Mol Biol Rep (2015) 42:603–609 DOI 10.1007/s11033-014-3805-2
Axonal regeneration in early stages of sciatic nerve crush injury is enhanced by a7nAChR in rats Dewei Wang • Xuming Wang • Shuo Geng Zhenggang Bi
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Received: 22 August 2014 / Accepted: 27 October 2014 / Published online: 5 November 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract This study investigated the role of alpha 7 nicotinic acetylcholine receptor (a7nAChR) in axonal regeneration after crush injury to the rat sciatic nerve. The time course of a7nAChR expression following injury was assessed by immunohistochemistry and western blotting, and local inflammation, as indicated by the expression of tumor necrosis factor (TNF)-a, was detected by enzymelinked immunosorbent assay. Axonal regeneration was evaluated by the pinch-test, morphometric analysis, and by measuring growth-associated protein 43 expressions. Local a7nAChR expression increased on day 1, peaked on day 3, and remained elevated on day 5 following nerve injuries. Prominent a7nAChR immunoreactivity was observed in Schwann cells, endothelial cells of the capillaries, and a small number of inflammatory cells. Application of the selective a7nAChR agonist PNU-282987 decreased TNF-a level and enhanced axonal regeneration, but this effect was blocked by concomitant treatment with methyllycaconitine, a a7nAChR antagonist. The results indicate that the local expression of a7nAChR is increased during the early stages of sciatic nerve injury, and application of a a7nAChR agonist promotes axonal regeneration by suppression of TNF-a-mediated inflammation. The a7nAChR can act as a neuroprotective agent and a7nAChR activation may be a useful therapeutic strategy to treat peripheral nerve injury. Keywords a7nAChR PNU-282987 TNF-a Axonal regeneration Crush injury
D. Wang X. Wang S. Geng Z. Bi (&) Department of Orthopedic Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin 150001, Heilongjiang, China e-mail: [email protected]
Introduction Peripheral nerve injury, particularly in the upper limb, can have devastating consequences, including chronic pain, restricted function and mobility, and diminished quality of life. In contrast to the brain and spinal cord, the peripheral nervous system has the ability to regenerate [1], and the repair process begins almost immediately after injury [2]. In axonotmesis, a 1–2-day latency during which axons cross the injury site is followed by regeneration at a steady rate along the distal nerve, aided by reactive Schwann cells and preserved endoneurial tubules that enhance axon elongation and facilitate adequate target reinnervation [3]. Surgical repair does not guarantee full functional recovery to damaged nerves [4], but therapeutic manipulation of the molecular response to injury can promote neuronal survival by stimulating the growth of axons across large gaps between nerve stumps, thereby maximizing the accuracy of target reinnervation and minimizing any potential neuropathic pain [5]. The distal segment of transected or crushed peripheral nerves undergoes Wallerian degeneration, which is essential for subsequent repair [6]; an important event during the early post-injury stages is the inflammatory response. Tumor necrosis factor (TNF)-a, a proinflammatory cytokine, is upregulated early and transiently at the site of nerve injury and plays a crucial role in Wallerian degeneration as an initiator of local inflammation [7]. While an overabundance of cytokine activity can impair organ function, induce shock, and aggravate tissue injury [8], immediate therapy with a TNFa antagonist enhances the rate of axon regeneration in injured peripheral nerves [9]. These data suggest that mitigating the inflammatory response after peripheral nervous injury could be a strategy for reducing damage and enhancing functional repair. In recent studies, agonists of
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the alpha 7 nicotinic acetylcholine receptor (a7nAChR)— ion channels that are expressed in neurons, astrocytes, epithelial cells, adipocytes, fibroblasts, keratinocytes, and immune cells were found to have anti-inflammatory and protective effects in various diseases [10–13]. However, the role of a7nAChR in post-injury repair of the rat sciatic nerve has not well been examined. This study characterized a7nAChR expression and activation of the cholinergic anti-inflammatory pathway in the early stages of sciatic nerve crush injury to assess whether a7nAChR can enhance axonal regeneration through attenuation of the inflammatory response. The spatio-temporal expression profile of a7nAChR was determined and the effects of a selective a7nAChR agonist on TNF-a levels and axon growth were evaluated.
Materials and methods Reagents and antibodies The following antibodies were used in this study: polyclonal rabbit anti-a7nAChR (Abcam, Cambridge, MA, USA); anti growth-associated protein (GAP)-43 (Beijing Bioss Technologies, Inc., Beijing, China); and monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The rat TNF-a Quantikine enzyme-linked immunosorbent assay (ELISA) kit was purchased from Sun Biomedical Technology Co., Ltd. (Beijing, China). Drug protocols [N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride] (PNU-282987) and methyllycaconitine (MLA), a7nAChR agonist and antagonist, respectively, were purchased from Abcam. Drug solutions were prepared in saline immediately before use and administered according to previously published protocols [14]. Both drugs were injected intraperitoneally at 12 mg/kg for PNU-282987 and 6 mg/kg for MLA at 45 and 60 min post-injury once a day for 4 days, with saline injected as a control. Thus, the experimental groups were saline ? saline (placebo, PLA), saline ? PNU-282987 (PNU), MLA ? PNU-282987 (MLA ? PNU), and MLA ? saline (MLA).
