|Contrast-enhanced ultrasound; Delayed onset muscle
soreness; Exercised-induced muscle damage
|Repeated contractions of skeletal muscle, as in exercise, have
been shown to increase local [1-6] and systemic blood flow [7,8]. This
increase in blood flow is proportional to the metabolic demands of
the muscle tissue being exercised . Even with relatively low force
contractions, blood flow is elevated . Microvascular perfusion
changes in an isolated muscle have been studied previously using
concentric, isometric, and eccentric contractions. Eccentric exercise
is often used as model for examining the effects of muscle damage
and inflammation [10,11]. Recently, we examined the effect of a
single bout of unilateral, eccentric exercise to the gastrocnemius.
We found that microvascular perfusion increased immediately after
exercise,  and this increase was sustained over 48 hours .
Understanding systemic microvascular perfusion characteristics in
a healthy population during eccentric exercise will help explain the
metabolic load of this type of exercise and quantifying the vascular
changes in healthy individuals is important prior to an examination
in pathological populations. Understanding normative values in
a healthy population will allow for deficiencies to be detected in
pathological individuals. If the deficiencies are vascular in nature,
treatments could be administered that affect vascular responses.
|Microvascular perfusion immediately following low force
isometric and concentric exercises have been examined in skeletal
muscle [2-4,14,15] more than eccentric exercise at the local level.
While blood volume and flow increase following exercise, it is
unclear what the vascular response in the unexercised limb is.
When injury occurs, particularly at a joint, deficits in strength and
muscle activation have been shown in the contralateral limb [16-18].
Clinicians aim to address these deficits during rehabilitation, but the
cause of these contralateral deficits remain unknown. It is possible
that during injury, blood flow increases systemically contributing to
some of the deficits present.
|There have been several measurements techniques to estimate
blood flow in the extremities of humans and animal models, however,
these tools monitor the arteriole function and total flow, but cannot
differentiate immediate or ongoing responses at the capillary level [19-
21]. Contrast-enhanced ultrasound (CEU) is a non-invasive method to
measure microvascular volume in muscle . Infused micro bubbles
act as the contrast agent through continuous intravenous infusion at
a constant rate and concentration . Based on the principle that
ultrasound does not transmit through air and media of differing
densities have greater contrast, the micro bubbles can be visualized
with a diagnostic ultrasound imager .
|While blood flow has been shown to increase during isometric
contractions of the lower limb using CEU,  and immediately
following eccentric exercise,  the systemic response in the
contralateral limb has not been assess using CEU. Therefore, the
purpose of our study was to examine the immediate effects of
unilateral, fatiguing eccentric exercise on microvascular perfusion in
the contralateral gastrocnemius and at 48 hours. Our hypothesis was
that blood flow and volume would increase immediately post-exercise
due to the systemic increase in perfusion from exercise. However, at
48 hours, microvascular perfusion would return to baseline levels,
since the exercised leg would only experience DOMS.
|This study was a descriptive laboratory study. The independent
variable was time (baseline, immediate post-exercise, and 48 hours
post exercise). The dependent variables were blood flow, blood volume,
and blood flow velocity.
|Six healthy volunteers participated in the study (1M, 5F; Age: 22.4
± 2.1 years; Height: 165.2 ± 16.6 cm; Weight: 64.5 ± 25.1 Kg) without
a lower extremity injury in the past six months or lower extremity
surgery in the past year. Other exclusion criteria were history of
cardiovascular disease, abnormal ECG, heart murmur, or pregnancy.
All participants read and signed informed consent and the study was
approved by the University’s investigational review board (IRB-HSR
#15227). Sample size was calculated at 9 subjects using blood flow data
from the exercised limb baseline to post-exercise, when power equals
.80 and alpha is set at p=0.05 . While our sample size is small, this was the control group to a larger study  and significant differences were found.
|CEU measurements were recorded using the SONOS 7500
ultrasound machine (Philips Medical Systems, Andover, MA). The S3
adult echo transducer was used to image the muscle with an imaging
ratio of 1.3:3.6 MHz. The mechanical index was set at 1.5.
|The contrast agent Definity® (Lantheus Medical Imaging,
N. Billerica, MA) is an FDA approved contrast agent containing
octafluorpropane gas-filled albumin. The definity microbubbles are
supplied in 1.5 ml volume vials and were mixed with 0.9% sodium
chloride in a ratio of 0.3 ml microbubbles per 7 ml saline. A maximum
of 2 vials (3.0 ml) can be safely infused per day. The solution was
infused intravenously at a rate of 1.5 ml.min-1.
