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Purpose: We investigated if oral ingestion of ibuprofen influenced leucocyte recruitment and infiltration following an acute bout of traditional resistance exercise Methods: Sixteen male subjects were divided into two groups that received the maximum over-the-counter dose of ibuprofen (1200mg d(-1)) or a similarly administered placebo following lower body resistance exercise. Muscle biopsies were taken from m.vastus lateralis and blood serum samples were obtained before and immediately after exercise, and at 3 and 24 h after exercise. Muscle cross-sections were stained with antibodies against neutrophils (CD66b and MPO) and macrophages (CD68). Muscle damage was assessed via creatine kinase and myoglobin in blood serum samples, and muscle soreness was rated on a ten-point pain scale. Results: The resistance exercise protocol stimulated a significant increase in the number of CD66b(+) and MPO(+) cells when measured 3 h post exercise. Serum creatine kinase, myoglobin and subjective muscle soreness all increased post-exercise. Muscle leucocyte infiltration, creatine kinase, myoglobin and subjective muscle soreness were unaffected by ibuprofen treatment when compared to placebo. There was also no association between increases in inflammatory leucocytes and any other marker of cellular muscle damage. Conclusion: Ibuprofen administration had no effect on the accumulation of neutrophils, markers of muscle damage or muscle soreness during the first 24 h of post-exercise muscle recovery.
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ORIGINAL RESEARCH
published: 29 March 2016
doi: 10.3389/fphys.2016.00086
Frontiers in Physiology | www.frontiersin.org 1 M
arch 2016 | Volume 7 | Article 86
Edited by:
Vincent Pialoux,
University Lyon 1, France
Reviewed by:
Gianluca Vernillo,
University of Milan, Italy
Scott Forbes,
Okanagan College, Canada
*Correspondence:
Luke Vella
lv280@bath.ac.uk
Specialty section:
This article was submitted to
Exercise Physiology,
a section of the journal
Frontiers in Physiology
Received: 12 December 2015
Accepted: 22 February 2016
Published: 29 March 2016
Citation:
Vella L, Markworth JF, Paulsen G,
Raastad T, Peake JM, Snow RJ,
Cameron-Smith D and Russell AP
(2016) Ibuprofen Ingestion Does Not
Affect Markers of Post-exercise
Muscle Inflammation.
Front. Physiol. 7:86.
doi: 10.3389/fphys.2016.00086
Ibuprofen Ingestion Does Not Affect
Markers of Post-exercise Muscle
Inflammation
Luke Vella
1
*
, James F. Markworth
2
, Gøran Paulsen
3
, Truls Raastad
3
, Jonathan M. Peake
4
,
Rod J. Snow
1
, David Cameron-Smith
2
and Aaron P. Russell
1
1
Centre for Physical Activity and Nutrition Research, School of Exercise and Nutrition Science, Deakin University, Burwood,
VIC, Australia,
2
Liggins Institute, University of Auckland, Auckland, New Zealand,
3
Department of Physical Performance,
Norwegian School of Sport Science, Oslo, Norway,
4
School of Biomedical Sciences and Institute of Health and Biomedical
Innovation, Queensland University of Technology, Brisbane, QLD, Australia
Purpose: We investigated if oral ingestion of ibuprofen influenced leucocyte recruitment
and infiltration following an acute bout of traditional resistance exercise.
Methods: Sixteen male subjects were divided into two groups that received the
maximum over-the-counter dose of ibuprofen (1200 mg d
1
) or a similarly administered
placebo following lower body resistance exercise. Muscle biopsies were taken from
m.vastus lateralis and blood serum samples were obtained before and immediately after
exercise, and at 3 and 24 h after exercise. Muscle cross-sections were stained with
antibodies against neutrophils (CD66b and MPO) and macrophages (CD68). Muscle
damage was assessed via creatine kinase and myoglobin in blood serum samples, and
muscle soreness was rated on a ten-point pain scale.
Results: The resistance exercise protocol stimulated a significant increase in the number
of CD66b
+
and MPO
+
cells when measured 3 h post exercise. Serum creatine kinase,
myoglobin and subjective muscle soreness all increased post-exercise. Muscle leucocyte
infiltration, creatine kinase, myoglobin and subjective muscle soreness were unaffected
by ibuprofen treatment when compared to placebo. There was also no association
between increases in inflammatory leucocytes and any other marker of cellular muscle
damage.
Conclusion: Ibuprofen administration had no effect on the accumulation of neutrophils,
markers of muscle damage or muscle soreness during the first 24 h of post-exercise
muscle recovery.
Keywords: exercise recovery, NSAID treatment, inflammation, resistance exercise, leucocyte
INTRODUCTION
Unaccustomed resistance exercise often results in tissue damage and inflammation, leading
to delayed onset muscle soreness (DOMS) and a consequent reduction in force production
(
Faulkner et al., 1993; Tidball, 1995). Animal models and i
n vitro studies have identified that local
and systemic inflammation exerts a regulatory influence during the different phases of muscle
recovery, including myofibrillar disruption, cellular necrosis, satellite cell activation, maturation
and subsequent regeneration, and ad aptation (Fridén et al., 1981; Armstrong et al., 1991). Thus,
Vella et al. Ibuprofen and Post-exercise Inflammation
post-exercise inflammation is intimately ne cessary and a key
feature of the normal process of tissue regeneration and
adaptation following acute muscle damage. However, excessive
inflammation is considered a potential cause of prolonged
post-exercise muscle soreness and may have a negative effect
on muscle recovery (Armstrong et al., 1991; Smith, 1991);
consequently strategies to reduce or counteract inflammation are
commonly implemented to aid in improving muscle re covery
after exercise (Urso, 2013).
Non-steroidal anti-inflammatory drugs (NSAIDs) are
commonly used as a treatment strategy in exercise and
sports medicine to assist with recovery from exercise-induced
inflammation, particularly following soft-tissue injury. NSAIDs
inhibit the cyclooxygenase (COX-1 and 2) enzymes and
consequently the formation of prostanoids (prostaglandins,
prostacyclins, and thromboxanes) that pla y a diverse role in
acute inflammation (Markworth et al., 2013). Prostanoids
stimulate an acute inflammatory process by controlling local
blood flow, vascular permeability, leucocyte infiltration, and
triggering sensations of pain (Markworth et al., 2013; Urso,
2013). In animal models of acute muscle d a mage, treatment
with NSAIDs blunts the infiltration of leucocytes into muscle
tissue (Lapointe et al., 2003; Bondesen et al., 200 4) and causes
a reduction in creatine kinase (CK) (Mishra et al., 1995).
