The Effect Of Hypoxia On Tau Protein Of Organotypic Hippocampal Slice In Rat

Objective 1. To establish a model for organotypic hippocampal slice culture of 4 week-old rats. 2.To observe the effect of hypoxia on tau protein of organotypic hippocampal slice in 4 week-old rats for building up a simulating dementia tissue model.Methods 1. Hippocampal slices from 4 week-old rats were incubated with insert of microporous membrane. Inverted phase contrast microscope, lactate dehydrogenase (LDH)kit, immunofluorescence histochemical method and confocal laser scanning microscope were used to observe the circumstance of the culture. 2. The hippocampal slices were cultured for seven days in normal condition. Then they were cultured for half an hour, an hour, one and half an hour, and two hours in the condition of hypoxia. To follow, they were cultured for six days in normal condition again. Inverted phase contrast microscope was used to observe the circumstance of the culture. The expression level of tau protein was observed and made some statistical analysis by immunofluorescence histochemical method and confocal laser scanning microscope.Results 1. With prolonged time of incubation, the cultured hippocampal slices got thinner, cell strap lightness got taper, neurons got fewer and astrocytes got more. Lactate dehydrogenase contents in the culture solution were stable and the activity of hippocampal slices was well for twelve days. 2. Fluorescence intensity ratio of phosphorylated-Tau(Ser214) and Tau-5 protein of an hour hypoxia group was greater than control group in the thirteenth day. Fluorescence intensity ratio of Tau(Ab-214) and Tau-5 protein of hypoxia group had no significant deviation with control group in the thirteenth day. 3. The expression amounts of phosphorylated-Tau(Ser396) protein in half an hour hypoxia group were heightened stably. Its expression amounts of an hour hypoxia group were heightened in the tenth day, and reduced in the thirteenth day with control group. 4. The expression amounts of phosphorylated- Tau(Thr231) protein of an hour hypoxia group were heightened in the tenth day, and its expression amounts of half an hour and an hour hypoxia group were both heightened in the thirteenth day with control group. The expression amounts of Tau(Ab-231) protein were heightened in an hour hypoxia group in the tenth day, and in half an hour hypoxia group in the thirteenth day with control group. 5. The expression amounts of phosphorylated- Tau(Thr205) protein of half an hour hypoxia group and an hour hypoxia group were more than control group in the tenth day and in the thirteenth day. 6. The expression amounts of phosphorylated-Tau(Thr181) protein of half an hour hypoxia group were more than control group in the thirteenth day.Conclusion 1. The model of organotypic hippocampal slice culture of 4 week-old rats could provide a way for research of the nervous system diseases in vitro. 2. The site Ser214 of tau protein had excess phosphorylation in an hour hypoxia model. 3. The site Ser396, Thr231, Thr205 and Thr181 of tau protein could have excess phosphorylation in this hypoxia model. 4. Organotypic hippocampal slice of half an hour hypoxia and an hour hypoxia were hopeful to be used as a simulating dementia tissue model.