Journal of Cosmetics, Dermatological Sciences and Applications, 2012, 2, 164-173
http://dx.doi.org/10.4236/jcdsa.2012.23032 Published Online September 2012 (http://www.SciRP.org/journal/jcdsa)
Semi-Quantitative Histological Analysis of the Effect of
Intense Pulsed Light (IPL) and Carbon Dioxide (CO2)
Intradermic Injection on Fibroblast and Collagen
Proliferation in the Skin of Wistar Rats
Tamara Lemos Maia-Figueiró1, Alexandre Nakao Odashiro2, Giovanna Padoa de Menezes3,
Lilian Rezende Coelho4, Ili Breda4, Bruno Areco de Souza4, Ernesto Antonio Figueiró-Filho1
1Post-Graduation Program, Faculty of Medicine, Federal University of Mato Grosso do Sul, Campo Grande, Brazil; 2Hospital
Enfant-Jesus of University Laval, Department of Pathology, Quebec, Canada; 3Medical Residents, University Hospital, Federal
University of Mato Grosso do Sul, Campo Grande, Brazil; 4Faculty of Medicine, Federal University of Mato Grosso do Sul, Campo
Grande, Brazil.
Received July 28th, 2012; revised August 26th, 2012; accepted September 9th, 2012
ABSTRACT
Background: In recent years, so-called “non-ablative rejuvenation” has been carried out with the use of lasers or in-
tense pulsed light (IPL) to stimulate collagen production by dermal fibroblasts. Intradermal infusion of CO2 stimulates
fibroblasts and the synthesis of collagen and elastin, contributing to the retraction of the skin and tissue rejuvenation.
Objectives: To evaluate the effects of IPL and the intradermal infusion of CO2 on fibroblast proliferation and collagen
in the skin of female rats. Methods: Sixteen adult female Wistar rats were divided into two groups of eight animals.
Group 1 underwent IPL and group 2 underwent intradermal CO2 infusion. There was a total of 8 weeks of treatment.
We conducted a punch in each animal before any procedure (T0), another punch in the middle of treatment at 4 weeks
post-procedure (T1) and a punch at the end of treatment at 8 weeks post-procedure (T2). The cells involved in inflam-
mation, fibrosis and vascularization of the injured tissue by histopathology were analyzed. Results: There was statisti-
cally significant fibroblast proliferation and collagen proliferation noted when analyzing all 16 animals together and
also when considering the two study groups separately. In both groups, the greatest proliferation of fibroblasts coin-
cided with periods of increased collagen production. Conclusion: Both IPL and intradermal CO2 infusion stimulated
fibroblast and collagen proliferation in the skin of the rats studied.
Keywords: Collagen; Cosmetic Dermatology; Rejuvenation; Pulsed Light; Intense Pulsed Light; Intradermal Injection
1. Introduction
Collagen is the most abundant protein in the human body
and is synthesized by several cell types, including fibro-
blasts. As people age, there is a reduction of the carrying
capacity of the skin and subsequent atrophy and loss of
elasticity secondary to the reduction in collagen produc-
tion [1,2]. Various proposed techniques for the rejuvena-
tion of skin involve increasing the proliferation of colla-
gen via laser-based methods, pulsed light and intradermal
injection of CO2 [3,4].
Intense pulsed light (IPL) produces a non-coherent
light beam that has a radiation spectrum that covers many
wavelengths simultaneously. A polychromatic beam is
captured by different chromophores associated with dif-
ferent wavelengths or colors. This, together with the
other aspects mentioned, increases the therapeutic spec-
trum covered by IPL [5-7]. The non-ablative photoreju-
venation with intense pulsed light works causing reversi-
ble thermal damage of collagen by light penetration into
the dermis and direct heating of these structures, sparing
the epidermis [5-7]. Thus, the contraction of collagen fi-
bers and proper remodeling of the fibers after the in-
flammatory period is obtained. A polychromatic beam is
captured by different chromophores associated with dif-
ferent wavelengths or colors. This, together with the
other aspects mentioned, increases the therapeutic spec-
trum covered by IPL [8].
In recent years, so-called “non-ablative rejuvenation”
involving the use of lasers or (IPL) to promote stimula-
tion of collagen production by dermal fibroblasts has
been increasingly conducted. The treatments typically are
repeated monthly, and at least four sessions are necessary

Semi-Quantitative Histological Analysis of the Effect of Intense Pulsed Light (IPL) and Carbon Dioxide (CO2)
Intradermic Injection on Fibroblast and Collagen Proliferation in the Skin of Wistar Rats 165
to obtain efficient results [4,5,8].
