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Egypt. J. Histol. Vol. 32, No.

1, June, 2009: 17 - 32

(ISSN: 1110 - 0559)

Original Article

Comparative Study on the Effect of Retinoic Acid, Glycolic Acid and


Salicylic Acid on the Thin Skin of Adult Female Guinea Pig. Light and
Electron Microscopic Study
Abeer 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 were
done.
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.

Key Words: Retinoic acid, glycolic acid, salicylic acid,


skin, guinea Pig.

Corresponding Author: Abeer A. Abd El Samad


Tel.: 0105223262

E-mail: abirmohsen@yahoo.com

INTRODUCTION
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 firstline 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

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 modify
keratinization 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.

3 (1128-2009)
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Comparative Study on the Effect of Retinoic Acid, Glycolic Acid and Salicylic Acid on the Thin Skin

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
authors10 noted 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 and
in photoaged skin.

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 was
prepared 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.

Alpha hydroxy acids (AHA), a group of naturallyoccurring compounds, have the potential to improve
a variety of skin disorders11. Some authors12 stated 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.

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 microscopic
studies.

Moreover, some authors14 stated that Glycolic


acid, 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 authors15 noted that
Glycolic acid was widely used as an agent for chemical
peeling and contributed to the recovery of photodamaged
skin through various actions.
Some scientists16 reported 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 free
access 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):

1.

For light microscopic study (LM): 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.

2.

For transmission electron microscopic study


(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 acetate18 followed by lead citrate19. Ultrathin 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.

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.

Mean

18

of each group was compared with the

Abeer A. Abd El Samad and Nagwa Kostandy Kalleny

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<0.05.

(Figs. 4&5). The reticular layer was composed mainly of


type I collagen fibers that was synthesized from dermal
fibroblasts (Fig. 7). A network of elastic fibers appearing
thicker in the reticular layer and became gradually thinner
as they progressed towards the papillary layer as shown
in sections stained by orcein stain (Fig. 8).

RESULTS
Light and Electron Microscopic Results:
Group I (Control group):

Ultra-thin sections also showed that the sebaceous


glands appeared to be formed of acini showing no lumen
but were filled with cells. The cells of the basal layer of
the acini appeared undifferentiated flattened epithelial
cells that rest on a basal lamina. The adjacent rounded
cells were more differentiated and appeared containing
abundant fat droplets in their cytoplasm. The cells at the
central part of acini showed shrunken and condensed
nuclei and their cytoplasm were filled with fat droplets
(Figs. 9&10).

The thin skin of guinea pig in H&E stained sections


and semi-thin sections stained by toluidine blue
appeared to be composed of epidermis and dermis.
The epidermis consisted mainly of stratified squamous
keratinized epithelium that was formed of keratinocytes
and also contained melanocytes. The stratum basale of
keratinocytes consisted of a single layer of basophilic
columnar cells with oval basal nuclei; in addition a
brownish melanin pigment was obvious. The stratum
spinosum consisted of polygonal cells with central
vesicular nuclei. The stratum granulosum consisted of
flattened cells whose cytoplasm appeared to be filled
with basophilic granules. The stratum corneum consisted
of many layers of flattened non-nucleated acidophilic
keratinized cells (Figs. 1,2&3).

Group II (Retinoic acid group):


The epidermis of thin skin sections of animals of this
group showed prominent rete pegs, apparent increased
thickness of viable keratinocytes (acanthosis) and reduced
melanin pigmentation as compared to control. The
keratinocytes showed enhanced epithelial proliferation,
in which the nuclei of cells of stratum spinosum appeared
vesicular and showed mitotic figures since anaphase
stage could be easily detected. The stratum granulosum
was apparently thickened, whereas the stratum corneum
was markedly thinned out as compared to control
(Figs. 11&12).

Ultra-thin sections of epidermis revealed melanocytes


that showed processes which extended into the interstices
between keratinocytes in attempt to transfer melanin
granules into the cytoplasm of keratinocytes of the
stratum basale. Moreover in ultra-thin sections, the
stratum basale showed active nuclei with extended
chromatin and apparent nucleoli (euchromatic). They
contained intermediate filaments and melanin granules.
The cytoplasm of the stratum spinosum showed bundles
of tonofilaments terminating in desmosomes, their nuclei
were euchromatic and showed apparent nucleoli. The
cytoplasm of the stratum granulosum appeared filled
with kerato-hyalin granules. The stratum corneum
showed flattened horny cells containing no nucleus
or cytoplasmic organelles, but was filled with keratin
filaments embedded in an amorphous matrix. The cells
of the lower layers of stratum corneun were closelyadherent and were attached by remnants of desmosomes,
but those in the outer layers appeared loosened and fully
keratinized (Figs. 4,5&6).

