Protective effect of melatonin against oxygen-induced retinopathy: a study based on the HMGB1/NF-κB/NLRP3 axis / 中国当代儿科杂志

OBJECTIVES@#To study the protective effect of melatonin (Mel) against oxygen-induced retinopathy (OIR) in neonatal mice and the role of the HMGB1/NF-κB/NLRP3 axis.@*METHODS@#Neonatal C57BL/6J mice, aged 7 days, were randomly divided into a control group, a model group (OIR group), and a Mel treatment group (OIR+Mel group), with 9 mice in each group. The hyperoxia induction method was used to establish a model of OIR. Hematoxylin and eosin staining and retinal flat-mount preparation were used to observe retinal structure and neovascularization. Immunofluorescent staining was used to measure the expression of proteins and inflammatory factors associated with the HMGB1/NF-κB/NLRP3 axis and lymphocyte antigen 6G. Colorimetry was used to measure the activity of myeloperoxidase.@*RESULTS@#The OIR group had destruction of retinal structure with a large perfusion-free area and neovascularization, while the OIR+Mel group had improvement in destruction of retinal structure with reductions in neovascularization and perfusion-free area. Compared with the control group, the OIR group had significant increases in the expression of proteins and inflammatory factors associated with the HMGB1/NF-κB/NLRP3 axis, the expression of lymphocyte antigen 6G, and the activity of myeloperoxidase (P<0.05). Compared with the OIR group, the OIR+Mel group had significant reductions in the above indices (P<0.05). Compared with the control group, the OIR group had significant reductions in the expression of melatonin receptors in the retina (P<0.05). Compared with the OIR group, the OIR+Mel group had significant increases in the expression of melatonin receptors (P<0.05).@*CONCLUSIONS@#Mel can alleviate OIR-induced retinal damage in neonatal mice by inhibiting the HMGB1/NF-κB/NLRP3 axis and may exert an effect through the melatonin receptor pathway.

An improved method for primary culture of neonatal mouse cardiomyocytes / 中国比较医学杂志

Objective To establish a stable and fast method for primary culture of mouse cardiomyocytes. Methods Dishes were coated with polylysine firstly.A two-step approach was used to isolate and digest mouse cardiomyocytes cells (0.25%trypsin in 4°C overnight and 0.5 mg/mL to 1.0 mg/mL collagenase +5 mg/mL albumin collagen digestion liquid in 37°C for short-time digestion), then the cardiomyocytes were purified through differential adhesion for 70 min and 5-bromodeoxyuridine ( BrdU) .The cell morphology was observed under an inverted microscope. The survival rate of cardiacmyocytes was detected by trypan-blue staining and their purity was identified by α-actinin immunofluorescence staining.Results The cardiomyocytes were in good shape and pulsed spontaneously.The survival rate of the cardiomyocytes reached 98%and the purity was 95%.Conclusions This method described in this study is an ideal method for primary culture of mouse cardiomyocytes with a high survival rate and high purity.

Isolation,culture and identification of neonatal mouse cardiomyocytes / 重庆医学

Objective To establish a method to isolate,culture and identify cardiomyocytes from neonatal mouse,estimate the cardiomyocytes survival rate,and identify the purity of cardiomyocytes.Methods Neonatal C5 7 mouse heart(within 3 days)was di-gested using trypsin and collagenase.Cardiomyocytes survival rate was estimated by trypan blue staining,and cardiomyocytes purity was identified byα-actin immunofluorescence staining.Results Mouse cardiomyocytes could be successfully isolated and cultured using neonatal mouse within 3 days.Trypan blue staining showed cardiomyocytes survival rate was>95%,and cardiomyocytes pu-rity was more than 95% demonstrated byα-actin immunofluorescence staining.Conclusion Under the strict experimental condi-tions,mouse cardiomyocytes can be successfully isolated and cultured with high survival rate and high purity.

