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. 2011 Aug 4;219(5):638–645. doi: 10.1111/j.1469-7580.2011.01419.x

Histomorphometric changes in repaired mouse sciatic nerves are unaffected by the application of a scar-reducing agent

Wei Cheong Ngeow 1,2, Simon Atkins 1, Claire R Morgan 1, Anthony D Metcalfe 3, Fiona M Boissonade 1, Alison R Loescher 1, Peter P Robinson 1
PMCID: PMC3222843  PMID: 21812777

Abstract

Microsurgical repair of transected peripheral nerves is compromised by the formation of scar tissue and the development of a neuroma, thereby limiting the success of regeneration. The aim of this study was to quantify histomorphometrically the structural changes in neural tissue that result from repair, and determine the effect of mannose-6-phosphate (M6P), a scar-reducing agent previously shown to enhance regeneration. In anaesthetised C57-black-6 mice, the left sciatic nerve was sectioned and repaired using four epineurial sutures. Either 100 μL of 600 mm M6P (five animals) or 100 μL of phosphate-buffered saline (placebo controls, five animals) was injected into and around the nerve repair site. A further group acted as sham-operated controls. After recovery for 6 weeks, the nerve was harvested for analysis using light and electron microscopy. Analysis revealed that when compared with sham controls, myelinated axons had smaller diameters both proximal and distal to the repair. Myelinated axon counts, axonal density and size all decreased across the repair site. There were normal numbers and densities of non-myelinated axons both proximal and distal to the repair. However, there were more Remak bundles distal to the repair site, and fewer non-myelinated axons per Remak bundle. Application of M6P did not affect any of these parameters.

Keywords: histomorphometry, nerve regeneration, nerve repair, scarring, sciatic nerve

Introduction

The aim of peripheral nerve repair is to allow regeneration of the damaged axons, with characteristics as close as possible to the pre-injury state. A common method to evaluate these characteristics is to undertake histomorphometric analysis of axon types, numbers and sizes. One factor that influences recovery is the formation of scar tissue at the repair site, and the subsequent development of a neuroma. The aim of the present study was to use histomorphometric methods to quantify the changes that occur in the neural tissues after nerve repair, and to determine whether application of a scar-reducing agent affects these changes.

Previous studies have shown that distal to a peripheral nerve injury the regenerating myelinated axons are fewer in number (Horch & Lisney, 1981; Holland et al. 1996a), have reduced diameters (Holland & Robinson, 1990; Holland et al. 1996a; Menovsky & Beek, 2001; Ayhan et al. 2003; Martins et al. 2005; Landegren et al. 2006) and hence slower conduction velocities (Cragg & Thomas, 1961; Aitken & Thomas, 1962; Devor & Govrin-Lippmann, 1986; Robinson, 1989; Smith & Robinson, 1995a,b;), and there may be a reduced axonal density (Nachemson et al. 1985; Atkins et al. 2007). The total non-myelinated axon counts may be unchanged (Holland & Robinson, 1990), but the number ensheathed within each Schwann cell may be altered (Holland et al. 1996a). Changes can also be detected proximal to the injury site due to a combination of retrograde Wallerian degeneration (Holland et al. 1996a) and the development of some axonal sprouts that become directed centrally due to an obstruction at the repair site (Holland et al. 1996a; McCallister et al. 1999).

The potential problems that can arise due to scar formation at a nerve injury or repair site have been recently reviewed (Koopmans et al. 2009; Ngeow, 2010), and include the creation of a mechanical barrier to the regenerating axons, shrinkage of the endoneurial sheaths that contain the axons (Sunderland & Bradley, 1959), and the development of multiple branched axonal terminals to form a neuroma (Lane et al. 1978). Thus, recovery after repair will be influenced by the extent of scar formation, and we have previously demonstrated an inverse relationship between the amount of collagen scar and the level of regeneration (Atkins et al. 2006a). This provides a therapeutic opportunity if an effective scar-reducing agent could be applied locally to a nerve repair site, and a series of studies has investigated that possibility (e.g. Graham et al. 1973; Adanali et al. 2003; Ozgenel & Filiz, 2003; Zou et al. 2006; Atkins et al. 2006b, 2007). Some investigations have manipulated the transforming growth factor-beta (TGF-β) pathway (Nath et al. 1998; Davison et al. 1999; Atkins et al. 2006b, 2007), as this plays a major role in wound healing and scar formation (O'Kane & Ferguson, 1997). Mannose-6-phosphate (M6P) is a TGF-β inhibitor that works by interfering with the conversion of latent forms of the TGF-βs into their active form, and has been shown to have a marked anti-scarring effect (McCallion & Ferguson, 1996). In recent studies on mice we used electrophysiological and histochemical techniques to screen the effect of a series of scar-reducing agents, including different concentrations of M6P, applied at a sciatic nerve repair site (Ngeow et al. 2011a). We demonstrated that intra-neural and peri-neural application of 600 mm M6P was the most effective agent for enhancing nerve regeneration (Ngeow et al. 2011a), and a subsequent study revealed benefits in early functional recovery (Ngeow et al. 2011b). We have now pursued those observations further to assess the detailed neural changes and to determine whether M6P would be an appropriate and safe agent to use clinically. We have used light and electron microscopical analysis to characterise the effects of M6P on neural tissues proximal and distal to a nerve repair site, 6 weeks after repair. We hypothesise that enhanced regeneration after application of a scar-reducing agent might be revealed by an increase in the number and/or diameter of axons distal to the repair site, and restoration of characteristics closer to that of an undamaged nerve.

