JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE RESEARCH ARTICLE J Tissue Eng Regen Med 2015; 9: 415–423. Published online 10 December 2013 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/term.1833
Motor function recovery during peripheral nerve multiple regeneration Shuai An, Peixun Zhang*, Jianping Peng, Lei Deng, Zhenwei Wang, Zhiyong Wang, Yanhua Wang, Xiaofeng Yin, Yuhui Kou, Na Ha and Baoguo Jiang* Peking University People’s Hospital, Beijing, China
Abstract Neuronal functional compensation and multiple regenerating axon sprouting occur during peripheral nerve regeneration. Sprouting nerve buds were quantitatively maintained and had matured when multiple injured distal nerves were anastomosed to smaller number of proximal nerve stumps; this has positive clinical significance for proximal stump damage. This study investigated whether sprouting axon buds would reinnervate the distal neuromuscular junction and maintain the function of the target organ under compensation conditions. The results showed that the sprouting axon buds maintained the numbers and morphology of motor end plates repaired by a smaller number of proximal nerve stumps, and recovered 80.0% tetanic muscle force compared with the normal side. Meanwhile, nerve conduction velocity, compound muscle action potential and diameter of muscular fibres declined 72.7%, 73.2% and 61.8%, respectively, compared with normal. This observation indicates the potential functional reserve of neurons and that it is feasible to repair nerve fibre injury through anastomosis of multiple distal nerve stumps with a smaller number of proximal nerve stumps, within the limits of compensation. © 2014 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd Received 8 January 2013; Revised 19 April 2013; Accepted 2 September 2013
Keywords
functional recovery; motor end plate; multiple regeneration
1. Introduction Peripheral nerve injury is common in clinical practice. Mangled nerve injury and avulsion often result in significant damage to proximal nerves, which render the nerve repair difficult (Lundborg et al., 1994; Allodi et al., 2012). Current repair methods, such as nerve transfer or nerve implantation, usually need sacrifice of another normal nerve as the donor nerve. Previous studies have revealed that regeneration and compensation occurs in the regeneration of peripheral nerves (Redett et al., 2005; Jiang et al., 2007; Wang et al., 2009). Furthermore, these studies have provided preliminary evidence for the maturation of sprouting nerve fibres (Redett et al., 2005; Yin et al., 2011). These findings provide a possible novel strategy for repairing serious damage to proximal nerve stumps through direct anastomosis to smaller number of proximal nerve stumps. This would avoid injury to donor nerve and not have the disadvantages * Correspondence to: B. Jiang and P. Zhang, Department of Orthopedics and Trauma, Peking University People’s Hospital, No. 11, South Xi-Zhi-Men Street, Beijing, China, 100044. E-mail:
[email protected]; E-mail:
[email protected]
of allograft transplant reactions. However, the recovery outcome has many contributing factors. Correlations have been observed between the functionality of repaired nerves and the maturity of nerve fibres, the quantity of motor end plates and the thickness of muscle fibres (Wood et al., 2011). In order to explore the possibilities of this repair method as a strategy, it should be considered from several aspects, such as nerve fibres, neuromuscular junctions and muscle fibres. A rat tibial nerve model was adopted in this study to further examine whether collateral axon buds could completely re-establish their control of motor end-plates and maintain the diameter and function of the original muscle fibres when multiple injured distal nerves were repaired through anastomosis to a smaller number of proximal nerve stumps.
2. Materials and methods 2.1. Animals Experiments were performed using 36 specific pathogenfree (SPF) female Sprague–Dawley rats with a body weight of 200 g. The animals were randomly divided into three
© 2014 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
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experimental groups, groups A, B and C, for the subsequent surgical treatments. Experimental procedures were reviewed and approved by the Ethics Committee of the People’s Hospital, Peking University.
2.2. Materials Biodegradable chitin conduits (People’s Hospital of Peking University and Chinese Textile Academy, Patent Number 01136314.2) with a length of 8 mm, an inner diameter of 1.5 mm and a wall thickness of 0.1 mm were used.
