Figure 5.

Substance P alters immunoreactivity for NGF and its receptors (TrkA, p75NTR) in organ-cultured human hair bulbs. Twenty-one human anagen scalp skin HFs derived from three different donors were cultured per group for 3 days in the presence of substance P as described in the Materials and Methods section. Basement membranes between dermal papilla and HF epithelia are indicated by white dotted lines. A: NGF expression in a control hair bulb is present throughout the HF epithelium. In HFs treated with substance P this expression is enhanced, and additional weak expression can be observed in the dermal papilla. Note the nuclear pronunciation of the staining pattern. B: p75 pan-neurotrophin receptor expression is present in the ORS as well as the connective tissue sheath (dotted arrow) of a control HF. After treatment with substance P, the expression of p75 is greatly enhanced in the ORS and connective tissue sheath (dotted arrows), and additional weak staining can be observed in the proximal matrix (arrows) and individual cells of the ORS (arrowheads). C: Expression of the high-affinity NGF receptor TrkA locates to the ORS in a control HF. After substance P treatment this expression is virtually absent and only a weak expression can be detected in a HF treated with 10⁻¹² mol/L substance P. D: The mean fluorescence intensity was measured at two previously defined reference areas as indicated in A–C. The observed effects are dose-dependent, and the differences to controls are significant by Mann-Whitney U-test for unpaired samples (*P < 0.05). E: NGF, p75NTR, and TrkA mRNA levels in cultured human HFs after 3 days of culture with or without SP. TaqMan RT-PCR data pooled from three different donors donating 3 to 12 HFs each are displayed Each extraction, containing three HFs, was measured in duplicate, and produced highly reproductive results. Amount of mRNA is presented in percentage of control levels, when control levels equal 100. Original magnifications, ×200.