Name: mechanisms underlying gut hormone secretion using the isolated perfused rat small intestine
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This work was supported by an unrestricted grant to Prof. Jens Juul Holst from the Novo Nordisk Center for Basic Metabolic Research (Novo Nordisk Foundation, Denmark), a separate grant from the Novo Nordisk Foundation for doing rodent perfusion studies (grant no. NNF15OC0016574), a grant to Prof. Holst from the European Research Council (Grant no.695069) and theEuropean Union&#39;s Seventh Framework Programme for Research, TechnologicalDevelopment, and Demonstration Activities (Grant No. 266408) as well as a postdoc grant to Rune E. Kuhre from the Lundbeck Foundation (R264-2017-3492). We thank Jenna E. Hunt and Carolyn F. Deacon for careful proofreading.

All studies were conducted with permission from the Danish Animal Experiments Inspectorate (2013-15-2934-00833) and the local ethical committee, in accordance with the guidelines of Danish legislation governing animal experimentation (1987) and the National Institutes of Health (publication number 85-23).
1. Experimental Animals
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	<li>Obtain male Wistar rats (250 g) and house 2 per cage, with ad libitum access to standard chow and water, and maintain in&#173;&#173;&#173; a 12:12 h light-...

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A., 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=Mechanistic+insights+into+the+detection+of+free+fatty+and+bile+acids+by+ileal+glucagon-like+peptide-1+secreting+cells.">Mechanistic insights into the detection of free fatty and bile acids by ileal glucagon-like peptide-1 secreting cells.</a> Molecular Metabolism. 7, 90-101 (2018).</li><li>Sun, E. W., 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=Mechanisms+Controlling+Glucose-Induced+Glp-1+Secretion+in+Human+Small+Intestine.">Mechanisms Controlling Glucose-Induced Glp-1 Secretion in Human Small Intestine.</a> Diabetes. , (2017).</li><li>Kuhre, R. E., 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=Peptide+production+and+secretion+in+GLUTag,+NCI-H716+and+STC-1+cells:+a+comparison+to+native+L-cells.">Peptide production and secretion in GLUTag, NCI-H716 and STC-1 cells: a comparison to native L-cells.</a> Journal of Molecular Endocrinology. 56, 11 (2016).</li><li>Reimann, F., 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=Glucose+sensing+in+L+cells:+a+primary+cell+study.">Glucose sensing in L cells: a primary cell study.</a> Cell Metabolism. 8, 532-539 (2008).</li><li>Reimann, F., 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=Characterization+and+functional+role+of+voltage+gated+cation+conductances+in+the+glucagon-like+peptide-1+secreting+GLUTag+cell+line.">Characterization and functional role of voltage gated cation conductances in the glucagon-like peptide-1 secreting GLUTag cell line.</a> The Journal of Physiology. 563, 161-175 (2005).</li><li>Kuhre, R. 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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=Comparison+of+Human+and+Murine+Enteroendocrine+Cells+by+Transcriptomic+and+Peptidomic+Profiling.">Comparison of Human and Murine Enteroendocrine Cells by Transcriptomic and Peptidomic Profiling.</a> Diabetes. , (2019).</li><li>Reimann, F., Gribble, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose-Sensing+in+Glucagon-Like+Peptide-1-Secreting+Cells.">Glucose-Sensing in Glucagon-Like Peptide-1-Secreting Cells.</a> Diabetes. 51, 2757-2763 (2002).</li><li>Drucker, D. J., Jin, T., Asa, S. L., Young, T. A., Brubaker, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+proglucagon+gene+transcription+by+protein+kinase-A+in+a+novel+mouse+enteroendocrine+cell+line.">Activation of proglucagon gene transcription by protein kinase-A in a novel mouse enteroendocrine cell line.</a> Molecular Endocrinology. 8, 1646-1655 (1994).</li><li>Svendsen, B., 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=GLP1-+and+GIP-producing+cells+rarely+overlap+and+differ+by+bombesin+receptor-2+expression+and+responsiveness.">GLP1- and GIP-producing cells rarely overlap and differ by bombesin receptor-2 expression and responsiveness.</a> The Journal of Endocrinology. 228, 39-48 (2016).</li><li>Bak, M. 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The isolated perfused rat small intestine is a powerful research tool that allows the dynamics and molecular mechanisms underlying gut hormone secretion to be studied in detail. The most critical step for the successful production of data with this model is the surgical operation. Handling of the gut will inevitably cause some damage to the intestine and should therefore be kept to an absolute minimum. Even more importantly, the speed of operation is key, particularly with regard to the time for the catheter placement in...

