Molecular biological effects of selective neuronal nitric oxide synthase inhibition in ovine lung injury

Abstract

Neuronal nitric oxide synthase is critically involved in the pathogenesis of acute lung injury resulting from combined burn and smoke inhalation injury. We hypothesized that 7-nitroindazole, a selective neuronal nitric oxide synthase inhibitor, blocks central molecular mechanisms involved in the pathophysiology of this double-hit insult. Twenty-five adult ewes were surgically prepared and randomly allocated to 1) an uninjured, untreated sham group (n = 7), 2) an injured control group with no treatment (n = 7), 3) an injury group treated with 7-nitroindazole from 1-h postinjury to the remainder of the 24-h study period (n = 7), or 4) a sham-operated group subjected only to 7-nitroindazole to judge the effects in health. The combination injury was associated with twofold increased activity of neuronal nitric oxide synthase and oxidative/nitrosative stress, as indicated by significant increases in plasma nitrate/nitrite concentrations, 3-nitrotyrosine (an indicator of peroxynitrite formation), and malondialdehyde lung tissue content. The presence of systemic inflammation was evidenced by twofold, sixfold, and threefold increases in poly(ADP-ribose) polymerase, IL-8, and myeloperoxidase lung tissue concentrations, respectively (each P < 0.05 vs. sham). These molecular changes were linked to tissue damage, airway obstruction, and pulmonary shunting with deteriorated gas exchange. 7-Nitroindazole blocked, or at least attenuated, all these pathological changes. Our findings suggest 1) that nitric oxide formation derived from increased neuronal nitric oxide synthase activity represents a pivotal reactive agent in the patho-physiology of combined burn and smoke inhalation injury and 2) that selective neuronal nitric oxide synthase inhibition represents a goal-directed approach to attenuate the degree of injury.

Fig. 1.

Mean total NOS enzyme activity and iNOS enzyme activity in lung tissue at death 24 h postinjury in pmol·mg protein⁻¹·min⁻¹ ± SE in the sham, control, and 7-nitroindazole (7-NI) groups. Within each group, the left column represents total NOS enzyme activity and the right column iNOS enzyme activity.

Fig. 2.

Malondialdehyde (MDA) (nmol/mg protein ± SE; A) and 3-nitrotyrosine (3-NT) (densitometry % sham; B) in lung tissue at death 24 h postinjury in the sham, control, and 7-NI groups. Data are expressed as means ± SE. *P < 0.05 vs. sham; †P < 0.05 vs. control.

Fig. 3.

PAR in airway (A), pulmonary glands (B), lung alveoli (C), and pulmonary vessels (D) at death 24 h postinjury, expressed in a mean score ± SE in the sham, control, and 7-NI groups. *P < 0.05 vs. sham; †P < 0.05 vs. control.

Fig. 4.

A: p65 in lung tissue at death 24 h after burn and smoke inhalation injury in densitometry % sham in the sham, control, and 7-NI groups. *P < 0.05 vs. sham; †P < 0.05 vs. control. B: IL-8 (pg/ml) in lung lymph at 12, 18, and 24 h postinjury. Data are expressed as means ± SE. At each time point, the left column represents the sham group, the middle column the control, and the right column the 7-NI group.

Fig. 5.

Trachea blood flow in ml/min/g % sham at 0, 3, 6, 12, 18, and 24 h after injury in the sham, control, and 7-NI groups. Data are presented as means ± SE. *P < 0.05 vs. sham; †P < 0.05 vs. control; ‡P < 0.05 vs. 7-NI.

Fig. 6.

Obstruction score in bronchi (A) and bronchioles (B), as well as histopathological score (C), in lung tissue at death 24 h postinjury in the sham, control, and 7-NI groups. Data are expressed as means ± SE. *P < 0.05 vs. sham; †P < 0.05 vs. control.

Fig. 7.

Peak (A) and pause (B) ventilatory pressures in cmH₂O ± SE at 0, 3, 6, 12, 18, and 24 h after injury in the sham, control, and 7-NI groups. *P < 0.05 vs. sham; †P < 0.05 vs. control; ‡P < 0.05 vs. 7-NI.

Fig. 8.

Pulmonary shunt fraction (Qs/Qt) (A) and PaO₂/FiO₂ ratio (B) at 0, 3, 6, 12, 18, and 24 h postinjury in the sham, control, and 7-NI groups. Data are expressed as means ± SE. *P < 0.05 vs. sham; †P < 0.05 vs. control; ‡P < 0.05 vs. 7-NI.