Campeanu2014 Article Near Infraredlow Levellasersti (1) [627422]

ORIGINAL ARTICLE
Near-infrared low-level laser stimulation of telocytes
from human myometrium
Razvan-Alexandru Campeanu &Beatrice Mihaela Radu &Sanda Maria Cretoiu &
Daniel Dumitru Banciu &Adela Banciu &Dragos Cretoiu &Laurentiu Mircea Popescu
Received: 15 January 2014 /Accepted: 24 April 2014 /Published online: 29 May 2014
#The Author(s) 2014. This article is published with open access at Springerlink.com
Abstract Telocytes (TCs) are a brand-new cell type frequent-
ly observed in the interstitial space of many organs (see www.
telocytes.com ) .T C sa r ed e f i n e db yv e r yl o n g( t e n so f
micrometers) and slender prolongations named telopodes. At
their level, dilations —called podoms (~300 nm), alternate
with podomers (80 –100 nm). TCs were identified in a
myometrial interstitial cell culture based on morphological
criteria and by CD34 and PDGF receptor alpha (PDGFR α)
immunopositivity. However, the mechanism(s) of telopodes
formation and/or elongation and ramification is not known.We report here the low-level laser stimulation (LLLS) using a
1,064-nm neodymium-doped yttrium aluminum garnet
(Nd:YAG) laser (with an output power of 60 mW) of thetelopodal lateral extension (TLE) growth in cell culture. LLLS
of TCs determines a higher growth rate of TLE in pregnant
myometrium primary cultures (10.3±1.0 μm/min) compared
to nonpregnant ones (6.6±0.9 μm/min). Acute exposure
(30 min) of TCs from pregnant myometrium to 1 μM
mibefradil, a selective inhibitor of T-type calcium channels,
determines a significant reduction in the LLLS TLE growth
rate (5.7±0.8 μm/min) compared to LLLS per se in same type
of samples. Meanwhile, chronic exposure (24 h) completely
abolishes the LLLS TLE growth in both nonpregnant and
pregnant myometria. The initial direction of TLE growthwas modified by LLLS, the angle of deviation being more
accentuated in TCs from human pregnant myometrium than in
TCs from nonpregnant myometrium. In conclusion, TCs frompregnant myometrium are more susceptible of reacting to
LLLS than those from nonpregnant myometrium. Therefore,
some implications are emerging for low-level laser therapy(LLLT) in uterine regenerative medicine.
Keywords 1064Nd:YAG laser .Low-level laser stimulation .
Telocytes .Telopodes .Human myometrium .Pregnancy
Introduction
Thorough knowledge of the structure of the uterine wall is
essential to contribute to the understanding of reproductivefunction. Alterations of normal function of human uterus are
reported in pregnant and nonpregnant state. Often these dis-
orders implicate the reproductive function and are difficult tomanage in the absence of a specific treatment.
Telocytes (TCs) were recently described as stromal/
interstitial cells found in many organs (for details, visitRazvan-Alexandru Campeanu and Beatrice Mihaela Radu contributed
equally to this work.
R.<A. Campeanu :B. M. Radu :D. D. Banciu :A. Banciu
Department of Anatomy Animal Physiology and Biophysics, Facultyof Biology, University of Bucharest, 050095 Bucharest, Romania
R.<A. Campeanu
Neuroscience Area, International School for Advanced Studies(SISSA), 34136 Trieste, Italy
B. M. Radu
Department of Neurological and Movement Sciences, University ofVerona, 37134 Verona, Italy
S. M. Cretoiu
:D. Cretoiu :L. M. Popescu ( *)
Department of Cellular and Molecular Medicine, Carol DavilaUniversity of Medicine and Pharmacy, 050474 Bucharest, Romania
e-mail: [anonimizat]
S. M. Cretoiu
Department of Ultrastructural Pathology, Victor Babe șNational
Institute of Pathology, 050096 Bucharest, Romania
D. Cretoiu
Department of Molecular Medicine, Victor Babe șNational Institute
of Pathology, 050096 Bucharest, Romania
L. M. Popescu
Division of Advanced Studies, Victor Babe șNational Institute of
Pathology, 050096 Bucharest, RomaniaLasers Med Sci (2014) 29:1867 –1874
DOI 10.1007/s10103-014-1589-1

www.telocytes.com ) including the human uterus [ 1,2].
