Single Local Application of TGF-β Promotes a Proregenerative State Throughout a Chronically Injured Nerve

Single Local Application of TGF-β Promotes a Proregenerative State Throughout a Chronically... Abstract BACKGROUND The lack of nerve regeneration and functional recovery occurs frequently when injuries involve large nerve trunks because insufficient mature axons reach their targets in the distal stump and because of the loss of neurotrophic support, primarily from Schwann cells (SCs). OBJECTIVE To investigate whether a single application of transforming growth factor-beta (TGF-β) plus forskolin or forskolin alone can promote and support axonal regeneration through the distal nerve stump. METHODS Using a delayed repair rat model of nerve injury, we transected the tibial nerve. After 8 wk, end-to-end repair was done and the repair site was treated with saline, forskolin, or TGF- β plus forskolin. After 6 wk, nerve sections consisting of the proximal stump, distal to the site of repair, and the most distal part of the nerve stump were removed for nerve histology, axon counts, and immunohistochemistry for activated SCs (S100), macrophages (CD68), cell proliferation (Ki67), p75NGFR, and apoptosis (activated caspase-3). RESULTS TGF-β plus forskolin significantly increased the numbers of axons regenerated distal to the repair site and the most distal nerve sections. Both treatments significantly increased the numbers of axons regenerated in the most distal nerve sections compared to saline treated. Both treatments exhibited extended expression of regeneration-associated marker proteins. CONCLUSION TGF-β plus forskolin treatment of chronically injured nerve improved axonal regeneration and increased expression of regeneration-associated proteins beyond the repair site. This suggests that a single application at the site of repair has mitogenic effects that extended distally and may potentially overcome the decrease in regenerated axon over long distance. Chronic nerve injuries, Nerve regeneration, Regeneration-associated genes/proteins, Transforming growth factor ABBREVIATIONS ABBREVIATIONS H&E hematoxylin and eosin RAGs regeneration-associated genes RT room temperature SCs Schwann cells TIB tibial TGF-β transforming growth factor-beta Injuries to the sciatic nerve and other large nerve trunks such as the brachial plexus often lead to suboptimal functional recovery.1-3 The capacity of injured nerves to regenerate is due mostly to the growth-supportive Schwann cells (SCs) in the distal stumps of the injured nerves.4,5 Animal studies have shown that suboptimal functional recovery is attributable to insufficient regenerating axons reaching the end target, and this deficiency can be attributed to the loss of the growth-supportive milieu provided by the SCs in the distal stump of injured nerves.6,7 After acute injury, SCs express regeneration-associated genes (RAGs) such as, neurotrophins and their receptors,8 adhesion molecules,9 and cytokines such as transforming growth factor-beta (TGF-β),10 that support growth of the regeneration units through the distal nerve stumps and reestablishment of neuromuscular junctions.11,12 SCs also proliferate in the distal nerve stumps and form cellular channels (bands of Bungner) through which regenerating axons traverse in the distal nerve stumps to get to the denervated muscle target.13 A second, proliferative phase of SCs seems to be initiated by their interaction with the growth cones.14 While the second phase of SC proliferation seems to be mediated by axonal contact and is not synchronous in all SCs of the distal nerve stump, it is unclear whether SC expression of RAGs in the distal nerve stumps is also dependent on axonal contact or is synchronous across the length of the distal nerve stumps once the growth cones cross the suture site. This is a critical question since it has potential effect on the efficacy of any therapeutic strategy to enhance axonal regeneration through the distal nerve stumps. Previously, we had shown that in vivo treatment of chronically denervated SCs with TGF-β plus forskolin “reversed the deleterious effect of chronic SC denervation” and improved their capacity to support axonal regeneration.15 One of the unanswered questions in the mitogen treatment of nerve repair is whether a treatment directed solely at the nerve repair site is sufficient to produce the mitogenic effects throughout the distal nerve stump, or if the positive effects would be lost as a function of distance from the repair and treatment site. Using our delayed repair rat model, we extended our examination both proximally and distally to the repair site by 15 to 25 mm and compared the responses of SCs to forskolin alone or with TGF-β treatment. We assessed SC activation, SC expression of regeneration-associated marker proteins and the numbers of regenerated axons along the length of the distal nerve stumps. METHODS We utilized our rat model of tibial (TIB) nerve injury and delayed repair as previously described15 (Figure 1). The Ochsner Institutional Animal Care and Use Committee approved the protocol. Eighteen adult female Sprague--Dawly rats (Harlan) weighing about 200 g were used. In the first surgery, we transected the TIB nerve, reflecting the proximal nerve stump backward and suturing to the femoris muscle to prevent regeneration. After 8 wk, the TIB nerve in all animals was re-exposed and repaired by direct coaptation with 6-0 Prolene sutures. Animals were divided into 3 treatment groups (n = 6 per group): (i) Saline only; (ii) Forskolin (2 ng/mL); and (iii) Forskolin plus TGF-β (0.5 μM). A 10 mm square piece of gelfoam was placed in the treatment solution to soak for 10 min and applied directly by wrapping the gelfoam around the TIB nerve repair site. Regeneration of TIB nerves was allowed for 6 wk. The animals were sacrificed and nerve sections were taken from (a) proximal stump (A section); (b) suture site and immediate distal stump (B section); and (c) most distal part of the distal nerve stumps (C section) for analysis (Figure 1). FIGURE 1. View largeDownload slide The TIB nerve was cut and reflected into muscle to prevent regeneration and then repaired, plus or minus treatment. Six weeks later, 2 10-mm sections and 1 15-mm section of the TIB nerve were harvested with the A section proximal, the B section containing the repair, and C section distal to the repair. The 15 to 25 mm analysis point of the most distal C section is indicated. FIGURE 1. View largeDownload slide The TIB nerve was cut and reflected into muscle to prevent regeneration and then repaired, plus or minus treatment. Six weeks later, 2 10-mm sections and 1 15-mm section of the TIB nerve were harvested with the A section proximal, the B section containing the repair, and C section distal to the repair. The 15 to 25 mm analysis point of the most distal C section is indicated. Nerve Histology and Axon Counts Nerve tissues were fixed in 4% paraformaldehyde and 5-μm transverse paraffin sections were used for hematoxylin and eosin (H&E) to stain for the myelin structures and nuclei as described.15 A minimum of 3 separate nerve sections A, B, and C were observed under a microscope. Photomicrographs were analyzed with Image J16 and axon counts were determined, and analyzed for statistical significance using the Kruskal–Wallis test. Immunohistochemistry Immunocytochemical analyses were carried out on 5-μm paraffin nerve sections as previously described.15 Primary antibodies included CD68 (1:200), Ki67 (1:500), S100 (1:1000), p75NGFR (1:300), and activated caspase-3 (1:200) and used overnight at 4°C. Fluorescent anti-rabbit secondary antibody (1:1000) was added and incubated at room temperature (RT) for 30 min, and mounted in media containing DAPI. For double-labeling studies, nerve sections were incubated in blocking buffer (Dako, Agilent, Santa Clara, California) at RT for 30 min then overnight at 4°C with a combination of anti-S100/p75NGFR or anti-CD68/caspase-3 to identify activated SCs and apoptotic macrophages, respectively. After 3 5-min washes, sections were incubated with Alexa Fluor 488/568 (Thermo Fischer Scientific, Waltham, Massachusetts) anti-rabbit/anti-mouse secondary antibody (1:500) combination for 30 min at RT and mounted. Sections without the primary antibody incubation were the negative control. Fluorescent-stained sections were viewed on a Zeiss Axiovert 200 M (Carl Zeiss, Oberkochen Germany) inverted microscope with fluorescence/phase imaging. RESULTS Axon Regeneration at Sections A, B, and C H&E stained transverse nerve sections were examined by light microscopy. No major anatomical and histological differences were observed among sections A, B, or C, although the saline-treated distal C section appeared to have an overall less organized array of axonal bundles (Figure 2A). The spatial distribution of the treatment effect on regenerated axon numbers at sections A, B, and C were counted both manually and by Image J16 software (Figure 2B). The saline control consistently had fewer regenerated axons