ARTICLE
Wrinkles
are modifications of the skin associated with cutaneous ageing and develop
preferentially on sun-exposed skin. Despite their importance in ageing,
very few studies have been specifically devoted to wrinkles, except for
those which form in some congenital or acquired skin disorders ([1, 2].
Wrinkles represent only one aspect of the modifications of the skin
surface occurring with age. Different names are used to refer to wrinkles,
which introduces some confusion about the type of skin modification [3].
Skin depressions are: folds, wrinkles and microrelief. Folds are anatomical
changes, which are easily seen, e.g. the naso-genial fold
and the postauricular fold. The skin microrelief comprises a number of
rectilinear grooves which can be classified according to their depth and
respective orientation, thus allowing a clinical appreciation of photoageing
[4, 5]. In contrast, there is no precise classification of the different
types of wrinkles, and different names (lines, furrows) are used as a
synonym for wrinkles in conjunction with the adjectives "coarse, fine,
small, thin" to better qualify them [5]. Concerning facial wrinkles, the
terminology is even more complex since frown lines and crow's feet lines
refer to frontal and peri-orbital wrinkles, respectively. The only proposed
definition of wrinkles comes from Griffith et al.: an extension
of the skin perpendicular to the axis of the wrinkle leaves a marked line
which represents the bottom of the wrinkle [6]. Controversial data have
been reported on the anatomy and histology of wrinkles with some authors
reporting major skin modifications specific for wrinkles [7], whereas
others report an almost normal histological aspect of the wrinkled skin
compared to the surrounding one [8]. This may be due to the semantic problems
detailed above, to the type of wrinkle studied and to the evolutionary
stage at which the wrinkle has been analyzed. The anatomical changes occurring
in the epidermis (thin and flattened) and in the dermis (interruption
of elastosis) which have been attributed to wrinkles [7, 9], refer to
permanent wrinkles or even to skin depressions other than wrinkles recovered
from cadaver skin [3].
The last area of interest in the studies on wrinkles is the fact that
they mainly develop on sun-exposed skin. The aspect and histology of the
skin is profoundly altered by chronic sun exposure, which induces epidermal
atrophy, an increase in melanocyte numbers, accumulation of truncated
elastic fibers (elastosis), decrease in collagen fibers and a dermal inflammatory
infiltrate [10]. Such findings specific to photo-damaged skin have also
frequently been reported to be associated with wrinkles.
The aim of the present study was to get better insight into the definition
of skin invaginations and into the aspect and the histology of wrinkles.
In order to avoid most of the above-described caveats in studies on wrinkles
we followed a precise and standardized protocol: 1) analysis of one type
of facial wrinkle developing on sun-exposed skin, i.e. the preauricular
wrinkle; 2) availability of control samples including: i) a sun-protected
skin sample recovered from the postauricular area for each individual;
ii) postauricular skin recovered from young individuals; 3) staining of
the wrinkle in vivo before surgery using indian ink in order to
precisely localize it after sampling, thus allowing the comparison of
the structure of the skin immediately below the wrinkle to that of the
surrounding skin; 4) both skin samples were recovered from middle aged
to elderly individuals undergoing plastic surgery, at the beginning of
the surgical procedure, and samples were immediately fixed for histology.
Our results show that the facial skin relief is characterized by four
types of depression: fold (including naso-genial fold and postauricular
fold); permanent wrinkle or reducible wrinkle (including frown lines,
crowlines around the corners of the eyes, and preauricular wrinkles) and
skin microrelief (non specific fine lines due to ageing). Whereas skin
fold and skin microrelief are recognized at the histological level, no
specific anatomical change is found under the permanent or reducible wrinkle
compared to the skin immediately adjacent to it. However, the accumulation
of basophilic fibers in the upper dermis, referred to as basophilic degeneration
or actinic elastosis, is markedly increased below the permanent wrinkle
compared to the reducible wrinkle.
Materials and methods
Skin specimens
Biopsies of normal skin were obtained from fold, pre-auricular or frown
wrinkles and postauricular zone, under general anesthesia, during surgical
lifting of sixteen Caucasian patients (aged 36 to 94 years). Control skin
was obtained from the postauricular zone of six young people (7-17 years
old) during ear surgery. Wrinkles were identified with indian ink (standarded
pen) before sampling and excised transversally to the principal axis,
including the skin on both sides (Fig.
1).
Tissue preparation
For histological study, a biopsy sample was fixed in Baker solution
(buffered formalin), and embedded in paraffin. 4 mum-thick cross sections
of the skin from the different samples were prepared and stained with
hematoxylin-eosin-safran (HES) and orcein according to standard procedures.
Photographs
Photographs were taken with Kodak Ektachrome ASA 400 daylight film on
a Leitz optical microscope.
Results and discussion
In this study which focused on facial skin invaginations, we first characterized
the morphological changes occurring in different types of skin relief
modifications. Then we showed that pre-auricular wrinkles develop on skin
sites with normal histological features but which could present local
alterations of the dermis.
The different types of skin surface modifications
In order to precisely define the different types of skin topography,
we first conducted a histological analysis of frown lines, preauricular
wrinkles and postauricular folds, recovered during surgical lifting. Wrinkles
were marked with indian ink before sampling and immediately processed
at the beginning of the surgical procedure (Fig.
