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

Laboratory investigation

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Object

Although peripheral nerves can regenerate after traumatic injury, functional recovery is often suboptimal, especially after injuries to large nerve trunks such as the sciatic nerve or brachial plexus. Current research with animal models suggests that the lack of functional recovery resides in the lack of sufficient mature axons reaching their targets due to the loss of neurotrophic support by Schwann cells in the distal stump of injured nerves. This study was designed to investigate the effect of one-time application of transforming growth factor–β (TGF-β) at the repair site of chronically injured nerve.

Methods

The authors used the rat tibial nerve injury and repair model to investigate the effects of application of physiological concentrations of TGF-β plus forskolin or forskolin alone in vivo at the repair site on gene and protein expression and axon regeneration at 6 weeks after nerve repair. They used gene expression profiling and immunohistochemical analysis of indicative activated proteins in Schwann cells to evaluate the effects of treatments on the delayed repair. They also quantified the regenerated axons distal to the repair site by microscopy of paraffin sections.

Results

Both treatment with forskolin only and treatment with TGF-β plus forskolin resulted in increased numbers of axons regenerated compared with saline-only control. There was robust activation and proliferation of both Schwann cells and macrophages reminiscent of the processes during Wallerian degeneration. The treatment also induced upregulation of genes implicated in cellular activation and growth as detected by gene array.

Conclusions

Addition of TGF-β plus forskolin to the repair after chronic nerve injury improved axonal regeneration, probably via upregulation of required genes, expression of growth-associated protein, and reactivation of Schwann cells and macrophages. Further studies are required to better understand the mechanism of the positive effect of TGF-β treatment on old nerve injuries.

Abbreviations used in this paper:BSA = bovine serum albumin; DAB = diaminobenzidine; GDNF = glial cell line–derived neurotrophic factor; HRP = horseradish peroxidase; RAG = regenerationassociated gene; RAGP = RAG product; TGF-β = transforming growth factor–β.

Abstract

Object

Although peripheral nerves can regenerate after traumatic injury, functional recovery is often suboptimal, especially after injuries to large nerve trunks such as the sciatic nerve or brachial plexus. Current research with animal models suggests that the lack of functional recovery resides in the lack of sufficient mature axons reaching their targets due to the loss of neurotrophic support by Schwann cells in the distal stump of injured nerves. This study was designed to investigate the effect of one-time application of transforming growth factor–β (TGF-β) at the repair site of chronically injured nerve.

Methods

The authors used the rat tibial nerve injury and repair model to investigate the effects of application of physiological concentrations of TGF-β plus forskolin or forskolin alone in vivo at the repair site on gene and protein expression and axon regeneration at 6 weeks after nerve repair. They used gene expression profiling and immunohistochemical analysis of indicative activated proteins in Schwann cells to evaluate the effects of treatments on the delayed repair. They also quantified the regenerated axons distal to the repair site by microscopy of paraffin sections.

Results

Both treatment with forskolin only and treatment with TGF-β plus forskolin resulted in increased numbers of axons regenerated compared with saline-only control. There was robust activation and proliferation of both Schwann cells and macrophages reminiscent of the processes during Wallerian degeneration. The treatment also induced upregulation of genes implicated in cellular activation and growth as detected by gene array.

Conclusions

Addition of TGF-β plus forskolin to the repair after chronic nerve injury improved axonal regeneration, probably via upregulation of required genes, expression of growth-associated protein, and reactivation of Schwann cells and macrophages. Further studies are required to better understand the mechanism of the positive effect of TGF-β treatment on old nerve injuries.

Injured nerves of the peripheral nervous system have the capacity to regenerate their axons and reinnervate denervated targets, especially after injuries to small nerve branches that are close to the target organs. This capacity for regeneration is supported mostly by the growth-permissive environment created by the Schwann cells of the peripheral nervous system located in the distal stumps of injured nerves. Injuries to larger nerve trunks, such as brachial plexus injuries, result in suboptimal functional recovery, even after excellent microsurgical repair, despite the growth-supportive capacity of the Schwann cells.13,41 Clinical experience and animal experiments have demonstrated that functional recovery is poor when injured nerves have to regenerate over a long distance to their targets and/or if any delay is incurred in the microsurgical repair. Our previous experiments showed that in both scenarios, injured neurons are denied access to their denervated targets (chronic axotomy) and Schwann cells do not interact with regenerating targets (chronic denervation), both of which lead to progressive loss of the capacity of injured neurons to regenerate their axons and Schwann cells to support axonal regeneration.15 In fact, when regeneration from the proximal nerve stump and through the distal nerve stumps is delayed in rat hindlimbs, the numbers of motor neurons that regenerate their axons through the distal nerve stumps and the numbers that reinnervate denervated muscle targets are reduced to 33% and 10%, respectively.12,36,37

We demonstrated in previous experiments that the capacity of injured neurons to regenerate and of Schwann cells to support regeneration is sustained if nerve repair is delayed for less than 4–6 weeks.36,37,40 Any delay beyond 4 weeks leads to progressive loss of regenerative capacity and functional recovery. Interestingly, the deterioration of the capacity of Schwann cells to support axonal regeneration after 4 weeks coincides with the decline in the number of infiltrated macrophages and the completion of Wallerian degeneration.22,27,28 Likewise, the expression of the markers of the growth-promoting, nonmyelinating phenotype of Schwann cells declines after 4 weeks.20 Thus, it appears that Schwann cells can maintain their growth-supportive phenotype only in the presence of active Wallerian degeneration.12,28 Macrophages' key roles in Wallerian degeneration include removing debris3 and producing cytokines that stimulate production of neurotrophic factors by nonneuronal cells of the distal nerve stumps,8,10,12,21 including transforming growth factor–β (TGF-β). After nerve injury, TGF-β is secreted into the injured nerves by invading macrophages2 and by Schwann cells themselves.5,8 Several lines of in vitro evidence have implicated TGF-β in the maintenance of the nonmyelinating, growth-promoting Schwann cell phenotype.6,8,9,27 TGF-β has also been shown to be essential for the neurotrophic effect of several neurotrophic factors, including a very potent neurotrophic factor for motor neurons, glial cell line–derived neurotrophic factor (GDNF).9,27,34 However, the role of TGF-β in axonal regeneration in vivo after nerve injury is poorly understood.

