A role for NT-3 in the hyperinnervation of neonatally wounded skin

Abstract

Neurotrophin-3 (NT-3) is a target-derived neurotrophic factor that regulates sensory neuronal survival and growth. Here we report that NT-3 plays a critical permissive role in cutaneous sensory nerve sprouting that contributes to pain and sensitivity following skin wounding in young animals. Sensory terminal sprouting in neonatally wounded dermis and epidermis is accompanied by increased NT-3 transcription, NT-3 protein levels, and NT-3 protein release 3-7 days post skin injury in newborn rats and mice. Functional blockade of NT-3 activity with specific antibodies greatly reduces sensory neurite outgrowth induced by wounded skin, but not by naïve skin, in dorsal root ganglion/skin co-cultures. The requirement for NT-3 for sensory terminal sprouting in vivo is confirmed by the absence of wound-induced hyperinnervation in heterozygous transgenic mice (NT-3(+/-)lacZ). We conclude that upregulation of NT-3 in neonatally wounded skin is a critical factor mediating the sensory nerve sprouting that underlies hypersensitivity and pain following skin injury.

Fig. 1

Neonatal wounding induces skin hyperinnervation in rat and mouse. (A) PGP9.5 immunohistochemistry in rat skin 7 days following P0 full-thickness skin wounding. The lightened area represents the extent of the skin wound. Dotted lines indicate the surface of the epidermis (upper) and epidermal/dermal boundary. Scale bar represents 200 μm. (B) PGP9.5 immunolabeling of cutaneous nerve fibres in mouse (left) and rat (right). Upper panels show cutaneous hyperinnervation 7 days following P0 wound. Lower panels show P7 naïve skin innervation. Scale bars represent 100 μm.

Fig. 2

Neonatal wounding induces the production and secretion of neurotrophin-3 (NT-3) in skin. (A) Enzyme-linked immunosorbent assay (ELISA) of NT-3 protein concentration in rat skin at the indicated times following P0 skin wounding (n = 6-8 for each age and condition). ^∗∗P < 0.001 (one-way analysis of variance). Data are represented as mean ± SEM. (B) ELISA of NT-3 protein levels secreted from P0 + 3 wounded or contralateral skin (n = 6 for each) following 24 hours in culture. ^∗∗∗P < 0001 (t-test). Data are represented as mean ± SEM.

Fig. 3

Neurotrophin-3 (NT-3) released from wounded skin selectively stimulates primary sensory neurite outgrowth. (A) Neurite outgrowth of newborn dorsal root ganglia (DRG) explants co-cultured for 24 hours with P3-naïve or P0 + 3-wounded skin (upper panels) and in the presence of a function blocking NT-3 antibody (lower panels). Scale bar represents 100 μm for higher-power images and 500 μm for lower-power images. (B) Quantification method for measuring neurite outgrowth. Red quadrant indicates the region within which neurite crossings were counted. (C) Number of DRG neurites growing towards skin explant (mean of 16 explants from 4 rats per condition). P < 0.0001 (one-way analysis of variance [ANOVA], Newman-Keuls multiple comparison test).

Fig. 4

Skin wounding leads to de novo lacZ expression in dermis and epidermis in NT-3^lacZneo/+ mice. LacZ expression (blue) in skin from NT-3^lacZneo/+ mice. P7 naïve expression (upper panel) and 7 days following P0 skin wounds (lower panel). (A, B) Higher power areas of epidermal and dermal LacZ expression. NT3 (NT-3) = neurotrophin-3.

Fig. 5

Absence of hyperinnervation in neurotrophin-3 (NT-3)-deficient skin following wounding. Comparison of wound-induced skin hyperinnervation in wild-type (left) and NT-3^lacZneo/+ mice (right). Cutaneous innervation is shown by PGP9.5 immunoreactivity (green) (n = 10 for each genotype and condition). P < 0.0001 (one-way analysis of variance [ANOVA], Newman-Keuls multiple-comparison test). Scale bar = 100 μm.

Fig. 6

Selective hyperinnervation of neonatally wounded skin by peptidergic afferents. Hyperinnervation of neonatally wounded skin by peptidergic afferents (A: calcitonin gene-related peptide [CGRP]) but not by nonpeptidergic afferents (B: P2X3 right); n = 10 for each condition and genotype. P < 0.001 (one-way analysis of variance, Newman-Keuls multiple-comparison test. (C) Pie chart representing the relative contribution of CGRP to the entire cutaneous innervation. WT = wild-type; NT3 = neurotrophin-3.