|The VAS was used as the primary tool for pain quantification. A
100 mm line, with no markings, except no pain at the left and worst pain at the right end of the continuum was used. Subjects were asked
to mark a vertical dash on the horizontal line indicating level of pain
relative to the continuum. A line without numerical markings was
used so the patient was less apt to remember previous markings.
|Eccentric exercise protocol
|There are several eccentric exercise protocols to induce DOMS
in the calf since this is a commonly utilized mechanism for creating
an inflammatory Model [25-28]. We chose to use a modified version
of the method described by Tegeder et al.  since this utilized a
unilateral exercise procedure for the calf. Subjects stood on one foot
on an aerobic step with the heel of the exercising leg off the edge of
the step. The subjects lifted the heel of the leg that was randomized to
do the exercise and then lowered the heel slowly for 3 seconds until
they could not lower the heel any further. A metronome was set at
60 beats per minute for uniform timing of the exercise. The control
leg was used to shift the weight distribution off the exercised leg, so it
could be returned to the starting position without doing a concentric
contraction on that extremity (Figure 1). A random number generator
was used to determine which leg was exercised. The exercise protocol
consisted of 2 sets of 50 eccentric contractions of the gastrocnemius
separated by a 5-minutes rest interval.
|Participants met with the lead investigator and physician for the
initial screening. Height, weight, blood pressure, pulse, respirations,
and heart sounds were recorded. A general health history and
lower extremity health history questionnaire were completed. If no
exclusion criteria were identified, the participant had a 12-lead ECG
assessment and metabolic blood draw. Female participants had a
serum pregnancy test to make sure they were not pregnant. Results
were obtained within 48 hours and were reviewed by a physician.
Once the physician cleared the participant, he or she was enrolled in
the exercise portion of the study.
|All participants stayed overnight at the GCRC the night before
testing began. Participants were instructed to stay in bed except to use
the bathroom and lights were turned off at 11 pm. between 5 and 6 am
the next morning, a registered nurse prepared the preferred arm for
an intravenous catheter for micro bubble infusion in an antecubital
vein using sterile techniques. A 3-lead ECG and pulse oxiometer was
attached to the participant for continuous monitoring of heart rhythm
and oxygen saturation until the subject was discharged. Subjects were positioned prone. The medial gastrocnemius of the contralateral limbs
was marked over the area of greatest girth with a 2.5 cm×2.5 cm square
(size of the transducer head). A solution of 0.9 ml of microbubbles
were mixed with 21 ml of saline and infused at a rate 1.5 ml/min.
After steady state was reached (about 2 min), CEU measurements
began. The ultrasound transducer was placed over the mark on the
gastrocnemius after applying an ample amount of ultrasound gel to
the transducer. Images were triggered to pulse intervals of the ECG,
with one image taken every 1 beat, 2 beats, 3 beats, 4 beats, 5 beats, 8
beats, 12 beats, 16 beats, and 20 beats, for three images at every pulse
interval, resulting in 27 images. Infusion of the micro bubbles stopped
after the last image was collected. Measurements took between 3-5
min, because of the dependence on heart rate.
|Participants were monitored for any cardiovascular effects or
symptoms associated with the micro bubble infusion for 5 minutes
before performing the eccentric exercise protocol. The examiner
taking the CEU measurements instructed the subject on how to
perform the exercise and allowed the subject to practice up to 10
repetitions with feedback. Once the exercise was finished, participants
were positioned prone and infusion of the micro bubbles began again.
CEU measurements were recorded as before.
|At 48 hours, the subject returned to the GCRC where the subject
was instructed to lay quietly for 2 hours to ensure a resting perfusion
level. The CEU measurements were captured as previously described.
The subject was monitored for 30 minutes before being discharged.
|Images were analyzed using specialized software developed for the
CEU procedures. QLAB (Philips Healthcare, Andover, MA) was used
to assess blood volume and blood flow velocity using replenishment
kinetics for a specific region of interest (Figure 2). Blood flow is
calculated as the product of blood volume and blood flow velocity.
