Development of chlorine-induced lung injury in the anesthetized, spontaneously breathing pig

Figures & data

Figure 1. Schematic of the chlorine gas exposure system. MFC: mass flow controller.

Table 1. Grades of epithelial injury scoring within trachea and bronchi.

	Loss of cilia & cell degeneration	Epithelial erosion	Ulceration
Normal (0)	None	None	None
Mild (1)	Present	Minimal	None
Moderate (2)	Present	Present	None
Severe (3)	Present	Present	Present

Table 2. Grades of injury scoring within bronchioles.

	Perivascular and peribronchiolar edema/fibrin	Neutrophil infiltration	Cell injury	Lumen debris
Normal (0)	None	None	None	None
Mild (1)	Mild	Few	Cell degeneration	Scant material in lumen
Moderate (2)	Moderate	Moderate	Occasional	Partial filling
		numbers	cell death	of lumen
Severe (3)	Marked	Large numbers	Overt necrosis	Filling of lumen

Table 3. Grades of injury scoring within parenchyma, alveoli and interstitium.

	Congestion	Interstitial / alveolar edema	Interstitial/ Alveolar neutrophils	Extravasated erythrocytes	Consolidation
Normal (0)	Absent	Absent	<1 per hpf	Absent	Absent
Mild (1)	Present	<50%	1–5 per hpf	Present	Absent
Moderate (2)	Present	>50%	>5 per hpf	Present	Absent
Severe (3)	Present	>50%	>5 per hpf	Present	Present

Hpf: high power field.

Figure 2. (A) A representative example of an exposure profile of an animal exposed to chlorine, where an inhaled dose was targeted by titrating the chlorine concentration [Cl₂] to the animal’s minute volume (Strategy 1). (B) A representative example of an exposure profile of an animal exposed to chlorine, where the exposure time was extended and chlorine concentration slowly increased (Strategy 2). (C) A representative example of an exposure profile of an animal exposed to chlorine, where the exposure time was short and chlorine concentration increased quickly (Strategy 3).

Table 4. Dosimetry metrics for Strategies 1, 2 and 3.

	Inhaled dose (mg·kg^−1)	Range (mg·kg^−1)	Mean Ct (mg·min·m^−3)	Mean Exposure Time (min)	Mean C (mg·m^−3)
Strategy 1 (n = 5)	1.81 ± 0.96	0.54 to 3.07	15931.3 ± 5766.0	17.6 ± 3.7	905.2
Strategy 2 (n = 5)	0.48 ± 0.30	0.24 to 0.99	4886.0 ± 3523.9	21.4 ± 4.9	228.3
Strategy 3 (n = 5)	0.28 ± 0.15	0.12 to 0.51	2781.9 ± 1145.4	8.6 ± 3.1	323.5

Inhaled dose is the sum of the product of minute volume and corresponding chlorine concentration throughout the exposure divided by the animal's body weight; mean Ct is derived from the AUC; mean C is calculated by dividing the mean Ct by the mean exposure time. Mean ± SD.

Table 5. Dosimetry metrics for low, medium and high inhaled dose groups.

	Inhaled dose (mg·kg^−1)	Range (mg·kg^−1)	Mean Ct (mg·min·m^−3)	Mean Exposure Time (min)	Mean C (mg·m^−3)
Low (n = 5)	0.22 ± 0.06	0.12 to 0.28	2435.8 ± 830.1	11.9 ± 7.7	204.7
Medium (n = 5)	0.45 ± 0.10	0.31 to 0.54	4409 ± 2528	15.2 ± 6.2	290.1
High (n = 5)	1.90 ± 0.82	0.99 to 3.07	16611 ± 5400	20.6 ± 3.6	806.4

Inhaled dose is the sum of the product of minute volume and corresponding chlorine concentration throughout the exposure divided by the animal's body weight; mean Ct is derived from the AUC; mean C is calculated by dividing the mean Ct by the mean exposure time. Mean ± SD.

Figure 3. Kaplan-Meier survival graph of air-exposed control animals and chlorine-exposed animals (n = 5/group). ID: inhaled dose.