Consequently, NSAIDs can also inhibit myofiber regeneration,
satellite cell proliferation and differentiation, and overload-
induced muscle hypertrophy (Mishra et al., 1995; Bondesen
et al., 2004, 2006). These findings provide preliminary evidence
that NSAIDs compromise the physiological link between
processes of acute muscle damage, inflammation and cellular
regeneration.
Research into t he effects of NSAIDs on exercise-induced
muscle damage and inflammation has produced equivocal
findings. In exercise models, NSAID administration has been
shown to attenuate post-exercise DOMS in some (
Baldwin et al.,
2001; Tokmakidis et al., 2003; Paulsen et al., 2010), but not
all studies (Trappe et al., 2002; Krentz et al., 2008; Mikkelsen
et al., 2009; Hyldahl et al., 2010). While a precise cellular
mechanism for an analgesic effect of NSAIDs remains unclear,
it has been largely as cribed to their effect on prostaglandin
synthesis and the capacity of NSAIDs to interfere with aspects
of inflammatory cell function (Hersh et al., 2000; Peterson
et al., 2003). Previous research has demonstrated that oral
consumption of both ibuprofen, a non-selective NSAID and
acetaminophen, an analgesic also known as paracetamol, had no
effect on macrophage infiltration at 24 h following an eccentric
exercise protocol (Peterson et al., 2003). Similarly treatment with
naproxen, another non-selective NSAID, had no effect on the
infiltration of leucocyte common antigen positive cells following
a unilateral, isotonic resistance exercise protocol (Bourgeois et al.,
1999). Interestingly, Paulsen et al. (2010) suggested a blunting
effect of the COX-2 specific celecoxib following maximum
eccentric muscle contractions (Paulsen et al., 2010). This research
identified a tendency for higher monocyte/macrophage numbers
in subjects within the placebo group, and those subjects who were
identified as “high-responders to the exercise protocol based on
the number of inflammatory leucocytes (
Paulsen et al., 2010).
Although this is not a partic ularly robust finding , it has led to the
hypothesis that NSAIDs may influence leucocyte infiltration in
skeletal muscle when a sufficiently strong and early inflammatory
reaction is present (
Paulsen et al., 2010). This hypothesis would
suggest that the intensity of the exercise stimulus and t he
consequent muscle-damage response would be largely influential
in determining the effect of a pharmacologically b ased anti-
inflammatory intervention.
Conflicting findings also exist with regard to the effect
of NSAIDs on the regenerative c a pacity of skeletal muscle
following exercise-induced muscle damage. The non-selective
COX inhibitor ibuprofen blunted skeletal muscle protein
synthesis (
Trappe et al., 2002) while local intramuscular
infusion of indomethacin (Mikkelsen et al., 2011) and the
oral administration of the COX-2 selective NSAID celecoxib
(Burd et al., 2010; Paulsen et al., 2010) had no such effect.
Similarly, treatment wit h indomethacin inhibited post-exercise
satellite cell proliferation (
Mikkelsen et al., 2009). Recent work
from our group demonstrated that treatment with ibuprofen
inhibited early translational signaling responses involved in post-
exercise muscle hypertrophy (Markworth et al., 2014). A clear
mechanistic pathway for NSAIDs to influence t he physiological
link between post-exercise inflammation and skeletal muscle
regeneration remains elusive.
These discrepancies in the research to date are likely due
to differences in the exercise protocol (concentric vs. eccentric
muscle contractions), the training status of subjects, the timing
of muscle biopsies, and the type of NSAID, the dosage
administered, and method of administration. Further research is
required to determine whether NSAID administration affects the
infiltration of leucocyte populations following exercise-induced
muscle damage and how this influences post-exercise adaptive
pathways. The aim of the present study was to investigate if
oral administration of the NSAID ibuprofen influenced skeletal
muscle leucocyte infiltration during the first 24 h after muscle-
damaging resistance exercises. We also aimed to explore how
any changes in leucocyte infiltration were related to markers
of muscle damage, including circulating muscle proteins CK
and myoglobin, and subjective markers of muscle soreness.
We hypothesized that the ingestion of ibuprofen following
acute resistance exercise would not influence the infiltration of
leucocytes, however it would attenuate sensations of DOMS.
MATERIALS AND METHODS
Participants
As described previously, 16 healthy male subjects were recruited
to participate in the study (Table 1;
Markworth et al., 2013). It
is important to note that the purpose of the original study by
Markworth et al. (2013) was to profile the human eicosanoid
response to acute exercise in blood plasma samples, as opposed
to the aim of the present study which was to determine the
effect of oral ingestion of ibuprofen on leucocyte recruitment
and infiltration following an acute bout of traditional resistance
exercise. While the subjects and experimental design are the same
in both studies the aims and dependent variables reported are
different. Exclusion criteria included participation in a lower
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Vella et al. Ibuprofen and Post-exercise Inflammation
TABLE 1 | Subject characteristics and strength testing data.
Characteristics Strength (1 RM)
Age (y) Height (m) Body mass (kg) BMI Squat (kg) Leg press (kg) Leg extension (kg)
PLA 23.9 ± 1.3 1.89 ± 0.1 86.9 ± 4.5 24.5 ± 1.2 94.9 ± 5.4 237 ± 17 236 ± 18
IBU 23 ± 0.5 1.89 ± 0.1 89.1 ± 4.4 24.8 ± 0.8 91.9 ± 6.0 240 ± 15 196 ± 22
Values are mean values ± SEM. No significant differences were observed between the two groups. PLA, placebo; IBU, Ibuprofen; BMI, body mass index.
body resist a nce exercise program within the last 6 months to
ensure a muscle damage response from the exercise stimulus,
and/or previous chronic treatments with anti-inflammatory
medication. Participants also completed a medical screening to
identify any potential risk factors for them to perform strenuous
physical activity.
Ethics Approval
All procedures involved in this study were approved by
the Deakin University Human Research Ethics Committee
(DUHREC 2010-019) and muscle sampling procedures were
performed in accordance with the Helsinki declaration. Each
participant was provided with written and oral details of the
nature and requirements of the study and provided written
consent to participate.
Familiarization
Each participant completed a familiarization and strength testing
session at least 7 days prior to completing the exercise protocol.
Details of the familiarization session have been described
previously (
Markworth et al., 2013). Briefly, subjects performed
repetition maximum testing for the Smith machine-assisted
squat, the leg press and the leg extension to determine their
experimental exercise load [80% of a 1 repetition maximum (1
RM)]. The maximum weight the subject could lift for 5–8 reps
was determined, and these data were entered into the Brzycki
equation to predict 1 RM (Whisenant et al., 2003). Subjects were
asked to abstain from any furt h er activity until the completion of
the tria l.
Experimental Procedures
The participants reported to th e laboratory on the morning
of the trial in an overnight fasted state. They were asked to
abstain from caffeine, tobacco and alcohol for the 24 h preceding
the trial. Participants rested in a supine position for 30 min,
following which the first muscle biopsy sample was taken.
