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Proteomic Analysis of the Spatio-temporal Based Molecular Kinetics of Acute Spinal Cord Injury Identifies a Time- and Segment-specific Window for Effective Tissue Repair

2016; Elsevier BV; Volume: 15; Issue: 8 Linguagem: Inglês

10.1074/mcp.m115.057794

1535-9484

Stéphanie Devaux, Dáša Čı́žková, Jusal Quanico, Julien Franck, Serge Nataf, Laurent Pays, Lena Hauberg‐Lotte, Peter Maaß, Jörg Kobarg, Firas Kobeissy, Céline Mériaux, Maxence Wisztorski, Lucia Slovinská, Juraj Blaško, V. Cigánková, Isabelle Fournier, Michel Salzet,

Signaling Pathways in Disease

Spinal cord injury (SCI) represents a major debilitating health issue with a direct socioeconomic burden on the public and private sectors worldwide. Although several studies have been conducted to identify the molecular progression of injury sequel due from the lesion site, still the exact underlying mechanisms and pathways of injury development have not been fully elucidated. In this work, based on OMICs, 3D matrix-assisted laser desorption ionization (MALDI) imaging, cytokines arrays, confocal imaging we established for the first time that molecular and cellular processes occurring after SCI are altered between the lesion proximity, i.e. rostral and caudal segments nearby the lesion (R1-C1) whereas segments distant from R1-C1, i.e. R2-C2 and R3-C3 levels coexpressed factors implicated in neurogenesis. Delay in T regulators recruitment between R1 and C1 favor discrepancies between the two segments. This is also reinforced by presence of neurites outgrowth inhibitors in C1, absent in R1. Moreover, the presence of immunoglobulins (IgGs) in neurons at the lesion site at 3 days, validated by mass spectrometry, may present additional factor that contributes to limited regeneration. Treatment in vivo with anti-CD20 one hour after SCI did not improve locomotor function and decrease IgG expression. These results open the door of a novel view of the SCI treatment by considering the C1 as the therapeutic target. Spinal cord injury (SCI) represents a major debilitating health issue with a direct socioeconomic burden on the public and private sectors worldwide. Although several studies have been conducted to identify the molecular progression of injury sequel due from the lesion site, still the exact underlying mechanisms and pathways of injury development have not been fully elucidated. In this work, based on OMICs, 3D matrix-assisted laser desorption ionization (MALDI) imaging, cytokines arrays, confocal imaging we established for the first time that molecular and cellular processes occurring after SCI are altered between the lesion proximity, i.e. rostral and caudal segments nearby the lesion (R1-C1) whereas segments distant from R1-C1, i.e. R2-C2 and R3-C3 levels coexpressed factors implicated in neurogenesis. Delay in T regulators recruitment between R1 and C1 favor discrepancies between the two segments. This is also reinforced by presence of neurites outgrowth inhibitors in C1, absent in R1. Moreover, the presence of immunoglobulins (IgGs) in neurons at the lesion site at 3 days, validated by mass spectrometry, may present additional factor that contributes to limited regeneration. Treatment in vivo with anti-CD20 one hour after SCI did not improve locomotor function and decrease IgG expression. These results open the door of a novel view of the SCI treatment by considering the C1 as the therapeutic target. Spinal cord injury (SCI) 1The abbreviations used are:SCISpinal cord injury. belongs to the serious, currently incurable disorders of the central nervous system (CNS), that are often accompanied by a permanent disability (1.Beattie M.S. Hermann G.E. Rogers R.C. Bresnahan J.C. Cell death in models of spinal cord injury.Prog. Brain Res. 2002; 137: 37-47Crossref PubMed Scopus (369) Google Scholar). Most SCI are related to traumatic spinal cord damages induced by road trauma, falls, or sport injuries (diving). Among the hallmark features of SCI is the axonal disruption in the spinal cord, which is often caused by fractured intervertebral disc or vertebrate. This primary event is followed by a progressive cascade of secondary deleterious reactions spreading to the adjacent spared tissue leading to a worsening of the neurological status (2.Tator C.H. Update on the pathophysiology and pathology of acute spinal cord injury.Brain Pathol. 