Stress inhibits hair growth in mice by induction of premature catagen development and deleterious perifollicular inflammatory events via neuropeptide substance P-dependent pathways

Abstract

It has been much disputed whether or not stress can cause hair loss (telogen effluvium) in a clinically relevant manner. Despite the paramount psychosocial importance of hair in human society, this central, yet enigmatic and controversial problem of clinically applied stress research has not been systematically studied in appropriate animal models. We now show that psychoemotional stress indeed alters actual hair follicle (HF) cycling in vivo, ie, prematurely terminates the normal duration of active hair growth (anagen) in mice. Further, inflammatory events deleterious to the HF are present in the HF environment of stressed mice (perifollicular macrophage cluster, excessive mast cell activation). This provides the first solid pathophysiological mechanism for how stress may actually cause telogen effluvium, ie, by hair cycle manipulation and neuroimmunological events that combine to terminate anagen. Furthermore, we show that most of these hair growth-inhibitory effects of stress can be reproduced by the proteotypic stress-related neuropeptide substance P in nonstressed mice, and can be counteracted effectively by co-administration of a specific substance P receptor antagonist in stressed mice. This offers the first convincing rationale how stress-induced hair loss in men may be pharmacologically managed effectively.

Figure 1.

In both mouse strains increased vulnerability of HFs toward an advanced catagen progression was detectable in stressed mice and could be mimicked by injection of SP in nonstressed mice and abrogated by injection of the highly specific NK1 receptor antagonist. The results for the CBA/J mice are shown on the left (A) and the data for C57BL/6 on the right (B). HCS was assessed as suggested by Maurer and colleagues. In brief the y axis depicts the mean ± 1 SEM of histometric score assessed on day 16 after anagen induction. For every mouse a minimum of 100 individual HFs was assigned to defined hair cycle stages. On the right of the graph, representative hair cycle stages for each HCS are depicted, ie, anagen VI is the dominant hair cycle stage with a score of 0.5. **, P < 0.01. The number of mice per group is given in the bars.

Figure 2.

The effect of stress on the hair cycle stage is depicted in A–D. A: A representative area of control mice 16 days after depilation with the majority of HFs in anagen VI (AVI). B mirrors the effect of stress on the hair cycle stage on day 16 after depilation with HFs in catagen IV (CIV) or catagen V (CV). C: HF of stressed mice that received injection of SP, which mimicked the effect of stress on the vulnerability of HFs toward catagen progression with HFs in catagen III to VI (CIII-VI). D: A representative example of mice exposed to stress and treated with the NK1-RA, in which the majority of HFs were scored as anagen VI, similar to the nonstressed control group. E–F: The effect of stress on immunocytes in murine skin. E: Distribution of MHC-II⁺ cells in parafollicular dermis of nonstressed mice or in similar distribution in stressed mice that received the NK1-RA (bright red staining), compared to F, increased number of MHC-II⁺ cells in the bulge region, forming cluster after stress exposure. Similar staining patterns were present after injection of SP. Original magnifications, ×200.

Figure 3.

The effect of stress on the percentage of HFs with TUNEL⁺ cells in bulge (A) and bulb (C) in CBA/J mice and TUNEL⁺ cells in bulge (B) and bulb (D) in C57BL/6 mice (mean ± SEM). Exposure to stress 2 days before assessment led to a significant increase of apoptotic cells/HF bulge (*, P < 0.05 for CBA/J; ***, P < 0.001 for C57BL/6) and bulb (***, P < 0.001 for C57BL/6). The effect of stress on HF apoptosis in bulge could be mimicked by injection of SP in both strains of nonstressed mice (A and B) and abrogated by treatment with NK1-RA in the bulge region of stressed mice (A and B). The number of mice per group is given in the bars.

Figure 4.

The effect of stress on HF keratinocyte apoptosis in bulge (A–D) and bulb (E and F) region in dermis of C57BL/6 mice. A and E: TUNEL staining in dermis of control mice, very few apoptotic cells can be observed in bulge (A) or bulb (E). HF keratinocyte nuclei appear in bright blue, resulting from DAPI counterstaining. B and F: Stress exposure 2 days before TUNEL staining caused an increase in apoptotic cells in the bulge (B) and bulb (F) region of the HF, as depicted by green fluorescence and pointed out in one case with an arrow. C and G: Injection of SP simulated the effect of stress in nonstressed C57BL/6 mice by causing an increase in apoptotic cells in the bulge (C) and bulb (G) region of the HF, as depicted by green fluorescence. D and H: HF from a stressed C57BL/6 mouse treated with NK1-RA. No signs of apoptosis in bulge (D) and bulb (H).

Figure 5.

The effect of stress on the percentage of HFs with MHC-II⁺ perifollicular cell cluster in CBA/J (A) and C57BL/6 (B) mice (mean ± SEM). Exposure to stress 2 days before assessment led to a significant increase of MHC-II⁺ cells cluster (***, P < 0.001 for both strains). Injection of SP in nonstressed mice mirrored the effect of stress in both strains and injection of NK1-RA abrogated the effect of stress on number of MHC-II⁺ cells cluster in CBA/J (A) and C57BL/6 (B) mice (***, P < 0.001 for both strains). The number of mice per group is given in the bars.

Figure 6.

The effect of stress on the percentage of degranulated (= activated) mast cells calculated from the total number of mast cells (mean ± SEM) in dermis (A and B) and subcutis (C and D) in CBA/J (A and C) or C57/BL6 (B and D) mice. Exposure to stress 2 days before assessment led to a significant increase of degranulated mast cells in murine dermis and subcutis in both strains. Injection of SP mimicked the effect of stress in nonstressed control mice. The number of mice per group is given in the bars.

Figure 7.

A: Mostly nonactivated mast cells in dermis of nonstressed C57BL/6 mice (dark purple staining). B: A magnification of the region marked in A with the stippled line. No extracellular granules are present in the vicinity of the resting mast cell, as pointed out by the white arrow. C: Stress exposure caused an increase in activated (degranulated) mast cells in dermis. The region magnification indicated in C and presented in D shows a representative example of an activated, degranulated mast cell with more than eight extracellular granules, as indicated by the purple arrow. E–H: Fluorescence immunohistochemistry of SP nerve fibers (delicate red lines) in the dermis (bulge area) of nonstressed mice is presented in E. A mast cell appears in bright green. F: SP nerve fibers in the dermis (bulge area) of a mouse exposed to stress. In this section, an increase in mast cell number can also be observed. Interestingly, we frequently observed SP-positive nerve fibers in the direct vicinity (<2 μm) of mast cells in dermis and subcutis (G) of stressed mice. H: Higher magnification of the stippled square in G.

Figure 8.

A: Stress exposure significantly up-regulates number of SP nerve fibers/visual field in dermis of C57BL/6 mice. B: A tendency of up-regulation could also be observed in the subcutis.