Enhanced axon outgrowth of spinal motor neurons in co-culturing with dorsal root ganglions antagonizes the growth inhibitory environment

Zi-Xing Xu; Dan Xu; Fang Fang; Ying-Juan Fan; Bing Wu; Yu-Fan Chen; Hao-En Huang; Xin-Hao Huang; Yue-Hong Zhuang; Wei-Hong Xu

doi:10.1016/j.reth.2023.11.013

Enhanced axon outgrowth of spinal motor neurons in co-culturing with dorsal root ganglions antagonizes the growth inhibitory environment

Regen Ther. 2023 Dec 10:25:68-76. doi: 10.1016/j.reth.2023.11.013. eCollection 2024 Mar.

Authors

Zi-Xing Xu^{1

2

3}, Dan Xu⁴, Fang Fang⁵, Ying-Juan Fan⁵, Bing Wu⁶, Yu-Fan Chen¹, Hao-En Huang¹, Xin-Hao Huang¹, Yue-Hong Zhuang⁴, Wei-Hong Xu^{1

2

3}

Affiliations

¹ Department of Spinal Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian Province, China.
² Department of Orthopedics, National Regional Medical Center, Binhai Campus of the First Affiliated Hospital, Fujian Medical University, Fuzhou, Fujian Province, China.
³ Fujian Provincial Institute of Orthopedics, Fuzhou, Fujian Province, China.
⁴ Fujian Key Laboratory of Brain Aging and Neurodegenerative Diseases, Fujian Medical University, Fuzhou, Fujian Province, China.
⁵ Department of Pharmacology, Fujian Medical University, Fuzhou, Fujian Province, China.
⁶ The Central Laboratory, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian Province, China.

Abstract

Introduction: Forming a bridge made of functional axons to span the lesion is essential to reconstruct the motor circuitry following spinal cord injury (SCI). Dorsal root ganglion (DRG) axons are robust in axon growth and have been proved to facilitate the growth of cortical neurons in a process of axon-facilitated axon regeneration. However, whether DRG transplantation affects the axon outgrowth of spinal motor neurons (SMNs) that play crucial roles in motor circuitry remains unclear.

Methods: We investigated the axonal growth patterns of co-cultured DRGs and SMN aggregates (SMNAs) taking advantage of a well-designed 3D-printed in vitro system. Chondroitin sulphate proteoglycans (CSPG) induced inhibitory matrix was introduced to imitate the inhibitory environment following SCI. Axonal lengths of DRG, SMNA or DRG & SMNA cultured on the permissive or CSPG induced inhibitory matrix were measured and compared.

Results: Our results indicated that under the guidance of full axonal connection generated from two opposing populations of DRGs, SMNA axons were growth-enhanced and elongated along the DRG axon bridge to distances that they could not otherwise reach. Quantitatively, the co-culture increased the SMNA axonal length by 32.1 %. Moreover, the CSPG matrix reduced the axonal length of DRGs and SMNAs by 46.2 % and 17.7 %, respectively. This inhibitory effect was antagonized by the co-culture of DRGs and SMNAs. Especially for SMNAs, they extended the axons across the CSPG-coating matrix, reached the lengths close to those of SMNAs cultured on the permissive matrix alone.

Conclusions: This study deepens our understanding of axon-facilitated reconstruction of the motor circuitry. Moreover, the results support SCI treatment utilizing the enhanced outgrowth of axons to restore functional connectivity in SCI patients.

Keywords: 3D printing; Axonal bridging; Central nervous system regeneration; Dorsal root ganglia transplantation; Experimental modeling; Spinal cord injury; Spinal motor neuron aggregates.