The porcine lung as a potential model for cystic fibrosis

Abstract

Airway disease currently causes most of the morbidity and mortality in patients with cystic fibrosis (CF). However, understanding the pathogenesis of CF lung disease and developing novel therapeutic strategies have been hampered by the limitations of current models. Although the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) has been targeted in mice, CF mice fail to develop lung or pancreatic disease like that in humans. In many respects, the anatomy, biochemistry, physiology, size, and genetics of pigs resemble those of humans. Thus pigs with a targeted CFTR gene might provide a good model for CF. Here, we review aspects of porcine airways and lung that are relevant to CF.

Fig. 1.

Strategy for generating pigs with targeted cystic fibrosis transmembrane conductance regulator (CFTR) gene. Fibroblasts were obtained from day 35 pig fetuses. Genomic DNA extracted from fetal fibroblasts was used as a template to generate a gene targeting vector that included the null CFTR allele (red X). Following introduction of the gene targeting vector and antibiotic selection, cells were screened for correctly targeted clones. Donor cells or donor nuclei were transferred to enucleated oocytes. Following activation, nuclear transfer embryos were implanted in a surrogate sow. After a 114-day gestation period, resulting piglets were born and found to be CFTR-null heterozygotes. NT, nuclear transfer.

Fig. 2.

Photo of 6 male CFTR+/− piglets taken at 1 day of age.

Fig. 3.

Porcine CFTR-ΔF508 produces mature band C protein. Human and porcine wild-type CFTR (WT) and CFTR-ΔF508 (ΔF) were expressed in COS cells, immunoprecipitated, and phosphorylated. Bands B and C are indicated by arrows. Adapted from Ref. with permission.

Fig. 4.

A and B: periodic acid-Schiff stain at ×400 magnification of bronchus from a porcine fetus ∼2 wk before term gestation (A) and from a piglet at 6 wk of age (B). A: developing submucosal glands form as downgrowths from the surface airway epithelium through the smooth muscle layer. B: submucosal glands are more numerous and with maturation increase cytoplasmic magenta staining.

Fig. 5.

Effect of luminal Cl⁻ substitution in a perfused small bronchiole on transepithelial voltage (V_t). The substitution of gluconate, an impermeant anion, for permeable Cl⁻ in the lumen markedly hyperpolarized the V_t. This response indicates a predominant conductance that appears to be constitutively active. Adapted from Ref. with permission. NaGlu, sodium gluconate.

Fig. 6.

Anatomic structure of a submucosal gland. Secretory products from each region are vectorally moved toward the surface airway epithelium (SAE). Mt, mucous tubule with mucous cells; St, serous tubule and acini containing serous cells; Cd, collecting duct.

Fig. 7.

Mucociliary transport and clearance in the pig. A: ex vivo mucociliary transport. Porcine trachea was excised and maintained in a warm-humidified chamber, and particle movement was recorded. Image is composite of 3 separate images taken at times indicated. Yellow arrows indicate white Teflon particles. A brown tantalum disc in close proximity is also visible. B: whole lung mucociliary clearance. Pigs received nebulized ^99mTc-sulfur colloid, and gamma camera was used to measure particle clearance. Images at 0 and 45 min are shown. L and R refer to left and right. C: automated airway tree construction from high-resolution computed tomography (CT) (posterior view). Note that the pig has a cranial lobe (top right) that is not present in humans. D: radiopaque particle movement in the porcine lung. Particles were deposited by insufflation. We then obtained serial CT images to track particles. Yellow line indicates path of movement of 1 particle from a distal airway to the trachea. For clarity, movements of other particles are not shown, and not all are visible on this composite.

Fig. 8.

Response of the porcine lung to instillation of P. aeruginosa. A: percentage of macrophages and neutrophils in bronchoalveolar lavage liquid before and 3 h after bronchoscopic instillation of P. aeruginosa strain O1 (PAO1) into a left lower lobe. B: example of cells from bronchoalveolar lavage stained with Wright stain. C: the chest CT revealed an area of consolation (increased lung density due to air space disease) in the left lower lobe. D: sections of lung showed leukocytes and sloughed epithelia in the airways.

Fig. 9.

Effect of NaCl on antimicrobial activity of human and porcine airway surface liquid (ASL). Relationship between salt concentration and antimicrobial activity of human and porcine ASL is shown. Samples were mixed with NaCl to indicated concentrations, and P. aeruginosa killing was measured using a luminescence assay. In the absence of ASL, there was no bacterial killing. Luminescence units (L.U.) are expressed as % of control values and correlate with bacterial viability.

Fig. 10.

Porcine airway tree analysis. A: a porcine airway tree extracted from the multi-row detector X-ray CT (MDCT) scan data using VIDA Diagnostics software. B: the blue pathway in A was selected for airway measurements and linearized using computer processing. The airway lumen can then be measured automatically, accurately, and reproducibly throughout its length. Measurements such as lumen diameter (inner and outer), area (inner and outer), wall thickness, and wall area can be generated at any point in the airway. This allows for the heterogeneity of the lung to be quantified. The linear scale on the lower part of the image is the same in horizontal and vertical directions.

Fig. 11.

Micro-optical bronchoscopy showing porcine lung alveolar connective tissue as visualized using a catheter-based confocal microscopy system through the subtending airway (Mauna Kea Technologies). In this image the native alveolar connective tissue can be seen because of strong autofluorescence. In the presence of endogenously administered fluorescein, alveolar size, alveolar wall thickness, and cells specifically labeled with a fluorescent signal can also be imaged.

Fig. 12.

Transverse (A) and 3-dimensionally rendered (B) views of the paranasal sinuses in the pig. Note in A the thickening of the mucosal lining in the sinus on the right. Using thresholding techniques, the volume of air in the sinuses (blue areas in B) can be calculated.

Fig. 13.

Air trapping in the porcine lung. A: CT images from a 4-wk-old pig before and after nebulization with methacholine. TLC indicates an image taken at total lung capacity; FRC indicates an image taken at functional residual capacity; PRE indicates before methacholine; POST indicates after methacholine. Note the heterogeneous air trapping (air trapping appears as darker areas in the lung fields) in the image taken at FRC after methacholine. B: CT lung density at FRC before (black line) and after (gray line) methacholine. Data are histograms of number of voxels (a voxel is a small volume element from the CT image) at each Hounsfield unit. The Hounsfield scale is a quantitative scale for describing radiodensity; a lower number indicate less density (e.g., air), and a higher number indicates greater density (e.g., water). In the histogram from the lung after methacholine, the broader distribution of the histogram and the presence of a greater proportion of voxels at low Hounsfield units indicate that the lung has not deflated uniformly and that air trapping is present.