ARTICLE
Auteur(s) :, W Meyer1,*,
NH Zschemisch1, H Lehmann2, R
Busche3, U Kunz2
1Institute for Anatomy, University of Veterinary
Medicine Hannover Foundation, Bischofsholer Damm 15, 30173
Hannover, GermanyFax: (+49) 511 856 7683.
2Institute for Animal Ecology and Cell Biology,
University of Veterinary Medicine, Hannover, Germany
3Institute for Physiological Chemistry, University of
Veterinary Medicine, Hannover, Germany
accepté le 16 Mars 2005
There is relatively little information on sulphur and its
derivatives in the epidermis of mammals, particularly on the
concentrations and intracellular distribution of such substances in
the stratum corneum. Preliminary studies on the distribution of
thiols and disulphides in the epidermis of the wild boar and
various breeds of the domestic pig have nevertheless shown that
keratinisation develops somewhat differently in the wild ancestor
species than in its domesticated descendants. Normally, thiol
groups are present in distinct amounts only in the stratum
granulosum [1, 2], as has also been shown for other mammals [3-7].
However, differences became obvious for disulphide groups in the
wild boar, in which keratinisation seems to proceed rather slowly
from the stratum basale to the stratum corneum, with a high level
of such groups in the keratinocytes of all epidermal layers, and
without a sharp increase in their contents from the stratum
granulosum to the stratum corneum conjunctum. In contrast, a clear
increase in -S-S- group content from the vital epidermis to the
inner layers of the stratum corneum was visible in several domestic
porcine breeds [1], and is known, likewise, for this skin layer in
humans or other mammals [3, 5, 7]. The feature is mainly due to the
fact that a distinct cornified cell envelope (CCE) does not develop
before the final stage of keratinocyte maturation, with -S-S-
groups found abundantly not only in the filaments and keratohyalin
granules but especially in the CCE [2, 8-12].The present study
relies on a broad spectrum of methods to verify both general as
well as intracellular variations in the distributions of sulphur,
thiols, and disulphides in the porcine stratum corneum, after
completion of the keratinisation process. It was especially of
importance to determine details of sulphur metabolism as high
sulphur levels, in particular, are indirectly involved in the
production of highly crosslinked proteins during cell envelope
keratinisation. The most important of these proteins is involucrin,
as supported by loricrin, filaggrin, and small proline-rich
proteins [11].Our findings may be of interest for the application
of the integument, or the skin layers, of different porcine breeds
as a model for the human skin, taking into consideration different
body regions, and the cost- and animal-saving use of porcine ear
skin [1, 13-15]. Our results may, additionally, help to bring more
insight into the efficacy of intrinsic epidermal protection
mechanisms of thiols against oxidative stress or UV radiation
[16-19].
Materials and methods
Demonstration of sulphur contents
Stratum corneum samples were obtained from freshly-killed animals
by controlled careful scraping of the SC from the body
(dorsolateral region, abdominal region) and from the outer side of
the ears (only this side is relevant for dermatological purposes,
see Meyer et al. [13, 14]). The study included the following
animals: 10 European wild boars (WB, Sus scrofa, 8 females, 2
males, 50-80 kg; total 20 ears), 10 large white domestic pigs (DP,
German landrace, all females, 40-60 kg, total 20 ears), and 10
Göttingen miniature pigs (GMP, all females, 40-50 kg) were used.
Each sample was inspected very carefully and cleared as far as
possible of any contamination (dirt, sand, etc.), bristles and wool
hairs. Sulphur contents were determined using a spectrometer
(ICP-OES Optima 3000) according to DIN EN ISO 11885 (E22), whereby
3-4 measurements (SD less than 1%) were made of pooled and
freeze-dried material that was subsequently decomposed in a
microwave oven.
