Wire Myography

Overview:

Myography (myo from ‘myocyte’) is the study of muscle contraction. A myograph is a device for quantifying contractile properties of muscles (e.g. force-length, force-velocity), generally through direct mechanical coupling to a force transducer.

Wire myography is an adaptation of this idea specific to smooth muscle tissue investigation and was first proposed in 1972 by Bevan and Osher. Further development of this idea by Mulvany and Halpern in 1977 resulted in a system that standardised the recording of mechanical contractile forces from isolated blood vessels ranging from 60 to 400 µm in diameter, such as postcapillary venules and precapillary ‘resistance arteries’ – so named according to their role in regulating peripheral circulatory resistance.

Three key features of wire myography set it apart from other forms of myography: 1) it is relatively atraumatic to the vessel under investigation. This is because the ends of the vessel segment are not clamped or tied as in other systems; 2) wire myography takes advantage of vessel smooth muscle natural plane of contraction, which is circular/cross-sectional, as opposed to longitudinal in the case of skeletal muscle tissue (for which the DMT820MS is suited); and 3) wire myography was developed specifically for the study of smaller resistance arteries – although in recent times this has now expanded to include larger vessels such as the small intestine (see DMT 620M).

Example applications of ADInstruments DMT myograph systems include:

• Lymphatic vessles, ureter, trachea, bronchii
• Primary, secondary, tertiary and resistance blood vessles (e.g., aorta, carotid artery, capillaries)
• Intestinal tract (e.g., duodenum, ileum, jejunum, colon)
• Skeletal muscle strips

Method:

ADInstruments offer Danish Myo Technology (DMT) wire myograph systems which feature two stainless steel jaws. Two mounting wires are threaded through the vessel lumen (not puncturing the walls) and each wire is secured to each of the jaws. One of the mounting jaws is attached to a micropositioner for the adjustment of vessel circumference and application of tension. The other jaw is attached to a built-in sensitive isometric force transducer. Isometric responses to chemical antagonist/agonists, pharmaceutical drugs, etc can then be investigated.

 A schematic view of the mounting jaws.

The rationale and theory behind normalizing elastic tissues such as microvessels has been extensively investigated by Professor Michael Mulvany. Mulvany determined that the optimal pre-tension conditions (i.e. internal circumference) for microvascular studies is best defined as the size when the vessel is fully relaxed and under a transmural pressure of 100 mmHg. Therefore the aim of the normalization procedure is to determine the internal circumference at which the vessel would be relaxed and under a transmural pressure of 100 mmHg. This index is denoted as IC100 and is calculated for each vessel mounted on the wire myograph.

The DMT Normalization Module is used to determine IC100. This is obtained by stepwise increases in internal circumference whilst measuring the isometric force. An equation is then used to determine when the vessel reaches an effective pressure of 100 mmHg. Following this, the vessel is set to 90% of IC100 (IC1) as this is the internal circumference at which the active force production of the vessel is maximal.

The calculation parameters (including eyepiece calibration, desired target pressure and online averaging times) may be set by the user depending on the particular type of tissue being used and the desired calculations.

Software:

The LabChart Advantage:

(may require additional Modules and Extensions)

• Units Conversion for easy calibration into appropriate units such as grams or milli Newtons
• The DMT Normalization Module provides an easy method for the calculation of optimal pretension conditions for microvascular or any small tubular tissue research.
• The Dose Response Module allows for the calculation of dose response type data from LabChart recordings, automatically or manually, offline or online.
• The Peak Analysis Module provides automatic detection and analysis of multiple, but not overlapping, signal waveforms from recordings, online or offline.
• Fast data extraction, analysis and export to other applications using Timed Add to Data Pad and Multiple Add to Data Pad (Note: export as .csv or .txt into Graph Pad Prism®)
• Comments can be added in real time, viewed or moved at a later stage
• The LabChart GLP Client and GLP Server software offers a comprehensive audit trail, secure signing and a centralized user authorization system required for GLP and 21 CFR Part 11 (FDA) compliant environments

LabChart

LabChart software (for Windows and Macintosh) together with a PowerLab data acquisition system offers up to 32 channels of real-time data acquisition, data integrity, easy selection of hardware settings, powerful online and offline analysis, procedure automation, seamless extraction of experimental data and flexible display options. Additional acquisition and analysis functionality is provided with the use of specialized LabChart Extensions and LabChart Modules. Modules are available as part of LabChart Pro while Extensions are free for download from the website for existing LabChart users.

DMT Normalization Module

The DMT Normalization Module (Windows & Macintosh) provides an easy method for the calculation of optimal pretension conditions for microvascular (or any small tubular tissue) research. It allows the user to define the pre-experimental conditions and standardize each piece of tissue.

This module:

• Automatically calculates tissue length
• Provides online and offline determination of effective pressure
• Calculates the pre-tension (micrometer setting) for normalization
• Determination of IC100 and IC1 (90% of IC100)

Dose Response Module

The Dose Response Module (Windows) provides easy analysis of dose response type data (response to stimulation by chemical, electrical or physical agonists) recorded in LabChart from various studies including myography.

