Authors:
Livia Assis, PhD
Thais Almeida
Luiz Paulo Milares, MS
Nayara dos Passos
Bruna Araújo
Caroline Bublitz, MS
Suellen Veronez, MS
Ana Claudia Muniz Renno, PhD
Affiliations:
From the Department of Bioscience,
Federal University of São Paulo,
Santos, Sao Paulo, Brazil.
Correspondence:
All correspondence and requests for
reprints should be addressed to: Livia
Assis, PhD, Department of Bioscience,
Federal University of São Paulo, Av.
Ana Costa, 95, Vila Mathias, Santos,
Sao Paulo, Brazil 11050-240.
Disclosures:
Financial disclosure statements have
been obtained, and no conflicts of
interest have been reported by the
authors or by any individuals in control
of the content of this article.
0894-9115/15/9408-0609
American Journal of Physical
Medicine & Rehabilitation
Copyright * 2014 Wolters Kluwer
Health, Inc. All rights reserved.
DOI: 10.1097/PHM.0000000000000219
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Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Sports Medicine
ORIGINAL RESEARCH ARTICLE
Musculoskeletal Atrophy in an
Experimental Model of Knee
Osteoarthritis
The Effects of Exercise Training and Low-Level Laser
Therapy
ABSTRACT
Assis L, Almeida T, Milares LP, dos Passos N, Araújo B, Bublitz C, Veronez S,
Renno ACM: Musculoskeletal atrophy in an experimental model of knee
osteoarthritis: the effects of exercise training and low-level laser therapy. Am J
Phys Med Rehabil 2015;94:609Y616.
Objective: The aim of this study was to evaluate the effects of an exercise
training protocol and low-level laser therapy (and the association of both treat-
ments) on musculoskeletal atrophy using an experimental model of knee osteo-
arthritis (OA).
Design: Fifty male Wistar rats were randomly divided into five groups: control
group, knee OA control group, OA plus exercise training group, OA plus low-level
laser therapy group, and OA plus exercise training associated with low-level laser
therapy group. The exercise training and the laser irradiation started 4 wks after
the surgery, 3 days per week for 8 wks. The exercise was performed at a speed of
16 m/min, 3 days per week, 50 mins per day, for 8 wks. Laser irradiation was
applied at two points of the left knee joint (medial and lateral), for 24 sessions.
Results: The results showed that both trained groups (irradiated or not) pre-
sented a significant increase in the muscle cross-sectional area and a decrease in
muscle fiber density compared with the knee OA control group. Moreover, both
trained and laser-irradiated groups demonstrated decreased muscle-specific ring-
finger protein 1 and atrogin-1 immunoexpression.
Conclusions: These results suggest that exercise training and low-level laser
therapy were effective in preventing musculoskeletal alterations related to atrophy
caused by the degenerative process induced by knee OA.
Key Words: Low-Level Laser Therapy, Exercise, Knee Injury, Atrophy, Rehabilitation
Exercise Training and LLLT on Musculoskeletal Atrophy 609

O steoarthritis (OA) is a chronic joint disease
characterized by progressive degeneration of the ex-
tracellular matrix of articular cartilage, subchondral
bone remodeling, and inflammation of periarticular
tissues.1 The knee is the most often affected joint, and
its incidence is rising because of the aging popula-
tion and the epidemic of obesity.2 It is estimated that
10% of the world’s population 60 yrs or older pre-
sent OA in the knees, having enormous public health
implications.3,4
Knee OA is directly associated with deleteri-
ous effects on the musculoskeletal system, such as
atrophy and weakness of periarticular muscles.5,6
The quadriceps muscle is deeply affected in the pres-
ence of OA of the knee, presenting a significant mus-
cle loss, which leads to physical disability, pain,
swelling, and morbidity.5,7 Clinical evidences found
a positive relationship between decreased quadriceps
muscle strength related to knee OA and pain and
functional joint disability.8Y10 Moreover, there are
strong evidences showing that the muscle weakness
related to OA contributes to accelerate joint degen-
eration, favoring the progression of knee OA.6,11,12
Thus, the development of therapeutic approaches
aiming to avoid skeletal muscle atrophy and, conse-
quently, improving physical function in the presence
of knee OA is of extreme clinical importance.
