Anti-inflammatory and anti-fibrotic treatment in a rodent model of acute lung injury induced by sulfur dioxide

Abstract

Context: Inhalation of sulfur dioxide (SO₂) affects the lungs and exposure to high concentrations can be lethal. The early pulmonary response after inhaled SO₂ involves tissue injury, acute neutrophilic lung inflammation and airway hyperresponsiveness (AHR). In rats, long-term pulmonary fibrosis is evident 14 days post-exposure as indicated by analysis of collagen deposition in lung tissue. Early treatment with a single dose of dexamethasone (DEX,10 mg/kg) significantly attenuates the acute inflammatory response in airways. However, this single DEX-treatment is not sufficient for complete protection against SO₂-induced injuries.

Methods: Female Sprague–Dawley rats exposed to SO₂ (2200 ppm, nose-only exposure, 10 min) were given treatments (1, 5 and 23 h after SO₂-exposure) with the anti-fibrotic and anti-inflammatory substance Pirfenidone (PFD, 200 mg/kg) or DEX (10 mg/kg) to evaluate whether the inflammatory response, AHR and lung fibrosis could be counteracted.

Results: Both treatment approaches significantly reduced the total leukocyte response in bronchoalveolar lavage fluid and suppressed pulmonary edema. In contrast to DEX-treatment, PFD-treatment reduced the methacholine-induced AHR to almost control levels and partially suppressed the acute mucosal damage whereas multiple DEX-treatment was the only treatment that reduced collagen formation in lung tissue.

Conclusions: To enable an accurate extrapolation of animal derived data to humans, a detailed understanding of the underlying mechanisms of the injury, and potential treatment options, is needed. The findings of the present study suggest that treatments with the capability to reduce both AHR, the inflammatory response, and fibrosis are needed to achieve a comprehensive mitigation of the acute lung injury caused by SO₂.

KEYWORDS::

• Sulfur dioxide
• chemical-induced lung injury
• animal models
• pirfenidone
• dexamethasone
• inflammation
• respiratory mechanics
• fibrosis

Introduction

Sulfur dioxide (SO₂) is extensively produced in numerous industrial applications and hence transported in high volumes as pressurized liquid which may be accidently released upon tanker disruption forming widespread clouds of highly concentrated gas. Inhalation to very high concentrations of SO₂ can be immediately dangerous to life or health (IDLH, 100 ppm (NIOSH)) [1]. The mechanisms by which SO₂ damages the lungs and the long-term consequences of this injury are not fully understood, but it has been shown that SO₂ can cause toxic effects both in the respiratory system (e.g., severe airway obstructions, bronchitis, sloughing of the mucosa of large and small airways) and in the cardiopulmonary system in animals and in humans [2–14].

In a previous study using Sprague–Dawley rats [15], the acute pulmonary response to inhaled SO₂ was shown to involve tissue injury, acute neutrophilic lung inflammation and hyperreactivity of large airways. The acute response was followed by a long-term inflammation dominated by macrophages and eosinophils, and an upregulation of the pro-fibrotic cytokine TGF-β1. Pulmonary fibrosis was evident 14 days post-exposure as indicated by histopathological evaluation and increased collagen deposition in lung tissue. Treatment with a single dose of dexamethasone (DEX, 10 mg/kg) to counteract SO₂-induced acute inflammation, AHR and long-term lung fibrosis was investigated showing significant down-modulation of the acute inflammatory response in airways. This treatment was, however, insufficient for complete protection against 1) the pulmonary toxicity (AHR and fibrosis), 2) animal body weight-loss and 3) cardiopulmonary effects (DEX-treatment did not reduce the increased heart-weight detected in the SO₂-exposed animals). The reducing effects of DEX on AHR were inconsistent, indicating that hyperreactive airways may only partly be linked to acute cellular inflammation [15].

The importance of early anti-inflammatory treatment of chemical-induced lung injury has been implicated in previous studies [16–20]. An ideal treatment against SO₂-induced lung injury includes protection against both acute and long-term effects. From the previous animal studies, it is clear that corticosteroids are generally beneficial in reducing adverse physiological effects induced by inflammatory responses, but also that alternative treatment approaches targeting other mechanisms such as pulmonary fibrosis in the pathogenesis are required for comprehensive protection [15,19,20]. It should also be emphasized that the lung injuries caused by toxic compounds depend on the specific interactions (due to physicochemical properties, e.g., water solubility, pH and oxidation strength) between the chemical, and biological tissues, i.e., the mechanisms leading to inflammation and hyperreactive airways are expected to differ between toxic irritant chemicals.

