Portosystemic shunting and persistent fetal vascular structures in aryl hydrocarbon receptor-deficient mice

Abstract

A physiological examination of mice harboring a null allele at the aryl hydrocarbon (Ah) locus revealed that the encoded aryl hydrocarbon receptor plays a role in the resolution of fetal vascular structures during development. Although the aryl hydrocarbon receptor is more commonly studied for its role in regulating xenobiotic metabolism and dioxin toxicity, a developmental role of this protein is supported by the observation that Ah null mice display smaller livers, reduced fecundity, and decreased body weights. Upon investigating the liver phenotype, we found that the decrease in liver size is directly related to a reduction in hepatocyte size. We also found that smaller hepatocyte size is the result of massive portosystemic shunting in null animals. Colloidal carbon uptake and microsphere perfusion studies indicated that 56% of portal blood flow bypasses the liver sinusoids. Latex corrosion casts and angiography demonstrated that shunting is consistent with the existence of a patent ductus venosus in adult animals. Importantly, fetal vascular structures were also observed at other sites. Intravital microscopy demonstrated an immature sinusoidal architecture in the liver and persistent hyaloid arteries in the eyes of adult Ah null mice, whereas corrosion casting experiments described aberrations in kidney vascular patterns.

Figure 1

Ah −/− mice have smaller hepatocytes than wild-type mice. Livers of 1-year-old mice were fixed in formalin, and 6-μm sections were examined after staining with hematoxylin/eosin. (A and B) Thin sections from wild-type (A) and age-matched Ah knockout (B) mice are shown, and results of morphometric analyses follow. (C) There is a significant decrease in the total area of the hepatocytes of Ah −/− mice. (D and E) Whereas the cytoplasmic area of Ah −/− hepatocytes is significantly decreased (D), the nuclear areas of Ah +/+ and Ah −/− hepatocytes are not different (E). Mean and standard errors generated from comparison of six 1-year-old male Ah +/+ and six age- and sex-matched Ah −/− mice are shown; asterisks indicate significance (P < 0.05).

Figure 2

(A) Livers of Ah −/− mice exhibit portosystemic shunting. With perfusion of colloidal carbon, livers of Ah +/+ mice become black (Left), whereas livers of Ah −/− mice remained pink (Right). The livers of male mice are shown above the livers of female mice. The figure represents livers of five male and five female mice from each group. (B) In Ah −/− mice, microspheres bypass liver sinusoids. The hepatic portal veins of six Ah +/+ and 6 Ah −/− mice were injected with microspheres and then livers and lungs were removed. The extent of portosystemic shunting was calculated as the number of microspheres found in lung divided by the number of microspheres found in both the lung and the liver. Mean and standard errors are shown; asterisks indicate significance (P < 0.05).

Figure 3

Angiography indicates patent ductus venosus. Continuous x-ray images were obtained over approximately 10 s as contrast was injected into the portal veins of Ah +/+ (A–F) and Ah −/− (G–L) mice. Angiograms are representative for five Ah +/+ and five Ah −/− mice. Serial radiographs are presented from left to right, showing the portal vein (PV), infrahepatic inferior vena cava (ihIVC), suprahepatic inferior vena cava (shIVC), ductus venosus (DV), and branching vessels (BV).

Figure 4

Corrosion casts provide a three-dimensional image of putative ductus venosus in Ah −/− mice. The portal vein (PV), inferior vena cava (IVC), and ductus venosus (DV) are shown in corrosion casts of wild-type (A) and congenic age-matched Ah −/− (B and C) livers. Corrosion casts are representative of at least six Ah +/+ and six Ah −/− mice. The cast in C shows the patent DV from an Ah −/− mouse after individual vessel branches were removed.

Figure 5

Sinusoids of Ah −/− mice are more anastomotic than those of Ah +/+ mice. Still images from 80× water immersion objectives were collected from videotapes of intravital microscopy of Ah +/+ (A) and Ah −/− (B) livers. Sinusoids are identified with dotted lines, as they not as readily visible in individual frames as compared with videotapes. A comparison of the internal diameter of individual sinusoids (ID), the number of flow-containing sinusoids (FCS), and the number of sinusoidal junctions per field (SJF) is shown (C–E, respectively) as mean and standard error. For ID and FCS, 28 and 18 fields were examined from four Ahr +/+ and three Ahr −/− livers, respectively. For SJF, 20 and 15 fields from three Ahr +/+ and three Ahr −/− livers were examined, respectively.

Figure 6

Ah −/− mice have a high incidence of persistent hyaloid artery. To provide histological sections defining the extended hyaloid artery, eyes of Ah +/+ (A) and Ah −/− (B) were fixed in 10% buffered formalin and 6-μm sections were stained with Alcian Blue PAS. The lens (L), vitreous (V), retina (R), and hyaloid artery (HA) are shown. Eyes of Ah +/+ and Ah −/− mice were examined by indirect and direct ophthalmoscopy for hyaloid structures; a graph of hyaloid artery persistence is shown (C). The difference between Ah +/+ and Ah −/− mice was significant (P < 0.005), and the percentage of mice with extensive hyaloid artery is shown with the 95% upper confidence internal. Results were statistically evaluated by using the two-tailed Fisher's exact test (25).

Figure 7

Ah −/− mice have expansion of limbal vessels and altered kidney vascular structure. (A and B) Limbal vessel structures of Ah +/+ (A) and Ah −/− (B) mice are shown. Limbal vessels were photographed after nine Ah +/+ and nine Ah −/− mice were injected with fluorescein isothiocyanate-dextran, and eyes were epi-illuminated at 488 nm. (C and D) Corrosion casts of kidney vasculature of Ah +/+ (C) and −/− (D) mice are also shown and are representative of at least four mice from each group.