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, 119 (2), 287-94

COX2 in CNS Neural Cells Mediates Mechanical Inflammatory Pain Hypersensitivity in Mice

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COX2 in CNS Neural Cells Mediates Mechanical Inflammatory Pain Hypersensitivity in Mice

Daniel Vardeh et al. J Clin Invest.

Abstract

A cardinal feature of peripheral inflammation is pain. The most common way of managing inflammatory pain is to use nonsteroidal antiinflammatory agents (NSAIDs) that reduce prostanoid production, for example, selective inhibitors of COX2. Prostaglandins produced after induction of COX2 in immune cells in inflamed tissue contribute both to the inflammation itself and to pain hypersensitivity, acting on peripheral terminals of nociceptors. COX2 is also induced after peripheral inflammation in neurons in the CNS, where it aids in developing a central component of inflammatory pain hypersensitivity by increasing neuronal excitation and reducing inhibition. We engineered mice with conditional deletion of Cox2 in neurons and glial cells to determine the relative contribution of peripheral and central COX2 to inflammatory pain hypersensitivity. In these mice, basal nociceptive pain was unchanged, as was the extent of peripheral inflammation, inflammatory thermal pain hypersensitivity, and fever induced by lipopolysaccharide. By contrast, peripheral inflammation-induced COX2 expression in the spinal cord was reduced, and mechanical hypersensitivity after both peripheral soft tissue and periarticular inflammation was abolished. Mechanical pain is a major symptom of most inflammatory conditions, such as postoperative pain and arthritis, and induction of COX2 in neural cells in the CNS seems to contribute to this.

