This work was supported by the Special Talent Program of Chengdu University of Traditional Chinese Medicine for &#34;Xinglin Scholars and Discipline Talents Research Promotion Plan&#34; (33002324).

This protocol is derived from previously published literature14,15,16,17. Male Sprague Dawley (SD) rats (260-300 g, 8-10 weeks old) were used for the present study. The animal protocol was reviewed and approved by the Management Committee from Chengdu University of Traditional Chinese Medicine (Record No. 2023017). Prior to the experiment, the rats were instructed to fast for 24 h. During the experiment, the rats were kept in an animal chamber and had free access to food and water.
1. Solution preparation
<ol>
	<li>Prepare physiological salt solution (PSS) containing 118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgCl2&#8729;6H2O, 25 mM NaHCO3, 11 mM D-glucose, and 5 mM HEPES (see the Table of Materials).</li>
	<li>Saturate the solutions and bubble with a mixed gas of 95% O2 and 5% CO2. Meanwhile, maintain the pH values of the solution between 7.38 and 7.42 with 2 mM NaOH.</li>
	<li>Precool 1/3 of the PSS to 4 &#176;C and prewarm the rest to 37 &#176;C for subsequent experiments. 
	NOTE: the PSS was originally prepared at room temperature in steps 1.1-1.2</li>
</ol>
2. Rat intestinal canal dissection
<ol>
	<li>Gather Petri dishes filled with 4&#176;C PSS, surgical tweezers, and scissors. Administer 2% isoflurane to anesthetize the rat through inhalation for approximately 5 min. Verify that the rat is deeply anesthetized by conducting a toe pinch test. If necessary, administer additional anesthetics. Next, expose the intestinal canal by opening the abdominal cavity on an operating table.</li>
	<li>Quickly place the stomach and intestine tubes in a Petri dish filled with 4 &#176;C PSS (pH 7.40) with 95% O2 and 5% CO2 saturated. Locate the duodenum, which is the beginning of the small intestine, in the pylorus of the stomach. Using tweezers, delicately lift the adjacent tissue and carefully trim it away from the intestine&#39;s edge with scissors. Subsequently, divide the intestine into 1-2 cm segments; this entire process is illustrated in Figure 1.</li>
</ol>
3. Suspension and fixation of the intestinal canal (Figure 2)
<ol>
	<li>Turn on the in vitro tissue perfusion system and adjust the bath temperature in the instrument to 37 &#176;C. Place the PSS (37 &#176;C) into the bath.</li>
	<li>Prepare a 15 cm surgical suture (see the Table of Materials) and soak it in 4 &#176;C PSS that is saturated with 95% O2 + 5% CO2. Using the suture, secure one end of the intestinal canal and use a steel needle hook to secure the other end.</li>
	<li>Install the intestinal tube. Mount the segment with the steel needle hook at the bottom of the bath and attach the other end of the surgical line to the transducer. Turn on the gas switch to allow bubbles to emerge in the bath.</li>
	<li>Open the data acquisition software (see the Table of Materials) and click on Start to ensure the corresponding path signal is being recorded.</li>
</ol>
4. Normalization
<ol>
	<li>Rotate the spiral axis of the bath counterclockwise to relax the intestinal tubes to their natural state. Click on Setup-Zero All Inputs to ensure that the initial tension of the intestinal tube is set to 0 g in the software.</li>
	<li>Rotate the spiral axis of the bath counterclockwise to pull the tension value to 1 g and stabilize it in the pH = 7.40, 95% O2 + 5% CO2 saturated 37 &#176;C PSS for 30 min. 
	NOTE: The normalization of the intestinal tube is to adjust its preload to an optimal state. For cavity samples, an optimal preload was necessary to maintain exceptional activity in vitro. The optimal preload of the rat intestinal tube was 1 g18.</li>
</ol>
5. Detection of reactivity
<ol>
	<li>Observe the rhythmic spontaneous contraction waves in the software and proceed to the next experiment as this indicates a sufficient response.</li>
</ol>
6. Experimental observation
<ol>
	<li>Add the test drug (such as acetylcholine, etc.) to the bath to study the effect of the drug on intestinal tube function. 
	NOTE: The drug&#39;s effect was assessed by comparing pre and post administration changes in the intestinal constriction curve. When the drug is added, it is appropriate to increase the bubble to mix the drug, and then adjust the bubble to normal after mixing.</li>
</ol>
7. Data analysis
NOTE: The in vitro tissue perfusion system has four channels that can simultaneously conduct tests on the effects of four identical or different drugs on four intestine tubes. Since the experimental parameters and analysis methods are the same for all channels, one channel is selected as an example for data analysis.
<ol>
	<li>Stop the data acquisition softwareand perform data analysis on this data acquisition software. Edit the data board and select the analysis parameters as follows: Click on Window-Data Pad and choose the average tension for the channel.</li>
	<li>Select the contraction curve before administration and click Add to Data pad; select the contraction curve after administration and click Add to data pad. The average tension value before and after administration will appear on the data pad in turn.</li>
	<li>Click on Window-Data Pad to copy data to other statistical analysis software (see the Table of Materials) for statistical analysis.</li>
	<li>Analyze other parameters, such as average amplitude, average frequency, and integral (area under the curve), simply replace the average tension with the respective parameter, and the operation is the same as steps 6.1-6.3.</li>
	<li>Save contraction curve: Select the contraction curve, click on Edit-Copy Labchart Data to copy data to drawing software (see the Table of Materials) to draw the contraction curve.</li>
</ol>
8. Postsurgical treatment
<ol>
	<li>After surgery, euthanize the animals following institutionally approved protocols. 
	NOTE: For the present study, the animals were euthanized by inhaling excess isoflurane.</li>
</ol>