Materials and methods

Surgical procedure

Fifteen C57 black-6 mice (8–16 weeks old) were used in this study. The animals were bred for experimental purposes by a supplier approved for the purpose by the UK Home Office. The experiments were carried out under appropriate UK Home Office project and personal licenses, with local ethical approval, in accordance with the UK Animals (Scientific Procedures) Act 1986. Under general anaesthesia (2–3% isoflurane; Abbott Laboratories, UK), the left sciatic nerve was exposed at a site approximately 12 mm distal to the sciatic foramen. The nerve was carefully freed from the surrounding tissues and, in five sham control animals, the wound was closed. The remaining 10 animals were divided in random order into two groups for administration of 100 μL of 600 mm M6P (Renovo, UK) or 100 μL phosphate-buffered saline (PBS, placebo control). The investigator was kept blind to the experimental group for this and subsequent parts of the study. Initially, an injection of approximately 20 μL of the agent was made beneath the epineurium of the intact nerve, using a microdialysis needle (0.164 mm, outside diameter; World Precision Instruments). The nerve was then sectioned transversely with microscissors in the middle of this area and immediately re-approximated using four 9/0 monofilament polyamide epineurial sutures (Ethilon®; Ethicon, UK). The remaining solution (80 μL) was then injected between the nerve ends and into the muscle and soft tissues surrounding the repair, and the surgical site closed. A single dose of analgesic was administered (0.01 mL buprenorphine hydrochloride 0.3 mg mL−1; Vetergesic®, Alstoe Animal Health, UK) subcutaneously, and the mice were allowed to recover for 6 weeks; this recovery period was chosen to be the same as used in our previous electrophysiological studies where M6P was shown to have enhanced regeneration (Ngeow et al. 2011a,b;).

Histomorphometric analysis

After the recovery period, under a second general anaesthetic (fentanyl/fluanisone 0.8 mL kg−1 and midazolam 4 mg kg−1, intraperitoneal; Janissen/Roche), the sciatic nerve was harvested in the region of the repair, rinsed in saline and immersed in 3% glutaraldehyde in 0.1 m phosphate buffer for 60 min. The nerve was then divided to allow transverse sections to be obtained from two sites; 1.5 mm proximal and 1.5 mm distal to the repair. The divided nerve was post-fixed in 3% glutaraldehyde in 0.1 m phosphate buffer at 4 °C for a further 4 h, and stored in PBS at 4 °C overnight.

Preparation and analysis of specimens using light microscopy

Semi-thin transverse sections (approximately 0.3–0.5 μm) were cut using glass knives, stained with 1% toluidine blue in 1% borax and mounted on glass slides. Under a 10 × objective, a single section was captured and the cross-sectional area measured using an image analysis system. Under a 40 × objective, the whole section of the nerve was scanned, and the captured images were montaged. Using Bioquant Life Science version 8.40.20 image analysis system (Bioquant Image Analysis, Nashville, TN, USA), the number, cross-sectional area (size) and diameter of the myelinated axons were determined using a randomised systematic sampling scheme (Mayhew, 1990). Initially a grid with sectors measuring 45 000 μm2 was superimposed on the cross-section of the nerve. An area measuring 10 420 μm2 in both the top left and right corner of each sector was analysed. The overall values for the whole nerve were derived from these counts and measurements. Population densities were calculated from the nerve cross-sectional area and the number of myelinated fibres.

Preparation and analysis of specimens using electron microscopy

Ultra-thin sections (70–90 nm) of nerve were collected on 200-mesh grids, and stained using uranyl acetate and lead citrate solutions. The 200-mesh grid provided the basis for a randomised systematic sampling scheme, similar to that described by Vora et al. (2005), and recommended by Mayhew & Sharma (1984). An area measuring 1575 μm2 in the bottom left corner of each mesh square was analysed at an original magnification of × 3000, and the number of non-myelinated axons, Remak bundles and number of non-myelinated axons in each Remak bundle counted (Remak bundles are defined as one or more unmyelinated axons ensheathed by Schwann cells and arranged within a single basal lamina; Murinson et al. 2005). The overall values for the whole nerve were derived from these counts, and population densities were calculated.