Previously, we demonstrated that in vitro incubation of long-term chronically denervated Schwann cells for 48 hours with TGF-β, and forskolin increased their capacity to support axonal regeneration of tibial motor neurons in vivo.38 In these experiments, nerve explants were taken from 6-month chronically denervated sciatic distal nerve stumps and were cultured with and without forskolin and TGF-β in vitro for 48 hours. The nerve explants were then used as a bridge. The mitogen exposure significantly increased the number of tibial motor neurons and showed that in vitro treatment of chronically denervated Schwann cells reversed the deleterious effect of chronic denervation on their capacity to support axonal regeneration in vivo. However, the exposure of the nerve stumps to both in vitro and in vivo environments made it difficult to ascertain the mechanism of the positive effects of exposure of the nerve explants to TGF-β. In the current experiments we investigated the effects of local application of TGF-β plus forskolin to the suture site after delayed repair of injured nerve. Our findings were similar to those of the in vitro experiments: The TGF-β-plus-forskolin treatment had a profound affect on gene and protein expression in the Schwann cells, driving them into the proliferative pathway, as well as improving regenerating axon numbers, suggesting that TGF-β applied to the repair site at the time of delayed nerve repair could be beneficial in a clinical setting.

Methods

Surgical Technique

Twenty-four Sprague-Dawley rats weighing about 200 g each were used in these experiments. All rats were housed in light-controlled (12 hours on) and temperaturecontrolled conditions with free access to food and water as per NIH guidelines. All procedures were approved and monitored by the Ochsner Animal Care and Use Committee following the National Research Council's Guide for the Care and Use of Laboratory Animals.

All rats were appropriately anesthetized (using ketamine and xylazine), and surgical procedures were performed using aseptic technique. To expose the sciatic nerve, an incision was made in the rat hindlimb and the tibial and common peroneal branches were exposed. The first surgery was transection of the tibial nerve, reflection of the proximal stump, and suturing into innervated muscle to prevent regeneration. At 8 weeks posttransection, the tibial nerve was reexposed in all animals and its proximal and distal stumps were repaired using 8–0 nylon suture. After microsurgical repair of the injured tibial nerve, we used a piece of Gelfoam as a carrier for our treatment solution. To apply the treatment directly at the suture site, we wrapped the Gelfoam around the tibial nerve repair. We divided the animals into 3 groups based on the treatment they received: 1) sham (saline control)—Gelfoam carrier containing physiological saline only; 2) forskolin—Gelfoam containing 2 ng/ml forskolin in saline; and 3) forskolin plus TGF-β—Gelfoam containing forskolin plus 0.5 μM TGF-β in saline. Regeneration was allowed for 6 weeks, and then the animals were again put in a state of general anesthesia and nerve sections were taken from the repaired tibial nerves. The nerve sections were processed depending on their final use, such that segments for RNA isolation were placed in RNAlater (Ambion, Inc.) or in 4% paraformaldehyde solution for subsequent immunohistochemical analysis. This last set of surgeries was terminal and all animals were humanely killed.

Axon Counts

Standard 5-μm paraffin sections were prepared, and selected sections were stained with hematoxylin and eosin to reveal the myelin structures and nuclei. Axon counts obtained from at least 3 sections from 2 animals from each treatment group were counted either from inherent fluorescence of the nerve or after staining.

Sections were observed at ×20 to ×40 magnification and photographed. Photomicrographs were analyzed with ImageJ,1 utilizing the particle-counting module with manual visual threshold adjustment. Counts were verified by manually counting selected images. Axon counts were analyzed by Kruskal-Wallis nonparametric ANOVA to determine statistical significance.

RNA Isolation

At least 3 animals from each treatment group and controls were randomly selected for RNA isolation. Nerve segments were quickly excised and immediately placed in RNAlater. RNA was isolated utilizing the small sample volume procedure from Qiagen Sciences. RNA was analyzed by NanoDrop spectroscopy and the quality was estimated by electrophoresis on an Agilent 2100 Bioanalyzer.

Preparation and Hybridization of cDNA and Data Acquisition and Analysis

All procedures were carried out by highly trained personnel of the Louisiana State University Health Science Center Microarray and Genome Bioinformatics Core utilizing Agilent Technologies Affymetrix gene chip technology. Procedures followed manufacturer's protocols for cDNA preparation and hybridization of rat genes using a 1.0 ST Array covering more than 27,000 gene-level probes. Arrays were analyzed on an Affymetrix GeneArray 7G scanner utilizing GeneChip RNA Expression Analysis Software. Two cutoff values were used, 2-fold and 6-fold, for both over- and underexpression. Activated genes were grouped by function, utilizing online DAVID software.24,25

Immunocytochemistry

Nerves from the distal nerve stumps were embedded in paraffin, and transverse 5-μm sections were cut and processed. Conditions for antibody incubation and washes were standard and consisted of a 2-hour blocking period with 1% bovine serum albumin (BSA) in Tris-buffered saline (TBS) plus 0.1% Triton X-100 (TBST) at room temperature. Incubation with primary antibody was overnight at 4° followed by three 5-minute washes in Tris-buffered saline with Tween (TBS-T) at room temperature. Primary antibodies were purchased from Abcam and included antibodies against CD68, Ki 67, S100, and activated caspase 3. Sections were reblocked after primary antibody washes with BSA due to the tendency of the secondary antibodies to nonspecifically bind to the nerve sections. Primary antibody visualization used Abcam secondary antibody coupled to horseradish peroxidase (HRP) with diaminobenzidine (DAB) as substrate. Mounting medium contained DAPI for visualization of DNA in nuclei.

Results

Gene Expression Profiles in the Reinnervated Distal Tibial Nerve Stumps

We used the gene array technique to determine the gene expression pattern in the distal nerve stumps of reinnervated tibial nerve stumps after 6 weeks of regeneration and treatment with saline or forskolin or TGF-β plus forskolin. While it is expected that the primary source of RNA from these segments derives from Schwann cells, infiltrating macrophages and support tissues such as fibroblasts were also present.

Gene profiling revealed a pattern of both gene activation and gene repression, even after 6 weeks of recovery (Table 1). We found that forskolin treatment led to 215 genes being activated at least 2-fold, with 77 activated over 6-fold. The addition of TGF-β resulted in an additional 222 activated genes, with 112 activated over 6-fold. Forskolin treatment also led to the downregulation of 586 genes at least 2-fold and 131 over 6-fold. The addition of TGF-β led to the downregulation of an additional 85 genes, with 18 of them showing over 6-fold reduction.

TABLE 1:

Effect of treatment on expression of regulated genes*

TreatmentUpregulatedUpregulated > 6-FoldDownregulatedDownregulated > 6-Fold
forskolin vs sham21577586131
TGF-β + forskolin vs sham476268710153
TGF-β + forskolin vs forskolin only2221128518

Values represent numbers of genes.

Effect of TGF-β.

Examination of forskolin-only treatment vs shamtreatment gene functional classification analysis by DAVID online software24,25 revealed 15 clusters of activated genes (Table 2). Functional groups consisted of genes indicative of the activated state, such as ribosomal-associated genes (15 genes), G protein–coupled receptor genes most numerously of the olfactory receptor family (65 genes), as well as activated-state genes involved mainly in metabolism and gene control.

