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. The differentially expressed genes were categorized to related canonical pathways based on IPKB. Wallerian degeneration in the distal nerve stump contributes to the construction of a permissible microenvironment for peripheral nerve regeneration. Learn how to navigate through the Canonical Pathway results in a QIAGEN Ingenuity Pathway Analysis (IPA) Core Analysis. Oncol. J. Neurosci. Gene expression profiling reveals that peripheral nerve regeneration is a consequence of both novel injury-dependent and reactivated developmental processes. Nerve injury is a common and difficult clinical problem worldwide with a high disability rate. Neurosci. Why is Wallerian degeneration in the CNS so slow? However, the up-regulation of MMP9 was largely compromised in mice with delayed Wallerian degeneration (Shubayev et al., 2006; Barrette et al., 2010). Sci. After peripheral nerve injury, axons and their myelin sheaths in the distal nerve stump are disrupted, and Wallerian degeneration takes place. Transcriptional profiling of the injured sciatic nerve of mice carrying the Wld(S) mutant gene: identification of genes involved in neuroprotection, neuroinflammation, and nerve regeneration. Muscle Nerve 50, 73–78. For instance, matrix metalloproteinase 9 (MMP9) and brain derived neurotrophic factor (BDNF) were drastically up-regulated starting from 6 h post injury and remained at high expression levels until 4 weeks post injury. Anat. 25, 1179–1187. In contrast, many of these biological processes were also detected in the distal nerve stump after peripheral nerve injury, as revealed by this study. During tissue remodeling, MMPs mediate the breakdown of the extracellular matrix (ECM), a key network containing physical and biochemical cues for tissue regeneration (Ravanti and Kähäri, 2000; Dubový, 2004; Chen et al., 2007). Keywords: 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. IPA allows you to compare analysis results from multiple experimental groups to identify similarities, differences, and trends. Why were not the myelin-related genes up-regulated until a relatively later stage post injury? For example, Gene Ontology analysis suggested that differentially expressed genes in the distal nerve stump could be mainly divided into functional groups with regulatory functions, including cell communication, cell transport, and transcriptional regulation (Bosse et al., 2006). FOIA The significance of the association between the dataset and a canonical pathway was determined 28:28-31:22 Diseases & Functions results. doi: 10.1007/s12031-012-9813-6, Frostick, S. P., Yin, Q., and Kemp, G. J. The examination for the genes involved in Nervous System Development and Function indicated that MMP9 and BDNF were significantly up-regulated starting from 6 h post nerve injury. doi: 10.1016/0022-510X(95)00315-S, Brown, M. C., Lunn, E. R., and Perry, V. H. (1991). 10, 1402–1408. J. Neuropathol. At 1 h post nerve transection, canonical pathways related to immune response and cytokine signaling were activated. doi: 10.1002/mus.24082, Li, S., Liu, Q., Wang, Y., Gu, Y., Liu, D., Wang, C., et al. Immun. doi: 10.1002/jnr.490400111, Xu, A., and Sun, S. (2015). 23:32-28:27 Upstream Analysis results. doi: 10.1007/s12264-013-1340-0, Yi, S., Yuan, Y., Chen, Q., Wang, X., Gong, L., Liu, J., et al. Rev. Small RNA Mcr11 requires the transcription factor AbmR for stable expression and regulates genes involved in the central metabolism of Mycobacterium tuberculosis. J Med Microbiol. However, the induction of top scoring networks varied among the strains. J. Neurosci. Orthop. Mol. Heatmaps for Canonical Pathways, Upstream Regulators, and Diseases & Functions allow you to easily and quickly view information from your analyses side-by-side. IPA will subdivide your data into slices based on molecule connectivity (networks), cellular functions, and involvement in canonical pathways Spend time surveying the information. Matrix metalloproteinases in wound repair (review). All categories of diseases and functions and their associated genes are listed in Supplementary Table 3. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/article/10.3389/fncel.2016.00274/full#supplementary-material. Analyzed the data: JY and SY. The authors thank Professor Jie Liu for his help in manuscript preparation. Protein expression profiling during Wallerian degeneration after rat sciatic nerve injury. All these results highlight the central roles of the MMP family in nerve regeneration. Ann. Despite these findings, it is still required to further investigate the molecular changes of Wallerian degeneration from a genetic perspective. Cell. (1998). Neural Regen. J. Neurochem. Privacy, Help Hierarchical clustering for enrichment of cholesterol biosynthesis and immune associated pathways revealed similar patterns for Beijing and Unique; F15/LAM4/KZN and F11; and, F28 and H37Rv strains, respectively. Differentially expressed genes in Nervous System Development and Function at 4 weeks post sciatic nerve transection. 