Statistical analysis

Comparisons between the three groups were undertaken using a Kruskal–Wallis test, followed by Conover–Inman pairwise comparisons where applicable (StatsDirect; StatsDirect, UK). Comparisons between the proximal and distal values within each group were undertaken using a Mann–Whitney U-test, and Pearson's correlation was used to detect if any association was present between the axon cross-sectional area and diameter. In all cases, statistical significance was accepted at the alpha level of 5% (P< 0.05), and all tests were two-tailed.

Results

Light microscopical analysis

The cross-sectional area of the nerve proximal to the repair site ranged from 0.144 to 0.273 mm2, and distally ranged from 0.131 to 0.262 mm2. There were no significant differences between the areas at the two sites, or between groups.

The typical appearance of myelinated axons in each group as seen under light microscopy is shown in Fig. 1. The area of the nerve that was analysed ranged from 34.0% to 51.4% (mean = 43.6%) for the proximal section of nerve, and from 31.1% to 61.8% (mean = 41.8%) for the distal section of nerve. These analysed areas were substantially more than the minimum of 18% recommended by Mayhew (1990), indicating that the samples should have been representative. The analysis results are shown in Table 1.

Fig. 1.

Fig. 1

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Examples of light micrographs showing myelinated axons in mouse sciatic nerve (top-proximal; bottom-distal). (A) Sections from an uninjured sham control nerve. (B) Sections from a repaired nerve injected with PBS. (C) Sections from a repaired nerve injected with 600 mm M6P (scale bar: 50 μm).

Table 1.

Results of light microscopical analysis of myelinated axons, showing median (interquartile range) values proximal (P) and distal (D) to the repair site, and statistical comparisons between groups (Kruskal–Wallis test)

Sham PBS M6P P-value
Axon count
 P 4083 (3439–4313) 4344 (3844–5634) 4262* (4000–4984) 0.403
 D 3555 (2711–4688) 3619 (3199–4315) 2717* (2498–3386) 0.289
Axon density (number per mm2)
 P 21 401 (20 729–23 304) 24 462† (19 055–26 181) 21 490‡ (20 792–26 286) 0.932
 D 21 587 (18 112–23 702) 18 378† (18 253–20 887) 18 301‡ (17 122–19 606) 0.185
Cross-sectional area (μm2)
 P 9.91 (8.69–11.5) 8.29§ (6.03–8.81) 5.55* (4.75–9.28) 0.08
 D 9.66 (8.53–10.47) 3.65§ (2.84–4.98) 3.80* (3.07–3.90) 0.009
Diameter (μm)
 P 2.97 (2.86–3.24) 2.60‡ (2.01–2.93) 2.26§ (2.02–2.59) 0.039
 D 3.07 (2.80–3.17) 1.77‡ (1.58–1.89) 1.78§ (1.67–2.03) 0.009
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Significant differences between proximal and distal values in the same group (Mann–Whitney U-test) were as follows:

*

P = 0.009

†

P = 0.047

‡

P = 0.016

§

P = 0.028.

M6P, mannose-6-phosphate; PBS, phosphate-buffered saline.

Comparisons between groups revealed no significant differences in myelinated axon counts or densities either proximal or distal to the repair site. Proximal to the repair site the axons in the repair groups had significantly smaller diameters than in sham controls. Distal to the repair site the axons in the repair groups had significantly smaller diameters and cross-sectional areas than in the sham controls. There were no significant differences between the two repair groups. Correlation analysis confirmed that a significant linear correlation existed between axon size and diameter (proximal, Pearson correlation r = 0.79, P = 0.001; distal, Pearson correlation r = 0.93, P = 0.0001).

Comparisons between results from the proximal and distal sites within the same group revealed that the myelinated axon counts were lower distally, although this difference only reached significance in the M6P group. The axon density was lower distally in both repair groups, and the myelinated axon diameter and cross-sectional area were also lower distally.

Electron microscopical analysis

Using our sampling protocol, the number of myelinated axons included in the area of analysis ranged from 168 to 649 axons, and was similar for the three groups. This equated to a percentage area of the section of 6.4–13.1% (mean = 9.2% proximally and 8.9% distally), exceeded the 6% recommended by Mayhew & Sharma (1984), and was similar to that achieved by Holland et al. (1996a,b); when using a similar protocol.