TABLE 2:

Functional gene clustering in sham-treated versus forskolin-treated nerves

Gene Group & AFFYX Exon IDGene Name
Gene Group 1 (enrichment score 2.92, 15 genes)
 10887809similar to hypothetical protein
 10836208similar to 60S ribosomal protein L29 (P23)
 10762590hypothetical gene X15098; ribosomal protein, large P2; similar to protein P2
 10778209mitochondrial ribosomal protein S24
 10705420ribosomal protein L28
 10855557similar to ribosomal protein L31; hypothetical LOC502772; ribosomal protein L31
 10886629similar to ribosomal protein L6; ribosomal protein L6; pseudogene 1
 10751237similar to ribosomal protein L27; ribosomal protein L27
 10824596ribosomal protein S27-like; ribosomal protein S27
 10934530similar to ribosomal protein L38; 5 others
 10839874similar to ribosomal protein L31
 10781730similar to RIKEN cDNA 3100001N19
 10933578similar to ribosomal protein S27a
 10781954similar to ribosomal protein S13; ribosomal protein S13
 10714743hypothetical LOC498755; similar to Rpl7a; ribosomal protein L7a; 7 others
Gene Group 2 (enrichment score 1.15, 65 genes)
 10750636olfactory receptor 1548
 10731938olfactory receptor 1373
 10781441purinergic receptor P2Y, G-protein coupled, 5
 10909866similar to RIKEN cDNA 1600029D21
 10798509olfactory receptor 1654
 10718466vomeronasal 2 receptor, 23
 10837443olfactory receptor 456
 10740171glucagon receptor
 10733084olfactory receptor 1393
 10838414olfactory receptor 756
 10742927olfactory receptor 1418
 10717293trace-amine-associated receptor 1
 10847246olfactory receptor 736
 10765818olfactory receptor 1583
 10709473olfactory receptor 143
 10901190vomeronasal 2 receptor, 56; vomeronasal 2 receptor, 57
 10893346olfactory receptor 954
 10916822chemokine (C-X-C motif) receptor 5
 10726628olfactory receptor 300; olfactory receptor 297
 10857206vomeronasal 1 receptor, A12
 10837463olfactory receptor 469
 10908237olfactory receptor 1186
 10733900olfactory receptor 1454
 10837476olfactory receptor 477
 10900043olfactory receptor 984
 10855798vomeronasal 1 receptor, C19; vomeronasal 1 receptor, C20
 10750647olfactory receptor 1559
 10742925olfactory receptor 1417
 10709362olfactory receptor 41
 10709467olfactory receptor 137
 10712070olfactory receptor 306
 10712076olfactory receptor 309
 10900857olfactory receptor 1077
 10723475vomeronasal 2 receptor, 43; 4 others
 10830832similar to olfactory receptor Olfr104
 10744793olfactory receptor 1479
 10718789vomeronasal 1 receptor, D26
 10712057olfactory receptor 298; olfactory receptor 292
 10735744olfactory receptor 1490
 10709450olfactory receptor 122
 10724595olfactory receptor 208
 10827647olfactory receptor 1738
 10830828olfactory receptor 1729
 10893315, 10931180, 10900004olfactory receptor 1845; 3 others
 10900087olfactory receptor 1060
 108128795-hydroxytryptamine (serotonin) receptor 1A
 10858140olfactory receptor 823
 10744815similar to olfactory receptor Olr1504; 3 others
 10724337olfactory receptor 130
 10846984olfactory receptor 502
 10724413olfactory receptor 172; olfactory receptor 176
 10891681G protein-coupled receptor 68
 10847199olfactory receptor 703
 10703856olfactory receptor 3
 10861124G protein-coupled receptor 85
 10906439solute carrier family 2 (facilitated glucose transporter), member 13
 10909285olfactory receptor 1321; olfactory receptor 1320
 10823483vomeronasal 2 receptor, 46; pseudogene 71
 10862285olfactory receptor 803
 10859277G protein-coupled receptor, family C, group 5, member A
 10770892, 10701643similar to vomeronasal 2 receptor, 1; 8 others
 10938592pyrimidinergic receptor P2Y, G-protein coupled, 4
Gene Group 3 (enrichment score 0.89, 8 genes)
 10726093protease, serine, 36
 10721292T-kininogenase
 10862160trypsin X5
 10772055transmembrane protease, serine 11c
 10780594mast cell protease 9
 10780538mast cell protease 4-like 1
 10784065granzyme B
 10740930similar to testis-specific serine protease 1
Gene Group 4 (enrichment score 0.66, 7 genes)
 10907448keratin 73
 10712445hypothetical protein LOC685544; LOC683138; RGD1561705
 10869695similar to hypothetical protein FLJ20276
 10840910TPX2, microtubule-associated, homolog (Xenopus laevis)
 10884075family with sequence similarity 110, member C
 10747288type I keratin KA11
 10747145high sulfur protein B2F
Gene Group 5 (enrichment score 0.57, 4 genes)
 10798501similar to histone H2B 291B; histone cluster 1, H2bm; histone cluster 1, H2bn
 10733940histone cluster 1, H2ai; similar to histone 2a; 4 others
 10780714poly (ADP-ribose) polymerase family, member 4; M-phase phosphoprotein 8
 10798473similar to histone 3, H2ba; histone cluster 3, H2ba
Gene Group 6 (enrichment score 0.53, 8 genes)
 10778588Meis homeobox 1
 10719200cone-rod homeobox
 10936270reproductive homeobox 11
 10892002goosecoid homeobox
 10843100myelin transcription factor 1
 10862578homeo box A10
 10899603homeo box C6; homeobox C9
 10914456zinc finger protein 167
Gene Group 7 (enrichment score 0.43, 4 genes)
 10867848cyclin C
 10771465heterogeneous nuclear ribonucleoprotein D-like
 10820419spermatogenic leucine zipper 1
 10775080down-regulator of transcription 1
Gene Group 8 (enrichment score 0.41, 11 genes)
 10719200cone-rod homeobox
 10756497GS homeobox 1
 10850949E2F transcription factor 1
 10911309general transcription factor IIA, 2
 10863275, 10863277transcription factor 3
 10740919transcription elongation factor B (SIII), polypeptide 2
 10837006homeo box D9
 10737262suppressor of Ty 4 homolog 1 (S. cerevisiae)
 10823109mastermind like 3 (Drosophila)
 10922096transcription factor AP-2 beta
Gene Group 9 (enrichment score 0.26, 6 genes)
 10878270translocase of outer mitochondrial membrane 7 homolog (yeast)
 10930790solute carrier family 25 (mitochondrial carrier; phosphate carrier), member 23
 10843907surfeit 1; surfeit 4
 10803995NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 2
 10888127cytochrome c oxidase subunit VIIa polypeptide 2 like
 10741239NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 10
Gene Group 10 (enrichment score 0.17, 5 genes)
 10708782potassium channel tetramerisation domain containing 14
 10865738potassium voltage gated channel, shaker related subfamily, member 6
 10842049potassium channel, subfamily K, member 15
 10734212potassium inwardly-rectifying channel, subfamily J, member 12
 10816037amiloride-sensitive cation channel 5, intestinal

