comparative study on the effect of retinoic acid, glycolic acid and salicylic acid
TRANSCRIPT
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Egypt. J. Histol. Vol. 32, No. 1, June, 2009: 17 - 32
Comparative Study on the Effect of Retinoic Acid, Glycolic Acid andSalicylic Acid on the Thin Skin of Adult Female Guinea Pig. Light and
Electron Microscopic StudyAbeer A. Abd El Samad and Nagwa Kostandy Kalleny
Histology Department, Faculty of Medicine, Ain Shams University
ABSTRACT
Introduction:Many products had been used as chemical peeling agents to renew thin skin. Retinoic, Glycolic and
Salicylic acids were used in many dermatological diseases with hyperkeratinization and/or hyperpigmentation.
Aim of the Work: To evaluate and compare the effects of these acids on the thin skin of adult female guinea pig.
Materials and Methods:Twenty animals were used and were divided into four equal groups. Group I served as the
control group, whereas the other treated groups were topically applicated daily on shaved area of back skin with
Retinoic (0.05%), Glycolic (12%) and Salicylic (10%) acids in Groups II, III and IV, respectively. Thin skin specimens
were processed for light and transmission electron microscopic studies. Morphometric and statistical studies weredone.
Results:Groups II, III and IV showed high significant increase in mean thickness of epidermal nucleated keratinocytes as
compared to Control group. Group II showed proliferation hyperkeratosis and acanthosis and shrinkage of the sebaceous
glands with decreased sebum production. Group III showed skin peeling by removing superficial layers of epidermis,
cytoplasmic and nuclear degeneration with disruption of intercellular junctions and degeneration in melanocytes with
marked decrease of melanin. Both Retinoic and Glycolic acids apparently increased the production of collagen and
elastic fibers as compared to control. Group IV showed skin peeling mainly by direct action on intercellular cement
substance and partially by inducing proliferation hyperkertosis.
Conclusion:Both Retinoic and Glycolic acids showed complementary actions in treatment of hyperpigmentation and
as chemical peeling agents. Salicylic acid also is a peeling agent, but its effects could be covered by either Retinoic or
Glycolic acids.
Recommendation:It is recommended to use combination of both Retinoic and Glycolic acids to give better effects on
various skin disorders with hyperkeratinization and hyperpigmentation.
Original Article
Key Words: Retinoic acid, glycolic acid, salicylic acid,skin, guinea Pig.
Corresponding Author:Abeer A. Abd El SamadTel.:0105223262 E-mail:[email protected]
(ISSN: 1110 - 0559)
3 (1128-2009)
INTRODUCTION
Hyperkeratinization is a fundamental event in a
majority of skin disorders. Hyperkeratinization is usually
the result of decreased desquamation due to increased
corneocyte cohesion. Agents that control or modifykeratinization can be useful in treatment of many skin
disorders1.
New methods to protect skin from sun exposure
are necessary if we need to overcome skin cancer and
photoaging. Sunscreens are useful, but their protection is
not ideal because of inadequate use, incomplete spectral
protection and toxicity2. Superficial chemical peeling
has become increasingly a popular method for facial
renewal3.
Some authors4 stated that photoaging
pathophysiology is characterized clinically by wrinkles,
mottled pigmentation, rough skin and loss of skin tone
with histologic changes in the dermal connective tissue.Other authors5 stated that the use of topical retinoids
were capable of repairing photoaged skin and treating
intrinsically aged skin. Moreover, some investigators6
noticed that topical retinoids were considered the first-
line therapy in the treatment of acne vulgaris. Isotretinoin
(retinoids) appeared to be the most potent agent that
affects all the pathogenic features of acne, which resulted
from the interplay of 4 factors: Increased production of
sebum by the sebaceous gland, altered keratinization of
follicular keratinocytes, activity of Propionibacterium
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acnes (P. acnes) and inflammation by P. acnes7. These
pathological effects of acne are not life threatening, but
it had significant physical and psychosocial morbidity8.
Acne is the most prevalent skin condition encountered
by dermatologists, affecting nearly 85% of the people
between the ages of 12 and 24 years9. In addition, some
authors10noted that retinoic acid and glycolic acid were
frequently used in the treatment of acne and skin aging,
as well as improving skin healing after dermabrasion andin photoaged skin.
Alpha hydroxy acids (AHA), a group of naturally-
occurring compounds, have the potential to improve
a variety of skin disorders11. Some authors12stated that
AHAs are derived from food sources; although many of
these compounds are available, glycolic acid has been the
most widely used. Hydroxy acids had been extensively
used in cosmetic and dermatologic formulations because
of their satisfactory results in terms of maintaining a
young and healthy skin and in recovering aged skin13.