To enable an accurate extrapolation of animal-derived data to humans, a detailed understanding of the underlying mechanisms of the injury may give important information about which treatments to test, with the aim to increase the therapeutic effect in humans exposed to high concentrations of toxic chemical gases. In this study, we employed a rat model of SO₂-induced lung injury suitable for studying treatment approaches and evaluated two different drug treatment protocols, both with a potential protection against bronchoconstriction, cellular inflammation and pulmonary fibrosis. An animal ventilator and a forced oscillation method were used to evaluate respiratory mechanics and airway reactivity in response to various treatments, and together with the pulmonary physiology we also described the histopathology, cellular pulmonary inflammation and inflammatory mediators in bronchoalveolar lavage fluid (BALF) and serum.

Materials and methods

Animals

Female Sprague–Dawley rats (8–9 weeks old, Envigo RMS B.V, Netherlands) were used in this study. The care of the animals and the experimental protocols were approved by the regional ethics committee on animal experiments in accordance to Swedish law. Food and water were provided ad libitum. All rats were weighed immediately before the experimental start, and their health condition was monitored during the experimental period. The experiment was divided into two studies:

Study 1. Short-term 24 h post-exposure

The experiment terminated at 24 h (n = 6 rats per group). Animals were allocated into four different groups: 1) control animals were breathing room air (Control (211 ± 5 g), n = 6), 2) SO₂-exposed animals given vehicle instead of treatment (SO₂ (214 ± 3 g), n = 6), 3) SO₂-exposed animals given dexamethasone (SO₂+DEX (214 ± 6 g), n = 6) and 4) SO₂-exposed animals given pirfenidone (SO₂+PFD (214 ± 4 g), n = 6). Data regarding body weight, cells in BALF, inflammatory cytokines, respiratory mechanics and histopathology are shown for study 1.

Study 2. Long-term 14 days post-exposure

The experiment terminated at 14 days (n = 4–6 rats per group). Animals were allocated into four different groups: 1) control animals were breathing room air (Control (211 ± 5 g), n = 6), 2) SO₂-exposed animals given vehicle instead of treatment (SO₂ (210 ± 4 g), n = 4, (n = 2 (221 ± 1 g) were terminated before 14 days), 3) SO₂-exposed animals given dexamethasone (SO₂+DEX (213 ± 6 g), n = 6) and 4) SO₂-exposed animals given pirfenidone (SO₂ + PFD (212 ± 4 g), n = 5). Data regarding body weight and collagen deposition are shown for study 2.

SO₂-exposure protocol

Animals were placed in individual nose-only containers (EMMS, UK) and coupled to an inhalation exposure tower (Battelle) providing equal and simultaneous exposure to SO₂ (AGA gas, Sweden; compressed gas in gas cylinders: 10 mol-% SO₂, 90 mol-% nitrogen). The compressed gas mixture was diluted with air to a final concentration of 2200 ppm. Rats were subjected to a single exposure of SO₂–gas mixture for 10 min. The SO₂ concentration in the inhalation tower was monitored and the experiments were conducted in a designated fume hood for toxic gas exposures [15]. In the meantime, control animals were retained in their cages while breathing room air for 10 min (not placed in individual containers).

Interventions in study 1 and study 2

The dose of DEX (dexamethasone 21-phosphate disodium salt, Sigma-Aldrich, St. Louis, MO) was selected based on the results of previous studies [15,21,22]. DEX was dissolved in NaCl (0.9% NaCl in water) and a volume of 500 μl was administered intraperitoneally (i.p.) (10 mg/kg) to the rats at three time-points (1 h, 5 h and 23 h) following exposure to SO₂. This DEX-dose has also been used in previous studies with rats [15,22].

The dose of PFD (Pirfenidone (Esbriet^®), Roche) was modified from Seifirad et al. [23]. PFD was dissolved in NaCl and a volume of 1000 μl was administered as an i.p. treatment (200 mg/kg) at three time-points (1 h, 5 h and 23 h) following exposure to SO₂.

The SO₂-animals treated with vehicle received 500 μl NaCl i.p. The animals included in the 14-days study, received the same treatment schedule.

Respiratory mechanics

Animals in the 24 h group were weighed and anesthetized with pentobarbital sodium (50 mg/kg, i.p.). Rats were tracheostomized with a gauge cannula and mechanically ventilated with a small animal ventilator (flexiVent™, SCIREQ^®) at a frequency of 1.5 Hz (90 breaths/min) and a tidal volume (V_T) of 10 ml/kg bw. Rats were paralyzed with pancuronium (0.1 mg/kg bw, i.p.) and a positive end-expiratory pressure of 3 cm H₂O was applied. In order to measure AHR, incremental doses of inhaled methacholine (MCh, acetyl-â-methylcholine chloride, Sigma-Aldrich) were given at 5-min intervals. The MCh, diluted in saline to a volume of 20 μl, was given during 10 s as an aerosol (Aeroneb™ PRO, SCIREQ^®). Each dose of MCh (0, 5 and 25 mg/ml) was aerosolized without any interference with the ventilation pattern. A more detailed description of the method can be found in previously published papers [15,24].

