Spontaneous liver tumors in aged germfree Wistar rats.

Liver tumors, ranging from benign nodules to carcinomas, developed spontaneously in 115 (87%) of 132 germfree Wistar rats beyond the age of 30 months. In addition, the rats developed a high incidence of benign adenomas of endocrine glands and of the breasts.     

Phytate hydrolysis by germfree and conventional rats.

Phytic acid is naturally occurring compound that reduces intestinal absorption of many metals. Early work suggests that some dietary phytate may be hydrolyzed in the large intestines by bacteria, but more recently nutritionists have suggested that a mucosal enzyme is responsible. This paper reports a study intended to resolve this controversy. The hydrolysis of dietary phytic acid was measured in germfree and conventional rats fed either of two diets that differed in their calcium content. Negligible phytate hydrolysis occurred in the germfree rats, whereas 22 and 56% of the phytic acid was hydrolyzed by conventional rats fed high- and low-calcium diets, respectively. We concluded that bacteria were responsible for the hydrolysis of phytate in these diets and that any activity of endogenous enzyme was negligible.     

Analysis of bile acids in conventional and germfree rats.

The well-known bile acid analysis technique used by us and others (Grundy, Ahrens, and Miettinen. 1965. J. Lipid Res. 6:397-410) does not allow for the detection of hyodeoxycholic acid, a product of quantitative importance in rodent feces. Using updated methodology, it was established that hyodeoxycholic acid and omega-muricholic acid, both apparent conversion products of beta-muricholic acid, occur in apppreciable amounts in intestinal contents and feces of conventional Wistar type Lobund rats. In conventional rats, these bile acids comprise about 50% of fecal bile acids; they are not found in intestinal contents or feces of germfree rats. Others have demonstrated that hyodeoxycholic acid if formed by combined action of gut flora and liver. A new method for the separation of conjugated and free bile acids in biological samples was developed. Results with this method confirmed the total conjugation of bile acids in the germfree rat, and the almost total deconjugation that takes place in the cecum of the conventional rat.     

Resistance of germfree rats to indomethacin-induced intestinal lesions.

Indomethacin given orally to conventional rats produced in three days a syndrome, often fatal, of intestinal lesions characterized by multiple ulcers and peritonitis. Male germfree rats were found to be resistant to this effect of indomethacin, while female germfree rats developed very mild lesions. Germfree rats became sensitive again to such lesions when monocontaminated with E. coli. In such animals, however, the lesions were less severe than in conventional animals, presumably because more than one microorganism is necessary for the full syndrome to develop. These results suggest that microorganisms are necessary for the development of indomethacin-induced intestinal lesions. Secondary bile acids, absent in germfree animals, may also be necessary. The prostaglandin deficiency caused by indomethacin appears to weaken the resistance of the intestinal mucosa to microorganisms and/or their toxins. The latter may then penetrate the mucosa, damage the cells and produce ulcers and perforations. Since several prostaglandins also protect against indomethacin-induced lesions, the hypothesis is advanced that certain prostaglandins may protect the mucosa ("cytoprotection") by preventing the spread of microorganisms and/or their toxin through the intestinal wall.     

The histochemistry of mucosaccharides in some organs of germfree rats.

In order to study the histochemical nature of mucosaccharides in germfree animals, the organs in natural contact with bacteria (stomach, small and large intestine) and those naturally remote from bacteria (tracheal and ear cartilage and aorta) were studied by means of light microscopic methods for mucosaccharides in germfree and conventional rats. In the stomach (surface and foveolar cells) of germfree rats the histochemical reactions for acid and neutral mucosaccharides were apparently less intense than in that of conventional rats, whereas in the small and large intestine (goblet cells) of germfree rats the reactions were significantly more intense than in those of conventional rats. In the cartilage (intercellular matrix, lacunar border and chondrocyte cytoplasm) and aorta (interelastic spaces) of germfree animals the reactions were less intense than in those of conventional animals. In addition, some differences in the histochemical nature of mucosaccharides between the organs of germfree and conventional rats were noted, as revealed by the effects of chemical modifications and digestions with enzymes upon the histochemical reactions studied.     

Glucose tolerance, insulin and catecholamine levels in germfree rats.

Glucose was administered intravenously to 50- and 100-day-old GF and CV rats. Fasting blood glucose levels in GF and CV rats were found to be comparable. Glucose tolerance tests showed that GF and CV rats clear glucose from the blood at a similar rate. Although insulin concentrations in 100-day-old GF rats tended to be somewhat lower than in CV rats, the percentage increase during the 30-min period after glucose administration was similar, and matched the increase in blood glucose. Levels of plasma catecholamines were analyzed fluorometrically and were found to be comparable in 100-day-old GF and CV rats. It was concluded that insulin insufficiency plays no role in the syndrome of metabolic anomalies demonstrated by the germfree rat.     

Endotoxin in germfree, gnotobiotic, or conventional-flora Sprague-Dawley rats.

