This article was originally published in a journal published by

Elsevier, and the attached copy is provided by Elsevier for the

author’s benefit and for the benefit of the author’s institution, for
non-commercial research and educational use including without
limitation use in instruction at your institution, sending it to specific
colleagues that you know, and providing a copy to your institution’s
administrator.
All other uses, reproduction and distribution, including without
limitation commercial reprints, selling or licensing copies or access,
or posting on open internet sites, your personal or institution’s
website or repository, are prohibited. For exceptions, permission
may be sought for such use through Elsevier’s permissions site at:

http://www.elsevier.com/locate/permissionusematerial

ORIGINAL ARTICLE
Heat Shock Protein Expression in Rat Skeletal Muscle After
Repeated Applications of Pulsed and Continuous Ultrasound
Ethne L. Nussbaum, PhD, PT, Marius Locke, PhD
ABSTRACT. Nussbaum EL, Locke M. Heat shock protein
expression in rat skeletal muscle after repeated applications
of pulsed and continuous ultrasound. Arch Phys Med Reha-
bil 2007;88:785-90.
Objective: To determine whether repeated ultrasound treat-
ments are capable of increasing the expression of heat shock
protein (HSP) 72 and HSP 25 in rat skeletal muscles.
Design: In vivo, experimental, controlled study.
Setting: Animal laboratory.
Animals: Male Sprague-Dawley rats (n⫽9).
Interventions: Ultrasound (1MHz, 15min, 2.0cm2 trans-
ducer) continuous at 1.0W/cm2 spatial average temporal aver-
age intensity (CONTUS) or pulsed at 2.0W/cm2 spatial average
temporal peak intensity 50% duty cycle (PULS50) was applied
on 4 consecutive days to the lower leg muscles of 1 hindlimb
in each rat (n⫽9).
Main Outcome Measures: Twenty-four hours after the final
ultrasound application, hindlimb muscles were removed,
weighed, and assessed for HSP 72 and HSP 25 content by
Western blotting. Bands from blots were quantified and data
were assessed using t tests (␣⫽.05).
Results: Ultrasound did not affect core or contralateral hind-
limb muscle temperature. Average muscle temperatures during
rs
the final day ultrasound treatments were 38.71°⫾0.30°C when
using PULS50 and 38.16°⫾0.57°C when using CONTUS.
PULS50 significantly increased HSP 25 content in the plantaris
and soleus muscles and HSP 72 content in the plantaris mus-
pe
cles. CONTUS significantly increased HSP 72 content in the
white gastrocnemius muscle.
Conclusions: HSPs can be induced in skeletal muscle when
ultrasound is used on a repeated basis to treat soft tissue.
Key Words: Heat shock proteins; Muscle, skeletal; Rats,
Sprague-Dawley; Rehabilitation; Ultrasound.
© 2007 by the American Congress of Rehabilitation Medi-
cine and the American Academy of Physical Medicine and
r's
Rehabilitation
HE BIOPHYSICAL EFFECTS OF ultrasound have been
T 1,2
well documented. However, the mechanism(s) by which
o
ultrasound provides beneficial effects to cells and tissues remains
th
From the Department of Physical Therapy, Mount Sinai Hospital and Faculty of
Medicine, (Nussbaum) and Faculty of Physical Education and Health (Locke), Uni-
Au
versity of Toronto, Toronto, ON, Canada.
Presented in part to the Canadian Physiotherapy Association, May 27-30 2004,
Quebec City, QC, Canada.
Supported in part by the Natural Science and Engineering Research Council of
Canada (grant no. OGP0183742).
No commercial party having a direct financial interest in the results of the research
supporting this article has or will confer a benefit upon the author(s) or upon any
organization with which the author(s) is/are associated.
Correspondence to Ethne L. Nussbaum, PhD, PT, Mount Sinai Hospital, Rm 1160,
600 University Ave, Toronto, ON M5G 1X5, Canada, e-mail: enussbaum@
mtsinai.on.ca. Reprints are not available from the author.
0003-9993/07/8806-11027$32.00/0
doi:10.1016/j.apmr.2007.03.020
785
a topic of investigation.3 Ultrasound treatment disrupts cellular
components and can also increase tissue temperature. Stressors
py
that perturb cellular homeostasis are known to induce proteins
known as stress proteins or heat shock proteins (HSPs). HSPs
are cytoprotective proteins expressed in all cells in response to
proteotoxic (protein damaging) stressors.4 As “molecular chap-
erones,” HSPs have been shown to play roles in protein for-
co
mation, maturation, and degradation, as well as in the transport
and assembly of newly formed proteins.5 It is thought that the
HSPs’ chaperonelike functions confer protection to cells and
tissues against damage by adverse physiologic conditions.6,7
For example, the transgenic overexpression of HSP 70 in
