Figure 1.
Biliary phenotype of genetically related BALB/cAnNTac (Tac) and BALB/cByJ (ByJ) and BALB/cJBomTac (Bom) and BALB/cJ (Jax) mice.
(A) Normalized gallbladder weight (B) mucin gel score (C) Liquid crystal phase separation (D) cholesterol monohydrate crystal formation, (E) sandy stone formation and (F) cholesterol gallstone formation were analyzed after 8 weeks of lithogenic diet feeding. (A) Normalized gallbladder weight significantly differed between Tac mice and both ByJ and Jackson mice (*P<0.05). (B) Mucin gel score for both BALB/cJ and BALB/cJBomTac mice was significantly elevated compared to either Tac or ByJ mice (P<0.05). (C) Liquid crystal phase separation occurred in all animals studied. (D) Cholesterol monohydrate crystal formation occurred significantly more in BALB/cJ mice compared to both ByJ and Tac mice (*P<0.05). (E) Both BALB/cJ and BALB/cJBomTac mice compared to either Tac or ByJ mice displayed significantly increased sandy stone formation (*P<0.05). (F) Cholesterol gallstone formation was significantly increased in BALB/cJ and BALB/cJBomTac mice compared to Tac mice (*P<0.05).
Figure 2.
Microbial colonization of 8 ASF species in the ceca of mice from different vendors.
(A) ASF 356, (B) ASF 361, (C) ASF 457, (D) ASF 492, (E) ASF 500, (F) ASF 502 and (G) ASF 519 were all evaluated by quantitative PCR and normalized to host tissue levels. Both ASF 361 (B) and ASF 457 (C) were significantly (*P<0.05) higher in mice from Taconic compared to Jackson origin mice. ASF 519 was significantly (*P<0.05) elevated in mice from one Taconic source (Tac) but not from the other Taconic source (Bom) (G). All other ASF species were colonized similarly among mice from both Jackson and Taconic sources.
Figure 3.
Components of the biliary phenotype may be altered by transferring flora to adult mice.
(A) Normalized gallbladder weight (B) mucin gel score (C) Liquid crystal phase separation (D) cholesterol monohydrate crystal formation, (E) sandy stone formation and (F) cholesterol gallstone formation were analyzed after 8 weeks of lithogenic diet feeding. (A) Both Jax and Tac mice colonized with Jax flora developed significantly smaller gallbladders when compared to Tac mice (*P<0.05) (B) Jackson origin mice regardless of flora produced higher mucin scores compared to control Taconic mice (*P<0.05). (D) Flora transferred Jax mice displayed significant increases in cholesterol monohydrate crystal formation compared to control Tac mice (*P<0.05) and control Jax mice displayed significant increases compared to both Taconic groups (**P<0.05). (E) Both groups of Jackson mice displayed significant increases in sandy stone formation compared to both groups of Taconic mice (P*<0.05). Control Taconic mice displayed significant increases in sandy stones when compared to Taconic mice that underwent crossfaunation (*P<0.05). (F) Jax mice displayed significant increases in cholesterol gallstone formation compared to both groups of Tac mice (*P<0.05).
Figure 4.
Transfer of Taconic flora to Jackson mice leads to significant increases in ASF457 (A) and 361.
ASF361 (A) and 457 (B) were quantified from a cohort of cross-faunated mice (n = 5) by real-time PCR. There were significant elevations in the colonization of these two bacteria in mice that were of Taconic origin or those which received Taconic flora (*P<0.05). Colonization of Taconic mice with Jackson flora did not significantly diminish the colonization by these organisms.
Figure 5.
Components of the biliary phenotype may be altered by crossfostering mice shortly after birth.
(A) Normalized gallbladder weight (B) mucin gel score (C) Liquid crystal phase separation (D) cholesterol monohydrate crystal formation, (E) sandy stone formation and (F) cholesterol gallstone formation were analyzed after 8 weeks of lithogenic diet feeding. (A) Both Tac and Jax fostered to Tac developed significantly larger gallbladders (*P<0.05) compared to Jax mice. (B) Jax mice displayed significant (*P<0.05) elevations in mucin gel accumulation compared to all other groups including Jax fostered to Tac. (D–E) Jax mice and Jax fostered to Tac developed significantly more (P<0.05) cholesterol monohydrate and sandy stones then either group from Taconic. (F) Tac mice developed significantly (P<0.05) less cholesterol gallstones then Jax mice and both fostered groups displayed intermediate and non-significantly different levels of cholesterol gallstone formation.
Figure 6.
Crossfostering of mice leads to significant alterations in ASF361 and 457.
ASF361 (A) and 457 (B) were quantified from a cohort (n = 5) of cross-fostered mice by real-time PCR. There were significant elevations in the colonization of these two bacteria in mice of Jackson origin crossfostered to Taconic mothers and significant decreases in Taconic pups crossfostered to Jackson mothers (*P<0.05) indicating effective alteration of the flora.
Figure 7.
Germ-free mice are more susceptible to cholesterol cholelithogenesis (A) Normalized gallbladder weight (B) mucin gel score (C) Liquid crystal phase separation (D) cholesterol monohydrate crystal formation, (E) sandy stone formation and (F) cholesterol gallstone formation were analyzed after 8 weeks of lithogenic diet feeding.
(A) Germ-free mice developed significantly increased normalized gallbladder weights compared to either conventional or ASF colonized mice (*P<0.05). Mucin gel score did not significantly differ among groups (all conventional and ASF mice developed mucin gel scores of “1”). (D–E) Germ-free mice developed significantly more cholesterol monohydrate (D, *P<0.05) and Sandy stones (E, *P<0.05) and had an non-significant increase in cholesterol gallstones (F).
Figure 8.
Germ-free mice develop significantly more severe gallbladder histopathological changes compared to ASF colonized mice.
(A, B) Low power and high power image of gallbladders from Germ-free mice demonstrate hyperplasia (arrowhead), inflammation (arrow) and the presence of eosinophilic luminal material which is consistent with mucin gel (asterisk). (C) Overall, gallbladder histopathology score which examines inflammation, edema, and hyperplasia was significantly increased (*P<0.05) in Germ-free mice compared to ASF colonized mice and nearly significant (P = 0.07) when compared to conventional mice. Both ASF colonized (D, low power, E, high power) and conventional (F, high power) mice display minimal inflammation and maintain a normal epithelial lining of a single layer of cuboidal epithelium.
Figure 9.
Germ-free mice do not exhibit a significantly increased gallbladder cholesterol saturation index.
(A, B) The concentration of gallbladder bile salts and phospholipids was not significantly altered between conventionally reared-mice and germ-free mice. (C) Gallbladder cholesterol concentration is mildly yet significantly decreased in germ-free mice compared to conventionally reared mice (P<0.05). (D) Overall the gallbladder CSI did not differ significantly between germ-free and conventionally reared mice.
Figure 10.
Mucin gene expression is significantly increased in germ-free mice compared to conventionally reared mice.
(A) Muc1, (B) Muc3, and (C) Muc4 are all significantly (P<0.05) increased (∼2.5–4.5 fold) in germ-free mice when compared to conventionally reared mice. Neither (D) Muc5ac nor (E) Muc5b displayed any differences and expression was highly variable among mice.