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A phenotypic screening platform for chronic pain therapeutics using all-optical electrophysiology

ABSTRACT: Chronic pain associated with osteoarthritis (OA) remains an intractable problem with few effective treatment options. New approaches are needed to model the disease biology and to drive discovery of therapeutics. Here, we present an in vitro model of OA pain, where dorsal root ganglion (DRG) sensory neurons were sensitized by a defined mixture of disease-relevant inflammatory mediators, here called Sensitizing PAin Reagent Composition or SPARC. OA-SPARC components showed synergistic or additive effects when applied in combination and induced pain phenotypes in vivo. To measure the effect of OA-SPARC on neural firing in a scalable format for drug discovery, we used a custom system for high throughput all-optical electrophysiology. This system enabled light-based membrane voltage recordings from hundreds of neurons in parallel with single cell resolution and a throughput of up to 500,000 neurons per day, with patch clamp-like single action potential resolution. A computational framework was developed to construct a multiparameter OA-SPARC neuronal phenotype and to quantitatively assess phenotype reversal by candidate pharmacology with different mechanisms of action. We screened ~3000 approved drugs and mechanistically focused compounds, yielding data from over 1.2 million individual neurons with detailed assessment of both functional OA-SPARC phenotype rescue and orthogonal “off-target” effects. Analysis of confirmed hits revealed diverse potential analgesic mechanisms including well-known ion channel modulators as well as less characterized mechanisms including MEK inhibitors and tyrosine kinase modulators, providing validation of the platform for pain drug discovery.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE237797 | GEO | 2023/08/02

REPOSITORIES: GEO

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Project description: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.

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A phenotypic screening platform for chronic pain therapeutics using all-optical electrophysiology

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