Serum sampling

Animals were euthanized through exsanguination of the abdominal aorta during anesthesia and blood samples were collected at 24 h or 14 days post-exposure. The blood was centrifuged (15 min, 20 °C, 3000 rpm) and the serum was stored in −70°C until analysis.

Differential cell count in BALF

The lungs were lavaged six times via a tracheal tube with a total volume of 2 ml +23 ml (4 × 5 ml +3 ml) Ca²⁺/Mg²⁺ free Hanks’ balanced salt solution (HBSS, Sigma-Aldrich (St. Louis, MO)) and the BALF was centrifuged (10 min, 4 °C, 1500 rpm). After removing the supernatant, the cell pellet was resuspended in 1 ml PBS. The supernatant (the first 2 ml recovered after lavage) was stored in −70°C until analysis of inflammatory mediators. Leukocytes were counted manually in a hemacytometer and 20,000 cells were stained with May-Grünwald-Giemsa reagents (Merck Millipore, VWR International, Sweden), and differential cell counts of pulmonary inflammatory cells (macrophages, neutrophils, lymphocytes and eosinophils) were performed in duplicates using standard morphological criteria and counting 200 cells per slide. A more detailed description of the method can be found in a previously published paper [15].

Inflammatory mediators in BALF and serum

Inflammatory mediators in BALF and serum were analyzed for the presence of IL-1â, IL-6 CINC-1 and MMP-9 using specific assay kits from R&D Systems, Inc. The inflammatory mediators MPO, PAI-1 and IL-18 were measured using specific assay kits from Nordic BioSites, Sweden. The amount of each analyte was analyzed with an ELISA reader (Thermo Scientific Multiskan FC, Thermo Fischer Scientific Oy, Vantaa, Finland) using the software program for the ELISA reader (SkanIt for Multiskan FC 3.1. Inc), referring to the standards added. The three isoforms of TGFβ (TGF-β1-3) were analyzed simultaneously in BALF using a multiplex kit (Bio-Plex Pro TGF-β 3-Plex Immunoassay) and detected with a Bio-Plex™ system (Bio-Rad).

Histopathologic analyses 24 hours post-exposure

Following lavage, the right lung lobe was fixed in 4% paraformaldehyde until paraffin embedding. After embedding, the tissue was cut into 3 μm thick sections and mounted on slides. To assess inflammatory cell infiltration, the sections were deparaffinized, dehydrated and stained with hematoxylin and eosin. Histopathological evaluations of stained sections were performed in a blinded manner using light microscopy for 4 randomly selected animals in each group (n = 4 rats/group). Specifically, scores of 0–4 (0 = normal histology, 4 = most severe injury) were assigned to each of the following characteristics: 1) Pulmonary congestion; 2) Bronchi (mucosal damage, leukocyte infiltration in the bronchi space and the regeneration of the epithelium); 3) Alveoli (leukocyte infiltration in the alveoli space); and 4) Interstitium (interstitial edema and leukocyte infiltration of the peribronchial and perivascular spaces, and interlobular septa).

Analysis of collagen deposition in lung tissue 14 days post-exposure

Following lavage, the left lung lobe was rinsed in HBSS, and frozen in liquid nitrogen and stored in −70°C until analysis. The collagen content of 500 mg lung tissue was measured by a spectrophotometric method, Sircol™ Collagen Assay kit for rats (Biocolor Ltd., Belfast, UK). The assay was performed in duplicate and the mean of two data was determined for each individual sample. A more detailed description of the method can be found in a previously published paper [15].

Statistical analysis

Data are presented as means ± standard error of means (SEM). Statistical significance was assessed by parametric methods using a two-way analysis of variance (ANOVA) or when appropriate, Student’s unpaired t-test or one-way ANOVA followed by Bonferroni post hoc test to determine differences between the untreated SO₂-group and the other groups (Control, SO₂ + DEX and SO₂ + PFD). A statistical result of p < .05 was considered significant. Statistical analysis was carried out and graphs were prepared with GraphPad Prism program (version 6.07 GraphPad software Inc., San Diego, CA).

Results

The observed health effects after SO₂-exposure

Rats exposed to SO₂ showed significant body weight-loss 24 h post-exposure (p < .001) (Figure 1A). They also displayed an increased number of total leukocytes, neutrophils and eosinophils in BALF compared to the control group (Figures 2A,B). A significant increase of MMP-9 was observed in BALF of SO₂-exposed animals compared to the control group. In contrast, a reduction of MMP-9 was observed in serum of the SO₂-exposed animals which differed significantly from the control group (Table 1). None of the other analyzed inflammatory mediators differed between the groups.