Carbon dioxide (CO2) treatment involves the subcuta-
neous or transcutaneous infusion of CO2 to induce thera-
peutic effects that benefit microcirculation and tissue
oxygenation [9,10]. Studies have demonstrated the bene-
ficial effects of subcutaneous carbon dioxide therapy for
various medical conditions, including functional disor-
ders of blood flow, peripheral artery disease, microcircu-
latory disorders, delayed healing, multiple symmetric lip-
omatosis, cellulite and adiposity [2,9-12].
There are no published reports of adverse effects or
complications associated with either local or systemic
intradermal and subcutaneous CO2 infusion [11]. The
possible side effects are limited to low-intensity pain dur-
ing application, small bruises of the punch and a local
crackling sensation [9]. Studies using carbon dioxide for
contrast angiography attest to the safety of this gas and
have shown that it is not likely to promote clot. CO2 can
be used with intravascular bolus injections of up to 100
ml and continuous flows between 20 and 30 ml/second
without adverse reactions [13-15].
When applied to the skin surface layer, carbon dioxide
stimulates fibroblast synthesis of collagen and elastin,
contributing to the retraction of the skin and resulting in
the rejuvenation and the reduction of tissue laxity [9,10,
16,17].
A review of the literature concerning IPL and intra-
dermal CO2 injection as applied to fibroblast and colla-
gen proliferation indicates existing gaps in knowledge
about this subject [2]. After consideration of the relevance
of the study of such techniques for the rejuvenation, pre-
vention and healing of skin aging, it is obvious that addi-
tional research in this area is imperative.
It is possible to experimentally evaluate the histologi-
cal effects of intense pulsed light and the intradermal
injection of CO2 into skin [2,4,5,8]. Therefore, in the pre-
sent study, our aim was to evaluate the effect of IPL and
intradermic CO2 injection on fibroblast proliferation and
collagen in the skin of female Wistar rats.
2. Methods
This study was an experimental, comparative, non-con-
trolled trial that provides a skin semi-quantitative histo-
logical analysis of 16 adult female Wistar rats with a
body mass ranging between 160 and 200 grams. The
animals received food and water ad libitum before and
during the experimental period and underwent alternating
cycles of 12 h light and 12 h darkness.
The animals were kept in collective cages with rec-
tangular dimensions of 49 × 34 × 16 cm (length × width ×
height) with eight animals in each. The environment was
quiet, and the temperature was maintained between 21˚C
and 25˚C, according with guidelines for the use of labo-
ratory animals [18,19].
The rats were divided into two groups, each consisting
of 8 animals, as described below:
• Group 1 (G1): underwent to intense pulsed light
(IPL);
• Group 2 (G2): underwent intradermal injection of
CO2.
Before any procedures, the animals were anesthetized
with 10% ketamine injectable solution at a dose of 10
mg/kg associated with a 1 mg/kg intramuscular xylazine
dose [18-21]. All animals were anesthetized and shaved
on the dorsal region to create a “punch” of approximately
6 mm in diameter to remove a circular piece of skin in
the left inferior-lateral portion of the back for histological
study. The skin piece was standardized as a control at
time zero (T0) that corresponded to intact skin from pre-
treatment and without intervention. These (T0) biopsies
were also called as controls non-treated skins.
After a week of completing the initial (T0) punch, the
animals were subjected to treatment according to the
group divisions:
• G1 (IPL): Eight animals in this group were treated
with intense pulsed light at a wavelength of 550 to
900 nanometers in the average pulse. Each animal
was submitted to six passages. The light was applied
to the right supero-lateral region of the animal’s back
just after shaving. The treatment was applied once
every two weeks for eight weeks;
• G2 (CO2): The eight animals in this group were
treated with an intradermal CO2 injection at a flow of
80 ml/min. The total volume infused by the applica-
tion of CO2, in milliliters, was limited to the formula
weight of the animal (in kg) × 5. The treatment was
applied in the right supero-lateral region of the ani-
mal’s back just after shaving once a week for eight
weeks.
After the fourth week of treatment, the animals under-
went a new punch of approximately 6 mm in diameter to
remove a circular piece of skin from the intervention site
(right superolateral portion of the back of the animal).
The piece was used for the histological analysis of the
middle portion of the treatment (T1).
At the end of eight weeks of treatment, the animals
underwent a new punch of approximately 6 mm in dia-
meter to remove circular piece of skin from the interven-
tion site (right superolateral portion of the back of the
animal) for the end treatment (T2) histological analysis.
