Artificial Cells, Nanomedicine, and Biotechnology, 2014; Early Online: 1–7 Copyright © 2014 Informa Healthcare USA, Inc. ISSN: 2169-1401 print / 2169-141X online DOI: 10.3109/21691401.2014.896372
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Effect of active Notch signaling system on the early repair of rat sciatic nerve injury Jin Wang1, Ke-Yu Ren2, Yan-Hua Wang3, Yu-Hui Kou3, Pei-Xun Zhang3, Jian-Ping Peng3, Lei Deng3, Hong-Bo Zhang3 & Bao-Guo Jiang3 1Department of Pathology, Medical College, Qing Dao University, Qing Dao, P. R. China, 2The Affiliated Hospital of Medical
College, Qing Dao University, Qing Dao, P. R. China, and 3Department of Trauma and Orthopedics, People’s Hospital, Peking University, Beijing, P. R. China
It has been known that Schwann cells (SCs), as structural and functioning cells in peripheral nerves, have the striking ability to recover the nerve continuity because SCs can dedifferentiate and revert back to an immature-like state following axonal loss (Jessen and Mirsky 2008), which is called dedifferentiated SCs. The dedifferentiated SCs support axonal regeneration by clearing myelin debris, proliferating quickly and steadly, and upregulating the synthesis of a number of neurotrophic factors. Therefore, dedifferentiated SCs play an important role in neural regeneration (Cao et al. 2012, Mosahebi et al. 2002). Using transgenic mice and cell cultures, it has been reported that Notch has complex and extensive regulatory functions in SCs (Woodhoo et al. 2009). Notch promoted the generation of SCs from Schwann cell precursors and regulated the size of the SC pool by controlling proliferation in the developing stage. Notch inhibited myelination, suggesting that myelination may be subject to Notch signaling system that opposes forward drives such as Krox20. Notably, in the adult nerve, Notch dysregulation results in demyelination, and this result identifies a signaling pathway that induces myelin breakdown in vivo. These findings are relevant for understanding the molecular mechanisms of Notch system that control SC dedifferentiation and underlie nerve repair (Mirsky et al. 2008). Therefore, how to further improve peripheral nerve regeneration by using Notch signaling becomes our focus. In this study, based on biodegradable conduit small gap tubulization, we tested the effect of transient active Notch signaling on peripheral nerve repair in order to lay the theoretical foundation for Notch signaling molecules strategy in future clinical application.
Abstract It is all known that dedifferentiated Schwann cells (SCs) play an important role in neural regeneration, and Notch signaling has complex and extensive regulatory functions in dedifferentiated SCs. So studies have focused on how to improve peripheral nerve repair by regulating proliferation and dedifferentiation in SCs with Notch signaling meloculars.We have found SCs can be activated when adding Recombinant rat jagged1/FC chimera (an activator of the Notch signaling system) in vivo. Compared with that of the control groups, at 4 weeks post-surgery nerve regeneration and functional rehabilitation in the Recombinant rat jagged1/FC chimera group were advanced significantly, and the expression of neurotrophic factors in the regenerated nerves was elevated largely. These results indicated that SCs activated by Notch signaling could promote nerve repair effectively in the early regenerative stage, suggesting the possible clinical application for the treatment of peripheral nerve defects. Keywords: nerve regeneration, Notch signaling, schwann cells, sciatic nerve
Introduction Peripheral nerve injury is a very common traumatic event in clinic. Now many researchers had focused on small gap bridging suture and its possible application (Mligiliche et al. 1999, Strauch 2000). Our labs have centered on the small gap sleeve bridging fields for about 30 years and has confirmed that the conduit method to recover nerve continuity is superior to the epineurium suture in previous studies (Zhang et al. 2008, 2013). Moreover, nerve conduit could offer not only the endo-luminal substructure so as to prevent neuroma formation at the anastomotic stoma, but also a relative closed microenvironment for adding diverse suitable seeding cells or neuro-growth factors (Tomita et al. 2007).
Materials and methods Animals and surgery Adult male Sprague–Dawley (SD) rats were used, with an average body weight of 250 g (225–275 g) provided by Peking
Correspondence: Bao-Guo Jiang, Department of Trauma and Orthopedics, People’s Hospital, Peking University, 11 South Xizhimen Street, Beijing 100044, P. R. China. Tel: ⫹ 86-10-88324570. Fax: ⫹ 86 1088324570. E-mail:[email protected] (Received 15 January 2014; revised 10 February 2014; accepted 11 February 2014)
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University People’s Hospital. Experiments were conducted in according with the Guide for the Care and Use of Laboratory Animals from the National Academy of Science. All the rats were anesthetized by an intraperitoneal injection of pentobarbital sodium (30 mg/kg body weight) during the whole surgical proceedings. The right sciatic nerves of rats were exposed and cut off at 1 cm above sciatic nerve crotch. Then the biological conduit small gap bridging suture (2 mm gap between the two stumps) was performed. The 4 mm long biological conduit was stitched to bridge both stumps of the nerve defects sutures. Then the rats were randomly divided into three groups. (n ⫽ 48 per group): Group A: Control group, a simple small gap for bridging the peripheral nerve was made. Group B: A total volume of 20 μl recombinant rat jagged1/FC chimera (10ug/ml) was injected into the conduit using a microinjector. Group C: A total volume of 20 μl DAPT(50 uM)was injected into the conduit. The conduit used in this experiment was invented by Peking University People’s Hospital and Chinese Spinning and Weaving Institute(ZL01136314.2), a kind of de-acetyl chitin biological conduit, with inner diameter 1.5 mm.
Method of drawing materials At 24 h, 72 h, 5 d, 7 d, 14 d, 21 d, 4 w, and12 w postoperatively, the proximal and distal sciatic nerve stumps (approximately a 5 mm length of nerve to the injured tip) as well as normal nerves were removed and washed with DEPC at 4°C for mRNA detection, and the distal sciatic nerve stumps (approximately a 2 mm length of nerve to the injured tip) as well as normal nerves were removed for histological studies.
period, wave amplitude, and nerve conduction velocity were calculated.
Immunofluorcscence staining All the samples from rats were fixed with 4% paraformaldehyde for 12 h, then transferred to 15%, 30% sucrose solution/PBS for cryoprotection. After embedded in optimum cutting temperature compound, 6-μm-thick sections were placed onto poly-L-lysine-coated slides. Primary antibody was rabbit polyclonal anti-NICD (Cell signaling, 1:100), and secondary antibody was fluorescein isothiocyanate (FITC) -conjugated goat anti-rabbit IgG. Nucleus was labeled by DAPI. In randomly five high-power fields, NICD/ DAPI-positive cells were counted and averaged.
Real-time PCR analysis For detecting the mRNA levels of NGF, BDNF, Notch1, and Hes1 in the injury nerve, quantitative real-time PCR was conducted. The primers were designed and synthesized by AOGCT Inc (China), and the oligonucleotide sequences of primers are detailed in Table I. Specimens were loaded into 1 ml of Trizol reagent (Invitrogen) and then homogenated. Total RNA was extracted. Purified RNA was diluted to 500 ng/ul and 3 μl RNA was utilized to synthesize cDNA with Prime-Script1 RT reagent kit (Promaga). After that, Real-time PCR was performed with SYBRGREEN PCR Master Mix (ABI) following the manufacturer’s instruction. The gene expression was calculated using the comparative CT (22DDCT) method, using β-acting as the control housekeeping gene for normalization of the amount of RNA added to the reverse transcription reactions.
