Expression of Toll-Like Receptors in the Lung Tissue of Mouse Fetuses Generated by in vitro Embryo Culture and Embryo Transfer.

Mouse fetuses generated by in vitro embryo culture and embryo transfer exhibit impaired lung development, altered composition of pulmonary epithelial cells associated with downregulation of several genes involved in lung development and toll-like receptor (TLR) signaling pathway. The aims of the present study were to determine the expression of all TLRs and to examine if the expression of TLRs, along with genes involved in TLR signaling pathway, is altered in the lung tissue of mouse fetuses generated through embryo culture and embryo transfer. Two experimental (EGs) and one control (CG) group were included in the study. Embryos cultured at 5% CO2-95% air for 95 h or less than 24 h were transferred to pseudo-pregnant females to obtain fetuses comprising EGin vitro (n = 18) and EGin vivo (n = 18), respectively. Fetuses obtained from naturally ovulating females on day 18 of pregnancy served as the CG (n = 18). Western blot and immunohistochemistry were used to determine the expression of TLR proteins. The expression of transcripts encoding TLRs, and the genes involved in TLR signaling pathway (Lbp, Pik3r1, Pik3cb, Nfkbia, and Fos), was determined using qRT-PCR. While all TLRs were expressed by cells lining the bronchial/bronchiolar epithelium of lung tissues in all groups, some of the TLRs were expressed in a specific pattern. When compared to CG, the expression of transcripts encoding TLR-2, -3, -4, -5, -7, -8, -9, -12, -13, Lbp, Pik3r1, Pik3cb, Nfkbia, and Fos was significantly downregulated in both EGs. It appears that stress imposed on embryos at preimplantation stages of development is associated with downregulation of TLRs, along with some of the genes involved in TLR signaling pathway, in the lung tissue during the perinatal period. It remains to be determined if downregulation of TLRs, along with the genes involved in TLR signaling pathway, has any functional consequences in the adult lung tissue.

Transfer of mouse blastocysts exposed to ambient oxygen levels can lead to impaired lung development and redox balance.

In vitro culture under atmospheric oxygen puts embryos under oxidative stress and impairs preimplantation development. However, to what extent this process alters the redox balance in the perinatal period remains largely unknown. The aim of the present study was to examine if the redox balance is altered in the lung tissue of fetuses generated through transfer of mouse embryos exposed to atmospheric oxygen at different stages of development and to determine if this has any effect on lung morphogenesis and gene expression. Two experimental groups (EGs) were generated by transferring in vitro- and in vivo-derived blastocysts to pseudo-pregnant females. In vivo-developed fetuses served as control. Enzymatic/nonenzymatic antioxidants, malondialdehyde (MDA) levels, total antioxidant capacity, stage of lung development and gene expression were evaluated on day 18 of pregnancy. Weight of fetuses was significantly less in both experimental cohorts (ANOVA, P < 0.001 versus control), associated with delayed lung development, higher amounts of MDA (ANOVA, P < 0.001 versus control) and altered expression of several genes in oxidative stress/damage pathways. Evidence gathered in the present study indicates that pre-implantation stress caused by culture under atmospheric oxygen, even for a short period of time, leads to fetal growth restriction, impaired lung development and redox balance along with dysregulation of several genes in oxidative stress response. Absence of an EG in which in vitro embryo culture was performed at 5% oxygen and the use of genetically heterogeneous F2 fetuses are the limitations of the study. In any case, the long-term impact of such dramatic changes in the developmental programming of resulting fetuses warrants further investigations.

Mice with diet-induced obesity demonstrate a relative prothrombotic factor profile and a thicker aorta with reduced ex-vivo function.

: Classical risk factors such as cholesterol and lipoproteins are currently not sufficient to explain all physiopathological processes of obesity-related vascular dysfunction as well as atherosclerosis and arteriosclerosis. Therefore, the discovery of potential markers involved in vascular dysfunction in the obese state is still needed. Disturbances in hemostatic factors may be involved in the developmental processes associated with obesity-related cardiovascular disorders. We hypothesized that alterations of several hemostatic factors in the obese state could correlate with the function and morphology of the aorta and it could play an important role in the development of vascular dysfunction. To test this, we fed mice with a high-fat diet for 18 weeks and investigated the relationships between selected hemostatic factors (in either plasma or in the liver), metabolic hormones and morphology, and ex-vivo function of the aorta. Here, we show that 18-week exposure to a high-fat diet results in a higher plasma fibrinogen and prolonged prothrombin time in diet-induced obese mice compared to the controls. In addition, liver levels or activities of FII, FX, activated protein C, AT-III, and protein S are significantly different in diet-induced obese mice as compared to the controls. Curiously, FII, FVIII, FX, activated protein C, PTT, and protein S are correlated with both the aorta histology (aortic thickness and diameter) and ex-vivo aortic function. Notably, ex-vivo studies revealed that diet-induced obese mice show a marked attenuation in the functions of the aorta. Taken together, aforementioned hemostatic factors may be considered as critical markers for obesity-related vascular dysfunction and they could play important roles in diagnosing of the dysfunction.

The short-term effects of different doses of dexamethasone on the numbers of some bacteria in the ileum.

Glucocorticoids are known to affect intestinal biota both directly or indirectly. The aim of the study reported here was to determine the short-term effects of different doses of dexamethasone on the numbers of various ileal bacteria populations. Rats were randomly put into groups, and each group was administered a single-dose injection of dexamethasone at either 0.1, 0.5, 1, 2.5, 5, or 10 mg/kg body weight. At 48-h post-injection, the numbers of total aerobe, anaerobe, lactobacilli and coliform bacteria in the ileum were determined. The numbers of total aerobes and lactobacilli were higher in the groups receiving 5 and 10 mg/kg dexamethasone than in the control and other dose groups (P < 0.01 and P < 0.001, respectively). The number of ileal anaerobic bacteria was higher in group receiving 5 mg/kg than in the other groups (P < 0.01). There were more coliform bacteria in the group receiving 0.1 mg/kg than in the groups receiving 0.5, 1 and 10 mg/kg (P < 0.05). In light of these results, the effects of dose-dependent increases in the number of different bacterial groups affecting gut functions have still to be determined in future studies.

The expression of matrix metalloproteinases 8 and 9 by neutrophils of Wistar albino rats with severe qualitative and quantitative protein malnutrition.

Neutrophils are the major cellular immune components in response to bacterial infections. Neutrophil enzymes are important in invasion, inflammation, and infection processes. In order to understand the basic effects of protein malnutrition on neutrophils we studied matrix metalloproteinases 8 and 9 (MMP-8 and MMP-9) production in severe quantitative and qualitative protein malnutrition in rats. Wistar rats (2 months old) were divided into four groups each with three subgroups and fed various protein-containing diets (24% protein, 20% gelatin-containing and N-free) for 7, 14, 21, and/or 28 days. Neutrophil enzyme expression was determined by Western blotting. Leukocytes decreased significantly due to malnutrition (p = 0.001 ) whilst the percentage of neutrophils increased (p = 0.02) in protein-deprived groups. Neutrophils of malnourished rats produced lower levels of MMP-8 at early stages of protein deprivation with an increase in the following weeks. MMP-9 production by neutrophils from N-free diet fed animals was highest after one week. Serum MMP-9 levels decreased in the qualitative but not in the quantitative protein malnutrition groups. Results suggest that neutrophils might be important in reuse of body cell proteins during fasting or malnutrition conditions and dietary manipulation might have profound effects on MMP-8 and -9 production in rats.