doi: 10.1016/j.jneumeth.2013.11.012, Vargas, M. E., and Barres, B. Epub 2019 Mar 7. Our study, from the genetic aspect, confirmed the critical roles of macrophages during Wallerian degeneration. Mucosal Immunol. Would you like email updates of new search results? The thermal setting was 5 min at 95°C; 40 cycles of 30 seconds at 95°C, 45 seconds at the annealing temperature, 30 seconds at 72°C; and 5 min at 72°C. Exp. Oncol. Neurobiol. doi: 10.3892/ol.2015.3472, Yao, D., Li, M., Shen, D., Ding, F., Lu, S., Zhao, Q., et al. Similar as the results from canonical pathway analysis, diseases and functions with a p-value less than 10−5 as well as involved representative genes are listed in Figure 3. doi: 10.1016/j.bbi.2010.07.249, Benowitz, L. I., and Popovich, P. G. (2011). Red color indicates up-regulation while green color indicates down-regulation. Expression changes and bioinformatic analysis of Wallerian degeneration after sciatic nerve injury in rat. eCollection 2021. IPA makes it easy to take this result even further by examining what biological processes, pathways, and diseases the transcriptional regulators and their targets may control, and how these upstream molecules may regulate one another. All categories of canonical pathways and their associated genes are listed in Supplementary Table 2. Gene networks and their related top diseases and functions are listed in Supplementary Table 4. Keywords: 536, 56–63. Rev. PLoS ONE 8:e57000. Cell Sci. The ratio is calculated as follows: The number of molecules in a given pathway that meet cutoff criteria, divided by total number of molecules that make up that pathway and that are in the reference set (see Defining the Reference Set). Neurol. Total RNA was extracted with Trizol reagent (Life technologies, Carlsbed, CA) and cleaned with RNeasy spin columns (Qiagen, Valencia, CA). Mol. doi: 10.1002/mus.24082, Li, S., Liu, Q., Wang, Y., Gu, Y., Liu, D., Wang, C., et al. Glia 64, 730–742. All these results highlight the central roles of the MMP family in nerve regeneration. Analyzed the data: JY and SY. Genes with a fold change greater than 2 (the absolute value of log2 fold change greater than 1) and an adjust p-value less than 0.05 were considered as differentially expressed and were investigated by IPA (Ingenuity Systems Inc., Redwood City, CA). 9, S33–S38. Cell. 2010 May;59(Pt 5):528-533. doi: 10.1099/jmm.0.016006-0. J. Neurochem. Why were not the myelin-related genes up-regulated until a relatively later stage post injury? The interferon signalling and hepatic fibrosis/hepatic stellate cell activation pathways were among the top 5 canonical pathways in all strains. Limited knowledge exists on pathways, networks and transcriptional factors regulated within epithelial cells by diverse Mycobacterium tuberculosis genotypes. J. Neurosci. 10:274. doi: 10.3389/fncel.2016.00274. The brightness of color is related to the fold change of differentially expressed gene and the darker the color, the higher fold change. Fortunately, the progress in bioinformatic analysis allows us to better interpret the gene expression profiles as revealed by microarray. From 4 days until 4 weeks post injury, less canonical pathways were activated. Int. Annu. Neurosci., 06 December 2016 Peripheral nerve injury and repair. Nihon Saikingaku Zasshi. Myelin is an important inhibiting factor for neurite growth and nerve repair (Bähr and Przyrembel, 1995; David et al., 1995). Figure 4. Neurosci. (1995). Surg. Sci. The inflammation and immune response, cytokine signaling, cellular growth and movement, and, especially, Nervous System Development and Function, were significantly activated in the distal nerve stump post peripheral nerve injury, suggesting their critical roles in Wallerian degeneration. National Library of Medicine Schwann cells and endoneurial extracellular matrix molecules as potential cues for sorting of regenerated axons: a review. This study aimed to elucidate these mechanisms induced in A549 epithelial cells by dominant clinical strains in KwaZulu-Natal, South Africa. 2021 Feb 4;12:612675. doi: 10.3389/fmicb.2021.612675. Expert Rev Proteomics. ＊PMCでIngenuity Pathway Analysisを検索をした結果 ナレッジ＆データベース 遺伝子、タンパク質、組織、細胞、ドラッグ、疾患などに関する、約760万（2020年10月現在）にもおよぶ生物学的機能、相互作用に関する情報が収集されたデータベースです。 