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Keywords:

  • SSeCKS;
  • sciatic nerve;
  • crush;
  • transect;
  • rat

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

SSeCKS (src suppressed C kinase substrate) functions in the control of cell signaling and cytoskeletal arrangement. It is expressed in brain and spinal cord, but little is known about its expression in peripheral nerves. In this study, in rats, real-time polymerase chain reaction and Western blot analysis showed that expression of SSeCKS in crushed sciatic nerve reached its highest level 6 hr after crushing, whereas in a transection model, SSeCKS peaked at 2 days in the proximal stump and 12 hr in the distal stump. Immunohistochemical analysis demonstrated up-regulation of SSeCKS protein surrounding the crush site and in the two stumps of the transected nerve. In addition, SSeCKS colocalized with growth-associated protein 43 and with S100, which also changed with time after injury. These findings support the idea that SSeCKS participates in the adaptive response to peripheral nerve injury and may be associated with regeneration. Anat Rec, 291:527–537, 2008. © 2008 Wiley-Liss, Inc.

Src suppressed C kinase substrate (SSeCKS) is a high molecular mass (290/280 kDa) heat-stable protein that was identified as a protein kinase C (PKC) substrate/PKC-binding protein. In vitro, SSeCKS mRNA and protein levels are down-regulated in transformed cells, suggesting a role in growth regulation (Chapline et al.,1996). In vivo, SSeCKS is expressed by fibroblasts, endothelial cells, and mesangial cells and has mitogenic regulatory activity, indicating a role in tumor suppression. During embryogenesis, SSeCKS is involved in controlling cytoskeletal structure and tissue architecture, forming the migratory process and cell migration (Gelman et al.,2000). The SSeCKS expression pattern is developmentally regulated in numerous cell types and tissues during perinatal growth, indicating a role in development and differentiation processes. In the nervous system of adult rats, SSeCKS is localized to central axonal collaterals of primary sensory neurons in the cerebellum, medulla, and sensory ganglia (including trigeminal ganglia) and in the dorsal horn at all spinal levels. In addition, SSeCKS is localized to C-fibers and is involved in nociception (Siegel et al.,2002). No prior studies have examined the involvement of SSeCKS in peripheral nerve injury; therefore, we examined the expression of SSeCKS in this condition, a complex process that remains poorly understood.

The cellular events that follow crushing or transection of a peripheral nerve have been extensively documented. To achieve successful nerve repair, axons have to re-grow and find their correct target cells. The first step is the removal of myelin debris, a process known as Wallerian degeneration, and is carried out by resident Schwann cells and infiltrating macrophages. The Schwann cells de-differentiate, proliferate, and align within basal lamina tubes (bands of Büngner), providing a guidance substrate for growing axons. The re-grown axons are then myelinated by re-differentiated Schwann cells (Kury et al.,2001).

In this study, we examined temporal changes of SSeCKS expression after crush and transection injury by real-time polymerase chain reaction (PCR) and Western blot analysis. Immunohistochemistry was used to detect the spatial expression of SSeCKS in proximal and distal portions of injured sciatic nerves. Double staining of SSeCKS with S100 and with growth-associated protein 43 (GAP43) was performed to determine its localization and possible significance. SSeCKS was shown to colocalize with the two markers, demonstrating that SSeCKS might correlate with nerve injury and regeneration.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Animals and Tissue Preparation

Adult (200–300 g) male/female Sprague-Dawley rats were obtained from the experimental animal center of Nantong University. Experiments were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Animals were maintained at constant temperature and humidity and kept on a 12-hr day/12-hr night schedule. Rats were anesthetized with pentobarbital (50 mg/kg, i.p.). All procedures were performed on animals in an unconscious state. For animals receiving axotomies, the sciatic nerve was exposed in the mid-gluteal region, crushed unilaterally at the mid-point with a small hemostat for 10 sec then unclamped for 10 sec. This procedure was repeated 3 times. In the transection model, the sciatic nerve was transected at the sciatic notch, and reflected caudally to prevent regeneration. Wounds were sutured and animals were allowed to recover. Water and food were provided ad libitum until animals were killed. The tissues of corresponding regions from three to four animals were pooled. All samples were kept on ice before protein/RNA isolation. For immunohistochemistry, rats were anesthetized and perfused intracardially with 0.9% saline, followed by fixative (4% paraformaldehyde). The sciatic nerves were immediately removed, immersion-fixed overnight at 4°C, in the same fixative, and subsequently processed for sectioning.

