Presentation By: Kenny DikeWorking Under: Dr' SakiyamaElbert

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Analyzing Sciatic Nerve Regeneration with Rodent
Paw Print Analysis
Presentation By Kenny Dike Working Under Dr.
Sakiyama-Elbert Group Members Kyle Mairose
David Working
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Background
For laboratory tests involving peripheral nerve
regeneration, rats are an excellent test subject
because rat peripheral nerves are biologically
indistinguishable from human peripheral nerves.
Additionally, they are inexpensive, easy to
house, and it is easy to obtain genetically
identical subjects.
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Background
For the study of nerve regeneration following a
crush injury (as opposed to a severance injury),
the sciatic nerve is most commonly studied. At
the injury site itself, the number of regenerated
nerve fibers may or may not correlate with the
subjects ability to regain sensory and motor
use. Regeneration must instead be measured using
tests that actually incorporate the subjects
ability to use the part of the body innervated by
the injured nerve. The simplest of these tests is
to observe the rat while walking.
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Background
An injury to the sciatic nerve affects many parts
of a rats normal walking gait, such as speed,
stride width, and stride length. Within a single
paw, the amount of toe spread narrows with
decreased nerve function. Some of these effects
can be quantified and used to calculate the
Sciatic Function Index, a combination of
measurable factors.
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Scope of Project
Dr. Sakiyama-Elberts lab currently does sciatic
nerve regeneration experiments, and they are
looking for a way to cut down the amount of time
and man-hours spent processing the results.
Ideally, they would like a method of data
collection that automates the process of
measuring the Paw Length, Toe Spread, and
Intermediary Toe spread from digital-video-collect
ed footprints and calculates the SFI. These
variable will be explained in detail in our
Preliminary analysis.
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Specific Design Requirements

• The design budget is 1000-2000
• The machine must be transportable, because it may
  be used in multiple labs at different
  locations.
• Specifically, our vendor wants the machine to be
  able to be transported on a basic lab cart, a 32
  foot base or a 6 square foot area.
• The machine can require assembly, but it must be
  simple and able to be assembled by one person and
  have a brief set up and break down time, on
  average 10 minutes.

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Specific Design Requirements

• The machine must also be relatively light in
  weight if it will be able to be handled
  completely by a single person. This puts a
  maximum weight limit of 30 lbs for any single
  component of the machine.
• We must use a transparent material for the
  walkway of our machine to maximizes the
  efficiency of the camera and image processing
  software to identify paw contact with the walkway
  surface from a ventral viewpoint.

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Specific Design Requirements
The walkway of our machine must be large and
strong enough to accommodate a lab mouse or rat
with sufficient room to walk at a natural gait.
The average adult body weight of a lab rat is
250-300g (female), and 450-520g (male). For lab
mice, the approximate values are 2.5g to 34g.
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Specific Design Requirements

• We will need a video camera with a minimum
  sampling rate of 40 fps in order ensure that we
  capture the image of the foot when it is
  completely flat against the walkway.
• We will also need a camera with a resolution
  great enough to distinguish a two .5 mm dots with
  2 mm of separation between them, because the
  average size of a mouse finger pad is .5 mm and
  their toes have on average 2 mm of separation
  between them.

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Existing Solutions
Gait analysis is the process of quantification
and interpretation of locomotion. In humans, gait
analysis is extensively used to quantify how a
patient walks, and to assist in the diagnosis and
treatment of movement disorders. Gait is
disturbed in numerous human conditions, including
neurodegenerative disease, stroke, and pain. Mice
and rats are widely used in the study of human
diseases, but methods for gait analysis in
rodents have been lacking.
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Existing Solutions
The classic method for characterizing gait in
mice is paw inking, where the mouse's paws are
dipped into ink and their paw prints are observed
as they walk on a piece of paper. The
investigator can then measure the distance
between paw prints. these measurements,
however, are often difficult to discern, and are
confounded by the variable speeds at which the
animals walk.
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Existing Solutions
In a research project titled, Gait dynamics in
mouse models of Parkinson's disease and
Huntington's disease, done at Harvard Medical
School, gait dynamics were recorded using ventral
plane videography. Briefly, they devised a
motor-driven treadmill with a transparent
treadmill belt. A high-speed digital video camera
was mounted below the transparent treadmill belt.
An acrylic compartment, 5 cm wide by 25 cm
long, was mounted on top of the treadmill to
maintain the mouse that was walking on the
treadmill belt within the view of the camera.
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Existing Solutions
Digital video images of the underside of mice
were collected at 80 frames per second. Each
image represents 12.5 ms the paw area indicates
the temporal placement of the paw relative to the
treadmill belt. The color images were converted
to their binary matrix equivalents, and the areas
(in pixels) of the approaching or retreating paws
relative to the belt and camera were calculated
throughout each stride.
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Existing Solutions
U.S. Patent 6,899,686 May 31, 2005
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Existing Solutions
In another study titled Automated Quantitative
Gait Analysis During Overground Locomotion in the
Rat Its Application to Spinal Cord Contusion and
Transection Injuries, researchers used a newly
developed gait analysis method that allows easy
quantization of a large number of locomotion
parameters during walkway crossing. The
following slides provide a description of their
method.
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Existing Solutions
Experimental setup side view