Each participant then completed a 10 min warm-up protocol
comprised of 5 min of low intensity cyc ling on a stationary
bike, and one low-intensity set of each exercise at a weig ht of
each subject’s own choice within the range of 30–50% 1 RM.
The resistance exercise session consisted of three sets of 8–10
repetitions performed on a Smith machine assisted squat, a 45
leg press and a leg extension at 80% of a predict ed 1 RM. The
exercises were performed as a circuit with 1 min rest permitted
between exercises and 3 min rest between sets. This protocol
has been used previously and has been a sufficient stimulus t o
activate inflammatory signaling pathways (
Vella et al., 2012) and
was implemented to replicate a commonly used exercise routine.
After exercise, the subjects rested while subsequent muscle biopsy
samples were collected. After the 3 h biopsy, participants were
provided a standardized meal and were allowed to go home.
The following morning, participants reported to the laboratory
in an over-night fasted state for a 24 h muscle biopsy and blood
sample.
Standardized Meals
Standardized meals were provided to participants on the night
before the trial, (carbohydrate 57%, fa t 22%, protein 21%),
immediately following t he 3 h muscle biopsy (carbohydrat e 71%,
fat 13%, protein 18%), and in the e vening (c ar bohydrate 64%, fat
27%, protein 18%) on the day of the exercise trial. Participants
were permitted to drink water ad libitum and were asked to report
if they could not finish their alloca ted meals. This nutrition plan
was included to ensure that each participant received the same
relative percentage of macro- and micro-nutrients.
NSAID Administration
Prior to exercise, participants were randomly assigned in
a double-blind method, to consume either the maximum
recommended dose of ibuprofen (IBU, n = 8) or a placebo
control (gelatin capsules identical in appearance containing
powdered sugar in place of ibuprofen) (PLA, n = 8). The IBU
group consumed three doses of 400 mg of ibuprofen throughout
the trial day. The first dose was administered immediately prior
to t he first muscle biopsy sample. Participants were instructed
to consume the following two doses at 6 and 12 h following
the exercise protocol. Follow-up phone calls from the research
team ensured compliance. To ensure this dosing structure was
appropriate to maintain biologically active levels of ibuprofen,
ibuprofen concentration was measured in blood serum samples
and these data are reported elsewhere (
Markworth et al.,
2013).
Sample Collection
Venous blood samples were drawn prior to exercise, within 5 min
post exercise (here-after referred to as 0 h post-exercise), and at 1,
2, 3, and 24 h post-exercise. Blood samples were drawn through
an indwelling cat h eter into VACUET T E serum tubes (Greiner,
Bio-One, Stonehouse UK). Whole blood was allowed to clot at
room temperature. Samples were then centrifuged at 10 00 × g
for 10 min. The serum layer was collected and stored at 80
C
for subsequent analysis.
Muscle biopsy samples were obtained from the vastus
lateralis muscle under local anesthesia (Xylocaine 1%) using
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Vella et al. Ibuprofen and Post-exercise Inflammation
a percutaneous needle biopsy technique modified to include
suction (Buford et al., 2009). To minimize interference from
the biopsy procedure, samples prior to exercise and at 24 h post
exercise were taken from the same leg, while samples taken at
0 and 3 h post-exercise were taken from the contralateral leg.
Samples obtained within the same leg were taken at le ast 5 cm
from the previous incision site. We have previously reported
that this technique is effective for minimizing the risk of any
inflammation arising from the biopsy procedure confounding
exercise-induced inflammation (Vella et al., 2012). Tissue-Tek
immersed tissue was rapidly frozen in isopentane cooled liquid
nitrogen before storage at 80
C.
Immunohistochemistry
Cross sections (8 µm) of muscle tissue were cut using a
microtome a t 20
C (CM3050, Leica, Germany) and mounted
on microscope slides (Superfrost Plus, Thermo Scientific,
MA, USA), air-dried and stored at 80
C. Each subjects
muscle sections from all time points were mounted on the
same microscope slide. Serial muscle cross-sections were
stained with three different leucocyte antibodies, MPO (#0398,
DakoCytomation, Glostrup, Denmark; dilution 1:2000), CD66b
(#CLB-B13.9, Sanquin Reagents, Amsterdam, The Netherlands;
dilution 1:500), and CD68 (#EBM-11, DakoCytomation,
Glostrup, Denmark; dilution 1:300). Furthermore, the sections
were also stained with dystrophin antibody (ab15277, Abcam,
Cambridge, UK) for visualizing borders of muscle fibers.
Sections were fixed in 4% paraformaldehyde solution in a
staining jar for 5 min at room temperature and rinsed twice
for 10 min in phosphate buffered saline (PBS) containing 0.5%
Tween 20 (Sigma-Aldrich). The microscope slides were moved
into a humidified chamber and non-specific binding sites were
blocked with 1% bovine serum albumin (BSA) on the section for
45 min at room temperature. Sections were incubated overnight
with leucocyte and dystrophin antibodies diluted in 1% BSA
at 4
C. After overnight incubation, the slides were washed
three times for 10 min in PBS-Tween in a staining jar. Slides
were moved back to the humidified chamber and se c tions
were incubated for 45 min with secondary antibodies diluted
1:200 in 1% BSA at room temperature. Alexa Fluor
R
594
F(ab
)2 fragment of goat anti-rabbit and Alexa Fluor
R
488 anti-
mouse IgG (Invitrogen, Eugene, Oregon, USA) were used as a
secondary antibodies. The fluorochrome-stained sections were
washed three times for 10 min in PBS-Tween. After the last
washing in PBS-Tween, the secti ons were mounted on ProLong
R
Gold Antifade reagent with DAPI (Invitrogen, Eugene, Orgeon,
USA).
Muscle sections were visualized using a high-resolution
camera (DP72, Olympus, Japan) mounted on a microscope
(BX61, Olympus, Japan) with a fluorescence light source (X-
Cite 120PCQ, EXFO, Canada). The number of MPO, CD66b,
and CD68 positive cells (as well as the total number of muscle
fibers from th e area included) was counted. Da ta was calculated
as percentage of MPO, CD66b, or CD68 positively stained cells
per 100 skeletal muscle fibers. Areas of sections that contained
freeze damage or were folded due to the cutting procedure were
not included in the analysis.
Biochemical Assays
Serum creatine kinase activity in pre- and post-exercise blood
samples was analyzed using an enzymatic assay (CK-NAC
kit, CDT14010, Thermo-Fisher Scientific Clinical Diagnostics,
Sydney, Australia) and an automated clinical analyser (Cobas
Mira, Roche Diagnostics, Germany). Serum myoglobin
concentration was also measured in these samples using an
immunoassay (Roche Diagnostics, Germany) and an automated
clinical analyzer (Cobas E411, Roche Diagnostics, Germany).