RNA Isolation and Reverse Transcription

Total RNA was isolated from 100 mg of sciatic nerve using Trizol. RNA concentration was determined by absorption at 260 nm, and the 260/280 nm absorption ratio of the samples was verified to be >1.9. Reverse transcription (RT) was performed on 5 μg RNA per sample using the ThermoScript RT System (Fermentas). cDNA was diluted 1:1 and 2 μl was used in each 20-μl PCR reaction.

Real-time PCR

Total RNA was extracted by Trizol. PCR primers for SSeCKS, and for β2-microglobulin (β2-M) were designed corresponding to coding regions, as follows: SSeCKS primers, sense 5′-AAGTGCTGGCTTCGGAGAAAG-3′ and antisense 5′-TGACTTCAGGAACTTCAAGGCTC-3′; β2-M primers, sense 5′-GTCTTTCTACATCCTGGCTCACA-3′ and antisense 5′-GACGGTTTTGGGCTCCTTCA-3′. TaqMan probes for SSeCKS, and β2-M were designed corresponding to coding regions, as follows: SSeCKS probe,5′(FAM)-AGCCTGTCCAGT- CTCAGAGCCCTGTG-(TAMRA)3′ and β2-M probe, 5′(FAM)-CACCCACCGA GACCGATGTATATGCTTGC-(TAMRA)3′. The reaction mixes included 1× PCR buffer, 20 mM magnesium chloride, 0.2 mM deoxyNTP, 10 nmol each TaqMan probe with a pair of 10 nmol SSeCKS primers or 10 nmol β2-M primers. Real-time PCR was performed in a Rotor Gene 3000 Detector (Perkin-Elmer/Applied Biosystems, Foster City, CA). The thermal cycling program consisted of 3 min at 94°C, followed by 40 cycles of 20 sec at 94°C and 1 min at 60°C. To account for variability in total RNA input, the expression of SSeCKS was normalized to β2-M.

Western Blot Analysis

Total protein was isolated from approximately 100 mg of sciatic nerve tissue from normal and injured rats at indicated time points. The length of the sciatic nerve was as follows: (1) in the crush model, the whole nerve was 1 cm and the crush site was in the middle; (2) in the transection model, the nerve was 1 cm to the stump; (3) the length of the normal sciatic nerve was 2 cm before the crotch. Samples were mixed with 2 × loading buffer and dithiothreitol (4:5:1), boiled in water for 5 to 10 min, then cooled on ice. Proteins were resolved on 6%/10% sodium dodecyl sulfate-polyacrylamide gel electropheresis gels and transferred to polyvinylidine difluoride (Millipore) membranes. The membranes were blocked with 5% dried skimmed milk in Tris-buffered saline with Tween (TBST). After 2 hr at room temperature, filters were washed three times in TBST and then incubated overnight at 4°C with appropriate antibody: sheep polyclonal anti-SSeCKS (Sigma, 1:2,000), mouse monoclonal anti-GAP43 (Sigma, 1:200), mouse monoclonal anti-S100 (Sigma, 1:200), or mouse monoclonal anti–β-actin (1:2,000). Finally, rabbit anti-sheep IgG, conjugated to horseradish peroxidase (Southern Biotech, 1:2,000), or goat anti-mouse IgG, conjugated to horseradish peroxidase (Pierce, 1:1,000), was added and incubated for an additional 2 hours. Blots were developed using enhanced chemiluminescence (Pierce Chemical Co., Rockford, IL).