1995; 5: 407-413Crossref PubMed Scopus (371) Google Scholar, 3.Schwab M.E. Bartholdi D. Degeneration and regeneration of axons in the lesioned spinal cord.Physiol. Rev. 1996; 76: 319-370Crossref PubMed Scopus (962) Google Scholar). Although axonal regeneration is initiated, it is hampered by a combination of local factors that include severe inflammation, lack of trophic support and development of an inhibitory scar-forming environment. In fact, the regenerative capacity of the central nervous system is particularly challenged in SCI as multiple cues converge to act as a chemical and physical barrier for the repair process (4.Schwab M.E. Bartholdi D. Degeneration and regeneration of axons in the lesioned spinal cord.Physiol. Rev. 1996; 76: 319-370Crossref PubMed Scopus (998) Google Scholar, 5.Rossignol S. Schwab M. Schwartz M. Fehlings M.G. Spinal cord injury: time to move?.J. Neurosci. 2007; 27: 11782-11792Crossref PubMed Scopus (216) Google Scholar). It is now acknowledged that inflammation is one of the major key player that determines abortive axonal repair in SCI. Thus, although the immune response is recognized as primordial to preserve tissue homeostasis, the spatio-temporal course of inflammation in SCI is not favorable to axonal regeneration. Spinal cord injury. Acute inflammation develops hours to days following initial spinal cord trauma and is triggered by the axonal damage and neuronal cell death at the lesion site. This is followed by a cellular and molecular inflammatory cascade that initiates the activation of resident glial cells (microglia, astrocytes), infiltration of blood-borne immune cells (lymphocytes, monocytes), and a massive release of chemokines/cytokines by microglia, macrophages and neuronal cells. During the acute inflammatory response, macrophages/microglia phagocyte cell debris and also release neurotoxic factors and stimulate the formation a glial scar that, on the long-term, prevents axonal regrowth. At this stage, a resolution of acute inflammation and a switch toward a neuroprotective cytokines/chemokines profile would favor tissue repair and limit glial scar extension. Instead, a chronic inflammatory process usually develops weeks after trauma and leads to both an aberrant tissue remodeling and a defective tissue repair. Interestingly, although there is currently no efficient therapy for SCI, one of the approved clinical treatments in the early phase of SCI is the administration of methylprednisolone treatment in order to prevent edema and to modulate inflammation (6.Leypold B.G. Flanders A.E. Schwartz E.D. Burns A.S. The impact of methylprednisolone on lesion severity following spinal cord injury.Spine. 2007; 32 (discussion 379–381): 373-378Crossref PubMed Scopus (43) Google Scholar). However, high-dose methylprednisolone is often associated with severe immunosuppression and side effects, such as pulmonary or urinary tract infections. Thus, there is an urgent need for identifying and testing novel neuro-therapeutic strategies that could prevent inflammation, limit scar extent, and stimulate tissue repair process. In this context, a large array of molecules and therapies has been tested experimentally with the goal of targeting the healthy tissue adjacent to spinal cord lesion. Such a strategy is aimed at not only protecting this spared tissue from secondary lesion but also stimulating its regenerative potential in order to promote neuronal networks connectivity and axonal outgrowth. Among these proposed therapeutic strategies, cellular therapy belongs to the promising candidate approaches. Ideally, cell therapy strategies may allow to: (1) bridge spinal cord segments over any cavities or cysts formed at the lesion site, (2) replace lost neurons, oligodendroglia, and (3) create a favorable environment for axonal regeneration (7.Rowland J.W. Hawryluk G.W. Kwon B. Fehlings M.G. Current status of acute spinal cord injury pathophysiology and emerging therapies: promise on the horizon.Neurosurg Focus. 