基于图像分析的周围神经Karnovsky-Roots法染色规律的效果观察

中国临床解剖学杂志 ›› 2011, Vol. 29 ›› Issue (4): 436-441.

基于图像分析的周围神经Karnovsky-Roots法染色规律的效果观察

戚　剑¹，　罗　鹏¹，　程思红²，　李增宏³，　张　毅⁴，　刘小林¹

中山大学 1.附属第一医院显微创伤外科, 2. 中山医学院, 3.附属第一医院耳鼻喉科,
4.附属第一医院整形修复外科，广州 510080

收稿日期:2010-08-30 发布日期:2011-07-26
通讯作者: 刘小林，教授，博士导师， E-mail: gzxiaolinliu@hotmail　com E-mail:speedsnow@126.com
作者简介:戚　剑（1974-）男，河南汝南县人，医学博士，主治医师，研究方向：显微外科，创伤骨科，Tel：13527797889
基金资助:
国家自然科学基金资助项目（30571913）；广东省自然基金（915008901000006）；广东省卫生厅科研基金资助项目（A2009173）

Karnovsky-Roots' stain of peripheral nerve based on image analysis

QI Jian1, LUO Peng1, CHENG Si-hong2, LI Zeng-hong3 , ZHANG Yi4, LIU Xiao-lin1

1. Department of Microsurgery and Orthopedics, 3. Department of Otolaryngology, 4. Department of Plastic Surgery, The first Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China; 2.Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China,

Received:2010-08-30 Published:2011-07-26

摘要/Abstract

摘要：

目的　基于图像分析探讨用于鉴别周围神经功能束的乙酰胆碱酯酶（AChE）染色Karnovsky-Roots法24小时内的染色规律，为基于乙酰胆碱酯酶染色的周围神经功能束三维重建确定标准化的孵育条件。方法　新鲜人体截肢腓总神经标本，连续横断冰冻切片，片厚10 μm，共切取56 张，随机分为7组，每组8张切片，采用Karnovsky-Roots法进行乙酰胆碱酯酶孵育，其中1～6 组分别为1、2、4、8、12和24 h，第7组切片为阴性对照组。采用Image-Pro Plus 6.0 图像分析软件对不同时间染色组图像进行图像分析，指标为：(1) AChE 反应阳性区域的面积；(2) AChE 反应阳性区域的平均光密度；(3) 　AChE 反应阳性区域的累积光密度。组间结果采用SPSS13.0进行t-test 统计检验，以P<0.05为有统计学差异。结果　8h 组的平均光密度值与12 h组有显著性差异，而12 h 与24 h 的平均光密度值无显著性差异；8 h、12 h 和24 h 间的染色面积、累计光密度值均存在显著性差异，但镜下可观察到24 h组存在过度染色。结论　孵育12～24 h间的染色效果趋于一致，染色结果稳定可靠，神经束功能性质鉴别容易，神经束膜与染色的神经纤维间有清晰的分界，利于轮廓获取，可作为基于乙酰胆碱酯酶染色的周围神经功能束三维重建的标准化的育条件。

关键词: 乙酰胆碱酯酶, Karnovsky-Roots法, 周围神经, 图像分析

Abstract:

Objective　To explore the feasibility of image analysis on the result of acetylcholinesterase stain (Karnovsky-Roots'method) of peripheral nerve, and provide the reference for the standard incubational conditions of the three-dimensional reconstruction of functional fascicles groups of peripheral nerves.　Methods　The samples came from fresh adult common peroneal nerve, which were serially horizontally sliced into 56 slices with the thickness of 10μm. All slices were randomly divided into seven groups with 8 slices in each group, including 1h, 2h, 4h, 8h, 12h, 24h and negative control group. After AChE histochemical staining, the stain characteristics of different nerve fiber in various stain phrases were observed under optic-microscope. The average area, density and integrated optical density (IOD) of AChE positive region of various stain period were analyzed by an image analytical software (Image-Pro Plus 6.0). T-test was adopted to distinguish the group differences through SPSS13.0.　Results　For the mean optical density value, there was notable diversity between 8h and 12h groups, but not for the 12h and 24h groups. The significant differences also appeared among 8h, 12h, and 24h groups for stain area and IOD. Furthermore, overstain was observed on slices of 24h group.　Conclusions　The staining results from 12h to 24h were stable and reliable, moreover, it is possible to identify different functional fascicles surrounding by perineurium. For three-dimensional reconstruction of functional fascicles groups of peripheral nerves, it can be taken as the standard stain incubational condition.