The typical electron microscopical appearance of myelinated and non-myelinated axons is shown in Fig. 2, and the analysis results are shown in Table 2. Comparisons between groups revealed no significant differences in non-myelinated axon counts, axon densities or ratios of non-myelinated axons to myelinated axons, either proximal or distal to the repair site. Proximal to the repair site there were no differences in the Remak bundle count or density, but there were significantly fewer non-myelinated axons per Remak bundle in the repaired nerves than in sham controls. Distal to the repair site there was an increase in number and density of Remak bundles in the repair groups, and a decrease in the number of non-myelinated axons per Remak bundle. There were no significant differences between the two repair groups.

Fig. 2.

Fig. 2

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Examples of electron micrographs showing myelinated and non-myelinated axons in mouse sciatic nerve. (A) A section from proximal to the repair site in a nerve injected with 600 mm M6P. (B) A section from distal to the repair site in a nerve injected with 600 mm M6P. (C) A higher power image showing groups of non-myelinated axons forming Remak bundles. Scale bars: 2 μm (A and B); 1 μm (C). Four scans were montaged for each image in (A) and (B).

Table 2.

Results of electron microscopical analysis of non-myelinated axons, showing median (interquartile range) values proximal (P) and distal (D) to the repair site, and statistical comparisons between groups (Kruskal–Wallis test)

Sham PBS M6P P-value
Axon count
 P 8163 (6966–9499) 7073 (6068–7367) 6150 (5198–8943) 0.174
 D 8982 (6670–12 864) 6611 (4603–10 149) 7108 (4681–14 481) 0.613
Axon density (number per mm2)
 P 45 060 (37 720–67 371) 36 880 (29 640–46 787) 32 784 0.112
 D 53 823 (40 943–75 416) 35 215 (25 430–53 041) 49 335 (30 277–64 957) 0.264
Non-myelinated : myelinated axon ratio
 P 1.72 (1.54–2.31) 1.50 (1.14–1.80) 1.38 (1.10–1.57) 0.093
 D 2.12 (1.69–2.38) 1.84 (1.39–2.38) 2.03 (1.46–2.18) 0.651
Remak bundle count
 P 955 (849–1105) 1534* (996–2490) 1448† (927–3152) 0.185
 D 1165 (584–1243) 3168* (2529–4482) 4241† (2836–6582) 0.007
Remak bundle density
 P 4878 (4546–6867) 7768* (5591–12 786) 7231* (5616–13 343) 0.114
 D 7025 (3616–7755) 15 557* (13 952–24 261) 28 863* (18 302–29 569) 0.005
Number of axons per Remak bundle
 P 8.4 (8.2–10.4) 4.5‡ (3.0–6.5) 4.3‡ (3.0–5.7) 0.018
 D 9.9 (8.2–13.4) 2.1‡ (1.8–2.3) 1.7‡ (1.6–2.2) 0.008
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Significant differences between proximal and distal values in the same group (Mann–Whitney U-test) were as follows:

*

P = 0.016

†

P = 0.032

‡

P = 0.008.

M6P, mannose-6-phosphate; PBS, phosphate-buffered saline.

Comparisons between results from the proximal and distal sites within the same group revealed that there were no significant differences between the proximal and distal non-myelinated axon counts, density or the ratio of non-myelinated axons to myelinated axons. However, the Remak bundle count and density was significantly higher distally in both repair groups, and the number of non-myelinated axons per Remak bundle decreased distally.

Discussion and Conclusions

Axon count and density

The range of axon counts in the present study is similar to that reported by Zou et al. (2006) and Atkins et al. (2007), who also studied the sciatic nerve in mice. In the sham controls the distal sample always had a lower myelinated axon count than the proximal sample, and this presumably results from some axons branching off to innervate peripheral targets. Similar patterns have been observed in studies on rats (Palatinsky et al. 1997; Martins et al. 2005, 2006).

There was substantial variability in both counts of myelinated and non-myelinated axons from different animals, and such inter-animal variation has been seen in other nerves (e.g. Jenq & Coggeshall, 1985a,b; Holland & Robinson, 1990). Taking this inherent variability into account, our study found no significant changes in the number or density of proximal or distal axons after repair, when compared with sham controls. However, when comparisons were made across the repair site within the same group, a significant reduction in myelinated axon count and density was seen distal to the repair. This could result from an inability of some axons to cross the repair site and/or the development of some ‘recurrent’ nerve sprouts whereby regenerating axons become redirected centrally, thereby increasing the proximal counts (Graham et al. 2007). This finding is in accord with observations on repaired lingual nerves (Holland et al. 1996a), but contrasts with the reports of Menovsky & Beek (2001) and Atkins et al. (2007), where it was found that the distal segment had a higher number of myelinated axons and it was assumed that this had resulted from axonal branching.