The same functional analysis of the TGF-β-plus-forskolin vs sham comparison identified 21 clusters of functional genes (Table 3). These clusters were similar to the pattern of gene activation seen with forskolin alone, with some modification. The highest enrichment scores were for genes involved in active cellular growth: 32 genes associated with ribosomes made up the highest enrichment score cluster, while the group of mitochondrial genes (22 genes) had the second highest enrichment score. Most other enriched clusters contained genes indicative of the active metabolic state, including genes that code for DNA-binding proteins (20 genes), histones (4 genes), and RNA-binding proteins (4 genes) as well as genes involved in potassium, sodium, and cation channels (8 genes). The third highest enrichment score was for a gene cluster consisting of 80 genes for olfactory receptors and a few other G-coupled receptor proteins such as the serotonin receptor.

TABLE 3:

Functional gene clustering in sham-treated versus TGF-β plus forskolin–treated nerves

Gene Group & AFFYX Exon IDGene Name
Gene Group 1 (enrichment score 5.95, 38 genes)
 10836208similar to 60S ribosomal protein L29 (P23)
 10851386similar to 60S ribosomal protein L13; ribosomal protein L13
 10752098similar to 60S ribosomal protein L12
 10802128ribosomal protein S23; similar to ribosomal protein S23
 10753533myc induced nuclear antigen; similar to myc induced nuclear antigen
 10886629similar to ribosomal protein L6; and 2 others
 10705420ribosomal protein L28
 10844946, 10723233similar to 40S ribosomal protein S17; ribosomal protein S17
 10933578similar to ribosomal protein S27a
 10847995ribosomal protein L35a; similar to ribosomal protein L35a
 10710846, 10806014, 10931410hypothetical gene NM_022504; ribosomal protein L36; and similar
 10839874similar to ribosomal protein L31
 10934530similar to ribosomal protein L38; and 5 others
 10911538similar to ribosomal protein L24-like; and 3 others
 10714743hypothetical LOC498755; similar to Rpl7a protein; and 9 others
 10781730similar to RIKEN cDNA 3100001N19
 10751237similar to ribosomal protein L27; ribosomal protein L27
 10787396similar to 60S ribosomal protein L18a; ribosomal protein L18A
 10933899ribosomal protein L5
 10890243similar to large subunit ribosomal protein L36a; & 4 others
 10742571, 10903216ribosomal protein L30
 10807017similar to 60S ribosomal protein L21
 10778209mitochondrial ribosomal protein S24
 10776064UTP3, small subunit (SSU) processome component, homolog (S. cerevisiae)
 10745607similar to FLJ34922 protein
 10758007mitochondrial ribosomal protein S17
 10820688similar to TGF beta-inducible nuclear protein 1 and 2 others
 10803676, 10729852similar to ribosomal protein S27a; ribosomal protein S27a
 10905605ribosomal protein L37a, pseudogene 1; 2; ribosomal protein L37a
 10764316, 10855557similar to ribosomal protein L31; hypothetical LOC502772
 10824596ribosomal protein S27-like; ribosomal protein S27
 10798841, 10716707similar to ribosomal protein L27a; and 2 others
Gene Group 2 (enrichment score 2.44, 23 genes)
 10861033NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4
 10888127cytochrome c oxidase subunit VIIa polypeptide 2 like
 10758978cytochrome c oxidase, subunit VIa, polypeptide 1
 10843907surfeit 1; surfeit 4
 10708775NADH dehydrogenase (ubiquinone) 1, subcomplex unknown, 2
 10844202, 10910338cytochrome c oxidase, subunit Va
 10930578COXII
 10861333NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 5
 10906544NADH dehydrogenase (ubiquinone) 1 beta subcomplex 4, pseudogene 1
 10803995NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 2
 107738914-nitrophenylphosphatase domain, non-neuronal SNAP25-like protein homolog 1
 10794935ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1
 10709093uncoupling protein 2 (mitochondrial, proton carrier)
 10930582cytochrome c oxidase subunit 3
 10818376similar to cytochrome c oxidase, subunit VIb polypeptide 1; VIb polypeptide 1
 10808426cytochrome c oxidase subunit IV isoform 1
 10917236succinate dehydrogenase complex, subunit D, integral membrane protein
 10839229, 10751348NADH dehydrogenase 1 beta subcomplex 4; similar to 3 others
 10930574cytochrome c oxidase subunit 1
 10741239NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 10
 10776873mitochondrial carrier triple repeat 1; & similar
 10788198solute carrier family 25 (mitochondrial carrier), member 4
Gene Group 3 (enrichment score 1.20, 8 genes)
 10712445, 10726938hypothetical protein LOC685544; LOC683138; RGD1561705
 10907448keratin 73
 10907468keratin 1
 10747214keratin 31
 10747288type I keratin KA11
 10726935hypothetical protein LOC682990; hypothetical protein LOC685347
 10747145high sulfur protein B2F
Gene Group 4 (enrichment score 0.88, 80 genes)
 10765818olfactory receptor 1583
 10712070olfactory receptor 306
 10709450olfactory receptor 122
 10901190vomeronasal 2 receptor, 56; vomeronasal 2 receptor, 57
 10938592pyrimidinergic receptor P2Y, G-protein coupled, 4
 10847088olfactory receptor 608
 10742954olfactory receptor 1436
 10717293trace-amine-associated receptor 1
 10857206vomeronasal 1 receptor, A12
 10893377olfactory receptor 932
 10733296olfactory receptor 1405
 10846984olfactory receptor 502
 10797469histamine receptor H 2
 10726628olfactory receptor 300; olfactory receptor 297
 10900045olfactory receptor 987
 10750647olfactory receptor 1559
 10916414olfactory receptor 1332
 10859277G protein-coupled receptor, family C, group 5, member A
 10894125olfactory receptor 1085; olfactory receptor 1084
 10855798vomeronasal 1 receptor, C19; vomeronasal 1 receptor, C20
 10723475vomeronasal 2 receptor, 43; and 4 others
 10900043olfactory receptor 984
 10847246olfactory receptor 736
 10712076olfactory receptor 309
 10900095olfactory receptor 1069
 10827647olfactory receptor 1738
 10750636olfactory receptor 1548
 10830832similar to olfactory receptor Olfr104
 10838414olfactory receptor 756
 10718780vomeronasal 1 receptor, D23; vomeronasal 1 receptor, D15
 10735744olfactory receptor 1490