Electrophysiological assay
Assessment of myelin density using luxol fast blue Staining
At the 4th week and 12th week postoperatively, electrophysiological study was conducted on the animals before sacrifice, using an electroneurogram device (Synergy, Oxford Inc). The rats were anesthetized and the right sciatic nerves were exposed thoroughly. After blotting up the electrolyte around the conduit with gauze, hooked silver needle electrodes were placed on the proximal and distal ends of the nerve to the injured tip. Electrical stimulus was applied with the intensity of 0.9mA to ensure the maximum waveform and prevent independent muscle contraction. The stimulus duration was 0.1 ms and stimulation frequency was 1 Hz. The evoked action potential responding to the stimulus was recorded, and then the latency
The frozen section in the distal nerve to the injury tip was made as previously describes for the assessment of myelin density. After dehydration sections were placed in 0.1% luxol fast blue (LFB) overnight at 37°C. Next day slides were rinsed in 95% ethanol and 70% ethanol and then de-stained in successive washes in 0.05% lithium carbonate, 70% ethanol, and water. By LFB staining, the mature myelin sheaths were staining blue and ringed. Myelinated fibers counting per unit vision field was conducted under a light microscope at 400 magnification. Three nerve transactions at every time point of eight groups were taken to counting. We got the center point of the vision field and chose up left, up right, down left, down right and the center as 5 unit vision fields.
Table I. Oligonucleotide sequences and product sizes of primers. cDNA Oligonucleotide primers NGF BDNF Notch1 Hes1 β-acting
F:5′-ACGCCCCTTCTCCTCTCACAATG-3′ R:5′-GGCTGTGTCAAGGGAATGCTAAG-3′ F: 5′-GTCACAGCGGCAGATAAA-3′ R: 5′-ATTGGGTAGTTCGGCATT-3′ F: ′-CACCCATGACCACTACCCAGTT-3′ R: 5′-CCTCGGACCAATCAGAGATGTT3′ F:5′- ACACCGGACAAACCAAAGAC-3′ R:5′- ATGCCGGGAGCTATCTTTCT-3 F:5′-CATTGCTGACAGGATGCAGAAG-3′ R:5′-GAGCCACCAATCCACACAGAGT-3′
Product size
Acc. No
331 bp
NM031523
196 bp
NM012513
186 bp;
Z11886
147 bp
NM024360
108 bp
NM031144
16.00 14.00
*
(a)
12.00
*
10.00
*
group group A group B group C
8.00
*
6.00 4.00 2.00 0.00
1
2
5
7
14
21
28
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Time (d)
Relative Hes1 mRNA expression
Relative Notch1 mRNA expression
The early repair of rat sciatic nerve injury 3 40.00
*
(b)
*
group group A group B group C
14
21
30.00
*
*
20.00 10.00 0.00
1
3
5
7
23
Time (d)
Figure 1. The expression of Notch1, Hes1 mRNA during nerve regeneration. Total RNA was isolated and qPCR was performed to quantify mRNA expression of Notch1 and Hes1 in the nerve tissues. (a) Notch1 mRNA levels in Group B were higher than those in Groups A and C during the early two weeks. As similar as the above result, (b) Hes1 mRNA levels were elevated significantly as compared to those in other groups. *p ⬍ 0.05.
Statistical analysis All values are expressed as mean ⫾ SD and analyzed using software SPSS 13.0. Difference between groups was examined for statistical significance using either the paired-t test or one-way factorial analysis of variance.
Results Evaluation of activated Notch signaling in SCs during nerve regeneration Notch1 mRNA level of normal sciatic nerves was less. After nerve injury, the expression of Notch1 mRNA in three groups all increased continuously and reached to peak at 2 weeks postoperatively, and then decreased (Figure 1a). Compared with other groups, Notch1 mRNA in Group B expressed at a higher level in the early two weeks (p ⬍ 0.05), indicating that Notch signaling in the nerve tissues may indeed be activated
not only after never injury but also by adding Recombinant rat jagged1/FC chimera in the conduit. In order to further demonstrate SCs could be activated by Recombinant rat jagged1/FC chimera, we detected the expression of Hes1 mRNA (Figure 1b) and NICD level using immunofluorcscence staining. There was very scant Hes1 expression in the normal nerve. After injury, for Group B Hes1 mRNA increased gradually and reached to peak at 7 days, and then gradually decreased until 4 weeks postoperatively. Consistent with the above result, Hes1 mRNA levels were elevated significantly as compared to those in other groups at 3 d, 5 d, 7 d, and 14 d post-operation (p ⬍ 0.05). The figures of immunofluorescence revealed that NICD expression increased immediately after injury (Figure 2a and b). From Figure 2c we can find NICD⫹/DAPI ⫹ SCs quantity in the distal sciatic nerve stumps increased after
Figure 2. The expression of NICD in the nerve distal stumps. (a) and (b) showed immunofluorescence staining of NICD in the cross-sections of nerves distal stumps in Groups A and B, respectively. Nuclei were stained with DAPI (blue). Green fluorescence signals of NICD in the crosssections of nerve revealed that NICD expression increased and reach to peak at 7d post-surgery in Group B (b1–b4), next gradually decreased until four weeks (b5–b7). Scale bar ⫽ 100 μm. (c) showed the change of number of NICD⫹/DAPI⫹ SCs in three groups. After injury, in agreement with the above finding, the change of NICD⫹/DAPI⫹ SCs quantity in Group B showed the same trend. Asterisk (*) indicates significant differences between Group B and the control groups (p ⬍ 0.05).
60.00
(a)
group A group B group C
50.00 40.00 30.00 20.00 10.00 0.00 1
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*
2
5
7
14
21
28
Time (d)
Relative BONF mRNA expression
J. Wang et al. Relative NGF mRNA expression
4
8.00
(b)
*
group A group B group C
6.00
4.00
*
*
1
3
2.00
0.00 5
7 14 Time (d)
21
28
Figure 3. The expression of NGF, BDNF mRNA during nerve regeneration. Total RNA was isolated and qPCR was performed to quantify mRNA expression of NGF and BDNF in the tissues. The figures (a) and (b) illustrated relative NGF and BDNF mRNA levels, respectively. * p ⬍ 0.05.
injury, reaching to a higher level at 3 days and peak at 7 days, and then decreased gradually until 4 weeks. And it is also showed that at 3 d, 5 d, 7 d, and 14 d post-operation NICD⫹/ DAPI⫹ SCs in Group B were elevated significantly compared with those of other groups (p ⬍ 0.05). We can also find that the trend of expression change of NICD was similar with Hes1mRNA expression.
