Skip to main content
NCBI home page
Neural Regeneration Research logoLink to Neural Regeneration Research
. 2020 Jan 28;15(8):1502–1509. doi: 10.4103/1673-5374.274343

Biological characteristics of dynamic expression of nerve regeneration related growth factors in dorsal root ganglia after peripheral nerve injury

Yin-Ying Shen 1,#, Xiao-Kun Gu 1,2,#, Rui-Rui Zhang 1,#, Tian-Mei Qian 1, Shi-Ying Li 1, Sheng Yi 1,*
PMCID: PMC7059586  PMID: 31997815

graphic file with name NRR-15-1502-g001.jpg

Keywords: axon growth, bioinformatic analysis, dorsal root ganglia, growth factors, Ingenuity Pathway Analysis, nerve regeneration, peripheral nerve injury, rat sciatic nerve crush injury, transcriptome sequencing, upstream regulators

Abstract

The regenerative capacity of peripheral nerves is limited after nerve injury. A number of growth factors modulate many cellular behaviors, such as proliferation and migration, and may contribute to nerve repair and regeneration. Our previous study observed the dynamic changes of genes in L4–6 dorsal root ganglion after rat sciatic nerve crush using transcriptome sequencing. Our current study focused on upstream growth factors and found that a total of 19 upstream growth factors were dysregulated in dorsal root ganglions at 3, 9 hours, 1, 4, or 7 days after nerve crush, compared with the 0 hour control. Thirty-six rat models of sciatic nerve crush injury were prepared as described previously. Then, they were divided into six groups to measure the expression changes of representative genes at 0, 3, 9 hours, 1, 4 or 7 days post crush. Our current study measured the expression levels of representative upstream growth factors, including nerve growth factor, brain-derived neurotrophic factor, fibroblast growth factor 2 and amphiregulin genes, and explored critical signaling pathways and biological process through bioinformatic analysis. Our data revealed that many of these dysregulated upstream growth factors, including nerve growth factor, brain-derived neurotrophic factor, fibroblast growth factor 2 and amphiregulin, participated in tissue remodeling and axon growth-related biological processes Therefore, the experiment described the expression pattern of upstream growth factors in the dorsal root ganglia after peripheral nerve injury. Bioinformatic analysis revealed growth factors that may promote repair and regeneration of damaged peripheral nerves. All animal surgery procedures were performed in accordance with Institutional Animal Care Guidelines of Nantong University and ethically approved by the Administration Committee of Experimental Animals, China (approval No. 20170302-017) on March 2, 2017.

Chinese Library Classification No. R459.9; R363; R364

Introduction

Peripheral nerve injury is a widespread medical problem worldwide (Sung et al., 2019). It is reported that around 2.8% of trauma patients suffer from peripheral nerve injury (Noble et al., 1998). In developed countries, peripheral nerve injury affects approximately 13 to 23 per 100,000 persons per year (Li et al., 2014). The peripheral nervous system has some regeneration ability after nerve injury. However, the regeneration rate of injured axons is slow and the functional recovery effects are generally not very satisfactory (Gordon et al., 2009; Chan et al., 2014; Gu, 2015). Achieving a better understanding of the genetic changes after peripheral nerve injury will help the discovery of essential factors for peripheral nerve regeneration and provide certain cues for the treatment of nerve injury.

Schwann cells in the peripheral nervous system contribute to the construction of a permissive and favorable microenvironment for peripheral nerve regeneration (Namgung, 2014; Jessen and Mirsky, 2016; Carr and Johnston, 2017). Schwann cells switch to a dedifferentiated state, proliferate and migrate to build the bands of Bungner, and then redifferentiate to form myelin sheath around regenerated axons (Ohara and Ikuta, 1988; Frostick et al., 1998). During this process, many growth factors, including nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF), stimulate the proliferation, migration, or re-myelination of Schwann cells that benefits the regeneration of injured peripheral nerves (Li et al., 2015; Qin et al., 2016; Yi et al., 2016). The significance of growth factors in modulating Schwann cell phenotypes led to the identification of those key growth factors that were differentially expressed in rat sciatic nerve stumps after sciatic nerve injury (Zhang et al., 2019).

Besides the essential roles in Schwann cells, growth factors are also involved in the physiological and pathological conditions of neurons, such as neuronal survival, death, differentiation, morphology, and outgrowth (Lowenstein and Arsenault, 1996; Zawada et al., 1996; Whitmire et al., 2011; Schwieger et al., 2015; Onger et al., 2017). Therefore, growth factors, especially differentially expressed growth factors in neurons after nerve injury, may regulate neuronal behaviors, activate the intrinsic growth capacity of neurons, and promote axon regrowth.

To identify essential growth factors during peripheral nerve degeneration and regeneration, we screened differentially expressed genes in dorsal root ganglia (DRG) after rat sciatic nerve injury using previously obtained transcriptome sequencing data (Gong et al., 2016). We explored upstream regulators of these differentially expressed genes with Ingenuity Pathway Analysis software, and screened dysregulated upstream growth factors. Gene Ontology (GO) category analysis and Kyoto Enrichment of Genes and Genome (KEGG) pathway analysis were then applied to identify biologically relevant pathways and processes.

