生物素化葡聚糖胺法顺行追踪大鼠颈段脊髓小脑束神经元向小脑中央核的投射

Projections from cervical spinocerebellar tract neurons to cerebellar nuclei in rats by the anterograde labeling of biotinylated dextran amine

目的:采用生物素化葡聚糖胺顺行追踪法观察大鼠颈髓投射纤维在小脑中央核中的分布情况。方法:实验于2005-03/09在首都医科大学解剖教研室完成。5只Wistar大鼠腹膜腔注射60g/L水合氯醛(5mL/kg)麻醉,打开各段脊髓相应部位的椎板,用注射针头刺破硬脊膜,暴露脊髓,通过连接在微量注射器上的微玻管(内径20～40μm)将溶于0.05mol/LTris-HCl缓冲液(pH7.6)的150g/L结合生物素的葡聚糖胺多点压力注射于一侧脊髓,每只大鼠注射至C3～6部位三四点,每点注射0.2～0.3μL,总量为0.6～1.2μL。其中2只大鼠在注射区尾侧加作脊髓外侧索横断。术后动物存活15～20d后,在60g/L水合氯醛深度麻醉下,经左心室插管至升主动脉进行灌注固定,制备连续切片,片厚50μm。其中小脑分别作矢状位和横切位切片,脊髓颈段作横切位切片。所有切片在光学显微镜下观察,观察结果用图像分析仪记录。标记终末依据Robertson的观察标准,即苔藓纤维的玫瑰花结样的结构特征。结果:①注射部位:生物素化葡聚糖胺注射部位均由深褐色密集区和棕色弥散区组成。注射部位的密集区基本限定于注射侧脊髓灰质后角基部、中间带和前角,覆盖了颈段脊髓小脑束的起始细胞。②标记终末在小脑横切片上的分布:主要分布于小脑内侧核头端背内侧、中段;在前间置核其主要分布于头端;而在后间置核的分布区集中在内侧;小脑外侧核未见标记纤维及终末。③标记终末在小脑矢状位上的分布:内侧核内侧部,中间部;少量分布于前间置核头端腹侧部;后间置核的分布区集中于尾端;外侧核未见标记终末。结论:大鼠脊髓颈段有向小脑中央核的投射,与腰髓的投射相比较,存在一定的定位关系。

AIM： Anterograde labeling with biotinylated dextran amine （BDA） is used to observe the distribution of projection fibers of cervical cord in cerebellar nuclei in rats. METHODS： The experiment was conducted at the Department of Anatomy, Capital Medical University from March to September 2005. Totally 5 Wistar rats were selected and anesthelized with 60 g/L chloral hydrate （5 mL/kg） by peritoneal injection. Vertebral plate at corresponding spinal cord segment was opened. Endorhachis was cut with needle to expose spinal cord. BDA dissolved in 0.05 mol/L Tris-HCL buffer （pH 7.6） 150 g/L was injected into one side of spinal cord （multipoint pressure） by mini-glass tube （inner diameter of 20-40 μm） connected in microinjector, three or four points at C3-6 regions in each rat, 0.2-0.3 μL in each point, totally 0.6-1.2 μL. Of them, 2 rats received lateral cord transsection at caudal injection region. After living for 15-20 days, post-operative animals received intubation from left ventricle to ascending aorta to fix by perfusion under deep anaesthesia with 60 g/L chloral hydrate, so as to prepare serial sections, with the depth of 50 μm. The cerebellum received sectioning at sagittal plane and transverse plane, respectively. Spinal cord at cervical segments received sectioning at transverse plane. All the sections were observed under light microscope. The observed result was recorded with image analysis. Labelling terminal was accorded with the Robertson observational criterion, which was roset-shape structural feature of mossy fiber. RESULTS： ① Distribution of injections： Distribution of injections of BDA included deep brown densely part and brown spread part, and deep brown densely part was limited to spinal cord gray matter： base of posterior horn, middle part, anterior horn, covered the original cells of cervical segment of the spinocerebellar tract. ② Distribution in the transverse plane： Most of terminals were located at the media-dorsal part of the rostal subdivision and the middle subdivision of the medial nucleus; Labeled terminals were also distributed at the rostal subdivision of the anterior interpositus nucleus and intensively at medial part of the posterior interpositus nucleus; no labeled fibers and terminals were seen in the lateral cerebellar nucleus. ③ Distribution in the sagittal plane： Labeled terminals were distributed at medial part and middle part of the medial nucleus; a few were located at ventral part of the rostal subdivision of the medial nucleus. Labeled terminals were distributed intensively in caudial subdivision of the posterior interpositus nucleus; no labeling terminal at lateral nucleus. CONCLUSION： The cervical segments of spinocerebellar tract project to the cerebellar nuclei in rats and locate somatotopically as compared with the lumbar spinal cord.