The expression of NGFand BDNF mRNA in the nerve during regeneration At the first day, the damaged nerve tissues in three groups all expressed NGF mRNA at a higher level (Figure 3a). Then NGF mRNA expression was transiently down-regulated at 3 days. For Group B NGF mRNA level was elevated significantly and reached to peak as compared to those in Groups A and C at 5d after injury (p ⬍ 0.05), but after 7 days, the mRNA expression of NGF in Group B decreased rapidly and almost achieved equivalent levels with other groups (p ⬎ 0.05). BDNF mRNA expression also increased significantly after nerve injury. Starting at 24 h and more prominent at 5 days, then BDNF level gradually down-regulated after 7 days (Figure 3b). Furthermore at any time points postsurgery, the mRNA expression of BDNF in Group B was higher than that in the other groups, and BDNF mRNA level was elevated significantly during 5 days postoperatively as compared to those in other two groups (p ⬍ 0.05). Both of these results indicated that the injured nerve tissues transiently treated with Recombinant rat jagged1/FC chimera may secrete more NGF and BDNF in the early stage of nerve repair.
slowest, which may be caused by the inhibitory effect of Notch signaling. Following LFB staining, the myelinated fiber number and shape in the cross-sections of regenerated nerves were assayed. Table II and Figure 5 showed that the change trend of the myelinated fiber number in three groups was similar and consistent with the result in the electrophysiological study. The myelinated fiber number decreased at 1 day post-surgery, and arrived to the minimum at the seventh day. Then the myelinated fiber number gradually increased and nearly returned to normal level at 3 months postoperation. Besides, demyelination in Group C treated with DAPT was inhibited in the early stage of injury, on the contrary, demyelination in Group B was promoted during 7 days post-surgery and then myelination was improved effectively. Additionally, at 14 d, 21 d, and 28 d postoperation, the myelinated fiber number of Group B was higher than those of Groups A and C (p ⬍ 0.05). Through observing Figure 5a and b, the change in morphology of myelin sheathes between Groups A and B was similar during 7 days after injury. The myelin sheathes swelled and demylinated at 1 day. And at 7 days there was no complete ring-shaped structure of myelin sheathes. However, after 2 weeks the ring-shaped structure of myelin sheathes in Group B gradually developed from single ring-shape to mature ring-shape, while there was still immature budding structure in Group A. After 3 months, the myelin sheath thickness among three groups was all near to normal.
Evaluation of nerve regeneration In the electrophysiological study (Figure 4), after surgery for 1 month, in comparison with either Group A or C, the nerve conduction velocity in Group B was improved markedly(p ⬍ 0.05). Although the parameters of neural electrophysiology in three groups had no apparent differences (p ⬎ 0.05) at 3 months post-surgery, the nerve conduction velocity in Group B (31.63 ⫾ 7.27 m/s) still show a little increase compared with that of the Group A (29.9 ⫾ 4.78 m/s) and Group C (27 ⫾ 5.82 m/s). That means the nerve conduction in Group B was superior to that in other two groups, and improved with a time-dependent manner. In contract, the nerve conduction velocity in Group C was
Figure 4. Electrophysiological studies in all groups. After surgery for 1 month, the nerve conduction velocity in Group B was improved markedly (p ⬍ 0.05), compared with other two groups. *p ⬍ 0.05.
The early repair of rat sciatic nerve injury 5 Table II. The counting of regenerated myelinated fiber (luxol fast blue staining) in all groups. Time Group 1d* 3d 5d 7d 14d* 21d* A B C
206 ⫾ 11.14 171 ⫾ 14.18 206 ⫾ 10.19
42 ⫾ 12.29 39 ⫾ 2.00 60 ⫾ 3.79
34 ⫾ 7.21 38 ⫾ 6.25 54 ⫾ 12.12
13 ⫾ 3.79 9 ⫾ 3.06 13 ⫾ 7.00
114 ⫾ 11.53 159 ⫾ 7.21 77 ⫾ 7.94
191.3 ⫾ 4.16 167 ⫾ 9.54 102 ⫾ 9.54
28d*
90d
Normal control
210.4 ⫾ 4.9 237 ⫾ 8.08 123 ⫾ 27.62
230 ⫾ 15.1 242 ⫾ 9.54 223 ⫾ 12.06
224 ⫾ 10.22
Values are depicted as mean ⫾ SD. *p ⬍ 0.05.
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Discussion A majority of researches have been currently focused on the construction of tissue-engineered nerve grafts, so as to provide suitable microenvironment for nerve regeneration. These biomaterials include bridging scaffolds, seed cells, growth factors, and so on (Bozkurt et al. 2009, Ribeiro-Resende et al. 2009, Jia et al. 2011, Stang et al. 2009). Consequently, many researchers have been studied the combination of the above methods (Fisher et al. 2001).
As one of the most promising seed cells, SCs play an important role in neural regeneration such as myelination and secretion of growth factors (Cai et al. 2004, Cao et al. 2012). However, because of the limited source of cell donors and the time-consuming amplification of cell culture in vitro, there are still some difficulties in the clinical application of SCs. One of the most striking features of SCs is their plasticity. When axons degenerate following injury, SCs dedifferentiate
Figure 5. Histopathological estimation of sciatic nerve regeneration during degeneration (luxol blue staining). The change in the shape of myelin sheathes between Group A (a1–4) and B (b1–4) was similar during the first week. The myelin sheathes swelled and demylinated, starting at 1 day, and appeared incomplete ring-shaped structure at 7 days. However, after 2 weeks the ring-shaped structure of myelin sheathes in Group B gradually developed (a5–7), while there was still immature budding structure in Group A (b5–7). After 3 months, the morphology of myelin sheaths among three groups was all near to normal (a8 and b8). Scale bar ⫽ 100 μm. (c) showed the myelinated fiber number in the cross-sections of regenerated nerves. The myelinated fiber number in three groups decreased at 1d post-surgery, and minimized at 7 days. Then the myelinated fiber number gradually increased and nearly returned to normal level at 3 months post-operation. Additionally, at 14d, 21d, and 28d post-operation, the myelinated fiber number of Group B in unit area was higher than those of Groups A and C (p ⬍ 0.05).
