The Watts Laboratory through the years, and Dr. Marlene Cohen and Kathy Schenck for teaching us this assay over two decades ago. NIGMS R25GM074119 supported development of this teaching module for a Short Course in Integrative and Systems Pharmacology held on the campus of MSU from 2005 to 2013.

NOTE: All procedures described in this paper are performed according to guidelines established by the Institutional Animal Care and Use Committee (IACUC) of Michigan State University.
1. System Preparation and Setup
<ol>
	<li>Make 5 L of a physiological salt solution (PSS), which is the amount needed for a tissue bath contraction experiment that uses up to 50 ml tissue baths; see Table 2 for PSS recipe. Calculate total required volume by multiplying number of tissue baths times the bath volume and then multiplying by the number of required tissue washes.
	<ol>
		<li>Use Table 2 as a guide to making PSS. Dissolve the salts in approximately 4 L of water. NOTE: HPLC &#8211; Type I water is recommended</li>
		<li>Add 8 ml of 1 M CaCl2 solution (147 g/L if using the dihydrate salt) to the solution, so the end solution is 1.6 mM Calcium.</li>
		<li>Quantum sufficit PSS solution to 5 L.</li>
	</ol>
	</li>
	<li>Preheat the tissue bath system to 37 &#176;C by turning on the recirculating heated water bath. Critical Step: Each component of the system is water-jacketed, ensure that they are connected in serial to one another. The direction of flow is critical &#8211; ensure that water flows into each component at the lowest barbed connection and out at the highest barbed connection.</li>
	<li>Turn on data acquisition system. Power on the force transducers at least 15 min prior the experiment to equilibrate temperature. 
	NOTE: Most force transducers employ strain gauges that are sensitive to variations in temperature and exhibit thermal drift initially after power is applied.</li>
	<li>Launch data acquisition software and ensure connection with data acquisition system. Please follow manufacture instructions for enabling data recording.</li>
	<li>Make sure the force transducers are calibrated before tissue is placed in the tissue bath and before data recording has started; follow manufacturer instructions for calibration.</li>
	<li>Connect the tissue bath system to a 95% O2 / 5% CO2 medical grade gas cylinder and check for gas leaks and then pressurize the system.</li>
	<li>Fill the tissue bath reservoirs with PSS and allow the solution time to reach optimal temperature. Prime the system and remove any air bubbles within the system and tubing.</li>
	<li>Check tissue bath aerators to ensure consistent solution aeration, which oxygenates the PSS buffer and provides Brownian motion to distribute drugs that will be introduced in the tissue bath during the experiment. Make sure aeration/bubbles does not cause tissue movement, which will disrupt data recordings; decrease gas flow as necessary. 
	NOTE: The tissue bath and data acquisition system are now ready to start the experiment.</li>
</ol>
2. Tissue Preparation
<ol>
	<li>Ideally, dissect tissues from the animal immediately prior to use and place directly into PSS. 
	NOTE: Some tissues can be saved in PSS overnight at 4 &#176;C, but appropriate controls must be done to validate tissue function after prolonged storage; see Section 6.</li>
	<li>Use the thoracic aorta as the tissue in this exercise.</li>
	<li>Anesthetize the rat in accordance with institutional guidelines and place the rat dorsal side down. Confirm anesthetization via a toe-pinch and the loss of reflex response to this painful stimulus. 
	NOTE: This protocol uses 70 mg/kg of pentobarbital delivered via an intraperitoneal injection. Institutional guidelines for rodent anesthesia may differ.</li>
	<li>Create a pneumothorax by creating an incision with scissors along the diaphragm at the bottom of the rib cage. Then, continue the incision from the bottom of the ribcage to the sternum and bisect.</li>
	<li>Dissect away or place aside those organs that are on top of the spinal column and locate the aorta, which lies directly along the spinal column. 
	NOTE: This step provides a clear working space to dissect out the aorta.</li>
	<li>Sever all connections to the aorta; from the esophagus, lung, etc. and cut the aorta perpendicular to the spine at the level of the diaphragm.</li>
	<li>Gently hold the aorta with forceps and use scissors to dissect the aorta from the spine. Start the dissection in the lower diaphragm area adjacent to the spine and work towards the heart. Make sure to take extra precaution to not pull or tug on the aortic tissue, which may damage the tissue.</li>
	<li>Immediately place the aorta in the prepared dissection dish containing PSS. 
