Degeneration and repair 429

Differential motor and sensory functional recovery in male but not female adult rats is associated with remyelination rather than axon regeneration after sciatic nerve crush Ling-Ling Tong*, You-Quan Ding*, Hong-Bo Jing, Xuan-Yang Li and Jian-Guo Qi Peripheral nerve functional recovery after injuries relies on both axon regeneration and remyelination. Both axon regeneration and remyelination require intimate interactions between regenerating neurons and their accompanying Schwann cells. Previous studies have shown that motor and sensory neurons are intrinsically different in their regeneration potentials. Moreover, denervated Schwann cells accompanying myelinated motor and sensory axons have distinct gene expression profiles for regenerationassociated growth factors. However, it is unknown whether differential motor and sensory functional recovery exists. If so, the particular one among axon regeneration and remyelination responsible for this difference remains unclear. Here, we aimed to establish an adult rat sciatic nerve crush model with the nonserrated microneedle holders and measured rat motor and sensory functions during regeneration. Furthermore, axon regeneration and remyelination was evaluated by morphometric analysis of electron microscopic images on the basis of nerve fiber classification. Our results showed that Aα fiber-mediated motor function was successfully recovered in both male and female rats. Aδ fiber-mediated sensory function was partially restored in male rats, but completely recovered in female littermates. For both male and female rats, the

Introduction In mammals, both axons and myelin sheaths are critical for the physiological functions of peripheral nerves. After peripheral nerve injuries, both axon regeneration and remyelination underlie peripheral nerve functional recovery [1]. Although axon regeneration is the prerequisite for peripheral nerve functional recovery, remyelination is crucial for the accomplishment of this functional regeneration. It has been reported that enhancement of remyelination improves functional recovery [2]. Both axon regeneration and remyelination require intimate interactions between regenerating neurons and their accompanying Schwann cells (SCs) [1]. In detail, the intrinsic responses of motor and sensory neurons to axotomy provide the primary force for axon regeneration. Furthermore, this regeneration process is robustly regulated by cellular and molecular environments from denervated SCs. For remyelination, the modified gene profiles of denervated SCs orchestrate this process upon re-contact with regenerating axons. In addition, axon-derived molecules, such as neuregulin-1, are also required for remyelination after peripheral nerve 0959-4965 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

numbers of regenerated motor and sensory axons were quite comparable. However, remyelination was diverse among myelinated motor and sensory nerve fibers. In detail, Aβ and Aδ fibers incompletely remyelinated in male, but not female rats, whereas Aα fibers fully remyelinated in both sexes. Our result indicated that differential motor and sensory functional recovery in male but not female adult rats is associated with remyelination rather than axon regeneration after sciatic nerve crush. NeuroReport 26:429–437 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. NeuroReport 2015, 26:429–437 Keywords: axons, sex differences, myelin sheaths, recovery of function, sciatic neuropathy Department of Histology, Embryology and Neurobiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, Sichuan, China Correspondence to Jian-Guo Qi, PhD, MD, Department of Histology, Embryology and Neurobiology, West China School of Preclinical and Forensic Medicine, Sichuan University, No. 17, Section 3, South Ren-min road, Chengdu, Sichuan 610041, China Tel: + 86 28 85503410; fax: + 86 28 85503204; e-mail: [email protected] *Ling-Ling Tong and You-Quan Ding contributed equally to the writing of this article. Received 14 January 2015 accepted 5 March 2015

injuries [3]. Therefore, both the inherent neuronal regeneration potential and the gene profiles of denervated SCs should be taken into account for peripheral nerve functional recovery. Previous studies have shown that motor and sensory neurons are intrinsically different in their regeneration potentials. In peripheral nerve neuromas, myelinated sensory axons grew out sooner than Aα motor axons, but motor axons regenerated to a greater quantity [4]. With the 10-mm gap repairs in collagen tubules, sensory neurons regenerated consistently better than motor neurons in the same environment [5]. Moreover, during in-vitro culture in a 3D collagen matrix, sensory neurons showed more robust axonal outgrowth and arborization than motor neurons under basal conditions [6]. Those motor and sensory neurons also responded differentially after the application of the same neurotrophic factor. These evidences support the contention that motor and sensory axons may respond differently in the same environment depending on their distinct intrinsic neuron regenerative capabilities. However, denervated SCs accompanying DOI: 10.1097/WNR.0000000000000366