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to assume a molecular phenotype similar to that of immature cells. This process can be viewed largely as a reversal of differentiation or dedifferentiation (Cheng and Zochodne 2002). This property is seen most obviously in SCs that occur after nerve cut or crush injury. Recent studies showed that dedifferentiation depends on activation of specific intracellular signaling molecules, which is called negative transcriptional regulators that should be expressed prior to myelination, downregulated as myelination starts but reactivated as SCs dedifferentiate following injury. Negative regulators are likely to have a major role during injury, because they promote the transformation of damaged nerves to an environment that fosters neuronal survival and axonal regrowth (Jessen and Mirsky 2005, 2008, Mirsky et al. 2008). Our work will determine whether control the transition of differentiated SCs to the dedifferentiated/ immature state can regulate SCs number and regenerative microenvironment so as to improve peripheral nerve repair. There is ample evidence that Notch signaling is potentially a powerful regulator of dedifferentiation in SCs. Notch is a transmembrane receptor protein that, following binding to a ligand, is cleaved to generate an intracellular fragment, the Notch intra-cellular domain (NICD) which acts in the nucleus as a transcriptional regulator and a reliable factor for assessing whether or not a cell is undergoing Notch signaling (Dontu et al. 2004, Schweisguth 2004, Thomas 2005). It is selectively downregulated in cells that start myelination and suppressed by positive regulators such as Krox-20 in vitro. Enforced NICD expression prevents myelination in co-cultures and myelin gene induction by cAMP (Arthur-Farraj et al. 2011). Also in vivo, myelination is delayed in mice in which NICD expression in SCs is transiently elevated around birth (Woodhoo et al. 2009). NICD levels also rise rapidly in injured nerves, and the resulting demyelination is slower if the NICD elevation is prevented. Conversely, demyelination is accelerated in injured nerves engineered to overexpress NICD. Even in uninjured nerves, activation of Notch is sufficient to induce rapid demyelination (Jessen and Mirsky 2010). All these indicated that Notch signaling is potentially a powerful regulator of dedifferentiation in myelinating SCs (Jessen and Mirsky 2008, 2010, Givogri et al. 2002). In additon, Notch1 is one of receptors of Notch signaling system, and Hes1 is a basic helix-loop-helix gene that is known to act downstream of notch signaling and belong to Notch target gene. In view of this, we describe the dramatic response of SCs activated by Notch signaling to injury in adult nerves. Immunolabeling of teased nerves and freshly dissociated SCs has confirmed that the Notch ligand Jagged-1 is expressed by axons and glia, whereas Notch1 is detected only on glial cells. Notch receptors in developing SCs can therefore be activated by Notch ligands on axons or on the glial cells themselves. Our study demonstrated there was increased expression of Notch1, Hes-1, and NICD in Group B compared with that of the other groups, suggesting that the expression of Notch signaling of injuried nerve significantly increased by adding Recombinant rat jagged1/FC chimera in the conduit. As the markers of active Notch signaling, the
change of NICD mRNA level was similar with Hes-1 level and the higher expression of these two factors mainly focused on the early regenerative stage, especially in the early 2 weeks. While the expression trend of Notch1 mRNA was not consistent with the above factors. Maybe the correlation between Hes1and Notch1 is complex (Fan et al. 2004). It has been reported that the canonical Notch pathway is mitogenic for SCs and that NICD elevation is part of the mechanism that controls SCs proliferation and number in developing nerves, for enforced NICD expression may upregulate the cell cycle markers cyclin D1 and cdk2 (Woodhoo et al. 2009) and the mitogenic effect of NICD on SCs also involved and depended on phosphorylation of the kinases ERK1/2 and JNK (Winseck and Oppenheim 2006, Woodhoo and Sommer 2008, Martensson et al. 2007). So in our study we hypothesized that addition of Recombinant rat jagged1/ FC chimera in the conduit may further increase SCs number to improve nerve regeneration. Another strong evidence that dedifferentiated SCs are conducive to axonal regeneration consisted in more secretion of various neurotrophic factors, such as NGF, BDNF, etc. Neurotrophic factors are known as a category of polypeptides or proteins released by neurons or nonneuron cells, and influence the both central and peripheral nervous system extensively. The factors facilitate neuron development, cell differentiation, neuron survival, synapse formation, and nerve regeneration (Fisher et al. 2001, Henderson 1996, Zhang et al. 2010). In order to show this directly, we have found that NGF and BDNF mRNA level both showed a marked increase in Group B, especially at 5 days post-surgery (p ⬍ 0.05), and at this time point, NICD expression also elevated significantly compared with that of other groups. All these results suggested that active Notch signaling may promote more secretion of NGF and BDNF during nerve repair, which play an important role on nerve regeneration. Although our data showed that there was not a completely one-to-one correspondence between NGF, BDNF mRNA expression, and NICD expression, we cannot deny the effect of Notch signal on promoting NGF and BDNF secretion. Because Notch signal is not the only factor to control the secretion of SCs, and NGF as well as BDNF secretion is also regulated by other factors. Compared with the Groups A and C, the parameters of neural electrophysiology in Group B were improved obviously, especially at 4 weeks post-surgery. Meanwhile, the quantities of myelinated nerve fibers increased significantly from 2 w to 4 w. Although the nerve conduction velocity and the myelinated fiber number at 3 months post-injury in Group B only showed a little increase, the present results in this work still indicated that the damaged nerve may recover more adequately in the early stage when treating with Recombinant rat jagged1/FC chimera. Through a series of beneficial actions such as secretion of neurotrophic factors and cell differentiation, nerve regeneration was promoted effectively. It is all known that early repair of nerve is very important, especially in clinc. This study provides a new method and experimental basis for engineering construction of peripheral nerve tissue. After transiently treating with an activator of the Notch signaling system, the results
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The early repair of rat sciatic nerve injury 7 of morphology and electrophysiology confirmed that the initial repair of the sciatic nerve and partial functional recovery was superior to that of simple biological conduit small gap bridging suture. It should be mentioned that, in the current study the impact of the active Notch signaling on the nerve regeneration was preliminarily investigated in peripheral nerve repairs for the complexity of mechanism in the Notch signaling system. In the other words, our data have verified that active Notch signaling play a beneficial role on SCs in the early regenerative stage, and the factors induced by Recombinant rat jagged1/FC chimera could be adjusted directly or indirectly via changing particular signaling molecules in the nerve stumps. However, to clarify the mechanism of nerve regeneration which may be improved outstandingly by the Notch signaling, the exploration of correlation factors in Notch signaling system at an extensive range of time points following the operation is still required.
Conclusion Our results showed that, based on biological conduit small gap bridging suture, addition of Recombinant rat jagged1/ FC chimera was effective in repairing defect of rat sciatic nerve, partially restored impaired nerve conduction function during the early stage of nerve regeneration. Although the scaffold with jagged1/FC chimera in the third month only had small increase in the number of regenerated axons and the nerve conduction velocity, this study still provides a new method and experimental basis for engineering construction of peripheral nerve tissue.
Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by grants from the National Natural Science Fund (No.30801169), China National Nature and Science Youth(No. 0625036)), and the Development Program of China (973 Program,2005CB522604). All authors have declared that no competing interest exists.