	NOTE: Dissection of the aorta into rings is best done in a dissecting dish that has a black silastic foundation. This allows for the use of wires that can be used to cannulate the aorta and be fastened to the dish, providing stability while in the dissection dish. The black background provides contrast that aids dissection. Care should be taken in use of cannulating wires as the endothelial cell layer can be removed with excess rubbing of the wire against the lumen of the vessel.</li>
	<li>Take a wire and make a small 90&#176; angle at one end and push the angled end into the silastic foundation to anchor the guide wire. Place the whole aorta in the dissection dish.</li>
	<li>Hold the end of the guide wire with forceps, and then gently thread the wire into the lumen of the aorta.</li>
	<li>Use forceps and slide the aorta onto the wire. When the free end of the guide wire is visible, curve the wire gently and place the free end into the silastic foundation of the dish to stabilize the tissue.</li>
	<li>Using small vannas scissors and forceps, remove the perivascular adipose tissue and all other extraneous tissue, blood clots, etc. until the thoracic aorta appears white and somewhat fibrous.</li>
	<li>Remove the cleaned aorta from wire and use scissors to cut the aorta in rings that are approximately 3-5 mm in width.</li>
	<li>Place an aortic ring on one of a pair of tissue hooks by holding the hook and using forceps to gently slide the ring onto hook. Repeat this process with the second hook. Take care to ensure the hooks do not tangle; see Figure 3.</li>
	<li>Check that each hook has a different silk suture attached. Check that one hook has a small knotted loop of silk that allows for attachment to a glass or stainless steel rod, while the other is a 10-4 cm long piece suture that will be hand-tied to the force transducer; see Figure 3. 
	NOTE: Once the aorta is secured to the hooks, the tissue is ready to be mounted in the tissue bath; see Figure 3.</li>
	<li>Repeat steps 2.14-2.15, to mount a second aortic ring in the second tissue bath chamber.</li>
</ol>
3. Tissue Placement in Bath
<ol>
	<li>Note that in this system, the tissue baths themselves are stationary. At this time, fill the tissue baths with warmed, aerated PSS and allow the solution to come to temperature.</li>
	<li>With the silk suture, tie one of the hooks on the tissue preparation to the peg on the stainless steel rod. Place this end into the tissue bath chamber. Connect the rod to a ring stand and place the other end in the staining dish filled with PSS to keep tissue immersed in buffer; see Figure 3.
	<ol>
		<li>Place the rod and tissue in the tissue bath chamber and make sure the tissue is fully immersed in PSS and the rod is secure; see Figure 3.</li>
		<li>Tie the other suture to the force transducer and make sure to leave slack in the suture between the tissue and the force transducer. 
		NOTE: This slack will be removed by adjusting the micrometer; see Figure 3.</li>
	</ol>
	</li>
	<li>Repeat these steps for the second aortic ring and tissue bath chamber.</li>
</ol>
4. Setting Passive Tension
NOTE: Each tissue has a length (Lo) at which smooth muscle cells respond optimally. Preliminary experiments to determine the optimal stretching tension which achieves this length must be performed for each individual tissue type to be examined. The rat thoracic aorta has an optimal passive stretching tension of 4 g.
<ol>
	<li>Use the micrometer/rack and pinion to increase tension to 2 g and wait for the tissue to reach plateau. Once plateau is reached, increase tension another 2 g and wait for the tissue to plateau. 
	NOTE: In the aortic rings, after the initial tension of 2 g has set and plateau reached, the tissue should then relax to ~1.6 g. A second application of 2 g would bring the tissue to ~3.6 g, from which the tissue will again relax and tension will decrease. Thus, after having added 4 g of total tension, the software should indicate roughly 3.2 g of tension.</li>
	<li>Repeat these steps for the second aortic ring and tissue bath chamber.</li>
</ol>
5. Equilibration and Drug Making
<ol>
	<li>Equilibrate the tissue for 60 min after applying passive tension to the tissue.</li>
	<li>During the equilibrium phase, wash the tissue every 15-20 min. Drain the tissues bath and replace the PSS with PSS from a warmed reservoir. 
	NOTE: During this time, the tissue will relax, which is indicated by the loss of passive tension.</li>
	<li>During this time, prepare drugs for the experiment, which need to be at least 1,000 times more concentrated than the actual concentration in the bath, so only a small volume of the drug stock is needed to achieve the desired concentration.</li>
</ol>
6. Initial Challenge
<ol>
	<li>At the end of the equilibration period, wash the tissue one final time, save data, and re-zero all the inputs.</li>
	<li>Pick an agonist (compound that will cause active contraction) to which the tissue responds. 