Figure 1. Measurement of body weight in treated (DEX: dexamethasone (10 mg/kg i.p) or PFD: Pirfenidone (200 mg/kg i.p)) and untreated rats (SO₂) exposed to a 10-min exposure to 2200 ppm sulfur dioxide (SO₂). Control animals were breathing room-air while the other rats were exposed to SO₂. Statistical significant differences between initial weight and weight on termination day within each group are shown. (A) 24 h post-exposure (n = 6 rats per group) and (B) 14-days post-exposure (n = 5–6 rats per group). ^#Two animals in the 14-day SO₂ group were excluded on day 13–14 from the study due to severe decline in general health status (last time-point n = 4) Values indicate means ± SEM, **p < .01, and ***p < .001.

Figure 2. Total cell counts in bronchoalveolar lavage fluid (BALF) from rats exposed to 2200 ppm sulfur dioxide (SO₂) at 24 h post-exposure, after treatment with dexamethasone (DEX, 10 mg/kg i.p) or pirfenidone (PFD, 200 mg/kg i.p) at 1 h, 5 h and 23 h. The numbers of (A) total leukocytes, macrophages and neutrophils and (B) eosinophils and lymphocytes are shown. Values indicate means ± SEM. Statistical significant differences compared to untreated SO₂-group (vehicle) are shown, *p < .05, **p < .01 and ***p < .001 (n = 6 rats per group).

Table 1. Cytokines in bronchoalveolar lavage fluid (BALF) and serum, measured 24 h after a 10-minute exposure of female rats to 2200 ppm sulfur dioxide (SO₂), with and without post-exposure treatment (1, 5 and 23 h after SO₂-exposure).

		Control	SO2	SO2 + DEX	SO2 + PFD
BALF	CINC-1	1.00 ± 0.09	0.56 ± 0.17	0.61 ± 0.25	0.47 ± 0.08
	IL-6	1.00 ± 0.06	0.95 ± 0.05	0.76 ± 0.03	0.95 ± 0.10
	IL-1β	1.00 ± 0.04	1.12 ± 0.07	1.01 ± 0.10	0.95 ± 0.08
	MMP-9	1.00 ± 0.08**	7.23 ± 2.8	5.73 ± 1.6	5.53 ± 1. 2
	MPO	1.00 ± 0.09	1.32 ± 0.10	1.04 ± 0.10	0.98 ± 0.09
	IL-18	1.00 ± 0.07	0.96 ± 0.22	0.96 ± 0.10	1.03 ± 0.15
	TGF-β1	1.00 ± 0.09	2.00 ± 0.68	0.90 ± 0.18	1.20 ± 0.13
Serum	CINC-1	1.00 ± 0.06	0.71 ± 0.21	0.17 ± 0.02	1.19 ± 0.29
	IL-6	1.00 ± 0.02	0.99 ± 0.03	1.04 ± 0.07	1.01 ± 0.03
	IL-1β	1.00 ± 0.11	0.85 ± 0.07	0.80 ± 0.14	1.00 ± 0.18
	MMP-9	1.00 ± 0.04**	0.70 ± 0.07	0.75 ± 0.06	0.75 ± 0.08
	PAI-1	1.00 ± 0.10	0.90 ± 0.16	0.81 ± 0.03	1.36 ± 0.05*

DEX: dexamethasone (10 mg/kg i.p.), PFD: Pirfenidone (200 mg/kg i.p). Data show fold increase compared to age-matched SO₂ animals. Values indicate means ± SEM, *p < .05, **p < .01 compared to SO₂ group.

Recordings of baseline respiratory parameters showed that hysteresivity (η) and tissue resistance (G) were the only baseline parameters significantly increased 24 h after the SO₂-exposure (Table 2A). Measurements of the MCh-induced changes in respiratory responses showed significant increases in respiratory resistance (R_RS), Newtonian resistance (R_N), tissue elastance (H), tissue resistance (G) and hysteresivity (η) in SO₂-exposed animals compared to control animals (Figure 3).

Figure 3. Respiratory mechanics in rats 24 h after exposure to 2200 ppm sulfur dioxide (SO₂) and treatment (1 h, 5 h and 23 h) with dexamethasone (DEX, 10 mg/kg i.p) or pirfenidone (PFD, 200 mg/kg i.p). Measurements of methacholine (MCh)-induced (A) respiratory resistance, R_RS (B) Newtonian resistance, R_N (C) tissue resistance, G (D) respiratory elastance, E_RS (E) tissue elastance, H and (F) hysteresivity (η) were performed using the Flexivent™. Values indicate means ± SEM. Statistical significant differences compared to untreated SO₂-group are shown, *^{, #}p < .05, **^{, # #, ¤¤}p < .01, and ***^{, # # #, ¤¤ ¤}p < .001 (*SO₂ vs. control, ^#SO₂ vs. PFD and ^¤SO₂ vs. DEX), (n = 6 rats per group).

