Gastrointestinal pathology in juvenile and adult CFTR-knockout ferrets

Abstract

Cystic fibrosis (CF) is a multiorgan disease caused by loss of a functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in many epithelia of the body. Here we report the pathology observed in the gastrointestinal organs of juvenile to adult CFTR-knockout ferrets. CF gastrointestinal manifestations included gastric ulceration, intestinal bacterial overgrowth with villous atrophy, and rectal prolapse. Metagenomic phylogenetic analysis of fecal microbiota by deep sequencing revealed considerable genotype-independent microbial diversity between animals, with the majority of taxa overlapping between CF and non-CF pairs. CF hepatic manifestations were variable, but included steatosis, necrosis, biliary hyperplasia, and biliary fibrosis. Gallbladder cystic mucosal hyperplasia was commonly found in 67% of CF animals. The majority of CF animals (85%) had pancreatic abnormalities, including extensive fibrosis, loss of exocrine pancreas, and islet disorganization. Interestingly, 2 of 13 CF animals retained predominantly normal pancreatic histology (84% to 94%) at time of death. Fecal elastase-1 levels from these CF animals were similar to non-CF controls, whereas all other CF animals evaluated were pancreatic insufficient (<2 μg elastase-1 per gram of feces). These findings suggest that genetic factors likely influence the extent of exocrine pancreas disease in CF ferrets and have implications for the etiology of pancreatic sufficiency in CF patients. In summary, these studies demonstrate that the CF ferret model develops gastrointestinal pathology similar to CF patients.

Figure 1

Histopathology in the CF ferret pancreas. Sections of pancreata from four CF and one non-CF animals ranging from 3 to 8 months of age are shown. Pancreas sections from a pair of 8-month-old non-CF (A and B) and CF (C–F) ferrets (CF-1). A predominantly normal pancreas in both genotypes with structurally intact islets is shown in B and D. Focal lobular destruction of part of the pancreas (arrow, E) is noted in CF-1 with ductular enlargement and plugging with eosinophilic secretions (F). Boxed regions in A, C, and E are enlarged in B, D, and F, respectively. Photomicrographs of pancreatic sections representing the most common histological findings in three additional CF animals older than 3 months (G–L). There is significant fibrosis with ductular enlargement and plugging with eosinophilic secretions. Epithelial-like cellular clusters predominate in the CF pancreas (examples marked with arrowheads in G–L). Insets in A, C, G, I, and K are gross images of the pancreas (arrows). All sections were stained with H&E. Scale bars: 500 μm (A, C, E, and G); 200 μm (I); 100 μm (F and K); 75 μm (H); and 50 μm (B, D, J, and L).

Figure 2

Islets are highly disorganized in the CF ferret and human pancreata. Sections of pancreata from non-CF and CF ferrets and humans are shown as marked. Each column of photomicrographs represents a different individual. Insulin immunostaining of pancreatic sections from non-CF ferret (A and E), CF ferret (B and F), non-CF human (C and G), and CF human (D and H). Glucagon immunostaining of pancreatic sections from non-CF ferret (I and M), CF ferret (J and N), non-CF human (K and O), and CF human (L and P). Photomicrographs in E–H and M–P emphasize islet structure. The non-CF patient in C and G died of head trauma. The CF patient in D and H died of respiratory failure and was not diagnosed with cystic fibrosis-related diabetes. CF ferret and human islets, identified by insulin and glucagon staining, were present within fibrotic regions of the pancreas and characterized by large clusters of endocrine cells with poorly defined islet boundaries (arrows in B, D, J, and L). By contrast, non-CF islets were smaller with well-defined boundaries and were more evenly dispersed throughout the pancreas (A, C, I, and K). For additional histological examples, see Supplemental Figure S2. Scale bars: 500 μm (A–D and I–L); 50 μm (E–H and M–P).

