The chemokine ligand fractalkine is found on neuronal membranes, and can be released during excitotoxic damage to interact with CX3CR1 on glial cells. This activation of CX3CR1 within the spinal cord has been suggested to be involved in the generation of hyperalgesia (Watkins et al, 2001). We investigated the role of CX3CR1 in models of inflammatory pain using spinal delivery of anti-CX3CR1 antibodies, alone or in combination with indomethacin.

Rats (male Sprague-Dawley, Charles River, U.K. 150-200g) were intrathecally (i.t.) cannulated using the method of Milligan et al(1999). The formalin test was performed based on the time-sampling method of Abbott et al (1999), with one hindpaw of the rat being injected with 50µl of 5% formalin. Rats received either anti-CX3CR1 antibody (100µg in 10µl, Torrey Pines TP501AF) or serum (i.t. via cannula) 5 min before formalin injection. The CFA model was initiated by the intraplantar injection of 100µl Complete Freund’s Adjuvant (Sigma) into one hindpaw. Measurements of thermal (plantar test) and mechanical sensitivity (von Frey hairs), and paw volume (plethysmometry) were made pre-CFA and 48h post-injection. Rats were then treated with indomethacin (10mgkg^-1, s.c.) or vehicle (10%DMSO / Labrafil, s.c.) and anti-CX3CR1 antibody or serum (as above), and re-tested 3h post drug administration. Data shown as percent reduction from 48h post-CFA measures, mean ± s.e.m., n=10-11, statistical analysis by ANOVA with Bonferroni’s post-test for thermal hyperalgesia and non-parametric ANOVA with Dunnett’s post test for mechanical hyperalgesia.

Administration of anti-CX3CR1 antibody inhibited second phase responses to formalin (mean total score 20-50min post-formalin 0.6 ± 0.1, compared with 2.0 ± 0.2 for serum-treated rats (n=7 for both groups), p<0.001 Mann-Whitney non-parametric t-test).

Significant increase in paw volume was seen 48h post-CFA (187 ± 3% of pre-CFA value, p<0.0001, paired t-test). Drug treatment had no significant effect. Administration of anti-CX3CR1 in combination with indomethacin produced a significant inhibition of thermal and mechanical hyperalgesia compared with serum / vehicle treated rats, (see Table1). Inhibition of mechanical hyperalgesia was also significantly greater with anti-CX3CR1 / Indomethacin group than with serum / indomethacin treatment.

Table1. Reduction in CFA-induced hyperalgesia. *p<0.05, ***p<0.001 compared to serum / vehicle, †p<0.05 compared to Serum / Indomethacin.

These results indicate that CX3CR1 activation in the spinal cord is involved in the generation of hyperalgesia in rat models of inflammatory pain, and that CX3CR1 antagonists may be useful as central analgesics.

Abbott F. V. et al., Pain, 83, 561-569 (1999)
Milligan E. D. et al., J Neurosci Methods, 90, 81-86 (1999)
Watkins L. R. et al., Trends Neurosci., 24, 450-455 (2001)