Histochemical and cytochemical preparations
For the light microscopical demonstration of SH- and -S-S- groups,
skin samples were taken from the back, flank, shoulder, buttocks,
abdomen, and auricle (central region of outer side) of the WB (3
females, 50-70 kg), the DP (6 females, 40-60 kg), and the GMP (6
females, 40-50 kg), and fixed at room temperature for 12 h in
4% phosphate-buffered formalin (pH 6.8) as well as for 48 h in
Bouin’s solution. The samples were then gradually dehydrated, and
embedded via Histosol (Shandon) in Paraplast (Shandon).
Additionally, skin samples were freshly stored in liquid nitrogen
for measurement of stratum corneum thickness.
Structural integrity of the epidermis was controlled light
microscopically using samples that had been embedded in the
relatively shrinkage-free and water-soluble plastic resin Technovit
7100 (Kulzer) [20]. Sections of this material were routinely
stained with haematoxylin-eosin (HE). For the different staining
purposes, 6 μm paraffin sections and 5 μm plastic
sections were cut with a rotation microtome (Autocut,
Reichert-Jung). TEM preparations are described in the section on
analytical EM.
LM densitometry of SH- and -S-S- groups
Two different methods were used to treat 6 μm paraffin
sections according to:
- A) The method of Sippel [21, 22], with the single-step
procedure and chromotropic acid (Sigma) as azo coupler;
N-(-4-aminophenyl)-maleimide (APM) was purchased from
Polysciences/Paesel and tri-n-butylphosphine (TBP) from Sigma.
-S-S- groups were demonstrated after reduction with TBP and prior
blocking of native SH- goups with NEM. Control slides were prepared
by preliminary blocking of thiol groups or TBP-reduced thiol groups
with N-ethylmaleimide (NEM) (Sigma) (0.1 M in 0.06 M phospate
buffer, pH 7.4, 24 h or overnight at room temperature) [22].
The quantitative evaluation was performed according to Broekaert et
al. [5] using a Zeiss microscope photometer (MPM 01 K) equipped
with a planachromatic objective (× 100). Measurements were made at
a wavelength of 520 nm with a circular diaphragm (0.1 and
0.25 mm diameter). Extinction measurements (60 per epidermal
layer, 80 steps) were recorded and the mean extinction and standard
deviation evaluated.
- B) The cyanine dye reagent for SH- groups,
IC3-PE-maleimide (Polysciences) was used (conc. 0.1-1 mg/mL), which
produces a highly fluorescent complex. Although the fluorescence of
IC3-PE-maleimide is suppressed by the maleimide group in the
structure, it is recovered by labelling with thiol compounds [23,
24]. -S-S- groups were demonstrated after reduction with TBP as
were control slides (see Sippel method above). All slides were
embedded in Mowiol (Sigma-Aldrich) for the visualisation and
densitometrical evaluation (image size 10 × 10 μm) of the
reaction on a Leica TCS SP2 confocal microscope (excitation 488 nm,
emission 580-590 nm).
TEM determination of sulphur (analytical EM)
Small skin samples were taken from the dorsolateral body region of
the WB (2 females, 50-70 kg), the DP (3 females, 40-60 kg) and the
GMP (3 females, 40-50 kg). The material was fixed in Karnovsky’s
fluid [25], washed several times in a sucrose-phosphate buffer
solution, and postfixed in buffered 1% osmium tetroxide [26]. After
dehydration in graded ethanol, the skin samples were embedded in
Durcupan (Serva) [27], and cut with a diamond knife on a Reichert
ultramicrotome (OM U2). Thin sections for conventional TEM
(< 100 nm) were stained with uranyl acetate [28] and lead
citrate [29] and viewed in a Zeiss EM10.
