Cryopreservation of rat embryos at all developmental stages by small-volume vitrification procedure and rapid warming in cryotubes.

Intracellular ice formation during cryopreservation is lethal to the cell, including during warming. Here, we examined the effect of sample volume and warming rate on the cryopreservation success of 1-cell rat embryos based on successful development into blastocysts in vitro and to term in vivo following embryo transfer. Embryos were equilibrated in 5% propylene glycol solution for 10 min, held for 40 s at 0 °C in cryopreservation solution (5%PG + PEPeS), and cooled by immersion in liquid nitrogen. When 1-cell embryos were cryopreserved in a volume of 30-100 µL at a cooling rate of 5830-7160 °C/min and warmed at 35,480-49,400 °C/min by adding 1 mL of 0.3 M sucrose solution at 50 °C, 17.3-28.8% developed into blastocysts, compared with 57.0% of untreated embryos. However, when 1-cell embryos were cryopreserved in a smaller volume of 15 µl at 7950 °C/min and warmed at 68,850 °C/min, 58.8 ± 10.6% developed into blastocysts and 50.0 ± 7.4% developed to term, comparable to that of non-treated embryos (57.0 ± 5.4% and 51.4 ± 3.1%, respectively). Cryopreserved embryos at other developmental stages also showed high in vitro culture potential similar to that of the control. Using a conventional cryotube and a small-volume vitrification procedure with rapid warming, we achieved high levels of subsequent rat embryonic development at all developmental stages.

Roxadustat and thyroid-stimulating hormone suppression.

Hypoxia-inducible factor prolyl-hydroxylase inhibitors belong to a new class of orally administered drugs for treating anemia in patients with chronic kidney disease (CKD). The prevalence of hypothyroidism is disproportionately high in patients with CKD on hemodialysis. We report a rapid suppression of thyroid-stimulating hormone (TSH) and decrease in free triiodothyronine (T3) and free tetraiodothyronine levels after switching from darbepoetin alfa to roxadustat in a hemodialysis patient with hypothyroidism on levothyroxine therapy. This was reversed after stopping roxadustat. Roxadustat has structural similarity with T3 and is a selective activating ligand for thyroid hormone receptor-ß possibly suppressing TSH release.

Renal cell carcinoma sharply captured by imaging technology at an early stage in a hemodialysis patient: Usefulness of noninvasive monochrome superb microvascular imaging.

It has been drawing much attention that type 2 diabetes mellitus is closely associated with increased incidence of numerous cancers and their poor prognosis. Consequently, malignancy has been recently recognized as one of diabetic complications in addition to various conventional complications. Furthermore, it is well known that the prevalence of renal cell carcinoma (RCC) is drastically increased in hemodialysis (HD) patients. Therefore, screening of RCCs in HD patients is a very important and urgent issue as there are no highly sensitive tumor markers for RCCs. Monochrome superb microvascular imaging (mSMI) is a relatively new Doppler ultrasound technique and is useful especially when evaluating very slow blood flow state, because this allows for imaging microvessels with low velocity in the absence of a contrast agent. Thus, mSMI might be also useful when contrast enhancement is not obvious on CT and/or contrast-enhanced ultrasonography using perflubutane or contrast agents are contraindicated. Moreover, it has been reported that mSMI could effectively detect vascularity of renal malignant tumor than benign renal mass in nondialysis patients. We propose that mSMI of ultrasonography could become one of the very useful methods for detecting RCCs at an early stage with high sensitivity in HD patients.

Small-volume vitrification and rapid warming yield high survivals of one-cell rat embryos in cryotubes†.

To cryopreserve cells, it is essential to avoid intracellular ice formation during cooling and warming. One way to achieve this is to convert the water inside the cells into a non-crystalline glass. It is currently believed that to accomplish this vitrification, the cells must be suspended in a very high concentration (20-40%) of a glass-inducing solute, and subsequently cooled very rapidly. Herein, we report that this belief is erroneous with respect to the vitrification of one-cell rat embryos. In the present study, one-cell rat embryos were vitrified with 5 µL of EFS10 (a mixture of 10% ethylene glycol (EG), 27% Ficoll, and 0.45 M sucrose) in cryotubes at a moderate cooling rate, and warmed at various rates. Survival was assessed according to the ability of the cells to develop into blastocysts and to develop to term. When embryos were vitrified at a 2613 °C/min cooling rate and thawed by adding 1 mL of sucrose solution (0.3 M, 50 °C) at a warming rate of 18 467 °C/min, 58.1 ± 3.5% of the EFS10-vitrified embryos developed into blastocysts, and 50.0 ± 4.7% developed to term. These rates were similar to those of non-treated intact embryos. Using a conventional cryotube, we achieved developmental capabilities in one-cell rat embryos by rapid warming that were comparable to those of intact embryos, even using low concentrations (10%) of cell-permeating cryoprotectant and at low cooling rates.

Characteristic features of newly established specific pathogen-free albino large rabbit (JW-AKT): Comparison with Japanese White and New Zealand White rabbits.