An integrated global view of gene expression patterns during Wallerian degeneration was obtained by performing IPA core analysis, overlaying microarray data to IPKB, and then assigning differentially expressed genes to specific canonical pathways, diseases and biological functions, and networks. This study was supported by the Natural Science Foundation of Jiangsu Province, China (Grant No. Biomaterials 35, 6143–6156. Muscle Nerve 50, 73–78. 2019 May;12(3):795-804. doi: 10.1038/s41385-019-0156-2. doi: 10.1093/nar/gkv007, Shubayev, V. I., Angert, M., Dolkas, J., Campana, W. M., Palenscar, K., and Myers, R. R. (2006). doi: 10.1002/jnr.490420417, Donnelly, D. J., and Popovich, P. G. (2008). Brain Behav. Bioinformatics analysis performed with the RNA-Seq Tuxedo pipeline identified differentially expressed genes. In this study, besides MMP9 and BDNF, myelin-related genes (MAG and MAL) were also found to be up-regulated starting from 4 days post injury. Table 2. |, https://www.frontiersin.org/article/10.3389/fncel.2016.00274/full#supplementary-material, Creative Commons Attribution License (CC BY), Jiangsu Key Laboratory of Neuroregeneration, Co-innovation Center of Neuroregeneration, Nantong University, Nantong, China. At 4 days to 4 weeks post injury, which represented a sub-chronic injury stage, less numerous categories of diseases and functions were involved. Adhesion to and invasion of pulmonary epithelial cells by the F15/LAM4/KZN and Beijing strains of Mycobacterium tuberculosis. 209, 378–388. doi: 10.1002/neu.480230507, Campbell, W. W. (2008). List of primer pairs for real-time quantitative qPCR. It is worth noting that cellular function Nervous System Development and Function was vastly involved in many gene networks at nearly all tested time points, except at 0.5 and 1 h post injury (Table 2). Anat. Small RNA Mcr11 requires the transcription factor AbmR for stable expression and regulates genes involved in the central metabolism of Mycobacterium tuberculosis. Consistent with the gene network diagram, MMP9 and BDNF were up-regulated post nerve injury while MAG and MAL were down-regulated. doi: 10.1097/00005072-199507000-00002, Lykissas, M. G., Batistatou, A. K., Charalabopoulos, K. A., and Beris, A. E. (2007). Bioinformatic analysis was performed to analysis differentially expressed genes at 0.5, 1, 6, 12, and 24 h, 4 days, and 1, 2, 3, and 4 weeks post sciatic nerve transection. Consistent with the results of canonical pathway analysis, following sciatic nerve transection, the number of categories of diseases and functions first increased and then progressively declined. At 0.5, 1, 6, 12, and 24 h, 4 days, and 1, 2, 3, and 4 weeks after surgery, rats were sacrificed by decapitation. doi: 10.1016/S0074-7742(09)87003-7, Gu, X., Ding, F., and Williams, D. F. (2014). To further identify key genes involved in this cellular function and establish the connections in-between these genes, the gene network diagrams were made based on IPKB. Exp. Up-regulated MMP9 stimulates the recruitment and migration of macrophages, mediates the differentiation of myelinating Schwann cells, and thus benefits nerve regeneration (Shubayev et al., 2006; Kim et al., 2012). QIAGEN Ingenuity Pathway Analysis helps you perform insightful data analysis and interpretation to understand your experimental results … Nerves are fragile tissues that are susceptible to traumatic injuries, such as penetration, crushing, and stretch tractions (Campbell, 2008). COVID-19 is an emerging, rapidly evolving situation. Top enriched canonical pathways following sciatic nerve transection. Neurobiol. Lett. A. Ingenuity pathway analysis indicated that inflammation and immune response, cytokine signaling, cellular growth and movement, as well as tissue development and function were significantly activated following sciatic nerve injury. The distal nerve stumps were removed and stored at −80°C. Ingenuity® Pathway Analysis (IPA) software was used to conduct pathway enrichment based on the recruited gene datasets mentioned above. All authors have read and approved the final manuscript. Copyright © 2016 Yu, Gu and Yi. Strain-specific behavior of Mycobacterium tuberculosis in A549 lung cancer cell line. BDNF is a well-known neurotrophic factor that promotes neuronal survival and activity and stimulates axon growth (Braun et al., 1996; Lykissas et al., 2007). Nerve injury is a common and difficult clinical problem worldwide with a high disability rate. Hadifar S, Mostafaei S, Behrouzi A, Fateh A, Riahi P, Siadat SD, Vaziri F. BMC Bioinformatics. doi: 10.1016/j.biomaterials.2014.04.064, Jang, S. Y., Shin, Y. K., Park, S. Y., Park, J. Y., Lee, H. J., Yoo, Y. H., et al. Canonical pathways with a p-value < 10−3 (1E-03) are labeled in red color while canonical pathways with a p-value > 10−3 (1E-03) are labeled in white color. 