Moreover, some authors14 stated that Glycolicacid, an alpha-hydroxy acid derived from fruit and
milk sugars, had been commonly used as a cosmetic
ingredient since it was known to have photo-protective,
anti-inflammatory effects and anti-oxidant effect in
UV-irradiated skin. Similarly, other authors15noted that
Glycolic acid was widely used as an agent for chemical
peeling and contributed to the recovery of photodamaged
skin through various actions.
Some scientists16reported that chemical peeling with
salicylic acid was an effective method for the renewal of
photo-damaged skin.
The aim of the present study was to evaluate and
compare the effect of Retinoic acid, Glycolic acid and
Salicylic acid on the thin skin of adult female guinea
pig.
MATERIALS AND METHODS
The current study was performed in the Medical
Research Center, Faculty of Medicine, Ain Shams
University. Twenty adult female guinea pigs were used,
with an average weight of 300 grams each. Animals
were fed on standard laboratory guinea pig diet with freeaccess to water. All animals were prepared by shaving the
hair over their back with a surface area measuring 2.5 x
2.5 cm. They were divided into the following four groups
(five animals each):
Group I (Control Group):Consisted of 5 untreated
guinea pigs.
Group II (Retinoic acid Group): Retinoic acid
cream (0.05%) was topically applied daily on the shaved
area, just to cover it, for three weeks. It was manufactured
by Cilag A.G. as Retin-A.
Group III (Glycolic acid Group): Glycolic acid
cream (12%) was topically applied daily on the shaved
area, just to cover it, for three weeks. It was manufactured
by ISIS Pharma as Glyco-A.
Group IV (Salicylic acid Group): Salicylic acid
ointment (10%) was topically applied daily on the
shaved area, just to cover it, for three weeks. It was
manufactured by a pharmacy. The Salicylic acid wasprepared in Vaseline base (vehicle), in which Salicylic
powder was added to equivalent amount of paraffin oil
and mixed well to form homogenous paste, then vaseline
was added gradually to get homogenous ointment. This
preparation is the lipophylic preparation, to be absorbed
through skin and it is the active form in contrast to the
aqueous formula.
All animals were sacrificed by decapitation under
anesthesia by Thiopental Sodium. The shaved areas
of skin of all animals were dissected out, then cut by
sharp surgical blade into slices and were prepared then
subjected to light and transmission electron microscopicstudies.
For light microscopic study (LM):1. Formalin (10%)
fixed skin slices were processed to form paraffin
blocks. Serial sections 5m in thickness were
prepared and subjected to Haematoxylin and Eosin
stain (H&E) and Orcein stain17.
For transmission electron microscopic study2.
(TEM): Phosphate buffered gluteraldehyde fixed
small pieces of the thin skin were processed to form
capsules. Semi-thin sections were cut at 1m in
thickness using glass knife, stained by 1% toluidine
blue in 1% borax and examined by light microscope.
Ultra-thin sections (50-60nm in thickness) were cut
using ultra-microtome. Then sections were mounted
on copper grids and stained with saturated solution
of uranyl acetate18followed by lead citrate19. Ultra-
thin sections were examined and photographed by
JEM-1200 EXII transmission electron microscope
in Faculty of Science, Ain Shams University.
Morphometric and Statistical studies:
The thicknesses of nucleated epidermal keratinocytes(m) in 5 fields from H&E sections from each animal of
all groups were measured.
The measurements were done by using the image
analyzer (Leica Q 500 MC program) in Histology
Department, Faculty of Medicine, Ain Shams University.
Data were entered on an IBM compatible PC and statistical
analysis was done using the Statistical Package of Social
Sciences (SPSS version 11). Descriptive statistics were
done in the form of mean and standard deviation.
Mean of each group was compared with the
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Abeer A. Abd El Samad and Nagwa Kostandy Kalleny
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others using the One Way Analysis of Variance (One
Way ANOVA) with Post hoc analysis. As regards
the probability, the least significant level used was at
P
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this group showed apparent increase in thickness of the
nucleated cells particularly in the cells of the stratum
spinosum and stratum granulosum as compared to control.
On the other hand, the stratum corneum was apparently
thinned out and peeled as noticed in H&E and toluidine
blue stained sections. The keratinocytes appeared
vacuolated and degenerated. Some keratinocytes showed
variable nuclear changes ranging from pyknosis to
karyorrhexis. The melanin was markedly decreased ascompared to control (Figs. 18,19&20).
