The DA rat strain is particularly susceptible to the induction of a number of chronic inflammatory diseases, such as models for rheumatoid arthritis and multiple sclerosis. Here we sequenced the genomes of two DA sub-strains and two disease resistant strains, E3 and PVG, previously used together with DA strains in genetically segregating crosses.
The data uncovers genomic variations, such as single nucleotide variations (SNVs) and copy number variations that underlie phenotypic differences between the strains. Comparisons of regional differences between the two DA sub-strains identified 8 genomic regions that discriminate between the strains that together cover 38 Mbp and harbor 302 genes. We analyzed 10 fine-mapped quantitative trait loci and our data implicate strong candidates for genetic variations that mediate their effects. For example we could identify a single SNV candidate in a regulatory region of the gene Il21r, which has been associated to differential expression in both rats and human MS patients. In the APLEC complex we identified two SNVs in a highly conserved region, which could affect the regulation of all APLEC encoded genes and explain the polygenic differential expression seen in the complex. Furthermore, the non-synonymous SNV modifying aa153 of the Ncf1 protein was confirmed as the sole causative factor.
This complete map of genetic differences between the most commonly used rat strains in inflammation research constitutes an important reference in understanding how genetic variations contribute to the traits of importance for inflammatory diseases.
Electronic supplementary material
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Rat genome; Whole-genome sequencing; QTL; Rheumatoid arthritis; Multiple sclerosis; Disease-associated SNVs
Using the ImmunoChip custom genotyping array, we analysed 14,498 multiple sclerosis subjects and 24,091 healthy controls for 161,311 autosomal variants and identified 135 potentially associated regions (p-value < 1.0 × 10-4). In a replication phase, we combined these data with previous genome-wide association study (GWAS) data from an independent 14,802 multiple sclerosis subjects and 26,703 healthy controls. In these 80,094 individuals of European ancestry we identified 48 new susceptibility variants (p-value < 5.0 × 10-8); three found after conditioning on previously identified variants. Thus, there are now 110 established multiple sclerosis risk variants in 103 discrete loci outside of the Major Histocompatibility Complex. With high resolution Bayesian fine-mapping, we identified five regions where one variant accounted for more than 50% of the posterior probability of association. This study enhances the catalogue of multiple sclerosis risk variants and illustrates the value of fine-mapping in the resolution of GWAS signals.
Parent-of-origin effects comprise a range of genetic and epigenetic mechanisms of inheritance. Recently, detection of such effects implicated epigenetic mechanisms in the etiology of multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system. We here sought to dissect the magnitude and the type of parent-of-origin effects in the pathogenesis of experimental neuroinflammation under controlled environmental conditions. We investigated inheritance of an MS-like disease in rat, experimental autoimmune encephalomyelitis (EAE), using a backcross strategy designed to identify the parental origin of disease-predisposing alleles. A striking 37–54% of all detected disease-predisposing loci depended on parental transmission. Additionally, the Y chromosome from the susceptible strain contributed to disease susceptibility. Accounting for parent-of-origin enabled more powerful and precise identification of novel risk factors and increased the disease variance explained by the identified factors by 2-4-fold. The majority of loci displayed an imprinting–like pattern whereby a gene expressed only from the maternal or paternal copy exerts an effect. In particular, a locus on chromosome 6 comprises a well-known cluster of imprinted genes including the paternally expressed Dlk1, an atypical Notch ligand. Disease-predisposing alleles at the locus conferred lower Dlk1 expression in rats and, together with data from transgenic overexpressing Dlk1 mice, demonstrate that reduced Dlk1 drives more severe disease and modulates adaptive immune reactions in EAE. Our findings suggest a significant epigenetic contribution to the etiology of EAE. Incorporating these effects enables more powerful and precise identification of novel risk factors with diagnostic and prognostic implications for complex disease.
Even with recent progress in determining the genetic basis of complex diseases, the issue of ‘missing heritability’ remains and its potential sources are frequently speculated about but rarely explained. Parent-of-origin effects might contribute to the ‘missing heritability’ and involve genetic and epigenetic mechanisms of inheritance. Our study is the first that establishes (i) the magnitude and (ii) the type of parent-of-origin effects in the pathogenesis of a multiple sclerosis-like disease, experimental autoimmune encephalomyelitis (EAE) in rat, using a strategy designed to identify genes that confer risk only when inherited from either mother or father. A striking 37-54% of all risk loci depended on parental origin. Accounting for parent-of-origin enabled more powerful and precise identification of novel risk factors for EAE, such as the imprinted Dlk1gene. Disease-predisposing alleles conferred lower Dlk1 expression in rats and transgenic Dlk1 mice demonstrated that lower Dlk1 drives more severe EAE and modulates adaptive immune responses. Because parental-origin effects are epigenetically regulated, our data implicate a contributory role for epigenetic mechanisms in complex diseases. Considering parent-of-origin effects in complex disease has enabled more powerful and precise identification of novel risk factors.
