周围神经损伤后免疫荧光染色鉴定感觉和运动神经束。
Identification of sensory and motor nerve fascicles by immunofluorescence staining after peripheral nerve injury.
机构信息
Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children'S Hospital of Wenzhou Medical University, Wenzhou, 325027, China.
Department of Neurosurgery, University of Maryland School of Medicine, 10 South Pine Street, MSTF Building 823, Baltimore, MD, 21201, USA.
出版信息
J Transl Med. 2021 May 13;19(1):207. doi: 10.1186/s12967-021-02871-w.
BACKGROUND
Inappropriate matching of motor and sensory fibers after nerve repair or nerve grafting can lead to failure of nerve recovery. Identification of motor and sensory fibers is important for the development of new approaches that facilitate neural regeneration and the next generation of nerve signal-controlled neuro-prosthetic limbs with sensory feedback technology. Only a few methods have been reported to differentiate sensory and motor nerve fascicles, and the reliability of these techniques is unknown. Immunofluorescence staining is one of the most commonly used methods to distinguish sensory and motor nerve fibers, however, its accuracy remains unknown.
METHODS
In this study, we aim to determine the efficacy of popular immunofluorescence markers for motor and sensory nerve fibers. We harvested the facial (primarily motor fascicles) and sural (primarily sensory fascicles) nerves in rats, and examined the immunofluorescent staining expressions of motor markers (choline acetyltransferase (ChAT), tyrosine kinase (TrkA)), and sensory markers [neurofilament protein 200 kDa (NF-200), calcitonin gene-related peptide (CGRP) and Transient receptor potential vanillic acid subtype 1 (TRPV1)]. Three methods, including the average area percentage, the mean gray value, and the axon count, were used to quantify the positive expression of nerve markers in the immunofluorescence images.
RESULTS
Our results suggest the mean gray value method is the most reliable method. The mean gray value of immunofluorescence in ChAT (63.0 ± 0.76%) and TRKA (47.6 ± 0.43%) on the motor fascicles was significantly higher than that on the sensory fascicles (ChAT: 49.2 ± 0.72%, P < 0.001; and TRKA: 29.1 ± 0.85%, P < 0.001). Additionally, the mean gray values of TRPV1 (51.5 ± 0.83%), NF-200 (61.5 ± 0.62%) and CGRP (37.7 ± 1.22%) on the motor fascicles were significantly lower than that on the sensory fascicles respectively (71.9 ± 2.32%, 69.3 ± 0.46%, and 54.3 ± 1.04%) (P < 0.001). The most accurate cutpoint occurred using CHAT/CRCP ratio, where a value of 0.855 had 100% sensitivity and 100% specificity to identify motor and sensory nerve with an area under the ROC curve of 1.000 (P < 0.001).
CONCLUSIONS
A combination of ChAT and CGRP is suggested to distinguish motor and sensory nerve fibers.
背景
神经修复或神经移植后，运动和感觉纤维的不匹配会导致神经恢复失败。识别运动和感觉纤维对于开发促进神经再生的新方法以及具有感觉反馈技术的下一代神经信号控制神经假肢至关重要。只有少数方法被报道可以区分感觉和运动神经束，并且这些技术的可靠性尚不清楚。免疫荧光染色是区分感觉和运动神经纤维最常用的方法之一，但准确性仍不清楚。
方法
本研究旨在确定常用的运动和感觉神经纤维免疫荧光标记物的效果。我们从大鼠中采集面神经（主要是运动束）和腓肠神经（主要是感觉束），并检查运动标记物（胆碱乙酰转移酶（ChAT），酪氨酸激酶（TrkA））和感觉标记物[神经丝蛋白 200kDa（NF-200），降钙素基因相关肽（CGRP）和瞬态受体电位香草酸亚型 1（TRPV1）]的免疫荧光染色表达。使用平均面积百分比，平均灰度值和轴突计数三种方法来量化免疫荧光图像中神经标记物的阳性表达。
结果
我们的结果表明，平均灰度值方法是最可靠的方法。运动束上 ChAT（63.0±0.76％）和 TRKA（47.6±0.43％）的免疫荧光平均灰度值明显高于感觉束（ChAT：49.2±0.72％，P<0.001;和 TRKA：29.1±0.85％，P<0.001）。此外，运动束上 TRPV1（51.5±0.83％），NF-200（61.5±0.62％）和 CGRP（37.7±1.22％）的平均灰度值分别明显低于感觉束（71.9±2.32％），69.3±0.46％和 54.3±1.04％（P<0.001）。使用 CHAT/CGRP 比值，最准确的截断点为 0.855，其对运动和感觉神经的灵敏度为 100％，特异性为 100％，ROC 曲线下面积为 1.000（P<0.001）。
结论
建议使用 ChAT 和 CGRP 联合区分运动和感觉神经纤维。
Zotero 插件