The document discusses muscle function assessment techniques using Aurora Scientific's 3-in-1 system. The system allows for in vivo, in situ, and in vitro muscle function assays from the whole animal to isolated muscle level using a single instrument. In vivo assays measure muscle function across a joint with intact neurovascular supply. In situ assays directly measure specific muscles with partial dissection. In vitro assays assess isolated muscle contractility independent of neurons. The system enables assays of contractile properties, fatigue, and injury susceptibility to characterize muscle phenotypes. Choosing the right assay depends on experimental goals and throughput needs.

1. Three Techniques,
One System:
How to Effectively
Characterize Complete
Muscle Function
Sponsored by:

2. Three Techniques, One System: How to Effectively Characterize
Complete Muscle Function
1. A Brief History Of Aurora Scientific And The 3-in-1 (M. Borkowski)
2. Muscle Function Assays: In-vivo, In-situ, In-vitro (R. Khairallah)
3. Understanding Functional Assessments (C. Ward)
4. How To Choose The Correct Assays Based On Your Objectives (C. Ward)
5. Q&A Session (Group)
Sponsored by:

3. InsideScientific is an online educational environment
designed for life science researchers. Our goal is to aid in
the sharing and distribution of scientific information
regarding innovative technologies, protocols, research
tools and laboratory services.

4. Three Techniques, One System:
How to Effectively Characterize
Complete Muscle Function
Matt Borkowski
Sales & Support Manager
Aurora Scientific

5. About Aurora Scientific
• Aurora has served the muscle
community for nearly 20 years.
• Test systems and solutions ranging
from single cells up to the whole
animal.
Cell
Whole
Animal
Fiber
Whole
Muscle

6. Ramzi Khairallah
Co-Founder, President• Founded in 2014
• Contract Research Organization providing:
 Neuromuscular and muscle phenotyping in pre-clinical models
 Assay development, study design
 Training
Myologica can validate your disease model, screen your
compounds, or provide insight into the mechanisms at work in
your system.
In partnership with:
Christopher Ward
Co-Founder, Scientific Director
Asst. Prof.
University of Maryland

7. Introduction: Neuromuscular Phenotyping
• In the rodent, measures of physical
performance are of growing interest.
• Many laboratories have adopted assays
that provide gross assessments of
functional performance.
• While these are informative screening
assays, the functional phenotypes are
not specific to neuromuscular function.
Voluntary Running Wheel
Forced Treadmill
Exercise Capacity
Grip-strength

8. Determinants of Neuromuscular Performance
Muscle Fibers
Assays with Behavioral
Component
• Treadmill
• Running Wheel
• Grip Strength
Aurora 3 in 1
Aurora Single Cell Setup

9. In situ In vitroIn vivo
The 3-in-1 System

10. How Does
it Work ?

11. • System is based around our Dual Mode Lever
• Single instrument for isolated muscle or whole
animal
– Lever arm for ex-vivo & in-situ
– Footplate for in-vivo.
• Goes light years beyond isometric
– Power, Force-Velocity, Work Loops and many other protocols
trivial to perform.

12. Experimental
Apparatus
• Temperature controlled
apparatus for various
animal models.
• Fine positioning of
transducer relative to
animal or muscle.
• Customized clamps and
accessories designed to
make switching easy.
Experimental apparatus for rat. In-vivo
measurement configuration shown.

13. System Control & Software

14. • All experiments performed with our customized acquisition
software (Dynamic Muscle Control).
• Stimulation, stretches, slacks and muscle tension all fully
controlled through software.
• Library of standard protocols allows for infinite customization.
• Straightforward to use. Once muscle/animal attached, load
protocol and press start.
System Control & Software

15. Data AnalysisData Analysis
• Software suite for performing
visualization and analysis
• Automatic High Throughput
data analysis module:
Automated analysis of fatigue,
force-frequency, force-velocity
• Available programming service
for customer specific features
and free updates released
regularly

16. Who is this system useful for?
 Muscle Physiologists
 Exercise Scientists
 Metabolic & Cardiovascular
Scientists
 Bioengineers &
Biologists
 Geneticists
 Neuroscientists
 Pharmacologists &
Biochemists
 Anyone studying muscle
mechanics

17. Three Techniques, One System:
How to Effectively Characterize
Complete Muscle Function
Ramzi Khairallah
Co-Founder, President
Myologica

18. The 3-in-1 System
In situ In vitroIn vivo

19. In vivo –
Ankle Torsion
Photo courtesy of Files Lab; Wake Forest University
Mouse Dorsiflexor Torque Assay
Ankle flexors (dorsiflexion)
• Tibialis anterior
• Extensor digitorum longus (EDL)
Ankle extensors (plantarflexion)
• Gastrocmenius
• Soleus
Note: Dr. Richard Lovering (Univ. Maryland School
of Medicine) has developed a method to evaluate
the quadriceps muscle (i.e. knee extension) with a
custom mechanical mounted to the torque sensor.