Materials and Methods

Animals

Thirty-six adult male Sprague-Dawley rats, weighing 180–220 g, were obtained from Experimental Animal Center of Nantong University, China (animal licenses No. SCXK [Su] 2014-0001 and SYXK [Su] 2012-0031). All animal surgery procedures were performed in accordance with Institutional Animal Care Guidelines of Nantong University and ethically approved by the Administration Committee of Experimental Animals, China (approval No. 20170302-017) on March 2, 2017.

Animal surgery and tissue collection

Sprague-Dawley rats were subjected to sciatic nerve crush injury according to a standardized surgical method (Luis et al., 2008) with modifications as previously described (Yi et al., 2015). Following anesthetization, rat sciatic nerve stumps were exposed at 10 mm above the bifurcation into the tibial and common fibular nerves and crushed three times, each for 10 seconds, with forceps. Rats were randomly divided into six groups and sacrificed by cervical dislocation at 0, 3, 9 hours, 1, 4, or 7 days after surgery. The lumbar 4, 5 and 6 (L4–6) DRGs were removed from each Sprague-Dawley rat in each groups, collected, and stored at –80°C.

Bioinformatic analysis

Transcriptome sequencing of DRGs at 0, 3, 9 hours, 1, 4, or 7 days after surgery was conducted as described previously (Gong et al., 2016). Raw data were stored in the online database (SRP200823, PRJNA547681) (https://www.ncbi.nlm.nih.gov/sra/SRP200823). Gene expression levels in DRGs at 3, 9 hours, 1, 4, or 7 days after rat sciatic nerve crush injury were determined by reads per kilobase transcriptome per million mapped reads method and compared with their expressions at zero hour. Relative gene expression patterns were core analyzed with Ingenuity Pathway Analysis software (Ingenuity Systems Inc., Redwood City, CA, USA) with a set cutoff of log2 ratio < –1 or > 1 and experimental false discovery rate (q-value) < 0.05.

Ingenuity Pathway Analysis upstream analysis was applied to identify the involved growth factors in DRGs at each time point after rat sciatic nerve crush injury by selecting growth factors among upstream regulators. Genes coding for growth factors with a log2 ratio < –1 or > 1 at 3, 9 hours, 1, 4, or 7 days as compared with zero hour were screened and considered as dysregulated upstream growth factors. These identified growth factors in DRGs at each time point were determined and enriched GO categories and KEGG pathways were identified using Database for Annotation, Visualization, and Integrated Discovery (DAVID) (https://david.ncifcrf.gov/).

RNA isolation and polymerase chain reaction validation

Total RNA samples were isolated from rat L4–6 DRGs and collected at each time point after surgery, with TRIzol reagent (Life Technologies, Carlsbad, CA, USA). RNA samples were reverse transcribed to cDNA using the Prime-Script reagent kit (TaKaRa, Dalian, Liaoning, China). The expression levels of the target genes were determined by polymerase chain reaction using SYBR Premix Ex Taq (TaKaRa) on an Applied Biosystems Step One System (Applied Biosystems, Foster City, CA, USA). The relative expression patterns of target genes in DRGs at different points after sciatic nerve crush injury were measured using the comparative 2–ΔΔCt method (Livak and Schmittgen, 2001) with Gapdh as the reference gene. The sequences of primer pairs of target genes and reference gene are listed in Table 1.

Table 1.

Primer sequences of validated genes

Gene Sequence (5′–3′) Product size (bp)
Ngf Forward CCA AGG ACG CAG CTT
TCT ATC		 186
Reverse CTG TGT CAA GGG AAT
GCT GAA G
Bdnf Forward AAC CAT AAG GAC GCG
GAC TT		 222
Reverse TGC AGT CTT TTT ATC
TGC CG
Fgf2 Forward ACT TCA AGG ATC CCA
AGC GG		 182
Reverse CAT AGC CAG GTA CCG
GTT CG
Areg Forward CCA ATG AGA ACT CCG
TCG CT		 157
Reverse AAA CCA CAA GTC CAC
CAG CA
Gapdh Forward ACA GCA ACA GGG TGG
TGG AC		 252
Reverse TTT GAG GGT GCA GCG
AAC TT
Open in a new tab

Areg: Amphiregulin; Bdnf: glial cell line-derived neurotrophic factor; Fgf2: fibroblast growth factor 2; Gapdh: glyceraldehyde-3-phosphate dehydrogenase; Ngf: nerve growth factor.

Statistical analysis

Polymerase chain reaction results were summarized from three paired experiments and presented as the mean ± SEM. One-way analysis of variance followed by Dunnett’s post hoc test was applied for statistical comparison using GraphPad Prism 6.0 software (GraphPad Software, Inc., San Diego, CA, USA). A P value < 0.05 was considered statistically significant.

Results

Discovery of dysregulated upstream growth factors in DRGs after sciatic nerve injury

Previously, the global gene expression in DRGs after peripheral nerve injury was determined by preparing rat models of sciatic nerve crush injury, collecting DRGs and performing transcriptome sequencing (Gong et al., 2016). In view of the importance of growth factors in regulating neuron activities and nerve regeneration, dysregulated genes with log2 ratio < –1 or > 1 and q-value < 0.05 were further core analyzed by Ingenuity Pathway Analysis software to screen upstream growth factors of dysregulated genes. Dysregulated upstream growth factors were then selected and identified.