Ultra-thin sections revealed that the melanocytes
appeared degenerated containing few melanin pigments.
Some cells of the stratum basale showed various stages
of degeneration, in which some showed vacuolization,
others appeared shrunken with deformed nuclei, whereas
others showed normal control appearance with few
melanin granules (Fig. 21). The cytoplasm of the stratum
spinosum showed marked decrease in tonofilaments
content as compared to control, with disruption of
desmosomes and intercellular disjunctions also showing
widening in the intercellular spaces. The nuclei showedchromatin pattern different than that of control with
unapparent nucleoli (Fig. 22).
The dermis showed active fibroblasts with marked
increase in the content of organized bundles of dense
collagen fibers as compared to control (Fig. 23).
Moreover, increased content of elastic fibers as compared
to control was noticed in orcein stained sections (Fig. 24).
No remarkable changes in the sebaceous glands of these
animals as compared to the control.
Group IV (Salicylic acid Group):
The epidermis of thin skin sections of animals of this
group showed apparent increased thickness of viable
keratinocytes showing areas of acanthosis, however
the thickness of the stratum corneum was decreased as
compared to the control. The cells of the stratum spinosum
showed vesicular nuclei. The melanin was dispersed in
the keratinocytes particularly those of the stratum basale
followed by the stratum spinosum (Figs. 25&26).
Ultra-thin sections revealed nearly normal and
viable cells of the stratum spinosum. They showed
apparent normal nuclear confi
guration with few melaningranules and well-defined bundles of tonofilaments in
their cytoplasm with preserved desmosomes between
cells. However, there was apparent separation between
cells due to widening in the intercellular space
(Figs. 27&28). The stratum granulosum showed
kerato-hyalin granules, whereas the stratum corneum
appeared less compact as compared to that of the control
(Fig. 29).
The content of dermal collagen fibers showed nearly
no remarkable difference as compared to control. In
addition, nearly no remarkable difference in content of
0
20
40
60
80
100
120
140
160
180
Group I
Control
Group II
Retinoic
acid
Group III
Glycolic
acid
Group IV
Salicylic
acid
Histogram 1: Showing the mean thickness of nucleated epidermal
keratinocytes in different studied groups.
elastic fibers in orcein stained sections as compared to
control (Fig. 30). Moreover, no remarkable changes in
the sebaceous glands of these animals as compared to the
control.
Morphometric and Statistical Results:
Table (1) and Histogram (1) showed the means
and standard deviations of thickness of epidermalkeratinocytes in different studied groups.
Table (2) showed high significant increase in mean
thickness of epidermal keratinocytes (p
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Abeer A. Abd El Samad and Nagwa Kostandy Kalleny
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Fig. 2: A photomicrograph of a section of thin skin of female guinea pig,
showing the stratum basale of keratinocytes (B), the stratum spinosum
(S) and the stratum granulosum (G). The stratum corneum consists of
many layers of flattened non-nucleated acidophilic keratinized cells (K).
Notice the brownish melanin pigment ().Group I H&E X 640.
Fig. 3: A photomicrograph of a section of thin skin of female guinea pig,
showing epidermis and dermis. The dermis shows sebaceous glands ()
and hair follicles ().
Group I Toluidine blue X 640.
Table 2: Showing the significance of difference in mean
thickness of nucleated epidermal keratinocytes in different
studied groups using one way ANOVA with Post hoc analysis:
(I) code (J) codeMean Difference
(I-J)Significance
Group I
(Control)
Group II -130.02200 p
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Fig. 4: An electron-micrograph of a section of thin skin of female guinea
pig, showing melanocyte in-between cells of the stratum basale. It
shows cell process containing melanin granules (). Cells of the stratum
spinosum show euchromatic nuclei and tonofilaments in their cytoplasm
(). The dermis shows collagen fibers (C).Group I TEM X 4000.
Fig. 5: An electron-micrograph of a section of thin skin of female
guinea pig, showing keratinocytes of the stratum basale with
euchromatic nuclei. Their cytoplasm contains intermediate filaments
() and melanin granules ().
Group I TEM X 3000.
Fig. 6: An electron-micrograph of a section of thin skin of female guinea
pig, showing cell of stratum spinosum with bundles of tonofilaments.
The cytoplasm of the stratum granulosum appears filled with kerato-
hyalin granules (G). Notice the desmosomes in-between cells of stratum
spinosum and stratum granulosum ().The cells of the stratum corneun
show keratin filaments embedded in an amorphous matrix.
Group I TEM X 3000.
