Results

LEI method can stably establish a mouse SWD-ALI model

Four methods were used to establish the C57BL/6 mouse SWD-ALI model, with the post-modeling status of mice shown in Figure 1A (n=7 per group). Mice in the control group (CON) were more active, with without dyspnea, choking and oronasal secretion overflow from the mouth and nose. The mice in the DD group showed severe dyspnea, and there were a lot of white foam discharge overflow, accompanied by obvious hypothermia and cyanosis. Mice in the ND group showed partial dyspnea, with no secretion overflow from the mouth and nose. Mice in the LEI group and the NEI group had obvious choking and dyspnea, and there were clear white foamy secretion overflowing from the mouth and nose, and mild to moderate cyanosis. In the transoral laryngoscopy, the vocal folds of the mice were seen to be white and inverted triangular (Figure 1B), opening and closing synchronously with respiration. Trypan‑blue staining in the LEI group precisely identified the injection site (Figure 1C). CON mice underwent the same isoflurane anesthesia procedure (without seawater injection) for micro-CT and lung coefficient detection, confirming that isoflurane anesthesia had no adverse effects on murine lungs and excluding anesthesia as a confounding factor.

Figure 1:: Different methods of preparation of SWD-ALI. Notes: A state of SWD-ALI mice prepared by different methods (the red line area is secretions); B laryngoscopic picture of the vocal cords in mice; C localization of trypan blue by LEI method. n=7 per group.

Model injury was verified through pathological examination and survival analysis (Figure 2A and B, n=7 per group). Lung tissues of CON group mice were pale pink, and there was no hemorrhage or edema in H&E staining. The lung tissues of DD group showed severe swelling and congestion changes, and H&E staining showed localized lung atelectasis, hemorrhage, edema, inflammatory cell exudation, with obvious alveolar rupture in some areas. The lung tissues of ND group showed local redness and swelling, and the pathological manifestations were focal relatively mild edema with less hemorrhage. NEI and LEI group lung tissues exhibited varying degrees of swelling and hemorrhage, H&E staining showed inflammatory cell exudation and pulmonary hyaline membrane formation – ”key pathological features of clinical SWD-ALI” [7]. Survival analysis of different modeling methods (Figure 2C, n=7 per group) suggested that five mice died in the DD group at 4 h, and one died at 20 h. There were no deaths in the ND group within 24 h. Two mice died in the NEI group at 4 h, one died in the LEI group at 4 h and one at 8 h. Differences in survival rates among the four groups were statistically significant (p<0.05), with the LEI group exhibiting a moderate mortality rate that avoided the excessively high mortality of the DD group and mild injury of the ND group, confirming model stability.

Figure 2:: Stabilization of modeling injury in a SWD-ALI mice model prepared by LEI. Notes: A representative lung tissues sections from each group of mice; B representative H&E-stained images from each group of mice; C 24 h survival analysis modeled by different methods. n=7 per group, *p<0.05 (log-rank test).

LEI of 4 mL/kg artificial seawater induced stable moderate ALI in mice

Based on the above results, the LEI method was selected for further exploration of different artificial seawater doses, with mice in different dose groups showing distinct dose-dependent pathological and clinical states (n=7 per group). Mice in the 2 mL/kg group were the most active, with the mildest respiratory distress and no obvious secretion from the mouth and nose. Mice in 8 mL/kg group had obvious choking and respiratory distress, with more white foamy secretion overflowing from the mouths and noses. The cyanosis was visible, and some of them showed hypothermia and even died. The performance of 4 mL/kg mice was between two groups, with moderate respiratory distress and foamy secretions, consistent with stable moderate ALI.

