Low-Volume Ex Situ Lung Perfusion System for Single Lung Application in a Small Animal Model Enables Optimal Compliance With " Reduction" in 3R Principles of Animal Research

Abstract

Ex situ lung perfusion (ESLP) is used for organ reconditioning, repair, and re-evaluation prior to transplantation. Since valid preclinical animal models are required for translationally relevant studies, we developed a 17 mL low-volume ESLP for double- and single-lung application that enables cost-effective optimal compliance "reduction" of the 3R principles of animal research. In single-lung mode, ten Fischer344 and Lewis rat lungs were subjected to ESLP and static cold storage using STEEN or PerfadexPlus. Key perfusion parameters, thermal lung imaging, blood gas analysis (BGA), colloid oncotic pressure (COP), lung weight gain, histological work-up, and cytokine analysis were performed. Significant differences between perfusion solutions but not between the rat strains were detected. Most relevant perfusion parameters confirmed valid ESLP with homogeneous lung perfusion, evidenced by uniform lung surface temperature. BGA showed temperature-dependent metabolic activities with differences depending on perfusion solution composition. COP is not decisive for pulmonary oedema and associated weight gain, but possibly rather observed chemokine profile and dextran sensitivity of rats. Histological examination confirmed intact lung architecture without infarcts or hemorrhages due to optimal organ procurement and single-lung application protocol using our in-house-designed ESLP system.

Keywords: ex situ lung perfusion/ESLP; ex vivo lung perfusion/EVLP; rat model; single lung; small animal model.

After lung procurement from Lewis and Fischer 344 rats, the left lung was perfused ex situ, while the right lung was kept statically cold as a control. In addition to thermal imaging, lung histology and analyses of lung weight, key perfusion parameters, blood gas analysis, cytokines, and colloidal oncotic pressure were performed. Figure was created with BioRender.com.

FIGURE 1

Schematic overview of the in-house-designed rat ESLP system. The left lung of Lewis and Fischer 344 rats was treated with ESLP for 120 min, while the right lung was treated with SCS for the same period of time. The circuit comprised the double-walled organ chamber, the reservoir with the corresponding pH control unit, the roller pump, the heat exchanger, and the water bath. Temperature and pressure were controlled via integrated three-way stopcocks, which were connected to the corresponding monitoring system.

FIGURE 2

ESLP monitoring. Applied pump flows (A), systolic pulmonary arterial pressures (B), pH (C), and perfusate temperatures (D) were recorded in the left lung during ESLP. Data are expressed as mean ± SD. Statistical significance is indicated if p < 0.05 or as “n.s” if there is no significance.

FIGURE 3

Thermographical data acquisition and analysis of the ex situ perfused lungs. Representative native photos and corresponding thermal images of the lungs are shown at various time points as well as the respective average temperature of the lung surface (A–G). Graphical representation shows the surface temperatures of the different test groups over time (H). Data are expressed as mean ± SD. Statistical significance is indicated if p < 0.05 or as “n.s” if there is no significance.

FIGURE 4

Blood gas analyses. Time-dependent representation of pO₂ (mmHg), pCO₂ (mmHg), sodium (mmol/L), potassium (mmol/L), and glucose (mmol/L) of left lungs under ESLP (A) and right lungs under SCS (B). Data are expressed as mean ± SD. Statistical significance is indicated if p < 0.05 or as “n.s” if there is no significance.

FIGURE 5

Colloid oncotic pressure and lung weight gain. Time-dependent representation of colloid oncotic pressures (COD) and lung weight gain (g) of left lungs under ESLP (A) and right lungs under SCS (B). Data are expressed as mean ± SD for COD and mean ± SEM for lung weight gain. Statistical significance is indicated if p < 0.05 or as “n.s” if there is no significance.

FIGURE 6

Overview of histological lung morphology and analysis of lung injury score. Representative images of hematoxylin-eosin-stained lungs (×50 magnification, scale bar 200 µm) and the classification of the lung injury score (0 = not present, 1 = present) by assessing the extent of atelectasis, oedema, widened alveolar spaces, and visible perfusate in the different test groups according to ESLP (A) and SCP (B). Data are expressed as mean ± SD. Statistical significance is indicated if p < 0.05 or as “n.s” if there is no significance.

FIGURE 7

Cytokine and chemokine analysis in the ESLP perfusate. Time-dependent representation of CXCL1/KC (pg/mL), CCL2/MCP-1 (pg/mL), CCL3/MIP-1α (pg/mL), CCL5/RANTES (pg/mL), and MIP-3α (pg/mL) of the left lung under ESLP. Data are expressed as mean ± SEM. Statistical significance is indicated if p < 0.05 or as “n.s” if no significance.