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate nutrient absorption and nutrient disposition, intestinal growth and modulate appetite1. Gut hormones are, therefore, involved in many fundamental physiological processes, and understanding these patterns of secretion and the molecular details that control secretion of the respective hormones is thus important for our basic physiological understanding and for addressing translational aspects of gut hormone actions; but how can one study the molecular sensing mechanisms underlying gut hormone secretion? In general, hormone secretion can be studied in intact organisms (humans or experimental animals), from isolated gut preparations or from gut hormone-secreting primary cell cultures or immortalized cell cultures2,3,4,5,6. Our preferred model is the isolated perfused rat small intestine, which is a physiologically relevant model that allows the secretion of gut hormones to be studied in detail with optimal time resolution (secretion rates can be determined with any time base down to the second), and the results are likely transferrable to an in vivo situation7. Here, we provide a detailed protocol on how to perform this procedure, but first we will discuss other methods for studying gut hormone secretion, including the benefits and limitations of these models compared to the isolated perfused rat small intestine.
If one wishes to establish whether a specific compound regulates the secretion of certain gut hormones, studies in humans are the ultimate goal. Thus, if a compound shows great effects on the secretion of one or several gut hormones in rodents (in vivo or perfusions) or from hormone secreting cells (cell lines or primary cells), this effect is only relevant to medicine and human physiology if it can be confirmed in humans. However, there are clear limits for the type of studies that can be performed in humans, and in vivo studies on experimental animals are, therefore, often the second-best option for such studies. Mice and rats are the most frequently used experimental animals presumably due to their convenient size, low cost and the option to genetically alter the genes suspected of being involved in the specific study questions (e.g., knock out of a certain transporter or receptor). In general, in vivo models benefit from being physiologically intact, but also have several limitations. Most importantly, the small size of rodents, particularly mice, is a limiting factor, as most assays for gut hormone quantification require at least 20 &#181;L of plasma (and often much more), meaning that at least 100 &#181;L of blood has to be withdrawn to make a duplicate quantification. Therefore, it is only possible to obtain very few samples corresponding to baseline samples and one or two post-stimulation samples (the total blood volume in a mouse of 20 g is ~1.4 mL). Consequently, potential secretory responses (e.g., rapidly or late-occurring responses) may therefore be missed.
In the perfusion model, this issue is overcome, as large sample volumes are obtained (flow rate: 7.5 mL/min) and the collection intervals can be adjusted as needed to ensure that the rapid and short-lasting responses are not missed (we collect samples every min)7. Another issue with in vivo studies in rodents is that most gut hormones are even more rapidly eliminated or metabolized than in humans8,9,10, which may complicate the subsequent biochemical analysis. For instance, we showed that GLP-1 is metabolized in mice at an even faster rate than in humans (where T1/2 is 1-2 min11) and, more importantly, that the cleavage of GLP-1 in mice involves, in addition to N-terminal cleavage by dipeptidyl-peptidase-4 (DPP4) (which is the major GLP-1 degrading enzyme in humans), further cleavage by the enzyme neutral endopeptidase 24.1112. Consequently, current commercial assays for the quantification of GLP-1, which are either based on the intact isoform of GLP-1 (7-36amide) or the DPP-4 cleaved isoform (9-39amide), vastly underestimate GLP-1 secretion in mice and result in misleading results12. In the isolated perfused rat small intestine, most of the metabolism of secreted hormones is eliminated or markedly reduced, since plasma-mediated degradation is avoided, and liver/kidney/lung extraction/degradation is prevented (because perfusate is collected as it leaves the gut).
Of course, important insight can be generated by the use of genetically modified animals, e.g., sodium-glucose transporter-1 knockout mice13, but a detailed assessment of the molecular sensors involved in secretion often requires consideration of multiple molecular sites, ranging from molecular transporters to ion channels and from different G-protein-coupled receptors to intracellular proteins. For instance, we targeted the activity of nine different molecular sites when unraveling the molecular sensors responsible for glucose-stimulated GLP-1 secretion7. A similar investigation would not be possible in vivo as some of the compounds used have unspecific or harmful/lethal effects. For instance, when using the perfused gut, it was possible to assess the role of intra-cellular glucose metabolism for the secretion of GLP-1 and neurotensin by blocking ATP formation with 2-4-dinitrophenol7,14 as well as the role of voltage-gated calcium channels for bile acid stimulated GLP-1, NT and PYY secretion3. Indeed, the highly toxic sodium channel blocker tetrodotoxin can be successfully applied in the perfusion studies. Finally, in the perfusion model it can be directly assessed where in the gut a certain compound stimulates secretion of a certain hormone, as the investigator can simply choose and prepare the desired region to perfuse, and at the same time it can be investigated whether a stimulus causes secretion by activation of molecular sensors from the luminal or vascular side of the intestine3,15,16.