Genetic variation in the major histocompatibility complex (MHC) affects CD4∶CD8 lineage commitment and MHC expression. However, the contribution of specific genes in this gene-dense region has not yet been resolved. Nor has it been established whether the same genes regulate MHC expression and T cell selection. Here, we assessed the impact of natural genetic variation on MHC expression and CD4∶CD8 lineage commitment using two genetic models in the rat. First, we mapped Quantitative Trait Loci (QTLs) associated with variation in MHC class I and II protein expression and the CD4∶CD8 T cell ratio in outbred Heterogeneous Stock rats. We identified 10 QTLs across the genome and found that QTLs for the individual traits colocalized within a region spanning the MHC. To identify the genes underlying these overlapping QTLs, we generated a large panel of MHC-recombinant congenic strains, and refined the QTLs to two adjacent intervals of ∼0.25 Mb in the MHC-I and II regions, respectively. An interaction between these intervals affected MHC class I expression as well as negative selection and lineage commitment of CD8 single-positive (SP) thymocytes. We mapped this effect to the transporter associated with antigen processing 2 (Tap2) in the MHC-II region and the classical MHC class I gene(s) (RT1-A) in the MHC-I region. This interaction was revealed by a recombination between RT1-A and Tap2, which occurred in 0.2% of the rats. Variants of Tap2 have previously been shown to influence the antigenicity of MHC class I molecules by altering the MHC class I ligandome. Our results show that a restricted peptide repertoire on MHC class I molecules leads to reduced negative selection of CD8SP cells. To our knowledge, this is the first study showing how a recombination between natural alleles of genes in the MHC influences lineage commitment of T cells.
Peptides from degraded cytoplasmic proteins are transported via TAP into the endoplasmic reticulum for loading onto MHC class I molecules. TAP is encoded by Tap1 and Tap2, which in rodents are located close to the MHC class I genes. In the rat, genetic variation in Tap2 gives rise to two different transporters: a promiscuous A variant (TAP-A) and a more restrictive B variant (TAP-B). It has been proposed that the class I molecule in the DA rat (RT1-Aa) has co-evolved with TAP-A and it has been shown that RT1-Aa antigenicity is changed when co-expressed with TAP-B. To study the contribution of different allelic combinations of RT1-A and Tap2 to the variation in MHC expression and T cell selection, we generated DA rats with either congenic or background alleles in the RT1-A and Tap2 loci. We found increased numbers of mature CD8SP cells in the thymus of rats which co-expressed RT1-Aa and TAP-B. This increase of CD8 cells could be explained by reduced negative selection, but did not correlate with RT1-Aa expression levels on thymic antigen presenting cells. Thus, our results identify a crucial role of the TAP and the quality of the MHC class I repertoire in regulating T cell selection.
Genetic mapping on fully sequenced individuals is transforming our understanding of the relationship between molecular variation and variation in complex traits. Here we report a combined sequence and genetic mapping analysis in outbred rats that maps 355 quantitative trait loci for 122 phenotypes. We identify 35 causal genes involved in 31 phenotypes, implicating novel genes in models of anxiety, heart disease and multiple sclerosis. The relation between sequence and genetic variation is unexpectedly complex: at approximately 40% of quantitative trait loci a single sequence variant cannot account for the phenotypic effect. Using comparable sequence and mapping data from mice, we show the extent and spatial pattern of variation in inbred rats differ significantly from those of inbred mice, and that the genetic variants in orthologous genes rarely contribute to the same phenotype in both species.
