Abstract: BACKGROUND: Studies have shown that tissue clearing technology enables the three-dimensional (3D) visualization of neurons in the spinal cord injury area, clearly presenting morphological changes of neurons, including soma atrophy, dendrite fragmentation, and axonal degeneration.
OBJECTIVE: To systematically evaluate the application potential of tissue clearing technology in a rat model of chronic spinal cord injury.
METHODS: Thirty-six female Sprague-Dawley rats were randomly and equally divided into a normal group (n=12), a sham surgery group (n=12), and a surgery group (n=12). The normal group received no treatment. The sham group underwent implantation and immediate removal of a poly(vinyl alcohol)/polyacrylamide interpenetrating network hydrogel into the C5-C7 spinal canal. The surgery group received implantation of the hydrogel to compress the spinal cord at C5-C7 to establish a chronic spinal cord injury model. Motor function was assessed using the Basso-Beattie-Bresnahan score and the modified Rivlin inclined plane test on postoperative days 1, 3, 7, and 14. On day 14, spinal cord tissue was harvested. Hematoxylin-eosin staining was used to observe tissue morphology. Tissue clearing combined with immunofluorescence labeling for neuron-specific nuclear protein (NeuN) was performed for 3D reconstruction analysis and cross-sectional view analysis of the spinal cord.
RESULTS AND CONCLUSION: (1) The Basso-Beattie-Bresnahan scores and inclined plane angles in the surgery group on postoperative days 1, 3, 7, and 14 were significantly lower than those in the normal and sham groups (P < 0.001). (2) Hematoxylin-eosin staining revealed significant spinal cord injury in the surgery group: neurons in the gray matter showed swelling and disruption, the white matter structure lost uniformity and stability, some nuclei disappeared, cell numbers decreased, extensive glial cell proliferation and aggregation occurred in the compression area, white matter structure was disordered, and numerous cavities formed. (3) 3D reconstruction and cross-sectional analyses of the spinal cord tissue showed that the spinal cord appeared continuous and full with uniformly distributed NeuN red fluorescence signal in the normal and sham groups. Neurons in the ventral horn of the gray matter were densely arranged in layers, and white matter fiber tracts were intact. In the surgery group, the spinal cord appeared sunken or even interrupted. NeuN fluorescence intensity was markedly weakened in the compressed segments, the layered structure of gray matter neurons was disrupted, and regional fluorescence interruption was observed. These findings indicate that tissue clearing technology can effectively reveal the structural changes of spinal cord tissue after injury, providing strong support for research into the pathological mechanisms of spinal cord injury.

Key words: tissue clearing technology, spinal cord injury, rat model, three-dimensional imaging, neuron-specific nuclear protein immunofluorescence labeling, tissue construction