Archives of Medical Research 44 (2013) 169e177

ORIGINAL ARTICLE
Accelerated Mice Skin Acute Wound Healing In Vivo by Combined Treatment
of Argon and Helium Plasma Needle
Elizabeth Garc
ıa-Alcantara,a,b R
egulo L

opez-Callejas,a Pedro R. Morales-Ram
ırez,a
a
Rosendo Pe~na-Eguiluz, Ra
ul Fajardo-Mu~ noz, Antonio Mercado-Cabrera,a Samuel R. Barocio,a
c

ul Valencia-Alvarado,a Benjam
ın G. Rodr
ıguez-M
endez,a Arturo E. Mu~

Ra
noz-Castro,a
b a,b
Anibal de la Piedad-Beneitez, and Israel A. Rojas-Olmedo
a
Plasma Physics Laboratory and Cell Radiobiology Laboratory, Instituto Nacional de Investigaciones Nucleares, M
exico D.F., Mexico
b
Instituto Tecnol
ogico de Toluca, Toluca, Mexico
c
Centro de Investigaci
on y Estudios Avanzados en Salud Animal, Facultad de Medicina Veterinaria y Zootecnia Universidad Aut
onoma del
Estado de M
exico, Toluca, Estado de M
exico, Mexico

Received for publication July 3, 2012; accepted January 29, 2013 (ARCMED-D-12-00341).

Background and Aims. The efficacy of a direct application of plasma needle to in vivo
wound healing was experimentally studied in mice. This kind of plasma has achieved
considerable success in blood coagulation and tissue restoration in mice. In the develop-
ment of the present study, an argon plasma needle was chosen for coagulation purposes,
whereas for healing purposes, a helium plasma needle was used.
Methods. Treatment was applied with a plasma needle produced by argon and helium to
a wound induced in laboratory mice. Tissue regeneration was carried out by three argon
plasma treatments with 0.5 SLPM flow for 1 min and three treatments of helium with
1.5 SLPM flow. Intervals between each treatment were 5 min and 60 min for argon
and helium plasmas, respectively, thus completing a total treatment time of 180 min.
Histological sections were performed to corroborate the internal bleeding and tissue
regeneration.
Results. After three treatments with argon plasma, the blood produced in the wound was
coagulated and protein material appeared. By means of treatment with helium plasma, an
approach of the wound edges was produced until the conclusion thereof. These results
were corroborated histologically.
Conclusions. This type of acceleration during the skin wound healing process can be
attributed to the formation of reactive species such as NO, which were increased in the
helium plasma needle with respect to the argon plasma needle. Ó 2013 IMSS. Published
by Elsevier Inc.
Key Words: Cold plasma, Plasma treatment, Tissue regeneration, Nitric oxide.

Introduction (1e3). Recently, the field of plasma physics has been

In biology and medicine, the term plasma refers to the
liquid component of the blood cells, whereas this same
word in physics refers to the fourth state of matter,
a partially ionized gas with a neutral electrical behavior
Address reprint requests to: R
egulo L
opez-Callejas, Instituto Nacional
de Investigaciones Nucleares, Plasma Physics Laboratory, Carretera
Mexico Toluca S/N, La Marquesa, Ocoyoacac, Estado de Mexico, Mexico,
Estado de Mexico 52750, Mexico; Phone: (þ52) (55) 5329-7200; FAX:
(þ52) (55) 5329-7234; E-mail: regulo.lopez@inin.gob.mx
significantly expanding with many applications, in partic-
ular, plasma in medicine and as an independent medical
field. It is emerging worldwide in a manner comparable
to the development several years ago of laser technology
(4). Research areas such as plasma surface modification
(5), plasma bio-decontamination, and therapeutic plasma
are often collectively known as plasma medicine (6). The
scientific basis of plasma medicine rests on a fundamental
knowledge of the plasma interaction mechanisms with
living cells and tissue (7).

