Improvement of Development of Vitrified Two-Cell Mouse Embryos by Vero Cell Coculture

Purpose: The purpose of this study was to determine the developmental potential of two-cell mouse embryos resulting from vitrification could increased using monolayer of Vero cells. Methods: Two-cell mouse embryos were divided into vitrified and nonvitrified groups. Embryos in the vitrified group were frozen with a combination of 10% ethylene glycol, 30% ficoll, and 0.5% M sucrose (EFS10) as cryoprotectants, and thawed rapidly with 0.5 M sucrose. The survived embryos were cultured either with Vero cells monolayer or in T6 medium. Accordingly the embryos of the nonvitrified group were also cultured. The rates of the development in all the groups were daily determined and statistically compared. At the end of the cultivation period, several expanded blastocysts from each group were stained with ethidium bromide and the mean number of the blastomers were counted and statistically compared. Results: After 4 days of culture, the developmental potential of vitrified-thawed embryos were significantly reduced in Vero cell-free medium, and the mean cell number of embryos reaching the expanded blastocyst stage were also lower than that of nonvitrified embryos. With exception of last day of culture, Vero cell coculture, resulted in a significant increase in the rate of development of vitrified-thawed embryos as well as improved the mean cell number of expanded blastocysts. On the other hand, the mean cell number of expanded blastocysts of nonvitrified group was significantly improved in coculture group. However, the rate of embryo development except for the first day of culture was similar to that of medium alone. Conclusions: The developmental potential of vitrified-thawed embryos appears to be retarded in conventional medium and Vero cell monolayer is capable to eliminate the postthaw deleterious effect of vitrification during the first 3 days of cultivation, but not for a longer period.     

Fusarochromanone-induced reactive oxygen species results in activation of JNK cascade and cell death by inhibiting protein phosphatases 2A and 5

Recent studies have shown that fusarochromanone (FC101), a mycotoxin, is cytotoxic in a variety of cell lines. However, the molecular mechanism underlying its cytotoxicity remains elusive. Here we found that FC101 induced cell death in COS7 and HEK293 cells in part by activating JNK pathway. This is evidenced by the findings that inhibition of JNK with SP600125 or expression of dominant negative c-Jun partially prevented FC101-induced cell death. Furthermore, we observed that FC101-activated JNK pathway was attributed to induction of reactive oxygen species (ROS). Pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger and antioxidant, suppressed FC101-induced activation of JNK and cell death. Moreover, we noticed that FC101 inhibited the serine/threonine protein phosphatases 2A (PP2A) and 5 (PP5) in the cells, which was abrogated by NAC. Overexpression of PP2A or PP5 partially prevented FC101-induced activation of JNK and cell death. The results indicate that FC101-induced ROS inhibits PP2A and PP5, leading to activation of JNK pathway and consequently resulting in cell death.     

Function and clustered expression of MaxiK channels in cerebral myocytes remain intact with aging

The incidence of stroke increases significantly in the aging population where stroke related deaths boost at >75 years and survivors are often permanently disabled. Aging is known to decrease cerebral blood flow likely due to an increase in arterial tone. Although MaxiK channels are key regulators of cerebral arterial tone their pattern of expression and function in cerebral blood vessels during aging is unknown. Using specific antibodies against the alpha-subunit of MaxiK channels and current recordings, we now demonstrate that in aging cerebral myocytes, MaxiK channels remain healthy. Furthermore, we show for the first time that in the vasculature, MaxiK channels are expressed in clusters. Clusters have an estimated radius of approximately 200 nm in young rats (3-5 month old Fisher 344 rats) which remains normal in old (25-30 month rats) cerebral myocytes. Consistent with a healthy MaxiK channel expression in old cerebral arteries, MaxiK current density, kinetics and Ca(2+) sensitivity were practically identical in young and old myocytes. Sensitivity to nanomolar concentrations of dehydrosoyasaponin-I that activates channels formed by alpha and beta subunits is also the same in young and old myocytes. These results demonstrate that MaxiK channels maintain normal expression during cerebral aging which is in sharp contrast to our previous finding of loss of expression in aging coronary arteries. It seems therefore, that cerebral myocytes have developed a protective anti-aging mechanism leading to the continued expression of MaxiK channels.     

