大鼠脑片的器官型培养及其中神经元的发育规律

Organotypic cerebral slice culture and the course of neuronal development in it

目的:建立器官型脑片培养的方法,证明体外培养到一定时间神经元发育成熟,可在此基础上制作神经系统变性疾病模型.方法:取出生24 h内SD大鼠的额叶皮质作器官型培养.取培养7～14 d内每天的及培养第3周、4周和8周时的脑片,固定、脱水、切片后行Nissl和抗神经丝重链(NFH)免疫组化染色.同时用正常生长大鼠的脑片作对照,观察与培养脑片染色结果的异同.结果:Nissl染色结果显示,培养脑片中的锥体细胞体积逐渐增大、染色变浅,1～4周内皮质分层清晰.抗NFH免疫组化染色显示,培养至第10天时,位于第V层的锥体细胞着色,第12天以后Ⅲ层和V层均着色.对照组第5天时V层着色,3周以后Ⅲ层和V层都着色.对位于Ml区的第V层锥体细胞进行计数,从培养第12天开始至2个月时,神经元数目保持恒定.结论:器官型脑片培养适用于出生后神经元发育的研究,并可用于制作神经系统变性疾病的体外模型.

Objective ：To establish a method of organotypic cerebral culture. So as to pave the way for building some neurodegenerative disease models. Methods： Organotypic cerebral cultures were prepared from prefrontal brain of neonatal SD rats. After culturing 7 to 14 days, 3 weeks, 4 weeks and 8 weeks, respectively, cerebral slices were fixed, dehydrated and sectioned in cryostat. The sections proceeded with Nissl staining and neurofilament high molecular weight （ NFH ） immunohistochemical staining. The difference was observed between controls and cultured slices using normal rats as controls. Results： Nissl staining showed that pyramidal neurons in cultured slices were increased in volume and lightened in staiping. The delaminating construction was clear from 1 to 4 weeks after culturing. In cultured slices, immunohistochemical staining showed that NFH positive pyramidal cells appeared on layer V on the tenth day and on both layers V and Ⅲ after culturing 12 days. In the control group, NFH positive pyramidal cells appeared on layer V in 5-day-old rats, and appeared on both layers V and Ⅲ in over 3-week-old rats. In cultured cerebral slices, the number of pyramidal neurons on layer V in M1 area was invariable from 12 days to 2 months. Conclusion ：Orgaotypic cerebral culture can be used to study postnatal development for neocortex and build some in vitro models for neurodegenerative diseases.