As compared with the zero hour control, 1, 5, 9, 12, and 13 dysregulated upstream growth factors were identified in DRGs at 3, 9 hours, 1, 4, and 7 days after sciatic nerve crush injury, respectively (Figure 1A). The majority of these differentially expressed upstream growth factors were up-regulated after sciatic nerve crush injury (Figure 1A). The Venn diagram shows that one specific growth factor, leptin (Lep), was persistently differentially expressed in DRGs at all tested time points after sciatic nerve crush injury (Figure 1B & C). Nodal growth differentiation factor (Nodal), Fgf2, and amphiregulin Areg were all dysregulated in DRGs at 9 hours, 1, 4, and 7 days after injury. Ngf and Bdnf were dysregulated in DRGs at 1, 4, and 7 days after injury. Gdnf, noggin (Nog), and angiotensinogen (Agt) were dysregulated in DRGs at both 4 and 7 days after injury. Nudix hydrolase 6 (Nudt6) was dysregulated in DRGs at both 1 and 7 days after injury. Other growth factors, including epiregulin (Ereg), inhibin subunit beta B (Inhbb), left-right determination factor (Lefty-1), fibroblast growth factor 19 (Fgf19), X chromosome linked amelogenin (Amelx), neuregulin 4 (Nrg4), fibroblast growth factor 23 (Fgf23), Vgf nerve growth factor inducible (Vgf), and dickkopf-1 (Dkk1), a WNT signaling pathway inhibitor, were only dysregulated in DRGs at one single time point after injury.

Figure 1.

Figure 1

Open in a new tab

Overview of dysregulated upstream growth factors in dorsal root ganglions at 3, 9 hours, 1, 4, and 7 days after sciatic nerve crush injury.

(A) Number of dysregulated upstream growth factors in dorsal root ganglions. Red shows up-regulated upstream growth factors and blue shows down-regulated upstream growth factors. (B) Overlap of dysregulated upstream growth factors. The different colors represent dysregulated growth factors at different days (3 h, 9 h, 1 d, 4 d, 7 d) in Venn diagram. (C) Gene list of dysregulated upstream growth factors. 1, 2, 3 indicate the number of dysregulated growth factors. Agt: Angiotensinogen; Amelx: amelogenin X-linked; Areg: amphiregulin; Bdnf: brain derived neurotrophic factor; Dkk1: dickkopf WNT signaling pathway inhibitor 1; Ereg: epiregulin; Fgf19: fibroblast growth factor 19; Fgf2: fibroblast growth factor 2; Fgf23: fibroblast growth factor 23; Gdnf: glial cell line-derived neurotrophic factor; Inhbb: inhibin subunit beta B; Lefty: left-right determination factor; Lep: leptin; Ngf: nerve growth factor; Nodal: nodal growth differentiation factor; Nog: noggin; Nrg4: neuregulin 4; Nudt6: nudix hydrolase 6; Vgf: Vgf nerve growth factor inducible.

Dynamic changes of representative dysregulated upstream growth factors

A precise determination of gene expression levels laid the foundation for subsequent bioinformatic analysis. To validate the veracity of sequencing outcomes, the DRGs of sciatic nerve crushed rats were collected and the expression levels of representative differentially expressed upstream growth factors were measured by polymerase chain reaction. Two representative upstream growth factors that were commonly dysregulated in DRGs at 1, 4 and 7 days after injury, Ngf and Bdnf, and two representative upstream growth factors that were commonly dysregulated in DRGs at 9 hours, 1, 4 and 7 days after injury, Fgf2 and Areg, were selected for validation (Figure 2).

Figure 2.

Figure 2

Open in a new tab

Dynamic changes of Ngf, Bdnf, Fgf2, and Areg in dorsal root ganglions after sciatic nerve crush injury.

(A–D) Gene abundances of Ngf, Bdnf, Fgf2, and Areg in dorsal root ganglions at 0, 4, and 7 days after sciatic nerve crush injury: the Y-axis indicates the relative expression levels of target genes Ngf, Bdnf, Fgf2, and Areg to that of Gapdh. The abundances of target genes were normalized with zero hour control. *P < 0.05, vs. zero hour. Areg: Amphiregulin; Bdnf: brain derived neurotrophic factor; Fgf2: fibroblast growth factor 2; Gapdh: glyceraldehyde-3-phosphate dehydrogenase; Ngf: nerve growth factor.

As compared with the zero hour control, the gene expression levels of Ngf in DRGs reached about 8-fold at 1 day after nerve injury and the expression levels remained high at later time points (Figure 2A). However, the gene expression levels of Bdnf only increased at 1 day after nerve injury (Figure 2B). The gene expression levels of Fgf2 slightly increased at 9 hours and then significantly increased at 1, 4 and 7 days (Figure 2C). The gene expression levels of Areg were also upregulated in DRGs after sciatic nerve crush injury ((Figure 2D). The polymerase chain reaction results were generally consistent with the transcriptome sequencing data, indicating the accuracy of transcriptome sequencing.