Lung tissues (Figure 3A and B, n=7 per group) of 2 mL/kg group mice showed local scattered redness and swelling, and the pathological manifestation showed focal and relatively mild edema with less hemorrhage. Lung tissues of 4 mL/kg group mice exhibited obvious alveolar and interstitial swelling, hemorrhage, and inflammatory cell infiltration – consistent with moderate ALI and stable injury characteristics suitable for subsequent experimental analysis. In contrast, 8 mL/kg group mice had swollen and congested lungs, H&E staining showed obvious atelectasis, pulmonary hyaline membrane formation, alveolar and interstitial inflammation and bleeding, and severe pulmonary edema. Survival curves of mice exposed to different doses of seawater inhalation were analyzed (Figure 3C, n=7 per group). No mortality was observed in the 2 mL/kg group during the observation period. In the 4 mL/kg group, 2 mice died within 4 h. Three mice in 8 mL/kg group died within 4 h period, one mouse died within 8 h, and the survival rate in the 24 h period was lower than 50 %. Differences in survival rate among the three groups were statistically significant (p<0.05), confirming the dose-dependent effect of artificial seawater on SWD-ALI induction via LEI.

Figure 3:: The LEI method of artificial seawater 4 mL/kg injury was relatively stable. Notes: A representative lung tissues images from each group of mice; B representative H&E-stained images from each group of mice; C 24 h survival analysis modeled by different doses. n=7 per group, *p<0.05 (log-rank test).

Early lung imaging damage can be easily detected in SWD-ALI mice after LEI artificial seawater

In order to observe the changes of lung injury at different times after seawater inhalation, the clinical manifestations, imaging, pathology, and BALF changes of mice after injury were compared (micro-CT: n=5 per time point; pathology/BALF: n=6 per time point). Firstly, the clinical manifestations of the mice in the 0.5 h and 2 h groups showed white or transparent secretions from the mouth and nose, accompanied by different degrees of choking and coughing, dyspnea, wet crackles, cyanosis, obvious reduction of activity, unkempt hair and lack of luster. Compared with two groups above, the mice in the 6 h group showed obvious reduction of secretions from the mouth and nose, but the dyspnea was more obvious, accompanied by varying degrees of cyanosis, and there was an obvious reduction of activity, reduced feeding, more unkempt hair and lack of luster. Mice in 1d group had no obvious oral and nasal secretions, still had dyspnea, less activity and feeding, unkempt fur and dull luster. Mice in 3d, 7d and 28d groups showed gradual clinical improvement but still had activity-induced dyspnea and poor motor flexibility – a recovery course consistent with clinical SWD-ALI patients [5].

Secondly, Micro-CT imaging was performed to assess lung injury, with similar expiratory phases selected for all mouse lung scans to ensure consistency (Figure 4A, 4C). CON group horizontal CT images showed uniform low-density shadows (gas-filled alveoli) with no exudation. In 0.5 h group had extensive exudative changes in the left lung with reduced gas content and mild exudation in the right lung. The 2 h group exhibited the “most severe exudative changes (imaging peak)”, with significantly aggravated left lung exudation and new small amounts of exudation in the right lung, consistent with early “white lung” changes in clinical ALI/ARDS [18]. The 3 h and 6 h groups showed gradual improvement in lung imaging with progressive absorption of exudate, and the 24 h group had near-normal lung imaging with minimal residual exudation. Damage area ratio and average gray value in horizontal (Figure 4B) and coronal (Figure 4D) CT images for all time points (0.5 h, 2 h, 3 h, 6 h, 24 h) were quantified; both indices were significantly increased in the 2 h group (p<0.05), and gradually decreased at 3 h and 6 h, returning to near normal at 24 h. A significant positive correlation was found between micro-CT damaged area ratio and pathological total injury score (Pearson r=0.64, p<0.001) (Figure 4E), validating the accuracy of micro-CT in quantifying SWD-ALI severity.

Figure 4:: Lung CT enables rapid assessment of SWD-ALI damage at an early stage. Notes: A representative micro-CT horizontal images of different times after modeling in mice (the red line area is the damage area); B damage area ratio and average gray value of horizontal lung CT, n=5, *p<0.05, **p<0.01, ***p<0.001; C representative Micro-CT coronal images of different times after modeling in mice (the red line area is the damage area); D damage area ratio and average gray value of coronal lung CT, n=5, *p<0.05, **p<0.01; E correlation scatter plot of micro-CT damaged area ratio and pathological total injury score (Pearson r=0.64, p<0.001).

