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Three Techniques, One System: How to Effectively Characterize Complete Muscle Function

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An essential resource for all muscle researchers interested in methodology, best-practices, and techniques for in-vivo, in-vitro, and in-situ muscle experimentation.


As muscle researchers know, functional measurements and assays are the foundation of successful research and are a growing necessity for publication. However, choosing the right experimental technique to answer a broad array of questions can be a challenge.

In this exclusive webinar sponsored by Aurora Scientific, Drs. Christopher Ward and Ramzi Khairallah will discuss methodology, best-practices, and show attendees how to perform basic in-vivo, in-situ, in-vitro experimentation. Discussions will focus on how these techniques can answer questions about animal phenotyping, compound screening, and be used to evaluate interventions and therapies longitudinally. In addition, presenters will review how these techniques can be integrated with other whole animal physiology measurements, biological assays, and genetic tests to provide greater insights into muscle function.

Published in: Science
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Three Techniques, One System: How to Effectively Characterize Complete Muscle Function

  1. 1. Three Techniques, One System: How to Effectively Characterize Complete Muscle Function Sponsored by:
  2. 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. 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. 4. Three Techniques, One System: How to Effectively Characterize Complete Muscle Function Matt Borkowski Sales & Support Manager Aurora Scientific
  5. 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. 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. 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. 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. 9. In situ In vitroIn vivo The 3-in-1 System
  10. 10. How Does it Work ?
  11. 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. 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. 13. System Control & Software
  14. 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. 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. 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. 17. Three Techniques, One System: How to Effectively Characterize Complete Muscle Function Ramzi Khairallah Co-Founder, President Myologica
  18. 18. The 3-in-1 System In situ In vitroIn vivo
  19. 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. 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. 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. 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. 23. In vitroIn vivo The 3-in-1 System In situ
  24. 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. 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. 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. 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. 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. 29. The 3-in-1 System In situIn vivo In vitro
  30. 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. 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. 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. 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. 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. 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. 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. 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. 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. 39. Force v. Frequency Cohen et. al., J Physiol, 2015; Wagner Lab. JHU Functional Assessments: Isometric Contraction
  40. 40. Force v. Frequency +dF/dT -dF/dT Contractile Kinetics Functional Assessments: Isometric Contraction Cohen et. al., J Physiol, 2015; Wagner Lab. JHU
  41. 41. Force v. Frequency * Functional Assessments: Isometric Contraction Contractile Kinetics +dF/dT -dF/dT Cohen et. al., J Physiol, 2015; Wagner Lab. JHU
  42. 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. 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. 44. in vivo Eccentric Contractions - Susceptibility to Injury Khairallah et al. Science Sig. 2012
  45. 45. 1 sec STIM 0.1N/cm in vivo Eccentric Contractions - Susceptibility to Injury Khairallah et al. Science Sig. 2012
  46. 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. 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. 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. 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.

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