The present study was conducted to reveal characteristic features of albino large rabbit (JW-AKT) which we formerly established a specific pathogen-free (SPF) colony. Body weights of JW-AKT rabbit at 52 weeks old was 5.7 ± 0.4 kg in males and 6.4 ± 0.4 kg in females. Weight of body, heart, lung and kidney in JW-AKT rabbit was significantly higher than in Japanese white and New Zealand white rabbits in both sexes. Though the body weight (BW) was rather lower in males, body length and brain weights tended to be higher in males than in females. Since body fat was significantly higher in females, what affects difference in BW is body fat, rather than the physical constitution of female JW-AKT rabbit. No critical sex difference was found in hematological parameters in JW-AKT rabbit. The results indicated that JW-AKT were about 1.5 times larger than the general laboratory rabbits with common properties in hematology. Thus, JW-AKT rabbit could be used as a novel SPF experimental animal model with some advantages in surgical experiments or collection of large amount of biological specimen.

Vitrification of one-cell mouse embryos in cryotubes.

Preventing intracellular ice formation is essential to cryopreserve cells. Prevention can be achieved by converting cell water into a non-crystalline glass, that is, to vitrify. The prevailing belief is that to achieve vitrification, cells must be suspended in a solution containing a high concentration of glass-inducing solutes and cooled rapidly. In this study, we vitrified 1-cell mouse embryos and examined the effect of the cooling rate, the warming rate, and the concentration of cryoprotectant on cell survival. Embryos were vitrified in cryotubes. The vitrification solutions used were EFS20, EFS30, and EFS40, which contained ethylene glycol (20, 30 and 40% v/v, respectively), Ficoll (24%, 21%, and 18% w/v, respectively) and sucrose (0.4 0.35, and 0.3 M, respectively). A 5-µl EFS solution suspended with 1-cell embryos was placed in a cryotube. After 2 min in an EFS solution at 23 °C, embryos were vitrified by direct immersion into liquid nitrogen. The sample was warmed at 34 °C/min, 4,600 °C/min and 6,600 °C/min. With EFS40, the survival was low regardless of the warming rate. With EFS30 and EFS20, survival was also low when the warming rate was low, but increased with higher warming rates, likely due to prevention of intracellular ice formation. When 1-cell embryos were vitrified with EFS20 and warmed rapidly, almost all of the embryos developed to blastocysts in vitro. Moreover, when vitrified 1-cell embryos were transferred to recipients at the 2-cell stage, 43% of them developed to term. In conclusion, we developed a vitrification method for 1-cell mouse embryos by rapid warming using cryotubes.

Prevalence of gastroesophageal reflux disease symptoms and effects of esomeprazole on the quality of life related to reflux and dyspepsia in patients on maintenance hemodialysis.

BACKGROUND: The prevalence of gastroesophageal reflux disease (GERD) symptoms has not been investigated in patients on maintenance hemodialysis in Japan, and few studies have reported the effect of proton pump inhibitors (PPIs) in hemodialysis patients with GERD symptoms. Here, we investigated the prevalence of GERD symptoms and the effects of the PPI esomeprazole on the quality of life related to reflux and dyspepsia in patients on maintenance hemodialysis. METHODS: This was a cross-sectional/cohort study of hemodialysis outpatients implemented in 10 Japanese medical facilities from October 2012 to March 2014. The trial was registered in the UMIN Clinical Trial Registry (UMIN000009124). RESULTS: Forty-one of 385 patients (11%) reported GERD symptoms on the Global Overall Symptom (GOS) questionnaire. Multivariate logistic regression analysis identified the independent prognostic factors for GERD symptoms as a history of gastric ulcer and use of sevelamer hydrochloride or calcium polystyrene sulfonate. Participants with GERD symptoms completed the Quality of Life in Reflux and Dyspepsia, Japanese version (QOLRAD-J) questionnaire and were assigned to receive 4-week esomeprazole treatment (20 mg/day). This PPI therapy significantly improved all QOLRAD-J domains in the full analysis set (n = 28) and improved the GERD symptoms listed in the GOS questionnaire. Significantly impaired disease-specific quality of life (QOL) in the QOLRAD-J domains was observed in 44.4-74.1% of patients who had symptoms before treatment. The mean GOS and QOLRAD-J scores correlated significantly. CONCLUSION: Therapy with 20 mg/day esomeprazole appears to be efficacious for improving disease-specific QOL and GERD symptoms in Japanese patients on maintenance hemodialysis.

Effects of slightly acidic electrolysed drinking water on mice.

Slightly acidic electrolysed (SAE) water is a sanitizer with strong bactericidal activity due to hypochlorous acid. We assessed the safety of SAE water as drinking water for mice at a 5 ppm total residual chlorine (TRC) concentration to examine the possibility of SAE water as a labour- and energy-saving alternative to sterile water. We provided SAE water or sterile water to mice for 12 weeks, during which time we recorded changes in body weight and weekly water and food intakes. At the end of the experiment, all of the subject animals were sacrificed to assess serum aspartate aminotransferase, alanine aminotransferase and creatinine levels and to examine the main organs histopathologically under a light microscope. In addition, we investigated the bacteria levels of both types of water. We found no difference in functional and morphological health condition indices between the groups. Compared with sterile water, SAE water had a relatively higher ability to suppress bacterial growth. We suggest that SAE water at 5 ppm TRC is a safe and useful alternative to sterile water for use as drinking water in laboratory animal facilities.