Sparing of Extraocular Muscles in Muscular Dystrophies: A Role for Retinoic Acid
Signaling
Sugandha Rajput, Sadie L. Hebert, and Linda K. McLoon
University of Minnesota, Department of Ophthalmology
Introduction
Duchenne muscular dystrophy (DMD) is a
muscular
degenerative
disease
that
is
characterized by an age-related loss of muscle
mass and function (Blake et al. 2012). It is caused
by a mutation in the dystrophin gene. This mutation
results in the absence of the dystrophin protein - a
cytoskeletal protein that plays an important role in
muscle function (Blake et al. 2012). DMD patients
experience decreased lower limb muscle strength
and joint contractures and most often die at an
early age of respiratory complications due to
intercostal muscle weakness (Blake et al. 2012).
Interestingly, the extraocular muscles (EOM) that
function in eye movement, primary gaze position
and
motor
fusion
are
functionally
and
morphologically spared in DMD patients (McLoon
EOM chapter, Kaminski et al. 1992, Karpati et al.
1988 and Kallestad et al. 2011). No studies have
been able to find why EOM are spared while limb
skeletal muscles degenerate in DMD patients. We
hypothesize that two differences between EOM and
limb muscle contribute to the sparing of EOM in
DMD. First, unlike limb muscles, EOM undergo
continuous myofiber remodelling(McLoon and
Wirtschafter 2002). Second, EOM and limb
muscles have different requirements for their
development. The transcription factor Pitx2 is
required for the development of EOM but not for
the development of limb muscle (Diehl et al. 2006
and Zhou et al. 2011). Retinoic acid, a derivative of
vitamin A, controls the expression of Pitx2 during
development. A loss of retinoic acid signaling
results in absence of EOM as well (Matt et al. 2008
and Duester 2012).
Conclusion: Immunostaining for RARα in EOM and TA was
successful. However, we can not make a count of RARα per
The aim of this project was to find the best optimized condition for myofiber in these tissue samples as the outline of each
staining for RARα and dystrophin.
myofiber is not specified.
Next, we doubled stained for RARα and dystrophin (myofiber
Method
membrane cytoskeleton) using flourescent
1. All tissue samples were prepared by following the same protocol. immunohistrochemistry.
The mouse globes with the EOM attached or tibialis anterior from the
limb were removed, embedded in tragacanth gum, and frozen in
Fluorescent IHC for RAR α and dystrophin
methylbutane chilled to slurry on liquid nitrogen.
2. The tissue samples were then subjected to different immunostaining 1. The EOM sections were incubated in primary antibodies
against dystrophin and RARα at a concentration of 1:500
conditions to find the best one.
and 1:100-1:1000, respectively.
Immunostaining for RARα in EOM
2. The sections were reacted with secondary antibodies,
anti-rabbit
AlexaFluor
488
and
Elite
ABC
reagents.
1. The EOM sections were incubated in primary antibody against
3.
The
peroxidase
was
developed
using
substrate
Amplex
RARα at a concentration of 1:100.
UltraRed.
The
staining
for
dystrophin
was
red,
and
the
2. The sections were reacted with secondary antibody, Elite ABC
RARα
should
be
green.
reagents.
Objective
3. The stained sections were developed using DAB (3,3’
diaminobenzidine tetrahydrochloride). The staining for the RARα
was black.
Figure 3: Myofiber from EOM double stained for RARα (picture not shown) and
dystrophin (red). Dystrophin staining defined the outline of each myofiber,. Staining
for RARα didn’t work.
Figure 1: Myofiber from EOM immunostained for RARα (black). No primary section
was subjected to the same procedure without primary antibody.
Conclusion: DAB staining was able to locate RARα in EOM.
Next, we stained for RARα in EOM and TA.
Conclusion: Fluorescent IHC gave no staining for RARα in
the subsequent staining procedures too. We tried to double
stain for Aldehyde dehydrogenase (ALDH, an enzyme
involved in converting retinol to retinoic acid), however, it
wasn’t successful either.
Summary
Immunostaining for RARα in EOM and TA
1. Similar procedure were followed for immunostaining as before,
using primary antibody concentration of 1:250.
One way to study the role of retinoic acid signaling
in the sparing of EOM could be to investigate the
number of the retinoic acid receptor alpha (RARα) positive nuclei per myofiber in EOM and tibialis
anterior (TA) limb muscle . We predict that EOM
Figure 2: Myofiber from EOM and TA Immunostained for RAR α (black).
would possess more RARα than limb muscle,
thereby indicating that retinoic acid signaling is I wish to express my gratitude to Dr. Linda McLoon, Sadie L. Hebert, the McLoon lab and
involved in the sparing of EOM in DMD.
the UROP department for the overall success of this project.
Immunostaining for RARα using DAB gave no information about the
number of RARα-positive nuclei per myofiber, which is critical for
understanding the role of RA signaling in the sparing of EOM in
DMD. Unfortunately, double staining for RARα (or ALDH) and
dystrophin using IHC was not successful either.
In the future, try different double staining conditions, such as varied
antibody concentrations or varied incubation times to find out the
best condition.
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