2008; 25: E2Crossref PubMed Scopus (548) Google Scholar). Different cell therapy approaches include embryonic stem cells, induced pluripotent stem cells (iPS) and different categories of adult stem cells and progenitors such as olfactory unsheathing stem cells, neural progenitor cells (NPC) and mesenchymal stem cells (MSCs). In addition, graft of activated macrophages and transplantation of peripheral or central nervous tissue have been also proposed as an alternative to these stem cells based treatments (8.Hawryluk G.W. Rowland J. Kwon B.K. Fehlings M.G. Protection and repair of the injured spinal cord: a review of completed, ongoing, and planned clinical trials for acute spinal cord injury.Neurosurg Focus. 2008; 25: E14Crossref PubMed Scopus (197) Google Scholar). Comparative to cell therapy, other approaches including the use of exogenously-delivered neuroprotective molecules that would protect neurons from deleterious secondary processes, promote axonal growth and/or enhance nerve conduction in the preserved or regenerating axons. Different classes of molecules were shown to afford variable levels of clinical recovery in animal models of SCI. These comprise anti-inflammatory compounds such as minocycline, neurotrophic factors (BDNF, GDNF, NGF, erythropoietin) and molecules that alleviate regenerating axons from the inhibitory effects of extracellular matrix molecules (9.Wells J.E. Hurlbert R.J. Fehlings M.G. Yong V.W. Neuroprotection by minocycline facilitates significant recovery from spinal cord injury in mice.Brain. 2003; 126: 1628-1637Crossref PubMed Scopus (347) Google Scholar, 10.Huang H. Fan S. Ji X. Zhang Y. Bao F. Zhang G. Recombinant human erythropoietin protects against experimental spinal cord trauma injury by regulating expression of the proteins MKP-1 and p-ERK.J. Int. Med. Res. 2009; 37: 511-519Crossref PubMed Scopus (20) Google Scholar). In particular, chondroitinase ABC eliminates chondroitin sulfate proteoglycans (CSPG) that interact with the major membranous component NG2 and inhibit the regeneration of damaged axons (11.Bradbury E.J. Moon L.D. Popat R.J. King V.R. Bennett G.S. Patel P.N. Fawcett J.W. McMahon S.B. Chondroitinase ABC promotes functional recovery after spinal cord injury.Nature. 2002; 416: 636-640Crossref PubMed Scopus (1901) Google Scholar). Also, Nogo-A is one of several neurite growth inhibitory receptors expressed by axons (12.Schwab M.E. Nogo and axon regeneration.Curr. Opin. Neurobiol. 2004; 14: 118-124Crossref PubMed Scopus (522) Google Scholar). Thereby Nogo neutralizing antibodies or blockers of the post-receptors components of RhoA are used to improve long-distance axon regeneration and sprouting (13.Schwab M.E. Repairing the injured spinal cord.Science. 2002; 295: 1029-1031Crossref PubMed Scopus (366) Google Scholar). Of note, Rho pathway is important to control the neuronal response after CNS injury and the RhoA inhibitor cethrin is actually in phase I/II a clinical trial (14.Fehlings M.G. Vawda R. Cellular treatments for spinal cord injury: the time is right for clinical trials.Neurotherapeutics. 2011; 8: 704-720Crossref PubMed Scopus (82) Google Scholar). However, although numerous therapies exhibit potentials to foster neuroprotection, stimulate neurite outgrowth and reduce inflammation, the translation to clinical side is still not crowned by success. Reasons for such a failure are multiple and reside notably in our relatively poor knowledge on the spatiotemporal kinetics of secondary molecular events that characterize the post-trauma phase. This holds particularly important with regard to inflammatory mechanisms that may greatly vary depending on the time point and spinal cord segment considered. Defining a time- and segment-specific window for effective treatment is a key knowledge for an appropriate neuro-therapeutic intervention. In this work, we present the first exhaustive spatio-temporal proteomic and biochemical analysis performed along the entire spinal cord axis in rat model of SCI. We combined this global proteomic analysis with 3D molecular mass spectrometry imaging study, time course analysis of immune cells infiltration and cytokine microarrays quantification. The whole spectrum of the data allowed us to depict a complete scheme along the spinal cord axis of the cellular and molecular sequel of events occurring through the time course of inflammatory process and abortive regeneration. We identified specific markers for each segment at different time points (3, 7, and 10 days) of the biochemical-pathophysiological