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This study evaluated the possible effects of ultrasound (US) on gene expression in brain tissue of rat embryos. Four groups (n = 5 each) of pregnant Wistar Han rats were exposed to US for different durations (55, 100, 145, and 195 seconds) via a multifrequency transducer in the 2-dimensional imaging mode with a pulse duration of 1.29 microseconds, a pulse repetition frequency of 1 kHz, and a derated spatial-peak pulse-average intensity of 222.4 W/cm(2) on day 5, 9, 7, or 13 of gestation. Gene expression profiling was performed in fetal brain tissue (n = 5 per group) by quantitative reverse transcription-polymerase chain reaction arrays. The results indicated substantial alterations in gene expression. The most differentially expressed genes were Adamts5, Gadd45a, Npy2r, and Chrna1, which are implicated in important developmental signaling pathways. On the basis of our findings, routine short US examinations for monitoring fetal development are not contraindicated, but prolonged exposures should be used only when needed to obtain important diagnostic information.
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Gene Expression Profiling of Rat Fetuses
Exposed to 2-Dimensional Ultrasound
ltrasound (US) is a crucial tool in clinical obstetrics and in
vitro fertilization (IVF) procedures. It allows assessment of
fetal anatomy, behavior, and function. Accordingly, diag-
nostic US use in obstetrics has been growing rapidly to become an
integral part of prenatal care today.1,2 Most epidemiologic studies
tend to support the safety of diagnostic US use during pregnancy.1,3
In a study by Stark et al,4a total of 425 children exposed to diag-
nostic US and 381 matched control children were studied for
adverse effects at birth and again at examination between 7 and 12
years of age. No biologically significant differences between exposed
and unexposed children were found. However, advances in US tech-
nology enabled development of high-power devices that are used
to scan the fetus, and increasingly, this procedure is performed early
in gestation, a time when the fetus is known to be particularly sen-
sitive to external influences.3,5,6 Current US technology has become
more powerful, and its safety profile is largely unknown.1,2 It has
been estimated that fetal exposure using modern equipment could be
up to 8 times greater than that with older US systems.3,7 Unfortu-
nately, most studies about US safety were conducted in the past, using
older equipment. Thus, there is a basis for concern, especially because
few studies have researched the bioeffects of modern US devices on
the molecular level.3Studies regarding US-tissue interactions have led
Zvonko Hocevar, MD, Janez Rozman, PhD, Alja Videtic Paska, PhD, Robert Frangez, PhD, DVM,
Tomaz Vaupotic, PhD, Petra Hudler, PhD
Received July 21, 2011, from the University Med-
ical Center Ljubljana, Ljubljana, Slovenia (Z.H.);
Center for Implantable Technology and Sensors,
Ljubljana, Slovenia (J.R.); Faculty of Medicine,
University of Ljubljana, Institute of Biochemistry,
Medical Center for Molecular Genetics, Ljubljana,
Slovenia (A.V.P., T.V., P.H.); and Veterinary Fac-
ulty, University of Ljubljana, Ljubljana, Slovenia
(R.F.). Revision requested August 29, 2011.
Revised manuscript accepted for publication
December 19, 2011.
This study was supported by the Ministry of
Higher Education and Science, Republic of Slovenia
(research project J3-0259).
Address correspondence to Petra Hudler,
PhD, Faculty of Medicine, University of Ljubljana,
Institute of Biochemistry, Medical Center for
Molecular Genetics, Vrazov Trg 2, 1000 Ljubljana,
CNS, central nervous system; PCR, polymerase chain reaction;
RT, reverse transcription; US, ultrasound
©2012 by the American Institute of Ultrasound in Medicine |J Ultrasound Med 2012; 31:923–932 |0278-4297 |
Objectives—This study evaluated the possible effects of ultrasound (US) on gene
expression in brain tissue of rat embryos.
Methods—Four groups (n = 5 each) of pregnant Wistar Han rats were exposed to US
for different durations (55, 100, 145, and 195 seconds) via a multifrequency transducer
in the 2-dimensional imaging mode with a pulse duration of 1.29 microseconds, a pulse
repetition frequency of 1 kHz, and a derated spatial-peak pulse-average intensity of
222.4 W/cm2on day 5, 9, 7, or 13 of gestation. Gene expression profiling was performed
in fetal brain tissue (n = 5 per group) by quantitative reverse transcription–polymerase
chain reaction arrays.
Results—The results indicated substantial alterations in gene expression. The most
differentially expressed genes were Adamts5, Gadd45a, Npy2r, and Chrna1, which are
implicated in important developmental signaling pathways.
Conclusions—On the basis of our findings, routine short US examinations for moni-
toring fetal development are not contraindicated, but prolonged exposures should be
used only when needed to obtain important diagnostic information.
Key Words—central nervous system; fetal development; gene expression profiling;
safety; ultrasound
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to a better understanding of its mechanisms and side effects.
As a result, these studies revealed the nature of the undesired
side effects of US therapy and imaging, but only a handful of
them searched for aberrant gene expression patterns in fetal
tissues exposed to US.
In some older studies, biological effects of US were
reported in animals,6but very few harmful effects have
been shown in humans.4,8 For example, in a study using
monkeys as a model and testing older equipment, Taran-
tal and Hendrickx6found significant differences in birth
weight, crown-rump length, and white blood cell counts.
Although it is possible that these findings were chance
effects, it is also plausible that frequent exposure to US
could influence fetal growth.1,9–14
The main goal of our study was to examine whether
the exposure of pregnant rats to US influences the gene
expression in the central nervous system (CNS) of the
embryos. We identified differences in gene expression
related to different levels of US exposure. We wanted to
gather the differences in gene expression with a focus on
genes that encode the crucial molecular components of the
CNS, such as extracellular matrix and adhesion molecules,
components of signal transduction pathways, neuropep-
tides and their receptors, and neurotransmitters and their
receptors, using novel and pathway-focused gene expres-
sion profiling technology.