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myelinated motor and sensory axons have distinct gene expression profiles for regeneration-associated growth factors [7]. Denervated sensory SCs in vivo strongly expressed mRNA for nerve growth factor (NGF), brainderived neurotrophic factor (BDNF), vascular endothelial growth factor, hepatocyte growth factor, and insulinlike growth factor-1, whereas denervated motor SCs in vivo significantly upregulated their expression of mRNA for pleiotrophin (PTN) and glial cell line-derived neurotrophic factor. Correspondingly, NGF and BDNF protein were significantly more abundant in denervated sensory SCs than in denervated motor SCs, but PTN protein was more abundant in denervated motor SCs. As peripheral nerve functional recovery requires the coordination of axotomized neurons and denervated SCs, the inherent difference in motor and sensory neuronal regeneration potential and the distinct gene profiles of denervated motor and sensory SCs may lead to differential motor and sensory functional recovery. Moreover, axonal regeneration and remyelination for injured motor and sensory neurons may be different. However, these predictions have not been studied before. In the present study, we established an adult rat sciatic nerve crush model with the nonserrated microneedle holders, measured motor and sensory functions in the regeneration period, and carried out a morphometric analysis of electron microscopic images. Our result indicated that differential motor and sensory functional recovery in male but not female adult rats is associated with remyelination rather than axon regeneration after sciatic nerve crush.

Materials and methods Animals

Twelve-week-old male and female littermates of adult Sprague–Dawley rats weighing 300–350 and 250–300 g, respectively, at the beginning of the study were obtained from the Laboratory Animal Center of Sichuan University and used in the study. The animals were housed with free access to standard chow and water in a room with an ambient temperature of 22 ± 1°C and a 12 : 12 h light/dark cycle. All animal manipulations were performed with the approval of the Animal Care and Ethics Committee of Sichuan University and in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (publication no. 85-23, revised 1985). Animal surgery

Adult rat sciatic nerve crush was performed as described previously, with some modifications (Fig. 1a) [8]. The nonserrated microneedle holder, rather than the nonserrated hemostatic forceps, was utilized to induce sciatic nerve crush in adult rats. In brief, after shaving and depilation of the right hindquarters of rats, right sciatic nerve crush was performed under 3.6% chloral hydrate

anesthesia (1 ml/100 g intraperitoneally) with animal temperature maintained at 37°C. The right sciatic nerve was exposed and gently freed of the surrounding connective tissues. Nerve crush was induced with a nonserrated microneedle holder (Lingqiao suture; Ningbo, Zhejiang, China) at the cross-border between the external obturator and the quadratus femoris. The microneedle holder was engraved with a mark at the 1 mm wide site of the tip. This would result in a 1 mm wide crush site. The outermost portion of the sciatic nerve was aligned with this mark before crush, which ensured a crush of uniform width. The crush was made perpendicular to the nerve once for the particular duration at standard pressure of the microneedle holder. For later identification of the crush site, a 9-0 suture was placed through the edge of the epineurium just distal to the crush. The muscles and skins were closed in separate layers with 4-0 and 2-0 silk sutures, respectively. The animals were placed on a heating pad at 37°C for recovery and then maintained in individual cages for postoperative care during the experimental period.

Immunofluorescence staining

Harvested 1 cm long sciatic nerve segments centered on the lesion sites, from animals used to determine the appropriate duration for nerve crush, were immediately postfixed in 4% paraformaldehyde in 0.1 M phosphate buffer with pH 7.4 for 2 h at 4°C. After gradient sucrose cryoprotection and OCT embedding, longitudinal slices were cut in a cryostat at 15 μm, thaw-mounted onto polyL-lysine-coated slides, and then dried for 30 min on a slide warmer at 37°C. For sequential immunofluorescence staining, sections were first permeabilized with 0.3% Triton X-100 in 0.01 M PBS for 20 min and blocked with 2% BSA and 5% goat serum in PBST (0.05% Tween 20 in 0.01 M PBS) for 1 h at room temperature (RT). Then, sections were incubated overnight at 4°C with mouse monoclonal antibodies against S100β (1 : 100; Abcam, Cambridge, UK) and then rinsed three times every 5 min in 0.01 M PBS. Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibodies (1 : 1000; Keygen, Beijing, China) were applied for 1 h at RT, and then rinsed five times every 10 min in 0.01 M PBS. Then, the sections were blocked again and incubated overnight at 4°C with mouse monoclonal antibodies against NF-200 kDa (1 : 100; Abcam), and then rinsed three times every 5 min in PBST. Alexa Fluor 568-conjugated goat anti-mouse IgG secondary antibodies (1 : 1000; Molecular Probes, Eugene, Oregon, USA) were applied for 1 h at RT and then rinsed five times every 10 min in PBST. Finally, the sections were mounted with the antifade mounting medium (Beyotime; Shanghai, China). All slices were imaged using an Olympus IX81 epi-fluorescence microscope (Olympus; Tokyo, Japan) with an Andor S-COMS camera (Andor; Belfast, Northern Ireland, UK).