muscles in mice reduces damage after cryoinduced lesions.8
Similarly, overexpression of HSP 25 in stably transfected myo-
blasts produces dose-dependent protection against hydrogen
al
peroxide–induced damage.9 Although HSPs are well known for
their chaperone abilities that control the quality of protein
folding and other protein-related functions, it has recently been
on
shown that HSPs are associated with a number of signaling
molecules.10 Thus, induction of HSPs may have other impor-
tant implications for influencing cell physiology. Given that
HSPs are known to be involved in protein processes and that
ultrasound may influence protein formation and function, it
seems relevant to investigate the effect of ultrasound on levels
of HSP.
Previous studies11,12 assessing HSP expression after ultrasound
have shown that 12 treatments of pulsed-mode ultrasound (1MHz;
2.5W/cm2 spatial average temporal peak intensity [ISATP], 20%
duty cycle) increased HSP expression in chondrocytes of arthritic
cartilage in the rat knee. Because ultrasound was applied in a 20°C
water bath, which is below physiologic temperature for the rat, it
may be assumed that thermal effects were not a factor in the HSP
induction. We previously investigated whether a single ultrasound
treatment might induce HSPs.13 Our work showed that HSP 72
content was not increased in rat skeletal muscles after a single
15-minute application of 1-MHz ultrasound performed by using
continuous-mode (1.0W/cm2 ISATP) or pulsed-mode ultrasound
(1.0W/cm2 or 2.0W/cm2 ISATP, 20% and 50% duty cycles, respec-
tively). Although some of these protocols significantly increased
local muscle temperatures to levels similar to those known to
induce HSPs, no increase in the major HSP (HSP 72) was detected
in the treated muscles. Although the exact reason for the lack of an
HSP response in our previous work remains unclear, either a
single treatment or the duration that tissues were exposed to
elevated temperatures may have been insufficient to induce an
HSP response.
In view of the fact that clinical applications of ultrasound for
soft-tissue conditions would typically involve repeated appli-
cations, it was of interest to determine if multiple treatments
over several days were capable of increasing HSP content in
skeletal muscle.
METHODS
Animals
Adult male (weight range, 437⫺688g) Sprague-Dawley rats
(n⫽9) were used in these experiments. All experiments and
Arch Phys Med Rehabil Vol 88, June 2007
786 ULTRASOUND INDUCES HEAT SHOCK PROTEINS IN RAT MUSCLE, Nussbaum
procedures were approved by the Animal Care Committee of
the University of Toronto. Animals were maintained on a
12-hour dark-light cycle, housed at 20°⫾1°C, 50% relative
humidity, and fed and watered ad libitum. Rats were anesthe-
tized with intraperitoneal sodium pentobarbital (65mg/kg) dur-
ing all procedures. Hair on both lower hindlimbs was removed
by using clippers followed by the application of a depilatory
cream. Twenty-four hours after the final ultrasound treatment,
rats were anesthetized with intraperitoneal sodium pentobarbi-
tal (65mg/kg), and the plantaris, soleus, and white gastrocne-
mius muscles were carefully removed, weighed, and frozen in
liquid nitrogen.
Ultrasound Treatments
Ultrasound treatments were applied to anesthetized animals
at 24-hour intervals on 4 consecutive days by using an Excel
Ultramax ultrasound unita recently calibrated by the manufac-
turer. Novel features of this device have been described previ-
ously.13 A sensing circuit continuously monitors the delivered
intensity of ultrasound and displays the amount as a percentage
of prescribed intensity. Thus, possible transmission loss can be
compensated for by increasing treatment time.
Nine animals received 1-MHz ultrasound for a duration of
15 minutes applied by using either (1) continuous mode at
1.0W/cm2 (spatial average temporal average intensity [ISATA])
(CONTUS) (n⫽4) or (2) pulsed mode at 2.0W/cm2 (ISATP) by
using a 50% duty cycle (PULS50) (n⫽5). Beam nonuniformity
ratio was 4 to 1; pulses of 2ms in duration were repeated at
100-Hz pulse-repetition frequency. In all cases, only 1 hind-
limb was treated, whereas the untreated contralateral limb
rs
muscles served as a control. The skin overlying the lower
hindlimb muscles was moistened with tap water and ultrasound
was applied in contact by using gel coupling. The 1.6-m
diameter ultrasound transducer head was moved in a circular
pe
fashion over an area of skin equal to twice the area of the
transducer, at an approximate rate of 4.0cm/s. Care was taken
throughout to ensure that incident ultrasound was normal to the