20. In vivo : Without surgical isolation of the
muscle or alteration of the neurovascular
supply.
 The mouse or rat is deeply anesthetized
 The hind limb is mechanically stabilized
 The foot is secured in a foot-plate mounted
to the torque sensor.
 Percutaneous or subcutaneous stimulation
of the motor nerve elicits muscle
contraction.
 Torque about the joint is measured
In vivo –
Ankle Torsion

21. Torque is often normalized to adjust
for muscle mass/animal size
• Animal weight
• Muscle mass
• Estimates of muscle cross-sectional area
• Direct measures of muscle cross-sectional
area
o MRI, CT scan
o Histology
Foot Plate
(torque arm)
Force causing
torque
τ = F x L
where τ is the torque
F is the force
L is the length of the lever arm
In vivo - Ankle Torsion

22. Strengths
 Most high-throughput assay
 Assay is across the joint which is the most
physiologically relevant
 No surgical isolation of the muscle or
alteration of the neurovascular supply.
 Repeated measures (days, weeks) is
possible.
Challenges
 Determination of “muscle specific”
contribution to function may be
challenging
 Dependent on intact neuromuscular
junction
 Placement of electrodes requires practice
to achieve consistency and can be
technically challenging
In vivo - Ankle Torsion

23. In vitroIn vivo
The 3-in-1 System
In situ

24. Photo courtesy of Granzier Lab; University of Arizona
Mouse TA muscle under fatigue test
Ankle Dorsiflexors
• Tibialis anterior
• Extensor digitorum longus (EDL)
Ankle Plantarflexors
• Gastrocnemius
• Soleus
Trapezius, diaphragm, etc…
As this an invasive technique, it is
best suited for terminal experiments.
In situ –
Intact Muscle

25. Mouse TA muscle
• The mouse or rat is deeply anesthetized.
• The hind limb is mechanically stabilized
and the skin retracted to expose the
muscle group.
• The muscle of choice is partially dissected,
the distal tendon is severed, and tied to
the force transducer with a silk suture.
• Percutaneous or subcutaneous
stimulation of the motor nerve - or –
muscle can be used to elicit muscle
contraction.
In situ –
Intact Muscle

26. Again, force is often normalized
to adjust for muscle mass or cross-sectional area.
• Muscle mass
• Estimates of muscle CSA
• Direct measures of fiber CSA
– Histology
In situ - Intact Muscle

27. Additional assays are possible
Motor Unit Number Estimation (MUNE)
Motor Unit: One neuron and all
the muscle fibers it innervates
• Sever the nerve, use suction
electrode to stimulate nerve evoked
muscle contractions.
• With small steps in voltage, assay
quantal increases in force production.
• Use predictive modeling to determine
motor unit number.
In situ - Intact Muscle

28. Strengths
 While surgically invasive, the
neurovascular supply is still preserved.
 Surgical isolation allows direct
measure of a specific muscle’s
contribution
 Determination of “muscle specific”
contribution to function is possible by
direct muscle stimulation.
Challenges
 Invasive
 Technically more difficult
 Surgical preparation makes
temporal repeated measures
challenging
In situ - Intact Muscle

29. The 3-in-1 System
In situIn vivo In vitro

30. • Muscle surgically excised from
the animal
• “Classical” isolated muscle
experiment
• Horizontal muscle chamber
• Tension & length recorded and
controlled by one instrument
• Field stimulation of the muscle -
or - nerve stimulation with
suction electrode
Courtesy of Wilkinson Lab; SJSU
In Vitro

31. Courtesy of Wilkinson Lab; SJSU
EDL Muscle with nerve attached
• Muscle surgically excised from
the animal
• “Classical” isolated muscle
experiment
• Horizontal muscle chamber
• Tension & length recorded and
controlled by one instrument
• Field stimulation of the muscle -
or - nerve stimulation with
suction electrode
In Vitro

32. Any small muscle whose
metabolic needs can be met
through diffusion.
 Extensor digitorum longus (EDL)
 Soleus
 Lumbricalis
 Diaphragm (hemi-diaphragm)
 Heart:
– Papillary
– Trabeculae
Photo courtesy of Wilkinson Lab; SJSU
In Vitro

33. Strengths:
 Surgical excision allows direct measure
of muscle contractility independent of
neuronal integrity.
 Dissection of the muscle with a portion
of the motor nerve allows ex vivo
nerve-muscle function to be assessed.
 Chemical/pharmacological
manipulation possible
Challenges:
 Surgical excision negates blood flow
support.
 Technically challenging
 Prep viability is limited by
oxygen/metabolic substrate diffusion to
inner muscle fibers and and CO2 diffusion
out from inner fibers.
 Preparation makes temporal repeated
measures impossible
In Vitro