Figure 3

A small subset of CF ferrets are pancreatic sufficient. A: Morphometric analysis of pancreatic histopathology in four CF animals that were pancreatic sufficient (PS) or pancreatic insufficient (PI) based on fecal EL-1 levels. The mean percentage of normal pancreas (±SEM) is shown for the analysis of three to four nonoverlapping sections from each animal. B: Fecal EL-1 levels at various ages from CF-14 and its age-matched non-CF control. C: Indicated amount of Viokase-V pancreatic enzymes were spiked into 1 g of CF feces lacking EL-1 activity or non-CF feces and the samples were then evaluated for EL-1 activity. D and E: Fecal EL-1 levels at various ages from the indicated CF animals and their age-matched non-CF control. These CF animals (CF-1 and CF-12) demonstrated near-normal pancreatic histology. The arrowhead (B and D) marks the age at which both the CF and non-CF animal were placed on Viokase-V pancreatic enzymes (ie, including all time points past the arrowhead). F: Summary of fecal EL-1 levels (means ± SEM) for N independent CF and non-CF control animals for three age groups as indicated. Comparison of non-CF fecal EL-1 levels between various age brackets demonstrated a significant increase in 0 to 30 versus 31 to 80 days (P < 0.001), but not 31 to 80 versus 81 to 150 days (P = 0.448), time points by Student's t-test. ∗P < 0.0001 between genotypes by Student's t-test. Dagger indicates two time points for which fecal EL-1 levels dipped below 200 μg/g feces (level considered to be pancreatic sufficient in CF humans).

Figure 4

Gallbladder abnormalities predominate in CF ferrets. Gross images of the liver and gallbladder and histological sections of the gallbladder from three CF animals and one non-CF animal, ranging from 3 to 8 months of age. Each row of photomicrographs represents a different animal. Gallbladders from non-CF (A–C) and CF (D–F) 8-month-old ferrets (CF-1). Gross appearance of the gallbladder in the non-CF animal (A) is indistinguishable from the CF animal (CF-1) (D). Histologically, the gallbladder between this non-CF (B and C) and CF animal (E–F) was also quite similar, with the only differences being decreased mucosal folds in the CF animal. Gallbladders from two additional CF animals older than 3 months of age represent the most commonly seen pathology (G–L). Gross appearance of the gallbladder is dark and blue in color in both CF animals (G and J). Multifocal hepatic necrosis (arrowheads, G). Histologically, the gallbladder in these CF animals (H, I, K, and L) demonstrates mucus accumulation (asterisks) and mucosal proliferation that formed cyst-like structures. All sections are stained with periodic acid-Schiff. Asterisks denote demonstrated mucus accumulation in CF gallbladders (H, I, K, and L). Scale bars: 200 μm (B, E, H, and K) and 100 μm (C, F, I, and L). gb, gallbadder.

Figure 5

Hepatic abnormalities are diverse and infrequent in CF ferrets. Liver sections from two non-CF animals (A, B, and M) and three CF animals (C–L and N). Unique animals are depicted in panels A and B, C and D, E and F, G, H, and N, I–L, and M. All animals range from 3 to 8 months in age, except for the 19-day-old CF animal in I–L. Arrowheads in E mark the boundaries of hepatic necrosis. Boxed regions in A and C are enlarged in B and D, respectively. Higher magnification photomicrographs of non-CF (M) and CF (N) littermates demonstrating lipid droplet accumulation in hepatocytes of the CF animal. The same CF animal is presented in G, H, and N. Insets are oil red O stained sections from the same animal demonstrating lipid droplets in red, non-CF (M) and CF (N). Hepatic triglycerides from CF and control non-CF littermates (O). Tissues from the animals marked by a red data point are shown in M and N. Data represents means ± SEM; N = 6 independent animals. All sections representing the main panels were stained with H&E. Scale bars: 1 mm (A and C); 200 μm (B, D, H, and I); 100 μm (E–G and J–L); and 25 μm for both main panel and inset (M and N). BDP, bile duct plugging; BF, biliary fibrosis; BH, biliary hyperplasia.