In this context, physical exercise is an effec-
tive, low-cost, and accessible therapeutic modality
and might play a crucial role in the prevention and
treatment of knee OA.13 Therapeutic exercise regi-
mens that focus on muscle strengthening or on
aerobic activity are recommended for people with
degenerative joint diseases.14,15 Previous studies have
demonstrated that moderate exercise improves mus-
cle function and joint disability in knee OA.13,16 Gomes
et al.17 demonstrated that an aerobic exercise pro-
gram (12 wks of training, consisting of a 50-min
walk three times per week) increased muscle strength
and improved function in patients with knee OA.
In addition, aerobic training (3 days per week for
12 wks) has been shown to reduce the plasmatic and
articular inflammatory cytokines, attenuating the
muscle proteolysis process and subsequent knee OA
progression.13,17
Similarly, low-level laser therapy (LLLT) has
been considered a promising therapeutic interven-
tion, mainly because of its stimulatory effect on tissue
metabolism and ability of modulating the inflam-
matory process after an injury.18,19 Several studies
have demonstrated, in experimental models of joint
inflammation, that LLLT can decrease the expression
of chemotactic factors and inflammatory cytokines
610 Assis et al. Am. J. Phys. Med. Rehabil. & Vol. 94, No. 8, August 2015
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
as well as increase antioxidant enzyme levels.20Y22
In addition, clinical trials have demonstrated that
LLLT has the ability to reduce pain, articular stiff-
ness, and knee swelling, increasing the functional
performance in knee OA patients.23,24
Although the positive effects of exercise train-
ing protocols and LLLT on degenerative joint disease
have already been demonstrated, their effects on skel-
etal muscle atrophy caused by knee OA were not
studied yet. On the basis of the promising effects of
the exercise training and LLLT on OA, it was hy-
pothesized that the association of both therapeutic
approaches may favor the metabolism of the cartilage
structure in the experimental model of OA in the
knee of rats, which may affect the muscles involved in
the knee joint, avoiding muscle atrophy and, conse-
quently, culminating in improved articular stability
and preventing disabilities. In this context, this study
had the aim of investigating the effects of a train-
ing protocol and LLLT (associated or not) on vastus
medialis (VM) muscle atrophy of rats in a model of
knee OA. For this purpose, muscle histomorphometry
and immunohistochemistry were used.
MATERIALS AND METHODS
Experimental Groups and Knee
OA Induction
Fifty adult male Wistar rats (Rattus norvegicus),
weighing T150 g, 6 wks old, were used in this study.
Good laboratory animal practice was observed accord-
ing to the international standards for animal experi-
mentation and after approval by the Animal Care
and Ethics Committee of the authors’ institution
(814715/2013).
The animals were randomly divided into five
groups (n = 10 per group) as follows: control group
(CG)Vanimals with no interventions, OA without
treatment (knee OA control group [OAC]), OA sub-
mitted to exercise training (OA plus exercise training
group [OAT]), OA submitted to laser irradiation treat-
ment (OA plus LLLT group [OAL]), and OA submitted
to exercise training and laser irradiation treatment
(OA plus exercise training associated with LLLT
group [OATL]).
The knee OA induction was performed on the
basis of those described by Galois et al., under in-
traperitoneal anesthesia with 40 mg/kg of ketamine
(Dopalen; Vetbrands, São Paulo, Brazil) and 20 mg/kg
of xylazine (Anasedan; Vetbrands, São Paulo, Brazil).