0188-4409/$ - see front matter. Copyright Ó 2013 IMSS. Published by Elsevier Inc.
http://dx.doi.org/10.1016/j.arcmed.2013.02.001
170 Garc
ıa-Alcantara et al./ Archives of Medical Research 44 (2013) 169e177
Various biomedical applications of nonthermal atmosph-
eric pressure plasma sources have been explored recently.
These have been focused on the treatment of medical
equipment and even living tissue. A major goal of tissue
treatment with plasmas is nondestructive surgery (8).
Provided that plasmas allow fast and efficient bacterial
inactivation, they seem suitable for the asepsis of surgical
tools and local tissue disinfection. However, as it is
expected that a novel approach to surgery will emerge
from plasma science, a great research effort must be made
before these techniques become common in medicine.
Examples of plasma applications in medicine are bacterial
decontamination of medical instruments (9), coating of
implants with biocompatible layers (10), surface modifica-
tion of substrates for cell culture (6), blood coagulation
by argon plasma (6,11e15), and dermatology (7,16e19).
The most frequently used plasma devices in medical
applications are the dielectric barrier discharge (DBD)
(8), the floating electrode dielectric barrier discharge (FE-
DBD) (6,12) and the plasma needle (16). The plasma nee-
dle device was originally designed for dental applications
(20). It produces a small-diameter low-power nonthermal
atmospheric pressure glow discharge. A radiofrequency
(RF) high-voltage signal is applied to a needle-shaped
electrode located inside a concentric gas flow nozzle with
a diameter of a few millimeters.
The plasma needle generates a multitude of reactive
oxygen species (ROS) together with reactive nitrogen
species (RNS) as well as ultraviolet (UV) radiation, and
these can reach the surface of the living tissue (6). These
reactive species can play a key role in proliferation
processes, for instance, involved in collagen production
of fibroblasts and synthesis of growth factors (21,22).
The small size of the plasma needle device allows focus-
ing a specific treatment on spots with a few-millimeter
diameter without applying excessive heat to the living
tissue. The aim of the present paper is to report the effect
of subsequent argon and helium plasma needle treatment
at room temperature and atmospheric pressure in regard
to the time required for skin wound healing in mice in vivo.
Experimental Set-up
Plasma Source
A system capable of generating plasma at atmospheric
pressure by means of a torch-like device known as a plasma
needle (16) constituted by an assembly of elements in
a coaxial configuration has been previously developed
and reported (23). A copper filament fulfills the role of axial
powered electrode; it is surrounded by a ceramic tube that
provides both mechanical rigidity and electric insulation.
The central electrode is visible only from the very tip of
the device where the encasing tube ends. The components
are assembled on a Nylamid SL frame, which includes
a gas nozzle and the electrode connection, producing
an |1-mm diameter plasma spot at low power levels. The
electrodes are fed by means of an RF voltage source at
13.56 MHz as reported in Reference (23). The helium
and argon gas fluxes are controlled by means of a commer-
cial MKS mass flow controller. The power density of
the argon and helium discharge has been measured at
|0.2 W/cm2. Considering that the tissue is heated during
the treatment by electric power dissipation, different collat-
eral effects can be induced. These effects depend not only
on the reached temperature, but also on the rate and dura-
tion of heating. Therefore, we measured the temperature
using an infrared (IR) thermometer directly in the wound.
Using this device we carried out the decontamination of
bacteria caused by S. mutans (24) and E. coli (25).
Plasma Exposure
Several series of plasma applications and different time
periods were tested at different power levels until approach-
ing the optimal combination of gases (data not shown) for
the treatment. All treatments were carried out at room
temperature, atmospheric pressure and according to the
same protocol. The experimentation was divided into two
phases: the first with argon gas and the second with helium
gas. In the first case, the exposure dose was 1.2 J/cm2 and in
the second case the exposure dose was 60 J/cm2. In the first
phase, an argon plasma needle operating at 0.5 SLPM flow
rate was projected onto the wound surface from a 3e5 mm
distance and, 1 min afterward, an incisive wound was
inflicted. After 20 min, the process was repeated twice with
the same pause time for 1 min each time. During this phase,
the mouse remained slightly sedated so that a degree of
coagulation, compatible with the specimen’s normal move-
ments, was attained. From this first treatment on, it was
decided to apply a coagulation procedure. Hemorrhage
must be avoided at this point (22) in order to carry out
the study. Thus, in the second phase, a helium plasma
needle at 1.5 SLPM was applied. After the respective
dose trials, a sequence of three applications, each of
5 min followed by a 60-min pause proved to be highly
successful. After each treatment the mice were released
immediately, allowing the mice normal movements and to
lick the wound.
Wound Production
Balb/c (28e32 g) specimens used in the experiment were
maintained in accordance with the Guide for the Care
and Use of Laboratory Animals (26). A wound (between
6 and 10 mm long and 4- to 5-mm in depth) was inflicted
on each rear leg of the specimen (Figure 1) with one of
the legs being used as a control sample. This was carried
out in six mice. The wounds were exposed to the plasma
needle under the previously mentioned conditions and were
exposed to the environment. The progress of the lesion was
 Plasma Needle Application on Mice Wound 171