Figure 6

Abnormalities in the small intestine of CF animals. Small intestinal sections from a non-CF animal (A–C) and three CF animals (D–L) ranging from 3 to 8 months of age are shown. Each row of photomicrographs represents a different animal. H&E (A, D, G, and J) and periodic acid-Schiff (PAS)-stained sections (B, C, E, F, H, I, K, and L). A reduction in the height of villi is most notable in the CF animals (D and G). Mucous plugging of crypts (arrows, F, I, and L) in CF animals but not in the non-CF control (C) is also evident by PAS staining. Scale bars: 500 μm (A, D, G, and J); 200 μm (B, E, H, and K); and 25 μm (C, F, I, and L).

Figure 7

Bacterial overgrowth occurs in the CF intestine. A: Intestinal tissue at the level of the ileum (with fecal material included) was harvested from six non-CF and CF animals at 3 to 4 months of age and quantitative culture was performed under aerobic and anaerobic conditions on blood agar. Results depict the means ± SEM colony forming unit per mg tissue for N = 6 independent animals of each genotype. B: The types of bacteria cultured from the non-CF and CF intestinal samples. Results for bacterial identification combined all types of bacteria cultured from the small intestine and colon. The pie charts depict a semiquantitative prevalence of each strain of bacteria in animals of each genotype. Semiquantitative prevalence was calculated by assigning a categorical value of 1 if the bacterial strain was detected in a culture and summing these values for all animals in each genotype. Gram-stained small intestine sections from one non-CF (C and D) and two CF (E–H) animals. Abundant bacterial colonies (arrows) are present within the lumen and occasionally adhered to the epithelial surface in CF intestine. Scale bars: 500 μm (C); 150 μm (E and G); 25 μm (D, F, and H). I: The fecal microbiota of three pairs of CF and non-CF ferrets. The bar graph depicts the relative abundance of bacteria within each sample identified by culture-independent sequence analysis of fecal samples from each animal. Bacteria with a relative abundance ≥1% are shown at the genus level. The identifying label of each pair is referenced by the CF animal number (CF-1, CF-2, and CF-3) given in Table 1; KO-CF animal and control-paired non-CF animal. The longest living CF animals and their non-CF paired control were chosen for the analysis. Fecal samples were harvested at the following ages for each pair (CF-1 pair, 102 days; CF-2 pair, 162 days, and CF-3 pair, 103 days). Both CF and non-CF animals within each pair were given the same antibiotics and reared on the same jill until approximately 6 weeks of age (approximately 42 days). ∗P < 0.05 by Student's t-test using log transformed colony forming unit titers. Dagger indicates unique bacterial strains found more than once in only non-CF samples; double dagger (‡) indicates unique bacterial strains found more than once in only CF samples.

Figure 8

Pathology of the colon and rectum in CF ferrets. Histological sections from the colon from a non-CF (A–C) and CF (D–F) ferret stained with H&E. Insets in A and D are gross images of the colon (arrow) demonstrating its white color in the CF animal. Boxed regions in B and E are enlarged in C and F, respectively, and show lymphangiectasia with dilation of lymphatic vessels in the CF animal (F). Rectal prolapse in a CF ferret at 28 days of age; gross image (G) and H&E-stained histology sections (H and I). Boxed region in H is enlarged in I. Scale bars: 1 mm (A and D); 500 μm (B, E, and H); and 100 μm (C, F, and I).

Figure 9

Histological abnormalities observed in the gastric mucosa of CF ferrets. Gross images and histological images of the stomach from a non-CF (A–D) and two CF animals (E–L). Each row of images represents a single animal, depicting the gross image of the stomach (A, E, and I) and various magnification of the gastric mucosa (B–D, F–H, and J–L). In this CF animal (E–H), gastric pathology was noted grossly by the multifocal dark areas on the serosal surface of the stomach (E, inset). The stomach contents contained digested blood indicative of mucosal ulceration. Histologically, there was erosion and ulceration of the mucosa (arrow, F) with marked submucosal edema (bracketed, F) and inflammation (arrowheads, H). A different CF animal (I–L), with less severe mucosal erosion and submucosal edema and gastric gland dilation (asterisk in L). Interstitial edema and gastric gland dilation were the most common changes in CF animals. Scale bars: 200 μm (B, F, and J); 100 μm (C, G, and K); and 50 μm (D, H, and L).