FIG. 1. (A) Principles of operation of the Cat
Walk setup light from a long fluorescent tube is
sent into the long edge of a normal glass sheet.
From a certain distance from the edge, all rays
strike the surface below the critical angle and
cannot escape (the same principle underlies
fiber-optics). At those planes where an object
touches the surface, light escapes, only to be
scattered by this object. As such, the place of
contact becomes clearly visible.
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Existing Solutions

FIG. 1. (B) Experimental setup front view
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Existing Solutions

FIG. 1. (C) Animals cross the corridor (width 7
cm, length 109 cm) from right to left. The floor
is monitored via a mirror placed at an angle of
45 under the floor. Data-acquisition starts as
the animal breaks the rightmost infrared beam and
stops as the animals breaks the leftmost one (90
cm apart).
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Existing Solutions
United States Patent 6,678,413 Liang et al.
January 13, 2004 The invention includes a system
with a video camera coupled to a computer in
which the computer is configured to automatically
provide object segmentation and identification,
object motion tracking (for moving objects),
object position classification, and behavior
identification. The invention is capable of
automatically monitoring a video image to
identify, track and classify the actions of
various objects and the object's movements within
the image. The image may be provided in real time
or from storage. The invention is particularly
useful for monitoring and classifying animal
behavior for testing drugs and genetic mutations,
but may be used in any of a number of other
surveillance applications.
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Existing Solutions
United States Patent 6,678,413 Liang et al.
January 13, 2004
System and method for object identification and
behavior characterization using video analysis
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Existing Solutions
Product Name DigGait Imaging System United
States Patent 6,899,686 Hampton , et al. May 31,
2005
Method and apparatus for monitoring locomotion
kinematics in ambulating animals
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Existing Solutions
Product DescriptionThe DigiGaitTM Imaging
System is a unique system that provides a more
accurate, faster, and much simpler means of
measuring gait indices than traditional paw
inking. The Gait System measures stride length,
stance width, stance time, braking time,
propulsion time, and swing time (in addition to
several other indices of gait) and can
demonstrate how genes and/or drugs affect
coordinated movement. As the rodent walks on the
motorized treadmill, a high-speed digital camera
captures the action from underneath the animal
software digitizes and scans the movie of paw
dynamics and generates a plot of paw placement
through sequential strides. The speed of the
motorized treadmill can be adjusted to examine a
running or walking gait. The software interprets
the dynamic gait plot to report the essential
temporal indices of locomotion.
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Preliminary Analysis
The sciatic function index (SFI) was developed by
de Medinaceli et al. in 1982 and was adapted by
Bain et al. in 1989. The Sciatic Function Index
quantifies and calculates a combination of
measurable factors of a rats toes. In 2000,
Bervar created the static sciatic index (SSI).
It uses the common method of walking track
analysis (foot prints) in which various toe
spreads are measured. They are simple,
non-invasive, and successful.
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Preliminary Analysis
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Preliminary Analysis
For all measures, E designates the experimental
(injured) side of the body, while the N
designates the normal (control) side. The
measures are the print length (PL), toe spread
(TS), and intermediary toe spread (IT). PL is
measured from the back of the heel to the tip of
the middle toe, TS is measured from the tip of
the first digit across to the tip of the fifth
digit, and IT is measured from the tip of the
second digit across to the fourth digit.
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Preliminary Analysis
Calculations are as Follows
1-5 (TS), 2-4 (ITS), Print length (PL) from heal
to third toe, Toe spread factor (TSF)
(ETS-NTS)/NTS Intermediate TSF (ITSF)
(EITS-NITS)/NITS Print length factor (PLF)
(EPL-NPL)/NPL
SSI (108.44 x TSF) (31.85 x ITSF) 5.49
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Design Schedule
9.19 Preliminary Oral Presentation 10.01
Preliminary Written Report 10.20 Target date for
hardware design 10.29 Progress Oral
Presentation, Progress Written Report 11.05
Target date for software design 11.26 Target
date for completed system 12.03 Final Oral
Report 12.05 Final Written Report 12.12 Poster
Judging
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Organization of Our Team
Responsibilities Kenny Hardware design,
hardware/software interface Kyle Software
development, software/user interface Dave
Software development, project administrator,
group representative
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References
Bain JR, Mackinnon SE, Hunter DA. Functional
evaluation of complete sciatic, peroneal and
posterior tibial nerve lesions in the rat. Plast
Reconstr Surg 83 (1989) 129-36. Dijkstra JR,
Meek MF, Robinson PH, Gramsbergen A. Methods to
evaluate functional nerve recovery in adult rats
walking track analysis, video analysis and the
withdrawal reflex. Journal of Neuroscience
Methods 96 (2000) 89-96. Luis AL, et. al.
Long-term functional and morphological assessment
of a standardized rat sciatic nerve crush injury
with a non-serrated clamp. Journal of
Neuroscience Methods 163 (2007) 92-104. Walker
JL, et. al. Gait-stance duration as a measure of
injury and recovery in the rat sciatic nerve
model. Journal of Neuroscience Methods 52 (1994)
47-52. Nichols CM, et. al. Choosing the correct
functional assay A comprehensive assessment of
functional tests in the rat. Behavioural Brain
Research 163 (2005) 143-158.
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Questions?