The secretory mechanisms underlying gut hormone secretion may also be studied by use of gut tissue pieces (including human tissue), primary intestinal cultures (usually from mice), immortalized hormone secreting cell lines (of mouse or human origin), by gut tissue mounted in Ussing chambers or by organoids (both most often from mice)2,3,4,5,6,17,18. Compared to intestinal perfusions, studies on human gut pieces, primary cell cultures and cell lines are technically easier to perform and are a faster and cheaper way of generating data, but of course the study of gut pieces requires access to fresh human gut specimens. However, in these models the normal cell polarization of the gut is inherently lost, meaning that these models cannot be used to assess normal activation of molecular sensors, and absorption processes also cannot be studied. Moreover, such studies usually employ static incubations (for up to several h) which is highly non-physiological and has nothing to do with the cells&#39; normal secretory dynamics, because the secreted product is not removed and thus may feedback influence the secretion of hormones. In contrast, in the perfused intestine, secreted and absorbed molecules are efficiently removed by the mucosal microcirculation as they are in vivo, ensuring that the transmucosal gradients are maintained so absorption and secretion can occur at a normal rate. Furthermore, cell cultures may have dedifferentiated from their native enteroendocrine cell origin, meaning that they are no longer representative of the native cells in terms of peptide content and expression of molecular sensors, although they may still secrete the hormone in question. This is, for instance, the case for GLP-1 secreting cell lines19.
It is, therefore, our opinion that primary cell cultures or cell line studies are most suited for screening purposes and for performing types experiments that cannot be performed in vivo or in the isolated perfused gut. For instance, a true strength of the primary cell cultures and cell line cultures is that intra-cellular secondary messengers (e.g. Ca2+, cAMP, NAD(P)H) can be monitored in real time, and electrical signaling of the hormone secreting cells can be investigated20,21,22. In addition, siRNA knockdown can be done, which is particularly useful if specific inhibitors are not available20,21,22,23,24. Gut tissue from mice mounted in Ussing chambers has recently been used for studying the molecular mechanisms underlying bile-acid stimulated GLP-1 secretion, while intestinal organoids (from mice) and human gut pieces have also been used for studying the molecular details of gut hormone secretion17,25. Whereas the former benefits from being polarized2 all of these models involve static incubations. Studies on human gut pieces, however, benefit from using human, rather than rodent, tissue which is important since species difference in tissue expression of 7TM receptors and molecular transporters may result in different molecular sensing pathways between species. In fact, most data in this field has been generated by studies on either pigs, mice or rats, and it remains elusive whether these findings can be transferred to humans. It is, however, reassuring that the molecular sensing mechanisms that underlie glucose-stimulated GLP-1 secretion appear to be similar between mouse, rat,&#160;man,&#160;and transcriptomic and peptidomic profiling of mouse and human L-cells&#160; revealed strong global similarities between the two species7,18,26,27.
The isolated perfused rat small intestine, however, also has some limitations that should be considered. Most importantly, it is impossible to determine whether a given secretory response results from direct activation by the test substance of the targeted hormone-producing cells or rather is caused by an indirect mechanism. For instance, KCl instantly increases GLP-1 secretion from the perfused intestine7, but it remains unknown whether this is a consequence of direct depolarization of the L-cell or results from depolarization of neurons close to the L-cells or effects of simultaneously released paracrine stimulators/inhibitors. Data arising from studies using the perfused intestine which aim to elucidate the molecular mechanisms underlying secretion should, therefore, always be put into context with data obtained from other more specific models to increase the ability to establish causality. For instance, glucose-stimulated GLP-1 secretion from the GLP-1 secreting cell line GLUTag28,29&#160;and from primary mouse L-cells depend on the activity of the glucose transporters (SGLT1 and GLUT2). Blocking these transporters in the perfused rat small intestine also attenuates secretion20, meaning that it is likely that glucose-stimulated GLP-1 secretion is largely mediated by direct actions of glucose on the L-cell. Another important limitation of the isolated perfused intestine is that some of the lipids are difficult to study due to their hydrophobicity. Although it is possible to investigate the final products of lipid digestion (fatty acids, diacyl glycerols, lysophosphatidylglycerols, etc.) and although the preparation may be able to re-esterify the lipids intra-cellularly and perhaps pack them into chylomicrons, the transport of the chylomicrons out of the cells and their subsequent uptake by the lacteals of the villi is disrupted, since the lymph flow in the isolated gut cannot be secured. Most likely, therefore, lipid absorption is halted once the absorbed products start to accumulate in the cells. The in vitro cell systems are even less suitable for lipid studies because of their lack of polarization. Obviously, this limitation is only relevant for lipids that are absorbed and transported via the lacteals, whereas those absorbed via the intestinal blood vessels are likely to be handled normally.