After anesthesia, the skin around the left knee was
shaved and cleaned. Then, a lateral parapatellar in-
cision (approximately 1 cm) of the skin was made, the
patella was then dislocated laterally to provide access
to the joint space, and the anterior cruciate ligament
(ACL) was isolated and transected in the flexed knee.
The Lachman testing confirmed complete transec-
tion of the ligament. The incision was sutured and
antiseptically treated. Further, the animals were
housed in single plastic cages and were given ap-
propriate postoperative care. All treatments were
performed after 4 wks after ACL transection.
Treatments
Exercise Training Protocol
Three weeks after OA induction, all animals were
familiarized on a motor-driver treadmill at a speed
of 10 m/min for 10 mins per day for 1 wk to decrease
their stress regarding the new environment. After the
adaptation period, the trained groups ran at a speed
of 16 m/min, 3 days per week, for 50 mins per day
for 8 wks.25
LLLT Protocol
Photobiostimulation also started 4 wks after the
surgery. A gallium-aluminum-arsenide (GaAlAs) diode
laser (Photon Laser II; DMC Equipment Ltda, Sao
Paulo, São Carlos, Brazil) was used in the following
parameters: continuous irradiation mode, 808-nm
wavelength, 50-mW power output, 28-sec irradiation
time, 0.028-cm2 spot area, dose of 50 J/cm2, irra-
diance of 1.7 W/cm2, and 1.4-J total energy per point
per section. Laser irradiation was applied 3 days per
week, at two points on the left knee joint (medial and
lateral side of the joint), through the punctual con-
tact technique, for 24 sessions. The optical fiber was
positioned perpendicularly to the skin. For the ani-
mals of the OATL, laser irradiation was performed im-
mediately after the exercise protocol.
Twelve weeks after the surgery, all animals were
euthanized individually by carbon dioxide asphyxia,
and the left VM muscles were removed for analysis.
Histology
Muscles were fixated in 10% formaldehyde for
1 day, and after, histologic sections were dehydrated
in a graded series of ethanol and embedded in par-
affin. Histologic transversal serial muscle cross sec-
tions were obtained (5 Km) using a microtome (Leica
Microsystems SP 1600, Nussloch, Germany), across
the middle of the VM muscle. For morphometric
evaluation of tissue, laminae were stained using he-
matoxylin and eosin staining (Merck). Moreover, two
laminae were obtained for the immunohistochemical
analysis.
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Morphometric Analysis
The muscle fiber cross-sectional area (CSA) was
examined under a light microscopy (40) (Leica
Microsystems AG, Wetzlar, Germany) using a computer-
based image analysis technique (AxioVision 4.7; Carl
Zeiss, Oberkochen, Germany). The CSA was obtained
by measuring the area of 100 fibers located in five
different areas of the section. A double-blind procedure
(L. Assis and T. Almeida) was used for measurements.26
Muscle Fiber Density
The same image analysis technique (Axiovision
4.7; Carl Zeiss, Oberkochen, Germany) was used to
count the number of fibers in six preselected areas
(150 Km2) to determine the fiber density. Two expe-
rienced observers (L. Assis and T. Almeida) performed
the scoring in a blinded manner.27
Immunohistochemistry Analysis
Muscle-Specific Ring-Finger Protein
1 and Atrogin-1 Expression
For muscle-specific ring-finger protein 1 (MuRF-1)
and atrogin-1 expression analysis, the paraffin was
removed with xylene from serial sections of 5 Km.
