Research On Method For Micrometer Scale Sectioning Of The Whole Mouse Brain

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.