<table>
	<tbody>
		<tr>
			<td>Chemicals for perfusion buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Merck</td>
			<td>1.12018.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate (CaCl2 x 2 H2O)</td>
			<td>Merck</td>
			<td>102382</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran 70</td>
			<td>Pharmacosmos</td>
			<td>40014</td>
			<td></td>
		</tr>
		<tr>
			<td>Fumaric acid disodium salt (C4H2Na2O4)</td>
			<td>Sigma Aldrich</td>
			<td>F9642</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose (C6H12O6)</td>
			<td>Merck</td>
			<td>108342</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate hepatahydrate (MgSO4)</td>
			<td>Merck</td>
			<td>105886</td>
			<td></td>
		</tr>
		<tr>
			<td>Potasium chloride (KCl)</td>
			<td>Merck</td>
			<td>104936</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate (KH2PO4)</td>
			<td>Merck</td>
			<td>104873</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyruvic acid sodium salt (C3H3NaO4)</td>
			<td>Merck</td>
			<td>106619</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (NaHCO3)</td>
			<td>Merck</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Merck</td>
			<td>106404</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium L-glutamate monohydrate (C5H8NNaO4 x H2O)</td>
			<td>Merck</td>
			<td>106445</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Perfusion equitment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Universial perfusion system</td>
			<td>Harvard Bioscience, Inc.</td>
			<td>732316</td>
			<td></td>
		</tr>
		<tr>
			<td>BASIC UNIT UNIPER UP-100, TYPE 834</td>
			<td>Harvard Bioscience, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Roller Pump, with four channels</td>
			<td>Harvard Bioscience, Inc.</td>
			<td>730100</td>
			<td></td>
		</tr>
		<tr>
			<td>Windkessel</td>
			<td>Harvard Bioscience, Inc.</td>
			<td>732068</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermostatic Circulator,Bath Volume 3L, 230V/50Hz</td>
			<td>Harvard Bioscience, Inc.</td>
			<td>730125</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating table, heated on tripod stand, type 873</td>
			<td>Harvard Bioscience, Inc.</td>
			<td>733776</td>
			<td></td>
		</tr>
		<tr>
			<td>Cannula with basked, OD= 2.0mm, ID= 1.0mm</td>
			<td>Harvard Bioscience, Inc.</td>
			<td>733313</td>
			<td></td>
		</tr>
	</tbody>
</table>

