Dissecting the contribution of knee joint NGF to spinal nociceptive sensitization in a model of OA pain in the rat D.R. Sagar, L. Nwosu, D.A. Walsh,

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Dissecting the contribution of knee joint NGF to spinal nociceptive sensitization in a model of OA pain in the rat D.R. Sagar, L. Nwosu, D.A. Walsh, V. Chapman Osteoarthritis and Cartilage Volume 23, Issue 6, Pages 906-913 (June 2015) DOI: 10.1016/j.joca.2015.01.010 Copyright © 2015 The Authors Terms and Conditions

Fig. 1 Intra-articular injection of MIA produces (A) weight bearing asymmetry and (B) decreased hindpaw withdrawal thresholds on the ipsilateral hind paw of MIA rats (n = 24 rats), compared to saline-injected rats (n = 24 rats). The effects of intra-articular MIA on the knee joint pathology were quantified in a sub-set of rats used in this study. There was a significant increase in the appearance of macroscopic lesions (C) on the articular joint surface of MIA-injected rats, compared to saline-injected rats (n = 12 MIA rats; n = 11 saline rats). Statistical analysis of weight bearing asymmetry and PWTs was performed using a 2-way ANOVA with a Bonferroni post-hoc test. Statistical test of knee joint pathology was performed using a Mann Whitney test. *P < 0.05, **P < 0.01, ***P < 0.001 MIA injection vs saline injection. Osteoarthritis and Cartilage 2015 23, 906-913DOI: (10.1016/j.joca.2015.01.010) Copyright © 2015 The Authors Terms and Conditions

Fig. 2 Effects of intra-articular injection of NGF or saline in MIA and saline-injected rats on spinal neuronal responses to (A,B) knee extension, (C–D)26 g receptive field size of spinal neurones innervating the knee joint, in MIA-injected and saline-injected rats. (E–F) A representative image of mapping the peripheral receptive field over the knee joint following stimulation with a 26 g von Frey hair (E) pre- and (F) 2 h post-NGF injection; the shaded area indicates areas over which stimulation was able to evoke single unit activity of a dorsal horn neurone in the spinal cord. Panels show effects of intra-articular injection of NGF or saline in MIA and saline-injected rats on spinal neuronal responses to (G–H) knee flexion (bend) and (I–J) 26 g von Frey punctate stimulation over the knee joint in MIA-injected and saline-injected rats. Data are normalised to baseline values and expressed as mean ± SEM. Statistical analyses comparing the area under curve were performed using a Kruskal Wallis test with a Dunn's post hoc test. *P < 0.05. MIA-NGF: n = 6 neurones in six rats; MIA-saline: n = 6 neurones in six rats, Saline-NGF: n = 6 neurones in six rats, Saline–saline: n = 7 neurones in seven rats. Osteoarthritis and Cartilage 2015 23, 906-913DOI: (10.1016/j.joca.2015.01.010) Copyright © 2015 The Authors Terms and Conditions

Fig. 3 Effects of intra-articular injection of NGF or saline in MIA or saline-injected rats on spinal neuronal responses to (A–B) 26 g von Frey stimulation and (C–D) 26 g receptive field size of neurones innervating the hind paw. Data are normalised to baseline values and expressed as mean ± SEM. Statistical analyses comparing the area under curve were performed using a Kruskal Wallis test. MIA-NGF: n = 6 neurones in six rats; MIA-saline: n = 6 neurones in six rats; saline-NGF: n = 6 neurones in six rats; saline–saline: n = 5 neurones in five rats. Osteoarthritis and Cartilage 2015 23, 906-913DOI: (10.1016/j.joca.2015.01.010) Copyright © 2015 The Authors Terms and Conditions

Supplementary Fig. 1 Representative traces from MIA-treated rats demonstrating spinal neuronal responses to extension of the knee joint within a physiological range before and 20 min post intra-articular injection with (A–B) saline or (C–D) NGF (10 μg/50 μl). Sorted trace = action potentials in raw trace sorted by Spike 2 Software. Osteoarthritis and Cartilage 2015 23, 906-913DOI: (10.1016/j.joca.2015.01.010) Copyright © 2015 The Authors Terms and Conditions

Supplementary Fig. 2 Effects of an intra-articular injection of NGF or saline on spinal neuronal responses to (A,B) knee bend (C–D) knee extension, (E–F) 26 g von Frey stimulation and (G–H) 26 g receptive field size over the knee joint in naive rats. Data are normalised to pre-intervention baseline values and expressed as mean ± SEM. Statistical analyses comparing the area under curve were performed using a Mann Whitney test. *P < 0.05, **P < 0.01: NGF versus saline. NGF: n = 7 neurones in seven rats; saline: n = 6 neurones in six rats. Osteoarthritis and Cartilage 2015 23, 906-913DOI: (10.1016/j.joca.2015.01.010) Copyright © 2015 The Authors Terms and Conditions