This study investigated the effects of passive manipulation on the fluorescent distribution of dextran injected into rat leg muscles. Rats were injected with fluorescent dextran and their muscles harvested. Some rats underwent passive ankle manipulation before harvesting. Image analysis of cross sections found lower average and median fluorescent intensities after manipulation. However, passive manipulation did not affect the area fraction of fluorescence or distribution of neuromuscular junctions compared to fluorescence. The results suggest passive manipulation may help create a more uniform fluorescent distribution in muscles.

1. Armando Gallegos Jr., James M. Love, Viviane Minamoto, Sam Ward, Sameer Shah
Department of Orthopaedic Surgery, University of California, San Diego
Passive manipulation and its effect on the fluorescent distribution of Dextran in leg muscles
Aim 1A: Rats are anesthetized with 2% Isoflurane. The Lateral Gastruc, Medial Gastruc, and Tibialis
Anterior are injected sequentially with Tetramethylrhodamine isothiocyanate-dextran. Passive manipulation
is performed on the left side of each rat by flexing the ankle for 10 minutes. Cross sections are then
harvested and frozen immediately after manipulation.
Aim 1B: Image analysis is performed by creating mask and background images in ImageJ (NIH,
Bethesda, MD) and inputting all images into MATLAB (Mathworks, Natick, MA). Area fraction of
fluorescence, mean fluorescence intensity, and median fluorescence intensity are outputted for comparison
between treatment and control. Ordinary Least Squares linear regression on scatter plots of mean
fluorescence intensity and median fluorescence to quantitatively analyze homogeneity caused by passive
manipulation
Aim 2: The neuromuscular junction distribution is ascertained by staining each muscle for α-bungarotoxin
to count the NMJ number. The distribution is then compared to that of the area fraction of fluorescence in
each cross section.
Results
Materials and MethodsAbstract
Background & Motivation
Hypotheses
Conclusions & Future Work
References & Acknowledgements
• By conducting passive manipulation on the Lateral Gastrocnemius
(LG), Medial Gastrocnemius (MG), and Tibialis Anterior (TA), it was
expected that a uniform distribution of fluorescent dye would result
• A lower amount of fluorescence intensity was expected to be seen
after performing passive manipulation to create the uniform
distribution
• A lower amount of area fraction of fluorescence was expected in
most slides as result of better
• On average, the mean and median fluorescence intensity was lower after
passive manipulation
• No effect on the area fraction of fluorescence was observed after passive
manipulation
• More rats are needed to confirm lower intensities are a result of passive
manipulation. A repeat experiment will help create a better statistical
inference on the effects of passive manipulation
• Injecting BTX-A in the same rat model and observing the distribution of
fluorescence in each cross section will help us understand if passive
manipulation can create an identical distribution compared to the number
of neuromuscular junctions
One of the greatest questions asked by clinicians today is how to maximize pharmaceutical drug
benefits while minimizing the risk of the side effects. Botulinum Toxin A, sometimes referred to as
BTX-A, is a drug that takes part in this dilemma. BTX-A is used to inhibit acetylcholine release at
peripheral neuromuscular junctions, thereby inhibiting muscle contraction.[1] It is composed of
one light chain and one heavy chain that are disulfide bonded with the light chain responsible for
its function.[1] This drug can then be used for neuromuscular disorders like muscle spasticity.
However, too much of BTX-A can cause adverse side effects and not enough can produce little
improvement.[2]
This study aims to understand the effect of passive manipulation on the distribution of
Tetramethylrhodamine isothiocyanate-dextran, a dye similar in molecular weight to the light
chain of BTX-A. A rat is anesthetized and injected with Dextran near the center of the muscle
sequentially in both the left and right muscles of the leg. The muscles are then cross sectioned
and analyzed. Analysis was conducted by creating mask and background images of the cross
sections and inputting them through a program based on the concepts of Otsu’s Law. We then
observed the distributions of area fraction of fluorescence, average fluorescence intensity, and
median fluorescence intensity. Distributions of neuromuscular junctions were also obtained by
staining for α-bungarotoxin in each of the rat muscles.