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protocol was reviewed and approved by the Institutional Animal Care Committee at the Harbin Medical University. Animals (n = 190) were assigned to the following groups: naı¨ve (n = 10), sham (n = 40), crush (n = 40), PLA (n = 25), PNU (n = 25), MLA ? PNU (n = 25) and MLA (n = 25). The sham and crush groups were used to evaluate a7nAChR and TNF-a expression (days 0.25, 1, 3, 5), while the remaining groups were used to examine changes in TNF-a (days 1 and 3) and axonal regeneration by pinch test (day 5), GAP-43 expression (day 5) and morphometric analysis (days 7 and 9). The model of sciatic nerve crush injury in rats was described in a previous study [15]. Ketamine (100 mg/kg) and xylazine (10 mg/kg) were administered by intramuscular injection. Animals were placed prone under sterile conditions, and the right sciatic nerve was unilaterally exposed through a skin incision extending from the greater trochanter to the mid-thigh followed by an incision that split the muscle. The nerve was crushed 5 cm distal to the sciatic notch using a 2-mm straight vascular clamp for 30 s to create a 2 mm-long crush injury, and the site was labeled by tying a 6-0 nylon suture to the adjacent muscle. The sham operation consisted of unilateral sciatic nerve exposure. Animals (n = 5 per group) were sacrificed by CO2 asphyxiation and sciatic nerves were harvested by cutting the nerve directly above the site of injury and 15 mm distally, or the comparable length of nerve in sham-operated animals. Western blotting Proteins were extracted from homogenized sciatic nerve samples with lysis buffer, and resolved by 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred to a polyvinylidene difluoride membrane and blocked with 5 % nonfat dry milk. The membrane was then incubated with primary antibodies overnight at 4 °C with gentle shaking and was exposed to anti-mouse immunoglobulin (Ig)G horseradish peroxidase-conjugated antibody for 1 h at room temperature, then treated with enhanced chemiluminescence solution (Millipore, Billerica, MA, USA) and exposed on Hyperfilm (GE Healthcare, Piscataway, USA). Quantitative analysis was performed and data were expressed as a mean ± SEM (n = 5). Immunohistochemistry
Animals and surgical procedure Male Wistar rats (250–300 g) were purchased from Changchun Yisi Experimental Animal Technology Co., Ltd. (Changchun, China). All animals were handled in compliance with the National Research Council’s Guide for the Care and Use of Laboratory Animals. The animal
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Rats were anesthetized and then transcardially perfused with 0.9 % saline solution followed by 4 % paraformaldehyde. Sciatic nerves were fixed with 4 % paraformaldehyde in PBS, and embedded in paraffin. Serial longitudinal sections were cut at a thickness of 4 lm, then deparaffinized and rehydrated. Nonspecific immunoreactivity was blocked by
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incubation with normal goat serum for 2 h at room temperature, and sections were incubated with anti-a7nAChR antibody (1:100) in a humid chamber at 4 °C overnight, followed by treatment with the Histostain-Plus streptavidinperoxidase kit according to the manufacturer’s instructions. Sections were counterstained with hematoxylin and eosin. As a control, a subset of sections was incubated with normal rabbit IgG or PBS instead of the primary antibody. The expression of a7nAChR, represented by the staining intensity at 9400 magnification, was assessed by two independent investigators who were blinded to the experimental groups and was categorized as no expression (-), or weak (?), moderate (??), or high (???) expression. ELISA The protocol for ELISA has been previously described [16]. Sciatic nerves were homogenized in ice-cold PBS containing a protease inhibitor cocktail (Roche, Mannheim, Germany). After centrifugation at 10,0009g and 4 °C for 10 min, the supernatant was removed and the pellet was rehomogenized in the same volume of homogenization buffer, with Triton X-100 added to a final concentration of 0.01 %. Samples were vortexed and centrifuged, and supernatants were assayed in duplicate using the TNF-a ELISA kit according to the manufacturer’s instructions. Data were expressed as a mean ± SEM (n = 5). Nerve pinch test The rate of axonal regeneration was evaluated using the nerve pinch test [9, 17]. Based on the predicted speed of sciatic nerve regrowth, the test was performed between 3 and 7 days after injury [17]. The sciatic nerve and its tibial nerve branch were exposed in lightly anesthetized rats. Consecutive 1-mm-long segments of the tibial nerve were pinched with a pair of fine forceps, starting from the distal end of the nerve and proceeding in the proximal direction until a reflex response consisting of a contraction of back muscles was observed. The distance between the most distal point of the nerve that produced a reflexive withdrawal response and the stitch marking the original crush site was measured and identified as the regeneration distance, and expressed as a mean ± SEM (n = 5). Morphometric evaluation Histological assessments were performed as previously described [18, 19]. On days 7 and 9 post-surgery, rats were anesthetized and perfused transcardially with freshly prepared 0.5 % glutaraldehyde in 0.1 M phosphate buffer at room temperature. And 3-mm segments of the right sciatic nerve were rapidly excised 5 mm below the distal portion
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of the crush site. For morphometric evaluation, a set of six images from each nerve was selected by a blinded investigator randomly sampling from a single slice, with three images each from the periphery and from the center of the nerve [20]. The analyses included assessment of the density of unmyelinated fibers by two independent blinded investigators (graded on a scale from - to ????) and measurement of number of myelinated fibers per unit area expressed as a mean ± SEM (n = 5). Statistical analysis Data are presented as the mean ± SEM. Mean differences were analyzed using Kruskal–Wallis test, Nemenyi test and Mann–Whitney U-test. P \ 0.05 was considered statistically significant.