Bioinformatic analysis of dysregulated upstream growth factors in DRGs

The global temporal expression patterns of differentially expressed upstream growth factors in DRGs after sciatic nerve injury were displayed in a heatmap and clustered (Figure 3A). The heatmap showed that these growth factors could be mainly grouped into two clusters: at 0–9 hours and 1–7 days (Figure 3A). Bioinformatic analysis using the DAVID database identified the top 10 significantly involved GO categories (Figure 3B) and KEGG pathways (Figure 3C).

Figure 3.

Figure 3

Open in a new tab

Bioinformatic analysis of dysregulated upstream growth factors in dorsal root ganglions after sciatic nerve crush injury.

(A) Heatmap of dysregulated upstream growth factors in dorsal root ganglions: Yellow shows up-regulated upstream growth factors and blue shows down-regulated upstream growth factors. (B) Enriched top 10 Gene Ontology (GO) categories of dysregulated upstream growth factors in dorsal root ganglions. (C) Enriched top 10 Kyoto Enrichment of Genes and Genome (KEGG) pathways of dysregulated upstream growth factors in dorsal root ganglions: Yellow shows up-regulated upstream growth factors and blue shows down-regulated upstream growth factors. Agt: Angiotensinogen; Amelx: amelogenin X-linked; Areg: amphiregulin; Bdnf: brain derived neurotrophic factor; Dkk1: dickkopf WNT signaling pathway inhibitor 1; Ereg: epiregulin; Fgf19: fibroblast growth factor 19; Fgf2: fibroblast growth factor 2; Fgf23: fibroblast growth factor 23; Inhbb: inhibin subunit beta B; Lefty: left-right determination factor; Lep: leptin; Ngf: nerve growth factor; Nodal: nodal growth differentiation factor; Nog: noggin; Nrg4: neuregulin 4; Nudt6: nudix hydrolase 6; Vgf: Vgf nerve growth factor inducible.

Given that our focus was axon regrowth during peripheral nerve repair and regeneration, GO biological processes that were highly related to organ remodeling and tissue regeneration were specifically investigated (Figure 4A). These tissue regeneration-related GO biological processes could be divided into two groups: before day one and after day one. This suggested that the biological processes occurring in the DRGs might be different in the acute phase and the sub-acute phase after nerve injury. As well as the overview of tissue regeneration-related GO biological processes, there were dynamic changes of growth factors in many essential biological processes. The following were displayed in Figure 4B; axon extension, axon guidance, axon target recognition, dendrite extension, cell migration involved in sprouting angiogenesis, brain-derived neurotrophic factor signaling pathway, glial cell proliferation, growth, negative regulation of neuron apoptotic process, nerve growth factor processing, nerve growth factor signaling pathway, neuron projection extension, regulation of axon extension, neuron recognition, and regulation of neurotransmitter secretion. It appeared that many essential biological processes also showed different trends during the first 24 hours after nerve injury.

Figure 4.

Figure 4

Open in a new tab

GO biological process categories in regeneration after sciatic nerve crush injury.

(A) Heatmap of GO biological process categories in regeneration: Orange shows up-regulated upstream growth factors and blue shows down-regulated upstream growth factors. (B) Temporal expression patterns of dysregulated upstream growth factors in specific GO biological process categories.

Discovery of dysregulated upstream growth factors related to axon growth

The axons of neurons are damaged following severe peripheral nerve injury. Therefore axon growth is crucial for peripheral nerve regeneration and subsequent target reinnervation. In this study we further investigated dysregulated growth factors in DRGs after nerve injury and focused on growth factors that were involved the biological processes related with axon growth. These included axonogenesis, axon extension, axon guidance, cell growth, proliferation, migration, differentiation, and extracellular matrix remodeling and assembly.

Fourteen dysregulated upstream growth factors participated in these biological processes (Figure 5A). Ten of these 14 upstream growth factors, including Fgf2, Bdnf, Fgf19, Nrg4, Fgf23, Amelx, Ngf, Nodal, Areg, and Gdnf, were up-regulated, while the other 4 upstream growth factors, including Lefty1, Nog, Inhbb, and Agt, were down-regulated (Figure 5A). The expressions of these upstream growth factors in DRGs at each time point after nerve injury were assessed and the details shown in Figure 5B–F. Many upstream growth factors showed altered expression at 1 day after nerve injury, indicating that this might be the important time point for initiating axon growth.

Figure 5.

Figure 5

Open in a new tab

Dysregulated upstream growth factors involved in axon growth after sciatic nerve crush injury.

(A) Heatmap of dysregulated upstream growth factors involved in axon growth. (B–F) Changes in expression of growth factors at (B) 3 hours, (C) 9 hours, (D) 1 day, (E) 4 days, and (F) 7 days after surgery. Red shows up-regulated upstream growth factors and green shows down-regulated upstream growth factors. Agt: Angiotensinogen; Amelx: amelogenin X-linked; Areg: amphiregulin; Bdnf: brain derived neurotrophic factor; Fgf19: fibroblast growth factor 19; Fgf2: fibroblast growth factor 2; Fgf23: fibroblast growth factor 23; Gdnf: glial cell line-derived neurotrophic factor; Inhbb: inhibin subunit beta B; Lefty: left-right determination factor; Ngf: nerve growth factor; Nodal: nodal growth differentiation factor; Nog: noggin; Nrg4: neuregulin 4.