Materials and Methods
Preparation of Animals
All procedures and protocols were approved by the
Veterinary Administration of the Republic of Slovenia,
Ministry of Agriculture, Forestry, and Food (number
34401-44/2008/2). Forty-eight 11-week-old outbred
Wistar Han rats (HsdRccHan:WIST) were included in the
study. Rats were housed individually and provided with
food and water ad libitum. The mean weight of the animals
± SD at 11 weeks was 190 ± 20.87 g. The female animals
were mated with 6 male animals, and forty female ani-
mals became pregnant. An acrylic US exposure chamber
was specifically designed for experimental bioeffects stud-
ies. The rats were trained to use this chamber. Unanes-
thetized pregnant rats were held in position within the
exposure chamber, and the lateral surfaces of the chamber
were cut to allow exposure of the abdominal wall to the US
transducer (Figure 1). The animals were divided into 8
groups with 5 animals in each group. Four groups (U1–
U4) were exposed to US in the 2-dimensional imaging
mode at gestational days 5, 7, 9, and 13 (described in detail
below and in Table 1), and 4 groups (C1–C4) were not
exposed to US. The animals in the control groups were
handled the same way as the animals exposed to US regard-
ing other procedures, such as depilation of the abdomen
and restraint in the US exposure chamber.
Before US exposure, the pregnant rats’ abdomens
were shorn. Afterward, a chemical depilatory cream (Veet;
Reckitt Benckiser, Hull, Yorkshire, England) was used.
The cream was tested on a small patch of skin before appli-
cation to avoid allergic reactions to the ingredients in the
product. The cream and dissolved hair were removed with
a spatula and sponge.
Ultrasound Exposure
For US exposure, we selected a portable all-digital soft-
ware-controlled broadband multifrequency US imaging
system, which has been routinely used in human medical
clinics (M-Turbo; SonoSite, Inc, Bothell, WA) and an
HFL38x transducer (13–6 MHz, linear array, 6-cm scan
depth). The intensity of the acoustic signal was calibrated
as recommended by the manufacturer on the US machine
without the transducer; therefore, we note that the values
given in the text are those reported by the manufacturer
for this device, transducer, and imaging mode. The system
configurations were 2-dimensional/tissue imaging, small
parts, and auto gain automatic image optimization with a
scan depth of 6 cm. The mechanical index in all of our
scans was 0.71. The transducer operates in the multifre-
quency mode. We used a 2.5-cm tissue standoff pad
(spacer) between the transducer and animals, filled with
an acoustic gel (Figure 1) to enable secure contact with the
animal skin and eliminate air pockets between the trans-
ducer and the skin. Because the fetal rat brain is small, a
small spread of the US beam perpendicular to the scan
plane is sufficient to include the entire brain. A water bag
was acoustically coupled to the side of the rat opposite the
transducer to minimize the possibility of standing waves
or reflections that might have affected the exposure. Both
the left and right sides of the animals were exposed to equal
durations of US to ensure exposure in both uterine horns.
An actual range of acoustic intensities to which fetuses are
exposed was provided by the producer of the US machine.
Instead of measurements performed in a water bath (free-
field measurements), the engineers of the US machine
defined the acoustic intensities experimentally using their
own reference MicroMaxx system and HFL US trans-
ducer, replicating our experimental setup. The testing of
the US system showed that the derated spatial-peak pulse-
average intensity was 222.4 W/cm2, and the derated spa-
tial-peak temporal-average intensity was 13.64 mW/cm2.
The acoustic output power was 16.21 mW.
Hocevar et al—Gene Expression in Rat Fetuses Exposed to Ultrasound
J Ultrasound Med 2012; 31:923–932924
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The exposure schemes were as follows: the first group
of pregnant rats was exposed to diagnostic US in one ses-
sion for a total of 55 seconds (group U1) per side on ges-
tational day 5; group U2 was exposed to US for 100
seconds per side on gestational days 5 and 13; group 3 was
exposed to US for a total of 145 seconds per side on gesta-
tional days 5, 9, and 13; and group 4 was exposed to US for
a total of 195 seconds per side on gestational days 5, 7, 9,
and 13. Detailed data on US exposures per session are pre-
sented in Table 1. The sessions were performed on differ-
ent days to simulate human scanning conditions. Day 5
was chosen because in humans and rats, the blastula
attaches to the uterus lining (5–8 days after fertilization
in humans and 5 days after fertilization in rats). Days 7, 9,
and 13 were chosen because the rat embryo brain is most
sensitive to teratogenic influences during this period. The
embryos were monitored on the screen, and the sono-
grams were stored. Four groups of pregnant animals, des-
ignated C1–C4, were used as controls, one control group
for the US-exposed group of the corresponding number;
control animals were not exposed to US.
Euthanasia of all animals was performed strictly by following
the “Recommendations for Euthanasia of Experimental Ani-
mals” (part 2, directive 86/609/EEC, number L358, ISSN
0378-6978). The animals from the treated and control
groups were euthanized with embutramide (T61; Merck
Animal Health, Summit, NJ) administered intraperitoneally
at the same day of pregnancy as shown in Table 1 to match
the day of gestation between these groups of animals.
Because embryos were to be removed, an increased amount
of embutramide (1 mL) was administered to the dam and
maintained for a longer period to ensure that the euthanasia
J Ultrasound Med 2012; 31:923–932 925
Hocevar et al—Gene Expression in Rat Fetuses Exposed to Ultrasound
Figure 1. a, Ultrasound wave transducer (HFL38x, 13–6 MHz). b, Ultrasound transducer with tissue spacer. c, Ultrasound transducer with tissue
spacer filled with acoustic gel. d, Chamber used for exposure of pregnant rats to ultrasound.
3106jumonline.qxp:Layout 1 5/22/12 3:32 PM Page 925
solution had crossed the placenta. Afterward, the animals
were placed in a box that was completely filled with inhala-
tional carbon dioxide from a commercially available gas
cylinder.15 Death was confirmed by extraction of the heart.
Extraction of Brain Tissue and Preparation of Samples
Immediately after euthanasia, a full-length median laparo-
tomy was performed in all groups of animals. The exposed
uterus with embryos was carefully extracted and opened.
The brains of the embryos were extracted using a micro-
surgical technique, stored in TRIzol reagent (Invitrogen,
Carlsbad, CA), and snap frozen in liquid nitrogen. The
frozen tissues were stored at –70°C. To preserve RNA in
brain tissue samples, which could be degraded by harmful
heat generated during microsurgical extraction by infrared
waves produced by the tungsten lamps used in ordinary
optic microscopes, white light-emitting diodes were used
for illumination. All carcasses were temporarily collected
and kept at –70°C until they were destroyed according to
valid internal regulations at the Veterinary Faculty of the
University of Ljubljana (V89, V155, and V171).