Figure 1. Incisions practiced on both rear legs of mice. (A color figure can be found in the online version of this article.)

followed with a microscope (50X) and photographs were Results and Discussion
taken immediately after the three argon plasma needle
Spectral Analysis
applications and after the three helium applications, with
reference to the time of the incision. Using the monochromator measurements within a 200e800
nm range, the single spectrum in Figure 2A, corresponding
Histopathological Analysis to argon flux of 0.5 SLPM, was taken. Likewise, Figure 2B
shows a typical spectrum recorded from a 1.5 SLPM of He
Tissue samples were extracted from slaughtered specimens flux, both experiments being conducted at room tempera-
in the form of slices normal to each incision trajectory, both ture. The ROS and RNS are well recognized for playing
from the treated and control mice. The slices were fixed in a dual role with both deleterious and beneficial species.
10% buffered formalin for 24 h and then processed by The respective spectra (Figure 2) shows the hydroxyl
conventional histopathological techniques. The tissue radical (OH) in both discharges, whereas nitric oxide
samples were hematoxylin-eosin stained (27) at the end, (NO) was found only in the helium discharge. NO, in
analyzed on an optical microscope and photomicrographed particular, is known to possess many important physiolog-
at 100X. In particular, for histopathological studies, six ical characteristics including, in the case of skin wounds,
mice in each test were used, i.e., six for each argon treat- that of healing by inhibiting the production of superoxide
ment time (1, 20 and 40 min) and six for each helium treat- anions (29e32).
ment time (60, 120, and 180 min). Figure 2A presents the emission bands from the argon
discharge in the wavelength range of 200e800 nm, mainly
Emission Spectroscopy of hydroxyl radical (OH) in the band of 306 nm; this is
We assembled the electromechanical spectroscopy diag-
nostic instrumentation around a controlled l-Minman mono-
chromator (Minuteman, model 305 SMP, l-Minuteman
Laboratories, Inc., Acton, MA) (28) endowed with a 0.5 m
focal length Czerny-Turner coma-corrected optical system
and interchangeable gratings. When a standard 1200
groove/mm grating is selected, the instrument exhibits
a reciprocal linear dispersion |1.67 nm/mm and a nominal
first-order half-width resolution of 0.03 nm. The grating
control consists of a DC engine-powered gearbox driving
a leadscrew, which provides 12 speeds (from 0.05e200
nm/min) and a reversible scanning mechanism. With this
grating control in operation, the monochromator range
covers from 105e1200 nm. According to the region of
energy aimed by our particular experiments (200e800
nm), an R955 photomultiplier tube (PM) with a compatible
spectral sensitivity was installed. Finally, a 300-cm long Figure 2. Emission spectra of the plasma revealing the radiation intensity
quartz optic fiber of 1-mm diameter core matched with the in the UV range from: (A) 0.5 SLPM argon flux, (B) 1.5 SLPM helium
monochromator is located in front of the plasma at |5 mm. flux. (A color figure can be found in the online version of this article.)
172 Garc
ıa-Alcantara et al./ Archives of Medical Research 44 (2013) 169e177