Transmission electron microscopy is considered to be the
most suited method for TCs identification [ 3,4]. TCs can
also be identified by CD34 and PDGF receptor alpha
(PDGFR α) immunohistochemistry [ 5–8]. The function of
TCs is not well understood yet; however, evidence pointstowards a role of telopodes in the coordination of the sur-
rounding cells by exosome/ectosome release [ 9–11]. TCs
display electrical activity [ 12] and have been observed to form
homo- and heterocellular junctions [ 4]. Currently, cell culture
has emerged as an important research method for studying the
TCs behavior [ 13]. Time-lapse microscopy revealed dynami-
cally moving telopodes which were supposed to serve as
guiding wires for other cells in coculture [ 12]. The process
standing behind this dynamics of telopodes is still to beunderstood, and information about the biophysical properties
of the telopodal plasma membrane would bring new insights.
To this purpose, we have decided to stimulate by near-
infrared (NIR) laser the telopodes for testing their ability to
grow and the possibility of stimulation of telopodal lateral
extension (TLE) growth. The ability of TCs to form homo-and heterocellular contacts w ith various cell types (e.g.,
myocytes, immune cells, stem cells, etc.) in different organs
[14–16] has a tremendous medical impact. The possibility of
influencing their dynamics in vitro and in vivo by means of
low-level NIR guidance can open new perspectives in uterineregenerative medicine.
The idea of optical stimulation and guidance was exten-
sively tested on primary neuronal cell cultures or neuronal celllines (for review, see [ 17–22] using low-level laser stimulation
(LLLS)). Moreover, other types of cells, such as Swiss 3T3
cells, extend pseudopodia towards NIR light sources [ 23].
Although TCs extend long telopodes with dynamic movement
[12] and are good candidates for optical stimulation by means
of LLLS, the topic is still uncovered. The goal of our studywas to identify, for the first time, the differences of TC
response to LLLS between nonpregnant and pregnant human
myometria.
Materials and methods
Tissue samples
Five biopsies of human myometrium were obtained from
different hysterectomy specimens (benign indications) of
nonmenopausal women (mean age 42.5 years). Other fivespecimens were obtained from the uteri of pregnant primipara
women (between 38 and 40 weeks of gestation, mean age
32.5 years), at the time of cesarean section. All patientsreceived information about the study and signed an informed
consent file. All experiments have been carried out in accor-
dance with the EU guidelines and approved by the BioethicsCommittee of “Carol Davila ”University of Medicine
Bucharest.
Cell cultures
Human myometrial samples were collected into sterile tubes
containing Dulbecco ’s modified Eagle medium (DMEM) sup-
plemented with fetal bovine serum (FBS) 2 %, HEPES
(1.5 mM), as well as 10,000 IU/ml penicillin, 0.2 mg/mlstreptomycin, and 0.50 mg/ml amphotericin (Sigma Chemi-
cal), placed on ice and transported to the cell culture labora-
tory. Samples were processed within 30 min from surgery.Cells were cultured using the procedure described in detail
elsewhere [ 12].
Immunofluorescence
Immunofluorescent staining was performed on cells cultured
on coverslips, at fourth passage. The cells were fixed in 2 %
paraformaldehyde for 10 min, washed in phosphate-buffered
saline (PBS), and then incubated in PBS containing 1 %bovine serum albumin (BSA) for another 10 min. Cells were
washed again and permeabilized in PBS containing 0.075 %
saponin for 10 min (all reagents were from Sigma Chemical,St. Louis, MO, USA). Incubation with the primary antibodies
was performed for 1 h, at room temperature, using antihuman
antibodies: CD34, goat polyclonal (sc-7045), 1:50 (SantaCruz Biotechnology, Inc., Heidelberg, Germany), and
PDGFR αrabbit polyclonal (sc-338), 1:100 (Santa Cruz Bio-
technology, Inc., Heidelberg, Germany). After three serialrinses, the bound primary antibodies were detected with sec-
ondary donkey anti-goat antibody conjugated to Alexa Fluor
546, 1:250, and goat anti-rabbit antibody conjugated toAlexa Fluor 488, 1:250; all were from Invitrogen Molecular
Probes, Eugene, OR, USA. Nuclei were finally counter-
stained with 1 μg/ml 4 ′,6-diamidino-2-phenylindole (DAPI)
(Sigma-Aldrich).
