Study And Preliminary Application On Cryofixation Of Energy Metabolism In Mouse Brain

Monitoring brain's energy metabolism plays an important role in revealing the mechanism of brain disease and function. Low-temperature microscopic imaging takes advantage of low temperature to acquire information of structure and metabolism simultaneously without limitation of imaging depth, which makes acquiring 3D structural information of large-size sample possible. Low-temperature microscopic imaging is an important method to monitor brain's energy metabolism information. However, its ability to precisely reveal the energy metabolism of tissue and organs is dependent on whether the energy metabolism status of tissues could be fixed exactly in vivo. Current effective cryofixation has been achieved on the rat model. Mouse is another widely-used animal model, but there is no feasible cryofixation method on mouse model yet. It is limited by mouse's easily mutability of physiological status, high sensitivity to environment temperature, and low tolerance of experimental operation. To this issue, a cryofixation method for mouse brain was proposed and demonstrated in regulation of oxygen supply to the brain and cortical spreading depression in this thesis.Improved progressive cryofixation had been chosen after analyzing the current cryofixation methods for rat model. According to the physiological feature of mouse brain, several possible factors in influencing cryofixation effect were analyzed. Experimental conditions of the cryofixation of mouse brain were optimized in this research. Finally, an efficient metabolism fixing method at low temperature for mouse brain was established.Cryofixation of brain oxygen supply regulation and cortical spreading depression(CSD) experiments were performed to evaluate the efficiency of the mouse brain fixing method. Energy metabolism states of whole mouse brain in hypoxia, normoxia and hyperoxia were captured to demonstrate the effectivity of the cryofixation method. The results indicated the correlation of redox ratio and oxygen supply. And the results show the responses of corpus callosum and hippocampus to hypoxic and hyperoxia were different. Moreover, the whole-brain metabolic status during CSD were shown, and the * This work was supported by Ph D Programs Foundation of Ministry of Education of China(Grant No. 20110142130006). changes of energy metabolism in CSD response areas were observed in the experiment. The preliminary research on these two model experiments demonstrates the validity of cryofixation method for mouse brain and its potential in brain function research.