Discussion

It is now possible to detect the expression levels of tens of thousands of transcripts in diverse physiological and pathological activities with the development of large-scale high-throughput data analysis methods, such as transcriptome sequencing and DNA array-based expression profiling (Nagalakshmi et al., 2010; Trapnell et al., 2010). To determine gene changes after peripheral nerve injury, the rat sciatic nerve injury model was chosen because rats have similar nerve trunk distributions as humans and their sciatic nerves are the largest peripheral nerve trunks in animals (Bryan et al., 1999; Magill et al., 2007; Savastano et al., 2014; Geuna, 2015). Gene expressions in DRGs after rat sciatic nerve injury, for example sciatic nerve chronic constriction injury and crush injury, have been comprehensively examined (Ha et al., 2001; Kim et al., 2001; Bosse et al., 2006; Martin et al., 2019). In a previous study, rat DRGs were collected at 0, 3, 9 hours, 1, 4, and 7 days after sciatic nerve crush injury for transcriptome sequencing to explore gene changes in DRGs and to screen altered genes during peripheral nerve degeneration and regeneration (Gong et al., 2016). In addition, differentially expressed transcription factors, especially transcription factors involved in cell death, have been discovered (Qin et al., 2018).

Growth factors are indispensable regulatory molecules for wound healing and tissue regeneration (Hajimiri et al., 2015; Park et al., 2017; Yamakawa and Hayashida, 2019). In the nervous system, growth factors play significant roles in modulating neuron behaviors and have been used for the treatment of many nervous system diseases, such as traumatic brain injury, amyotrophic lateral sclerosis and familial frontotemporal dementia (Suzuki and Svendsen, 2008; Seib et al., 2013; Petkau and Leavitt, 2014; Zhou et al., 2018). In the present study, 19 dysregulated upstream critical growth factors in DRGs were identified by re-analyzing previously obtained transcriptome sequencing data.

Many growth factors, including Nudt6, Gdnf, Nog, Agt, Ngf, Bdnf, Nodal, Fgf2, Areg, and Lep were dysregulated in DRGs at multiple time points after nerve injury. Many of these differentially expressed growth factors had been closely linked with neuron functions. For instance, NGF and BDNF promoted the neurite outgrowth as well as the survival of DRG neurons (Tosaki et al., 2008; Li et al., 2016; Castillo et al., 2018; Kaval Oguz, 2018). Besides these growth factors, the biological effects of many other commonly differentially expressed upstream growth factors on neurons were not fully investigated. It has been found that Nog expression inhibited BMP4-WNT1 signaling and neural crest cells delamination (Osorio et al., 2009). AGT was expressed in both astrocytes and neurons in the central nervous system of mice (Yang et al., 1999) and could affect the firing rates of neurons (Modgil et al., 2012). AREG regulated the autocrine survival of adult sensory neurons (Nilsson and Kanje, 2005). Our study showed that these growth factors were robustly changed after nerve injury and indicated that these growth factors might regulate neuron activities and contribute to peripheral nerve repair and regeneration. However, it was worth noting that DRGs contain multiple cell types besides neurons, such as perineuronal satellite glial cells (Castillo et al., 2013). Therefore, these dysregulated growth factors might not only be associated with DRG neurons but also with glial cells in DRGs. To evaluate the specific changes of genes in DRG neurons, single cell sequencing could be conducted in future studies. Moreover, our current investigation focused on the early period after peripheral nerve injury. To obtain a better view of dynamic changes of genes during the whole degeneration and regeneration, gene profiling at longer time points, for instance 14, 21, or 28 days after sciatic nerve injury, should be further determined.

In conclusion, we have provided a general view of the dynamic profiling of dysregulated expressed upstream growth factors and discovered critical growth factors that might be potential targets for peripheral nerve injury treatment.

Footnotes

Conflicts of interest: The authors declare that there are no conflicts of interest associated with this manuscript.

Financial support: This work was supported by the Natural Science Foundation of Jiangsu Higher Education Institutions of China (Major Program), No. 16KJA310005 (to SYL); the Natural Science Foundation of Nantong City in China, No. JC2018058 (to TMQ); the Priority Academic Program Development of Jiangsu Higher Education Institutions in China. The funding sources had no role in study conception and design, data analysis or interpretation, paper writing or deciding to submit this paper for publication.

Institutional review board statement: All animal surgery procedures were performed in accordance with Institutional Animal Care guidelines of Nantong University, China and ethically approved by the Administration Committee of Experimental Animals, Jiangsu, China (approval No. 20170302-017) on March 2, 2017. The experimental procedure followed the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996).

Copyright license agreement: The Copyright License Agreement has been signed by all authors before publication.

Data sharing statement: Datasets analyzed during the current study are available from the corresponding author on reasonable request.

Plagiarism check: Checked twice by iThenticate.

Peer review: Externally peer reviewed.

Funding: This work was supported by the Natural Science Foundation of Jiangsu Higher Education Institutions of China (Major Program), No. 16KJA310005 (to SYL); the Natural Science Foundation of Nantong City of China, No. JC2018058 (to TMQ); the Priority Academic Program Development of Jiangsu Higher Education Institutions of China.