After eight weeks of treatment and the acquisition of
all of the punches for histological analysis, the animals
were euthanized by chemical methods (overdose of pen-
tobarbital) [18-21].
The specimens were fixed in 10% buffered formalin

Semi-Quantitative Histological Analysis of the Effect of Intense Pulsed Light (IPL) and Carbon Dioxide (CO2)
Intradermic Injection on Fibroblast and Collagen Proliferation in the Skin of Wistar Rats
166
for 24 hours. For the routine histopathology techniques,
the specimens were embedded in paraffin and 4 μm thick
sections were taken with a rotating microtome. We ana-
lyzed the cells involved in inflammation, fibrosis and the
vascularity of the injured tissue histopathologically. For
the analysis of inflammatory cells, we used universal his-
tochemical staining (hematoxylin-eosin). For the analysis
of fibrosis, a Masson’s trichrome stain was used.
To assess fibroblast and collagen proliferation, a semi-
quantitative analysis was carried out estimating the amount
of newly formed collagen and fibroblasts in an organized
fashion as seen on the blades. The slides were classified
into four grades (G0, GI, GII and GIII) according to the
percentage of the slide on which new fibroblasts and or-
ganized collagen [22] had formed (Table 1). The semi-
quantitative analysis was proposed in accordance with
the classic technique of quantitative morphometry [22-
27].
The slides were coded so as to not identify the treat-
ment groups or time of biopsy. A pathologist evaluated
the slides at random, not knowing the groups or the cor-
responding treatment times. The fibroblast and collagen
proliferation ratings were recorded in a spreadsheet,
along with the identity of the blade used, and were then
subsequently decoded.
The data is presented as absolute values, percentages
and proportions. A nonparametric analysis of variance test
(Kruskal-Wallis) was used to compare the study groups.
When the test indicated a significant difference, we used
a Dunn post-test to compare the two groups. As a meas-
ure of accuracy, 95% confidence intervals were employed.
A p < 0.05 was considered significant. All statistical
analysis was performed using the GraphPad Prism, ver-
sion 5.0.1 statistical software package (GraphPad Soft-
ware, San Diego, CA, USA). This study was approved by
the Ethics Committee on Animal Use, protocol 241/2009.
3. Results
In the eight animals subjected to IPL treatment, when
fibroblast proliferation between the times of onset (T0),
the middle (T1) and the end (T2) of treatment was com-
pared, it initially appeared that non-treated skin exhibited
a grade 0 classification in 100% of the animals. By the
middle of treatment (T1), five of the eight animals
(62.5%) presented grade II and III fibroblast proliferation,
and at the end of treatment (T2), six of the eight (75%) of
the animals presented grade I and II proliferation. This
evolution of the histological proliferation of fibroblasts
was statistically significant when the beginning and the
end of treatment were compared (T0 vs. T2, p < 0.05,
Table 2).
For the non-treated skin controls, grade 0 fibroblast
proliferation was also observed in 100% of the animals
that received the intradermal injections of CO2. After 4
weeks of treatment, all animals in this group presented
no changes in histological grade, as they maintained
100% presentation of grade 0 fibroblast proliferation.
However, by the end of treatment (T2), all animals (100%)
exhibited grade I and II fibroblast proliferation. The evo-
lution of histological fibroblast proliferation was also
statistically significant in the CO2 group when the begin-
ning and the end of treatment were compared (T0 vs. T2,
p < 0.001, Table 2).
In regards to collagen proliferation, both study groups
exhibited grade 0 proliferation in non-treated control skin
at time zero in 100% of the samples. By the middle of
treatment (T1), 87.5% (7/8) of the animals in the IPL
group exhibited proliferation of grade I and grade II col-
lagen, and 87.5% (7/8) the CO2 group animals exhibited
histological grade I collagen proliferation. At the end of
treatment (T2), in the intense pulsed light-treated animals,
62.5% (5/8) exhibited grade II and III proliferation, and
25% (2/8) exhibited grade I collagen proliferation. In the
CO2 group, 50% (4/8) exhibited grade I proliferation and
50% (4/8) grade II collagen proliferation after the treat-
ment (Table 2).
There was a statistically significant difference in the
degree of collagen proliferation between the time points,
both for the animals in the light-pulse treated group (T0
vs. T2, p < 0.001) and the animals in the CO2 group (T0
vs. T2, p < 0.001). These data are summarized in Table
2.