Some differences between studies will be purely methodological; in our investigation the proximal and distal sampling sites were only 1.5 mm from the repair, and a different outcome might have been found if the samples had been obtained at more distant points. The recovery period before harvesting the samples is also important, and at 6 weeks there may have been ongoing changes resulting from the nerve injury. For example, Siironen et al. (1994) reported that the number of axonal sprouts distal to a site of nerve injury increased to their highest level after 42 days, only to return to control levels 2 weeks later.

The number and density of non-myelinated axons was similar on each side of the repair site, but our methods do not allow us to establish the proportion of proximal axons that had crossed over to produce the distal population, and the number that were branched. Previous studies have shown a reduction in the number of non-myelinated axons both proximal and distal to an unrepaired nerve injury (Carter & Lisney, 1987), or distal to some forms of entubulation (Jenq & Coggeshall, 1985b), but have revealed a substantial increase distally after epineurial suture repair (Holland et al. 1996a). Thus, it appears that the method of repair has a substantial impact upon outcome.

The ratio of non-myelinated to myelinated axons was similar in the repaired nerves to the values in the sham controls, suggesting that the injury and the M6P or PBS injection did not hinder the Schwann cells involved in remyelination, or the transformation of non-myelinated axons to myelinated axons.

Axon size

Significantly smaller cross-sectional areas and diameters of the myelinated axons were found in the repaired nerves treated with either M6P or PBS, when compared with sham controls. Proximal to the repair the size reduction results from a combination of retrograde degenerative changes and the potential inclusion of small recurrent sprouts within the analysis (Cragg & Thomas, 1961; Aitken & Thomas, 1962; Holland & Robinson, 1990; Graham et al. 2007). A reduction in axon size distal to a nerve injury is a well-recognised change and has been reported in numerous studies, such as those by Menovsky & Beek (2001), Ayhan et al. (2003) and Martins et al. (2005). After initial reinnervation of the distal stump there is a progressive increase in axonal size over time, and the duration of distal nerve stump denervation has an effect (Sulaiman & Gordon, 2000); in our study the repair was immediate.

Remak bundle characteristics

The envelopment of several fine axons within a single Schwann cell to form a Remak bundle is a characteristic of non-myelinated nerve fibres (Murinson et al. 2005). Murinson & Griffin (2004) found that in rats the number of non-myelinated axons within a Remak bundle differed in different nerves, and in different regions along a single nerve. The median number of axons per Remak bundle in the sciatic nerve of rats was six (range 1–26), while more distally in the plantar nerve the number had reduced to three (range 1–14). Our mouse sciatic values were just slightly higher than those reported in the rat sciatic nerve.

The proliferation of Schwann cells in response to a nerve injury (Siironen et al. 1994) would have led to the significant increase in Remak bundle counts and density distal to the repair, seen in the present study. When associated with a normal number of non-myelinated axons, this results in a significant reduction in the number of axons per Remak bundle. This latter change was also seen proximal to the repair site, presumably due to the small (but not significant) increase in Remak bundle numbers within the area of retrograde Wallerian degeneration (Pellegrino & Spencer, 1985; Siironen et al. 1994).

Few previous studies have evaluated Remak bundle characteristics after nerve injury. However, Holland et al. (1996b) found that when compared with uninjured nerves, there was a 50% increase in axons per Schwann cell distal to a lingual nerve section in the cat, and a similar increase after epineurial suture repair (Holland et al. 1996a); in each case this was associated with an increase in non-myelinated axon numbers. In contrast, Carter & Lisney (1987) described a reduction in the size of ‘Schwann cell units’ after saphenous nerve regeneration in the rat, but there was a concurrent reduction in non-myelinated axon numbers. The administration of M6P does not seem to have had any detrimental effect on Schwann cell proliferation, even though it will have temporarily blocked the effect of TGF-β1, which is mitogenic for Schwann cells (Ridley et al. 1988).

The effect of a scar-reducing agent

This present histomorphometric study did not reveal any difference between the repaired nerves treated with the scar-reducing agent M6P or the PBS placebo. As the results of our previous electrophysiological studies have shown that M6P treatment can enhance nerve regeneration (Ngeow et al. 2011a,b;), we hypothesised that we would see an increase in the number and/or size of the myelinated axons distal to the repair site. However, electrophysiological recordings are heavily biased towards activity in the large myelinated fibres and, if changes were confined to a specific subgroup of axons, they may not have been detected in the present study. In addition, a reduction in scarring at the repair site might have been expected to reduce the number of non-myelinated axon sprouts. It could be argued that our repairs were too successful to allow some differences between groups to be seen, as histologically regeneration of the repaired groups had reached a level comparable to the non-operated nerves. Equally, it is possible that the high level of variability in some of the parameters measured, or the mixed population of regenerated, recurrent and branched axons, may have masked any effect. However, it seems reasonable to conclude that local treatment with the scar-reducing agent M6P is well tolerated, with no negative effects on any of the histomorphometric parameters that we have studied, and in particular did not appear to affect Schwann cell proliferation.