43:e47. Epub 2017 Oct 9. Neurosci. These top networks were mainly connected to the functions: Cellular Movement and Cell-To-Cell Signaling. The gene list was loaded, and the source of the organism was filtered as only “Homo Sapiens”, so that the analysis of interactions and pathways was limited to humans. Ingenuity Pathway Analysis (IPA) was used to investigate canonical pathways and immunohistochemical staining was performed to validate protein expression levels in samples. Notably, a cellular function called Nervous System Development and Function was drastically stimulated at 6, 12, and 24 h post injury, and was enriched during nearly whole post-injury period. Nerve injury disturbs signal transmission, causes loss or alteration of sensation, impairs the power and function of target organs, and leads to disability and even mortality of victims. Different from the central nervous system, the peripheral nervous system is able to regenerate after injury. Nerves are fragile tissues that are susceptible to traumatic injuries, such as penetration, crushing, and stretch tractions (Campbell, 2008). Notably, a cellular function highly related to nerve regeneration, which is called Nervous System Development and Function, was continuously activated from 4 days until 4 weeks post injury. doi: 10.1016/j.expneurol.2007.06.009, Dubový, P. (2004). Differential gene expression profiling and biological process analysis in proximal nerve segments after sciatic nerve transection. 25, 1179–1187. Mycobacterium tuberculosis strains induce strain-specific cytokine and chemokine response in pulmonary epithelial cells. Among the transcriptional factors, only EHL, IRF7, PML, STAT1, STAT2 and VDR were induced by all clinical strains. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/article/10.3389/fncel.2016.00274/full#supplementary-material. Allué-Guardia A, García JI, Torrelles JB. 36:51-37:19 My Lists, My Pathways, and Molecules tab Epub 2010 Jan 28. The application of IPA analysis helps to decipher dynamic molecular changes in the distal part of injured sciatic nerve, especially in Schwann cells and macrophages, two important types of cells in the distal stump, and thus may provide some new knowledge about Wallerian degeneration. Bähr, M., and Przyrembel, C. (1995). B., Archer, D. R., Li, Y., and Griffin, J. W. (2005). The relative expression levels of (A) MMP9, (B) BDNF, (C) MAG, (D) MAL, (E) SHH, (F) SLC6A17, and (G) RET were calculated using comparative Ct with GAPDH as the reference gene. Myelin from peripheral and central nervous system is a nonpermissive substrate for retinal ganglion cell axons. Rats were randomly divided into 11 groups according to different observation points and went through surgical transection of sciatic nerves as previously described (Yu et al., 2012). Neurosci. Microarray data analysis was performed by the R software platform (v.2.13.0) and the limma (linear regression model) package (Ritchie et al., 2015; Xu and Sun, 2015). Activation of the different pathways, networks and transcriptional factors revealed in the current study may be an underlying mechanism that results in the differential host response by clinical strains of M. tuberculosis. To further validate the gene expression profiles identified by microarray analysis, some representative differentially expressed genes involved in Nervous System Development and Function were selected for qRT-PCR examination (Figure 6). The quality of qPCR was examined by a single peak melt curve corresponding to single PCR product. f4-ijmm-35-06-1511: Ingenuity Pathway Analysis (IPA) canonical pathways most significantly changed in third-trimester peripheral blood NK (pNK) cells compared to first-trimester pNK cells. IPA results provided us with some new insights into Wallerian degeneration. Over the last 160 years, however, most studies on Wallerian degeneration have been limited to morphological descriptions while molecular changes during Wallerian degeneration have not been fully elucidated (Lee and Wolfe, 2000; Zochodne, 2000; Geuna et al., 2009; Sta et al., 2014). Am. 2020 Feb;113(2):504-520. doi: 10.1111/mmi.14436. 