When all 16 animals were analyzed together (G1 and
G2), there was a statistically significance different in
fibroblast and collagen proliferation when comparing the
beginning and the end of treatment (T0 vs. T2, p < 0.001),
Table 1. Classifications for the semi-quantitative analysis of fibroblast and collagen proliferation.
Grade Fibroblastic proliferation (% of blade) Collagen proliferation (% of blade)
Grade 0 0% to 5% 0% to 5%
Grade I 5% to 25% 5% to 25%
Grade II 25% to 50% 25% to 50%
Grade III More than 50% More than 50%

Semi-Quantitative Histological Analysis of the Effect of Intense Pulsed Light (IPL) and Carbon Dioxide (CO2)
Intradermic Injection on Fibroblast and Collagen Proliferation in the Skin of Wistar Rats 167
Table 2. Expression of fibroblast proliferation and collagen proliferation according to the period analyzed and separated by
study group.
Time 0: T0
(Onset treatment)
Time 1: T1
(4 weeks of treatment)
Time 2: T2
(8 weeks of treatment)
Grade 0
0% - 5% 5% - 25%
Grade IGrade II
25% - 50%
Grade III
>50%
Grade 0
0% - 5%
Grade I
5% - 25%
Grade II
25% - 50%
Grade III
>50%
Grade 0
0% - 5% 5% - 25%
Grade I
25% - 50%
Grade IIGrade III
>50%
N
(%)
N
(%)
N
(%)
N
(%)
N
(%)
N
(%)
N
(%)
N
(%)
N
(%)
N
(%)
N
(%)
N
(%)
Intense pulsed
light (G1)
(N = 8 animals)
Fibroblastic
proliferation
8
(100%) - - -
3
(37.5%)
2
(25%) - 3 (37.5%)
2
(25%)
3
(37.5%)
3
(37.5%) -
Collagen
proliferation
8
(100%) - - -
1
(12.5%)
5
(62.5%)
2
(25%) - 1 (12.5%)
2
(25%)
3
(37.5%)
2
(25%)
CO2 Intradermic
Injection (G2)
(N = 8 animals)
Fibroblastic
proliferation
8
(100%) - - -
8
(100%) - - - -
5
(62.5%)
3
(37.5%) -
Collagen
proliferation
8
(100%) - - -
1
(12.5%)
7
(87.5%) - - -
4
(50%)
4
(50%) -
Comparisons using the Kruskal-Wallis with Dunn post-test:
G1: Pulsed light (fibroblastic proliferation)
T0 vs. T1 p < 0.05 G2: CO2 (fibroblastic proliferation) T0 vs. T1 p > 0.05
T0 vs. T2 p < 0.05 T0 vs. T2 p < 0.001
T1 vs.T2 p > 0.05 T1 vs. T2 p < 0.001
G1: Pulsed light (collagen proliferation)
T0 vs. T1 p < 0.05 G2: CO2 (Collagen proliferation) T0 vs. T1 p < 0.05
T0 vs. T2 p < 0.001 T0 vs. T2 p < 0.001
T1 vs. T2 p > 0.05 T1 vs. T2 p > 0.05
as shown in Table 3. Figures 1 - 4 illustrate the prolif-
eration of collagen at different time points (T0, T1 and
T2) in the IPL group animals and the CO2 group animals.
The Figures 4-7 demonstrate the evaluation of colla-
gen in group intense pulsed light (G1) and CO2 (G2) at
three time points analyzed: onset (T0), middle (T1) and
final (T2) treatment.
4. Discussion
Intense pulsed light (IPL) is a source of light energy that
has many applications. IPL is composed of different wave-
length, i.e., all or part of the light spectrum, while the
laser has a single wavelength [3]. IPL reaches the skin
surface and allows, through the principle of selective
photothermolysis, the correction of various skin lesions
and facial blemishes resulting from photoaging, as well
as stains and pigmentation issues [5].
As IPL is defined by not being composed of coherent
light, it can interact with a variety of chromophores, and
its energy can, therefore, be more quickly dissipated.
Thus, IPL heating is more superficial compared to simi-
lar use of lasers. The difference provides better security
and safety, especially in most advanced skin types [28].
A recent study evaluated the action of IPL on stimu-
lating the proliferation of collagen in human skin dam-
aged by sun and concluded that, in addition to collagen
deposition, the clinical improvement observed after treat-
ment may be secondary to the reduction of perifolicullar
inflammatory infiltrate [4,8]. Other reports indicate some
degree of the appearance of newly formed collagen in the
upper dermis after IPL treatment, thus suggesting the
possibility that stimulated dermal fibroblasts are the
source of this increased collagen expression [6,29].
