Late-stage Mice Research Articles

Gene expression of connective tissue growth factor (CTGF/CCN2) in calcifying tissues of normal and cbfa1-null mutant mice in late stage of embryonic development

Connective tissue growth factor (CTGF/CCN2), one of the most recently described growth factors, is produced by chondrocytes, vascular endothelial cells, and transforming growth factor (TGF)-beta-stimulated fibroblasts. CTGF was isolated from a chondrosarcoma-derived chondrocytic cell line, HCS-2/8, and found to be normally expressed in cartilage tissues, especially in hypertrophic chondrocytes, and also to stimulate both the proliferation and the differentiation of chondrocytes in vitro. Therefore, CTGF is thought to be one of the most important regulators of endochondral ossification in vivo. Herein we describe the expression pattern of the ctgf gene in the calcifying tissues of normal developing mouse embryos in comparison with that in core binding factor a1 (Cbfa1)-targeted mutant (cbfa1-null) mouse embryos, in which impaired development and growth were characteristically observed in the skeletal system. After 15 days of development (E15), the expression of ctgf was detected in the zone of hypertrophy and provisional calcification, in which ossification proceeds toward the epiphysis during the skeletal development of the mouse embryo. Furthermore, ctgf was expressed in developing molar and incisal tooth germs around the perinatal stage. However, no expression of the gene was found in the cbfa1-null mouse embryos. These results indicate that CTGF may have certain important roles in the development of the calcifying tissues in the mouse embryo.

Local and systemic control of the osteoblast.

Local and systemic control of the osteoblast.

Glutamate Augments Retrovirus-Induced Immunodeficiency Through Chronic Stimulation of the Hypothalamic-Pituitary- Adrenal Axis

The mechanisms for activating the hypothalamic-pituitary-adrenal (HPA) axis and the roles glucocorticoids play in the pathogenesis of chronic infectious disease are largely undefined. Using the LP-BM5 model of retrovirus-induced immunodeficiency, we found alterations in HPA axis function, manifested as an increase in circulating levels of adrenocorticotropic hormone and corticosterone, beginning after only 3 mo of infection. These changes occurred contemporaneously with a shift in the profile of circulating cytokines from a Th1-dominant (IFN-gamma) to Th2-dominant (IL-4, IL-10) phenotype. No significant changes in either circulating IL-1beta, IL-6, or TNF-alpha levels were observed in infected mice. Administering the N-methyl-D-aspartate receptor antagonist MK-801 to infected mice normalized plasma adrenocorticotropic hormone and corticosterone levels, indicating that glutamate was a major activator of the HPA axis. Moreover, MK-801 treatment of late-stage mice also reversed the type 1 to type 2 cytokine shift to a degree comparable or superior to treatment with the glucocorticoid receptor antagonist RU-486. These findings indicate that HPA axis activation during LP-BM5 retrovirus infection is mediated by the chronic hyperactivation of glutamatergic pathways in the hypothalamus. Through this mechanism, the degree of peripheral immunodeficiency observed in the late-stage disease is profoundly augmented.

Genetic expression of hexokinase and glucose phosphate isomerase in late-stage mouse preimplantation embryos: transcription activities in glucose/phosphate-containing HTF and glucose/phosphate-free P1 media.

In mouse and human preimplantation development, pyruvate is consumed preferentially during early embryogenesis; however, during the morula and blastocyst stages, glucose is the preferred energy substrate. Studies have suggested that the glycolytic enzymes, hexokinase and glucose phosphate isomerase, are important enzymes in glucose metabolism during these later stages of human and mouse preimplantation development. In order to investigate the genetic activities of these enzymes in late-stage mouse embryos developing in vitro, we analysed hexokinase and glucose phosphate isomerase transcription activities by qualitative RNA assays using reverse transcriptase-nested polymerase chain reaction amplification of individual mouse morulae and early blastocysts incubated in glucose/phosphate-free preimplantation stage one (P1) medium and glucose/phosphate-containing human tubal fluid (HTF) medium. We observed an increased incidence of hexokinase transcripts in the population of blastocysts compared with morulae, and differences in transcript incidence between early blastocysts developing in HTF medium and in P1 medium. In contrast, glucose phosphate isomerase transcripts were consistantly present in all embryos analysed, and appear to be constitutively expressed during late-stage mouse embryogenesis. The different activity patterns of the two glycolytic genes may reflect different mechanisms of gene regulation or differential transcript stability during the later stages of mouse preimplantation development.