Protective effects of N-acetylcysteine on 3, 4-methylenedioxymethamphetamine-induced neurotoxicity in male Sprague–Dawley rats

Exposure to 3, 4-methylenedioxymethamphetamine (MDMA) leads to spatial memory impairment and hippocampal cell death. In the present study we have examined the protective effects of N-acetyl-L-cysteine (NAC) on MDMA-induced neurotoxicity. A total of 56 male Sprague Dawley rats (200–250 g) received twice daily intraperitoneal (IP) injections of 5, 10 or 20 mg/kg MDMA plus NAC (100 mg/kg). Rectal temperatures were recorded before and after daily treatment. We used a Morris water maze (MWM) to assess spatial learning and memory. At the end of the study rats’ brains were removed, cells were counted and the level of Bcl-2, Bax and caspase-3 expression in the hippocampi were measured. NAC pretreatment significantly reduced MDMA-induced hyperthermia. In the MWM, NAC significantly attenuated the MDMA-induced increase in distance traveled; however the observed increase in escape latency was not significant. The decrease in time spent in the target quadrant in MDMA animals was significantly attenuated (p?<?0.001, all groups). NAC protected against MDMA-induced cell death and the up -regulation of Bax and Caspase-3, in addition to the down-regulation of Bcl-2. This data suggested a possible benefit of NAC in the treatment of neurotoxicity among those who use MDMA.     

Comparison of 1,2-propanediol and ethylene glycol for cryopreservation of slow-cooled mouse zygotes and their subsequent development

Purpose : This study was conducted on cryoprotective activity of ethylene glycol (ETG) and propanediol (PROH) on cleavage rate of mouse zygotes. Methods : Mouse oocytes were excised from fallopian tube of gonadotropin-treated mice, then inseminated with spermatozoa. After 16.5–17.5 h, zygotes were randomly allocated into three groups; control, toxicity, and frozen. In the latter, zygotes were slowly cooled with ETG and PROH similar to those used for human embryo cryopreservation. The survived zygotes cultured for 120 h and their later stages of development were compared with nonfrozen embryos. Results : The toxicity test showed that no differences were observed in cleavage rate between exposed and nonexposed embryos. The survival and expanded hatching blastocyst rate of embryos frozen with PROH was significantly better than with ETG (92.8 vs. 58.2% and 68.2 vs. 39.1%, respectively). Conclusions : ETG does not appear to be a good alternative to the classical PROH for freezing of mouse zygotes.     

Use of continuous subcutaneous insulin infusion (insulin pump) therapy in the hospital setting: proposed guidelines and outcome measures

PURPOSE: Individuals whose diabetes is being treated in the outpatient setting via an insulin pump often wish to maintain this therapy during hospitalization. The authors propose guidelines for management of patients on insulin pumps who require a hospital admission. METHODS: A collaborative interinstitutional task force reviewed current available information regarding the use of insulin pumps in the hospital. RESULTS: There was little information in the medical literature on how to manage individuals on established insulin pump therapy during a hospital stay. The task force believed that a policy that promotes patient independence through continuation of insulin pump therapy while ensuring patient safety was possible. A set of contraindications for continued use of pump therapy in the hospital are proposed. A sample patient consent form and order set are presented. Finally, measures that can be used to assess effectiveness of an inpatient insulin pump policy are outlined. CONCLUSIONS: Patients on established insulin pump therapy do not necessarily have to discontinue treatment while hospitalized. However, clear policies and procedures should be established at the institutional level to guide continued use of the technology in the acute care setting.