Endobronchial perfluorocarbon reduces inflammatory activity before and after lung transplantation in an animal experimental model

Abstract

Background: The aim of this study was to evaluate the use of liquid perfluorocarbon (PFC) as an adjuvant substance for lung preservation and assess its role in pulmonary protection after transplantation.

Methods: Seventy-two rat lungs were flushed with low-potassium dextran (LPD) solution and randomized into three main groups: control with LPD alone and experimental with 3 (PFC3) and 7 mL/kg (PFC7) of endobronchial PFC instilled just after harvest. Each group was divided into four subgroups according to preservation time (3, 6, 12, and 24 hours). Afterwards, we performed lung transplantation using rat lungs preserved for 12 hours with LPD alone or with 7 mL/kg of endobronchial PFC.

Results: There was a significant increase in oxidative stress in the control group at 6 h of cold ischemic time compared with the PFC3 and PFC7 groups. The apoptotic activity and NF-κB expression were significantly higher in the control group compared with the PFC groups at 3, 12, and 24 h of cold preservation. After transplantation, the NF-κB, iNOS, and nitrotyrosine expression as well as caspase 3 activity were significantly lower in the PFC groups.

Conclusion: The use of endobronchial PFC as an adjuvant to the current preservation strategy improved graft viability.

Figure 1

Apoptosis and inflammatory activity during lung preservation. (a) Effects of different periods of lung preservation on pulmonary lipid peroxidation. Using the TBARS assay, there was a significant decrease in lipid peroxidation at 3 hours of preservation in the PFC7 group (*P > 0.05). The control group showed a significant increase in lipid peroxidation at 6 h (^# P > 0.05). Data are means ± standard deviation of the mean (P > 0.05). (b) Caspase 3 activity at different times of lung preservation. There is a significant increase in caspase 3 activity in the control group at 6, 12, and 24 hours (*P < 0.001). Data are means ± SD of the mean. (c) NF-κB expression (subunit p65) in the lung tissue at different times of lung preservation. A significant increase in the expression of p65 was observed in the control group at 3, 12, and 24 hours compared with the animals that used PFC (*P < 0.05). PFC7 reduced the expression of p65 at 24 hours of preservation compared with PFC3 (^# P < 0.05).

Figure 2

TUNEL staining after the different periods of cold ischemia. DAPI and FITC staining: DAPI-positive cells (nucleated cells) are blue and FITC-positive cells (TUNEL-positive) are green. There is predominance of TUNEL-positive cells in PFC 3 group at 12 h of cold preservation.

Figure 3

Apoptosis and inflammatory activity after lung transplantation. (a) Expression of NF-κB (subunit p65) in the lung tissue after transplantation. There was an increased expression of NF-κB subunit p65 in the LTx group compared with the LTx/PFC group (*P < 0.05). The images are representative samples of the groups. (b) Caspase 3 expression in the lung tissue after transplantation. There was a significant reduction in the expression of caspase 3 in the LTx/PFC group (*P < 0.05) compared with the LTx group. The images are representative samples of the groups. (c) Effect of lung transplantation on iNOS activity. iNOS activity was significantly higher in the LTx group (*P < 0.05). These results indicate that LTx induced the overexpression of iNOS, and the PFC treatment reduced iNOS expression. The images are representative samples of the groups. (d) Expression of nitrotyrosine in the lung tissue after transplantation. Nitrotyrosine expression was significantly lower in the LTx/PFC group compared with the LTx group (*P < 0.05). The images are representative samples of the groups.

Figure 4

Immunofluorescence staining of caspase 3 (a) and IκB (b). Cy3-positive cells (nucleated cells) are in red. There was an increased number of apoptotic cells by caspase 3 staining in the LTx group (A1) and an inhibition of phosphorylation of the inhibitor IκB in the LTx/PFC group (B2). ((A1) and (B1)—LTx group; (A2) and (B2)—LTx/PFC group.)

Figure 5

Histological score (HIS) after the lung transplantation. Lung injury was characterized by perivascular edema, intra-alveolar hemorrhage, and interstitial and intra-alveolar leukocyte infiltration in the LTx group (a) compared with the LTx/PFC group (b). The HIS demonstrated significantly more lung injury in the Ltx group (*P < 0.05) (200x magnification).