Predictive criteria to study the pathogenesis of malaria-associated ALI/ARDS in mice

Abstract

Malaria-associated acute lung injury/acute respiratory distress syndrome (ALI/ARDS) often results in morbidity and mortality. Murine models to study malaria-associated ALI/ARDS have been described; we still lack a method of distinguishing which mice will develop ALI/ARDS before death. This work aimed to characterize malaria-associated ALI/ARDS in a murine model and to demonstrate the first method to predict whether mice are suffering from ALI/ARDS before death. DBA/2 mice infected with Plasmodium berghei ANKA developing ALI/ARDS or hyperparasitemia (HP) were compared using histopathology, PaO2 measurement, pulmonary X-ray, breathing capacity, lung permeability, and serum vascular endothelial growth factor (VEGF) levels according to either the day of death or the suggested predictive criteria. We proposed a model to predict malaria-associated ALI/ARDS using breathing patterns (enhanced pause and frequency respiration) and parasitemia as predictive criteria from mice whose cause of death was known to retrospectively diagnose the sacrificed mice as likely to die of ALI/ARDS as early as 7 days after infection. Using this method, we showed increased VEGF levels and increased lung permeability in mice predicted to die of ALI/ARDS. This proposed method for accurately identifying mice suffering from ALI/ARDS before death will enable the use of this model to study the pathogenesis of this disease.

Figure 1

Infection of DBA/2 mice with P. berghei ANKA constitutes a rodent model for malaria-associated ALI/ARDS. (a) Survival and (b) parasitemia curves from the ALI/ARDS and HP mice over time. The red line was the mice that died with ALI/ARDS. The gray area represents the period when the mice die of ALI/ARDS. The data presented are representative of 13 independent experiments; n = 10–12 mice/experiment. (c) The lungs of mice that died with ALI/ARDS weighed 40% more than the lungs of mice that died with HP (**P ≤ 0.005; Mann-Whitney test of lung weights are representative of four separate experiments). Data ((b) and (c)) represent means and SEM; n = 10–12 mice/experiment. (d) Representative pictures of a NI mouse, (e) an infected mouse that died with ALI/ARDS showing hemorrhagic lungs and a large amount of pleural effusion, and (f) a mouse that died with HP showing pale and grayish lungs and no pleural effusion. Representative histopathological images of lungs from (g) NI mice and infected DBA/2 mice that died with (h) ALI/ARDS and (i) HP on the 10th and 21st days after infection, respectively. The arrow points to the hyaline membranes in the lungs of the DBA/2 mice that died with (j) ALI/ARDS stained with hematoxylin-eosin and (k) stained with phosphotungstic acid hematoxylin. The bar corresponds to 100 μm. HP: hyperparasitemia; ALI/ARDS: acute lung injury/acute respiratory distress syndrome.

Figure 2

Radiography of the lungs, hypoxemia, and body temperature over time. (a) From left to right, X-rays from NI and infected DBA/2 mice that died with ALI/ARDS and HP showing different lung opacification scores on the 7th DAI. (b) Lung opacification scores on the 7th DAI. Mice that will later die with ALI/ARDS have a higher lung opacification score compared with mice that will die with HP (n = 12; *P ≤ 0.05, Mann-Whitney test of scores taken from two separate experiments). NI mice do not have any lung opacification and are assigned score zero. (c) PaO₂/FiO₂ values in P. berghei ANKA-infected mice on the 7th DAI. Results from three grouped experiments (n = 13 mice, *P ≤ 0.05; Mann-Whitney test). (d) Body temperatures in DBA/2 mice infected with P. berghei ANKA slightly increased on the 5th DAI from 37.1°C in the NI mice to 37.3 in the ALI/ARDS mice and 37.4°C in the HP mice). However, the temperatures dropped and the mice became hypothermic (especially the ALI/ARDS mice), with mean temperatures of 33.0°C on the 7th DAI and 32.8°C on the 9th DAI. Results are from three grouped experiments (n = 31 mice; ***P ≤ 0.001, two-way ANOVA with Bonferroni post test). Data (d) represents means and SEM. The dashed line represents the mean value of NI mice. NI: noninfected mice; ALI/ARDS: acute lung injury/acute respiratory distress syndrome; HP: hyperparasitemia.

