Impaired cholecystokinin-induced gallbladder emptying incriminated in spontaneous "black" pigment gallstone formation in germfree Swiss Webster mice

Abstract

"Black" pigment gallstones form in sterile gallbladder bile in the presence of excess bilirubin conjugates ("hyperbilirubinbilia") from ineffective erythropoiesis, hemolysis, or induced enterohepatic cycling (EHC) of unconjugated bilirubin. Impaired gallbladder motility is a less well-studied risk factor. We evaluated the spontaneous occurrence of gallstones in adult germfree (GF) and conventionally housed specific pathogen-free (SPF) Swiss Webster (SW) mice. GF SW mice were more likely to have gallstones than SPF SW mice, with 75% and 23% prevalence, respectively. In GF SW mice, gallstones were observed predominately in heavier, older females. Gallbladders of GF SW mice were markedly enlarged, contained sterile black gallstones composed of calcium bilirubinate and <1% cholesterol, and had low-grade inflammation, edema, and epithelial hyperplasia. Hemograms were normal, but serum cholesterol was elevated in GF compared with SPF SW mice, and serum glucose levels were positively related to increasing age. Aged GF and SPF SW mice had deficits in gallbladder smooth muscle activity. In response to cholecystokinin (CCK), gallbladders of fasted GF SW mice showed impaired emptying (females: 29%; males: 1% emptying), whereas SPF SW females and males emptied 89% and 53% of volume, respectively. Bilirubin secretion rates of GF SW mice were not greater than SPF SW mice, repudiating an induced EHC. Gallstones likely developed in GF SW mice because of gallbladder hypomotility, enabled by features of GF physiology, including decreased intestinal CCK concentration and delayed intestinal transit, as well as an apparent genetic predisposition of the SW stock. GF SW mice may provide a valuable model to study gallbladder stasis as a cause of black pigment gallstones.

Keywords: black pigment gallstones; cholecystokinin; germfree mice; impaired gallbladder motility.

Fig. 1.

Gallbladders of germfree (GF) Swiss Webster (SW) mice were markedly enlarged, and 75% contained gallstones grossly and microscopically consistent with “black” pigment gallstones. A: twelve-month-old female GF SW mouse with dilated gallbladder containing gallstones (arrow). B: excised gallbladder with gallstones from a 12-mo-old female GF SW mouse. Gallstones were present in varying number, size, and color, but were often dark brown to black, as pictured here. Black bar indicates 1 cm. C: Eppendorf tube containing gallstones and gallbladder bile from a 12-mo-old female GF SW mouse. D and E: gallbladder volumes (μl) and gallbladder bile pHs of SW mice were reported as adjusted mean ± standard error, with age and body weight fixed at their means (n = 41; mean age: 11.4 mo; mean body wt: 44.9 g). Asterisks indicate level of significance of differences in gallbladder volume and bile pH, related to microbial status, with ***P < 0.001 and **P < 0.01; note that the gallbladder bile samples of GF SW mice were acidic. Statistically significant differences in analytes related to sex in the overall model were noted with #, and if also found significant when stratified by microbial status, were marked by a difference in letters (a, b; c, d) (gallbladder volume GF SW mice: P < 0.01; SPF SW mice: P < 0.001). F: gallstones and sediment in gallbladder bile from a 15-mo-old female GF SW mouse at ×100 magnification, under direct light. G: direct light microscopy of a gallstone from a 15-mo-old female specific pathogen-free (SPF) SW mouse viewed at ×200 magnification. H: polarized light microscopy at ×40 magnification of gallstones present on the mucosal surface of the gallbladder of an 11-mo-old GF SW mouse; one gallstone appears to be broken. I: high-magnification view (×100) of a gallstone in gallbladder bile from an 11-mo-old GF SW mouse viewed under polarized light.

Fig. 2.

Electron paramagnetic resonance (EPR) spectra of gallstones and gallbladder bile. Top: bilirubin (A) and gallstone samples from GF (B) and SPF (C) SW mice, highlighting the organic radical observed at g = 2. A: a sample of commercial bilirubin (10 μM in Chelex-treated Milli-Q water) contained a derivative EPR signal centered at g = 2.00. Additional features were observed at g = 2.04 and g = 1.98. B: EPR spectrum of gallstones obtained from GF SW mice. Spectrum B contained a derivative feature centered at g = 2.0 attributed to bilirubin radicals. An additional feature was observed at g = 1.98. Multiple additional features that display weak signal intensities were observed at lower field and may indicate the presence of additional EPR-detectable species in the sample. C: EPR spectrum of gallstones from one SPF SW mouse. The spectrum is scaled by 5× to facilitate comparison with spectra A and B. A derivative feature centered at g = 2.00 was also observed, and multiple weak features were present in the baseline. Instrument conditions: temperature, 5 K; microwaves, 20.1 μW at 9.4 GHz; modulation amplitude, 1 mT. Middle: EPR spectra of gallstones and gallbladder bile from GF SW mice. D: spectrum of twice-washed gallstones. E: spectrum of undiluted gallbladder bile. F: spectrum of gallstones washed 5 times. Instrument conditions: temperature, 20 K; microwaves, 0.2 mW at 9.4 GHz. Bottom: expanded view of the EPR signals in the g = 2 region from spectrum E. The Mn²⁺ and Cu²⁺ signals were obtained from standards of each metal ion (prepared in Milli-Q water). The Mn²⁺ + Cu²⁺ spectrum was generated through a linear combination of the Mn²⁺ and Cu²⁺ standard spectra. Because it was necessary to record the spectra under nonideal spectroscopic conditions (higher power) to observe and maximize signals for the transition metal ions, the radical signal at g ∼ 2 is saturated, resulting in loss of the characteristic derivative signal that is apparent under ideal spectroscopic conditions in the panels at top and middle. Instrument conditions: temperature, 20 K; microwaves, 0.2 mW at 9.4 GHz.