Results Expression of a7nAChR is upregulated in the early stages of sciatic nerve crush injury The expression of the a7nAChR in the sciatic nerve was examined at various time points after crush injury and shamoperation. The extent of a7nAChR-positive staining increased significantly on days 1–5, which was maximum on day 3 after crush injury by immunohistochemistry. Immunoreactivity was observed in the myelin sheath, in the cytoplasm of Schwann cells, endothelial cells of the capillaries, and inflammatory cells (Fig. 1a). In injured specimens, sections of the sciatic nerve distal to the crush site showed extensive Wallerian degeneration. By day 1 after injury, endoneurial edema, swelling of axons, myelin abnormalities, and hypertrophy of Schwann cell cytoplasm were present. By day 3, endoneurial edema, the collapse of myelin, irregularities in axon morphology, and a greater number of Schwann cells were observed as part of the early changes associated with peripheral degeneration. By day 5, axons had collapsed and more Schwann cells were detected, indicating that the repair process was activated. An increase in a7nAChR protein expression was also observed by western blotting on days 1–5 following injury, which peaked on day 3 (P \ 0.05 by Kruskal–Wallis, Nemenyi test and Mann–Whitney U-test); however, there were slight but statistically not significant differences in a7nAChR expression in the sham-operated rats at various time points (P [ 0.05 by Kruskal–Wallis test; Fig. 1b). Injury-induced local inflammation in the nerve is reversed by a7nAChR agonist treatment An early and transient increase in TNF-a protein level was induced in the sciatic nerve by injury. In the naı¨ve nerve, the
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Fig. 1 a7nAChR expression in the rat sciatic nerve following crush injury. a The level of a7nAChR in (a) naı¨ve specimens (day 0), and (b– e) sham-operated or (f–i) injured animals was examined at the indicated time points by immunohistochemistry. A high level of expression was observed in the myelin sheath and in the cytoplasm of Schwann cells (filled triangles), endothelial cells of the capillaries (stars), and inflammatory cells (slanted down pointing arrow). b Temporal profile of a7nAChR level in the injured sciatic nerve. Sciatic nerves were removed 0.25 (i.e., 6 h), 1, 3, and 5 days after rats were subjected to crush injury (C0.25, C1, C3, and C5) or sham surgery (S0.25, S1, S3, and S5), and the time course of a7nAChR expression was evaluated by western blotting. Densitometric quantification of a7nAChR/GAPDH is shown. *P \ 0.05 versus Sham
TNF-a content was 38.9 ± 2.8 pg/mg protein; a similar amount was detected in the sham-operated groups at various time points (P [ 0.05 by Kruskal–Wallis test). After crush, a rapid increase of TNF level was observed on days 0.25–3, reaching its maximum by about 1.8-fold higher than that of the sham control (89.1 ± 4.7 vs. 49.7 ± 2.4 pg/mg protein; P \ 0.05 by Kruskal–Wallis test, Nemenyi test and Mann– Whitney U-test) on day 1 (Fig. 2a). Previous studies have shown that early treatment with the selective a7nAChR agonist PNU-282987 attenuates brain injury [14]; thus, the effects of PNU-282987 treatment on TNF-a expression and axonal regeneration were
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evaluated. PNU decreased TNF-a level relative to the placebo group on days 1 and 3 after injury (P \ 0.05). However, the a7nAChR antagonist MLA reversed this effect, but only when co-administered with PNU (P [ 0.05 MLA ? PNU vs. PLA; Fig. 2b, by Kruskal–Wallis test and Mann–Whitney U-test). Axonal regeneration is enhanced by treatment with PNU-282987 Nerve regeneration was evaluated with the nerve pinch test. PNU-282987 treatment improved the distance of regeneration
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GAP-43 expression is induced in regenerating nerve fibers and is therefore a marker for axonal regeneration [9, 21]. The expression level of GAP-43 in the sciatic nerve after administration of PNU-282987 was increased relative to placebo-treated controls on day 5 after injury (P \ 0.05 by Kruskal–Wallis test and Mann–Whitney U-test); this effect was abolished by MLA (P [ 0.05 MLA ? PNU vs. PLA; Fig. 3c). Taken together, these results indicate that cholinergic activity promotes peripheral nerve regrowth in rats by suppressing local inflammation.
Discussion
Fig. 2 TNF-a level in the injured rat sciatic nerve and treatment with an a7nAChR agonist. a An early and transient increase in TNF-a protein level was induced in the sciatic nerve by injury. In the naı¨ve nerve, the TNF-a content was 38.9 ± 2.8 pg/mg protein; a similar amount was detected in the sham-operated groups at various time points (P [ 0.05 by Kruskal–Wallis test). After crush, a rapid increase of TNF level was observed on days 0.25–3, reaching its maximum by about 1.8-fold higher than that of the sham control (89.1 ± 4.7 vs. 49.7 ± 2.4 pg/mg protein; P \ 0.05). b Rats were subjected to crush injury to the sciatic nerve, and were injected 45 and 60 min later. Treatment groups were PLA, PNU, MLA ? PNU and MLA. PNU decreased TNF-a level relative to the placebo group on days 1 and 3 after injury (P \ 0.05). However, the a7nAChR antagonist MLA reversed this effect, but only when co-administered with PNU (P [ 0.05 MLA ? PNU vs. PLA) *P \ 0.05
compared to the PLA group on day 5 (P \ 0.05 by Kruskal–Wallis test and Mann–Whitney U-test). However, the protective effect was blocked by MLA (P [ 0.05 MLA ? PNU vs. PLA), although MLA applied alone did not inhibit regeneration (Fig. 3a). A histological examination of the distal portion of the sciatic nerve—which is typically used to evaluate recovery from nerve injuries— revealed more unmyelinated, regenerating nerve fibers in the PNU than in the PLA on day 7. Myelinated nerve fibers were seldom observed on day 7 after injury; however, a greater number of myelinated nerve fibers were seen in the PNU than in the PLA on day 9 (P \ 0.05). The effect was abolished by MLA (P [ 0.05 MLA ? PNU vs. PLA; Fig. 3b, by Kruskal–Wallis test and Mann–Whitney U-test).