Table 2A. Acute effects of sulfur dioxide (SO₂) at 24 h post-exposure. Measurement of baseline respiratory mechanics (without methacholine) in female rats exposed for 10 minutes to 2200 ppm SO₂, with and without treatments (1, 5 and 23 h after SO₂-exposure).

	RRS cmH2O s ml^−1	ERS ml cmH2O^−1	RN cmH2O s ml^−1	G cmH2O s ml^−1	H cmH2O s ml^−1	η
Control	0.17 ± 0.01	3.00 ± 0.17	0.11 ± 0.01	0.61 ± 0.04**	3.20 ± 0.19	0.19 ± 0.01***
SO2	0.24 ± 0.05	3.43 ± 0.33	0.12 ± 0.02	0.93 ± 0.08	3.23 ± 0.41	0.30 ± 0.04
SO2 + DEX	0.17 ± 0.03	3.25 ± 0.18	0.06 ± 0.00***	0.57 ± 0.03**	3.60 ± 0.22	0.16 ± 0.00***
SO2+ PFD	0.14 ± 0.01*	2.96 ± 0.14	0.07 ± 0.01**	0.61 ± 0.01**	3.08 ± 0.16	0.20 ± 0.01***

DEX: dexamethasone (10 mg/kg i.p), PFD: Pirfenidone (200 mg/kg i.p). Values indicate means ± SEM, *p < .05, **p < .01, ***p < .001 compared to SO₂ group.

Based on previous results [15] and results in this study, respiratory mechanics were only measured in a few animals (belonging to the 14-day group) before further analysis was canceled due to the fact that many animals died during anesthesia and surgery, even before being connected to the ventilator. The animals in the 14-day group that could be anesthetized and mechanically ventilated showed no MCh-induced AHR and had equivalent R_RS response to control animals (data not shown). This reaction was not seen in animals belonging to the 24 h-group.

Histopathological analysis of the lung showed severe damage of the bronchial mucosa with epithelial degeneration, necrosis and erosion/ulceration 24 h post-exposure (Table 3, Figure 4). The epithelium was extensively disrupted, and in some animals there were no remaining epithelial cells. The large airways exhibited rich fibrin exudation and cellular infiltration but the intensity of the cellular exudate varied between rats, ranging from no infiltration to severe infiltration. The SO₂-group at 24 h showed a marked cell infiltration in the large airways (bronchi) with numerous polymorphs, monocytes, macrophages but low numbers of lymphocytes in the exudates. The alveoli of the SO₂-animals showed mild emphysema and leukocytes and there were also signs of mild to severe edema in the interstitium 24 h post-exposure (Table 3, Figure 4). After 14-days, there was an on-going fibrosis which was confirmed by quantitative measurement of collagen deposition in lung tissue (Table 2B). Two animals were excluded from the study due to severe decline in general health status; one was terminated on day 11 and the other was terminated on day 13, both rats belonging to the 14-day SO₂-group without treatment. The rationale for excluding these animals was that they lost too much weight according to the permission given by the regional ethics committee on animal experiments in accordance to Swedish law. The permission admits studies that induce a lung damage that could only be reconstituted by careful physiological measurements and avoid evoking an extreme lung damage in animals which could be life-threatening or deterioration in visible general functions. Symptoms such as body weight-loss, reduced mobility are expected to occur after exposure within the first days but be of a transitory nature.

Figure 4. Lung tissue sections from (A) healthy control and (B–D) rats 24 h post-exposure to 2200 ppm sulfur dioxide (SO₂), (B) without treatment (vehicle) and with (C) dexamethasone (DEX, 10 mg/kg i.p)-treatment, or (D) pirfenidone (PFD, 200 mg/kg i.p)-treatment. Tissue sections were stained with hematoxylin-eosin and photos were taken at 20 x magnification using a light microscope. AL = airway lumen.

Table 2B. Analysis of collagen deposition in female rats at 14 days post-exposure for 10 minutes to 2200 ppm SO₂, with and without treatments (1, 5 and 23 h after SO₂-exposure).

Control	SO2	SO2 + DEX	SO2 + PFD
1.05 ± 0.08*	1.49 ± 0.16	1.00 ± 0.17*	1.09 ± 0.17

Data show fold increase compared to the SO₂ group. DEX: dexamethasone (10 mg/kg i.p), PFD: Pirfenidone (200 mg/kg i.p). Values indicate means ± SEM, *p < .05 compared to SO₂ group.

