Electromagnetic field (EMF) induces cell death, therefore it seems to be an option for non-invasive treatment with low side-effect risks and without problems of drug interactions in chronic inflammatory diseases (1). Pulsating electromagnetic field (PEMF) stimulation showed anti-inflammatory effects in Crohn's disease patients by induction of peripheral blood mononuclear cells apoptosis and changes in cytokine profile (2). In the last decade, extracorporeal EMF stimulation of pelvic muscle floor has been introduced for treating urge and/or stress urinary incontinence, and also overactive bladder (OAB) (3, 4).
The urothelium as an "active barrier" plays an important role in storage and
voiding
via detrusor muscle motor activity modulation, as well as seeming
to contribute to OAB and LUT (lower urinary tract) symptoms (5). Neurogenic
and myogenic mechanisms, and also changes in the urothelium underlying overactive
bladder (OAB) have been described (6). In response to different stimuli, urothelial
cells can release many substances including prostaglandins (PG), adenosine triphosphate
(ATP), nitric oxide (NO), acetylcholine (Ach),
etc. which affect urinary
bladder compartments (smooth muscle, afferent and efferent nerve endings, interstitial
and immune cells). Afferent nerve fibres stimulation (
via vanilloid TRPV1-6
and ankyrin TRPA1 receptors) may release substance P (SP), calcitonin gene-related
peptide (CGRP) and interleukins generating blood vessels, mastocytes and lymphocytes
response, and, in a consequence, alter the urinary bladder sensory and/or motor
activity due to neurogenic inflammation (7-9). Overstimulation of afferent unmyelinated
C-fibres and its local effector function (
via neurogenic inflammation)
by substances released from urothelium may induce detrusor overactivity leading
to OAB symptoms and/or urge incontinence (10, 11). Diminishing the release of
the urothelial cells' mediators to reduce afferent nerve overstimulation seems
to be crucial in urinary bladder sensory and motor activity control. Moreover,
there is still no evidence on the influence of PEMF on the urothelium.
Therefore, the objective of our study was to examine the effect of pulsating electromagnetic field (PEMF) stimulation on rat urothelial cultured cells (RUCC).
MATERIAL AND METHODS
Animals
Urothelial cells were isolated from 15 adult female Wistar rats (weight: 200-250 g). Rats were housed individually per cage. The animal room was maintained at a constant temperature of 23°C, humidity and a 12:12 h alternating light-dark cycle. They were fed with animal food (Labofeed; Kcynia, Poland) without water restraint. The study has been approved by the Regional Animals Ethical Committee.
Urothelial cells isolation and culture
Rat urothelial cell cultures were prepared according to the procedure described
by Birder
et al. (12), with slight modification of the experimental protocol.
15 female Wistar rats were anesthetized with intraperitoneal injection of 1.2
g/kg urethane (Sigma-Aldrich, St. Louis, USA), and the urinary bladders were
removed and placed in cold Dulbecco's phosphate buffered saline (Sigma, Germany)
containing penicillin/streptomycin/fungizone (PSF; 1%; Sigma, Germany). The
bladder was cut open to expose the urothelium and incubated in dispase (2.5
mg/ml; Sigma, Germany) overnight at 4°C. Urothelial cells were gently scraped
from the underlying tissue, placed in trypsin - EDTA solution (0.25% wt/vol;
Sigma, Germany) for 10-15 min. at 37°C, and dissociated by trituration. Cells
were suspended in Dulbecco's modified Eagle's medium (DMEM; Sigma, Germany)
containing 10% fetal bovine serum - FBS (GibcoBRL; Invitrogen, Grand Island,
NY) to arrest the trypsin reaction and centrifuged at 416 g for 10 min. The
supernatant was removed and cells were suspended in keratinocyte basal medium
(Invitrogen) with 1% PSF, counted with a hemacytometer and seeded on 96-well
plate at density 0.5×10
6 cells/ml in fourfold
repetition separately each bladder cells and cultured at 37°C in a 5% CO
2
incubator of 90% humidity. The medium was changed every day. Cells were used
for experiments after 48 hours of culture.
