LIRI is due primarily to mechanisms that cause alveolar–capillary barrier, especially alveolar epithelial cell damage, and that increase pulmonary vascular permeability, which is associated with the formation of reactive oxygen intermediates, endothelial cell injury, cytokine activity, neutrophil activation, complement activation, and inflammation response [
18,
19].
Reactive oxygen species (ROS) generation
It is commonly believed that reperfusion lung injury is primarily based on the generation of ROS on reperfusion, which may cause cellular damage and apoptosis [
20,
21]. In hypoxic conditions, the inducible nitric oxide synthetase (iNOS) can be enhanced, which may lead to ROS-type cellular injury and increase oxidant radical byproducts, including the peroxynitrite anion [
22]. ROS have diverse actions on pulmonary tissue, including cell proliferation, gene transcription, smooth muscle contraction, and interaction with redox enzymes [
23]. The array of inflammatory mediators released into the circulation, in turn, governs the chemoattraction of various nonresident leukocytes that initiate the production of ROS [
24]. Of the chemoattracted cell types, neutrophils and monocytes possess the greatest ROS-generating potential [
25]. Therefore, during LIRI, the damage and apoptosis in the lung may be more serious.
The inflammatory response in our LIRI model
The inflammatory response plays a pivotal role in the development of LIRI after transplantation. This inflammatory process is characterized by the infiltration of PMNs and other inflammatory cells such as macrophages that release inflammatory mediators, including TNF-α.
Roles of macrophages and TNF-α
during LIRI in the PTE model Macrophages play a key role in the response to LIRI [
26]. They are known as “the defenders of human health” by phagocyting bacteria, viruses, foreign bodies, damaged cells, and necrotic tissue. In our study, the lung ultrastructural architecture showed that swelling of lamellar bodies occurred in some type II pneumocytes in the reperfusion group. Additionally, macrophages with long pseudopodia phagocytosed the swelling and vacuolar lamellar bodies. Moreover, disintegrated naked nuclei and other structures of the amorphous necrosis material were also observed in the alveolar cavity.
Macrophages may also promote the production of inflammatory mediators [
26]. Damaged cells spill cytoplasmic and nuclear components into the extracellular milieu, which activate macrophages, leading to the production of pro-inflammatory cytokines and chemokines, including TNF-α [
27]. TNF-α is a pro-inflammatory factor that is released by lung macrophages in early stages and plays an important role in initiating lung inflammation [
28]. Another study showed that there was a close correlation between pro-inflammatory factors and LIRI in rabbit models after the left pulmonary artery occlusion was released. TNF-α level was continuously elevated in the reperfusion group [
29]. In our experimental model, serum TNF-α concentrations in the reperfusion group increased significantly 6 h after reperfusion as compared with the baseline value and the value at 2 h after reperfusion. TNF-α concentrations were also much higher than those of the ischemia and sham groups. TNF-α released by macrophage is an objective index to evaluate the severity of inflammatory response during LIRI in PTE. Therefore, we can attenuate acute LIRI by reducing TNF-α level [
29]. TNF-α produced by macrophages can also lead to the recruitment of PMNs to the injured tissue [
27], which may be responsible for serious lung damage during LIRI in PTE.
Roles of PMN and MPO during LIRI in the PTE model After ischemia–reperfusion, PMN infiltration into the alveolar cavity increased significantly and PMNs are responsible for tissue damage [
30]. In our experimental model, alveolar PMN infiltration in the reperfusion group was much higher than that of the ischemia group, resulting in the destruction of alveolar structure as observed by H&E staining, including an incompletely destructed alveolar structure with a large number of exudative cells and exudation. MPO concentrations in the lung tissue are related to neutrophil activation [
31]. In this study, MPO concentrations from lung homogenates in the reperfusion group were much higher than those of the ischemia and sham groups. Meanwhile, the amount of alveolar PMNs in the lower lobar lung was positively correlated with the lung MPO, indicating PMN recruitment and progressive activation in the lung tissue. One study demonstrated that PMNs play an important role in LIRI in a model of rat lung transplantation and that the gas exchange of the transplanted lung could be improved by reducing alveolar PMN infiltration [
32]. In this study, PMN infiltration was also negatively correlated with the arterial blood PaO
2/FiO
2. Therefore, it is vital to control PMN activation to avoid excessive tissue damage during PTE reperfusion.
Interaction between PMNs and macrophages during LIRI in the PTE model The inflammatory mediators produced by macrophages can also stimulate the recruitment of neutrophils to the injured tissue. It has been shown in experimental models of inflammation and also in clinically relevant models that macrophage-derived chemokines (TNF-α) promote neutrophils’ egress from the vasculature [
27]. In this study, electron microscopy showed that PMNs were in close contact with the alveolar epithelial cells with vacuoles degeneration in the reperfusion group, and the amount of alveolar PMNs in the lower lobar lung was positively correlated with serum TNF-α concentrations. Thus, PMN recruitment to the injured lung tissue may be due to the increased concentrations of TNF-α produced by macrophages during LIRI in the chronic PTE model.
Apoptotic pneumocytes after ischemia–reperfusion When a cell is sufficiently injured, cell death occurs either through necrosis or apoptosis. Apoptosis is morphologically characterized by nuclear condensation and shrinkage followed by fragmentation of nuclear chromatin without typical inflammation. Apoptosis was determined by TUNEL in this study. Studies indicated that RPE clinical manifestations after interventions for PTE are similar to those of lung transplantation [
5‐
8]. A significant number of pneumocytes undergo apoptosis after reperfusion in the transplanted rat lung [
33]. In our experimental model, the number of apoptotic pneumocytes increased after reperfusion similarly to our previous study [
34]. The amount of apoptotic pneumocytes in the lower lobar lung was also negatively correlated with the arterial blood PaO
2/FiO
2 in the reperfusion and NO inhalation groups. Therefore, during LIRI in PTE, pneumocyte apoptosis may be attributed to the low PaO
2/FiO
2, which is similar to triggering apoptosis by exposure to certain environmental conditions such as hypoxic conditions [
20] and may result from the increased number of alveolar PMNs after reperfusion.