Acknowledgments

We thank Dr Emma Bird and Mr David Thompson for their help during the course of these studies, and Renovo Group plc, for funding the project.

References

  1. Adanali G, Verdi M, Tuncel A, et al. Effects of hyaluronic acid-carboxymethylcellulose membrane on extraneural adhesion formation and peripheral nerve regeneration. J Reconstr Microsurg. 2003;19:29–36. doi: 10.1055/s-2003-37188. [DOI] [PubMed] [Google Scholar]
  2. Aitken JT, Thomas PK. Retrograde changes in fibre size following nerve section. J Anat. 1962;96:121–129. [PMC free article] [PubMed] [Google Scholar]
  3. Atkins S, Smith KG, Loescher AR, et al. The effect of antibodies to TGF-beta1 and TGF-beta2 at a site of sciatic nerve repair. J Peripher Nerv Syst. 2006a;11:286–293. doi: 10.1111/j.1529-8027.2006.00100.x. [DOI] [PubMed] [Google Scholar]
  4. Atkins S, Smith KG, Loescher AR, et al. Scarring impedes regeneration at sites of peripheral nerve repair. Neuroreport. 2006b;17:1245–1249. doi: 10.1097/01.wnr.0000230519.39456.ea. [DOI] [PubMed] [Google Scholar]
  5. Atkins S, Loescher AR, Boissonade FM, et al. Interleukin-10 reduces scarring and enhances regeneration at a site of sciatic nerve repair. J Peripher Nerv Syst. 2007;12:269–276. doi: 10.1111/j.1529-8027.2007.00148.x. [DOI] [PubMed] [Google Scholar]
  6. Ayhan S, Markal N, Siemionow K, et al. Effect of subepineurial dehydroepiandrosterone treatment on healing of transected nerves repaired with the epineurial sleeve technique. Microsurg. 2003;23:49–55. doi: 10.1002/micr.10088. [DOI] [PubMed] [Google Scholar]
  7. Carter DA, Lisney SJ. The numbers of unmyelinated and myelinated axons in normal and regenerated rat saphenous nerves. J Neurol Sci. 1987;80:163–171. doi: 10.1016/0022-510x(87)90152-3. [DOI] [PubMed] [Google Scholar]
  8. Cragg BG, Thomas PK. Changes in conduction velocity and fibre size proximal to peripheral nerve lesion. J Physiol. 1961;157:315–327. doi: 10.1113/jphysiol.1961.sp006724. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Davison SP, McCaffrey TV, Porter MN, et al. Improved nerve regeneration with neutralization of transforming growth factor-beta1. Laryngoscope. 1999;109:631–635. doi: 10.1097/00005537-199904000-00021. [DOI] [PubMed] [Google Scholar]
  10. Devor M, Govrin-Lippmann R. Retrograde slowing of conduction in sensory axons central to a sciatic nerve neuroma. Exp Neurol. 1986;92:522–532. doi: 10.1016/0014-4886(86)90294-3. [DOI] [PubMed] [Google Scholar]
  11. Graham WP, III, Pataky PE, Calabretta AM, et al. Enhancement of peripheral nerve regeneration with triamcinolone after neurorrhaphy. Surg Forum. 1973;24:457–459. [PubMed] [Google Scholar]
  12. Graham JB, Neubauer D, Xue Q-S, et al. Chondroitinase applied to peripheral nerve repair averts retrograde axonal regeneration. Exp Neurol. 2007;203:185–195. doi: 10.1016/j.expneurol.2006.08.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Holland GR, Robinson PP. The number and size of axons central and peripheral to inferior alveolar nerve injuries in the cat. J Anat. 1990;173:129–137. [PMC free article] [PubMed] [Google Scholar]
  14. Holland GR, Andrade D, Smith KG, et al. A quantitative morphological comparison of cat lingual nerve repair using epineurial sutures and entubulation. J Dent Res. 1996a;75:942–948. doi: 10.1177/00220345960750031201. [DOI] [PubMed] [Google Scholar]
  15. Holland GR, Robinson PP, Smith KG, et al. A quantitative morphological study of the recovery of cat lingual nerves after transection or crushing. J Anat. 1996b;188:289–297. [PMC free article] [PubMed] [Google Scholar]
  16. Horch KW, Lisney SJ. On the number and nature of regenerating myelinated axons after lesions of cutaneous nerves in the cat. J Physiol. 1981;313:275–286. doi: 10.1113/jphysiol.1981.sp013664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jenq CB, Coggeshall RE. Numbers of regenerating axons in parent and tributary peripheral nerves in the rat. Brain Res. 1985a;326:27–40. doi: 10.1016/0006-8993(85)91381-2. [DOI] [PubMed] [Google Scholar]