Urothelial cell culture exposure to pulsating electromagnetic field
The generator which produces low energy pulsating electromagnetic field (PEMF) of 50 Hz and 45±5 mT peak was designed to generate such PEMF inside the cell culture incubator. The rationale for choosing such frequency of PEMF was as follows: firstly, frequency of magnetic stimulation is higher than the range, which directly depolarizes autonomic fibers; and secondly, the heating effect is minimal (13). The 96-well plate with urothelial cells was placed in a pocket of the generator. The PEMF was applied three times, for 4 hours each stimulation per day, with 24-hour intervals between stimulations. The control RUCC was in the same incubator, but in a distance of 35 cm from the PEMF generator to avoid the influence of magnetic field on the control culture.
In vitro animal model of rat urothelial cell culture (control group)
The control urothelial cultures (seeded in fourfold repetitions per animal)
at density 0.5×10
6 cells/ml/well were in the same
incubator, but in a distance of 35 cm from the PMS generator.
Cell death evaluation by flow cytometric analysis
Twenty four hours after last PEMF stimulation, the urothelial cells were harvested
from culture plates by trypsinization and washed three times with cold PBS (Sigma,
Germany) then stained according to the manufacturer's procedure for FACS analysis.
APC-conjugated annexin V (BD, Pharmingen
TM, USA)
was used to determine the percentage of cells within the population that were
undergoing apoptosis. Propidium iodide (PI) (BD, Pharmingen
TM,
USA) was used as standard flow cytometric
viability probe to distinguish
necrotic cells from
viable ones. Annexin V-APC positive cells were analysed
as apoptotic, Annexin V-APC and PI positive were either in the end stage of
apoptosis or undergoing necrosis and analysed as already dead, and PI positive
cells were necrotic. In apoptosis the cell membrane alterations consist of the
translocation of phosphatidylserine (PS) from the inner side of the membrane
to the outer layer, by which PS becomes exposed at the external surface of the
cell. Annexin V is a Ca
2+ dependent phospholipid-binding
protein with high affinity for PS. Therefore the measurement of annexin V binding
to the cell membrane surface is indicative for apoptosis cells (14). PI accumulation
inside necrotic cells distinguishes between apoptotic and necrotic cells. Apoptotic
cells can be distinguished from AnV- living cells by using flow cytometric procedure.
PI allows a further distinction of necrotic (AnV+/PI+), apoptotic (AnV+/PI-)
cells. Furthermore, when the cells are incubated with annexin V prior to harvesting,
the former cell populations can be separated from cells damaged during isolation
(AnV-/PI+).
For staining, urothelial cells were washed twice with cold PBS and resuspended
in 1× binding buffer (BD, Pharmingen
TM, USA) at
a concentration 1×10
6 cells/ml. Then 100 µl of
solution was transferred to a 5 ml culture tube and 5 µl of annexin V-APC and
5 µl of PI were added. Cells were gently vortexed and incubated in dark for
15 minutes at RT. Prior to flow cytometric analysis 400 µl of 1× binding buffer
was added and cells were analyzed on a FACS calibur flow cytometer (Becton Dickinson,
San Jose, CA) using Cell-Quest software. The Cell-Quest software provided the
percentage calculation of the cell types in RUCC. Controls to set up compensation
and quadrants included unstained cells, cells stained with annexin V-APC alone
(for FL-4 fluorescence) and cells stained with PI alone (detected in FL-3).
A minimum 10,000 events were collected on each sample.
Statistical analysis
All data was expressed as mean and (±) standard de
viation (SD) and compared using the Student t-test considering p<0.05 defined as significantly different.
RESULTS
in vitro rat urothelial cells culture (control RUCC)
The density of RUCC was 0.5×10
6 cells/ml. The
RUCC characterized high urothelial cells
viability (about >75% of
viable
urothelial cells in the culture). The rat urothelial cell culture was characterized
by 1.24% ±0.34% of apoptotic cells (AnV+ cells) and 11.03% ±1.55% of necrotic
cells (PI+ cells). The percentage of AnV+PI+ positive cells was 12.43% ±1.96%
(
Table 1;
Fig. 1).