in all nerve sections while both treatment groups showed an increase in the number of axons. However, only the TGF-β plus forskolin treated nerves showed statistically significant increases over the saline control, at least in sections A and C (Figure 3). The effect of TGF-β and forskolin was additive with significantly more regenerated axons in both the A and C sections. Interestingly the combination of TGF-β and forskolin was significant when compared to the forskolin only treatment, especially in the B section. Furthermore, nerves that were exposed to TGF-β appeared to have better organized axonal regeneration. FIGURE 2. View largeDownload slide A, H&E stained transverse TIB nerve sections showed a more organized array of axonal bundles with treatments. Columns are, left to right: (i) saline control, (ii) forskolin only, (iii) forskolin plus TGF-β. A, B, C refers to nerve segment region where A is proximal stump, B is suture site and immediate distal stump, and C is most distal part of distal nerve stump; B, A grey scale representation of a H&E stained transverse TIB nerve section (i) and an binary (black & white) image depicting particle counting of axons in Image J16 (ii). FIGURE 2. View largeDownload slide A, H&E stained transverse TIB nerve sections showed a more organized array of axonal bundles with treatments. Columns are, left to right: (i) saline control, (ii) forskolin only, (iii) forskolin plus TGF-β. A, B, C refers to nerve segment region where A is proximal stump, B is suture site and immediate distal stump, and C is most distal part of distal nerve stump; B, A grey scale representation of a H&E stained transverse TIB nerve section (i) and an binary (black & white) image depicting particle counting of axons in Image J16 (ii). FIGURE 3. View largeDownload slide Histogram of axon nerve counts from each of 3 nerve segment regions surrounding and including the repair site. TGF-β plus forskolin treatment leads to a significant increase in the number of regenerated axons in segments B and C as compared to saline control. FIGURE 3. View largeDownload slide Histogram of axon nerve counts from each of 3 nerve segment regions surrounding and including the repair site. TGF-β plus forskolin treatment leads to a significant increase in the number of regenerated axons in segments B and C as compared to saline control. Immunohistochemistry of RAGs Our previous work has shown that under the same treatment regimen,15 the region near the suture site of the saline control and treated groups exhibited a striking dichotomy in the expression of SC RAGs and the presence or absence of activated macrophages. We have extended those findings to regions both proximal and distal to the repair and treatment site. Apoptosis All sections, A, B, and C, from the treated groups exhibited many cells undergoing active apoptosis as indicated by positive staining for activated caspase-3 (Figure 4A). No particular pattern was observed as apoptotic cells were detected throughout the nerve sections. Some nuclear localization of activated caspase-3 were detected in the nerve sections of the treated groups. Interestingly, all SCs, even those not undergoing apoptosis, also contained reactive material localized to punctate bodies throughout the cell body (Figure 4B). No activated caspase-3 was detected in any of the sections in the saline control (data not shown). FIGURE 4. View largeDownload slide Immunohistochemistry showed increased levels of activated caspase-3 (green) and DAPI (blue) in A, TGF-β plus forskolin treated nerve segments B; and B, TGF-β plus forskolin treated nerve segment C. (100x mag). FIGURE 4. View largeDownload slide Immunohistochemistry showed increased levels of activated caspase-3 (green) and DAPI (blue) in A, TGF-β plus forskolin treated nerve segments B; and B, TGF-β plus forskolin treated nerve segment C. (100x mag). Activated Macrophage All nerve sections of the treated groups showed immunoreactivities to CD68, indicating the presence of activated macrophages throughout the nerve bundle (Figure 5A). Only a few scattered staining for CD68 were found in each of the saline-control sections (data not shown). Furthermore, double staining for CD68 + cells and apoptotic cells found cells with positive staining for activated caspase-3 in the cytoplasm as well as punctate nuclear staining in close proximity to activated CD68+ macrophages (Figure 5B), suggesting that these macrophages may be activated. FIGURE 5. View largeDownload slide Immunofluorescent double staining of CD68+ macrophages (red) and caspase-3 (green) in A, TGF-β plus forskolin treated nerve segments B, and B, TGF-β plus forskolin treated nerve segments C. Activated macrophages were detected (arrow) as well as CD68+ macrophages colocalized in close proximity to caspase-3 (*) positive cells (400× mag). FIGURE 5. View largeDownload slide Immunofluorescent double staining of CD68+ macrophages (red) and caspase-3 (green) in A, TGF-β plus forskolin treated nerve segments B, and B, TGF-β plus forskolin treated nerve segments C. Activated macrophages were detected (arrow) as well as CD68+ macrophages colocalized in close proximity to caspase-3 (*) positive cells (400× mag). Cell Proliferation Immunostaining for Ki67 was used to ascertain for active cell proliferation. Similar to the immunostaining pattern of CD68, staining for Ki67 was seen in all sections of the treated groups suggesting active cell proliferation (Figure 6). In the saline control, Ki67 was not detected in either the proximal A section or the repair site B section, while sporadic Ki67 staining was detected in the distal C section (data not shown). FIGURE 6. View largeDownload slide Immunohistochemical analysis of Ki-67 (green) and DAPI (blue) in TGF-β plus forskolin nerve section B. A, 100× mag; and B, 400× mag. FIGURE 6. View largeDownload slide Immunohistochemical analysis of Ki-67 (green) and DAPI (blue) in TGF-β plus forskolin nerve section B. A, 100× mag; and B, 400× mag. Activated SCs To determine the presence of activated SCs in nerve sections, expression of S100 was used. As shown, all SCs in the B sections of the mitogen-treated nerves were positive for S100 (Figure 7). Furthermore, staining for p75NGFR was also found colocalized to S100-positive SC (Figure 7B). In contrast, no S100 staining was detected in the A or B sections of the saline control. Only the distal C nerve section of the saline control contained S100 expressing SCs, primarily near the perineurium (data not shown). FIGURE 7. View largeDownload slide Immunofluorescent double staining of S-100 (red) and NGFR p75 (green) proteins, markers of activated Schwann cells (arrow) in A, TGF-β plus forskolin treated nerve segment B, and B, TGF-β plus forskolin treated nerve segment C. (400× mag). FIGURE 7. View largeDownload slide Immunofluorescent double staining of S-100 (red) and NGFR p75 (green) proteins, markers of activated Schwann cells (arrow) in A, TGF-β plus forskolin treated nerve segment B, and B, TGF-β plus forskolin treated nerve segment C. (400× mag). Relative Abundance of Expressed Marker Proteins To determine the relative abundance of the various marker proteins in each of the nerve segments, positive cells were counted manually and normalized to 100 nuclei, as detected with DAPI (Table). There was a striking dichotomy between the saline control and the 2 treatments. The saline control exhibited little to no positive staining for the various marker proteins, whereas both treatments exhibited numerous positive cells for the various marker proteins. Generally, the forskolin only treatment exhibited the greatest number of positive cells, irrespective of the marker protein examined. For example, forskolin treatment resulted in the expression of S100 protein in all SCs in all segments, whereas the TGF-β plus forskolin nerves showed some SCs with detectable S100 protein in the proximal (A section) and the most distal (C section) segments. Only the TGF-β plus forskolin treated site of nerve injury section B showed extensive S100 staining of SCs. Interestingly, the most distal segment C of the saline control exhibited some sparse S100-positive SCs located near the perineurium. TABLE. Positive Cell Counts of the Various Marker Proteins (Caspase-3, CD68, Ki67, S100) by Treatment and Nerve Section. Values Represent the Numbers of DAPI-stained Nuclei per mm2 From 3 Areas per Cross Section and the Numbers of Positive Cell Counts for the Marker Proteins per 100 Nuclei Marker Proteins Treatment # DAPI-labeled nuclei per mm2 Positive cell count per 100 nuclei A B C A B C Caspase-3 Saline control 5230 4409 2670 0 0 0 Forskolin only 5181 5512 3982 5.7 4.5 8.8 TGF-β plus forskolin 1946 1789 2102 5.6 2.9 3.2 CD68 Saline control 4861 3978 3500 <1 < 1 <1 Forskolin only 6482 7235 13 036 5.6 9.8 8.8 TGF-β plus forskolin 16 398 12 040 15 000 5.4 7.7 4.6 Ki67 Saline control 6467 5475 4578 <1 <1 <1 Forskolin only 9271 5952 5861 4.0 5.3 8.8 TGF-β plus forskolin 30 373 29 904 30 373 6.0 7.7 6.7 S100 Saline control 4142 7036 3433 0 0 10.5 Forskolin