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 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.
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.
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
2. Preparation of Buffers
3. Collection and Preparation of Vasculature
4. Experiment
The blood vessels used to generate the included results were collected from 6 Holstein steers (425 ± 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.
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.
Figure 2: Example of a concentration response of a mesenteric vein to increasing concentrations of norepinephrine (1×10-7 to 1×10-4 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. Please click here to view a larger version of this figure.
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 – bottom) / (1 + 10(logEC50 – 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.
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 – bottom) / (1 + 10(logEC50 – 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.
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.
The authors have nothing to disclose.
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.
Name of the Material/Equipment | Company | Catalog Number | Comment/Description (optional) |
Multi Myograph | Danish Myo Technologies | 610M | A myograph is critical to this bioassay, but there are other platforms available for use that will suffice. |
Powerlab 8/sp | ADIntruments | ML785 | |
LabChart 7 | ADInstruments | Version 7 | |
Force Calibration Kit | Danish Myo Technologies | 100055 | Specific to DMT myographs |
Bottle-top Filter | Nalgene | 595-4520 | 0.22 um pore size; 45 mm neck size |
#5 Jewler’s Forceps | Miltex | 555008FT | Any brand of forceps can be used |
Noyes Iris Scissors | Miltex | 18-1510 | Any brand of scissors can be used |
Dissecting Scope | Zeiss | Stemi 2000-C | Any brand of dissecting light microscope will suffice |
Adjustable Tissue Matrice | Braintree Scientific | TM C12 | This is not critical to the assay, but greatly reduces section to section variation in length and speeds up the slicing process greatly |
Krebs-Hensleit Buffer | Sigma-Aldrich | K3753-10x1L | It is not necessary to buy Krebs, this can be made in house |
Calcium chloride dehydrate | Sigma-Aldrich | C7902-500G | |
Sodium bicarbonate | Sigma-Aldrich | S5761-500G | |
Desipramine-HCl | Sigma-Aldrich | D3900-5G | |
Propranolol-HCl | Sigma-Aldrich | P0884-1G | |
KCl | Sigma-Aldrich | P9333-500G | |
95% O2/5% CO2 | Scott Gross | UN3156 |