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
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.
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<ol>
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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 border="0" cellpadding="0" cellspacing="0" width="611">
	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:105px;">Name of the Material/Equipment</td>
			<td style="width:127px;">Company</td>
			<td style="width:120px;">Catalog Number</td>
			<td style="width:259px;">Comment/Description (optional)</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:105px;">Multi Myograph</td>
			<td style="width:127px;">Danish Myo Technologies</td>
			<td style="width:120px;">610M</td>
			<td style="width:259px;">A myograph is critical to this bioassay, but there are other platforms available for use that will suffice.&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:105px;">Powerlab 8/sp</td>
			<td style="width:127px;">ADIntruments</td>
			<td style="width:120px;">ML785</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:105px;">LabChart 7</td>
			<td style="width:127px;">ADInstruments</td>
			<td style="width:120px;">Version 7</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:105px;">Force Calibration Kit</td>
			<td style="width:127px;">Danish Myo Technologies</td>
			<td style="width:120px;">100055</td>
			<td style="width:259px;">Specific to DMT myographs</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:105px;">Bottle-top Filter</td>
			<td style="width:127px;">Nalgene</td>
			<td style="width:120px;">595-4520</td>
			<td style="width:259px;">0.22 um pore size; 45 mm neck size</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:105px;">#5 Jewler&#8217;s Forceps</td>
			<td style="width:127px;">Miltex</td>
			<td style="width:120px;">555008FT</td>
			<td style="width:259px;">Any brand of forceps can be used</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:105px;">Noyes Iris Scissors</td>
			<td style="width:127px;">Miltex</td>
			<td style="width:120px;">18-1510</td>
			<td style="width:259px;">Any brand of scissors can be used</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:105px;">Dissecting Scope</td>
			<td style="width:127px;">Zeiss</td>
			<td style="width:120px;">Stemi 2000-C</td>
			<td style="width:259px;">Any brand of dissecting light microscope will suffice</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:105px;">Adjustable Tissue Matrice</td>
			<td style="width:127px;">Braintree Scientific</td>
			<td style="width:120px;">TM C12</td>
			<td style="width:259px;">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 height="43">
			<td height="43" style="height:43px;width:105px;">Krebs-Hensleit Buffer</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:120px;">K3753-10x1L</td>
			<td style="width:259px;">It is not necessary to buy Krebs, this can be made in house</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:105px;">Calcium chloride dehydrate</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:120px;">C7902-500G</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:105px;">Sodium bicarbonate</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:120px;">S5761-500G</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:105px;">Desipramine-HCl</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:120px;">D3900-5G</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:105px;">Propranolol-HCl</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:120px;">P0884-1G</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:105px;">KCl</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:120px;">P9333-500G</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:105px;">95% O2/5% CO2</td>
			<td style="width:127px;">Scott Gross</td>
			<td style="width:120px;">UN3156</td>
			<td style="width:259px;"></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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