The differential effects of inspiratory, expiratory, and combined resistive breathing on healthy lung

Figures & data

Figure 1 IRB deranged respiratory system mechanics.

Notes: IRB load dependently increased lung elasticity, as measured by the force oscillation technique (A), and decreased static compliance of the respiratory system, calculated at the expiratory part of the pressure–volume curve (B), compared to ctr. White column, ctr; light gray column, IRB 20%; dark gray column, IRB 30%; black column, IRB 40%. (C) Likewise, a right and downward shift of the static pressure–volume curve of the respiratory system was noticed, as a result of IRB, suggestive of the occurrence of acute lung injury. Data are presented as mean ± SEM. *P<0.05 to ctr; ^#P<0.05 to IRB 20%; ^$P<0.05 to IRB 30%; n=7–8 per group.
Abbreviations: ctr, control; IRB, inspiratory resistive breathing; SEM, standard error of the mean.

Figure 2 IRB caused lung inflammation.

Notes: (A) IRB load dependently increased BALF cellularity compared to ctr. Differential cell counting revealed significantly increased macrophages, when the major load of 40% was applied. Neutrophils were elevated both at the major and intermediate loads of 40% and 30%. Lymphocytic infiltration was negligible. (B) Consistently, MPO activity measurement showed neutrophilic infiltration, as a result of IRB. White column, ctr; light gray column, IRB 20%; dark gray column, IRB 30%; black column, IRB 40%. Data are presented as mean ± SEM. Total count, n=5–10 per group; differential count, n=5–8 per group; MPO, n=6–7 per group. (C) IL-1β and IL-6 protein levels in lung tissue were load dependently increased following IRB 30% and IRB 40%. White column, IRB 20%; gray column, IRB 30%; black column, IRB 40%. Data are presented as mean ± SEM fold increase relative to ctr. n=5–8 per group. *P<0.05 to ctr; ^#P<0.05 to IRB 20%; ^$P<0.05 to IRB 30%.
Abbreviations: BALF, bronchoalveolar lavage fluid; ctr, control; IL, interleukin; IRB, inspiratory resistive breathing; MPO, myeloperoxidase; OD, optical density; SEM, standard error of the mean.

Figure 3 IRB increased lung permeability and induced lung injury.

Notes: (A) IRB increased alveolar capillary barrier permeability compared to unloaded breathing, as assessed by total protein concentration in BALF. The effect was load dependent. (B) Total lung injury score was increased after IRB 30% and IRB 40% compared to ctr and lower inspiratory loading of 20%. White column, ctr; light gray column, IRB 20%; dark gray column, IRB 30%; black column, IRB 40%. Data are presented as mean ± SEM. *P<0.05 to ctr; ^#P<0.05 to IRB 20%; ^$P<0.05 to IRB 30%. BALF protein, n=6–12 per group; histology n=7–10 per group.
Abbreviations: BALF, bronchoalveolar lavage fluid; ctr, control; IRB, inspiratory resistive breathing; SEM, standard error of the mean.