Biochemical Procedures for Quantitative Gene
Expression Profiling
RNA was isolated with TRIzol reagent according to the
manufacturer’s protocol. Briefly, 5 embryo brains (from 1
selected maternal animal) per group were homogenized
in 1 mL of TRIzol reagent. After the addition of 200 µL of
chloroform and centrifugation, the RNA was precipitated
with isopropanol and washed twice with ethanol. Air-dried
RNA was dissolved in 10 µL of RNase-free water (Invitro-
gen). RNA was then purified using an RNeasy Mini kit
(Qiagen, Valencia, CA). The 260:280 ratio of RNA was in
the range from 1.8 to 2.0. In each tested and control group,
RNA was isolated in duplicate (5 brains from 2 selected
maternal animals per group) to obtain biological duplicates.
Synthesis of complementary DNA was performed from 1 µg
of total RNA using an RT2first-strand kit (SABiosciences,
Valencia, CA) according to the manufacturer’s instructions.
Pathway-focused RT2Profiler quantitative reverse
transcription–polymerase chain reaction (RT-PCR)
arrays (SABiosciences) were used to determine gene
expression profiles. Each array represents 84 genes
involved in specific pathways, 5 internal controls/house-
keeping genes, and 3 controls for detecting DNA con-
tamination, RNA quality, and general PCR performance.
The following RT Profiler quantitative PCR arrays were
used: (1) rat extracellular matrix and adhesion molecule
array (PARN-013A-12), (2) rat signal transduction Path-
wayFinder array (PARN-014A-12), (3) rat neurotrophin
and receptor array (PARN-031A-12), and (4) rat neuro-
transmitter receptors and regulator array (PARN-060A-12).
Quantitative PCR was performed using the RT Profiler
arrays and RT SYBR Green/ROX Fast PCR MasterMix
(SABiosciences) in a real-time PCR 7500 instrument
(Applied Biosystems, Foster City, CA). Real-time PCR
signals were evaluated using SDS version 1.4 software
(Applied Biosystems). Melting curve analysis was performed
(1°C/s increases from 60°C to 95°C, with continuous fluo-
rescence readings) at the end of the run to ensure that single
PCR products were obtained. Gene expression was nor-
malized to 5 internal controls/housekeeping genes
prespotted on the RT Profiler arrays: (1) ribosomal pro-
tein P1, large subunit (MGC72935), (2) hypoxanthine
guanine phosphoribosyl transferase (Hprt1), (3) riboso-
mal protein L13A (Rpl13a), (4) lactate dehydrogenase A
(Ldh), and (5) β-actin (Actx), using the RT2Profiler array
data analysis software (SABiosciences). The relative
expression of each RNA was calculated by the compara-
tive Ct (Ct) method where Ct = Ct (tested group
gene) – Ct (control group gene). The fold change in
expression of the gene of interest between the two samples
is then equal to 2^(–Ct). The relative expression ratios
of the target genes normalized to internal controls and rel-
Hocevar et al—Gene Expression in Rat Fetuses Exposed to Ultrasound
J Ultrasound Med 2012; 31:923–932926
Table 1. Ultrasound Exposure Scheme for the Pregnant Wistar Han Rats
Time of Exposure per Session Day of
(Total Time of Day of Exposure Pregnancy at
Animal Group Animals, n Exposure Per Side), s (Day of Gestation) Euthanasia
U1 5 55 (55) 9 16
U2 5 55, 45 (100) 5, 13 17
U3 5 55, 45, 45 (145) 5, 9, 13 15
U4 5 55, 45, 45, 50 (195) 5, 7, 9, 13 14
C1 5 NA NA 16
C2 5 NA NA 17
C3 5 NA NA 15
C4 5 NA NA 14
C indicates control animals; NA, not applicable, and U, ultrasound-exposed animals.
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ative to expression of genes in the control groups are pre-
sented in Figures 2–5. This relative expression is reported
as fold regulation, which is the inverse of the fold change in
the case of upregulated genes and the negative inverse of
the fold change in the case of downregulated genes.
We gathered qualitative and quantitative information on
differential gene expression in brains of rat embryos
exposed to US compared to control groups. We found sub-
stantial differences in expression of genes belonging to 4
functional gene groups, namely, (1) extracellular matrix and
adhesion molecules, (2) signal transduction pathways, (3)
neuropeptides and their receptors, and (4) neurotransmit-
ters and their receptors.
Altogether, 336 different genes belonging to 4 func-
tional groups of 84 genes were tested. A total of 64 genes
were assigned as differentially expressed, showing a greater
than 2-fold difference in their transcript levels in any of ani-
mal groups U1–U4. The 5 most differentially expressed
genes from each of the functional groups and their levels of
expression based on CNS-isolated RNA from embryos of
different US-treated animal groups (U1–U4) relative to
nontreated animal groups (C1–C4) are graphically pre-
sented in Figures 2–5. Genes were assigned their protein
products and physiological functions together with fold
regulation as detected by the quantitative PCR arrays.
Because of the fold regulation axis adjustment, the most
differentially expressed gene from each of the functional
groups is shown in a separate chart.
Among genes coding for extracellular matrix and
adhesion molecules (Figure 2) 45-fold upregulation was
observed for Adamts5 in the CNS of embryos extracted
from animal group U4, with the longest exposure to US.
In the CNS of embryos from groups U1, U2, and U3, it
was almost undetectable. Four other differentially expressed
genes, namely, Postn, Tgfbi, Thbs2, and Mmp1a, were all
found to be substantially downregulated on exposure to US,
with Postn and Thbs2 showing the strongest downregula-
tion in the CNS of embryos from group U3, Tgfbi showing
the strongest downregulation in the CNS of embryos from
groups U1 and U3, and Mmp1a showing the strongest
downregulation in the CNS of embryos from group U1.
