【作者】 张斌；

【导师】 骆清铭；

【作者基本信息】 华中科技大学 ， 生物医学工程， 2011， 博士

【摘要】 神经解剖学在揭示脑的复杂结构和功能方面占有重要的地位。近年来众多科学家都在研究新的方法来获取全脑的显微结构信息,以便对脑进行跨层次的、系统性的研究。面对该需求,我们实验室开发了显微光学切片断层成像系统,它能够对标本进行连续微米切片,并实时对切片成像。通过这种成像方法可以在微米水平获取小鼠全脑标本的显微结构信息。要基于该成像方法获取小鼠全脑显微结构信息,就需要制备适于微米切片的小鼠全脑染色标本。但是,传统标本制备方法中尚无对小鼠全脑尺度的大标本进行整体染色,同时标本适于微米切片的标本制备方法。为此本文开展了适于微米切片的小鼠全脑染色标本制备方法的研究。研究了Nissl法染色小鼠全脑标本制备方法,以显示完整的小鼠脑中的神经元胞体的形态和分布。主要实验步骤为对完整的小鼠脑依次进行固定、染色、分色、脱水和包埋。小鼠全脑在Nissl染液中的浸染时间为10天,在梯度酒精-丙酮中进行分色、脱水的时间为9天,使用Spurr树脂包埋全脑标本,制备出了适于微米切片的小鼠全脑Nissl法染色标本。研究了Golgi-Cox法染色小鼠全脑标本制备方法,以显示完整的小鼠脑中的神经元的形态、突起的行向以及神经元在脑中的空间位置。主要实验流程为对完整的小鼠脑依次进行固定、浸染、黑化、脱水和包埋。小鼠全脑在Cox液中的浸染时间约为半年,用氢氧化锂黑化全脑,黑化时间为1天,脱水时间为1天,使用Spurr树脂包埋全脑标本,制备出了适于微米切片的小鼠全脑Golgi-Cox法染色标本。研究了标本的微米厚度切片质量评价方法。通过测量“切片贴紧刀面滑行的距离”来评价研制的标本的硬度是否适于微米切片,并确保获取到完好的切片图。经过多次实验,确定了合适的Spurr树脂包埋方法,结果显示所研制的标本制备方法可满足切片和成像的要求。基于研制的小鼠全脑Nissl法和小鼠全脑Golgi-Cox法制备了相应的小鼠全脑标本,标本形态完整。利用显微光学切片断层成像系统对制备的标本进行了厚度1μm的连续切片和成像,分别获得了小鼠全脑经嗅球、端脑-间脑、中脑-脑桥、小脑-延髓完整冠状面的图像。结果表明,从脑的表层到深层均有大量神经元被染色,能清晰显示神经元的形态、结构和分布以及核团的结构等,同时证明研制的小鼠全脑标本制备方法能满足微米切片和成像的要求。更多还原

【Abstract】 Neuroanatomy plays an important role in demonstrating the complex structures and functions of the brain. To systematically investigate the brain across multiple scales, various methods were developed to get the microstructures of the whole brain in the recent decade. To achieve the aim, our lab has developed the micro-optical sectioning tomography (MOST) which sections the whole brain continuously in micrometers and simultaneously performs imaging on the sections in real time. The microstructures of the whole mouse brain at the micron level can be obtained using MOST.The stained whole mouse brain specimen is required for MOST to perform micron sectioning. However, preparing the specimen as large as a whole mouse brain for micron sectioning is difficult and no such method has been reported in the literature. Hence, the present study aims to tackle this issue.An improved Nissl method for preparing stained whole mouse brain specimen has been developed for demonstrating the morphology and the spatial distribution of somas in the whole mouse brain. The protocol has been changed to:fixation, staining, differentiation and dehydration, infiltration and embedding. In the staining procedure, we stained the whole mouse brains by impregnating them in the Nissl solution and prolonging the staining time to 10 days. In the differentiation and dehydration procedure, we prolonged the differentiation and dehydration time to 9 days. Finally, the specimen of whole mouse brain was ready for micron sectioning after we embedded the Nissl-stained whole brain with Spurr resin.An improved Golgi-Cox method for preparing stained whole mouse brain specimen has been developed for demonstrating the morphology of neurons, the trace and direction of neurites, and the spatial location of neurons in the whole mouse brain. The protocol has been changed to:fixation and impregnating, darkening, dehydration, infiltration and embedding. In the impregnating procedure, we prolonged the time of impregnating the whole mouse brain in Cox solution to half year. In the darkening procedure, we darkened the whole mouse brain with LiOH solution and prolonged the darkening time to 1 day. In the dehydration procedure, we prolonged the dehydration time to 1 day. Finally, the specimen of whole mouse brain was ready for micron sectioning after we embedded the Golgi-Cox stained whole brain with Spurr resin.A method to evaluate the specimen in terms of the sectioning and imaging performance has been developed. To obtain a complete section image in micron sectioning, the hardness of specimen was determined by the sliding distance of the section along the knife edge. The suitable parameters for embedding with Spurr resin were discovered after lots of trials. The results have shown that the embedded specimen prepared by our method meets the demand for sectioning and real-time imaging.The intact whole mouse brain specimens were prepared correspondingly by the improved Nissl method and the improved Golgi-Cox method. The continuous 1μm sectioning and simultaneous imaging of the prepared specimens was performed using MOST. The whole coronal images through the olfactory bulb, telencephalon-diencephalon, midbrain-pons and cerebellum-medulla oblongata of the mouse brains were obtained. The results have showed that stained neurons can be found across the superficial and the deep areas of the brain. The morphology and spatial distribution of stained neurons were clearly demonstrated, as well as the structure of nuclei. Our results also demonstrate that the proposed methods for preparing the whole mouse brain meet the requirement of micron sectioning and real time imaging.更多还原