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Dontu G, Jackson KW, Mcnicholas E, Kawamura MJ, Abdallah WM, Wicha MS. 2004. Role of Notch signaling in cell-fate determination of human mammary stem/progenitor cells. Breast Cancer Res. 6: R605–R615. Fan X, Mikolaenko I, Elhassan I, Ni X, Wang Y, Ball D, et al. 2004. Notch1 and notch2 have opposite effects on embryonal brain tumor growth. Cancer Res. 64:7787–7793. Fisher J, Levkovitch-Verbin H, Schori H, Yoles E, Butovsky O, Kaye JF, et al. 2001. Vaccination for neuroprotection in the mouse optic nerve: implications for optic neuropathies. J Neurosci. 21: 136–142. Givogri MI, Costa RM, Schonmann V, Silva AJ, Campagnoni AT, Bongarzone ER. 2002. Central nervous system myelination in mice with deficient expression of Notch1 receptor. J Neurosci Res. 67:309–320. Henderson CE. 1996. Role of neurotrophic factors in neuronal development. Curr Opin Neurobiol. 6:64–70. Jessen KR, Mirsky R. 2005. The origin and development of glial cells in peripheral nerves. Nat Rev Neurosci. 6:671–682. Jessen KR, Mirsky R. 2008. Negative regulation of myelination: relevance for development, injury, and demyelinating disease. Glia. 56:1552–1565. Jessen KR, Mirsky R. 2010. Control of Schwann cell myelination. F1000 Biol Rep. 2. Jia H, Wang Y, Tong XJ, Liu GB, Li Q, Zhang LX, Sun XH. 2011. Biocompatibility of acellular nerves of different mammalian species for nerve tissue engineering. Artif Cells Blood Substit Immobil Biotechnol. 39:366–375. Martensson L, Gustavsson P, Dahlin LB, Kanje M. 2007. Activation of extracellular-signal-regulated kinase-1/2 precedes and is required for injury-induced Schwann cell proliferation. Neuroreport. 18:957–961. Mirsky R, Woodhoo A , Parkinson DB, Arthur-Farraj P, Bhaskaran A , Jessen KR. 2008. Novel signals controlling embryonic Schwann cell development, myelination and dedifferentiation. J Peripher Nerv Syst. 13:122–135. Mligiliche NL, Tabata Y, Ide C. 1999. Nerve regeneration through biodegradable gelatin conduits in mice. East Afr Med J. 76:400–406. Mosahebi A , Fuller P, Wiberg M, Terenghi G. 2002. Effect of allogeneic Schwann cell transplantation on peripheral nerve regeneration. Exp Neurol. 173:213–223. Ribeiro-Resende VT, Koenig B, Nichterwitz S, Oberhoffner S, Schlosshauer B. 2009. Strategies for inducing the formation of bands of Bungner in peripheral nerve regeneration. Biomaterials. 30:5251–5259. Schweisguth F. 2004. Regulation of notch signaling activity. Curr Biol. 14: R129–R138. Stang F, Keilhoff G, Fansa H. 2009. Biocompatibility of different nerve Tubes. Materials. 2:1480–1507. Strauch B. 2000. Use of nerve conduits in peripheral nerve repair. Hand Clin. 16:123–130. Thomas BJ. 2005. Cell-cycle control during development: taking it up a notch. Dev Cell. 8:451–452. Tomita K, Kubo T, Matsuda K, Hattori R, Fujiwara T, Yano K , Hosokawa K. 2007. Effect of conduit repair on aberrant motor axon growth within the nerve graft in rats. Microsurgery. 27:500–509. Winseck AK , Oppenheim RW. 2006. An in vivo analysis of Schwann cell programmed cell death in embryonic mice: the role of axons, glial growth factor, and the pro-apoptotic gene Bax. Eur J Neurosci. 24:2105–2117. Woodhoo A , Alonso MB, Droggiti A , Turmaine, M, D’antonio, M, et al. 2009. Notch controls embryonic Schwann cell differentiation, postnatal myelination and adult plasticity. Nat Neurosci. 12: 839–847. Woodhoo A , Sommer L. 2008. Development of the Schwann cell lineage: from the neural crest to the myelinated nerve. Glia. 56: 1481–1490. Zhang LX, Tong XJ, Yuan XH, Sun XH, Jia H. 2010. Effects of 660-nm gallium-aluminum-arsenide low-energy laser on nerve regeneration after acellular nerve allograft in rats. Synapse. 64:152–160. Zhang P, Han N, Wang T, Xue F, Kou Y, Wang Y, et al. 2013. Biodegradable conduit small gap tubulization for peripheral nerve mutilation: a substitute for traditional epineurial neurorrhaphy. Int J Med Sci. 10:171–175. Zhang P, Yin X, Kou Y, Wang Y, Zhang H, Jiang B. 2008. The electrophysiology analysis of biological conduit sleeve bridging rhesus monkey median nerve injury with small gap. Artif Cells Blood Substit Immobil Biotechnol. 36:457–463.
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Effect of active Notch signaling system on the early repair of rat sciatic nerve injury Jin Wang1, Ke-Yu Ren2, Yan-Hua Wang3, Yu-Hui Kou3, Pei-Xun Zhang3, Jian-Ping Peng3, Lei Deng3, Hong-Bo Zhang3 & Bao-Guo Jiang3 1Department of Pathology, Medical College, Qing Dao University, Qing Dao, P. R. China, 2The Affiliated Hospital of Medical
College, Qing Dao University, Qing Dao, P. R. China, and 3Department of Trauma and Orthopedics, People’s Hospital, Peking University, Beijing, P. R. China
It has been known that Schwann cells (SCs), as structural and functioning cells in peripheral nerves, have the striking ability to recover the nerve continuity because SCs can dedifferentiate and revert back to an immature-like state following axonal loss (Jessen and Mirsky 2008), which is called dedifferentiated SCs. The dedifferentiated SCs support axonal regeneration by clearing myelin debris, proliferating quickly and steadly, and upregulating the synthesis of a number of neurotrophic factors. Therefore, dedifferentiated SCs play an important role in neural regeneration (Cao et al. 2012, Mosahebi et al. 2002). Using transgenic mice and cell cultures, it has been reported that Notch has complex and extensive regulatory functions in SCs (Woodhoo et al. 2009). Notch promoted the generation of SCs from Schwann cell precursors and regulated the size of the SC pool by controlling proliferation in the developing stage. Notch inhibited myelination, suggesting that myelination may be subject to Notch signaling system that opposes forward drives such as Krox20. Notably, in the adult nerve, Notch dysregulation results in demyelination, and this result identifies a signaling pathway that induces myelin breakdown in vivo. These findings are relevant for understanding the molecular mechanisms of Notch system that control SC dedifferentiation and underlie nerve repair (Mirsky et al. 2008). Therefore, how to further improve peripheral nerve regeneration by using Notch signaling becomes our focus. In this study, based on biodegradable conduit small gap tubulization, we tested the effect of transient active Notch signaling on peripheral nerve repair in order to lay the theoretical foundation for Notch signaling molecules strategy in future clinical application.
Abstract It is all known that dedifferentiated Schwann cells (SCs) play an important role in neural regeneration, and Notch signaling has complex and extensive regulatory functions in dedifferentiated SCs. So studies have focused on how to improve peripheral nerve repair by regulating proliferation and dedifferentiation in SCs with Notch signaling meloculars.We have found SCs can be activated when adding Recombinant rat jagged1/FC chimera (an activator of the Notch signaling system) in vivo. Compared with that of the control groups, at 4 weeks post-surgery nerve regeneration and functional rehabilitation in the Recombinant rat jagged1/FC chimera group were advanced significantly, and the expression of neurotrophic factors in the regenerated nerves was elevated largely. These results indicated that SCs activated by Notch signaling could promote nerve repair effectively in the early regenerative stage, suggesting the possible clinical application for the treatment of peripheral nerve defects. Keywords: nerve regeneration, Notch signaling, schwann cells, sciatic nerve
Introduction Peripheral nerve injury is a very common traumatic event in clinic. Now many researchers had focused on small gap bridging suture and its possible application (Mligiliche et al. 1999, Strauch 2000). Our labs have centered on the small gap sleeve bridging fields for about 30 years and has confirmed that the conduit method to recover nerve continuity is superior to the epineurium suture in previous studies (Zhang et al. 2008, 2013). Moreover, nerve conduit could offer not only the endo-luminal substructure so as to prevent neuroma formation at the anastomotic stoma, but also a relative closed microenvironment for adding diverse suitable seeding cells or neuro-growth factors (Tomita et al. 2007).
Materials and methods Animals and surgery Adult male Sprague–Dawley (SD) rats were used, with an average body weight of 250 g (225–275 g) provided by Peking
Correspondence: Bao-Guo Jiang, Department of Trauma and Orthopedics, People’s Hospital, Peking University, 11 South Xizhimen Street, Beijing 100044, P. R. China. Tel: ⫹ 86-10-88324570. Fax: ⫹ 86 1088324570. E-mail:[email protected] (Received 15 January 2014; revised 10 February 2014; accepted 11 February 2014)
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University People’s Hospital. Experiments were conducted in according with the Guide for the Care and Use of Laboratory Animals from the National Academy of Science. All the rats were anesthetized by an intraperitoneal injection of pentobarbital sodium (30 mg/kg body weight) during the whole surgical proceedings. The right sciatic nerves of rats were exposed and cut off at 1 cm above sciatic nerve crotch. Then the biological conduit small gap bridging suture (2 mm gap between the two stumps) was performed. The 4 mm long biological conduit was stitched to bridge both stumps of the nerve defects sutures. Then the rats were randomly divided into three groups. (n ⫽ 48 per group): Group A: Control group, a simple small gap for bridging the peripheral nerve was made. Group B: A total volume of 20 μl recombinant rat jagged1/FC chimera (10ug/ml) was injected into the conduit using a microinjector. Group C: A total volume of 20 μl DAPT(50 uM)was injected into the conduit. The conduit used in this experiment was invented by Peking University People’s Hospital and Chinese Spinning and Weaving Institute(ZL01136314.2), a kind of de-acetyl chitin biological conduit, with inner diameter 1.5 mm.