The intra-assay coefficient of variation was 10.4% for creatine
kinase and 1.7% for myoglobin.
Muscle Soreness Assessment
Upon arrival at the laboratory and prior to the first muscle
biopsy procedure, as well as 24 h following exercise prior to
the fourth and final muscle biopsy, subjects were asked to rate
their subjective muscle soreness on a 0–10 visual analog scale.
The subjects were instructed to contract, stretch, and palpate
the quadriceps muscle to assess general muscle soreness. In
both instances 0 was considered to represent no pain and
correspondingly a rating of 10 was considered to represent
intense pain.
Statistics
Data are expressed as means ± SEM. Prior to analysis the
data displaying a lack of normality were log transformed to
stabilize variance. Data were analyzed using a two-way ANOVA
with repeated measures for time. The sphericity adjustment
was checked, and if required, a Greenhouse-Geisser epsilon
correction was applied. Where no significant effect for treatment
was observed, we explored pair-wise comparisons between
individual time-points using the Least Significant Difference
(LSD) of means. Bivariate relationships were examined with
a Spearman rank correlation test. These statistic al analyses
were per formed using GenStat for Windows 16th Edition (VSN
International, Hemel Hempstead, UK). Confidence intervals
were calculated at 90% using a bootstrapping technique for non-
normally distributed data using SPSS Statistics Version 22 (IBM,
Portsmouth Hampshire, UK). Statistical significance was set at
P < 0.05.
RESULTS
Muscle Soreness
Muscle soreness showed a main effect for time (p < 0.01),
suggesting that the exercise protocol was sufficient to induce a
muscle stress response. No effect of ibuprofen treatment was
obser ved (Figure 1).
Immunohistochemistry
The number of MPO
+
cells per 100 myofibers analyzed showed a
main effect for time (P < 0.01). No main effect for treatment
(P = 0.250) or time × treatment (P = 0.709) was observed
(Figure 2A). LSD pairwise comparisons indicated a significant
increase in MPO
+
cells at all sampling time points post-exercise.
The greatest increase in MPO
+
cells was observed at 3 h post-
exercise (Figure 2B). Similarly, the number of CD66b
+
cells per
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Vella et al. Ibuprofen and Post-exercise Inflammation
FIGURE 1 | Subjective rating for delayed onset muscle soreness
(DOMS) Values depicted are mean values ± SEM. *denotes statistical
significance from pre-exercise values (p < 0.05). White bars, PLA group; black
bars, IBU group. This figure has been adapted from
Vella et al. (2014).
100 myofibers showed a main effect for time (P < 0.05), with
no main effect for treatment (P = 0.149) or time × treatment
interaction (P = 0.907; Figure 2C). LSD pairwise comparisons
indicated a significant increase in the number of CD66b
+
cells
at 3 h post exercise (Figure 2D ). The number of CD68
+
cells
per 100 myofibers analyzed showed no effect for time (P =
0.061), treatment (P = 0.530) or time × treatment interaction
(P = 0.688; Figure 2E). MPO
+
, CD66b
+
, and CD68
+
cells were
obser ved within the endomysium and the perimysium, with no
cellular infiltration occurring at any time point. Representative
images for each cell sur face marker are presented in Figure 3.
Serum Proteins
Serum CK activity demonstrated a main effect for time (P <
0.01) with no effect for treatment (P = 0.843) or time ×
treatment interaction (P = 0.494; Figure 4A). LSD comparisons
revealed that serum CK increased from pre-exercise values at
2 h post-exercise and peaked at 24 h post-exercise (Figure 4B).
Similarly, serum myoglobin concentration showed a main effect
for time (P < 0.01) with no effect for treatment (P =
0.710) or time × treatment interaction (P = 0.666; Figure 4C).
LSD comparisons demonstrated that the biggest increase in
serum myoglobin concentrations occurred at 1 h post-exercise
and remained significantly elevated up to 3 h post exercise
(Figure 4D).
Correlation Analysis
In general, large inter-individual differences were seen in the
appearance of all of the leucocyte cell types. Results from the
Spearman rank correlation test showed no correlation between
the histochemical appearance of inflammatory leucocytes,
biological markers of muscle damage, and subjective markers
of muscle soreness (Table 2). Anecdot ally, three subjects were
identified as the highest responders in the appearance of cell
surface markers applicable for localization of neutrophils (one
subject within the placebo group, and two subjects within the
ibuprofen group). However, these subjects showed no significant
increase in CK, myoglobin or subjective markers of muscle
damage when compared with the other subjects. Interestingly,
subject 5 recorded the highest number of CD66b
+
cells and
MPO
+
cells, whereas he presented with the lowest subjective
rating for DOMS.
DISCUSSION
The main finding of this study was that ibuprofen, a non-selective
COX inhibitor, had no effect on the histological detection of
leukocytes following an acute bout of traditional resistance
exercise. Serum CK and myoglobin, and muscle soreness
also did not change in response to ibuprofen. Furthermore,
no correlation was observed between the accumulation of
inflammatory leucocytes, increases in CK or myoglobin, and
sensations of DOMS. These observations suggest that the
intramuscular infiltration of inflammatory white blood cells, or
an increase in serum levels of intramuscular proteins, are not
predictive of post-exercise muscle soreness.
The current st udy focused on the acute local inflammatory
response to exercise-induced muscle damage. Our protocol was
effective in inducing muscle soreness as shown in Figure 1. It is
important to note t hat we have pre viously reported this exercise-
induced stress response (
Vella et al., 2014). However, we felt it
was necessary to re-emphasize this response here. In the present
study we identified an increase in the histological appearance
of CD66b
+
and MPO
+
cells that peaked at 3 h post exercise
and found no effect of NSAID treatment. CD66b is a highly
specific cell surface marker for detecting human neutrophils, and
this is the first paper to show a small but significant increase
in the intramus cular number of CD66b
+
cells following acute
resistance exercise.
Paulsen et al. (2013) were unable to identify
any change in the number of CD66b
+
cells in the elbow flexors
at 1, 2, 4, or 7 days following 70 maximal e c centric contractions
(Paulsen et al., 2013). Differences in the timing of muscle biopsy
samples may explain the discrepancy between the two trials, and
suggest that neutrophils may be involved in the acute phase
inflammatory response to resistance exercise. We also identified
a small but significant increase in t h e number of MPO
+
cells
at all sampled time points, with a peak in expression at 3 h
post exercise. MPO has typically been used to detect changes
in neutrophils, and an increased number of MPO
+
cells have
been reported following high-force eccentric muscle contractions
(Mahoney et al., 2008; MacNeil et al., 2011). However, MPO
is also expressed on the lysosomes of monocytes a nd has been
detected on basophils and eosinophils, perhaps providing a
reason why there were a higher number of MPO
+
cells when
compared to CD66b
+
cells detected in the present study (Paulsen
et al., 2013).
