The evolution of isolated bilateral lung contusion from blunt chest trauma in rats: cellular and cytokine responses

Abstract

Lung contusion is the leading cause of death from blunt thoracic trauma in adults, but its mechanistic pathophysiology remains unclear. This study uses a recently developed rat model to investigate the evolution of inflammation and injury in isolated lung contusion. Bilateral lung contusion with minimal cardiac trauma was induced in 54 anesthetized rats by dropping a 0.3-kg hollow cylindrical weight onto a precordial shield (impact energy, 2.45 Joules). Arterial oxygenation, pressure-volume (P-V) mechanics, histology, and levels of erythrocytes, leukocytes, albumin, and inflammatory mediators in bronchoalveolar lavage (BAL) were assessed at 8 min, at 4, 12, 24, and 48 h, and at 7 days after injury. The role of neutrophils in the evolution of inflammatory injury was also specifically studied by depleting these cells with intravenous vinblastine before lung contusion. Arterial oxygenation was severely reduced at 8 min to 24 h postcontusion, but became almost normal by 48 h. Levels of erythrocytes, leukocytes, and albumin in BAL were increased at <or=24 h, and returned toward normal by 48 h. Deficits in P-V mechanics were most apparent at 24 h postcontusion. Levels of macrophage inflammatory polypeptide-2, cytokine-induced neutrophil chemoattractant-1, and interleukin 6 in BAL peaked at 24 h, whereas monocyte chemoattractant protein-1 and interleukin 1beta peaked at 24 to 48 h postcontusion. Histology showed early hemorrhagic injury (8 min-12 h), with neutrophilic infiltration at 24 h and areas of bronchiolitis obliterans organizing pneumonia-associated fibrosis at 7 days. Vinblastine-treated neutropenic rats had significantly reduced lung injury based on total lung volume at 4 h and on BAL albumin levels at 24 h postcontusion. Inflammatory injury from isolated bilateral lung contusion in rats is most severe in the acute period (8 min-24 h) after initial blunt trauma, and includes a component of neutrophil-dependent pathology.

Fig. 1. Histopathology of tissue injury in rats with lung contusion

(A) Hematoxylin and eosin-stained lung tissue section at 48 h (10×) showing neutrophils and fluid in the air spaces and thickening of the alveolar lining. (B) Masson's trichrome staining of lung tissue at 7 days postcontusion (40×). Alveolar injury has largely resolved, with repair accompanied by increased collagen deposition around respiratory bronchioles and evidence of fibroblastic plugs (features similar to BOOP, arrowhead). (C) Hematoxylin and eosin-stained sections of the left atrium at 48 h postinjury (10×). Hemorrhagic congestion is present but cardiac muscle is not significantly disrupted. No histological evidence of significant trauma to cardiac muscle was observed at any time point studied (8 min-7 days). See text for details.

Fig. 2. Arterial oxygenation in rats with evolving bilateral lung contusion injury

Arterial oxygenation was assessed as the ratio of PaO₂/FiO₂ (mmHg, mean ± SEM) in aortic blood after a 5-min period of breathing 98% oxygen. Blood samples were drawn just before sacrifice at 8 min to 168 h (7 days) after induction of lung contusion from a falling weight with a chest impact energy of 2.45 J. Numbers of rats were n = 9 at each time point except 12 h (n = 7) and 7 days (n = 8). The PaO₂/FiO₂ ratio in uninjured control rats was 452 ± 14 mmHg (indicated by the dotted line, n = 9). *P < 0.001 compared with uninjured controls.

Fig. 3. Albumin concentrations in BAL from rats with evolving lung contusion injury

Albumin levels (μg/ml, mean ± SEM) were measured by ELISA in BAL fluid at the times shown. Numbers of rats were n = 9 at each time point except at 12 h (n = 7) and 7 days (n = 8). The albumin concentration in BAL from uninjured control rats was 25.3 ± 2.6 μg/mL (indicated by the dotted line, n = 9). *P < 0.001 compared with uninjured controls. See text for details.

Fig. 4. Blood cell numbers in BAL from rats with evolving lung contusion injury

(A) Erythrocytes; (B) total leukocytes (WBC); (C) neutrophils. Data are shown at absolute cell numbers (mean ± SEM), with other details as in the legends to Figures 2 and 3. Cell numbers in BAL from uninjured control rats (dotted lines, n = 9) were red blood cells (7.3 ± 1.4 × 10⁵), total WBC (8.8 ± 1.2 × 10⁵), and neutrophils (9.2 ± 8.5 × 10⁴). See text for details. *P < 0.001 compare with uninjured controls.

Fig. 5. Concentrations of chemokines in BAL from rats with evolving lung contusion injury

(A) MIP-2 (pg/mL); (B) CINC-1 (pg/mL); (C) MCP-1 (pg/mL). Chemokine concentrations were measured by ELISA, with other details the same as in the legends to Figures 2 and 3. Chemokine values for uninjured control rats (dotted lines, n = 9) were CINC-1 (13.1 ± 1.5 pg/mL), MIP-2 (undetectable), and MCP-1 (28.0 ± 3.0 pg/mL). *P < 0.001 compared with uninjured controls. See text for details.

Fig. 6. Levels of cytokines IL-6 and IL-1β in BAL from rats after lung contusion

(A) IL-6; (B) IL-1β. Cytokine levels (pg/mL) were measured by ELISA, and other details are as in the legends to Figures 2 and 3. Levels of IL-6 and IL-1β in uninjured controls (dotted lines, n = 9) were at or below the lower limit of assay detection. *P < 0.001 compared with uninjured controls.

Fig. 7. Quasistatic P-V inflation and deflation curves from rats after lung contusion

P-V curves were measured at 8 min, and at 4, 12, 24, 48, and 168 h (7 days) after lung contusion compared with uninjured control rats. Pressures are in centimeters per water and volumes are in milliliters per kilogram of body weight. Points along each curve represent the mean ± SEM for nine animals at each time point except for 12 h (n = 8) and 7 days (n = 8). Abnormalities in P-V mechanics compared with uninjured control rats are most apparent at 24 h postcontusion. See text for details.

Fig. 8. Effects of neutrophil depletion on the severity of lung contusion injury

(A) Albumin in BAL; (B) PaO₂/FiO₂ (after breathing 98% O₂ for 5 min); (C) quasistatic P-V curves. Neutrophils were depleted by intravenous Vinblastine, and injury was subsequently evaluated at 4 and 24 h after contusion (see “Materials and Methods”). Neutrophil depletion significantly reduced lung injury based on BAL albumin at 24 h and total lung volume at 4 h as described in the text. Data are mean ± SEM for n = 6 at each time point. ^#P <0.05, *P < 0.001 compared with undepleted, contused rats.