Characteristic Multiorgan Pathology of Cystic Fibrosis in a Long-Living Cystic Fibrosis Transmembrane Regulator Knockout Murine Model
2004; Elsevier BV; Volume: 164; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)63234-8
ISSN1525-2191
AutoresPeter R. Durie, Geraldine Kent, M. J. Phillips, Cameron Ackerley,
Tópico(s)Congenital Ear and Nasal Anomalies
ResumoThe lack of an appropriate animal model with multiorgan pathology characteristic of the human form of cystic fibrosis has hampered our understanding of the pathobiology of the disease. We evaluated multiple organs of congenic C57BL/6J cystic fibrosis transmembrane regulator (Cftr)−/− and Cftr+/+ mice maintained from weaning on a liquid diet then sacrificed between 1 and 24 months of age. The lungs of the Cftr−/− animals showed patchy alveolar overdistention, interstitial thickening, and fibrosis, with progression up to 6 months of age. The proximal and distal airway surface was encased with mucus-like material but lacked overt evidence of chronic bacterial infections or inflammation. All Cftr−/− animals showed progressive liver disease, with hepatosteatosis, focal cholangitis, inspissated secretions, and bile duct proliferation; after 1 year of age there was progression to focal biliary cirrhosis. The intercalated, intralobular and interlobular ducts and acinar lumina of the exocrine pancreas, the parotid and submaxillary glands of the Cftr−/− animals were dilated and filled with inspissated material, as well as mild inflammation and acinar cell drop out. Quantitative measurements of the pancreas showed significant acinar atrophy and increased acinar volume in comparison with age-matched Cftr+/+ littermates. The ileal lumen and crypts were filled with adherent fibrillar material. After 3 months of age the vas deferens of the Cftr−/− animals could not be identified. None of the aforementioned pathological changes were observed in the Cftr+/+ littermates fed the same liquid diet. We show, for the first time, that long-lived C578L/6J Cftr−/− mice develop manifestations of cystic fibrosis-like disease in all pathologically affected organs in the human form of cystic fibrosis. The lack of an appropriate animal model with multiorgan pathology characteristic of the human form of cystic fibrosis has hampered our understanding of the pathobiology of the disease. We evaluated multiple organs of congenic C57BL/6J cystic fibrosis transmembrane regulator (Cftr)−/− and Cftr+/+ mice maintained from weaning on a liquid diet then sacrificed between 1 and 24 months of age. The lungs of the Cftr−/− animals showed patchy alveolar overdistention, interstitial thickening, and fibrosis, with progression up to 6 months of age. The proximal and distal airway surface was encased with mucus-like material but lacked overt evidence of chronic bacterial infections or inflammation. All Cftr−/− animals showed progressive liver disease, with hepatosteatosis, focal cholangitis, inspissated secretions, and bile duct proliferation; after 1 year of age there was progression to focal biliary cirrhosis. The intercalated, intralobular and interlobular ducts and acinar lumina of the exocrine pancreas, the parotid and submaxillary glands of the Cftr−/− animals were dilated and filled with inspissated material, as well as mild inflammation and acinar cell drop out. Quantitative measurements of the pancreas showed significant acinar atrophy and increased acinar volume in comparison with age-matched Cftr+/+ littermates. The ileal lumen and crypts were filled with adherent fibrillar material. After 3 months of age the vas deferens of the Cftr−/− animals could not be identified. None of the aforementioned pathological changes were observed in the Cftr+/+ littermates fed the same liquid diet. We show, for the first time, that long-lived C578L/6J Cftr−/− mice develop manifestations of cystic fibrosis-like disease in all pathologically affected organs in the human form of cystic fibrosis. Cystic fibrosis (CF) is an autosomal recessive condition that is caused by mutations in the cystic fibrosis transmembrane regulator gene (CFTR).1Rommens JM Iannuzzi MC Kerem B Drumm ML Melmer G Dean M Rozmahel R Cole JL Kennedy D Hidaka N Identification of the cystic fibrosis gene: chromosome walking and jumping.Science. 