Isotonic Contraction

Overview:

Dose response studies are typically conducted to assess concentration-response relationships in isolated tissue preparations. Traditionally, tissue-organ baths are used for in vitro dose response experiments to investigate the physiology and pharmacology of tissue preparations from various species (e.g. chick, toad, rabbit, rat, guinea-pig, etc.). Tissue-organ baths are used to maintain the integrity of the tissue for several hours, in a temperature-controlled environment, while physiological measurements are performed. Typical experiments involve the addition of drugs to the organ bath or direct/field stimulation of the tissue. The tissue reacts by contracting/relaxing and an isometric or isotonic transducer is used to measure force or displacement, respectively. From the experimental results dose-response curves are generated (tissue response against drug dosage or stimulus potency).

 

Some of the more common tissues that are studied with an organ bath system include:

Cardiovascular

  • Aorta rings
  • Heart tissue (papillary muscle, left ventricles)
  • Arteries (mesenteric arteries)

Gastrointestinal

  • Ileum
  • Colon
  • Gastric antral muscle
  • Sphincter

Respiratory

  • Tracheal rings
  • Phrenic diaphragm
  • Pulmonary arterial smooth muscle
  • Lung parenchyma

Skeletal Muscle

  • Soleus
  • Gastrocnemius

Other isolated tissue preparations include urinary bladder, penile muscle strips and prostate.

Method:

Isolated tissue-organ experiments can generally be run in groups of 2, 4, 8 or more sample preparations, thereby enabling a high throughput in most laboratories. These in vitro preparations are also more readily instrumented and can be easily subjected to controlled changes in perfusate, oxygen availability, drug administration etc than is otherwise possible in the intact animal.

A water-jacketed organ bath provides a stable and easily adjustable way of temperature control. Substrates and other nutrients that are required to sustain tissue function are provided via a physiological solution, which allows the study of evoked tissue responses to:

  • Pharmacological drug/agents (Dose Response studies)
  • Electrical stimulation
  • Both pharmacological and electrical stimulation

Tissues are usually prepared in a petri-dish containing physiological solution (i.e. Kreb’s solution). The ends of the tissue are then attached to the mounting hook and transducer using silk or fine wire (non-compliant) to mount the tissue. The tissue placement may vary depending on the tissue type such as:

  • Ring/Vessel Preparation
  • Strip Preparation

 

Organ Bath Apparatus

ADInstruments offer the following range of organ baths manufactured by Panlab and Radnoti.

Compact “all-in-one” Organ Bath systems by Panlab which allow;

  • Immersion of all tissue chambers and perfusate warming coils in a single temperature controlled bath
  • Individual or simultaneous filling and emptying of tissue chambers
  • Compact design to save on bench space
  • Bath capacity options of 5 to 50 mL

Modular Organ Bath systems by Radnoti which feature;

  • Water-jacketed reservoirs and tubing to ensure perfusate temperature stability
  • Easy addition and substitution of components
  • Modular design allows easy customization of glassware placement and type
  • Bath capacity options of 5 to 300 mL

The recorded responses are the contraction/relaxation of muscle in the tissue in the form of:

  • Force (isometric)
  • Displacement (isotonic)

ADInstruments Single, Quad or Octal Bridge Amplifier models are software controlled preamplifiers, providing a range of filter and amplification settings for isometric or isotonic transducers. A range of Isometric and Isotonic Transducers of varying grades and specifications for recording tension are also available.

Electrical Stimulation

The electrical stimulus can either be constant current or constant voltage and can be used in:

  • Field Stimulation - The response evoked by electromagnetic field generating membrane depolarization and Ca2+ influx resulting in contraction and/or relaxation
  • Point Stimulation - The response evoked by local charge stimulus spreading from one excitable cell to another through the sarcolemma or adjacent cell membranes

For experiments where stimulation is required, the PowerLab output can be used to provide the stimulation or a trigger signal that controls the frequency and pulse duration of an external stimulator. ADInstruments also supplies a variety of External Stimulators for use with isolated tissue studies that supply voltage and/or current stimulation.

A range of Stimulating Electrodes for various types of tissues and experimental studies are also available for use in organ baths larger than 10 ml. They include:

  • Single or double ring stainless steel or platinum electrodes for tissue strips
  • Straight or zig-zag electrodes for tissue strips and spiral samples
  • Coil and flat electrodes for use with very small tissue
  • L-shaped electrodes for vascular (i.e. blood vessel) ring preparations

PowerLab can also control perfusion or syringe pumps or auto-injectors via eight TTL compatible digital outputs.