Method of drawing materials At 24 h, 72 h, 5 d, 7 d, 14 d, 21 d, 4 w, and12 w postoperatively, the proximal and distal sciatic nerve stumps (approximately a 5 mm length of nerve to the injured tip) as well as normal nerves were removed and washed with DEPC at 4°C for mRNA detection, and the distal sciatic nerve stumps (approximately a 2 mm length of nerve to the injured tip) as well as normal nerves were removed for histological studies.
period, wave amplitude, and nerve conduction velocity were calculated.
Immunofluorcscence staining All the samples from rats were fixed with 4% paraformaldehyde for 12 h, then transferred to 15%, 30% sucrose solution/PBS for cryoprotection. After embedded in optimum cutting temperature compound, 6-μm-thick sections were placed onto poly-L-lysine-coated slides. Primary antibody was rabbit polyclonal anti-NICD (Cell signaling, 1:100), and secondary antibody was fluorescein isothiocyanate (FITC) -conjugated goat anti-rabbit IgG. Nucleus was labeled by DAPI. In randomly five high-power fields, NICD/ DAPI-positive cells were counted and averaged.
Real-time PCR analysis For detecting the mRNA levels of NGF, BDNF, Notch1, and Hes1 in the injury nerve, quantitative real-time PCR was conducted. The primers were designed and synthesized by AOGCT Inc (China), and the oligonucleotide sequences of primers are detailed in Table I. Specimens were loaded into 1 ml of Trizol reagent (Invitrogen) and then homogenated. Total RNA was extracted. Purified RNA was diluted to 500 ng/ul and 3 μl RNA was utilized to synthesize cDNA with Prime-Script1 RT reagent kit (Promaga). After that, Real-time PCR was performed with SYBRGREEN PCR Master Mix (ABI) following the manufacturer’s instruction. The gene expression was calculated using the comparative CT (22DDCT) method, using β-acting as the control housekeeping gene for normalization of the amount of RNA added to the reverse transcription reactions.
Electrophysiological assay
Assessment of myelin density using luxol fast blue Staining
At the 4th week and 12th week postoperatively, electrophysiological study was conducted on the animals before sacrifice, using an electroneurogram device (Synergy, Oxford Inc). The rats were anesthetized and the right sciatic nerves were exposed thoroughly. After blotting up the electrolyte around the conduit with gauze, hooked silver needle electrodes were placed on the proximal and distal ends of the nerve to the injured tip. Electrical stimulus was applied with the intensity of 0.9mA to ensure the maximum waveform and prevent independent muscle contraction. The stimulus duration was 0.1 ms and stimulation frequency was 1 Hz. The evoked action potential responding to the stimulus was recorded, and then the latency
The frozen section in the distal nerve to the injury tip was made as previously describes for the assessment of myelin density. After dehydration sections were placed in 0.1% luxol fast blue (LFB) overnight at 37°C. Next day slides were rinsed in 95% ethanol and 70% ethanol and then de-stained in successive washes in 0.05% lithium carbonate, 70% ethanol, and water. By LFB staining, the mature myelin sheaths were staining blue and ringed. Myelinated fibers counting per unit vision field was conducted under a light microscope at 400 magnification. Three nerve transactions at every time point of eight groups were taken to counting. We got the center point of the vision field and chose up left, up right, down left, down right and the center as 5 unit vision fields.
Table I. Oligonucleotide sequences and product sizes of primers. cDNA Oligonucleotide primers NGF BDNF Notch1 Hes1 β-acting
F:5′-ACGCCCCTTCTCCTCTCACAATG-3′ R:5′-GGCTGTGTCAAGGGAATGCTAAG-3′ F: 5′-GTCACAGCGGCAGATAAA-3′ R: 5′-ATTGGGTAGTTCGGCATT-3′ F: ′-CACCCATGACCACTACCCAGTT-3′ R: 5′-CCTCGGACCAATCAGAGATGTT3′ F:5′- ACACCGGACAAACCAAAGAC-3′ R:5′- ATGCCGGGAGCTATCTTTCT-3 F:5′-CATTGCTGACAGGATGCAGAAG-3′ R:5′-GAGCCACCAATCCACACAGAGT-3′
Product size
Acc. No
331 bp
NM031523
196 bp
NM012513
186 bp;
Z11886
147 bp
NM024360
108 bp
NM031144
16.00 14.00
*
(a)
12.00
*
10.00
*
group group A group B group C
8.00
*
6.00 4.00 2.00 0.00
1
2
5
7
14
21
28
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Time (d)
Relative Hes1 mRNA expression
Relative Notch1 mRNA expression
The early repair of rat sciatic nerve injury 3 40.00
*
(b)
*
group group A group B group C
14
21
30.00
*
*
20.00 10.00 0.00
1
3
5
7
23
Time (d)
Figure 1. The expression of Notch1, Hes1 mRNA during nerve regeneration. Total RNA was isolated and qPCR was performed to quantify mRNA expression of Notch1 and Hes1 in the nerve tissues. (a) Notch1 mRNA levels in Group B were higher than those in Groups A and C during the early two weeks. As similar as the above result, (b) Hes1 mRNA levels were elevated significantly as compared to those in other groups. *p ⬍ 0.05.
Statistical analysis All values are expressed as mean ⫾ SD and analyzed using software SPSS 13.0. Difference between groups was examined for statistical significance using either the paired-t test or one-way factorial analysis of variance.
Results Evaluation of activated Notch signaling in SCs during nerve regeneration Notch1 mRNA level of normal sciatic nerves was less. After nerve injury, the expression of Notch1 mRNA in three groups all increased continuously and reached to peak at 2 weeks postoperatively, and then decreased (Figure 1a). Compared with other groups, Notch1 mRNA in Group B expressed at a higher level in the early two weeks (p ⬍ 0.05), indicating that Notch signaling in the nerve tissues may indeed be activated
not only after never injury but also by adding Recombinant rat jagged1/FC chimera in the conduit. In order to further demonstrate SCs could be activated by Recombinant rat jagged1/FC chimera, we detected the expression of Hes1 mRNA (Figure 1b) and NICD level using immunofluorcscence staining. There was very scant Hes1 expression in the normal nerve. After injury, for Group B Hes1 mRNA increased gradually and reached to peak at 7 days, and then gradually decreased until 4 weeks postoperatively. Consistent with the above result, Hes1 mRNA levels were elevated significantly as compared to those in other groups at 3 d, 5 d, 7 d, and 14 d post-operation (p ⬍ 0.05). The figures of immunofluorescence revealed that NICD expression increased immediately after injury (Figure 2a and b). From Figure 2c we can find NICD⫹/DAPI ⫹ SCs quantity in the distal sciatic nerve stumps increased after
Figure 2. The expression of NICD in the nerve distal stumps. (a) and (b) showed immunofluorescence staining of NICD in the cross-sections of nerves distal stumps in Groups A and B, respectively. Nuclei were stained with DAPI (blue). Green fluorescence signals of NICD in the crosssections of nerve revealed that NICD expression increased and reach to peak at 7d post-surgery in Group B (b1–b4), next gradually decreased until four weeks (b5–b7). Scale bar ⫽ 100 μm. (c) showed the change of number of NICD⫹/DAPI⫹ SCs in three groups. After injury, in agreement with the above finding, the change of NICD⫹/DAPI⫹ SCs quantity in Group B showed the same trend. Asterisk (*) indicates significant differences between Group B and the control groups (p ⬍ 0.05).
