Neutrophils Mediate Pulmonary Artery Thrombosis In Situ

Abstract

Pulmonary embolism is a life-threatening condition, which can result in respiratory insufficiency and death. Blood clots occluding branches of the pulmonary artery (PA) are traditionally considered to originate from thrombi in deep veins (usually in legs). However, growing evidence suggests that occlusion of the vessels in the lungs can develop without preceding deep vein thrombosis (DVT). In this work, we used an inferior vena cava (IVC) complete ligation model of DVT in Wistar rats to explore the possibility and mechanisms of PA thrombosis under the conditions where all routes of thrombotic mass migration from peripheral veins are blocked. We demonstrate that rats both with normal and reduced neutrophil counts developed thrombi in the IVC, although, neutropenia caused a substantial decrease in thrombus size and a shift from fresh fibrin toward mature fibrin and connective tissue inside the thrombus. Massive fibrin deposition was found in the PA branches in the majority of DVT rats with normal neutrophil counts, but in none of the neutropenic animals. Neutrophil ablation also abolished macroscopic signs of lung damage. Altogether, the results demonstrate that thrombi in the lung vasculature can form in situ by mechanisms that require local neutrophil recruitment taking place in the DVT setting.

Keywords: deep vein thrombosis; neutrophils; pulmonary embolism.

Figure 1

Thrombosis in the IVC: Volume (A), area (B) and weight (C) of thrombi in normal (Control) and neutropenic (NP) conditions. Horizontal line represents median. (D,E), sections of thrombi in the IVC of non-neutropenic and neutropenic rats, respectively, stained by the Martius Scarlet Blue (MSB) method. Red, fresh fibrin; blue, organized fibrin/connective tissue. Scale bar 1 mm. Representative images out of 10–15 experiments.

Figure 2

Macroscopic changes in the rat lungs: (A), normal intact rat lung without macroscopic changes. (B), diffuse (arrowheads) and local (arrows) sites of cyanosis; rat with normal neutrophil count after 48 h IVC ligation. (C), no signs of lung injury/malfunction in a neutropenic rat after 48 h IVC stenosis; (D), sporadic local site of cyanosis (arrow) near lung root in the sham-operated rat. RL, right lung; LL, left lung; H, heart. Representative images out of 10–15 experiments.

Figure 3

Microscopic changes in the branches of the PA (A–D) and area of fibrin in PA branches (E): (A), PA branch in an intact rat without thrombus inside. (B), PA branches of a non-neutropenic rat after 48 h IVC ligation, with firmly packed young fibrin meshwork adjacent to the vessel wall and occluding most of the vessel lumen. (C), PA branch of a neutropenic rat after 48 h IVC ligation, free of fibrin deposition, with yellow erythrocytes inside the vessel. (D), Separate loose young fibrin fibers in the PA branch of a sham-operated rat. (E), Area of fibrin in PA branches was measured by ImageJ in random fields of view (one per section). Statistical comparison was performed by Kruskal–Wallis test. NS, non-significant. N = 10 (intact), 15 (48 h IVC stenosis/DVT), 10 (48 h IVC stenosis/DVT + neutropenia), 10 (sham). VW, vessel wall; VL, vessel lumen; Leu, leukocytes; Fib—fibrin, Er—erythrocytes. Scale bar 200 μm. Representative images out of 10–15 experiments.