Sulphur distribution was studied in unstained thin sections
(< 50 nm) with an energy-filtering transmission electron
microscope (EFTEM, Zeiss CEM 902). The basic aspect of such a
procedure is that monoenergetic electrons of the primary electron
beam passing through a sample in a TEM can be scattered
non-elastically by collision with atomic shell electrons of the
sample, whereby they lose a part of their energy. The value of
energy loss (ΔE) depends on the atomic number of the element, and
ΔE is thus specific for each element. Therefore, the poly-energetic
electrons that have passed through a sample contain all the
information on its chemical composition. The EFTEM can separate the
non-elastically scattered electrons from each other, producing an
‘electron energy loss spectrum’ (EELS). The characteristic electron
energy loss used for detecting sulphur was 165-200 eV
(SL). The sites for taking EELS had a diameter of
150-370 nm depending on magnifications (30 000-85 000). In
6-10 cells of the stratum corneum conjunctum of the representatives
of each porcine group studied, the cell envelope (marginal band)
and the central cytoplasm regions were evaluated. The net
distribution of sulphur was obtained by subtracting the background
from the peak [30]. With regard to the use of fixated material for
EELS, it has to be emphasized that in contrast to the bulk loss of
endogenous elements from animal tissues during chemical fixation,
predominantly structural elements such as sulphur or phosphorus,
are less readily, or to a lesser extent, lost during EM processing
[31].
Thickness measurements
The thickness of the stratum corneum was measured using 10 μm
frozen sections of fresh skin samples (back, flank, shoulder,
buttocks, abdomen, central region of outer side of auricle; for
animals used see Materials and methods) that had been stored in
liquid nitrogen. After preparation with a Bright 5030 freeze
microtome, the sections were carefully embedded in glycerin, one
part of the sections was also postfixed in 10% formalin in order to
prevent the desquamation of loose corneal lamellae that usually
occurs after the relatively long histological fixation, embedding,
and staining procedures. Moreover, HE-stained 5 μm plastic
sections (Technovit 7100; see Materials and methods) were used for
thickness evaluation. Tissue shrinkage was prevented by embedding
the material without any organic solvents, using only aqua dest. as
Technovit solvent. Between 50 and 100 measurements of intact
stratum corneum were recorded and the mean thickness and standard
deviation evaluated, using the computer-assisted CUE 3 system
(Olympus, Image Analysis, version 4.5, 1993).
Results
Sulphur contents
The amount of sulphur found in the stratum corneum (SC) samples of
the three pig types studied was in the range between 0.48 and
1.76 % DW in the WB, 0.46 and 0.90 in the DP, and between 2.25
and 2.66 in the GMP. From these findings and the averages shown in
( figure 1 ), it
became clear that sulphur concentrations were lowest in the ears of
all animals except for the GMP. It was also very obvious that
concentrations were extraordinarily high in all body regions of the
GMP. It must be noted that in this animal the contamination of the
samples was very low due to the very hygienic husbandry conditions
and to the fact that the bristles were very fine and that wool
hairs were very short or missing.
Thiol and disulphide histochemistry and densitometry
The application of the Sippel method, as developed by the single
step reactions, and the IC3-PE-maleimide fluorescence reaction for
the demonstration of thiols and disulphides in the stratum corneum
of the three pig types generated very distinct light microscopical
results for all body regions studied, also regarding the controls.
In most cases the thiol reactions were relatively weak, although
they were somewhat more intense in the SC of the DP and the GMP
than in the WB, and were strongest in the stratum granulosum. In
contrast, the staining for disulphides produced very strong
reaction intensities, especially in the miniature pig, in which the
vital epidermis was unstained and clearly distinguishable from the
stratum corneum conjunctum and the lamellae of the stratum corneum
disjunctum. The disulphide colouring of the stratum corneum was
very homogeneous and uniform throughout the entire layer (( figure 2 )).
The densitometric measurements generally confirmed the light
microscopical picture described above, although there were
variations in the staining intensity in the different body regions.
As in the normal light microscopical picture, the densitometrical
results demonstrated a very uniform reaction intensity for
disulphides, without any specific increase or decrease in the
staining course towards the surface within the SC cell lamellae.