 10724297olfactory receptor 120
 10701643similar to vomeronasal 2, receptor, 1; and 8 others
 10858618vomeronasal 2 receptor, 50
 10846912olfactory receptor 441
 108128795-hydroxytryptamine (serotonin) receptor 1A
 10865388vomeronasal 2 receptor, 49; vomeronasal 2 receptor, 51
 10742927olfactory receptor 1418
 10823483vomeronasal 2 receptor, 46; pseudogene 71
 10823462solute carrier family 33 (acetyl-CoA transporter), member 1
 10925687family with sequence similarity 174, member A
 10838449olfactory receptor 796
 10909285olfactory receptor 1321; olfactory receptor 1320
 10900067olfactory receptor 1024
 10733084olfactory receptor 1393
 10909190olfactory receptor 1244
 10779782olfactory receptor 1619
 10893346olfactory receptor 954
 10753563olfactory receptor 1555
 10900857olfactory receptor 1077
 10709467olfactory receptor 137
 10709362olfactory receptor 41
 10909109olfactory receptor 1199
 10848138olfactory receptor 785
 10703443formyl peptide receptor 2; formyl peptide receptor 2-like
 10849632inositol 1,4,5-triphosphate receptor interacting protein-like 1
 10724307olfactory receptor 127
 10718381similar to vomeronasal V1r-type receptor V1re22; E22
 10859296epithelial membrane protein 1
 10862285olfactory receptor 803
 10754201G protein-coupled receptor 156
 10798509olfactory receptor 1654
 10731938olfactory receptor 1373
 10781441purinergic receptor P2Y, G-protein coupled, 5
 10708535frizzled homolog 4 (Drosophila)
 10718466vomeronasal 2 receptor, 23
 10724337olfactory receptor 130
 10733900olfactory receptor 1454
 10837463olfactory receptor 469
 10900087olfactory receptor 1060
 10709511olfactory receptor 167; olfactory receptor 162
 10724413olfactory receptor 172; olfactory receptor 176
 10893381olfactory receptor 1012
 10847051olfactory receptor 550
 10830828olfactory receptor 1729
 10712057olfactory receptor 298; olfactory receptor 292
 10704306vomeronasal 1 receptor, J3
 10714200olfactory receptor 384
 10900055olfactory receptor 1002
 10868186cannabinoid receptor 1 (brain)
Gene Group 5 (enrichment score 0.83, 4 genes)
 10815795arginine/serine-rich coiled-coil 1
 10828823splicing factor, arginine/serine-rich 3; splicing factor
 10872836SYF2 homolog, RNA splicing factor (S. cerevisiae)
 10773809splicing factor 3a, subunit 1
Gene Group 6 (enrichment score 0.82, 4 genes)
 10896943similar to H3 histone, family 3B; 3A; H3.3; H2ai
 10798501similar to histone H2B 291B; histone cluster 1, H2bm; H2bn
 10733940histone cluster 1, H2ai; 2a; H2an; H2a; H2af
 10798473similar to histone 3, H2ba; histone cluster 3, H2ba
Gene Group 7 (enrichment score 0.60, 4 genes)
 10837056RNA binding motif protein 45
 10757786eukaryotic translation initiation factor 4H
 10845130RNA binding motif protein 43
 10774438zinc finger (CCCH type), RNA binding motif and serine/arginine rich 1
Gene Group 8 (enrichment score 0.56, 27 genes)
 10881824hexose-6-phosphate dehydrogenase (glucose 1-dehydrogenase)
 10909368, 10813721, 10787301, 10748800, 10769885, 10776679, 10905817, 10870014, 10787069, 10751599, 10840272, 10937400, 10905815, 10865622, 10754880, 10759492, 10778888, 10787808, 10935263, 10916014, 10919555, 10754991, 10876834, 10799940similar to glyceraldehyde-3-phosphate dehydrogenase & others
 10778661malate dehydrogenase 1, NAD (soluble)
 10865527triosephosphate isomerase; similar to triosephosphate isomerase & others
Gene Group 9 (enrichment score 0.46, 5 genes)
 10733039guanine nucleotide binding protein (G protein), beta polypeptide 2 like 1
 10750391chromatin assembly factor 1, subunit B (p60)
 10836794dynein cytoplasmic 1 intermediate chain 2
 10896269WD repeats and SOF1 domain containing
 10773205WD repeat domain 1
Gene Group 10 (enrichment score 0.37, 6 genes)
 10771465heterogeneous nuclear ribonucleoprotein D-like
 10867848cyclin C
 10798169forkhead box Q1
 10820419spermatogenic leucine zipper 1
 10845087MYC associated factor X
 10775080down-regulator of transcription 1
Gene Group 11 (enrichment score 0.31, 10 genes)
 10885050AT rich interactive domain 4A (Rbp1 like)
 10742740, 10725155Y box binding protein 1
 10891594forkhead box N3
 10734910hairy and enhancer of split 7 (Drosophila)
 10863277transcription factor 3
 10737262suppressor of Ty 4 homolog 1 (S. cerevisiae)
 10772194HOP homeobox
 10782454thyroid hormone receptor beta
 10708809remodeling and spacing factor 1
Gene Group 12 (enrichment score 0.30, 8 genes)
 10708782potassium channel tetramerisation domain containing 14
 10710669calcium channel, voltage-dependent, gamma subunit 3
 10718561calcium channel, voltage-dependent, gamma subunit 8
 10932726transient receptor potential cation channel, subfamily C, member 5
 10925228transient receptor potential cation channel, subfamily M, member 8
 10865738potassium voltage gated channel, shaker related subfamily, member 6
 10734212potassium inwardly-rectifying channel, subfamily J, member 12
 10816037amiloride-sensitive cation channel 5, intestinal

Axonal Regeneration 6 Weeks After Nerve Repair

Six weeks after microsurgical repair and treatment with either saline, forskolin alone, or TGF-β plus forskolin, transverse paraffin sections distal to the nerve repair were examined by microscopy to determine the number of regenerated myelinated axons.

Treatment with either forskolin or TGF-β plus forskolin increased the number of axons compared with saline control (Fig. 1), although the increase was significant only in sections from the TGF-β-plus-forskolin group (p = 0.007, Kruskal-Wallis nonparametric ANOVA).

Fig. 1.
Fig. 1.

Axon counts of transverse sections of tibial distal nerve stump after 8 weeks of chronic denervation followed by 6 weeks of regeneration with and without addition of forskolin and TGF-β. The mean values were as follows: for saline control (cont), 1288 ± 151 axons; for forskolin alone (for), 1729 ± 214 axons; for forskolin plus TGF-β (for + TGF), 2108 ± 224 axons. The difference between the saline control and forskolin plus TGF-β groups was statistically significant (p = 0.007, Kruskal-Wallis nonparametric ANOVA). Error bars indicate SDs.

