Three Dimensional Reconstruction Of Microstructures In Rat Central Nervous System Based On Serial Sections

Objective:"Visible Human"plan becomes popular in medical morphological research in 21st century, with the development of modern physical technology, radiological methods such as CT and MRI have been used in the research proverbially, but histological methods are occasionally used, a critical problem which obstructing the development of histological method is the rotation and distartion of the slices. According to the three axises in three dimensional (3D) space, a new align method—three axises localizing system(X, Y, Z)was used in this experiment to explore its application and effects in the 3D reconstruction models of the nerve fibers, nucleus and nervous pathways in the rat central nervous system based on serial sections.Methods: (1) Serial sections of the rat brain were directly mounted, then stained with Luxol Fast Blue (LFB). The images were collected by Leica microscope under the 2.5 objective lens. The images were stitched and alligned according three axises(X, Y, Z) in Photoshop7.0. Pyramidal tracts were identified according to the positive fibers and stereotaxis atlas of the rat brain. The surface and volume reconstructions were executed in the 3D-DOCTOR4.0, and the transverse diameteres of pyramidal tracts at different site of the brain were measured. The models of reconstructions were exported as .3ds files, and then imported into the 3DMAX8.0 software. The new materials and ID numbers were given to render the models, the .avi files were exported at last. (2) Serial sections of the rat brain stem were directly mounted, and histochemically stained with NADPH. The images were collected by Leica microscope under 5 objective lens. The images were stitched and alligned according the three axises(X, Y, Z) in Photoshop7.0, surface and volume reconstructions were executed in 3D-DOCTOR4.0. The size, volume and distribution of NOS positvie nucleus were observed and measured. Reconstruction models were exported as .3ds files. (3) The reconstruction model of nucleus was merged with the model of pyramidal tracts. The new materials, opacities and ID number were reset to render the models. The animations were exported to explore the relationship between these NOS positive nuclei and pyramidal tracts.Results: (1) Pyramidal tracts can be located by Luxol Fast Blue stained slices with the assistence of stereotaxis atlas of the rat brain. The pyramidal tracts originate from the motor area of cortex, and pass down through the internal capsule, the cerebral peduncle, the basilar part of pons, the pyramid of ventral medulla oblongata, the pyramidal decussation, and crossed to the deepest part of the dorsal fasciculus of the cervical cord. (2) Using 3D-DOCTOR4.0 software, the brain surface and pyramidal tracts of rat can be reconstructed, and the pyramidal tracts originate from the motor area, and then pass through internal capsule, cerebral peduncle, the basliar part of pons, and the pyramid of ventral medulla oblongata, the pyramidal decussation, and crossed to the deepest part of the dorsal fasciculus of the cervical cord. The whole length of pyramidal tract was measured and the changes of transverse diameters in internal capsule, midbrain, pons, medulla oblongata and cervical cord were measured and compared. The length of pyramidal tract from motor area to 1st cervical segment is 15,690μm. (3) Using 3D-DOCTOR4.0 software, the brain stem and NOS positive nuclei were reconstructed. NOS positive nuclei distribute in the ventral part of midbrain duct(LDTg), the basliar part of pons(LDTg,LPB,MPB), and so on. Moreover, the names of these nuclei were identified according to the stereotaxic atlas, and their distribution, shape and volumes were measured and calculated. The biggest nucleu is LDTg, the volume is 0.6747mm3. (4) Using 3DMAX8.0 software, the pyramidal tracts and the NOS positive nucleus were matched together to explore the spacial relationship between the fibers and nuclei.Conclusion: (1) With the three axises localizing system, three dimensional reconstruction of pyramidal tract can be made based on serial sections. The length and transverse diameters can be measured in the 3D-DOCTOR4.0 software. (2) Three dimensional reconstruction of NOS positive nuclei in brain stem can be made based on serial sections after the slices were histochemically stained by NADPH, the volume of the nucleus can be calculated.(3) The spacial relationship between these NOS positive nuclei and the pyramidal tracs can be revealed by the 3DMAX8.0 software.