mechanisms underlying gut hormone secretion using the isolated perfused rat small intestine

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, nutrient absorption and distribution, and post-prandial glucose excursions. Understanding the molecular mechanisms that regulate gut hormone secretion is fundamental for understanding and translating gut hormone physiology. Traditionally, the mechanisms underlying gut hormone secretion are either studied in vivo (in experimental animals or humans) or using gut hormone-secreting primary mucosal cell cultures or cell lines. Here, we introduce an isolated perfused rat small intestine as an alternative method for studying gut hormone secretion. The virtues of this model are that it relies on the intact gut, meaning that it recapitulates most of the physiologically important parameters responsible for the secretion in in vivo studies, including mucosal polarization, paracrine relationships and routes of perfusion/stimulus exposure. In addition, and unlike in vivo studies, the isolated perfused rat small intestine allows for almost complete experimental control and direct assessment of secretion. In contrast to in vitro studies, it is possible to study both the magnitude and the dynamics of secretion and to address important questions, such as what stimuli cause secretion of different gut hormones, from which side of the gut (luminal or vascular) is secretion stimulated, and to analyze in detail molecular sensors underlying the secretory response. In addition, the preparation is a powerful model for the study of intestinal absorption and details regarding the dynamics of intestinal absorption including the responsible transporters.

The ability to determine whether a given stimulus causes secretion of the gut hormone of interest relies on a steady baseline secretion. Furthermore, if no response to the stimulus is observed, a robust secretory response to the positive control must be evident to exclude that the lack of response to the test stimulus does not reflect a general lack of responsiveness. Figure 2A and 2B shows an example of good quality data; GLP-1 secretion fro...

Watch this Scientific Journal Video about Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine at JoVE.com

Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine

Here, we present a powerful and physiological model to study the molecular mechanisms underlying gut hormone secretion and intestinal absorption &#8212; the isolated perfused rat small intestine.

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, ...

The method we've developed here has turned out to be extremely useful in our work, trying to find out the molecular mechanisms of secretion of hormones from the gut, but also absorption of nutrients, and the coupling between the two. The main advantage of this highly physiological technique is that the gut is left in situ, while allowing detailed studies that are usually only performed within In Vitro models. First place the rat on a heated operating table, confirm a lack of response to toe pinch, and make a skin and peritoneal excision to expose the abdominal cavity.Move the small intestine to the side to expose the terminal region of the colon. And ligate the supplying vasculature to the colon. Gradually extract the colon, starting from the terminal region, and moving towards the small intestine, hydrating the tissue with isotonic saline, and using cotton swabs to remove the connective tissue as necessary.When all of the colon has been removed, insert a 15 centimeter piece of plastic tubing into the proximal end of the small intestinal lumen, and secure the tubing with sutures. Then use a 10-milliliter syringe and a syringe pump to carefully flush the small intestine with room temperature isotonic saline at a 25-milliliter per minute flow rate. When the tissue has been emptied of chyme, adjust the tissues so that the upper mesenteric artery is accessible and remove the connective tissue and fat tissue to expose the artery.Use fine point forceps to place two sutures under the mesenteric artery and two sutures under the portal vein. Fill a plastic 22-gauge catheter attached to a rolling pump with perfusion buffer and use a pair of surgical scissors to cut a small hole in the mesenteric artery, immediately inserting the catheter into the incision. Insert the catheter within two minutes of cutting a hole in the artery, otherwise the gut will likely suffer from ischemic damage.As soon as the catheter has been secured with a suture, start the rolling pump to initiate perfusion at a 7.5 milliliter per minute flow rate. When the blood vessels in the gut and portal vein turn pale, cut a hole in the portal vein. Insert a metal catheter, and secure the metal catheter with another suture.Take care when inserting the catheter into the portal vein, as the vein is thin walled. However, as the guard is now being perfused, quick insertion is not crucial. After collecting the perfusion output for one minute, measure the volume and click execute experiment in the pressure recording program to initiate the perfusion pressure acquisition recordings.Then cover the gut with a moistened lab tissue to keep it from drying out, and confirm that the distal end of the intestine is not blocked, and a luminal haploid can exit. To measure the partial pressure of oxygen and carbon dioxide, collect perfusion buffer through a three-way stop-cock valve immediately before it enters the organ, after approximately three minutes of perfusion, and from the catheter inserted into the portal vein. Place the samples on ice, and use an automated blood gas analyzer to measure the samples soon after collection.Use a fraction collector to obtain the first baseline samples. After 10 to 15 minutes of baseline collection, use a syringe pump to administer the first intraarterial test stimulant through a three-way stop-cock at a 35 milliliters per minute flow rate. Perform luminal stimulations by an initial stimulation boas injection delivered at a 2.5 milliliter per minute flow rate over the first five minutes of the stimulation, followed by administration of the test solution at 5 milliliter per minute flow rate.As soon as a luminal stimulation period is over, flush the lumen with isotonic saline at a 2.5 milliliter per minute flow rate for five minutes. Then collect baseline samples at 5 milliliters per minute for 15 to 30 minutes before the next test substance is administered. At the end of the experiment, administer a suitable, positive control to test for the responsiveness of the preparation for five to 10 minutes, collecting and additional 10 to 15 minutes of baseline samples at the end of the positive control stimulation period.Here is shown an example of good quality data, demonstrating Glucagon-like peptide-1 or GLP-1, secretion from an isolated perfused rat small intestine collected at one minute intervals. Under basal conditions, the secretion was steady while both before and after administration of the test stimulus and the positive control, a robust secretory response was evident. These GLP-1 secretion data obtained from an isolated perfused rat small intestine are of poor quality with an unsteady secretion measured at basal conditions that drifted upward throughout the testing in a manner that appeared to be unrelated to the test substance administrations or to the positive control.It is therefore impossible to conclude whether the increased GLP-1 secretion observed at the end of the vascular fructose stimulation was a fructose mediated response. While attempting this procedure to care to secure the catheters properly and to thoroughly clean the perfusion equipment as the perfusion buffer provides an excellent medium for bacterial growth. Following this procedure, other methods like In Vitro studies on hormone-secreting cell lines of primary intestinal cell cultures, can be performed to directly investigate the intracellular production of cyclic AMP or intracellular calcium.So we've already used the method now for elucidation of a number of processes behind absorption of nutrients and the mechanism of secretion of the hormones and it has turned out to be extremely useful. Don't forget that working with blockers or activators of molecular sites can be extremely hazardous and precautions such as using a lab coat, gloves, and safety glasses, a fume hood should always be taken while performing this procedure.