Myography

In some isolated tissue-organ bath studies, a myograph is used. A myograph is a device used for determining and recording the intensity of muscular contraction (force) or velocity generated at the different stages of a contracting muscle. Typical myography techniques include:

  • Wire Myography- an in vitro technique that examines the functional responses and vascular reactivity of isolated small vessels and other tubular tissues with internal diameters of 60 - 400 µm.
  • Muscle Strip Myography- for measuring the myo-mechanical properties of strips of muscle including skeletal and cardiac muscle and larger pieces of smooth muscle.

Refer to the Wire Myography application page for more information.

Related Areas of Interest

  • Radnoti Tissue/Organ Bath Principles Technique Note: Information about various techniques and principles related to isolated tissue/organ research applications. Download (95 KB)
  • Glassware Maintenance and Post-Experimental Cleaning Technique Note: Information about maintaining and cleaning glassware and other components manufactured by Radnoti Technology Inc. Download (65 KB)
  • MLT0015 Isotonic Transducer Calibration and Operation Technique Note: Information about adjusting preload, measuring units of force and measuring units of displacement. Download (119 KB)

Software:

The LabChart Advantage:

(may require additional Modules and Extensions)

  • Units Conversion for easy calibration into appropriate units such as grams or milli Newtons
  • Comments can be added in real time, viewed or moved at a later stage
  • 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.
  • The DMT Normalization Module provides an easy method for the calculation of optimal pretension conditions for microvascular or any small tubular tissue research.
  • 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 GraphPad Prism®)
  • 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. 

 

For most experiments the degree of muscle contraction can be logged into the LabChart's Data Pad. The results in the Data Pad can be exported as a .csv or .txt file to GraphPad Prism® (Windows and Macintosh) for further analysis and to prepare dose response graphs (including Schild plots).

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:

  • Muscle contraction
  • Enzyme activity
  • Hormone secretion
  • Heart rate
  • Blood pressure
  • Membrane potential

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 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
    • Evoked Response, Population Spike, Synaptic, Action Potential and Cardiac Action Potential
  • Instant tabulation of extracted parameters that can be exported to other applications
    • Height, width, area of the peak and time to peak

DMT Normalization Module

The DMT Normalization Module (Windows and Macintosh) is designed for use with PowerLab data acquisition units, LabChart software and DMT Wire Myographs, and calculates the optimal pretension conditions for microvascular or any small tubular tissue research.

It provides:

  • Real-time and offline data recording and analysis including;
    • Tension
    • Stimulation amplitude
    • Temperature
    • Flow
  • Easy method of determining optimal microvascular pretension when using DMT myographs

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:

Research Systems

Compact "All-in-One" Organ Bath Systems

Modular Organ Bath Systems

Myograph Systems

  • 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. not supplied by ADInstruments.

Instruments

Organ Baths (not available as pre-configured research systems)

  • ML0126/C-V Panlab Two Chamber Organ Bath – includes 2 tissue chambers, pre-heating reservoir coils, gas diffusers, tissue holders, micropositioners, a water pump, and thermostat controller. Note: force transducers are not supplied.

Muscle Strip Myograph (not available as pre-configured research systems)

Stimulators

Signal Conditioners

Bridge Amplifiers

Transducers and Accessories

Isometric Force Transducers

For measuring small changes in force:

Research-grade, suitable for use where larger forces are expected:

Wide-range, semi-isometric strain-gauge transducers:

Isotonic Force Transducers

For measuring displacement against a constant force:

Stimulating Electrodes

Tissue Supports

Antihypertensive, antioxidant, antidyslipidemic and endothelial modulating activities of a polyherbal formulation (POL-10)
N Aziz, M H Mehmood, S R Mandukhal, S Bashir, S Raoof and A H Gilani, Vascular Pharmacology, 57-64, 2009

Characterization of Serotonin Receptors in Pregnant Human Myometrium
Cordeaux Y, Pasupathy D, Bacon J, Charnock-Jones D S, and Smith G C S, Journal of Pharmacology and Experimental Therapeutics, 682–691, 2009

Adipose triacylglycerol lipase deletion alters whole body energy metabolism and impairs exercise performance in mice
Huijsman E, van de Par C, Economou C, van der Poel C, Lynch GS, Schoiswohl G, Haemmerle G, Zechner R, Watt MJ, American Journal of Physiology: Endocrinology and Metabolism, E505–E513, 2009

Citations Database


The material on this page is provided in good faith and believed accurate at the time of writing. No responsibility will be taken, or liability accepted, for damages arising from the use of information herein. Readers are urged to check with respective manufacturers the accuracy of all product related information.

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