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目的:探讨应用组织学方法对大鼠脑干内一氧化氮合酶(nitric oxide synthase,NOS)阳性神经核团进行三维重建的技术。方法:以4只成年雄性SD大鼠为材料,标本固定后取中脑至脊髓颈1节段组织。3只于冰冻切片机上行冠状面连续切片,所得切片行NADPH-黄递酶(nicotinamide aenine inucleotide phosphate-diaphorase)染色,每张切片厚度为30μm。1只行正中矢状切片。所得切片行LFB(Luxol fast blue)染色,5倍物镜下行显微照相。应用Photoshop7.0软件拼接图像,并以三轴法将图片配准;应用3D-DOCTOR 4.0软件对连续图像中NOS阳性核团进行三维可视化重建,参照大鼠脑立体定位图谱确定核团名称,并利用软件自带工具计算核团体积。结果:三维重建的NOS阳性神经核团分布于中脑背侧、中脑导水管周围、桥脑基底部外侧、延脑腹侧外部。脑干内体积最大的NOS阳性神经核团为被盖背外侧核(laterodorsal tegmentum,LDTg),平均体积为0.6747±0.0026mm3。结论:利用组织学方法可以对大鼠脑干内NOS阳性神经核团进行三维可视化重建;借助3D-DOCTOR 4.0软件可对重建核团进行测量并计算出其体积。
Objective: To investigate the technique of three-dimensional reconstruction of nitric oxide synthase (NOS) positive neural nuclei in rat brainstem by histological method. Methods: Four adult male Sprague-Dawley rats were used as materials. The specimens were fixed and taken from the midbrain to the spinal cord. Three frozen sections of the coronal plane were sliced serially. The resulting sections were stained with NADPH-diaphorase with a thickness of 30 μm. 1 only a median sagittal slice. The resulting slices were stained with Luxol fast blue (LFB) and photographed with a 5x objective lens. Application of Photoshop7.0 software stitching images, and three-axis method will be the picture registration; application of 3D-DOCTOR 4.0 software NOS positive nuclei in three-dimensional visualization of the continuous reconstruction of the image, refer to the rat brain stereotactic map to determine the name of the nucleus and Use the software comes with tools to calculate the volume of the nucleus. Results: Three-dimensional reconstruction of NOS-positive nerve nuclei distributed in the dorsal midbrain, midbrain aqueduct, pontine basal lateral, the ventral lateral ventral. The NOS positive nucleus, the largest volume in the brain stem, was laterodorsal tegmentum (LDTg) with an average volume of 0.6747 ± 0.0026 mm3. CONCLUSION: Three-dimensional visualization of NOS-positive neural nuclei in rat brainstem can be performed by histological methods. The reconstructed nuclei can be measured and the volume calculated by 3D-DOCTOR 4.0 software.