2.3. Animal model preparation The surgery was performed under a microscope. The rats were anaesthetized through intraperitoneal (i.p.) injection of sodium pentobarbital (30 mg/kg). Following complete anaesthesia, skin preparation and disinfection were carried out in the right hind limb. The right sciatic nerve and its two main branches (common peroneal nerve and tibial nerve) were isolated until fully exposed. The tibia nerve was severed at 5 mm below the bifurcation site of the sciatic nerve. The animals in each group (n = 12) were treated with the following procedures (Figure 1): Group A: The severed tibial nerve was repaired through anastomosis with the small-gap conduits. The distance between stumps was 2 mm. Group B: The common peroneal nerve was severed at the same level, and the proximal stump was anastomosed to the distal stump of the severed tibial nerve through a conduit (with a gap of 2 mm). The other two stumps were sutured to the muscle in opposite directions to avoid self-repair. Group C: The tibial nerve was severed, and the resulting two stumps were sutured to the muscle in opposite directions to avoid self-repair. Nerve anastomosis through conduits was carried out with 10–0 nylon suture, and the incision was subsequently closed with 4–0 suture.
2.4. Observed parameters 2.4.1. Gross morphology and behavioural observation The animals (n = 36) were observed to evaluate wound healing, muscle morphology of the hind limb, and behavioural changes at different time-points (weeks 4, 6, 8 and 12).
2.4.2. Neuroelectrophysiological examination A Medlec Synergy electrophysiological system (Oxford Instrument Inc., Oxford, UK) was used for the examination. The repaired sciatic nerve was exposed at week 12 after the surgery. The stimulating electrodes were placed on the distal and proximal nerve trunks, on the anastomotic
Figure 1. (a) Proximal tibial nerve and distal tibial nerve at a nerve fibre ratio of 1:1; repairing the nerve through anastomosis with small-gap conduits. (b) Proximal common peroneal nerve and distal tibial nerve at a nerve fibre ratio of about 1:3 to 1:2; repairing the nerve through anastomosis with small-gap conduits. (c) The two stumps of the severed tibial nerve were sutured to the muscle in opposite directions to avoid self-repair, which led to denervation of the gastrocnemius. Tp, proximal tibial nerve stump; Td, distal tibial nerve stump; CPp, proximal common peroneal nerve stump
plane in the sciatic nerve, while the recording electrode was inserted into the middle of gastrocnemius; the reference electrode was placed in the thigh muscle on the same side. Paraffin oil was applied around the nerve trunk to reduce bypass conduction through the liquid. The stimulation signal was a square wave, with an intensity of 0.9 mA, a wave width of 0.1 ms and a frequency of 1 Hz. The conduction velocity of the regenerated nerve fibres was recorded by measuring the latent period. The stimulation intensity was gradually strengthened until the amplitude of the compound muscle action potential (CMAP) wave ceased to progressively increase and a generally identical shape for the CMAP wave was formed from the stimulation at both the distal and proximal stumps. The amplitude of the distal CMAP was recorded, which was the distance from the initiation point to the negative peak of the wave.
2.4.3. Tetanic muscle contraction strength The rats were fully anaesthetized before sample collection at week 12 after surgery; this involved dissection and
© 2014 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd
J Tissue Eng Regen Med 2015; 9: 415–423. DOI: 10.1002/term
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isolation of the gastrocnemius. The hind limb was fixed on a specially made holding frame, with the distal end of the gastrocnemius connected to a tension sensor and then using the holding frame kept the gastrocnemius and the tension sensor aligned. The initial tension was maintained at a chosen level (0 < F < 0.1 N). An electrophysiological system was used to generate an initial electric stimulation with an intensity of 0.9 mA, a wavelength of 0.1 ms and a frequency of 1 Hz. The stimulation electric current was subsequently strengthened until the waveform of the tetanic contraction induced stopped increasing. A PCLAB-UE biomedical signal acquisition and processing system (Beijing Microsignal star Inc., Beijing, China) was used to record the waveform of the tetanic contraction of the gastrocnemius on both sides. The amplitudes of the waves were measured and the ratio of the wave amplitudes of experimental side to the untreated normal control side was used as the overall recovery rate of muscle strength (Shin et al., 2008).