skin surface. The sensing circuit of the device showed that
during application the transmission rate of ultrasound was
consistently greater than 80% of the set intensity.
Core and Muscle Temperature
r's
During all ultrasound treatments, rectal temperature was
recorded by using a thermistor TSD 102C probe.b The ther-
mistor was attached to a Biopac data-acquisition systemc linked
to a Power Macintosh G3 computer equipped with Acknowl-
o
edge MP-100 software system (version 3.2.1)b that allowed for
continuous recording of rectal temperatures. During ultra-
th
sound, rectal temperature was carefully maintained as close to
37°C as possible by using a warming pad under the animal.
After ultrasound treatment, animals were revived by the oral
administration of water. Twenty-four hours after the final ul-
Au
trasound treatment, rats were anesthetized, and the plantaris,
soleus, and white gastrocnemius muscles were excised, quickly
weighed, and frozen in liquid nitrogen until further analyses.
During the final ultrasound treatment, local muscle temper-
ature was measured bilaterally by using 3-cm sterile needle
thermistors inserted along the Achilles’ tendons into the deep
portion of the triceps surae muscle group. The tip of the
thermistor was positioned at a tissue depth of about 1cm and
within the ultrasound treatment area. Muscle thermistors were
attached to a Physiotemp (model TH-8)c connected to a Biopac
data-acquisition system as described for rectal temperature.
Arch Phys Med Rehabil Vol 88, June 2007
Polyacrylamide Gel Electrophoresis and Immunoblotting
The separation of muscle proteins by sodium dodecyl sulfate
polyacrylamide gel electrophoresis followed by HSP identifi-
cation by Western blotting was performed as previously de-
scribed.13 Briefly, muscles or muscle portions were homoge-
nized in 600mmol/L of NaCl and 15mmol/L of Tris pH 7.5,
and protein concentration was determined by using the method
of Lowry et al.14 One-dimensional sodium dodecyl sulfate
polyacrylamide gel electrophoresis was performed according to
the method described by Laemmli,15 except that the separating
gel (0.15⫻4.5⫻8cm) consisted of a 10% polyacrylamide slab
py
gel. Equal amounts of protein (100␮g/lane for the white gas-
trocnemius and plantaris, 50␮g/lane for the soleus muscle)
from homogenized muscles (control, CONTUS, and PULS50
treatment) were separated. After electrophoretic separation,
co
proteins were transferred to nitrocellulose membranes (thick-
ness, 0.22␮m)d as described by Towbin et al16 by using the
Bio-Rad miniprotean II gel transfer system.d After protein
transfer, blots were reacted with antibodies specific for HSP 72
or HSP 25e diluted 1:2500 in Tris-buffered saline with 2%
nonfat dried milk as previously described.17 Immunoblots were
scanned by using an Agfa Arcus II scanner,f and quantification
of bands from immunoblots was performed by using Kodak 1D
2.0 Image Analysis Software.g Standard curves were con-
al
structed to assure linearity.
Analyses
on
Data obtained from ultrasound treated and contralateral (un-
treated) muscles were analyzed by using Student t tests. In all
cases, the level of significance was set at ␣ equal to .05.
RESULTS
Muscle Mass
Absolute muscle mass for the soleus, plantaris, and gastroc-
nemius muscles after 4 consecutive days of either CONTUS or
PULS50 ultrasound treatments are shown in table 1. When
compared with respective contralateral controls by using the
Student t test, there were no significant changes in muscle mass
after treatment (P range, .15⫺.74). To correct for differences in
muscle mass because of body mass, muscle mass was also
expressed relative to body mass. Again, no significant differ-
ences in relative muscle mass were detected (P⬎.05). These
results show that significant swelling did not occur in treated
muscles as a consequence of CONTUS or PULS50 after 4
consecutive days of treatment.
Table 1: Body Mass Values on Day 1 and Muscle Mass Values
After the Final Treatment on Day 4
Body Plantaris Gastrocnemius
Ultrasound Mass (g) Soleus (g) (g) (g)
Continuous
ultrasound
Control
645⫾13
0.23⫾0.04 0.51⫾0.02 2.65⫾0.08
Treated 0.26⫾0.03 0.57⫾0.05 2.64⫾0.05
Pulsed
ultrasound
Control
517⫾53
0.21⫾0.02 0.45⫾0.03 2.29⫾0.17
Treated 0.23⫾0.01 0.50⫾0.03 2.29⫾0.16
NOTE. Values are mean ⫾ standard error of the mean (SEM). No
statistically significant differences were detected between the con-
trol and treated muscles in the continuous or pulsed ultrasound
groups.
 ULTRASOUND INDUCES HEAT SHOCK PROTEINS IN RAT MUSCLE, Nussbaum
Table 2: Rectal and Muscle Temperatures Before and During the Final Treatment of Either Continuous or Pulsed Ultrasound on Day 4
Rectal Temperature (°C)