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Peripheral functional regeneration Tong et al. 431

(a) A1

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S100β/NF-200 400 μm

Establishment of an adult rat sciatic nerve crush model with the nonserrated microneedle holders. (a) Stereomicroscopic images of surgical procedures for adult rat sciatic nerve crush. A1, before nerve crush; A2, during nerve crush; A3, after nerve crush. (b) The translucency of the sciatic nerves at the crush sites when the microneedle holders are reopened. At least 30 s of crush duration made the nerves at the crush sites translucent. (c) Percentage of rats positive to the pinch stimulus 1 day after sciatic nerve crush for different crush durations. (d) Representative images of immunofluorescent staining of the injured nerves after a particular crush duration with S100β and NF-200 kD. All the experiments were independently repeated five times, with consistent results.

Walking track analysis test

Ten male and female littermates of adult rats were first used to assess motor function recovery by the walking track analysis test following the De Medinacelli methods and modified by Varejao and Alrashdan before surgery and every other week from 1 to 5 weeks after surgery [9]. Before surgery, three conditioning trials were conducted in all rats. In general, after surgery, each rat was allowed to walk two or three times to obtain measurable prints. The footprints were evaluated for three different parameters: print length, the longitudinal distance between the tip of the third toe and the heel; toe spread, the distance between first and fifth toes; and intermediate toe spread, the distance between the second and fourth toes. Measurements of all the parameters were made for the right (experimental) and the left (normal) footprints.

On the basis of these parameters, the sciatic functional index (SFI) was calculated according to the formula suggested by Bain: SFI = − 38.3 × [(EPL − NPL)/NPL] + 109.5 × [(ETS − NTS)/NTS] + 13.3 × [(EIT − NIT)/ NIT] − 8.8. The SFI values were designed to be an indicator of motor function, with values around − 100 indicating total loss of function and values around 0 indicating normal function. Pinprick test

Another five male and female littermates of adult rats were used to evaluate sensory function recovery by the pinprick test. As the pinprick test may damage the plantar skin of the hindpaws and impair the accomplishment of the walking track analysis test, we used an independent cohort of animals for sensory function

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evaluation. The pinprick test was performed as described previously [10] every other day from 3 to 29 days after surgery. The most lateral part of the plantar surface of the hindpaw, the sural nerve territory, was divided into five areas. The insect pin was gently applied to those plantar skin areas from the most lateral toe to the heel. Stimuli were applied at intervals of 5–8 s, allowing for apparent resolution of any behavioral responses to previous stimuli. A positive response was identified if the paw was sharply withdrawn or the rat licked its foot. The saphenous territory of the same paw was used as a positive control. The positive response in every area was recorded as 1 and the summation of the scores of all the areas was the score of the pinprick test. Electron microscopy and morphometry

Additional three male and female littermates of adult rats were used for the morphometric analysis of electron microscopic images of regenerated nerve segments 28 days after operation. Harvested sciatic nerve segments were fixed in 3% glutaric dialdehyde and postfixed in 1% osmium tetroxide for 2 h. The segments were dehydrated in serial acetone solutions and then embedded in Eponate 812 resin (Ted Pella Inc., Redding, California, USA). Both semithin (0.5 μm) and ultrathin (100 nm) transverse sections were made 3 mm distal to the lesion site with an ultrathin microtome. The semithin sections were stained with toluidine blue and examined using a bright-field light microscope. The ultrathin sections were stained with uranyl acetate and lead citrate and then analyzed using a transmission electron microscope (H-600IV; Hitachi, Tokyo, Japan). Morphometric analysis was carried out on four random visual fields for each animal. Software ImageJ (NIH; Bethesda, Maryland, USA) was used to measure axon diameter and myelinated nerve fiber diameter for the calculation of the g-ratio (axon diameter/myelinated nerve fiber diameter). Statistical analysis

All data were presented as mean ± SEM or mean. For behavioral tests, the differences between groups at serial time points were analyzed by the two-way repeatedmeasures analysis of variance, followed by Dunn’s posttest. For electron microscopy and morphometric analysis, all the comparisons between injured and uninjured nerves were made using independent sample tests or Mann–Whitney U-tests. For all data analyses, a P value less than 0.05 was considered to be statistically significant.