Defining genetic variants that predispose for diseases is an important initiative that can improve biological understanding and focus therapeutic development. Genetic mapping in humans and animal models has defined genomic regions controlling a variety of phenotypes known as quantitative trait loci (QTL). Causative disease determinants, including single nucleotide polymorphisms (SNPs), lie within these regions and can often be identified through effects on gene expression. We previously identified a QTL on rat chromosome 4 regulating macrophage phenotypes and immune-mediated diseases including experimental autoimmune encephalomyelitis (EAE). Gene analysis and a literature search identified lysine-specific demethylase 3A (Kdm3a) as a potential regulator of these phenotypes. Genomic sequencing determined only two synonymous SNPs in Kdm3a. The silent synonymous SNP in exon 15 of Kdm3a caused problems with quantitative PCR detection in the susceptible strain through reduced amplification efficiency due to altered secondary cDNA structure. Shape Probability Shift analysis predicted that the SNP often affects RNA folding; thus, it may impact protein translation. Despite these differences in rats, genetic knockout of Kdm3a in mice resulted in no dramatic effect on immune system development and activation or EAE susceptibility and severity. These results provide support for tools that analyze causative SNPs that impact nucleic acid structures.
The experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease of the central nervous system commonly used to study multiple sclerosis (MS). We combined clinical EAE phenotypes with genome-wide expression profiling in spleens from 150 backcross rats between susceptible DA and resistant PVG rat strains during the chronic EAE phase. This enabled correlation of transcripts with genotypes, other transcripts and clinical EAE phenotypes and implicated potential genetic causes and pathways in EAE. We detected 2285 expression quantitative trait loci (eQTLs). Sixty out of 599 cis-eQTLs overlapped well-known EAE QTLs and constitute positional candidate genes, including Ifit1 (Eae7), Atg7 (Eae20-22), Klrc3 (eEae22) and Mfsd4 (Eae17). A trans-eQTL that overlaps Eae23a regulated a large number of small RNAs and implicates a master regulator of transcription. We defined several disease-correlated networks enriched for pathways involved in cell-mediated immunity. They include C-type lectins, G protein coupled receptors, mitogen-activated protein kinases, transmembrane proteins, suppressors of transcription (Jundp2 and Nr1d1) and STAT transcription factors (Stat4) involved in interferon signaling. The most significant network was enriched for T cell functions, similar to genetic findings in MS, and revealed both established and novel gene interactions. Transcripts in the network have been associated with T cell proliferation and differentiation, the TCR signaling and regulation of regulatory T cells. A number of network genes and their family members have been associated with MS and/or other autoimmune diseases. Combining disease and genome-wide expression phenotypes provides a link between disease risk genes and distinct molecular pathways that are dysregulated during chronic autoimmune inflammation.
MicroRNAs (miRNAs) are known to regulate most biological processes and have been found dysregulated in a variety of diseases, including multiple sclerosis (MS). In this study, we characterized miRNAs that associate with susceptibility to develop experimental autoimmune encephalomyelitis (EAE) in rats, a well-established animal model of MS. Using Illumina next-generation sequencing, we detected 544 miRNAs in the lymph nodes of EAE-susceptible Dark Agouti and EAE-resistant Piebald Virol Glaxo rats during immune activation. Forty-three miRNAs were found differentially expressed between the two strains, with 81% (35 out of 43) showing higher expression in the susceptible strain. Only 33% of tested miRNAs displayed differential expression in naive lymph nodes, suggesting that a majority of regulated miRNAs are EAE dependent. Further investigation of a selected six miRNAs indicates differences in cellular source and kinetics of expression. Several of the miRNAs, including miR-146a, miR-21, miR-181a, miR-223, and let-7, have previously been implicated in immune system regulation. Moreover, 77% (33 out of 43) of the miRNAs were associated with MS and other autoimmune diseases. Target genes likely regulated by the miRNAs were identified using computational predictions combined with whole-genome expression data. Differentially expressed miRNAs and their targets involve functions important for MS and EAE, such as immune cell migration through targeting genes like Cxcr3 and cellular maintenance and signaling by regulation of Prkcd and Stat1. In addition, we demonstrated that these three genes are direct targets of miR-181a. Our study highlights the impact of multiple miRNAs, displaying diverse kinetics and cellular sources, on development of pathogenic autoimmune inflammation.
