COX-2 expression and function in the hyperalgesic response to paw inflammation in mice

Abstract

Peripheral inflammation and edema are often accompanied by primary and secondary hyperalgesia which are mediated by both peripheral and central mechanisms. The role of cyclooxygenase-2 (COX-2)-mediated prostanoid production in hyperalgesia is a topic of substantial current interest. We have established a murine foot-pad inflammation model in which both pharmacologic and genetic tools can be used to characterize the role of COX-2 in hyperalgesia. Zymosan, an extract from yeast, injected into the plantar surface of the hindpaw induces an edema response and an increase in COX-2 expression in the hindpaw, spinal cord and brain. Zymosan-induced primary hyperalgesia, measured as a decrease in hindpaw withdrawal latency in response to a thermal stimulus, is long-lasting and is not inhibited by pre-treatment with the systemic COX-2 selective inhibitor, parecoxib (20 mg/kg). In contrast, the central component of hyperalgesia, measured as a reduction in tail flick latency in response to heat, is reduced by parecoxib. Zymosan-induced primary hyperalgesia in Cox-2-/- mice is similar to that of their Cox-2+/+ littermate controls. However, the central component of hyperalgesia is substantially reduced in Cox-2-/- versus Cox-2+/+ mice, and returns to baseline values much more rapidly. Thus pharmacological data suggest, and genetic experiments confirm, (i) that primary hyperalgesia in response to zymosan inflammation in the mouse paw is not mediated by COX-2 function and (ii) that COX-2 function plays a major role in the central component of hyperalgesia in this model of inflammation.

Fig. 1

(A) Zymosan (0.5, 1.0 and 2% w/v) induces paw edema following intraplantar injection in C57BL/6 mice. Zymosan suspensions (30 μl) were injected into the right (ipsilateral) hindpaw and PBS was injected into the left (contralateral) hindpaw. Paw thickness was measured at the times shown following zymosan injection. Data are expressed as percent increase in paw edema and are presented as means ± SEM for 6 mice per group. (B) Western blots were performed on homogenates prepared from mouse hindpaws at the times shown following intraplantar zymosan injection (30 μl). 100 μg of total protein was loaded in each well. Antibody to 14-3-3 protein was used as a loading control. (C) Quantification of the data from panel B. Data are expressed as the ratios of COX-2/14-3-3 intensities from optical density analyses, and are presented as means ± SEM for three mice at each time point. (D) RT-PCR for COX-2 mRNA was performed on homogenates prepared from mouse hindpaws at the times shown following intraplantar zymosan A injection (2%, 30 μl). GAPDH mRNA was analyzed as a control. (E) Histological appearance of mouse hindpaws 24 hrs following intraplantar zymosan (2%, 30 μl) and contralateral PBS (30 μl) administration. H&E staining and COX-2 immunostaining (brown color) are shown for the contralateral paw (left two panels) and the ipsilateral paw (right two panels). K; keratin layer, E; epidermal layer, Ec; eccrine tubular glands, I; infiltrating inflammatory cells. Scale bar: 100 μm.

Fig. 2

(A) Zymosan-induced thermal hyperalgesia in C57BL/6 mice. Zymosan suspensions (0.5, 1.0 and 2%; 30 μl) were injected into the right (ipsilateral) hindpaw and PBS was injected as a control treatment into the left (contralateral) hindpaw. Hindpaw (HWL) withdrawal latency data are expressed as time (in seconds) to paw withdrawal, and are presented as means ± SEM for 4–6 mice per group. *p<0.01, ipsilateral HWL compared to contralateral HWL (not shown) at each time point. (B) Tail-flick latency (TFL) following zymosan-induced paw inflammation, for the mice described in Panel A. TFL data are expressed as means ± SEM for 4–6 mice per group. *p<0.01 for each time point, compared to TFL prior to zymosan/saline injection.

Fig. 3

COX-2 protein accumulation in the brain and spinal cord following intraplantar zymosan administration in the paws of C57BL/6 mice. Western blots were performed on homogenates prepared from the brains and spinal cords of mice 24 hrs after intraplantar zymosan injection (2.0%; 30 μl). 100 μg of total protein was loaded in each well. Antibody to 14-3-3 protein was used as a loading control.

Fig. 4

Effect of parecoxib on (A) hindpaw edema, (B) hindpaw withdrawal latency and (C) tail-flick latency following intraplantar zymosan administration in the ipsilateral hindpaw and PBS in the contralateral hindpaw. Parecoxib (20 mg/kg, i.p., 30 min prior to the zymosan challenge) or vehicle was administered. Data are presented as means ± SEM (6 mice per group). *p<0.01 compared to vehicle-treated group.

Fig. 5

Comparison of (A) hindpaw edema, (B) hindpaw withdrawal latency and (C) tail-flick latency in Cox-2^−/− and Cox-2^+/+ C57BL6/129 mice, following intraplantar zymosan administration. (A) Intraplantar zymosan (2%, 30 μl) induced paw edema. Zymosan suspension (2%) was injected into the right (ipsilateral) hindpaw and PBS was injected as a control treatment into the left (contralateral) hindpaw. Data are presented as means ± SEM (5–6 mice per group). *p<0.01 for all values for Cox-2^−/− mice when compared to the same time points for Cox-2^+/+ mice. (B) Intraplantar zymosan (2%)-induced Hindpaw withdrawal latency (HWL) for Cox-2^−/− and Cox-2^+/+ mice. Data are presented as means ± SEM for 5–6 mice per group. *p<0.01 for ipsilateral HWLs of Cox-2^−/− when compared to ipsilateral HWLs of Cox-2^+/+ mice. (C) Intraplantar zymosan (2%)-induced tail-flick latency (TFL) in Cox-2^−/− and Cox-2^+/+ mice. Data are presented as means ± SEM (5–6 mice per group). *p<0.01 for all time points for Cox-2^−/− mice TFL values, when compared to Cox-2^+/+ mice. The inset in panel B shows Western blots for COX-2 and 14-3-3 proteins in the hindpaws of Cox-2^−/− mice and Cox-2^+/+ mice 20 hrs after zymosan injection. Note the complete absence of the COX-2 protein signal in the Cox-2^−/− mouse.