Table 3. Scoring the main histopathology features in female rats 24 h after a 10-minute exposure to 2200 ppm sulfur dioxide (SO₂), with and without post-exposure treatments (1, 5 and 23 h after SO₂-exposure).

Groups	Rat no.	Pulmonary congestion	Bronchi			Alveoli	Interstitium
			Mucosal damage	Cells	Regeneration (mitoses in epithelium)	Cells	Edema	Cells
Control	1	0	0	0	0	0	0	0
	2	0	0	0	0	0	0	0
	3	0	0	0	0	0	0	0
	4	0	0	0	0	0	0	0
SO2	1	1	4	4	1	0	3	3
	2	1	4	1	0	0	2	1
	3	1	4	3	1	2	1	3
	4	1	4	1	1	0	1	0
SO2 + DEX	1	2	4	2	0	0	0	0
	2	1	4	0	0	0	0	0
	3	0	4	1	0	0	0	0
	4	1	4	1	1	0	0	0
SO2 + PFD	1	1	3	1	0	0	0	0
	2	1	4	1	0	0	0	0
	3	2	0	1	2	0	0	0
	4	0	2	0	2	0	0	0

Grade of lesions: 4 very severe, 3: severe, 2 moderate, 1: mild or low numbers, 0: significant lesions not observed.

^aPeribronchial and perivascular spaces, and interlobular septa. Cells: leukocytes, DEX: dexamethasone (10 mg/kg i.p), PFD: Pirfenidone (200 mg/kg i.p).

Multiple DEX-treatments

The initial animal body weight of the DEX-treatment group was not significantly different from the animal body weight at 24 h and 14 days post SO₂-exposure (Figures 1A,B). The accumulation of leukocytes in BALF was significantly reduced after DEX-treatment compared to SO₂-exposed rats (Figure 2A). Cell counts showed a decrease in the total number of macrophages and eosinophils, and non-significant decrease of neutrophils in BALF at 24 h post-exposure (Figures 2A,B).

The baseline alteration in lung configuration at 24 h, which showed heterogeneity after SO₂-exposure (hysteresivity, η), was significantly decreased after DEX-treatment, reaching baseline hysteresivity similar to that of control animals (Table 2A). Other baseline parameters such as the Newtonian resistance (R_N) and tissue resistance (G) were significantly decreased after DEX-treatment compared to untreated SO₂-exposed rats (Table 2A). The respiratory recordings showed a significantly decreased MCh-induced hysteresivity (η) and tissue resistance (G) after DEX-treatment, but the other MCh-induced parameters compared to untreated SO₂-exposed rats were not different (Figure 3).

The histopathology evaluation of the lung tissue after DEX-treatment showed that the cellular response in the alveolar region and the interstitium was suppressed despite the severe damage to the bronchial mucosa of similar magnitude to that seen in SO₂-animals without treatment 24 h post-exposure. The edema observed in the interstitium of the lung in the untreated SO₂-group was undetectable after DEX-treatment at 24 h (Table 3, Figure 4). DEX-treatment was efficient in the reduction of long-term fibrosis 14 days post-exposure, as shown by quantitative measurement of collagen deposition in lung tissue (Table 2B).

Multiple PFD-treatments

The animal body weight at 24 h and 14 days post-exposure in the PFD-treatment group was significantly lower than the initial body weight before exposure (p < .001, and p < .01 respectively, Figures 1A,B).

The PFD-treatment decreased the total numbers of leukocytes in BALF in comparison to SO₂-exposed rats without treatment at 24 h (Figure 2A). The number of eosinophils was significantly reduced while macrophages and neutrophils in BALF showed a non-significant decline after PFD-treatment (Figure 2).

Compared to DEX-treatment, the alteration in baseline lung configuration, which showed heterogeneity after SO₂-exposure, the hysteresivity (ç), was significantly decreased to a level similar to the level of control animals at 24 h (Table 2A). In addition to hysteresivity, the baseline levels of Newtonian resistance (R_N), respiratory resistance (R_RS) and tissue resistance (G) were significantly declined after PFD-treatment (Table 2A). Recordings of the MCh-induced changes in respiratory responses showed significant decreases of the respiratory resistance (R_RS), Newtonian resistance (R_N), tissue resistance (G) and hysteresivity (η) in the PFD-treated animals compared to untreated SO₂-exposed animals at 24 h (Figure 3).

The histopathology evaluation of the lung tissue at 24 h after PFD-treatment showed a tendency towards less mucosal damage in bronchi compared to SO₂-animals without treatment, in addition to suppressed cellular response in alveoli region and the interstitium. The pulmonary edema observed in the SO₂-group was undetectable also after PFD-treatment 24 h post-exposure (Table 3, Figure 4) and there was a tendency towards decreased collagen deposition in lung tissue from PFD-treated rats 14 days post-exposure (Table 2B).

