Application of In Vivo Cryotechnique for Morphological Visualization of Nerve and Vascular Structures in a Sciatic Nerve Crush Model.

Abstract

After a crush injury in sciatic nerve fibers, dynamic changes in blood circulation and immune-cell mobilization occur during axonal regeneration. High-resolution visualization under near-physiological conditions is crucial for understanding these mechanisms. Conventional histological techniques introduce perfusion- and dehydration-induced artifacts that obscure circulation. We employed the in vivo cryotechnique (IVCT) to visualize blood flow within sciatic nerve fibers and assess temporal changes during regeneration. In uninjured mice, IVCT preserved native tissue architecture with minimal shrinkage compared to perfusion fixation, with superiority quantitatively shown by fractal analysis. In the crush model, hematoxylin-eosin, Luxol fast blue, and immunohistochemical staining of IVCT-prepared, freeze-substituted sections revealed axonal degeneration and regrowth. The close association between regenerating fibers and vascular structures, along with erythrocyte distribution, indicates a morphological link between nerves and blood vessels. Electrophysiological assessment using compound muscle action potentials and functional recovery measured by the sciatic functional index demonstrated restored nerve function at 28 days, consistent with histology. These findings suggest that IVCT is a useful method for analyzing peripheral nerve regeneration and vascular dynamics, thereby highlighting its potential as a novel approach in peripheral nerve research.