Mechanisms of Peripheral Sensitization in Inflammatory Knee Pain

Arthritis affects millions of people and costs billions to the global economy with painful, inflamed joints being the major clinical symptoms. Knee joints are innervated by the distal ends of dorsal root ganglion (DRG) neurons and hyperexcitability of knee-innervating DRG neurons (knee neurons), through a process called peripheral sensitization, underlies arthritic knee pain. My research goal was to elucidate the mechanisms of knee neuron peripheral sensitization by developing and utilizing several in vitro and in vivo disease models. Since experimental animal models of arthritis do not fully recapitulate the human disease, I developed a novel, in vitro translational model of arthritic pain by incubating mouse knee neurons with human synovial fluid obtained from osteoarthritic patients. Results from this disease model provide proof-of-concept that synovial fluid is a key modulator of arthritic pain. In order to reconcile the behavioral and neural correlates of arthritic pain, I utilized the mouse model of complete Freund’s adjuvant (CFA)-induced knee inflammation to establish digging behavior as an ethologically relevant spontaneous pain measure. I observed that digging is reduced after knee inflammation and that it occurs concomitant with an increase in knee neuron excitability. After inflammation, knee neurons also showed increased expression of the nociceptive ion channel transient receptor potential vanilloid 1 (TRPV1), possibly mediated by nerve growth factor, and systemic administration of a TRPV1 antagonist normalized digging behavior in mice. Interactions between non-neuronal cells and neurons are critical in peripheral sensitization and I examined the role of fibroblast-like synoviocytes (FLS, non-neuronal joint cell) in mediating knee neuron hyperexcitability by establishing a mouse FLS/DRG neuron co-culture system. A pro-inflammatory phenotype was produced in FLS after stimulation with tumor necrosis factor-α (TNF-α a cytokine that is upregulated in CFA mouse models, TNF-FLS); and in co-culture with TNF-FLS or their secreted mediators knee neurons became hyperexcitable and showed altered TRP channel function. These results suggest that specifically controlling the excitability of knee neurons could provide pain relief in arthritis, one possibility for achieving such control is to deliver excitatory or inhibitory genes via adeno-associated virus (AAV). However, delivering genes into DRG neurons by injection of AAVs into the peripheral organs has had limited success due to the large distances involved. Here I show that the newly engineered serotype, AAV-PHP.S, can deliver functional excitatory (Gq) and inhibitory (Gi) designer receptors activated by designer drugs (DREADDs) into knee neurons to bi-directionally control excitability in vitro and that Gi- DREADD activation in vivo can reverse a knee inflammation-induced decrease in digging behavior. Findings from this thesis highlight multiple ways to identify drivers of inflammatory knee pain and open the door for peripheral organ targeted gene therapy to alleviate arthritic pain.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2019

Author dc:creator

• Chakrabarti, Sampurna

Advisor dc:contributor.advisor

• Smith, Ewan St John

• Pain
• Inflammation
• Knee
• arthritis
• Neuron
• AAV
• synovial fluid

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/c365ec4d-fde9-4c58-ae26-b34fdc274eac/download
• https://creativecommons.org/licenses/by-nc-sa/4.0/

Language dc:language

en

Author Identifier

0000-0002-2750-3877
0000-0002-2699-1979

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/304465