Autoclaved Diet with Inactivated Spores of Bacillus spp. Decreased Reproductive Performance of Muc2-/- and Muc2+/- Mice.

Within barrier facilities, autoclaved diet and bedding are used for husbandry of laboratory rodents. Bacillus spp. are ubiquitous in nature and some of them are known as probiotics. Inactivation of the Bacillus spores and reduction of the diet nutritional value due to autoclavation could be especially critical for immunodeficient mice. We studied the effect of the autoclaved and non-autoclaved diets on the reproductive performance and the age of prolapse manifestation in Muc2-/- mice with impaired gut barrier function and, therefore, sensitive to change of microbiota. We found that the non-autoclaved diet led to enhancement of the fertility index of Muc2-/- and Muc2+/- female mice. The non-autoclaved diet affected the prolapse of Muc2-/- mice that occurred later in comparison with females eating the autoclaved diet. We showed that Bacillus spp. was present in the non-autoclaved diet and feces of mice on the non-autoclaved diet. Bacterial strains of the non-autoclaved diet and feces belonged to B. amyloliquefaciens, B. thuringiensis, B. subtilis, Lysinibacillus macrolides, B. cereus, and other representatives of Bacillus spp. Moreover, autoclavation of the diet affected on the percent of the blood and spleen immune cells, the bacterial composition of the intestine, and increased the level of methionine in the thigh muscle of mice. Enhanced reproductive performance and delayed prolapse manifestation in Muc2-/- mice could be due to improved digestion, as Bacillus spp. from diet and feces had enzymatic activity.

Colitis-associated intestinal microbiota regulates brain glycine and host behavior in mice.

Inflammatory bowel diseases (IBD) are chronic and relapsing inflammatory disorders of the gastrointestinal tract with complex etiology and no strategies for complete cure. IBD are often complicated by mental disorders like anxiety and depression, indicating substantial shifts in the microbiota gut-brain axis. However, the mechanisms connecting IBD to mental diseases are still under debate. Here we use Muc2 knockout mouse model of chronic colitis to uncouple the effects of the intestinal microbiota on host behavior from chronic inflammation in the gut. Muc2 knockout male mice exhibit high exploratory activity, reduced anxiety-related behaviors, impaired sensorimotor gating, and altered social preference towards males and females. Microbial transfer to wild-type mice via littermate co-housing shows that colitis-associated microbiota rather than inflammation per se defines behavioral features in Muc2 colitis model. Metagenomic profiling and combination of antibiotic treatments revealed that bacterial species Akkermansia muciniphila is associated with the behavioral phenotype in mutants, and that its intestinal abundance correlates with social preference towards males. Metabolomic analysis together with pharmacological inhibition of Gly and NMDA receptors helped us to determine that brain glycine is responsible for the behavioral phenotype in Muc2 mice. Blood and brain metabolic profiles suggest that microbiota-dependent changes in choline metabolism might be involved in regulation of central glycine neurotransmission. Taken together, our data demonstrates that colitis-associated microbiota controls anxiety, sensorimotor gating and social behavior via metabolic regulation of the brain glycinergic system, providing new venues to combat neurological complications of IBD.

Fat of the Gut: Epithelial Phospholipids in Inflammatory Bowel Diseases.

Inflammatory bowel diseases (IBD) comprise a distinct set of clinical symptoms resulting from chronic inflammation within the gastrointestinal (GI) tract. Despite the significant progress in understanding the etiology and development of treatment strategies, IBD remain incurable for thousands of patients. Metabolic deregulation is indicative of IBD, including substantial shifts in lipid metabolism. Recent data showed that changes in some phospholipids are very common in IBD patients. For instance, phosphatidylcholine (PC)/phosphatidylethanolamine (PE) and lysophosphatidylcholine (LPC)/PC ratios are associated with the severity of the inflammatory process. Composition of phospholipids also changes upon IBD towards an increase in arachidonic acid and a decrease in linoleic and a-linolenic acid levels. Moreover, an increase in certain phospholipid metabolites, such as lysophosphatidylcholine, sphingosine-1-phosphate and ceramide, can result in enhanced intestinal inflammation, malignancy, apoptosis or necroptosis. Because some phospholipids are associated with pathogenesis of IBD, they may provide a basis for new strategies to treat IBD. Current attempts are aimed at controlling phospholipid and fatty acid levels through the diet or via pharmacological manipulation of lipid metabolism.

Mucin-2 knockout is a model of intercellular junction defects, mitochondrial damage and ATP depletion in the intestinal epithelium.

The disruption of the protective intestinal barrier-the 'leaky gut'-is a common complication of the inflammatory bowel disease. There is limited data on the mechanisms of the intestinal barrier disruption upon low-grade inflammation characteristic of patients with inflammatory bowel disease in clinical remission. Thus, animal models that recapitulate the complexity of chronic intestinal inflammation in vivo are of particular interest. In this study, we used Mucin-2 (Muc2) knockout mice predisposed to colitis to study intestinal barrier upon chronic inflammation. We used 4-kDa FITC-Dextran assay and transmission electron microscopy to demonstrate the increased intestinal permeability and morphological defects in intercellular junctions in Muc2 knockout mice. Confocal microscopy revealed the disruption of the apical F-actin cytoskeleton and delocalization of tight junction protein Claudin-3 from the membrane. We further demonstrate mitochondrial damage, impaired oxygen consumption and the reduction of the intestinal ATP content in Muc2 knockout mice. Finally, we show that chemically induced mitochondrial uncoupling in the wild type mice mimics the intestinal barrier disruption in vivo and causes partial loss of F-actin and membrane localization of Claudin-3. We propose that mitochondrial damage and metabolic shifts during chronic inflammation contribute to the leaky gut syndrome in Muc2 knockout animal model of colitis.