only 7036 7994 2880 100 100 100 TGF-β plus forskolin 5946 8458 12 680 67 100 60 Marker Proteins Treatment # DAPI-labeled nuclei per mm2 Positive cell count per 100 nuclei A B C A B C Caspase-3 Saline control 5230 4409 2670 0 0 0 Forskolin only 5181 5512 3982 5.7 4.5 8.8 TGF-β plus forskolin 1946 1789 2102 5.6 2.9 3.2 CD68 Saline control 4861 3978 3500 <1 < 1 <1 Forskolin only 6482 7235 13 036 5.6 9.8 8.8 TGF-β plus forskolin 16 398 12 040 15 000 5.4 7.7 4.6 Ki67 Saline control 6467 5475 4578 <1 <1 <1 Forskolin only 9271 5952 5861 4.0 5.3 8.8 TGF-β plus forskolin 30 373 29 904 30 373 6.0 7.7 6.7 S100 Saline control 4142 7036 3433 0 0 10.5 Forskolin only 7036 7994 2880 100 100 100 TGF-β plus forskolin 5946 8458 12 680 67 100 60 View Large TABLE. Positive Cell Counts of the Various Marker Proteins (Caspase-3, CD68, Ki67, S100) by Treatment and Nerve Section. Values Represent the Numbers of DAPI-stained Nuclei per mm2 From 3 Areas per Cross Section and the Numbers of Positive Cell Counts for the Marker Proteins per 100 Nuclei Marker Proteins Treatment # DAPI-labeled nuclei per mm2 Positive cell count per 100 nuclei A B C A B C Caspase-3 Saline control 5230 4409 2670 0 0 0 Forskolin only 5181 5512 3982 5.7 4.5 8.8 TGF-β plus forskolin 1946 1789 2102 5.6 2.9 3.2 CD68 Saline control 4861 3978 3500 <1 < 1 <1 Forskolin only 6482 7235 13 036 5.6 9.8 8.8 TGF-β plus forskolin 16 398 12 040 15 000 5.4 7.7 4.6 Ki67 Saline control 6467 5475 4578 <1 <1 <1 Forskolin only 9271 5952 5861 4.0 5.3 8.8 TGF-β plus forskolin 30 373 29 904 30 373 6.0 7.7 6.7 S100 Saline control 4142 7036 3433 0 0 10.5 Forskolin only 7036 7994 2880 100 100 100 TGF-β plus forskolin 5946 8458 12 680 67 100 60 Marker Proteins Treatment # DAPI-labeled nuclei per mm2 Positive cell count per 100 nuclei A B C A B C Caspase-3 Saline control 5230 4409 2670 0 0 0 Forskolin only 5181 5512 3982 5.7 4.5 8.8 TGF-β plus forskolin 1946 1789 2102 5.6 2.9 3.2 CD68 Saline control 4861 3978 3500 <1 < 1 <1 Forskolin only 6482 7235 13 036 5.6 9.8 8.8 TGF-β plus forskolin 16 398 12 040 15 000 5.4 7.7 4.6 Ki67 Saline control 6467 5475 4578 <1 <1 <1 Forskolin only 9271 5952 5861 4.0 5.3 8.8 TGF-β plus forskolin 30 373 29 904 30 373 6.0 7.7 6.7 S100 Saline control 4142 7036 3433 0 0 10.5 Forskolin only 7036 7994 2880 100 100 100 TGF-β plus forskolin 5946 8458 12 680 67 100 60 View Large DISCUSSION One of the unanswered questions in the mitogen treatment of nerve repair is whether a treatment solely at the nerve repair site could overcome the effects of chronic denervation throughout the distal nerve stump, or the positive effects would be lost as a function of distance from the repair and treatment site. In this study, we show that the positive mitogenic effects of forskolin or TGF-β plus forskolin treatment from the application point at the repair site extend both proximally as well as distally to enhance nerve regeneration. Effects were noted up to 25 mm from the repair site, the furthest distance away that was tested (Table). Mitogenic treatment at the time of delayed repair enhances SC growth-supportive phenotype and improved axonal regeneration at the site of repair.15,17 Axonal Regeneration after Chronic Axotomy and Denervation Prior works by many have firmly established that in the rat TIB nerve regeneration model, 8 wk of axotomy and denervation would result in at least a 50% reduction in the number of regenerating axons compared to an immediate repair.5 Although we did not conduct an immediate repair in this study, treatment with TGF-β plus forskolin led to a near doubling of regenerated axons distal to the repair site when compared to saline alone (1288 ± 151 vs 2108 ± 200, P = .006). This level of increase would suggest that these mitogens had the effect of returning chronic axotomized and denervated nerves back to the state comparable to immediate repair. The number of regenerating axons near the repair site as well as up to 25 mm proximal and distal to the repair site was also higher than the saline control. The TGF-β plus forskolin treated nerves had significantly more regenerated axons, but did not double them (Table, Figure 3). Forskolin also led to an increase in regenerated axons in the proximal region compared to the saline control, but the increase did not reach statistical significance. The modulating effect of TGF-β would appear to enable the survival of greater numbers of axons and/or axonal sprouts. This is borne out by comparisons of axonal numbers among the 3 sections, which reveal that the B sections, nerve repair site and the regions immediately surrounding it, exhibited fewer axons than either the proximal A region or the distal C region in the 2 treatment groups (Table, Figure 3). The lower number of axon counts in the repair site nerve segment B may be due to staggered axonal regeneration across the suture site and pruning that occur at the injury site. Although the difference between the forskolin plus TGF-β and saline control did not reach significance level, it does show that the treatment has some positive effect on increasing axon counts in the B sections. Hence, treatment with forskolin plus TGF-β seem to promote axon regeneration across the injured and sutured site, perhaps by augmenting axonal sprouts from the proximal nerve stump (section A) as demonstrated by a higher number of axons in the A section after forskolin plus TGF-β treatment. The sections of the saline alone show consistent decreases in axon counts the more distal the region. This suggests that the treatments may support axonal sprouting, growth, and survival. Expression of Regeneration-Associated Marker Proteins We previously showed that TGF-β treatment at the time of delayed nerve repair could lead to the extended expression of RAGs.15 The treatments supported the continued expression of the growth supporting phase as shown by continued S100 expression of SCs,18 Ki67-positive proliferative cells,19 apoptotic cells detected by nuclear associated activated caspase-3,20 and activated CD68-macrophages21 at the site of repair. In this study, we examined the spatial expression of regeneration-associated marker proteins after nerve injury and repair, specifically proximal (A), repair site and immediate distal nerve stump (B), and more distal parts of the distal nerve stump (C). Similar to our previous findings,15 the saline control exhibited little to no expression of the 4 protein markers at the site of repair B. These protein markers were also not detected in either the proximal A section or distal C section, with the exception of S100, which was found in a subset of SCs near the perineurium in a few sections. Although we cannot conclusively determine that these are the most distal sections, the observed expression pattern of the regeneration-associated marker proteins is consistent with the transient expression that is detected upon SC interaction with the advancing axonal growth cone. The S100 expression as compared between the 2 treatments reveals an interesting pattern. All SCs that were treated with forskolin strongly expressed S100 in all 3 regions. The addition of TGF-β significantly reduced the expression of S100 in sections A and C, suggesting a modulatory role of TGF-β in SC activation. Although both treatments resulted in a prolonged expression of Ki67 suggesting that these cells are in a proliferative state, there are noted differences in Ki67 expression upon the addition of TGF-β to forskolin. The combination of TGF-β and forskolin resulted in fewer cells undergoing apoptosis, especially in the B and C sections when compared to forskolin only (Table). This reduction is matched by a similar decrease of CD68-activated macrophages. It is of interest to note that in both treatment groups, nearly all SCs expressed activated caspase-3 that is localized to small punctuate bodies within the cell body. Clearly these cells are not undergoing apoptotic changes, yet they express significant amounts of activated caspase-3. Similar expression of activated caspase-3 has been noted in other cell types that are not undergoing apoptosis22,23 including neurons where it was hypothesized to be involved in axon pruning.24 The exact function of activated caspase-3 in SCs, if any, is unknown at this time. Our results are consistent with prior in vitro works where TGF-β plus forskolin added to cultures of chronically denervated isolated SCs for 48 h were sufficient to lead to increased axon survival when used in vivo as a nerve bridge17 and lend credence to the SCs being “refreshed” to a more receptive fully activated state. This fully activated and receptive state is further evident by the expression of p75NGFR by SCs. It is also consistent with the hypothesis that while forskolin is sufficient to activate cAMP functions in SCs,25 and hence mimic the action of various growth factors such as GDNF,26 the modulatory action of TGF-β is important for full effects to be seen. In fact, the effect of several growth factors requires the presence of TGF-β.27-29 Although TGF-β has been shown to act as a