34. Three Techniques, One System:
How to Effectively Characterize
Complete Muscle Function
Christopher Ward
Co-Founder, Scientific Officer
Myologica
Asst. Professor
University of Maryland

35. Basic Assumptions
Optimal muscle length (i.e., resting
length: L0) for each preparation.
Supra-maximal stimulation to ensure
recruitment of all muscle fibers with
single action potential (i.e., twitch)
stimulation.
Note: Both L0 and stimulation intensity
are determined for each preparation.
• Contractile Function
o Isometric Contractions
o Concentric Contractions
• Fatigue
• Injury Susceptibility
o Eccentric
o Isometric
Functional assays enabled by the 3-in-1 system

36. Functional assays enabled by 1300A
Functional assays enabled by the 3-in-1 system
• Contractile Function
o Isometric Contractions
o Concentric Contractions
• Fatigue
• Injury Susceptibility
o Eccentric
o Isometric
Variety of variables can be examined
to gain insight into muscle function
• Peak torque/force
• Kinetics
 +df/dt
 –df/dt
 Time to max
 Time to min

37. The most common functional
assessment is isometric contraction
– force produced with no change in
muscle length.
Force vs. Stimulation Frequency
• Trains of action potentials at
increasing frequency until there is no
further force increase.
• The peak force responses for each
stimulation frequency is determined.
Functional Assessments:
Isometric Contraction
Force vs. Stimulation Frequency
Relationship for EDL in vitro
0.2 0.4 0.6
0
5
10
15
20
25
30
35
IsometricTension(g)
Time (sec)
250 msec.Train

38. Pratt et. al., J Physiol, Volume 591, Issue 2 2012; Lovering Lab. UMB
Nerve Stimulation
• The femoral nerve is stimulated
every second for 2 mins
• (330-ms tetanic)
Muscle Stimulation
• The electrodes are placed directly
over the muscle
• A direct muscle tetanic contraction
is superimposed every 15 s
Fatigue in situ
Functional Assessments: Isometric Contraction

39. Force v. Frequency
Cohen et. al., J Physiol, 2015; Wagner Lab. JHU
Functional Assessments: Isometric Contraction

40. Force v. Frequency
+dF/dT
-dF/dT
Contractile Kinetics
Functional Assessments: Isometric Contraction
Cohen et. al., J Physiol, 2015; Wagner Lab. JHU

41. Force v. Frequency
*
Functional Assessments: Isometric Contraction
Contractile Kinetics
+dF/dT
-dF/dT
Cohen et. al., J Physiol, 2015; Wagner Lab. JHU

42. Muscles of locomotion transmit force across a
joint resulting in a change in muscle length and
articulation of the joint.
Concentric contraction – Muscles actively shortening
Eccentric Contraction – Muscles actively lengthening
Popular type of contraction to study
• Much of a muscle's normal activity occurs while it is actively lengthening.
• Muscle injury and soreness are selectively associated with eccentric contraction
Functional Assessments

43. in vivo Eccentric Contractions - Susceptibility to Injury
mdxWT
0.1N/CM
100 msec
STIM
STRETCH
1st
20th
Khairallah et al. Science Sig. 2012

44. in vivo Eccentric Contractions - Susceptibility to Injury
Khairallah et al. Science Sig. 2012

45. 1 sec
STIM
0.1N/cm
in vivo Eccentric Contractions - Susceptibility to Injury
Khairallah et al. Science Sig. 2012

46. How to choose the best assay(s)?
• The 1300A ‘3-in-1’ System provides the
scientist with many options for the functional
assessment of neuromuscular phenotype
• While it is tempting run all the assays possible, this
is rarely feasible, time efficient, or cost-effective

47. What is the phenotyping goal?
Neuro Muscular
In vivo
• Moderate throughput
• Assay is across the joint which is the most
physiologically relevant
• Repeated measures (days, weeks) is possible
In situ
• Direct measure of a specific muscle
• Motor nerve or muscle stimulation
In vitro
• An ex vivo prep allowing direct measure of muscle
contractility independent of neuronal integrity.
1300A ‘3-in-1’ System

48. Thank You!
For additional information on the Aurora 3-in-1 system, muscle
measurement assays, and other products related to the study of
muscle function please visit:
http://www.AuroraScientific.com/

49. InsideScientific is an online educational environment
designed for life science researchers. Our goal is to aid in
the sharing and distribution of scientific information
regarding innovative technologies, protocols, research
tools and laboratory services.