Stereological assessment of the blood-air barrier and the surfactant system after mesenchymal stem cell pretreatment in a porcine non-heart-beating donor model for lung transplantation

Abstract

More frequent utilization of non-heart-beating donor (NHBD) organs for lung transplantation has the potential to relieve the shortage of donor organs. In particular with respect to uncontrolled NHBD, concerns exist regarding the risk of ischaemia/reperfusion (IR) injury-related graft damage or dysfunction. Due to their immunomodulating and tissue-remodelling properties, bone-marrow-derived mesenchymal stem cells (MSCs) have been suspected of playing a beneficial role regarding short- and long-term survival and function of the allograft. Thus, MSC administration might represent a promising pretreatment strategy for NHBD organs. To study the initial effects of warm ischaemia and MSC application, a large animal lung transplantation model was generated, and the structural organ composition of the transplanted lungs was analysed stereologically with particular respect to the blood-gas barrier and the surfactant system. In this study, porcine lungs (n = 5/group) were analysed. Group 1 was the sham-operated control group. In pigs of groups 2-4, cardiac arrest was induced, followed by a period of 3 h of ventilated ischaemia at room temperature. In groups 3 and 4, 50 × 10⁶ MSCs were administered intravascularly via the pulmonary artery and endobronchially, respectively, during the last 10 min of ischaemia. The left lungs were transplanted, followed by a reperfusion period of 4 h. Then, lungs were perfusion-fixed and processed for light and electron microscopy. Samples were analysed stereologically for IR injury-related structural parameters, including volume densities and absolute volumes of parenchyma components, alveolar septum components, intra-alveolar oedema, and the intracellular and intra-alveolar surfactant pool. Additionally, the volume-weighted mean volume of lamellar bodies (lbs) and their profile size distribution were determined. Three hours of ventilated warm ischaemia was tolerated without eliciting histological or ultrastructural signs of IR injury, as revealed by qualitative and quantitative assessment. However, warm ischaemia influenced the surfactant system. The volume-weighted mean volume of lbs was reduced significantly (P = 0.024) in groups subjected to ischaemia (group medians of groups 2-4: 0.180-0.373 μm³) compared with the sham control group (median 0.814 μm³). This was due to a lower number of large lb profiles (size classes 5-15). In contrast, the intra-alveolar surfactant system was not altered significantly. No significant differences were encountered comparing ischaemia alone (group 2) or ischaemia plus application of MSCs (groups 3 and 4) in this short-term model.

Keywords: lung transplantation; mesenchymal stem cells; non-heart-beating donor; stereology; surfactant system.

Figure 1

Light micrographs of lung sections of sham‐operated control group (a), non‐heart‐beating donor (NHBD) group (b), mesenchymal stem cell (MSC)vasc group (c) and MSCbronch group (d). Toluidine blue staining. Images show intact lung parenchyma with air‐filled alveoli free from oedema and slender alveolar septa. Peribronchovascular space was narrow without oedema fluid accumulation. AL, alveolar lumen (i.e. central lumina of ductus alveolares or sacculi alveolares or lumina of alveoli); AS, alveolar septum; BL, bronchiolar lumen; PBV, peribronchovascular space.

Figure 2

Transmission electron micrographs showing details of the alveolar septum. Sham‐operated control group (a, e), non‐heart‐beating donor (NHBD) group (b, f), mesenchymal stem cell (MSC)vasc group (c, g), MSCbronch group (d, h). (a–d) Alveolar septum with two adjacent alveoli. The alveoli are free from oedema fluid. The alveolar septum contains an open capillary, which is separated from the alveolar air space by the blood–air barrier (bab). Both the thin and the thick part of the bab are continuous. (e–h) Thin part of the bab. No swelling is seen in the epithelium, basal lamina or endothelium. AL, alveolar lumen; CL, capillary lumen; EC, endothelial cell (nucleus); 1, thin part of bab; 2, thick part of bab; I, interstitial layer of bab; F, fibroblast (nucleus); EF, elastic fibre; E, erythrocyte; M, intravascular macrophage; EP, alveolar epithelium; BL, basal lamina; EN, capillary endothelium.

Figure 3

Transmission electron micrographs of cellular and intra‐alveolar surfactant system. (a) Type II cell, sham group. The cytoplasm contains numerous lamellar bodies (lbs). (b–d) Intra‐alveolar surfactant subtypes. AL, alveolar lumen; CL, capillary lumen; TII, type II cell; LB, lamellar body; LBL, lamellar body‐like form; TM, tubular myelin; MLV, multilamellar vesicle; ULV, unilamellar vesicle.

Figure 4

Volume weighted mean volume of lamellar bodies (lbs). Individual data points and group medians. The sham control group differed significantly from all other groups (P = 0.024). Sham, sham control group; NHBD, non‐heart‐beating donor group; MSCvasc, pulmoarterial application of mesenchymal stem cells; MSCbronch, endobronchial application of mesenchymal stem cells.

Figure 5

Profile size distribution of lamellar bodies (lbs). Lb size was analysed using the point sampled intercepts method. For all sampled lbs, the profile intercept length was measured and assigned to one of 15 profile size classes. The group medians of observation numbers in class 1, sum of classes 2–4 and sum of classes 5–15 are depicted. Sum of classes 5–15 differed significantly between sham control group and all other groups (P = 0.031). Sham, sham control group; NHBD, non‐heart‐beating donor group; MSCvasc, pulmoarterial application of mesenchymal stem cells; MSCbronch, endobronchial application of mesenchymal stem cells.