End-to-side neurorrhaphy repairs peripheral nerve injury: sensory nerve induces motor nerve regeneration

Qing Yu, She-hong Zhang, Tao wang, Feng Peng, Dong Jan, Yu-dong Gu,

1 Department of Hand Surgery, Huashan Hospital of Fudan University, Key Laboratory of Hand Reconstruction, the Ministry of Health, Key

Laboratory of Peripheral Nerve and Microsurgery, Shanghai, China

2 Department of Rehabilitation Medicine, Huashan Hospital of Fudan University, Shanghai, China

How to cite this article: Yu Q, Zhang SH, Wang T, Peng F, Han D, Gu YD (2017) End-to-side neurorrhaphy repairs peripheral nerve injury:sensory nerve induces motor nerve regeneration. Neural Regen Res 12(10):1703-1707.

End-to-side neurorrhaphy repairs peripheral nerve injury: sensory nerve induces motor nerve regeneration

Qing Yu1, She-hong Zhang2, Tao wang1, Feng Peng1, Dong Jan1, Yu-dong Gu1,*

1 Department of Hand Surgery, Huashan Hospital of Fudan University, Key Laboratory of Hand Reconstruction, the Ministry of Health, Key

Laboratory of Peripheral Nerve and Microsurgery, Shanghai, China

2 Department of Rehabilitation Medicine, Huashan Hospital of Fudan University, Shanghai, China

How to cite this article: Yu Q, Zhang SH, Wang T, Peng F, Han D, Gu YD (2017) End-to-side neurorrhaphy repairs peripheral nerve injury:sensory nerve induces motor nerve regeneration. Neural Regen Res 12(10):1703-1707.

End-to-side neurorrhaphy is an option in the treatment of the long segment defects of a nerve. It involves suturing the distal stump of the disconnected nerve (recipient nerve) to the side of the intimate adjacent nerve (donor nerve). However, the motor-sensory specificity aer end-to-side neurorrhaphy remains unclear.is study sought to evaluate whether cutaneous sensory nerve regeneration induces motor nerves aer end-to-side neurorrhaphy.irty rats were randomized into three groups: (1) end-to-side neurorrhaphy using the ulnar nerve(mixed sensory and motor) as the donor nerve and the cutaneous antebrachii medialis nerve as the recipient nerve; (2) the sham group:ulnar nerve and cutaneous antebrachii medialis nerve were just exposed; and (3) the transected nerve group: cutaneous antebrachii medialis nerve was transected and the stumps were turned over and tied. At 5 months, acetylcholinesterase staining results showed that 34% ±16% of the myelinated axons were stained in the end-to-side group, and none of the myelinated axons were stained in either the sham or transected nerve groups. Retrograde fluorescent tracing of spinal motor neurons and dorsal root ganglion showed the proportion of motor neurons from the cutaneous antebrachii medialis nerve of the end-to-side group was 21% ± 5%. In contrast, no motor neurons from the cutaneous antebrachii medialis nerve of the sham group and transected nerve group were found in the spinal cord segment.ese results confirmed that motor neuron regeneration occurred aer cutaneous nerve end-to-side neurorrhaphy.

nerve regeneration; peripheral nerve injury; end-to-side neurorrhaphy; motor-sensory specificity; rat; dorsal root ganglion; motor neuron; axon; cutaneous antebrachii medialis nerve; ulnar nerve; acetylcholinesterase staining; retrograde neuron tracing; neural regeneration

Introduction

Peripheral nerve injury is a common result of trauma (Reyes et al., 2005; Chen et al., 2007; Sullivan et al., 2016).e incidence of peripheral nerve injury in patients with multiple injuries was 2–2.8% (Selecki et al., 1982; Noble et al., 1998),and this injury may cause severe disability. End-to-side neurorrhaphy is an alternative procedure for some nerve injury repairs (Lutz et al., 2000; Hanna and Dempsey, 2013; Isaacs,2013; Gao et al., 2015; Li et al., 2015). It consists of suturing the distal stump of the disconnected nerve (recipient nerve)to the side of the intimate adjacent nerve (donor nerve)(Viterbo et al., 1998; Al-Qattan, 2001; Yan et al., 2002; Ka-kibuchi et al., 2004; Baltzer et al., 2016).is technique was used a long time ago, but was recently revived by Viterbo et al. (1992) in an experimental study of rats. Although the value of end-to-side nerve repair is still controversial, experimental evidence suggests that it promotes axonal growth and nerve regeneration in the receptors of motor or sensory nerves(Yuksel et al., 1999; De Sa et al., 2004; Hayashi et al., 2004;Haastert et al., 2010; Gao et al., 2012; Zheng et al., 2012; Hanna and Dempsey, 2013; Kettle et al., 2013; Civi et al., 2017).