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Research

JoVE Journal

Medicine

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an extended perfusion period is challenging. We have added the kidney to our previous ex vivo perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

Study of the animal organ physiology can be achieved by performing an experimental ex vivo perfusion system. The addition of a porcine kidney, as a homeostatic organ to our previously developed ex vivo liver perfusion model can be a principal step to achieve a better physiological environment.

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an ...

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

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

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

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

Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal vascular occlusion, retinal detachment, and diabetic retinopathy. It was the object of this study to introduce a standardized stress model for future preclinical therapeutic testing.
Bovine retinas were prepared and perfused with an oxygen saturated standard solution, and the ERG was recorded. After recording stable b-waves, hypoxia (pure N2) or glutamate stress (250 &#181;m glutamate) was exerted for 45 min. To investigate the effects on photoreceptor function alone, 1 mM aspartate was added to obtain a-waves. ERG-recovery was monitored for 75 min.
For hypoxia, a decrease in a-wave amplitude of 87.0% was noted (p &#60;0.01) after an exposition time of 45 min (decrease of 36.5% after the end of the washout p = 0.03). Additionally, an initial decrease in b-wave amplitudes of 87.23% was recorded, that reached statistical significance (p &#60;0.01, decrease of 25.5% at the end of the washout, p = 0.03).
For 250 &#181;m glutamate, an initial 7.8% reduction of a-wave amplitudes (p &#62;0.05) followed by a reduction of 1.9% (p &#62;0.05). A reduction of 83.7% of b-wave amplitudes (p &#60;0.01) was noted; after a washout of 75 min the reduction was 2.3% (p = 0.62). In this study, a standardized stress model is presented that may be useful to identify possible neuroprotective effects in the future.

With this study, we introduce a standardized stress model for the isolated superfused bovine retina for future preclinical therapeutic testing. The effect of either hypoxia (pure N2) or glutamate stress (250 &#181;M glutamate) on retinal function represented by a- and b-wave amplitudes was evaluated.

