Topical substance P increases inflammatory cell density in genetically diabetic murine wounds

Abstract

The neuropeptide substance P (SP) is a known inflammatory mediator released from cutaneous peripheral nerve terminals. SP effects on cellular composition in the cutaneous response to injury remain unclear. Based on our previous observations about SP effects on wound repair, we hypothesized that topical SP increases inflammatory cell density infiltration early after injury. A full-thickness 1.5 x 1.5 cm(2) wound was created on the dorsum of 8-9-week-old C57BL/6J-m+Lepr(db) mice (db/db). Wounds were treated daily with 300 muL of either normal saline (0.9% NaCl) or 10(-9) M SP for 7 days. Three wounds from each group were harvested at 2, 3, 7, 14, and 28 days. Samples underwent enzymatic digestion and were incubated with fluorescent-labeled antibodies. Using flow cytometry, cellular content and density for each sample was derived. Masson Trichrome stained histology specimens were prepared to confirm results. Cell density in the SP-treated wounds (11.3 x 10(7) cells/g tissue, standard deviation [SD]+/-1.5 x 10(7)) was greater than in NaCl-treated wounds (7 x 10(7) cells/g tissue, SD+/-2.3 x 10(7), p<0.05) at day 7 postwounding. SP significantly increased the density of leukocytes (2.1 x 10(7), SD +/-3.6 x 10(6) vs. 1.8 x 10(7), SD+/-4.9 x 10(5), p<0.02) 3 days after wounding and the density of macrophages (2.9 x 10(7), SD+/-7.5 x 10(6) vs. 1.3 x 10(7), SD+/-1.4 x 10(6), p<0.05) 7 days after wounding. There were no significant differences in endothelial cell, leukocyte, or macrophage density at later time points. Topical SP treatment increases early inflammatory density in the healing wounds of db/db mice. These data support a role for nerve-mediated inflammation in cutaneous wound repair.

Figure 1

Overall cell density (# cells/gram of wound tissue) is described in the y axis of the graph below. This represents the number of cells quantified per gram of tissue harvested. Cell density is compared in cutaneous wounds following treatment with substance P (10⁻⁹M) or NaCl. Cell density is transiently increased on days 3 and 7 following injury compared to normal saline treatment, with day 7 reaching statistical significance (p<0.05). “Day 0” represents the skin harvested during the wounding, with neither SP nor NaCl treatment. N=36 for this experiment. Error bars represent standard deviation.

Figure 2

Leukocyte density was increased at all time points in cutaneous wounds treated with substance P (10⁻⁹M) compared to NaCl. A statistically significant difference was observed at day 3 (p<0.05). N=36 for this experiment. Error bars represent standard deviation.

Figure 3

Macrophage density was higher in cutaneous wounds following treatment with substance P (10⁻⁹M) compared to normal saline treated wounds at all time points. A statistically significant difference was observed at day 7 (p<0.05). N=36 for this experiment. Error bars represent standard deviation.

Figure 4

Endothelial cell density increased on day 7 and 14 in both substance P-treated and saline-treated wounds. No statistically significant differences were identified at any time points. N=36 for this experiment. Error bars represent standard deviation.

Figure 5

Figure 5a and 5b. Histology at day 3 in SP-treated wounds (5a) revealed a marked inflammatory cell infiltrate when compared with NaCl treated wounds (5b), confirming the flow cytometry results. Note that the magnification bar in the lower right is equal to 100 microns.