The cholinergic anti-inflammatory pathway has a central role in neuroimmunomodulation and involves many nAChR subtypes, which are composed of various combinations of a (1–10), b (1–4), c, d, and e subunits [22]. The a7 subunit of nAChR is most abundantly expressed in the nervous system and a7nAChR agonists have analgesic, anti-hyperalgesic and anti-inflammatory effects [23, 24]. The present study confirmed that a7nAChR expression was increased following sciatic nerve crush injury in rats, and determined that cholinergic activity enhanced axonal regeneration by attenuating the local proinflammatory response in the early stages of recovery. Crush injury to peripheral nerves is a well-established model in regeneration studies for investigating the molecular mechanisms of regeneration as well as the impact of various pharmacological treatments [25]. Here, crush injury induced Wallerian degeneration of the portion of the nerve distal to the injury site (Fig. 1a). The widespread expression of the homomeric a7nAChR throughout the central nervous system suggests that these ion channels contribute to essential physiological functions. In the peripheral nervous system, a7nAChR is expressed in the dorsal root ganglion neurons [24] and sciatic nerve [23]. One study found that after traumatic brain injury in rats, receptor density initially decreased in most brain regions over the first 24 h relative to controls, before increasing up until 72 h [26]. In mice subjected to partial sciatic nerve ligation, a7nAChR was upregulated over the subsequent 3 days, although the peak occurred within 24 h [23]. Emerging evidence highlights a key role for a7nAChR in the regulation of inflammation and neuroprotection. The a7nAChR agonist PNU-282987 decreased neuronal cell death and brain edema, and improved neurological status in a rat perforation model of subarachnoid hemorrhage, that effects were reversed by MLA treatment [14]. In the rat sciatic nerve injured by loose ligation, cholinergic transmission was shown to be involved in neuropathic pain and neuroprotection by application of PNU-282987. Moreover,
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Fig. 3 Effect of a7nAChR agonist treatment on axonal regeneration. a The nerve pinch test was used to assess regeneration distance from the crush site (mm) on day 5 in PLA, PNU, MLA ? PNU, and MLA groups. PNU-282987 treatment improved the distance of regeneration compared to the PLA group on day 5 (P \ 0.05). However, the protective effect was blocked by MLA (P [ 0.05), although MLA applied alone did not inhibit regeneration. b Digitized micrographs of sections from regenerating sciatic nerves 9 days after crush injury.
A greater number of myelinated nerve fibers were seen in the PNU than in the PLA on day 9 (P \ 0.05). The effect was abolished by MLA (P [ 0.05). c After treatment with PLA, PNU, MLA ? PNU or MLA, nerves were removed on day 5 and GAP-43 level was evaluated by western blotting. The expression level of GAP-43 in the sciatic nerve after administration of PNU-282987 was increased relative to placebo-treated controls on day 5 after injury (P \ 0.05); this effect was abolished by MLA (*P \ 0.05)
a7nAChR activation in microglia potentiated their protective effects and suppressed inflammation, suggesting that ACh controls inflammation in nervous tissue, similar to its demonstrated function in peripheral tissues [27]. Here it is reported for the first time that a7nAChR expression is increased for the first 5 days following sciatic nerve injury in rats, with the peak occurring on day 3 (Fig. 1b). It is worth noting that the TNF-a level was elevated within the first 24 h, but decreased thereafter and returned to baseline by day 5 (Fig. 2a), while the a7nAChR level was still elevated. These results suggest that the initial inflammatory response is suppressed by the upregulation of a7nAChR—which is shown to prevent cytokine release upon activation [8]— possibly providing additional binding sites for a7nAChR agonists that further suppress neuroinflammation. The functional consequences of post-injury cholinergic activation were demonstrated by the nerve pinch test. Enhanced distance of regeneration was observed following treatment with PNU-282987 (Fig. 3a). Based on a previously reported regeneration rate of approximately 4 mm/day over 3.4 days, the new nerve growth after 5 days was predicted to be 13.6 mm [17], which is similar to the value of about 13.2 mm observed here upon administration of saline. Notably, the regeneration distance in the PNU group was
considerably larger at 18.6 mm, and a morphometric evaluation revealed a greater number of unmyelinated and myelinated regenerated fibers per unit area in these animals, accompanied by an upregulation of GAP-43 level (Fig. 3b, c). The specificity of these effects to a7nAChR activation was demonstrated by their reversal upon application of MLA. These findings suggest that a7nAChR activation is a useful strategy for the immediate treatment of peripheral nerve injury, although additional experiments are required to determine the long-term effects as well as the capacity for functional recovery using this approach. In summery, the local expression of a7nAChR is increased during the early stages of sciatic nerve injury, and a7nAChR activation promotes axonal regeneration by suppressing TNF-a-mediated inflammation. These findings indicate that a7nAChR can potentially serve as a neuroprotective agent and that a7nAChR activation may be a useful therapeutic strategy to treat peripheral nerve injury.