Ultrasound Pre Post
Continuous ultrasound 36.91⫾0.19 37.10⫾0.16
Pulsed ultrasound 36.96⫾0.16 37.18⫾0.07
NOTE. Values are mean ⫾ SEM.
*Significant differences (P⬍.05) from preultrasound values.
Core Temperature
Rectal temperature was measured before and during each
ultrasound treatment (table 2). The average rectal temperature
of PULS-treated animals was 36.96°⫾0.16°C before the ultra-
sound and 37.18°⫾0.07°C after ultrasound; for CONTUS-
treated animals, the average rectal temperature was 36.91°⫾
0.19°C before ultrasound and 37.10°⫾0.16°C after ultrasound.
These changes were not statistically significant (P⫽.44, P⫽.19,
respectively). Furthermore, these temperatures were well be-
low body temperatures known to activate the stress response in
rat skeletal muscle.17
Muscle Temperature
To avoid repeated trauma to the muscles from the insertion
on trasound is thought to promote the healing of soft-tissue inju-
of the needle thermistor, muscle temperature was only recorded
during the last ultrasound treatment. Before both continuous
and pulsed ultrasound, the nontreated and the ultrasound-
treated muscles showed similar muscle temperatures (see table 2).
rs
As expected, in nontreated contralateral control limbs, there
was no increase in muscle temperature during the 15-minute
continuous-ultrasound (36.17°⫾0.21°C to 37.03°⫾0.60°C, P⫽
.12) or pulsed-ultrasound treatment (36.45°⫾0.32°C to 36.81°⫾
pe
0.08°C, P⫽.25) to the treated hindlimb. In contrast, in the
treated limbs, the temperature increased significantly in the
CONTUS-treated muscle (36.24°⫾0.35°C to 38.16°⫾0.57°C,
P⬍.04) and similarly in the PULS-treated muscles (36.79°⫾
0.31°C to 38.71°⫾0.30°C, P⫽.002). Although both pulsed-
and continuous-ultrasound treatments increased muscle tem-
perature, the temperature achieved was below temperatures
known to activate the stress response in rat skeletal muscle.17
r's
HSP 25 Expression After Ultrasound Treatment
A visual inspection of Western blots showed an increase of
HSP 25 content in the plantaris, soleus, and white gastrocne-
mius muscles after pulsed-ultrasound treatments (fig 1A). The
o
results are shown graphically in figure 1B. The changes in the
plantaris and soleus muscles were significant (P⫽.025, P⫽
.007, respectively). There was a 4-fold increase in HSP 25
th
content in the white gastrocnemius muscle by using pulsed
ultrasound, which proved to be marginally significant (P⫽.05).
Visually, there was marginal change in HSP 25 content in the
Au
white gastrocnemius muscle after CONTUS; the 50% increase
was not statistically significant (P⫽.16). There was no statis-
tically significant increase in HSP 25 content in the soleus or
plantaris muscles after continuous ultrasound.
HSP 72 Expression After Ultrasound Treatment
Western blots showed increases in HSP 72 content in the white
gastrocnemius muscle when using PULS50 and CONTUS and in
the plantaris muscle when using PULS50 (fig 2A). The change
in HSP 72 content in the white gastrocnemius muscle after
CONTUS and in the plantaris muscle when using PULS50
787
Muscle Temperature (°C)
Pre Post
Control 36.17⫾0.21 37.03⫾0.60
Treated 36.24⫾0.35 38.16⫾0.57*
Control 36.45⫾0.32 36.81⫾0.08
Treated 36.79⫾0.31 38.71⫾0.30*
py
proved to be significant (P⫽.02, P⫽.004, respectively). There
was a 4-fold increase in HSP 72 content in the white gastroc-
co
nemius muscles after pulsed ultrasound (fig 2B); however, this
result was not statistically significant (P⫽.14). There were no
statistically significant changes in HSP 72 content in the soleus
muscles after CONTUS or PULS50 ultrasound (P⫽.20,
P⫽.99, respectively). These results suggest that repeated ultra-
sound treatments to mammalian muscle may increase the con-
tent of certain HSPs.
al
DISCUSSION
Therapeutic ultrasound is usually applied on a repeated basis
to allow for possible cumulative effects of treatment.18,19 Ul-
ries yet the mechanism of action remains uncertain. HSPs are
induced in cells after perturbations to cellular protein ho-
meostasis and have been shown to repair damaged or denatured
proteins, thereby protecting cells from normally lethal stres-
sors. Although temperature is a primary inducer of HSPs, other
stressors such as metabolic or mechanical stresses are also
capable of inducing certain HSPs. The novel finding of this
study is that repeated daily treatments of pulsed and to a lesser
extent, continuous ultrasound, increased HSP content in certain
mammalian skeletal muscles. Our failure to find statistical
significance in the increased HSPs in white gastrocnemius
muscles in this study may be related to the small sample size.
We investigated 2 modes of ultrasound in an attempt to
