Timeline of Multi-Organ Plasma Extravasation After Bleomycin-Induced Acute Lung Injury

Abstract

Acute lung injury (ALI) is characterized by the abrupt onset of clinically significant hypoxemia in the context of non-hydrostatic pulmonary edema. Acute lung injury is associated with cytokine release and plasma extravasation (PEx) that can cause pulmonary edema and subsequently acute respiratory distress syndrome (ARDS). Therefore, it is critical we understand the relationship between ALI and lung PEx. In addition, it is also important to assess PEx in the lungs and other organs post-ALI since ALI/ARDS often causes multi-organ failure. We hypothesized that ALI induces time-dependent lung PEx, which promotes extravasation in the heart, liver, kidney, spleen, pancreas, and gastrointestinal (GI) tract, in a time-dependent manner. To test our hypothesis, we administered bleomycin or saline via tracheal intubation in 8-week-old Sprague Dawley rats. At the terminal experiments, Evans Blue was injected (IV) through the femoral vein to allow for the visualization of PEx. Plasma extravasation of desired organs was evaluated at 3-, 7-, 14-, 21-, and 28-days after bleomycin or saline treatment by evaluating Evans Blue concentrations calorimetrically at fluorescence excitation wavelength of 620 nm (bandwidth 10 nm) and an emission wavelength of 680 nm (bandwidth 40 nm). Data show that ALI induces lung PEx beginning at day 3 and peaking between 7 and 21 days. Extravasation was also seen in all organs at varying degrees beginning at day 3 and peaking between days 7 and 14. Resolution appears to start after day 21 and continues past day 28. We conclude that ALI caused by bleomycin incites a time-dependent PEx of the lungs and multiple other organs.

Keywords: ARDS; COVID-19; bleomycin; inflammation; multi-organ failure.

FIGURE 1

Bleo instillation induces plasma extravasation (PEx) visualized by the tissue content of Evans Blue. PEx begins to occur around day 3 (B) and peaks around day 7 (C). PEx subsequently plateaus at day 14 (D) and begins to resolve at day 21 (E) with resolution seen at day 28 (F). Day 7 control is shown in panel (A).

FIGURE 2

Body weight (A) and lung weight (B) for Bleo and sham rats (A). Lung edema was indirectly estimated by obtaining lung weight and body weight ratio (C). Bleo induces plasma extravasation within the lung at 7-, 14-, and 21-days (D). Mean ± SD. n = 5–7 per group. Comparing bleomycin with control group *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001.

FIGURE 3

Arterial blood gases in control, 1-week and 2-week post-Bleo rats. pCO₂, partial pressure of carbon dioxide; pO₂, partial pressure of oxygen; BEecf, base excess; TCO₂, total CO₂; sO₂, Oxygen saturation; Lac, lactate. Values are mean ± SD. n = 6 per group. *P < 0.05 vs. control. ^#P < 0.05 vs. Bleo 1w.

FIGURE 4

Bleo reduced organ weights at 7-days (A–D). Liver weight was reduced at day 3 (B). Heart weight was increased 28 days after Bleo (A). Mean ± SD. n = 5–7 per group. Comparing Bleo with control group *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001.

FIGURE 5

Bleo instillation caused plasma extravasation starting at day 3 for all organs except the duodenum (A–F). Resolution of plasma extravasation occurred after day 21 for all organs except the heart and pancreas (A–F). Mean ± SD. n = 5–7 per group. Comparing bleomycin with control group *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001.