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

Abstract

Background: 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. Methods: In 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. Results: Comparisons 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. Conclusions: These 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.