2.4.4. Wet muscle weight measurement and diameter of muscle fibres by haematoxylin and eosin (H&E) staining The gastrocnemius was isolated by severing it at its starting and ending point immediately after the aforementioned parameters were measured, and the weight of the muscle was measured. Transverse sectioning of the muscle samples was performed for H&E staining after fixing with paraformaldehyde, dehydrating with graded ethanol and embedding in paraffin wax. The crosssections of the muscle fibres were photographed under a magnification of 10 × 20 and five fields were selected in the upper left, lower left, upper right, lower right and centre of the cross-section of the muscle fibres for quantification of the diameter of muscular fibres in each field and for measurements using IMAGE PRO PLUS 6.0 software (Media Cybernetics Inc., Rockville, MD, USA).
2.4.5. Osmium tetroxide staining of the tibial nerve and quantification of nerve fibres Twelve samples of each group were post-fixed in 1% osmium tetroxide for 1 day, after which the specimen was sliced into 2 μm cross-sections. The cross-section of the nerve was photographed under a magnification of 10 × 20 and five fields were selected in the upper left, lower left, upper right, lower right and centre of the nerve for quantification of the myelinated nerve fibres in each field and the measurement of the area of each field using a combination of manual measurements and measurements using IMAGE PRO PLUS 6.0 software. The numbers of myelinated nerve fibres in each field were calculated manually and the area of the field and total area were measured using IMAGE PRO PLUS. The average number of myelinated nerve fibres per unit area were then calculated. The total number of myelinated nerve fibres (N) = the number of myelinated nerve fibres per unit area (n/ds) × area of the cross-section (s). The total number of myelinated nerve fibres was calculated using this above equation.
2.4.6. Immunohistochemical staining of motor end plates Using the cupric-ferricyanide staining method developed by Kamovsky and Roots, acetylthiocholine iodide was added to freshly prepared incubation buffer as the enzyme’s substrate, which was hydrolysed into thiocholine by acetylcholinesterase (AChE) in tissue (Karnovsky and Roots 1964). Thiocholine reduced the ferricyanide in the incubation buffer into ferrocyanide and this reacted with copper ions to form cupric ferrocyanide, which was deposited as a brown precipitate at sites with AChE activity. The entire muscle was divided into three equal parts along its longitudinal axis, and consecutive sagittal cryosectioning (10 μm) was performed. One section was collected every 100 sections for staining. The selected sections were washed with phosphate-buffered saline (PBS) three times for 20 min each time, followed by incubation in Kamovsky–Roots (KR) solution for 6 h. The sections were then washed with PBS three more times for 20 min each (Ma et al., 2002). The sections were finally mounted and observed for quantification of the motor end plates under a 10 × 20 light microscope. The total number of motor end-plates (N) = the sum of motor end-plates per section (n1 + n2 + …… + n) × 100.
2.4.7. Immunofluorescence staining of acetylcholine receptors Sagittal cryosectioning was performed with the muscle specimen, and one section was collected every 100 sections. The sections were 20 μm thick. After being air dried, the sections were washed with PBS three times for 20 min each time and blocked with serum. The sections then underwent specific staining of the acetylcholine receptor using tetramethylrhodamine-labelled α-bungarotoxin (T-BTX). The T-BTX stock solution (1 mg/ml in PBS) was diluted 1:400. The sections were incubated with diluted T-BTX at 4°C overnight (12 h) and then were washed with PBS three times for 20 min each time. The sections were mounted with anti-quenching mounting media. The morphology of the motor end plates was observed and recorded under a magnification of 10 × 40 with fluorescence excitation at 620 nm (Ma et al., 2002; Magill et al., 2007). Five fields were observed in the upper left, lower left, upper right, lower right and centre areas for each section, and the perimeter and area of a motor end plate were measured by drawing the maximal smooth perimeter around the image of each endplate according to their grey level and then calculated under image scale using IMAGE-PRO PLUS automatically.
2.5. Statistical analysis Statistical analyses was performed using SPSS 11.0 (SPSS Inc., Chicago, IL, USA). The measurement data are expressed as mean ± SD. An independent t-test was adopted for two-group comparison, and analysis of variance (ANOVA) was used for multi-group comparison. Comparison between the groups was made by analysing data with a post-hoc
© 2014 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd
J Tissue Eng Regen Med 2015; 9: 415–423. DOI: 10.1002/term
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method Student-Newman-Keuls (S-N-K). The S-N-K test is a pairwise comparison of every combination of group pairs. This test calculates a q test statistic for each pair, and displays the P value for that comparison. Enumeration data were analysed using a chi-square test. Statistical significance was established as p < 0.05.