Table 1. The percentages
of rat urothelial cells types (AnV+, PI+, AnV+PI+) in rat urothelial cells
culture (RUCC) unstimulated and stimulated with pulsating electromagnetic
field (PEMF). |
|
* vs. control
RUCC (p<0.05); Abbreviations: PEMF - pulsating electromagnetic
field;
RUCC - rat urothelial cells culture |
|
Fig. 1. The percentage of
apoptotic (AnV+ cells), necrotic (PI+ cells), as well as AnV+PI+ positive
urothelial cells in rat urothelial cells culture (RUCC) unstimulated and
stimulated with pulsating electromagnetic field (PEMF). |
Effect of pulsating electromagnetic field stimulation on rat urothelial cells culture
The flow cytometry analysis of rat urothelial cell culture was performed after
the last pulsating electromagnetic field (PEMF) stimulation of the cells culture.
PEMF stimulation significantly induced the apoptosis (an increase of 16.9%,
as compared to PEMF-unstimulated culture), and inhibited the necrosis (a decrease
of 36.3%, as compared to PEMF-unstimulated culture) of the urothelial cells
in rat urothelial cells culture (RUCC) (
Fig. 2). The percentages of apoptotic
(AnV+) and necrotic (PI+) urothelial cells obtained after PEMF stimulation were
1.45% ±0.16% (p=0.027) and 7.03% ±1.76% (p<0.001), respectively. Additionally,
AnV+PI+ positive urothelial cells measured as a percentage of double stained
cells - annexin V and propidium iodide positive in RUCC, upon PEMF exposure,
achieved lower level (9.48% ±3.40%, p=0.003), as compared to control unstimulated
RUCC (a decrease of 23.7%, as compared to PEMF-unstimulated culture) (
Table
1,
Fig. 1, 2).
|
Fig. 2. The percentage changes
of rat urothelial cells in RUCC (AnV+, PI+, AnV+PI+) after pulsating electromagnetic
field (PEMF) stimulation, as compared to unstimulated RUCC (control).
The percentage of increment (positive values) or decrement (negative values)
of the amount of different types of rat urothelial cells were compared
to the values of normal RUCC (set at 100%). For example, in RUCC after
PEMF stimulation there is increased amount of AnV+ cells, as compared
to control (PEMF unstimulated) RUCC. The increase is about 16.9% of the
mean value of PEMF-unexposed RUCC. |
DISCUSSION
The potential mechanisms of electromagnetic field (EMF) action on lower urinary tracts, especially urinary bladder are poorly described in the literature. Irrespective of this, the influence of EMF on urothelium is still unknown. Therefore, we explored the effect of pulsating EMF stimulation on rat urothelial cell culture (RUCC). Our main findings revealed that low-frequency PEMF stimulation (50Hz, 45±5 mT) induces apoptosis (increase of AnV+ cells) and inhibits necrosis (decrease of PI+ cells) of urothelial cells in culture.
Apoptosis (programmed process) and necrosis (incidental process, induced by
extracellular conditions) are two different ways of cell death (15). Independent
molecular pathways regulate apoptosis, such a [1] extrinsic
via Fas-R
or TNF-R death receptor, and [2] intrinsic or mitochondrial: the stress mediated
by pro-apoptotic members of Bcl-2 family
e.g. via drugs, PEMF,
etc. (16-18). However, the potential influence of PEMF on apoptosis and
necrosis of urothelial cells is still unknown. PEMF affects cell proliferation
in vitro, which depends on the cell's type, the magnetic field's physical
parameters (frequency, strength, waveform), and also time exposure. Additionally,
DNA damage, signal transduction pathway alterations, and changes in immune system
cells (
e.g. lymphocytes) function after PEMF exposure was described (19,
20). Apoptotic cells are phagocytosed and digested by macrophages, preventing
the release of intracellular substances to the extracellular space. Apoptosis
of the urothelium is at a low rate in naive conditions (21). Contrary to apoptosis,
urothelium necrosis distributes an extensive release of substances from dead
cells, which activates afferent nerve fibres and induces neurogenic inflammation
leading to detrusor overactivity or exacerbation of chronic urinary bladder
illness.