In vitro development of bovine demi-embryos bisected at early-, mid- and late- blastocyst stages

The purpose of this experiment was to determine the loss of cells during the splitting of embryos. The experiment was carried out on early, middle and late stage mouse blastocysts as well as on late stage sheep and cow blastocysts. The embryos were bisected with a metal mioroscalpel. Manipulation of the embryos was done in phosphate buffered saline (PBS) solution with 20% fetal calf serum (FCS) at +37°C or in PBS solution free of Ca++ and Mg++ ions. To determine the number of cells present, intact embryos and demi-embryos were placed in acridine orange solution and examined under a fluorescent microscope.The average number of cells in early, middle and late stage mouse blastocysts was 39.4, 54.1 and 71.9, respectively. The loss of cells caused by splitting was 6.4, 7.6 and 9.9, respectively. The average number of cells in late stage sheep blastocysts was 115.3 and in late stage cow blastocysts it was 138.5. In sheep and cows, the loss of cells due to splitting the embryos in a PBS solution free of Ca++ and Mg++ ions did not differ from that observed in a PBS solution with 20% FCS. Similarly, bisection carried out at +37°C did not result in lower cell losses, than that done at room temperature.

Splitting cattle embryos commercially: The effects of sucrose, embryo stage and the duration between embryo recovery and bisection

The purpose of this experiment was to determine the loss of cells during the splitting of embryos. The experiment was carried out on early, middle and late stage mouse blastocysts as well as on late stage sheep and cow blastocysts. The embryos were bisected with a metal mioroscalpel. Manipulation of the embryos was done in phosphate buffered saline (PBS) solution with 20% fetal calf serum (FCS) at +37°C or in PBS solution free of Ca++ and Mg++ ions. To determine the number of cells present, intact embryos and demi-embryos were placed in acridine orange solution and examined under a fluorescent microscope.The average number of cells in early, middle and late stage mouse blastocysts was 39.4, 54.1 and 71.9, respectively. The loss of cells caused by splitting was 6.4, 7.6 and 9.9, respectively. The average number of cells in late stage sheep blastocysts was 115.3 and in late stage cow blastocysts it was 138.5. In sheep and cows, the loss of cells due to splitting the embryos in a PBS solution free of Ca++ and Mg++ ions did not differ from that observed in a PBS solution with 20% FCS. Similarly, bisection carried out at +37°C did not result in lower cell losses, than that done at room temperature.

Cytoplasmic inclusions in spinal neurons of the motor neuron degeneration ( Mnd) mouse : I. Light microscopic analysis

The motor neuron degeneration ( Mnd) mutation in the mouse is a late onset, autosomal dominant, neurodegenerative disease in which ventral horn neurons have been shown to contain numerous, large cytoplasmic inclusions. Histochemical and immunocytochemical studies performed on spinal cord from Mnd Mnd mice in late stages of the disease showed the inclusions to contain protein, lipid and carbohydrate moieties. Spinal neurons, especially those in spinal lamina IX, contained increased β-glucuronidase activity in the form of large cytoplasmic inclusions. Such inclusions also contained increased acid phosphatase and trimetaphosphatase activity. When immunostained with antiubiquitin antibodies, intracellular ubiquitin deposits were present as accumulations of varying size; some were amorphous while others contained small granules. Extraneuronal ubiquitin deposits were detected in the neuropil. Immunostaining with monoclonal antibody ML30, used here to assay for the presence of a mitochondrial epitope in the inclusions, was widespread and punctate in white and grey matter from Mnd Mnd and age-matched control spinal cords. The overall pattern of staining was similar for both tissue sources and did not correspond to any of the other probes which reacted with the inclusions in Mnd neurons. The presence of increased levels of lysosomal hydrolases and ubiquitinated molecules suggests that the two general systems for intracellular digestion are activated in Mnd Mnd spinal neurons.

A Quantitative Histological Study of Strain-Dependent Differences in the Effects of Irradiation on Mouse Lung during the Intermediate and Late Phases

Strain differences in the intermediate and late phases of the radiation response of mouse lung were investigated histologically. The proportion of lung impairment in mice at 28 and 52 weeks postirradiation and in mice dying of respiratory insufficiency was assessed by scoring lung acini as nonfunctional due to lesions which obstructed airflow, or open and presumably functional. The nine strains tested were divided into three groups on the basis of the late fibrotic response. Group 1 mice, three C57 strains, developed extensive contracted fibrosis and usually showed enough damage to explain late deaths. Group 2, SWR, A, and BALB/c strains, developed foci of contracted fibrosis. Group 3, CBA and two C3H strains, did not form fibrotic scars. Mice in Groups 2 and 3 that died with no pleural effusions appeared to have insufficient late lung damage to account for respiratory distress. Problems with pulmonary blood flow were indicated by evidence of loss of fine vasculature and right ventricular hypertrophy. In nondistressed, late-stage mice in Groups 2 and 3, loss of capillary perfusion in lung parenchyma free of obvious lesions was demonstrated by infusion of colloidal carbon. In one strain, A, an estimate of the proportion of nonperfused lung was made on distressed late-stage mice. Almost 50% of lung acini were nonfunctional as a result of nonperfusion, and an additional 9% of acini were nonfunctional due to lesions obstructing ventilation. It is suggested that nonperfusion of apparently normal lung acini is a major factor in late-phase deaths in those mouse strains which show little or no fibrosis.

