Name: <meta charset="utf8"></head><body>Autor im Rampenlicht: Zuverlässige und reproduzierbare In-vitro-Bewertung der Auswirkungen von Medikamenten auf Darmröhrchen von Ratten
Uploaded: 2024-07-26T12:40:00
Description: Gastrointestinal diseases, which have a high incidence, pose considerable challenges for humans. The small intestine is integral to food and drug ...

<meta charset="utf8"></head><body>Protokoll zur Untersuchung der gastrointestinalen Dynamik und der Auswirkungen von Arzneimitteln auf die Motilität

<ol>
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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. 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<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetylcholine&#160;</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>CaCl2</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>&#8212;</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>KH2PO4</td>
			<td>Sangon Biotech Co., Ltd., Shanghai, China</td>
			<td>A100781</td>
			<td></td>
		</tr>
		<tr>
			<td>LabChart Professional version 8.3&#160;</td>
			<td>ADInstruments, Australia</td>
			<td>&#8212;</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2&#183;6H2O</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>NaHCO3</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>&#8212;</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical sutures</td>
			<td>Johnson, USA</td>
			<td>&#8212;</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, 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.

<meta charset="utf8"></head><body>Autor im Rampenlicht: Zuverlässige und reproduzierbare In-vitro-Bewertung der Auswirkungen von Medikamenten auf Darmröhrchen von Ratten

Verwendung eines In-vitro-Gewebeperfusionssystems zum Nachweis der funktionellen Aktivitäten isolierter Darmschläuche in Echtzeit

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. The current experimental challenge is maintaining the consistency and the stability of technical methods to ensure reliable and reproducible scientific research outcomes, particularly considering the sensitivity and the variability inherent in biological systems.This protocol offers advantages such as simplicity, economical feasibility, easy control of experimental conditions, minimal influencing factors, high reproducibility, and accurate results.

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

Research

JoVE Journal

Medicine

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.

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.

Forschung

Medizin

Isolation of Rat Intestinal Tubes to Study Gastrointestinal Dynamics

To begin, prepare a physiological salt solution, or PSS. Saturate the solution by bubbling with a mixed gas of 95%oxygen and 5%carbon dioxide. Maintain the pH values of the solution between 7.38 and 7.42, with two millimolar sodium hydroxide.Pre-cool one-third of the PSS to four degrees Celsius. Then, pre-warm the remaining solution to 37 degrees Celsius for subsequent experiments. Now gather Petri dishes filled with four degrees Celsius PSS, surgical tweezers, and scissors.Quickly place the stomach and intestine tubes in a Petri dish filled with four degrees Celsius PSS saturated with 95%oxygen and 5%carbon dioxide. Locate the duodenum in the pylorus of the stomach. Using tweezers, delicately lift the adjacent tissue and carefully trim it away from the intestine's edge with scissors.Divide the intestine into one to two centimeter segments.

Isolierung von Darmschläuchen von Ratten zur Untersuchung der gastrointestinalen Dynamik

Fixation of the Intestinal Canal in the Perfusion System and Analyzing its Functional Activity 

To begin, turn on the in vitro tissue perfusion system. Adjust the instrument's bath temperature to 37 degrees Celsius. Then, place the 37 degrees Celsius physiological salt solution, or PSS, into the bath.Prepare a 15 centimeter surgical suture and soak it in four degrees Celsius PSS saturated with 95%oxygen and 5%carbon dioxide. Secure one end of the intestinal canal with the suture and use a steel needle hook to secure the other end. Next, 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. Now, open the data acquisition software and click on Start to ensure the corresponding path signal is being recorded.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 software sets the intestinal tubes'initial tension to zero grams. Then, rotate the spiral axis of the bath clockwise to pull the tension value to one gram and stabilize it in the 37 degrees Celsius PSS for 30 minutes.Observe the rhythmic spontaneous contraction waves in the software as this indicates a sufficient response. Next, add the test drug to the bath to study its effect on intestinal tube function. Afterward, stop the data acquisition software and perform data analysis using the software.To edit the data board and select the analysis parameters, click on Window, Data Pad, and choose the average tension for the channel. Select the contraction curve before administration and click add to data pad. Then, 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. Then click on Window, Data Pad to copy data to other statistical analysis software for further analysis. Analyze other parameters such as average amplitude, average frequency, and integral area under the curve.Replace the average tension with the respective parameter. Afterward, select the curve. Click on Edit, Copy LabChart Data to copy the data to drawing software for visualization.The study examined the effects of various agents on the tension of isolated intestinal tube rings. Acetylcholine significantly enhanced tension in these tubes. Conversely, atropine inhibited the tension caused by acetylcholine.Similarly, barium chloride also enhanced tension. On the other hand, both epinephrine and nifedipine inhibited tension in the isolated intestinal tube rings.