The results of this study concluded that on average, the fluorescent intensity of the dextran in rat
muscle diminished as a result of passive manipulation. Passive manipulation did not have an
effect on the area fraction of fluorescence in each cross section. Distributions of area fraction
compared with neuromuscular junctions did not show significant similarities after manipulation.
1. Aoki, K. Roger. "Evidence of Antinociceptive Activity of Botulinum Toxin Type A in Pain Management." Headache 43.1 (2003): 9-15. Wiley
Online Library. Web. 4 Mar. 2014.
2. Mancini, F., G. Sandrini, A. Moglia, G. Nappi, and C. Pacchetti. "A Randomised, Double-blind, Dose-ranging Study to Evaluate Efficacy and
Safety of Three Doses of Botulinum Toxin Type A (Botox) for the Treatment of Spastic Foot." Neurological Sciences 26.1 (2004): 26-31.
Springer Link. Web. 4 Mar. 2014.
3. Lieber, Richard L., Viviane Minamoto, Samuel R. Ward, Jonah B. Hulst, Michael Lim, William J. Peace, Shannon N. Bremner. "Increased
Efficacy and Decreased Systemic-Effects of Botulinum Toxin A Injection After Active or Passive Muscle Manipulation." Developmental
Medicine & Child Neurology 49.12 (2007): 907-14. Wiley Online Library. Web. 4 Mar. 2014.
4. Otsu, Nobuyuki. "A Threshold Selection Method from Gray-Level Histograms." IEEE Transactions on Systems, Man, and Cybernetics 9.1
(1979): 62-66. Print.
I would like to thank Dr. Shah, Matt, Brian, Ken, Armando D., and all members of the Orthopaedic Surgery department for offering me a fulfilling
experience under the FMP as well as their guidance for constructing this poster.
• Botulinum Toxin A, commonly known as Botox, is a neurotoxin that
blocks the release of acetylcholine, a neurotransmitter responsible for
muscle contraction[1]
• Higher dosages of BTX-A creates adverse effects in the long run[2]
• Passive muscle manipulation is seen to increase efficacy, keeping
dosage constant[3]
• By utilizing the concepts developed by Otsu, we can determine which
pixels can be considered fluorescent, mathematically[4]
Figure 1: The
number of pixels
considered
fluorescent over
the total number
of pixels,
comparing the
effect of
manipulation
Rat
Slope
(stretched)
Slope
(unstretched)
R squared
(stretched)
R squared
(unstretched)
1 -0.07 -1.44 0.060 0.541
2 -0.69 0.05 0.638 0.003
3 -0.09 -0.22 0.143 0.220
Rat
Slope
(stretched)
Slope
(unstretched)
R squared
(stretched)
R squared
(unstretched)
1 0.16 -0.93 0.064 0.510
2 -0.81 0.04 0.590 0.002
3 -0.17 -0.32 0.183 0.187
Figure 2: The distribution of
Neuromuscular Junctions along
the Tibialis Anterior. Muscle
Length Percentage
corresponds measuring the
muscle from the proximal end
to the distal end.
Figure 4:
Comparing the
mean intensity of
fluorescent pixels
in manipulated
and control
muscles. OLS
linear regression
was used to
compare
distributions
Figure 5: The
median intensity
of fluorescent
pixels in the
image, comparing
manipulated and
control muscles.
OLS linear
regression was
used to compare
distributions
Cross
sections
outlined on a
rat muscle
with around a
1.4 mm
spacing
𝑖=1
𝑘
𝑖𝑝𝑖
𝜔0
= 𝜇0𝑝𝑖 = 𝑛𝑖/𝑁
𝑛𝑖 = no. of pixels at level i
𝑖=𝑘+1
𝐿
𝑖𝑝𝑖
𝜔1
= 𝜇1
𝑘 = threshold level to be considered fluorescent
𝜔0 = probability of not being fluorescent
𝜔1 = probability of being fluorescent
Cross section of LG
Figure 3: Comparing the effect of passive
manipulation on the average fluorescence
intensity and median fluorescence intensity in
each rat muscle. Each value corresponds to the
expected value of mean or median fluorescence
intensity with all cross sections in one muscle for
one rat.