	NOTE: In the rat thoracic aorta, the arterial smooth muscle will actively contract to an alpha adrenergic receptor agonist due to the innervation of the tissue by the sympathetic nervous system. An adrenergic agonist would not be useful in intestinal tissues given that adrenergic agonists cause relaxation. In this case a receptor agonist such as acetylcholine (cholinergic receptor) could be used. An alternative to both phenylephrine and acetylcholine is to use a non-receptor agonist such as high potassium (K) as the first challenge. In smooth muscle, high K operates to indirectly open calcium channels and thus is viewed as a general index of smooth muscle integrity.
	<ol>
		<li>Perform the first challenge or the wake-up challenge by adding 10-5 M phenylephrine to the tissue bath. To achieve this concentration in 50 ml tissue bath, add 50 &#956;l&#160;of a 10-2 M phenylephrine solution. 
		NOTE: 50 &#956;l&#160;into 50 ml of PSS is a 1/1,000 dilution, so the final concentration is 1,000x less than stock, or 10-5 M. Knowledge of volume and consistent maintenance of the volume within the tissue bath chamber is critical to determining the final drug concentration.</li>
	</ol>
	</li>
	<li>Add 10-5 M phenylephrine to the tissue bath and allow the contraction to peak and then plateau, when the slope of the data becomes zero. Make sure to record each experimental event to aid post-experimental analysis.</li>
	<li>After plateau, wash the agonist out thoroughly by emptying and refilling the tissue bath chamber with new PSS. Make sure to record these events as well. 
	NOTE: A rule of thumb is that the concentration of agonist in the bath is reduced 10 fold with each wash. Although, more than 3-4 washes may be necessary to bring the tissue back down to its equilibrium baseline tone (tension).</li>
	<li>Let the tissue rest at baseline tone for ~10 min before proceeding.</li>
</ol>
7. Experiment
NOTE: Prazosin, an alpha adrenergic receptor antagonist, will be introduced to the tissue bath chamber to shift the concentration response curve to phenylephrine (PE); this is a demonstrable antagonism.
<ol>
	<li>To one bath, add the vehicle (H2O) in which prazosin was dissolved. To another, add the appropriate concentration of prazosin. In this instance, add 25 &#181;l&#160;of vehicle to one bath and 25 &#181;l&#160;of 10-5 M concentration of prazosin to the other to achieve a 5 x 10-9 M or a 5 nM concentration in the bath. 
	NOTE: While the addition of vehicle seems unnecessary in this example, it is imperative to do so when using other vehicles that have a potential to be vasoactive (e.g., DMSO, ethanol, acetate). Add the corresponding vehicle even if little evidence exists for it to be vasoactive.</li>
	<li>Leave vehicle/prazosin in the baths and do not wash the tissue for 1 hr.
	<ol>
		<li>While tissues are equilibrating, prepare multiple concentrations of agonist (PE). Include a wide range of concentrations, from concentrations eliciting no response (e.g., 10-9 M PE) to concentrations that surpass the maximal response (e.g., 10-4 M PE). Since concentration-response curves are logarithmic in nature, make six separate stock solutions of PE (10-6-10-1 M) through serial dilutions from the 10-1 M stock solution. Make sure the volume needed for each concentration is slightly greater than triple the total volume needed for all baths (i.e. &#62;300 &#181;l).</li>
	</ol>
	</li>
	<li>Proceed to adding agonist, as described in Table 1. 
	NOTE: This experiment will generate a cumulative concentration response curve to PE; each iteration takes into account the amount of substance already added to the bath. Additions occur until they no longer increase contraction, or the whole curve has plateaued.
	<ol>
		<li>Add each drug concentration individually until the tissue threshold is reached.</li>
		<li>If no change occurs, add the next concentration in the series; see Table 2. 
		NOTE: The timing of these additions depends on the agonist used. For example, norepinephrine and phenylephrine contraction develops rapidly, and should plateau within minutes. By contrast, endothelin-1 contraction develops slowly and may not plateau for close to 45 min. Other experimentation can be done on the tissue after construction of a curve such as this, as long as (1) the substance washes out; and (2) it can be demonstrated that the tissue returns to normal contractile behavior and is not affected by the previous stimulations.</li>
	</ol>
	</li>
</ol>
8. Data Analysis
<ol>
	<li>Make sure to save data immediately prior to and after an intervention (e.g., wake up or full curve).</li>
	<li>Find and set the tissue baseline for each tissue bath chamber/input channel, which will aid in data analysis.</li>
	<li>Once the baseline is set, find the maximal response for each concentration, and record the change from baseline. Sequentially move through each of the concentrations and record the baseline shift.</li>
	<li>Graph the resulting data. Do this in any graphing program. 
	NOTE: Graph Pad Prism is designed for pharmacologists, and this is ideal for the type of experiment presented here.</li>
</ol>