Discussion

Limited studies in animals have addressed the effects of a single inhaled exposure to high concentrations of SO₂. The concentration of SO₂ used in this study was considered to be relevant for a scenario of a chemical disaster e.g., at an industrial plant or during transportation, which can result in life-threatening injuries. Rats were subjected to a single exposure of SO₂-gas for 10 min (367 ppm·h). The concentration used in this study was non-lethal and lower than a rat study performed by Cohen et al., who showed a 4-h LC₅₀ of 1057 ppm (4227 ppm·h) and a BMCL₀₅ (95% of the exposed mice are expected to survive) of 573 ppm SO₂ (2292 ppm·h) [6]. Fortunately, SO₂ accidents are rare events but in case of a release of sufficient amounts, an exposure may cause both acute- and long-term health effects in humans [15,25]. It has been reported by us and others that the main acute injuries after SO₂-exposure are located to larger bronchi and only minor changes are observed peripherally [6,7,15]. In this study, we examined the protective effects of two different drug treatments in Sprague–Dawley rats after nose-only exposure to SO₂, specifically addressing treatments that could provide protection against weight-loss, bronchoconstriction (AHR), cellular inflammation and pulmonary fibrosis.

We have previously demonstrated a rapid recruitment of inflammatory cells and release of inflammatory mediators in BALF and serum following SO₂-exposure [15]. These mediators could be used as possible biomarkers to identify the risk of inducing long-term lung injuries. From that study, it is also evident that SO₂-induced acute inflammation can develop into prolonged respiratory symptoms similar to those that have been observed in the late-resolving phases of acute respiratory distress syndrome (ARDS) [26] as well as symptoms associated with severe pathological airway obstruction in chronic obstructive pulmonary disease (COPD) patients [27]. A concern with this study is that two out of six animals in the SO₂ group had to be terminated before day 14, since those animals showed a severe decline in general health condition observed as major body weight-loss and air entrapment in the GI-tract, and thereby skewing the results at day 14. BALF, serum and lungs of those animals are not included in the analyses and for ethical reasons the investigation of lethal effects are not further studied. The underlying mechanism of how SO₂ exerts its toxic effects is, however, unclear [15].

A single dose of DEX administered early after chemical exposure seems to provide some protection against acute symptoms in animals, such as reduced number of inflammatory cells in BALF, diminished collagen deposition and MCh-induced AHR [15], and here we show that multiple early treatments with DEX (within one day) also can protect against weight-loss and reduction in tissue resistance. In comparison with a previous study, these results show that even with multiple DEX-treatments, there is no significant improvement in protection against the AHR caused by SO₂ in the central larger airways but a reduction in tissue resistance in peripheral airways. DEX does not have the complete ability to prevent the acute response after SO₂-exposure such as AHR, but the insufficient therapeutic effect may to a large extent depend on the dose of DEX. Since DEX has been tested in other animal models with significant results, we used similar doses for easier comparisons of results [15,21–23]. Contrary to our findings, high-dose single treatment with DEX (100 mg/kg) on other chemical-induced lung injuries has proven ineffective, e.g., in phosgene-induced lung injury [28].

Previously, we have shown that a single dose of DEX could reduce pulmonary fibrosis in lung tissue to some extent [15]. In the present study, multiple treatment with DEX significantly counteracted also the long-term effects such as the pulmonary fibrosis shown 14 days post-exposure. Similar result of DEX-treatment has been shown to reduce the level of pulmonary fibrosis following inhalation exposure to chlorine in mice and following exposure to an alkylating nitrogen mustard analog (melphalan) in mice [18,19]. Altogether these exposure models support the notion that corticosteroids are generally beneficial, at least to some extent, in the protection against adverse physiological effects induced by inflammatory responses, but also that alternative treatment approaches targeting other mechanisms in the pathogenesis are required for complete elimination of adverse effects such as infiltration of leukocytes, AHR and pulmonary fibrosis. Based on clinical data it has previously been pointed out that the efficiency of corticosteroid treatment following human exposure to lung-damaging agents are inconclusive and that there is a lack of controlled studies following high-dose exposure to lung-damaging agents in humans [29]. Although results from animal studies cannot be directly extrapolated to humans, these studies are of utmost importance to finding pathways and understanding the mechanism behind the complex interactions in the body.