mitogen on SCs in culture in the absence of co-cultured neurons,30 in the normal milieu of the nerve, activated macrophages,31 neurons,32 as well as SCs33 secrete TGF-β. The effects of TGF-β on SCs in the presence of regenerating axons are modulatory as demonstrated in our study and are in part responsible for the switch from a quiescent myelinating phenotype to a more active, proliferative growth-supportive phenotype in the absence of regenerating axons34 as evidenced during Wallerian degeneration.14 CONCLUSION Our study demonstrates that 1-time local application of forskolin plus TGF-β at the nerve repair site reactivates SCs and macrophages, induced expression of regeneration-associated marker proteins and promoted axonal sprouting, elongation, and regeneration across the whole length of the injured nerve, even after delayed repair. This suggests that treatment at the repair site is sufficient to propagate a synchronous response of SCs of the distal nerve stumps similar to what is seen during normal nerve regeneration after a nerve crush or immediate repair, although nerve crush will have better regenerative capacity than direct repair.35,36 Although the release and biodistribution of TGF-β from the gel foam sponge was not analyzed, we believe that the effect of TGF-β is time-limited at the time of application after repair which is enough to induce the bio-molecular changes beyond the site of repair. Moreover, this suggests that incorporation of mitogenic or neurotrophic factors into guidance channels may be sufficient to improve nerve regeneration along the distal nerve stump without having to treat whole length of the nerve. Further studies along these lines including functional outcomes will be needed to address the problem of delayed repair. Disclosures This study was supported in parts by ASPN/PSEF Combined Pilot Research Grant from the American Society for Peripheral Nerve and Plastic Surgery Educational Foundation and Ochsner Clinic Foundation (WS). Research reported in this publication/presentation is supported by the Ochsner Translational Medicine Research Initiative sponsored by the Ochsner Health System (WS). The authors have no personal, financial, or institutional interest in any of the drugs, materials, or devices described in this article. REFERENCES 1. Fu SY , Gordon T . Contributing factors to poor functional recovery after delayed nerve repair: prolonged axotomy . J Neurosci . 1995 ; 15 ( 5 ): 3876 - 3885 . Google Scholar CrossRef Search ADS PubMed 2. Kline DG. Nerve surgery: where we are and where we might go . Neurosurg Clin N Am . 2008 ; 19 ( 4 ): 509 - 516 . Google Scholar CrossRef Search ADS PubMed 3. Sulaiman W , Gordon T . 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Effect of local application of transforming growth factor-β at the nerve repair site following chronic axotomy and denervation on the expression of regeneration associated genes . J Neurosurg. 2014 ; 121 ( 4 ): 859 - 874 . Google Scholar CrossRef Search ADS PubMed 16. Abramoff MD , Magalhaes PJ , Ram SJ . Image processing with imageJ . Biophotonics Int . 2004 ; 11 ( 7 ): 36 - 42 . 17. Sulaiman OAR , Gordon T . Transforming growth factor-beta and forskolin attenuate the adverse effects of long-term Schwann cell denervation on peripheral nerve regeneration in vivo . Glia . 2002 ; 37 ( 3 ): 206 - 218 . Google Scholar CrossRef Search ADS PubMed 18. Gonzalez-Martinez T , Perez-Piñera P , Díaz-Esnal B , Vega JA . S-100 proteins in the human peripheral nervous system . Microsc Res Technol . 2003 ; 60 ( 6 ): 633 - 638 . Google Scholar CrossRef Search ADS 19. Kramer F , Stöver T , Warnecke A , Diensthuber M , Lenarz T , Wissel K . 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Caspase-3 is transiently activated without cell death during early antigen driven expansion of CD8(+) T cells in vivo . PLoS One . 2010 ; 5 ( 12 ): e15328 . Google Scholar CrossRef Search ADS PubMed 24. Hyman BT . Caspase activation without apoptosis: insight into Aβ initiation of neurodegeneration . Nat Neurosci . 2011 ; 14 ( 1 ): 5 - 6 . Google Scholar CrossRef Search ADS PubMed 25. Iacovelli J , Lopera J , Bott M et al. Serum and forskolin cooperate to promote G1 progression in Schwann cells by differentially regulating cyclin D1, cyclin E1, and p27Kip expression . Glia . 2007 ; 55 ( 16 ): 1638 - 1647 . Google Scholar CrossRef Search ADS PubMed 26. Höke A , Gordon T , Zochodne DW , Sulaiman OA . A decline in glial cell-line-derived neurotrophic factor expression is associated with impaired regeneration after long-term Schwann cell denervation . Exp Neurol . 2002 ; 173 ( 1 ): 77 - 85 . Google Scholar CrossRef Search ADS PubMed 27. Morgan L , Jessen KR , Mirsky R . Negative regulation of the P0 gene in Schwann cells: suppression of P0 mRNA and protein induction in cultured Schwann cells by FGF2 and TGF beta 1, TGF beta 2 and TGF beta 3 . Development. 1994 ; 120 ( 6 ): 1399 - 1409 . Google Scholar PubMed 28. Krieglstein K , Henheik P , Farkas L et al. Glial cell line-derived neurotrophic factor requires transforming growth factor-beta for exerting its full neurotrophic potential on peripheral and CNS neurons . J Neurosci . 1998 ; 18 ( 23 ): 9822 - 9834 . Google Scholar CrossRef Search ADS PubMed 29. Schober A , Hertel R , Arumäe U et al. Glial cell line-derived neurotrophic factor rescues target-deprived sympathetic spinal cord neurons but requires transforming growth factor-beta as cofactor in vivo . J Neurosci . 1999 ; 19 ( 6 ): 2008 - 2015 . Google Scholar CrossRef Search ADS PubMed 30. Ridley AJ , Davis JB , Stroobant P , Land H . Transforming growth factors-beta 1 and beta 2 are mitogens for rat Schwann cells . J Cell Biol . 1989 ; 109 ( 6 Pt 2 ): 3419 - 3424 . Google Scholar CrossRef Search ADS PubMed 31. Assoian RK , Fleurdelys BE , Stevenson HC et al. Expression and secretion of type beta transforming growth factor by activated human macrophages . Proc Natl Acad Sci USA . 1987 ; 84 ( 17 ): 6020 - 6024 . Google Scholar CrossRef Search ADS PubMed 32. Einheber S , Hannocks MJ , Metz CN , Rifkin DB , Salzer JL . Transforming growth factor-beta 1 regulates axon/Schwann cell interactions . J Cell Biol . 1995 ; 129 ( 2 ): 443 - 458 . Google Scholar CrossRef Search ADS PubMed 33. Böttner M , Krieglstein K , Unsicker K . The transforming growth factor-betas: structure, signaling, and roles in nervous system development and functions . J Neurochem . 2000 ; 75 ( 6 ): 2227 - 2240 . Google Scholar CrossRef Search ADS PubMed 34. Guénard V , Rosenbaum T , Gwynn LA , Doetschman T , Ratner N , Wood PM . Effect of transforming growth factor-beta 1 and -beta 2 on Schwann cell proliferation on neurites . Glia . 1995 ; 13 ( 4 ): 309 - 318 . Google Scholar CrossRef Search ADS PubMed 35. Magnaghi V , Procacci P , Tata AM . Novel pharmacological approaches to Schwann cells as neuroprotective agents for peripheral nerve regeneration . Int Rev Neurobiol. 2009 ; 87 : 295 - 315 . Google Scholar CrossRef Search ADS PubMed 36. Camara-Lemarroy CR , Guzman-de la Garza FJ , Fernández-Garza NE . Molecular inflammatory mediators in peripheral nerve degeneration and regeneration . Neuroimmunomodulation . 2010 ; 17 ( 5 ): 314 - 324 . Google Scholar CrossRef Search ADS PubMed COMMENT The paper revolves around mitogen treatment of peripheral nerve repair to enhance axon growth. It presents as a well-conducted study with clear design and results, which are well discussed (already established rat model with transection of tibial nerve, 18 rats, 3 groups, 6 per group; histology, axon counts, immunohistochemistry; 1 time application of TGF-ß versus TGF-ß plus forskolin via soaked gel foam at repair site). Overall, the paper suggests that single application of TGF-ß plus forskolin at the repair site leads to a significant higher amount of axon counts distal to the repair site. The underlying theory of its influence on switching Schwann cells from their quiescent myelinating phenotype to their proliferative phenotype is discussed and supported by the literature referenced.As such, this animal research is part of an ongoing series of research that, step-by-step, has expanded the role of mitogens, and in particular TGF-ß in delayed nerve repair. Other aspects (S100, activated macrophages, cell proliferation, apoptosis and caspase-3) are also covered. This is a significant contribution to our knowledge about nerve regeneration and repair. It also reminds us about the time dependent changes in expression of regenerative factors. Thomas Kretschmer Oldenburg, Germany Copyright © 2017 by the Congress of Neurological Surgeons This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Neurosurgery Oxford University Press

Single Local Application of TGF-β Promotes a Proregenerative State Throughout a Chronically Injured Nerve

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Oxford University Press
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Copyright © 2017 by the Congress of Neurological Surgeons
ISSN
0148-396X
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1524-4040
D.O.I.