Protocol for Studying Gastrointestinal Dynamics and Drug Effects on Motility

<ol>
	<li>Jiang, Z., 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=Therapeutic+role+of+wuda+granule+in+gastrointestinal+motility+disorder+through+promoting+gastrointestinal+motility+and+decreasing+inflammatory+level.">Therapeutic role of wuda granule in gastrointestinal motility disorder through promoting gastrointestinal motility and decreasing inflammatory level.</a> Front Pharmacol. 14, 1-15 (2023).</li><li>Talley, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+causes+functional+gastrointestinal+disorders?+A+proposed+disease+model.">What causes functional gastrointestinal disorders? A proposed disease model.</a> Am J Gastroenterol. 115 (1), 41-48 (2020).</li><li>Frazer, C., Hussey, L., Bemker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+motility+problems+in+critically+ill+patients.">Gastrointestinal motility problems in critically ill patients.</a> Crit Care Nurs Clin North Am. 30 (1), 109-121 (2018).</li><li>Waclawikova, B., Codutti, A., Alim, K., El, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut+microbiota-motility+interregulation:+insights+from+in+vivo,+ex+vivo+and+in+silico+studies.">Gut microbiota-motility interregulation: insights from in vivo, ex vivo and in silico studies.</a> Gut Microbes. 14 (1), 1997296 (2022).</li><li>Kwak, D. S., 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=Short-term+probiotic+therapy+alleviates+small+intestinal+bacterial+overgrowth,+but+does+not+improve+intestinal+permeability+in+chronic+liver+disease.">Short-term probiotic therapy alleviates small intestinal bacterial overgrowth, but does not improve intestinal permeability in chronic liver disease.</a> Eur J Gastroenterol Hepatol. 26 (12), 1353-1359 (2014).</li><li>Tang, A. T., 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=Endothelial+Tlr4+and+the+microbiome+drive+cerebral+cavernous+malformations.">Endothelial Tlr4 and the microbiome drive cerebral cavernous malformations.</a> Nature. 545 (7654), 305-310 (2017).</li><li>Mayer, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut+feelings:+the+emerging+biology+of+gut-brain+communication.">Gut feelings: the emerging biology of gut-brain communication.</a> Nature Rev Neurosci. 12 (8), 453-466 (2011).</li><li>Singh, R., Zogg, H., Ghoshal, U. C., Ro, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+treatment+options+and+therapeutic+insights+for+gastrointestinal+dysmotility+and+functional+gastrointestinal+disorders.">Current treatment options and therapeutic insights for gastrointestinal dysmotility and functional gastrointestinal disorders.</a> Front Pharmacol. 13, 1-20 (2022).</li><li>Sanger, G. J., Alpers, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+drugs+for+gastrointestinal+motor+disorders:+Translating+science+to+clinical+need.">Development of drugs for gastrointestinal motor disorders: Translating science to clinical need.</a> Neurogastroenterol Motil. 20 (3), 177-184 (2008).</li><li>Valentin, N., Acosta, A., Camilleri, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+investigational+therapeutics+for+gastrointestinal+motility+disorders:+From+animal+studies+to+phase+ii+trials.">Early investigational therapeutics for gastrointestinal motility disorders: From animal studies to phase ii trials.</a> Expert Opin Investig Drugs. 24 (6), 769-779 (2015).</li><li>Shoor, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Athletes,+nonsteroidal+anti-inflammatory+drugs,+coxibs,+and+the+gastrointestinal+tract.">Athletes, nonsteroidal anti-inflammatory drugs, coxibs, and the gastrointestinal tract.