108, 173–206. The microenvironment of injured and regenerating peripheral nerves. Only a signaling pathway, called Agranulocyte Adhesion and Diapedesis, was activated during nearly whole post-injury period. Supplementary Table 1. Int. In the current study, we made rat sciatic nerve transection, collected the distal nerve stump samples at different time points, re-analyzed previous obtained microarray data (Yao et al., 2012, 2013) by using the R software and the limma package and uploaded the massive microarray data into the IPA program for a systematic bioinformatic analysis. One of advanced bioinformatic tools is the Ingenuity pathway analysis (IPA) software program, which can analyze the gene expression patterns using a build-in scientific literature based database (according to IPA Ingenuity Web Site, www.ingenuity.com). (2016). Genes that were first up-regulated and then down-regulated are boxed in the red frame while genes that were first down-regulated and then up-regulated are boxed in the green frame. IPA Canonical Pathways Canonical pathways are constructed, curated, IPA pathway diagrams for well established signaling and metabolic pathways Vary in size May contain one or more pathway branches or paths –Shows the canonical pathway in a large context Canonical Pathways can be modified and saved Ann. At 6 to 24 h post injury, diseases and cellular functions related to organ development, tissue and organ morphology, tissue disorder, as well as organismal survival and function were significantly activated. The heapmap suggested that nearly 2/3 of genes involved in this cellular function were firstly up-regulated and then down-regulated while a little bit less than 1/3 of differentially genes were first down-regulated and then up-regulated. Neural Regenerat. Med. Int. 15:25-22:35 Core Analysis Canonical Pathway results. Cell. Res. Ratio Calculations for Pathways Go Back Ratio Calculations for Pathways. Top enriched networks following sciatic nerve transection. Further in-depth studies are needed to determine which genes and functions represent the key molecular elements for peripheral nerve regeneration. doi: 10.1016/j.aanat.2011.03.001, Ravanti, L., and Kähäri, V. M. (2000). BMC Genomics. Learn how to navigate through the Canonical Pathway results in a QIAGEN Ingenuity Pathway Analysis (IPA) Core Analysis. The authors thank Professor Jie Liu for his help in manuscript preparation. (2014). At 1 h post nerve transection, canonical pathways related to immune response and cytokine signaling were activated. This product is not intended for the diagnosis, prevention, or treatment of a disease. Degeneration of myelinated efferent fibers prompts mitosis in Remak Schwann cells of uninjured C-fiber afferents. Red color indicates the increased expression whereas green color indicates the decreased expression as compared to control (expression at 0 h). Anat. Data are summarized from 3 independent experiments and values are shown as the means ± SEM. 6, 391–407. (1998). 9, 1186–1194. Res. 10, 1402–1408. Deep sequencing and bioinformatic analysis of lesioned sciatic nerves after crush injury. Neural tissue engineering options for peripheral nerve regeneration. (2014). Rev. Microsurgery 18, 397–405. Macrophages, monocytes, and Schwann cells collectively remove axon and myelin debris and contribute to the construction of a favorable microenvironment for nerve regeneration (Brown et al., 1991, 1992; Vargas and Barres, 2007; Chen et al., 2015). There has been a previous study which compared the differentially gene expressions in proximal and distal nerves after nerve injury (Jiang et al., 2014). Eur. As indicated by all diagrams, IL-6 was immediately up-regulated at 0.5 h post nerve injury and highly expressed at all tested time points; with the passage of time, more genes were differentially expressed and the molecular network seemed more complex. PLoS ONE 10:e0143491. 40, 99–107. 