Results An adult rat sciatic nerve crush model was established successfully with the nonserrated microneedle holders

In our study, we chose an adult rat sciatic nerve crush model, in which axonal regeneration, remyelination, and functional recovery are not impaired by the obstacles imposed by the injury sites found in nerve transection.

After nerve crush, all axons are interrupted, whereas SC basal laminae are preserved. Currently, adult rat sciatic nerve crush is frequently established by the nonserrated hemostatic forceps [11,12]. However, the nonserrated hemostatic forceps are not easy to obtain. Instead, we used the nonserrated microneedle holders because of their widespread availability. The appropriate duration of adult rat sciatic nerve crush by the nonserrated microneedle holders with standard pressure was determined among a set of nerve crush durations (15, 30, 45, and 60 s) for both sexes. Four littermate rats of the same sex were assigned individually to each duration group. Then, three male and two female rats in each duration group were subjected to adult rat right sciatic nerve crush. After sciatic nerve crush for at least a 30-s crush duration, the entire nerves became translucent at the crush sites (Fig. 1b). Both 30- and 45-s crush durations resulted in complete loss of motor functions in all crushed animals, indicated by the toe spreading reflex test performed as described previously [13] 1 day after surgery (data not shown). However, the pinch test performed as described previously [13] showed that two rats among five rats withdrew their right hindpaws after a 30-s nerve crush, whereas no rat showed this behavior after a 45 s nerve crush (Fig. 1c). Immunofluorescence staining of longitudinal frozen nerve sections showed that the 60 s crush duration resulted in the disruption of the overall tissue structure of the nerves, whereas the 15 s crush duration did not completely interrupt all the axons (Fig. 1d). It seemed that the 30- and 45-s crush durations can completely interrupt all the axons (Fig. 1d). However, we could not examine the integrity of SC basal laminae. This task requires electronic microscopy analysis. Therefore, a duration of 45 s was appropriate for adult rat sciatic nerve crush by the nonserrated microneedle holders with standard pressure. The recovery patterns of sensory and motor functions after crushing for 45 s (Fig. 2) also suggested the successful establishment of an adult rat sciatic nerve crush model. The myelinated motor nerve functions in both male and female adult rats recovered completely

To take the possible sexual dimorphism of our addressing issue into account, male and female littermates were used in our study. Motor functional recovery was shown as dynamic changes of SFIs (Fig. 2a). Male and female rats showed SFIs of − 5.93 ± 1.72 and − 7.65 ± 2.86, respectively, before nerve crush. This indicated that all the animals used for motor function recovery analysis had normal sciatic nerve motor functions. One week following surgery, SFIs for male and female rats decreased markedly (− 74.47 ± 7.63 and − 85.12 ± 3.72, respectively, P < 0.001, for both sexes compared with the values before surgery), which suggested complete loss of motor functions. This state persisted at the second week after nerve crush (− 86.45 ± 4.73 for male rats and − 86.34 ± 3.23,

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Peripheral functional regeneration Tong et al. 433

Fig. 2

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Dynamic recovery of Aα nerve fiber-mediated motor function and Aδ nerve fiber-mediated sensory function. (a) Scores of the walking track analysis test. Results were expressed as the sciatic functional index. Ten rats for each sex were used. (b) Scores of the pinprick test. Five rats of each sex were used. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the values before surgery. #P < 0.05, ###P < 0.001, compared with the values 3 days after surgery.