Ultra-thin sections showed that the stratum spinosum


cells exhibited active euchromatic nuclei with extended
chromatin. Their cytoplasm contained tonofilaments
terminating in desmosomes (Fig. 13). The cells of the
stratum granulosum showed apparent increase in their
number as compared to control. They were enriched
with large kerato-hyalin granules, whereas the stratum
corneum was thinner and more compact as compared to
control (Fig. 14).
The dermis showed active fibroblast depositing new
dermal collagen fibers leading to increase in the collagen
content as compared to control (Fig. 15). Moreover,
orcein stained sections demonstrated massive deposition
of elastic fibers in the dermis as compared to control
(Fig. 16). The sebaceous glands appeared shrunken
and showed marked decrease in the sebum content
as compared to control. The nuclei of the cells at the
central part of acini appeared condensed and shrunken.
They contained altered fat droplets as compared to
control. On the other hand, the cells at the periphery
and those at the middle part of the acini were mostly
undifferentiated (Fig. 17).

By LM, the dermis showed irregular surface that had


dermal papillae interdigitated with epidermal ridges. It had
rich network of blood vessels, hair follicles, in addition
to sebaceous and sweat glands. The dermis consisted of
two layers with indistinct boundaries; the outermost thin
papillary layer and the deeper thick reticular layer. The
papillary layer was composed of loose connective tissue
(Figs. 1,2&3).
From the papillary layer, ultra-thin sections
showed that special anchoring collagen fibrils inserted
into the basal lamina and extended into the dermis

Group III (Glycolic acid group):


The epidermis of thin skin sections of animals of

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Comparative Study on the Effect of Retinoic Acid, Glycolic Acid and Salicylic Acid on the Thin Skin

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 as
compared to control (Figs. 18,19&20).

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.

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 showed
chromatin pattern different than that of control with
unapparent nucleoli (Fig. 22).

Table (2) showed high significant increase in mean


thickness of epidermal keratinocytes (p<0.001) in
Retinoic acid, Glycolic acid and Salicylic acid Groups
when compared to Control Group using One Way ANOVA
with Post hoc analysis. A high significant increase in
mean thickness of epidermal keratinocytes (p<0.001)
in Retinoic acid Group, was detected as compared to
either Glycolic acid or Salicylic acid Groups. On the
other hand, a high significant decrease in mean thickness
of epidermal keratinocytes (p<0.001) was detected in
Glycolic acid Group, when compared to either Retinoic
acid or Salicylic acid Groups. However, a high significant
decrease in mean thickness of epidermal keratinocytes
(p<0.001) was detected in Salicylic acid Group when
compared to Retinoic acid Group, on the other hand, a
high significant increase in mean thickness of epidermal
keratinocytes (p<0.001) was detected in Salicylic acid
Group when compared to Glycolic acid Group.

Morphometric and Statistical Results:


Table (1) and Histogram (1) showed the means
and standard deviations of thickness of epidermal
keratinocytes in different studied groups.

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.

Table 1: Showing the mean thickness of nucleated epidermal


keratinocytes in different studied groups:

Group IV (Salicylic acid Group):

Nucleated epidermal keratinocytes


Mean SD (m)

Groups

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).

Group I (Control)

30.575.69

Group II (Retinoic acid)

160.595.43

Group III (Glycolic acid)

86.4613.53

Group IV (Salicylic acid)

117.4011.11

SD = standard deviation.
180
160
140

Ultra-thin sections revealed nearly normal and


viable cells of the stratum spinosum. They showed
apparent normal nuclear configuration with few melanin
granules 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).

120
100
80
60
40
20
0

Group I
C ontrol

The content of dermal collagen fibers showed nearly


no remarkable difference as compared to control. In
addition, nearly no remarkable difference in content of

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.

20

Abeer A. Abd El Samad and Nagwa Kostandy Kalleny

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

Group I
(Control)

Group II
(Retinoic acid)

Group III
(Glycolic acid)

Group IV
(Salicylic acid)

(J) code

Mean Difference
(I-J)

Significance

Group II

-130.02200

p<0.001 (H.S.)

Group III

-55.88800

p<0.001 (H.S.)

Group IV

-86.83300

p<0.001 (H.S.)

Group I

130.02200

p<0.001 (H.S.)