Figure 3

Breathing patterns and parasitemia from ALI/ARDS and HP mice over time. (a) and (b) Breathing patterns and (c) parasitemia from DBA/2 mice infected with P. berghei ANKA that developed ALI/ARDS and HP over time. (a) There was no evidence on the 5th and 9th DAI that the ALI/ARDS and HP mice had different breathing patterns. However, on the 7th DAI, there was evidence that the ALI/ARDS mice had a higher enhanced pause (Penh) (a) and a lower respiratory frequency (b) than the HP mice. Parasitemia increased over time in both groups (c). Results are representative from three independent experiments (n = 11 mice/experiment; *P ≤ 0.05, two way ANOVA with Bonferroni post test). The dashed line represents the mean value of NI mice; NI: noninfected mice; ALI/ARDS: acute lung injury/acute respiratory distress syndrome; HP: hyperparasitemia.

Figure 4

Breathing patterns and parasitemia could be used to group mice into two main clusters. Ward's linkage cluster analysis illustrates the distance between the physiological cluster patterns in DBA/2 mice infected with P. berghei ANKA that developed ALI/ARDS and HP. Values were measured on the 7th DAI. The data are from 13 independent experiments; n = 142 mice. Group 1 = 88.46% of individuals with ALI/ARDS and 11.53% with HP; group 2 = 57.77% of individuals with ALI/ARDS and 42.22% with HP; group 3 = 19.35% individuals with ALI/ARDS and 80.64% with HP; group 4 = 21.42% of individuals with ALI/ARDS and 78.57% with HP. ALI/ARDS: acute lung injury/acute respiratory distress syndrome; HP: hyperparasitemia.

Figure 5

A murine model to predict malaria-associated ALI/ARDS. (a) Penh (enhanced pause), (b) respiratory frequency, and (c) parasitemia measured on the 7th DAI. The sacrificed mice were classified according predictive model, using the parameter cut-offs measured from the survival mice and applied to the sacrificed mice. DBA/2 mice infected with P. berghei ANKA and their breathing parameters were measured in plethysmograph chambers (BUXCO Electronics, USA). Note that these three parameters are similar between the survival group and sacrificed group; (n = 11 mice/group; *P < 0.05, **P < 0.005, Mann-Whitney test). ALI/ARDS: acute lung injury/acute respiratory distress syndrome; HP: hyperparasitemia; bpm: beats per minute. Results are representative of more than 5 independent experiments.

Figure 6

Increased vascular permeability and serum VEGF protein confirmed the predictive criteria for malaria-associated ALI/ARDS. (a) Serum VEGF protein in DBA/2 mice infected with P. berghei ANKA on the 7th DAI was measured by ELISA. The VEGF levels are higher in the mice classified as likely to die with ALI/ARDS compared with HP mice (according to the proposed predictive criteria). The data represent fold increases in relation to NI mice taken from three experiments; (n = 28 mice; **P < 0.005, Mann-Whitney test). (b) Lung vascular permeability in DBA/2 mice infected with P. berghei ANKA 7th DAI, assessed using Evans Blue. The vascular permeability is higher in the mice classified as likely to die with ALI/ARDS compared with the HP mice (according to the proposed predictive criteria). The data represent fold increased in relation to NI mice taken from three experiments; (n = 51 mice; *P < 0.05, Mann-Whitney test). Bars represent means and SEM. The images represent (c) NI: noninfected mice, (d) ALI/ARDS: acute lung injury acute respiratory distress syndrome, and (e) HP: hyperparasitemia.