Comparisons of acute inflammatory responses of nose-only inhalation and intratracheal instillation of ammonia in rats

Figures & data

Figure 1. (A–D) Acute effects in rats, 24 h post-exposure, of anhydrous ammonia (NH₃) exposure via nose-only inhalation (9000–13 000 ppm during 10–20 min); (A) recordings of body weight loss (weight before and after exposure using registered weight); (B) the level of fibrinogen in serum; (C) total cell counts (cells/ml) in bronchoalveolar lavage fluid (BALF); (D) the number of neutrophils (cells/ml) in BALF. The figure bars show increasing concentrations (ppm) of NH₃ regardless of exposure time and the values indicate means ± SEM, n = 5–9 rats per group. Statistical significances of NH₃-exposed compared to age-matched control rats are indicated (*p < 0.05 and **p < 0.01).

Figure 2. (A–C) Effects in rats subjected to ammonia (1% NH₃) via intratracheal instillation on (A) body weight at 5 h–28 days after exposure, (B) spleen weight (g) and (C) heart weight (g) at 5 h and 24 h after exposure. Values indicate means ± SEM, n = 6–8 rats per group. Statistical significances of NH₃-exposed animal compared to age-matched control rats (*p < 0.05 and **p < 0.01), and control groups at 14 and 28 days compared to control rats at 5 h–24 h are indicated (

p < 0.001).

Figure 3. Total cell counts in bronchoalveolar lavage fluid (BALF) in rats at 5 h and 24 h after intratracheal instillation of ammonia (1% NH₃). Values indicate means ± SEM, n = 5–8 rats per group. Statistical significances of NH₃-exposed compared to age-matched control rats are indicated (*p < 0.05 and ***p < 0.001).

Table 1. In female rats exposed to i.t. instillation of ammonia (1% NH₃) (A) the number of thrombocytes (×10⁹/L) in blood was measured at 24 h, (B) inflammatory mediators in bronchoalveolar lavage fluid (BALF) and serum were measured at 5 h and 24 h after exposure and (C) the amount of collagen was quantified at 14 and 28 days after exposure.

(A) The number of thrombocytes per liter blood^a
	Control	NH3 24 h
Thrombocytes (×10^9/l)	872.5 ± 15.7	1029 ± 62.0

Table

(B) Inflammatory mediators in BALF and serum^b
	Control	NH3 5 h	NH3 24 h	Control 14 d	NH3 14 d
BALF
CINC-1	1.00 ± 0.09	0.89 ± 0.20	0.69 ± 0.11	NA	NA
IL-6	1.00 ± 0.06	0.84 ± 0.08	0.77 ± 0.02*	NA	NA
IL-1β	1.00 ± 0.04	0.70 ± 0.03***	1.05 ± 0.05	NA	NA
MMP-9	1.00 ± 0.27	1.05 ± 0.38	8.30 ± 0.58***	NA	NA
MPO	1.00 ± 0.09	0.91 ± 0.14	1.00 ± 0.09	1.00 ± 0.22	1.46 ± 0.05
IL-18	1.00 ± 0.07	1.72 ± 0.38*	1.38 ± 0.28	1.00 ± 0.21	0.80 ± 0.09
TGFβ1	1.00 ± 0.11	0.33 ± 0.05**	1.90 ± 0.17***	1.00 ± 0.41	0.98 ± 0.43
TGFβ2	1.00 ± 0.13	0.80 ± 0.13	7.08 ± 1.41***	1.00 ± 0.15	1.10 ± 0.16
TGFβ3	1.00 ± 0.07	0.34 ± 0.08**	0.50 ± 0.04*	1.00 ± 0.09	0.81 ± 0.12
Serum
CINC-1	1.00 ± 0.06	0.81 ± 0.04	0.50 ± 0.06***	NA	NA
IL-6	1.00 ± 0.02	1.01 ± 0.02	1.06 ± 0.03	NA	NA
IL-1β	1.00 ± 0.11	0.89 ± 0.05	1.98 ± 0.48*	NA	NA
MMP-9	1.00 ± 0.04	0.86 ± 0.09**	1.60 ± 0.20***	NA	NA
Fibrinogen	1.00 ± 0.43	0.41 ± 0.23	4.51 ± 1.38*	1.00 ± 0.51	6.47 ± 1.71*
PAI-1	1.00 ± 0.11	0.69 ± 0.08	1.76 ± 0.41*	1.00 ± 0.11	0.90 ± 0.23

Table

(C) Analysis of collagen in lung tissue^b
		Control 14 d	NH3 14 d	Control 28 d	NH3 28 d
Tissue	Collagen	1.00 ± 0.05	1.25 ± 0.03**	1.00 ± 0.02	1.23 ± 0.20

^aValues indicate means ± SEM.

^bData of NH3 exposed animals show fold change from age-matched control animals. Values indicate means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control animals. NA = not analyzed.

Figure 4. (A–F) Respiratory mechanics in rats at 24 h after exposure intratracheal instillation of ammonia (1% NH₃). Measurements of methacholine (MCh)-induced (A) R_RS: respiratory resistance, (B) R_N: Newtonian resistance, (C) G: tissue resistance, (D) E_RS: respiratory elastance, (E) H: tissue elastance, and (F) η: hysteresivity were performed using the Flexivent™. Values indicate means ± SEM, n = 6 rats per group. Statistical significances of NH₃-exposed compared to age-matched control rats are indicated (**p < 0.01 and ***p < 0.001).

Figure 5. (A–F) Lung tissue sections stained with Masson’s Trichrome in order to visualize fibrin (red stain) and collagen deposition (blue stain) in small-bronchial regions (top panel C and E) and alveolar regions (lower panel, D and F) of rats subjected to ammonia (1% NH₃) via intratracheal instillation evaluated in comparison to (A,B) healthy controls. Representative sections are shown from (C,D) 5 h and (E,F) 24 h post-exposure. Photos were taken at 100× magnification using light microscopy. The figure shows representative tissue sections from each group of 4 animals.

Figure 6. (A–H) Histopathological changes in alveolar (top panel C, E, and G) and bronchial regions (lower panel, D, F, and H) of rats subjected to ammonia (1% NH₃) via intratracheal instillation evaluated in comparison to (A,B) healthy controls. Representative sections are shown from (C,D) 5 h, (E,F) 24 h, and (G,H) 14 days and 28 days post-exposure. Sections were stained with hematoxylin-eosin and evaluated with light microscopy using 40× magnification (n = 4 rats per group).

Table 2. Histopathology, scoring the main features of lung lesions at different time-points after ammonia (1% NH₃) i.t. instillation exposure.

Time-points	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
5 h	1	0	+	0	+	0	+	0
	2	0	0	0	+	0	+	0
	3	0	++	+	+	0	++	0
	4	0	+++	+++	0	0	+++	++
24 h	1	+	+	++	+	0	0	0
	2	0	0	0	+	0	0	+
	3	0	+	+	+	+	+	+
	4	++	+++	++	+	++(+)	++	++
14 d	1	0	0	0	0	0	0	+
	2	0	0	0	0	0	0	+
	3	0	0	0	0	0	0	++
	4	0	0	0	0	0	0	0
28 d	1	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

Grade of lesions: +++: severe, ++ moderate, +: mild or low numbers.

0: Significant lesions not observed. Exposed rats: n = 4 analyzed from each time-point. Controls: n = 3 from each time-point: 24 h, 14 d and 28 d (Control animals from 14 d and 28 d are not shown). *Peribronchial and perivascular spaces, and interlobular septa. Cells**: leukocytes.