10.1093/neuros/nyx362
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Abstract

Abstract BACKGROUND The lack of nerve regeneration and functional recovery occurs frequently when injuries involve large nerve trunks because insufficient mature axons reach their targets in the distal stump and because of the loss of neurotrophic support, primarily from Schwann cells (SCs). OBJECTIVE To investigate whether a single application of transforming growth factor-beta (TGF-β) plus forskolin or forskolin alone can promote and support axonal regeneration through the distal nerve stump. METHODS Using a delayed repair rat model of nerve injury, we transected the tibial nerve. After 8 wk, end-to-end repair was done and the repair site was treated with saline, forskolin, or TGF- β plus forskolin. After 6 wk, nerve sections consisting of the proximal stump, distal to the site of repair, and the most distal part of the nerve stump were removed for nerve histology, axon counts, and immunohistochemistry for activated SCs (S100), macrophages (CD68), cell proliferation (Ki67), p75NGFR, and apoptosis (activated caspase-3). RESULTS TGF-β plus forskolin significantly increased the numbers of axons regenerated distal to the repair site and the most distal nerve sections. Both treatments significantly increased the numbers of axons regenerated in the most distal nerve sections compared to saline treated. Both treatments exhibited extended expression of regeneration-associated marker proteins. CONCLUSION TGF-β plus forskolin treatment of chronically injured nerve improved axonal regeneration and increased expression of regeneration-associated proteins beyond the repair site. This suggests that a single application at the site of repair has mitogenic effects that extended distally and may potentially overcome the decrease in regenerated axon over long distance. Chronic nerve injuries, Nerve regeneration, Regeneration-associated genes/proteins, Transforming growth factor ABBREVIATIONS ABBREVIATIONS H&E hematoxylin and eosin RAGs regeneration-associated genes RT room temperature SCs Schwann cells TIB tibial TGF-β transforming growth factor-beta Injuries to the sciatic nerve and other large nerve trunks such as the brachial plexus often lead to suboptimal functional recovery.1-3 The capacity of injured nerves to regenerate is due mostly to the growth-supportive Schwann cells (SCs) in the distal stumps of the injured nerves.4,5 Animal studies have shown that suboptimal functional recovery is attributable to insufficient regenerating axons reaching the end target, and this deficiency can be attributed to the loss of the growth-supportive milieu provided by the SCs in the distal stump of injured nerves.6,7 After acute injury, SCs express regeneration-associated genes (RAGs) such as, neurotrophins and their receptors,8 adhesion molecules,9 and cytokines such as transforming growth factor-beta (TGF-β),10 that support growth of the regeneration units through the distal nerve stumps and reestablishment of neuromuscular junctions.11,12 SCs also proliferate in the distal nerve stumps and form cellular channels (bands of Bungner) through which regenerating axons traverse in the distal nerve stumps to get to the denervated muscle target.13 A second, proliferative phase of SCs seems to be initiated by their interaction with the growth cones.14 While the second phase of SC proliferation seems to be mediated by axonal contact and is not synchronous in all SCs of the distal nerve stump, it is unclear whether SC expression of RAGs in the distal nerve stumps is also dependent on axonal contact or is synchronous across the length of the distal nerve stumps once the growth cones cross the suture site. This is a critical question since it has potential effect on the efficacy of any therapeutic strategy to enhance axonal regeneration through the distal nerve stumps. Previously, we had shown that in vivo treatment of chronically denervated SCs with TGF-β plus forskolin “reversed the deleterious effect of chronic SC denervation” and improved their capacity to support axonal regeneration.15 One of the unanswered questions in the mitogen treatment of nerve repair is whether a treatment directed solely at the nerve repair site is sufficient to produce the mitogenic effects throughout the distal nerve stump, or if the positive effects would be lost as a function of distance from the repair and treatment site. Using our delayed repair rat model, we extended our examination both proximally and distally to the repair site by 15 to 25 mm and compared the responses of SCs to forskolin alone or with TGF-β treatment. We assessed SC activation, SC expression of regeneration-associated marker proteins and the numbers of regenerated axons along the length of the distal nerve stumps. METHODS We utilized our rat model of tibial (TIB) nerve injury and delayed repair as previously described15 (Figure 1). The Ochsner Institutional Animal Care and Use Committee approved the protocol. Eighteen adult female Sprague--Dawly rats (Harlan) weighing about 200 g were used. In the first surgery, we transected the TIB nerve, reflecting the proximal nerve stump backward and suturing to the femoris muscle to prevent regeneration. After 8 wk, the TIB nerve in all animals was re-exposed and repaired by direct coaptation with 6-0 Prolene sutures. Animals were divided into 3 treatment groups (n = 6 per group): (i) Saline only; (ii) Forskolin (2 ng/mL); and (iii) Forskolin plus TGF-β (0.5 μM). A 10 mm square piece of gelfoam was placed in the treatment solution to soak for 10 min and applied directly by wrapping the gelfoam around the TIB nerve repair site. Regeneration of TIB nerves was allowed for 6 wk. The animals were sacrificed and nerve sections were taken from (a) proximal stump (A section); (b) suture site and immediate distal stump (B section); and (c) most distal part of the distal nerve stumps (C section) for analysis (Figure 1). FIGURE 1. View largeDownload slide The TIB nerve was cut and reflected into muscle to prevent regeneration and then repaired, plus or minus treatment. Six weeks later, 2 10-mm sections and 1 15-mm section of the TIB nerve were harvested with the A section proximal, the B section containing the repair, and C section distal to the repair. The 15 to 25 mm analysis point of the most distal C section is indicated. FIGURE 1. View largeDownload slide The TIB nerve was cut and reflected into muscle to prevent regeneration and then repaired, plus or minus treatment. Six weeks later, 2 10-mm sections and 1 15-mm section of the TIB nerve were harvested with the A section proximal, the B section containing the repair, and C section distal to the repair. The 15 to 25 mm analysis point of the most distal C section is indicated. Nerve Histology and Axon Counts Nerve tissues were fixed in 4% paraformaldehyde and 5-μm transverse paraffin sections were used for hematoxylin and eosin (H&E) to stain for the myelin structures and nuclei as described.15 A minimum of 3 separate nerve sections A, B, and C were observed under a microscope. Photomicrographs were analyzed with Image J16 and axon counts were determined, and analyzed for statistical significance using the Kruskal–Wallis test. Immunohistochemistry Immunocytochemical analyses were carried out on 5-μm paraffin nerve sections as previously described.15 Primary antibodies included CD68 (1:200), Ki67 (1:500), S100 (1:1000), p75NGFR (1:300), and activated caspase-3 (1:200) and used overnight at 4°C. Fluorescent anti-rabbit secondary antibody (1:1000) was added and incubated at room temperature (RT) for 30 min, and mounted in media containing DAPI. For double-labeling studies, nerve sections were incubated in blocking buffer (Dako, Agilent, Santa Clara, California) at RT for 30 min then overnight at 4°C with a combination of anti-S100/p75NGFR or anti-CD68/caspase-3 to identify activated SCs and apoptotic macrophages, respectively. After 3 5-min washes, sections were incubated with Alexa Fluor 488/568 (Thermo Fischer Scientific, Waltham, Massachusetts) anti-rabbit/anti-mouse secondary antibody (1:500) combination for 30 min at RT and mounted. Sections without the primary antibody incubation were the negative control. Fluorescent-stained sections were viewed on a Zeiss Axiovert 200 M (Carl Zeiss, Oberkochen Germany) inverted microscope with fluorescence/phase imaging. RESULTS Axon Regeneration at Sections A, B, and C H&E stained transverse nerve sections were examined by light microscopy. No major anatomical and histological differences were observed among sections A, B, or C, although the saline-treated distal C section appeared to have an overall less organized array of axonal bundles (Figure 2A). The spatial distribution of the treatment effect on regenerated axon numbers at sections A, B, and C were counted both manually and by Image J16 software (Figure 2B). The saline control consistently had fewer regenerated axons in all nerve sections while both treatment groups showed an increase in the number of axons. However, only the TGF-β plus forskolin treated nerves showed statistically significant increases over the saline control, at least in sections A and C (Figure 3). The effect of TGF-β and forskolin was additive with significantly more regenerated axons in both the A and C sections. Interestingly the combination of TGF-β and forskolin was significant when compared to the forskolin only treatment, especially in the B section. Furthermore, nerves that were exposed to TGF-β appeared to have better organized axonal regeneration. FIGURE 2. View largeDownload slide A, H&E stained transverse TIB nerve sections showed a more organized array of axonal bundles with treatments. Columns are, left to right: (i) saline control, (ii) forskolin only, (iii) forskolin plus TGF-β. A, B, C refers to nerve segment region where