1989; 245: 1059-1065Crossref PubMed Scopus (2478) Google Scholar Although other genetic and environmental factors almost certainly contribute to disease pathobiology, CF disease arises from impaired ion conductance on epithelial cell surfaces, which increases the concentration and alters the viscosity of intraluminal contents.2Anderson MP Rich DP Gregory RJ Smith AE Welsh MJ Generation of cAMP-activated chloride currents by expression of CFTR.Science. 1991; 251: 679-682Crossref PubMed Scopus (425) Google Scholar Multiple organs are affected but the phenotype is extremely heterogeneous.3Oppenheimer EH Esterly JR Pathology of cystic fibrosis review of the literature and comparison with 146 autopsied cases.Perspect Pediatr Pathol. 1975; 2: 241-278PubMed Google Scholar, 4Scriver CR Fujiwara TM Cystic fibrosis genotypes and views on screening are both heterogeneous and population related.Am J Hum Genet. 1992; 51: 943-950PubMed Google Scholar CF may present in the neonate with bowel obstruction because of meconium ileus, whereas older patients may be plagued by intermittent chronic recurrent ileo-cecal obstruction from adherent mucofeculent material. In the airways, mucus clearance is impaired by the inability of the surface liquid to serve as a medium for ciliary boating of the mucigel. Obstruction of the airways results in chronic bacterial infection and rampant inflammation leads to end-stage lung disease and death. In its severest form, exocrine pancreatic disease begins in utero.5Imrie JR Fagan DG Sturgess JM Structural and developmental abnormalities of the exocrine pancreas in cystic fibrosis.Am J Pathol. 1979; 95: 697-707PubMed Google Scholar, 6Waters DL Dorney SF Gaskin KJ Gruca MA O'Halloran M Wilcken B Pancreatic function in infants identified as having cystic fibrosis in a neonatal screening program.N Engl J Med. 1990; 322: 303-308Crossref PubMed Scopus (115) Google Scholar Intraluminal obstruction by inspissated material within the acinar lumina and small ducts leads to progressive acinar atrophy7Cleghorn G Benjamin L Corey M Forstner G Dati F Durie P Age-related alterations in immunoreactive pancreatic lipase and cationic trypsinogen in young children with cystic fibrosis.J Pediatr. 1985; 107: 377-381Abstract Full Text PDF PubMed Scopus (26) Google Scholar and replacement by fat and fibrous tissue.3Oppenheimer EH Esterly JR Pathology of cystic fibrosis review of the literature and comparison with 146 autopsied cases.Perspect Pediatr Pathol. 1975; 2: 241-278PubMed Google Scholar Similar alterations occur in the small ducts of the parotid gland, but the pathological effects are less severe.8Blomfield J Rush AR Allars HM Brown JM Parotid gland function in children with cystic fibrosis and child control subjects.Pediatr Res. 1976; 10: 574-578Crossref PubMed Scopus (23) Google Scholar, 9Kolberg H Danielsson A Glitterstam K Henriksson R Marklund S Studies on parotid saliva in cystic fibrosis.Acta Paediatr Scand. 1982; 71: 321-322Crossref PubMed Scopus (11) Google Scholar The biliary canaliculi and small bile ducts are obstructed with inspissated material.3Oppenheimer EH Esterly JR Pathology of cystic fibrosis review of the literature and comparison with 146 autopsied cases.Perspect Pediatr Pathol. 1975; 2: 241-278PubMed Google Scholar, 10di Sant'Agnese PA Blanc WA A distinctive type of biliary cirrhosis of the liver associated with cystic fibrosis of the pancreas.Pediatrics. 1956; 188: 387-409Google Scholar, 11Craig JM Haddad H Shwachman H The pathologic changes in the liver in cystic fibrosis of the pancreas.Am Dis Child. 1957; 93: 357-369PubMed Google Scholar, 12Sokol RJ Durie P Recommendations for management of liver and biliary tract disease in cystic fibrosis.J Pediatr Gastroenterol Nutr. 1999; 28: S1-S13Crossref PubMed Scopus (196) Google Scholar, 13Griscom NT Capitanio MA Wagoner ML Culham G Morris L The visibly fatty liver.Radiology. 1975; 117: 385-389PubMed Google Scholar Cholangitis develops and the biliary ductules proliferate; with progression most patients develop focal biliary cirrhosis and in some cases extensive multilobular cirrhosis ensues. Some patients develop focal or diffuse hepatic steatosis.13Griscom NT Capitanio MA Wagoner ML Culham G Morris L The visibly fatty liver.Radiology. 