Semi-Quantitative Histological Analysis of the Effect of Intense Pulsed Light (IPL) and Carbon Dioxide (CO<sub>2</sub>) Intradermic Injection on Fibroblast and Collagen Proliferation in the Skin of Wistar Rats

doi:10.4236/jcdsa.2012.23032

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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.

Email: tamarafigu[email protected]

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

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%

(%)

Intense pulsed

light (G1)

(N = 8 animals)

Fibroblastic

proliferation

(100%) - - -

(37.5%)

(25%) - 3

(37.5%)

(25%)

(37.5%)

(37.5%) -

Collagen

proliferation

(100%) - - -

(12.5%)

(62.5%)

(25%) - 1

(12.5%)

(25%)

(37.5%)

(25%)

CO2 Intradermic

Injection (G2)

(N = 8 animals)

Fibroblastic

proliferation

(100%) - - -

(100%) - - - -

(62.5%)

(37.5%) -

Collagen

proliferation

(100%) - - -

(12.5%)

(87.5%) - - -

(50%)

(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%

(%)

Intense pulsed

light (G1) +

CO2 intradermic

injection (G2)

(N = 16 animals)

Fibroblastic

proliferation 16 (100%) - - - 11

(68.75%)

(12.5%)- 3

(18.75%)

(12.5%)

(50%)

(37.5%)-

Collagen

proliferation 16 (100%) - - - 2

(12.5%)

(75%)

(12.5%)- 1

(6.25%)

(37.5%)

(43.75%)

(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.

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