A is proximal stump, B is suture site and immediate distal stump, and C is most distal part of distal nerve stump; B, A grey scale representation of a H&E stained transverse TIB nerve section (i) and an binary (black & white) image depicting particle counting of axons in Image J16 (ii). FIGURE 2. View largeDownload slide A, H&E stained transverse TIB nerve sections showed a more organized array of axonal bundles with treatments. Columns are, left to right: (i) saline control, (ii) forskolin only, (iii) forskolin plus TGF-β. A, B, C refers to nerve segment region where A is proximal stump, B is suture site and immediate distal stump, and C is most distal part of distal nerve stump; B, A grey scale representation of a H&E stained transverse TIB nerve section (i) and an binary (black & white) image depicting particle counting of axons in Image J16 (ii). FIGURE 3. View largeDownload slide Histogram of axon nerve counts from each of 3 nerve segment regions surrounding and including the repair site. TGF-β plus forskolin treatment leads to a significant increase in the number of regenerated axons in segments B and C as compared to saline control. FIGURE 3. View largeDownload slide Histogram of axon nerve counts from each of 3 nerve segment regions surrounding and including the repair site. TGF-β plus forskolin treatment leads to a significant increase in the number of regenerated axons in segments B and C as compared to saline control. Immunohistochemistry of RAGs Our previous work has shown that under the same treatment regimen,15 the region near the suture site of the saline control and treated groups exhibited a striking dichotomy in the expression of SC RAGs and the presence or absence of activated macrophages. We have extended those findings to regions both proximal and distal to the repair and treatment site. Apoptosis All sections, A, B, and C, from the treated groups exhibited many cells undergoing active apoptosis as indicated by positive staining for activated caspase-3 (Figure 4A). No particular pattern was observed as apoptotic cells were detected throughout the nerve sections. Some nuclear localization of activated caspase-3 were detected in the nerve sections of the treated groups. Interestingly, all SCs, even those not undergoing apoptosis, also contained reactive material localized to punctate bodies throughout the cell body (Figure 4B). No activated caspase-3 was detected in any of the sections in the saline control (data not shown). FIGURE 4. View largeDownload slide Immunohistochemistry showed increased levels of activated caspase-3 (green) and DAPI (blue) in A, TGF-β plus forskolin treated nerve segments B; and B, TGF-β plus forskolin treated nerve segment C. (100x mag). FIGURE 4. View largeDownload slide Immunohistochemistry showed increased levels of activated caspase-3 (green) and DAPI (blue) in A, TGF-β plus forskolin treated nerve segments B; and B, TGF-β plus forskolin treated nerve segment C. (100x mag). Activated Macrophage All nerve sections of the treated groups showed immunoreactivities to CD68, indicating the presence of activated macrophages throughout the nerve bundle (Figure 5A). Only a few scattered staining for CD68 were found in each of the saline-control sections (data not shown). Furthermore, double staining for CD68 + cells and apoptotic cells found cells with positive staining for activated caspase-3 in the cytoplasm as well as punctate nuclear staining in close proximity to activated CD68+ macrophages (Figure 5B), suggesting that these macrophages may be activated. FIGURE 5. View largeDownload slide Immunofluorescent double staining of CD68+ macrophages (red) and caspase-3 (green) in A, TGF-β plus forskolin treated nerve segments B, and B, TGF-β plus forskolin treated nerve segments C. Activated macrophages were detected (arrow) as well as CD68+ macrophages colocalized in close proximity to caspase-3 (*) positive cells (400× mag). FIGURE 5. View largeDownload slide Immunofluorescent double staining of CD68+ macrophages (red) and caspase-3 (green) in A, TGF-β plus forskolin treated nerve segments B, and B, TGF-β plus forskolin treated nerve segments C. Activated macrophages were detected (arrow) as well as CD68+ macrophages colocalized in close proximity to caspase-3 (*) positive cells (400× mag). Cell Proliferation Immunostaining for Ki67 was used to ascertain for active cell proliferation. Similar to the immunostaining pattern of CD68, staining for Ki67 was seen in all sections of the treated groups suggesting active cell proliferation (Figure 6). In the saline control, Ki67 was not detected in either the proximal A section or the repair site B section, while sporadic Ki67 staining was detected in the distal C section (data not shown). FIGURE 6. View largeDownload slide Immunohistochemical analysis of Ki-67 (green) and DAPI (blue) in TGF-β plus forskolin nerve section B. A, 100× mag; and B, 400× mag. FIGURE 6. View largeDownload slide Immunohistochemical analysis of Ki-67 (green) and DAPI (blue) in TGF-β plus forskolin nerve section B. A, 100× mag; and B, 400× mag. Activated SCs To determine the presence of activated SCs in nerve sections, expression of S100 was used. As shown, all SCs in the B sections of the mitogen-treated nerves were positive for S100 (Figure 7). Furthermore, staining for p75NGFR was also found colocalized to S100-positive SC (Figure 7B). In contrast, no S100 staining was detected in the A or B sections of the saline control. Only the distal C nerve section of the saline control contained S100 expressing SCs, primarily near the perineurium (data not shown). FIGURE 7. View largeDownload slide Immunofluorescent double staining of S-100 (red) and NGFR p75 (green) proteins, markers of activated Schwann cells (arrow) in A, TGF-β plus forskolin treated nerve segment B, and B, TGF-β plus forskolin treated nerve segment C. (400× mag). FIGURE 7. View largeDownload slide Immunofluorescent double staining of S-100 (red) and NGFR p75 (green) proteins, markers of activated Schwann cells (arrow) in A, TGF-β plus forskolin treated nerve segment B, and B, TGF-β plus forskolin treated nerve segment C. (400× mag). Relative Abundance of Expressed Marker Proteins To determine the relative abundance of the various marker proteins in each of the nerve segments, positive cells were counted manually and normalized to 100 nuclei, as detected with DAPI (Table). There was a striking dichotomy between the saline control and the 2 treatments. The saline control exhibited little to no positive staining for the various marker proteins, whereas both treatments exhibited numerous positive cells for the various marker proteins. Generally, the forskolin only treatment exhibited the greatest number of positive cells, irrespective of the marker protein examined. For example, forskolin treatment resulted in the expression of S100 protein in all SCs in all segments, whereas the TGF-β plus forskolin nerves showed some SCs with detectable S100 protein in the proximal (A section) and the most distal (C section) segments. Only the TGF-β plus forskolin treated site of nerve injury section B showed extensive S100 staining of SCs. Interestingly, the most distal segment C of the saline control exhibited some sparse S100-positive SCs located near the perineurium. TABLE. Positive Cell Counts of the Various Marker Proteins (Caspase-3, CD68, Ki67, S100) by Treatment and Nerve Section. Values Represent the Numbers of DAPI-stained Nuclei per mm2 From 3 Areas per Cross Section and the Numbers of Positive Cell Counts for the Marker Proteins per 100 Nuclei Marker Proteins Treatment # DAPI-labeled nuclei per mm2 Positive cell count per 100 nuclei A B C A B C Caspase-3 Saline control 5230 4409 2670 0 0 0 Forskolin only 5181 5512 3982 5.7 4.5 8.8 TGF-β plus forskolin 1946 1789 2102 5.6 2.9 3.2 CD68 Saline control 4861 3978 3500 <1 < 1 <1 Forskolin only 6482 7235 13 036 5.6 9.8 8.8 TGF-β plus forskolin 16 398 12 040 15 000 5.4 7.7 4.6 Ki67 Saline control 6467 5475 4578 <1 <1 <1 Forskolin only 9271 5952 5861 4.0 5.3 8.8 TGF-β plus forskolin 30 373 29 904 30 373 6.0 7.7 6.7 S100 Saline control 4142 7036 3433 0 0 10.5 Forskolin only 7036 7994 2880 100 100 100 TGF-β plus forskolin 5946 8458 12 680 67 100 60 Marker Proteins Treatment # DAPI-labeled nuclei per mm2 Positive cell count per 100 nuclei A B C A B C Caspase-3 Saline control 5230 4409 2670 0 0 0 Forskolin only 5181 5512 3982 5.7 4.5 8.8 TGF-β plus forskolin 1946 1789 2102 5.6 2.9 3.2 CD68 Saline control 4861 3978 3500 <1 < 1 <1 Forskolin only 6482 7235 13 036 5.6 9.8 8.8 TGF-β plus forskolin 16 398 12 040 15 000 5.4 7.7 4.6 Ki67 Saline control 6467 5475 4578 <1 <1 <1 Forskolin only 9271 5952 5861 4.0 5.3 8.8 TGF-β plus forskolin 30 373 29 904 30 373 6.0 7.7 6.7 S100 Saline control 4142 7036 3433 0 0 10.5 Forskolin only 7036 7994 2880 100 100 100 TGF-β plus forskolin 5946 8458 12 680 67 100 60 View Large TABLE. Positive Cell Counts of the Various Marker Proteins (Caspase-3, CD68, Ki67, S100) by Treatment and Nerve Section. Values Represent the Numbers of DAPI-stained Nuclei per mm2 From 3 Areas per Cross Section and the Numbers of Positive Cell Counts for the Marker Proteins per 100 Nuclei Marker Proteins Treatment # DAPI-labeled nuclei per mm2 Positive cell count per 100 nuclei A B C A B C Caspase-3 Saline control 5230 4409 2670 0 0 0 Forskolin only 5181 5512 3982 5.7 4.5 8.8 TGF-β plus forskolin 1946 1789 2102 5.6 2.9 3.2 CD68 Saline control 4861 3978 3500 <1 < 1 <1 Forskolin only 6482 7235 13 036 5.6 9.8 8.8 TGF-β plus forskolin 16 398 12 040 15 000 5.4 7.7 4.6 Ki67 Saline control 6467 5475 4578 <1 <1 <1 Forskolin only 9271 5952 5861 4.0 5.3 8.8 TGF-β plus forskolin 30 373 29 904 30 373 6.0 7.7 6.7 S100 Saline control 4142 7036 3433 0 0 10.5 Forskolin only 7036 7994 2880 100 100 100 TGF-β plus forskolin 5946 8458 12 680 67 100 60 Marker