ABSTRACT
ObjectiveTo explore the effect of different hypoxic duration on the brain white matter injury.Methods ewborn rats were randomly divided into two groups, normal group (n=24) and model group (n=45). The model group was di-vided into 3 subgroups (n=15) according to the time of hypoxia (50 min, 70 min, and 90 min). The animal model of white matter injury was established by unilateral carotid artery ligation in model group. After different duration of hypoxia, the mortality rate was recorded, the morphological changes of brain pathology was observed by hematoxylin eosin (HE) staining, myelin basic pro-tein (MPB) of white matter was detected by immunolfuorescence staining and motor function was evaluated by climbing slope test.ResultsThe mortality rates signiifcantly increased with prolonged hypoxia. The mortality rate was as high as 60% in 90 min subgroup. The HE staining showed that there were no obvious injury in 50 min subgroup, selective white matter injury on the operative side appeared in 70 min subgroup, and a wide range of infarction of white matter, hippocampus, and cortex appeared in 90 min subgroup. MBP semi-quantitative scores of white matter injury were higher in 70 min subgroup (3.89 ± 0.47) and 90 min subgroup (4.72 ± 0.57) than that in the normal group (0.06 ± 0.24), the difference was statistically signiifcant (P <0.05). In climb-ing slope test, the subgroups had different degrees of motor dysfunction on affected side with 90 min subgroup being the most serious.ConclusionsWhite matter injury model could be established by unilateral carotid artery ligation, and different hypoxic duration signiifcantly affects the range and degree of injury.
ABSTRACT
Objective:To remove murine embryonic stem cells(mESC)from the differentiating cell culture and purify the differentiated cells by Magnetic Activated Cell Sorting(MACS).Methods:Neural differentiation of mESC was induced by a 5-stage method.The specific cell surface marker,SSEA-1,was used to identify ES cells in the differentiating cells.The optimal dilutions of mouse anti mouse SSEA-1 IgM primary antibody and FITC conjugated goat anti mouse secondary antibody were determined before the flow cytometry test.The incubation time and incubation temperature of primary antibody were all optimized to make the cytometry test accurate.After the optimization,stage 4 cells were dissociated into single cell suspension,incubated with antibody of SSEA-1 and microbeads conjugated goat anti mouse IgM,and then sorted through the magnetic field.The rate of SSEA-1 positive cells in pre-and post-separation groups was assessed by flow cytometry,and the viability of cells was evaluated by trypan blue staining counting under light microscopy.Results:The proportion of SSEA-1 positive cells in the separated cells can be reduced from(7.19?1.36)% to(1.34?0.80)%.The survival rate of sorted cells was more than 92%,similar to that of pre-separation cells.Conclusions:The MACS system we used can effectively remove mESC from the differentiated cells.The sorted cells will be well provided for the subsequent studies about transplantation therapy.