This module provides:

• Real-time or offline analysis
• Automated or manual modes of analysis
• Fast analysis of raw data to dose response parameters
  • Fast comment detection and conversion to dose response markers
  • Easy options for calculating different response parameters
  • Instant single or multiple dose response curves (Hill-curves) generation
  • Instant calculation of EC50 and Hill slopes  
• Export options to other software applications for further analysis 

Peak Analysis Module

The Peak Analysis Module (Windows) provides automatic detection and analysis of multiple, but not overlapping, signal peaks in acquired waveforms. It can be applied to a wide range of waveforms including recorded signals from myography:

• Peak isometric force
• Sustained isometric force (area under curve)
• Time to peak isometric force development (and slope)

It provides:

• Real-time or offline analysis
• Fast parameter extraction and analysis from single or multiple peaks
• Automatic peak detection of an entire channel or a selection within a channel
• Detection of positive or negative peaks
• Provides several analysis presets suitable for general and specific types of waveforms
• Instant tabulation of extracted parameters that can be exported to other applications

GLP and 21 CFR Part 11

For those researchers working within a laboratory requiring GLP and 21 CFR Part 11 compliance the GLP Client and GLP Server are available for use with LabChart (Windows only) and PowerLab data acquisition systems. For more information, visit the Good Laboratory Practice application page or contact your nearest ADInstruments representative.

Hardware:

PowerLab Data Acquisition Systems

The PowerLab is a high-performance data acquisition unit capable of recording at speeds of up to 400,000 samples per second continuously to disk (aggregate). PowerLab units are compatible with instruments, signal conditioners and transducers supplied by ADInstruments, as well as many other third-party companies. In addition to standard single-ended BNC inputs, 4 differential Pod ports are also available for direct connection of Pod signal conditioners and appropriate transducers. Research PowerLab units include:

• PL3504 PowerLab 4/35 – 4 Channels
• PL3508 PowerLab 8/35 – 8 Channels
• PL3516 PowerLab 16/35 – 16 Channels

Research Systems

Each of these research systems includes a PowerLab 8/35 and LabChart Pro (includes the DMT Normalization Module). All of the DMT units feature built-in temperature control, ports for filling and injection of drugs, ports for rapid draining (a vacuum pump is required for drainage and is not supplied with the systems) and ports for gas supply. Myograph systems include:

• PL3508B21 Single Wire Myograph System - Single Wire Myograph (maximum chamber volume = 10 ml) suitable for isometric experimentation.
• PL3508B22 Dual Wire Myograph System - Dual Wire Myograph and is ideal for investigating two isolated tissues simultaneously in isometric conditions (the 10 ml chamber can be divided to give 2 x 5 ml).
• PL3508B23 Auto Dual Wire Myograph System - Auto Dual Wire Myograph which is similar to the dual chamber wire myograph; however, this system has motorized micropositioners (the 10 ml chamber can be divided to give 2 x 5 ml).
• PL3508B24/TS Multi Chamber Myograph w/Touch Screen System – Multi Chamber Myograph with four chambers (maximum chamber volume = 8 ml) that allow the user to conduct up to 4 separate experiments. It is ideal for work requiring high throughput such as drug screening. This system also includes Tissue Pins as well as the jaws allowing larger tissues such as aorta to be easily mounted.
• PL3508B25 Confocal Wire Myograph System - Single Confocal Wire Myograph that is specifically designed for use with confocal microscopic techniques. Enables highly resolved images of fluorescent dyes in living tissues to be obtained with a laser-scanning confocal microscope (LSCM). The chamber has a maximum volume of 10 ml. Note: Microscope is not supplied by ADInstruments.

Instruments

Individual myograph units include:

• DMT310A Single Wire Myograph
• DMT410A Dual Wire Myograph
• DMT510A Auto Dual Wire Myograph
• DMT120CW Confocal Wire Myograph
• DMT620M Multi Chamber Myograph w/Touch Screen
• DMT720MO Tissue Bath System w/Touch Screen - Multi Chamber Myograph with four chambers for recording tension in isolated larger blood vessels (>500 μm) and other larger vessels or tubular structures (up to 10 mm), such as trachea or gut mounted as large ring preparation
• DMT820MS Muscle Strip Myograph w/Touch Screen - Multi Chamber Myograph with four chambers for recording tension in isolated skeletal or other muscle preparations, mounted as a strip (15 mm in length ) between clamp supports
• DMT100192 Automated Buffer Filler System 2 (625FS) 
  • For use with the current Multi Chamber Myographs including DMT620M, DMT720MO and DMT820MS.
  • Provides an automatic, reliable and consistent way to replace buffer in the bath chambers
  • Easily attached to allow filling of a selected chamber or all 4 bath chambers with a single touch of a button
  • Two standard volumes of buffer can be applied, which are 6 mL and 8 mL

Additional required equipment (not supplied by ADInstruments) include:

• Binocular dissecting microscope with at least 40x magnification
• Optical Fiber Light Source

Accessories and Spare Parts

• DMT100128 Wire (Tungsten, 25µm OD, 25m) Tungsten wire (25 μm) for use with wire myographs
• DMT100112 Wire (Stainless steel, 40µm OD,1500m) A 40 μm stainless steel wire for wire myography
• DMT100111 Screws (x12) and Screwdriver Microscrews for wire myographs
• DMT100129 Grease (High Vacuum, 50g) High vacuum grease
• DMT100087 Grease (for Linear Slides) Micometer linear slide lubrication grease
• DMT100092 Myograph Mounting Jaws - Plastic Plastic mounting jaws used for field stimulation studies
• DMT100065 Chamber Cover for DMT620M/720MO (Nerve Field Stimulation) Wire myograph chamber cover used for nerve field stimulation
• DMT100055 Myograph Calibration Kit A calibration kit which includes a bridge, balance and weight
• DMT100091 Myograph Mounting Jaws - Stainless steel Myograph Mounting Jaws - Stainless steel

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