Immunohistochemistry

Immunohistochemistry was performed on 14-μm sections of sciatic nerve, 6 hr after crushing, or on transected nerve, 2 days (proximal) and 12 hr (distal) after transection. The standard two-step staining method was adopted. Briefly, sections were immersed in 0.3% (v/v) H2O2/methanol for 30 min at 37°C to inhibit endogenous peroxidase. Sections were washed three times with 0.01 mM phosphate-buffered saline (PBS) and then incubated in 10% normal serum blocking solution (from the same species as the secondary antibody), containing 3% (w/v) bovine serum albumin (BSA) and 0.1% Triton X-100. Sections were then incubated with sheep polyclonal anti-SSeCKS (Sigma, 1:50) in a moist chamber for 6 hr at 37°C. Incubation of primary antibody was omitted from several sections for negative control. The sections were washed three times in PBS and incubated for 2 hr with rabbit anti-sheep IgG, conjugated to horseradish peroxidase (Southern Biotech, 1:200) at 37°C. After washing three times, sections were stained in diaminobenzidine containing 0.5% H2O2 for approximately 3 min, then counterstained with hematoxylin, dehydrated, cleared, and mounted in Permount.

For double immunofluorescent staining, sections were first blocked with 10% normal serum blocking solution (from the same species as the secondary antibody), containing 3% (w/v) BSA and 0.1% Triton X-100 and 0.05% Tween 20, overnight at 4°C. Sections were then incubated with both sheep polyclonal anti-SSeCKS (Sigma, 1:50) and mouse monoclonal anti-GAP43 (Serotec, 1:500) for 48–60 hr at 4°C. Mouse monoclonal anti-S100 (Sigma, 1:100) was added 48 hr after anti-SSeCKS and incubation continued at 4°C overnight. After washing for 10 min in PBS three times, the secondary antibodies (fluorescein isothiocyanate-rabbit-anti-sheep, Santa Cruz, 1:50; tetramethyl rhodamine isothiocyanate-donkey-anti-mouse, SBA, 1:50/1:100) were added and incubated in the dark for 2–3 hr at 4°C. Fluorescence was detected by a Leica fluorescence microscope (Germany).

Statistical Analysis

For all experiments, relative expression values were calculated using Stata 7.0 statistics software. Data are expressed as mean ± SD. One-way analysis of variance, followed by Tukey's post hoc multiple comparison tests, was used for statistical analysis. A difference was accepted as significant if P < 0.05. Each experiment consisted of at least three independent samples per protocol.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Expression of SSeCKS in Injured Rat Sciatic Nerve

To reveal any change in the expression of SSeCKS in postinjury rat sciatic nerve, we chose real-time PCR to analyze mRNA levels. Our time points included 6 hr, 12 hr, 1 days, 2 days, 1 week, 2 weeks, and 4 weeks after injury. Sciatic nerves from normal rats were used as a control. Total RNA samples were freshly isolated from nerves of the two different models. As shown in Figure 1, the relative quantity of SSeCKS changed with time after injury. In the crush model, it was significantly higher at 6hr after injury. In the transection model, the highest level was at 2 days in the proximal stump; whereas in the distal stump, its peak was at 12 hr after injury.

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Figure 1. Expression of src suppressed C kinase substrate (SSeCKS) after sciatic nerve injury by real-time polymerase chain reaction analysis. High levels of expression of SSeCKS were observed at different times in the two models. The ratios acquired from the quantities of SSeCKS to β2-M were defined as the relative SSeCKS mRNA levels. #P < 0.01.

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To corroborate our findings, Western blot analysis was used to determine the abundance of the protein. In crushed sciatic nerve, it reached its highest level at6 hr post injury, and then decreased gradually (Fig. 2A). In the proximal stump of the transected nerve, SSeCKS was almost undetectable at 6 hr after the operation, and gradually increased and reached its peak at 2 days (Fig. 2B). Thereafter, the level decreased. In the distal stump (Fig. 2C), SSeCKS peaked at a relative early stage, 12 hr after transection, and then levels declined. These results were in good agreement with those from real-time PCR analysis, and together indicated that the expression of SSeCKS changed with time after sciatic nerve injury, and in different models, the pattern of expression was different.