PFD and Nintedanib are two approved medications used for the treatment of idiopathic pulmonary fibrosis (IPF). In this study PFD was used, as it has both anti-inflammatory and anti-fibrotic properties and acts as an important regulator of synthesis of cytokines, growth factors and collagen, and also attenuates pulmonary fibrosis by slowing functional decline and disease progression [30–36]. However, the management of IPF with PFD-treatment does not cure the disease but slow down the natural course of the disease. IPF has some similarities with the acute effects of SO₂-induced ALI, both appear to be driven by a mechanism that promote fibroblast recruitment and proliferation leading to a diffuse parenchymal lung disease, resulting in loss of lung function [15,34]. PFD-treatment has shown to have protective effects in other experimental lung disease-models induced by e.g., paraquat, bleomycin and allergen-induced asthma [30,37–39]. In addition to reducing pulmonary fibrosis, PFD-treatment has also been shown to attenuate fibrosis in the liver, heart and kidney [34].

In the present study, it was found that PFD-treatment could reduce the AHR to levels similar to control-levels and suppressed the lung tissue inflammation and especially reduced the observed mucosal damage in larger airways, however, multiple PFD-treatment could not counteract the long-term effects such as the pulmonary fibrosis (14 days post-exposure) or protect against animal body weight-loss as a result of SO₂-exposure. In the previous SO₂-study, animals had on-going fibroblastic activity and fibrosis 14 days after exposure and after 28 days, animals were almost recovered. The findings in the animal model of long-term SO₂-induced lung injury, however, show more resemblance with the late resolving phases in ARDS rather than IPF [15,21,26,40].

Combination therapy has proven to be successful in clinical studies and patients with advanced IPF had favorable outcomes after combined treatment with e.g., PFD and N-acetylcysteine (NAC) [41]. PFD-treatment has also been proven to produce synergistic therapeutic effects together with inhalation of the antioxidant lecithinized superoxide dismutase in a bleomycin-induced pulmonary fibrosis mouse model [42]. However, there are studies that provide evidence against the use of combination treatment, e.g., increased risks of death and hospitalization were observed in IPF-patients treated with a combination of prednisone, azathioprine, and NAC [43]. The therapeutic effects of combining multiple treatments with NAC (200 mg/kg i.p) and DEX (10 mg/kg i.p) in the SO₂ rat model described in the present study was not sufficient to reduce the SO₂-induced lung injury any further than DEX alone (unpublished, data not shown). When given in combination, a protective effect on AHR and a significant reduction in inflammatory cells (neutrophils) has previously been observed in a Cl₂-induced ALI mouse model [20].

It must be emphasized that care should be taken when designing drug-treatment protocols. The method of administering treatments in this study has the advantage that it is simple and rapid but a limitation was that both DEX and PFD were administrated systemically via i.p. injection when there are oral and intravenous alternatives. DEX has been used systemically in previous studies and has proven to have a beneficial effect when given as an i.p injection within one hour after exposure in e.g., chlorine, endotoxin, or melphalan exposure models [18,19,44]. A rapid absorption of the active substance is of importance and PFD was therefore given via the i.p route [23] to enable comparisons with the effects of DEX-treatment. The injuries after exposure to toxic industrial chemicals are rapidly induced since the chemicals immediately react to the lung tissue and start an immune response [45,46], thus the most optimal solution was to treat SO₂-induced ALI by i.p. administration. The rationale of using different time-points was that previous studies have shown that early treatment with a fast-acting drug is important to adequately treat chemical-induced ALI. The animals then received further maintenance doses after four hours (5 h time-point) and 23 hours similar to the recommendations in the medical guidelines for treatment of chemical-induced lung injury in humans.

Conclusions

Both treatment-protocols reduced the acute SO₂-induced inflammatory cellular response in airways, but only PFD-treatment diminished the MCh-induced AHR while DEX was the only treatment that showed efficacy at reducing the long-term effects monitored 14 days post-exposure.

Due to lack of effective therapies that have the capability to stop the progression of chemical-induced ALI, there is a need for innovative treatment strategies and novel therapies. Few drugs may have the ability to completely stop the aggravating ALI and the therapeutic effects are highly dependent on the dose and timing of the treatment administered. It should also be emphasized that the lung injuries caused by toxic industrial chemicals are dependent on the specific chemical reactivity and that the mechanisms leading to inflammation and hyperreactive airways most likely differ depending on chemical agent. Given the different mechanisms, it cannot be assumed that one certain treatment protocol beneficially applies to all types of chemical-induced ALI. Collectively, taking the findings of the present study into consideration, we suggest treatments that have the capability to reduce both the AHR, the inflammatory response, and the fibrosis caused by SO₂ or other industrial gases with pulmonary toxicity.

Acknowledgements

Karin Wallgren is gratefully acknowledged for invaluable help with the animal experiments and Dr. Ulrika Bergström and Dr. Åsa Gustafsson for thankful suggestions while writing the manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by grants from the Swedish Center for Disaster Toxicology at the National Board of Health and Welfare, the Swedish Ministry of Defence and the Swedish Armed Forces.