Role of nerve growth factor in cutaneous wound healing: accelerating effects in normal and healing-impaired diabetic mice

Abstract

Four full-thickness skin wounds made in normal mice led to the significant increase in levels of nerve growth factor (NGF) in sera and in wounded skin tissues. Since sialoadenectomy before the wounds inhibited the rise in serum levels of NGF, the NGF may be released from the salivary gland into the blood stream after the wounds. In contrast, the fact that messenger RNA and protein of NGF were detected in newly formed epithelial cells at the edge of the wound and fibroblasts consistent with the granulation tissue produced in the wound space, suggests that NGF was also produced at the wounded skin site. Topical application of NGF into the wounds accelerated the rate of wound healing in normal mice and in healing-impaired diabetic KK/Ta mice. This clinical effect of NGF was evaluated by histological examination; the increases in the degree of reepithelialization, the thickness of the granulation tissue, and the density of extracellular matrix were observed. NGF also increased the breaking strength of healing linear wounds in normal and diabetic mice. These findings suggested that NGF immediately and constitutively released in response to cutaneous injury may contribute to wound healing through broader biological activities, and NGF improved the diabetic impaired response of wound healing.

Figures

Figure 1

Serum NGF levels in male SJL/J mice after cutaneous wounding. Each point represents the mean ± SE of three separate experiments using duplicate samples.

Figure 2

NGF levels at the wounded skin site (closed) and control unwounded skin site (open) of male SJL/J mice after cutaneous wounding. Each point represents the mean ± SE of three separate experiments using duplicate samples.

Figure 3

Cellular localization of mRNA and protein of NGF in newly formed epithelium at edge of the wound (B and F, original magnification: 240) and granulation tissue produced in the wound space (D and H, original magnification: 550). All specimens were obtained from male SJL/J mice 3 d after cutaneous wounding. Basal cells of the epidermis show positive reaction for mRNA expression of NGF (B) and superficial epithelial cells show positive reaction for protein of NGF (F). Positive reaction for both the mRNA (D) and protein (H) of NGF is observed in fibroblasts in the granulation tissue. No positive reactions are observed in the sections treated with the sense primer (A and C) and the irrelevant Ab instead of anti-NGF Ab (E and G).

Figure 4

Production of NGF by 3T3 fibroblasts stimulated with various cytokines and histamine. NGF levels in culture medium were measured by a sandwich ELISA as described in Materials and Methods. Each point represents the mean ± SE of three separate experiments using duplicate samples.

Figure 5

Production of NGF by PAM 212 keratinocytes stimulated with various cytokines and histamine. NGF levels in culture medium were measured by a sandwich ELISA as described in Materials and Methods. Each point represents the mean ± SE of three separate experiments using duplicate samples.

Figure 6

Wound healing accelerated by topical applications of NGF in control C57BL/6 mice and diabetic KK/Ta mice. NGF (1 μg) or vehicle solution alone was applied to the wound space each day for 3 d beginning with the day of wounding as described in Materials and Methods, and a healing term was examined macroscopically. Each histogram represents the mean ± SE of six mice per group. *P <0.02; ‡P <0.01; when compared with diluent alone.

Figure 7

Histological features of wound specimens from control C57BL/6 mice (A and B) and diabetic KK/Ta mice (C and D) 8 d after cutaneous wounding. NGF (1 μg; B and D) or vehicle solution alone (A and C) was applied to the wound space each day for 3 d beginning with the day of wounding as described in Materials and Methods. Sections were stained with hematoxylin and eosin. Original magnification: 60. Arrow heads, the original wound margin.

Figure 8

Effects of NGF on wound tear strength in control C57BL/6 mice and diabetic KK/ Ta mice. NGF (2 μg) or vehicle solution alone was applied to the wound space shortly after the wounding, and wound tear strength was measured as described in Materials and Methods. Each histogram represents the mean ± SE of four mice per group. *P <0.01; when compared with diluent alone.