We have concentrated our investigations on the motor-sensory specificity after end-to-side neurorrhaphy. In previous studies, we found that the motor or sensory specificity was not clear when a mixed nerve was the recipient nerve (Yu et al., 2011, 2013). In this study, we used a rat ulnar nerve (mixed sensory and motor) as the donor nerve and the cutaneous antebrachii medialis nerve as the recipient sensory nerve.e aim of this study was to assess whether a cutaneous nerve can induce motor nerve regeneration after end-to-side neurorrhaphy, and explore the sensory/motor specificity aer end-to-side suturing.

Materials and Methods

Animals and surgery procedures

Study protocols were approved by the Animal Ethics Committee of Wenzhou Medical University of China (approval No. 201601-005). The experimental procedure followed the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85–23, revised 1986).

Rats were randomly divided into end-to-side group (n =12), sham group (n = 12) and the transected nerve group (n =6). Surgery was performed as described previously (Yu et al.,2011). Briefly, rats were anesthetized by intraperitoneal injection of ketamine (40 mg/kg, Shanghai Ziyuan Pharmaceutical Co., Ltd., Shanghai, China) and atropine (0.04 mg/kg, Shanghai Ziyuan Pharmaceutical Co., Ltd.). The right cutaneous antebrachii medialis nerve and ulnar nerves were exposed.For rats in the end-to-side group, we cut a window (diameter,1 mm) in the ulnar nerve epineurium.e cutaneous antebrachii medialis nerve was transected at the root level, and the distal stump was then sutured to the ulnar nerve medial window with 10/0 non-invasive monofilament polyamide(Ethicon, San Angelo, TX, USA) to form an end-to-side junction. The proximal stump of the cutaneous antebrachii medialis nerve was ligated tightly and turned back to prevent spontaneous innervation (Figure 1). In the sham group, we exposed the cutaneous antebrachii medialis nerve and ulnar nerve, and then closed the skin. In the transected nerve group, we cut the cutaneous antebrachii medialis nerve, ligated the two ends, and turned them back to prevent spontaneous innervation. Aer regaining consciousness, the rats were housed separately in boxes in an approved animal house facility.

Acetylcholinesterase staining

Application of neuron tracer

Five months after surgery, the fluorescence retrograde axon tracer Fluoro-Gold (Sigma, St. Louis, MO, USA) was used to demonstrate axonal growth after end-to-side neurorrhaphy(McBride et al., 1990; Byers and Lin, 2003; Chiu et al., 2008;Chen et al., 2012). As described above, the animals were re-anesthetized, and 1 μL 3% Fluoro-Gold dyes were injected into the cutaneous antebrachii medialis nerve (end-to-side and sham groups, n = 6) using a Hamilton syringe. Retrograde neuron tracing was not used in the transected nerve group because the nerve had been cut off and could not transport the tracer retrogradely. To avoid contamination of adjacent tissues or leakage of dyes, the surrounding tissue was covered with filter paper infused by Vaseline (Sigma) and the filter paper was removed aer injection.e wound was closed with 5/0 sutures.e retrograde transport of the dyes took 6 days.

Retrogradely labeled neurons

Animals survived for 6 days and were sacrificed by pentobarbital overdose.e rats were then perfused through the heart with 500 mL of warm Ringer solution + heparin 1,000 IE/kg/rat, followed by 500 mL of ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer. The spinal cord segments(C5-T1) and the corresponding dorsal root ganglion (DRG)were removed and placed in 30% sucrose/4% paraformaldehyde solution for 24 hours. Continuous frozen sections (50 μm for motor neurons in the spinal cord segment and 40 μm for DRG neurons) were cut with a cryostat and mounted on slides. Specimens were observed under a fluorescence microscope (DMLB2) with a filter (Leica G/R; Leica, Wetzlar,Germany) to observe Fluoro-Gold fluorescence staining.Neurons marked with yellow-white fluorescence with clearly visible nuclei were counted in the ventral horn of the spinal cord segment (C5-T1) and the corresponding DRG.

Statistical analysis

All data are consistent with the normal distribution and presented as the mean ± SD. One-way analysis of variance followed by the least significant difference post hoc test were used to analyze the data statistically with STATA 7.0 software (StataCorp, TX, USA). A value of P less than 0.05 was considered statistically significant.

