Application of tissue clearing technology in a rat model of chronic spinal cord injury

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. At postoperative days 1, 3, 7, and 14, motor function was assessed using the Basso, Beattie, and Bresnahan (BBB) score and the modified Rivlin inclined plane test. At day 14, spinal cord tissue was harvested for hematoxylin-eosin staining to observe morphology, and tissue clearing combined with neuron-specific nuclear protein immunofluorescence labeling was used for three-dimensional reconstruction and cross-sectional view analysis. RESULTS AND CONCLUSION: (1) The BBB scores and inclined plane test angles in the surgery group were significantly lower than those in the normal and sham groups at all time points (P < 0.001). (2) Hematoxylin-eosin staining showed significant spinal cord injury in the surgery group, with swelling and destruction of nerve cells in the gray matter, loss of uniformity in white matter structure, disappearance of some nuclei, reduced cell number, massive glial cell proliferation and aggregation in the compression area, disordered white matter structure, and formation of numerous cavities. (3) Three-dimensional reconstruction and cross-sectional analysis of the spinal cord showed that in the normal and sham groups, the spinal cord appeared continuous and full, with uniform distribution of neuron-specific nuclear protein red fluorescence, dense layered arrangement of neurons in the anterior horn of the gray matter, and intact white matter fiber tracts. In the surgery group, the spinal cord appeared depressed or even interrupted, with significantly reduced fluorescence intensity of neuron-specific nuclear protein in the compressed segment, disrupted gray matter neuronal layer structure, and regional fluorescence interruption. These results indicate that tissue clearing technology can effectively display structural changes after spinal cord injury, providing strong support for studying the pathological mechanisms of spinal cord injury.

1. Introduction

Spinal cord injury (SCI) is typically triggered by an initial mechanical insult, followed by a cascade of pathophysiological processes that exacerbate the damage and lead to further neurological deterioration, resulting in temporary or permanent functional changes [1-2]. SCI is characterized by high morbidity, high disability rates, and a trend toward younger onset [3-5]. Globally, the incidence of SCI ranges from 10.4 to 83 cases per million population, with a rapidly increasing trend [6-9]. The pathological mechanisms of SCI are complex and difficult to repair, especially chronic compressive injury, which can lead to irreversible neuronal disconnection and disruption of neural circuits [10-12]. Traditional evaluation methods, such as behavioral scoring, can reflect functional deficits but fail to reveal the spatial heterogeneity of injury [13]. Histological staining can observe local morphological changes, but due to the limitations of two-dimensional sections, it cannot provide a panoramic view of three-dimensional structural damage [14-15]. This cognitive gap between structure and function severely restricts in-depth analysis of SCI mechanisms and precise assessment of therapeutic strategies.

In recent years, breakthroughs in tissue clearing technology have provided new approaches for neuroscience research [16-17]. Tissue clearing technology, combined with various fluorescence labeling techniques such as transgenic animals, viral tracing, fluorescent dyes, and in situ hybridization, enables the acquisition of three-dimensional structural information of biological tissues at the organ or even whole-body level [18]. By using chemical agents to eliminate light scattering in tissues, combined with immunolabeling and three-dimensional imaging, it achieves high-resolution, non-destructive visualization of intact organs [19]. The 'panoramic imaging' capability of tissue clearing technology allows researchers to break through the physical limitations of traditional two-dimensional sections and resolve long-distance neural projection trajectories, neuron-glia interaction networks, and spatial topological relationships of vascular-neural units at subcellular resolution [20]. In the field of musculoskeletal repair, tissue clearing technology helps improve the depth and resolution of bone and muscle research [21-22]. Studies have shown that tissue clearing technology enables dynamic observation of astrocytes and activated host anti-inflammatory microglia in promoting wound repair after spinal cord injury [23]. After SCI, neuronal survival, death, and neural circuit remodeling are key factors affecting functional recovery [24-25]. Tissue clearing technology can perform three-dimensional visualization of neurons in the injured area, clearly presenting morphological changes of neurons, including soma atrophy, dendrite fragmentation, and axonal degeneration.