The proper identification of differentially methylated CpGs is central in most epigenetic studies. The Illumina HumanMethylation450 BeadChip is widely used to quantify DNA methylation; nevertheless, the design of an appropriate analysis pipeline faces severe challenges due to the convolution of biological and technical variability and the presence of a signal bias between Infinium I and II probe design types. Despite recent attempts to investigate how to analyze DNA methylation data with such an array design, it has not been possible to perform a comprehensive comparison between different bioinformatics pipelines due to the lack of appropriate data sets having both large sample size and sufficient number of technical replicates. Here we perform such a comparative analysis, targeting the problems of reducing the technical variability, eliminating the probe design bias and reducing the batch effect by exploiting two unpublished data sets, which included technical replicates and were profiled for DNA methylation either on peripheral blood, monocytes or muscle biopsies. We evaluated the performance of different analysis pipelines and demonstrated that: (1) it is critical to correct for the probe design type, since the amplitude of the measured methylation change depends on the underlying chemistry; (2) the effect of different normalization schemes is mixed, and the most effective method in our hands were quantile normalization and Beta Mixture Quantile dilation (BMIQ); (3) it is beneficial to correct for batch effects. In conclusion, our comparative analysis using a comprehensive data set suggests an efficient pipeline for proper identification of differentially methylated CpGs using the Illumina 450K arrays.
technical variability; DNA methylation; microarray; Illumina 450K; normalization
Multiple sclerosis (MS) is a polygenic disease characterized by inflammation and demyelination in the central nervous system (CNS), which can be modeled in experimental autoimmune encephalomyelitis (EAE). The Eae18b locus on rat chromosome 10 has previously been linked to regulation of beta-chemokine expression and severity of EAE. Moreover, the homologous chemokine cluster in humans showed evidence of association with susceptibility to MS. We here established a congenic rat strain with Eae18b locus containing a chemokine cluster (Ccl2, Ccl7, Ccl11, Ccl12 and Ccl1) from the EAE- resistant PVG rat strain on the susceptible DA background and utilized myelin oligodendrocyte glycoprotein (MOG)-induced EAE to characterize the mechanisms underlying the genetic regulation. Congenic rats developed a milder disease compared to the susceptible DA strain, and this was reflected in decreased demyelination and in reduced recruitment of inflammatory cells to the brain. The congenic strain also showed significantly increased Ccl11 mRNA expression in draining lymph nodes and spinal cord after EAE induction. In the lymph nodes, macrophages were the main producers of CCL11, whereas macrophages and lymphocytes expressed the main CCL11 receptor, namely CCR3. Accordingly, the congenic strain also showed significantly increased Ccr3 mRNA expression in lymph nodes. In the CNS, the main producers of CCL11 were neurons, whereas CCR3 was detected on neurons and CSF producing ependymal cells. This corresponded to increased levels of CCL11 protein in the cerebrospinal fluid of the congenic rats. Increased intrathecal production of CCL11 in congenic rats was accompanied by a tighter blood brain barrier, reflected by more occludin+ blood vessels. In addition, the congenic strain showed a reduced antigen specific response and a predominant anti-inflammatory Th2 phenotype. These results indicate novel mechanisms in the genetic regulation of neuroinflammation.
A single nucleotide polymorphism causing constitutive activation of Vav1 results in increased natural Treg generation and is responsible for the imbalance between Vav1 GEF and adaptor functions.
CD4+ regulatory T cells (Treg cells) expressing the transcription factor Foxp3 play a pivotal role in maintaining peripheral tolerance by inhibiting the expansion and function of pathogenic conventional T cells (Tconv cells). In this study, we show that a locus on rat chromosome 9 controls the size of the natural Treg cell compartment. Fine mapping of this locus with interval-specific congenic lines and association experiments using single nucleotide polymorphisms (SNPs) identified a nonsynonymous SNP in the Vav1 gene that leads to the substitution of an arginine by a tryptophan (p.Arg63Trp). This p.Arg63Trp polymorphism is associated with increased proportion and absolute numbers of Treg cells in the thymus and peripheral lymphoid organs, without impacting the size of the Tconv cell compartment. This polymorphism is also responsible for Vav1 constitutive activation, revealed by its tyrosine 174 hyperphosphorylation and increased guanine nucleotide exchange factor activity. Moreover, it induces a marked reduction in Vav1 cellular contents and a reduction of Ca2+ flux after TCR engagement. Together, our data reveal a key role for Vav1-dependent T cell antigen receptor signaling in natural Treg cell development.
