The authors acknowledge Ryan Chaplin and Dr. Gregg Rentfrow of the University of Kentucky Meats Lab and Department of Animal and Food Sciences for providing opportunities to collect experimental tissues utilized herein.

Procedures used in this study did not require approval from the University of Kentucky Animal Care and Use Committee because no live animals were used. Prior to collection of any sample used herein, all animals were stunned with a captive bolt and exsanguinated. This was conducted at a federally inspected abattoir facility at the University of Kentucky. An official representative of the USDA Food Safety and Inspection Service observed all activities that dealt with the live animal and handling of the carcass.
1. Preparation of Instrumentation
<ol>
	<li>To minimize the amount of time between collections of tissue samples to the start of an experiment, set up equipment and prepare buffers prior to collecting experimental tissue (or if additional personnel are available, these tasks can occur simultaneously). 
	NOTE: This not only provides the greatest amount of time that the tissue remains viable to conduct experiments, but some experiments can run for long periods of time (or there is a desire to complete multiple experiments in a day), therefore it is usually desirable to get started as early as feasible.</li>
	<li>Power up the associated computer, data acquisitions equipment, water bath (for buffer(s)) and myograph unit(s). Place the calibration equipment on the myograph unit(s) and turn on myograph heat (preset to 37.5 &#176;C) and allow units and calibration equipment to warm to preset temperatures.
	<ol>
		<li>Open a previously created Chart software settings file on the computer and start a run (but not acquiring data).</li>
	</ol>
	</li>
	<li>Warm up the equipment for 10 min.
	<ol>
		<li>Start acquiring data.</li>
		<li>Zero all channels.</li>
		<li>Check, and conduct calibration (if necessary) on each channel (4 channels per multimyograph) to standardize the electric signal supplied from the respective force transducer associated with that channel to a 2-g force supplied by a certified weight.</li>
		<li>After calibration and a units conversion are completed, store all subsequent data as grams. Change accordingly based on desires of each separate laboratory; the equipment and software permit the use of other units, such as milliNewtons and volts.</li>
	</ol>
	</li>
	<li>Make sure that the gas lines are clear of blockages and turn on gas supply (95% O2/5% CO2) to myograph(s) units. Continuously gas all buffers (in the myograph chambers) during the entire procedure.</li>
	<li>Fill all myograph chambers with 70% ethanol and soak for 10 min, remove ethanol, and repeat for a second 10 min soak.
	<ol>
		<li>After removing the ethanol, rinse three times with deionized water, followed by three rinses with prepared buffer (see Section 2 for buffer preparation), leaving the third addition of buffer in the chambers. 
		NOTE: At this point the equipment can remain idle in this state until buffer and tissue are prepared and staff is ready to proceed with the experiment.</li>
	</ol>
	</li>
</ol>
2. Preparation of Buffers
<ol>
	<li>Prepare a 1 L Krebs-Henseleit buffer solution for use in transportation and processing of tissue samples to achieve final concentrations of 11.1 mM D-glucose; 1.2 mM MgSO4; 1.2 mM KH2PO4; 4.7 mM KCl; 118.1 mM NaCl; 3.4 mM CaCl2; 24.9 mM NaHCO3.
	<ol>
		<li>Mix 9.6 g of Krebs salts into approximately 900 ml of deionized water on a stir plate.</li>
		<li>Mix in 0.373 g calcium chloride dehydrate followed by 2.1 g sodium bicarbonate per L of buffer desired.</li>
		<li>Gas buffer solution for 20 min with 95% O2/5% CO2.</li>
		<li>After gassing, adjust pH to 7.05 (filtration of buffer increases pH; final target pH should be 7.4) and adjust volume to 1 L.</li>
		<li>Filter sterilize buffer into a clean autoclaved 1 L media bottle and store buffer at 4 &#176;C until ready for use.</li>
	</ol>
	</li>
	<li>Prepare separate Krebs-Henseleit buffer solution for use in myograph experiments as described in step 2.1 for the transportation Krebs-Henseleit buffer with additional compounds that pertain to the contractility experiments. 