Effects of Murine Dermal Cells on the Regulation of Hair Growth Is Dependent on the Cell Number and Post-Natal Age of Newborn Mice

Dermal cells from neonatal mice can initiate the formation of hair follicles (HFs) when combined with adult mouse epidermal cells and transplanted subcutaneously into athymic mice. In the present study, the effects of dermal cells on HF formation were tested in terms of total cell number and the time course of cell harvest. Results demonstrated that the number of dermal cells is critical to the formation of HF. Furthermore, hair forming ability is rapidly decreasing as the neonatal mice age. To examine potential differences in gene expression, cDNA array was performed. Results demonstrate that numerous molecules which are directly involved in receptor and signaling correlated with decreased hair inductivity in early time points after delivery. It is reported that bone morphogenic protein (BMP)-6 and Wnt3a treatment increased hair inductivity of dermal papilla cells. But in our study, no changes were observed in the expression levels of BMP-6 and Wnt3a. However, several Wnt related genes demonstrate increased or decreased expression levels. Thus, our results suggest that co-ordinated regulation of these molecules will be important in hair neogenesis within our model system.

The Effects of Glutamate Receptor Antagonists on Cultured Cerebral Cortical Neurons of Neonatal Mouse Damaged by Oxidative Stress

PURPOSE: To evaluate neurotoxic effects induced by oxygen-radicals, which were generated by adding xanthine oxidase(XO) and hypoxanthine(HX), and protective effects of glutamate receptor antagonist such as MK-801 and 6-cyano-7-nitroquinoxaline(CNQX). METHODS: Dissociated cell cultures were prepared from cerebrum of neonatal mouse. Tissues were dissected and diced into small pieces in phosphate buffered saline and were incubated at 37degrees C. Isolated cells were resuspended in Eagle's minimum essential medium and plated poly-L-lysine coated plastic coverslips in 96 well multichambers at a cell density of 3x105 cells/well. Cells were grown in a 5% CO2/95% air atmosphere at 37degrees C. Cytotoxic effects were examined in cerebral cortical neurons cultured for 3 hours in media containing various concentration of XO and HX. The protective effects of glutamate receptor antagonist were also examined by MTT assay and neurofilament enzymeimmunoassay(EIA). Microscopic examinations were also done. RESULTS: Oxygen radicals markedly induced decrement of the cell viability of cultured mouse cerebral cortical neurons in a dose-dependent manner. Midpoint cytotoxicity value was 30mU/ml XO/0.1mM HX, when mouse cerebral cortical neurons were incubated for 3 hours with various concentrations of XO and HX. The number of cells and neurites was decreased when cerebral cortical neurons were cultured for 3 hours in a medium containing 30mU/ml XO/0.1mM HX. MK- 801 was very effective in blocking oxidant-induced neurotoxicity, while CNQX falied to show any protective effect in these cultures. CONCLUSION: It is suggested that oxygen radicals are neurotoxic, and selective N-methyl-D-aspartate antagonists such as MK-801 are very effective in protecting neurotoxicity induced by oxygen radicals in cultured cerebral cortical neurons of neonatal mouse.

The Protective Effect of Vitamin E and Desferrioxamine on Cultured Cerebral Neurons of Neonatal Mouse Damaged by Ischemic Condition

PURPOSE: Perinatal asphyxia is an important cause of neurologic morbidity. Experiments in animal models of hypoxic-ischemic brain injury demonstrate that brain damage starts during hypoxia-ischemia. In order to evaluate the ischemic condition-induced neurotoxic effect in view of oxi-dative stress, we examined the cytotoxic effect in cultured cerebral neurons of neonatal mouse. METHODS: Dissociated cell cultures were prepared from cerebrum of neonatal mouse. Tissues were diced into small pieces and were incubated in phosphate buffered saline at 37degrees C. Isolated cells were resuspended in the medium and plated in poly-L-lysine coated 96 well multichambers at a cell density of 5x104cells/well. Cells were grown in a 5% CO2/95% air atmosphere at 37degrees C. Cytotoxic effects were examined in the cultured cerebral neurons with time interval in the ischemic condition with a 95% nitrogen/5% CO2. And the protective effect of vitamin E and desferrioxamine as an antioxidant was examined by MTT assay and neurofilament enzymeimmunoassay(EIA). Microscopic examinations were also done. RESULTS: Ischemic condition markedly decreased the cell viability in a time-dependent manner in cultured cerebral neurons. MTT50 value was estimated at 10 minutes, when cerebral neurons were incubated for various time intervals in ischemic condition. Under light microscopy, the number of cells and neurites were decreased when cerebral neurons were cultured for 10 minutes in the ischemic condition. Vitamin E was an effective antioxidant in blocking ischemic condition-induced neurotoxicity, while desferrioxamine was not in these cultures. CONCLUSION: It is suggested that ischemic conditions are neurotoxic and selective antioxidant such as vitamin E is effective in protecting against the neurotoxicity induced by ischemic condition in cultured cerebral neurons of neonatal mouse.