Figure 3. Coagulation process without and with argon plasma needle: (A), (B) start, (C), (E), (G) control at 1 min, 20 min and 40 min after wound, respec-
tively; (D), (F), (H) wound treated for 1 min at 1, 20 and 40 min after wound infliction, respectively, without treatment (control): (A) (C) (E) (G). With
treatment (argon): (B) (D) 0, (H). (A color figure can be found in the online version of this article.)

attributed to water vapor. Because the process is carried out
in the atmosphere, the bands of the N2 second positive
system at 316 nm, 337 nm and 380 nm are attributed
to the nitrogen that is present in the discharge. The N2þ first
negative system is at 358 nm, O at 777 nm and Ar in
404 nm, 675 nm, 696 nm, 706 nm, 727 nm, 738 nm,
751 nm, 763 nm, 772 nm, and 794 nm; these bands
are in good agreement with previously reported data
(33e36).
As to the helium discharge (Figure 2B) in the same
wavelength range (200e800 nm), the emission bands are
as follows: Nog third positive system at 258e259 nm,
hydroxyl radical (OH) in the band of 306 nm, the bands
of the N2 second positive system at 295 nm, 316 nm,
337 nm, 375 nm, and 380 nm, the N2þ first negative system
at 358 nm, 391 nm, 427 nm, O 777 nm, as well as He at 396
nm, 400 nm, 437 nm, 447 nm, 587 nm, 667 nm, 706 nm,
729 nm and 781 nm. These bands are in agreement with
other studies (37e40). From Figures 2A and 2B, it is noted
on which band NOg in helium plasmas is generated; this
situation can promote the wound healing process (29e41).
Temperature Monitoring
The mice wound temperature was measured with an IR
thermometer and without treatment was close to 28 C.
During the application of the plasma needle, the wound
temperature increased almost instantaneously and reached
its maximum in |8e10 sec at 3e5 mm of distance between
the plasma source and the tissue surface. In the experi-
ments, the plasma needle was in relative motion, and the
wound temperature increased typically to 41 C and 33 C
for the argon and helium gases, respectively. Lower temper-
atures were measured with increasing scan velocities. In
 Plasma Needle Application on Mice Wound 173

all experiments, the exposure dose with argon (1.2 J/cm2)
and helium (60 J/cm2) plasma treatments is !135 J/cm2,
a dose that produces tissue damage as reported by Dobrynin
et al. (42).
Treatment without and with Argon Plasma Needle
We varied the experimental conditions until an optimal
condition of the wound healing was determined. Thus,
Figures 3A and 3B display incisions immediately after they
are carried out. The untreated control cut on the right-hand
side rear leg of each specimen exhibits profuse bleeding for
|1 min after the incision (Figure 3C). At about 20 min, the
bleeding restricts itself to the wound borders (Figure 3E);
a similar condition is maintained at t 5 40 min (Figure 3G).
After the first 1-min treatment with argon, the blood spot
acquires a darker coloration and produces a thin dry crust of
clotted blood that seals the wound (Figure 3D). Following
the second argon treatment for 1 min (Figure 3F), the dry
crust becomes thicker and shrinks while exuding some
droplets of a reddish lymphatic fluid. At the third appli-
cation of 1 min, the dry crust becomes more contracted
and covered by a number of discrete exudation points
suggesting the initiation of the tissue repairing process
(Figure 3H).
Treatment without and with Helium Plasma Needle
Evolution of the untreated control wound is represented
in Figures 4A, 4C, 4E and 4G taken at 60, 120, 180
and 240 min, respectively, after the wound infliction.
Figure 4A reveals the presence of a thin blood clot layer
covered with discrete exudation points. A more abundant
lymph secretion becomes detectable on Figure 4C at that