P < 0.001, for both sexes compared with the values before surgery). Both male and female rats showed a robust improvement, but still a significant decrease in the SFIs (− 28.98 ± 4.96 and − 30.21 ± 5.83, respectively, P = 0.004, for male rats and P = 0.001 for female rats compared with the values before surgery) at the third week. At the fourth and fifth weeks after surgery, all the animals showed almost the same walking track patterns as those before surgery(P > 0.05). SFI values of male and female rats along serial time points showed no significant differences in motor functional recovery (P = 0.282, P > 0.05). In summary, the myelinated motor nerve functions for both male and female adult rats recovered completely. The myelinated sensory nerve functions were fully restored in female rats, but recovered partially in male rats

Functional recovery of the myelinated sensory nerves was shown as dynamic changes in the scores of the pinprick test (Fig. 2b). It has been reported that Aδ myelinated sensory axons are responsible for the positive response in the pinprick test [14–16]. The pattern of sensory function recovery is similar to that previously described [17]. Moreover, the scores of the pinprick test showed a statistically significant difference between male and female rats during the regeneration period (P = 0.007, P < 0.05). In detail, the response to the pinprick test was completely lost 3 days after nerve crush in all animals (0 for both sexes, P < 0.001 for both sexes compared with the values before surgery). This state persisted until 19 days after nerve crush in both male and female rats (P > 0.05 for both sexes compared with the values 3 days after surgery). Then, the scores of the pinprick test began

to increase significantly on the 21st day after nerve injuries (1.4 ± 0.5, P = 0.017 for male rats; 2.6 ± 0.4, P < 0.001 for female rats; compared with the values 3 days after surgery). The response to the pinprick test was robustly recovered in both male and female adult rats 23 and 28 days after sciatic nerve crush (P < 0.05 for both sexes at both time points compared with the values 3 days after surgery). The scores of the pinprick test in female rats 23 and 28 days after surgery were comparable with those before the operation (3.8 ± 0.2, P = 0.123 for 23 days after surgery; 4.4 ± 0.2, P = 0.788 for 28 days after surgery; compared with the values before surgery). However, the response to the pinprick test was not completely restored in male rats at the same time (1.4 ± 0.6, P < 0.001 for 23 days after surgery; 3.4 ± 0.2, P = 0.003 for 28 days after surgery; compared with the values before surgery). In summary, the myelinated sensory nerve functions were fully restored in female rats but were partially recovered in male rats. In other words, the myelinated sensory nerve functions differentially regained in male and female rats. Remyelination rather than axonal regeneration is distinct between myelinated motor and sensory neurons in male but not female adult rats

Results from behavioral tests suggested that motor and sensory functions restored considerably 4 weeks after nerve crush. Therefore, we chose this time point for analysis of axonal regeneration and remyelination of peripheral nerves. Toluidine blue staining of the semithin sections was examined using a bright-field light microscope and showed no visible differences in axons and myelin sheaths between injured and intact nerves in

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434 NeuroReport 2015, Vol 26 No 7

both sexes (data not shown). Electron microscopic analysis of the ultrathin sections showed that remyelination deficiency existed in both male and female rats 28 days after surgery (Fig. 3a). We further applied morphometry on images of electron microscopy for quantitative analysis of axonal regeneration and remyelination using parameters such as the axon diameter and the g-ratio. Our results showed that there was no difference in the number of axons in all the specific ranges of axon diameter between regenerated and intact nerves in both sexes (Fig. 3b and c). This suggested that there were no overt deficits of axonal regeneration in both sexes. Moreover, axonal regeneration for myelinated motor and sensory neurons is quite comparable for both male and female adult rats. To study remyelination of regenerated motor and sensory axons, we quantitatively analyzed remyelination on the basis of nerve fiber classification. Nerve fiber classification was ideally performed by electrophysiology; however, electrophysiological identification of nerve fibers did not allow quantitative analysis of myelin sheath. As there was no visible deficiency in axonal regeneration in both sexes, we classified the nerve fibers on the basis of axonal diameters. As described previously [18], nerve fibers in intact sciatic nerves from adult rats were divided into four types according to the diameter of the nerve fibers as follows: Aα-fiber (>6.76 μm); Aβ-fiber (3.64–7.17 μm); Aγ-fiber (1.00–6.76 μm); and Aδ-fiber (< 3.64 μm). To classify nerve fibers in practice, we modified the criterion for Aα-, Aβ-, and Aδ-nerve fiber classification as follows: Aα-fiber (>7.17 μm); Aβ-fiber (3.64–6.76 μm); and Aδ-fiber (< 3.64 μm). Aγ-fibers were not considered in the morphometric analysis of particular nerve fiber class remyelination because of the rare involvement of those nerve fibers in the neural reflexes for those two behavioral tests. According to mathematical modeling in adult intact rat sciatic nerves [19], the correlation between axon diameter (x1) and the g-ratio (y1) is expressed by the correlation coefficient (r1) of the logarithmic regression curve as follows: y1 = 0.220 log (x1) + 0.508 (r1 = 0.702). On the basis of these data, we calculated the following criteria for Aα-, Aβ-, and Aδnerve fiber classification on the basis of the axon diameters: Aα-fiber (>4.70 μm); Aβ-fiber (2.11–4.39 μm); and Aδ-fiber (< 2.11 μm). On the basis of the above criterion, in both male and female rats, Aα motor axons completely remyelinated in comparison with those of the intact contralateral sciatic nerves (P > 0.05 for both sexes) (Fig. 3d and e). This is consistent with the full recovery of motor functions in both sexes. However, remyelination of regenerated Aδ sensory axons was incomplete in male, but not female adult rats (P = 0.019 for male rats, P = 0.355 for female rats), whereas regenerated Aβ sensory axons partially remyelinated in both male and female rats (P < 0.001 for male rats, P = 0.001 for female rats) (Fig. 3d and e). This may indicate sexual