No change was identified in the number of CD68
+
cells. CD68
cell counts are widely used as a marker of monocyte/macrophage
infiltration. However, CD68 may a lso be expressed on other cell
types including satellite cells and fibroblasts (Paulsen et al., 2013).
Other researchers have been able to identify a change in CD68
+
cells following exercise-induced muscle damage (
Peterson et al.,
2003; Mahoney et al., 2008; MacNeil et al., 2011
). Each of
these studies have used a high-force maximal e c c entric muscle
contraction protocol, which may suggest that the detection of
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Vella et al. Ibuprofen and Post-exercise Inflammation
FIGURE 2 | Immunohistochemistry data for muscle cells staining positive for myeloperoxidase (MPO), showing group specific data (A) and collapsed
data (B), CD66b showing group specific data (C), and collapsed data (D), and CD68 showing group data only (E). Data represent the mean number of
positively stained cells per 100 fibers analyzed ± SEM. **denotes statistical significance from pre-exercise values (p < 0.01). *denotes statistical significance from
pre-exercise values (p < 0.05).
denotes statistical significance from 0 h post-exercise (p < 0.05). White bars, PLA group; black bars, IBU group.
CD68
+
cells is dependent on the extent of skeletal muscle
damage.
Previous research exploring the effect on NSAIDs on
post-exercise inflammation has produced equivocal findings.
Earlier research from in-vivo rodent trials demonstrated a
blunted inflammatory response to eccentric muscle contractions
following the administra tion of both selective and non-selective
NSAIDs (
Lapointe et al., 2002a,b; Bondesen et al., 2004, 2006).
However, t hese findings are yet to be supported in human
trials (
Bourgeois et al., 1999; Pet erson et a l., 2003; Tokmakidis
et al., 2003). Peterson et al. (2003) found no effect of oral
ibuprofen administration on the appearance of CD68
+
cells 24 h
following 100 eccentric muscle contractions (
Peterson et al.,
2003). Similarly, Tokmakidis showed no effect of ibuprofen
on circulating white blood cells 24 h following an eccentric
exercise protocol (Tokmakidis et al., 2003). Paulsen et al. (2010)
proposed the concept of an inflammatory threshold, suggesting
that NSAIDs may only influence post-exercise inflammation in
response to a sufficiently strong inflammatory stimulus (Paulsen
et al., 2010). It was suggested that the absence of an intramuscular
prostaglandin response to exercise, specifically PGE
2
, could
explain the lack of an NSAID effect on leucocyte recruitment
(
Trappe et al., 2001; Paulsen et al., 2010). Interestingly, our
group recently reported an increase in serum PGE
2
in samples
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Vella et al. Ibuprofen and Post-exercise Inflammation
that were obtained during the same exercise trials as those
currently reported (Markworth et al., 2013). This change in
serum PGE
2
was blunted by the administration of ibuprofen
FIGURE 3 | Immunohistochemical analysis of skeletal muscle samples
following about of resistance exercise. Sections were probed with
antibodies raised against leucocyte cell surface markers (green; A—MPO,
B—CD66b, C—CD68) and dystrophin (red), while DAPI was used to stain
nuclei (blue). Scale bars = 50 µm.
and suggests that in t he presence of a blunted prostaglandin
response to exercise, NSAID treatment had no effect on leucocyte
recruitment within skeletal muscle. Although this finding does
not refute the possibility of an inflammatory threshold, based
on our previous findings (
Markworth et al., 2013), it is
reasonable to suggest that the presence of an exercise-induced
serum prostaglandin response is not the required underlying
mechanism for neutrophil infiltration of muscle during the early
hours of post-exercise recovery.
In accordance with previous studies, resistance exercise
resulted in an increase in serum CK and myoglobin, and
subjective muscle soreness (Nosaka et al., 2006). Although large
inter-individual variations in the human response to exercise
can make it difficult to establish causal relationships, there
was no relationship between the appearance of leucocytes
and blood-derived markers of muscle d a mage or sensations
of muscle soreness. Interestingly, the subject with the highest
number of CD66b
+
and MPO
+
cells presented with the lowest
subjective rating of DOMS. These findings provide support for
the assumption that leucocyte recruitment and inflammation do
not consistently cause DOMS.
From animal studies it appears that cellular events in post-
exercise inflammation, occur concomitantly with the onset of
DOMS (Armstrong et al., 1991; Faulkner et al., 1993). The
mechanistic link has been proposed to be a leucocyte-mediated
FIGURE 4 | Blood derived proteins creatine kinase (A,B) and myoglobin (C,D). Values depicted are mean values ± SEM. (A,C) represent data divided into two
treatment groups; (B,D) represent collapsed data. *denotes statistical significance from pre-exercise values (p < 0.05). **denotes statistical significance from
pre-exercise values (p < 0.01).
denotes statistical significance from 1, 2, and 3 h post exercise (p < 0.05). White bars, PLA; black bars, IBU.
Frontiers in Physiology | www.frontiersin.org 7 March 2016 | Volume 7 | Article 86
Vella et al. Ibuprofen and Post-exercise Inflammation
TABLE 2 | Spearman rank correlation analysis.
CK MYO CD68
+
CD66b
+
MPO
+
DOMS
CK 0.59 (0.01 0.91) 0.46 (0.88 0.8) 0.15 (0.42 0.61) 0.36 (0.14 0.73) 0.01 (0.60 0.62)
MYO 0.01 (0.55 0.52) 0.14 (0.42 0.69) 0.35 (0.21 0.67) 0.33 (0.74 0.24)
CD68
+
0.05 (0.46 0.56) 0.27 (0.75 0.33) 0.37 (0.82 0.25)
CD66b
+
0.41 (0.13 0.84) 0.37 (0.78 0.16)
MPO
+
0.08 (0.49 0.56)
DOMS
Values depicted are the correlation coefficient with 90% confidence intervals in parenthesis. Statistical significance was set at p < 0.05. No statistically significant correlations were
observed. CK, creatine kinase; MYO, myoglobin; MPO, Myeloperoxidase; DOMS, delayed-onset muscle soreness.
release of cytotoxic substances and reactive oxygen species
as a bi-product of phagocytosis (
Connolly et al., 2003).
Furthermore, the prostaglandin response to exercise has been
shown to occur concurrently to the onset of DOMS, and
is known to influence both the recruitment of inflammatory
leucocytes and neural afferents to pain (Markworth et a l., 2013).
