Effect of gallbladder hypomotility on cholesterol crystallization and growth in CCK-deficient mice

Abstract

We investigated the effect of gallbladder hypomotility on cholesterol crystallization and growth during the early stage of gallstone formation in CCK knockout mice. Contrary to wild-type mice, fasting gallbladder volumes were enlarged and the response of gallbladder emptying to a high-fat meal was impaired in knockout mice on chow or the lithogenic diet. In the lithogenic state, large amounts of mucin gel and liquid crystals as well as arc-like and tubular crystals formed first, followed by rapid formation of classic parallelogram-shaped cholesterol monohydrate crystals in knockout mice. Furthermore, three patterns of crystal growth habits were observed: proportional enlargement, spiral dislocation growth, and twin crystal growth, all enlarging solid cholesterol crystals. At day 15 on the lithogenic diet, 75% of knockout mice formed gallstones. However, wild-type mice formed very little mucin gel, liquid, and solid crystals, and gallstones were not observed. We conclude that lack of CCK induces gallbladder hypomotility that prolongs the residence time of excess cholesterol in the gallbladder, leading to rapid crystallization and precipitation of solid cholesterol crystals. Moreover, during the early stage of gallstone formation, there are two pathways of liquid and polymorph anhydrous crystals evolving to monohydrate crystals and three modes for cholesterol crystal growth.

Figure 1

(A) Representative photographs and (B) gross observations of gallbladders show that fasting gallbladder volumes are significantly enlarged in CCK KO mice compared with CCK WT mice. (C) Postprandial gallbladder sizes in response to the high fat meal. Duodenal infusion of corn oil could stimulate the release of CCK from the upper part of small intestine. As a result, the secreted CCK induces gallbladder emptying in CCK WT mice but not in CCK KO mice. These results indicate that gallbladder contractile function is impaired in CCK KO mice.

Figure 2

Gallbladder sizes as functions of time on the lithogenic diet. At day 0, the gallbladder volumes are significantly larger in CCK KO mice than in CCK WT mice. Gallbladder sizes are enlarged significantly over time in CCK KO mice challenged to the lithogenic diet, whereas in CCK WT mice, gallbladder volumes increased slightly. In the lithogenic state, the gallbladders of CCK KO mice (black circles) double in size compared to those of CCK WT mice (white squares).

Figure 3

The relative lipid compositions of pooled gallbladder biles from CCK KO and WT mice at each time point are plotted on condensed phase diagrams for average total lipid concentrations (~8.0 g/dL) of the bile samples (from Table 1). The one-phase micellar zone at bottom is enclosed by a solid curved line. Above the micellar zone, two solid lines divide the two-phase zones from a central three-phase zone. Based upon the solid and liquid crystallization sequences present in the biles, the left two-phase and central three-phase regions are divided by dashed lines into Regions A to E. With passage of time, the relative lipid compositions of gallbladder bile shift upward and to the right in both CCK KO and WT mice. Only the lipid compositions of biles in CCK KO mice pass through Region B. In contrast, the lipid compositions of bile in CCK WT mice enter crystallization pathway C directly from the one-phase micellar zone. Black symbols represent CCK KO mice and white symbols are for CCK WT mice.

Figure 4

Representative photomicrographs of mucin gel as well as habits of liquid crystals, solid cholesterol crystals, and gallstones as observed in fresh gallbladder biles by polarizing light microscopy: (A) non-birefringent amorphous mucin gel; (B) arc-like (possible anhydrous cholesterol) crystal; (C) tubular crystal; (D) tubular crystal fracturing at the end to produce plate-like cholesterol monohydrate crystals; (E) numerous aggregated non-birefringent liquid crystals and few fused liquid crystals; (F) agglomerates of typical cholesterol monohydrate crystals, with 79.2° and 100.8° angles, and often a notched corner; (G) disintegrable amorphous sandy stones surrounded by mucin gel, with individual plate-like cholesterol monohydrate crystals projecting from the edges; (H) true gallstones displaying rounded contours and black centers from light scattering/absorption. All magnifications are ×800, except Figure 4(F and G) ×400 and Figure 4H ×200, by polarizing light microscopy.

Figure 5

Ongoing cholesterol crystallization within mucin gel. (A and B) Very tiny solid cholesterol crystals are detected first within the mucin gel. Also, a lot of small single classic cholesterol monohydrate crystals embedded in mucin gel are often found. Both magnifications are ×800, by polarizing light microscopy.

Figure 6

Three modes of cholesterol crystal growth habits in CCK KO mice on the lithogenic diet for 15 days: (A and B) proportional enlargement patterns, (C and D) spiral dislocation growth patterns, and (E and F) twin crystal growth patterns, all of which induce cholesterol crystals enlarged in size. The twin crystals grow upright and perpendicular to the surface. See text for further description. All magnifications are ×800, by polarizing light microscopy.

Figure 7

Schematic presentation of the crystal habits in the cholesterol crystallization sequence from fresh mouse gallbladder bile as functions of days on the lithogenic diet. The vertical axes represent arbitrary numbers of crystals and liquid crystals per high power microscopic field, all normalized to the same maximum. The panels show the time sequences as means for each group of CCK WT (top panel) and KO mice (bottom panel). The arrow indicates the first appearance of true gallstones. Abbreviations: ACh, anhydrous cholesterol crystals, including arc-like and transitional tubular crystals; ChM, classic plate-like cholesterol monohydrate crystals; LC, liquid crystals, including small, aggregated and fused (multilamellar) varieties; SS, sandy stones; GS, true gallstones. See text for further description.