Gene expression profile and regulatory networks at the onset of clinical pain following tissue injury
Using the pathway-focused microarray, we found that 25 genes associated with the inflammatory cascade were up-regulated following tissue injury, including cytokines, chemokines and their receptors (). Of these, IL-6 (4.4-fold, p=0.04), CCL2 (3.7-fold, p=0.02) and CXCL2 (3.3-fold, p=0.01) were significantly up-regulated after tissue injury (n = 9 per group of pre- and post-surgery). Gene expression of CXCL3 (3.4-fold), IL8 (2.12-fold), TNF (1.9-fold), TNFRSF1B (2.0-fold, tumor necrosis factor receptor superfamily, member 1B), and C3 (1.7-fold, complement component 3) were also increased following tissue injury but did not reach statistical significance due to the large range of inter-individual variations.
Figure 1 Gene expression profile of inflammatory cytokines and receptors after tissue injury in a clinical model of acute inflammatory pain. The data derived from analysis using GE SuperArray are expressed as median (n = 9). *, p < 0.05 for comparison (more ...)
HG U133 Plus 2.0 arrays were then used to further study gene expression beyond the inflammatory cascade response to tissue injury as well as the effect of ketorolac, a widely used NSAID, on these gene expression responses. Consistent with the data from the pathway focused microarray, tissue injury induced a significant up-regulation on the gene expression of IL-6 (3.3 fold, p=0.01), IL-8 (4.2 fold, p=0.04), CCL2 (4.4 fold, p=0.01), CXCL2 (7.1 fold, p=0.002) 3 hrs following tissue injury (). Furthermore, the gene expression of CXCL1 (2.6 fold, p=0.03), ANXA1 (22-fold, p=0.001) and CYR61 (2.0-fold, p=0.04, encoding cysteine-rich, angiogenic inducer 61) were also increased at 3 hrs post-surgery. Ketorolac treatment showed modulatory effects on the tissue injury induced up-regulation of the gene expression of CXCL1, CXCL2, IL-8 and CYR61, but not on the gene expression of IL-6, CCL2, or ANXA1 ().
Participants demographic characteristics
Verification of changes in gene expression by quantitative RT-PCR
The changes in expression of the genes selected from microarray were further verified using qRT-PCR. The tissue injury-induced up-regulation on the gene expression of IL-6 (63.3-fold, p<0.0001), IL-8 (8.1-fold, p<0.001), CCL2 (8.9-fold, p< 0.0001), CXCL1 (30.5-fold, p<0.001), CXCL2 (26-fold, p<0.0001) and ANXA1 (12-fold, p<0.0001) was consistent with the microarray data in the direction of change. Consistent with the microarray results, ketorolac treatment did not change the gene expression of IL-6, CCL2 and ANXA1 following acute inflammation as compared to that in the placebo-treated group (). The changes in gene expression of IL-8, CXCL1 and CXCL2 after treatment of ketorolac were consistent with the results from microarray in the direction of change but did not reach statistical significance.
Figure 2 The changes in gene expression following acute inflammation and ketorolac treatment as assessed by qRT-PCR. The gene expression level is expressed as the average threshold cycle after normalization using 18 rRNA expression (Average Delt Ct). The open (more ...)
CYR61 as well was up-regulated by tissue injury (3.1-fold, p< 0.0005), with no significant effect by keterolac treatment (data not shown). On the other hand, the gene expression of peroxisome proliferator activated receptor alpha (PPARA) was not significantly affected by tissue injury or ketorolac treatment (data not shown).
Correlation between pro-inflammatory cytokine gene expression and clinical pain intensity
To examine whether the up-regulation in the gene expression of inflammatory cytokines is associated with the patient-reported pain intensity, the association between the gene expression and pain on the VAS over the first 3 hours post-surgery was examined using Pearson correlation. Interestingly, the tissue injury induced up-regulation on the gene expression of IL6 (r=0.68, p=0.005), IL8 (r=0.52, p=0.049) and CCL2 (r=0.64, p=0.014) were positively correlated to the pain intensity reported from patients in the placebo group (). This correlation was not observed in the ketorolac treatment group, as expected, since ketorolac significantly lowered the pain scores reported by these patients.
Correlation between gene expression level (RQ) and pain intensity (VAS). The association between the gene expression and pain scale was examined using Pearson correlation at the time of onset of clinical pain and was adjusted for age, sex and race.
Correlation among the changes in expression of genes related to inflammatory cytokines
To further examine whether these genes encoding inflammatory cytokines have a similar expression profile and whether they participate in a common or intersecting biological pathway in this acute inflammatory model, the Pearson correlation coefficients were calculated among these genes. Because there was no significant difference in gene expression level between the treatments (placebo and ketorolac), the two treatment groups were pooled to calculate the correlation coefficient for each gene. As shown in , the up-regulation of IL-6 gene expression was highly correlated to the up-regulation of the gene expression of IL-8 (r=0.57, p=0.001), CCL2 (r=0.74, p<0.001), CXCL1 (0.48, p=0.014) and CXCL2 (r=0.63, p=0.001). Moreover, the up-regulation of CCL2 gene expression was significantly correlated with that of CXCL1 (r=0.52, p=0.006) and CXCL2 (r=0.68, p<0.0001), with the up-regulation of CXCL1 expression being highly correlated with the up-regulation of CXCL2 (r=0.79, p<0.0001).
Chanes in gene expression following acute inflammation and ketorolac treatment