Intratracheal administration of influenza virus is superior to intranasal administration as a model of acute lung injury

Abstract

Infection of mice with human or murine adapted influenza A viruses results in a severe pneumonia. However, the results of studies from different laboratories show surprising variability, even in genetically similar strains. Differences in inoculum size related to the route of viral delivery (intranasal vs. intratracheal) might explain some of this variability. To test this hypothesis, mice were infected intranasally or intratracheally with different doses of influenza A virus (A/WSN/33 [H1N1]). Daily weights, a requirement for euthanasia, viral load in the lungs and brains, inflammatory cytokines, wet-to-dry ratio, total protein and histopathology of the infected mice were examined. With all doses of influenza tested, intranasal delivery resulted in less severe lung injury, as well as smaller and more variable viral loads in the lungs when compared with intratracheal delivery. Virus was not detected in the brain following either method of delivery. It is concluded that compared to intranasal infection, intratracheal infection with influenza A virus is a more reliable method to deliver virus to the lungs.

Keywords: Influenza A infection; Inoculation; Intranasal; Intratracheal; Lung injury; Mice.

Fig. 1

Mortality is higher and weight loss less variable when identical doses of influenza A are administered intratracheally compared with intranasally. (A, D) C57BL/6 mice were inoculated intratracheally and intranasally with influenza A virus 350 pfu/mouse, (B, E) 750 pfu/mouse, and (C, F) 1500 pfu/mouse and the need for euthanasia (mortality) was measured at least daily in the same mice. For all groups the initial number of mice for each treatment condition is between 8 and 10 animals, however, in D–F the number of mice in each group falls over the duration of the experiment by a number corresponding to the rate of mortality. Error bars indicate standard errors of the mean. *p < 0.05; **p < 0.001; ***p < 0.0001.

Fig. 2

Viral replication is higher and less variable when identical doses of influenza A are administered intratracheally compared with intranasally. C57BL/6 mice were treated with influenza A virus (500 pfu/mouse) intratracheally and intranasally. Four days after influenza A infection, the lungs were harvested and viral titers were measured using viral plaque assays. n = 10 for intranasal infection, n = 9 for intratracheal infection. *p < 0.05. Error bars indicate standard deviations, the standard deviation is 134% of the mean for intranasal infection and 61% of the mean for intratracheal infection.

Fig. 3

Measures of lung injury and lung inflammation are higher and less variable when identical doses of influenza A are administered intratracheally compared with intranasally. C57BL/6 mice were inoculated intratracheally and intranasally with influenza A virus (500 pfu/mouse) and harvested 4 days later for assessment of lung inflammation and the severity of lung injury. (A) Total leukocyte counts and (B) neutrophil counts in BAL fluid, (C) wet-to-dry ratio of both lungs, a measure of total lung water, (D) total protein levels in the BAL fluid, (E, F) inflammatory cytokine levels, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in BAL fluid were measured. (G) Lung injury was assessed histologically by examination of hematoxylin and eosin stained lung sections obtained using a TissueGnostics imaging system (100×). The left panels show a representative montage image from the intranasally (top) or intratracheally (bottom) infected mice. For all experiments, there were five mice in each group. *p < 0.05. Error bars indicate standard deviations.