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,1
* Cutaneous Neuroimmunology, Biomedical Research Center, University Medicine Charité, Virchow and Mitte Campus;
Psychoneuroimmunology, Biomedical Research Center, University Medicine Charité, Virchow Campus; and
Internal Medicine, Psychosomatics, University Medicine Charité, Mitte Campus, Humboldt-University of Berlin, Berlin, Germany; and
Dermatology, Medical School, University of Newcastle, UK
2Correspondence: Biomedical Research Center, Rm. Nr. 2.0549, University Medicine Charité, Virchow Campus, Humboldt University of Berlin, Augustenburger Platz 1, Berlin 13353, Germany. E-mail: eva.peters{at}charite.de or frl_peters{at}yahoo.com
SPECIFIC AIMS
Graying is a spectacular and intriguing phenomenon often discussed in the context of stress and aging. In our society, fixed on eternal youth, the loss of pigmented hair has great socio-economic as well as psychosomatic impact. Despite these pressing interests, the phenomenon has not yet been satisfactorily explained and a scientifically sound therapeutic strategy remains missing. From animal experiments and mutation-reports we know about predetermined breaking points in the function of the hair follicle pigmentary-unit. This unit, formed between the pigment-producing melanocytes and the hair shaft-producing keratinocytes of the hair bulb, produces melanin continuously over many years. Melanin-synthesis generates H2O2 and other free radicals, placing hair follicle melanocytes under high oxidative stress. Moreover, oxidative stress appears to be a common theme in melanocyte dysfunction and results in impairment of growth factors, antioxidative enzyme and cofactor functions, DNA-repair mechanisms, and antiapoptotic signals such as BclII. Ultimately, this may lead to permanent melanocyte damage and the exhaustion of a melanocyte stem cell pool residing in the bulge region of the hair follicle. Psychoemotional and inflammatory-induced oxidative stress may add to the endogenous oxidative stress, overwhelm the hair follicle melanocyte antioxidant capacity, and speed up terminal damage to hair bulb melanocytes. However, conclusive proof that any of the above braking points of melanocyte function are active in and relevant to graying has not been brought forward to date. This encouraged us to propose a "free radical theory of graying" in analogy to the "free radical theory of aging". In support of this theory, we performed experiments to answer the following questions: 1) Are decreased numbers, morphology and melanocyte apoptosis in hair follicles of aging individuals associated with oxidative stress in the pigmentary-unit? 2) Do graying hair follicles have higher levels of oxidative stress induced permanent damage (e.g., mitochondrial DNA damage)? 3) Is oxidative stress leading to apoptosis preferably in hair follicle melanocytes?
PRINCIPAL FINDINGS
1.1 Morphological and immunohistochemical analysis indicate accumulating and deleterious oxidative stress damage in hair follicle melanocytes of graying hair follicles
We microdissected pigmented, graying and unpigmented human scalp skin hair follicles from healthy aging donors (elective face-lift) under sterile conditions and analyzed melanocyte presence, morphology and distribution by macroscopic (inverted microscopy on free-floating isolated hair follicles) and immunohistochemical (NKI-beteb for melanocytes; TUNEL for apoptotic nuclei) analysis. We found, that melanocytes in graying hair bulbs, when compared to pigmented and unpigmented hair follicles harvested from the same donors, were reduced in number and showed features of oxidative stress and apoptosis by morphometric analysis (Table 1
). We also found the occasional hair bulb with TUNEL-positive labeling of pigment-producing melanocytes in graying individuals but never in samples from nongraying individuals. Compared to other hair follicles within the same donor, hair follicles with apoptotic melanocytes consistently contained an intermediate number of melanocytes in their pigmentary-unit and were thus classified as graying (Table 1)
. Hair follicles with maximal melanocyte numbers never contained TUNEL+ melanocytes, while pigment-producing melanocytes were absent from unpigmented hair bulbs. This observation demonstrates loss of melanocytes by apoptosis selectively from the graying hair bulbs. Moreover, outside the pigmentary-unit, melanocytes in the stem cell niche in the bulge region of the outer root sheath were only rarely detected in graying or unpigmented hair follicles, which indicates a diminished stem cell pool selectively in these hair follicles (Table 1
).
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1.2 Immunohistochemistry confirms presence of oxidative stress in the pigmentary-unit of graying individuals
The oxidatively damaged oligonucleotide 8OHdG could be detected in the pigmentary-unit of anagen hair follicles in the scalp skin of graying individuals (Table 1)
. In the hair bulb, strongest expression localized to melanocytes, indicating high oxidative stress selectively in this cell population.