1975; 117: 385-389PubMed Google Scholar Approximately 20 percent of patients present with bowel obstruction because of meconium ileus.14Kerem E Corey M Kerem B Durie P Tsui LC Levison H Clinical and genetic comparisons of patients with cystic fibrosis, with or without meconium ileus.J Pediatr. 1989; 114: 767-773Abstract Full Text PDF PubMed Scopus (116) Google Scholar Almost all affected men are infertile because of obstructive azoospermia. The sweat duct, which lacks intraluminal macromolecules, is the only CFTR-expressing ductular system that is not affected by obstructive pathological changes. The first murine model of CF was established by gene targeting of embryonic stem cells to disrupt the murine cftr gene.15Snouwaert JN Brigman KK Latour AM Malouf NN Boucher RC Smithies O Koller BH An animal model for cystic fibrosis made by gene targeting.Science. 1992; 257: 1083-1088Crossref PubMed Scopus (754) Google Scholar Other murine models have been developed to model human mutations, including the most common CFTR gene mutation ΔF508 (confers loss of phenylalanine) and models carrying common missense mutations (G551D, R117H).16Dorin JR Dickinson P Alton EW Smith SN Geddes DM Stevenson BJ Cystic fibrosis in the mouse by targeted insertional mutagenesis.Nature. 1992; 359: 211-215Crossref PubMed Scopus (237) Google Scholar, 17Delaney SJ Alton EW Smith SN Lunn DP Farley R Lovelock PK Thomson SA Hume DA Lamb D Porteous DJ Dorin JR Wainwright BJ Cystic fibrosis mice carrying the missense mutation G551D replicate human genotype-phenotype correlations.EMBO J. 1996; 15: 955-963Crossref PubMed Scopus (99) Google Scholar, 18Colledge WH Abella BS Southern KW Ratcliff R Jiang C Cheng SH MacVinish LJ Anderson JR Cuthbert AW Evans MJ Generation and characterization of a delta F508 cystic fibrosis mouse model.Nat Genet. 1995; 10: 445-452Crossref PubMed Scopus (190) Google Scholar, 19Zeiher BG Eichwald E Zabner J Smith JJ Puga AP McCray Jr, PB Capecchi MR Welsh MJ Thomas KR A mouse model for the delta F508 allele of cystic fibrosis.J Clin Invest. 1995; 96: 2051-2064Crossref PubMed Scopus (248) Google Scholar, 20Dickinson P Generation of a CF mutant mouse possessing the G480C mutation.in: 22nd European CF Conference. Berlin Book of Abstracts, Berlin1998: 143Google Scholar Most of these animals develop a range of intestinal pathology that may cause fatal complications after bowel obstruction at birth or when weaned to solid chow. However, pathological changes in other CF-affected organs, including the lungs, pancreas, and liver, were reported to be either mild or absent. Rozmahel and colleagues,21Rozmahel R Wilschanski M Matin A Plyte S Oliver M Auerbach W Moore A Forstner J Durie P Nadeau J Bear C Tsui LC Modulation of disease severity in cystic fibrosis transmembrane conductance regulator deficient mice by a secondary genetic factor.Nat Genet. 1996; 12: 280-287Crossref PubMed Scopus (358) Google Scholar used an exon-1 knockout CF mouse model (CftrM1HSC/CftrM1HSC) to demonstrate that the severity of intestinal disease was influenced by the genetic background, and that modifier genes, could, in part, compensate for the lack of functional intestinal CFTR. Subsequently, Kent and colleagues22Kent G Iles R Bear CE Huan LJ Griesenbach U McKerlie C Frndova H Ackerley C Gosselin D Radzioch D O'Brodovich H Tsui LC Buchwald M Tanswell AK Lung disease in mice with cystic fibrosis.J Clin Invest. 1997; 100: 3060-3069Crossref PubMed Scopus (144) Google Scholar demonstrated that Cftr−/−mice bred into a congenic C57BL/6J consistently developed spontaneous lung disease. Features of the lung disease included defective mucociliary transport, alveolar distention, and interstitial thickening with fibrosis and inflammation. In this report, we demonstrate that, with further aging, a CF-knockout murine model bred into a congenic C57BL/6J background consistently develops progressive pathology in multiple organs that are strikingly similar to the human manifestations of CF disease. All experimental protocols were conducted after review and approval by the institutional Animal Care Committee. Congenic C57BL/6J heterozygous breeding pairs were maintained on regular mouse chow and continuously bred. To maintain congenic status and prevent genetic drift, each new generation of mice