3. Results 3.1. General observation of the animals Claudication of the right hind limb, and gradual muscular atrophy in the lower leg were observed in the right hind limb in each group after the surgery. At 10 days after repair, the rats in all groups exhibited discoloration of the toenails, sparse and dull hair coat on the lower leg and foot and an insensitivity to pain stimuli on the experimental side. Foot ulcers were observed at 6 weeks postoperatively in the untreated group (n = 2), without significant improvement of gait, while the other two groups displayed nearly normal gait and gradual improvement coordination.
3.2. Quantification of nerve fibres and electrophysiological examination The number of normal tibial nerve fibres was 3313 ± 204 at 12 weeks after the surgery. The numbers of proximal and distal nerve fibres were 3384 ± 273 and 3197 ± 242, respectively, in group A and 1159 ± 151 and 2909 ± 189, respectively, in group B. The distal nerve fibres in group C showed evident degeneration, and some even disappeared (Table 1). No statistically significant difference was observed in the number of distal nerve fibres between group A and group B. Electrophysiological examination was performed with fully anaesthetized rats. The nerve conduction velocity of a normal tibial nerve was 49.9 ± 7.1 m/s. The nerve conduction velocity was 43.4 ± 11.7 m/s and 36.3 ± 8.1 m/s in group A and group B, respectively. The nerve conduction velocity in group B was lower than that of the intact nerve; the difference was statistically significant. The compound muscle action potential indicated that the wave amplitude of the experimental side was 88.4 ± 5.6% and 73.2 ± 18.9% of the contralateral side in group A and group B, respectively, but this was not statistically significant.
3.3. Muscle morphology and strength Haematoxylin and eosin staining showed that the crosssectional diameter of a normal muscle fibre was approximately 31.9 ± 5.6 μm, with distinct borders and uniform staining (Figure 2). The muscle fibres of group C displayed marked atrophy caused by prolonged denervation, with indistinct borders and uneven staining. The muscle fibre diameters of the three groups were 21.2 ± 6.9 μm, 19.7 ± 6.4 μm and 13.6 ± 4.8 μm for groups A, B and C, respectively. The muscle fibre diameter of the untreated normal control group was greate than those of the experimental three groups; that of group C was significantly lower than those of the other two groups. There were no significant differences among the body weights of rats of all groups before the surgery and there were also no significant differences in the body weights of the rats among all groups 4 weeks after the surgery. At 12 weeks after the surgery, the body weights of the rats in group C were slightly greater than those of the other two groups, but this was not significantly different. The gastrocnemius was collected for wet weight measurement after the rats were euthanized. The wet weight ratios of the experimental side to the normal side were 64.75 ± 13.5%, 54.66 ± 12.5% and 28.27 ± 10.9% in groups A, B, and C, respectively; the ratio of group C was significantly lower than those of the other two groups. Under anaesthesia, the ratio of the tetanic contractility of the experimental side to the contralateral side was 93.7 ± 21.4% and 80.0 ± 10.1% for groups A and B, respectively. The muscles from group C did not show significant tetanic contraction when the reverse sutured distal nerve was stimulated.
3.4. Morphology and quantity of neuromuscular junctions After the staining of AChE, the normal neuromuscular junction appeared as spherical or clostridial form with a brown colour and the centre was shallow while the peripheral region was saturated (Figure 3). The numbers of motor end-plates in the gastrocnemius nerve fibres of the normal control and the three experimental groups (A, B and C) were 27400 ± 6698, 22950 ± 8817, 20433 ± 7187 and 9283 ± 1653, respectively; the numbers in group C were significantly lower than those of the other groups.
Table 1. Comparison of Myelinated axon numbers, MCV, Peak of CAMP, muscle force and diameter of muscular fibers for all group Group
Myelinated axon numbers Proximal
Normal Group A Group B Group C
MCV (m/s)
Peak of CAMP (%)
Muscle force (%)
Diameter of muscular fibers (μm)
49.2±7.1 43.4±11.7 36.3±8.1* NA
NA 88.4±5.6 73.2±18.9 NA
NA 93.7±21.4 80.0±10.1 NA
31.9±5.6 21.2±6.9* 19.7±6.4* 13.6±4.8*
Distal
3313±204 3384±273 3197±242 1159±151* 2909±189 NA NA
*p