The most remarkable absolute values for thiols were found in the SC
of the GMP, which were very distinct in the shoulder (( figure 3 ) above left). The
disulphide concentrations in the GMP were also very high in all
body regions, but likewise very high in some body parts of the WB
(shoulder, buttock, abdomen) (( figure 3 ) above right).
The relative concentrations of thiols and disulphides, shown as a
percentage of results from the stratum basale, indicated a
relatively homogeneous intensity of staining for thiols in the SC
of most of the body regions, except for the shoulder in the DP and
GMP, and a very strong reaction for disulphides, particularly, in
all of the body regions of the DP, except for the ear (( figure 3 ) below left
and below right). Regression analysis, including the absolute and
relative thickness values of the stratum corneum, showed that only
the relative disulphide concentrations in the GMP seemed to be
correlated with SC thickness (r = 0.7181 for absolute thickness
values; r = 0.7956 for relative thickness values; in both cases p
< 0.001; ( figure
4 ) above right). There was a negative correlation between
absolute -S-S- extinctions and relative SC thickness in the DP ((
figure 4 ) below
left; SH correlation results not shown).
Absolute SC thicknesses (table 1)( Table
1 ) were generally greatest in the GMP (min. 25 μm –
max. 120 μm), and very high in the back and shoulder of the
other animals (min. 55 μm – max. 97 μm). The lowest
thicknesses (about 20 μm) were found for the ear skin of the
DP. Although somewhat higher in the GMP (about 65% in shoulder,
flank, and back), the relative SC thickness values (as % of total
epidermis thickness, including vital epidermis and stratum
corneum), were generally more homogeneous in the overall view
(between 65 and 50%, with the highest values in the shoulder); and
in the ear (about 30% in DP and GMP and 40% in WB).
Table 1 Absolute and relative thickness of the porcine
stratum corneum (SC)
|
|
Back
|
Shoulder
|
Flank
|
Buttock
|
Abdomen
|
|
Absolute SC thickness (μm):
|
|
Wild boar (WB)
|
33.0 (± 3.8)
|
65.0 (± 6.6)
|
66.8 (± 15.4)
|
48.0 (± 4.7)
|
26.1 (± 8.1)
|
47.3 (± 7.8)
|
|
Domestic pig (DP)
|
20.8 (± 2.2)
|
68.8 (± 21.2)
|
75.3 (± 20.1)
|
47.3 (11.5)
|
37.9 (± 14.4)
|
34.8 (± 9.4)
|
|
Goettingen miniature pig (GMP)
|
38.5 (± 10.2)
|
80.0 (± 34.9)
|
76.7 (± 6.9)
|
71.3 (21.9)
|
62.5 (10.7)
|
63.5 (± 10.6)
|
|
Relative SC thickness (% total epidermis thickness):
|
|
Wild boar (WB)
|
40.2 (± 4.0)
|
60.0 (± 6.2)
|
63.8 (± 12.3)
|
54.5 (± 6.6)
|
45.2 (± 10.1)
|
52.2 (± 5.5)
|
|
Domestic pig (DP)
|
31.3 (± 2.8)
|
52.1 (± 10.4)
|
64.3 (± 5.9)
|
52.6 (± 11.5)
|
43.9 (± 10.5)
|
41.3 (17.9)
|
|
Goettingen miniature pig (GMP)
|
30.3 (± 4.0)
|
64.3 (± 13.8)
|
65.4 (± 5.4)
|
65.9 (14.7)
|
54.2 (± 4.1)
|
53.1 (± 6.8)
|
TEM demonstration of sulphur
Conventional TEM exhibited a slight variation in the ultrastructure
of the corneal cells (Str. corneum conjunctum, SC cells) when the
WB was compared to the two porcine domestic breeds studied. In the
wild animal, the SC cells were very flattened with a fine
filamentous network within an amorphous ground substance, so that
the cytoplasm appeared extraordinarily dense and homogenously dark.