</a> Curr Sports Med Rep. 1 (2), 107-115 (2002).</li><li>Lacy, B. 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=Effects+of+antidepressants+on+gastric+function+in+patients+with+functional+dyspepsia.">Effects of antidepressants on gastric function in patients with functional dyspepsia.</a> Am J Gastroenterol. 113 (2), 216-224 (2018).</li><li>Lin, R. K., 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=The+effects+of+ginsenosides+on+contractile+activity+of+antibiotic-treated+isolated+small+intestinal+smooth+muscle+in+mice.">The effects of ginsenosides on contractile activity of antibiotic-treated isolated small intestinal smooth muscle in mice.</a> Lishizhen Medicine and Materia Medica Research. 33 (04), 790-793 (2022).</li><li>Jespersen, B., Tykocki, N. R., Watts, S. W., Cobbett, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+smooth+muscle+function+in+the+isolated+tissue+bath-applications+to+pharmacology+research.">Measurement of smooth muscle function in the isolated tissue bath-applications to pharmacology research.</a> J Vis Exp. (95), e52324 (2015).</li><li>Park, K. H., 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=Ex+vivo+assessment+of+contractility,+fatigability+and+alternans+in+isolated+skeletal+muscles.">Ex vivo assessment of contractility, fatigability and alternans in isolated skeletal muscles.</a> J Vis Exp. (69), e4198 (2012).</li><li>Semenov, I., Herlihy, J. T., Brenner, R. <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+measurements+of+tracheal+constriction+using+mice.">In vitro measurements of tracheal constriction using mice.</a> J Vis Exp. (64), e3703 (2012).</li><li>Han, J., Chen, X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+overview+of+invisible+spectro+intestine+experiment.">Study overview of invisible spectro intestine experiment.</a> Guiding Journal of Traditional Chinese Medicine and Pharmacy. 14 (3), 94-96 (2008).</li><li>Hao, F. F., Liu, W. Q., Wang, J. N., Nie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+study+on+the+effect+of+Forsythia+suspensa+water+extract+on+the+movement+of+isolated+intestinal+tract+in+rats.">Experimental study on the effect of Forsythia suspensa water extract on the movement of isolated intestinal tract in rats.</a> Shandong Journal of Traditional Chinese Medicine. 37 (1), 63-66 (2018).</li><li>Ruoff, H. J., Fladung, B., Demol, P., Weihrauch, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+receptors+and+drugs+in+motility+disorders.">Gastrointestinal receptors and drugs in motility disorders.</a> Digestion. 48 (1), 1-17 (1991).</li><li>Foong, D., Zhou, J., Zarrouk, A., Ho, V., O'connor, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+biology+of+human+interstitial+cells+of+cajal+in+gastrointestinal+motility.">Understanding the biology of human interstitial cells of cajal in gastrointestinal motility.</a> Int J Mol Sci. 21 (12), 4540 (2020).</li><li>M&#246;ssner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motilit&#228;tsst&#246;rungen+des+gastrointestinaltrakts.">Motilit&#228;tsst&#246;rungen des gastrointestinaltrakts.</a> Der Internist. 56 (6), 613-614 (2015).</li><li>Yin, J., Chen, J. D. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+motility+disorders+and+acupuncture.">Gastrointestinal motility disorders and acupuncture.</a> Autonomic Neuroscience. 157 (1-2), 31-37 (2010).</li></ol>

The authors have no conflicts of interest to disclose.

