Urine collection in the common marmoset (Callithrix jacchus) and its applicability to endocrinological studies

We investigated a new method of urine collection in the common marmoset. We entered the cage as soon as the light cycle started in the breeding room and collected urine from the animal directly without any restraint. We were able to take separate samples from completely different individuals housed together for behavioral studies in the same cage. Urine and blood samples were taken from individuals from late pregnancy through postpartum nursing period. Cortisol and prolactin concentrations measured in urine were compared to those measured in blood to evaluate this collection method. LH/CG level in the urine samples was also measured. Urine data in females indicated a tendency toward high cortisol values during late pregnancy, a sharp drop before parturition, and increase after delivery. In females cortisol levels measured in blood closely resembled concentrations measured in urine. Urine cortisol in males clearly indicated an increase postpartum, but the increase was not indicated in plasma. Plasma and urine prolactin concentrations in females made a similar increase during lactation. Male's plasma prolactin clearly indicated an increase directly proportional to strong behavioral contact with the infant. We also confirmed hormonal changes during pregnancy, and postpartum ovulation and subsequent pregnancies, from urine LH/CG data. We found this method extremely useful because of the high correlation between cortisol, prolactin and LH/CG data from blood and urine. Additionally, we collected urine samples with little stress to the animal from fear, irritation, pain, etc.     

Distribution of γ-Aminobutyric Acid and Glutamate Decarboxylase in the Layers of Rat Oviduct

An enzymatic microassay method for glutamate decarboxylase (GAD) and gamma-aminobutyric acid (GABA) was improved to yield a high sensitivity and a low blank. The 20-microns thick freeze-dried sections (0.2-1.5 micrograms dry weight) were prepared from the oviduct and ovary of rat. The analysis of these microsamples by the improved method showed that, contrary to the previous observations, the rat ovary is devoid of GAD activity and contains a trace amount of GABA. Both are present abundantly in the oviduct. In the oviduct mucosa, significant GAD activity was found in the estrous phase, whereas the activity was nearly null during other phases of the estrous cycle. GABA concentration in the oviduct mucosa was 10-fold higher than in the cerebral cortex; its variation during the estrous cycle was not remarkable. In the muscle layer of oviduct, GAD activity had a low peak in the estrous phase and GABA concentration was almost constant during the estrous cycle. The denervation experiment showed that GAD is present in the nerve terminals innervating the oviduct.     

Differential Effect of Cerebral Ischemia on Monoamine Content of Discrete Brain Regions of the Mongolian Gerbil (Meriones unguiculatus)

The effect of bilateral cerebral ischemia on noradrenaline, dopamine, and serotonin concentrations in six brain regions (i.e., the cerebral cortex, striatum, hippocampus, midbrain-diencephalon, cerebellum, and pons-medulla oblongata) was examined in the gerbil stroke model. The relative changes in regional cerebral blood flow after bilateral common carotid occlusion were also assessed using the radioactive microsphere technique. At 1 h after bilateral carotid occlusion, a significant decrease of monoamine concentration was observed in the cerebral cortex, striatum, hippocampus, and midbrain-diencephalon whereas no significant change was detected in the cerebellum and pons-medulla oblongata. The fall in NA content was most prominent in the cerebral cortex and hippocampus and percentage reductions of dopamine and serotonin were greatest in the striatum and cerebral cortex, respectively. These results suggest that the monoamine neurons in various brain regions might have different vulnerabilities to ischemic insult and show no evidence of transtentorial diaschisis.     