The Limulus assay for bacterial endotoxin was performed on serum and (or) plasma from animals monoassociated with Clostridium species, Staphylococcus aureus, Escherichia coli, Proteus mirabilis, Enterobacter agglomerans, Bacteroides fragilis, Klebsiella pneumoniae, or Candida albicans. Plasma from animals monoassociated with the gram-negative bacteria or C. albicans consistently showed a positive Limulus test while conventional-flora controls, germfree rats, and gnotobiotic animals monoassociated with gram-positive bacteria or E. agglomerans were negative. Germfree and conventional rats were injected (intraperitoneal (i.p.)) with Salmonella typhosa lipopolysaccharide (LPS). Although no endotoxin was detectable in either group prior to the injection, by 1 h post injection endotoxin was in the plasma of all groups. The germfree rats appeared to clear the LPS quicker than their conventional-flora counterparts. Generally, LPS-injected rats (conventional and germfree) showed clumping and decreased number of platelets, a decrease in their lymphocyte counts, and increased polymorphonuclear leukocyte (PMN) counts.     

Catabolism and elimination of cholesterol in germfree rats

Three-month old germfree and conventional male rats were maintained on a complete steam-sterilized, semisynthetic diet. After intravenous injection of cholesterol-26-(14)C the animals were housed in a plastic metabolism chamber for 72 hr. Expired CO(2) was collected throughout the period. The conventional rats released 50% more (14)C as (14)CO(2) than the germfree animals. The total amount of the label recovered as (14)CO(2) during the 72 hr period amounted to 30% and 19% respectively, of the original dose. In both conventional and germfree rats the release of (14)CO(2) accounted for approximately 75% of the (14)C recovered in forms other than the original cholesterol-26-(14)C; 15-20% was found incorporated in water-soluble and fat-soluble fractions other than 3Beta-OH sterol of liver and carcass while the remainder was excreted with feces and urine. After the 72 hr period the specific activities of the cholesterol in plasma and liver were lower in conventional than in germfree animals. The data express the accelerating effect of the intestinal microflora on systemic cholesterol catabolism. They demonstrate that the release of (14)CO(2) from cholesterol-26-(14)C in the intact rat is a suitable and convenient indicator of the oxidative catabolism of cholesterol.     

Exteriorization of the bile duct in germfree rats

A modified T cannula and a cannula housing receptacle was employed to cannulate the bile duct of germfree rats. Bile was collected without anesthesia or sedation. During collection, the rat was restrained in a plastic holder where it was free to eat.     

Ultrastructure of the enlarged cecum in germfree rats

Summary The cecum of germfree rats, as studied by light microscopy and scanning and transmission electron microscopy, differs in many respects from the cecum of conventional rats. Epithelial cells in germfree rats are taller and have larger nuclei and longer microvilli than similar cells in conventional rats. The cecal mucosa of germfree rats shows a larger variability in the arrangement of the crypts of Lieberkühn than does the mucosa of conventional rats. Some crypts are funnel-shaped and connected close to the mucosal surface with adjacent similar crypts to form long valleys. Less wide crypts open on elevated regions of the mucosal surface. The lamina propria of germfree animals is devoid of plasma cells but rich in mast cells. Germfree animals show hypertrophy of the tunica muscularis externa.In conventional rats the cecal lumen contains a large variety of morphologically different bacteria. However, the lumen of the crypts of Lieberkühn contains only one type of elongated bacteria, which are present in large amounts. This finding suggests that symbiotic relations may be of particular importance in the crypts of Lieberkühn in the cecum.Supported by research grants from the Swedish Medical Research Council (206), Knut och Alice Wallenbergs Stiftelse and Stiftelsen Therese och Johan Anderssons Minne.     

Effects of cholestyramine on 1,2-dimethylhydrazine-induced enteric carcinoma in germfree rats.

Oral administration of 1,2-dimethylhydrazine (DMH) induced intestinal neoplasms in germfree rats. A supplement of 2% cholestyramine resin in the diet increased the frequency of DMH-induced intestinal tumors and accelerated malignant transformation. Bile acids in the cecal content were determined with and without cholestyramine in order to obtain a correlation between the bile acid metabolism and the enteric carcinogenesis.     

Ammonia in the intestinal contents from germfree rats

The values of pH and the concentrations of nitrogen (N) and ammonia in the middle part of the small intestinal and cecal contents of germfree (GF) and conventionalized (CVZ) seven-week-old rats were compared. The pH of the small intestinal and cecal contents of GF rats was higher than that of CVZ rats. There was no difference in total N per fresh weight in contents from the middle part of the small intestine between GF and CVZ, whereas total N per fresh weight of the cecal contents was higher in CVZ than in GF rats. The ammonia concentrations per fresh weight or per total N in the intestinal and cecal contents of CVZ rats were higher than those of GF rats.     

Sex-linked differences in bile acid metabolism of germfree rats

The bile acid composition was investigated in male and female germfree rats. β-Muricholic acid and cholic acid were the major bile acids in both sexes; in addition, 3β-hydroxy-5-cholenoic acid, chenodeoxycholic acid, α-muricholic acid, allochenodeoxycholic acid and allocholic acid were present. Important sex-linked differences in the relative amounts and the sulfation of these substances were observed. β-Muricholic and cholic acid accounted for 61.4 % and 27.7 % of total bile acids in the small intestine of males; females had 38.9 % of β-muricholic acid and 50 % of cholic acid. In females, the bile acid sulfate fraction increased from 1.1 % in the small intestine to 22.3 % in the large intestine; in males these values were 0.2 % and 1.7 %, respectively. A considerable increase in the relative amounts of allochenodeoxycholic and allocholic acid was observed in the cecum and large intestine of the female rat, where more than 70 % of these substances was in the bile acid sulfate fraction. In males these allo-bile acids were mainly in the unsulfated fraction and their relative amounts did not increase in the large intestine.