After this procedure, the sections were rehydrated in
graded ethanol and pretreated in a microwave with
0.01-M citric acid buffer (pH 6) for three cycles of
5 mins each at 850 W for antigen retrieval. Subse-
quently, the material was preincubated with 0.3%
hydrogen peroxide in phosphate-buffered saline (PBS)
solution for 5 mins to inactivate the endogenous
peroxidase and then blocked with 5% normal goat
serum in PBS solution for 10 mins. The specimens
were incubated with antiYMuRF-1 polyclonal primary
antibody (MURF1C-20; Santa Cruz Biotechnology,
United States) at a concentration of 1:100 and anti-
MAFbx polyclonal primary antibody (MAFbx H-300;
Santa Cruz Biotechnology, United States) at a con-
centration of 1:100. Incubation was performed over-
night at 4-C in the refrigerator and followed by two
washes in PBS for 10 mins. Afterward, the sections
were incubated with biotin conjugated secondary
antibody antirabbit immunoglobulin G (Vector Lab-
oratories, Burlingame, CA) at a concentration of 1:200
in PBS for 1 hr. The sections were washed two times
with PBS followed by the application of avidin biotin
complex conjugated to peroxidase (Vector Labora-
tories) for 45 mins. The visualization of the bound
complexes was realized by the application of a 0.05%
solution of 3-3¶-diaminobenzidine and counterstain-
ing with Harris hematoxylin. Finally, for control
analyses of the antibodies, the serial sections were
treated with rabbit immunoglobulin G (Vector
Exercise Training and LLLT on Musculoskeletal Atrophy 611
 Laboratories) at a concentration of 1:200 instead of
the primary antibody. Furthermore, internal positive
controls were performed with each staining bath.
Digital images of the 100 magnification were cap-
tured by optical microscope (Leica Microsystems AG,
Wetzlar, Germany). Nucleus fibers marked brown
were considered positive for MuRF-1 and atrogin-1
expression. Two experienced observers (L. Assis and
C. Bublitz) performed the scoring in a blinded manner.
Statistics
Data are expressed as mean T standard error of
the mean. Shapiro-Wilk and Levene test were ap-
plied to evaluate the normality and homogeneity
of the results, respectively. Comparisons between
the experimental groups were performed by one-
way analysis of variance, and the Tukey posttest was
used to compare individual groups. A P value of less
than 0.05 was considered significant. All analyses were
performed using the GraphPad Prism 4 (GraphPad
Software, San Diego, CA).
RESULTS
General Observation of the
Experimental Animals
From the 50 animals available for this study,
2 animals were lost because of an anesthesia-induced
respiratory depression. The remaining animals re-
covered uneventfully from the OA induction, train-
ing, and laser procedure. No signs of macroscopic
adverse tissue responses were observed during the
experimental period.
Morphometry of CSA
Figure 1 shows the data of CSA evaluation. A
significant decrease in muscle fiber CSA was ob-
served in all injured groups (F4,45 = 18.20; OAC,
1459 T 36.12, P G 0.0001; OAL, 1680 T 42.38,
P G 0.0001; OAT, 1834 T 36.42, P = 0.0029; OATL,
1810 T 70.3, P = 0.009) compared with the CG
(F4,45 = 18.20; 2163 T 92.36). In addition, the OAT
(F4,45 = 18.20; 1834 T 36.42, P = 0.0002) and the
OATL (F4,45 = 18.20; 1810 T 70.3, P = 0.0005) pro-
duced a significant increase in the muscle fiber CSA
compared with the OAC (F4,45 = 18.20; 1459 T
36.12). No difference was observed between the OAL
and the OAC (F4,45 = 18.20; OAL, 1680 T 42.38; OAT,
1834 T 36.42; P = 0.054).
Muscle Fiber Density
Muscle fiber density analysis (Fig. 2) revealed
that all injured groups (F4,45 = 11.74; OAC, 19.59 T
0.64, P G 0.0001; OAL, 17.86 T 0.9, P G 0.0001; OAT,
612 Assis et al. Am. J. Phys. Med. Rehabil. & Vol. 94, No. 8, August 2015
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
16.87 T 0.50, P = 0.003; OATL, 16.88 T 0.41, P = 0.003)
produced a significant increase in fiber density
compared with the CG (F4,45 = 11.74; 13.03 T 0.75).
