大鼠脑干连续冰冻切片在NOS阳性神经核团三维重建中的应用

Application of serial sections of rat brain stem to three dimensional reconstruction of NOS-positive nuclei

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 explore the histological method of 3D reconstruction of nitric oxide synthase （NOS） positive nuclei in rat brain stem. Methods： Specimens from midbrain to C1 segment of spinal cord were obtained from four adult male SD rats after perfusion, and fixation Slices in 30~m thickness were continuously sectioned in coronary plane from 3 specimens, the slices were stained with nicotinamide aenine inucleotide phosphate-diaphorase diaphorase （NADPH-d） histoehemistry. One specimen was sectioned in sagittal plane. All slices were stained with Luxol fast blue （LFB）. Images were stitched and aligned via three-axes locating system in PhotoShop7.0 after photographed under 5 x objective lens. Then images were imported into 3D-DOCTOR4.0 to reconstruct NOS-positive nuclei. The nuclei were identified based on stereotaxie atlas of rat brain, the volumes were calculated in 3 D-DOCTOR4.0. Results： The distribution of NOS-positive nuclei were observed in areas of dorsal midbrain, around the midbrain duct, and basolateral parts of pons and ventrolateral parts of medulla obolongada. The largest nucleus with an average vulume of 0.6747 ± 0.0026 mm3 was observed to be lateroorsal tegmentum（LDTg）. Conclusions： The NOS-positive nuclei can be renconstrueted by histological method and the sizes of the nuclei can be measured and the volumes of the nuclei can be calculated with 3D-DOCTOR4.0.