Fig. 7: An electron-micrograph of a section of the reticular layer of
dermis thin skin of female guinea pig, showing collagen fibers (C) and
dermal fibroblasts ().
Group I TEM X 6000.
Fig. 8: A photomicrograph of a section of thin skin of female guinea pig,
showing network of elastic fibers in the reticular layer (E) and few thinner
fibers in the papillary layer () of the dermis.
Group I Orcein X 250.
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Fig. 12: A photomicrograph of a section of thin skin of female guinea
pig, showing vesicular nuclei of stratum spinosum cells that showed
mitotic figures with prominent anaphase stage (). Notice the reduced
melanin pigmentation as compared to control.
Group II H&E X 640.
Fig. 13: An electron-micrograph of a section of thin skin of female
guinea pig, showing active euchromatic nuclei with extended chromatin
in stratum spinosum cells. Their cytoplasm contained tonofilaments ()
terminating in desmosomes.
Group II TEM X 3000.
Fig. 9: An electron-micrograph of a section of thin skin of female guinea
pig, showing acinus of sebaceous gland. The cells of the basal layer of
the acinus appear flattened () resting on a basal lamina. The adjacent
rounded cells contain abundant fat droplets in their cytoplasm.
Group I TEM X 2000.
Fig. 10: An electron-micrograph of a section of thin skin of female guinea
pig, showing cells at the central part of a sebaceous gland acinus with
shrunken and condensed nuclei and their cytoplasm appear filled with
fat droplets.
Group I TEM X 3000.
Fig. 11: A photomicrograph of a section of thin skin of female guinea pig,
showing prominent rete pegs with apparent increased thickness of viable
keratinocytes. Whereas, the stratum corneum was markedly thinned out
as compared to control.
Group II H&E X 250.
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Fig. 14: An electron-micrograph of a section of thin skin of female
guinea pig, showing increased number of cells of the stratum granulosum
rich with large kerato-hyalin granules. Whereas the stratum corneum was
apparently thinner and more compact as compared to control.
Group II TEM X 3000.
Fig. 15: An electron-micrograph of a section of thin skin of female
guinea pig, showing dermal collagen fibers deposited from fibroblast.
Group II TEM X 6000.
Fig. 16: A photomicrograph of a section of thin skin of female guinea
pig, showing massive deposition of elastic fibers () in the dermis as
compared to control.
Group II Orcein X 250.
Fig. 17: An electron-micrograph of a section of thin skin of female guinea
pig, showing undifferentiated cells of the peripheral part of a sebaceous
gland. However the cells at the central part of acini appeared condensed
and shrunken containing altered fat droplets as compared to control.
Group II TEM X 2000.
Fig. 18:A photomicrograph of a section of thin skin of female guinea
pig, showing apparent increase in thickness of epidermal keratinocytes
as compared to control. Notice the detached stratum corneum.
Group III H&E X 250.
Fig. 19: A photomicrograph of a section of thin skin of female guinea pig,
showing vacuolated and degenerated keratinocytes. Notice pyknosis ()
and karyorrhexis () in some keratinocytes. The melanin was markedly
decreased as compared to control.
Group III H&E X 640.
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Fig. 22: An electron-micrograph of a section of thin skin of female guinea
pig, showing widening in the intercellular spaces of stratum spinosum cells
with disruption of desmosomes and intercellular disjunctions (). Notice
the marked decrease in tonofilaments content as compared to control.
The nuclei showed chromatin pattern different than that of control withunapparent nucleoli.
Group III TEM X 3000.
Fig. 23: An electron-micrograph of a section of thin skin of female guinea
pig, showing active fibroblasts () with marked increase in the content of
organized bundles of dense collagen fibers as compared to control.
Group III TEM X 6000.
Fig. 20: A photomicrograph of a section of thin skin of female guinea
pig, showing apparent increase in thickness of the cells of the stratum
spinosum (S) and stratum granulosum (G) as compared to control. Notice
the thinned out and peeled stratum corneum.
Group III Toluidine blue X 250.
Fig. 21: An electron-micrograph of a section of thin skin of female
guinea pig, showing various stages of degeneration of stratum basal
cells, in which vacuolization (V), shrunken cells with deformed nuclei
(S) appear. Whereas other cells show normal control appearance with few
melanin granules (C). Notice the degenerated melanocyte containing few
melanin pigments (M).
Group III TEM X 4000.
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Fig. 24: A photomicrograph of a section of thin skin of female guinea
pig, showing increased content of elastic fibers () in the dermis as
compared to control.
Group III Orcein X 250.
