ARTICLE
Auteur(s) : Jingjun
Zhao1, Jianxin Chen2,
Yinghong Yang1, Shuangmu Zhuo2, Xingshan
Jiang2, Wei Tian1, Xiaoyin Ye1,
Lihang Lin1, Shusen Xie2
2Department of Dermatology, Affiliated Union
Hospital Fujian Medical University, Fuzhou 350001, China
1Institute of Laser and Optoelectronics
Technology, Fujian Provincial Key Laboratory for Photonics
Technology, Key Laboratory of OptoElectronic Science
and Technology for Medicine of Ministry
of Education, Fujian Normal University, Fuzhou 350007,
China
accepté le 22 Juillet 2009
Anetoderma is clinically characterized by localized areas of
flaccid or herniated sack-like skin [1]. Currently it is usually
classified into two clinical groups: primary anetoderma, which
arises from previously normal skin and secondary anetoderma, which
occurs at sites of skin diseases such as syphilis, acne, lupus, or
varicella [2]. Primary anetoderma can be divided into
Schweninger-Buzzi type (no preceding erythema) and
Jadassohn-Pellizzari type (preceded by macular erythema or papular
urticaria). The histopathological features and prognosis of these
two types are identical. Up to now, the clinical/pathological
diagnosis of anetoderma still heavily relies on histology [3]. The
ultrastructural techniques, electron microscopy has also been used
to investigate the etiology of anetoderma [4]. Both histological
methods and electron microscopy have vital drawbacks in their
invasive natures. It is completely impossible to carry out
non-invasive studies of dermatological dynamic processes because of
their fixation and processing procedures. With the rapid
development of optical imaging techniques, non-invasive or
minimally invasive optical methods may help the clinician and
researcher to better understand the pathophysiology of this
disease.
Multiphoton microscopy based on TPEF and SHG is one of the most
important inventions of biomedical imaging in the
20th century [5]. Because of its unique merits,
such as lower photobleaching and photodamage, imaging unstained
tissue samples as well as enhanced penetration depth, it has become
a powerful non-invasive optical method in investigating
physiological and pathological states of clinical dermatology. For
example, this technique has been used to study normal human skin
[6], assess skin aging [7], study scars [8, 9], and diagnose basal
cell carcinoma and evaluate photoaging [10]. In this paper,
Jadassohn-Pellizzari anetoderma was investigated using multiphoton
microscopy in order to understand its pathological process.
Materials and methods
Case report
A 21-year-old Chinese female presented with progressive generalized
wrinkled patches on her face, trunk, arms and legs since she was 3
years old. The lesions initially began with itchy erythematous
patches over her legs and trunk. After scratching, the patches
enlarged. About 2~3 weeks later the erythematous patches
resolved, leaving finely wrinkled, bulging areas. More and more
similar lesions were found on her face and trunk. To date, the
lesions have spread to her face, trunk, arms and legs. New lesions
are still continually emerging. She had no history of drug allergy
and a family history of cutaneous or joint laxity was negative.
Physical examination revealed 5- to 30-mm-diameter, round,
finely wrinkled patches which appeared sunken, atropic or flaccid
on her face, trunk and upper extremities, a few sack-like patches
on both sides of the shoulders and a few erythematous patches on
the back. Because of a slack and wrinkled face, her appearance was
that of a woman older than her age (figure 1). The remainder
of her skin appeared to be normal. Joints were not hyper-extensible
and apart from the cutaneous manifestations, her clinical
examination was normal and her general status was good.
Routine laboratory tests were normal or negative, including
complete blood cell counts, glucose, serum urea and creatinine,
urinalysis, cholesterol, triglycerides, serum protein
electrophoresis, erythrocyte sedimentation rate, antinuclear
antibody, serum copper, ceruloplasmin, C3, C4, aspartate
aminotransferase, alanine aminotransferase, sodium, potassium,
serological reactions for syphilis (TRUST, TPPA), thyroxine, T3,
T4, TSH. Serum elastase, serum elastase inhibitor and assays to
measure lysyl oxidase activity were not available. Chest and spine
X-ray, electrocardiogram, echocardiogram of the heart and bone
density were all normal.
An elastic tissue staining of the unaffected skin (Gomori’s
aldehyde-fuchsin method) revealed normal elastic fibers in upper,
mid, and deep dermis (figure 2A). But the
elastic fibers in the erythema showed a marked reduction of elastic
fibers in all layers of the dermis. In the mid-dermis and deep
dermis, elastic fibers were almost completely absent (figure 2B). An elastic
tissue staining in the affected area indicated that the elastic
fibres were fragmented in all layers of the dermis and even
granular (figure
2C). These findings were consistent with the diagnosis of
Jadassohn-Pellizzari anetoderma.