processes and observed that, surprisingly, segments caudal to the lesion site host a robust inflammatory process accompanied by the local synthesis of neuroprotective and regenerative molecules. In particular, we demonstrated that the caudal segment immediately adjacent to the lesion site possesses, at least temporarily, all the intrinsic components/features that may allow axonal regeneration to occur. Such a caudal-to-rostral altered regenerative potential is likely hampered by inhibitory signals such as glycans that are abundantly detected or even secreted at the lesion site. Finally, we provided evidence that immunoglobulins (IgGs) are present at the lesion site only 3 days after injury and that in vivo treatment of anti-CD20 did not diminished the presence of these antibodies and did not ameliorate the BBB score of the treated animals. Dulbecco's modified Eagle's medium (DMEM) media, phosphate buffer saline (PBS), fetal calf serum (FCS) were purchased from Invitrogen Life Technologies (Milan, Italy). Rat Cytokine Array Panel A was from R&D Systems (Minneapolis, MN). All chemicals were of the highest purity obtainable. Water, formic acid (FA), trifluoroacetic acid (TFA), acetonitrile (AcN) were purchased from Biosolve B.V. (Valkenswaard, The Netherlands). dl-dithiothreitol (DTT), Thiourea and iodoacetamide (IAA) were purchased from SIGMA (Saint-Quentin Fallavier, France). Trypsin/Lys-C Mix, Mass Spec Grade was purchased from Promega (Charbonnieres, France) and Dynabeads® Protein A from Novex (Life Technologies, France). Fluorescence conjugated secondary antibodies and DAPI were purchased from MolecularProbes (Eugene, OR). The study was performed with approval and in accordance to the guidelines of the Institutional Animal Care and Use Committee of the Slovak Academy of Sciences and with the European Communities Council Directive (2010/63/EU) regarding the use of animals in Research, Slovak Law for Animal Protection No. 377/2012 and 436/2012. For the collection of the conditioned media n = 3 rats control (no balloon inflation, 0 day) were sacrificed, n = 3 rats after 3 days SCI, n = 3 rats after 7 days post injury and n = 3 rats after 10 days post SCI. For the control group all segments are in triplicate. For the group 3 days after SCI, R3 and C3 segments include 2 replicates and n = 3 for the other segments. For the group 7 days after SCI R3 and C3 segments include 2 replicates and n = 3 for the other segments. For the group 10 days post SCI all segments are in triplicate. For the cytokines arrays experiments n = 1 rat were sacrificed per condition. The experiments were performed in experimental replicates. For the IgG purification n = 3 rats per condition (control, 3, 7, and 10 days) were sacrificed. The experiments were performed in biological replicates. For the immunohistochemistry experiments n = 3 rats per condition were sacrificed. The analyses were performed in biological triplicates. For MALDI imaging experiment n = 1 rat was sacrificed 3 days post injury. Twenty-five sections for the complete 3D MALDI imaging experiments have been performed from R1 to C1. Statistical analysis: For the proteomic statistical analysis of conditioned media, only proteins presenting as significant by the ANOVA test were used with FDR 5%. Normalization was achieved using a Z-score with a matrix access by rows. The immunohistochemistry statistical analyses were based on one-way ANOVA followed by Tukey Kramer test, significant values were marked * p < 0.05, ** p < 0.01, *** p < 0.001. Quantification analyses of immunofluorescence staining for Iba1, FoxP3 and neutrophil elastase were performed on six sections from rostral and caudal/per condition (n = 3 each). Error bars represent the S.E. BBB score analysis was based on one-way ANOVA test. Values of p < 0.05 were considered statistically significant. For the cytokines array panel, the statistical analyses were performed using Student's t test *p < 0.05, **p < 0.01, ***p < 0.001. Error bars represent the S.E. The SCI was induced using the modified balloon compression technique in adult male Wistar rats, according to our previously published study (15.Vanicky I. Urdzikova L. Saganova K. Cizkova D. Galik J. A simple and reproducible model of spinal cord injury induced by epidural balloon inflation in the rat.J. Neurotrauma. 