Immunohistochemical Detection of Regeneration-Associated Marker Proteins

To determine the effect of a one-time addition of physiological concentrations of forskolin and TGF-β at the nerve repair suture site on the metabolic status of the Schwann cells and infiltrating macrophages after 6 weeks of regeneration, immunohistochemical analysis was carried out on transverse paraffin sections of distal tibial nerve stumps (Fig. 2). Antibodies to specific cellular and metabolic markers were used to detect Schwann cell activation (anti-S100), apoptosis (anti–activated caspase 3), activated macrophages (anti-CD68), and a general cell proliferation marker (anti-Ki 67) and were visualized using HRP-conjugated secondary antibody and DAB as substrate (all Abcam products).

Fig. 2.
Fig. 2.

Photomicrographs of sections of distal tibial nerve stumps obtained 6 weeks after repair of previously transected nerve and application of Gelfoam impregnated with saline (control), 2 ng/ml forskolin in saline, or forskolin and 0.5 μM TGF-β in saline. Sections were immunohistochemically stained for the presence of caspase 3, Ki 67, S100, and CD68. Antibody dilutions were 1:500 to 1:1000, and bound antibody was visualized by HRP-coupled secondary antibody with DAB as substrate. Original magnifications ×200.

A striking dichotomy between treated and nontreated nerve bundles was revealed after 6 weeks of regeneration. The animals that received forskolin or TGF-β plus forskolin all showed strong staining for S100, CD68, activated caspase 3, and Ki 67 (Fig. 2). However, the animals in the saline-only group did not express detectable S100 in their Schwann cells (Fig. 2, panel 3A), and sections from this group did not contain activated macrophages (as evident by lack of CD68 staining; Fig. 2, panel 4A) or contain cells undergoing apoptosis (caspase-3 staining; Fig. 2, panel 1A) or undergoing proliferation (Ki-67 staining; Fig. 2, panel 2A). There were sporadic instances where sections of saline control animal nerve did show isolated cells with activated caspase-3 and Ki 67, both within the axon bundles and in supporting cell types, indicating that lack of staining was due to the absence of the proteins and not nonspecific antibody failure (not shown).

All animals treated with TGF-β plus forskolin expressed S100 in Schwann cells associated with axons, indicative of an activated state, even after 6 weeks of regeneration. Also, there were activated macrophages observed in the distal tibial nerve stumps of the treated animals that were generally spread evenly throughout the axons (Fig. 2, panels 4B and 4C). Interestingly, in addition to Schwann cell activation and proliferation, we also observed cells actively undergoing apoptosis, as evidenced by nucleus-associated active caspase-3 (Fig. 2, panels 1B and 1C).

No cells within saline-only nerve bundles were undergoing mitosis, as shown by an absence of Ki 67 staining (Fig. 2, panel 2A); in the treatment group there were many mitotic cells both associated with axons (Fig. 2, panels 2B and 2C) and with supporting cell types (not shown) in the perineurium.

Discussion

In this study, we extended our observations of the effect of forskolin and TGF-β plus forskolin from our previous “in vitro to in vivo” experiments to a direct in vivo application of TGF-β plus forskolin at the nerve repair site using a rat nerve injury model that mimics clinic scenarios. We used a delayed nerve repair paradigm that allowed us to evaluate the effects of our treatment under conditions of chronic axotomy and denervation that mimic conditions after injury to large nerve trunks or delayed nerve repair. We not only characterized the regenerative effect of the treatment with TGF-β but also investigated the cellular and molecular changes after our treatments compared with saline control.

Injuries to large nerve trunks such as the brachial plexus often result in poor functional recovery despite excellent microsurgical repair.13,14,17,36,37,39,42 Management of the most common type of nerve injuries, namely stretch injuries, has a built-in delay by way of expectant observation. Patients are observed for a period of 3–4 months both clinically and with electrodiagnostic studies to check for return of function. Microsurgical repair is offered only if there is no evidence of spontaneous recovery after 3–4 months. Carefully designed experimental studies that mimic the clinical scenarios as described have established that poor functional recovery is due to chronic Schwann cell denervation, chronic neuronal axotomy, and misdirection of regenerating axons.13,14,36,37 The delay in surgical repair subjects the distal stumps of the injured nerves to chronic denervation and the injured neurons to chronic axotomy, which deleteriously affect axonal regeneration and functional recovery. Another factor that contributes to chronic denervation and chronic axotomy is the very sluggish rate of axonal regeneration (1–3 mm/day). After a proximal nerve injury, such as a brachial plexus injury, it can therefore take years to reinnervate muscles of the hand, and during this time provision of neurotrophic support for the injured neurons and support of regenerating axons (by Schwann cells) may be lost.14,36,37 In fact, experimental studies have demonstrated that upregulation of regeneration-associated genes (RAGs) is short-lived, such that 4–6 weeks after a nerve injury the expression of RAGs is lost while axons are still regenerating.8,17,18,28 This mismatch in the duration of expression of RAGs and rate of axonal regeneration presents a significant challenge. Addressing this problem requires the development of new molecular strategies for 1) sustaining the duration of expression of RAGs, 2) reintroducing RAGs, or 3) accelerating the rate of axonal regeneration.18

Previous work has shown that in vitro Schwann cells respond to TGF-β in the presence of forskolin synergistically.20,26 In our experiments, we used TGF-β plus forskolin applied at the suture site immediately after microsurgical repair of chronic nerve injuries in an attempt to reactivate the expression of RAGs and promote axonal regeneration. We used a rat model of nerve injury and repair to mimic the clinical scenarios and tested the efficacy of direct application of Schwann cell mitogens at the suture site to promote nerve regeneration despite chronic Schwann cell denervation and neuronal axotomy. As demonstrated in our results, these mitogens evoke a series of molecular events that lead to 1) activation and repression of RNA transcription as revealed by RNA profiling, 2) reactivation of Schwann cells, and 3) recruitment of activated macrophages to the injury site, a critical step in the removal of myelin and axonal debris2,31 during Wallerian degeneration and in the production of neurotrophic factors and other RAGs essential for nerve regeneration. They also induced Schwann cell proliferation and to a lesser degree apoptosis, probably related to control of the Schwann cell population.

Gene Profile Change During Axonal Regeneration With or Without TGF-β Treatment

After nerve injury, Schwann cells change their pattern of gene expression from a myelinating to a nonmyelinating phenotype by downregulating myelination-associated genes and upregulating regeneration-associated genes.4,12 Gene profiling revealed a pattern of both gene activation and downregulation, as expected from nerves undergoing Wallerian degradation and subsequent Schwann cell activation.