These cells were surrounded by a plasmalemma and a so-called
envelope (marginal band), which together had a thickness of 40-45
nm. The envelope was as dark as or a little lighter than the
cytoplasm. In the domesticated animals, the cytoplasm of the SC
cells was distinctly less concentrated and less homogenous than in
the same cell type of the WB. In addition to areas with filament
bundles, there were more or less regularly large electron-lucent
regions of the cell. Furthermore, the plasmalemma and/or the
envelope of the SC cells were clearly delineated from the cytoplasm
as a dark electron-dense structure. This feature was very obvious
in the SC cells of the DP but less so in the GMP (for pictures see
Meyer [1] and Meyer and Neurand [39]).
The results of the EELS ( (figure 5) ) generally
corroborated the structural peculiarities of the cells of the
porcine stratum corneum conjunctum as described above, in
particular as to the differences between the wild animal and its
domesticated descendants, including differences in sulphur
distribution in the envelope of the SC cells and in the central
cytoplasm. In the WB, the sulphur concentrations were rather low
and differences between the two cell parts were inconspicuous, with
values somewhat lower or varying in the periphery. In contrast,
there were very clear differences in the SC cells of the DP between
cell periphery and cytoplasm, with particularly high relative
sulphur concentrations occurring in the cell envelope. On the other
hand, no differences in sulphur concentrations between peripheral
and central cellular regions became obvious in the GMP, and
concentrations were generally as great as in the former porcine
breed.
Discussion
Our results underline first of all the fact that relevant
interpretations of findings of dermatological investigations on
pigs should always be based on exact information on the breeds
used.
The concentration of sulphur in the porcine stratum corneum was
found to be generally comparable with that from the very rare
measurements of this element in human skin – but only for the DP
(German landrace, deriving from White Yorkshire pig lines). The
sulphur concentration in this breed, especially from the ear skin,
was about 0.50% DW and is between 0.30 and 0.50% DW in humans [32].
Distinctly higher concentrations were found in both the wild animal
as well as in the miniature breed, and these findings conform to
our cyto- and histochemical measurements. In case of the WB it must
be added that the results may be affected by the fact that the
stratum corneum of the WB is very densely structured or packed
(‘concentrated’), respectively, as it is protected from abrasion by
a distinct hair coat [1].
Histochemical analysis corroborated the findings for sulphur
contents in the stratum corneum, in that the absolute values for
both thiols and disulphides were in accordance with high and
constant amounts of the element sulphur, as is also known from
rodents [33]. The increase in sulphur per unit volume from the
inner to the outer layers of the stratum corneum, as detected by
electron probe analysis in humans, is largely due to cytoplasmic
water loss as cells migrate to the surface [34]. The relative
concentration of thiols and disulphides (percentage of staining
intensity of the stratum basale) was surprisingly homogeneous and
uniform in most of the animals and body regions studied. It thus
appears likely that all pig types share a basic keratinisation
which changes according to domestication (breed) effects during the
intraepidermal course of keratinisation towards the epidermal
surface. Support for this hypothesis may be the very high relative
disulphide values in the DP, as these indicate a rapid, effective
and intensive keratinisation development from the stratum basale to
the stratum corneum. This feature can be locally more intense due
to frequent rubbing, especially at the shoulder and buttocks in
animals of the large breed, for which husbandry conditions are
often not optimal so that the animals may exhibit hyperactivity
[35]. In the GMP, keratinisation development also seems very
intensive but possibly starts earlier to produce the
extraordinarily high sulphur (and disulphide) amounts found in the
final corneal layer. However, husbandry conditions are better for
these animals, particularly as this pig breed is used for
laboratory or scientific purposes, and behavioural disorders are
rare under such circumstances [36]. Thus, extreme keratinisation in
the GMP may be due to hyper-parakeratosis as a result of epidermal
domestication defects [1]. High absolute and low relative
disulphide concentrations in the stratum corneum of the WB could be
due to the fact that the stratum basale of this animal already
contains very high amounts of -S-S- as a normal basis for a slow
but intensive keratinisation process [1, 2]. In the wild animal,
the rough hair coat protects against SC abrasion, which is not the
case in the domesticated breeds that have lost their wool hairs and
produce only fine bristles [1]. Such a view may be supported by the
finding that the relative thiol and disulphide concentrations
measured first of all in the GMP, i.e. those calculated on the
basis of the results from the stratum basale, correlated more or
less with the thickness of the SC, indicating a relationship
between strong epidermal keratinisation and the SC produced. On the
other hand, the negative correlation between absolute disulphide
extinctions and (relative) SC thickness in the DP, may be an
indication of a decrease of -S-S- groups from the inner to the
outer SC parts, but are better explained as an increasingly more
loosely structured SC.