Evaluation of a new approach that inject bilirubin solution into cerebellomedullary cistern to establish bilirubin encephalopathy model in neonatal mouse / 重庆医科大学学报

Objective:To evaluate the feasibility of animal model of bilirubin encephalopathy by injecting bilirubin solution into cerebellomedullary cistern of neonatal full-term and premature mice.Methods:Thirty 7-day-old mice were randomly divided into 5 groups:control group,model group 1,model group2,model group3,and model group4.Bilirubin solution was injected into the cerebellomedullary cistern in model groups(bilirubin 20?g/g bodyweight),while equal volume of normal saline was injected into the cerebellomedullary cistern in control group.The mice in model groups were sacrificed at the time of 12h,24h,48 and 72h after injection,respectively,and those in control group were sacrificed at 24h after injection.The apoptosis and necrosis of neurocytes,the expressions of Bcl-2 and Bax proteins were detected by HE stain,immunohistochemical method and TUNEL in the hippocampus,respectively.Preterm mice were delivered by cesarean section at 21 days gestation and fed by surrogate mother mouse until 7 days old.Bilirubin solution was injected into their cerebellomedullary cistern.The mice's abnormal behaviors were observed.Results:The mice of model groups and preterm group appeared abnormal behaviors in different degree.Under light microscope,the neurocytes in their hippocampus appear apoptosis and necrosis.The nissl body in pyramidal cell body and dendron decreased,even dissolved to disappear.TUNEL showed that the apoptosis rate in the hippocampus increased obviously at 12h post-injection,reached the peak at 24h post-injection.The positive rate of Bax protein in the hippocampus reached the peak at 24h post-injection,while the positive rate of Bcl-2 protein decreased to the lowest.Conclsions:The method that injecting bilirubin solution into neonatal mice' cerebellomedullary cistern to set up the animal model of bilirubin encephalopathy is simple and feasible.The peak time of neurocytes' apoptosis in hippocampus was 24h post-injection.It's crucial to prevent bilirubin encephalopathy as early as possible.

Functional study of spermatozoa derived from neonatal mouse testes grafted into immunodeficient mice / 西安交通大学学报(医学版)

Objective To investigate the fertilization ability of spermatozoa derived from neonatal mouse testes grafted into immunodeficient mice by intracytoplasmic sperm injection(ICSI).Methods Neonatal Kun-ming mouse testes containing primitive spermatogenic cells as the only germ cells were grafted under the back skin of castrated BALB/c-nu/nu mice.Grafts were taken out after 8 weeks.The initiation and restoration of spermatogenesis in grafts was documented by histological analyses.The ultrastructure of spermatogenic cells in grafts was observed using transmission electron microscopy.The activity of acrosomal enzyme of spermatozoa was analyzed with gelatin substrate test.The fertilization ability of spermatozoa was determined by using intracytoplasmic sperm injection.Results Histological analysis showed complete restoration of spermatogenesis in the grafts after 8 weeks.1?10~(3) spermatozoa with normal morphological appearance in 1mg suspended graft tissue were counted.Spermatogonia and spermatocytes with normal ultrastructure were observed.Gelatin substrate test positive spermatozoa were found implicating the activity of acrosomal enzyme of the spermatozoa.Cleavages were observed both in the ICSI group and the ICSI plus electric stimulation group.Conclusion Spermatozoa with normal morphology and fertilization ability can be developed by allografting neonatal mouse testes under the back skin of immunodeficient mice.