Gastrointestinal motility is accomplished by a series of precisely coordinated smooth muscle contractions and relaxations. This process involves rhythmic contraction of one group of muscle groups, coordinated contraction of multiple groups, and special propulsive contraction20,21. The occurrence of gastrointestinal motility disorders may be associated with dysfunctions at different levels, such as the central nervous system, autonomic nervous system, enteric nervous system, and gastrointestinal smooth muscle. These tissue structures function together through the release of various neurotransmitters and humoral factors, which bind to corresponding receptors to carry out different physiological functions22. Therefore, studying the local mechanism of drug action is essential, and conducting in vitro small intestine experiments can provide more accurate insights by excluding interference from foreign substances and the central nervous system regulation.
Isolated intestinal tube specimens of guinea pig ileum, rabbit jejunum, and duodenum are commonly used in experimental animals. In addition, the ileum, duodenum, and jejunum of rats and cats can also be used for in vitro experiments. This method is suitable for isolated tissues. The intestinal smooth muscle has the characteristics of automatic rhythmic contraction and is affected by the nerve plexus in the intestinal wall. The isolated intestinal tubes of many animals can maintain their spontaneous motor function in a suitable survival environment. In vitro intestinal tube experiment has the advantages of simple operation, easy control of experimental conditions, few influencing factors, good reproducibility, accurate and reliable results, and it is more conducive to the discussion of the mechanism of drug action. Additionally, this method is considered economical and feasible.
The in vitro intestinal tube experiment also has its limitations, such as using unpurified crude Chinese medicine preparations that contain impurities, inorganic ions, saponins, and tannins, which can affect the reliability of the experiment. Therefore, it is necessary to fully evaluate the influence of impurities and physicochemical factors on the experimental results, including the pH of the liquid and various electrolytic and impurity particles. Furthermore, improving the stability of the in vitro experimental instrument and the preservation technique for the in vitro intestinal tube is necessary. When designing in vitro experiments, it is essential to consider whether the drugs or preparations used meet the requirements of these experiments (e.g., levels of impurities).
In this method, there are several precautions that need to be observed. To begin with, the intestinal tube should be removed from the rat's abdominal cavity and soaked in a cold PSS prior to the experiment. Furthermore, it is important to cut the entire tube into multiple pieces and store them in the refrigerator, minimizing any stimulation to the intestines. During the dosing process, it is crucial to ensure that the dosing tube does not come into contact with the line on which the intestinal tube is suspended. Additionally, the quantity of drug added to the bath should be accurate, and the drug should not be added to the wall of the bath or directly to the intestinal tube to avoid influencing the results. When rinsing the intestinal tube, a gentle approach should be used, and high-pressure rinsing should be avoided to prevent tissue contracture. It is recommended to handle the intestinal tube with tweezers and avoid exposing it to air for extended periods to prevent any loss of activity.
This paper presents a demonstration of the experimental methodology for isolated intestinal tube experiments, using the effects of various traditional drugs acting on intestinal receptors to illustrate their impact on intestinal tube movement, which provides an effective approach to investigate the mechanism of action of drugs that regulate gastrointestinal motility on the gastrointestinal tract.

Gastrointestinal diseases, a prevalent condition, gravely impact human life and health1. Gastrointestinal motility disorder is an important part of functional gastrointestinal diseases, manifesting primarily in debilitating symptoms, delayed gastric emptying, and severe gastric issues2. It can disrupt gastrointestinal coordination, hinder gastric emptying, impact intestinal food intolerance, and even cause functional obstruction in the small or large intestine3. For patients undergoing gastrointestinal surgery, this disorder can directly lead to intestinal failure. Moreover, intestinal disorder is not only related to gastrointestinal diseases but also to the pathogenic factors of various other diseases, such as hepatitis and central nervous system diseases. Intestinal microbial communities play a crucial regulatory role in intestinal physiology, including motility, which subsequently influences colonization within the microbial ecosystem4. As hepatitis B virus infection progresses to chronic hepatitis B, there are varying degrees of changes in the intestinal flora. Modulating the intestinal flora has demonstrated benefits in hepatitis B virus treatment5. Additionally, the central nervous system can influence the intestine and alter its microbial composition. Recent advancements in microflora sequencing technology have uncovered bidirectional interactions between gut microflora and central nervous system function, closely associated with the occurrence and progression of central nervous system diseases6,7.
With the aging of society, the incidence of gastrointestinal motility disorder is escalating, linked to the decline or loss of neuronal function in the enteric nervous system and bowel&#39;s intrinsic innervation8. As our comprehension of gastrointestinal diseases broadens, numerous novel ideas and approaches emerge, potentially leading to novel drug developments. However, many of these ideas are still hypothetical or await positive clinical trial outcomes to materialize9,10. Effective research methods are crucial in overcoming gastrointestinal diseases. In recent years, extensive research has focused on gastrointestinal drugs and motility regulation. Gastrointestinal drugs and gastrointestinal dynamics are inseparable, and many other systemic drugs have varying effects on gastrointestinal dynamics. For instance, non-steroidal anti-inflammatory drugs (NSAIDs) are used for pain and inflammation and slow gastrointestinal movement, heightening peptic ulcer risk11. On the other hand, some antidepressants may affect gastrointestinal motility12. Currently, the main in vitro pharmacological experiment studying the effects of gastrointestinal drugs and other systemic drugs on gastrointestinal motility is the in vitro intestine movement assay13. By simulating physiological conditions, they observe drugs&#39; direct impact on intestinal smooth muscle contraction and relaxation, evaluating their gastrointestinal effects. However, the precise cause of gastrointestinal motility disorders remains unclear, likely a complex interplay of genetic, environmental, dietary, and neuroendocrine factors. Consequently, the treatment of gastrointestinal motility disorders continues to pose significant challenges.
The small intestine, being a crucial site for digestion, absorption, and drug metabolism, holds significance in gastrointestinal function. As a result, the isolated intestinal tube movement test is an essential tool for studying gastrointestinal diseases. This involves preparing and placing the animal&#39;s isolated intestinal tube in a bath, connecting it to an energy exchanger, utilizing a transducer to convert mechanical movements into electrical signals for amplification, and recording by a physiological recorder. Various parameters such as frequency, average amplitude of vibration, tension, and area under the curve can be measured to evaluate the motor function of the intestinal tube. This method offers advantages such as simplicity, economic feasibility, easy control of experimental conditions, minimal influencing factors, high reproducibility, and accurate and reliable results. Moreover, it is particularly useful for investigating the mechanism of drug action. However, there are notable challenges in the operation of the isolated intestinal tube experiment, for example, intestinal activity is difficult to maintain for a long time. To address these issues and draw from experiences in in vitro experiments, this paper will provide a detailed introduction to the key problems in experimental operation and present a valuable reference experimental protocol for studying drugs that regulate gastrointestinal motility.