elucidate the mechanism by which HSPs might be increased in
muscle tissue after repeated applications of ultrasound. One
mode comprised continuous ultrasound to ascertain whether
HSP induction was dependent mainly on the thermal compo-
nent of the treatment, whereas a second mode was comprised
of pulsed ultrasound. The latter mode was used to account for
the possibility that HSP induction might be dependent on the
peak intensity applied, rather than the amount of local heat
generated. The temporal average intensities of ultrasound were
equivalent for PULS50 and CONTUS (1.0W/cm2 ISATA), and
they produced equal increases in muscle temperature (1.92°C).
This was expected because it is known that the heating rate of
ultrasound is directly related to temporal average intensity of
the power used.20 It should be noted that no significant thermal
shock was experienced in our model because muscle temper-
atures never exceeded 39°C for any substantial length of time.
From this, we conclude that any effects of ultrasound in this
study were not primarily mediated by local heat but by the
mechanical effects of ultrasound on the tissue. The temporal
peak intensity of the PULS50 protocol (2.0W/cm2 ISATP) was
twice that of CONTUS (1.0W/cm2 ISATP) and HSP increased in
muscles to a greater extent when using the pulsed mode. It is
known that the athermal effects of ultrasound and the likeli-
hood of producing cavitation in the tissues depend largely on
the temporal peak intensity of power in the sound field.20 This
Arch Phys Med Rehabil Vol 88, June 2007
788 ULTRASOUND INDUCES HEAT SHOCK PROTEINS IN RAT MUSCLE, Nussbaum
rs
pe
r's
suggests that mechanical perturbation is the major factor in
HSP induction by ultrasound. Further investigation is required
to determine the effects on HSP induction of repeated ultra-
o
sound applications by using temporal peak intensity values
greater or less than 2.0W/cm2 ISATP 50% duty cycle.
In the present study, significant induction of HSPs was
th
observed without evidence of the development of tissue edema.
Notwithstanding the absence of overt signs of tissue damage, it
is possible that mechanical effects of repeated ultrasound treat-
Au
ments, and especially ultrasound delivered at high temporal
peak intensity, perturbed cells and tissues to the extent that the
stress response was activated followed by HSPs accumulation.
It is of interest to note that in preliminary unpublished work we
observed significant induction of HSPs after a single 15-minute
application of continuous mode ultrasound at considerably
higher intensity than that used in the present study (1.5W/cm2
ISATA). However, treatment at that intensity was associated
with intense swelling of the treated hindlimb. In the presence of
obvious damage, increased expression of HSPs might be con-
sidered a “marker” of cell and tissue damage to the muscles.
Arch Phys Med Rehabil Vol 88, June 2007
py
co
al
on
Fig 1. HSP 25 content is increased
after pulsed-ultrasound treatment.
(A) Portions of Western blots re-
acted with HSP 25 antibody. (B)
Graphical representation of HSP
content obtained from densitomet-
ric scanning of the Western blots.
NOTE. Values are mean ⴞ standard
error of the mean (SEM) and ex-
pressed as a percentage of their re-
spective contralateral control.
*Statistical significance (P<.05)
from their respective contralateral
control.
We cannot determine whether the HSPs induced in the present
study represent a marker of damage or induction of a protective
response. However, given that no increase in muscle mass (ie,
no swelling) was observed, it appears that the induction was
not in response to damage. We hypothesize that a hormesis
effect may have occurred whereby ultrasound provided a mild
subinhibitory stress on tissues, which induced HSPs in an effort
to withstand subsequent perturbations from ultrasound.
Other studies investigating effects of ultrasound on soft
tissue healing have shown the benefit of multiple exposures.
For example, Karnes and Burton21 applied continuous-mode
ultrasound to rat beginning on day 1 in a model of exercise-
induced muscle injury; progressively increasing benefit was
observed from daily ultrasound. However, the difference be-
tween treated and untreated muscles only reached a significant
level on day 7. In contrast, Fisher et al22 found that daily pulsed
ultrasound (1.0W/cm2 ISATP) increased protein content in rat
gastrocnemius muscle on day 7 after impact trauma when
compared with continuous-mode ultrasound but not when com-
pared with untreated injured muscles. Fisher22 applied ultra-
 ULTRASOUND INDUCES HEAT SHOCK PROTEINS IN RAT MUSCLE, Nussbaum 789