	NOTE: Typically 2 L will suffice for experiments described in this article, but this volume may need to be increased or reduced based on the length of the experiment, number of myograph chambers, and number buffer replacements (relative to treatment additions).
	<ol>
		<li>Prior to the gassing step (step 2.1.3), add 9.1 mg (per L of buffer being prepared) of desipramine-HCl to buffer.</li>
		<li>Prepare a 1.0 &#956;M solution of propranolol-HCl and add 1 ml (per L of buffer being prepared) of this solution to the Krebs-Henseleit buffer solution. Make this buffer on the day of use and keep at myograph operating temperatures (a temperature that yields a 37.5 &#176;C temperature in the myograph). 
		NOTE: The desipramine is added to inhibit the biogenic amine reuptake mechanisms and allow clearing of experimental additions from the buffer bathing the blood vessels to occur more rapidly. The addition of propranolol prevents the non-specific binding of treatment compounds to &#946;-adrenergic receptors.</li>
	</ol>
	</li>
</ol>
3. Collection and Preparation of Vasculature
<ol>
	<li>As soon as possible, remove the gastrointestinal tract from the carcass. Once the animal no longer exhibits any involuntary reflex, remove the head and hide and then raise the carcass vertically. Removal of the gastrointestinal viscera (esophagus to anus) from the carcass should be completed by approved abattoir personnel (&#60;20 min from time of stunning). 
	NOTE: How soon, is generally limited by the location/facility where the intestinal tract is being collected from and standard operating procedures or federally mandated procedures followed by the facility personnel.
	<ol>
		<li>In most facilities, conduct manipulations of the gastrointestinal tract at a separate location from the processing of the carcass that is destined to enter the food supply. Use a nearby convenient location where the gastrointestinal tract can be taken and spread out for examination.</li>
	</ol>
	</li>
	<li>Once the gastrointestinal tract is spread out, identify the small intestine, the ileocecal fold connecting the small intestine to cecum, and the ileal flange extending proximal to the ileocecal fold (Figure 1A).
	<ol>
		<li>Using a scalpel or knife, very lightly make an incision in the mesenteric membrane in the center of the ileal flange. Using two index fingers, bluntly dissect away the fat and connective tissue exposing the mesenteric vasculature (Figure 1B).</li>
	</ol>
	</li>
	<li>From this flange or bulge in the intestinal mesentery, dissect out multiple branches (~2 cm in length) of the exposed mesenteric artery and vein bundles that support this portion of the ileum (Figure 1C).
	<ol>
		<li>If using forceps to grasp tissue, take care to not grasp directly or pull on the blood vessels at all while removing them as any sort of stretching can damage and negatively effect tissue performance14. Blood vessel removal is best done, by cutting across each end of the section to be removed and then cutting alongside or parallel to the now isolated section. For ease, remove some of the surrounding tissue along with vessels to provide some tissue to grasp with a pair of forceps.</li>
		<li>With a pair of forceps, submerge tissue sample in a tube containing ice cold Krebs-Henseleit buffer and store on ice until processing can occur in the laboratory.</li>
	</ol>
	</li>
	<li>Place tissue sample on a cutting surface or in a petri dish and partially submerge in ice cold Krebs-Henseleit.
	<ol>
		<li>Using #5 jewelers forceps, Noyes iris scissors, and magnifying lamp or dissecting scope (2.5 to 5.0X magnification is sufficient), carefully dissect away the surrounding fat and connective tissues and separate the artery and vein. Identifying the vessel opening at one end of the section and carefully grasp the fascia surrounding the vessel with the forceps.</li>
		<li>Make a cut with the scissors parallel to the vessel by sliding the tip of the scissor underneath the raised fascia. After the initial incision, the fat and connective tissues can be further detached from the vessel by cutting down either side with the scissors. Ensure that the vessels are as clean as possible while minimizing the amount of time the vessels spend on the bench.</li>