Cell loss in bisected mouse, sheep and cow embryos

The purpose of this experiment was to determine the loss of cells during the splitting of embryos. The experiment was carried out on early, middle and late stage mouse blastocysts as well as on late stage sheep and cow blastocysts. The embryos were bisected with a metal mioroscalpel. Manipulation of the embryos was done in phosphate buffered saline (PBS) solution with 20% fetal calf serum (FCS) at +37°C or in PBS solution free of Ca ++ and Mg ++ ions. To determine the number of cells present, intact embryos and demi-embryos were placed in acridine orange solution and examined under a fluorescent microscope. The average number of cells in early, middle and late stage mouse blastocysts was 39.4, 54.1 and 71.9, respectively. The loss of cells caused by splitting was 6.4, 7.6 and 9.9, respectively. The average number of cells in late stage sheep blastocysts was 115.3 and in late stage cow blastocysts it was 138.5. In sheep and cows, the loss of cells due to splitting the embryos in a PBS solution free of Ca ++ and Mg ++ ions did not differ from that observed in a PBS solution with 20% FCS. Similarly, bisection carried out at +37°C did not result in lower cell losses, than that done at room temperature.

Distribution of halothane and the metabolites trifluoroacetic acid and bromide in the conceptus after halothane inhalation by pregnant mice.

Mice in late stage of gestation were exposed to halothane at various concentrations for 1 hr, and were killed at different time intervals after discontinuance of inhalation. The concentration of halothane in maternal plasma decreased rapidly, and in the amniotic fluid the halothane never reached more than 20% of maternal plasma levels. Trifluoroacetic acid (TFA) and bromide, formed mainly by maternal metabolism of halothane, accumulated in foetus and amniotic fluid with time, and reached plateau levels in amniotic fluid between 4 and 24 hrs. TFA infused intravenously to the mother reached higher levels in amniotic fluid after long survival times, than in maternal plasma. Equilibrium dialysis experiments showed that TFA and trichloroacetic acid (TCA) (previously shown to accumulate in amniotic fluid) were bound to amniotic fluid macromolecules only to approximately 20-30 percent. This was at the same magnitude (or lower) as compared to binding in maternal plasma, suggesting that such binding did not contribute to the observed retention in the amniotic fluid. Other possible explanations for the slow accumulation and long-term retention in amniotic fluid are transport by bulk flow via foetus, excretion via foetal urine, or paraplacentally through endometrium and foetal membranes, followed by trapping in the amniotic fluid. The significance of this accumulation of metabolites of halogenated organic solvents and halothane for their foetotoxicity is not clear.

Correlation between metabolism of betamethasone 17,21-dipropionate and adrenal hypertrophy in rat fetuses

The effects of metabolites of betamethasone 17,21-dipropionate (BMDP) on the hypothalamo-pituitary-adrenocortical axis were assessed by measurements of adrenal weights, after studying the metabolism of BMDP in vivo and in vitro in pregnant rats and mice. After BMDP was injected intravenously at a dose of 5 mg/kg into rats and mice in late stages of pregnancy, it disappeared rapidly from the plasma and brain in both mothers and fetuses while betamethasone 17-propionate (BMP) was detected as the main metabolite followed by betamethasone (BM). In vitro studies demonstrated that BMDP was metabolized to BMP in maternal and fetal tissues (plasma, liver, brain and placenta) of both species. The subcutaneous administration of BMP to rats in the late stages of pregnancy induced adrenal hypertrophy in fetuses, though the adrenals of the mothers became atrophic. In the case of mice, both maternal and fetal adrenals became atrophic. Administration of BM produced adrenal atrophy in mothers and fetuses of both species. The subcutaneous administration of 6β-hydroxybetamethasone 17-propionate (6β-OH-BMP) to rat fetuses in utero produced adrenal hypertrophy and 6β-hydroxybetamethasone (6β-OH-BM) showed no effect. These data suggest that BMP is transferred across the placental barrier to produce marked adrenal hypertrophy in rat fetuses.