Effects of brain-derived and glial cell line-derived neurotrophic factors on startle response and disrupted prepulse inhibition in mice of DBA/2J inbred strain.

Prepulse inhibition (PPI), the reduction in acoustic startle reflex when it is preceded by weak prepulse stimuli, is a measure of critical to normal brain functioning sensorimotor gating. PPI deficit was shown in a variety of psychiatric disorders including schizophrenia, and in DBA/2J mouse strain. In the current study, we examined the effects of brain-derived (BDNF) and glial cell line-derived (GDNF) neurotrophic factors on acoustic startle response and PPI in DBA/2J mice. It was found that BDNF (300 ng, i.c.v.) significantly increased amplitude of startle response and restored disrupted PPI in 7 days after acute administration. GDNF (800 ng, i.c.v.) did not produce significant alteration neither in amplitude of startle response nor in PPI in DBA/2J mice. The reversal effect of BDNF on PPI deficit was unusually long-lasting: significant increase in PPI was found 1.5 months after single acute BDNF administration. Long-term ameliorative effect BDNF on disrupted PPI suggested the implication of epigenetic mechanism in BDNF action on neurogenesis. BDNF rather than GDNF could be a perspective drug for the treatment of sensorimotor gating impairments.

Ameliorative effect of BDNF on prenatal ethanol and stress exposure-induced behavioral disorders.

Brain-derived neurotrophic factor (BDNF) plays critical role in neuronal development, function, survival and plasticity of mature neurons. The present experiments investigated whether BDNF ameliorates the damaging effect of prenatal ethanol and stress exposure on behavior in offspring. Prenatal exposure of ethanol and stress combined during gestation inverted sexual partner preference of male offspring, increased social contacts with juvenile male mouse and stereotypic burying activity in the marble-burying test suggesting predisposition to homosexuality and to obsessive-compulsive disorder. Centrally administered BDNF (300ng i.c.v.) restored sexual female preference of male adult offspring and decreased marble-burying activity. Ameliorative effect was shown in 7-10 days after BDNF administration. The results provide the first evidence that BDNF improves epigenetic impairment of behavior and may have profound implications in the treatment of neurologic disorders induced by early environmental challenges.

Prenatal stress and ethanol exposure produces inversion of sexual partner preference in mice.

The presence of a sexually receptive female behind perforated transparent partition induced sexual arousal and specific behavior in male mice so they spent more time near partition in an attempt to make their way to the female. Three-chambered free-choice model was used to evaluate sexual partner preference. The main pattern of sexual preference was the time spent by a male mouse at the partition dividing female (F-partition time) versus a partition dividing male (M-partition time). Pregnant mice were given ethanol (11vol.%) for 1-21 gestational days, and were exposed to restraint stress (2h daily for 15-21 day of the gestation). Control pregnant mice had free access to water and food and were not stressed. Adult male offspring of ethanol and stress exposed dams (E+S) showed decreased F-partition time and increased M-partition time. Whereas F-partition time in all control mice prevailed over M-partition time, 78% E+S mice demonstrated prevailed M-partition time. E+S mice were more active in social interaction with juvenile male. No significant differences between E+S and control mice in the open field and novelty tests were revealed. Therefore, E+S exposure during dam gestation inverted sexual partner preference in male offspring, suggesting that stress and alcohol in pregnancy produces predisposition to homosexuality.

Automated analysis of antidepressants' effect in the forced swim test.

The forced swim test (FST) is a commonly used procedure of preclinical screening of drugs for the antidepressant activity. It has high predictive validity for a large group of antidepressant drugs blocking serotonin and noradrenaline reuptakes and improvement of immobility time evaluation in the FST is an important problem of preclinical psychopharmacology. Here a new automated version of the FST was developed. This version includes 4 inventions: (1) transmitted lighting instead of reflected lighting, (2) mouse silhouette tracking, (3) automated choice of immobility threshold and (4) the permutation test of drug's effect. Experiment was carried out on adult males of C57BL/6J and BALB/cJ mouse strains. The mice were treated with tricyclic antidepressant imipramine (15 and 30 mg/kg, i.p.). Mouse was placed in water tank, its movements were recorded by a rater and the silhouette alterations were automatically tracked. The sequence of silhouette alterations was scanned for immobility bouts with a threshold algorithm. Threshold was gradually altered and the value which maximized the difference between control and treated groups was chosen. The immobility values obtained with the procedure were compared with the permutation test. The data obtained with this procedure did not differ from those obtained by the rater. Imipramine dose dependently attenuated immobility time in C57BL/6 mice without any effect on BALB/c mice. The new procedure has been implemented in the EthoStudio software. It provides an objective automated evaluation of immobility time in the FST.