was bred to wild-type C57BL/6J mice, obtained from the Jackson Laboratories (Bar Harbor, ME). This created the production stock from which experimental animals were obtained. Male and female wild-type animals were used in alternate breedings. Offspring were genotyped at 14 days of age using polymerase chain reaction analysis of tail clip DNA. To minimize bowel obstruction and optimize long-term viability, 20- to 23-day-old Cftr−/− mice and their Cftr+/+ littermates were weaned to a liquid diet (Liquidet F3107; Bioserve, Frenchtown, NJ) using glass liquid mouse feeders, prepared in sterile water according to the manufacturer's instructions.23Kent G Oliver M Foskett JK Frndova H Durie P Forstner J Forstner GG Riordan JR Percy D Buchwald M Phenotypic abnormalities in long-term surviving cystic fibrosis mice.Pediatr Res. 1996; 40: 233-241Crossref PubMed Scopus (67) Google Scholar Fresh diet and feeders, sterilized by autoclave, were replaced daily. Mice and their offspring were housed in a nonsterile conventional housing unit in microisolators cages, with corncob bedding changed daily, and provided with sterile water in addition to the liquid diet. The colony was maintained at a pathogen-free status by serological screening at a commercial laboratory. Mice were kept in a 12-hour light-dark cycle. Groups of Cftr−/− animals and their wild-type littermates were sacrificed at varying ages [1 to 2 months (Cftr−/−, n = 6; Cftr+/+, n = 6), 3 to 5 months (Cftr−/−, n = 13; Cftr+/+, n = 8), 9 to 12 months (Cftr−/−, n = 8; Cftr+/+, n = 3), and 15 to 24 months (Cftr−/−, n = 5; Cftr+/+, n = 5)]. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg/kg) and the abdominal cavity was opened. The inferior vena cava was cut and 1% glutaraldehyde and 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4) was perfused through the left ventricle. The airways were perfused through the larynx with the same fixative and the tissues were maintained at a constant pressure. The trachea, lungs, liver, pancreas, ileum parotid, and submaxillary and sublingual glands were harvested and immersed in the same fixative. Tissue from the reproductive tract of males older than 3 months of age, including the intact testis, epididymis, and vas deferens, was collected and fixed in formalin. Portions of the harvested organs were minced into 1-mm3Oppenheimer EH Esterly JR Pathology of cystic fibrosis review of the literature and comparison with 146 autopsied cases.Perspect Pediatr Pathol. 1975; 2: 241-278PubMed Google Scholar pieces, fixed for an additional 4 hours in the same fixative and then postfixed for 1 hour in phosphate-buffered 2% osmium tetroxide. Samples were then dehydrated before infiltration, in ascending concentrations of ethanol, embedded in Epon Araldite (JEOL, Peabody, MA), and polymerized at 60°C overnight. Areas of interest were selected by light microscopic examination of 0.5-μm-thick sections, cut by ultra-microtome and stained with toluidine blue. Ultrathin sections exhibiting a pale gold interference color from selected blocks were cut, mounted on grids, and stained with ethanol uranyl acetate and lead citrate before TEM examination. Portions of the trachea, bronchi, bronchioles, and lungs were prepared for SEM. Median longitudinal sections of lung as well as segments of the airways were postfixed with osmium tetroxide, dehydrated in ethanol, critical point-dried, mounted on stubs, and rendered conductive with a thin layer of sputter-coated gold palladium and then examined under SEM. All remaining tissues were fixed in formalin and prepared for routine histology. Slides were stained with hematoxylin and eosin (H&E), and either Masson's trichrome, diastase-digested periodic acid-Schiff reagent (PASD), or reticulin stain. Care was taken to orient the entire male reproductive tract, before embedding and cutting, to ensure correct alignment of all section. As a result, midline sections of the testes and cross sections of epididymis were consistently observed in all animals. Sections were evaluated for the presence or absence of the vas deferens; and if present for evidence of pathological changes in the vas deferens. We also assessed the epididymis and its luminal content, and staging of the seminiferous tubules. Random digital images of the lung