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Microarray analyses of the dorsal root ganglia support a role for innate neuro-immune pathways in persistent pain in
R E Miller1, P B Tran1, S Ishihara1
1Division of Rheumatology, Department of Internal Medicine, Rush University Medical Center, Chicago, IL, USA.
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.
Insights
Distinct molecular pathways in dorsal root ganglia (DRG) differentiate early from persistent osteoarthritis (OA) pain. Neuroimmune interactions in the DRG contribute to OA pain initiation and maintenance.
Area of Science:
- Neuroscience
- Molecular Biology
- Immunology
Background:
- Osteoarthritis (OA) pain is a complex condition with distinct phases.
- Understanding the molecular mechanisms underlying OA pain is crucial for developing effective treatments.
Purpose of the Study:
- To investigate gene expression changes in knee-innervating dorsal root ganglia (DRG) following destabilization of the medial meniscus (DMM) in mice.
- To identify molecular pathways associated with distinct pain phenotypes: post-surgical, early-stage OA, and persistent OA pain.
Main Methods:
- Mice underwent DMM or sham surgery, with DRG harvested at 4, 8, and 16 weeks post-surgery.
- RNA sequencing (Affymetrix Mouse Transcriptome Array 1.0) was performed on DRG samples.
- Pathway analysis (Ingenuity Pathway Analysis) and qPCR were used to identify differentially expressed genes and pathways.
Main Results:
- Distinct sets of differentially expressed genes were identified for each pain phenotype.
- Genes associated with immune cell recruitment and activation, including NGF, CX3CL1, CCL2, and TLR1, were upregulated in persistent OA pain.
- Elevated Ngf levels and increased F4/80+ macrophages were confirmed in DRG after DMM.
Conclusions:
- Transcriptomic analysis reveals distinct molecular pathways underlying early and persistent OA pain.
- Neuroimmune interactions within the DRG play a significant role in the initiation and maintenance of OA pain.