py
co
al
on
Fig 2. HSP 72 content is increased
rs

in fast muscle after ultrasound

treatment. (A) Portions of Western
blots reacted with HSP 72 anti-
body. (B) Graphical representation
pe

of HSP content obtained from den-

sitometric scanning of the Western
blots. NOTE. Values are mean ⴞ
SEM and expressed as a percent-
age of their respective contralat-
eral control. *Statistical signifi-
cance (P<.05) from their respective
contralateral control.
r's

sound by using a temporal peak intensity that was half of that
used in the present study, which might explain the lack of
benefit. Cumulative effects of other stressors have been re-
ported; muscle from rats that were exercised only once (60min)
o
stressed soleus muscle already expresses relatively high HSP
72 levels, it may be better equipped to endure cellular pertur-
bations caused by ultrasound and thus may not synthesize
additional HSP 72. In agreement with this, the soleus muscle

did not result in elevated HSP 72 content. However, a signif- showed little, if any, increase in HSP 72 content 24 hours after
icant increase was noted when rats were exercised on 3 con- a 15-minute 42°C heat stress.17 In contrast, faster muscles, such
secutive days.23 Thus, although a single exposure to a stressor
th

as the white gastrocnemius and plantaris, which are known to

may, in some cases, be insufficient to elicit an HSP response, contain relatively lower basal HSP 72 levels, may be less well
repetitive exposures to the same stressor may allow for signif- protected and thus rapidly synthesize additional HSP 72 to
icant accumulation of HSPs. Because we only examined HSP cope with any perturbations caused by ultrasound. Thus, this
Au

