Development of a 3D-immunofluorescence analysis for sensory nerve endings in human ligaments.

The analysis of ligamentous mechanoreceptors is difficult due to a high amount of unclassifiable mechanoreceptors, which result from incomplete visualization through limited microscopic techniques. The method was developed using dorsal intercarpal ligaments and dorsal regions of the scapholunate interosseous ligament from human cadaver wrists. Consecutive 70 µm thick cryosections were stained with immunofluorescence markers for protein S100B, neurotrophin receptor p75 (p75), protein gene product 9.5 (PGP 9.5) and 4',6-diamidino-2-phenylindole (DAPI). 3D images of sensory nerve endings were obtained using a confocal laser scanning microscope. Experimental point spread functions (PSF) were used to deconvolve images. Sensory nerve endings were localised in each section plane and classified according to Freeman and Wyke. Finally, confocal data was visualized as 3D-images. The method produced excellent image quality, revealing detailed three-dimensional structures. The created 3D-model of sensory nerve endings could be analyzed in all three dimensions, augmenting visualization of the form and immunoreactive pattern of sensory nerve endings. Deconvolution with experimentally measured PSFs aided in enhancing image quality. Using a triple immunofluorescent staining method allows to visualize the structure of sensory nerve endings more precisely than techniques with serial analysis of different monostaining of neural markers. Imaging in three dimensions enhances morphologic details, which are limited in 2D-microscopy. 3D-triple immunofluorescence produces high quality visualization of mechanoreceptors, thereby improving their analysis. As an elaborate technique, it is ideal for defined research questions concerning the microstructure of sensory nerve endings.