dimorphism of the recovery of Aδ fiber-mediated sensory functions. In summary, we showed that remyelination rather than axonal regeneration is distinct between myelinated Aα motor and Aδ sensory neurons in male, but not female adult rats.

Discussion In this study, we first successfully established an adult rat sciatic nerve crush model with the nonserrated microneedle holders. Then, we showed in this model that motor function recovered successfully in both male and female rats. In contrast, Aδ fiber-mediated sensory function was partially restored in male rats, whereas it was completely recovered in female rats. In other words, differential motor and sensory functional recovery was present in male, but not female adult rats. Furthermore, for both male and female rats, axon regenerations for axotomized motor and sensory neurons are quite comparable. However, remyelination deficiency of regenerated Aδ sensory axons was present in male, but not female adult rats, whereas regenerated Aα sensory axons remyelinated completely in both male and female rats. That is, remyelination but not axonal regeneration for myelinated Aα motor and Aδ sensory neurons is different in male, but not female adult rats. Our results suggested that differential motor and sensory functional recovery in male but not female adult rats is associated with remyelination rather than axon regeneration after sciatic nerve crush. Sexual dimorphism of differential sensory and motor functional recovery

Surprisingly, our results showed sexual dimorphism of differential sensory and motor functional recovery. In detail, both Aδ fiber-mediated sensory functions and Aα fiber-mediated motor functions restored completely in female adult rats. In contrast, Aδ fiber-mediated sensory functions rather than Aα fiber-mediated motor functions recovered partially in male adult rats. Accumulating evidences suggested that axotomized motor and sensory neurons have different inherent regeneration potentials. Moreover, denervated SCs accompanying myelinated motor and sensory axons distinctly express regenerationassociated growth factors. Peripheral nerve functional recovery requires the cooperation of axotomized neurons and denervated SCs. Hence, the inherent difference in the motor and sensory neuronal regeneration potential and the distinct gene profiles of denervated motor and sensory SCs may lead to differential motor and sensory functional recovery. In our study, we found that differential sensory and motor functional recovery is specifically present in male, but not female rats. Consistently, the fastest growth and maturation rates that can be achieved during regeneration have been shown to be similar for motor and sensory myelinated fibers in adult female cats [20]. To the best of our knowledge, this is the first time that the phenomenon of differential motor and

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Peripheral functional regeneration Tong et al. 435

Fig. 3

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Morphometric analysis of axonal regeneration and remyelination 4 weeks after sciatic nerve crush of male and female adult rats. (a) Representative electron microscopy images of ipsilateral regenerated nerves and contralateral intact nerves for both sexes. Axons for Aα-, Aβ-, and Aδ-nerve fibers were marked with red, green, and yellow colors. All the experiments were independently repeated three times, with consistent results. (b, c) The distribution of axon diameters in ipsilateral regenerated nerves and contralateral intact nerves from three adult male (b) and female (c) rats. (d, e) g-ratio of pooled Aα-, Aβ-, and Aδ-nerve fibers from ipsilateral regenerated nerves and contralateral intact nerves of three animals in adult male (d) and female (e) rats. The numbers of particular subtype of nerve fibers for injured and uninjured nerves are indicated in brackets below the boxes.*P < 0.05, **P < 0.01, ***P < 0.001.