<table><tbody><tr><td>Acetylcholine&amp;nbsp;</td><td>Sigma, USA</td><td>A6625</td><td></td></tr><tr><td>atropine</td><td>Sangon Biotech Co., Ltd., Shanghai, China</td><td>IA06501</td><td></td></tr><tr><td>Barium chloride</td><td>Macklin Biochemical Co.,Ltd.,Shanghai, China</td><td>B861682</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sangon Biotech Co., Ltd., Shanghai, China</td><td>A501330</td><td></td></tr><tr><td>D-glucose</td><td>Sangon Biotech Co., Ltd., Shanghai, China</td><td>A610219</td><td></td></tr><tr><td>drawing software</td><td>GraphPad Software, San Diego, California, USA</td><td>&amp;mdash;</td><td></td></tr><tr><td>Epinephrine</td><td>Sigma, USA</td><td>E4642</td><td></td></tr><tr><td>HEPES</td><td>Xiya Reagent Co., Ltd., Shandong, China</td><td>S3872</td><td></td></tr><tr><td>In vitro tissue perfusion system</td><td>PowerLab, ADInstruments, Australia</td><td>ML0146</td><td></td></tr><tr><td>KCl</td><td>Sangon Biotech Co., Ltd., Shanghai, China</td><td>A100395</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sangon Biotech Co., Ltd., Shanghai, China</td><td>A100781</td><td></td></tr><tr><td>LabChart Professional version 8.3&amp;nbsp;</td><td>ADInstruments, Australia</td><td>&amp;mdash;</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sangon Biotech Co., Ltd., Shanghai, China</td><td>A100288</td><td></td></tr><tr><td>NaCl</td><td>Sangon Biotech Co., Ltd., Shanghai, China</td><td>A100241</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sangon Biotech Co., Ltd., Shanghai, China</td><td>A100865</td><td></td></tr><tr><td>nifedipine</td><td>Macklin Biochemical Co.,Ltd.,Shanghai, China</td><td>N5087</td><td></td></tr><tr><td>statistical analysis software</td><td>GraphPad Software, San Diego, California, USA</td><td>&amp;mdash;</td><td></td></tr><tr><td>Surgical sutures</td><td>Johnson, USA</td><td>&amp;mdash;</td><td></td></tr></tbody></table>

using an in vitro tissue perfusion system to detect the functional activities of isolated intestinal tubes in real time

Gastrointestinal diseases, which have a high incidence, pose considerable challenges for humans. The small intestine is integral to food and drug digestion and absorption and plays a crucial role in treating these diseases. The intestinal tube movement experiment, a common and essential in vitro method, is utilized to study gastrointestinal dynamics. This includes the preparation of the isolated intestinal tube, as well as the suspension of the prepared intestinal tube in the bath and its connection to a signal detector. This is followed by the recording and analysis of a series of parameters, such as tension, which can be used to assess intestinal motor function, as well as considerations for keeping the intestinal tube active in vitro. The standardized program from sampling to data collection greatly improves the repeatability of the experimental data and ensures the authenticity of the recording of intestinal tension after physiological, pathological, and drug intervention. Here we present the key problems in experimental operation and a valuable reference experimental protocol for studying drugs that regulate gastrointestinal motility.