J Ultrasound Med 2012; 31:923–932 927
Hocevar et al—Gene Expression in Rat Fetuses Exposed to Ultrasound
Figure 2. Differentially expressed genes coding for extracellular matrix and adhesion molecules (RT² Profiler quantitative polymerase chain reaction
array PARN-013A-12). The left chart represents the most differentially expressed gene, and the right chart represents other highly differentially
expressed genes in the group. U1a–U4a and U1b–U4b represent biological replicates.
3106jumonline.qxp:Layout 1 5/22/12 3:32 PM Page 927
Among genes coding for signal transduction path-
way molecules (Figure 3), the strongest upregulation, at
several hundred-fold, was observed for Gadd45a in the
CNS of embryos from group U3, whereas it was unde-
tectable in the CNS of embryos from groups U1, U2,
and U4. Approximately 7-fold upregulation was
observed for Birc1b in the CNS of embryos from groups
U3 and U4. Nos2, Tnf, and Cxcl1 were substantially
downregulated in groups U1, U2, and U4, respectively.
Among genes coding neuropeptides and their recep-
tors (Figure 4), the strongest upregulation was observed
for Npy2r in the CNS of embryos extracted from group
U2 and Maged1 in the CNS of embryos from group U4.
The strongest downregulation was observed for Gfra3 in
the CNS of embryos extracted from group U3. Bdnf was
most evidently downregulated in group U4. Ppyr1
showed a promiscuous profile of upregulation and down-
regulation between groups U1 and U2–U4.
Among genes coding neurotransmitters and their
receptors (Figure 5), most of the differentially expressed
genes were downregulated, with Chrna1, Chrnd, and Glra1
showing greater than 10-fold downregulation in the CNS
of embryos extracted from groups U3 and U4. Chat was
also substantially downregulated in group U4, whereas
Brs3 was evidently downregulated only in group U1.
This study was intended to provide new insight for the
medical/scientific community regarding the effects of US
on the gene expression patterns in the CNS of rat fetuses.
More precisely, it was aimed at making experimental con-
tributions to the research on US effects at the molecular level.
Although it is generally assumed that prenatal US use
is safe, very few studies have focused on possible side effects
at the molecular level, and much data have been obtained
using older US machines with far less output potential.1,2,7,9
Hocevar et al—Gene Expression in Rat Fetuses Exposed to Ultrasound
J Ultrasound Med 2012; 31:923–932928
Figure 3. Differentially expressed genes coding for signal transduction molecules (RT² Profiler quantitative polymerase chain reaction array
PARN-014A-12). The left chart represents the most differentially expressed gene, and the right chart represents other highly differentially expressed
genes in the group. U1a–U4a and U1b–U4b represent biological replicates.
3106jumonline.qxp:Layout 1 5/22/12 3:32 PM Page 928
The introduction of Doppler US, the use of more power-
ful equipment in obstetrics, and the trend of increased
use of US for nonmedical purposes requires continued
scrutiny of the safety issues of US.1,7 Recently, Ang et al10
showed that exposure of embryonic mice to US could
affect neuronal migration in the cerebral cortex. They
determined that a small, but statistically significant,
number of neurons failed to reach their proper positions
and remained scattered within inappropriate cortical layers
or in the subjacent white matter. They also emphasized the
fact that the cellular and molecular mechanisms of this
effect are unknown.
In our study, we found different gene expression pro-
files in the brains of fetuses exposed to US. In the group of
extracellular genes and adhesion molecules, we observed
45-fold upregulation of Adamts5, which was found to be
implicated in the destruction of the cartilage proteoglycan
aggrecan in arthritis (Figure 2). Its expression profile during
embryogenesis was recently studied in mice and was found
to have a role in the development of nervous system, head,
and neck structures and limbs, although its exact physio-
logic functions remain unclear.11 Nevertheless, the extra-
cellular matrix is a network that provides a substrate for cell
anchorage, serves as a tissue scaffold, guides cell migration
during embryonic development and wound repair, and has
a key role in tissue morphogenesis. The extracellular matrix
is also responsible for modulating signal transduction path-
ways, which ultimately affects cell proliferation, differentia-
tion, and death. Other differentially expressed genes from
this group, Postn and Thbs2, belong to cell adhesion mole-
cules, which are involved in cell-cell and cell-extracellular
matrix binding. Thbs2, for example, was shown to be impli-
cated in the inhibition of angiogenesis and promotion of
CNS synaptogenesis.12,13 Christopherson et al12 suggested
that human homologs thrombospondin 1 and 2 (rat Thbs2)
could act as permissive switches that time CNS synapto-
genesis by enabling neuronal molecules to assemble into
synapses within a specific window of CNS development.
J Ultrasound Med 2012; 31:923–932 929
Hocevar et al—Gene Expression in Rat Fetuses Exposed to Ultrasound
Figure 4. Differentially expressed genes coding neuropeptides and their receptors (RT² Profiler quantitative polymerase chain reaction array
PARN-031A-12). The left chart represents the most differentially expressed gene, and the right chart represents other highly differentially expressed
genes in the group. U1a–U4a and U1b–U4b represent biological replicates.
3106jumonline.qxp:Layout 1 5/22/12 3:32 PM Page 929
Clearly, the disruption of these molecular mechanisms
could have prominent effects on developing fetuses.
Gadd45a probably induces DNA repair, and the
upregulation of the corresponding gene in our study could
indicate that US exposure caused DNA damage in rat
fetuses (Figure 3). Interestingly, Birc1b, which is impli-
cated in inhibition of apoptosis, was also upregulated in
our study. This finding contradicts the action of Gadd45a,
thereby indicating that US exposure could disrupt normal
signaling in cells. Cxcl1 plays a fundamental role in cell traf-
ficking of various leukocytes and development of immune
system and has also effects on cells of the CNS as well as
endothelial cells involved in angiogenesis or angiostasis.
Tsai et al14 showed a role for rodent Cxcl1 in patterning the
developing spinal cord, namely, signaling through Cxcl1
and Cxcr2 inhibited oligodendrocyte precursor migration.
