DISCUSSION 2

 

A NEW APPROACH FOR THE UNDERSTANDING OF MELANOCYTES: STEREOLOGICAL QUANTIFICATION OF MELANIZATION BY IMAGE ANALYSIS

Eric Donois and Jean-Etienne Surleve-Bazeille

Département de Biologie Cellulaire et Microscopie Electronique, Université Bordeaux I

351, cours de la Libération, 33405 Talence CEDEX

Tel: 56 84 63 88 / Fax: 56 84 66 70

 

Pigmentation of human skin results from the transport of melanized melanosomes (MMs) in melanocytes dendrites which developpement is controled by keratinocytes [1,2], their transfer to the surrounding undifferentiated keratinocytes according to a mechanism still debated today [3, 4, 5, 6], and finally from the more or less efficient degradation of melanosomes in keratinocytes [7] which ascend in the epidermis to finish their differentiation as corneocytes fated to desquamation.

The macroscopic cutaneous degree of pigmentation depends on several factors playing a critical role at various levels in this process: phototype (heredity, race), solar exposition, hormonal background [8, 9], etc. However, the initial requirement for variations of skin pigmentation is the potential variations of melanin biosynthesis by melanocytes themselves, which modulation may be reproduced in vitro by direct action of artificial ultra violet radiations [10,11,12].

Routine techniques for the quantification of melanin pigments are used for a long time. Spectrophotometrical dosages are the most commonly utilized and consist in the comparison of the optical densities obtained at 415 nm after alkaline extraction, with those of standards (e.g. techniques used in [10,1,13]); Nonetheless, natural (from Sepia officinalis), synthetic (chemical oxidation of L-tyrosine) or biosynthetic (oxidation of L-tyrosine or L-DOPA by purified tyrosinase) eumelanin may be used as standard [14].

Physically deposited melanins to be measured are extracted by alkaline solutions, which may induce alterations in the polymer with release of colorless precursors [14] and may be influenced by the percentage of pheomelanic and eumelanic monomers in the case of mixed type melanin [15].

More recently, High Performance Liquid Chromatography (HPLC) procedures have been ellaborated [16,17] and now permit to estimate pheomelanin and eumelanin contents separately, which allows to understand how certain bioactive molecules change the pheomelanin to eumelanin ratio, e.g. a-MSH in mammals [18, 19, 20]. Nevertheless, HPLC methods or like spectrophotometrical methods require to be applied, a great number of melanocytes.

With the aim to understand the fine mechanisms of stimulation and inhibition of pigment production at the organite level, we developped in our laboratory a quantitative method based on the analysis of melanocytes images obtained by transmission electron microscopy [21, 22].

Melanin profiles are automatically extracted from melanocytes images with the aid of an image analysis system which generates melanization primary data. Those primary data are introduced in stereological formula, stereology being the body of mathematical methods relating three-dimensional parameters defining the structure to two-dimensional measurements obtainable on sections of the structure [23,24,25]. Those formulae enables us to estimate various meaningful melanization parameters. Thus, we are able to estimate concomitantly the melanocyte volume, the melanin volume density in the cytoplasm, the mean melanin volume per melanocyte, the numerical density of MMs in the cytoplasm and the mean number of MMs per melanocyte.

Additionally, two parameters, the mean melanin volume per MM and an indicator of melanosomal maturation, i.e. NN(mp,m), the mean number of detected melanin particles per MM [26], can be calculated in order to quantitatively appraise the level of melanosomal maturation.

Only two studies on the melanocyte have used morphometrical techniques, including one relating to the manual morphometrical measurement of melanosomes observed by transmission electron microscopy [19] and the other, dealing with a quantitative image analysis of Fontana Masson-stained 1 µm plastic sections of human skin samples observed by light microscopy [27].

Our stereological computerized tool permits to quantify different aspects of the intracytoplasmic melanization, which have never been performed at the ultrastructural level with automatic methods, and never approached from a stereological angle.

Works are carried out at the present time in our laboratory, with the aim to validate this original method by comparing morphometrical data and results obtained from a classical melanin assay [28].

Moreover, the Inazu's alkali elution method, which permits to dissolve pheomelanin on ultrathin sections with an acceptable specificity [29], is actually applied to our quantitative method in order to measure pheomelanin and eumelanin like only HPLC methods allow.

At least, the last appreciable advantage of our method is that it requires a low number of pigment cells. As a consequence, more complex experiments with human epidermal melanocytes cultured physiologically might be perfomed with small experimental units of short term cultures.

In conclusion, this original approach, by using new type of data will contribute to a better understanding of intracytoplasmic events underlying stimulation or repression of human melanogenesis by physical or chemical agents or in pathological conditions.

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