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). Res. C-fos expression, bFGF production, phagocytosis and proliferation during Wallerian degeneration. Following sciatic nerve transection, a diverse array of biological processes, including stimulus detection and response, inflammatory and immune response, cell migration, cell proliferation, cell death, axonal regeneration and guidance, and myelination, were significantly activated in the proximal nerve stump (Li S. et al., 2013). 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. Neurovasc. Ann. The representative differentially expressed genes in the disease and function are listed to the right. Supplementary Table 2. Inflammation and its role in neuroprotection, axonal regeneration and functional recovery after spinal cord injury. 11, 1165–1171. Expression changes and bioinformatic analysis of Wallerian degeneration after sciatic nerve injury in rat. J. The expression levels of genes were indicated by the color bar (attached to the raster plot). Cell. Build custom pathways and gene or chemical list libraries Create custom pathways with My Pathways and gene or chemical list libraries from a range of input data: gene lists from IPA search results, existing IPA networks or canonical pathways, uploaded lists of targets or biomarkers, or imported pathways using XGMML, BioPax, SBML, or GPML. Briefly, rats were anesthetized by an injection of mixed narcotics (85 mg/kg trichloroacetaldehyde monohydrate, 42 mg/kg magnesium sulfate, and 17 mg/kg sodium pentobarbital). The interferon signalling and hepatic fibrosis/hepatic stellate cell activation pathways were among the top 5 canonical pathways in all strains. Schwann cells, neurotrophic factors, and peripheral nerve regeneration. Differential gene expression profiling and biological process analysis in proximal nerve segments after sciatic nerve transection. 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). Glia 64, 730–742. The quality of qPCR was examined by a single peak melt curve corresponding to single PCR product. Rat sciatic nerve was exposed through an incision on the lateral aspect of the mid-thigh of the left hind limb and a 10-mm nerve segment was excised. Rev. RNA for sequencing was extracted from epithelial cells at 48 h post-infection with 5 strains at a multiplicity of infection of approximately 10:1. 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. Expert Rev Proteomics. Our results may help to illuminate the molecular aspects of Wallerian degeneration and to develop potential therapeutic targets for peripheral nerve repair. Total RNA was extracted with Trizol reagent (Life technologies, Carlsbed, CA) and cleaned with RNeasy spin columns (Qiagen, Valencia, CA). doi: 10.1016/j.aanat.2011.03.001, Ravanti, L., and Kähäri, V. M. (2000). The asterisk indicates significant difference: *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001 as compared to contrsol (0 h post injury). At 0.5, 1, 6, 12, and 24 h post nerve injury, top networks had scores of 21, 23, 23, 27, and 29, respectively. No use, distribution or reproduction is permitted which does not comply with these terms. 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. Regulation of Schwann cell proliferation and migration by miR-1 targeting brain-derived neurotrophic factor after peripheral nerve injury. Figure 1. Am. Top enriched canonical pathways following sciatic nerve transection. Res. A p-value less than 0.05 was considered as significantly different. The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. Exp. Wallerian degeneration: gaining perspective on inflammatory events after peripheral nerve injury. IPA読み取り専用ライセンス 【解析データの内容】 Canonical Pathway インプットデータと既知のパスウェイを比較し、p-value の低いパスウェイを抽出します。 Upstream Analysis 4, 143–151. 134, 87–93. The Wallerian degeneration process, since its first observation by Augustus Volney Waller in 1850, has been widely studied. Lett. doi: 10.1016/B978-0-12-410499-0.00007-1, Faroni, A., Calabrese, F., Riva, M. A., Terenghi, G., and Magnaghi, V. (2013). 