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Figure 2. Immunoblot analysis of src suppressed C kinase substrate (SSeCKS) expression in rat sciatic nerve. Western blot analysis of tissue lysates (45 of μg protein loaded/lane), prepared from sciatic nerve collected from uninjured rats and animals after axotomy, were probed with antibodies specific for SSeCKS. The protein of the expected 290/280-kD size is detectable in nerve lysates and shows variable alterations in its level of expression after injury. Arrows at left of each panel indicate its size (in kD). β-actin was used as a control. A: Crush model. B: Proximal stump of the transected nerve. C: Distal stump of the transected nerve. D: Each lane was scanned by laser densitometry and the absorbance of the appropriate bands was calculated. The relative level of SSeCKS was from its expression normalized to actin at each time point. Data are representative of the results of three independent experiments and are presented as mean ± SD (standard deviation). #P < 0.01.

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Immunohistochemical Localization of SSeCKS in Injured Rat Sciatic Nerve

To observe the localization of SSeCKS in the sciatic nerve, we used immunohistochemistry. In the crush model, no immunoreactivity was found at the crush site, immediately after crushing. Six hours after crush injury, striking staining of SSeCKS was found on both proximal and distal sides of the crush site (Fig. 3A:a,b). While in the contralateral side, the staining of SSeCKS was weaker and its distribution was relatively uniform (Fig.3A:c). After 2 weeks, the sciatic nerve was undergoing repair and had become thicker and the crush site was not apparent. At this time, expression was uniform on both sides of the crush site and the corresponding contralateral side (Fig. 3A:d–f). The expression level 2 weeks after crush injury was lower than that after 6 hr.

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Figure 3. Immunohistochemical localization of src suppressed C kinase substrate (SSeCKS) in rat sciatic nerve. A: Crush model: a and b are the proximal and distal sides of the crush site from the nerve 6 hr after crush injury, d and e are the two sides from the nerve 2 w after crush injury; c and f are the corresponding contralateral sides; g and h, negative control = primary antibody (−); i, normal nerve. B,C: Transection model: B, proximal stump; C, distal stump; a, time point of high expression level; d, time point of low expression level; b and e were the corresponding contralateral sides; c, negative control; f, normal nerve. We used time point of high SSeCKS expression as a negative control. In nerves 6 hr after crushing, and 2 days (proximal stump) and 12 hr (distal stump) after transection, strong SSeCKS staining was observed in the two sides of the crush site and in the stumps. Scale bar = 50 μm.

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In the transection model, we investigated the expression pattern of SSeCKS in the proximal and distal stumps (Fig. 3B,C). Intensive staining of SSeCKS was observed in the proximal stump of the sciatic nerve 2 days after transection (Fig. 3B:a), and it was in a cluster distribution. In the same position of the nerve 6 hr after transection (Fig. 3B:d), much weaker signals were detected. In the contralateral side of both models, and in normal sciatic nerve, the level of SSeCKS was low and distribution was relatively uniform (Fig. 3B:b,e,f). The expression pattern of SSeCKS in the distal stump of the sciatic nerve, 12 hr and 4 weeks after transection, was similar to that of the proximal stump.

Double Immunofluorescent Staining for SSeCKS and S100 or GAP43 in Injured Sciatic Nerve

To further investigate the localization of SSeCKS in crushed or transected sciatic nerve and to investigate possible function, we carried out double immunofluorescent staining to detect whether SSeCKS colocalizes with S100 or with GAP43. Little colocalization between SSeCKS and S100, a marker for Schwann cells, was found in longitudinal sections of sciatic nerve 6 hr after crush or from cross-sections of sciatic nerve 12 hr (distal stump) after transection (Figs. 4A:g,h; Fig. 5f). But in the proximal stump of the nerve 2 days after transection, striking colocalization was detected in the cross-sections (Fig. 5c). Next, we labeled GAP43, a growth-associated protein, in the injured sciatic nerve. We found SSeCKS prominently colocalized with GAP43, both in crushed and transected nerve (Figs. 4B, 6). In the sections of all corresponding contralateral nerves, no colocalization was observed in either model. We, therefore, only show the contralateral side of the crush model (Fig. 4c,f,i) as a control.

