基于连续切片的大鼠中枢神经显微结构的三维重建研究
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摘要
目的:“虚拟人”的研究已经成为21世纪医学形态学研究的一大热点,借助于现代的影像学设备,通过CT、MRI等影像学方法能快速准确地对各组织进行三维可视化重建,而基于传统的组织学切片图像进行三维重建的研究却为数不多,其关键问题在于组织学切片处理过程中发生切片的旋转、位移和分散,给三维重建过程中图像的对齐和配准带来了困难。基于上述原因,本实验探索一种新的配准方法——三轴定位系统(X、Y、Z)应用于组织学切片的对齐和配准,探讨三轴定位法在大鼠脑内神经纤维束、神经核团以及神经通路等显微结构三维重建中的应用和效果。
方法:(1)大鼠全脑进行连续冰冻切片,Luxol Fast Blue(LFB)染色显示锥体束,利用德国Leica正置显微镜采集切片图像。Photoshop7.0软件对图像进行拼接、按三轴定位系统(X、Y、Z轴)校准和对齐,利用3D-DOCTOR4.0软件进行表面重建(surface reconstruction)和体重建(volume reconstruction),测量重建模型中锥体束的横径变化。重建模型以.3ds文件输出,利用3DMAX8.0软件对重建模型进行动画制作并实现可视化。(2)大鼠脑干横断面连续冰冻切片,进行NADPH组织化学染色显示NOS阳性神经核团在中枢神经系统的表达和分布,利用德国Leica正置显微镜采集切片图像,Photoshop7.0软件对图像进行拼接、按三轴法(X、Y、Z轴)校准和对齐,3D-DOCTOR4.0软件进行表面重建和体重建,测量重建模型中NOS阳性核团的大小和分布范围,重建模型以.3ds文件输出。(3)利用3DMAX8.0软件对重建模型进行材质重贴、透明度调整、ID值(渲染组值)设置和动画制作并将重建的锥体束与NOS神经核团进行等比拟合,以探讨这些核团和锥体束的位置关系。
结果:(1) LFB染色结合立体定位图谱显示锥体束自运动中枢发出,行向内囊顶部经内囊、大脑脚腹侧、桥脑基底部、锥体和延脑上部腹侧经锥体交叉,行向背侧并在颈髓后索下行。(2)利用3D-DOCTOR4.0软件重建出SD大鼠脑外形、脑内锥体束,可以观察到锥体束行程与LFB染色相一致。测量出锥体束自运动中枢至脊髓颈1节段全长为15 690μm,根据锥体束在内囊、中脑、桥脑、延脑和颈髓的横径变化和形态学观察,可见锥体束在内囊处最集中,桥脑处最分散。(3)利用3D-DOCTOR4.0软件重建出SD大鼠脑干外形、脑干内NOS阳性神经核团,根据立体定位图谱确定核团,观察并测量核团分布、形状和体积大小,NOS阳性核团主要分布于中脑导水管腹侧为被盖背外侧核(LDTg),桥脑基底部为被盖背外侧核(LDTg)、臂旁外侧核(LPB)、臂旁内侧核(MPB),菱形窝腹侧为被盖背后核(PDTg)、桥脑中央灰质(CGPn)、前庭内侧核小细胞部(MvePC),其中最大的阳性核团为被盖背外侧核(LTDg),体积为0.6747mm3。(4)利用3DMAX8.0软件将大鼠脑锥体束、NOS阳性神经核团进行拟合,并能清楚地显示NOS阳性神经核团与锥体束的位置关系。
结论: (1)利用三轴定位系统可以对大鼠全脑连续切片图像以及LFB显示的锥体束进行三维重建并对锥体束进行形态学测量。(2)根据三轴定位系统可以对大鼠脑干内NOS阳性神经核团进行三维重建和测量,并计算出有关核团的体积。(3) 3DMAX8.0可以将经3DDOCTOR4.0重建出的模型进行三维拟合,并能显示锥体束与脑干内NOS阳性核团的空间位置关系。
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.
方法:(1)大鼠全脑进行连续冰冻切片,Luxol Fast Blue(LFB)染色显示锥体束,利用德国Leica正置显微镜采集切片图像。Photoshop7.0软件对图像进行拼接、按三轴定位系统(X、Y、Z轴)校准和对齐,利用3D-DOCTOR4.0软件进行表面重建(surface reconstruction)和体重建(volume reconstruction),测量重建模型中锥体束的横径变化。重建模型以.3ds文件输出,利用3DMAX8.0软件对重建模型进行动画制作并实现可视化。(2)大鼠脑干横断面连续冰冻切片,进行NADPH组织化学染色显示NOS阳性神经核团在中枢神经系统的表达和分布,利用德国Leica正置显微镜采集切片图像,Photoshop7.0软件对图像进行拼接、按三轴法(X、Y、Z轴)校准和对齐,3D-DOCTOR4.0软件进行表面重建和体重建,测量重建模型中NOS阳性核团的大小和分布范围,重建模型以.3ds文件输出。(3)利用3DMAX8.0软件对重建模型进行材质重贴、透明度调整、ID值(渲染组值)设置和动画制作并将重建的锥体束与NOS神经核团进行等比拟合,以探讨这些核团和锥体束的位置关系。
结果:(1) LFB染色结合立体定位图谱显示锥体束自运动中枢发出,行向内囊顶部经内囊、大脑脚腹侧、桥脑基底部、锥体和延脑上部腹侧经锥体交叉,行向背侧并在颈髓后索下行。(2)利用3D-DOCTOR4.0软件重建出SD大鼠脑外形、脑内锥体束,可以观察到锥体束行程与LFB染色相一致。测量出锥体束自运动中枢至脊髓颈1节段全长为15 690μm,根据锥体束在内囊、中脑、桥脑、延脑和颈髓的横径变化和形态学观察,可见锥体束在内囊处最集中,桥脑处最分散。(3)利用3D-DOCTOR4.0软件重建出SD大鼠脑干外形、脑干内NOS阳性神经核团,根据立体定位图谱确定核团,观察并测量核团分布、形状和体积大小,NOS阳性核团主要分布于中脑导水管腹侧为被盖背外侧核(LDTg),桥脑基底部为被盖背外侧核(LDTg)、臂旁外侧核(LPB)、臂旁内侧核(MPB),菱形窝腹侧为被盖背后核(PDTg)、桥脑中央灰质(CGPn)、前庭内侧核小细胞部(MvePC),其中最大的阳性核团为被盖背外侧核(LTDg),体积为0.6747mm3。(4)利用3DMAX8.0软件将大鼠脑锥体束、NOS阳性神经核团进行拟合,并能清楚地显示NOS阳性神经核团与锥体束的位置关系。
结论: (1)利用三轴定位系统可以对大鼠全脑连续切片图像以及LFB显示的锥体束进行三维重建并对锥体束进行形态学测量。(2)根据三轴定位系统可以对大鼠脑干内NOS阳性神经核团进行三维重建和测量,并计算出有关核团的体积。(3) 3DMAX8.0可以将经3DDOCTOR4.0重建出的模型进行三维拟合,并能显示锥体束与脑干内NOS阳性核团的空间位置关系。
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.
引文
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