Comprehensive Multi-Variable Controlled Examination of Sex Differences in Acute Lung Injury: Respiratory, Histological, and Molecular Changes in a Bleomycin Rat Model

Introduction: Acute lung injury (ALI) is characterized by rapid-onset respiratory failure stemming from widespread inflammation and increased pulmonary vascular permeability. Despite advances in critical care, ALI presents significant challenges with high mortality rates. Sex-based differences have been observed in ALI incidence and outcomes, with males often showing a more severe phenotype. While previous research has primarily focused on sex differences in the later fibrotic phase of ALI, this study explores sex differences during the acute/exudative phase and its progression. Using a rigorously controlled rat model of bleomycin-induced ALI, we examine the dynamics of sex-specific responses and offer a detailed characterization of how sex influences the ALI phenotype. Methods: We thoroughly assessed sex differences in a bleomycin ALI rat model, controlling for body weight, age, drug concentration, and volume to isolate sex-specific effects. Seven days post-intratracheal bleomycin (2.5 mg/kg), we investigated alterations in respiratory mechanics and inflammation using whole-body plethysmography (WBP), invasive resistance/compliance (RC) measurements, arterial blood gas analysis, cytokine profiling, and immunohistochemistry (IHC). Statistical significance was determined by two-way ANOVA (P < 0.05). Results: Significant sex-specific differences were observed one week post-bleomycin-induced ALI concerning WBP-derived respiratory parameters. Males displayed higher respiratory rates (P < 0.0001; N = 26, 38) and mid-expiratory volume (EF50) (P = 0.0019). Airway resistance (RI) and dynamic compliance (Cdyn) were both significantly altered in male and female animals at one week post-bleomycin-induced ALI compared to sham (Male RI: P = 0.0023, N = 5, 5; Male Cdyn: P < 0.0001; Female RI: P < 0.0001; Female Cdyn: P = 0.0016), but there was no significant difference between the sexes. Males showed a trend toward lower partial pressure of arterial oxygen (PaO2) compared to females (P = 0.0793; N = 6, 8) and significantly lower blood oxygen saturation (sO2%) versus females (P = 0.0390). Lung infiltration of CD68 + cells was elevated in both sexes post-bleomycin (P < 0.001; N = 5, 5), with females being significantly higher than males (P = 0.001). Males had increased CD3 + cells post-bleomycin vs sham (P = 0.001), whereas females did not (P = 0.10), resulting in lower CD3 + cells in females compared to males post-ALI (P = 0.031). MPO + cells increased in males and females following bleomycin ALI (P < 0.05). Collagen deposition was significantly elevated in females compared to males following bleomycin ALI (P = 0.0463). Lung tissue levels of IL-1b were significantly higher in males vs females post-bleomycin (P = 0.0068; N = 5, 5). Females had higher IL-10 levels than males in sham and bleomycin groups (P < 0.01). Additionally, TNF-α (P = 0.0036) and IL-6 (P = 0.0447) were elevated only in males post-bleomycin. Conclusions: By controlling for body weight, age, drug concentration, and volume, our study identifies significant sex-specific differences in respiratory function, immune cell infiltration, collagen deposition, and cytokine profiles within a bleomycin-induced ALI model. These findings enhance our understanding of sex as a biological variable in ALI and underscore the necessity for sex-specific therapeutic approaches. This study was supported by NIH grant R01 HL-152160-01 and in part, by NIH grants R01 HL171602-01, HL-169205-01, HL126796-01, HL172029-01A1 and R21 HL170127-01 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.