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

The Inverted Heart Model for Interstitial Transudate Collection from the Isolated Rat Heart

The present protocol describes a unique approach that enables the collection of cardiac transudate (CT) from the isolated, saline-perfused rat heart. After isolation and retrograde perfusion of the heart according to the Langendorff technique, the heart is inverted into an upside-down position and is mechanically stabilized by a balloon catheter inserted into the left ventricle. Then, a thin latex cap &#8211; previously cast to match the average size of the rat heart &#8211; is placed over the epicardial surface. The outlet of the latex cap is connected to silicon tubing, with the distal opening 10 cm below the base level of the heart, creating slight suction. CT continuously produced on the epicardial surface is collected in ice-cooled vials for further analysis. The rate of CT formation ranged from 17 to 147 &#181;L/min (n = 14) in control and infarcted hearts, which represents 0.1-1% of the coronary venous effluent perfusate. Proteomic analysis and high performance liquid chromatography (HPLC) revealed that the collected CT contains a wide spectrum of proteins and purinergic metabolites.

This protocol describes a method to collect cardiac interstitial fluid from the isolated, perfused rat heart. To physically separate interstitial transudate from coronary venous effluent perfusate, the Langendorff perfused heart is inverted, and the transudate (interstitial fluid) formed on the cardiac surface is collected using a soft latex cap.

The present protocol describes a unique approach that enables the collection of cardiac transudate (CT) from the isolated, saline-perfused rat heart. ...

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 ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

A Model of Long-Term Ventricular Fibrillation in Isolated Rat Hearts

Ventricular fibrillation (VF) is a fatal arrhythmia with a high incidence in cardiac patients, but VF arrest under perfusion is a neglected method of intraoperative arrest in the field of cardiac surgery. With recent advances in cardiac surgery, the demand for prolonged VF studies under perfusion has increased. However, the field lacks simple, reliable, and reproducible animal models of chronic ventricular fibrillation. This protocol induces long-term VF through alternating current (AC) electrical stimulation of the epicardium. Different conditions were used to induce VF, including continuous stimulation with a low or high voltage to induce long-term VF and stimulation for 5 min with a low or high voltage to induce spontaneous long-term VF. The success rates of the different conditions, as well as the rates of myocardial injury and recovery of cardiac function, were compared. The results showed that continuous low-voltage stimulation induced long-term VF and that 5 min of low-voltage stimulation induced spontaneous long-term VF with mild myocardial injury and a high rate of recovery of cardiac function. However, the low-voltage, continuously stimulated long-term VF model had a higher success rate. High-voltage stimulation provided a higher rate of VF induction but showed a low defibrillation success rate, poor recovery of cardiac function, and severe myocardial injury. On the basis of these results, continuous low-voltage epicardial AC stimulation is recommended for its high success rate, stability, reliability, reproducibility, low impact on cardiac function, and mild myocardial injury.

This protocol presents a model of long-term ventricular fibrillation in rat hearts induced by continuous stimulation with low-voltage alternating current. This model has a high success rate, is stable, reliable, and reproducible, has a low impact on cardiac function, and causes only mild myocardial injury.

Ventricular fibrillation (VF) is a fatal arrhythmia with a high incidence in cardiac patients, but VF arrest under perfusion is a neglected method of ...

Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation

Heart transplantation is the most effective therapy for end-stage heart failure. Despite the improvements in therapeutic approaches and interventions, the number of heart failure patients waiting for transplantation is still increasing. The normothermic ex situ preservation technique has been established as a comparable method to the conventional static cold storage technique. The main advantage of this technique is that donor hearts can be preserved for up to 12 h in a physiologic condition. Moreover, this technique allows resuscitation of the donor hearts after circulatory death and applies required pharmacologic interventions to improve donor function after implantation. Numerous animal models have been established to improve normothermic ex situ preservation techniques and eliminate preservation-related complications. Although large animal models are easy to handle compared to small animal models, it is costly and challenging. We present a rat model of normothermic ex situ donor heart preservation followed by heterotopic abdominal transplantation. This model is relatively cheap and can be accomplished by a single experimenter.

Here, we present an assessment protocol of a heterotopically implanted heart after normothermic ex situ preservation in the rat model.

Heart transplantation is the most effective therapy for end-stage heart failure. Despite the improvements in therapeutic approaches and interventions, the ...