  18. Jenq CB, Coggeshall RE. Nerve regeneration through holey silicone tubes. Brain Res. 1985b;361:233–241. doi: 10.1016/0006-8993(85)91294-6. [DOI] [PubMed] [Google Scholar]
  19. Koopmans G, Hasse B, Sinis N. Chapter 19. The role of collagen in peripheral nerve repair. Int Rev Neurobiol. 2009;87:363–379. doi: 10.1016/S0074-7742(09)87019-0. [DOI] [PubMed] [Google Scholar]
  20. Landegren T, Risling M, Brage A, et al. Long-term results of peripheral nerve repair: a comparison of nerve anastomosis with ethyl-cyanoacrylate and epineural sutures. Scand J Plast Reconstr Surg Hand Surg. 2006;40:65–72. doi: 10.1080/02844310500513030. [DOI] [PubMed] [Google Scholar]
  21. Lane JM, Bora FW, Pleasure D. Neuroma scar formation in rats following peripheral nerve transection. J Bone Joint Surg Am. 1978;60:197–203. [PubMed] [Google Scholar]
  22. Martins RS, Siqueira MG, Da Silva CF, et al. Overall assessment of regeneration in peripheral nerve lesion repair using fibrin glue, suture, or a combination of 2 techniques in a rat model. Which is the ideal choice? Surg Neurol. 2005;64(Suppl 1):S10–16. doi: 10.1016/j.surneu.2005.04.022. [DOI] [PubMed] [Google Scholar]
  23. Martins RS, Siqueira MG, da Silva CF, et al. Correlation between parameters of electrophysiological, histomorphometric and sciatic functional index evaluations after rat sciatic nerve repair. Arq Neuropsiquiatr. 2006;64:750–756. doi: 10.1590/s0004-282x2006000500010. [DOI] [PubMed] [Google Scholar]
  24. Mayhew TM. Efficient and unbiased sampling of nerve fibres for estimating fiber number and size. In: Conn PM, editor. Quantitative and Qualitative Microscopy. London: Academic Press; 1990. pp. 172–187. [Google Scholar]
  25. Mayhew TM, Sharma AK. Sampling schemes for estimating nerve fibre size. I. Methods for nerve trunks of mixed fascicularity. J Anat. 1984;139:45–58. [PMC free article] [PubMed] [Google Scholar]
  26. McCallion RL, Ferguson MWJ. Fetal wound healing and the development of antiscarring therapies for adult wound healing. In: Clark RAF, editor. The Molecular and Cell Biology of Wound Repair. 2nd edn. New York: Plenum Press; 1996. pp. 561–599. [Google Scholar]
  27. McCallister WV, Tang P, Trumble TE. Is end-to-side neurorrhaphy effective? A study of axonal sprouting stimulated from intact nerves. J Reconstr Microsurg. 1999;15:597–603. doi: 10.1055/s-2007-1000144. [DOI] [PubMed] [Google Scholar]
  28. Menovsky T, Beek JF. Laser, fibrin glue, or suture repair of peripheral nerves: a comparative functional, histological, and morphometric study in the rat sciatic nerve. J Neurosurg. 2001;95:694–699. doi: 10.3171/jns.2001.95.4.0694. [DOI] [PubMed] [Google Scholar]
  29. Murinson BB, Griffin JW. C-fiber structure varies with location in peripheral nerve. J Neuropathol Exp Neurol. 2004;63:246–254. doi: 10.1093/jnen/63.3.246. [DOI] [PubMed] [Google Scholar]
  30. Murinson BB, Hoffman PN, Banihashemi MR, et al. C-fibre (Remak) bundles contain both isolectin B4-binding and calcitonin gene-related peptide-positive axons. J Comp Neurol. 2005;484:392–402. doi: 10.1002/cne.20506. [DOI] [PubMed] [Google Scholar]
  31. Nachemson AK, Lundborg G, Myrhage R, et al. Nerve regeneration after pharmacological suppression of the scar reaction at the suture site. An experimental study on the effect of estrogen-progesterone, methylprednisolone-acetate and cis-hydroxyproline in rat sciatic nerve. Scand J Plast Reconstr Surg. 1985;19:255–260. doi: 10.3109/02844318509074512. [DOI] [PubMed] [Google Scholar]