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). Ingenuity Pathways Analysis (IPA) IPA is a web-based bioinformatics application that allows researchers to upload data analysis results from high-throughput experiments such as microarray and next generation sequencing for functional analyze, integration, and further understanding. Epub 2010 Jan 28. Role of inflammation and cytokines in peripheral nerve regeneration. Canonical pathways; Epithelial cells; Mycobacterium tuberculosis; Networks; RNA sequencing; Transcriptional factors. IPA, Ingenuity pathway analysis; SD, Sprague-Dawley; IPKB, Ingenuity pathway knowledge base; qPCR, quantitative real time polymerase chain reaction; IL, Interleukin; MMP, matrix metalloproteinase; BDNF, brain derived neurotrophic factor; SHH, sonic hedgehog; MAG, myelin-associated glycoprotein; MAL, myelin and lymphocyte protein; SLC6A17, solute carrier family 6 member 17; ECM, extracellular matrix. J. Neurosci. This product is not intended for the diagnosis, prevention, or treatment of a disease. 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. J. Neurosci. Nihon Saikingaku Zasshi. Res. Data are summarized from 3 independent experiments and values are shown as the means ± SEM. 2016 Dec;65:321-329. doi: 10.1016/j.dci.2016.07.022. The rats in the 0 h group received sham-surgery on their left sciatic nerves were used as controls. Annu. Epub 2019 Mar 7. Supplementary Table 5. From 4 days until 4 weeks post injury, less canonical pathways were activated. Sci. At 0.5 h post sciatic nerve transection, only Interleukin-17A (IL-17A) Signaling in Airway Cells (an IL-17 cytokine signaling) and TREM1 Signaling (a proinflammatory immune response) are significantly activated. Top enriched diseases and functions following sciatic nerve transection. doi: 10.1007/s00401-015-1482-4, Chen, Z. L., Yu, W. M., and Strickland, S. (2007). 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). Our results may provide further understanding of Wallerian degeneration from a genetic perspective, thus aiding the development of potential therapies for peripheral nerve injury. 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. List of primer pairs for real-time quantitative qPCR. Therefore, it is a common and severe clinical problem worldwide. 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. Microarray analysis was performed by an Affymetrix GeneChip Hybridization Oven 640 and Gene Array Scanner 3000. (2013). 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. These top networks are generally associated with cellular morphology, assembly, organization, and organ development. Starting from 6 h post injury, a larger number of canonical pathways were activated, which were mainly related to inflammation and immune response, cytokine signaling, diseases, nuclear receptor signaling, and intracellular and second messenger signaling. 136, 17–23. IPA provides two powerful tools to understand the pathway analysis results: the networks and canonical pathways that showed several pathways related to an adaptative immune response. Would you like email updates of new search results? Detected global gene expressions at each time point following sciatic nerves transection were compared with the control group. 193, 267–275. The quality and quantity of isolated RNA were checked by Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA) and NanoDrop ND-1000 spectrophotometer (Infinigen Biotechnology Inc., City of Industry, CA), respectively. The human lung mucosa drives differential Mycobacterium tuberculosis infection outcome in the alveolar epithelium. To investigate peripheral nerve injury-induced molecular changes during Wallerian degeneration, microarray analysis was performed to detect the expression levels of more than 30,000 genes at 0, 0.5, 1, 6, 12, and 24 h, 4 days, and 1, 2, 3, and 4 weeks in the distal nerve stumps post sciatic nerve transection. 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. Consequences of slow Wallerian degeneration for regenerating motor and sensory axons. 31, 407–415. Red color indicates up-regulation while green color indicates down-regulation. eCollection 2021. canonical-pathway Sizes: 150x83 / 300x166 / 600x332 / 900x498 / 2362x1306 / Meanwhile, immune signal molecules and cytokines were also activated rapidly after injury. doi: 10.1016/S0074-7742(09)87003-7, Gu, X., Ding, F., and Williams, D. F. (2014). Signal flow and pathways in response to early Wallerian degeneration after rat sciatic nerve injury. 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). Neural tissue engineering options for peripheral nerve regeneration. 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). 2019 May;12(3):795-804. doi: 10.1038/s41385-019-0156-2. QIAGEN Ingenuity Pathway Analysis helps you perform insightful data analysis and interpretation to understand your experimental results … Microsurgery 18, 397–405. 