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Figure 4. Longitudinal sections of sciatic nerve 6 hr after crush injury stained for src suppressed C kinase substrate (SSeCKS) and S100/growth-associated protein 43 (GAP43). Significant colocalization between SSeCKS and GAP43 was detected in proximal and distal sides of the crush site (B: a, b, d, e, g, h), while in the corresponding contralateral side, no colocalization existed (B: c, f, i). For S100, few positively stained cells expressed SSeCKS (A). Green, SSeCKS; red, S100/GAP43. Scale bar = 50 μm.

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Figure 5. Proximal stump 2 days after transection and distal stump 12 hr after transection stained for src suppressed C kinase substrate (SSeCKS) and S100. a–f: Striking colocalization of SSeCKS with S100 was observed in cross-sections of proximal stump (arrows in a–c) while in the distal stump, few cells that stained positively for S100 expressed SSeCKS (d–f). Green, SSeCKS; red, S100. Scale bar = 20 μm.

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Figure 6. Proximal stump 2 days after transection and distal stump 12 hr after transection stained for src suppressed C kinase substrate (SSeCKS) and growth-associated protein 43 (GAP43). a–c: Striking colocalization of SSeCKS with GAP43 was observed in longitudinal sections of the proximal stump (arrows). d–f: In the distal stump, partial colocalization of SSeCKS with GAP43 was also found (arrows). Green, SSeCKS; red, GAP43. Scale bar = 50 μm.

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GAP43 and S100 Also Changed in Injured Rat Sciatic Nerve

Because the expression of SSeCKS changed after injury and we found it colocalized with GAP43/S100 at different time points in different models, we investigated whether GAP43 and/or S100 were also up-regulated at the selected time points. Therefore, Western blot analysis was performed to observe the expression pattern of the two molecules. The results showed that, in the crush model, the level of GAP43 was high 12 hr after injury, and in later stages the protein also had a high level. In the transection model, levels peaked at 12 hr in the proximal stump, while in the distal stump, the high level was at 2 days and this level was maintained up to 2 weeks after injury. The highest level of S100 was observed at 2 weeks in the crush model. In the proximal stump, the protein was relatively high 2 days after injury and persisted to the end of our experiment. In the distal stump, the highest level was at 4 weeks (Fig. 7).

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Figure 7. Immunoblot analysis of growth-associated protein 43 (GAP43) and S100 expression in sciatic nerve. Western blot of tissue lysates (45 μg of protein loaded/lane), prepared from sciatic nerve collected from uninjured rats and animals after axotomy, were probed with antibodies specific for GAP43 and S100. The proteins of the expected size are detectable in nerve lysates and show variable alterations in levels of expression after injury. β-actin was used as a control. A: Crush model. B: Proximal stump of the transected nerve. C: Distal stump of the transected nerve. D,E: Each lane was scanned by laser densitometry, and the absorbance of the appropriate bands was calculated. The relative level of GAP43(D)/S100(E) was from its expression normalized to actin at each time point. Data are representative of the results of three independent experiments and presented as mean ± SD (standard deviation). #P < 0.01.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

In the present study, we characterized the alterations of SSeCKS expression at gene and protein levels in two different injury models of the sciatic nerve, and also described its localization. The main findings can be listed as follows: (1) High SSeCKS levels are observed soon after injury, contrasting with relatively low SSeCKS levels in normal nerves or in nerves several weeks after injury. (2) Differences in the time of SSeCKS expression in the two models: in the crush model, the highest SSeCKS level was at 6 hr after injury; while in the transection model, SSeCKS levels peaked at 2 days in the proximal stump and at 12 hr in the distal stump. (3) SSeCKS colocalized with S100 and GAP43. (4) Expression of GAP43 and S100 also changed with time after nerve injury.