C-Editor: Zhao M; S-Editors: Wang J, Li CH; L-Editors: Dawes EA, Haase R, Qiu Y, Song LP; T-Editor: Jia Y

References

  • 1.Bosse F, Hasenpusch-Theil K, Kury P, Muller HW. Gene expression profiling reveals that peripheral nerve regeneration is a consequence of both novel injury-dependent and reactivated developmental processes. J Neurochem. 2006;96:1441–1457. doi: 10.1111/j.1471-4159.2005.03635.x. [DOI] [PubMed] [Google Scholar]
  • 2.Bryan DJ, Wang KK, Summerhayes C. Migration of schwann cells in peripheral-nerve regeneration. J Reconstr Microsurg. 1999;15:591–596. doi: 10.1055/s-2007-1000143. [DOI] [PubMed] [Google Scholar]
  • 3.Carr MJ, Johnston AP. Schwann cells as drivers of tissue repair and regeneration. Curr Opin Neurobiol. 2017;47:52–57. doi: 10.1016/j.conb.2017.09.003. [DOI] [PubMed] [Google Scholar]
  • 4.Castillo C, Norcini M, Martin Hernandez LA, Correa G, Blanck TJ, Recio-Pinto E. Satellite glia cells in dorsal root ganglia express functional NMDA receptors. Neuroscience. 2013;240:135–146. doi: 10.1016/j.neuroscience.2013.02.031. [DOI] [PubMed] [Google Scholar]
  • 5.Castillo X, Melo Z, Varela-Echavarria A, Tamariz E, Arona RM, Arnold E, Clapp C, Martinez de la Escalera G. Vasoinhibin suppresses the neurotrophic effects of vegf and ngf in newborn rat primary sensory neurons. Neuroendocrinology. 2018;106:221–233. doi: 10.1159/000477768. [DOI] [PubMed] [Google Scholar]
  • 6.Chan KM, Gordon T, Zochodne DW, Power HA. Improving peripheral nerve regeneration: from molecular mechanisms to potential therapeutic targets. Exp Neurol. 2014;261:826–835. doi: 10.1016/j.expneurol.2014.09.006. [DOI] [PubMed] [Google Scholar]
  • 7.Frostick SP, Yin Q, Kemp GJ. Schwann cells neurotrophic factors and peripheral nerve regeneration. Microsurgery. 1998;18:397–405. doi: 10.1002/(sici)1098-2752(1998)18:7<397::aid-micr2>3.0.co;2-f. [DOI] [PubMed] [Google Scholar]
  • 8.Geuna S. The sciatic nerve injury model in pre-clinical research. J Neurosci Methods. 2015;243:39–46. doi: 10.1016/j.jneumeth.2015.01.021. [DOI] [PubMed] [Google Scholar]
  • 9.Gong L, Wu J, Zhou S, Wang Y, Qin J, Yu B, Gu X, Yao C. Global analysis of transcriptome in dorsal root ganglia following peripheral nerve injury in rats. Biochem Biophys Res Commun. 2016;478:206–212. doi: 10.1016/j.bbrc.2016.07.067. [DOI] [PubMed] [Google Scholar]
  • 10.Gordon T, Chan KM, Sulaiman OA, Udina E, Amirjani N, Brushart TM. Accelerating axon growth to overcome limitations in functional recovery after peripheral nerve injury. Neurosurgery. 2009;65:A132–144. doi: 10.1227/01.NEU.0000335650.09473.D3. [DOI] [PubMed] [Google Scholar]
  • 11.Gu X. Progress and perspectives of neural tissue engineering. Front Med. 2015;9:401–411. doi: 10.1007/s11684-015-0415-x. [DOI] [PubMed] [Google Scholar]
  • 12.Ha SO, Kim JK, Hong HS, Kim DS, Cho HJ. Expression of brain-derived neurotrophic factor in rat dorsal root ganglia, spinal cord and gracile nuclei in experimental models of neuropathic pain. Neuroscience. 2001;107:301–309. doi: 10.1016/s0306-4522(01)00353-0. [DOI] [PubMed] [Google Scholar]
  • 13.Hajimiri M, Shahverdi S, Kamalinia G, Dinarvand R. Growth factor conjugation: strategies and applications. J Biomed Mater Res A. 2015;103:819–838. doi: 10.1002/jbm.a.35193. [DOI] [PubMed] [Google Scholar]
  • 14.Jessen KR, Mirsky R. The repair Schwann cell and its function in regenerating nerves. The J Physiol. 2016;594:3521–3531. doi: 10.1113/JP270874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Kaval Oguz E. Neuronal survival of DRG neurons after neurite transection in vitro promotes by nerve growth factor and brain derived neurotrophic factor. Cell Mol Biol (Noisy-le-grand) 2018;64:41–46. [PubMed] [Google Scholar]
  • 16.Kim DS, Lee SJ, Park SY, Yoo HJ, Kim SH, Kim KJ, Cho HJ. Differentially expressed genes in rat dorsal root ganglia following peripheral nerve injury. Neuroreport. 2001;12:3401–3405. doi: 10.1097/00001756-200110290-00050. [DOI] [PubMed] [Google Scholar]
  • 17.Li L, Li Y, Yang C. Chemical filtration of Cr (VI) with electrospun chitosan nanofiber membranes. Carbohydr Polym. 2016;140:299–307. doi: 10.1016/j.carbpol.2015.12.067. [DOI] [PubMed] [Google Scholar]