Group III

74.13400

p<0.001 (H.S.)

Group IV

43.18900

p<0.001 (H.S.)

Group I

55.88800

p<0.001 (H.S.)

Group II

-74.13400

p<0.001 (H.S.)

Group IV

-30.94500

p<0.001 (H.S.)

Group I

86.83300

p<0.001 (H.S.)

Group II

-43.18900

p<0.001 (H.S.)

Group III

30.94500

p<0.001 (H.S.)

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.

H.S. = High Significant.

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.

Fig. 1: A photomicrograph of a section of thin skin of female guinea pig,


showing epidermis (E) of stratified squamous keratinized epithelium
and connective tissue dermis (D).
Group I
H&E X 250.

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Comparative Study on the Effect of Retinoic Acid, Glycolic Acid and Salicylic Acid on the Thin Skin

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 keratohyalin 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. 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. 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. 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. 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.

22

Abeer A. Abd El Samad and Nagwa Kostandy Kalleny

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. 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. 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.

23

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.

Comparative Study on the Effect of Retinoic Acid, Glycolic Acid and Salicylic Acid on the Thin Skin

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. 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. 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.

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.

24

Abeer A. Abd El Samad and Nagwa Kostandy Kalleny

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 with
unapparent nucleoli.
Group III
TEM X 3000.

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.

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.

25

Comparative Study on the Effect of Retinoic Acid, Glycolic Acid and Salicylic Acid on the Thin Skin

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. 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. 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. 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.

26

Abeer A. Abd El Samad and Nagwa Kostandy Kalleny

The later clarified that; keratinocyte proliferation might


provide an explanation for the acanthosis induced by
topical retinoids in aged and steroid-damaged skin.
Moreover, other investigators24 observed that epidermal
acanthosis with correction of atypia were some of the
effects typical for tretinoin (Retinoic acid) that occurred
to induce rapid re-epithelialization. Thus tretinoin could
dramatically accelerate wound healing in photodamaged
skin.
In group II in the present study, Retinoic acid topical
application induced an apparent increase in the number
of stratum granulosum cells and they became enriched
with large keratohyalin granules. This was in agreement
with some authors21, who noticed that the granular layer
became thicker and contained large kerotohyalin granules
and the stratum corneum was thinner in retinoic acid
treated sites. Moreover, other investigators22,24 noticed
compaction of stratum corneum. This coincided with
thinning out and compaction of the stratum corneum in
group II in the present study as compared to control.

Fig. 29: An electron-micrograph of a section of thin skin of female


guinea pig, showing kerato-hyalin granules in stratum granulosum cells.
The stratum corneum appears less compact as compared to the control.
Group IV
TEM X 4000.

Moreover, the epidermal melanocytes in group II in


the present study showed apparent reduction in melanin
pigmentation as compared to control coinciding with
the findings of some investigators20,22 who noticed that
topical tretinoin (retinoic acid) reduced number and
activity of melanocytes. They also observed deposition
of new dermal collagen fibers. This observation was
seen in the dermis of group II in the present study and
was confirmed by the appearance of active fibroblast
depositing new dermal collagen fibers leading to
increase in the collagen content as compared to control.
Moreover, some investigators4,25 noticed that application
of topical tretinoin to human skin, remedied any collagen
deficiency existing in photoaged skin. Retinoids,
therefore, had become essential in the treatment and
prevention of photoaging. Coinciding, some authors5
stated that although a number of surgical procedures
could improve the clinical appearance of photoaged
skin, the only medical therapy with proved benefit
was the use of topical retinoids. In addition there was
evidence that topical retinoids could be beneficial in the
treatment of intrinsically aged skin. In addition, other
investigators26 noticed that topical retinol improved fine
wrinkles associated with natural aging by significant
increased collagen production. They added that
retinol-treated aged skin was more likely to withstand
skin injury and ulcer formation along with improved
appearance. Moreover, other investigator20 noticed
massive elastosis due to deposition of elastic fibers in
the dermis after topical tretinoin, thus it was concluded
that topical tretinoin could be also used for therapy for
chronological skin ageing, which is characterized by
atrophy of the skin with loss of elasticity. This finding
appeared as marked deposition of elastic fibers in
orcein-stained sections of the dermis of Group II in the
present study.

Fig. 30: A photomicrograph of a section of thin skin of female guinea


pig, showing elastic fibers () in the dermis with nearly no remarkable
difference as compared to control.
Group IV
Orcein X 250.