The first part of the study focuses on the process of separating isolated intestinal tubes from the body and converting them into 2 cm tubes in vitro. This process is illustrated in detail in Figure 1. The second part involves the suspension and standardization of the isolated intestinal tube ring. The success of this process is demonstrated in Figure 2, which shows the automatic rhythmic contraction of a normal tube. Lastly, the study examines the effects of representative contractile agents and relaxants on the isolated intestinal tube ring. It was found that acetylcholine (0.3 &#181;mol/L) and barium chloride (1.2 mmol/L) enhanced tension in these tubes, while atropine (300 &#181;mol/L), epinephrine (1 &#181;mol/L) and nifedipine (10 &#181;mol/L) inhibited their tension, as illustrated in Figure 3.
This method mainly introduces the determination of the motor function of the isolated intestinal tube. Intestinal movements are affected by a number of factors, and most of the effects are exerted through receptors in the intestine. In the gastrointestinal tract, there are various receptors, including cholinergic M receptors, adrenergic &#945; and &#946; receptors, 5-HT receptors, histamine receptors, and gastrointestinal hormone receptors19. The spasm in the isolated intestine can be caused by the action of acetylcholine on M receptors. This effect can be blocked by atropine, which acts as a blocking agent for M receptors (Figure 3A). However, the spasm in the smooth muscle of the intestinal segment, which is caused by the entry of barium ions in barium chloride and their binding with calmodulin, cannot be blocked by atropine (Figure 3B). Epinephrine functions as an agonist for both &#945; and &#946; receptors (Figure 3C). It excites the &#945; receptor and &#946;2 receptor of the small intestine, resulting in the relaxation of the smooth muscle and a weakened contraction of the small intestine. As a result, the peristalsis is slowed down and the absorption rate is reduced. In contrast, nifedipine prevents the internal flow of Ca2+ and promotes intestinal relaxation (Figure 3D). This helps in relieving intestinal spasm, decreasing intestinal pressure, and facilitating the absorption of water in the intestine.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66243/66243fig01.jpg" /> 
Figure 1: The isolation of intestinal tubes. (A) intestinal tubes in rats; (B) Intestinal tubes were removed from the body and placed in a Petri dish containing 4 &#176;Cphysiological salt solution; (C) intestinal tube after removal of surrounding tissue; (D) 2 cm long intestinal tube used in the experiment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66243/66243fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66243/66243fig02.jpg" /> 
Figure 2: The process of suspension and standardization of isolated intestinal tube rings. (A) In vitro tissue perfusion system (see the Table of Materials); (B) fixing both ends of the intestinal tube with an iron hook and a surgical line in a Petri dish containing 4 &#176;Cphysiological salt solution; (C) fixing the intestinal tube in the one of baths containing 37 &#176;Cphysiological salt solution; (D) giving preload and recording normal waveform. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66243/66243fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66243/66243fig03.jpg" /> 
Figure 3: Effects of drugs on isolated intestinal tubes. Effects of (A) acetylcholine (0.3 &#181;mol/L) and atropine (300 &#181;mol/L), (B) barium chloride (1.2 mmol/L), (C) epinephrine (1 &#181;mol/L), and (D) nifedipine (10 &#181;mol/L) on isolated intestinal tubes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66243/66243fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Author Spotlight: Reliable and Reproducible In Vitro Assessment of Drug Impact on Rat Intestinal Tubes

We present a method for isolating rat intestinal tubes and assessing the impact of drugs on their tension, frequency, and amplitude in vitro. This method offers a valuable approach for researchers investigating intestinal tubes.

