Discussion

In the present study, we found that the laryngoscopic endotracheal injection (LEI) method was able to establish a SWD-ALI mouse model with moderate mortality, controllable injury severity, and good clinical simulation; micro-CT can accurately identify the lesion area and evaluate the severity of SWD-ALI in the early stage (as early as 0.5 h post-injury), and the quantitative indices of micro-CT are significantly correlated with pathological injury scores, which can provide a reliable basis for early clinical treatment guidance. The sample size and experimental design of this study are consistent with our previously published SWD-ALI research [17], with core results (inflammatory factor upregulation, lung coefficient elevation, micro-CT imaging changes) successfully reproduced, further supporting the validity and reproducibility of the experimental findings. SWD-ALI is a complex pathological process caused by direct hypertonic injury of seawater to the lung tissue and subsequent severe inflammatory response [7], with an extremely high mortality rate due to the lack of effective early assessment and treatment methods [6]. We systematically explored the effects of various modeling approaches, artificial seawater doses and injury time on SWD-ALI induction in mice, and clarified the value of micro-CT in early injury assessment, which provides an important experimental basis for in-depth study of SWD-ALI pathogenesis and clinical translation.

Based on previous studies, we selected three commonly used methods to prepare ALI models and innovatively applied the LEI method for comparison [7], [8], [9, 20]. Compared with the control mice, the mice of all four modeling methods showed different degrees of choking, shortness of breath, white foam overflow, and abnormalities in respiratory rate, activity, and diet. Among them, the ND method was easy to operate, but the performance of mice was the lightest, and the dose of seawater inhalation was not controllable, leading to poor model stability. The DD method could simulate the real drowning situation well, but the mortality rate was extremely high (85.7 % within 24 h), which not only leads to small effective sample size but also increases animal suffering, which is violated the 3R principles the 3R principle. The NEI method has a relatively stable injury effect but requires invasive neck incision, which causes severe systemic inflammatory response and long anesthesia time, leading to secondary injury and inconsistent with the clinical characteristics of seawater drowning. In this study, we proposed the LEI method for the first time to prepare SWD-ALI models, which highly simulated the real drowning scenarios (seawater inhalation via the upper respiratory tract), avoided the neck incision, and the operation was faster and easier by comparison (Table 1). In addition, this method uses isoflurane inhalation anesthesia with short duration and rapid awakening, avoiding the effect of prolonged anesthesia on mice, and the moderate mortality rate (28.6 % within 24 h) ensures sufficient effective sample size for subsequent mechanistic and therapeutic studies – ” a key advantage for studies of pathologic mechanisms and for use with genetically altered animals”.

Methods | Operation steps | Advantages | Disadvantages
Direct drowning (DD) | 1. Place the mouse in a plastic container filled with seawater, allowing the mouse to be immersed in seawater; 2. Immerse for 25 s, then pat dry and observe | Simulates a real sense of suffocation, relatively quick and convenient | Significant individual differences in mouse, extremely high mortality rate
Nasal drip (ND) | 1. Inject a certain amount of seawater into the mouse’s nasal cavity; 2. Hang the mouse for 15 s and observe the drowning process | Convenient operation | Relatively poor reliability of inhaled dose, some may be swallowed
Neck incision endotracheal injections (NEI) | 1. Perform anterior tracheotomy; 2. Inject seawater into the incision; 3. Hang for 15 s and observe the drowning process | Highly realistic, relatively stable | Slightly difficult operation, slightly longer anesthesia time, higher systemic inflammatory response
Oral laryngoscope endotracheal injection (LEI) | 1. Lift the epiglottis under a laryngoscope and observe the opening and closing of the glottis with breathing; 2. Inject a certain amount of seawater with a microliter syringe; 3. Hang for 15 s, and observe the drowning process | Highly realistic, requires short anesthesia time, relatively stable, moderate mortality | Slightly difficult operation, repeated operations may cause passive injury to mouse

To verify the compliance with the criteria after modeling, we based our criteria on the latest animal ALI model proposed by the official workshop report of the American Thoracic Society [19], which is featured in four aspects: tissue damage, alteration of alveolar-capillary barrier, inflammatory response, and physiological dysfunction. In this study, the lungs of the model group revealed obvious tissue damage including alveolar cell injury, alveolar septal thickening/edema, intra-alveolar hemorrhage, and alveolar structure destruction, with clear evidence of histological damage [21], [22], [23]. The marked elevation in lung coefficient and BALF total protein concentration suggested impaired alveolar-capillary barrier integrity [24], [25], [26]. The percentage of neutrophils in BALF and peripheral blood of mice was significantly elevated, revealing a severe inflammatory response after seawater drowning [7], 27], 28]. Clinical symptoms such as cyanosis, bradypnea and reduced activity in SWD-ALI mice suggested obvious physiological dysfunction [5], 18], 29], 30]. In summary, SWD-ALI mouse model prepared by LEI fully conforms to the international standard of animal ALI models, with good validity and clinical relevance.