Multiple sclerosis (OMIM 126200) is a common disease of the central nervous system in which the interplay between inflammatory and neurodegenerative processes typically results in intermittent neurological disturbance followed by progressive accumulation of disability.1 Epidemiological studies have shown that genetic factors are primarily responsible for the substantially increased frequency of the disease seen in the relatives of affected individuals;2,3 and systematic attempts to identify linkage in multiplex families have confirmed that variation within the Major Histocompatibility Complex (MHC) exerts the greatest individual effect on risk.4 Modestly powered Genome-Wide Association Studies (GWAS)5-10 have enabled more than 20 additional risk loci to be identified and have shown that multiple variants exerting modest individual effects play a key role in disease susceptibility.11 Most of the genetic architecture underlying susceptibility to the disease remains to be defined and is anticipated to require the analysis of sample sizes that are beyond the numbers currently available to individual research groups. In a collaborative GWAS involving 9772 cases of European descent collected by 23 research groups working in 15 different countries, we have replicated almost all of the previously suggested associations and identified at least a further 29 novel susceptibility loci. Within the MHC we have refined the identity of the DRB1 risk alleles and confirmed that variation in the HLA-A gene underlies the independent protective effect attributable to the Class I region. Immunologically relevant genes are significantly over-represented amongst those mapping close to the identified loci and particularly implicate T helper cell differentiation in the pathogenesis of multiple sclerosis.
multiple sclerosis; GWAS; genetics
To elucidate mechanisms involved in multiple sclerosis (MS), we studied genetic regulation of experimental autoimmune encephalomyelitis (EAE) in rats, assuming a conservation of pathogenic pathways. In this study, we focused on Eae23, originally identified to regulate EAE in a (LEW.1AV1xPVG.1AV1)F2 cross. Our aim was to determine whether one or more genes within the 67 Mb region regulate EAE and to define candidate risk genes.
We used high resolution quantitative trait loci (QTL) analysis in the 10th generation (G10) of an advanced intercross line (AIL) to resolve Eae23 into two QTLs that independently regulate EAE, namely Eae23a and Eae23b. We established a congenic strain to validate the effect of this region on disease. PVG alleles in Eae23 resulted in significant protection from EAE and attenuated CNS inflammation/demyelination. Disease amelioration was accompanied with increased levels of Foxp3+ cells in the CNS of the congenic strain compared to DA. We then focused on candidate gene investigation in Eae23b, a 9 Mb region linked to all clinical phenotypes. Affymetrix exon arrays were used to study expression of the genes in Eae23b in the parental strains, where none showed differential expression. However, we found lower expression of exon 4 of ZEB1, which is specific for splice-variant Zfhep1. ZEB1 is an interleukin 2 (IL2) repressor involved in T cell development. The splice-specific variance prompted us to next analyze the expression of ZEB1 and its two splice variants, Zfhep1 and Zfhep2, in both lymph node and spleen. We demonstrated that ZEB1 splice-variants are differentially expressed; severity of EAE and higher IL2 levels were associated with down-regulation of Zfhep1 and up-regulation of Zfhep2.
We speculate that the balance between splice-variants of ZEB1 could influence the regulation of EAE. Further functional studies of ZEB1 and the splice-variants may unravel novel pathways contributing to MS pathogenesis and inflammation in general.
Poly(ADP-ribosyl)ation of the conserved multifunctional transcription factor CTCF was previously identified as important to maintain CTCF insulator and chromatin barrier functions. However, the molecular mechanism of this regulation and also the necessity of this modification for other CTCF functions remain unknown. In this study, we identified potential sites of poly(ADP-ribosyl)ation within the N-terminal domain of CTCF and generated a mutant deficient in poly(ADP-ribosyl)ation. Using this CTCF mutant, we demonstrated the requirement of poly(ADP-ribosyl)ation for optimal CTCF function in transcriptional activation of the p19ARF promoter and inhibition of cell proliferation. By using a newly generated isogenic insulator reporter cell line, the CTCF insulator function at the mouse Igf2-H19 imprinting control region (ICR) was found to be compromised by the CTCF mutation. The association and simultaneous presence of PARP-1 and CTCF at the ICR, confirmed by single and serial chromatin immunoprecipitation assays, were found to be independent of CTCF poly(ADP-ribosyl)ation. These results suggest a model of CTCF regulation by poly(ADP-ribosyl)ation whereby CTCF and PARP-1 form functional complexes at sites along the DNA, producing a dynamic reversible modification of CTCF. By using bioinformatics tools, numerous sites of CTCF and PARP-1 colocalization were demonstrated, suggesting that such regulation of CTCF may take place at the genome level.
Multiple Sclerosis (MS) is a chronic inflammatory disease causing demyelination and nerve loss in the central nervous system. Experimental autoimmune encephalomyelitis (EAE) is an animal model of MS that is widely used to investigate complex pathogenic mechanisms. Transcriptional control through isoform selection and mRNA levels determines pathway activation and ultimately susceptibility to disease.