Negative controls were obtained following the same pro-
tocol, but omitting the primary antibodies. Samples were
examined under a Nikon TE300 microscope equipped with a
Nikon DS-Qi1 camera, Nikon PlanApo ×20 and ×40 objec-tives, and the appropriate fluorescence filters.
Near-infrared low-level laser stimulationThe optical stimulation of the TLE growth was done by means
of a MicroTweezers Module twinflex Rel. 4.2 system (CarlZ e i s s ,G e r m a n y )m o u n t e do na ni n v e r t e dm i c r o s c o p e
AxioObserver D1 (Carl Zeiss, Germany). We used a diode-
pumped solid-state IR neodymium-doped yttrium aluminumgarnet (Nd:YAG) laser (Ventus 1064-3000, Carl Zeiss), con-
tinuous wave (cw) mode, wavelength 1,064 nm, power
3,000 mW, transverse mode TEM
00, beam divergence1868 Lasers Med Sci (2014) 29:1867 –1874

<1 mrad, beam diameter 2.5 mm. The parameters of the laser
beam (e.g., output power, spot size, position) were controlled
by the RoboSoftware 4.3 Pro SP2 (Carl Zeiss, Germany). Thebeam was focused on the cells through a Plan-Neofluar ×100/
1.3 oil objective. During telopodal stimulation, the laser out-
put power was set to 60 mW, and the spot size was 2 μm. The
laser spot size was controlled and done by optically de-
focusing the beam to rich the desired size, similar to [ 17].
The beam was applied on the telopode surface as pulses of 1 slength with a frequency of 0.1 Hz for appropriate periods of
stimulation. The whole optical setup is placed on top of a
vibration-isolated table (Thorlabs, USA).
The experiments of optical stimulation of TCs consist in
exposing a viable telopode to a laser beam (as described
above) by placing approximately half of the laser beam on aTLE. The laser beam position is continuously adjusted as the
telopode expands laterally. The TLE growing speed has been
calculated from the moment it started to grow after beingstimulated with the laser to the moment it stopped growing
and started to retract; considering the growing distance in a
given time, the average growing speed was estimated.
TCs were continuously perfused using a MPS-2 multichan-
nel perfusion system with a micromanifold of 100 μm( W o r l d
Precision Instruments, USA) at a rate flow of 1 μl/s. In the
acute experiment, LLLS is performed before (control) and
after 30 min of mibefradil (1 μM) (from Sigma-Aldrich, St.
Louis, MO, USA) continuous perfusion on two different
telopodes of the same TC. TCs were chronically (24 h) ex-
posed to mibefradil (1 μM) by overnight incubation at 37 °C
in a humidified atmosphere (5 % CO
2in air), and LLLS was
performed in the next day. In both types of treatments,
mibefradil is prepared into DMEM supplemented with FBS10 %.
Statistical analysisData are analyzed and plotted using Excel (Microsoft, Red-
mond, WA, USA). The values of growth rate are reported asmean ± SD. Unpaired Student ’sttest was employed to com-
pare the growth rates upon LLLS on TCs from nonpregnant
vs. pregnant myometrium. Meanwhile, paired Student ’sttest
was used to compare growth rates upon LLLS on TCs from
pregnant myometrium before and after mibefradil treatment.
Results
In this study, we identified TCs in myometrial cell culture,
using accepted criteria: morphology under phase contrast
microscopy and immunocytochemistry criteria in fluores-cence microscopy. TCs were seen as cells with very long
telopodes in phase contrast microscopy (Fig. 1a). In fluores-
cence microscopy, CD34-positive cells were seen (Fig. 1b)having approximately the same morphology with PDGFR α-
positive cells (Fig. 1c).