Figure 4. Changes in temperature adjusted PaO₂ (A) and PaCO₂ (B) in animals grouped as air-exposed controls, chlorine-exposed low ID, chlorine-exposed medium ID and chlorine-exposed high ID. * Significant linear trend (p < 0.05) between increasing doses of chlorine and the air controls. ‘d’ represents the death of an animal. Change in temperature adjusted arterial blood PaO₂ (C) and PaCO₂ (D) in animals grouped as air-exposed controls, chlorine survivors and chlorine non-survivors. Dashed line (- - -) displays a significant difference across 12 h between the chlorine-exposed survivors and the air-exposed controls. ** Percentage change from baseline AUC survivors vs air controls (p = 0.0049). Mean ± SD.

Table 6. Dosimetry metrics for survivors vs non-survivors.

	Inhaled dose (mg·kg^−1)	Range (mg·kg^−1)	Mean Ct (mg·min·m^−3)	Mean Exposure Time (min)	Mean C (mg·m^−3)
Survivors (n = 9)	0.94 ± 0.74	0.21 to 2.31	9403.2 ± 6986.6	18.1 ± 6.4	519.5
Non-survivors (n = 6)	0.74 ± 1.15	0.12 to 3.07	5561.2 ± 6973.6	12.6 ± 6.2	441.3

Inhaled dose is the sum of the product of minute volume and corresponding chlorine concentration throughout the exposure divided by the animal's body weight; mean Ct is derived from the AUC; mean C is calculated by dividing the mean Ct by the mean exposure time. Mean ± SD.

Figure 5. Changes in temperature adjusted arterial blood lactate (A), pH (B), base excess (C) and bicarbonate (D). Mean ± SD.

Figure 6. Changes in percentage shunt fraction. Dashed line (- - -) displays a significant difference across 12 h between the chlorine-exposed survivors and the air-exposed controls. Percentage change from baseline AUC survivors vs air controls (p = 0.049). Mean ± SD.

Figure 7. Differential WBC count analysis in terminal lavage fluid. The air-exposed control group for BAL fluid WBC counts were reduced to 3 animals due to lung abnormalities or difficulty collecting a full sample of BAL fluid at post mortem. * indicates significant difference for neutrophils (p = 0.02) and alveolar macrophages (p = 0.04) between the chlorine-exposed survivors when compared to air-exposed controls (Kruskal-Wallis One-Way ANOVA). Mean ± SD.

Figure 8. Hematoxylin and eosin stained sections (A) Air control, trachea, no injury (score 0), pseudostratified, ciliated, columnar epithelium with goblet cells. (B) Medium dose, trachea, moderate injury (score 2), erosion and degeneration of epithelium, edema, mixed inflammatory cell infiltration, and cellular debris, mucus, and erythrocytes in the lumen. (C) Air control, bronchiole, no injury (score 0). (D) Medium dose, bronchiole, moderate injury (score 2), erosion and degeneration of epithelium, mixed inflammatory cell infiltration, and sloughed epithelial cells, cellular debris, mucus and erythrocytes in the lumen. The surrounding parenchyma exhibits congestion, edema, fibrin exudation and polymorphonuclear cell infiltration. (E) Air control, lung parenchyma, no injury (score 0), (F) Medium dose, lung parenchyma, moderate injury (score 2), diffuse filling of alveolar spaces by eosinophilic fluid (edema) and fibrin strands, with congestion of alveolar capillaries and larger vessels, and numerous neutrophils within alveolar spaces and the interstitium.

Figure 9. Mean histopathology score comparing air-exposed controls with animals surviving to 12 h along the length of the respiratory tract (A – trachea and bronchi; B – Bronchioles; C – Parenchyma, alveoli and interstitium; D - Parenchyma, alveoli and interstitium using scoring system of Matute-Bello et al., 2011 (M-B)). Air-exposed control = 3, chlorine-exposed survivors = 9, chlorine-exposed non-survivors = 5. * indicates significant difference between air-exposed controls and chlorine-exposed survivors (p < 0.0001, Tukey’s multiple comparison, one way ANOVA). Mean ± SD.

Matute-Bello G, Downey G, Moore B, Groshong S, Matthay M, Slutsky A, Kuebler W. 2011. An official American Thoracic Society workshop report: features and measurements of experimental acute lung injury in animals. Am J Respir Cell Mol Biol. 44(5):725–738.

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