Bosse et al.4 used an acute nerve crush injury model and found that the pattern of gene activation/repression was a combination of novel injury-dependent gene activation and reactivation of developmental genes. Our results are from a “snapshot” of gene activity after 6 weeks of posttreatment regeneration, and like Bosse et al.,4 we found both activated and repressed transcription. Also, the genes activated and repressed were similar in function to the ones Bosse et al.4 identified and were primarily metabolic genes as well as genes involved with cell signaling and transcriptional and translational control.

Li et al.29 recently completed a study of gene expression following sciatic nerve transection in which they examined the gene expression profile for 14 days posttransection in the proximal stump. They found a set of genes being activated that was similar to the set we found; for some of the gene types, the number of genes affected and the extent of activation were significantly higher than the values we obtained, but their observations were made during active Wallerian degeneration, whereas our results were obtained after 8 weeks of denervation and 6 weeks after repair plus treatment. Interestingly, they also found activation of many olfactory receptor genes and showed that they were expressed in the Schwann cells.29 Olfactory receptor expression has been reported previously in nonolfactory tissues such as germ cells19 and heart.7 The function of this expression of olfactory receptors in the Schwann cells remains to be elucidated.

Comparison of the gene expression patterns between the two treatments, forskolin and TGF-β plus forskolin, utilizing the DAVID set of online functions, revealed that while there were some differences in the genes differentially expressed, the interesting differences are in the enrichment score and the number of genes in each group, as well as some differentially expressed groups (Tables 13). For example, the highest enrichment score in both treatment groups includes primarily ribosomal protein components, while in the group treated with TGF-β plus forskolin the enrichment score is twice that of the forskolin-only group, and the TGF-β-plus-forskolin group had over twice the number of activated genes.

Overall, the addition of TGF-β led to more genes remaining active at higher levels (compared with sham treatment), in keeping with the known effect of TGF-β as a modulator of gene expression. Gene groups activated in the TGF-β-plus-forskolin group and not in the forskolinonly group include mitochondrial proteins as well as transcriptional and translational proteins. As it is expected that gene activity affected by the treatments would be in decline due to the single act of treatment at the time of repair and observation 6 weeks later, the activation of genes in the TGF-β-plus-forskolin group that were not active in the forskolin-only group could be due to a direct effect of TGF-β treatment, the modulatory effect of TGF-β, or a combination of both. Further times of observation could distinguish among the possibilities, but in any case, TGFβ did affect the gene expression patterns, and only with the addition of TGF-β to the forskolin treatment was the increase of axons over sham treatment significant.

In light of the powerful ability of forskolin to affect gene expression, the modulatory effects of TGF-β secreted by both activated macrophages and activated Schwann cells would appear to be important in order for significant axon regeneration to proceed.3,11

Effect of TGF-β Treatment on Axonal Regeneration, Schwann Cell Activation, and Expression of RAGs

After nerve injury, the distal nerve stump of the injured nerve undergoes Wallerian degeneration, during which the Schwann cells switch from a myelinated, quiescent phenotype to a metabolically and proliferative activated state,12 and this is mirrored in the change of expressed proteins, shifting from the mature myelinated phenotype to expression of growth-associated products and activated Schwann cell products such as S100,16 glial fibrillary acidic protein (GFAP),43 and neural cell adhesion molecule (NCAM).35 As we had previously shown, in vitro treatment of Schwann cells with forskolin and TGF-β enhanced their capacity to support regeneration of injured axons when used as a graft between freshly injured nerves. However, the experimental paradigm used did not mimic the scenarios we often encounter in patients with nerve injuries. Most patients sustain injuries to large nerve trunks or present with chronic injuries. In both scenarios, the injured neurons and Schwann cells are subjected to chronic axotomy and chronic denervation, respectively. Moreover, our study did not explore in detail the molecular mechanisms of the positive effect of TGFβ treatment.

Therefore, we designed the current experiments to test the ability of TGF-β to promote axonal regeneration after chronic nerve injuries, using the well-established rat model of nerve injury and repair, and to investigate the mechanism of action of TGF-β treatment by examining expression of RAGs. In these experiments, we applied the mitogens to the suture site at the time of repair, but after 8 weeks of chronic Schwann cell denervation and neuronal axotomy. We found that treatment with TGFβ plus forskolin at the repair site resulted in statistically significant increased numbers of regenerated axons at 6 weeks postrepair compared with the saline control (Fig. 1). Although the numbers of axons found in the sections of nerves treated with forskolin alone exhibited increased axon numbers, the increase was not statistically significant. The animals treated with TGF-β plus forskolin had the greatest numbers of axons that crossed the repair site (Fig. 1), suggesting that the combination of the two mitogens did result in increased numbers of axons that could reinnervate their targets. Since forskolin and TGF-β would have their greatest effect on the Schwann cells and not on the growing axonal cone, reactivation of the Schwann cells at the time of repair suggests that Schwann cell activation is required for effective axonal growth cones to cross the repair site. Xu et al.44 found that while the proximal nerve stump, even when subjected to chronic axotomy, retained a normal complement of axons, the number of axons reaching target endpoints was nevertheless much reduced. They suggested that exogenous neuroprotective mitogens would have their greatest effect on the repair site and distal nerve trunks where Schwann cells are deprived of the mitogen milieu provided by the axonal growth cones.44

The increases in axon numbers distal to the repair site in the TGF-β–treated nerves were mirrored in their immense metabolic differences, as revealed by expression patterns of products of RAGs—regeneration-associated gene products (RAGPs). There are clear differences in the expression patterns of RAGPs between saline control nerves and TGF-β–treated nerves. The saline controls expressed very little to no RAGPs, suggesting lack of Schwann cell or macrophage activation (Fig. 2, panels 1A–4A). In contrast, both forskolin-treated groups expressed within their nerve bundles RAGPs indicative of an activated state and the presence of activated macrophages (Fig. 2, panels 1B–4B and 1C–4C). Also, cell proliferation as measured by Ki 67 detection indicated that significant cell division, presumably of Schwann cells, was still occurring, even after 6 weeks of regeneration (Fig. 2, panels 2B and 2C). The mitogens would be expected to immediately begin to diffuse and be degraded within a few days at most.23 However, their effects on the metabolic state of the nerve bundles were still present after 6 weeks, suggesting that one-time treatment at the time of repair can reactivate Schwann cells and sustain their activated state and hence may influence the influx and activation of macrophages in the distal nerve stump, since Schwann cells are known to secrete macrophage chemoattractive factor.30,31 This sustained interaction between macrophages and Schwann cells may play a key role in the maintenance of Schwann cell activation in the distal nerve stump even after 6 weeks of regeneration, which may, in turn, be responsible for the robust regenerative response to TGF-β treatment.32,33

Numerous cells, presumably Schwann cells, were observed undergoing active apoptosis as detected by nucleus-associated active caspase-3 in sections from the forskolin and forskolin plus TGF-β groups (Fig. 2, panels 1B and 1C), in contrast to sections from the saline controls, where few actively dividing or apoptotic cells were observed (Fig. 2, panels 1A and 2A). Schwann cells that are not involved in myelinating axons and are nevertheless in an activated state undergo apoptosis and are cleared by macrophages.3,21,45 The activated macrophages are attracted to factors released from the cellular debris produced by the presence of active apoptotic cells, such that as long as apoptotic cells are present, activated macrophages will be also.