The mean duration of TLE stimulation was around 30 min
or above, and it was chosen depending on the moment TLE
stopped growing or retraction. The period of TLE exposure to
LLLS and the beam laser characteristics are comparable to theprevious reports on in vitro neuronal stimulation [ 17,18].
Reactivity of telopodes to LLLS varies in pregnant and
nonpregnant myometrium. There is a net difference betweenthe reactivities to optical stimulation of telopodes originating
from pregnant (Fig. 2b(a–c)) or nonpregnant uterus
(Fig. 2a(a–c)). Telopodes from pregnant uterus are more prone
to extend upon LLLS compared to those from nonpregnant
uterus. The maximal length of TLE upon LLLS in telopodes
from pregnant myometrium was 7.4 μm, while only a maxi-
mal growth of 2.2 μm was attained in telopodes from pregnant
myometrium. It should be also taken into account the diffi-
culty to obtain an LLLS TLE growth in TCs from nonpreg-nant myometrium, the required time of stimulation sometimes
being three times higher than that in pregnant myometrium.
We have found a speed rate of TLE growth of 10.3±
1.0μm/min ( n=6) in TCs from pregnant myometrium, which
is significantly higher than 6.6±0.9 μm/min ( n=5) in TCs
from nonpregnant myometrium, p<0.01, unpaired Student ’s
ttest (Fig. 3).
In both preparations, telopodes seem to accumulate a big
part of their resources near the stimulation area, and the laser
beam with finger-like structures was probed. In some exper-
iments, the telopode looks like it is breaking off its old
connection, maintaining only thin “anchors ”beyond the point
of stimulation.
We tested whether we could deviate by LLLS the direction
of TLE growth by at least 30° away from the original direction
of TC growth, following a protocol previously described on
NG108 neuroblastoma cell line [ 24]. The black arrows in
Fig.2a, b indicate the direction of the TLE. As we are working
on human tissue, it was impossible to accumulate a large
number of data as in previous studies on neuronal guidance.We have obtained a deviation below or up to 30° in the TCs
from nonpregnant myometrium (Fig. 2a), while a deviation
above 30° was attained in one preparation or even 72° in TCsfrom pregnant myometrium (Fig. 2b).
Twenty-five percent of TCs from pregnant uterus present
local thickening of the telopode upon LLLS (Fig. 2c(a–c)).
The local thickening phenomenon was directly correlated with
a delayed telopodal response to stimulation (>1,000 s). The
great variability of response to LLLS in pregnant myometriummust be considered as very important, probably being corre-
lated with distinct uterine morphological characteristics in
each patient.
We found that mibefradil modulates LLLS effect on TLE
from pregnant myometrium. It is already known that
mibefradil inhibits the bioelectrical signal and uterineLasers Med Sci (2014) 29:1867 –1874 1869

contractile forces [ 25], and we have tested the combined effect
of mibefradil and LLLS on TCs. TCs from pregnant
myometrium have been exposed to mibefradil (1 μM), a
selective antagonist of T-type calcium channels [ 26].
Acute (30 min) and chronic (24 h) exposure to mibefradil
was done, and the LLLS effect on TLE growth rate was
measured. In pregnant myometrium, the LLLS effect wastested on TCs per se (control; Fig. 4a(a–c)) and on TCs
exposed to mibefradil (1 μM; Fig. 4b(a–c)).
The chronic exposure to mibefradil determined a decrease
in the growth rate, 5.7±0.8 μm/min ( n=3), that is significantly
lower than that in control conditions (9.7±0.4 μm/min, n=3;
p<0.05, paired Student ’sttest; Fig. 5). The control value of
the growth rate for pregnant myometrium was found to be
different from the above-reported values. It should be noted
that the LLLS growth rate after acute mibefradil treatment inpregnant myometrium is below the growth rate for control
nonpregnant myometrium ( p<0.05, unpaired Student ’sttest).
After chronic exposure to mibefradil, LLLS performed onTCs from pregnant myometrium indicated an inhibition of
the growth process.