60.00
(a)
group A group B group C
50.00 40.00 30.00 20.00 10.00 0.00 1
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*
2
5
7
14
21
28
Time (d)
Relative BONF mRNA expression
J. Wang et al. Relative NGF mRNA expression
4
8.00
(b)
*
group A group B group C
6.00
4.00
*
*
1
3
2.00
0.00 5
7 14 Time (d)
21
28
Figure 3. The expression of NGF, BDNF mRNA during nerve regeneration. Total RNA was isolated and qPCR was performed to quantify mRNA expression of NGF and BDNF in the tissues. The figures (a) and (b) illustrated relative NGF and BDNF mRNA levels, respectively. * p ⬍ 0.05.
injury, reaching to a higher level at 3 days and peak at 7 days, and then decreased gradually until 4 weeks. And it is also showed that at 3 d, 5 d, 7 d, and 14 d post-operation NICD⫹/ DAPI⫹ SCs in Group B were elevated significantly compared with those of other groups (p ⬍ 0.05). We can also find that the trend of expression change of NICD was similar with Hes1mRNA expression.
The expression of NGFand BDNF mRNA in the nerve during regeneration At the first day, the damaged nerve tissues in three groups all expressed NGF mRNA at a higher level (Figure 3a). Then NGF mRNA expression was transiently down-regulated at 3 days. For Group B NGF mRNA level was elevated significantly and reached to peak as compared to those in Groups A and C at 5d after injury (p ⬍ 0.05), but after 7 days, the mRNA expression of NGF in Group B decreased rapidly and almost achieved equivalent levels with other groups (p ⬎ 0.05). BDNF mRNA expression also increased significantly after nerve injury. Starting at 24 h and more prominent at 5 days, then BDNF level gradually down-regulated after 7 days (Figure 3b). Furthermore at any time points postsurgery, the mRNA expression of BDNF in Group B was higher than that in the other groups, and BDNF mRNA level was elevated significantly during 5 days postoperatively as compared to those in other two groups (p ⬍ 0.05). Both of these results indicated that the injured nerve tissues transiently treated with Recombinant rat jagged1/FC chimera may secrete more NGF and BDNF in the early stage of nerve repair.
slowest, which may be caused by the inhibitory effect of Notch signaling. Following LFB staining, the myelinated fiber number and shape in the cross-sections of regenerated nerves were assayed. Table II and Figure 5 showed that the change trend of the myelinated fiber number in three groups was similar and consistent with the result in the electrophysiological study. The myelinated fiber number decreased at 1 day post-surgery, and arrived to the minimum at the seventh day. Then the myelinated fiber number gradually increased and nearly returned to normal level at 3 months postoperation. Besides, demyelination in Group C treated with DAPT was inhibited in the early stage of injury, on the contrary, demyelination in Group B was promoted during 7 days post-surgery and then myelination was improved effectively. Additionally, at 14 d, 21 d, and 28 d postoperation, the myelinated fiber number of Group B was higher than those of Groups A and C (p ⬍ 0.05). Through observing Figure 5a and b, the change in morphology of myelin sheathes between Groups A and B was similar during 7 days after injury. The myelin sheathes swelled and demylinated at 1 day. And at 7 days there was no complete ring-shaped structure of myelin sheathes. However, after 2 weeks the ring-shaped structure of myelin sheathes in Group B gradually developed from single ring-shape to mature ring-shape, while there was still immature budding structure in Group A. After 3 months, the myelin sheath thickness among three groups was all near to normal.
Evaluation of nerve regeneration In the electrophysiological study (Figure 4), after surgery for 1 month, in comparison with either Group A or C, the nerve conduction velocity in Group B was improved markedly(p ⬍ 0.05). Although the parameters of neural electrophysiology in three groups had no apparent differences (p ⬎ 0.05) at 3 months post-surgery, the nerve conduction velocity in Group B (31.63 ⫾ 7.27 m/s) still show a little increase compared with that of the Group A (29.9 ⫾ 4.78 m/s) and Group C (27 ⫾ 5.82 m/s). That means the nerve conduction in Group B was superior to that in other two groups, and improved with a time-dependent manner. In contract, the nerve conduction velocity in Group C was
Figure 4. Electrophysiological studies in all groups. After surgery for 1 month, the nerve conduction velocity in Group B was improved markedly (p ⬍ 0.05), compared with other two groups. *p ⬍ 0.05.
The early repair of rat sciatic nerve injury 5 Table II. The counting of regenerated myelinated fiber (luxol fast blue staining) in all groups. Time Group 1d* 3d 5d 7d 14d* 21d* A B C
206 ⫾ 11.14 171 ⫾ 14.18 206 ⫾ 10.19
42 ⫾ 12.29 39 ⫾ 2.00 60 ⫾ 3.79
34 ⫾ 7.21 38 ⫾ 6.25 54 ⫾ 12.12
13 ⫾ 3.79 9 ⫾ 3.06 13 ⫾ 7.00
114 ⫾ 11.53 159 ⫾ 7.21 77 ⫾ 7.94
191.3 ⫾ 4.16 167 ⫾ 9.54 102 ⫾ 9.54
28d*
90d
Normal control
210.4 ⫾ 4.9 237 ⫾ 8.08 123 ⫾ 27.62
230 ⫾ 15.1 242 ⫾ 9.54 223 ⫾ 12.06
224 ⫾ 10.22
Values are depicted as mean ⫾ SD. *p ⬍ 0.05.
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Discussion A majority of researches have been currently focused on the construction of tissue-engineered nerve grafts, so as to provide suitable microenvironment for nerve regeneration. These biomaterials include bridging scaffolds, seed cells, growth factors, and so on (Bozkurt et al. 2009, Ribeiro-Resende et al. 2009, Jia et al. 2011, Stang et al. 2009). Consequently, many researchers have been studied the combination of the above methods (Fisher et al. 2001).
As one of the most promising seed cells, SCs play an important role in neural regeneration such as myelination and secretion of growth factors (Cai et al. 2004, Cao et al. 2012). However, because of the limited source of cell donors and the time-consuming amplification of cell culture in vitro, there are still some difficulties in the clinical application of SCs. One of the most striking features of SCs is their plasticity. When axons degenerate following injury, SCs dedifferentiate
Figure 5. Histopathological estimation of sciatic nerve regeneration during degeneration (luxol blue staining). The change in the shape of myelin sheathes between Group A (a1–4) and B (b1–4) was similar during the first week. The myelin sheathes swelled and demylinated, starting at 1 day, and appeared incomplete ring-shaped structure at 7 days. However, after 2 weeks the ring-shaped structure of myelin sheathes in Group B gradually developed (a5–7), while there was still immature budding structure in Group A (b5–7). After 3 months, the morphology of myelin sheaths among three groups was all near to normal (a8 and b8). Scale bar ⫽ 100 μm. (c) showed the myelinated fiber number in the cross-sections of regenerated nerves. The myelinated fiber number in three groups decreased at 1d post-surgery, and minimized at 7 days. Then the myelinated fiber number gradually increased and nearly returned to normal level at 3 months post-operation. Additionally, at 14d, 21d, and 28d post-operation, the myelinated fiber number of Group B in unit area was higher than those of Groups A and C (p ⬍ 0.05).