Specimen preparation
To investigate the pathological process of this disease, three
pieces of skin biopsies were taken from unaffected normal skin, far
from affected skin and erythema (for the study of unaffected normal
skin), from the back with erythema (for the study of the
erythematous phase), and from the affected left arm (for the study
of an affected abnormal specimen), respectively. Informed consent
was obtained from this patient who participated in the study. We
strictly conformed to the institutional rules governing clinical
investigation of human subjects in biomedical research. Immediately
after being excised, tissue samples were snap-frozen and kept in
liquid nitrogen (– 196 °C) until use. The ex vivo skin samples
were cut into 120 μm thickness, perpendicular to the epidermal
layer so that each section comprised a complete transverse
cross-section of the epidermal and dermal layers, and sandwiched
between the microscope slide and a piece of the cover glass. To
avoid dehydration or shrinkage during the imaging process, a little
phosphate-buffered saline (PBS) solution was dripped into the
tissue specimen.
Multiphoton microscopic system
The multiphoton microscopic system used in this study has been
described in detail previously [6, 11]. The multiphoton microscopic
image was performed on a Zeiss LSM 510 META equipped with a
mode-locked femtosecond Ti: sapphire laser (110 fs,
76 MHz), tunable from 700 nm to 980 nm (Coherent
Mira 900-F). In this system, the META detector has eight detecting
channels and each channel covers a spectral window of approximately
340 nm, ranging from 377 nm to 716 nm. Two different
channels were used to obtain large area images from the epidermis
to the dermis of the skin biopsy. One channel corresponded to the
wavelength range of 398-410 nm, to show the microstructure of
collagen fiber in the dermis arising from SHG signals, whereas
another channel covered the wavelength range from 430 to 714 nm, in
order to image elastin fibers in the dermis, keratins and cells in
the epidermis, from TPEF signals. For high-resolution imaging, a
high-numerical-aperture, oil immersion objective (Plan-Apochromat
63×, N.A.1.4, Zeiss) was employed in all experiments. All images
have a 12-bit pixel depth.
Results
Figures 3A to C
present multiphoton microscopic images of large areas from the
epidermis to the dermis of the tissues examined. During the
experiments, we found that the morphology and content of collagen
and elastin fibers at the boundary of affected and unaffected skin
showed apparent differences. In comparison, figure 3D also gives an
image of the boundary of affected and unaffected skin. To better
illustrate variations in elastin and collagen in the dermis during
the developing process of anetoderma, the images of small areas in
the upper and deep dermis were extracted from figure 3, shown in figures 4 and 5,
respectively.
In the unaffected normal skin, there were unremarkable changes
in the epidermis and dermis compared with control skin from a
person aged 32 years who had had no anetoderma lesion before
we obtained his skin specimen (the image is not shown). The
basement membrane zone clearly separated the epidermis from the
dermis and there was no inflammatory cell infiltration. The
oxytalan fibers and elaunin fibers in the papillary and upper
dermis were perpendicular to the basement membrane of
dermo-epidermal junction. And the arranging direction of mature
elastic fibers in the mid and deep dermis was mostly parallel to
the epidermis. They formed a distinct elastic network to give the
skin elasticity and toughness. The network collagen in the
papillary and upper dermis and thick collagen bundles in the mid
and deep dermis had no abnormalities.
The erythematous skin tissue revealed an apparent variation. The
cells of the epidermis showed remarkable activity and infiltrated
into the papillary dermis. The number of elastic fibers in the
dermis was markedly reduced, even in areas of mid and deep dermis,
they were almost completely absent. There were very few elastic
fibers in the papillary and upper dermis and they presented
irregular granules, there were no oxytalan fibers, elaunin fibers
and mature elastic fibers, as compared with unaffected skin.
Network collagen in the papillary and upper dermis was
significantly reduced and was replaced by thick collagen bundles.
Compared with the deep dermis in unaffected skin, the collagen
bundles in the mid and deep dermis had become thicker.
In affected skin, the quantity of elastic fibers was increased,
as compared with erythematous skin. However, the structure of the
elastic fibers was still irregular and abnormal as compared with
unaffected skin, showing a lot of granular morphology both in the
upper dermis and deep dermis. Collagen orientation was completely
different from that found in erythematous skin tissue, being
random.
Skin tissue at the boundary of affected and unaffected skin
presented an interesting structure. From figures 3A to C, the
dermo-epidermal interface had no apparent fluctuation. But in figure 3D, the
dermo-epidermal junction was very wavy. And the diameter and
quantity of elastic fibers had obviously increased in the upper and
deep dermis. However, the collagen fiber content was reduced when
compared with unaffected normal skin (figures 3A and D).