However, in line with the current findings, the accumulation
of inflammatory leucocytes in human skeletal muscle tissue
during exercise recovery appears to be both spatially a nd
temporally out of phase with the development of DOMS
(
Paulsen et al., 2010). Malm et al. (2004) explored the
concept that the activation of local leucocytes present in the
epimysium may be involved in the regulation of DOMS.
This hypothesis has since received support from both rodent
(Gibson et al., 2009) and human trials (Crameri et al., 2007;
Raastad et al., 2010) and presents a promising area for future
research.
CONCLUSION
This is the first study to demonstrate that ibuprofen
administration has no effect on the histological appearance
of inflammatory white blood cells following an acute bout
of traditional resistance exercise. We also found no effect
of ibuprofen administration on blood markers of muscle
damage or subjective muscle soreness, and no significant
correlations between leucocyte numbers and post-exercise
muscle damage or soreness. Future research needs to explore
the possibility of an inflammatory threshold above which
NSAID administration influences post-exercise inflammation.
The underlying cause of post-exercise muscle soreness also
remains largely unknown. Future investigations a re needed to
determine the role that inflammation plays in exercise-induced
DOMS.
AUTHOR CONTRIBUTIONS
LV—Research design, sample acquisition and analysis, data
analysis, drafting and formatting manuscript. JM—Research
design, data analysis, revising manuscript, final approval of
version to be published. GP—Data collection and analysis,
revising manuscript, final approval of version to be published.
TR—Data collection and analysis, interpret at ion of data, revising
manuscript, final approval of version to be published. JP
Research design, data analysis, interpretation of data, revising
manuscript, final approval of version to be published. RS—
Research design, interpretation of data, revising manuscript,
final approval of version to be published. DC—Research design,
interpretation of data, revisi ng manuscript, final approval of
version to be published. AR—Data analysis, interpretation of
data, revising manuscript, final approval of version to be
published.
ACKNOWLEDGMENTS
The authors would like to acknowledge to assista nce of Associate
Professor John Reynolds of Deakin University, and Dr. Sean
Williams of University of Bath, for their contributions to the
statistical analysis of the dataset. We further thank Dr. Andrew
Garnham of Deakin University for all muscle biopsy procedures,
and the participants for volunteering their time to be involved in
the tria l.
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2016 Vella, Markworth, Paulsen, Raastad, Peake, Snow, Cameron-
Smith and Russell. This is an open-access article distributed under the terms
of the Creative Commons Attribution License (CC BY). The use, distribution or
reproduction in other forums is permitted, provided the original author(s) or licensor
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Frontiers in Physiology | www.frontiersin.org 9 March 2016 | Volume 7 | Article 86
... On the other hand, the use of naproxen did not alter tissue in ltration of in ammatory cells after experimental muscle damage protocol [8]. Vella et al. (2016) propose that NSAIDs decreases the intensity of the in ammatory response and leukocyte in ltration in skeletal muscle. Their hypothesis reinforces that the intensity of exercise and tissue responses in uence the clinical and side effects of anti-in ammatory drugs used to treat DOMS [9]. ...
... Vella et al. (2016) propose that NSAIDs decreases the intensity of the in ammatory response and leukocyte in ltration in skeletal muscle. Their hypothesis reinforces that the intensity of exercise and tissue responses in uence the clinical and side effects of anti-in ammatory drugs used to treat DOMS [9]. ...
... United Kingdom [33][34][35]; Germany [36,37]; Greece [11]; Denmark [18,38]; Belgium [39];Africa (3.8%): South Africa [40] and Oceania (3.8%): Australia [9]. ...
Preprint
Full-text available
Objective: To investigate the effects of pharmacological interventions in the treatment of Delayed Onset Muscle Soreness (DOMS). Design: Systematic review and meta-analysis of randomised controlled clinical trials (RCTs). Data sources: The PubMed / MEDLINE, EMBASE, SPORTDiscus, Scielo and CENTRAL (Cochrane Central Register of Controlled Trials) databases were searched from the oldest records to August 3, 2020. Eligibility criteria: 1) Tue used a RCTs design; 2) Evaluate the effects of Steroidal or Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) for treatment DOMS; and 3) Therapeutically used drugs, after exercise. Results: In total, 26 studies (patients = 934) were eligible for qualitative analysis on the treatment of DOMS. The results of the meta-analysis showed no superiority between the use or not of NSAIDs, in the improvement of late muscle pain, since statistically significant differences were not verified (21 studies, n= 955; SMD= 0.02; 95% CI -0.58, 0.63; p=0.94; I2=93%). The quality of the synthesized evidence was very low according to the criteria of Evaluation, Development and Evaluation of the Classification of Recommendations, associated with the significant heterogeneity among the included studies. Conclusion: The results demonstrate that the use of NSAIDs is not a superior treatment to the control / placebo on DOMS improvement. The variation between dose-response and exercise protocol used in the studies may have influenced the results. In addition, the high risk of identified bias characterizes limitation to be considered in profound interpretations.
... 30 In contrast, some studies suggested that NSAIDs were not effective, particularly on large muscles. 31,34,35 Probably it may be due to deficiency of drug absorption in muscle tissues. A study with prophylactic use of NSAIDs failed to reduce pain and muscle damage linked with muscle soreness in running athletes after 48 hours. ...
... 28 The prophylactic utility of NSAIDs is still ambiguous as there are a number of studies concluding no effect on symptoms of DOMS. 31,34 This disagreement in data may exist because of a variety of methods incorporated for assessment, identification and inducing muscle soreness. Moreover, it is difficult to generalise the result due to variation in the intensity of muscle soreness, eccentric mode or variation in type, dosage and duration of NSAID administration. ...
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Eccentric exercises are being used widely as a strategy to manage sarcopenia. However, eccentric exercises are linked to increased risk of myofibre damage and delayed recovery. There is elevated muscle soreness, decrease muscle strength and increased levels of muscle-specific circulatory protein. There is a huge variation in the severity of the symptoms after eccentric exercise. Several factors affect the degree and severity of muscle soreness. It includes exercise intensity, duration, mode, muscle group, age, gender, genetics and nutritional status. Therefore, designing a specific individual exercise plan is required to overcome injuries, myofibre damage and muscle soreness. At present, we still do not have enough knowledge about the exact sources and factors that trigger muscle soreness linked with strenuous exercise. Deep insight and identification of the risk factors which predispose individuals at an increased risk of muscle soreness after unaccustomed exercises may be a key to help them by prescribing personalised exercise therapy to speed up recovery and adaptation. Non-steroidal anti-inflammatory drugs are being used widely to manage muscle soreness, pain and tenderness linked with post exercise complications. But there is more to it than just treating pain. Is there any substantive gain besides pain relief? Can they improve muscle function? Could they prevent muscle soreness or speed up recovery? The current narrative review was planned to discuss the sources and factors that trigger exercise-induced muscle damage. Furthermore, it also provides a comprehensive analysis of the literature concerning the effectiveness of non-steroidal anti-inflammatory drugs in reducing symptom and improving muscle function in exercise induced muscle soreness.
