 3). Lactate was recorded at 4.9–6.4 mmol/L and potassium was 5.3–6.1 mmol/L. The Horowitz index was at 110 mmHg, indicating moderately severe lung injury. The graph depicting Horowitz index changes is illustrated below (Figure 4).

At the end of the study, the lungs were inspected and post-mortem lung tissue were obtained. The tissue was stained with H&E, which revealed diffuse alveolar damage, inflammatory infiltrate and edema, consistent with early ARDS (Figure 5).

This study demonstrates a swine model of ARDS, implementing IRI by interrupting the blood supply to the hindlimb (Figure 6). This model has been shown to be easily performed, cost-effective and replicable. Furthermore, it serves as a valuable tool for studying ARDS, as it mimics physiological and sterile conditions, like those seen in a clinical setting. We have used a porcine model, as the anatomy, physiology, immune system and metabolism are more similar to humans compared to rodents, and the size allows for a broader spectrum of therapeutic testing [10].

Traditional animal ARDS models are important tools used by physicians and researchers to study the mechanisms and potential treatment options for this condition. While they provide valuable insights into the pathophysiological processes, none of them perfectly replicates all aspects of human ARDS [8]. The most used models are the lavage (LAV), OA and LPS models [6,9]. The LAV model, induced by repeatedly flushing the lungs with saline, primarily focuses on surfactant depletion and its consequences, including alveolar collapse [9]. This model is useful for studying the impact of different ventilation strategies and evaluating the potential therapies aimed at restoring surfactant friction. However, the LAV model does not inherently replicate the robust inflammatory response and permeability changes seen in ARDS [8,11]. In the OA model, OA is injected intravenously, leading to direct damage of the capillary endothelium and triggering the cascade of inflammatory response leading to ARDS. However, the main limitation lies in the distinct underlying cause, as ARDS very rarely results from fat embolism, the primary injury simulated in this model [8,12].

The third most popular model is created by injecting LPS, a component of bacterial cell walls and the aim is to replicate the pathophysiology of sepsis-induced ARDS, one of the most common ARDS etiologies [8,9]. LPS triggers a widespread inflammatory response, leading to lung injury, making this model valuable for exploring the inflammatory processes in ARDS and evaluating potential treatment targets. However, the LPS model often produces milder alveolar inflammation and permeability changes compared to those in ARDS. Additionally, the LPS model provides an incomplete representation of the effects of live bacteria, as it lacks direct cellular damage [8,9,13,14]. A slightly different approach was described where, instead of injecting LPS, mitochondrial damage-associated molecular patterns (DAMPs) were isolated and injected, replicating an inflammatory response comparable in severity to that seen in the LPS model [15]. It has also been shown that succinate, a marker of shock, global hypoxia and failure in energy production, can contribute to endotheliopathy in the gut, causing the release of LPS that further results in ARDS [16,17].

Pulmonary IRI models stimulate ARDS scenarios where lung injury arises from blood flow restoration after a period of oxygen deprivation, as seen in lung transplants or thoracoabdominal aortic surgery. Pulmonary IRI involves clamping the pulmonary artery or the hilum of the lung for a defined period, followed by the restoration of blood flow. While this model helps examine injury mechanisms, it requires precise surgical skills and is time-consuming [6,18,19].

Our current approach, opting for an EIA and MSA occlusion IRI model over a pulmonary IRI model to induce ARDS, comes with unique advantages and disadvantages. Some of the former include the ability to investigate the systemic inflammatory cascade triggered by peripheral ischemia, mirroring human ARDS cases that occur with distal injuries. Because it mimics real-life pathophysiology, it might cause multi-system organ dysfunction from reperfusion injury in addition to ARDS, which contrasts with models that only target lung dysfunction [9]. Unlike pulmonary IRI, non-pulmonary IRI does not directly impair lung function, minimizing hypoxemia and hemodynamic instability within the lungs. This method offers a sterile approach, without the necessity of introducing systemic bacteria or DAMPs [15,20].

This model was designed with a focus on clinically relevant components, which is why the evaluation of biochemical markers was not included in the scope of this project. The animals presented symptoms typical of a lung injury, such as impaired gas exchange demonstrated by a dramatic decrease in the Horowitz index, but also confirmed with CT, ultrasound and histology. The sample size was limited to two animals, although the results remain promising. While combat-related trauma often involves polytrauma with extensive soft tissue and skeletal injuries, our model isolates IRI to establish a controlled platform for studying its direct role in ARDS pathogenesis. We do, however, acknowledge this limitation and see our model as complementary to more complex trauma models, providing foundational insights into isolated IRI contributions to ARDS. Further replication of this model in future experiments will help to validate the technique and enhance future experimental use. Extending the duration of critical care periods will facilitate a more comprehensive progression of ARDS due to systemic reperfusion injury, providing greater insight into its pathophysiology and enhancing the model’s relevance for future experimental applications.

Within the constraints of these limitations, this methodology for creating an ARDS model by inducing non-pulmonary IRI demonstrates promising results for further study. This model can be used in future studies to examine the pathophysiology and pathomechanism of ARDS.