Key words: Acetylcholinesterase, Karnovsky-Roots, Peripheral nerve, Image analysis

中图分类号:

R329.33
R602

戚　剑, 罗　鹏, 程思红, 李增宏, 张　毅, 刘小林. 基于图像分析的周围神经Karnovsky-Roots法染色规律的效果观察[J]. 中国临床解剖学杂志, 2011, 29(4): 436-441.

CU 　Jian, LUO 　Feng, CHENG Sai-Gong, LI Ceng-Hong, ZHANG 　Yi, LIU Xiao-Lin. Karnovsky-Roots' stain of peripheral nerve based on image analysis[J]. Chinese Journal Of Clinical Anatomy, 2011, 29(4): 436-441.

参考文献

[1] He YS, Zhong SZ. Acetylcholinesterase: a histochemical identification of motor and sensory fascicles in human peripheral nerve and its use during operation
[J]. Plast Reconstr Surg, 1988,82(1):125-132.

[2] Stewart JD. Peripheral nerve fascicles: anatomy and clinical relevance
[J]. Muscle Nerve, 2003,28(5):525-41.

[3] 任高宏, 裴国献. 周围神经三维重建与可视化研究进展
[J]. 中国修复重建外科杂志, 2009,23(02):239-244.

[4] Sun K, Zhang J, Chen T, et al. Three-dimensional reconstruction and visualization of the median nerve from serial tissue sections
[J]. Microsurgery, 2009,29(7):573-577.

[5] Xie S, Xiang B, Bu S, et al. Rapid identification of anterior and posterior root of cauda equina nerves by near-infrared diffuse reflectance spectroscopy
[J]. J Biomed Opt, 2009,14(2):0240051-0240057.

[6] Wang H, Lu L, Liu D. Identification of property of nerve fascicles by physical and histochemistry methods
[J]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi, 2008,22(1):80-83.

[7] Riley DA, Lang DH. Carbonic anhydrase activity of human peripheral nerves: a possible histochemical aid to nerve repair
[J]. J Hand Surg Am, 1984,9(1):112-120.

[8] Kawasaki Y, Yoshimura K, Harii K, et al. Identification of myelinated motor and sensory axons in a regenerating mixed nerve
[J]. J Hand Surg Am, 2000,25(1):104-111.

[9] 朱家恺，罗永湘，陈统一. 现代周围神经外科学
[M]. 上海:上海科学技术出版社,2007:86-94.

[10]Karnovsky MJ, Roots L. A "direct-coloring" thiocoline method for cholinesterase
[J]. J Histochem Cytochem, 1964,12:219-221.

[11]凌启波. 实用病理特殊染色和组化技术
[M] 广州：广东高等教育出版社，1989:288-294.

[12]Gruber H, Zenker W, Hohberg E. Studies on the specificity of cholinesterases in the peripheral nervous system of the rat
[J]. Histochemie, 1971,27(1):78-86.

[13]Gruber H. Identification of motor and sensory funiculi in cut nerves and their selective reunion
[J]. Br J Plast Surg, 1976,29(1):70-73.

[14]Kanaya F, Ogden L, Breidenbach WC, et al. Sensory and motor fiber differentiation with Karnovsky staining
[J]. J Hand Surg Am, 1991,16(5):851-858.

[15]Liu XL, Zhu JK. The histochemical study of rat sciatic nerve cholinesterase in degeneration and regeneration
[J]. J Reconstr Microsurg, 1990,6(1):43-47.