The results of the present study indicate that eight
weeks after the application of intense pulsed light once
every other week, there were significant differences on
fibroblast proliferation and collagen in the skin of the
animals studied. The moments of the greatest prolifera

Semi-Quantitative Histological Analysis of the Effect of Intense Pulsed Light (IPL) and Carbon Dioxide (CO2)
Intradermic Injection on Fibroblast and Collagen Proliferation in the Skin of Wistar Rats
168
Table 3. Expression of fibroblast proliferation and collagen proliferation according to the period analyzed and separated by
study group.
Time 0: T0
(onset treatment)
Time 1: T1
(4 weeks treatment)
Time 2: T2
(8 weeks treatment)
Grade 0
0% - 5%
Grade I
5% - 25% 25% - 50%
Grade II Grade III
>50%
Grade 0
0% - 5%
Grade I
5% - 25%
Grade II
25% - 50%
Grade III
>50%
Grade 0
0% - 5% 5% - 25%
Grade I
25% - 50%
Grade IIGrade III
>50%
N
(%)
N
(%)
N
(%)
N
(%)
N
(%)
N
(%)
N
(%)
N
(%)
N
(%)
N
(%)
N
(%)
N
(%)
Intense pulsed
light (G1) +
CO2 intradermic
injection (G2)
(N = 16 animals)
Fibroblastic
proliferation 16 (100%) - - - 11 (68.75%)
2
(12.5%)- 3 (18.75%)
2
(12.5%)
8
(50%)
6
(37.5%)-
Collagen
proliferation 16 (100%) - - - 2 (12.5%)
12
(75%)
2
(12.5%)- 1 (6.25%)
6
(37.5%)
7
(43.75%)
2
(12.5%)
Comparisons using the Kruskal-Wallis with Dunn post-test:
Fibroblastic proliferation
T0 vs. T1 p > 0.05
T0 vs. T2 p < 0.001
T1 vs. T2 p < 0.05
Collagen proliferation
T0 vs. T1 p < 0.001
T0 vs. T2 p < 0.001
T1 vs. T2 p > 0.05
Figure 1. Experimental procedures on Wistar Rats. A “punch”
of approximately 6 mm in diameter to remove a circular
piece of skin in the left inferior-lateral portion of the back,
designed as (T0) that corresponded to intact skin from pre-
treatment and without intervention.
Figure 2. Experimental procedures on Wistar Rats. The CO2
intradermal infusion treatment was applied in the right su-
pero-lateral region of the animal’s back just after shaving
once a week for eight weeks.

Semi-Quantitative Histological Analysis of the Effect of Intense Pulsed Light (IPL) and Carbon Dioxide (CO2)
Intradermic Injection on Fibroblast and Collagen Proliferation in the Skin of Wistar Rats 169
Figure 3. Experimental procedures on Wistar Rats. Each
animal was submitted to six passages of IPL. The light was
applied to the right supero-lateral region of the animal’s
back just after shaving. The treatment was applied once
every two weeks for eight weeks. The bottom image shows
the final aspect of the treated skin just before the sacrifice.
tion of fibroblasts coincide with the periods of increased
collagen production. These results support the hypothesis
that the source of the increased expression of collagen is
the stimulation of dermal fibroblasts, probably due to the
principle of selective photothermolysis. The concept un-
derlying the principle is that the absorption of light by
water causes a photothermal effect and a consequent in-
flammatory response that stimulates the fibroblastic ac-
tivity [3].
With respect to the intradermal injection of CO2, the
hypothesis of action is that carbon dioxide, when applied
subcutaneously, results in the mechanical destruction of
fat cells. In addition, the CO2 promotes local vasodilata-
tion and a subsequent increase in tissue oxygenation and,
when applied in the most superficial layer of the skin,
will stimulate fibroblasts and the synthesis of elastin and
collagen. These processes contribute to the retraction
ofthe skin and result in skin tissue rejuvenation and a re-
duction in sagging [2,9-12].
The easily recognized skin undergoes changes with
Figure 4. Evaluation of collagen in group intense pulsed
light (G1) at three time points analyzed: baseline (T0), half
(T1) and final (T2) treatment, from up to down, respectively.
Hematoxylin-eosin (100×).
advancing age, as the appearance of furrows, atrophy,
ptosis and laxity alters its appearance. Changes in the
connective tissue, which acts as the structural foundation
for the epidermis, outline these changes externally and

Semi-Quantitative Histological Analysis of the Effect of Intense Pulsed Light (IPL) and Carbon Dioxide (CO2)
Intradermic Injection on Fibroblast and Collagen Proliferation in the Skin of Wistar Rats
170
Figure 5. Evaluation of collagen in group intense pulsed
light (G1) at three time points analyzed: baseline (T0), half
(T1) and final (T2) treatment, from up to down, respectively.