alveolar wall from all animals were acquired at ×10,000 magnification with a digital charge-coupled device camera in the TEM (AMT Corp., Danvers, MA). A minimum of 50 fields was collected from each animal and the thickness of the interstitium was measured from five points within each image. Linear measures were expressed as mean and SE and significance was determined by multivariate analysis of variance analysis. Quantitative morphometry was performed on images captured by a digital charge-coupled device camera (Cool Snaps; Roper Scientific) attached to a light microscope. Images were all captured at a nominal magnification (×250) on at least four different slides of toluidine blue-stained pancreas using three different blocks from each animal. Pancreatic acinar volume (mm3), volume density (vol of acini/vol of exocrine pancreas), and numerical density (number of acini/mm3) were measured according to Weible's principles of stereology.24Weible ER Point counting methods. Methods for Biological Morphometry. Academic Press, New York1979: 101-159Google Scholar An image analysis program (Image Pro Plus; Iowa) used the following three formulae: 1. Acinar volume V=4/3·πmidsdot;r3where V is the average volume of an acinus and r is the average acinar radius. 2. Acinar numerical density NA=Na((4/π)·d)+t-2hwhere NA is the number of acini per unit volume of tissue (number/mm3), Na is the number of acini per area, d is the average diameter measured, t is the section thickness (0.5 μm), and h is the smallest recognizable acinar diameter (15 μm). 3. Acinar volume density percent VA=NAV·100where VA is the total volume of the acini in a unit volume of tissue (mm3/mm3), NA is the acinar numerical density, and V is the average volume of a single pancreatic acinar. Data from each animal group was expressed as a mean and SE and significance was calculated using multivariate analysis of variance analysis. As previously reported in younger animals,22Kent G Iles R Bear CE Huan LJ Griesenbach U McKerlie C Frndova H Ackerley C Gosselin D Radzioch D O'Brodovich H Tsui LC Buchwald M Tanswell AK Lung disease in mice with cystic fibrosis.J Clin Invest. 1997; 100: 3060-3069Crossref PubMed Scopus (144) Google Scholar sections of lung demonstrated widespread pathology. In comparison with age-matched wild-type littermates, alveolar architecture of the Cftr−/− mice was compromised (Figure 1, A and B). Patchy areas of acinar dilation, typical of obstructive lung disease, were observed in all Cftr−/− animals at all ages. Interstitial disease, which was detected as early as 1 month of age, showed progression up to 6 months of age. Sections stained with either Masson's trichrome or reticular stain demonstrated focal areas of intense interstitial staining indicative of an increase in collagen (Figure 1, A and B). After 6 months of age, there was little progression of lung pathology, although many of the alveoli showed greater variation in size. Inflammatory cells were frequently seen in the interstitium in animals less than 6 months of age, as were alveolar macrophages. When compared to wild-type animals of the same age, interstitial connective tissue was markedly increased. Immunoperoxidase staining for vimentin of some sections demonstrated an increase in the number of interstitial fibroblasts in the Cftr−/− animals up to 5 months of age. In older animals a marked decrease in the number of these cells was observed. Surprisingly, the number of inflammatory cells and macrophages in both the proximal and distal airways of the affected animals appeared to decrease with age. All of the affected animals showed positive PASD staining for acidic mucosubstances throughout the surface of the bronchioles and some alveoli also showed increased intensity and distribution. As previously demonstrated in young Cftr−/− mice,23Kent G Oliver M Foskett JK Frndova H Durie P Forstner J Forstner GG Riordan JR Percy D Buchwald M Phenotypic abnormalities in long-term surviving cystic fibrosis mice.Pediatr Res. 1996; 40: 233-241Crossref PubMed Scopus (67) Google Scholar the entire proximal and distal airways were diffusely encrusted in a thick coating of mucus-like material at all ages, which completely enveloped the ciliated surface. From 1 month of age, SEM of the surface of the