Nitric oxide is negatively correlated to pain during acute inflammation

Abstract

Background: The role that nitric oxide (NO) plays in modulating pain in the periphery is unclear. We show here, the results of two independent clinical studies (microdialysis and gene expression studies) and a pilot dose finding study (glyceryl trinitrate study), to study the role of NO in the early phase of acute inflammatory pain following oral surgery. The effect of ketorolac on NO production and nitric oxide synthase (NOS) gene expression was also studied.

Results: Microdialysis samples showed significantly higher levels of NO at the first 100 min compared to the last 80 minutes in the placebo treated group. In the ketorolac group, on the other hand, NO levels gradually decreased over the first 60 min but were similar to placebo over the later 100-180 min, with no significant change in NO level over time. The levels of NO were negatively correlated to pain intensity scores. Local infusion of the NO donor glyceryl trinitrate at the site of surgery, showed a small analgesic effect that did not reach statistical significance in the sample size used. While the gene expression of iNOS and eNOS were not up-regulated, 3 hours after surgery, nNOS was downregulated in both treatment groups and eNOS gene expression was significantly lower in the ketorolac group compared to the placebo group. Further, there was a positive correlation between the change in gene expression of nNOS and eNOS in the placebo group but not in the ketorolac group.

Conclusion: We suggest that at this early stage of inflammatory pain in man, NO is analgesic in the periphery. Further, ketorolac down-regulates eNOS gene expression.

Figure 1

Microdialysis Study. (a) Effect of tissue injury and ketorolac treatment on pain intensity following third molar tooth extraction (p = 0.018; repeated measure two way ANOVA, n = 24 & 20 for placebo and ketorolac treatment groups, respectively). (b) Effect of tissue injury and ketorolac treatment on NO production at the site of oral surgery. Data are presented as mean ± SEM; n = 19 & 10 for placebo and ketorolac treatment groups, respectively; *indicates the first five time points were significantly different from the last four time points, p < 0.0001; contrast analysis under the mixed linear model. Raw data were not normally distributed so were transformed to ln(VAS+1) and statistical analysis were conducted on transformed data.

Figure 2

Glyceryl trinitrate Study. (a) Effect of glyceryl trinitrate (100, 150 or 200 μg) on pain intensity when infused into the surgical site of third molar tooth extraction over 20 min (n = 18-20 per group). (b) Effect of glyceryl trinitrate (150 or 200 μg) on pain intensity when infused into the surgical site of third molar tooth extraction over 20 min n = 20 & 37 for the placebo and glyceryl trinitrate groups respectively; p = 0.038; repeated measure 2 way ANOVA. (c) Dose response curve of glyceryl trinitrate's effect on pain intensity when infused into the surgical site of third molar extraction. Linear regression shows a slope significantly different from zero (n = 18-20 per group).

Figure 3

Gene expression study. (a) Tissue injury resulted in the down-regulation of gene expression of nNOS 2.4-fold in the placebo group and 3.4-fold in the ketorolac group. eNOS was insignificantly up-regulated in the placebo group, but down-regulated in the ketorolac group, with significant difference between the two treatment groups. The changes in gene expression following tissue injury were assessed by qRT-PCR. The gene expression level is expressed as fold change relative to pre-surgery level. *indicates p = 0.02; **indicates p = 0.0003 compared to pre-surgery group (paired t-test); #indicates p = 0.026 compared to the placebo group (independent-sample t test). (b & c) The relative changes in gene expression (RQ) from qRT-PCR of eNOS and nNOS were correlated in the placebo group but not in the ketorolac treated group. The association was examined using Pearson correlation at 3 hours post-surgery.

Figure 4

Tissue injury increases NO production possibly via eNOS or nNOS in the early phase following surgery. This causes a small increase in NO level that lead to an analgesic effect. However, in the late phase iNOS isoform is up-regulated, producing a marked increase in NO level that would have a hyperalgesic effect. cNOS, constitutive NOS.