Neurotropin® ameliorates chronic pain associated with scar formation in a mouse model: A gene expression analysis of the inflammatory response

Abstract

Background: Scar formation after trauma and surgery involves an inflammatory response and can lead to the development of chronic pain. Neurotropin^® (NTP) is a nonprotein extract of inflamed skin of rabbits inoculated with vaccinia virus. It has been widely used for the treatment of chronic pain. However, the in vivo effects of NTP on painful scar formation have not been determined. To investigate the molecular mechanisms underlying the effects of NTP on the inflammatory response, we evaluated gene expression in the scar tissues and dorsal root ganglions (DRGs) of mice administered NTP and control mice. Methods and results: Mice injected with saline or NTP were used as controls; other mice were subjected to surgery on the left hind paw to induce painful scar formation, and then injected with saline or NTP. Hind paw pain was evaluated by measuring the threshold for mechanical stimulation using the von Frey test. The paw withdrawal threshold gradually returned to pre-operative levels over 4 weeks post-operation; NTP-treated mice showed a significantly shortened recovery time of approximately 3 weeks, suggesting that NTP exerted an analgesic effect in this mouse model. Total RNA was extracted from the scarred hind paw tissues and DRGs were collected 1 week post-operation for a microarray analysis. Gene set enrichment analysis revealed that the expression of some gene sets related to inflammatory responses was activated or inhibited following surgery and NTP administration. Quantitative real-time reverse transcription-polymerase chain reaction analysis results for several genes were consistent with the microarray results. Conclusion: The administration of NTP to the hind paws of mice with painful scar formation following surgery diminished nociceptive pain and reduced the inflammatory response. NTP inhibited the expression of some genes involved in the response to surgery-induced inflammation. Therefore, NTP is a potential therapeutic option for painful scar associated with chronic pain.

Keywords: Chronic pain; gene expression; inflammatory response; neurotropin®; painful scar formation.

Figure 1.

Experimental system. Painful scars were generated in the left hind paws of mice by puncturing a hole at the proximal end of the sole and stripping the subcutaneous tissue of the entire sole (operation group n = 16). Mice subjected to puncture but no stripping served as controls (sham group n = 16). The mice were divided into two groups to receive injections of NTP (sham/NTP and operation/NTP) or saline (sham/saline and operation/saline) (n = 8 per group). The right hind paw was left untreated in all mice. NTP or saline was administered to all mice at a dose of 4 μL/g of body weight. The injections were scheduled at 2 h post-operation, every 24 h during the 1 week post-operation, and at 2, 3, and 4 weeks post-operation. NTP: Neurotropin^®.

Figure 2.

Body weight changes in mice. All mice were weighed before injection. Data are presented as mean values. No significant differences in weight among the four groups were determined using the Mann–Whitney U test.

Figure 3.

Changes in the appearance of the operated heel in the mice. (a) The circumference was measured at 5 mm from the heel of the mice. Data are presented as mean values. To compare the circumference of the operated paw in each group more directly, the Part 2.5 mm below the ordinate was removed. Significant differences in operation/saline versus sham/saline (*: p < 0.05, **: p < 0.01) and operation/NTP versus operation/saline (+: p < 0.05, ++: p < 0.01) were determined using the Mann–Whitney U test. The measurement times were the same as those for body weight. (b) Changes in the appearance of the operated hind paw at 2 h, 2 days, and 1 week post-operation.

Figure 4.

Von Frey test. The von Frey test was used to measure the withdrawal threshold of the operative side hind paw (a) and nonoperative side hind paw (b) in response to mechanical stimuli. Each measurement was performed prior to all injections. Data are presented as mean values. Significant differences in operation/saline versus sham/saline (*: p < 0.05, **: p < 0.01) and operation/NTP versus operation/saline (+: p < 0.05, ++: p < 0.01) were determined using the Mann–Whitney U test.

Figure 5.

GSEA of paw samples. Gene expression levels are presented as a gradient from high (red) to low (blue). Only core enriched genes in the gene sets are listed. (a) Cytokine–cytokine receptor interaction was activated by the surgery (operation/saline vs sham/saline) and NTP treatment (operation/NTP vs operation/saline). (b) Extracellular matrix receptor interaction was activated by the surgery (operation/saline vs sham/saline) but suppressed by NTP treatment (operation/NTP vs operation/saline). FDR: false discovery rate; NES: normalized enrichment score (|NES| > 1 and FDR <0.25 were considered significant).

Figure 6.

GSEA of DRG samples. (a) The transforming growth factor-β signaling pathway was activated by the surgery (operation/saline vs sham/saline) but suppressed by NTP treatment (operation/NTP vs operation/saline) in the DRGs. (b) The neurotrophin (neurotrophins are a family of proteins that are distinct from NTP) signaling pathway was activated by the surgery (operation/saline vs sham/saline) but suppressed by NTP treatment (operation/NTP vs operation/saline) in the DRGs. FDR: false discovery rate; NES: normalized enrichment score (|NES| > 1 and FDR <0.25 were considered significant).