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. Muscle Nerve Suppl. Curr. Microarray technique provides an easy way to screen many thousands of genes or proteins in one assay, and is widely used to detect expression change patterns under various physiological and pathological conditions. (2016). Neurosci. (2015). J. Neuroinflammation 8:110. doi: 10.1186/1742-2094-8-110, Geuna, S., Raimondo, S., Ronchi, G., Di Scipio, F., Tos, P., Czaja, K., et al. doi: 10.1097/00005072-199507000-00002, Lykissas, M. G., Batistatou, A. K., Charalabopoulos, K. A., and Beris, A. E. (2007). 43:e47. 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) Only cellular functions of Cellular Growth and Proliferation and Molecular Transport were considerably activated at 0.5 h post injury. Mvubu NE, Pillay B, McKinnon LR, Pillay M. Cytokine. 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. Different from the central nervous system, the peripheral nervous system is able to regenerate after injury. 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). The QIAGEN Ingenuity Pathway Analysis (IPA) is intended for molecular biology applications. IPA results provided us with some new insights into Wallerian degeneration. Chapter 3: histology of the peripheral nerve and changes occurring during nerve regeneration. Canonical Pathways The canonical pathway analysis identifies pathways that were significantly enriched in the QIAGEN IPA dataset. doi: 10.1016/j.neulet.2013.01.008, Li, M., Zhang, P., Guo, W., Li, H., Gu, X., and Yao, D. (2014). 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. 34:58-36:16 Network results. 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). This session focus on formatting your own data and the best practices to analyze them in IPA. The editor and reviewers' affiliations are the latest provided on their Loop research profiles and may not reflect their situation at the time of review. doi: 10.1002/glia.22957, Jiang, N., Li, H., Sun, Y., Yin, D., Zhao, Q., Cui, S., et al. 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). With the development of high-throughput genomic tools, such as microarray analysis and deep sequencing, it is now possible and preferable to detect the gene expression changes during Wallerian degeneration in order to identify the molecular basis of the morphological changes. Rev. doi: 10.1002/neu.480230507, Campbell, W. W. (2008). QIAGEN Ingenuity Pathway Analysis 5 Glossary of Terms . Int. A. B., Archer, D. R., Li, Y., and Griffin, J. W. (2005). Changes in pathways, networks and transcriptional factors were identified using Ingenuity Pathway Analysis (IPA). 2010 May;59(Pt 5):528-533. doi: 10.1099/jmm.0.016006-0. The interferon signalling and hepatic fibrosis/hepatic stellate cell activation pathways were among the top 5 canonical pathways in all strains. Red color indicates the increased expression whereas green color indicates the decreased expression as compared to control (expression at 0 h). 9, S33–S38. All animal procedures were ethically approved by the Administration Committee of Experimental Animals, Jiangsu Province, China and were used in accordance with Institutional Animal Care guideline of Nantong University. doi: 10.4103/1673-5374.187059, Raimondo, S., Fornaro, M., Tos, P., Battiston, B., Giacobini-Robecchi, M. G., and Geuna, S. (2011). This study was supported by the Natural Science Foundation of Jiangsu Province, China (Grant No. Acad. 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. Conceived and designed the experiments: JY, XG and SY. miR-221 and miR-222 promote Schwann cell proliferation and migration by targeting LASS2 after sciatic nerve injury. Mol Microbiol. 125, 2675–2683. Gene expression patterns at each time point following sciatic nerve transection. Therefore, the up-regulated BDNF in the distal nerve stump would augment axonal regrowth and promote nerve regeneration. [Analysis of molecular mechanisms of the virulence and growth coordination of Mycobacterium tuberculosis]. 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). Med. 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. Neurol. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Cell Sci. doi: 10.5435/00124635-200007000-00005, Li, M., Guo, W., Zhang, P., Li, H., Gu, X., and Yao, D. (2013). Bull. Neurol. Allué-Guardia A, García JI, Torrelles JB. Ingenuity Pathway Analysis (IPA) was used to investigate canonical pathways and immunohistochemical staining was performed to validate protein expression levels in samples. 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). IPA University: Examining Canonical Pathway results in QIAGEN Ingenuity Pathway Analysis software video tutorial video Active Link Making your RNAseq, microarray, proteomics and metabolomics datasets work for you webpage Inactive Link Wrote the manuscript: SY. 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).
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