  • 18.Li R, Liu Z, Pan Y, Chen L, Zhang Z, Lu L. Peripheral nerve injuries treatment: a systematic review. Cell Biochem Biophys. 2014;68:449–454. doi: 10.1007/s12013-013-9742-1. [DOI] [PubMed] [Google Scholar]
  • 19.Li S, Wang X, Gu Y, Chen C, Wang Y, Liu J, Hu W, Yu B, Wang Y, Ding F, Liu Y, Gu X. Let-7 microRNAs regenerate peripheral nerve regeneration by targeting nerve growth factor. Mol Ther. 2015;23:423–433. doi: 10.1038/mt.2014.220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402–408. doi: 10.1006/meth.2001.1262. [DOI] [PubMed] [Google Scholar]
  • 21.Lowenstein DH, Arsenault L. The effects of growth factors on the survival and differentiation of cultured dentate gyrus neurons. J Neurosci. 1996;16:1759–1769. doi: 10.1523/JNEUROSCI.16-05-01759.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Luis AL, Rodrigues JM, Geuna S, Amado S, Simoes MJ, Fregnan F, Ferreira AJ, Veloso AP, Armada-da-Silva PA, Varejao AS, Mauricio AC. Neural cell transplantation effects on sciatic nerve regeneration after a standardized crush injury in the rat. Microsurgery. 2008;28:458–470. doi: 10.1002/micr.20524. [DOI] [PubMed] [Google Scholar]
  • 23.Magill CK, Tong A, Kawamura D, Hayashi A, Hunter DA, Parsadanian A, Mackinnon SE, Myckatyn TM. Reinnervation of the tibialis anterior following sciatic nerve crush injury: a confocal microscopic study in transgenic mice. Exp Neurol. 2007;207:64–74. doi: 10.1016/j.expneurol.2007.05.028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Martin SL, Reid AJ, Verkhratsky A, Magnaghi V, Faroni A. Gene expression changes in dorsal root ganglia following peripheral nerve injury: roles in inflammation, cell death and nociception. Neural Regen Res. 2019;14:939–947. doi: 10.4103/1673-5374.250566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Modgil A, Zhang Q, Pingili A, Singh N, Yao F, Ge J, Guo L, Xuan C, O’Rourke ST, Sun C. Angiotensin-(1-7) attenuates the chronotropic response to angiotensin II via stimulation of PTEN in the spontaneously hypertensive rat neurons. Am J Physiol Heart Circ Physiol. 2012;302:H1116–1122. doi: 10.1152/ajpheart.00832.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Nagalakshmi U, Waern K, Snyder M. RNA-Seq: a method for comprehensive transcriptome analysis. Curr Protoc Mol Biol. 2010 doi: 10.1002/0471142727.mb0411s89. Chapter 4:Unit 4111-13. [DOI] [PubMed] [Google Scholar]
  • 27.Namgung U. The role of Schwann cell-axon interaction in peripheral nerve regeneration. Cells Tissues Organs. 2014;200:6–12. doi: 10.1159/000370324. [DOI] [PubMed] [Google Scholar]
  • 28.Nilsson A, Kanje M. Amphiregulin acts as an autocrine survival factor for adult sensory neurons. Neuroreport. 2005;16:213–218. doi: 10.1097/00001756-200502280-00002. [DOI] [PubMed] [Google Scholar]
  • 29.Noble J, Munro CA, Prasad VS, Midha R. Analysis of upper and lower extremity peripheral nerve injuries in a population of patients with multiple injuries. J Trauma. 1998;45:116–122. doi: 10.1097/00005373-199807000-00025. [DOI] [PubMed] [Google Scholar]
  • 30.Ohara S, Ikuta F. Schwann cell responses during regeneration after one or more crush injuries to myelinated nerve fibres. Neuropathol Appl Neurobiol. 1988;14:229–245. doi: 10.1111/j.1365-2990.1988.tb00884.x. [DOI] [PubMed] [Google Scholar]
  • 31.Onger ME, Delibas B, Turkmen AP, Erener E, Altunkaynak BZ, Kaplan S. The role of growth factors in nerve regeneration. Drug Discov Ther. 2017;10:285–291. doi: 10.5582/ddt.2016.01058. [DOI] [PubMed] [Google Scholar]
  • 32.Osorio L, Teillet MA, Catala M. Role of noggin as an upstream signal in the lack of neuronal derivatives found in the avian caudal-most neural crest. Development. 2009;136:1717–1726. doi: 10.1242/dev.028373. [DOI] [PubMed] [Google Scholar]
  • 33.Park JW, Hwang SR, Yoon IS. Advanced growth factor delivery systems in wound management and skin regeneration. Molecules. 2017;22:E1259. doi: 10.3390/molecules22081259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Petkau TL, Leavitt BR. Progranulin in neurodegenerative disease. Trends Neurosci. 2014;37:388–398. doi: 10.1016/j.tins.2014.04.003. [DOI] [PubMed] [Google Scholar]