PEMF anti-inflammatory influence was described in Crohn's disease by induction
of peripheral blood mononuclear cells apoptosis and changes in cytokine profile
(2). Anti-inflammatory PEMF backgrounds may be due to free radicals profile
by the radical pair mechanisms. EMF inhibits reactive oxygen production by neutrophils
in inflammatory process in mice (22). Contrarily, a rise in the formation of
reactive oxygen species by macrophages derived from mouse bone marrow, indicate
the induction of an oxidative burst (23). The formation of reactive oxygen species
in different pathological conditions (
e.g. inflammation) may affect urinary
bladder function leading to OAB (24). Christmas (25) and Harrington
et al.
(26) studies revealed different T cells sub-populations within urinary bladder
mucosa, as follows: CD8
+ T cells are sparsely
scattered in the urothelium and present in lamina propria, while CD4
+
T cells are within the urothelium to a lower extent than CD8
+
T cells. Moreover, Liu
et al. (27) indicted that urothelial Ag-specific
CD4
+ T cells can function as direct effector cells
to induce bladder autoimmune inflammation independent of CD8
+
T cells. Apoptosis plays a main regulatory role in lymphocyte development and
homeostasis. However, the molecular mechanisms of PEMF-induced apoptosis are
inconsistent, EMF may reduce apoptosis
via Ca
2+
influx modulation and/or alteration of c-myc transcription rate (28). Moreover,
EMF induces free radical production and changes the redox homeostasis, and consequently
may lead to DNA-damage and apoptosis
via direct interaction between reactive
oxygen species (29). Also, Lai
et al. (30) revealed that the DNA strand
breaks after EMF in rat brain cells, and that effect was diminished by melatonin
(a radical scavenger).
Several probable mechanisms of PEMF action affect urinary bladder activity,
as follows: [1] changes in urothelium apoptosis/necrosis balance, and secondarily
in cellular substance release regulating the nerves, interstitial cells, and
immune cells (lymphocytes,
etc.), [2] direct effect on nerve fibres (depolarization)
and sub-population of lymphocytes in urinary bladder mucosa, [3] changes in
the formation of reactive oxygen species.
Recently, magnetic therapy (MT) has become an alternative option for the treatment
of urge and/or stress urinary incontinence, and overactive bladder (especially
in the case of neurogenic detrusor overactivity). The advantages of such treatment
are the completely non-invasive nature, excellent safety and tolerability (31).
However, the evidence based on randomized controlled trials for evaluation of
the efficacy of MT in urinary incontinence and OAB treatment is generally poor
(32). Morris
et al. (4) observed that MT in women with idiopathic detrusor
overactivity diminishes detrusor contractility in the acute term of MT, and
also reduces urge episodes in prolonged mode. The potential mechanisms of electromagnetic
field action on lower urinary tracts (LUT) are only partially described, especially
due to neuromuscular compartments. It is believed that nerves (afferent, efferent,
and autonomic fibres) are particularly sensitive to the effects of PEMF-induced
eddy currents causing its depolarization, and, in a consequence, may regulate
local blood flow and other factors (still unknown). PEMF stimulation activates
efferent nerves and motor end plates of pelvic-floor muscle, providing better
muscle strength and endurance. On the other hand, PEMF may affect the somatic
nerve firing rate responsible for pelvic muscle and sphincter tone (3). Moreover,
the imbalance between apoptosis and necrosis of the urothelium affects the detrusor
muscle activity (11, 33). The 'pro-apoptotic' and 'anti-necrotic' action of
PEMF on urothelial cells may be potentially applied in the therapy of functional
disorders of urinary bladder (
e.g. overactive bladder, urge incontinence,
etc.). Nevertheless, further experimental and clinical studies are strongly
required for the evaluation of the potential role of PEMF actions on urinary
bladder.
A low-frequency pulsating electromagnetic field (PEMF) induces apoptosis and diminishes necrosis of urothelial cells. However, further evaluation is strongly required to explain the potential molecular mechanism of pulsating electromagnetic field application on urothelium.
Conflict of interests: None declared.
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