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 morphometric
and statistical study where the mean thickness of
nucleated epidermal keratinocytes was high significantly
increased (p<0.001) as compared to either Control,
Glycolic acid or Salicylic acid Groups. It measured the
highest mean thickness. This coincided with the results
of some investigators20,21. They found that Retinoic
acid induced keratinocyte proliferation in aged and
photodamaged skin. Coinciding in the present study,
the epidermis of Group II showed enhanced epithelial
proliferation and the nuclei of cells of stratum spinosum
appeared vesicular and exhibited markedly obvious
mitotic figures. This was observed by some scientists22,23.

27

Comparative Study on the Effect of Retinoic Acid, Glycolic Acid and Salicylic Acid on the Thin Skin

Similarly, some authors32 noted that glycolic acid worked


by removing superficial portions of the epidermis.

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 sebaceous
gland 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 sebaceous
glands 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.

An investigator33 noticed that alpha hydroxy acids


resulted in epidermolysis and upper dermal changes,
producing a vibrant, less wrinkled and more uniformly
colored skin and a decrease in corneocyte cohesion.
Moreover, other author34 stated that alpha-hydroxy acids
reduced the calcium ion concentration in the epidermis
and removed calcium ions from the cell adhesions by
chelation. This caused loss of calcium ions from the
cadherins of the desmosomes and adherens junctions, from
the tight junctions and possibly also from other divalent
metallic cation-dependent cell adhesion molecules.
The cell adhesions are thereby disrupted, resulting in
desquamation. Desquamation is enhanced by cleavage
of the endogenous stratum corneum chymotryptic
enzyme on the cadherins, which are otherwise protected
from proteolysis by conjugation with calcium ions. The
decrease of calcium ion level so brought about in the
epidermis also tends to promote cell growth and retard
cell differentiation, giving rise to a younger-looking skin.
This might explain the marked decrease in tonofilaments
content in the cytoplasm of the stratum spinosum as
compared to control, with disruption of desmosomes
and intercellular dysjunctions. It also might explain
the vacuolization and degeneration in the keratinocytes
showing nuclear changes ranging from pyknosis to
karyorrhexis by LM and different nuclear chromatin
pattern than that of control with unapparent nucleoli of
the keratinocytes by EM showed in epidermis of group
III in the present study. Coinciding, some investigators12
found that Glycolic acid treatment on rat skin induced
cytoplasmic vacuolization in epidermal keratinocytes and
intercellular dysjunctions. They also found that Glycolic
acid treatment resulted in an increase in numbers of active
fibroblasts and in the amount of dense collagen on rat
skin. This explained the appearance of active fibroblasts
with marked increase in the content of organized bundles
of dense collagen in skin of animals of group III in the
present study treated with topical Glycolic acid. Some
authors15 demonstrated that Glycolic acid, not only
directly accelerated collagen synthesis by fibroblasts, but
also modulated matrix degradation and collagen synthesis
through keratinocyte-released cytokines. Moreover, other
authors32 noted that glycolic acid worked by stimulating
dermis regeneration during treating photoaged skin. This
also coincided with the increased content of elastic fibers
in dermis of skin of group III of the present study.

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<0.001)
when compared to either Retinoic acid or Salicylic acid
groups. However, it showed high significant increase in
the mean epidermal thickness in the nucleated epidermal
cells as compared to control (p<0.001) particularly in the
cells of the stratum spinosum and stratum granulosum.
This coincided with the results of some investigators31
on hairless guinea pig epidermis treated with 5% and
10% glycolic acid. They observed a twofold increase
in epidermal thickness. Similarly, other scientists13
noticed that formulations containing glycolic acid caused
thickening of the epithelium of hairless mouse, where the
basal and spinous layers also showed increased thickness.
The cells reached the largest volume and the nuclei were
also more voluminous. They concluded that hydroxy
acids had satisfactory results in terms of maintaining
a young and healthy skin and in recovering aged skin,
thus they could be used in cosmetic and dermatologic
formulations. Moreover, some investigators12 found that
Glycolic acid treatment on rat skin resulted in statistically
significant increased thickness of the epidermis; however
they added that AHA had an important role in treating
dermatologic problems with widespread keratinization.
These effects coincided with the peeling and thinning of
the stratum corneum in group III of the present study.