Figure 4. Healing asepsis process without (left-hand side) and with (right-hand side) helium plasma needle: (A), (C), (E), (G) control at 60, 120, 180 and 240
min after wound, respectively; (B), (D), (F) and (H) wound treated with helium needle plasma for 5 min at the same times after wound infliction. Without
treatment (control): (A), (C), (E), (G). With treatment (argon): (B), (D), (F), (H). (A color figure can be found in the online version of this article.)
174 Garc
ıa-Alcantara et al./ Archives of Medical Research 44 (2013) 169e177

time (t 5 120 min). From t 5 180 min on (Figures 4E and
4G), little significant change is apparent in the wound. Fluid
exudates are present in all cases, although no significant
tissue restoration becomes visible.
The helium-treated wound (Figure 4B) shows, after
60 min of its infliction, no evident evolution in tissue resto-
ration in comparison with the last argon treatment
(Figure 3H). The second helium application (Figure 4D)
makes the wound surface ostensibly shorter and encrusted
with a thin dark blood layer. The wound width is shrunk
by healing from its borders towards the central zone.
During the third helium plasma treatment (Figure 4F), the
wound borders have practically joined together with little
traces of serous exudates. The tissue looks practically
restored, leaving only a narrow visible line even after the
mouse leg has been flexed and extended several times.
From the aforementioned results, it is evident that
the helium plasma needle accelerates the wound healing
process in all the treated mice. It has been shown by exper-
imentation that there are effects that can modify wound heal-
ing: a) ROS promotes angiogenesis that is the production of
new blood vessels or repair of existing ones (30,31,41); b)
the effect of plasma screening, which promotes blood clot-
ting allowing activation of platelets and formation of fibrin
filaments in blood (6,12,13). Recent research indicates that
NO promotes the acceleration of the healing process and
wound closure (29,32,42). It is possible that NO generated
by helium plasma needle has a beneficial effect on the heal-
ing process. Future plans are to investigate this effect.
Histological Tests with and without Argon Plasma Needle
We used six mice in each of these particular tests, either in
argon or helium treatments. The histopathology of the
control wounds presented a blood clot relevant evolution
between min 1 and 40 (Figures 5A and 5C) measured with
respect to the time of the incision; 33% of the mice popu-
lation showed signs of blood coagulation from the first
minute on. This proportion increased to 66% at min 40,
although 100% of the cases developed hemorrhagic foci
under the blood clots. An acute inflammatory process char-
acterized by the presence of neutrophils took place next to
the wound borders in 86% of the population. In all cases,
the edges of the wounds remained constant without indica-
tion of any small junction thereof.
In the case of argon plasma-treated wounds and in strict
synchronic correspondence to the previously discussed
images, 100% of the treated mice showed a degree of coag-
ulation (Figures 5B and 5D). The same percentage pre-
sented hemorrhagic foci under the clots. A mild
inflammatory reaction around the lesions occurred in 86%
of the population. In all treated and control cases, muscle
fiber fragmentation was always present.

Figure 5. Histologic slices: (A) and (B) at 1 min, (C) and (D) at 40 min. Without treatment (control): (A) A bulging blood clot fills the cavity, separates the
edges and covers the wound (8). Under the blood clot, a small hemorrhagic point is observed (/). (C) A flat blood clot covers part of the wound surface
(8) separating the edges of it (/). With treatment (argon): (B) A concave thin blood clot covers the lesion cavity (8), which joins together the wound
borders (/). (D) A blood clot lies at the bottom of the wound (8). The wound edges are collapsed into the wound cavity coming closer across it (/).
(A color figure can be found in the online version of this article.)
 Plasma Needle Application on Mice Wound 175