  • 35.Qin J, Wu JC, Wang QH, Zhou SL, Mao SS, Yao C. Transcription factor networks involved in cell death in the dorsal root ganglia following peripheral nerve injury. Neural Regen Res. 2018;13:1622–1627. doi: 10.4103/1673-5374.237183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Qin J, Wang L, Sun Y, Sun X, Wen C, Shahmoradi M, Zhou Y. Concentrated growth factor increases Schwann cell proliferation and neurotrophic factor secretion and promotes functional nerve recovery in vivo. Int J Mol Med. 2016;37:493–500. doi: 10.3892/ijmm.2015.2438. [DOI] [PubMed] [Google Scholar]
  • 37.Savastano LE, Laurito SR, Fitt MR, Rasmussen JA, Gonzalez Polo V, Patterson SI. Sciatic nerve injury: a simple and subtle model for investigating many aspects of nervous system damage and recovery. J Neurosci Methods. 2014;227:166–180. doi: 10.1016/j.jneumeth.2014.01.020. [DOI] [PubMed] [Google Scholar]
  • 38.Schwieger J, Warnecke A, Lenarz T, Esser KH, Scheper V. Neuronal survival morphology and outgrowth of spiral ganglion neurons using a defined growth factor combination. PLoS One. 2015;10:e0133680. doi: 10.1371/journal.pone.0133680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Seib DR, Corsini NS, Ellwanger K, Plaas C, Mateos A, Pitzer C, Niehrs C, Celikel T, Martin-Villalba A. Loss of Dickkopf-1 restores neurogenesis in old age and counteracts cognitive decline. Cell Stem Cell. 2013;12:204–214. doi: 10.1016/j.stem.2012.11.010. [DOI] [PubMed] [Google Scholar]
  • 40.Sung K, Yang W, Wu C. Uncoupling neurotrophic function from nociception of nerve growth factor: what can be learned from a rare human disease? Neural Regen Res. 2019;14:570–573. doi: 10.4103/1673-5374.247442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Suzuki M, Svendsen CN. Combining growth factor and stem cell therapy for amyotrophic lateral sclerosis. Trends Neurosci. 2008;31:192–198. doi: 10.1016/j.tins.2008.01.006. [DOI] [PubMed] [Google Scholar]
  • 42.Tosaki T, Kamiya H, Yasuda Y, Naruse K, Kato K, Kozakae M, Nakamura N, Shibata T, Hamada Y, Nakashima E, Oiso Y, Nakamura J. Reduced NGF secretion by Schwann cells under the high glucose condition decreases neurite outgrowth of DRG neurons. Exp Neurol. 2008;213:381–387. doi: 10.1016/j.expneurol.2008.06.017. [DOI] [PubMed] [Google Scholar]
  • 43.Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010;28:511–515. doi: 10.1038/nbt.1621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Whitmire W, Al-Gayyar MM, Abdelsaid M, Yousufzai BK, El-Remessy AB. Alteration of growth factors and neuronal death in diabetic retinopathy: what we have learned so far. Mol Vis. 2011;17:300–308. [PMC free article] [PubMed] [Google Scholar]
  • 45.Yamakawa S, Hayashida K. Advances in surgical applications of growth factors for wound healing. Burns Trauma. 2019;7:10. doi: 10.1186/s41038-019-0148-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Yang G, Gray TS, Sigmund CD, Cassell MD. The angiotensinogen gene is expressed in both astrocytes and neurons in murine central nervous system. Brain Res. 1999;817:123–131. doi: 10.1016/s0006-8993(98)01236-0. [DOI] [PubMed] [Google Scholar]
  • 47.Yi S, Zhang H, Gong L, Wu J, Zha G, Zhou S, Gu X, Yu B. Deep sequencing and bioinformatic analysis of lesioned sciatic nerves after crush injury. PLoS One. 2015;10:e0143491. doi: 10.1371/journal.pone.0143491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Yi S, Yuan Y, Chen Q, Wang X, Gong L, Liu J, Gu X, Li S. Regulation of Schwann cell proliferation and migration by miR-1 targeting brain-derived neurotrophic factor after peripheral nerve injury. Sci Rep. 2016;6:29121. doi: 10.1038/srep29121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Zawada WM, Kirschman DL, Cohen JJ, Heidenreich KA, Freed CR. Growth factors rescue embryonic dopamine neurons from programmed cell death. Exp Neurol. 1996;140:60–67. doi: 10.1006/exnr.1996.0115. [DOI] [PubMed] [Google Scholar]
  • 50.Zhang R, Zhang Y, Yi S. Identification of critical growth factors for peripheral nerve regeneration. RSC Adv. 2019;9:10760–10765. doi: 10.1039/c9ra01710k. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Zhou S, Yin DP, Wang Y, Tian Y, Wang ZG, Zhang JN. Dynamic changes in growth factor levels over a 7-day period predict the functional outcomes of traumatic brain injury. Neural Regen Res. 2018;13:2134–2140. doi: 10.4103/1673-5374.241462. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Neural Regeneration Research are provided here courtesy of Wolters Kluwer -- Medknow Publications

RESOURCES