An author34 stated that alpha-hydroxy acids might


also possess anti-inflammatory capacities. Similarly,
other authors35 noted that beta-lipohydroxy acid had
antimicrobial, anti-inflammatory and anticomedogenic
properties, which made it effective against acne. Its
antifungal and exfoliating properties were also likely to
prove useful in combating dandruff. This might explain
the use of Glycolic acid in the treatment of acne, since
topical application of glycolic acid to skin of animals of

28

Abeer A. Abd El Samad and Nagwa Kostandy Kalleny

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.

the use of Salicylic acid in the treatment of acne, since


topical application of Salicylic acid to skin of animals of
group IV in this study did not show remarkable changes
in the sebaceous glands as compared to the control,
moreover this coincided with the marked dispersion of
melanin in the keratinocytes in skin of animals of group
IV in the present study treated with topical Salicylic acid.
In addition, this group showed that the content of dermal
collagen fibers were nearly not different as compared
to control. Moreover, nearly no difference in content of
elastic fibers was noticed in orcein stained sections as
compared to control.

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.

Some authors42 stated that although ageing has been


thought to be irreversible, studies made during the last
decade had shown that a few topical compounds may
limit or improve the age-related skin damages. On their
comparative study of some topical agents, they noticed
that tretinoin induced enhanced epidermal proliferation
and thickness; whereas salicylic acid-treated areas had an
aspect similar to untreated control sites. On the other hand,
some investigators21 on their study on some antiaging
compounds, noticed that Retinoic acid had a beneficial
impact upon the aging epidermis in which it exhibited
improvement in the various epidermal compartments,
similarly the Lipophilic derivative of salicylic acid also
exhibited similar positive changes, although to a lesser
degree; whereas Glycolic acid showed no significant
effect and appeared almost inactive.

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<0.001) was noticed
when compared to Retinoic acid group. Whereas, a high
significant increase in mean thickness of nucleated
epidermal keratinocytes (p<0.001) was noticed when
compared to either Control or Glycolic acid groups. The
cells of the stratum spinosum showed vesicular nuclei
with preserved well-defined bundles of tonofilaments
in their cytoplasm and preserved desmosomes between
cells, however there was apparent separation between
cells most probably due to widening in the intercellular
space. Some investigators38 noticed that cell proliferation
and acanthosis were barely enhanced by salicylic acid and
they concluded that the mode of peeling by salicylic acid
was different than classical peeling agents as resorcinol,
crystalline sulfur, which peeled via proliferation
hyperkeratosis. They noticed that the clinically known
keratolytic effect of salicylic acid might be due to a direct
action on the intercellular cement substance of the horny
cells. Some authors39 noted that use of salicylic acid as a
peeling agent might produce systemic toxic effects. On
the other hand, other scientists40 noticed that chemical
peeling with salicylic acid was a well tolerated and safe
treatment modality in many superficial facial dermatoses
(melasma, acne vulgaris, freckles, post-inflammatory
scars/pigmentation, actinic keratoses and plane facial
warts). Similarly, some authors41 stated that salicylic
acid peels had effectiveness for both of acne and postinflammatory hyper-pigmentation that were common
in patients with skin of color. Salicylic acid peels were
beneficial in whitening the face of patients with acne. The
whitening effect would be an important factor in choosing
the superficial peeling agent for them. This might explain

It is concluded that topical application of Retinoic


acid-induced peeling by causing proliferation
hyperkeratosis and acanthosis proved by presence of
viable, mitotic keratinocytes. The mean thickness of
nucleated epidermal keratinocytes measured highest
mean thickness and it showed high significant increase
as compared with each of other studied groups. Whereas,
topical application of Glycolic acid-induced peeling by
removing superficial layers of epidermis (epidermolysis),
cytoplasmic and nuclear degeneration, disrupting
intercellular junctions. However, topical application of
Salicylic acid-induced peeling mainly by direct action on
intercellular cement substance and partially by inducing
proliferation hyperkertosis.
As regards their use in Acne, it was found that topical
application of Retinoic acid acted directly on the sebaceous
glands, inducing their shrinkage and decreasing sebum
production. Whereas, Glycolic acid had anti-inflammatory
effect, however Salicylic acid dispersed basal melanin
in their use as a topical application in treating postinflammatory hyperpigmentation in acne. Moreover,
Glycolic acid-induced degeneration in melanocytes
with marked decrease of melanin, also Retinoic acid
showed apparent reduction in melanin pigmentation.
In addition both Retinoic and Glycolic acids-induced
dermal fibroblastic stimulation to increase production of
collagen and elastic fibers, thus both could be used in
treatment of wrinkled skin. On the other hand Salicylic

29

Comparative Study on the Effect of Retinoic Acid, Glycolic Acid and Salicylic Acid on the Thin Skin

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 known
systemic side effects.

14.

15.

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