Previous work has shown Wallerian degeneration takes place after nerve damage, and during this process Schwann cells switch from a myelinated, quiescent phenotype to a metabolically activated and proliferative state.4,12,17 Macrophages also infiltrate the injured nerve and secrete many cytokines, including TGF.3,12 Wallerian degeneration is essentially complete after 4 weeks, and previous studies have determined that after 4 weeks of chronic denervation axotomy, activated Schwann cells progressively lose their ability to support regeneration of axons over a further period of several weeks. Our past work and that of others has established that the loss of numbers of regenerated axons as a function of duration of chronic denervation is due, in large extent, to the loss of the ability of the Schwann cells to support the growing axonal growth cones.13,14,36,37 The Schwann cell loss of response may be in part due to the decline in the growth factors secreted by the activated macrophages such as TGF-β.3 TGF-β has many effects throughout development as well as in wound healing and carcinogenesis.23,32,35 While most cell types express TGF-β receptors,9 the effect of TGF-β depends on the metabolic status of the cell, as well as the presence of other growth factors with effects that are modulated by TGF-β.12,32,34 For instance, Schwann cells in culture respond to TGF-β in the presence of serum by suppressing the myelinating phenotype and promote the expression of the growth supportive state.6,8

Our results suggest that application of either forskolin alone or TGF-β plus forskolin at the time of repair of an 8-week old nerve injury leads to the activation of at least some of the remaining Schwann cells in the distal nerve stumps, as shown by the continued expression of activated Schwann cell markers even after 6 weeks of regeneration. Both treatments induced Schwann cell activation, as shown by expression of S100 and proliferation as demonstrated by Ki 67 staining, both of which are crucial for the growthsupportive state of Schwann cells in the distal nerve stumps. These findings are in sharp contrast to the lack of Schwann cell activation or proliferation in saline controls.

The exact mechanism of Schwann cell activation by forskolin and TGF-β has not been fully elucidated; however, results from in vitro Schwann cell culture studies suggest that the switch from the quiescent myelinating phenotype to the proliferative activated state is key and that these mitogens are sufficient to elicit this switch.11,12,38 Products of genes activated in the proliferative condition include those proteins that are responsible for adhesion and Schwann cell–axon interactions, such as the neurotrophin receptor, p75, neuregulin, and their Erb family of receptors as well as the potent neurotrophic factor GDNF.6 Interestingly, the effects of GDNF have been found to require the presence of TGF-β both in vitro and in vivo.34 Our past in vitro results with TGF-β treatment of explants reintroduced to nerve repair sites coupled with the lasting effects of a one-time treatment of the repair site with TGF-β and forskolin suggest that this treatment regimen could have clinical relevance for increasing the number of neurons that regenerate across the repair site and into the distal nerve stumps even in chronic injuries. We are investigating the roles of other RAGPs to further elucidate the effects of our treatment on Schwann cells. Further analysis of the gene products of the gene array will also shed more light on the molecular mechanism of the positive effects of TGF-β on Schwann cells after chronic injuries.

Conclusions

The results of our current study strongly suggest that treatment of chronically injured nerves with forskolin and TGF-β at the time of microsurgical repair is sufficient to reactivate Schwann cells and maintain the activated state for at least 6 weeks. The reactivation of Schwann cells results in improved regeneration of axons, which is essential for improved return of function after nerve injuries. This has important clinical relevance because most patients present to the clinic with chronic nerve injuries, and the chronicity may be contributing to the observed poor functional recovery.

Our results demonstrate that that the positive effects of TGF-β treatment may be due to strong Schwann cell activation and proliferation and recruitment of macrophages to the injury site of distal nerve stumps. In essence, the TGF-β effect may be due to a “re-creation” of the Wallerian degeneration milieu in the distal nerve stumps where Schwann cells and macrophages can interact and promote axonal regeneration. Further studies are required to better understand the nature of the interactions between Schwann cells and macrophages after TGF-β treatment that leads to robust axonal regeneration.

Disclosure

This study was funded by the American Society for Peripheral Nerve/Plastic Surgery Educational Foundation combined pilot research grant # 175839. The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

Author contributions to the study and manuscript preparation include the following. Conception and design: Sulaiman. Acquisition of data: Dreesen. Analysis and interpretation of data: both authors. Drafting the article: Dreesen. Critically revising the article: both authors. Reviewed submitted version of manuscript: both authors. Approved the final version of the manuscript on behalf of both authors: Sulaiman. Statistical analysis: Dreesen.

This article contains some figures that are displayed in color online but in black-and-white in the print edition.

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Article Information

Address correspondence to: Wale Sulaiman, M.D., Ph.D., Department of Neurosurgery, Ochsner Health Systems, 1514 Jefferson Hwy., New Orleans, LA 70121. email: wsulaiman@ochsner.org.

Please include this information when citing this paper: published online July 18, 2014; DOI: 10.3171/2014.4.JNS131251.

© AANS, except where prohibited by US copyright law.

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    Axon counts of transverse sections of tibial distal nerve stump after 8 weeks of chronic denervation followed by 6 weeks of regeneration with and without addition of forskolin and TGF-β. The mean values were as follows: for saline control (cont), 1288 ± 151 axons; for forskolin alone (for), 1729 ± 214 axons; for forskolin plus TGF-β (for + TGF), 2108 ± 224 axons. The difference between the saline control and forskolin plus TGF-β groups was statistically significant (p = 0.007, Kruskal-Wallis nonparametric ANOVA). Error bars indicate SDs.

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    Photomicrographs of sections of distal tibial nerve stumps obtained 6 weeks after repair of previously transected nerve and application of Gelfoam impregnated with saline (control), 2 ng/ml forskolin in saline, or forskolin and 0.5 μM TGF-β in saline. Sections were immunohistochemically stained for the presence of caspase 3, Ki 67, S100, and CD68. Antibody dilutions were 1:500 to 1:1000, and bound antibody was visualized by HRP-coupled secondary antibody with DAB as substrate. Original magnifications ×200.

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