The equation of y=A (1-exp-βt), where y equals video intensity (VI) at
pulse interval t, VI plateau (blood volume) is A, and β is the rate of rise
in VI (blood flow velocity), is used .
|Three separate 1×3 repeated measures ANOVAs (SPSS 17, Chicago,
IL) were used to analyze microvascular perfusion over 48 hours. ADD
is VAS planned effect size calculations were performed on significant
|There was a significant main effect for time for blood volume
(p=0.023) and blood flow (p=0.010), with no significant difference in blood flow velocity (p=0.316). There were significant increases in
blood volume (p=0.001) and blood flow (p<0.001) immediately postexercise
(9.77 ± 3.19 dB and 3.53 ± 0.86 dB/sec), respectfully in the
contralateral limb compared to baseline (6.18 ± 2.05 dB and 2.40 ±
0.69), with no change in blood flow velocity (p=0.487). The effect
size for blood volume was 1.34 (0.09, 2.60) and blood flow was 1.41
(0.15, 2.68). The increases in contra lateral blood volume (p=0.002)
and blood flow (p=0.003) were maintained at 48 hours (9.41 ± 1.90 dB
and 3.51 ± 0.47 dB/sec) compared to baseline, with again no change
in blood flow velocity (p=0.411). The effect size for blood volume was
1.62 (0.32, 2.92) and blood flow was 1.86 (0.51, 3.22). There were no
changes in blood volume (p=0.814), blood flow (p=0.962), or blood
flow velocity (p=0.493) between post-exercise and 48 hours for the
contra lateral limb.
|VAS scores for the exercised limb were significantly different at
24 and 48 hours from baseline (p<.001), as well as between 24 and 48
hours (p<0.001). The effect sizes are all above 5. Baseline was 0.0 ± 0.0
mm, 24 hours was 18.38 ± 5.01 mm and 48 hours was 52.5 ± 5.37 mm.
|Following eccentric exercise to a single limb, the contralateral
limb resulted in increased blood volume and blood flow immediately
after exercise and at 48 hours post exercise. From previous research in
our lab  immediately after eccentric exercise, blood volume and
blood flow increased in the exercise leg by 42% and 80%, respectfully.
From this study, the contra lateral leg increased 17% and 35% for blood
volume and blood flow, respectfully. This finding supports earlier
work by Seals  and Taylor et al.  that identified vasodilatation of
the contra lateral limb after exercise initiation. Blood flow velocity did
not change in the contra lateral limb after exercise and at 48 hours.
Since this limb was not exercised, recruitment of capillaries is not
necessary, as would be in exercised muscle .
|The increases in blood volume and blood flow of the contra lateral
limb were maintained over the 48 hour period. Since eccentric exercise
was performed in our study, we feel that the sustained increase in
micro vascular perfusion was from an inflammatory response in
the form of DOMS from the exercised leg. This local inflammatory
response leads into a systemic response called the acute-phase
response, which is mediated by circulating cytokines from the injured
tissue, including IL-6 and TNF-α . This may lead to a systemic
response in increased blood flow. Pain scores were also elevated over
48 hours, indicating DOMS was achieved in the exercised leg and a
form of muscle damage occurred.
|Another explanation could be from the added work performed by
the contra lateral limb for locomotion. Several of our subjects could not
bear full weight on the exercised limb and had limited dorsiflexionin
the exercised limb. While this was not directly measured, side-toside
differences were noted. Subjects reported having to limp because
they could not lower the heel all the way to the ground due to pain.
Loss of function occurs immediately after eccentric exercise, where
the greatest deficits in force production and range of motion are
noted . The reduced range of motion may allow for a position of
optimal healing when new capillaries and new connective tissue are
forming 48 hours after injury . There were no functional deficits
or pain observed in the contralateral limb. Examining micro vascular
perfusion of the upper extremity may have been a better model for
assessing systemic blood flow. The mechanisms behind micro vascular
perfusion increases systemically remain unknown.
|While we controlled for ice applications and analgesic use, a limitation of our study was that we could not control for activities of
daily living within the 48 hours between CEU assessments. They were
told to go about activities of daily living, but to refrain from exercise
or taking over the counter pain medication. Some subjects walked
to and from class, potentially accumulating up to 30 minutes, while
other subjects worked desk jobs. We asked each subject to report use
of NSAIDs or additional treatments, but all reported none were used.
|Eccentric exercise increased microvascular perfusion immediately
after exercise in the contralateral limb, which had not been examined
before. The increased perfusion was maintained over 48 hours, so the
prolonged increased in perfusion of the contralateral limb may have
been due to an inflammatory response or the extra demands placed on
the contralateral limb for support during walking.
|Supported by NIH Grant RR00847 and the University of Virginia Curry
School of Education.
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