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Microarray Analyses of the Dorsal Root Ganglia Support a Role for Innate Neuro-Immune Pathways in Persistent Pain in Experimental Osteoarthritis

ABSTRACT: Objective – Following destabilization of the medial meniscus (DMM), mice develop experimental osteoarthritis (OA) and associated pain behaviors that are dependent on the stage of disease. We aimed to describe changes in gene expression in knee-innervating dorsal root ganglia (DRG) after surgery, in order to identify molecular pathways associated with three pre-defined pain phenotypes: “post-surgical pain”, “early-stage OA pain”, and “persistent OA pain”. Design – We performed DMM or sham surgery in 10-week old male C57BL/6 mice and harvested L3-L5 DRG 4, 8, and 16 weeks after surgery or from age-matched naïve mice (n=3/group). RNA was extracted and an Affymetrix Mouse Transcriptome Array 1.0 was performed. Three pain phenotypes were defined: “post-surgical pain” (sham and DMM 4-week vs. 14-week old naïve), “early OA pain” (DMM 4-week vs. sham 4-week), and “persistent OA pain” (DMM 8- and 16-week vs. naïve and sham 8- and 16-week). ‘Top hit’ genes were defined as p<0.001. Pathway analysis (Ingenuity Pathway Analysis) was conducted using differentially expressed genes defined as p<0.05. In addition, we performed qPCR for Ngf and immunohistochemistry for F4/80+ macrophages in the DRG. Results – For each phenotype, top hit genes identified a small number of differentially expressed genes, some of which have been previously associated with pain (7/67 for “post-surgical pain”; 2/14 for “early OA pain”; 8/37 for “persistent OA pain”). Overlap between groups was limited, with 8 genes differentially regulated (p<0.05) in all three phenotypes. Pathway analysis showed that in the persistent OA pain phase many of the functions of differentially regulated genes are related to immune cell recruitment and activation. Genes previously linked to OA pain (CX3CL1, CCL2, TLR1, and NGF) were upregulated in this phenotype and contributed to activation of the neuroinflammation canonical pathway. In separate sets of mice, we confirmed that Ngf was elevated in the DRG 8 weeks after DMM (p=0.03), and numbers of F4/80+ macrophages were increased 16 weeks after DMM (p=0.002 vs. Sham). Conclusion- These transcriptomics findings support the idea that distinct molecular pathways discriminate early from persistent OA pain. Pathway analysis suggests neuroimmune interactions in the DRG contribute to initiation and maintenance of pain in OA. We grouped samples based on pain phenotype in the DMM mouse model of osteoarthritis. Group 1: post-surgical pain (DMM and sham +4 week samples); Group 2: post-surgical control (Naïve +4 week samples); Group 3: Early osteoarthritis pain (DMM +4 week samples); Group 4: Early controls (sham+4 and naive+4 week samples); Group 5: Persistent pain (DMM+8 and DMM+16 week samples); Group 6: Persistent controls (sham+8, naive+8, sham+16, and naive +16 samples). We compared Group 1 vs Group 2; Group 3 vs Group 4; and Group 5 vs Group 6 to draw the conclusions presented in our manuscript.

