Background/Purpose: Nerve growth factor (NGF) is under investigation as a promising target for osteoarthritis (OA) pain. NGF exerts its potent pro-algesic effects through sensitization of nociceptors. Sensitization is a key process in chronic pain states, including OA, characterized by exaggerated responses to innocuous stimuli. However, the precise biological mechanisms underlying the pain-producing effects of NGF in OA are incompletely understood. Here, we tested whether nociceptors become sensitized to NGF in experimental OA.

Methods: DMM surgery was performed in the right knee of 10-week old male Na_V1.8 cre-GCaMP6s loxp mice. In vivo calcium imaging:  Na_V1.8-GCaMP6s loxp mice 20 weeks after DMM (n=8) or age-matched naïve control (n=6) were used. These mice express the fluorescent calcium indicator, GCaMP6s, in nociceptors. In vivo calcium imaging of the L4-DRG was performed as described (Miller RE et al, Arthr Rheum 2018, PMID: 28992367). A series of compounds were injected IA into the right knee of anesthetized mice while imaging was performed: (1) Saline; (2) CCL2 (100 mg/mL); (3) 2.5S NGF (100 mg/mL); (4) Pam3CSK4 (1 mg/mL); (5) LPS (5 mg/mL); (6) capsaicin (10 mM). For each injection, a 30 G needle was inserted in the joint cavity, imaging was started, the compound was injected from frame 15-20, and imaging continued until frame 100. For each mouse, change in fluorescence over time was calculated using a custom ImageJ macro in order to identify responding sensory neurons. The area under the peak was calculated using GraphPad Prism. Knee hyperalgesia: 3 mL NGF (100 ng/mL, n=4 mice) or 3 mL of vehicle, n=4 mice) was injected IA in the right knee of 10-week old naïve mice. Knee hyperalgesia was assessed prior to injection and 30 mins, 2, 4, and 24 hours after injection using a Pressure Application Measurement device by a person blinded to treatment.

Results: Injection of NGF into the knee joints of anesthetized Na_V1.8-GCaMP6s mice elicited intracellular calcium increases in a similar number of neurons in naïve and DMM mice (mean±SEM; naïve: 1.7±1.0%; DMM: 2.6±0.7%; p=0.47), suggesting that the NGF receptor is expressed by a similar number of cells at this time point after DMM. As a comparison, we have previously shown that 15% of L4-DRG neurons innervate the knee joint in healthy mice.

Examining the intracellular calcium responses in each neuron, we found that responses were greater after DMM, as assessed by peak area under the curve (naïve: 0.9±0.2 peak AUC; DMM: 1.7±0.2 peak AUC; p=0.02), suggesting that neurons expressing the NGF receptor have become sensitized to NGF after DMM. Neurons that responded to NGF after DMM also responded more to CCL2 and LPS compared to NGF-responsive neurons in naïve mice (p=0.04 for both NGF-CCL2 and NGF-LPS), further suggesting that NGF-responsive neurons are sensitized in OA.

The neuronal response to NGF was reflected in the behavioral response, where IA administration of NGF, but not vehicle, induced knee hyperalgesia in naïve wild-type mice (NGF: 311±16 g; vehicle: 439±5 g; p=0.01).

Conclusion: These findings suggest that NGF directly binds to neuronal receptors in the IA space, and sensitizes neurons to mediators present in the OA joint.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/ngf-responsive-neurons-are-sensitized-in-experimental-osteoarthritis/