Co-induction of cyclooxygenase-2 [correction of cyclooxyenase-2] and early growth response gene (Egr-1) in spinal cord in a clinical model of persistent inflammation and hyperalgesia

Abstract

Background: This study characterised the effects of persistent peripheral inflammation of the foot on pain and spinal cord expression of cyclooxygenase-1 and -2 (COX-1 and COX-2) and early growth response gene 1 (Egr-1), known markers of neuronal plasticity, in a clinical model of naturally-occurring inflammatory disease and hyperalgesia in sheep ('footrot'), before and after routine treatment (parenteral treatment with antibiotics and antiseptic footbathing). The temporal pattern of expression of COX-1, COX-2 and Egr-1 mRNA and protein were analysed using real-time PCR and Western blotting.

Results: Animals affected with persistent peripheral inflammation displayed significant hyperalgesia and lameness (a proxy indicator of spontaneous pain) restricted to the inflamed limb. Hyperalgesia and lameness were significantly attenuated 1 day after treatment, and resolved further by day 7 and day 3, respectively. COX-2 but not COX-1, protein expression was up-regulated in spinal cord from lame animals on day 0, before treatment. Following treatment and attenuation of pain behaviours, levels of COX-2 returned to control levels. Significant induction of Egr-1 mRNA and protein were observed in spinal cord from lame animals. Three days after treatment, levels of Egr-1 mRNA returned to control levels, however, Egr-1 protein remained elevated.

Conclusion: Elevated levels of spinal COX-2 and Egr-1 protein correlate with the presence of pain and hyperalgesia, and may underlie persistent pain, although a direct causal link has still to be established. Understanding the temporal pattern of expression of key mediators in clinical pain states may lead to better strategies to manage pain.

Figure 1

Resolution of lameness and hyperalgesia after treatment. Photograph of a clinically lame animal and close-up of the hindfeet (a). The animal was given a lameness score of 10 (non-weight bearing). Inflammation can be seen in the right foot and tracking up the lower limb. The magnitude of hyperalgesia (Emax%) on the lame limb on day 0 prior to treatment, and up to 7 days post-treatment is represented as the percentage change from 3 non-affected limbs according to the formula: ((mean response threshold of non-affected limbs - response threshold of lame limb)/mean response threshold of non-affected limbs) × 100. In control animals, the mean percentage change in response threshold measured on one limb relative to the three other limbs was calculated (b). Mean lameness score for animals affected by unilateral lameness on day 0 prior to treatment for lameness, and up to 7 days post-treatment (c). Data are presented as mean values; s.e.d. represents the standard error of the difference. ^{a, b, c} Means with disparate letters are significantly different (p < 0.05).

Figure 2

Lymph node hyperplasia in infected sheep. Photograph of popliteal lymph nodes ipsilateral and contralateral to inflamed limb collected from a clinically lame animal (a). Mean lymph node size (each lymph node represented as proportion (%) of combined weight) in control animals (left and right prescapular and popliteal lymph nodes) and animals affected by unilateral hindlimb (popliteal lymph nodes) and forelimb (prescapular lymph nodes) lameness on day 0 prior to treatment, and 3 days post-treatment (b). Data are presented as mean values; s.e.d. represents the standard error of the difference. ^{a, b, c} Means with disparate letters are significantly different (p < 0.001).

Figure 3

Expression of COX-1, COX-2 and Egr-1 mRNA in spinal cord. Real-time semi-quantitative measurement of COX-1 (a), COX-2 (b) and Egr-1 (c) mRNA in spinal cord from control sheep (n = 6) and sheep affected by unilateral lameness on day 0 (pre-treatment) (n = 6) and 3 days post-treatment (n = 6). Spinal cords were hemisected into ipsilateral and contralateral portions (data is pooled for control animals). Levels of mRNA are expressed relative to the endogenous reference gene β-actin. Significant increase from control: * p < 0.05.

Figure 4

Expression of COX-1, COX-2 and Egr-1 proteins in spinal cord. Western blot analyses of COX-1 (a), COX-2 (b) and Egr-1 (c) protein in spinal cord. Photomicrographs show expression of COX-1, COX-2 and Egr-1 at 70, 72 and 60 kDa, respectively, as expected in a representative control sample (con) and ipsilateral (i) and contralateral (c) spinal cord from an animal affected by unilateral lameness on day 0. Graphs show densitometric quantification of levels of COX-1 (a), COX-2 (b) and Egr-1 (c) protein in ipsilateral and contralateral spinal cord tissues from animals affected by unilateral lameness on day 0 before treatment (n = 6) and 3 days after treatment (n = 6). Bars represent mean ± SEM protein expression (as %) relative to control levels (100%). Significant increase from control: * p < 0.05; significant decrease from day 0: * p < 0.05.