... 30 In contrast, some studies suggested that NSAIDs were not effective, particularly on large muscles. 31,34,35 Probably it may be due to deficiency of drug absorption in muscle tissues. A study with prophylactic use of NSAIDs failed to reduce pain and muscle damage linked with muscle soreness in running athletes after 48 hours. ...
... 28 The prophylactic utility of NSAIDs is still ambiguous as there are a number of studies concluding no effect on symptoms of DOMS. 31,34 This disagreement in data may exist because of a variety of methods incorporated for assessment, identification and inducing muscle soreness. Moreover, it is difficult to generalise the result due to variation in the intensity of muscle soreness, eccentric mode or variation in type, dosage and duration of NSAID administration. ...
... However, such training can also bring about tissue damage and inflammation, resulting in delayed onset muscle soreness (DOMS) and a consequent decrease in muscle strength (9). Despite these detrimental effects, it has been proposed that the acute inflammatory response may be a key element in beneficial post-exercise tissue adaptations (10). ...
... Evidence regarding use of NSAIDs to alleviate DOMS remains equivocal, with some studies showing little to no efficacy of ibuprofen (4,10,23). For naproxen, Bourgeois et al. showed that a 500-mg dose taken pre-and postexercise did not decrease perceived DOMS (24). ...
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This study investigated whether naproxen has an ergogenic effect on neuromuscular performance. A randomized, double-blind, placebo-controlled, crossover trial was conducted on 11 resistance-trained men who performed one strength-training session after taking 500 mg of naproxen and another session after taking a placebo. Participants performed three sets of the horizontal bench press with a load of 90% of repetition maximum (RM) to concentric failure. Outcome variables included number of repetitions, workload, fatigue index (FI), and delayed onset muscle soreness (DOMS). Results showed a statistically insignificant reduction in the number of repetitions for placebo when compared to naproxen, amounting to a relative difference of 44.89%. DOMS was lower in the naproxen group, but differences between conditions were not statistically significant. A statistically significant treatment effect was found for workload, favoring naproxen treatment. A statistically significant difference was found for FI between the second and third sets compared to the first set, with results favoring naproxen. We concluded that naproxen helps enhance neuromuscular outcomes in an acute high-intensity strength training bout.
... Apart from the classical blood or saliva samples, that are usually collected before and several time points after the exercise trial this type of experiments allow you to take samples of the inflamed tissue, namely muscle biopsies. This technique has been used in many studies, investigating either the training-induced adaptations on muscle fibers (for example [89,122,[257][258][259][260][261][262][263]), or muscle damage-inflammation (for example [63,122,128,231,248,[264][265][266][267][268][269][270][271]. Muscle samples for such type of studies are usually obtained with Bergstrom needles from vastus lateralis of lower extremities, under local anesthesia, easy and quite safe for the volunteers, while in the majority of the countries, this is well accepted from the bioethics committees. ...
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Subclinical, low-grade, inflammation is one of the main pathophysiological mechanisms underlying the majority of chronic and non-communicable diseases. Several methodological approaches have been applied for the assessment of the anti-inflammatory properties of nutrition, however, their impact in human body remains uncertain, because of the fact that the majority of the studies reporting anti-inflammatory effect of dietary patterns, have been performed under laboratory settings and/or in animal models. Thus, the extrapolation of these results to humans is risky. It is therefore obvious that the development of an inflammatory model in humans, by which we could induce inflammatory responses to humans in a regulated, specific, and non-harmful way, could greatly facilitate the estimation of the anti-inflammatory properties of diet in a more physiological way and mechanistically relevant way. We believe that exercise-induced muscle damage (EIMD) could serve as such a model, either in studies investigating the homeostatic responses of individuals under inflammatory stimuli or for the estimation of the anti-inflammatory or pro-inflammatory potential of dietary patterns, foods, supplements, nutrients, or phytochemicals. Thus, in this review we discuss the possibility of exercise-induced muscle damage being an inflammation model suitable for the assessment of the anti-inflammatory properties of diet in humans.
... Tais células atuam seja no processo de formar novas fibras musculares, seja no processo de juntar-se com as fibras danificadas, promovendo, em ambos os casos, o reparo tecidual (YANG; HU, 2018; ZAMMIT; PARTRIDGE; YABLONKA-REUVENI, 2006), que acontece em função da lesão aguda do músculo (PEREIRA-NETO et al., 2019). Dessa forma, na literatura, são encontrados estudos que utilizaram os AINES como recurso ergogênico no TF, porém os resultados envolvendo esses fármacos permanecem conflitantes quanto aos protocolos de administração e respostas ao TF agudo e crônico (BREWER et al., 2015;KRENTZ et al., 2008;LILJA et al., 2017;LUNDBERG;HOWATSON, 2018;VELLA et al., 2016). ...
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... Some of these techniques have been used based on scientifically hypothesized mechanisms, but the results have been inconsistent. Some have reported that ibuprofen had no major effects on inflammatory response following resistance exercise [6,7], whereas others [8] supported its benefits in lowering CK levels and reporting lower muscle soreness after eccentric leg curl exercises, though without benefits in restoring muscle function. A recent metanalysis supports NSAID use as a means to lower strength loss, soreness, and blood creatine kinase level after an acute muscle injury [9] and ibuprofen has been shown to mitigate fatigue in competitive male runners [10]. ...
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... In response to these chemical signals, white blood cells (leukocytes) are mobilized into the systemic circulation (20) and recruited to the exercised musculature (55). Neutrophils accumulate within muscle in the early hours of recovery (56,60,84). This is followed by the migration of blood monocytes, which (depending on the extent of tissue damage inflicted) may infiltrate within muscle and differentiate locally into tissue macrophages (52). ...
... 47 Moreover, the administration of ibuprofen (1,200 mg/d) did not significantly reduced the infiltration of neutrophils or macrophages following exercise-induced muscle damage. 48,49 Thus, it is important to keep in mind that many therapeutic modalities have a limited ef-fect on inflammation and act mostly on pain reduction. Since pain is a frequently reported symptom, the analgesic versus anti-inflammatory effect of a given modality must be fully understood by physical therapists to determine the best therapeutic approach in order to promote patient recovery. ...