1).
Analysis of the different samples showed four types of skin relief:
- the fold, defined by an invagination of the skin structures leading
to a depression of roughly 250 to 400 micrometers. The main histological
changes concerned the epidermis which appeared thin and was composed of
only 3 to 4 epidermal layers in the deepest portion of the fold (Fig.
2 a-d) ;
- permanent wrinkle is defined as a wrinkle observed in vivo
and persisting after processing of the skin sample for routine histology.
These wrinkles correspond to depressions of skin structures of roughly
100 micrometers in depth which are easily found on the skin section below
the indian ink marker. Such wrinkles include frown lines and eight out
of sixteen preauricular wrinkles of this study (36, 47, 54, 55, 57, 74,
77 and 94 years old) (Fig.
2 e-h) ;
- reducible wrinkles are those seen in vivo but which cannot
be observed after sampling and processing for routine histology. These
wrinkles could only be localized by the indian ink marker (Fig.
2 i-l). Such wrinkles correspond to eight out sixteen preauricular
wrinkles of the study (39, 48, 53, 56, 58, 59, 60 and 79 years old) ;
It is noteworthy that the natural evolution of wrinkles starts from
a reducible to a permanent skin invagination with differences among individuals
depending on age, coefficient of developed skin surface, severity of photo-damage
and influence of gravity [11] ;
- skin microrelief corrresponds to shallow depressions at the surface
of the epidermis, involving the horny and granular layer (Fig.
2 k-l); these depressions (10 to 30 micrometers) are seen on any type
of skin and are not associated with sun-exposed or wrinkled skin.
Thus, the four types of skin relief modifications are characterized
by depressions of the skin surface of different depths. The skin fold
and the skin microrelief are easily recognized at the histological level.
Conversely, the wrinkles give only minor or no microrelief modifications
which makes them difficult to detect microscopically, unless the depression
has been marked in vivo.
Wrinkles lack specific histological
features
In order to test for the possibility that wrinkles could be associated
with modifications of skin structures at the site of the wrinkle formation,
we next analyzed the histological features of the skin of sixteen preauricular
wrinkles (8 reducible, 8 permanent), compared to that of the patient's
sun-protected postauricular skin. The width of the wrinkle (300-400 mum)
was precisely defined using the ink marker and comparison was made between
the skin immediately below the wrinkle and the skin adjacent to the wrinkle.
In contrast to the skin from folds, analysis of the epidermis and dermis
of the wrinkled skin revealed a normal histology in all nine cases, identical
to that of the skin adjacent to the wrinkle (Fig.
2 e-l, i-j). This was particularly striking for reducible wrinkles
which could not be individualized in the absence of in vivo staining.
Thus, in agreement with Kligman [3] and Pierard [8], the histology of
wrinkles appears similar to that of normal adjacent skin. Histology of
the hypodermis is also noted to be unaltered below the wrinkle. The number,
size and distribution of skin appendages as well as the general organization
of the extracellular matrix components was similar below the wrinkle and
in the surrounding skin. No histological differences were observed between
the permanent and reducible wrinkles.
The major histological changes in actinically damaged skin are seen
in the superficial dermis, and concern microfibrils and collagen fibers
resulting in two findings: 1) actinic elastolysis also known as basophilic
degeneration which corresponds to the accumulation of altered elastic
fibers [12, 13], and 2) disappearance of the network of elastin microfibrils,
which in young skin lies perpendicular to the DJE. Both changes were observed
in all patients' skin samples, but were more pronounced in sun-exposed
wrinkled skin than in sun protected skin (Fig.
3). Furthermore, in sun-exposed skin, these modifications were not
specifically located at the site of the wrinkle but were uniformly observed
through the section. Thus no specific histologic feature could be assigned
to the wrinkle skin depression. Interestingly, in the skin of the eight
permanent wrinkles, the heavy accumulation of elastotic material involved
the entire depth of the superficial dermis (Fig.
4d, arrow). In contrast, in the skin of the eight reducible wrinkles,
elastosis was less severe and did not involved the uppermost superficial
dermis (Fig. 4d, arrow).
These observations, showing that the wrinkles develop on sun-damage skin
with a gradation of histological changes from the reducible to the permanent
wrinkles, are in keeping with studies demonstrating major alterations
in the dermal matrix of individuals with clinically severe photo-ageing.
These changes include reduction in the number of anchoring fibrils at
the DEJ [14], loss of fibrillin-rich microfibrils in this region [15]
and significant reductions in the major collagens (type I and type II)
[16].
Taken together, our results show that there is no specific histologic
feature characterizing the wrinkle, since no modification was specifically
found under the wrinkle compared to the skin surrounding it. A diminished
skin resistance at the level of the DEJ and the upper superficial dermis
due to sun damage is a prerequisite for wrinkle formation. Among all the
alterations induced by UV light, elastosis appears primordial since permanent
wrinkles are always found in skin severely affected by elastosis. Future
studies will address the issue of the role of other factors, especially
molecules of the DEJ, in the formation of wrinkles.
CONCLUSION
Acknowledgements
We thank Dr B. Chouvet, Dr M. Faisant and Dr J. Kanitakis for their
collaboration and helpful expertise in histological analysis and J. Messenger
for English correction of the manuscript.
Article accepted on 14/3/02
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