		<li>Return blood vessels to tubes of fresh Krebs-Henseleit buffer and store at 4 &#176;C (samples can remain stored for up to 24 hr after collection and still produce valid contractility data).</li>
		<li>Using a razor blade, slice vessel to be used in myograph into desired number of 2-mm sections (it is helpful to use a tissue slicer to obtain consistent cross-sections).</li>
		<li>Examine each section under a dissecting scope (12.5X magnification) to ensure that each section has no abnormalities, branches, valves, or superficial damage inadvertently done during dissection and cleaning. Replace the vessel section if there is any abnormality, branch, valve, or superficial damage.</li>
	</ol>
	</li>
	<li>Store acceptable sliced vessel sections (submerged in Krebs-Henseleit buffer) on ice or at 4 &#176;C until ready for mounting in myograph chambers (usually &#60;30 min).</li>
	<li>Gently mount the vessel on the myograph by inserting the supports through the lumen and increase the tension (using the micropositioner on the myograph) being careful to not stretch the vessels above a 2 to 3 g reading.</li>
	<li>Once all chambers are covered, vacuum the buffer from all chambers and refill with 5.0 ml of buffer and start a 15 min timer to begin the equilibration period and the experiment.</li>
</ol>
4. Experiment
<ol>
	<li>Equilibrate all vessel sections for 1.5 hr to achieve a stable resting tension of 1.0 g.
	<ol>
		<li>Replace the Krebs-Henseleit incubation buffer every 15 min.</li>
		<li>During the equilibration, constantly adjust the tension on the blood vessel sections up to 2 g and then allow to relax down to approximately 0.80 g. Try not to allow vessels to relax too much (they may slip off the mounts). Try to achieve a steady baseline tension, where the vessel holds 1 g tension without requiring adjustment.</li>
	</ol>
	</li>
	<li>During the equilibration, prepare an aqueous 1.32 M solution of KCl to be used as a reference.</li>
	<li>Following the completion of a satisfactory equilibration, add 500 &#956;l of the 1.32 M KCl solution to result in a 0.12 M solution in the 5.5 ml solution bathing the vessel.
	<ol>
		<li>Following the addition of the 1.32 M KCl, do not adjust the tension manually as the maximal response from this addition is used to evaluate tissue viability and can be used to normalize treatment data (e.g. concentration responses to an agonist).</li>
		<li>Replace the buffer in 15-min intervals until tension has returned to the baseline value of 1.0 g.</li>
	</ol>
	</li>
	<li>Once baseline is reached, change the buffer and simultaneously begin a 1 min timer. Do this for all chambers simultaneously to avoid a staggering of start times.
	<ol>
		<li>Vortex the standard to be added and prepare a comment for chamber 1 during the 1 min countdown.</li>
		<li>Add the standards in 25 &#956;l aliquots to each chamber as the experiment dictates (this keeps the treatment below 0.5% of the total volume).</li>
		<li>When the last standard has been added, start a 9 min timer.</li>
		<li>At the end of the 9 min standard incubation, remove the treatment-containing buffer from the chambers and add 5.0 ml of fresh buffer, and start the 2.5 min timer.</li>
		<li>Repeat step 4.4.4.</li>
		<li>At the end of the second 2.5 min rinse, vacuum the chambers and add fresh buffer, and start a 1 min timer to countdown the commencement of the second standard addition.</li>
	</ol>
	</li>
	<li>Continue this cycle for all of the day&#8217;s standard additions.</li>
	<li>During the final standard addition and subsequent 9 min incubation prepare the 1.32 M KCl reference compound for an end of run reference dose addition of 500 &#956;l.</li>
	<li>Following the 1 min interval after the final treatment addition, add the KCl and start a 9 min timer. 
	NOTE: The KCl addition is to confirm tissue viability at the conclusion of the experiment and is helpful if administering a treatment that results in negligible response.</li>
	<li>At the conclusion of the final 9 min reference compound incubation, vacuum or remove buffer from all of the chambers. Do not add fresh buffer. Conclude the experiment.</li>
	<li>Save Chart file, remove blood vessel sections and dispose of properly, and follow the clean up protocol (provided by manufacturer and can be lab specific) for myograph equipment.</li>
</ol>