Using An In Vitro Tissue Perfusion System to Detect the Functional Activities of Isolated Intestinal Tubes in Real Time

Gastrointestinal diseases, which have a high incidence, pose considerable challenges for humans. The small intestine is integral to food and drug ...

using-an-vitro-tissue-perfusion-system-to-detect-functional

Nanyang Technological University

Research

JoVE Journal

Medicine

434 Views.  Chengdu University of Traditional Chinese Medicine. We presenteren een methode voor het isoleren van darmbuizen van ratten en het beoordelen van de impact van geneesmiddelen op hun spanning, frequentie en amplitude in vitro. Deze methode biedt een waardevolle aanpak voor onderzoekers die onderzoek doen naar darmbuisjes. De huidige experimentele uitdaging is het handhaven van de consistentie en de stabiliteit van technische methoden om betrouwbare en reproduceerbare wetenschappelijke onderzoeksresultaten te gar...

Video: Auteur in de schijnwerpers: betrouwbare en reproduceerbare in-vitrobeoordeling van de impact van geneesmiddelen op de darmbuizen van ratten

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

This protocol describes the measurement of epithelial barrier permeability in real-time following pharmacologic treatment in human intestinal organoids using fluorescent microscopy and live cell microscopy.

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

Developmental Biology

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Bioengineering

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 ...

A Novel 3D Printed Bioreactor for Ex Vivo Culture of Blood Vessels in Perfusion

Ex Vivo Perfusion Culture of Large Blood Vessels in a 3D Printed Bioreactor

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious surgical and pharmacological interventions to prevent and treat vascular disease are urgently needed. In part, the shortage of translational models limits the understanding of the cellular and molecular processes involved in vascular disease. Ex vivo perfusion culture bioreactors provide an ideal platform for the study of large animal vessels (including humans) in a controlled dynamic environment, combining the ease of in vitro culture and the complexity of the live tissue. Most bioreactors are, however, custom manufactured and therefore difficult to adopt, limiting the reproducibility of the results. This paper presents a 3D printed system that can be easily produced and applied in any biological lab, and provides a detailed protocol for its setup, enabling users&#39; operation. This innovative and reproducible ex vivo perfusion culture system enables the culture of blood vessels for up to 7 days in physiological conditions. We expect that adopting a standardized perfusion bioreactor will support a better understanding of physiological and pathological processes in large animal blood vessels and accelerate the discovery of new therapeutics.

Author Spotlight: EasyFlow - An Economical and Adaptable Perfusion Bioreactor for Large Blood Vessel Culture 

This protocol presents the setup and operation of a newly developed, 3D printed bioreactor for the ex vivo culture of blood vessels in perfusion. The system is designed to be easily adopted by other users, practical, affordable, and adaptable to different experimental applications, such as basic biology and pharmacological studies.

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious ...

Canine Intestinal Organoids in a Dual-Chamber Permeable Support System

The permeable support system is typically used in conjunction with traditional two-dimensional (2D) cell lines as an in vitro tool for evaluating the oral permeability of new therapeutic drug candidates. However, the use of these conventional cell lines has limitations, such as altered expression of tight junctions, partial cell differentiation, and the absence of key nuclear receptors. Despite these shortcomings, the Caco-2 and MDCK models are widely accepted and validated for the prediction of human in vivo oral permeability.
Dogs are a relevant translational model for biomedical research due to their similarities in gastrointestinal anatomy and intestinal microflora with humans. Accordingly, and in support of parallel drug development, the elaboration of an efficient and accurate in vitro tool for predicting in vivo drug permeability characteristics both in dogs and humans is highly desirable. Such a tool could be the canine intestinal organoid system, characterized by three-dimensional (3D), self-assembled epithelial structures derived from adult stem cells.
The (1) Permeable Support Seeding Protocol describes the experimental methods for dissociating and seeding canine organoids in the inserts. Canine organoid isolation, culture, and harvest have been previously described in a separate set of protocols in this special issue. Methods for general upkeep of the canine intestinal organoid monolayer are discussed thoroughly in the (2) Monolayer Maintenance Protocol. Additionally, this protocol describes methods to assess the structural integrity of the monolayer via transepithelial electrical resistance (TEER) measurements and light microscopy. Finally, the (3) Permeability Experimental Protocol&#160;describes the tasks directly preceding an experiment, including in vitro validation of experimental results.
Overall, the canine organoid model, combined with a dual-chamber cell culture technology, overcomes limitations associated with 2D experimental models, thereby improving the reliability of predictions of the apparent oral permeability of therapeutic drug candidates both in the canine and human patient.

Here, we present a protocol describing the culture of canine intestinal organoids in a dual-chamber, permeable support system. Organoid seeding in the permeable supports, the monolayer maintenance, and subsequent drug permeability experiments are described.