Neuropeptides directly or indirectly modulate synap-
tic activity and may also function as primary neurotrans-
mitters. In our study, we detected abnormally upregulated
Npy2r, a neuropeptide receptor (Figure 4). Its role in
embryogenesis is unknown, although it was reported that
it could exert inhibition of cyclic adenosine monophosphate
production, an important second messenger of several sig-
naling pathways (Gene; National Center for Biotechnology
Information; We also
found downregulation of Gfra3, which promotes the sur-
vival and maintenance of different neuronal cell types and
could be responsible for cell survival in the inner ear.16 In
addition, we detected reduced levels of Bdnf, a member of
the nerve growth factor family. Among several other func-
tions, Bdnf is necessary for survival of striatal neurons, and
it was also shown that Bdnf could be effective in preserving
photoreceptors from the cell death that normally accom-
panies retinal degeneration.17,18 Because the bioeffects of
more powerful modern US devices have not been exten-
sively studied, it would be interesting to assess exposure
to modern US systems and vision in infants and children.
We also found upregulation of Maged1, which is involved in
Hocevar et al—Gene Expression in Rat Fetuses Exposed to Ultrasound
J Ultrasound Med 2012; 31:923–932930
Figure 5. Differentially expressed genes coding neurotransmitters and their receptors (RT² Profiler quantitative polymerase chain reaction array
PARN-060A-12). The left chart represents the most differentially expressed gene, and the right chart represents other highly differentially expressed
genes in the group. U1a–U4a and U1b–U4b represent biological replicates.
3106jumonline.qxp:Layout 1 5/22/12 3:32 PM Page 930
mediating nerve growth factor–dependent apoptosis.
Whole-mount in situ hybridization analysis showed low-
level expression of this gene throughout embryonic day 11.5
in rat embryos.19 In our study, its expression was 8-fold
higher in group U4, which was exposed to US 4 times
longer than in embryos that were not exposed to US.
These results could suggest impairment of neuronal devel-
opment in US-exposed embryos.
Among neurotransmitters, we found aberrant expres-
sion of Chrna1, which encodes the α subunit of the acetyl-
choline receptor that plays a role in acetylcholine binding
and channel gating (Figure 5). In a study by Scheffer et al,20
this gene was active in vestibular and cochlear hair cells
during early development of the inner ear innervations.
Although several studies have been performed in the past,
none associated US with impaired hearing.1,21 Interest-
ingly, Chrnd and Chat, the first gene coding another
subunit of the acetylcholine receptor and the second impli-
cated in biosynthesis of acetylcholine, were both underex-
pressed in fetal rat tissue exposed to US. Mutations in
Chrnd and Chrna1 and, consequently, impaired function
of their protein products are associated with congenital
myasthenic and multiple pterygium syndromes.22,23
Glra1 encodes the α1 subunit of the glycine receptor, a
ligand-gated chloride channel. This inhibitory glycine
receptor mediates postsynaptic inhibition in the spinal
cord and other regions of the CNS.24 Hirzel et al25 showed
that mutant mice with a homozygous deletion in this
gene had a phenotype that was similar to human hyper-
ekplexia, with increased neuromuscular tone, inducible
tremors, and an abnormal gait. In our study, this gene was
downregulated; thus, it could be implicated in abnormal
development of the CNS.
Our results show that US could probably affect expres-
sion of highly important genes implicated in embryonic
development. The exact mechanisms and the consequences
of this aberrant expression remain to be elucidated.
Our study also showed on the molecular level that US should
be used with caution, particularly in early pregnancy and in
vitro fertilization procedures. Repeated prenatal US imag-
ing and Doppler flow examinations should be restricted to
those women for whom the information is likely to be of
clinical benefit. Because there are no set rules for determin-
ing the correct exposure for every situation, it is reasonable
for the US user to use the ALARA (as low as reasonably
achievable) principle and follow the information concern-
ing the US procedures prudently.26 The qualified US user
should determine the most appropriate way to keep the
exposure low and bioeffects to a minimum while obtaining
a diagnostic examination. The nonmedical use of powerful
2-dimensional, Doppler, 3-dimensional, and 4-dimensional
US for keepsake imaging should be discouraged. Our results
show that assessment of the safety of diagnostic US should
be approached in two directions: by conducting long-term
epidemiologic studies of exposed infants and by studying the
interactions of US in tissues of animal models and attempt-
ing to extrapolate the results to clinical practice.
1. Houston LE, Odibo AO, Macones GA. The safety of obstetrical ultra-
sound: a review. Prenat Diagn 2009; 29:1204–1212.
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... trichostatin A, PCI-247810), non-steroidal anti-inflammatory drugs (e.g. indomethacin, sulindac), and various environmental stressors (e.g., hypoxia, ischemia, UV light, osmotic stress, X-rays, seizures and ultrasound) have been shown to induce Gadd45a expression [20], [30], [31], [32], [33], [34], [35]. In humans, blood levels of Gadd45a have been positively correlated with increasing levels of radiation exposure [14]. ...
... Since the Gadd45 protein family is highly conserved across most cell types and has been shown to be upregulated in the brain in response to various stimuli (e.g. ultrasound [20]) it is very likely that Gadd45a is present in neurons. In addition, a recent study found that Gadd45a (endogenous or tagged) is localized to the nuclei of RKO and HEK293 cells where it colocalizes and interacts with other RNA-binding proteins [7]. ...
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To better understand the short and long-term effects of stress on the developing cerebral cortex, it is necessary to understand how early stress response genes protect or permanently alter cells. One family of highly conserved, stress response genes is the growth arrest and DNA damage-45 (Gadd45) genes. The expression of these genes is induced by a host of genotoxic, drug, and environmental stressors. Here we examined the impact of altering the expression of Gadd45alpha (Gadd45a), a member of the Gadd45 protein family that is expressed throughout the developing cortices of mice and humans. To manipulate levels of Gadd45a protein in developing mouse cortex, we electroporated cDNA plasmids encoding either Gadd45a or Gadd45a shRNA to either overexpress or knockdown Gadd45a levels in the developing cortices of mice, respectively. The effects of these manipulations were assessed by examining the fates and morphologies of the labeled neurons. Gadd45a overexpression both in vitro and in vivo significantly impaired the morphology of neurons, decreasing neurite complexity, inducing soma hypertrophy and increasing cell death. Knockdown of Gadd45a partially inhibited neuronal migration and reduced neurite complexity, an effect that was reversed in the presence of an shRNA-resistant Gadd45a. Finally, we found that shRNA against MEKK4, a direct target of Gadd45a, also stunted neurite outgrowth. Our findings suggest that the expression of Gadd45a in normal, developing brain is tightly regulated and that treatments or environmental stimuli that alter its expression could produce significant changes in neuronal circuitry development.
