Application of second near infrared fluorescence imaging to trace CelTrac1000-labeled hair follicle epidermal neural crest stem cells in repairing rat facial nerve defects

Abstract

Background Tissue engineering based on stem cells has achieved satisfactory results in repairing facial nerve defects. However, the in vivo process of the transplanted cells has not been fully clear until now, although it is critical to understand the process and the underlying mechanism of regeneration for better therapeutic outcomes. Recently, second near-infrared window (NIR-II) fluorescence imaging has emerged as a rapidly evolving bio-imaging technique capable of visualizing and quantifying biological processes at the cellular level of living organisms. Methods Firstly, rat hair follicle epidermal neural crest stem cells (EPI-NCSCs) were isolated, cultured and identified by expression of SOX10 and Nestin, and then labeled with CelTrac1000. Rat acellular nerve allografts (ANAs) were prepared by chemical extraction. Secondly, 30 adult male rats were randomly and equally assigned into three groups: ANA + cells group, ANA group, and autograft group. The buccal branch of the facial nerve on right side was exposed and a 10-mm-long gap was bridged by ANA laden with CelTrac1000-labeled EPI-NCSCs, ANA laden with CelTrac1000 dye, and autologous nerve, respectively. Thirdly, CelTrac1000-labeled EPI-NCSCs were detected by NIR-II optical imaging system to visualize the behavior of the transplanted cells in vivo postoperatively. Finally, vibrissa movement, compound muscle action potentials (CMAPs) of vibrissal muscle, facial motoneurons retrotraced by Fluorogold, morphology and histology of the regenerated nerves in three groups were analyzed after surgery, respectively. Results Through 14 weeks of dynamic observation, we found that EPI-NCSCs successfully survived in the ANAs in vivo. Meanwhile, the region of the NIR-II fluorescence signals was gradually limited to be consistent with the route of the regenerative segment of the facial nerve. Furthermore, the degree of the vibrissa movement, the recovery value of the onset latency and amplitude of CMAPs, the number of Fluorogold-labeled cells, the CD31 positive area/total area, the mean gray value of S100 and β-tubulin III, the number and the diameter of the myelinated nerve fibers in the ANA group were lower than the other two groups (P < 0.05), and the other two groups had similar values (P > 0.05). Additionally, the thickness of the myelin sheaths was the thinnest in the ANA group, and the thickest in the autograft group (P< 0.05). Conclusions The migration map of local CelTrac1000-labeled EPI-NCSCs was successfully monitored by the NIR-II fluorescence imaging system when EPI-NCSCs within the ANAs were applied to treat rat facial nerve defects. Additionally, EPI-NCSCs promoted the ANAs to repair facial nerve defects in a small animal model.