Proteins Treatment # DAPI-labeled nuclei per mm2 Positive cell count per 100 nuclei A B C A B C Caspase-3 Saline control 5230 4409 2670 0 0 0 Forskolin only 5181 5512 3982 5.7 4.5 8.8 TGF-β plus forskolin 1946 1789 2102 5.6 2.9 3.2 CD68 Saline control 4861 3978 3500 <1 < 1 <1 Forskolin only 6482 7235 13 036 5.6 9.8 8.8 TGF-β plus forskolin 16 398 12 040 15 000 5.4 7.7 4.6 Ki67 Saline control 6467 5475 4578 <1 <1 <1 Forskolin only 9271 5952 5861 4.0 5.3 8.8 TGF-β plus forskolin 30 373 29 904 30 373 6.0 7.7 6.7 S100 Saline control 4142 7036 3433 0 0 10.5 Forskolin only 7036 7994 2880 100 100 100 TGF-β plus forskolin 5946 8458 12 680 67 100 60 View Large DISCUSSION One of the unanswered questions in the mitogen treatment of nerve repair is whether a treatment solely at the nerve repair site could overcome the effects of chronic denervation throughout the distal nerve stump, or the positive effects would be lost as a function of distance from the repair and treatment site. In this study, we show that the positive mitogenic effects of forskolin or TGF-β plus forskolin treatment from the application point at the repair site extend both proximally as well as distally to enhance nerve regeneration. Effects were noted up to 25 mm from the repair site, the furthest distance away that was tested (Table). Mitogenic treatment at the time of delayed repair enhances SC growth-supportive phenotype and improved axonal regeneration at the site of repair.15,17 Axonal Regeneration after Chronic Axotomy and Denervation Prior works by many have firmly established that in the rat TIB nerve regeneration model, 8 wk of axotomy and denervation would result in at least a 50% reduction in the number of regenerating axons compared to an immediate repair.5 Although we did not conduct an immediate repair in this study, treatment with TGF-β plus forskolin led to a near doubling of regenerated axons distal to the repair site when compared to saline alone (1288 ± 151 vs 2108 ± 200, P = .006). This level of increase would suggest that these mitogens had the effect of returning chronic axotomized and denervated nerves back to the state comparable to immediate repair. The number of regenerating axons near the repair site as well as up to 25 mm proximal and distal to the repair site was also higher than the saline control. The TGF-β plus forskolin treated nerves had significantly more regenerated axons, but did not double them (Table, Figure 3). Forskolin also led to an increase in regenerated axons in the proximal region compared to the saline control, but the increase did not reach statistical significance. The modulating effect of TGF-β would appear to enable the survival of greater numbers of axons and/or axonal sprouts. This is borne out by comparisons of axonal numbers among the 3 sections, which reveal that the B sections, nerve repair site and the regions immediately surrounding it, exhibited fewer axons than either the proximal A region or the distal C region in the 2 treatment groups (Table, Figure 3). The lower number of axon counts in the repair site nerve segment B may be due to staggered axonal regeneration across the suture site and pruning that occur at the injury site. Although the difference between the forskolin plus TGF-β and saline control did not reach significance level, it does show that the treatment has some positive effect on increasing axon counts in the B sections. Hence, treatment with forskolin plus TGF-β seem to promote axon regeneration across the injured and sutured site, perhaps by augmenting axonal sprouts from the proximal nerve stump (section A) as demonstrated by a higher number of axons in the A section after forskolin plus TGF-β treatment. The sections of the saline alone show consistent decreases in axon counts the more distal the region. This suggests that the treatments may support axonal sprouting, growth, and survival. Expression of Regeneration-Associated Marker Proteins We previously showed that TGF-β treatment at the time of delayed nerve repair could lead to the extended expression of RAGs.15 The treatments supported the continued expression of the growth supporting phase as shown by continued S100 expression of SCs,18 Ki67-positive proliferative cells,19 apoptotic cells detected by nuclear associated activated caspase-3,20 and activated CD68-macrophages21 at the site of repair. In this study, we examined the spatial expression of regeneration-associated marker proteins after nerve injury and repair, specifically proximal (A), repair site and immediate distal nerve stump (B), and more distal parts of the distal nerve stump (C). Similar to our previous findings,15 the saline control exhibited little to no expression of the 4 protein markers at the site of repair B. These protein markers were also not detected in either the proximal A section or distal C section, with the exception of S100, which was found in a subset of SCs near the perineurium in a few sections. Although we cannot conclusively determine that these are the most distal sections, the observed expression pattern of the regeneration-associated marker proteins is consistent with the transient expression that is detected upon SC interaction with the advancing axonal growth cone. The S100 expression as compared between the 2 treatments reveals an interesting pattern. All SCs that were treated with forskolin strongly expressed S100 in all 3 regions. The addition of TGF-β significantly reduced the expression of S100 in sections A and C, suggesting a modulatory role of TGF-β in SC activation. Although both treatments resulted in a prolonged expression of Ki67 suggesting that these cells are in a proliferative state, there are noted differences in Ki67 expression upon the addition of TGF-β to forskolin. The combination of TGF-β and forskolin resulted in fewer cells undergoing apoptosis, especially in the B and C sections when compared to forskolin only (Table). This reduction is matched by a similar decrease of CD68-activated macrophages. It is of interest to note that in both treatment groups, nearly all SCs expressed activated caspase-3 that is localized to small punctuate bodies within the cell body. Clearly these cells are not undergoing apoptotic changes, yet they express significant amounts of activated caspase-3. Similar expression of activated caspase-3 has been noted in other cell types that are not undergoing apoptosis22,23 including neurons where it was hypothesized to be involved in axon pruning.24 The exact function of activated caspase-3 in SCs, if any, is unknown at this time. Our results are consistent with prior in vitro works where TGF-β plus forskolin added to cultures of chronically denervated isolated SCs for 48 h were sufficient to lead to increased axon survival when used in vivo as a nerve bridge17 and lend credence to the SCs being “refreshed” to a more receptive fully activated state. This fully activated and receptive state is further evident by the expression of p75NGFR by SCs. It is also consistent with the hypothesis that while forskolin is sufficient to activate cAMP functions in SCs,25 and hence mimic the action of various growth factors such as GDNF,26 the modulatory action of TGF-β is important for full effects to be seen. In fact, the effect of several growth factors requires the presence of TGF-β.27-29 Although TGF-β has been shown to act as a mitogen on SCs in culture in the absence of co-cultured neurons,30 in the normal milieu of the nerve, activated macrophages,31 neurons,32 as well as SCs33 secrete TGF-β. The effects of TGF-β on SCs in the presence of regenerating axons are modulatory as demonstrated in our study and are in part responsible for the switch from a quiescent myelinating phenotype to a more active, proliferative growth-supportive phenotype in the absence of regenerating axons34 as evidenced during Wallerian degeneration.14 CONCLUSION Our study demonstrates that 1-time local application of forskolin plus TGF-β at the nerve repair site reactivates SCs and macrophages, induced expression of regeneration-associated marker proteins and promoted axonal sprouting, elongation, and regeneration across the whole length of the injured nerve, even after delayed repair. This suggests that treatment at the repair site is sufficient to propagate a synchronous response of SCs of the distal nerve stumps similar to what is seen during normal nerve regeneration after a nerve crush or immediate repair, although nerve crush will have better regenerative capacity than direct repair.35,36 Although the release and biodistribution of TGF-β from the gel foam sponge was not analyzed, we believe that the effect of TGF-β is time-limited at the time of application after repair which is enough to induce the bio-molecular changes beyond the site of repair. Moreover, this suggests that incorporation of mitogenic or neurotrophic factors into guidance channels may be sufficient to improve nerve regeneration along the distal nerve stump without having to treat whole length of the nerve. Further studies along these lines including functional outcomes will be needed to address the problem of delayed repair. Disclosures This study was supported in parts by ASPN/PSEF Combined Pilot Research Grant from the American Society for Peripheral Nerve and Plastic Surgery Educational Foundation and Ochsner Clinic Foundation (WS). Research reported in this publication/presentation is supported by the Ochsner Translational Medicine Research Initiative sponsored by the Ochsner Health System (WS). The authors have no personal, financial, or institutional interest in any of the drugs, materials, or devices described in this article. REFERENCES 1. Fu SY , Gordon T . Contributing factors to poor functional recovery after delayed nerve repair: prolonged axotomy . J Neurosci . 