bronchiolar epithelial cells of affected animals demonstrated that the cilia were embedded in this material, as were the alveolar walls (Figure 1; A to D). Morphometric determination of alveolar interstitial thickness demonstrated a significant age-related increase in the affected animals (Figure 2) up to 5 months of age. After this age no further changes occurred in the interstitium except for the lack of fibroblasts. After 3 to 5 months of age, alveolar interstitial thickening of the Cftr−/− animals was significantly greater than their age-matched wild-type littermates (Figure 2B). Type II pneumocytes were flatter than their wild-type counterparts and many lacked normal looking lamellar bodies. All affected animals showed focal and progressive hepatobiliary disease (Figure 3, Figure 4, Figure 4, Figure 6, Figure 7). Varying degrees of progressive bile duct proliferation, typical of obstruction, was observed in all Cftr−/− animals but not in their wild-type littermates. Focal portal tracts, in animals as young as 1 month of age, showed acute and chronic cholangitis. By 2 months of age polymorphonuclear leukocytes and chronic inflammatory cells were seen both within and surrounding focal biliary canaliculi and bile ducts. By 3 months of age there were varying degrees of periportal and bridging fibrosis, which progressed with age (Figure 3, B and C). Most animals sacrificed at 12 to 24 months of age developed focal biliary cirrhosis and some animals showed areas of advanced lobular cirrhosis (Figure 3D). Hepatic steatosis, which was common in the Cftr−/− animals, increased in severity with age. These changes could not be attributed to the liquid diet because steatosis was not evident in the wild-type littermates fed the same diet. Surprisingly little inspissating material was detectable in the common bile duct, the interlobular bile ducts, or biliary canaliculi when evaluated by light microscopy using H&E or PAS staining.Figure 4A: TEM of a portal tract from a 22-month-old wild-type animal containing a bile duct. The lumen (asterisk) appears void of any material. B: TEM of a portal tract from a 12-month-old Cftr−/− animal that has developed severe cholangitis showing macrophages and polymorphs both within the lumen of one of the ducts (asterisks) and in the periportal spaces. One of the ducts is filled with inspissated material. C: TEM of several hepatocytes from a 3-month-old Cftr−/− animal filled with lipid droplets, which is consistent with steatosis. D: Periportal fibrosis and duct proliferation. Bundles of collagen fibrils (arrowheads) and several bile ducts are seen in the field (asterisks). Original magnifications: ×2500 (A, B, D); ×5000 (C).View Large Image Figure ViewerDownload (PPT)Figure 6A: Toluidine blue-stained section of pancreas from a 12-month-old Cftr−/− animal showing periacinar and periductal (asterisk) inflammation (arrowheads) and acinar loss and atrophy. B: Histogram of acinar volume. Acinar volume increased with age in the Cftr−/− age groups. C: Histogram of percentage of acinar volume to tissue volume. Unlike the acinar volumethere was a progressive decrease. D: Histogram of acinar numerical density. Density also decreased, which is suggestive of acinar cell drop-out in the older Cftr−/− animals. Original magnification, ×400.View Large Image Figure ViewerDownload (PPT)Figure 7A: Toluidine blue-stained section from a 12-month-old Cftr−/− submaxillary gland. Note the periductal inflammation (arrowheads). The duct is filled with inspissated material (asterisk). B: TEM of material found in the lumen of a parotid duct from a 5-month-old Cftr−/− animal. In all of the affected animals many of the acinar and ductular lumina were filled with this material. Original magnifications: ×800 (A); ×10,000 (B).View Large Image Figure ViewerDownload (PPT) Many of the affected portal tracts contained inflammatory cells. These were found surrounding and within the bile duct lumina (Figure 4, B and C). Some of the bile ducts contained fibrillar material (Figure 4B). By 2 months of age, the majority of hepatocytes in the Cftr−/− animals contained lipid droplets consistent with steatosis (Figure 4C). Most biliary canaliculi of animals older than 9 months contained the same material and there was evidence of severe