2001; 18: 1399-1407Crossref PubMed Scopus (167) Google Scholar). Manual bladder expression, 2 times a day, was required for 3–10 days after the injury. In the control group a 2-French Fogarty catheter was inserted at the same level of spinal cord, but the balloon was not inflated and no lesion was performed. Experimental SCI rats at 3, 7, and 10 days and sham-operated-control rats were sacrificed by isoflurane anesthesia followed by decapitation. The spinal cord was pressure expressed by injecting sterile saline (10 ml) throughout the vertebrate canal, along the caudo-rostral axis. Each spinal cord was macroscopically observed to check that lesion was well centered at the Th8-Th9 level on the longitudinal axis. Entire spinal cord was divided into transversally sectioned slides (∼1.0 cm thick each) obtained from the lesion site (Th7-Th11) and from the segments rostral (C1-Th6) and caudal (Th12-L6) to the site of injury. Slides were then chopped into 0.5 cm thick sections (2 sections per segment) and deposited into a 12-well culture plate containing 1 ml DMEM without FCS. After 24 h incubation in a humidified atmosphere with 5% CO2 at 37 °C, 1 ml of SCI-derived conditioned media CM (CM-SCI) were collected (rostral, lesion, caudal segments) and centrifuged 30 min at 15,000 rpm at 4 °C. The same procedure was performed for obtaining CM from control spinal cord tissue. Samples were stored at −80 °C. To address the degree of cell-to cell integrity and viability, additional cryostat sections were cut from incubated segments for 24 h and immersed into tissue-tek®. Afterward cryostat sections were processed to standard IHC with NeuN and GFAP antibodies. The data confirmed the cyto- architecture of neurons and glial cells (supplemental Fig. S1) and confirmed the well preserved neuro-glial integrity within cultured spinal cord segments 24 h in vitro. This confirms that the collected molecules are products of vital cells processes. A volume of 150 μl of tissue supernatants were denatured with 2 m urea in 10 mm HEPES pH 8 by ultrasonication on ice. Protein reduction is realized by incubation with 10 mm DTT for 40 min at 56 °C followed by alkylation with 55 mm IAA for 40 min in the dark. IAA was quenched with 100 mm thiourea. Protein digestion was performed with 30 μg/ml LysC/Trypsin mix, overnight, at 37 °C. Digestion was stopped with 0.5% TFA. The solution was dried in a SpeedVac to reduce the volume. Peptides were desalted using C18 ziptips (Millipore). Elution peptides were dried in a SpeedVac and resuspended in 0.1% FA before injecting into nanoLC. Samples were separated by online reversed-phase chromatography using a Thermo Scientific Proxeon Easy-nLC1000 system equipped with a Proxeon trap column (100 μm ID × 2 cm, Thermo Scientific) and a C18 packed-tip column (Acclaim PepMap, 75 μm ID × 15 cm, Thermo Scientific). Peptides were separated using an increasing amount of acetonitrile (5–35% over 120 min) at a flow rate of 300 nL/min. The LC eluent was electrosprayed directly from the analytical column and a voltage of 1.7 kV was applied via the liquid junction of the nanospray source. The chromatography system was coupled to a Thermo Scientific Q-exactive mass spectrometer programmed to acquire in a data-dependent mode Top 10 most intense ion method. The survey scans were done at a resolving power of 70,000 FWHM (m/z 400), in positive mode and using an AGC target of 3e6. Default charge state was set at 2, unassigned and +1 charge states were rejected and dynamic exclusion was enabled for 25 s. The scan range was set to 300–1600 m/z. For ddMS2, the scan range was between 200–2000 m/z, 1 microscan was acquired at 17,500 FWHM and an isolation window of 4.0 m/z was used. All the MS data were processed with MaxQuant (17.Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9150) Google Scholar) (version 1.5.1.2) using the Andromeda (18.Cox J. Neuhauser N. Michalski A. Scheltema R.A. Olsen J.V. Mann M. Andromeda: a peptide search engine integrated into the MaxQuant environment.J. Proteome Res. 2011; 10: 1794-1805Crossref PubMed Scopus (3448) Google Scholar) search engine. Proteins were identified by searching MS and MS/MS data against Decoy version of the complete proteome for Rattus norvegicus of the UniProt database (19.UniProt C. Reorganizing the protein space at the Universal Protein Resource (UniProt).Nucleic Acids Res. 2012; 40: D71-D75Crossref PubMed Scopus (1099) Google Scholar) (Release June 2014, 33,675 entries) combined with 262 commonly detected contaminants. Trypsin specificity was used for the digestion mode with N-terminal acetylation and methionine oxidation selected as the variable. Carbarmidomethylation of cysteines was set as a fixed modification, with up to two missed cleavages. For MS spectra, an initial mass accuracy of 6 ppm was selected, and the MS/MS tolerance was set to 20 ppm for HCD data. For identification, the FDR at the peptide spectrum matches (PSMs) and protein level was set to 1%. Relative, label-free quantification of proteins was performed using the MaxLFQ algorithm (20.Cox J. Hein M.Y. Luber C.A. Paron I. Nagaraj N. Mann M. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ.Mol. Cell Proteomics. 2014; 13: 2513-2526Abstract Full Text Full Text PDF PubMed Scopus (2687) Google Scholar) integrated into MaxQuant with the default parameters. The data sets and the Perseus result files used for analysis were deposited at the ProteomeXchange Consortium (21.Vizcaino J.A. Deutsch E.W. Wang R. Csordas A. Reisinger F. Rios D. Dianes J.A. Sun Z. Farrah T. Bandeira N. Binz P.A. Xenarios I. Eisenacher M. Mayer G. Gatto L. Campos A. Chalkley R.J. Kraus H.J. Albar J.P. Martinez-Bartolome S. Apweiler R. Omenn G.S. Martens L. Jones A.R. Hermjakob H. ProteomeXchange provides globally coordinated proteomics data submission and dissemination.Nat. Biotechnol. 2014; 32: 223-226Crossref PubMed Scopus (2070) Google Scholar) (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (22.Vizcaino J.A. Cote R.G. Csordas A. Dianes J.A. Fabregat A. Foster J.M. Griss J. Alpi E. Birim M. Contell J. O'Kelly G. Schoenegger A. Ovelleiro D. Perez-Riverol Y. Reisinger F. Rios D. Wang R. Hermjakob H. The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013.Nucleic Acids Res. 2013; 41: D1063-1069Crossref PubMed Scopus (1594) Google Scholar) with the data set identifier PXD003375. Analysis of the proteins identified was performed using Perseus software (http://www.perseus-framework.org/) (version 1.5.0.31). The file containing the information from identification was used with hits to the reverse database, and proteins only identified with modified peptides and potential contaminants were removed. Then, the LFQ intensity was logarithmized (log2[x]). Categorical annotation of rows was used to defined different groups after grouping replicates: (1) Temps (Control, 3, 7 and 10 days), (2) Segments (R3, R2, R1, L, C1, C2, and C3). Multiple-samples tests were performed using ANOVA test with a FDR of 5% and preserving grouping in randomization. Normalization was achieved using a Z-score with a matrix access by rows. For the statistical analysis, only proteins presenting as significant by the ANOVA test were used for statistical analysis. Hierarchical clustering depending on time or segment was first performed using the Euclidean parameter for distance calculation and average option for linkage in row and column trees using a maximum of 300 clusters. For visualization of the variation of proteins expression depending to the condition, the profile plot tool was used with a reference profile and an automatic selection of the 10 or 15 correlated profiles. To quantify fold changes of proteins across samples, we used MaxLFQ. To visualize these fold changes in the context of individual protein abundances in the proteome, we projected them onto the summed peptide intensities normalized by the number of theoretically observable peptides. Specifically, to compare relative protein abundances between and within samples, protein lengths normalized to log 2 protein intensities (termed "iBAQ" value in MaxQuant) were added to the MaxLFQ differences. Functional annotation and characterization of identified proteins were obtained using PANTHER software (version 9.0, http://www.pantherdb.org) and STRING (version 9.1, http://string-db.org). Using the GeneMANIA Cytoscape plugin (23.Montojo J. Zuberi K. Rodriguez H. Bader G.D. Morris Q. GeneMANIA: Fast gene network construction and function prediction for Cytoscape.F1000Res. 2014; 3: 153Crossref PubMed Scopus (183) Google Scholar). 