免疫组化和乙醛酸诱发荧光法研究人颈椎关节囊交感神经纤维的分布

Use of immunohistochemical staining and ethylaldehyde acid-induced biomonoamine fluorescence for determining the distribution of sympathetic nerve terminals in human cervical capsule tissues

目的应用神经肽Y(neuropeptide Y, NPY)免疫组化标记法及乙醛酸激发荧光法,观察交感神经纤维在人颈椎关节囊的分布,比较2种方法的优异,探讨染色方法的改进. 方法 17例颈椎C3～7手术切除的颈椎间关节囊组织,分别进行石蜡包埋NPY免疫组化染色,冰冻切片乙醛酸诱发生物单胺递质荧光反应染色.以1%KMnO4预处理载玻片,采用15～20 μm的切片厚度,100 ℃ 5 min,然后80 ℃ 2 min染色时间和温度.大鼠肾上腺标本作为阳性对照.激发光范围380～420 nm高压汞灯为源的荧光显微镜观察. 结果 NPY免疫组化法显示,关节囊的部分细小动脉壁和神经束上具有较粗大的阳性物质;乙醛酸激发荧光法诱发生物单胺递质产生黄绿色荧光, 多存在于血管壁、滑膜基底层、致密结缔组织中,多为呈网状或放射状分布的较细碎纤维.NPY免疫组化法各颈椎节段阳性率分别为C3～4 70.6%(12/17), C4～5 42.9%(6/14), C5～6 57.1%(8/14), C6～7 50.0%(5/10),总阳性率56.4%(31/55);乙醛酸荧光染色法各颈椎节段阳性率分别为C3～4 70.6%(12/17), C4～5 93.8%(15/16), C5～6 66.7%(10/15), C6～7 80.0%(8/10),总阳性率77.6 %(45/58).乙醛酸荧光染色法颈椎节段染色阳性率明显高于NPY免疫组化法,统计学上具有显著性差异(χ2=5.774,P=0.016),尤其在C4～5颈椎节段差异显著(P=0.004). 结论 2种方法均可进行交感神经末梢的定位,提示人颈椎关节囊存在交感神经纤维末梢分布.经过改进的乙醛酸激发荧光法特异性更强.

Objective By using immunohistochemical staining of neuropeptide Y （NPY） and fluorescence of ethylaldehyde acid - induced biomonoamine mediators, to observe the distribution of sympathetic nerve terminals in human cervical capsule tissues,and to compare the two methods for further improvement. Methods Surgically removed joint capsule tissues from C3-7 vertebrae of 17 cases were treated by paraffin imbedding with NPY immunohistochemical staining and frozen - section with fluorescence staining of ethylaldehyde acid - induced biomonoamine mediators. Slides were preconditioned by 1% KMnO4 and sections 15 -20μm in thickness were made. The staining was conducted at 100℃ for 5 min firstly, and then at 80℃ for 2 min, We utilized adrenal glands of rats as positive control, The samples were observed under fluorescence microscope at 380 -420 nm excitation wavelengths from a highvoltage mercury light source, Results NPY immunostaining findings indicated bulky positive materials in some arteriolar walls and nerve tracts of the joint capsules; biomonoamine mediators gave off fluorescence in green - yellow color under the induction of ethylaldehyde acid, which presented mostly as reticular or radial finely -broken fibers in vascular walls, basal laminae of the synovial membrane and dense connective tissues. The positive rates of NPY immunohistochemical staining were 70, 6% （ 12/17 ） at C3-4 intervertebral segment, 42.9% （6/14） at C4-5, 57. 1% （8/14） at C5-6, and 50, 0% （5/10） at C6-7, respectively, the total positive rate being 56.4% （31/55 ）. When using the ethylaldehyde acid - induced biomonoamine fluorescence, the positive rates were 70, 6% （12/17） at C3-4 intervertebral segment, 93.8% （15/16） at C4-5, 66.7% （10/15） at C5-6, and 80.0% （8/10） at C6-7,respectively, the total positive rate being 77.6% （45/58）, The positive rate was remarkably higher in ethylaldehyde acid -induced biomonoamine fluorescence than in NPY immunohistochemical staining, with statistically significant difference （X^2 = 5. 774, P =0, 016）, especially at C4-5 intervertebral segment （P = 0, 004）. Conclusions Both the two methods can demonstrate the distribution of sympathetic nerve terminals, suggesting the presence of the terminals in human cervical capsule tissues, Modified ethylaldehyde acid - induced biomonoamine fluorescence offers a greater specificity.