Hematoxylin-eosin (400×).
Figure 6. Evaluation of collagen in group CO2 injection (G2)
at three time points analyzed: baseline (T0), half (T1) and
final (T2) treatment, from up to down, respectively. Hema-
toxylin-eosin (100×).

Semi-Quantitative Histological Analysis of the Effect of Intense Pulsed Light (IPL) and Carbon Dioxide (CO2)
Intradermic Injection on Fibroblast and Collagen Proliferation in the Skin of Wistar Rats 171
Figure 7. Evaluation of collagen in group CO2 injection (G2)
at three time points analyzed: baseline (T0), half (T1) and
final (T2) treatment, from up to down, respectively. Hema-
toxylin-eosin (400×).
are reflected in the corneum stratum [1]. The modifica-
tions to the lifelong unit-elastic collagen establish a sub-
stantial morphological basis for the understanding of the
biochemical and biomechanical changes of the skin with
age [1]. Thus, the intradermal injection of CO2 provides a
greater exchange rate (increase in blood flow) and im-
proves tissue oxygenation; in addition, it may stimulate
dermal fibroblasts and may increase collagen and elastin
synthesis [9-12].
A recent study evaluated CO2 injection into the skin of
ten Wistar male rats. The results of the study indicated a
marked increase in collagen after infusion of carbon di-
oxide into the skin of animals. Furthermore, intradermal
injections appeared more effective than subcutaneous in-
jections in reducing wrinkles [2].
Based on those results, we studied the stimulation of
fibroblast and collagen proliferation via the application
of intense pulse light and intradermal CO2 in the skin of
sixteen rats. It should be emphasized that the prolifera-
tion of collagen is a dynamic process and depends mainly
on the stimulation of fibroblasts. Therefore, it is possible
that a longer period of exposure to the treatments could
lead to a greater activation of dermal fibroblasts and even
greater collagen proliferation.
The results of the current study allow us to conclude
that both the intradermal injection of CO2 and intense
pulsed light promotes fibroblast and collagen prolifera-
tion in the skin of animals. However, other questions are
raised by these results. Is it possible that various me-
chanical or traumatic stimuli (punctures, local heat) can
also stimulate fibroblast and collagen proliferation? Can
the results observed in rat skin be repeated in human
skin?
Because of the paucity of data (especially experimen-
tal study data) published in medical journals about this
subject, this work represents a milestone in rejuvenating
dermatopathology procedures. Thus, further studies in
this area utilizing similar methodologies are needed. Der-
matology now offers unlimited possibilities in the use of
skin-rejuvenating procedures. The gathering of scientific
evidence is the best way to establish new methods for
cosmetic dermatology.
REFERENCES
[1] R. B. Oriá, F. V. A. Ferreira, É. N. Santana, M. R. Fernan-
des and G. A. C. Brito, “Study of Age-Related Changes
in Human Skin, Using Methods of Histo-Morphometry
and Autofluorescence,” Anais Brasileiros de Dermatologia,
Vol. 78, No. 4, 2003, pp. 425-434.
[2] J. C. Ferreira, A. Haddad and S. A. Tavares, “Increase in
Collagen Turnover Induced by Intradermal Injection of
Carbon Dioxide in Rats,” Journal of Drugs in Dermatol-
ogy, Vol. 7, No. 3, 2008, pp. 201-206.

Semi-Quantitative Histological Analysis of the Effect of Intense Pulsed Light (IPL) and Carbon Dioxide (CO2)
Intradermic Injection on Fibroblast and Collagen Proliferation in the Skin of Wistar Rats
172
[3] M. G. Catorze, “Laser: Bases and Use in Dermatology,”
Medicina Cutánea Ibero-Latino-Americana, Vol. 37, No.
1, 2009, pp. 5-27.
[4] V. G. Prieto, A. H. Diwan, C. R. Shea, P. Zhang and N. S.