22:36-23:31 Overlapping Canonical Pathway results. The involvement of Nervous System Development and Function at different time points. Sonic hedgehog (SHH) was first up-regulated at 6 h post injury and then down-regulated at later time points. Besides the predominant pathways and cellular functions, gene networks were built to connect key genes and enriched categories of diseases and functions based on the correlation between differentially expressed genes. Differential temporal expression of matrix metalloproteinases following sciatic nerve crush. Conceived and designed the experiments: JY, XG and SY. Perspectives in regeneration and tissue engineering of peripheral nerves. doi: 10.2174/156720207780637216, Murinson, B. Consequences of slow Wallerian degeneration for regenerating motor and sensory axons. doi: 10.1007/s12031-012-9813-6, Frostick, S. P., Yin, Q., and Kemp, G. J. doi: 10.5435/00124635-200007000-00005, Li, M., Guo, W., Zhang, P., Li, H., Gu, X., and Yao, D. (2013). Macrophage recruitment in different models of nerve injury: lysozyme as a marker for active phagocytosis. Sci. 193, 334–340. At relatively later time points post peripheral nerve injury, the most significant canonical pathways and cellular functions were found to be tissue development and function. miR-221 and miR-222 promote Schwann cell proliferation and migration by targeting LASS2 after sciatic nerve injury. doi: 10.3969/j.issn.1673-5374.2012.17.001, Yao, D., Li, M., Shen, D., Ding, F., Lu, S., Zhao, Q., et al. J. Neurosci. Hu W, Yang S, Shimada Y, Münch M, Marín-Juez R, Meijer AH, Spaink HP. [Analysis of molecular mechanisms of the virulence and growth coordination of Mycobacterium tuberculosis]. doi: 10.1002/(SICI)1098-2752(1998)18:7<397::AID-MICR2>3.0.CO;2-F, Gaudet, A. D., Popovich, P. G., and Ramer, M. S. (2011). 30, 153–179. doi: 10.1016/j.mcn.2005.10.011, Sta, M., Cappaert, N. L., Ramekers, D., Baas, F., and Wadman, W. J. Orthop. No use, distribution or reproduction is permitted which does not comply with these terms. Different from the central nervous system that can hardly regenerate by itself, the peripheral nervous system has a certain ability to regenerate on its own (Raimondo et al., 2011; Gu et al., 2014). doi: 10.1006/exnr.1995.1039, PubMed Abstract | CrossRef Full Text | Google Scholar, Barrette, B., Calvo, E., Valliéres, N., and Lacroix, S. (2010). At 0.5, 1, 6, 12, and 24 h post nerve injury, top networks had scores of 21, 23, 23, 27, and 29, respectively. IPA allows you to compare analysis results from multiple experimental groups to identify similarities, differences, and trends. FOIA Inflammation reaction and immune response are protective reactions against injury stimuli (Donnelly and Popovich, 2008; Benowitz and Popovich, 2011; Dubový et al., 2013; Li S. et al., 2013). Sci. doi: 10.1002/1097-4598(2000)999:9<::AID-MUS7>3.0.CO;2-F, Keywords: sciatic nerve transection, distal nerve stump, microarray, bioinformatics, Ingenuity pathway analysis, Citation: Yu J, Gu X and Yi S (2016) Ingenuity Pathway Analysis of Gene Expression Profiles in Distal Nerve Stump following Nerve Injury: Insights into Wallerian Degeneration. The representative differentially expressed genes in the disease and function are listed to the right. (2007). List of the gene networks at 0.5, 1, 6, 12, and 24 h, 4 days, and 1, 2, 3, and 4 weeks post sciatic nerve transection. Why is Wallerian degeneration in the CNS so slow? It may be because that at an early stage after nerve injury only non-myelinating Schwann cells, but not myelinating Schwann cells, enter into the cell cycle to avoid negative factors for nerve regeneration (Murinson et al., 2005), but later on myelinating Schwann cells start to proliferate and to form myelin sheaths (Liu et al., 1995; Vargas and Barres, 2007). RNA samples were converted to cDNA using the Prime-Script reagent Kit (TaKaRa, Dalian, China). Heatmaps for Canonical Pathways, Upstream Regulators, and Diseases & Functions allow you to easily and quickly view information from your analyses side-by-side. 130, 605–618. It has been known that after peripheral nerve injury, Schwann cells not only proliferate to form the bands of Bungner, but also produce a range of neurotrophic factors, including BDNF (Frostick et al., 1998; Faroni et al., 2013; Jang et al., 2016). Timely inflammatory and immune reactions are highly relevant with Wallerian degeneration, and