Results

Acetylcholinesterase stain

Retrograde neuron tracing

Discussion

In the present study, motor and sensory nerve fibers were differentiated by Karnovsky and Roots’ histochemical method (Engel et al., 1980; He and Zhong, 1988). In the sham group, the myelinated nerve fibers were unstained, but a great many unmyelinated fibers were stained for acetylcholine esterase in amongst the unstained myelinated fibers.With end-to-side neurorrhaphy, we found significantly increased numbers of myelinated axons showing acetylcholinesterase activity. A total of 34% of the myelinated fibers of the recipient cutaneous antebrachii medialis nerve were stained, and percentages were similar to the donor ulnar nerve demonstrated by our previous report (Yu et al., 2011).e results confirmed that the regenerated axons showed the same acetylcholinesterase activity as the donor ulnar nerve aer end-to-side neurorrhaphy.

Retrograde neuron tracing demonstrated that no motor neurons for the cutaneous antebrachii medialis nerve were found in the spinal cord segment in the sham group. In contrast, the proportion of motor neurons for the cutaneous antebrachii medialis nerve of the end-to-side group was 21%.The result is consistent with the acetylcholinesterase stain findings.e Fluoro-Gold tracer was injected into the nerve and would be incorporated into the axon when the axon was damaged during injection (Prodanov and Feirabend, 2008).As a result, the cell population labeled and quantified at DRGs and ventral horns would be underestimated.

End-to-side nerve repair is an alternative to the treatment of certain nerve damage, especially for surgeons confronted with a defect of a long segment of nerve (Koh et al., 2002;Kovacic et al., 2003, 2012; Barbour et al., 2012; Celis-Aguilar et al., 2013; Civi et al., 2017). Experimental studies published by Viterbo et al. (1992, 1994) and Lundborg et al. (1994) are generally considered to represent the rediscovery and development of the concept of end-to-side neurorrhaphy. They used the rat model to demonstrate that the denervated nerve stem connected to the donor nerve in an end-to-side manner stimulates the collateral sprouting of the axons from the intact donor nerve.

Consistent with our findings, Kalliainen et al. (1999) reported a good motor recovery after end-to-side suture. In their model, the distal stump of the peroneal nerve in the rat was sutured to the side of the tibial nerve. Despite the lower muscle mass and the higher percentage of denervated fibers,these authors did not observe any significant differences between the end-to-end and end-to-side groups associated with force contraction.

治療前西醫治療組、中西醫聯合治療組癥狀積分水平、生存質量水平相近，P＞0.05；治療后中西醫聯合治療組癥狀積分水平、生存質量水平變化幅度更大，P＜0.05。如表2。

A good motor recovery after peripheral nerve injury requires the precise regeneration of the motor axons to its original target tissue and structure (Tessier-Lavigne and Goodman, 1996; Hoke et al., 2006; Wright et al., 2014).Brushart et al. (1988, 1998, 2005) found that motor axons regenerating aer transection of mixed nerve preferentially reinnervate distal muscle branches and termed it preferential motor reinnervation. Madison et al. (1996) suggest that preferential motor reinnervation is potentially regulated by distal nerve pathways, terminal organs and motor neurons.e motor-sensory specificity aer end-to-side neurorrhaphy is currently unclear. Our experiments show that cutaneous nerve can induce motor nerve regeneration aer end-to-side neurorrhaphy and confirmed that motor-sensory specificity following end-to-side neurorrhaphy is not significant.

In conclusion, end-to-side nerve repair of a sensory nerve gives rise to successful motor nerve reinnervation. We also show no marked motor-sensory specificity aer end-to-side neurorrhaphy of a sensory nerve recipient.

Figure 2 Cutaneous antebrachii medialis nerve motor axons stained for acetylcholinesterase utilizing Karnovsky and Roots’ histochemical method, viewed through a Leica DM3000 microscope.

Figure 3 Regeneration of motor neuron cells in nerve tissue proximal to the site of end-to-side neurorrhaphy.

Figure 1 Surgical operation of the end-to-side neurorrhaphy.

Author contributions: YDG conceived and designed the study. QY and DH performed the experiments. QY and TW wrote the paper. FP and SHZ reviewed and edited the paper. All authors read and approved the final version of the paper.

Conflicts of interest:None declared.

Research ethics:The study protocol was approved by Animal Ethics Committee of Wenzhou Medical University of China (approval No.201601-005). The experimental procedure followed the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1986).

Data sharing statement: Datasets analyzed during the current study are available from the corresponding author on reasonable request.

Plagiarism check:Checked twice by ienticate.

Peer review:Externally peer reviewed.

Open access statement:is is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under identical terms.

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Graphical Abstract

Cutaneous nerve induces motor nerve regeneration aer end-to-side neurorrhaphy

＊Correspondence to:

Yu-dong Gu,yudonggu@hotmail.com.

orcid:

0000-0003-3143-8307

(Yu-dong Gu)

10.4103/1673-5374.217350

Accepted: 2017-09-17

Copyedited by Wang J, Li CH, Qiu Y, Song LP, Zhao M