... W cm -2 ) for from 1 to 4 sessions of 45 to 55 seconds each on different days of gestation exhibited changes in the expression of some genes, most notably those implicated in developmental signaling pathways. 110 Prenatal exposure of rat fetuses to B--mode ultrasound (2.89 MHz, MI = 1.1, spatial--average pulse--average intensity [I SAPA ] = 157 mW cm -2 ) for 1 or 2 hours per day for 9 days reportedly increased the permeability of the blood--brain barrier on postnatal day 10 but not later. 111 The acoustic mechanism responsible for these bioeffects is not yet understood. ...
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The mechanical index (MI) has been used by the US Food and Drug Administration (FDA) since 1992 for regulatory decisions regarding the acoustic output of diagnostic ultrasound equipment. Its formula is based on predictions of acoustic cavitation under specific conditions. Since its implementation over 2 decades ago, new imaging modes have been developed that employ unique beam sequences exploiting higher-order acoustic phenomena, and, concurrently, studies of the bioeffects of ultrasound under a range of imaging scenarios have been conducted. In 2012, the American Institute of Ultrasound in Medicine Technical Standards Committee convened a working group of its Output Standards Subcommittee to examine and report on the potential risks and benefits of the use of conditionally increased acoustic pressures (CIP) under specific diagnostic imaging scenarios. The term "conditionally" is included to indicate that CIP would be considered on a per-patient basis for the duration required to obtain the necessary diagnostic information. This document is a result of that effort. In summary, a fundamental assumption in the MI calculation is the presence of a preexisting gas body. For tissues not known to contain preexisting gas bodies, based on theoretical predications and experimentally reported cavitation thresholds, we find this assumption to be invalid. We thus conclude that exceeding the recommended maximum MI level given in the FDA guidance could be warranted without concern for increased risk of cavitation in these tissues. However, there is limited literature assessing the potential clinical benefit of exceeding the MI guidelines in these tissues. The report proposes a 3-tiered approach for CIP that follows the model for employing elevated output in magnetic resonance imaging and concludes with summary recommendations to facilitate Institutional Review Board (IRB)-monitored clinical studies investigating CIP in specific tissues.
Ultrasound safety is of particular importance in fetal and neonatal scanning. Fetal tissues are vulnerable and often still developing, the scanning depth may be low, and potential biological effects have been insufficiently investigated. On the other hand, the clinical benefit may be considerable. The perinatal period is probably less vulnerable than the first and second trimesters of pregnancy, and ultrasound is often a safer alternative to other diagnostic imaging modalities. Here we present step-by-step procedures for obtaining clinically relevant images while maintaining ultrasound safety. We briefly discuss the current status of the field of ultrasound safety, with special attention to the safety of novel modalities, safety considerations when ultrasound is employed for research and education, and ultrasound of particularly vulnerable tissues, such as the neonatal lung. This CME is prepared by ECMUS, the safety committee of EFSUMB, with contributions from OB/GYN clinicians with a special interest in ultrasound safety.
Objectives Ultrasound users are advised to observe the ALARA (as low as reasonably achievable) principle, but studies have shown that most do not monitor acoustic output metrics. We developed an adaptive ultrasound method that could suggest acoustic output levels based on real‐time image quality feedback using lag‐one coherence (LOC). Methods Lag‐one coherence as a function of the mechanical index (MI) was assessed in 35 healthy volunteers in their second trimester of pregnancy. While imaging the placenta or the fetal abdomen, the system swept through 16 MI values ranging from 0.15 to 1.20. The LOC‐versus‐MI data were fit with a sigmoid curve, and the ALARA MI was selected as the point at which the fit reached 98% of its maximum. Results In this study, the ALARA MI values were between 0.35 and 1.03, depending on the acoustic window. Compared to a default MI of 0.8, the pilot acquisitions suggested a lower ALARA MI 80% of the time. The contrast, contrast‐to‐noise ratio, generalized contrast‐to‐noise ratio, and LOC all followed sigmoidal trends with an increasing MI. The R² of the fit was statistically significantly greater for LOC than the other metrics (P < .017). Conclusions These results suggest that maximum image quality can be achieved with acoustic output levels lower than the US Food and Drug Administration limits in many cases, and an automated tool could be used in real time to find the ALARA MI for specific imaging conditions. Our results support the feasibility of an automated, LOC‐based implementation of the ALARA principle for obstetric ultrasound.
Objectives: The objective of this study was to investigate if the on-screen displayed thermal index for bone (TIB) was an adequate predictor for the derated spatial-peak temporal-average (ISPTA .3 ) and spatial-peak pulse-average (ISPPA .3 ) acoustic intensity in a selection of clinical diagnostic ultrasound machines and transducers. Methods: Five clinical diagnostic ultrasound scanners and 10 transducers imaging in 2D greyscale, color Doppler, and pulsed wave Doppler, both close to and far from the transducer, with a thermal index for bone between 0.1 and 4.0, were calibrated resulting in 103 unique measurements. Acoustic measurements were performed in a bespoke 3-axis computer-controlled scanning tank using a 200-µm diameter calibrated needle hydrophone. Results: Significant but poor correlation was observed between the acoustic intensities and the on-screen TIB. At TIB 0.1, the ISPTA .3 range was 0.51 - 50.49 mW/(cm)(2) and the ISPPA .3 range was 0.01-207.29 W/(cm)(2) . At TIB of 1.1, the ISPTA .3 range was 19.02 - 309.44 mW/(cm)(2) and the ISPPA .3 range was 3.87-51.89 W/(cm)(2) . Conclusions: The TIB is a poor predictor for spatial-peak temporal-average intensity, spatial-peak pulse-average acoustic intensity and potential bioeffects from clinical diagnostic ultrasound scanners.