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Axonal regeneration in early stages of sciatic nerve crush injury is enhanced by a7nAChR in rats Dewei Wang • Xuming Wang • Shuo Geng Zhenggang Bi
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Received: 22 August 2014 / Accepted: 27 October 2014 / Published online: 5 November 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract This study investigated the role of alpha 7 nicotinic acetylcholine receptor (a7nAChR) in axonal regeneration after crush injury to the rat sciatic nerve. The time course of a7nAChR expression following injury was assessed by immunohistochemistry and western blotting, and local inflammation, as indicated by the expression of tumor necrosis factor (TNF)-a, was detected by enzymelinked immunosorbent assay. Axonal regeneration was evaluated by the pinch-test, morphometric analysis, and by measuring growth-associated protein 43 expressions. Local a7nAChR expression increased on day 1, peaked on day 3, and remained elevated on day 5 following nerve injuries. Prominent a7nAChR immunoreactivity was observed in Schwann cells, endothelial cells of the capillaries, and a small number of inflammatory cells. Application of the selective a7nAChR agonist PNU-282987 decreased TNF-a level and enhanced axonal regeneration, but this effect was blocked by concomitant treatment with methyllycaconitine, a a7nAChR antagonist. The results indicate that the local expression of a7nAChR is increased during the early stages of sciatic nerve injury, and application of a a7nAChR agonist promotes axonal regeneration by suppression of TNF-a-mediated inflammation. The a7nAChR can act as a neuroprotective agent and a7nAChR activation may be a useful therapeutic strategy to treat peripheral nerve injury. Keywords a7nAChR PNU-282987 TNF-a Axonal regeneration Crush injury
D. Wang X. Wang S. Geng Z. Bi (&) Department of Orthopedic Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin 150001, Heilongjiang, China e-mail: [email protected]
Introduction Peripheral nerve injury, particularly in the upper limb, can have devastating consequences, including chronic pain, restricted function and mobility, and diminished quality of life. In contrast to the brain and spinal cord, the peripheral nervous system has the ability to regenerate [1], and the repair process begins almost immediately after injury [2]. In axonotmesis, a 1–2-day latency during which axons cross the injury site is followed by regeneration at a steady rate along the distal nerve, aided by reactive Schwann cells and preserved endoneurial tubules that enhance axon elongation and facilitate adequate target reinnervation [3]. Surgical repair does not guarantee full functional recovery to damaged nerves [4], but therapeutic manipulation of the molecular response to injury can promote neuronal survival by stimulating the growth of axons across large gaps between nerve stumps, thereby maximizing the accuracy of target reinnervation and minimizing any potential neuropathic pain [5]. The distal segment of transected or crushed peripheral nerves undergoes Wallerian degeneration, which is essential for subsequent repair [6]; an important event during the early post-injury stages is the inflammatory response. Tumor necrosis factor (TNF)-a, a proinflammatory cytokine, is upregulated early and transiently at the site of nerve injury and plays a crucial role in Wallerian degeneration as an initiator of local inflammation [7]. While an overabundance of cytokine activity can impair organ function, induce shock, and aggravate tissue injury [8], immediate therapy with a TNFa antagonist enhances the rate of axon regeneration in injured peripheral nerves [9]. These data suggest that mitigating the inflammatory response after peripheral nervous injury could be a strategy for reducing damage and enhancing functional repair. In recent studies, agonists of
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the alpha 7 nicotinic acetylcholine receptor (a7nAChR)— ion channels that are expressed in neurons, astrocytes, epithelial cells, adipocytes, fibroblasts, keratinocytes, and immune cells were found to have anti-inflammatory and protective effects in various diseases [10–13]. However, the role of a7nAChR in post-injury repair of the rat sciatic nerve has not well been examined. This study characterized a7nAChR expression and activation of the cholinergic anti-inflammatory pathway in the early stages of sciatic nerve crush injury to assess whether a7nAChR can enhance axonal regeneration through attenuation of the inflammatory response. The spatio-temporal expression profile of a7nAChR was determined and the effects of a selective a7nAChR agonist on TNF-a levels and axon growth were evaluated.
Materials and methods Reagents and antibodies The following antibodies were used in this study: polyclonal rabbit anti-a7nAChR (Abcam, Cambridge, MA, USA); anti growth-associated protein (GAP)-43 (Beijing Bioss Technologies, Inc., Beijing, China); and monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The rat TNF-a Quantikine enzyme-linked immunosorbent assay (ELISA) kit was purchased from Sun Biomedical Technology Co., Ltd. (Beijing, China). Drug protocols [N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride] (PNU-282987) and methyllycaconitine (MLA), a7nAChR agonist and antagonist, respectively, were purchased from Abcam. Drug solutions were prepared in saline immediately before use and administered according to previously published protocols [14]. Both drugs were injected intraperitoneally at 12 mg/kg for PNU-282987 and 6 mg/kg for MLA at 45 and 60 min post-injury once a day for 4 days, with saline injected as a control. Thus, the experimental groups were saline ? saline (placebo, PLA), saline ? PNU-282987 (PNU), MLA ? PNU-282987 (MLA ? PNU), and MLA ? saline (MLA).