Figures

Figure 1
Figure 1. Generation of a neural-specific Cox2-deficient mouse.
(A) loxP sites were inserted in introns 5 and 8 to enable deletion of exons 6–8. COX2 is functional and undisrupted after being flanked with loxP sites. β-Actin levels were used for loading control. (B) Genotyping of nCOX2–/– (Nes-Cre;Cox2fl/fl) and WT (Cox2+/+) animals showing WT COX2 (COX2+; ii), floxed COX2 (COX2fl; i), and Cre recombinase (bottom; iii) alleles. The asterisk indicates a Nes-Cre;Cox2fl/fl (nCOX2–/–) mouse. (C) Sixteen hours after LPS exposure (5 μg/ml), WT and Cox2fl/fl macrophages show similar expression of both COX1 and COX2. (D) Detection of Nes-Cre–mediated deletion of Cox2 exons 6–8 by PCR, using primers targeted against introns 5 and 8. The shorter fragment represents the recombined COX2 allele (COX2recomb; ii), whereas the longer one represents the nonrecombined allele (COX2Exon6–8; i). (E) Real-time quantitative PCR showing similar basal expression of COX2 in the spinal cord of WT C57BL/6 and Cox2fl/fl controls, while nCOX2–/– animals exhibit significantly lower Cox2 mRNA levels (*P < 0.05, n = 4–5, Student’s t test; mean ± SEM).
Figure 2
Figure 2. COX2 expression and development of peripheral inflammation in nCOX2–/– mice.
(A) Paw swelling after intraplantar CFA injection. nCOX2–/– and control mice show similar increases in paw diameter 24 and 48 hours after intraplantar CFA injection (**P < 0.01, n = 6, 1-way ANOVA with Dunnett’s post test; mean ± SEM). (B) Immunohistochemistry depicting, for both nCOX2–/– and control mice, little or no COX2 expression in the naive paw but strong COX2 induction after CFA injection. H&E staining reveals a comparable infiltration of inflammatory cells in both animals. Scale bars: 50 μm. (C) Quantitative real-time PCR results showing similar increases in Cox2 mRNA in the paw in nCOX2–/– and control mice (*P < 0.05, **P < 0.01, n = 5–6, 1-way ANOVA with Dunnett’s post test; mean ± SEM). (D and E) Western blots showing induction of COX2 in the paw in both animals in response to intraplantar CFA (***P < 0.001, n = 3, Student’s t test; mean ± SEM). GAPDH was used as internal control.
Figure 3
Figure 3. COX2 expression in the spinal cord of nCOX2–/– mice after peripheral inflammation.
(A) After peripheral inflammation, spinal Cox2 mRNA levels are significantly increased in control animals (*P < 0.05, **P < 0.01, n = 4–5, 1-way ANOVA with Dunnett’s procedure). In contrast, a smaller but nonsignificant increase in spinal Cox2 mRNA expression is detected in nCOX2–/– mice (P > 0.05, n = 4–5; mean ± SEM). (B) Immunohistochemical study of COX2 expression in the dorsal horn of L4–L5 from control and nCOX2–/– mice. After peripheral inflammation (24 hours after CFA), COX2 expression is induced in the ipsilateral dorsal horn (dotted line) of control but not nCOX2–/– mice. NeuN is used as a neuronal marker in the bottom row. Scale bars: 50 μm. (C and D) Western blots show induction of COX2 in the spinal cord of control animals after CFA injection (**P < 0.01, n = 3, Student’s t test), whereas little or no COX2 protein is found in nCOX2–/– mice. GAPDH is used as internal control (mean ± SEM).
Figure 4
Figure 4. Basal and acute pain hypersensitivity in nCOX2–/– mice.
(A) No significant difference in basal mechanical or thermal sensitivity was detected between nCOX2–/– mice and littermate controls (n = 11–12; mean ± SEM). (B) Formalin test: Cumulative paw licking in 5-minute intervals after intraplantar formalin injection. nCOX2–/– and control mice both show the typical biphasic response (n = 6; mean ± SEM). (C) Formalin test: nCOX2–/– and control mice show a similar response in phase 1 (5–15 minutes) and early phase 2 (15–30 minutes) of the formalin reaction, whereas a slightly earlier recovery is apparent in late phase 2 (30–45 minutes) in the mutants (*P < 0.05, Student’s t test, n = 6; mean ± SEM).
Figure 5
Figure 5. Mechanical and heat hypersensitivity in nCOX2–/– mice after peripheral inflammation and after administration of the selective COX2 inhibitor SC-58236.
(A) After hind paw inflammation, mechanical threshold is significantly reduced in littermate control mice and remains decreased after 10 days. However, no significant drop in mechanical threshold is observed in nCOX2–/– mice (**P < 0.01, n = 9–10; mean ± SEM). (B) Thermal sensitivity threshold decreased in both nCOX2–/– and control littermates after paw inflammation (**P < 0.01, ***P < 0.001, 1-way ANOVA with Dunnett’s procedure, n = 9–10; mean ± SEM). (C) After i.p. administration of the COX2-selective inhibitor SC-58236 three days after CFA-induced inflammation (CFA + COX2 inhib), both littermate controls and nCOX2–/– animals exhibited increased heat thresholds (*P < 0.05 and **P < 0.01, Student’s t test, n = 6; mean ± SEM). (D) SC-58236 i.p. administration 3 days after CFA-induced inflammation significantly increases mechanical threshold in control animals (***P < 0.001, Student’s t test, n = 6; mean ± SEM).
Figure 6
Figure 6. Mechanical and heat hypersensitivity in nCOX2–/– mice after induction of periarticular inflammation and fever response to LPS.
(A) Swelling of the tibiotarsal joint after periarticular injection of CFA in nCOX2–/– and control mice (**P < 0.01 and ***P < 0.001, 1-way ANOVA with Dunnett’s procedure, n = 12; mean ± SEM). (B) The joint inflammation induced a significant drop in hind paw mechanical threshold in the littermate control over 21 days but failed to do so in the nCOX2–/– mice (*P < 0.05, **P < 0.01, 1-way ANOVA with Dunnett’s procedure, n = 12; mean ± SEM). (C) Thermal hypersensitivity in the hind paw was present in both littermate control and nCOX2–/– mice (*P < 0.05 and **P < 0.01, 1-way ANOVA with Dunnett’s procedure, n = 11–12; mean ± SEM). (D) Effect of i.p. LPS on body temperature in nCOX2–/– mice. The mice were injected with LPS (1 mg/kg) or 0.9% saline at time 0. Between 90 and 180 minutes, fever in nCOX2–/– mice did not differ from that in littermate control mice (P > 0.05, n = 8; mean ± SEM).

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