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IZVLEČEK Ta študija obravnava možne učinke ultrazvočnega valovanja (UZ) na izražanje genov v osrednjem živčevju podganjih zarodkov. Štiri skupine (n = 5 v vsaki) brejih neinbridiranih Wistar Han (HsdRccHanTM:WIST) podganjih samic so bile izpostavljene UZ valovanju v različnem časovnem trajanju (55, 100, 145 in 195 sekund) s pomočjo multifrekvenčne UZ sonde v 2–dimenzionalnem slikovnem načinu, trajanjem impulza UZ 1,29 mikrosekund, s ponavljajočo frekvenco UZ impulza 1 kHz in znižano maksimalno jakostjo UZ signala 222,4 W/ cm2 5., 7., 9. ali 13. dan brejosti. Profiliranje izražanja genov je bilo izvedeno na tkivu osrednjega živčevja (OŽ) podganjih zarodkov (n = 5 na skupino) z matrikami za kvantitativno obratno transkripcijo in verižno reakcijo s polimerazo. Rezultati so pokazali precejšne spremembe v izražanju genov. Najbolj odstopajoče izraženi geni so bili Adamts5, Gadd45a, Npy2r in Chrna1, ki so udeleženi v pomembnih razvojnih signalnih poteh. Na podlagi naših izsledkov lahko sklepamo, da rutinske kratkotrajne UZ preiskave za spremljanje razvoja zarodka niso kontraindicirane in da so dolgotrajne izpostavitve UZ valovanju indicirane samo kadar je potrebno pridobiti pomembne informacije za diagnosticiranje. ABSTRACT This study evaluated the possible effects of ultrasound (US) on gene expression in brain tissue of rat embryos. Four groups (n = 5 each) of pregnant outbred Wistar Han (HsdRccHanTM:WIST) rats were exposed to US waves for different durations (55, 100, 145, and 195 seconds) via a multifrequency transducer in the 2–dimensional imaging mode with a pulse duration of 1.29 microseconds, a pulse repetition frequency of 1 kHz, and a derated spatial–peak pulse–average intensity of 222.4 W/cm2 on day 5, 9, 7, or 13 of gestation. Gene expression profiling was performed in fetal brain tissue (n = 5 per group) by quantitative reverse transcription–polymerase chain reaction arrays. The results indicated substantial alterations in gene expression. The most differentially expressed genes were Adamts5, Gadd45a, Npy2r, and Chrna1, which are implicated in important developmental signaling pathways. On the basis of our findings, routine short US examinations for monitoring fetal development are not contraindicated, but prolonged exposures should be used only when needed to obtain important diagnostic information.
To investigate the effect of daily exposure in utero to static magnetic fields during prenatal development on germ cell development and fertility of exposed offspring in adulthood. Mice were exposed daily in utero to different static magnetic field strengths at the bore entrance or in the isocenter of 1.5 T and 7 T MRI systems during the entire course of prenatal development. In utero-exposed male mice revealed no effect of magnetic field strength on weight of testes and epididymis or on sperm count, sperm morphology, or fertility. Exposed pregnant female mice showed no reduced fertility in terms of pregnancy rates and litter size, pointing to a normal ovarian function. However, a reduced placental weight of offspring of intrauterine exposed female mice was observed that correlated with a decrease in embryonic weight in those animals exposed at the strongest magnetic field. This effect seemed to be parent-dependent, since it was not observed in those embryos fathered by in utero-exposed male mice. Repetitive in utero exposure to strong static magnetic fields does not impair fertility but may have a parental-dependent effect on fetal programming with regard to placental development and fetal growth.J. Magn. Reson. Imaging 2013. © 2013 Wiley Periodicals, Inc.
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Recent evidence suggests a potential role for thrombospondin-2 (TSP-2), a matricellular glycoprotein, in the regulation of primary angiogenesis. To directly examine the biological effect of TSP-2 expression on tumor growth and angiogenesis, human A431 squamous cell carcinoma cells, which do not express TSP-2, were stably transfected with a murine TSP-2 expression vector or with vector alone. A431 cells expressing TSP-2 did not show an altered growth rate, colony-forming ability, or susceptibility to induction of apoptosis in vitro. However, injection of TSP-2-transfected clones into the dermis of nude mice resulted in pronounced inhibition of tumor growth that was significantly stronger than the inhibition observed in A431 clones stably transfected with a thrombospondin-1 (TSP-1) expression vector, and combined overexpression of TSP-1 and TSP-2 completely prevented tumor formation. Extensive areas of necrosis were observed in TSP-2-expressing tumors, and both the density and the size of tumor vessels were significantly reduced, although tumor cell expression of the major tumor angiogenesis factor, vascular endothelial growth factor, was maintained at high levels. These findings establish TSP-2 as a potent endogenous inhibitor of tumor growth and angiogenesis.
purpose. To assess the capacity of a retrovirus-engineered Schwann cell line (SCTM41), transfected with either a glial cell line–derived neurotrophic factor (GDNF) construct or a brain-derived neurotrophic factor (BDNF) construct, to sustain visual function in the dystrophic Royal College of Surgeons (RCS) rat. methods. Cell suspensions were injected into the subretinal space of the right eye of 3-week-old dystrophic RCS rats through a transscleral approach. The left eye remained as an unoperated control. Sham-surgery animals received injections of carrier medium plus DNase to the right eye. All animals were placed on oral cyclosporine. At 8, 12, 16, and 20 weeks of age, animals were placed in a head-tracking apparatus and screened for their ability to track square-wave gratings at various spatial frequencies (0.125, 0.25, and 0.5 cyc/deg). At the end of the experiment, the animals were perfused and processed for histologic assessment of photoreceptor survival. results. Animals with SCTM41-GDNF–secreting cells, on average, head tracked longer than animals with SCTM41-BDNF–secreting cells, and both performed better than those injected with the parent SCTM41 line. All tracked longer than sham-surgery or nonsurgical dystrophic eyes. Each cell type demonstrated preservation of photoreceptors up to at least 4 months of age, over and above the sham-surgery control. conclusions. Engineered Schwann cells sustain retinal structure and function in the dystrophic RCS rat. Cells overexpressing GDNF or BDNF had a greater effect on photoreceptor survival than the parent line or sham surgery. This study demonstrates that ex vivo gene therapy and subsequent cell transplantation can be effective in preserving photoreceptors from the cell death that normally accompanies retinal degeneration.
