Background: We reported that intra-articular implantation of a hydrogel containing human Agrin resulted in cartilage regeneration in mice and sheep (Eldridge, 2020). In our long term study in sheep, Agrin was implanted soon after the generation of an osteochondral defect. Six months later, the animals receiving Agrin had significant bone and cartilage regeneration. What was intriguing, however, was that starting very soon after implantation, the sheep receiving Agrin were spending more time playing and less resting compared to the control animals receiving GFP as measured with accelerometers. This rapid symptomatic relief is inconsistent with the long time required for cartilage regeneration and suggested that Agrin may have a direct analgesic effect.

Objectives: In this study we investigated whether Agrin has a direct analgesic effect and what is its mechanism.

Methods: Osteoarthritis was induced by resecting the medial collateral ligament and the anterior horn of the medial meniscus. Acute osteochondral defects were generated surgically on the weight bearing area of the lateral femoral condyle as previously reported (Eldridge,). Pain was measured using incapacitance or von Frey filaments. Transgenic mice overexpressing human non-neuronal agrin in cartilage were generated by crossing mice expressing cre recombinase fused with the estrogen receptor driven by the Aggrecan promoters with mice expressing agrin preceded by a floxed stop codon under the control of a ubiquitous promoter. Mice overexpressing neuronal agrin in the dorsal root ganglia (DRG) were generated by crossing mice expressing cre recombinase fused to the estrogen receptor driven by the advillin promoters with mice ubiquitously expressing human neuronal agrin preceded by a floxed stop codon. DRG neurons were cultured in microfluidic chambers for functional testing.

Results: Overexpression of human Agrin either within the cartilage or within the DRG 4 weeks following joint destabilization resulted in reduced pain on weightbearing in mice. The injection of femtomolar concentrations of Agrin in the knee of mice with advanced osteoarthritis or with acute pain following the generation of an osteochondral defect resulted in strong analgesia (incapacitance and von Frey filaments) as early as 2 hours after injection. In DRG cultures in microfluidic chambers, agrin reduced calcium mobilization induced by KCl. Mining a publicly available single cell RNAseq dataset (Usoskin, 2015), we identified subsets of DRG neurons expressing the agrin receptor LRP4 and characterized their phenotype.

Conclusion: In addition to its previously reported chondrogenic effect, Agrin reduces pain relief in osteoarthritis and after acute osteochondral defects. Agrin has a direct inhibitory effect on DRG neurons in in vitro models of pain.

REFERENCES: [1] Eldridge SE, Barawi A, Wang H, Roelofs AJ, Kaneva M, Guan Z, Lydon H, Thomas BL, Thorup A-S, Fernandez BF, et al (2020) Agrin induces long-term osteochondral regeneration by supporting repair morphogenesis. Sci Transl Med 12: eaax9086.

[2] Usoskin D, Furlan A, Islam S, Abdo H, Lönnerberg P, Lou D, Hjerling-Leffler J, Haeggström J, Kharchenko O, Kharchenko PV, et al (2015) Unbiased classification of sensory neuron types by large-scale single-cell RNA sequencing. Nature Neuroscience 18: 145–153.

Acknowledgements: We are grateful for funding the following bodies: FOREUM (1016807), Versus Arthritis (22628; 21515); MRC (MR/R000956/1).

Disclosure of Interests: Suzanne Eldridge: None declared, Federico Dajas-Bailador: None declared, Victoria Chapman: None declared, Sabah Bharde: None declared, Shafaq Sikandar GSK, Daniela Cici: None declared, Francesco Dell’Accio UCB pharma and Biosplice, UCB Pharma.