Table 1 Differential effect of resistive loading on lung histology

	ctr	IRB			ERB				CRB
		20%	30%	40%	40%	50%	60%	70%	30%/50%	40%/50%	40%/60%
Focal thickness	0.4 (0.16)	0.43 (0.20)	1 (0.22)	1.14* (0.14)	0.43 (0.20)	0.38 (0.18)	0.63 (0.18)	0.86 (0.34)	1 (0.22)	1* (0.0)	1.3* (0.26)
Capillary congestion	0.6 (0.22)	0.86 (0.34)	1.57* (0.20)	1.29 (0.18)	1 (0.0)	1.75*,† (0.16)	1.75*,† (0.16)	1.71*,† (0.18)	1.29 (0.18)	1.43* (0.20)	1.5* (0.17)
Hemorrhage	0.2 (0.13)	0.29 (0.18)	0.29 (0.18)	0.43 (0.30)	0.14 (0.14)	0.25 (0.16)	0.25 (0.16)	0.29 (0.18)	0.29 (0.18)	0.43 (0.20)	1*,† (0.15)
Interstitial neutrophils	0.3 (0.15)	0.57 (0.20)	1.29* (0.18)	1.71*,† (0.18)	1.29* (0.18)	1.5* (0.19)	1.63* (0.18)	1.86* (0.14)	1.43* (0.20)	2* (0.22)	2.5*,† (0.17)
Intraalveolar neutrophils	0.1 (0.1)	0.29 (0.18)	0.71* (0.18)	1.43*,† (0.20)	0.86* (0.14)	0.75* (0.16)	0.75* (0.16)	1* (0.0)	1* (0.22)	1.43* (0.20)	1.8*,† (0.2)
Total lung injury score	1.6 (0.56)	2.43 (0.57)	4.86*,† (0.51)	6*,† (0.49)	3.71* (0.52)	4.63* (0.50)	5* (0.53)	5.71*,† (0.36)	5* (0.58)	6.29* (0.57)	8.1*,† (0.67)

Notes: Data are presented as mean ± SEM.

* P<0.05 to ctr;

† P<0.05 to the lower load of the same type of loading. n=7–10 per group.

Abbreviations: CRB, combined resistive breathing; ctr, control; ERB, expiratory resistive breathing; IRB, inspiratory resistive breathing; SEM, standard error of the mean.

Figure 4 ERB did not affect lung mechanics.

Notes: ERB did not significantly alter lung tissue elasticity (A) and static compliance of the respiratory system (B). White column, ctr; light gray column, ERB 40%; gray column, ERB 50%; dark gray column, ERB 60%; black column, ERB 70%. Data are presented as mean ± SEM fold increase relative to ctr. n=6–8 per group.
Abbreviations: ctr, control; ERB, expiratory resistive breathing; SEM, standard error of the mean.

Figure 5 ERB produced neutrophilic lung inflammation.

Notes: (A) ERB led to prominent BALF neutrophilia, irrespective of the applied load. Of notice, ERB was also associated with significantly decreased macrophage count compared to ctr. (B) Lung MPO activity measurement confirmed an increased neutrophilic infiltration in the ERB groups compared to ctr. White column, ctr; light gray column, ERB 40%; gray column, ERB 50%; dark gray column, ERB 60%; black column, ERB 70%. Data are presented as mean ± SEM. Total count, n=7–8 per group Differential count, n=5–8 per group; MPO, n=5–7 per group. (C) Lung IL-1β protein levels were significantly and load dependently elevated compared to ctr in all ERB groups, whereas only higher expiratory loads of 60% and 70% significantly increased lung IL-6 protein levels compared to ctr and lower loads. White column, ctr; light gray column, ERB 40%; gray column, ERB 50%; dark gray column, ERB 60%; black column, ERB 70%. Data are presented as mean ± SEM fold increase relative to ctr. n=5–8 per group. *P<0.05 to ctr; ^#P<0.05 to ERB 40%; ^$P<0.05 to ERB 50%.
Abbreviations: BALF, bronchoalveolar lavage fluid; ctr, control; ERB, expiratory resistive breathing; IL, interleukin; MPO, myeloperoxidase: OD, optical density; SEM, standard error of the mean.

Figure 6 ERB effect on lung permeability and histology.

Notes: (A) Total protein content in BALF was significantly increased only in response to the major expiratory load of 70% compared to ctr. (B) Total lung injury score was significantly increased in all ERB groups compared to ctr. White column, ctr; light gray column, ERB 40%; gray column, ERB 50%; dark gray column, ERB 60%; black column, ERB 70%. Data are presented as mean ± SEM. *P<0.05 to ctr; ^#P<0.05 to ERB 40%; ^$P<0.05 to ERB 50%. BALF protein, n=5–12 per group; histology, n=7–10 per group.
Abbreviations: BALF, bronchoalveolar lavage fluid; ctr, control; ERB, expiratory resistive breathing; SEM, standard error of the mean.