The permeable support system is typically used in conjunction with traditional two-dimensional (2D) cell lines as an in vitro tool for evaluating the oral ...

Immunology and Infection

Investigating Intestinal Barrier Breakdown in Living Organoids

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was not possible in classical cell culture monolayers. Moreover, monolayer cell cultures are good in vitro model systems but do not represent the complex cellular differentiation processes and functions that rely on 3D structure. This has so far only been possible in animal experiments, which are laborious, time consuming, and hard to assess by optical techniques. Here we describe an assay to quantitatively determine the barrier integrity over time in living small intestinal mouse organoids. To validate our model, we applied interferon gamma (IFN-&#947;) as a positive control for barrier destruction and organoids derived from IFN-&#947; receptor 2 knock out mice as a negative control. The assay allowed us to determine the impact of IFN-&#947; on the intestinal barrier integrity and the IFN-&#947; induced degradation of the tight junction proteins claudin-2, -7, and -15. This assay could also be used to investigate the impact of chemical compounds, proteins, toxins, bacteria, or patient-derived probes on the intestinal barrier integrity.

Here we describe a technique to quantify the barrier integrity of small intestinal organoids. The fact that the method is based on living organoids enables the sequential investigation of different barrier integrity modulating substances or combinations thereof in a time-resolved manner.

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was ...

Establishing In Situ Closed Circuit Perfusion of Lower Abdominal Organs and Hind Limbs in Mice

Ex vivo perfusion is an important physiological tool to study the function of isolated organs (e.g. liver, kidneys). At the same time, due to the small size of mouse organs, ex vivo perfusion of bone, bladder, skin, prostate, and reproductive organs is challenging or not feasible. Here, we report for the first time an in situ lower body perfusion circuit in mice that includes the above tissues, but bypasses the main clearance organs (kidney, liver, and spleen). The circuit is established by cannulating the abdominal aorta and inferior vena cava above the iliac artery and vein and cauterizing peripheral blood vessels. Perfusion is performed via a peristaltic pump with perfusate flow maintained for up to 2 h. In situ staining with fluorescent lectin and Hoechst solution confirmed that the microvasculature was successfully perfused. This mouse model can be a very useful tool for studying pathological processes as well as mechanisms of drug delivery, migration/metastasis of circulating tumor cells into/from the tumor, and interactions of immune system with perfused organs and tissues.

A protocol is described for in situ perfusion of the mouse lower body, including the bladder, the prostate, sex organs, bone, muscle and foot skin.

Ex vivo perfusion is an important physiological tool to study the function of isolated organs (e.g. liver, kidneys). At the same time, due to the small ...

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

Biology

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, and membrane potential. All of these approaches depend on measurement of activity at one or multiple locations along the gut simultaneously which are then interpreted to provide a sense of overall motility patterns. Recently, the development of video recording and spatiotemporal mapping (STmap) techniques have made it possible to observe and analyze complex patterns in ex vivo whole segments of colon and intestine. Once recorded and digitized, video records can be converted to STmaps in which the luminal diameter is converted to grayscale or color [called diameter maps (Dmaps)]. STmaps can provide data on motility direction (i.e., stationary, peristaltic, antiperistaltic), velocity, duration, frequency and strength of contractile motility patterns. Advantages of this approach include: analysis of interaction or simultaneous development of different motility patterns in different regions of the same segment, visualization of motility pattern changes over time, and analysis of how activity in one region influences activity in another region. Video recordings can be replayed with different timescales and analysis parameters so that separate STmaps and motility patterns can be analyzed in more detail. This protocol specifically details the effects of intraluminal fluid distension and intraluminal stimuli that affect motility generation. The use of luminal receptor agonists and antagonists provides mechanistic information on how specific patterns are initiated and how one pattern can be converted into another pattern. The technique is limited by the ability to only measure motility that causes changes in luminal diameter, without providing data on intraluminal pressure changes or muscle tension, and by the generation of artifacts based upon experimental setup; although, analysis methods can account for these issues. When compared to previous techniques the video recording and STmap approach provides a more comprehensive understanding of gastrointestinal motility.

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Recently available video recording and spatiotemporal mapping (STmap) techniques make it possible to visualize and quantify both propagating and mixing patterns of intestinal motility. The goal of this protocol is to explain the generation and analysis of STmaps using the GastroIntestinal Motility Monitoring (GIMM) system.

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Using An In Vitro Tissue Perfusion System to Detect the Functional Activities of Isolated Intestinal Tubes in Real Time

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