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Authors have nothing to disclose.

Measurement of isometric force as a research tool is over 150 years old, but it continues to be the prototypical technique for receptor characterization in contractile tissues2. The power of this technique is in its simplicity and versatility: by recording responses elicited by increasing concentrations of an agonist in the presence or absence of an antagonist, a myriad of information can be derived about the pharmacological characteristics of each drug and the receptor to which it binds3-5. Experiments of this type also garner information about the competitive versus non-competitive nature of the antagonists used, as well as receptor heterogeneity and non-specific drug effects6-8. Thus, with simple permutations to this isolated tissue bath experiment, a relatively complete pharmacological profile of the receptors that mediate agonist-induced muscle contraction can be generated.
In terms of agonism, the relative efficacy (EMAX) and potency (EC50) of an agonist in a given tissue can be calculated and compared to responses of other agonists in the same tissue1. In our experiment, the rat thoracic aorta was incubated with either vehicle or the &#945;1 adrenergic receptor antagonist prazosin (5 nM) for one hr prior to adding the &#945;1 adrenergic agonist phenylephrine to the tissue bath and generating contraction.&#160; Figure 2 presents modified results of Figure 1. The green responses have been marked with the maximum contraction achieved by PE marked as max, the &#189; maximum contraction marked as such and then associated with the PE concentration that caused that response. Identifying these values is identifying the effective concentration of an agonist that achieves a half max (50%) response or EC50 value.
Unfortunately, using agonist-dependent parameters alone to determine receptor binding characteristics can be complicated9. Ideally, additional experiments using receptor antagonists allow the calculation of two important parameters that are key to defining the interaction between a drug and a receptor: the -log10 of the antagonist dissociation constant (pKB) and the -log10 of the molar antagonist concentration necessary to elicit two-fold rightward shift in the concentration-response curve (pA2)10. Both KB and pA2 gain their usefulness from the fact that they are agonist-independent values, and remain constant even between different tissues11. From the data in Figure 2, pKB can be calculated using the following equation and based on EC50 values calculated elsewhere:
EC50 in the absence of prazosin&#160; = 2 x 10-8 M 
EC50 in the presence of prazosin = 7 x 10-6 M
Solve for KB in the following equation: 
log (dr-1) = log [B] - log KB 
Where: 
[B] = antagonist concentration, or 5 x 10-9 M.&#160; log (5 x 10-9 M) = -8.3 
dr = dose ratio of EC50 value in presence of antagonist/EC50 in absence.&#160; If there is a rightward shift, this value will be greater than one. Thus: 
dr = 7 x 10-6 M/2 x 10-8 M or 700 x 10-8 M/2 x 10-8 M = 700/2 = 350 
Substituting for [B] and dr: 
log (350-1) = -8 - log KB 
2.54 = -8 - log KB 
pKB = 10.54
This value for prazosin is consistent with the values obtained when prazosin interacts with an &#945;1adrenergic receptor12,13, suggesting that the adrenergic receptor mediating contraction to phenylephrine is the &#945;1 adrenergic receptor.&#160;
Several steps are critical to the success of these experiments. Tissues must remain in PSS after dissection to prevent loss of tissue viability. Any isometric muscle preparation has an optimal length-tension relationship that generates maximal force against passive tension14,15. For the experiment described in this protocol, optimal passive tension was previously determined by briefly adding increments of 0.5 g of passive tension to the tissue. The tissue should begin without tone and then after 30 min of equilibration, the tissue was challenged with a maximal concentration of KCl (80 mM), which generated active tone. Then, the tissue was washed and allowed to return to baseline tone. After 15 min at baseline, another 0.5 g of passive tension was placed and this process repeated until a plateau of active tension was achieved with additional passive tension. In preliminary experiments, 4 g total of passive tension was determined to achieve maximal active tension generation in aortic rings, and thus this total amount of tension is placed on the rings prior to equilibration. Procedurally, it&#8217;s best to zero prior passive tension application, but can be done at any time prior the experiment. Over-stretching tissues, at any point in the experiment or dissection, will negatively impact tissue viability and experimental outcomes. This is most imperative when placing tissues in the bath, as this step has the highest likelihood of excessive stretch. Adequate washing, in number and duration is required for reproducible effects. A second challenge too soon after an initial challenge, or before tissues have returned to baseline tension, will result in aberrant responses. If studying reversible antagonists/inhibitors, tissues should not be washed prior to agonist addition, as the inhibitor concentration will be decreased.
There are notable advantages and disadvantages to the isolated tissue bath assays.
Disadvantages: Tissues may experience different degrees of damage during surgical removal or placement of rings on hooks. Since the endothelial cell is the inner lining of the aortic ring, care must be taken on the placement of the ring on hooks so as not to damage this cell layer. Tissues may also have distinctly different lengths of time in which they are viable in the tissue bath, and this has to be determined; not all tissues are the same. Along these lines, tissues may change in their responsiveness throughout the day such that time controls become a necessary control for every experiment. A good example of this is the guinea pig trachea that improves in contractility maximum by 100% during a typical 8 hr experiment. Drugs that are poorly soluble in water may precipitate out in the PSS. Finally, cumulative and non-cumulative additions of a drug that is an agonist could result in different outcomes if receptor desensitization to the agonist occurs; angiotensin II is one such drug which shows rapid tachyphylaxis.
Advantages: One of the primary advantages of tissue bath experiments is that it is real time; one can see the experiment as it unfolds and can rapidly make conclusions and plan next steps, as well as troubleshoot during an experiment. An experiment takes a day to do. Multiple tissues can typically be prepared from one animal such that an animal can serve as its own control, and that adds strength to an experiment. One also can isolate the tissue from other factors so as to test a relatively pure response of the tissue to the drug. In vitro experiments such as the tissue bath system also allow use of a small amount of drug compared to an in vivo experiment.
The basic experimental design described herein can be extensively modified to allow for the recording of additional parameters or the introduction of other external stimuli. For example, addition of electrodes allow for electric field stimulation of innervating nerves16,17. With the addition of thermal or pH probes, the effects of temperature and pH on contractile responses can also be measured18,19. Similarly, oxygen can be substituted partially or in full with N2 to evaluate hypoxia induced effects. Furthermore, the same basic principles of isometric contractility measurement used in this video can be used to develop systems that allow concomitant measurement of isometric tension development and changes in intracellular calcium20. Signal transduction is also readily studied, since systems exist that can rapidly freeze a tissue sample during a response, such that the activity of a signal transduction pathway system can be verified biochemically.
The variation of equipment that can be used to do this is enormous. This whole system, either hand constructed or automated, can be purchased from multiple different companies. The tissue baths and tissue holders used in this protocol were hand-blown (tissue baths) and hand constructed (holders) by an in-house Michigan State University machine shops.

The discipline of pharmacology has used the isolated tissue bath system for over 150 years. The versatility of this system has allowed scientists across the world to characterize receptors and receptor signal transduction, with this knowledge forming the basis of therapies that have treated millions of individuals with diseases or disorders such as hypertension, heart failure, diabetes, gastrointestinal disease, bladder dysfunction, asthma, and swallowing disorders, to name just a few. To this day, the isolated tissue bath remains an important facet of drug development and basic research, as it allows the tissue to function as a tissue. In this JoVE lesson, a formal protocol is shared to demonstrate a visual and virtual experiment utilizing the data from an isolated tissue bath experiment that measures isometric contraction, which permits receptor characterization.
The primary advantage of this technique is that the tissue is living and functions as a whole tissue, with a physiological outcome (contraction or relaxation) that is relevant to the body. It is a synthesis of steps (drug-receptor interaction, signal transduction, second messenger generation, change in smooth muscle excitability, and change in tissue function). While other techniques allow study of each of these steps (e.g. radioligand binding for drug affinity, measurement of second messengers), the isolated tissue bath technique allows for integration of all these steps1. Another advantage is that retaining tissue function permits calculation of important pharmacological variables that are more meaningful in a tissue vs a cellular setting; it comes closer to how the drugs examined would work in the body as a whole.

