Endocrine pathology in young rabbits with cystic fibrosis

Abstract

Background: Cystic fibrosis (CF) is an autosomal recessive genetic disorder caused by loss-of-function mutations in the CF transmembrane conductance regulator gene. CF-related pancreatic lesions are known to cause exocrine dysfunctions such as pancreatic insufficiency, and endocrine dysfunctions, including CF related diabetes. In a previous study, we generated CF rabbits using CRISPR/Cas9-mediated gene editing.

Methods: CF rabbits were subjected to histological analysis with a focus on CF associated pancreatic lesions. Endocrine function related assays were conducted to evaluate CF related pancreatic endocrine disorders in these animals.

Results: We report that CF rabbits develop spontaneous pancreatic lesions at a young age, characterised by pancreatic inflammation and fibrosis, vacuolar degeneration, epithelium mucus-secretory cell metaplasia, and pancreatic duct dilation. The size of the pancreatic islets in the CF rabbits is significantly smaller than that of the wild type animals. Consistent with these pathological findings, young CF rabbits exhibited signs of pancreatic endocrine related disorders such as lower insulin levels and impaired glucose metabolism.

Conclusions: Our results suggest that the CF rabbit could serve as a valuable model for translational research on CF related pancreatic endocrine dysfunction.

Keywords: Abnormal pancreatic endocrine function; CF rabbits; Cystic fibrosis.

Figure 1. Representative histological sections of the pancreas of rabbits with cystic fibrosis (CF). Histological analysis was conducted on pancreas samples from wild-type (WT) rabbits (n=3) and rabbits with CF (n=5). Representative images are shown here. (A) H&E-stained sections of WT pancreas. A1: zoomed-in view of the area labelled in panel A. (B and C) H&E-stained sections of CF pancreas. B1, B2, B3 and C1: zoomed-in views of the areas labelled in panels B and C. B1 shows exocrine pancreatic tissue fibrosis (five-pointed star); B2 depicts dilated pancreatic ducts filled with eosinophilic material (*); B3 presents proliferation of the epithelium in the pancreatic duct (empty arrow); C1 shows infiltration of inflammatory cells (#). (D) Representative vacuolar degeneration indicated by the ‘$’ sign. (E) Representative acinar atrophy and decreased zymogen granules indicated by arrowheads. (F) Epithelium mucus secretory cell metaplasia indicted by arrows in Alcian blue (AB) stain. (G) Epithelium mucus secretory cell metaplasia indicated by arrows in periodic acid-Schiff (PAS) stain. Scale bar: 200 μm.

Figure 2. Morphometric analysis of pancreatic islets in wild-type (WT) rabbits and rabbits with cystic fibrosis (CF). (A) Insulin immunostaining of pancreatic sections from WT animals (n=3) and animals with CF (n=3) was conducted. Representative images are shown here. The high-power magnification in the right panel shows the boxed region in the left panel, demonstrating islet size. The scale bar in the left panel is 50 μm; the scale bar in the right panel is 25 μm. (B) Total β-cell area per pancreas for WT animals (n=3) and animals with CF (n=3). (C) Quantification of islet size in WT (n=3) and CF (n=3) pancreas. (D) Frequency distribution of individual islet sizes in WT (n=3) and CF (n=3) pancreas. The pancreas area, β-cell area and islet size were measured using the ImageJ program. The β-cell area (%) was calculated as the ratio of (sum of β-cell area)/(total pancreas area). IHC, immunohistochemistry.

Figure 3. Compromised glucose metabolism in a subset of rabbits with cystic fibrosis (CF). (A) Fasting blood glucose and insulin levels in rabbits with CF (n=10) and wild-type (WT) (n=10) rabbits. Haemoglobin A1c (HbA1c) levels in rabbits with CF (n=6) and WT (n=6) rabbits (B) Intravenous glucose tolerance test (IVGTT) curves and corresponding areas under the curve (AUCs) for rabbits with CF (n=10) and WT (n=10) rabbits. (C) IVGTT curves and corresponding AUCs of rabbits with CF with signs of indeterminate glucose tolerance (INDET) (n=4) compared with rabbits with CF without signs of INDET (n=6). (D) IVGTT curves and corresponding AUCs of female rabbits with CF (n=6) and male rabbits with CF (n=4). ns, not significant.