<ol><li>Matheson, P. J., Wilson, M. A., Garrison, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+intestinal+blood+flow%5BTitle%5D)+AND+%22The+Journal+of+surgical+research%22%5BJournal%5D)">Regulation of intestinal blood flow.</a> The Journal of surgical research. 93, 182-196 (2000).</li>
<li>Dobson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blood+flow+and+absorption+from+the+rumen%5BTitle%5D)+AND+%22Quarterly+journal+of+experimental+physiology%22%5BJournal%5D)">Blood flow and absorption from the rumen.</a> Quarterly journal of experimental physiology. 69, 599-606 (1984).</li>
<li>Mulvany, M. J., Halpern, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanical+properties+of+vascular+smooth+muscle+cells+in+situ%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Mechanical properties of vascular smooth muscle cells in situ.</a> Nature. 260, 617-619 (1976).</li>
<li>Nielsen-Kudsk, F., Poulsen, B., Ryom, C., Nielsen-Kudsk, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+strain-gauge+myograph+for+isometric+measurements+of+tension+in+isolated+small+blood+vessels+and+other+muscle+preparations%5BTitle%5D)+AND+%22Journal+of+pharmacological%22%5BJournal%5D)">A strain-gauge myograph for isometric measurements of tension in isolated small blood vessels and other muscle preparations.</a> Journal of pharmacological. 16, 215-225 (1986).</li>
<li>Mulvany, M. J., Aalkjaer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure+and+function+of+small+arteries%5BTitle%5D)+AND+%22Physiological+reviews%22%5BJournal%5D)">Structure and function of small arteries.</a> Physiological reviews. 70, 921-961 (1990).</li>
<li>Klotz, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessment+of+vasoconstrictive+potential+of+D-lysergic+acid+using+an+isolated+bovine+lateral+saphenous+vein+bioassay%5BTitle%5D)+AND+%22Journal+of+animal+science%22%5BJournal%5D)">Assessment of vasoconstrictive potential of D-lysergic acid using an isolated bovine lateral saphenous vein bioassay.</a> Journal of animal science. 84, 3167-3175 (2006).</li>
<li>Klotz, J. L., Bush, L. P., Strickland, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+vascular+contractility+bioassay+using+bovine+right+ruminal+artery+and+vein%5BTitle%5D)+AND+%22Journal+of+animal+science%22%5BJournal%5D)">A vascular contractility bioassay using bovine right ruminal artery and vein.</a> Journal of animal science. 89, 1944-1951 (2011).</li>
<li>Westendorf, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+and+in+vivo+ruminal+and+physiological+responses+to+endophyte-infected+tall+fescue%5BTitle%5D)+AND+%22Journal+of+dairy+science%22%5BJournal%5D)">In vitro and in vivo ruminal and physiological responses to endophyte-infected tall fescue.</a> Journal of dairy science. 76, 555-563 (1993).</li>
<li>Strickland, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Board-invited+review%3A+St.+Anthony%26%2334%3Bs+Fire+in+livestock%3A+causes%2C+mechanisms%2C+and+potential+solutions%5BTitle%5D)+AND+%22Journal+of+animal+science%22%5BJournal%5D)">Board-invited review: St. Anthony&#34;s Fire in livestock: causes, mechanisms, and potential solutions.</a> Journal of animal science. 89, 1603-1626 (2011).</li>
<li>Eckert, H., Kiechel, J. R., Rosenthaler, J., Schmidt, R., Schreier, E. Ch. 11. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aEckert%2C+H+%22Ergot+Alkaloids+and+Related+Compounds%22">Ergot Alkaloids and Related Compounds</a>. B, B. erde, HO, S. child , Springer-Verlag. (1978).</li>
<li>Rhodes, M. T., Paterson, J. A., Kerley, M. S., Garner, H. E., Laughlin, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reduced+blood+flow+to+peripheral+and+core+body+tissues+in+sheep+and+cattle+induced+by+endophyte-infected+tall+fescue%5BTitle%5D)+AND+%22Journal+of+animal+science%22%5BJournal%5D)">Reduced blood flow to peripheral and core body tissues in sheep and cattle induced by endophyte-infected tall fescue.</a> Journal of animal science. 69, 2033-2043 (1991).</li>
<li>Foote, A. P., Harmon, D. L., Strickland, J. R., Bush, L. P., Klotz, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+ergot+alkaloids+on+contractility+of+bovine+right+ruminal+artery+and+vein%5BTitle%5D)+AND+%22Journal+of+animal+science%22%5BJournal%5D)">Effect of ergot alkaloids on contractility of bovine right ruminal artery and vein.</a> Journal of animal science. 89, 2944-2949 (2011).</li>
<li>Foote, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ergot+alkaloids+from+endophyte-infected+tall+fescue+decrease+reticuloruminal+epithelial+blood+flow+and+volatile+fatty+acid+absorption+from+the+washed+reticulorumen%5BTitle%5D)+AND+%22Journal+of+animal+science%22%5BJournal%5D)">Ergot alkaloids from endophyte-infected tall fescue decrease reticuloruminal epithelial blood flow and volatile fatty acid absorption from the washed reticulorumen.</a> Journal of animal science. 91, 5366-5378 (2013).</li>
<li>Hocking, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detrimental+effects+of+mechanical+stretch+on+smooth+muscle+function+in+saphenous+veins%5BTitle%5D)+AND+%22Journal+of+vascular+surgery%22%5BJournal%5D)">Detrimental effects of mechanical stretch on smooth muscle function in saphenous veins.</a> Journal of vascular surgery. 53, 454-460 (2011).</li>
<li>Smith, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bovine+intestinal+surgery%5BTitle%5D)+AND+%22Modern+veterinary+practice%22%5BJournal%5D)">Bovine intestinal surgery.</a> Modern veterinary practice. 65, 705-710 (1984).</li>
<li>Budras, K. D., Habel, R. E. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBudras%2C+KD+%22Bovine+Anatomy+An+Illustrated+Text%22">Bovine Anatomy An Illustrated Text</a>. , first, Schlütersche GmbH and Co. KG, Verlag and Druckerei. (2003).</li>
<li>Klotz, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antagonism+of+lateral+saphenous+vein+serotonin+receptors+from+steers+grazing+endophyte-free%2C+wild-type%2C+or+novel+endophyte-infected+tall+fescue%5BTitle%5D)+AND+%22Journal+of+animal+science%22%5BJournal%5D)">Antagonism of lateral saphenous vein serotonin receptors from steers grazing endophyte-free, wild-type, or novel endophyte-infected tall fescue.</a> Journal of animal science. 91, 4492-4500 (2013).</li>
<li>Egert, A. M., Kim, D. H., Schrick, F. N., Harmon, D. L., Klotz, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dietary+exposure+to+ergot+alkaloids+decreases+contractility+of+bovine+mesenteric+vasculature%5BTitle%5D)+AND+%22Journal+of+animal+science%22%5BJournal%5D)">Dietary exposure to ergot alkaloids decreases contractility of bovine mesenteric vasculature.</a> Journal of animal science. 92, 1768-1779 (2014).</li>
</ol>

Mention of trade name, proprietary product, or specified equipment does not constitute a guarantee or warranty by the USDA and does not imply approval to the exclusion of other products that may be available.

The initial challenge in the development of this bioassay was the establishment of a repeatable collection site for mesenteric vasculature. Sample site consistency is critical, as some of the functions of the small intestine change during the progression from the jejunum through the ileum and consequently the mesentery vary in a similar pattern. The ileal branches of mesenteric artery and vein were the most easily identified through anatomic landmarks. By locating the cecum and following the ileocecal fold to its terminus on the left side of the mesentery, this is near the termination of the cranial mesenteric artery15. The area of longer mesentery (termed the flange) and the extension of the ileocecal fold (unique to the bovine animal)16 made sampling across numerous animals very repeatable. One of the larger pitfalls that can be made is at this phase of the bioassay. Mechanical stretching has a large negative impact on subsequent vessel performance14 and the researcher will notice that vessels that are excessively manipulated or stretched during removal and subsequent cleaning will not respond to the reference compound being used. This unfortunately is the only way to truly evaluate the quality of the vascular preparation at a functional level and vessels that do not respond to KCl should not be considered further for generation of reliable data.
Two other method validation steps (not shown) that led to the final method presented herein were the use of other chemicals as reference compounds and testing the viability of blood vessels following 24 hr storage. Norepinephrine and serotonin were evaluated as reference compounds, but responses by the mesenteric artery and vein were too variable across vessel type and across animals for them to be successfully used. Conversely, the addition of 120 mM KCl resulted in a very reproducible response across both artery and vein preparations. Also, artery and vein responses were evaluated the day of collection and reevaluated 24 hr post collection and there were no differences in agonist response by either the artery or the vein. This was an important aspect of the bioassay, as there are often a large number of treatments (or various treatment combinations) that can be applied, but researchers are limited by space on the myograph and time. By extending the period of viability out 24 hr from collection, the time for additional experimentation is increased greatly.
It is important for the researcher to understand the flexibility of the treatment structure applied to this bioassay. A researcher can add any compound to the incubation buffer that he or she is interested in chronically exposing the blood vessels to in order to control something of biologic relevance or as a treatment (in the case of Klotz et al.17, this was done with lateral saphenous veins continuously exposed to ketanserin, which antagonized a serotonin receptor of interest). The mesenteric blood vessels could be pretreated with a compound of interest prior to conducting a contractile response experiment. The bioassay, as presented, was designed to conduct a cumulative concentration response experiment looking at ergot alkaloids as agonists and whether prior dietary exposure to ergot alkaloids affected the vascular response18. The representative results demonstrate that this method can produce a measurable vascular response and only four concentrations were used to achieve this. In the case of Egert et al.18, there were up to 10 different concentrations used to construct a response curve. This method is a way to evaluate a large number of different compounds or receptors on blood vessels from a single animal in a small window of time.

Alterations in blood flow to a tissue bed can have a large impact on organ function. A primary function of the small intestine is nutrient absorption. Arterial blood flow to the absorptive surface of the gut is required for nutrient absorption and blood flow increases to aid in nutrient absorption as digesta moves along the surface1. A decrease in blood flow can cause a reduction in nutrient absorption due to a decrease in the transepithelial gradient2. In addition to nutrients, the small intestine can also be exposed to secondary metabolites, drugs, or toxins that exert an effect on localized blood flow in the mesentery. In the case of the ruminant animal, compounds can be liberated from a feedstuff (e.g., nutrients such as amino acids, or toxins such as ergot alkaloids) through fermentative processes of the foregut. If these compounds survive the microbial metabolism of ruminal fermentation, they are now available for absorption or interaction as they travel through the gastrointestinal tract of the animal.
There are a number of different methods available to measure blood flow in vivo (e.g., Doppler ultrasound, indwelling blood flowmeters, radiolabeled microspheres, and indicator-dilution techniques) that permit evaluation of various experimental scenarios or treatments. However, to obtain information regarding the mechanical or pharmacological properties of vascular smooth muscle, methods remained limited to large vessels until Mulvany and Halpern3 published an article describing a technique using wire mounted vascular ring preparations in a myograph. Since the development of this technique, modifications continue to be made to the associated myograph systems that permit a variety of different applications for evaluation of tubular structures. The system has also been adapted to utilize fixed rods for mounting larger vessels4 where perfusion techniques are not desired.
Because of dissimilarities in vessels from different anatomical origins and distinctions in the same vessels from different species of animal, data from vessel and animal type cannot easily be extrapolated across different vessels or the same vessel in different animal types5. Consequently, separate bioassays must be developed and validated anytime these aspects are changed. Recently several bioassays have been developed with these technologies for use in cattle lateral saphenous vein and right ruminal artery and vein6,7.
This bioassay was developed to specifically investigate the effects that ergot alkaloids have on vasculature supporting the small intestine. It was reported that 50-60% of fed alkaloids appear in abomasal contents, but only 5% are recovered in feces8. Strickland et al.9 stated in a review of ergot alkaloids, that available data suggest that the small intestine may be the most important site for ergopeptine absorption. Eckert et al.10 reviewed biopharmaceutical aspects of ergot alkaloids and stated that once they cross the epithelial barrier, ergot alkaloids are transported either by lymphatic system to the subclavian vein or via mesenteric vein and into portal blood. Rhodes et al.11 reported a decrease in blood flow to duodenum and colon in steers consuming a high endophyte-infected (high ergot alkaloid) diet. Using the right ruminal artery and vein bioassay, Foote et al.12 demonstrated that ergot alkaloids are vasoactive in ruminal vasculature. Foote et al.13 subsequently demonstrated in vivo that ruminal exposure to ergot alkaloids results in a decreased rumen epithelial blood flow. This decrease in blood flow to the absorptive surface of the rumen concomitantly caused a reduction in nutrient (volatile fatty acid) flux. Given the quantity of ergot alkaloids passing on to the small intestine from the foregut; it was hypothesized that a similar effect on small intestinal vasculature and nutrient absorption would occur. This necessitated the development of the bovine proximal ileal mesenteric artery and vein bioassay.

<table><tbody><tr><td>Multi Myograph</td><td>Danish Myo Technologies</td><td>610M</td><td>A myograph is critical to this bioassay, but there are other platforms available for use that will suffice.&amp;nbsp;</td></tr><tr><td>Powerlab 8/sp</td><td>ADIntruments</td><td>ML785</td><td></td></tr><tr><td>LabChart 7</td><td>ADInstruments</td><td>Version 7</td><td></td></tr><tr><td>Force Calibration Kit</td><td>Danish Myo Technologies</td><td>100055</td><td>Specific to DMT myographs</td></tr><tr><td>Bottle-top Filter</td><td>Nalgene</td><td>595-4520</td><td>0.22 &amp;#956;m pore size; 45 mm neck size</td></tr><tr><td>#5 Jewler&amp;rsquo;s Forceps</td><td>Miltex</td><td>555008FT</td><td>Any brand of forceps can be used</td></tr><tr><td>Noyes Iris Scissors</td><td>Miltex</td><td>18-1510</td><td>Any brand of scissors can be used</td></tr><tr><td>Dissecting Scope</td><td>Zeiss</td><td>Stemi 2000-C</td><td>Any brand of dissecting light microscope will suffice</td></tr><tr><td>Adjustable Tissue Matrice</td><td>Braintree Scientific</td><td>TM C12</td><td>This is not critical to the assay, but greatly reduces section to section variation in length and speeds up the slicing process greatly</td></tr><tr><td>Krebs-Hensleit Buffer</td><td>Sigma-Aldrich</td><td>K3753-10x1L</td><td>It is not necessary to buy Krebs, this can be made in house</td></tr><tr><td>Calcium chloride dehydrate</td><td>Sigma-Aldrich</td><td>C7902-500G</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S5761-500G</td><td></td></tr><tr><td>Desipramine-HCl</td><td>Sigma-Aldrich</td><td>D3900-5G</td><td></td></tr><tr><td>Propranolol-HCl</td><td>Sigma-Aldrich</td><td>P0884-1G</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9333-500G</td><td></td></tr><tr><td>95% O&lt;sub&gt;2&lt;/sub&gt;/5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Scott Gross</td><td>UN3156</td><td></td></tr></tbody></table>