Furthermore, the OAT (F4,45 = 11.74; 16.87 T 0.50,
P = 0.02) and the OATL(F4,45 = 11.74; 16.88 T 0.41,
P = 0.02) showed a significant reduction in fiber
density compared with the OAC (F4,45 = 11.74; 19.59 T
0.6 ). Similar findings were observed between the OAL
and the OAC (F4,45 = 11.74; OAL, 17.86 T 0.9; OAC,
19.59 T 0.64; P = 0.2).
Immunohistochemistry
MuRF-1 Expression
Immunohistochemistry evaluation demonstrated
that MuRF-1 expression was observed mainly in
the nucleus of the muscle cells in the OAC and the
OAL (Fig. 3). In addition, this analysis showed that a
higher MuRF-1 expression was observed in the OAC
compared with OAL. The CG, the OAT, and the OATL
did not present any expression of MuRF-1.
Atrogin-1 Expression
Atrogin expression was also detected in the nu-
cleus of the muscle cells in the animals of the OAC
and the OAL (with a higher atrogin expression in
the OAC compared with the OAL, Fig. 4). However,
no immunoexpression of atrogin was observed in the
CG, the OAT, and the OATL.
DISCUSSION
This study aimed to evaluate the in vivo mus-
culoskeletal tissue response to an exercise training
protocol and LLLT (associated or not), in an expe-
rimental model of OA in the knee of rats. The main
findings showed that both trained groups (irradiated
or not with LLLT) demonstrated a higher muscle CSA
and lower values of muscle fiber density. Further-
more, both interventions (exercise training and LLLT)
produced a decrease in the immunoexpression of
MuRF-1 and atrogin-1.
Muscle atrophy and decreased skeletal muscle
strength are commonly related to OA. It is suggested
that the inflammatory mediators present in the ar-
ticular cartilage in the presence of the degenerative
disease stimulate muscle catabolism mainly by the
activation of the ubiquitin/proteasome pathway, an
important pathway for selective myofibrilar protein
degradation related to muscle atrophy.28,29
The morphometric analysis revealed that the
animals with OA showed a significant decrease in
the VM CSA and increase in fiber density. The ex-
ercise protocol used in this study has been shown to
be able to attenuate muscle proteolysis and increase
FIGURE 1 Morphometry of CSA. OAC, ACL transection; OAL, ACL transection plus LLLT; OAT, ACL transection
plus exercise training; OATL, ACL transection plus exercise training associated with LLLT. *P e 0.05 vs.
CG; #P e 0.05 vs. OAC.

muscle mass in different experimental models.29Y31
These findings corroborate those of Al-Nassan et al.,29
who demonstrated that an endurance exercise pro-
tocol significantly attenuated atrophy and increased
muscle mass in a model of muscle atrophy in rats.
In addition, it is well known that LLLT has
stimulatory effects on muscle tissue, on cartilage
metabolism, and on the modulation of the inflam-
matory process.20,22 On the basis of these statements,
it was hypothesized that, in this study, laser irradia-
tion would modulate the cartilage degeneration
and the inflammatory processes related to OA,
preventing muscle atrophy. Interestingly, the results
of this study revealed that LLLT, applied alone, did
not have any effect in CSA and muscle density in the
studied animals. One of the explanations could be
related to the laser parameters used in the pre-
sent study, which may have not been sufficient to
offer a sufficient stimulus to the cartilage tissue and,
consequently, did not affect VM muscle tissue. In
addition, previous studies have already demonstrated
that physical exercise associated with phototherapy

FIGURE 2 Muscle fiber density. OAC, ACL transection; OAL, ACL transection plus LLLT; OAT, ACL transection plus
exercise training; OATL, ACL transection plus exercise training associated with LLLT. *P e 0.05 vs. CG;
#P e 0.05 vs. OAC.

www.ajpmr.com Exercise Training and LLLT on Musculoskeletal Atrophy 613

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 FIGURE 3 Representative sections of MuRF-1 immunohistochemistry. Immunolabeled muscle cell (arrow) (600).