expression after 4 days, the precise temporal pattern of HSP

accumulation and any conferred benefit on healing remains to
be determined.
The constitutive or basal expression of HSP 72 appears to
follow a fiber-type–specific pattern of expression such that
slow twitch muscles (soleus) express a relatively high level in
the “unstressed” state.24 An increased HSP 72 expression was
detected in the white gastrocnemius muscle after continuous
ultrasound and in the fast plantaris muscle after both pulsed and
continuous ultrasound. Although the exact reasons for this
pattern of expression remain undetermined, because the un-
fiber-type–specific expression of HSP 72 may explain the pat-
tern of HSP 72 expression observed in response to ultrasound
in the present study.
We observed significant increases in HSP 25 content in the
soleus and plantaris as well as large (4-fold) but nonsignificant
increases in the white gastrocnemius muscles after only pulsed
ultrasound. HSP 72 followed a different pattern of expression,
which suggests that the different ultrasound treatments (pulsed
vs continuous) may work to increase HSP content by different
HSP inducing pathways.

Arch Phys Med Rehabil Vol 88, June 2007

790 ULTRASOUND INDUCES HEAT SHOCK PROTEINS IN RAT MUSCLE, Nussbaum
Study Limitations
The present work should be replicated by using a larger
sample size because the number of animals used in this study
was insufficient to conclude that pulsed ultrasound did not
produce a significant increase in HSPs in the white gastrocne-
mius muscle (ie, a beta effect). Notwithstanding, this study is
the first to link repetitive applications of ultrasound and ex-
pression of the protective HSPs in muscle. Given that HSPs
have been shown to protect cells and tissues from a variety of
stresses and serve as molecular chaperones that are known to
repair damaged proteins, any increased HSP content after ul-
trasound treatments might be beneficial in tissue repair, which
suggests exciting possibilities for ultrasound.
CONCLUSIONS
The results show that 15-minute treatments of either CONTUS
or PULS50 modes of ultrasound on 4 consecutive days in-
creased HSP expression in certain rat hindlimb muscles. Pulsed
ultrasound performed by using a high temporal peak and a low
temporal average intensity appeared to be optimal for activat-
ing the stress response. These findings may implicate HSPs as
a mechanism by which ultrasound treatment enhances tissue
repair. Exactly how ultrasound increased HSP expression
might influence the recovery of damaged tissue remains to be
determined, and the present study may provide incentive for
further investigations into effective use of ultrasound.
Acknowledgment: We thank James Ostoya for help with techni-
cal aspects of the study.
References
1. Barnett SB, ter Haar GR, Ziskin MC, Nyborg WL, Maeda K,
Bang J. Current status of research on biophysical effects of ultra-
rs
sound. Ultrasound Med Biol 1994;20:205-18.
2. Gallo JA, Draper DO, Brody LT, Fellingham GW. A comparison
of human muscle temperature increases during 3-MHz continuous
and pulsed ultrasound with equivalent temporal average intensi-
pe
ties. J Orthop Sports Phys Ther 2004;34:395-401.
3. Barnett S. Free-radical production: its biological consequences.
Ultrasound Med Biol 1998;24:S29-34.
4. Morimoto RI, Jurivich D, Kroeger P, et al. Regulation of heat
shock gene transcription by a family of heat shock factors. In:
Morimoto RI, Tissieres A, Georgopoulos C, editors. The biology
of heat shock proteins and molecular chaperones. Cold Spring
Harbor: Cold Spring Harbor Laboratory Pr; 1994. p 417-55.
r's
5. Morimoto RI. Regulation of the heat shock transcriptional re-
sponse: cross talk between a family of heat shock factors, molec-