High SSeCKS levels were observed in sciatic nerves within a few hours after crushing, whereas in the transection model, the highest levels were at 12 hr in the distal stump and 2 days in the proximal stump. The increased SSeCKS protein levels during the first step of peripheral nerve regeneration suggest time-restricted synthesis of SSeCKS within the injured nerve. Increase in SSeCKS expression emerges earlier in crushed nerve compared with transected nerve, which may be because the axolemma structure is relatively complete in the crush model, while in the transection model, the structure of the nerve is destroyed. We found that SSeCKS peaked earlier in the distal transected stump compared with the proximal one. This finding may be because this scaffolding protein participates in retrograde transport, or in both anterograde and retrograde transport. It is also known that the axonal transport machinery conveys not only structural components and organelles, but also molecules used to transmit information about injury events. Indeed, SSeCKS was initially identified as a substrate of PKC, which plays an important role in signal transmission. Hence, we presume that SSeCKS may be involved in this process, but further studies are required to verify this view.

After nerve injury, damaged nerve tissue must be eliminated. In particular, myelin debris, which inhibits axon growth, has to be removed. Active Schwann cell division occurs in the distal stump after nerve injury, where Schwann cells act as phagocytes to digest axons and their myelin sheaths (Nishio et al.,2002). At the same time, in response to nerve injury, Schwann cells divide and form long chains of cells, known as bands of Büngner, which provide a substrate for axonal regeneration. It is well known that denervated Schwann cells increase production of several neurotrophic molecules, including nerve growth factor (Heumann et al.,1987) and brain-derived neurotrophic factor (Meyer et al.,1992). Axon–Schwann cell interaction, mediated by neurotrophic factors, may play a pivotal role in peripheral nerve regeneration (Liu et al.,1995). Increased production of trophic factors by Schwann cells may substitute for the usual target-derived trophic factors. Using anti–S-100 antibody for Schwann cell identification, we found that few Schwann cells showed SSeCKS immunostaining within the first few hours after injury, but strong SSeCKS staining in Schwann cells was seen in cross-sections of the proximal stump 2 days after transection. This finding is consistent with the early stage of sciatic nerve injury, when most Schwann cells undergo apoptosis and are not in their activated state. However, some time after injury, Schwann cells start to divide, proliferate, and play related biologic activity. The strong SSeCKS immunostaining observed in Schwann cells at this time indicated that SSeCKS might contribute to the mechanism of these complicated changes that are induced by different signals.

Normally, successful axonal regeneration is accompanied by the appearance of numerous, functionally diverse families of molecules that regulate surface-cytoskeletelal interaction (Makwana and Raivich,2005). Zheng and others suggested that intra-axonal translation of cytoskeletal components was required for sustaining growth cones in regenerating axons (Zheng et al.,2001). GAP43 is a membrane- and cytoskeletal-associated phosphoprotein (Benowitz and Routtenberg,1987; Skene,1989; Coggins and Zwiers,1991) that is expressed at high levels in neurons during development and is concentrated in axonal growth cones (Biffo et al.,1990; Fitzgerald et al.,1991). After neural injury in the adult, however, GAP43 is re-expressed and is rapidly transported along the axons of those neurons where there is successful regeneration (Bisby,1988; Tetzlaff et al.,1989; Van der Zee et al.,1989; Woolf et al.,1990). The correlation between the presence of GAP43 and the growth state of neurons has led to the use of GAP43 as a marker for axonal growth, and evidence from in vitro studies suggest that GAP43 is directly involved in such growth (Zuber et al.,1989; Yanker et al.,1990; Shea et al.,1991; Jap Tjoen San et al.,1992; Aigner and Caroni,1993; Widemer and Caroni,1993). Thus, the colocalization of SSeCKS with GAP43 was performed to determine whether SSeCKS associated with regeneration. Our results showed that the two molecules colocalized in crushed nerve and in the two transected stumps. This finding indicates that SSeCKS may have a function in the regeneration process of the injured nerve. For example, it may participate in the transmission of some signals, effecting the reformation of the cystoskeleton and so on. The observation raises the potential role of SSeCKS in regenerative tissue. However, the demonstration of a direct role of SSeCKS in nerve regeneration requires further investigation.

Taken together, these findings indicate that SSeCKS participates in the adaptive response to peripheral nerve injury and may play a role in nerve regeneration. However, its exact implication has yet to be defined. Maybe, it participates in part by means of partial replication of the molecular and cellular mechanisms that operate during development. A better understanding of its contribution may generate new strategies for enhancing peripheral nerve regeneration.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED