Name: isolating and using sections of bovine mesenteric artery and vein as a bioassay to test for vasoactivity in the small intestine
Uploaded: 2014-10-07T15:00:00.000+00:00
Duration: 7 min 32 s
Description: 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

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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+intestinal+blood+flow.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+flow+and+absorption+from+the+rumen.">Blood flow and absorption from the rumen.</a> Quarterly journal of experimental physiology. 69, 599-606 (1984).</li><li>Mulvany, M. 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J., Aalkjaer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+small+arteries.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+vasoconstrictive+potential+of+D-lysergic+acid+using+an+isolated+bovine+lateral+saphenous+vein+bioassay.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+vascular+contractility+bioassay+using+bovine+right+ruminal+artery+and+vein.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+ruminal+and+physiological+responses+to+endophyte-infected+tall+fescue.">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. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+blood+flow+to+peripheral+and+core+body+tissues+in+sheep+and+cattle+induced+by+endophyte-infected+tall+fescue.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ergot+alkaloids+on+contractility+of+bovine+right+ruminal+artery+and+vein.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ergot+alkaloids+from+endophyte-infected+tall+fescue+decrease+reticuloruminal+epithelial+blood+flow+and+volatile+fatty+acid+absorption+from+the+washed+reticulorumen.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detrimental+effects+of+mechanical+stretch+on+smooth+muscle+function+in+saphenous+veins.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bovine+intestinal+surgery.">Bovine intestinal surgery.</a> Modern veterinary practice. 65, 705-710 (1984).</li><li>Budras, K. D., Habel, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bovine Anatomy An Illustrated Text. , (2003).</li><li>Klotz, J. 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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 terminu...

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 a...

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 ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

9.6K Views.  USDA-Agricultural Research Service. 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. 

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 ...

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

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

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  ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

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 ...

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.

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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Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography

An in vivo Assay to Test Blood Vessel Permeability

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

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

Rat Mesentery Angiogenesis Assay

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

Assessing Murine Resistance Artery Function Using Pressure Myography

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

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