<table><tbody><tr><td>LabChart Software</td><td>ADInstruments</td><td></td><td>7.2</td></tr><tr><td>PowerLab (4 channel)&amp;nbsp;</td><td>ADInstruments</td><td>ML760</td><td></td></tr><tr><td>QuadBridge (4 channel)</td><td>ADInstruments</td><td>ML112</td><td>ML112</td></tr><tr><td>Grass Adapter Cable</td><td>ADInstruments</td><td>MLAC11</td><td>MLAC11</td></tr><tr><td>Grass Force-displacement Transducer</td><td>Grass Instrument Co</td><td>FT03</td><td>FT03</td></tr><tr><td>Grass Transducer Cable</td><td>Grass Instrument Co</td><td>TAC-7 REV-1</td><td></td></tr><tr><td>BNC to BNC Cable</td><td>ADInstruments</td><td>MLAC01</td><td></td></tr><tr><td>IsoTemp 2100</td><td>Fisher Scientific</td><td></td><td>IC-2100</td></tr><tr><td>Tissue Bath</td><td>Multiple Sources&amp;nbsp;</td><td></td><td></td></tr><tr><td>Physiological Salt Solution</td><td></td><td></td><td>PSS</td></tr><tr><td>Braided Silk Suture</td><td>Harvard Apparatus</td><td>51-7615</td><td>SP104</td></tr><tr><td>Ring Stand</td><td>Humboldt MFG Co</td><td>H-2122 7</td><td></td></tr><tr><td>Dissecting Dishes</td><td>Handmade with Silicone</td><td></td><td></td></tr><tr><td>Tygon Tubing</td><td>VWR Scientific</td><td>63010-100</td><td>R-3603</td></tr><tr><td>Hose Clamps</td><td>Cole-Parmer Instrument Co</td><td>06832-08</td><td>SNP-8</td></tr><tr><td>50 ml Muscle Bath</td><td>Eberhartglass Blowing</td><td>Custom</td><td></td></tr><tr><td>250 ml Warming Chambers</td><td>Eberhartglass Blowing</td><td>Custom</td><td></td></tr><tr><td>Gas Dispersion Tube</td><td>Ace Glass</td><td>7202-06</td><td>7202-02</td></tr><tr><td>Micrometer</td><td></td><td></td><td></td></tr><tr><td>Custom Stands</td><td></td><td></td><td></td></tr><tr><td>Three-prong Clamps</td><td>VWR International</td><td></td><td>Talon</td></tr><tr><td>S-connector</td><td>VWR International</td><td></td><td>Talon</td></tr><tr><td>Tissue Hooks</td><td>Hand Made in House</td><td>Custom</td><td></td></tr><tr><td>Tissue Dissection</td><td></td><td></td><td></td></tr><tr><td>Leica Stereomicroscope MZ6</td><td>Leica</td><td></td><td>10447254</td></tr><tr><td>Stereomaster Microscope Fiber-optic Light Source</td><td>Fisher Scientific</td><td>12562-36</td><td>12562-36</td></tr><tr><td>Culture Petri Dish</td><td>Pyrex</td><td></td><td>7740 Glass</td></tr><tr><td>Sylgard Silicone Elastomer</td><td>Dow Corning</td><td></td><td>Sylgard 170 Kit</td></tr><tr><td>Vannas Scissors</td><td>George Tiemann &amp;amp; Co</td><td>160-150</td><td>160-150</td></tr><tr><td>Splinter &amp;amp; Fixation Forceps</td><td>George Tiemann &amp;amp; Co</td><td>160-55</td><td>160-55</td></tr></tbody></table>

measurement of smooth muscle function in the isolated tissue bath-applications to pharmacology research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