  32. Nath RK, Kwon B, Mackinnon SE, et al. Antibody to transforming growth factor beta reduces collagen production in injured peripheral nerve. Plast Reconstr Surg. 1998;102:1100–1106. [PubMed] [Google Scholar]
  33. Ngeow WC. Scar less: a review of methods of scar reduction at sites of peripheral nerve repair. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2010;109:357–366. doi: 10.1016/j.tripleo.2009.06.030. [DOI] [PubMed] [Google Scholar]
  34. Ngeow WC, Atkins S, Morgan CR, et al. A comparison between the effects of three potential scar-reducing agents applied at a site of sciatic nerve repair. Neuroscience. 2011a;181:271–277. doi: 10.1016/j.neuroscience.2011.02.054. [DOI] [PubMed] [Google Scholar]
  35. Ngeow WC, Atkins S, Morgan CR, et al. The effect of mannose-6-phosphate on recovery after sciatic nerve repair. Brain Res. 2011b;1394:40–48. doi: 10.1016/j.brainres.2011.04.021. [DOI] [PubMed] [Google Scholar]
  36. O'Kane S, Ferguson MWJ. Transforming growth factor βs and wound healing. Int J Biochem Cell Biol. 1997;29:63–78. doi: 10.1016/s1357-2725(96)00120-3. [DOI] [PubMed] [Google Scholar]
  37. Ozgenel GY, Filiz G. Effects of human amniotic fluid on peripheral nerve scarring and regeneration in rats. J Neurosurg. 2003;98:371–377. doi: 10.3171/jns.2003.98.2.0371. [DOI] [PubMed] [Google Scholar]
  38. Palatinsky EA, Maier KH, Touhalisky DK, et al. ADCON-T/N reduces in vivo perineural adhesions in a rat sciatic nerve reoperation model. J Hand Surg Br. 1997;22:331–335. doi: 10.1016/s0266-7681(97)80397-x. [DOI] [PubMed] [Google Scholar]
  39. Pellegrino RG, Spencer PS. Schwann cell mitosis in response to regenerating peripheral axons in vivo. Brain Res. 1985;341:16–25. doi: 10.1016/0006-8993(85)91467-2. [DOI] [PubMed] [Google Scholar]
  40. Ridley AJ, Davis JB, Stroobant P, et al. Transforming growth factors beta 1 and beta 2 are mitogens for rat Schwann cells. Z Cell Biol. 1988;84:739–752. doi: 10.1083/jcb.109.6.3419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Robinson PP. The reinnervation of the tongue and salivary glands after lingual nerve injuries in cats. Brain Res. 1989;483:259–271. doi: 10.1016/0006-8993(89)90170-4. [DOI] [PubMed] [Google Scholar]
  42. Siironen J, Collan Y, Röyttä M. Axonal reinnervation does not influence Schwann cell proliferation after rat sciatic nerve transection. Brain Res. 1994;654:303–311. doi: 10.1016/0006-8993(94)90492-8. [DOI] [PubMed] [Google Scholar]
  43. Smith KG, Robinson PP. The reinnervation of the tongue and salivary glands after two methods of lingual nerve repair in the cat. Arch Oral Biol. 1995a;40:373–383. doi: 10.1016/0003-9969(94)00189-i. [DOI] [PubMed] [Google Scholar]
  44. Smith KG, Robinson PP. An experimental study of lingual nerve repair using epineurial suture or entubulation. Br J Oral Maxillofac Surg. 1995b;33:211–219. doi: 10.1016/0266-4356(95)90002-0. [DOI] [PubMed] [Google Scholar]
  45. Sulaiman OA, Gordon T. Effects of short- and long-term Schwann cell denervation on peripheral nerve regeneration, myelination, and size. Glia. 2000;32:234–246. doi: 10.1002/1098-1136(200012)32:3<234::aid-glia40>3.0.co;2-3. [DOI] [PubMed] [Google Scholar]
  46. Sunderland S, Bradley KC. Endoneurial tube shrinkage in the distal segment of a severed nerve. J Comp Neurol. 1959;93:411–420. doi: 10.1002/cne.900930305. [DOI] [PubMed] [Google Scholar]
  47. Vora AR, Loescher AR, Boissonade FM, et al. Ultrastructural characteristics of axons in traumatic neuromas of the human lingual nerve. J Orofacial Pain. 2005;19:22–33. [PubMed] [Google Scholar]
  48. Zou T, Ling C, Xiao Y, et al. Exogenous tissue plasminogen activator enhances peripheral nerve regeneration and functional recovery after injury in mice. J Neuropathol Exp Neurol. 2006;65:78–86. doi: 10.1097/01.jnen.0000195942.25163.f5. [DOI] [PubMed] [Google Scholar]

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