Figure 6. Histologic slices: (A) and (B) at 60 min, (C) and (D) at 180 min, (E) and (F) at 240 min. Without treatment (control): (A) The borders are still
separated by (/) a small blood clot that fills the wound cavity (8). (C) The wound surface is covered by abundant pale proteinaceous exudate that fills the
wound cavity and still separates the wound borders. One of them is partly collapsed toward the wound cavity (/). (E) Proteinaceous exudate without eryth-
rocytes covers the wound surface and fills the wound cavity, still separating the wound borders (/). With treatment (helium) (B) The wound borders are
partly collapsed into the wound cavity which joins them together (/). Inside the cavity, a blood clot is disintegrating (8). (D) The borders collapse
completely to the bottom of the wound cavity (/) by approximating the wound edges into the wound cavity, resulting in a macroscopic appearance of a thin
line. (F) The wound borders are completely joined, forming a flat surface and demonstrating very good tissue regeneration. (A color figure can be found in the
online version of this article.)

Histological Test without and with Helium Plasma Needle restoration (Figure 6F), which suggest that the incisions
were completely healed. Plasma needle doses of argon
The helium-treated mice exhibited, from the start, only clot
and helium have together proven to promote and/or hasten
traces in the wounds (Figures 6B, 6D, and 6F), which remain
the coagulation and healing physiological processes with
during the ensuing two applications. The occurrence of
respect to the untreated samples. Similar effects have been
hemorrhagic points decreased from 86e0%, and inflamma-
observed on FE-DBD devices (11,16,42).
tion abated along with treatment progress. A visible increase
In conclusion, we have developed a methodology for
of serous exudates lining the wounds reached 66.7% of the
in vivo coagulation (by argon plasma) and healing (by
cases 2 h after the treatment and 100% in 240 min. Only
helium plasma) of wounded mouse tissue by means of
66.7% exhibited phagocytosis of fragmentary muscle fiber.
a plasma needle, in a very short time and without any
Furthermore, the wound borders of 50% of the population
collateral damage of the tissue (Figure 4H). Wound healing
had joined together by that time while coming closer in
is a complex biological and biochemical process with
the rest of the cases. After 240 min the treated specimens
a multitude of variables involved. However, complicated
showed an almost complete lip joining, while the untreated
as the healing process may be, some facts are clear: for
ones remained without any appreciable improvement.
wound healing to occur, tissue damage during treatment
The control wounds presented (at least) one clot, one
must be minimized or eliminated while wounds must be de-
hemorrhagic point and one mild inflammatory reaction
contaminated in order to prevent any bacterial invasion.
after 240 min (Figures 6A, 6C, and 6E). There were no
Our results with sequenced argon and helium plasma nee-
cases of wound borders being reunited and 67% of the pop-
dles are consistent with previous observations using
ulation exhibited the presence of opportunistic bacterial
different plasma technologies in order to treat wounds, indi-
colonies. At the same points in time, the treated wounds
cating that non-thermal plasmas decontaminate the zone of
presented joined wound edges and normal pigmentation
176 Garc
ıa-Alcantara et al./ Archives of Medical Research 44 (2013) 169e177
application and allow a rapid coagulation, stimulating the
healing processes. This may be mainly due to action of
reactive species and nitric oxide. A similar conclusion
has been reported by different investigators (4,7,11,12).
Additionally, mice tolerate the argon and helium plasma
treatment well and show no signs of distress even 1 day
post-needle treatment. This combined treatment of plasma
devices promises great applicability in the medical field
where tissue coagulation is recognized as a property of
non-thermal plasmas. The efficiency of the process could
be summarized as an ability to decontaminate wounded
tissue while stopping bleeding and inducing the formation
of a protective clot. Further studies, however, are required
in order to clinically prove this hypothesis.
To summarize, the present methodology represents the
basis for a procedure with multiple surgical applications
entailing a substantial clinical benefit. Although the meth-
odology is repeatable, studies have yet to be done,
especially with regard to the effects it may cause the NO
gamma in real time.
Acknowledgments
This study received financial support from CONACYT, Mexico.
The authors are grateful to the technical collaboration of C.
Alejandr
ı Cort
es, A. Reyes Pozos, P. Aguilar Vargas, M.T. Torres
Mart
ınez, P. Angeles Espinoza and I. Contreras Villa.
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