An enhanced staining method K-B-2R staining for three-dimensional nerve reconstruction

Peng Luo,Xiaolin Liu,Jian Qi,Liping Wang,Jianghui Dong,Yi Zhang,Cory J Xian,Yingchun Zhong

doi:10.1186/s12868-019-0515-7

Abstract

BackgroundThree-dimensional (3D) reconstruction of human peripheral nerves, as a useful tool to understand the nerve internal information and functional basis, has become an important area of research in the peripheral nerve field.MethodsIn this study, we proposed a two-dimensional (2D) Karnovsky–Roots toluidine blue ponceau 2R (K-B-2R) staining method based upon conventional Karnovsky–Roots staining. It significantly improved the ability to display nerve fascicles, motor and sensory nerve fiber textures. In this method, Karnovsky–Roots staining was carried out, followed by toluidine blue counterstain and ponceau 2R counterstain.ResultsComparisons were conducted between the three methods in staining of median nerve sections, which showed similar distribution characters in acetylcholinesterase-positive sites. The additional counterstaining did not change the basis of Karnovsky–Roots staining. However, the resulting images from this new method significantly facilitated the subsequent 3D nerve reconstruction and 3D printing.ConclusionsThese results show that the new staining method significantly enhanced the display qualities of nerve fascicle edges and fiber textures of motor and sensory nerves and facilitated 3D nerve reconstruction.

Highlights

Three-dimensional (3D) reconstruction of human peripheral nerves, as a useful tool to understand the nerve internal information and functional basis, has become an important area of research in the peripheral nerve field
3D reconstruction is often performed on the basis of acetylcholinesterase staining [16,17,18] and image partition of region of interest (ROI) is manually conducted on each stained image [19]
1 Sample preparation of peripheral nerve sections; 2 Acetylcholinesterase staining, including Karnovsky–Roots staining, K-toluidine blue (K-B) counterstaining, and Karnovsky–Roots toluidine blue ponceau 2R (K-B-2R) counterstaining; 3 2D image acquisition, including image recognition, image mergence and image partition; 4 2D image processing consisting of edge acquisition and functional recognition; 5 3D reconstruction based on 2D images; 6 3D printing based on standard triangulated language (STL) data of the 3D nerve model

Summary

Introduction

Three-dimensional (3D) reconstruction of human peripheral nerves, as a useful tool to understand the nerve internal information and functional basis, has become an important area of research in the peripheral nerve field. Detailed layer information of internal structures and textural properties in the section images are the basis of image processing and the key information ensuring the precision of 3D nerve reconstruction. Karnovsky–Roots staining [9,10,11,12,13] is the only acetylcholinesterase staining method for 3D peripheral nerve reconstruction. 3D reconstruction is often performed on the basis of acetylcholinesterase staining (which is believed to have a high quantity of sections) [16,17,18] and image partition of region of interest (ROI) is manually conducted on each stained image [19]. Karnovsky–Roots acetylcholinesterase staining (that is currently employed for 3D nerve reconstruction) has several limitations [20, 21], such as arduous workload for image partition and low accuracy in 3D reconstruction outcomes [9]

Methods

Results

Conclusion