OAC, ACL transection; OAL, ACL transection plus LLLT; OAT, ACL transection plus exercise training;
OATL, ACL transection plus exercise training associated with LLLT.

can prevent sarcopenia and increase the muscle vol-
ume in different experimental models.26,32 In this
study, the association of exercise training and LLLT
was not able to optimize the beneficial effects of the
exercise on muscle atrophy. Similarly, it can be as-
sumed that laser energy was not sufficient to produce
any extra positive effects in the trained animals.
To further investigate the action of the exercise
training protocol and LLLT on muscle metabolism
in the knees of OA rats, two proteins considered
crucial for regulation of muscle atrophy were evalu-
ated. MuRF-1 and atrogin-1 (or muscle atrophy F-box)
are ubiquitin E3 ligases, activated by an increase in
the concentration of inflammatory cytokines and

FIGURE 4 Representative sections of atrogin-1 immunohistochemistry. Immunolabeled muscle cell (arrow) (600).
OAC, ACL transection; OAL, ACL transection plus LLLT; OAT, ACL transection plus exercise training;
OATL, ACL transection plus exercise training associated with LLLT.

614 Assis et al. Am. J. Phys. Med. Rehabil. & Vol. 94, No. 8, August 2015

Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

which contribute to the selective myofibrilar protein
degradation and muscle atrophy.33 In the current
study, a similar reduced expression of MuRF-1 and
atrogin-1 was observed for all experimental groups
(both trained and irradiated group). Likewise, several
studies have been demonstrated that endurance ex-
ercise training attenuates muscle protein degrada-
tion signals, including atrogin-1 and MuRF-1, and
enhances muscle protein synthesis signals.34 More-
over, LLLT has been used in an attempt to minimize
or prevent muscle atrophy in many conditions, such
as denervated muscle and muscle myopathies. Shen
et al.35 demonstrated that LLLT was able to inhibit
muscle fiber atrophy and molecular changes in a
denervated rat model. In addition, Leal-Junior et al.36
observed that LLLT has a potential to prevent muscle
atrophy, decreasing progression of Duchenne mus-
cular dystrophy. Thus, a lower expression of MuRF-1
and atrogin-1 observed in the laser group led the
authors to infer that LLLT treatment may reduce the
protein degradation. Furthermore, the association of
exercise and LLLT produced a similar response in the
MuRF-1 and atrogin-1 expression compared with the
other experimental groups, suggesting that no ad-
ditional stimulus was offered by the association of
the two interventions in regulating the expression of
myofibrilar protein degradation during knee OA.
In this context, the exercise training prevented
musculoskeletal atrophy in the OA rats. In addition,
LLLT used alone was able to attenuate the expres-
sion of atrophy markers, preventing muscle protein
degradation in a model of knee OA. Consequently,
these data highlight the potential of both interven-
tions to be used as a therapeutic approach to stimu-
late cartilage metabolism, which may culminate in
the prevention of muscle atrophy of muscle tissue
in compromised conditions, such as OA. However,
the association of both therapies was not able to offer
additional stimulus to the skeletal muscle tissue in
the OA rats. Because this study was limited to rela-
tively short-term evaluation of the effects of physical
exercise and only one laser fluence, information on the
long-term performance of these therapies and higher
amount of laser energy remains to be provided.
CONCLUSIONS
In conclusion, the exercise training improved
muscle tissue metabolism, culminating in the de-
crease in muscle atrophy. In addition, both interven-
tions were able to decrease muscle atrophy markers,
preventing muscle protein degradation. The associ-
ation of exercise training and LLLT was not able to
supply an extra stimulus to muscle tissue in the
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treatment of chronic joint disease. Consequently,
these data highlight the potential of physical exercise
and LLLT in the management of muscle loss caused
by OA. Further long-term studies should be carried
out to provide additional information concerning
tissue interaction in the late stages of therapies and
other laser parameters.
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