2.1 As a result of increased oxidative stress, graying hair follicles show increased mitochondrial DNA deletion
Mitochondrial deletions such as the so called "common" deletion are the result of excessive cellular oxidative stress and lead to defects in the respiratory chain, more oxidative stress, hydrops, and ultimately apoptosis of the damaged cells. We found an increased number of deletions in hair follicles that were macroscopically classified as graying (8 out 20 donors=40%) and in unpigmented hair follicles (4 out of 20 donors=20%) harvested from the same donors. These later hair follicles may still contain some unpigmented, macroscopically undetectable melanocytes. In macroscopically pigmented hair follicles, we found only one sample with a deletion (1 out of 20 donors=5%). The presence of mitochondrial DNA deletions was independent of age within the sample group (all donors older than 50 yr) and only depended on pigmentation-status.
2.2 Unpigmented hair follicles retain full viability and grow better in culture than pigmented hair follicles
Over a culture period of 8 d unpigmented hair follicles showed significantly higher hair shaft elongation as a measure of in vitro hair growth than pigmented hair follicles (Table 1)
. Both groups maintained an anagen-like morphology of the hair bulb and did not enter into a catagen-like stage over this culture period.
3.1 Pigmented hair follicles show melanocyte apoptosis in the pigmentary-unit on additional exogenous oxidative stress
Pigmented hair follicles cultured over a period of 3 d in the presence of exogenous oxidative stress (hydroquinone at concentrations between 10–3 to 10–7 M) showed a dose dependent increase of TUNEL+ apoptotic melanocytes in their pigmentary-unit (Table 1)
. 10–7 M hydroquinone appeared to be without effect and showed no significant differences to control in TUNEL+ melanocyte number. 10–4 and 10–3 M hydroquinone showed toxic effects on the hair follicle, with increased apoptosis in the hair follicle keratinocyte population in the hair matrix and the inner root sheath. 10–5 to 10–6 M hydroquinone showed apoptosis of melanocytes selectively in the pigmentary-unit by TUNEL-labeling (Table 1)
.
CONCLUSION
Here we provide unique evidence for oxidative stress induced loss of melanocytes from the human hair follicle during aging. In detail, we show for the first time that: 1) A decreased number of viable melanocytes in the aging hair follicle bulge and bulb and an increased incidence of hair bulb melanocyte apoptosis in aging individuals are associated with oxidative stress in the pigmentary-unit; 2) a higher frequency of oxidative stress associated permanent mitochondrial DNA damage occurs in graying hair follicles, while unpigmented hair follicles retain their growth capacity and proof to be not generally "older" than pigmented hair follicles; and 3) melanocytes of the pigmentary-unit are highly and selectively susceptible to additional exogenous oxidative stress-damage.
Together, we provide evidence that the major route, by which oxidative stress leads to permanent and selective melanocyte damage, passes by the mitochondria. Mitochondrial DNA is not so well protected as genomic DNA. The accumulation of mutations therefore correlates with age and is indicative of general oxidative stress levels, caused for example by psycho-emotional stress, inflammation, UV-light, etc. The highly susceptible and easily accessible hair follicle melanocytes can thus be utilized to indicate general oxidative stress-exposure and -damage, later to be expected in other cell-types and organs.
Moreover, to our knowledge, this is first report that proves the long-suspected and often anecdotally described accelerated growth of white scalp hair. Interestingly, keratinocytes and fibroblasts reportedly have higher antioxidant enzyme activities than melanocytes. This observation further supports that keratinocytes and fibroblasts in unpigmented hair follicles are fully viable and that melanocytes have aged selectively. Clinical observations support the premature and selective loss of hair bulb melanocytes by oxidative stress as an indicator for overall oxidative stress levels (Fig. 1
).
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Future investigations may reveal whether interindividual differences in hair graying derive from genetic differences in antioxidative capacity, as suggested by the premature graying of mice with BclII deficiency in the absence of general features of premature aging. The graying hair follicle offers a unique model–system to study oxidative stress effects and aging and to test antioxidants and other anti-aging pharmaceuticals in their ability to slow down or even stop this process. Supporting this line of thought, one study in mice has already shown, that topical application of an antioxidant enzyme can protect hair follicles from UV-light induced graying.
In summary, our findings support the proposed hypothesis of a "free radical theory of graying" and suggest, that melanocytes in the hair follicle are highly susceptible to endogenous oxidative stress. Psychoemotional stress and inflammatory stress may add to the endogenous oxidative stress-load of the hair bulb melanocyte (Fig. 1)
. Oxidative stress damage in hair follicle melanocytes may be viewed as the tip of the iceberg and can be used as an indicator and model for the systematic search for oxidative stress-reducing strategies. These strategies will involve endo- and exogenous protection from oxidative stress-induced apoptosis by stimulation of the endogenous antioxidative capacity or by treatment with exogenous antioxidants.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4039fje
1 These authors contributed equally to this work. 
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