In terms of agonism, the relative efficacy (EMAX) and potency (EC50) of an agonist in a given tissue can be calculated and compared to responses of other agonists in the same tissue1. In our experiment, the rat thoracic aorta was incubated with either vehicle or the &#945;1 adrenergic receptor antagonist prazosin (5 nM) for one hour prior to adding the &#945;1 adrenergic agonist phenylephrine to the tissue bath and generating contraction (Figure 1).&#160;
Figure 2 presents modified results of Figure 1. The green responses have marked the maximum contraction achieved by PE marked as max, the &#189; maximum contraction marked as such and then associated with the PE concentration that caused that response. Identifying these values is identifying the effective concentration of an agonist that achieves a half max (50%) response or EC50 value. This was donegraphically in this example.Computer software that uses logistic functions for sigmoidal curves can be used for curve fitting and calculation of EC50 values.
In generating and interpreting these results, it is important to use both low and high concentrations of agonist to create the concentration-response curve. Without enough points to show a stable baseline and maximum plateau, EC50 and EMAX can only be estimated. This was done graphically in this example. Computer software that uses logistic functions for sigmoidal curves can be used for curve fitting and calculation of EC50 values.
<img alt="Figure 1" src="/files/ftp_upload/52324/52324fig1highres.jpg" /> 
Figure 1: Tissue Bath Schematic. Cartoon representing a ring of tissue placed in the double-wall, water jacketed tissue bath. Buffer inflow and outflow are regulated by a 3-way glass stopcock integrated into the base of the chamber. O2/CO2 is bubbled in through an aerator that is sealed with a gasket to prevent leak of PSS or gas. Recirculation input is below the output to maintain proper recirculation and prevent formation of air pockets that would cause temperature disregulation.
<img alt="Figure 2" src="/files/ftp_upload/52324/52324fig2highres.jpg" /> 
Figure 2: Rat Thoracic Aorta + Endothelium. The figure above depicts four separate experiments (different animals and done by different investigators on different set ups as represented by different colors) to test the ability of prazosin to shift a PE-induced contraction. Prazosin (5 nM) clearly shifted PE-induced contraction curve rightward in a parallel fashion.
<img alt="Figure 3" src="/files/ftp_upload/52324/52324fig3highres.jpg" /> 
Figure 3: Rat Thoracic Aorta + Endothelium. Graphical estimation of EC50 values for PE in the absence (vehicle) and presence (prazosin) of antagonist. Green line (group C) only is marked.
<table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Salt</td>
			<td>MW (g/mole)</td>
			<td>5 Liters</td>
			<td>FINAL mM</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (grams)</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>58.45</td>
			<td>37.99</td>
			<td>130</td>
		</tr>
		<tr>
			<td>KCl&#160;</td>
			<td>74.56</td>
			<td>1.75</td>
			<td>4.7</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>136.1</td>
			<td>0.8</td>
			<td>1.18</td>
		</tr>
		<tr>
			<td>MgSO4&#8226;7H20</td>
			<td>246.5</td>
			<td>1.45</td>
			<td>1.17</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>84.21</td>
			<td>6.25</td>
			<td>14.9</td>
		</tr>
		<tr>
			<td>Dextrose&#160;</td>
			<td>180.16</td>
			<td>5</td>
			<td>5.5</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>380</td>
			<td>0.05</td>
			<td>0.03</td>
		</tr>
	</tbody>
</table>
Table 1: Normal Physiological Salt Solution (PSS) Recipe. Contents of the bicarbonate-buffered PSS used in this experiment. Included are the molecular weights of all salts and the amount added to 5 L of dH2O to achieve the desired concentration.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Concentration in bath</td>
			<td>Addition of agonist made to bath</td>
		</tr>
		<tr>
			<td>1 x 10-9 M</td>
			<td>50 ml 1 x 10-6 M</td>
		</tr>
		<tr>
			<td>3 x 10-9 M</td>
			<td>100 ml 1 x 10-6 M</td>
		</tr>
		<tr>
			<td>1 x 10-8 M</td>
			<td>35 ml 1 x 10-5 M</td>
		</tr>
		<tr>
			<td>3 x 10-8 M&#160;</td>
			<td>100 ml 1 x 10-5</td>
		</tr>
		<tr>
			<td>1 x 10-7 M</td>
			<td>35 ml 1 x 10-4 M</td>
		</tr>
		<tr>
			<td>3 x 10-7 M</td>
			<td>100 ml 1 x 10-4 M</td>
		</tr>
		<tr>
			<td>1 x 10-6 M</td>
			<td>35 ml 1 x 10-3 M</td>
		</tr>
		<tr>
			<td>3 x 10-6 M</td>
			<td>100 ml 1 x 10-3 M</td>
		</tr>
		<tr>
			<td>1 x 10-5 M</td>
			<td>35 ml 1 x 10-2 M</td>
		</tr>
		<tr>
			<td>3 x 10-5 M</td>
			<td>100 ml 1 x 10-2 M</td>
		</tr>
		<tr>
			<td>1 x 10-4 M</td>
			<td>35 ml 1 x 10-1 M</td>
		</tr>
	</tbody>
</table>
Table 2: Tissue Bath Additive Agonist Concentrations. The amount of agonist to be added to each bath to properly construct cumulative concentration-response curves. The desired bath concentration is achieved through addition of sequential amounts of increasing concentrations of agonist. Each addition takes into account the concentration of agonist already present in the bath. This is done to minimize the increase in volume in the tissue bath that would result from using increasing volumes of a single concentration of agonist.

Watch this Scientific Journal Video about Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research at JoVE.com

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

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Drugs Acting on Autonomic Nervous System: Adrenergic Agonists and Antagonists Agents

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137.5K Views.  Michigan State University. This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition. 

Video: Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between 100-400 &mu;m in diameter function primarily in directing blood flow to various organs according to the overall requirements of the body. The rat mesenteric artery has a diameter greater than 100 &mu;m. The myography technique, first described by Mulvay and Halpern1, was based on the method proposed by Bevan and Osher2. The technique provides information about small vessels under isometric conditions, where substantial shortening of the muscle preparation is prevented. Since force production and sensitivity of vessels to different agonists is dependent on the extent of stretch, according to active tension-length relation, it is essential to conduct contraction studies under isometric conditions to prevent compliance of the mounting wires. Stainless steel wires are preferred to tungsten wires because of oxidation of the latter, which affects recorded responses3.The technique allows for the comparison of agonist-induced contractions of mounted vessels to obtain evidence for normal function of vascular smooth muscle cell receptors.
We have shown in several studies that isolated mesenteric arteries that are contracted with phenylyephrine relax upon addition of cumulative concentrations of extracellular calcium (Ca2+e). The findings led us to conclude that perivascular sensory nerves, which express the G protein-coupled Ca2+-sensing receptor (CaR), mediate this vasorelaxation response. Using an automated wire myography method, we show here that mesenteric arteries from Wistar, Dahl salt-sensitive(DS) and Dahl salt-resistant (DR) rats respond differently to Ca2+e. Tissues from Wistar rats showed higher Ca2+-sensitivity compared to those from DR and DS. Reduced CaR expression in mesenteric arteries from DS rats correlates with reduced Ca2+e-induced relaxation of isolated, pre-contracted arteries. The data suggest that the CaR is required for relaxation of mesenteric arteries under increased adrenergic tone, as occurs in hypertension, and indicate an inherent defect in the CaR signaling pathway in Dahl animals, which is much more severe in DS.
The method is useful in determining vascular reactivity ex vivo in mesenteric resistance arteries and similar small blood vessels and comparisons between different agonists and/or antagonists can be easily and consistently assessed side-by-side6,7,8.