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to assume a molecular phenotype similar to that of immature cells. This process can be viewed largely as a reversal of differentiation or dedifferentiation (Cheng and Zochodne 2002). This property is seen most obviously in SCs that occur after nerve cut or crush injury. Recent studies showed that dedifferentiation depends on activation of specific intracellular signaling molecules, which is called negative transcriptional regulators that should be expressed prior to myelination, downregulated as myelination starts but reactivated as SCs dedifferentiate following injury. Negative regulators are likely to have a major role during injury, because they promote the transformation of damaged nerves to an environment that fosters neuronal survival and axonal regrowth (Jessen and Mirsky 2005, 2008, Mirsky et al. 2008). Our work will determine whether control the transition of differentiated SCs to the dedifferentiated/ immature state can regulate SCs number and regenerative microenvironment so as to improve peripheral nerve repair. There is ample evidence that Notch signaling is potentially a powerful regulator of dedifferentiation in SCs. Notch is a transmembrane receptor protein that, following binding to a ligand, is cleaved to generate an intracellular fragment, the Notch intra-cellular domain (NICD) which acts in the nucleus as a transcriptional regulator and a reliable factor for assessing whether or not a cell is undergoing Notch signaling (Dontu et al. 2004, Schweisguth 2004, Thomas 2005). It is selectively downregulated in cells that start myelination and suppressed by positive regulators such as Krox-20 in vitro. Enforced NICD expression prevents myelination in co-cultures and myelin gene induction by cAMP (Arthur-Farraj et al. 2011). Also in vivo, myelination is delayed in mice in which NICD expression in SCs is transiently elevated around birth (Woodhoo et al. 2009). NICD levels also rise rapidly in injured nerves, and the resulting demyelination is slower if the NICD elevation is prevented. Conversely, demyelination is accelerated in injured nerves engineered to overexpress NICD. Even in uninjured nerves, activation of Notch is sufficient to induce rapid demyelination (Jessen and Mirsky 2010). All these indicated that Notch signaling is potentially a powerful regulator of dedifferentiation in myelinating SCs (Jessen and Mirsky 2008, 2010, Givogri et al. 2002). In additon, Notch1 is one of receptors of Notch signaling system, and Hes1 is a basic helix-loop-helix gene that is known to act downstream of notch signaling and belong to Notch target gene. In view of this, we describe the dramatic response of SCs activated by Notch signaling to injury in adult nerves. Immunolabeling of teased nerves and freshly dissociated SCs has confirmed that the Notch ligand Jagged-1 is expressed by axons and glia, whereas Notch1 is detected only on glial cells. Notch receptors in developing SCs can therefore be activated by Notch ligands on axons or on the glial cells themselves. Our study demonstrated there was increased expression of Notch1, Hes-1, and NICD in Group B compared with that of the other groups, suggesting that the expression of Notch signaling of injuried nerve significantly increased by adding Recombinant rat jagged1/FC chimera in the conduit. As the markers of active Notch signaling, the
change of NICD mRNA level was similar with Hes-1 level and the higher expression of these two factors mainly focused on the early regenerative stage, especially in the early 2 weeks. While the expression trend of Notch1 mRNA was not consistent with the above factors. Maybe the correlation between Hes1and Notch1 is complex (Fan et al. 2004). It has been reported that the canonical Notch pathway is mitogenic for SCs and that NICD elevation is part of the mechanism that controls SCs proliferation and number in developing nerves, for enforced NICD expression may upregulate the cell cycle markers cyclin D1 and cdk2 (Woodhoo et al. 2009) and the mitogenic effect of NICD on SCs also involved and depended on phosphorylation of the kinases ERK1/2 and JNK (Winseck and Oppenheim 2006, Woodhoo and Sommer 2008, Martensson et al. 2007). So in our study we hypothesized that addition of Recombinant rat jagged1/ FC chimera in the conduit may further increase SCs number to improve nerve regeneration. Another strong evidence that dedifferentiated SCs are conducive to axonal regeneration consisted in more secretion of various neurotrophic factors, such as NGF, BDNF, etc. Neurotrophic factors are known as a category of polypeptides or proteins released by neurons or nonneuron cells, and influence the both central and peripheral nervous system extensively. The factors facilitate neuron development, cell differentiation, neuron survival, synapse formation, and nerve regeneration (Fisher et al. 2001, Henderson 1996, Zhang et al. 2010). In order to show this directly, we have found that NGF and BDNF mRNA level both showed a marked increase in Group B, especially at 5 days post-surgery (p ⬍ 0.05), and at this time point, NICD expression also elevated significantly compared with that of other groups. All these results suggested that active Notch signaling may promote more secretion of NGF and BDNF during nerve repair, which play an important role on nerve regeneration. Although our data showed that there was not a completely one-to-one correspondence between NGF, BDNF mRNA expression, and NICD expression, we cannot deny the effect of Notch signal on promoting NGF and BDNF secretion. Because Notch signal is not the only factor to control the secretion of SCs, and NGF as well as BDNF secretion is also regulated by other factors. Compared with the Groups A and C, the parameters of neural electrophysiology in Group B were improved obviously, especially at 4 weeks post-surgery. Meanwhile, the quantities of myelinated nerve fibers increased significantly from 2 w to 4 w. Although the nerve conduction velocity and the myelinated fiber number at 3 months post-injury in Group B only showed a little increase, the present results in this work still indicated that the damaged nerve may recover more adequately in the early stage when treating with Recombinant rat jagged1/FC chimera. Through a series of beneficial actions such as secretion of neurotrophic factors and cell differentiation, nerve regeneration was promoted effectively. It is all known that early repair of nerve is very important, especially in clinc. This study provides a new method and experimental basis for engineering construction of peripheral nerve tissue. After transiently treating with an activator of the Notch signaling system, the results
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The early repair of rat sciatic nerve injury 7 of morphology and electrophysiology confirmed that the initial repair of the sciatic nerve and partial functional recovery was superior to that of simple biological conduit small gap bridging suture. It should be mentioned that, in the current study the impact of the active Notch signaling on the nerve regeneration was preliminarily investigated in peripheral nerve repairs for the complexity of mechanism in the Notch signaling system. In the other words, our data have verified that active Notch signaling play a beneficial role on SCs in the early regenerative stage, and the factors induced by Recombinant rat jagged1/FC chimera could be adjusted directly or indirectly via changing particular signaling molecules in the nerve stumps. However, to clarify the mechanism of nerve regeneration which may be improved outstandingly by the Notch signaling, the exploration of correlation factors in Notch signaling system at an extensive range of time points following the operation is still required.
Conclusion Our results showed that, based on biological conduit small gap bridging suture, addition of Recombinant rat jagged1/ FC chimera was effective in repairing defect of rat sciatic nerve, partially restored impaired nerve conduction function during the early stage of nerve regeneration. Although the scaffold with jagged1/FC chimera in the third month only had small increase in the number of regenerated axons and the nerve conduction velocity, this study still provides a new method and experimental basis for engineering construction of peripheral nerve tissue.
Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by grants from the National Natural Science Fund (No.30801169), China National Nature and Science Youth(No. 0625036)), and the Development Program of China (973 Program,2005CB522604). All authors have declared that no competing interest exists.
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