benefit subsequent nerve repair and functional recovery (Dubový, 2011; Gaudet et al., 2011). (2015). Matrix metalloproteinases in wound repair (review). Schwann cells, neurotrophic factors, and peripheral nerve regeneration. In addition to canonical pathways, differentially expressed genes were also categorized to related diseases and functions. Obtained cDNA was used as a template for amplification and qPCR was performed using SYBR Green Premix Ex Taq (TaKaRa) with specific primer pairs on an Applied Biosystems Stepone real-time PCR System. Cell. Exp. The functional and morphological characteristics of sciatic nerve degeneration and regeneration after crush injury in rats. 79, 198–208. Inflammation and axon regeneration. IPA was performed to identify canonical pathways, diseases and functions, and gene networks that are most significant to microarray outcomes and to categorize differentially expressed genes in specific diseases and functions. The IPA system (version 42012434, Ingenuity Systems; Qiagen China Co., Ltd.) was used for subsequent bioinformatics analysis, which included canonical pathway analysis, disease and function, regulator effects, upstream regulators and molecular networks. Autophagic myelin destruction by Schwann cells during Wallerian degeneration and segmental demyelination. In this study we used microarray and bioinformatic analysis to examine dynamic molecular changes in the distal nerve stump following peripheral nerve injury. However, the induction of top scoring networks varied among the strains. Canonical pathways; Epithelial cells; Mycobacterium tuberculosis; Networks; RNA sequencing; Transcriptional factors. Neural Regenerat. Heatmap and hierarchical clustering of genes involved in Nervous System Development and Function. Differentially expressed genes in Nervous System Development and Function at 4 weeks post sciatic nerve transection. Protein expression profiling during Wallerian degeneration after rat sciatic nerve injury. Bethesda, MD 20894, Copyright Acad. Regulation of Schwann cell proliferation and migration by miR-1 targeting brain-derived neurotrophic factor after peripheral nerve injury. The top enriched categories of canonical pathways with a p-value less than 10−3 as well as representative differentially expressed genes in each canonical pathway are listed in Figure 2. The expression levels of genes were indicated by the color bar above the heatmap. doi: 10.1007/s00401-015-1482-4, Chen, Z. L., Yu, W. M., and Strickland, S. (2007). Poor growth of Mammalian motor and sensory axons into intact proximal nerve stumps. Curr. Different from the central nervous syst… 134, 87–93. canonical-pathway Sizes: 150x83 / 300x166 / 600x332 / 900x498 / 2362x1306 / Among these gene network diagrams, a diagram related to 4 weeks post nerve injury is shown in Figure 5, and other diagrams related to other time points are shown in Supplementary Figure 1. The number of differentially expressed genes increased starting from 6 h post injury and remained at a high level at 4 weeks post injury (Figure 1). Front Microbiol. 34:58-36:16 Network results. Received: 12 September 2016; Accepted: 15 November 2016; Published: 06 December 2016. (2012). This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). An Overview of the Canonical Pathways in IPA Causal Networks Ingenuity Pathway Analysis (IPA) | Advanced Analytics - Generate virtual upstream regulators which are networks of regulators Upstream Regulators Analysis Tutorial Ingenuity Pathway Analysis (IPA) doi: 10.1016/j.neulet.2013.01.008, Li, M., Zhang, P., Guo, W., Li, H., Gu, X., and Yao, D. (2014). 2016 Dec;65:321-329. doi: 10.1016/j.dci.2016.07.022. Immediately after peripheral nerve injury, the most significant pathways and cellular functions were found to be inflammatory and immune response, which remained to be activated until 4 weeks post nerve injury. (2015). Neurovasc. Figure 3. The expression patterns of SHH, solute carrier family 6 member 17 (SLC6A17), and ret proto-oncogene RET were also in agreement with microarray results. doi: 10.1002/glia.22957, Jiang, N., Li, H., Sun, Y., Yin, D., Zhao, Q., Cui, S., et al. Epub 2016 Aug 3. limma powers differential expression analyses for RNA-sequencing and microarray studies.
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