Objective: To test a hypothesis of no association between ultrasound exposure in early fetal life and growth or impaired vision or hearing during childhood. Design: Follow up of eight to nine year old children born to women who participated in a randomised controlled trial on ultrasound screening during pregnancy. Setting: Nineteen antenatal care clinics run by three central hospitals in Sweden from 1985 to 1987. Population and methods: Of 4637 eligible singleton pregnancies, 3265 (71%) were followed up through a questionnaire sent to their mothers. Analyses were performed both according to randomised groups and to ultrasound exposure. Main outcome measures: Parents' report of vision and hearing tests as recorded on child's record card. Parents' report of their child's weight and height at 1, 4 and 7 years of age. Results: Reduced hearing was reported by 3.4% in the screening group compared with 3.5% in the nonscreening group (odds ratio [OR] 1.0; 95% confidence interval [CI] 0.67-1.41). The same prevalences were found when analysed according to ultrasound exposure (OR 1.0; 95% CI 0.67-1.42). Reduced vision was reported by 6.3% in the screening group compared with 7.8% in the nonscreening group (OR 0.8; 95% CI 0.60-1.03). Corresponding figures for ultrasound exposed and unexposed were 6.2% and 8.0%, respectively (OR 0.8; 95% CI 0.58-1.00). No statistically significant differences in body weight or height at 1, 4 or 7 years of age between screened and not screened children or between exposed and unexposed were found. Conclusion: This study found no association between ultrasound exposure in early fetal life and growth or impaired vision or hearing during childhood.
The development and maintenance of the vertebrate nervous system depends upon neuronal survival proteins known as neurotrophic factors. Nerve growth factor (NGF) remains the best characterized neurotrophic molecule. Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are two recently cloned neurotrophic factors that are homologous to NGF. Here we describe the molecular cloning of the human and rat genes encoding BDNF, as well as the isolation of the human NT-3 gene. On the basis of comparison of our genomic and cDNA clones with those of previously isolated BDNF and NT-3 genes and cDNAs, we make inferences about the structures of processed transcripts derived from the neurotrophin genes and the protein precursors they encode. We demonstrate that the mature form of BDNF is identical in all mammals examined, and that the same is true of the mature form of NT-3. Furthermore, the respective tissue-distributions and neuronal specificities of NT-3 and BDNF are also conserved among mammals. Finally, we localize the gene encoding human BDNF (gene symbol designated BDNF) to chromosome 11, band p13, and the gene encoding human NT-3 (gene symbol designated NTF3) to chromosome 12, band p13.
Despite that epidemiological studies did not indicate negative effects on neurological development of offspring when exposed to diagnostic ultrasound, possible association with nonright-handedness in males could not be excluded. In addition, an experimental study on fetal mice suggested that prolonged ultrasound exposure may cause mild disturbance in neuronal migration. No doubt, further studies are warranted. The present knowledge of the potential bioeffects of ultrasound suggests that, when using ultrasound for examinations in pregnancy, fetal scanning without medical indication should be avoided and that adherence to ALARA principle (use of energy "as low as reasonably achievable") is compulsory.
Ultrasound is a commonly employed imaging modality in obstetrics and is generally regarded as safe to the fetus. Current ultrasound technology, however, has significantly higher output potential than older machines used in most clinical studies, and the safety profile for the increasing use of Doppler, 3-dimensional (D) and 4-D ultrasound with modern machines is unknown. This article reviews the current status of ultrasound safety within obstetrics, including proposed mechanisms of harm, existing scientific and clinical evidence regarding those mechanisms, and considerations of safety for the clinical user.
Obstetric ultrasound is the well-recognized prenatal test used to visualize and determine the condition of a pregnant woman and her fetus. Apart from the clinical application, some businesses have started promoting the use of fetal ultrasound machines for nonmedical reasons. Non-medical fetal ultrasound (also known as 'keepsake' ultrasound) is defined as using ultrasound to view, take a picture, or determine the sex of a fetus without a medical indication. Notwithstanding the guidelines and warnings regarding ultrasound safety issued by governments and professional bodies, the absence of scientifically proven physical harm to fetuses from this procedure seems to provide these businesses with grounds for rapid expansion. However, this argument is too simplistic because current epidemiological evidence is not synchronous with advancing ultrasound technology. As non-medical fetal ultrasound has aroused very significant public attention, a thorough ethical analysis of this topic is essential. Using a multifaceted approach, we analyse the ethical perspective of non-medical fetal ultrasound in terms of the expectant mother, the fetus and health professionals. After applying four major theories of ethics and principles (the precautionary principle; theories of consequentialism and impartiality; duty-based theory; and rights-based theories), we conclude that obstetric ultrasound practice is ethically justifiable only if the indication for its use is based on medical evidence. Non-medical fetal ultrasound can be considered ethically unjustifiable. Nevertheless, the ethical analysis of this issue is time dependent owing to rapid advancements in ultrasound technology and the safety issue. The role of health professionals in ensuring that obstetric ultrasound is an ethically justifiable practice is also discussed.
In the context of the planned International Society of Ultrasound in Obstetrics and Gynecology-World Health Organization multicenter study for the development of fetal growth standards for international application, we conducted a systematic review and meta-analysis to evaluate the safety of human exposure to ultrasonography in pregnancy. A systematic search of electronic databases, reference lists and unpublished literature was conducted for trials and observational studies that assessed short- and long-term effects of exposure to ultrasonography, involving women and their fetuses exposed to ultrasonography, using B-mode or Doppler sonography during any period of pregnancy, for any number of times. The outcome measures were: (1) adverse maternal outcome; (2) adverse perinatal outcome; (3) abnormal childhood growth and neurological development; (4) non-right handedness; (5) childhood malignancy; and (6) intellectual performance and mental disease. The electronic search identified 6716 citations, and 19 were identified from secondary sources. A total of 61 publications reporting data from 41 different studies were included: 16 controlled trials, 13 cohort and 12 case-control studies. Ultrasonography in pregnancy was not associated with adverse maternal or perinatal outcome, impaired physical or neurological development, increased risk for malignancy in childhood, subnormal intellectual performance or mental diseases. According to the available clinical trials, there was a weak association between exposure to ultrasonography and non-right handedness in boys (odds ratio 1.26; 95% CI, 1.03-1.54). According to the available evidence, exposure to diagnostic ultrasonography during pregnancy appears to be safe.