We have studied the role of alternative splicing and differential expression in lymph node cells from EAE-susceptible Dark Agouti (DA) and EAE-resistant Piebald Virol Glaxo.AV1 (PVG) inbred rat strains using Affymetrix Gene Chip Rat Exon 1.0 ST Arrays. Comparing the two strains, we identified 11 differentially spliced and 206 differentially expressed genes at day 7 post-immunization, as well as 9 differentially spliced and 144 differentially expressed genes upon autoantigen re-stimulation. Functional clustering and pathway analysis implicate genes for glycosylation, lymphocyte activation, potassium channel activity and cellular differentiation in EAE susceptibility.
Our results demonstrate that alternative splicing occurs during complex disease and may govern EAE susceptibility. Additionally, transcriptome analysis not only identified previously defined EAE pathways regulating the immune system, but also novel mechanisms. Furthermore, several identified genes overlap known quantitative trait loci, providing novel causative candidate targets governing EAE.
The imprinted insulin-like growth factor 2 (IGF2) gene is expressed predominantly from the paternal allele. Loss of imprinting (LOI) associated with hypomethylation at the promoter proximal sequence (DMR0) of the IGF2 gene was proposed as a predisposing constitutive risk biomarker for colorectal cancer. We used pyrosequencing to assess whether IGF2 DMR0 methylation is either present constitutively prior to cancer or whether it is acquired tissue-specifically after the onset of cancer. DNA samples from tumour tissues and matched non-tumour tissues from 22 breast and 42 colorectal cancer patients as well as peripheral blood samples obtained from colorectal cancer patients [SEARCH (n=case 192, controls 96)], breast cancer patients [ABC (n=case 364, controls 96)] and the European Prospective Investigation of Cancer [EPIC-Norfolk (n=breast 228, colorectal 225, controls 895)] were analysed. The EPIC samples were collected 2–5 years prior to diagnosis of breast or colorectal cancer. IGF2 DMR0 methylation levels in tumours were lower than matched non-tumour tissue. Hypomethylation of DMR0 was detected in breast (33%) and colorectal (80%) tumour tissues with a higher frequency than LOI indicating that methylation levels are a better indicator of cancer than LOI. In the EPIC population, the prevalence of IGF2 DMR0 hypomethylation was 9.5% and this correlated with increased age not cancer risk. Thus, IGF2 DMR0 hypomethylation occurs as an acquired tissue-specific somatic event rather than a constitutive innate epimutation. These results indicate that IGF2 DMR0 hypomethylation has diagnostic potential for colon cancer rather than value as a surrogate biomarker for constitutive LOI.
The aim of the present study was to examine inflammatory responses during Wallerian degeneration in rat peripheral nerve when the regrowth of axons was prevented by suturing.
Transected rat sciatic nerve was sutured and ligated to prevent reinnervation. The samples were collected from the left sciatic nerve distally and proximally from the point of transection. The endoneurium was separated from the surrounding epi- and perineurium to examine the expression of cytokines in both of these compartments. Macrophage invasion into endoneurium was investigated and Schwann cell proliferation was followed as well as the expression of cytokines IL-1β, IL-10, IFN-γ and TNF-α mRNA. The samples were collected from 1 day up to 5 weeks after the primary operation.
At days 1 to 3 after injury in the epi-/perineurium of the proximal and distal stump, a marked expression of the pro-inflammatory cytokines TNF-α and IL-1β and of the anti-inflammatory cytokine IL-10 was observed. Concurrently, numerous macrophages started to gather into the epineurium of both proximal and distal stumps. At day 7 the number of macrophages decreased in the perineurium and increased markedly in the endoneurium of both stumps. At this time point marked expression of TNF-α and IFN-γ mRNA was observed in the endo- and epi-/perineurium of the proximal stump. At day 14 a marked increase in the expression of IL-1β could be noted in the proximal stump epi-/perineurium and in the distal stump endoneurium. At that time point many macrophages were observed in the longitudinally sectioned epineurium of the proximal 2 area as well as in the cross-section slides from the distal stump. At day 35 TNF-α, IL-1β and IL-10 mRNA appeared abundantly in the proximal epi-/perineurium together with macrophages.
The present studies show that even during chronic denervation there is a cyclic expression pattern for the studied cytokines. Contrary to the previous findings on reinnervating nerves the studied cytokines show increased expression up to 35 days. The high expressions of pro-inflammatory and anti-inflammatory cytokines in the proximal epi-/perineurial area at day 35 may be involved in the formation of fibrosis due to irreversible nerve injury and thus may have relevance to the formation of traumatic neuroma.