Sadick, “Effects of Intense Pulsed Light and the 1064 nm
Nd:YAG Laser on Sun-Damaged Human Skin: Histolo-
gic and Immunohistochemical Analysis,” Dermatologic
Surgery, Vol. 31, No. 5, 2005, pp. 522-525.
doi:10.1111/j.1524-4725.2005.31154
[5] R. C. R. Patriota, C. J. Rodrigues and L. C. Cucé, “Intense
Pulsed Light in Photoaging: Clinical, Histopathological
and Immunohistochemical Evaluation,” Anais Brasileiros
de Dermatologia, Vol. 86, No. 6, 2011, pp. 1129-1133.
doi:10.1590/S0365-05962011000600010
[6] D. J. Goldberg, “New Collagen Formation after Dermal
Remodeling with an Intense Pulsed Light Source,” Jour-
nal of Cutaneous Laser Therapy, Vol. 2, No. 2, 2000, pp.
59-61.
[7] D. J. Goldberg and K. B. Cutler, “Nonablative Treatment
of Rhytids with Intense Pulsed Light,” Lasers in Surgery
and Medicine, Vol. 26, No. 2, 2000, pp. 196-200.
doi:10.1002/(SICI)1096-9101(2000)26:2<196::AID-LSM
10>3.0.CO;2-9
[8] V. G. Prieto, P. S. Zhang and N. S. Sadick, “Evaluation of
Pulsed Light and Radiofrequency Combined for the Treat-
ment of Acne Vulgaris with Histologic Analysis of Facial
Skin Biopsies,” Journal of Cosmetic and Laser Therapy,
Vol. 7, No. 2, 2005, pp. 63-68.
[9] C. Brandi, C. D’Aniello, L. Grimaldi, B. Bosi, I. Dei, P.
Lattarulo, et al., “Carbon Dioxide Therapy in the Treat-
ment of Localized Adiposities: Clinical Study and Histo-
pathological Correlations,” Aesthetic Plastic Surgery, Vol.
25, No. 3, 2001, pp. 170-174.
doi:10.1007/s002660010116
[10] C. Brandi, L. Grimaldi, G. Nisi, A. Brafa, A. Campa, M.
Calabrò, et al., “The Role of Carbon Dioxide Therapy in
the Treatment of Chronic Wounds,” In Vivo, Vol. 24, No.
2, 2010, pp. 223-226.
[11] T. Brockow, A. Dillner, A. Franke and K. L. Resch, “An-
algesic Effectiveness of Subcutaneous Carbon-Dioxide
Insufflations as an Adjunct Treatment in Patients with
Non-Specific Neck or Low Back Pain,” Complementary
Therapies in Medicine, Vol. 9, No. 2, 2001, pp. 68-76.
doi:10.1054/ctim.2001.0434
[12] A. Sönmez, M. Yaman, O. Yalçin, B. Ersoy, M. Serin and
A. Sav, “Carbon Dioxide Therapy Increases Capillary
Formation on Random Pedicled Skin Flaps in the Rat,”
Journal of Plastic, Reconstructive & Aesthetic Surgery,
Vol. 62, No. 7, 2009, pp. e236-e237.
doi:10.1016/j.bjps.2009.01.067
[13] D. Kummer-Kloess, W. Kloess, N. Marienhoff, R. M.
Schütz, M. Zwaan and H. D. Weiss, “Angiography during
Interventional Procedures with Carbon Dioxide (CO2)
(Carbo-Angiography) in Patients with Increased Contrast
Media Risk,” Zentralblatt für Chirurgie, Vol. 122, 1997,
pp. 725-729.
[14] M. Zwaan, W. Kloess, C. Kagel, D. Kummer-Kloess, S.
Matthies-Zwaan, R. M. Schütz, et al., “Carbon Dioxide as
an Alternative Contrast Medium in Peripheral Angiogra-
phy,” Rofo, Vol. 164, No. 5, 1996, pp. 445-448.
doi:10.1055/s-2007-1015687
[15] M. Zwaan, J. Steinhoff, L. Fricke, C. Kagel, H. Lorch and
H. D. Weiss, “The Angiography of Kidney Transplant
Arteries Using Carbon Dioxide,” Dtsch Med Wochenschr,
Vol. 122, 1997, pp. 1133-1136.
doi:10.1055/s-2008-1047738
[16] B. R. Hartmann, E. Bassenge and M. Pittler, “Effect of
Carbon Dioxide-Enriched Water and Fresh Water on the
Cutaneous Microcirculation and Oxygen Tension in the
Skin of the Foot,” Angiology, Vol. 48, No. 4, 1997, pp.
337-343. doi:10.1177/000331979704800406
[17] B. R. Hartmann, E. Bassenge and M. Hartmann, “Effects
of Serial Percutaneous Application of Carbon Dioxide in
Intermittent Claudication: Results of a Controlled Trial,”
Angiology, Vol. 48, No. 11, 1997, pp. 957-963.
doi:10.1177/000331979704801104
[18] K. Field, M. Bailey, L. L. Foresman, R. L. Harris, S. L.