4 coexpression networks were generated from the proteomic values obtained by the analysis of control samples and caudal, rostral and lesion segments respectively. Each segment-specific coexpression network was calculated from data obtained at all-time points following SCI. Such an approach allowed then to perform a supervised clustering in order to identify functionally-relevant subnetworks that would constitute a segment-specific and time-independent molecular signature. In particular, to identify "Inflammation" subnetworks, Fibrinogen alpha (FgA) was chosen as a reference protein and, for each group of samples, networks were built that gathered the 100 proteins whose values were the most closely correlated with those of FgA. Lists of proteins and their encoding genes were then submitted to an enrichment analysis based on gene ontology (GO) annotations, using the open source tool EnrichR (24.Chen E.Y. Tan C.M. Kou Y. Duan Q. Wang Z. Meirelles G.V. Clark N.R. Ma'ayan A. Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool.BMC Bioinformatics. 2013; 14: 128Crossref PubMed Scopus (3021) Google Scholar). Finally, coexpressed proteins whose encoding genes were annotated with the same GO term of interest (for instance "complement activation"), were extracted and subnetworks visualized on cytoscape. The same method was applied to identify "axon guidance" and "neuron differentiation regulation" subnetworks from the networks of proteins coexpressed with neurofilament proteins Nfl, Nfm and Nfh in each group of samples. For presentation purposes, nodes were assigned equal weights and subnetworks were slightly distorted to avoid node superimposition. The Elsevier's Pathway Studio version 9.0 (Ariadne Genomics/Elsevier) was used to deduce relationships among differentially expressed proteomics protein candidates using the Ariadne ResNetdatabase (25.Yuryev A. Kotelnikova E. Daraselia N. Ariadne's ChemEffect and Pathway Studio knowledge base.Expert Opin. Drug Discov. 2009; 4: 1307-1318Crossref PubMed Scopus (48) Google Scholar, 26.Bonnet A. Lagarrigue S. Liaubet L. Robert-Granie C. Sancristobal M. Tosser-Klopp G. Pathway results from the chicken data set using GOTM, Pathway Studio and Ingenuity softwares.BMC Proc. 2009; 3: S11Crossref PubMed Google Scholar). "Subnetwork Enrichment Analysis" (SNEA) algorithm was selected to extract statistically significant altered biological and functional pathways pertaining to each identified set of protein hits (C1, C2, C3, R1, R2, and R3 sets). SNEA utilizes Fisher's statistical test used to determine if there are non-randomized associations between two categorical variables organized by specific relationship. SNEA starts by creating a central "seed" from all relevant entities in the database, and retrieving associated entities based on their relationship with the "seed" (i.e. binding partners, expression targets, protein modification targets, regulation). The algorithm compares the subnetwork distribution to the background distribution using one-sided Mann-Whitney U-Test, and calculates a p value indicating the statistical significance of difference between two distributions. In our analysis, "GenBank" ID and gene symbols from each set were imported to the software to form an experimental data set. For the reconstruction of networks of pathways, biological processes and molecular function were evaluated for each single protein hit and its associated targets (networks and pathways) (27.Pyatnitskiy M. Mazo I. Shkrob M. Schwartz E. Kotelnikova E. Clustering gene expression regulators: new approach to disease subtyping.PLoS ONE. 2014; 9: e84955Crossref PubMed Scopus (26) Google Scholar, 28.Daraselia N. Wang Y. Budoff A. Lituev A. Potapova O. Vansant G. Monforte J. Mazo I. Ossovskaya V.S. Molecular signature and pathway analysis of human primary squamous and adenocarcinoma lung cancers.Am. J. Cancer Res. 2012; 2: 93-103PubMed Google Scholar). Integrated Venn diagram analysis was performed using "the InteractiVenn"; a web-based tool for the analysis of complex data sets. Cytokines and chemokines expression of CM from control, 3, 7, and 10 days for the segments R1 and C1 was performed by using a Rat Cytokine Array Panel A according to the manufacturer's instructions. Briefly, the array membranes were first incubated in the blocking buffer for 1 h. In the meantime, 200 μl of CM were mixed w