Immunohistochemical study of mouse sciatic nerves under various stretching conditions with "in vivo cryotechnique".

Abstract Background In living animal bodies, some morphological changes of nerve fibers will probably occur when peripheral nerves are stretched or not stretched during various joint exercises. We aimed to capture the dynamic structures of nerves under various stretching conditions and to keep soluble serum proteins in their tissue sections. New method Morphological changes of stretched or non-stretched sciatic nerve fibers were examined with “in vivo cryotechnique” (IVCT). Fibers were directly frozen with liquid isopentane-propane cryogen (−193 °C). Immunolocalizations of protein 4.1G and albumin were also examined in the fibers. Results The structures of IVCT-prepared sciatic nerves under the stretched condition showed a beaded appearance. By immunostaining for membrane skeletal protein 4.1G, Schmidt-Lanterman incisures (SLIs) were clearly identified, and the heights of their circular truncated cones were increased at narrow sites of the nerve fibers under the stretched condition, compared to those of non-stretched nerve fibers. Albumin was immunolocalized in blood vessels and also along endoneurium including regions near the node of Ranvier. Comparison with existing methods With the conventional perfusion-fixation method (PF), it was difficult to keep stable postures of living mouse limbs for tissue preparation. In nerve fibers after PF, the structures of SLI were easily modified, and albumin was heterogeneously immunolocalized due to diffusion artifacts. Conclusions IVCT revealed (1) the structures of peripheral nerve fibers under dynamically different conditions, indicating that the morphological changes of SLIs play a functional role as a bumper structure against mechanical forces, and (2) accurate immunolocalization of serum albumin in the sciatic nerve fibers.