ular chaperones, and negative regulators. Genes Dev 1998;12:
3788-96.
6. Garramone RR, Winters RM, Das DK, Deckers PJ. Reduction of
o
skeletal injury through stress conditioning using the heat-shock
response. Plast Reconstr Surg 1994;93:1242-7.
th
7. Marber MS, Mestril R, Chi SH, Sayen MR, Yellon DM, Dillmann
WH. Overexpression of the rat inducible 70-kD heat stress protein
in a transgenic mouse increases the resistance of the heart to
ischemic injury. J Clin Invest 1995;95:1446-56.
Au
8. Miyabara EH, Martin JL, Griffin TM, Moriscot AS, Mestril R.
Overexpression of inducible 70-kDa heat shock protein in mouse
attenuates skeletal muscle damage induced by cryolesioning.
Am J Physiol Cell Physiol 2006;290:C1128-38.
Arch Phys Med Rehabil Vol 88, June 2007
9. Escobedo J, Augustina MP, Koh T. HSP25 protects skeletal
muscle cells against oxidative stress. Free Radic Biol Med 2004;
37:1455-62.
10. Nollen EA, Morimoto RI. Chaperoning signaling pathways: mo-
lecular chaperones as stress-sensing ‘heat shock’ proteins. J Cell
Sci 2002;115:2809-16.
11. Huang MH, Ding HJ, Chai CY, Huang YF, Yang RC. Effects of
sonication on articular cartilage in experimental osteoarthritis.
J Rheumatol 1997;24:1978-84.
12. Huang M, Yang R, Ding H, Chai C. Ultrasound effect on level of
py
stress proteins and arthritic histology in experimental arthritis.
Arch Phys Med Rehabil 1999;80:551-6.
13. Locke M, Nussbaum EL. Continuous and pulsed ultrasound do not
increase heat shock protein 72 content. Ultrasound Med Biol
2001;27:1413-9.
co
14. Lowry OH, Rosebrough A, Farr L, Randall RJ. Protein measure-
ment with the folin phenol reagent. J Biol Chem 1951;193:265-75.
15. Laemmli UK. Cleavage of structural proteins during the assembly
of the head of bacteriophage T4. Nature 1970;227:680-5.
16. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer from
polyacrylamide gels to nitrocellulose sheets: procedure and some
applications. Proc Natl Acad Sci U S A 1979;76:4350-4.
17. Locke M, Tanguay R. Increased HSF activation in muscles with a
al
high constitutive Hsp70 expression. Cell Stress Chaperones 1996;
1:189-96.
18. Rubin C, Bolander M, Ryaby J, Hadjiargyrou M. The use of
on
low-intensity ultrasound to accelerate the healing of fractures.
J Bone Joint Surg Am 2001;83:259-70.
19. Knight CA, Rutledge CR, Cox ME, Acosta M, Hall SJ. Effect of
superficial heat, deep heat, and active exercise warm-up on the
extensibility of the plantar flexors. Phys Ther 2001;81:1206-14.
20. Barnett S. Thresholds for nonthermal bioeffects: theoretical and
experimental basis for a threshold index. Ultrasound Med Biol
1998;24:S41-9.
21. Karnes JL, Burton HW. Continuous therapeutic ultrasound accel-
erates repair of contraction-induced skeletal muscle damage in
rats. Arch Phys Med Rehabil 2002;83:1-4.
22. Fisher BD, Hiller CM, Rennie SG. A comparison of continuous
and pulsed ultrasound on soft tissue injury markers in the rat.
J Phys Ther Sci 2003;15:65-70.
23. Locke M, Tanguay RM, Klabunde RE, Ianuzzo CD. Enhanced
postischemic myocardial recovery following exercise induction of
HSP 72. Am J Physiol 1995;269(1 Pt 2):H320-5.
24. Locke M, Atkinson BG, Tanguay RM, Noble EG. Shifts in type I
fiber proportion in rat hindlimb muscle are accompanied by
changes in HSP72 content. Am J Physiol 1994;266(5 Pt 1):
C1240-6.
Suppliers
a. Excel Tech Ltd, 2568 Bristol Cir, Oakville, ON L6H 5S1, Canada.
b. Biopac Systems Inc, 42 Aero Camino, Goleta, CA 93117.
c. Physiotemp Instruments Inc, 154 Huron Ave, Clifton, NJ 07013.
d. Bio-Rad Laboratories, 1000 Alfred Nobel Dr, Hercules, CA 94547.
e. Product nos. SPA-812, SPA-801; Stress-Gen Biotechnologies, Vic-
toria, Canada.
f. Agfa Canada, 77 Belfield Rd, Toronto, ON M9W 1G6, Canada.
g. Kodak Scientific Imaging Systems, 343 State St, Rochester, NY
14650.