Fig. 25:A photomicrograph of a section of thin skin of female guinea
pig, showing apparent increase in thickness of keratinocytes, however
the stratum corneum appears thinner than that of the control.
Group IV H&E X 250.
Fig. 26: A photomicrograph of a section of thin skin of female guinea
pig, showing dispersed melanin in the keratinocytes particularly those of
the stratum basale followed by the stratum spinosum.
Group IV H&E X 640.
Fig. 27: An electron-micrograph of a section of thin skin of female
guinea pig, showing apparent separation between stratum spinosum cells
due to widening in the intercellular space. They show apparent normal
nuclear configuration and few melanin granules.
Group IV TEM X 4000.
Fig. 28: An electron-micrograph of a section of thin skin of female guinea
pig, showing well-defined bundles of tonofilaments in their cytoplasm
with preserved desmosomes between cells of stratum spinosum.
Group IV TEM X 4000.
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Abeer A. Abd El Samad and Nagwa Kostandy Kalleny
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DISCUSSION
The epidermis of thin skin sections of animals of
Group II showed that topical application of Retinoic acid
increased the thickness of viable keratinocytes forming
prominent rete pegs. This was proved by morphometricand statistical study where the mean thickness of
nucleated epidermal keratinocytes was high significantly
increased (p
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Topical application of Retinoic acid in animals of
Group II in the present study induced undifferentiation
in acinar cells and shrinkage in the sebaceous glands
with altered and reduced content of sebum as compared
to control, explained as hypo-activity in the sebaceous
gland. In addition, the nuclei of the sebocytes appeared
shrunken and condensed. This was in agreement with
some scientists27 who stated that isotretinoin (retinoic
acid) is the most effective drug in reducing sebaceousgland size (up to 90%) by decreasing proliferation of basal
sebocytes, suppressing sebum production and inhibiting
sebocyte differentiation in vivo. The molecular basis for
its anti-sebotrophic activity had not been fully elucidated.
Isotretinoin also exhibited anti-inflammatory activities.
Systemic isotretinoin is considered to be the regimen of
choice in severe seborrhoea, since it reduced sebocyte
lipid synthesis by 75%. Moreover, other scientists28
clarified that isoterinoin induced apoptosis in sebocytes in
addition to inhibiting cell-cycle progression and reduced
sebaceous lipid production. Coinciding, some scientists29
noticed that isotretinoin is the only therapeutic agent that
drastically reduced the size and secretion of sebaceousglands and it is the most potent agent available for
treatment of acne by inducing apoptosis in the cells of
the sebaceous glands. The ability of isotretinoin to induce
apoptosis is specific and selective to sebocytes, but not
keratinocytes; nor dermal fibroblasts30. This coincided
with the results of Group II of the present study, in which
the keratinocytes exhibited euchromatic active nuclei
and the dermal fibroblasts showed active synthesis of
collagen.
Topical application of Glycolic acid in group III in the
present study, showed a high significant decrease in mean
thickness of nucleated epidermal keratinocytes (p
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group III in this study did not show remarkable changes
in the sebaceous glands as compared to the control.
Coinciding, some investigators36 noticed that glycolic
acid did not affect sebum secretion of the facial skins
of patients with facial acne however glycolic acid was a
popular superficial chemical peel agent for the treatment
of facial acne and increased sebum secretion which is
one of the major aetiological factors of acne.
Some scientists37 clarified that alpha-hydroxy acid
(AHA) peels had been recognized as important adjunctive
therapy in a variety of conditions including photodamage,
melasma, hyper-pigmentation disorders and acne. It had
been demonstrated that AHAs improve these disorders by
thinning the stratum corneum, promoting epidermolysis,
dispersing basal layer melanin and increasing collagen
synthesis within the dermis. These effects coincided
with the peeling and thinning of the stratum corneum,
in addition to the marked decrease of melanin with
degeneration of the melanocytes and increase in collagen
fibers in group III of the present study.
The epidermis of thin skin sections of animals that
received topical application of Salicylic acid (Group IV)
in the present study showed apparent decrease thickness
of the stratum corneum as compared to the control. In
addition, high significant decrease in mean thickness of
nucleated epidermal keratinocytes (p
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acid did not show dermal fibroblastic stimulation.
It is recommended to apply topical combination
of both Retinoic and Glycolic acids as they showed
complementary actions as chemical peeling agents and
in the treatment of acne, however Salicylic acid better
not to be used as a peeling agent and in treatment of acne,
mostly because its effects could be covered by either
Retinoic or Glycolic acids and also because of its knownsystemic side effects.
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