The LLLS-induced deviation in telopodal growth direction
was also monitored. Acute mibefradil treatment accentuatesthe angle of deviation above 30° (Fig. 4b). However, due to
the large variability of responses of the TCs from pregnant
myometrium to LLLS, it is difficult to estimate the exactincrease in the angle of deviation due to acute mibefradil
exposure.
The same experiment was performed on TCs from non-
pregnant myometrium, and both acute and chronic mibefradil
exposures completely abolished the TLE growth rate upon
LLLS.
Discussion
This study provides evidence for the presence of TCs in
myometrial interstitial cell cultures, identified by their
Fig. 1 TCs in myometrial cell
culture (fourth passage, day 3). a
Phase contrast microscopy of
typical a TC with very long
telopodes. bDistribution of CD
34 immunopositivity in the sameTC.cCells that display the TC
morphology express PDGFR α.
Scale bar =50μm1870 Lasers Med Sci (2014) 29:1867 –1874

morphology and CD34 and PDGFR αpositivity. Our findings
are in correlation with recent data suggesting that PDGFR α-
positive and CD34-positive cells are the same cell type —the
TCs [ 6–8].
Low-level laser therapy (LLLT) has an extensive medical
use, and the idea of using sub-thermal doses of laser light
dated from the early 1970s (see reviews [ 27,28]). Althoughthe medical use of high power NIR lasers in endometrial laser
intrauterine thermotherapy [ 29,30] or endometrial resection
a n da b l a t i o n[ 31,32] is a clinical routine, uterine LLLT
interventions are still pioneering. Therefore, our data on cel-
lular mechanisms underlying in vitro LLLS of TCs are push-ing forward this domain.
The differences in TC reactivity to LLLS highlighted by
our study in human nonpregnant and pregnant myometriumare not surprising since previous morphological studies have
proved significant differences in telopodal width and in
podomic thickness and gauge in human nonpregnant andpregnant myometrium [ 12]. A possible explanation of TC
differences in reactivity to LLLS might be related to distinct
cytoskeleton characteristics of TCs in pregnant uterus. Arecent study indicates that the expression of integrins (ITGA5,
ITGA7, ITGAV, and ITGB3) increases in pregnant
myometrium, and there is important colocalization with focal
adhesion proteins in human myometrium at term, and it was
also emphasized that mechanical signals are transmitted fromthe extracellular matrix through focal adhesions in pregnant
human myometrium [ 33].
Frequency domain NIR spectroscopy has proved that op-
tical properties of the human uterine cervix are influenced by
the hormonal status depending on the phases of menstrual
cycle [ 34] or by pregnancy [ 35]. Therefore, we might suppose
that the uterine remodeling in pregnancy is correlated with
changes in cellular dynamics and morphology, and TCs are
actively participating in this 3D network reorganization. All
Fig. 2 Comparative LLLS effect
on TCs from nonpregnant andpregnant myometrium cell
cultures (fourth passage, day 3). a
TLE growth of TCs fromnonpregnant myometrium. Theangle of the TLE deviation is
≤30°. The time course of this
effect is 20 s. bTLE growth of
TCs from pregnant myometrium.
The angle of the TLE deviation is
between 30° and 72°, while thetime course of this effect islonger —1 min and 6 s. Scale
bar=10μm.cTelopodal local
thickening upon optical tweezerstimulation was obtained in 25 %of TCs from pregnant myometrial
cell culture. Scale bar =40μm.
Yellow arrows indicate the TLE
subjected to LLLS. The black
arrows indicate the direction of
the TLE. Each red square
evidences the region of interestfor the LLLS effect
Fig. 3 The comparative average of telopodal growth rate upon LLLS
between TCs from nonpregnant and pregnant myometrium. * p<0.01,
unpaired Student ’sttestLasers Med Sci (2014) 29:1867 –1874 1871

these optical properties can be distinctly exploited in LLLS on
human pregnant and nonpregnant myometrium.