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protocol was reviewed and approved by the Institutional Animal Care Committee at the Harbin Medical University. Animals (n = 190) were assigned to the following groups: naı¨ve (n = 10), sham (n = 40), crush (n = 40), PLA (n = 25), PNU (n = 25), MLA ? PNU (n = 25) and MLA (n = 25). The sham and crush groups were used to evaluate a7nAChR and TNF-a expression (days 0.25, 1, 3, 5), while the remaining groups were used to examine changes in TNF-a (days 1 and 3) and axonal regeneration by pinch test (day 5), GAP-43 expression (day 5) and morphometric analysis (days 7 and 9). The model of sciatic nerve crush injury in rats was described in a previous study [15]. Ketamine (100 mg/kg) and xylazine (10 mg/kg) were administered by intramuscular injection. Animals were placed prone under sterile conditions, and the right sciatic nerve was unilaterally exposed through a skin incision extending from the greater trochanter to the mid-thigh followed by an incision that split the muscle. The nerve was crushed 5 cm distal to the sciatic notch using a 2-mm straight vascular clamp for 30 s to create a 2 mm-long crush injury, and the site was labeled by tying a 6-0 nylon suture to the adjacent muscle. The sham operation consisted of unilateral sciatic nerve exposure. Animals (n = 5 per group) were sacrificed by CO2 asphyxiation and sciatic nerves were harvested by cutting the nerve directly above the site of injury and 15 mm distally, or the comparable length of nerve in sham-operated animals. Western blotting Proteins were extracted from homogenized sciatic nerve samples with lysis buffer, and resolved by 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred to a polyvinylidene difluoride membrane and blocked with 5 % nonfat dry milk. The membrane was then incubated with primary antibodies overnight at 4 °C with gentle shaking and was exposed to anti-mouse immunoglobulin (Ig)G horseradish peroxidase-conjugated antibody for 1 h at room temperature, then treated with enhanced chemiluminescence solution (Millipore, Billerica, MA, USA) and exposed on Hyperfilm (GE Healthcare, Piscataway, USA). Quantitative analysis was performed and data were expressed as a mean ± SEM (n = 5). Immunohistochemistry
Animals and surgical procedure Male Wistar rats (250–300 g) were purchased from Changchun Yisi Experimental Animal Technology Co., Ltd. (Changchun, China). All animals were handled in compliance with the National Research Council’s Guide for the Care and Use of Laboratory Animals. The animal
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Rats were anesthetized and then transcardially perfused with 0.9 % saline solution followed by 4 % paraformaldehyde. Sciatic nerves were fixed with 4 % paraformaldehyde in PBS, and embedded in paraffin. Serial longitudinal sections were cut at a thickness of 4 lm, then deparaffinized and rehydrated. Nonspecific immunoreactivity was blocked by
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incubation with normal goat serum for 2 h at room temperature, and sections were incubated with anti-a7nAChR antibody (1:100) in a humid chamber at 4 °C overnight, followed by treatment with the Histostain-Plus streptavidinperoxidase kit according to the manufacturer’s instructions. Sections were counterstained with hematoxylin and eosin. As a control, a subset of sections was incubated with normal rabbit IgG or PBS instead of the primary antibody. The expression of a7nAChR, represented by the staining intensity at 9400 magnification, was assessed by two independent investigators who were blinded to the experimental groups and was categorized as no expression (-), or weak (?), moderate (??), or high (???) expression. ELISA The protocol for ELISA has been previously described [16]. Sciatic nerves were homogenized in ice-cold PBS containing a protease inhibitor cocktail (Roche, Mannheim, Germany). After centrifugation at 10,0009g and 4 °C for 10 min, the supernatant was removed and the pellet was rehomogenized in the same volume of homogenization buffer, with Triton X-100 added to a final concentration of 0.01 %. Samples were vortexed and centrifuged, and supernatants were assayed in duplicate using the TNF-a ELISA kit according to the manufacturer’s instructions. Data were expressed as a mean ± SEM (n = 5). Nerve pinch test The rate of axonal regeneration was evaluated using the nerve pinch test [9, 17]. Based on the predicted speed of sciatic nerve regrowth, the test was performed between 3 and 7 days after injury [17]. The sciatic nerve and its tibial nerve branch were exposed in lightly anesthetized rats. Consecutive 1-mm-long segments of the tibial nerve were pinched with a pair of fine forceps, starting from the distal end of the nerve and proceeding in the proximal direction until a reflex response consisting of a contraction of back muscles was observed. The distance between the most distal point of the nerve that produced a reflexive withdrawal response and the stitch marking the original crush site was measured and identified as the regeneration distance, and expressed as a mean ± SEM (n = 5). Morphometric evaluation Histological assessments were performed as previously described [18, 19]. On days 7 and 9 post-surgery, rats were anesthetized and perfused transcardially with freshly prepared 0.5 % glutaraldehyde in 0.1 M phosphate buffer at room temperature. And 3-mm segments of the right sciatic nerve were rapidly excised 5 mm below the distal portion
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of the crush site. For morphometric evaluation, a set of six images from each nerve was selected by a blinded investigator randomly sampling from a single slice, with three images each from the periphery and from the center of the nerve [20]. The analyses included assessment of the density of unmyelinated fibers by two independent blinded investigators (graded on a scale from - to ????) and measurement of number of myelinated fibers per unit area expressed as a mean ± SEM (n = 5). Statistical analysis Data are presented as the mean ± SEM. Mean differences were analyzed using Kruskal–Wallis test, Nemenyi test and Mann–Whitney U-test. P \ 0.05 was considered statistically significant.