Micro-CT is a specialized type of CT that is commonly used for imaging examinations of small animals. This study also used CT, a common clinical imaging examination [31], 32], to evaluate early lung injury in SWD-ALI mice, and found that it could quickly identify and assess the severity of injury in the early stage. Marked exudative changes were observed as early as 0.5 h after injury, and exudation peaked at 2 h, with significantly increased average gray value and percentage of damaged area in lung CT, and even “white lung” like changes in severe cases – consistent with the early imaging characteristics of clinical ALI/ARDS [18]. We further validated the accuracy of micro-CT by Pearson correlation analysis, and found a significant positive correlation between micro-CT damaged area percentage and pathological total injury score (r=0.64, p<0.001), which verified that micro-CT can accurately quantify the severity of SWD-ALI.

However, the results of pathological and molecular tests of lung tissue injury were not consistent with imaging results, with the pathological peak lagging behind the imaging peak (imaging peak at 2 h, pathological peak at 6 h). This experiment suggested that the early stage of SWD-ALI in mice is dominated by the exudation of small molecules such as water and electrolytes, due to hypertonic stress, which leads to a sharp increase in imaging damage (CT is highly sensitive to liquid exudation [32]). With the aggravation of the disease, the exudate is gradually transformed into proteins and inflammatory cells, while the water is reabsorbed by the body, so the imaging results suggest an improvement, but the pathological results show that inflammatory cells and other substances are continuously exuding into the alveolar space, leading to the continuous aggravation of pathological injury. Most SWD-ALI mice were significantly aggravated or died in the early stage, so early treatment is the key to affect the prognosis [6]. Micro-CT can quickly identify SWD-ALI in the early stage, and with the clinical application of mobile CT detection means, timely CT detection can directly guide early treatment options (such as reducing exudates, removing airway secretions, or emphasizing lung ventilation) and affect the early mortality and prognosis of drowning patients.

The artificial seawater used in this study was formulated based on the ionic composition of seawater along the southeast coast of China, with a total salinity of 35 g/L (consistent with the average global ocean salinity of 33–37 g/L) [3]. Regional differences in seawater composition are mainly reflected in the slight variation of major cation/anion concentrations (e.g., higher Mg2+/Ca2+ in the Pacific Ocean vs. the Atlantic Ocean [3]) and the content of trace elements (e.g., iodine, strontium) and organic matter, while the core hypertonic components (NaCl, Mg2+, Ca2+ salts) that induce SWD-ALI remain consistent across global oceans. The hypertonic stress caused by 3.5 % NaCl is the primary driver of alveolar capillary fluid shift and pulmonary edema in SWD-ALI [7], and the Mg2+/Ca2+ in the artificial seawater mimics the pro-inflammatory and endothelial damage effects of natural seawater [16] – these core pathogenic factors are retained in our formulation. Although seawater collected from different regions may exhibit slight variations in trace element composition, previous studies have confirmed that trace components have no significant effect on the overall pathological process of SWD-ALI, and the hypertonic-induced ALI is the dominant pathological change [7]. Therefore, our LEI-based SWD-ALI model, established with southeast China coastal seawater components, can reflect the core pathological characteristics of SWD-ALI in global marine regions. For studies focusing on regional seawater-specific injury mechanisms (e.g., high trace metal content in coastal industrial areas), the artificial seawater formulation can be slightly adjusted based on local seawater ion detection data, and the LEI modeling method remains applicable with good reproducibility.

The LEI model established in this study has good translational relevance to clinical ALI, as it mimics the clinical characteristics of seawater inhalation via the upper respiratory tract, avoids invasive surgery, and the injury severity and recovery course are consistent with clinical patients [5]. This model can be used to study the core pathogenic mechanisms of SWD-ALI (e.g., hypertonic stress-induced ferroptosis [16], inflammatory response [7]) and screen potential therapeutic drugs. Bora SE et al. [33]. found that octreotide has a short-term protective effect on sepsis-induced lung injury in rats by inhibiting inflammatory response and reducing lung vascular permeability, which provides an important reference for the treatment of SWD-ALI. Since SWD-ALI and sepsis-induced lung injury share similar pathological processes (severe inflammatory response and alveolar-capillary barrier damage [28]), octreotide may also have a protective effect on SWD-ALI, which is worthy of further study using the LEI model. In addition, our study found that micro-CT can accurately evaluate early lung injury in SWD-ALI mice, which is consistent with the clinical application of CT in the diagnosis of drowning-induced lung injury [32], and provides an experimental basis for the clinical use of mobile CT for early bedside assessment of SWD-ALI patients.

Long-term observations in this study showed that model mice exhibited bronchiolar epithelial shedding (3 days), blunt rupture (7 days), and subpleural alveolar fusion (28 days) post-injury, which are potential pathological changes leading to chronic lung injury [22]. It is not clear whether these changes are related to bronchiolar remodeling [34], 35], and further studies are needed to determine whether SWD-ALI mice develop chronic changes such as emphysema [36], 37]. Therefore, we need to adopt effective evaluation tools (such as micro-CT) during the golden period of rescue to guide targeted therapy, which would not only reduce the severity of lung injury in the acute phase, but also reduce the occurrence of chronic lung changes and improve the long-term prognosis of SWD-ALI patients.