Effects of portal free fatty acid elevation on insulin clearance and hepatic glucose flux

We tested the hypothesis that, due to greater hepatic free fatty acid (FFA) load, portal delivery of FFAs, as in visceral obesity, induces hyperinsulinemia and increases endogenous glucose production to a greater extent than peripheral FFA delivery. For 5 h, 10 microeq.kg(-1).min(-1) portal oleate (n = 6), equidose peripheral oleate (n = 5), or saline (n = 6) were given intravenously to conscious dogs infused with a combination of portal and peripheral insulin to enable calculation of hepatic insulin clearance during a pancreatic euglycemic clamp. Peripheral FFAs were similar with both oleate treatments and were threefold greater than in controls. Portal FFAs were 1.5- to 2-fold greater with portal than with peripheral oleate. Peripheral insulin concentrations were greatest with portal oleate, intermediate with peripheral oleate (P < 0.001 vs. portal oleate or controls), and lowest in controls, consistent with corresponding reductions in plasma insulin clearance and hepatic insulin clearance. Although endogenous glucose production did not differ between the two routes of oleate delivery, total glucose output (endogenous glucose production plus glucose cycling) was greater with portal than with peripheral oleate (P < 0.001) despite the higher insulin levels. In conclusion, during euglycemic clamps in dogs, the main effect of short-term elevation in portal FFA is to generate peripheral hyperinsulinemia. This may, in the long term, contribute to the metabolic and cardiovascular risk of visceral obesity.     

Effects of atorvastatin and aspirin combined therapy on inflammatory responses in patients undergoing coronary artery bypass grafting

This study was conducted to compare the effects of atorvastatin plus aspirin combined therapy on inflammatory responses, endothelial cell function, and blood coagulation system in patients undergoing coronary artery bypass grafting (CABG) to aspirin monotherapy. The patients were randomized into atorvastatin plus aspirin combined therapy group and aspirin monotherapy group. Reduced total cholesterol in the combined therapy group was found in a short term of medication for 14 days. On postoperative day (POD)-14, inhibitory effects of the combined therapy on whole blood aggregation as well as platelet activation assessed by flow cytometry were stronger than those of the monotherapy. Furthermore, cytokine, cytokine receptors, c-reactive protein and alpha1-acid glycoprotein in the combined therapy group were down-regulated on POD-14. At the same time, circulating levels of thromboxane A(2), vascular endothelial growth factor and thrombin-antithrombin III complex as well as P-selectin, L-selectin and intercellular adhesion molecule-1 were down-regulated, while E-selectin and transforming growth factor-beta1 was up-regulated. Atorvastatin plus aspirin combined therapy may improve inflammatory responses, accelerated platelet function, vascular endothelial cell function, blood coagulation system at the early stage such as 14th day after CABG. In conclusion, atorvastatin and aspirin combined therapy may bring beneficial effects to the patient after CABG.     

Isolation and characterization of embryonic stem-like cells from canine blastocysts

Embryonic stem (ES) cells are pluripotent cells with the capacity to generate any type of cell. Here we describe the isolation of ES-like cells from canine blastocysts. Canine embryos were collected from beagle bitches at day 11-16 of first estrus. A total of 80 normal embryos were obtained from 15 dogs. Of the embryos, 13 were at the morulae stage, 39 at the blastocyst stage, and 28 at the hatched blastocyst stage. The inside of morulae or inner cell masses (ICMs) of blastocysts were isolated mechanically, and cultured onto mouse embryonic fibroblasts (MEF) as feeder layers. Primary cell colonies were formed in 0% (0/13) of morulae, 25.6% (10/39) of blastocysts, and 67.9% (19/28) of hatched blastocysts. These colonies were separated either by enzymatic dissociation or by mechanical disaggregation. Dissociation with collagenase resulted in immediate differentiation, but with mechanical disaggregation these cells remained undifferentiated, and two ES-like cell lines (cES1, cES2) continued to grow in culture after eight passages. These cells had typical stem cell-like morphology and expressed specific markers such as alkaline phosphatase activity, stage specific embryonic antigen-1 and Oct-4. These cells formed embryoid bodies (EBs) in a suspension culture; extended culture of EBs resulted in the formation of cystic EBs. When the simple EBs were cultured on tissue culture plates, they differentiated into several types of cells including neuron-like, epithelium-like, fibroblast-like, melanocyte-like, and myocardium-like cells. These observations indicate that we successfully isolated and characterized canine ES-like cells.