Motzel, R. A. Rockar, et al., “Medical Records for Ani-
mals Used in Research, Teaching, and Testing: Public
Statement from the American College of Laboratory Ani-
mal Medicine,” Institute of Laboratory Animal Resources
Journal, Vol. 48, 2007, pp. 37-41.
[19] D. F. Kohn, T. E. Martin, P. L. Foley, T. H. Morris, M. M.
Swindle, G. A. Vogler, et al., “Public Statement: Guide-
lines for the Assessment and Management of Pain in Ro-
dents and Rabbits,” Journal of American Association for
Laboratory Animal Science, Vol. 46, No. 2, 2007, pp. 97-
108.
[20] ACLAM, “ACLAM Position Statement on Animal Ex-
perimentation,” Comparative Medicine, Vol. 53, No. 5,
2003, p. 472.
[21] J. Artwohl, P. Brown, B. Corning, S. Stein and A. T.
Force, “Report of the ACLAM Task Force on Rodent
Euthanasia,” Journal of American Association for Labo-
ratory Animal Science, Vol. 45, No. 1, 2006, pp. 98-105.
[22] A. L. Domingos, S. Tucci, S. B. Garcia, J. de Bessa, A. J.
Cologna and A. C. Martins, “Use of a Latex Biomem-
brane for Bladder Augmentation in a Rabbit Model: Bio-
compatibility, Clinical and Histological Outcomes,” In-
ternational Journal of the Brazilian Society of Urology,
Vol. 35, No. 2, 2009, pp. 217-224.
[23] J. J. S. Gonçalves, L. E. V. Leão, R. G. Ferreira, R. Olivei-
ra, L. H. Ota and R. S. D. Santos, “Semiquantitative Ana-
lysis of Surgical Biopsies of Different Lung Lobes of Pa-
tients with Usual Interstitial Pneumonia/Idiopathic Pul-
monary Fibrosis,” Jornal Brasileiro de Pneumologia, Vol.
35, No. 7, 2009, pp. 676-682.
[24] D. M. Hyde, T. E. King, T. McDermott, J. A. Waldron, T.
V. Colby, W. M. Thurlbeck, et al., “Idiopathic Pulmonary
Fibrosis. Quantitative Assessment of Lung Pathology. Com-
parison of a Semiquantitative and a Morphometric Histo-
pathologic Scoring System,” American Review of Respi-
ratory Disease, Vol. 146, No. 4, 1992, pp. 1042-1047.
[25] M. F. Y. Maeda, C. D. Silva, L. S. Harima, L. F. F. Silva,
B. Ctenas and V. A. F. Alves, “Vascularization in Hepatic
Cirrhosisbased on Autopsies,” Archives of Gastroentero-

Semi-Quantitative Histological Analysis of the Effect of Intense Pulsed Light (IPL) and Carbon Dioxide (CO2)
Intradermic Injection on Fibroblast and Collagen Proliferation in the Skin of Wistar Rats
173
logy, Vol. 45, 2008, pp. 38-45.
[26] D. Araf, M. Moura, C. J. Rodrigues and L. A. Figueiredo,
“Homologous Scleral and Dura Mater Grafts in Rabbit
Eyes,” Arquivos Brasileiros de Oftalmologia, Vol. 70, No.
6, 2007, pp. 981-987.
doi:10.1590/S0004-27492007000600018
[27] W. C. Cavazana, M. L. P. B. de Simões, S. O. Yoshii, C.
A. B. Amado and R. K. N. Cuman, “Sugar (Sucrose) and
Medium Chain Triglycerides with Essential Fatty Acids
in the Treatment of Wounds: An Experimental Study in
Rats,” Brazilian Annals of Dermatology, Vol. 84, No. 3,
2009, pp. 229-236.
doi:10.1590/S0365-05962009000300003
[28] D. Laury, “Intense Pulsed Light Technology and Its Im-
provement on Skin Aging from the Patients’ Perspective
Using Photorejuvenation Parameters,” Dermatology Online
Journal, Vol. 9, No. 1, 2003, p. 5.
[29] D. J. Goldberg and S. Silapunt, “Q-Switched Nd:YAG La-
ser: Rhytid Improvement by Non-Ablative Dermal Re-
modeling,” Journal of Cutaneous Laser Therapy, Vol. 2,
No. 3, 2000, pp. 157-160.