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 blood vessels used to generate the included results were collected from 6 Holstein steers (425 &#177; 8 kg) within a 3 week interval. An example of a typical mesenteric vein contractile response to KCl and treatment additions increasing in concentration is presented in Figure 2. The magnitude of response will vary some with the size of the vessel (correlated with the size of the donor animal), but can also be influenced (negatively) by improper handling (stretching) of the vessels during collection and cleaning. Therefore, it is important to observe a significant contractile response in the vessels exposure to KCl. If a negligible response is observed at this point, it is not advisable to proceed any further into the procedure with that vessel. As seen in the 9 min incubation periods in Figure 2, there are incremental increases in the response that correspond with increasing concentrations. Although desirable for this particular experimental design, it may not always occur or be sought after with other treatments. The KCl addition at the conclusion of the experiment, although not used in the processing of data collected, is very important as it confirms the viability of the tissue at the conclusion of the experimental period (becomes more important if the vessel is not responding to any of the treatment additions).
The data presented in Figure 2 are recorded in grams of tension. It is important to normalize these data to minimize any animal-to-animal and vessel-to-vessel variation. Thus, in addition to being used as an indicator of viability, the maximum KCl response is used to normalize all of the treatment additions. This generates contractile response data as a percentage of the KCl maximum. This is how mesenteric artery and vein response curves to increasing concentration of 5-hydroxytryptamine (serotonin; Figure 3) and norepinephrine (Figure 4) are presented. The data in these figures have been fit sigmoidal concentration response curve using a non-linear regression.
The mesenteric vein response to both 5-hydroxytryptamine (Figure 3) and norepinephrine (Figure 4) started out as negative values. This is a result of correcting the maximum tension recorded during each 9 min incubation period by the baseline tension recorded prior to the initial KCl addition. This baseline correction ensures that any slight differences in tension are not included in the analyses and allow the data presented to reflect only a change in tension. The negative value results from the blood vessel relaxing and dropping below pre-KCl baseline levels before the subsequent treatment addition. This is especially common in venous samples where the initial concentrations of the treatment or agonist are too low to cause a contractile response. This was not the case for the mesenteric artery response to either biogenic amine, which held tension above baseline until the vessel began to respond to the treatment additions. This difference exemplifies the differences in vessels on either side of the capillary bed and why they were both considered as part of this bioassay.
This bioassay can be used to evaluate vasoactivity of compounds on blood vessels supplying nutrients to the small intestine as well as blood vessels carrying absorbed nutrients away from the small intestine. Data in Figures 3 and 4 are simple examples of how this may be accomplished. In the case of 5-hydroxytryptamine (Figure 3), there was not a difference in the arterial or venous contractile response. Conversely, the contractile response by the mesenteric vein to increasing concentrations of norepinephrine was greater than that of the mesenteric artery.
<img alt="Figure 1" fo:content-width="3in" src="/files/ftp_upload/52020/52020fig1highres.jpg" width="300" /> 
Figure 1: An example of bovine intestinal tract used as source of mesenteric blood vessels. The red asterisk indicates the visible portion of the ileocecal fold in all three panes as a point of reference. (A) The whole tract with the red oval indicating the ileal flange where sampling of vasculature occurred. (B) Exposure of mesenteric vascular bed. (C) Iris scissors are pointing towards an exposed branch of mesenteric vein (visible) and artery (not visible) just prior to collection.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/52020/52020fig2highres.jpg" width="500" /> 
Figure 2: Example of a concentration response of a mesenteric vein to increasing concentrations of norepinephrine (1&#215;10-7 to 1&#215;10-4 M). The large events at the beginning and end of the trace are responses to the 0.12 M KCl additions. The red rectangle signifies the data collection region corresponding with the 9 min incubation period for each treatment addition. The 3 slender spikes that follow this are artifacts generated by the buffer replacements and are not included in the analysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52020/52020fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/52020/52020fig3highres.jpg" width="500" /> 
Figure 3: Mean contractile responses of mesenteric artery (MA) and vein (MV; n = 6 steers) to increasing concentrations of 5-hydroxytryptamine (serotonin). Lines shown were generated using non-linear regression analysis to fit the data to a sigmoidal concentration response curve which utilized the following equation: y = bottom + [(top &#8211; bottom) / (1 + 10(logEC50 &#8211; x))], where top and bottom are the percentage of 120 mM KCl maximum contractile response at the plateaus, and the EC50 is the molar concentration of alkaloid producing 50% of the KCl maximum response.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/52020/52020fig4highres.jpg" width="500" /> 
Figure 4: Mean contractile responses of mesenteric artery (MA) and vein (MV; n = 6 steers) to increasing concentrations of norepinephrine. Lines shown were generated using non-linear regression analysis to fit the data to a sigmoidal concentration response curve which utilized the following equation: y = bottom + [(top &#8211; bottom) / (1 + 10(logEC50 &#8211; x))], where top and bottom are the percentage of 120 mM KCl maximum contractile response at the plateaus, and the EC50 is the molar concentration of alkaloid producing 50% of the KCl maximum response.

Watch this Scientific Journal Video about Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine at JoVE.com

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

isolating-using-sections-bovine-mesenteric-artery-vein-as-bioassay-to

Nanyang Technological University

Research

JoVE Journal

Medicine

9.7K Views.  USDA-Agricultural Research Service.  De dunne darm wordt vaak blootgesteld aan giftige stoffen die de bloedstroom kunnen beïnvloeden en een negatieve invloed op de opname van voedingsstoffen. Met een multimyograph en mesenteriale slagader en ader isolaten, verbindingen of toxinen plaats kunnen worden gescreend vasoactiviteit. 