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Intense resistance exercise causes a significant inflammatory response. NF-κB has been identified as a prospective key transcription factor mediating the postexercise inflammatory response. The purpose of this study was to determine whether a single bout of intense resistance exercise regulates NF-κB signaling in human skeletal muscle. Muscle biopsy samples were obtained from the vastus lateralis of five recreationally active, but not strength-trained, males (21.9 ± 1.3 yr) prior to, and at 2 and 4 h following, a single bout of intense resistance exercise. A further five subjects (4 males, 1 female) (23 ± 0.89 yr) were recruited as a nonexercise control group to examine the effect of the muscle biopsy protocol on key markers of skeletal muscle inflammation. Protein levels of IκBα and phosphorylated NF-κB (p65), as well as the mRNA expression of inflammatory myokines monocyte chemoattractant protein 1 (MCP-1), IL-6, and IL-8 were measured. Additionally, NF-κB (p65) DNA binding to the promoter regions of MCP-1, IL-6, and IL-8 was investigated. IκBα protein levels decreased, while p-NF-κB (p65) protein levels increased 2 h postexercise and returned to near-baseline levels by 4-h postexercise. Immunohistochemical data verified these findings, illustrating an increase in p-NF-κB (p65) protein levels, and nuclear localization at 2 h postexercise. Furthermore, NF-κB DNA binding to MCP-1, IL-6, and IL-8 promoter regions increased significantly 2 h postexercise as did mRNA levels of these myokines. No significant change was observed in the nonexercise control group. These novel data provide evidence that intense resistance exercise transiently activates NF-κB signaling in human skeletal muscle during the first few hours postexercise. These findings implicate NF-κB in the transcriptional control of myokines known to be central to the postexercise inflammatory response.
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SMITH, L. L. Acute inflammation: the underlying mechanism in delayed onset muscle soreness? Med. Sci. Sports Exerc., Vol. 23, No. 5, pp. 542-551, 1991. It is well documented in animal and human research that unaccustomed eccentric muscle action of sufficient intensity and/or duration causes disruption of connective and/or contractile tissue. In humans, this appears to be associated with the sensation of delayed onset muscle soreness (DOMS). During the late 1970's, it was proposed that this sensation of soreness might be associated with the acute inflammatory response. However, subsequent research failed to substantiate this theory. The present article suggests that the results of much of the research concerning DOMS reflect events typically seen in acute inflammation. Similarities between the two events include: the cardinal symptoms of pain, swelling, and loss of function; evidence of cellular infiltrates, especially the macrophage; biochemical markers such as increased lysosomal activity and increased circulating levels of some of the acute phase proteins; and histological changes during the initial 72 h. In the final section of this paper, a theoretical sequence of events is proposed, based on research involving acute inflammation and DOMS. (C)1991The American College of Sports Medicine
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Objective: To determine if duration and time of onset of treatment with diclofenac sodium influence force recovery after exercise-induced muscle damage in rats. Design: Randomized placebo-controlled trial. Setting: Animal laboratory. Animals: A total of 217 female adult Wistar rats. Intervention: Rats were submitted to a protocol consisting of 450 eccentric contractions of the ankle dorsiflexors. Treatment by gavage with diclofenac sodium (1 mg/kg, twice daily) was started at different times pre- and postprotocol or for various treatment durations. Main outcome measures: In vitro contractile properties. Results: When treatment was initiated shortly postprotocol, force recovery was roughly proportional to treatment duration during the first 3 days but not at 7 and 28 days postprotocol. A 7-day treatment was no more effective than 1- or 2-day treatments when force was measured at 7 and 28 days; however, such prolonged treatment had no deleterious effect on muscle force at either time. A single-dose prophylactic treatment was as effective as a 2-day treatment initiated soon after the protocol when force was assessed 2 days postprotocol; on the other end, a treatment delayed for 3 days had no effect when force was measured at 7 days. Conclusions: Treatment with diclofenac sodium extending past the acute inflammatory phase was no more effective than short and timely treatment in this model of skeletal muscle damage.
17β-estradiol (E2) attenuates exercise-induced muscle damage and inflammation in some models. Eighteen men completed 150 eccentric contractions after random assignment to placebo (Control group) or E2 supplementation (Experimental group). Muscle biopsies and blood samples were collected at baseline, following 8-day supplementation and 3 h and 48 h after exercise. Blood samples were analyzed for sex hormone concentration, creatine kinase (CK) activity and total antioxidant capacity. The mRNA content of genes involved in lipid and cholesterol homeostasis [forkhead box O1 (FOXO1), caveolin 1, and sterol regulatory element binding protein-2 (SREBP2)] and antioxidant defense (SOD1 and -2) were measured by RT-PCR. Immunohistochemistry was used to quantify muscle neutrophil (myeloperoxidase) and macrophage (CD68) content. Serum E2 concentration increased 2.5-fold with supplementation (P < 0.001), attenuating neutrophil infiltration at 3 h (P < 0.05) and 48 h (P < 0.001), and the induction of SOD1 at 48 h (P = 0.02). Macrophage density at 48 h (P < 0.05) and SOD2 mRNA at 3 h (P = 0.01) increased but were not affected by E2. Serum CK activity was higher at 48 h for both groups (P < 0.05). FOXO1, caveolin 1 and SREBP2 expression were 2.8-fold (P < 0.05), 1.4-fold (P < 0.05), and 1.5-fold (P < 0.001) and higher at 3 h after exercise with no effect of E2. This suggests that E2 attenuates neutrophil infiltration; however, the mechanism does not appear to be lesser oxidative stress or membrane damage and may indicate lesser neutrophil/endothelial interaction.
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Unaccustomed exercise leads to satellite cell proliferation and increased skeletal muscle protein turnover. Several growth factors and cytokines may be involved in the adaptive responses. Non-steroidal anti-inflammatory drugs (NSAIDs) negatively affect muscle regeneration and adaptation in animal models, and inhibit the exercise-induced satellite cell proliferation and protein synthesis in humans. However, the cellular mechanisms eliciting these responses remain unknown. Eight healthy male volunteers performed 200 maximal eccentric contractions with each leg. To block prostaglandin synthesis locally in the skeletal muscle, indomethacin (NSAID) was infused for 7.5 h via microdialysis catheters into m. vastus lateralis of one leg. Protein synthesis was determined by the incorporation of 1,2-(13) C(2) leucine into muscle protein from 24 to 28 h post-exercise. Furthermore, mRNA expression of selected genes was measured in muscle biopsies (5 h and 8 days post-exercise) by real-time reverse transcriptase PCR. Myofibrillar and collagen protein synthesis were unaffected by the local NSAID infusion. Five hours post-exercise, the mRNA expression of cyclooxygenase-2 (COX2) was sixfold higher in the NSAID leg (P=0.016) compared with the unblocked leg. The expression of growth factors and matrix-related genes were unaffected by NSAID. Although NSAIDs inhibit the exercise-induced satellite cell proliferation, we observed only limited effects on gene expression, and on post-exercise protein synthesis.