Fig. 3.

Normal glucose tolerance testing results from 9-mo-old GF SW and SPF SW mice. The mean area under the curves of all groups compared were statistically the same, including not pictured SPF SW mice with or without gallstones, and SPF SW females or males. A: GF SW mice (n = 11; 79.8 ± 6.9) compared with SPF SW mice (n = 12; 93.8 ± 6.9). B: GF SW mice with gallstones (n = 9; 80.5 ± 7.9) compared with GF SW mice without gallstones (n = 2; 77.0 ± 15.0). C: GF SW females (n = 6; 84.2 ± 9.8) compared with GF SW males (n = 5; 74.6 ± 9.9). Mean baseline blood glucose values were significantly higher in GF SW male mice compared with GF SW female mice; *P < 0.05.

Fig. 4.

Hematoxylin and eosin images of the range of gallbladder lesions in GF SW (A–D) compared with SPF SW (E and F) mice. A: gallbladder of an 8-mo-old male GF SW mouse with gallstones, showing mild subepithelial inflammation, edema, and epithelial hyalinosis (intensely eosinophilic granular hyaline-like cytoplasmic alteration). B: gallbladder of an 8-mo-old female GF SW mouse without gallstones showing moderate mixed (lymphocytic and granulocytic) inflammation of the epithelium and stroma with minimal papillary epithelial projections. C: low-magnification image of a gallbladder of an 8-mo-old male GF SW mouse without gallstones showing prominent papillomatous epithelial hyperplasia, scattered inflammatory cells, and edema in the subepithelial space/stroma. D: higher magnification of C, showing hyperplastic long columnar epithelium with mostly basal oval nuclei, abundant eosinophilic to vacuolated (mucous) cytoplasm, and an intraglandular protein cast (arrow). E and F: low- and high-magnification images of a gallbladder of a 10-mo-old male SPF SW mouse with sparse inflammatory cells and mild papillary epithelial hyperplasia. Bars: A, B, and F = 80 μm; C and E = 160 μm; D = 40 μm.

Fig. 5.

Gallbladder smooth muscle activity is disrupted in aged GF and SPF SW mice. Ca²⁺ transient recordings from pairs of gallbladder smooth muscle cells (gray and black) showing an age-related disruption in spontaneous activity. Gallbladder smooth muscle cells in young SPF SW mice exhibit synchronized rhythmic Ca²⁺ flashes (panel at top left). Ca²⁺ flash activity is absent in 10-mo-old GF and SPF SW mice (panels at middle and bottom left), where only Ca²⁺ waves were detected. Carbachol (3 μM) induced Ca²⁺ flashes in all three groups of mice once peak frequency was reached (panels at right; time point indicated above each trace).

Fig. 6.

GF SW mice showed impaired cholecystokinin (CCK)-induced gallbladder emptying, compared with SPF SW mice. Gallbladder volumes (μl) of SW mice were reported as adjusted mean ± standard error, with age and body weight fixed at their means (control mice: n = 34; mean age: 8.0 mo; mean body wt: 55.0 g; experimental mice: n = 34; mean age: 8.0 mo; mean body wt: 55.7 g). Asterisks indicate level of significance of differences in gallbladder volumes of control and experimental mice, related to microbial status, with ****P < 0.0001. Statistically significant differences in gallbladder volume related to sex in the overall model were noted by #, and if also found significant when stratified by microbial status, were marked by a difference in letters (a, b) (SPF SW mice: P < 0.0001). A difference in numbers (1, 2) denotes a statistically significant difference in gallbladder volume between SPF SW control and experimental mice (P < 0.0001).

Fig. 7.

GF SW and SPF SW mice were comparable in concentration, secretion rate and % of unconjugated bilirubin (UCB) in hepatic bile. Bilirubin concentrations (μM) (A), secretion rates (nmol/h) (B), and % UCB of hepatic bile (C) of SW mice were reported as adjusted mean ± standard error, with age and body weight fixed at their means (n = 49; mean age: 11.2 mo; mean body wt: 54.8 g). Asterisks indicate level of significance of difference in conjugated bilirubin concentration, related to microbial status, with **P < 0.01. Statistically significant differences in analytes related to sex in the overall model were noted by #, and if also found significant when stratified by microbial status, were marked by a difference in letters (a, b; c, d) (conjugated bilirubin concentration GF SW mice: P < 0.01; SPF SW mice: P < 0.05; % UCB SPF SW mice: P < 0.05).