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436 NeuroReport 2015, Vol 26 No 7

sensory functional recovery in male rats has been observed directly. This also suggested that sex-associated factors such as sex hormones might be responsible for the sexual dimorphism of differential sensory and motor functional recovery. After peripheral nerve injuries, both axon regeneration and remyelination are required for peripheral nerve functional recovery. As all the myelinated axons regenerate almost completely across the ranges of axon diameters in both male and female rats, motor and sensory axons might regenerate in the same manner. Therefore, it is unlikely that differential regeneration of myelinated motor and sensory axons underlies differential sensory and motor functional recovery in male rats. In contrast, our results showed that remyelination deficiency of regenerated Aδ sensory axons is specifically present in male, but not female adult rats. This indicated that differential motor and sensory functional recovery in male but not female adult rats is associated with remyelination rather than axon regeneration after sciatic nerve crush. Both axon regeneration and remyelination require intimate interactions between regenerating neurons and their accompanying SCs. Although the inherent differences in Aα motor neurons and Aδ sensory neurons during regeneration might contribute toward their differential remyelination, it is more likely that the intrinsic differences in denervated sensory and motor SCs might have been responsible for their differential remyelination. This prediction was supported by the fact that denervated motor and sensory SCs have different gene expression profiles of remyelination-associated neurotrophic factors [7]. It can also be inferred that sex-specific factors such as sex hormones might underlie differential remyelination in Aα motor axons and Aδ sensory axons of male, but not female rats. Furthermore, sensory SCs, rather than motor SCs, may have particular molecular elements that are subjected to regulation by sex-associated factors such as sex hormones. Identifications of these sex-associated SC phenotype-specific molecules would enable the discovery of novel biomarkers for the identification of sensory and motor nerve fibers, which is very important for basic research and clinical practice.

remyelination was unaffected by the removal of gonadal sources of sex steroid hormones [21]. The underlying mechanism for sexual dimorphism of remyelination of regenerated Aδ sensory axons in young adult animals in the PNS might be related to the sex steroid hormones. It has been reported that female sex hormones can promote myelin formation both in vitro and in vivo. In vitro, cultured primary SCs can produce progesterone and express its corresponding receptors [22]. However, whether these cultured primary SCs were derived from sensory nerve fibers requires further characterization. Progesterone can induce Krox-20 and Sox-10 gene expression through the nonclassical progesterone response elements in the promoter region of these genes [23]. In vivo, SCs in adult sciatic nerve also produce progesterone and express its corresponding receptors [22]. Exogenously added progesterone promotes remyelination while blocking either the synthesis of progesterone or the receptor-mediated action-inhibited remyelination [22]. However, male sex hormones, such as androgen, were also shown to promote the expression of myelin-associated genes, such as glycoprotein P0 and peripheral myelin protein 22 (PMP22), in vitro and in vivo [24]. Our in-vivo data suggested that female sex hormones might promote myelin formation of sensory nerve fibers more powerfully than male sex hormones as remyelination of regenerated Aδ sensory axons in females is significantly more efficient than that in males. This might explain why the vast majority of researchers focused on the therapeutic effects of progesterone rather than androgen on myelin formation.

Acknowledgements The work reported here was supported by a grant (0040105301230) from the Science and Technology Department of Sichuan Province, China. Conflicts of interest

There are no conflicts of interest.

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Sexual dimorphism of remyelination of regenerated Aδ sensory axons

In our study, we also found that for both female and male rats, regenerated Aα motor axons remyelinate fully. In contrast, regenerated Aδ sensory axons incompletely remyelinate in male, but not female rats. In our study, young adult rats were used. Therefore, remyelination of Aδ sensory but not Aα motor axons after nerve crush is sexually dimorphic in young adult rats. In contrast, remyelination in the CNS is more efficient in females than that in males following demyelination in aged but not young adult rats, and sexual dimorphism of

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Differential motor and sensory functional recovery in male but not female adult rats is associated with remyelination rather than axon regeneration after sciatic nerve crush.

Peripheral nerve functional recovery after injuries relies on both axon regeneration and remyelination. Both axon regeneration and remyelination requi...
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