It was suggested that the TC network may be involved in
mechanical modulations and remodeling in various organs
[36]. In particular, this issue is very interesting in the uterus,
as the mechanical modulation exerted by TCs on other celltypes can be distinct in pregnant and nonpregnant
myometrium. The differences founded in TC reactivity to
LLLS can be related to extensive changes in cell-cell commu-nication residing in exosomes trafficking through the
telopodes. A very recent challenging idea considers the cross
talk between TCs-exosomes-gap junctions-cytoskeleton to bethe equivalent of the primitive nervous system [ 11].Cytoskeleton elements have had also being implicated as a
substrate responsible for guidance of neuronal growth upon
optical stimulation [ 19]. Telopodes also contain cytoskeleton
elements, proved very recently, when the cellular TC prote-
ome was analyzed and revealed the presence of proteins from
intermediate filaments (56 %), actin cytoskeleton (19 %), andmicrotubule (6 %) [ 37]. The higher growth plasticity of the
telopodes with respect to the neuronal growth cone should be
taken into account since this parallelism can be furtherexploited, as the growth rate reported in our study is signifi-
cantly higher than the previous data on neuronal optical
guidance [ 21,22].
The contribution of TCs in mechanical stretching during
uterine contraction is still to be understood; nevertheless,
membrane stiffness properties tested by LLLS can revealinteresting molecular mechanisms. It is of particular interest
the fact that the telopodes of TCs from nonpregnant
myometrium are intensively positive for vimentin [ 38], a
cytoskeleton protein. A recent study proved that vimentin is
very important in cytoplasmic stiffness of optically trapped
mouse embryonic fibroblasts using a 1,064-nm Nd:YAG laser
at a power of 200 mW [ 39]. The differences that we observed
between pregnant and nonpregnant myometrium can possiblybe explained in terms of telopodal stiffness due to vimentin-
based anchoring mechanisms. Additionally, TCs from the
human myometrium are positive for PDGFR, and PDGFRsignaling might also modulate the telopodal membrane me-
chanical properties, in an analog manner, as described in
vascular smooth muscle cells [ 40].
In conclusion, taking into consideration the profound phys-
iological myometrial remodeling during pregnancy, which
involves extensive structural and molecular changes, our
Fig. 4 Mibefradil effect on TLE upon LLLS in pregnant myometrium
(myometrial interstitial cell culture at fourth passage, day 3). aUntreated
TCs exposed to LLLS were considered as control. The time course ofLLLS effect in these images ( a–c) is 36 s. We can observe how a TLE
grows ( yellow arrow ).bMibefradil (1 μM) was perfused for 30 min, and
afterwards, TCs were re-exposed to LLLS. Comparison of the TLEgrowth rate reveals that in 1 min and 4 s, the length of TLE is approxi-
mately the same as that in control and that the angle of the deviation isslightly above 30°. Yellow arrows indicate the TLE subjected to LLLS.
The black arrows indicate the direction of the TLE. Each red square
evidences the region of interest for the LLLS effect. Scale bar =10μm
Fig. 5 The comparative average growth rate of TLE upon LLLS between
TCs from pregnant myometrium before and after acute mibefradil
(30 min) treatment. * p<0.05, paired Student ’sttest1872 Lasers Med Sci (2014) 29:1867 –1874

findings suggest that the molecular mechanisms activated in
TCs by means of LLLS are distinct in human nonpregnant and
pregnant myometrium. Therefore, LLLT in human uterusshould consider these cellular differences in pregnant and
nonpregnant myometrium, and the laser power and exposure
time should be adequately chosen.
Acknowledgments This work was supported by a grant from the
Romanian National Authority fo r Scientific Research, CNCS –
UEFISCDI, project number 82/2012 PN-II-PT-PCCA-2011-3.1-0553.
Dr. B.M. Radu is financed by the Italian ministerial fellowship D.M.198/2003. We are grateful to Dr. Laura C. Ceafalan and T. Regalia fortheir discussion and expert technical assistance. We also thank Dr. Mihai
Radu for his useful comments and suggestions that helped to improve this
manuscript.
Open Access This article is distributed under the terms of the Creative
Commons Attribution License which permits any use, distribution, and
reproduction in any medium, provided the original author(s) and the
source are credited.
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