Results Expression of a7nAChR is upregulated in the early stages of sciatic nerve crush injury The expression of the a7nAChR in the sciatic nerve was examined at various time points after crush injury and shamoperation. The extent of a7nAChR-positive staining increased significantly on days 1–5, which was maximum on day 3 after crush injury by immunohistochemistry. Immunoreactivity was observed in the myelin sheath, in the cytoplasm of Schwann cells, endothelial cells of the capillaries, and inflammatory cells (Fig. 1a). In injured specimens, sections of the sciatic nerve distal to the crush site showed extensive Wallerian degeneration. By day 1 after injury, endoneurial edema, swelling of axons, myelin abnormalities, and hypertrophy of Schwann cell cytoplasm were present. By day 3, endoneurial edema, the collapse of myelin, irregularities in axon morphology, and a greater number of Schwann cells were observed as part of the early changes associated with peripheral degeneration. By day 5, axons had collapsed and more Schwann cells were detected, indicating that the repair process was activated. An increase in a7nAChR protein expression was also observed by western blotting on days 1–5 following injury, which peaked on day 3 (P \ 0.05 by Kruskal–Wallis, Nemenyi test and Mann–Whitney U-test); however, there were slight but statistically not significant differences in a7nAChR expression in the sham-operated rats at various time points (P [ 0.05 by Kruskal–Wallis test; Fig. 1b). Injury-induced local inflammation in the nerve is reversed by a7nAChR agonist treatment An early and transient increase in TNF-a protein level was induced in the sciatic nerve by injury. In the naı¨ve nerve, the
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Fig. 1 a7nAChR expression in the rat sciatic nerve following crush injury. a The level of a7nAChR in (a) naı¨ve specimens (day 0), and (b– e) sham-operated or (f–i) injured animals was examined at the indicated time points by immunohistochemistry. A high level of expression was observed in the myelin sheath and in the cytoplasm of Schwann cells (filled triangles), endothelial cells of the capillaries (stars), and inflammatory cells (slanted down pointing arrow). b Temporal profile of a7nAChR level in the injured sciatic nerve. Sciatic nerves were removed 0.25 (i.e., 6 h), 1, 3, and 5 days after rats were subjected to crush injury (C0.25, C1, C3, and C5) or sham surgery (S0.25, S1, S3, and S5), and the time course of a7nAChR expression was evaluated by western blotting. Densitometric quantification of a7nAChR/GAPDH is shown. *P \ 0.05 versus Sham
TNF-a content was 38.9 ± 2.8 pg/mg protein; a similar amount was detected in the sham-operated groups at various time points (P [ 0.05 by Kruskal–Wallis test). After crush, a rapid increase of TNF level was observed on days 0.25–3, reaching its maximum by about 1.8-fold higher than that of the sham control (89.1 ± 4.7 vs. 49.7 ± 2.4 pg/mg protein; P \ 0.05 by Kruskal–Wallis test, Nemenyi test and Mann– Whitney U-test) on day 1 (Fig. 2a). Previous studies have shown that early treatment with the selective a7nAChR agonist PNU-282987 attenuates brain injury [14]; thus, the effects of PNU-282987 treatment on TNF-a expression and axonal regeneration were
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evaluated. PNU decreased TNF-a level relative to the placebo group on days 1 and 3 after injury (P \ 0.05). However, the a7nAChR antagonist MLA reversed this effect, but only when co-administered with PNU (P [ 0.05 MLA ? PNU vs. PLA; Fig. 2b, by Kruskal–Wallis test and Mann–Whitney U-test). Axonal regeneration is enhanced by treatment with PNU-282987 Nerve regeneration was evaluated with the nerve pinch test. PNU-282987 treatment improved the distance of regeneration
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GAP-43 expression is induced in regenerating nerve fibers and is therefore a marker for axonal regeneration [9, 21]. The expression level of GAP-43 in the sciatic nerve after administration of PNU-282987 was increased relative to placebo-treated controls on day 5 after injury (P \ 0.05 by Kruskal–Wallis test and Mann–Whitney U-test); this effect was abolished by MLA (P [ 0.05 MLA ? PNU vs. PLA; Fig. 3c). Taken together, these results indicate that cholinergic activity promotes peripheral nerve regrowth in rats by suppressing local inflammation.
Discussion
Fig. 2 TNF-a level in the injured rat sciatic nerve and treatment with an a7nAChR agonist. a An early and transient increase in TNF-a protein level was induced in the sciatic nerve by injury. In the naı¨ve nerve, the TNF-a content was 38.9 ± 2.8 pg/mg protein; a similar amount was detected in the sham-operated groups at various time points (P [ 0.05 by Kruskal–Wallis test). After crush, a rapid increase of TNF level was observed on days 0.25–3, reaching its maximum by about 1.8-fold higher than that of the sham control (89.1 ± 4.7 vs. 49.7 ± 2.4 pg/mg protein; P \ 0.05). b Rats were subjected to crush injury to the sciatic nerve, and were injected 45 and 60 min later. Treatment groups were PLA, PNU, MLA ? PNU and MLA. PNU decreased TNF-a level relative to the placebo group on days 1 and 3 after injury (P \ 0.05). However, the a7nAChR antagonist MLA reversed this effect, but only when co-administered with PNU (P [ 0.05 MLA ? PNU vs. PLA) *P \ 0.05
compared to the PLA group on day 5 (P \ 0.05 by Kruskal–Wallis test and Mann–Whitney U-test). However, the protective effect was blocked by MLA (P [ 0.05 MLA ? PNU vs. PLA), although MLA applied alone did not inhibit regeneration (Fig. 3a). A histological examination of the distal portion of the sciatic nerve—which is typically used to evaluate recovery from nerve injuries— revealed more unmyelinated, regenerating nerve fibers in the PNU than in the PLA on day 7. Myelinated nerve fibers were seldom observed on day 7 after injury; however, a greater number of myelinated nerve fibers were seen in the PNU than in the PLA on day 9 (P \ 0.05). The effect was abolished by MLA (P [ 0.05 MLA ? PNU vs. PLA; Fig. 3b, by Kruskal–Wallis test and Mann–Whitney U-test).