Figure 7 CRB modified lung mechanical parameters.

Notes: Increased lung elasticity (A), decreased static compliance (B), and downward shifted P–V curve (C) were witnessed in the CRB groups of 40%/50% and 40%/60% compared to ctr; alterations consistent with the presence of high-permeability pulmonary edema. White column, ctr; light gray column, CRB 30%/50%; dark gray column, CRB 40%/50%; black column, CRB 40%/60%. Data are presented as mean ± SEM. *P<0.05 to ctr; ^#P<0.05 to CRB 30%/50%; ^$P<0.05 to CRB 40%/60%. n=6–8 per group.
Abbreviations: ctr, control; CRB, combined resistive breathing; P–V, pressure–volume; SEM, standard error of the mean.

Figure 8 CRB provoked lung inflammation.

Notes: (A) CRB led to prominent total BALF cellularity increase compared to ctr, when the intermediate and major loads of 40%/50% and 40%/60% were applied. Macrophage and lymphocyte counts were significantly elevated only in the CRB 40%/60% group, while considerable neutrophilia was present in response to all loads. (B) Similarly, measured MPO activity in lung tissue was load dependently elevated compared to ctr. White column, ctr; light gray column, CRB 30%/50%; dark gray column, CRB 40%/50%; black column, CRB 40%/60%. Data are presented as mean ± SEM. Total count, n=5–10 per group; differential count, n=5–8 per group; MPO, n=6–7 per group. (C) All CRB groups, independent of loading magnitude, produced significant lung IL-1β protein levels augmentation compared to ctr. Increased IL-6 protein levels in lung tissue were witnessed only when intermediate and higher combined loads of 40%/50% and 40%/60% were applied. White column, CRB 30%/50%; gray column, CRB 40%/50%; black column, CRB 40%/60%. Data are presented as mean ± SEM fold increase relative to ctr. n=5–10 per group. *P<0.05 to ctr; ^#P<0.05 to CRB 30%/50%; ^$P<0.05 to CRB 40%/60%.
Abbreviations: BALF, bronchoalveolar lavage fluid; ctr, control; CRB, combined resistive breathing; IL, interleukin; MPO, myeloperoxidase; OD, optical density; SEM, standard error of the mean.

Figure 9 CRB increased lung permeability and induced lung injury.

Notes: (A) Total protein content in BALF calculation revealed load-dependent lung barrier permeability augmentation after CRB of 40%/50% and 40%/60% compared to ctr. (B) ALI indicative histological alterations, as assessed by total lung injury score, were found in all CRB groups but were more pronounced when the highest load of 40%/60% was applied. White column, ctr; light gray column, CRB 30%/50%; dark gray column, CRB 40%/50%; black column, CRB 40%/60%. Data are presented as mean ± SEM. *P<0.05 to ctr; ^#P<0.05 to CRB 30%/50%; ^$P<0.05 to CRB 40%/60%. BALF protein, n=5–12 per group; histology, n=7–10 per group.
Abbreviations: ALI, acute lung injury; BALF, bronchoalveolar lavage fluid; ctr, control; CRB, combined resistive breathing; SEM, standard error of the mean.

Figure 10 Representative histological sections (magnification ×400) from control (A), IRB 30% (B) and IRB 40% (C), ERB 40% (D) and ERB 70% (E), and CRB 30%/50% (F) and CRB 40%/60% (G) groups. Neutrophilic infiltration, capillary congestion, and focal thickening of the alveolar membrane are evident in lower resistive loads but are significantly more pronounced in the highest load of each group, especially CRB 40%/60%.

Abbreviations: CRB, combined resistive breathing; ERB, expiratory resistive breathing; IRB, inspiratory resistive breathing.