1995 ; 15 ( 5 ): 3876 - 3885 . Google Scholar CrossRef Search ADS PubMed 2. Kline DG. Nerve surgery: where we are and where we might go . Neurosurg Clin N Am . 2008 ; 19 ( 4 ): 509 - 516 . Google Scholar CrossRef Search ADS PubMed 3. Sulaiman W , Gordon T . Neurobiology of peripheral nerve injury, regeneration, and functional recovery: from bench top research to bedside application . Ochsner J. 2013 ; 13 ( 1 ): 100 - 108 . Google Scholar PubMed 4. Gaudet AD , Popovich PG , Ramer MS . Wallerian degeneration: gaining perspective on inflammatory events after peripheral nerve injury . J Neuroinflamm . 2011 ; 8 ( 1 ): 110 - 123 . Google Scholar CrossRef Search ADS 5. Sulaiman OAR , Gordon T , Role of chronic Schwann cell denervation in poor functional recovery after nerve injuries and experimental strategies to combat it . Neurosurgery. 2009 ; 65 ( 4 Suppl ): A105 - A114 (Abstract) . Google Scholar CrossRef Search ADS PubMed 6. Gordon T , Sulaiman OAR , Boyd JG . Experimental strategies to promote functional recovery after peripheral nerve injuries . J Peripher Nerv Syst. 2003 ; 8 ( 4 ): 236 - 250 . Google Scholar CrossRef Search ADS PubMed 7. Sulaiman OAR , Boyd J , Gordon T . Axonal regeneration in the peripheral nervous system of mammals . In: Kettermann H , Ransom B , eds. Neuroglia . 2nd ed . Oxford, UK : Oxford University Press . 2005 : 454 - 466 . Google Scholar CrossRef Search ADS 8. Tomita K , Kubo T , Matsuda K et al. The neurotrophin receptor p75NTR in Schwann cells is implicated in remyelination and motor recovery after peripheral nerve injury . Glia . 2007 ; 55 ( 11 ): 1199 - 1208 . Google Scholar CrossRef Search ADS PubMed 9. Avari P , Huang W , Averill S et al. The spatiotemporal localization of JAM-C following sciatic nerve crush in adult rats . Brain Behav . 2012 ; 2 ( 4 ): 402 - 414 . Google Scholar CrossRef Search ADS PubMed 10. Feng Z , Ko CP . Schwann cells promote synaptogenesis at the neuromuscular junction via transforming growth factor-beta1 . J Neurosci . 2008 ; 28 ( 39 ): 9599 - 9609 . Google Scholar CrossRef Search ADS PubMed 11. Gordon T. The role of neurotrophic factors in nerve regeneration . Neurosurg Focus . 2009 ; 26 ( 2 ): E3 . Google Scholar CrossRef Search ADS PubMed 12. Kingham PJ , Mantovani C , Terenghi G . Stem cell and neuron co-cultures for the study of nerve regeneration . Methods Mol Biol . 2011 ; 695 : 115 - 127 . Google Scholar CrossRef Search ADS PubMed 13. Li H , Terenghi G , Hall SM . Effects of delayed re-innervation on the expression of c-erbB receptors by chronically denervated rat Schwann cells in vivo . Glia . 1997 ; 20 ( 4 ): 333 - 347 . Google Scholar CrossRef Search ADS PubMed 14. Yang DP , Zhang DP , Mak KS , Bonder DE , Pomeroy SL , Kim HA . Schwann cell proliferation during Wallerian degeneration is not necessary for regeneration and remyelination of the peripheral nerves: axon-dependent removal of newly generated Schwann cells by apoptosis . Mol Cell Neurosci . 2008 ; 38 ( 1 ): 80 - 88 . Google Scholar CrossRef Search ADS PubMed 15. Sulaiman W , Dreesen TD . Effect of local application of transforming growth factor-β at the nerve repair site following chronic axotomy and denervation on the expression of regeneration associated genes . J Neurosurg. 2014 ; 121 ( 4 ): 859 - 874 . Google Scholar CrossRef Search ADS PubMed 16. Abramoff MD , Magalhaes PJ , Ram SJ . Image processing with imageJ . Biophotonics Int . 2004 ; 11 ( 7 ): 36 - 42 . 17. Sulaiman OAR , Gordon T . Transforming growth factor-beta and forskolin attenuate the adverse effects of long-term Schwann cell denervation on peripheral nerve regeneration in vivo . Glia . 2002 ; 37 ( 3 ): 206 - 218 . Google Scholar CrossRef Search ADS PubMed 18. Gonzalez-Martinez T , Perez-Piñera P , Díaz-Esnal B , Vega JA . S-100 proteins in the human peripheral nervous system . Microsc Res Technol . 2003 ; 60 ( 6 ): 633 - 638 . Google Scholar CrossRef Search ADS 19. Kramer F , Stöver T , Warnecke A , Diensthuber M , Lenarz T , Wissel K . BDNF mRNA expression is significantly upregulated in vestibular schwannomas and correlates with proliferative activity . J Neurooncol . 2010 ; 98 ( 1 ): 31 - 39 . Google Scholar CrossRef Search ADS PubMed 20. Abraham MC , Shaham S . Death without caspases, caspases without death . Trends Cell Biol . 2004 ; 14 ( 4 ): 184 - 193 . Google Scholar CrossRef Search ADS PubMed 21. Martini R , Fischer S , López-Vales R , David S . Interactions between Schwann cells and macrophages in injury and inherited demyelinating disease . Glia . 2008 ; 56 ( 14 ): 1566 - 1577 . Google Scholar CrossRef Search ADS PubMed 22. Ayyash M , Tamimi H , Ashhab Y . Developing a powerful in silico tool for the discovery of novel caspase-3 substrates: a preliminary screening of the human proteome . BMC Bioinform . 2012 ; 13 : 1 - 14 . Google Scholar CrossRef Search ADS 23. McComb S , Mulligan R , Sad S . Caspase-3 is transiently activated without cell death during early antigen driven expansion of CD8(+) T cells in vivo . PLoS One . 2010 ; 5 ( 12 ): e15328 . Google Scholar CrossRef Search ADS PubMed 24. Hyman BT . Caspase activation without apoptosis: insight into Aβ initiation of neurodegeneration . Nat Neurosci . 2011 ; 14 ( 1 ): 5 - 6 . Google Scholar CrossRef Search ADS PubMed 25. Iacovelli J , Lopera J , Bott M et al. Serum and forskolin cooperate to promote G1 progression in Schwann cells by differentially regulating cyclin D1, cyclin E1, and p27Kip expression . Glia . 2007 ; 55 ( 16 ): 1638 - 1647 . Google Scholar CrossRef Search ADS PubMed 26. Höke A , Gordon T , Zochodne DW , Sulaiman OA . A decline in glial cell-line-derived neurotrophic factor expression is associated with impaired regeneration after long-term Schwann cell denervation . Exp Neurol . 2002 ; 173 ( 1 ): 77 - 85 . Google Scholar CrossRef Search ADS PubMed 27. Morgan L , Jessen KR , Mirsky R . Negative regulation of the P0 gene in Schwann cells: suppression of P0 mRNA and protein induction in cultured Schwann cells by FGF2 and TGF beta 1, TGF beta 2 and TGF beta 3 . Development. 1994 ; 120 ( 6 ): 1399 - 1409 . Google Scholar PubMed 28. Krieglstein K , Henheik P , Farkas L et al. Glial cell line-derived neurotrophic factor requires transforming growth factor-beta for exerting its full neurotrophic potential on peripheral and CNS neurons . J Neurosci . 1998 ; 18 ( 23 ): 9822 - 9834 . Google Scholar CrossRef Search ADS PubMed 29. Schober A , Hertel R , Arumäe U et al. Glial cell line-derived neurotrophic factor rescues target-deprived sympathetic spinal cord neurons but requires transforming growth factor-beta as cofactor in vivo . J Neurosci . 1999 ; 19 ( 6 ): 2008 - 2015 . Google Scholar CrossRef Search ADS PubMed 30. Ridley AJ , Davis JB , Stroobant P , Land H . Transforming growth factors-beta 1 and beta 2 are mitogens for rat Schwann cells . J Cell Biol . 1989 ; 109 ( 6 Pt 2 ): 3419 - 3424 . Google Scholar CrossRef Search ADS PubMed 31. Assoian RK , Fleurdelys BE , Stevenson HC et al. Expression and secretion of type beta transforming growth factor by activated human macrophages . Proc Natl Acad Sci USA . 1987 ; 84 ( 17 ): 6020 - 6024 . Google Scholar CrossRef Search ADS PubMed 32. Einheber S , Hannocks MJ , Metz CN , Rifkin DB , Salzer JL . Transforming growth factor-beta 1 regulates axon/Schwann cell interactions . J Cell Biol . 1995 ; 129 ( 2 ): 443 - 458 . Google Scholar CrossRef Search ADS PubMed 33. Böttner M , Krieglstein K , Unsicker K . The transforming growth factor-betas: structure, signaling, and roles in nervous system development and functions . J Neurochem . 2000 ; 75 ( 6 ): 2227 - 2240 . Google Scholar CrossRef Search ADS PubMed 34. Guénard V , Rosenbaum T , Gwynn LA , Doetschman T , Ratner N , Wood PM . Effect of transforming growth factor-beta 1 and -beta 2 on Schwann cell proliferation on neurites . Glia . 1995 ; 13 ( 4 ): 309 - 318 . Google Scholar CrossRef Search ADS PubMed 35. Magnaghi V , Procacci P , Tata AM . Novel pharmacological approaches to Schwann cells as neuroprotective agents for peripheral nerve regeneration . Int Rev Neurobiol. 2009 ; 87 : 295 - 315 . Google Scholar CrossRef Search ADS PubMed 36. Camara-Lemarroy CR , Guzman-de la Garza FJ , Fernández-Garza NE . Molecular inflammatory mediators in peripheral nerve degeneration and regeneration . Neuroimmunomodulation . 2010 ; 17 ( 5 ): 314 - 324 . Google Scholar CrossRef Search ADS PubMed COMMENT The paper revolves around mitogen treatment of peripheral nerve repair to enhance axon growth. It presents as a well-conducted study with clear design and results, which are well discussed (already established rat model with transection of tibial nerve, 18 rats, 3 groups, 6 per group; histology, axon counts, immunohistochemistry; 1 time application of TGF-ß versus TGF-ß plus forskolin via soaked gel foam at repair site). Overall, the paper suggests that single application of TGF-ß plus forskolin at the repair site leads to a significant higher amount of axon counts distal to the repair site. The underlying theory of its influence on switching Schwann cells from their quiescent myelinating phenotype to their proliferative phenotype is discussed and supported by the literature referenced.As such, this animal research is part of an ongoing series of research that, step-by-step, has expanded the role of mitogens, and in particular TGF-ß in delayed nerve repair. Other aspects (S100, activated macrophages, cell proliferation, apoptosis and caspase-3) are also covered. This is a significant contribution to our knowledge about nerve regeneration and repair. It also reminds us about the time dependent changes in expression of regenerative factors. Thomas Kretschmer Oldenburg, Germany Copyright © 2017 by the Congress of Neurological Surgeons This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

NeurosurgeryOxford University Press

Published: Jul 21, 2017

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