The cholinergic anti-inflammatory pathway has a central role in neuroimmunomodulation and involves many nAChR subtypes, which are composed of various combinations of a (1–10), b (1–4), c, d, and e subunits [22]. The a7 subunit of nAChR is most abundantly expressed in the nervous system and a7nAChR agonists have analgesic, anti-hyperalgesic and anti-inflammatory effects [23, 24]. The present study confirmed that a7nAChR expression was increased following sciatic nerve crush injury in rats, and determined that cholinergic activity enhanced axonal regeneration by attenuating the local proinflammatory response in the early stages of recovery. Crush injury to peripheral nerves is a well-established model in regeneration studies for investigating the molecular mechanisms of regeneration as well as the impact of various pharmacological treatments [25]. Here, crush injury induced Wallerian degeneration of the portion of the nerve distal to the injury site (Fig. 1a). The widespread expression of the homomeric a7nAChR throughout the central nervous system suggests that these ion channels contribute to essential physiological functions. In the peripheral nervous system, a7nAChR is expressed in the dorsal root ganglion neurons [24] and sciatic nerve [23]. One study found that after traumatic brain injury in rats, receptor density initially decreased in most brain regions over the first 24 h relative to controls, before increasing up until 72 h [26]. In mice subjected to partial sciatic nerve ligation, a7nAChR was upregulated over the subsequent 3 days, although the peak occurred within 24 h [23]. Emerging evidence highlights a key role for a7nAChR in the regulation of inflammation and neuroprotection. The a7nAChR agonist PNU-282987 decreased neuronal cell death and brain edema, and improved neurological status in a rat perforation model of subarachnoid hemorrhage, that effects were reversed by MLA treatment [14]. In the rat sciatic nerve injured by loose ligation, cholinergic transmission was shown to be involved in neuropathic pain and neuroprotection by application of PNU-282987. Moreover,
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Fig. 3 Effect of a7nAChR agonist treatment on axonal regeneration. a The nerve pinch test was used to assess regeneration distance from the crush site (mm) on day 5 in PLA, PNU, MLA ? PNU, and MLA groups. PNU-282987 treatment improved the distance of regeneration compared to the PLA group on day 5 (P \ 0.05). However, the protective effect was blocked by MLA (P [ 0.05), although MLA applied alone did not inhibit regeneration. b Digitized micrographs of sections from regenerating sciatic nerves 9 days after crush injury.
A greater number of myelinated nerve fibers were seen in the PNU than in the PLA on day 9 (P \ 0.05). The effect was abolished by MLA (P [ 0.05). c After treatment with PLA, PNU, MLA ? PNU or MLA, nerves were removed on day 5 and GAP-43 level was evaluated by western blotting. The expression level of GAP-43 in the sciatic nerve after administration of PNU-282987 was increased relative to placebo-treated controls on day 5 after injury (P \ 0.05); this effect was abolished by MLA (*P \ 0.05)
a7nAChR activation in microglia potentiated their protective effects and suppressed inflammation, suggesting that ACh controls inflammation in nervous tissue, similar to its demonstrated function in peripheral tissues [27]. Here it is reported for the first time that a7nAChR expression is increased for the first 5 days following sciatic nerve injury in rats, with the peak occurring on day 3 (Fig. 1b). It is worth noting that the TNF-a level was elevated within the first 24 h, but decreased thereafter and returned to baseline by day 5 (Fig. 2a), while the a7nAChR level was still elevated. These results suggest that the initial inflammatory response is suppressed by the upregulation of a7nAChR—which is shown to prevent cytokine release upon activation [8]— possibly providing additional binding sites for a7nAChR agonists that further suppress neuroinflammation. The functional consequences of post-injury cholinergic activation were demonstrated by the nerve pinch test. Enhanced distance of regeneration was observed following treatment with PNU-282987 (Fig. 3a). Based on a previously reported regeneration rate of approximately 4 mm/day over 3.4 days, the new nerve growth after 5 days was predicted to be 13.6 mm [17], which is similar to the value of about 13.2 mm observed here upon administration of saline. Notably, the regeneration distance in the PNU group was
considerably larger at 18.6 mm, and a morphometric evaluation revealed a greater number of unmyelinated and myelinated regenerated fibers per unit area in these animals, accompanied by an upregulation of GAP-43 level (Fig. 3b, c). The specificity of these effects to a7nAChR activation was demonstrated by their reversal upon application of MLA. These findings suggest that a7nAChR activation is a useful strategy for the immediate treatment of peripheral nerve injury, although additional experiments are required to determine the long-term effects as well as the capacity for functional recovery using this approach. In summery, the local expression of a7nAChR is increased during the early stages of sciatic nerve injury, and a7nAChR activation promotes axonal regeneration by suppressing TNF-a-mediated inflammation. These findings indicate that a7nAChR can potentially serve as a neuroprotective agent and that a7nAChR activation may be a useful therapeutic strategy to treat peripheral nerve injury.
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