ORGANISM(S): Mus musculus

PROVIDER: GSE140785 | GEO | 2019/11/22

REPOSITORIES: GEO

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Project description:The purpose of this study was to characterize the histologic development of OA in a mouse model where OA is induced by destabilization of the medial meniscus (DMM model) and to identify genes regulated during different stages of the disease, using RNA isolated from the joint M-bM-^@M-^\organM-bM-^@M-^] and analyzed using microarrays.427 genes from the microarrays passed consistency and significance filters. There was an initial up-regulation at 2 and 4 weeks of genes involved in morphogenesis, differentiation, and development, including growth factor and matrix genes, as well as transcription factors including Atf2, Creb3l1, and Erg. Most genes were off or down-regulated at 8 weeks with the most highly down-regulated genes involved in cell division and the cytoskeleton. Gene expression increased at 16 weeks, in particular extracellular matrix genes including Prelp, Col3a1 and fibromodulin.The results support a phasic development of OA with early matrix remodelling and transcriptional activity followed by a more quiescent period that is not maintained. A group of 9 mice was used for collection of RNA at time 0 (before surgery) when the animals were 12 weeks old. For the other time points, 9 DMM and 9 sham controls were sacrificed at 2, 4, 8, and 16 weeks after surgery for RNA isolation. The tissue included tibial plateau and femoral condyle articular cartilage, subchondral bone with any osteophytes, meniscus, and the joint capsule with synovium was used for RNA isolation. The tissue was treated with RNAlaterM-BM-. (Invitrogen) prior to freezing and storage at -800 C. RNA was extracted by homogenization using the Precellys 24 tissue homogenizer (Bertin Technologies purchased from MO BIO) and the amount and quality of the RNA was determined using an Agilent 2100 Bioanalyzer. RNA was pooled prior to microarray analysis such that 3 randomly selected samples from each surgical group and time point were pooled to create each biological replicate. Because 9 mice were used for each experimental group, a total of three biological replicates per group were analyzed using the Affymetrix Mouse Genome 430 2.0 oligonucleotide arrays as described. One replicate pool, which was from week two DMM mice, did not meet the RNA integrity level needed for microarray analysis; thus, this pool was not analyzed further, leaving two pools for the week two DMM mice.

Project description:Mechanical Stimuli are arguably the most important aetiolgical factors in osteoarthritis (OA) development. Not only do we see disease arising from joints where the cartilage has sustained direct (e.g. intraarticular fracture) or indirect (e.g. meniscal injury) trauma, but mechanical factors are considered, at least partly, to explain the disease associations with aging and obesity. It is now well established that OA is not simply due to repeated wear and tear, leading to attrition of the articular surfaces, but that it requires activation of a number of inflammatory genes, which drive catabolic protease activity in the joint. These enzymes lead to breakdown of the major extracellular matrix components of cartilage, namely type II collagen, and the proteoglycan, aggrecan. Although it is unclear precisely which enzymes are responsible for matrix breakdown in human OA, Glasson et al showed that deletion of the aggrecan degrading enzyme, ADAMTS5 substantially protected the joint from surgically induced murine OA suggesting that it is a major aggrecanase in the mouse. This study was part of a wider study using the surgical model of murine OA, induced by destabilising the medial meniscus (DMM), to confirm the dependence of joint loading on disease progression and to reveal mechanosensitive genes within the joint which may be involved in the development of OA. Using this model we, and others, have shown that there is a robust degradation of articular cartilage over 4-12wks in male C57B/6 mice. We identified the gene response in joints early following induction of OA, prior to cartilage degradation, by extracting RNA from whole joints (after skin and muscle had been removed) 6hrs, 3 days and 7 days following sham and DMM surgery. An Affymetrix ST1 gene array was performed. Significantly regulated genes (>1.4 fold above naive and sham samples at any timepoint), or those considered to have a putative role in OA pathogenesis were selected for quantitative validation using Taqman low density microfluidic card arrays (TLDA). Gene expression in whole knee joints at 3 early time points post DMM surgery (before cartilage loss), 6hrs, 3days and 7days was examined. Two controls were identified, the first control consisted of age matched naïve mice that had undergone 15 minutes of anaesthesia but were not operated on (0hrs post surgery). The second control included mice which had undergone sham surgery (capsulotomy alone). For this RNA was taken at the same time points post surgery as DMM samples (6hrs, 3days and 7days). There were three biological replicates per condition (total of 21 samples). The right (ipsilateral) knee joint was taken and RNA extracted and processed for microarray analysis using the Affymetrix Mouse Gene 1.0 st v1 platform.

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Microarray Analyses of the Dorsal Root Ganglia Support a Role for Innate Neuro-Immune Pathways in Persistent Pain in Experimental Osteoarthritis

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