Discussion
Variations in elastic fibers, collagen and inflammatory cell
infiltration are the main features of anetoderma. From figure 3 to figure 5, it was found that
normal elastic fibers in unaffected skin were almost completely
absent in erythematous skin tissue, then replaced by a lot of
elastic fibers with granular morphology in affected skin, which was
consistent with the histopathological results (figure 2). These results
suggested that a deficiency of elastin leads to the development of
the cutaneous lesions of anetoderma and that the variation of
elastin in the inflammatory phase (erythema) was the main
characteristic for anetoderma. Previous reports only focused on
variations in elastic fibers during the process of cutaneous
lesions of anetoderma [2, 3]. Even if only a few reports considered
the changes in collagen, their results showed that collagen in the
dermis had no abnormalities [4]. Our study suggests that the
obvious change of collagen fibers, which appears in erythematous
tissue, should also be noted as another important characteristic of
Jadassohn-Pellizzari anetoderma, and which has been ignored by
previous investigations [2, 3].
The dermo-epidermal junction changes may be further features of
Jadassohn-Pellizzari anetoderma. We found weaker fluctuation and
wavier changes. This trend was very similar to that during skin
aging, collagen fibers in the dermis were gradually replaced by
elastic fibers with increasing age, giving the skin the clinical
features of dryness and roughness, irregular pigmentation and deep
wrinkling [10].
Compared with histological methods and electron microscopy,
multiphoton microscopy is based on TPEF and SHG of the intrinsic
fluorophores of tissue. Without fixation and staining procedures,
structural and biochemical information of tissue can be obtained.
As the above data present, keratin in the upper layer of the
epidermis can produce a TPEF signal. And cellular NAD(P)H and FAD
in the epidermis and elastin in the dermis can also be effectively
imaged from their TPEF signals. Moreover, collagen fibers in the
skin dermis can generate strong second-harmonic signals because of
their non-centrosymmetric structure. So, using multiphoton
microscopy, one can simultaneously obtain the morphological
variations, concentration and distribution of keratin, cells,
elastic fibers and collagen in skin tissue to understand the
physiological processes of tissues or distinguish between abnormal
and normal tissues. This means that multiphoton microscopy has a
potential to carry out the observation of dynamic dermatological
processes for living specimens. The most important finding was that
the results of multiphoton microscopy were extraordinarily
consistent with those of histological methods. This further
indicated that once this technology becomes sufficiently compact
and widespread, it will serve as a useful adjunct to conventional
histology in analyzing the pathological variations of skin
diseases.
On the other hand, previous reports have indicated that an
increase in cellular NAD(P)H fluorescence and a decrease in
cellular FAD fluorescence show higher metabolic rates of cells
[12]. The structural alterations of collagen and elastin
corresponded to changes in SHG and elastin TPEF signal intensity
[8]. These variations were closely related to the developing
processes of tissue pathology. In this work, we do not show the
alterations in TPEF and SHG signal intensity of intrinsic
fluorophores in tissue. But we found apparent variations in the
morphology and distribution of collagen and elastic fibers and cell
contents, which must result in changes to their spectral signal
intensities. So, multiphoton microscopy can be combined with
spectral measurement technology, based on tissue TPEF and SHG, to
comprehensively reflect the physiological and pathological states
of tissues [11, 13].
In conclusion, we have demonstrated that multiphoton microscopy
can show the development of anetoderma lesions based on the
variations of elastic fibers, collagen, and inflammatory cell
infiltration at nearly histological resolution. And this method has
potential for the observation of dermatopathological dynamic
processes for living specimens. Some skin diseases are closely
related to variations of collagen and elastic fibers in skin dermis
and cells in skin epidermis, such as skin photoaging, chronological
aging, skin wound healing, scar, morphea, systemic scleroderma,
elastolysis, skin cancer, melanoma, basal cell carcinoma, etc.
Variations of collagen and elastic fibers are also involved in
other genetic diseases of connective tissue, such as Ehlers-Danlos
syndrome, osteogenesis imperfecta (OI) and Marfan syndrome. Our
results in this study give an insight to non-invasive study of the
above diseases. With the development of multiphoton tomography
DermaInspect [14], this technique will have wider applications in
in vivo skin biology research and clinical diagnosis and
assessments.
Acknowledgements
We would like to thank the patient who participated in this study.
The project was supported by Program for New Century Excellent
Talents in Fujian Province University (NCETFJ-0706), the Grant from
Education Hall of Fujian Province (NO. JA06013), the Grant from
Fujian Union Hospital (No. XH 200502), the Science and Technology
Planning Key Program of Fujian Province (2008Y0037), Program for
New Century Excellent Talents in University (NCET-07-0191), Natural
Science Funds for Distinguished Young Scholar in Fujian Province
(2009J06031).
Conflict of interest: none.
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