An automated myography method for force measurements in isolated mesenteric arteries is described. It employs a Mulvany-Halpern Auto Dual Wire Myograph 510A to determine responses to phenylephrine and extracellular calcium. The method allows consistent determination of isometric responses to agonists in small vessels of diameters of 60 - 300 &mu;m, independently.

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between ...

Medicine

Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e. smooth muscle, mucosa, nerves) in healthy and pathological conditions.
The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.
The in vitro smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.

This manuscript presents a simple, yet powerful, in vitro method for evaluating smooth muscle contractility in response to pharmacological agents or nerve stimulation. Main applications are drug screening and understanding tissue physiology, pharmacology,&#160;and pathology.

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties ...

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that system. Changes in blood flow to the small intestine can result in effects on the absorptive functions of the organ. Particular interest in toxins liberated from feedstuffs through fermentative and digestive processes has developed in ruminants as an area where productive efficiencies could be improved. The video associated with this article describes an in vitro bioassay developed to screen compounds for vasoactivity in isolated cross-sections of bovine mesenteric artery and vein using a multimyograph. Once the blood vessels are mounted and equilibrated in the myograph, the bioassay itself can be used: as a screening tool to evaluate the contractile response or vasoactivity of compounds of interest; determine the presence of receptor types by pharmacologically targeting receptors with specific agonists; determine the role of a receptor with the presence of one or more antagonists; or determine potential interactions of compounds of interest with antagonists. Through all of this, data are collected real-time, tissue collected from a single animal can be exposed to a large number of different experimental treatments (an in vitro advantage), and represents vasculature on either side of the capillary bed to provide an accurate picture of what could be happening in the afferent and efferent blood supply supporting the small intestine.

The small intestine is frequently exposed to toxins that can influence blood flow and negatively impact nutrient absorption. Using a multimyograph and mesenteric artery and vein isolates, compounds or toxins of interest can be screened for vasoactivity.

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that ...

Contractility Measurements of Human Uterine Smooth Muscle to Aid Drug Development

Discovery and characterization of novel pharmaceutical compounds or biochemical probes rely on robust and physiologically relevant assay systems. We describe methods to measure ex vivo myometrium contractility. This assay can be used to investigate factors and molecules involved in the modulation of myometrial contraction and to determine their excitatory or inhibitory actions, and hence their therapeutic potential in vivo. Biopsies are obtained from women undergoing cesarean section delivery with informed consent. Fine strips of myometrium are dissected, clipped and attached to a force transducer within 1 mL organ baths superfused with physiological saline solution at 37 &#176;C. Strips develop spontaneous contractions within 2&#8211;3 h under set tension and remain stable for many hours (&#62;6 h). Strips can also be stimulated to contract such as by the endogenous hormones, oxytocin and vasopressin, which cause concentration-dependent modulation of contraction frequency, force and duration, to more closely resemble contractions in labor. Hence, the effect of known and novel drug leads can be tested on spontaneous and agonist-induced contractions.
This protocol specifically details how this assay can be used to determine the potency of known and novel agents by measuring their effects on various parameters of human myometrial contraction. We use the oxytocin- and V1a receptor antagonists, atosiban and SR49059 as examples of known compounds which inhibit oxytocin- and vasopressin-induced contractions, and demonstrate how this method can be used to complement and validate pharmacological data obtained from cell-based assays to aid drug development. The effects of novel agonists in comparison to oxytocin and vasopressin can also be characterized. Whilst we use the example of the oxytocin/ vasopressin system, this method can also be used to study other receptors and ion channels that play a role in uterine contraction and relaxation to advance the understanding of human uterine physiology and pathophysiology.

This article describes experimental protocols to study ex vivo contractions of human myometrium and their application in drug discovery. This technique is used to improve the understanding of myometrial physiology and pathophysiology as well as to validate pharmacological data from novel research probes or drug leads.

Discovery and characterization of novel pharmaceutical compounds or biochemical probes rely on robust and physiologically relevant assay systems. We ...

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+]cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.

Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

Assessing Murine Resistance Artery Function Using Pressure Myography

Pressure myograph systems are exquisitely useful in the functional assessment of small arteries, pressurized to a suitable transmural pressure. The near physiological condition achieved in pressure myography permits in-depth characterization of intrinsic responses to pharmacological and physiological stimuli, which can be extrapolated to the in vivo behavior of the vascular bed. Pressure myograph has several advantages over conventional wire myographs. For example, smaller resistance vessels can be studied at tightly controlled and physiologically relevant intraluminal pressures. Here, we study the ability of 3rd order mesenteric arteries (3-4 mm long), preconstricted with phenylephrine, to vaso-relax in response to acetylcholine. Mesenteric arteries are mounted on two cannulas connected to a pressurized and sealed system that is maintained at constant pressure of 60 mmHg. The lumen and outer diameter of the vessel are continuously recorded using a video camera, allowing real time quantification of the vasoconstriction and vasorelaxation in response to phenylephrine and acetylcholine, respectively.
	To demonstrate the applicability of pressure myography to study the etiology of cardiovascular disease, we assessed endothelium-dependent vascular function in a murine model of systemic hypertension. Mice deficient in the &alpha;1 subunit of soluble guanylate cyclase (sGC&alpha;1-/-) are hypertensive when on a 129S6 (S6) background (sGC&alpha;1-/-S6) but not when on a C57BL/6 (B6) background (sGC&alpha;1-/-B6). Using pressure myography, we demonstrate that sGC&alpha;1-deficiency results in impaired endothelium-dependent vasorelaxation. The vascular dysfunction is more pronounced in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice, likely contributing to the higher blood pressure in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice. 
	Pressure myography is a relatively simple, but sensitive and mechanistically useful technique that can be used to assess the effect of various stimuli on vascular contraction and relaxation, thereby augmenting our insight into the mechanisms underlying cardiovascular disease.

In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3rd order mesenteric arteries in c57 and sGC&alpha;1-/- mice using pressure myography.

Pressure myograph systems are  exquisitely useful in the functional assessment of small arteries, pressurized  to a suitable transmural pressure. The  ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic diseases. Endothelial dysfunction represents a major culprit for the reduced vasodilatation and enhanced vasoconstriction of arteries. Adipose (fat) tissues surrounding the arteries play important roles in the regulation of endothelium-dependent relaxation and/or contraction of the vascular smooth muscle cells. The cross-talks between the endothelium and perivascular adipose tissues can be assessed ex vivo using mounted blood vessels by a wire myography system. However, optimal settings should be established for arteries derived from animals of different species, ages, genetic backgrounds and/or pathophysiological conditions.

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic ...

Protocol for Studying Gastrointestinal Dynamics and Drug Effects on Motility

Using An In Vitro Tissue Perfusion System to Detect the Functional Activities of Isolated Intestinal Tubes in Real Time

Gastrointestinal diseases, which have a high incidence, pose considerable challenges for humans. The small intestine is integral to food and drug digestion and absorption and plays a crucial role in treating these diseases. The intestinal tube movement experiment, a common and essential in vitro method, is utilized to study gastrointestinal dynamics. This includes the preparation of the isolated intestinal tube, as well as the suspension of the prepared intestinal tube in the bath and its connection to a signal detector. This is followed by the recording and analysis of a series of parameters, such as tension, which can be used to assess intestinal motor function, as well as considerations for keeping the intestinal tube active in vitro. The standardized program from sampling to data collection greatly improves the repeatability of the experimental data and ensures the authenticity of the recording of intestinal tension after physiological, pathological, and drug intervention. Here we present the key problems in experimental operation and a valuable reference experimental protocol for studying drugs that regulate gastrointestinal motility.

Author Spotlight: Reliable and Reproducible In Vitro Assessment of Drug Impact on Rat Intestinal Tubes

We present a method for isolating rat intestinal tubes and assessing the impact of drugs on their tension, frequency, and amplitude in vitro. This method offers a valuable approach for researchers investigating intestinal tubes.

Gastrointestinal diseases, which have a high incidence, pose considerable challenges for humans. The small intestine is integral to food and drug ...

Measurement of Endothelium-Dependent Vasorelaxation in the Mouse Thoracic Aorta Using Tensometric Small Volume Chamber Myography

Small volume chamber tensometric myography is a commonly used technique to evaluate the vascular contractility of small and large blood vessels in laboratory animals and small arteries isolated from human tissue. The technique allows researchers to maintain isolated blood vessels in a tightly controlled and standardized (near-physiological) setting, with the option of adjusting to various environmental factors, while challenging the isolated vessels with different pharmacological agents that can induce vasoconstriction or vasodilation. The myograph chamber also provides a platform to measure vascular reactivity in response to various hormones, inhibitors, and agonists that may impact the function of smooth muscle and endothelial layers separately or simultaneously. The blood vessel wall is a complex structure consisting of three different layers: the intima (endothelial layer), media (smooth muscle and elastin fibers), and adventitia (collagen and other connective tissue). To gain a clear understanding of the functional properties of each layer, it is critical to have access to an experimental platform and system that would allow for a combinational approach to study all three layers simultaneously. Such an approach demands access to a semi-physiological condition that would mimic the in vivo environment in an ex vivo setting. Small volume chamber tensometric myography has provided an ideal environment to evaluate the impact of environmental cues, experimental variables, or pharmacological agonists and antagonists on vascular properties. For many years, scientists have used the tensometric myograph technique to measure endothelial function and smooth muscle contractility in response to different agents. In this report, a small volume chamber tensometric myograph system is used to measure endothelial function in the isolated mouse aorta. This report focuses on how small volume chamber tensometric myography can be used to evaluate the functional integrity of the endothelium in small segments of a large artery such as the thoracic aorta.

The present protocol describes the concepts and technical application of the tensometric myograph technique using a multi-chamber myograph system in the experimental ex vivo assessment of mouse aortic endothelial function.