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

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 ...

Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic response is a key regulator of local blood flow. In isobaric conditions small resistance arteries develop sustained constriction known as myogenic tone (MT), which is a major determinant of systemic vascular resistance (SVR). Hence, ex vivo&#160;pressurized preparations of small resistance arteries are major tools to study microvascular function in near-physiological states. To achieve this, a freshly isolated intact segment of a small resistance artery (diameter ~260 &#956;m) is mounted onto two small glass cannulas and pressurized. These arterial preparations retain most in vivo characteristics and permit assessment of vascular tone in real-time. Here we provide a detailed protocol for assessing vasoactivity in pressurized small resistance mesenteric arteries from rats; these arteries develop sustained vasoconstriction - approximately 25% of maximal diameter - when pressurized at 70 mmHg. These arterial preparations may be used to study the effect of investigational compounds on relationship between intra-arterial pressure and vasoactivity and determine changes in microvascular function in animal models of various diseases.

Pressure myography is used to assess vasoactivity of small arteries that develop sustained constriction when pressurized. This manuscript provides a detailed protocol to assess in isolated segments of small mesenteric arteries from rats, vasoactivity and the effect of intraluminal pressure on vascular diameter.

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic ...

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using &#34;intestinal sacs&#34; are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (Papp) of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

Biology

Targeting the Rat's Small Bowel: Long-Term Infusion into the Superior Mesenteric Artery

The superior mesenteric artery can be cannulated in humans through minimally invasive radiological catheterization of the femoral or axillary artery. SMA cannulation is more difficult in rats due to small anatomical dimensions. The aim of the study is to describe a surgical technique for cannulation of the SMA in rats to perform long-term infusion of drugs into the SMA vascular bed in unrestricted animals, which will result in a high rate of catheter patency after the post surgical recovery for 24 hours. 
To avoid the risk of SMA thrombosis or bleeding from direct access, a proximal branch of the SMA is isolated, ligated distally and cannulated with a 0.25 mm polyurethane capillary tube whose tip is advanced close to the origin of the SMA from the aorta. The cannula is then tunnelled subcutaneously to the back of the animal's neck and through the skin via an artificial valve. The external portion of the cannula is inserted in a semi-rigid support system and connected to the continuous infusion pump outside the cage where the rat is free to move. 
Correct positioning of the cannula was demonstrated by post-surgical angiography and autopsy findings. Catheter patency after 24 hours of saline infusion into the SMA region was assured in most rats by the total discharge of the pump and recognition of a functional cannula for blood sampling or saline infusion.

Access for long-term infusion in the superior mesenteric artery (SMA) of rats is a surgical procedure that consists of cannulation of a proximal branch of the SMA. The cannula exits from the abdominal wound and is tunneled through the subcutaneous space back to the interscapular fold.

The superior mesenteric artery can be cannulated in humans through minimally invasive radiological catheterization of the femoral or axillary artery. SMA ...

Bioengineering

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

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 ...

Invasive Hemodynamic Characterization of the Portal-hypertensive Syndrome in Cirrhotic Rats

This is a detailed protocol describing invasive hemodynamic measurements in cirrhotic rats for the characterization of portal hypertensive syndrome. Portal hypertension (PHT) due to cirrhosis is responsible for the most severe complications in patients with liver disease. The full picture of the portal hypertensive syndrome is characterized by increased portal pressure (PP) due to the increased intrahepatic vascular resistance (IHVR), hyperdynamic circulation, and increased splanchnic blood flow. Progressive splanchnic arterial vasodilation and increased cardiac output with elevated heart rate (HR) but low arterial pressure characterizes the portal hypertensive syndrome.
Novel therapies are currently being developed that aim to decrease PP by either targeting IHVR or increased splanchnic blood flow &#8212; but side effects on systemic hemodynamics may occur. Thus, a detailed characterization of portal venous, splanchnic, and systemic hemodynamic parameters, including measurement of PP, portal venous blood flow (PVBF), mesenteric arterial blood flow, mean arterial pressure (MAP), and HR is needed for preclinical evaluation of the efficacy of novel treatments for PHT. Our video article provides the reader with a structured protocol for performing invasive hemodynamic measurements in cirrhotic rats. In particular, we describe the catheterization of the femoral artery and the portal vein via an ileocolic vein and the measurement of portal venous and splanchnic blood flow via perivascular Doppler-ultrasound flow probes. Representative results of different rat models of PHT are shown.

Here we describe a detailed protocol for invasive measurements of hemodynamic parameters including portal pressure, splanchnic blood flow, and systemic hemodynamics in order to characterize the portal hypertensive syndrome in rats.

This is a detailed protocol describing invasive hemodynamic measurements in cirrhotic rats for the characterization of portal hypertensive syndrome. ...

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  ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

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

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