Furthermore, it is very interesting to note that the relative
developments in thiol and disulphide production in the ear
epidermis are the same in the three pig types studied, and that
this represents a more or less normal keratinisation course. The
porcine auricle is not subjected so much to severe mechanical
hazards, as for example is possible for other body regions. In the
WB, the outer side of the ear is protected by a relatively dense
hair coat [37], while the hair coat is sparse in the other two pig
types studied. However, ear biting may occur, especially in
densely-housed large domestic breeds (e.g. German landrace); but in
this case only the ear tips are affected [38].
In the cytological approach, sulphur was detected and localised
on the basis of an electron energy loss spectrum (EELS). This
specific methodical approach made it obvious that sulphur
distribution in the cells of the stratum corneum conjunctum of the
porcine epidermis varied according to peripheral or central
cellular regions, particularly in the domesticated animals. In that
group, the structurally distinct envelope (marginal band in TEM) of
the SC cells [1, 9, 39] contained relatively high amounts of
sulphur in comparison to the cytoplasm. This was especially true of
the DP, and indicates high keratinisation intensity in the cell
periphery. Such a cellular envelope appears to provide a rigid
‘exoskeleton’ as it were, the physical and chemical stability of
which may critically govern the integrity of the protective system
of the whole stratum corneum [2, 40, 41]. The development of the
envelope seems very important in the domesticated pigs in view of
the fact that the insulating and protecting (wool) hair coat has
been lost and/or refined with regard to the primary hairs
(bristles) [1], so that the direct and constant mechanical load of
the outer epidermal layer has increased enormously. Such
differences in intracellular sulphur distribution of corneal cells
were less obvious in the GMP, although sulphur concentrations were
also high in the central cell part. However, keratinisation
intensity of the epidermis appears enhanced in this breed, possibly
in association with hyper- and/or parakeratosis. This implies that
the stratum corneum is more or less loosely structured and very
easily lost [1]. The few publications on the spatial distribution
of sulphur in cornifying epithelia as on the basis of analytical
electron microscopy, generally confirm our findings in the pig,
where high sulphur levels in the cell envelope of corneal cells,
are also found, for example, in the very thin murine epidermis [42,
43], and seem to be correlated with the presence of loricrin and
fillagrin, which are rich in the amino acids glycine and glutamic
and aspartic acid [44-47].
The present study demonstrates that breed-related domestication
effects on keratinisation in the stratum corneum of the porcine
epidermis must be taken into consideration when using the epidermis
of different porcine breeds as a model system for human epidermis,
e.g. for the testing of transdermal systems [1, 13, 14, 48-50].
Only the porcine ear skin model appears to have a definite
scientific value and is thus to be recommended, and only the large
pig breed (German landrace, White Yorkshire lines), and only for
the central region of the outer side of the auricle.
Acknowledgements
The authors gratefully acknowledge the excellent technical
assistance of I. Blume, M. Gähle, and G. Wirth. We also very much
appreciate the support of F. Cipra (LUFA Nord-West Hameln) and the
help given with the animal material by G. Hempfler (Wild Animal
Collection Centre, Schülern), Dr. U. Viek (Office for Meat Hygiene,
Gleidingen), and A. Andreae (Medimplant GmbH, Hannover).
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