periportal fibrosis (Figure 4D). As young as 2 months of age the intercalated, intralobular, and interlobular pancreatic ducts of the Cftr−/− animals were increased in diameter in comparison with their wild-type littermates. Intraluminal obstruction with inspissated material that progressed with age was observed in the Cftr−/− animals but not in the wild-type littermates (Figure 5, A and C). After 6 months, foci of inflammatory cells and macrophages were evident in the exocrine pancreas of the Cftr−/− mice (Figure 6A). Affected animals older than 12 months of age showed subjective evidence of acinar atrophy. Pathological changes were not observed in the islets of Langerhans of the affected mice or in the wild-type animals. Furthermore none of the affected animals showed clinical evidence of diabetes mellitus. The results of the morphometric studies of the pancreas are summarized in (Figure 6; B, C, and D). Acinar volume of the wild-type animals, which reflects intraluminal volume, showed no significant alteration with aging. However, there was a modest age-related increase in mean acinar volume in the 3- to 6-month-old Cftr−/− animals and by 15 to 24 months of age, the acinar volume was significantly greater than that determined in the 1- to 2-month-old Cftr−/− animals (Figure 6B). Percentage of acinar volume expressed per unit pancreatic tissue volume showed no age-related changes in the Cftr+/+ mice. Also at 1 to 2 and 3 to 5 months of age the percentage of acinar to tissue volume of the exocrine pancreas in the Cftr−/− mice did not differ significantly from measurements in the wild-type, age-matched littermates. In contrast, the percentage of acinar volume to tissue volume in the Cftr−/− mice demonstrated a progressive decrease from 9 to 12 months of age, which became statistically significant at 15 to 24 months of age(Figure 6C). These morphometric results reflect intraluminal distention. The number of acini, expressed per unit volume of pancreatic tissue, was also determined. Although the wild-type animals showed no age-related change in acinar number, the Cftr−/− animals showed an age-dependent decrease that was statistically significant from 3 to 5 months of age in comparison with the 1- to 2-month-old Cftr−/− mice (Figure 6D). The number of pancreatic acini of the Cftr−/− animals was also significantly lower than that determined in the 9- to 12-month-old and 15- to 24-month-old wild-type littermates. These data provide objective evidence of progressive age-related acinar cell drop out in the Cftr−/− mice. Acinar lumina of the affected animals were filled with fibrillar material as young as 1 month of age and the zymogen granules were not as numerous or dense in comparison with acini from the wild-type littermates. The majority of the acinar lumina in the Cftr−/− animals were dilated and plugged with inhomogenous material (Figure 5D). Unlike, the wild-type animals the intralobular and interlobular ducts of the Cftr−/− animals were plugged with inspissated material (Figure 5, B and E) and the epithelial cells were flattened (Figure 4E). The parotid and submaxillary glands of the Cftr−/− animals showed histological abnormalities, whereas the sublingual glands were normal in appearance. The pathological features of the parotid and submaxillary glands were similar to those observed in the exocrine pancreas (Figure 6A and Figure 7). Unlike the exocrine pancreas of the Cftr−/− mice, the light microscopic appearance of the serous-type acini and ducts showed obvious inspissation with fibrillar material and greatly dilated lumina. This was particularly evident with toluidine blue-stained sections (Figure 7A). In the 9 to 12 month and older Cftr−/− animals, foci of acute and chronic inflammatory cells were present surrounding the ducts and progressive acinar loss was observed. Mineralized sludge was seen in several of the ducts from the parotids of the 15 to 24 month Cftr−/− animals. Lumina of the acini and ducts of the parotid and submaxillary glands were filled with material that was more amorphous and electron opaque than the typical secretions found in other pathologically affected organs (Figure 7B). As was observed with electron microscopy images of the plugged ex
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