Thirdly, the lung tissues (Figure 5A, n=6 per time point) of mice in the 0.5 h group showed obvious edema in the lungs, with relatively little hemorrhage. However, the central part of the lung was bleeding (bright red), and there was still significant edema in the 2 h group. The edema in the lung tissues of mice in the 6 h group was slightly improved, but obvious hemorrhagic manifestations were seen, which were mainly centrally distributed (“pathological peak”). The hemorrhage and edema were partially absorbed in the lung tissues of mice in 1d group. Mice lung tissues of 3d group showed edema was absorbed, but stale hemorrhage was visible, and the damage site was dark red, mainly centrally distributed.

Figure 5:: The SWD-ALI mouse model was prepared by LEI and the damage was obvious at 6 h. Notes: A The representative lung tissue images at different time points after modeling; B representative H&E staining images at different time points after modeling; C compared with the CON group, the total cell counts and total protein concentration in BALF were determined at different time points after modeling, n=6, nsP>0.05, *p<0.05, **p<0.01, ***p<0.001.

Then, the mice inhaled seawater for different periods of time showed different lung pathological changes (Figure 5B, n=6 per time point). The lungs of mice in the 0.5 h group showed local edema, increased inflammatory cells, local alveolar structure disappeared. The lungs of mice in the 2 h group showed part of the interstitial edema, hemorrhage, alveolar collapse, inflammatory cells and erythrocytes had aggregation. The lungs of mice in the 6 h group showed obvious alveolar collapse, disappearance of local alveolar structures, alveolar and interstitial edema, hemorrhage, and severe inflammatory cell exudation. In the 1d group, alveolar and interstitial hemorrhage and edema were slightly improved, the alveoli were partially expanded, and different degrees of inflammatory cells were exudated. In the lungs of mice in the 3d group, some epithelial cells in the small bronchus were detached and a few macrophages were observed. The lung injury was recovered in the 7d group, but the small bronchial blunt rupture was obviously dispersed, and the lumen contents were significantly reduced compared with those in the 3d group. Some of the subpleural lung tissues in the 28d group showed alveolar rupture and hyperventilation. Finally, total protein concentration and total cell number (Figure 5C, n=6 per time point) in BALF were raised post-inhalation seawater, and those in the 6 h group were the highest among the time points measured, consistent with the pathological peak of lung injury.

The SWD-ALI model in mice conformed the criteria for animal ALI

From the above results, it was confirmed that the model mice showed obvious lung histologic damage (n=6 per group). We then examined cell and protein exudates in BALF (Figure 6A) and calculated lung coefficients (Figure 6B) indicating altered permeability of the lung air-blood barrier. It was found that the mice in the model group showed significant air-blood barrier permeability damage (p<0.05). The neutrophil count in BALF was markedly elevated in the model group (p<0.05) (Figure 6C), which represented an enhanced inflammatory response. A significant increase in peripheral blood neutrophils was also observed in the model group (Figure 6D), combined with clinical symptoms such as cyanosis and dyspnea, which were consistent with physiological dysfunction [19]. Therefore, the mouse SWD-ALI model, prepared by LEI of 4 mL/kg artificial seawater after 6 h, fully meets the “four core criteria for animal ALI” proposed by the American Thoracic Society official workshop report [19].

Figure 6:: The LEI method of preparing a mouse SWD-ALI model conforms to the latest standards. Notes: A Total cell counts and total protein concentrations in the BALF; B mouse lung coefficient; C H&E staining images of BALF cells, and quantitative analysis of the NE% in BALF; D quantitative analysis of the NE% in blood. n=6, **p<0.01, ***p<0.001.