神经丝组织形态计量学在评估周围神经损伤后未髓鞘轴突再生中的比较：电子显微镜的替代方法。

Neurofilament-histomorphometry comparison in the evaluation of unmyelinated axon regeneration following peripheral nerve injury: An alternative to electron microscopy.

机构信息

Division of Plastic and Reconstructive Surgery, The Hospital for Sick Children, 555 University Avenue, Toronto, ON, M5G 1X8, Canada; Department of Surgery, University of Toronto, 149 College Street, 5th floor, Toronto, ON, M5T 1P5, Canada; Institute of Medical Science, University of Toronto, 1 King's College Circle, Room 2374, Toronto, ON, M5S 1A8, Canada.

Division of Plastic and Reconstructive Surgery, The Hospital for Sick Children, 555 University Avenue, Toronto, ON, M5G 1X8, Canada; Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street, Room 407, Toronto, ON, M5S 3G9, Canada.

出版信息

J Neurosci Methods. 2019 May 15;320:37-43. doi: 10.1016/j.jneumeth.2019.03.006. Epub 2019 Mar 7.

DOI:10.1016/j.jneumeth.2019.03.006

PMID:30851340

Abstract

BACKGROUND

Currently, assessment of unmyelinated axon regeneration is limited to electron microscopy (EM), which is expensive, time consuming and not universally available. This study presents a protocol to estimate the number of unmyelinated axons in a regenerating peripheral nerve without the need for electron microscopy.

NEW METHOD

The common peroneal nerve of Sprague-Dawley rats was transected, repaired and regenerated for 4 weeks. Two distal adjacent segments of the regenerating nerve were then processed for either conventional histomorphometry using toluidine blue or immunolabeling of neurofilament protein. Myelinated axon and total axon counts were obtained, respectively, to generate estimates of unmyelinated axon numbers, which were then compared to unmyelinated axon counts using EM from the same specimens. For comparison, unmyelinated axons were counted in an uninjured rat laryngeal nerve.

RESULTS

After 4 weeks of regeneration, the estimated number of regenerating unmyelinated axons was 4044 ± 232 using this technique, representing 81.3% of the total axonal population. By comparison, the proportion of unmyelinated axons in the uninjured laryngeal nerve was 55% of the total axonal population.

COMPARISON WITH EXISTING METHOD

These estimates correlate with electron microscopy measurements, both in terms of the proportion of unmyelinated axons and also by linear regression analysis.

CONCLUSIONS

The neurofilament staining method correlates with electron microscopy estimates of the same nerve sections. It is useful for the efficient counting of unmyelinated axons in the regenerating peripheral nerve and can be used by laboratories that do not have access to EM facilities.

摘要

背景

目前，未髓鞘轴突再生的评估仅限于电子显微镜（EM），这种方法费用昂贵、耗时且并非普遍适用。本研究提出了一种无需电子显微镜即可评估再生周围神经中未髓鞘轴突数量的方案。

新方法

切断、修复和再生 Sprague-Dawley 大鼠的腓总神经 4 周。然后，对再生神经的两个远端相邻节段进行常规甲苯胺蓝组织形态计量学处理或神经丝蛋白免疫标记处理。分别获得有髓轴突和总轴突计数，以生成未髓鞘轴突数量的估计值，然后将这些估计值与同一标本的 EM 获得的未髓鞘轴突计数进行比较。为了比较，在未受伤的大鼠喉神经中计数未髓鞘轴突。