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 nerv...

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 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, 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 effe...

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 ...

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362 Views.  Chengdu University of Traditional Chinese Medicine. 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.

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

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion

There is currently a severe shortage of liver grafts available for transplantation. Novel organ preservation techniques are needed to expand the pool of donor livers. Machine perfusion of donor liver grafts is an alternative to traditional cold storage of livers and holds much promise as a modality to expand the donor organ pool. We have recently described the potential benefit of subnormothermic machine perfusion of human livers. Machine perfused livers showed improving function and restoration of tissue ATP levels. Additionally, machine perfusion of liver grafts at subnormothermic temperatures allows for objective assessment of the functionality and suitability of a liver for transplantation. In these ways a great many livers that were previously discarded due to their suboptimal quality can be rescued via the restorative effects of machine perfusion and utilized for transplantation. Here we describe this technique of subnormothermic machine perfusion in detail. Human liver grafts allocated for research are perfused via the hepatic artery and portal vein with an acellular oxygenated perfusate at 21 &#176;C.

We describe a method of ex vivo machine perfusion of human liver grafts at subnormothermic temperature (21 &#176;C).

There is currently a severe shortage of liver grafts available for transplantation.  Novel organ preservation techniques are needed to expand the pool of ...

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily and highly reproducible MI induction procedure is required to obtain further insight and better understanding of the underlying pathological changes. This procedure can also be used to evaluate the effects or potency of new and promising treatments (as drugs or interventions) in acute MI, subsequent remodeling and heart failure (HF). After intubation and pre-operative preparation of the animal, an anesthetic protocol with isoflurane was performed, and the surgical procedure was conducted&#160;quickly. Using a minimally invasive approach, the left anterior descending artery (LAD) was located and occluded by a ligature. The occlusion can be performed acutely for subsequent reperfusion (ischemia/reperfusion injury). Alternatively, the vessel can be ligated permanently to investigate the development of chronic MI, remodeling or HF. Despite common pitfalls, the drop-out rates are minimal. Various treatments such as remote ischemic conditioning can be examined for their cardioprotective potential pre-, peri- and post-operatively. The post-operative recovery was quick as the anesthesia was precisely controlled and the duration of the operation was short. Post-operative analgesia was administered for three days. The minimally invasive procedure reduces the risk of infection and inflammation. Furthermore, it facilitates rapid recovery. The &#8220;working heart&#8221; measurements were performed ex vivo and enabled precise control of preload, afterload and flow. This procedure requires specific equipment and training for adequate performance. This manuscript provides a detailed step-by-step introduction for conducting these measurements.

This article presents an efficient method to perform myocardial ischemia and subsequent chronic reperfusion in rats using a minimally invasive approach. In addition, left ventricular hemodynamic function of rats is assessed by echocardiography and isolated working heart methods.&#160;

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...

Laser Doppler Perfusion Imaging in the Mouse Hindlimb

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is a common, noninvasive, repeatable method for assessing blood flow recovery. The technique calculates overall blood flow in the sampled tissue from the Doppler shift in frequency caused when a laser hits moving red blood cells. Measurements are expressed in arbitrary perfusion units, so the contralateral non-intervened upon leg is usually used to help control measurements. Measurement depth is in the range of 0.3-1 mm; for hindlimb ischemia, this means that dermal perfusion is assessed. Dermal perfusion is dependent on several factors&#8212;most importantly skin temperature and anesthetic agent, which must be carefully controlled to result in reliable readings. Furthermore, hair and skin pigmentation can alter the ability of the laser to either reach or penetrate to the dermis. This article demonstrates the technique of LDPI in the mouse hindlimb.

Here, we present a protocol that demonstrates the technique and necessary controls for Laser Doppler perfusion imaging to measure blood flow in the mouse hindlimb.

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is ...

Isolated Lung Perfusion System in the Rabbit Model

The isolated lung perfusion system has been widely used in pulmonary research, contributing to elucidate the lungs&#39; inner workings, both micro and macroscopically. This technique is useful in the characterization of pulmonary physiology and pathology by measuring metabolic activities and respiratory functions, including interactions between circulatory substances and the effects of inhaled or perfused substances, as in drug testing. While in vitro methods involve the slicing and culturing of tissues, the isolated ex vivo lung perfusion system allows to work with a complete functional organ making possible the study of a continuous physiological function while recreating ventilation and perfusion. However, it should be noted that the effects of the absence of central innervation and lymphatic drainage still have to be fully assessed. This protocol aims to describe the assembly of the isolated lung apparatus, followed by the surgical extraction and cannulation of lungs and heart from experimental lab animals, as well as to display the perfusion technique and signal processing of data. The average viability of the isolated lung ranges between 5-8 h; during this period, the pulmonary capillary permeability increases, causing edema and lung injury. The functionality of preserved pulmonary tissue is measured by the capillary filtration coefficient (Kfc), used to determine the extent of pulmonary edema through time.

The isolated rabbit lung preparation is a gold standard tool in pulmonary research. This publication aims to describe the technique as developed for the study of physiological and pathological mechanisms involved in airway reactivity, lung preservation, and preclinical research in lung transplantation and pulmonary edema.

The isolated lung perfusion system has been widely used in pulmonary research, contributing to elucidate the lungs&#39; inner workings, both micro and ...

Assessing Blood Hypercoagulation of EV-Rich Plasma

Determination of the Procoagulant Activity of Extracellular Vesicle (EV) Using EV-Activated Clotting Time (EV-ACT)

The role of extracellular vesicles (EV) in various diseases is gaining increased attention, particularly due to their potent procoagulant activity. However, there is an urgent need for a bedside test to assess the procoagulant activity of EV in clinical settings. This study proposes the use of thrombin activation time of EV-rich plasma as a measure of EV's procoagulant activity. Standardized procedures were employed to obtain sodium-citrated whole blood, followed by differential centrifugation to obtain EV-rich plasma. The EV-rich plasma and calcium chloride were added to the test cup, and the changes in viscoelasticity were monitored in real-time using an analyzer. The natural coagulation time of EV-rich plasma, referred to as EV-ACT, was determined. The results revealed a significant increase in EV-ACT when EV was removed from plasma obtained from healthy volunteers, while it significantly decreased when EV was enriched. Furthermore, EV-ACT was considerably shortened in human samples from preeclampsia, hip fracture, and lung cancer, indicating elevated levels of plasma EV and promotion of blood hypercoagulation. With its simple and rapid procedure, EV-ACT shows promise as a bedside test for evaluating coagulation function in patients with high plasma EV levels.

Author Spotlight: Deciphering Coagulation Disorders in Traumatic Brain Injury Patients 

This protocol investigates the use of extracellular vesicle (EV)-rich plasma as an indicator of the coagulative ability of EV. EV-rich plasma is obtained through a process of differential centrifugation and subsequent recalcification.

The role of extracellular vesicles (EV) in various diseases is gaining increased attention, particularly due to their potent procoagulant activity. ...

Monitoring Cardiac Function of Rats Using the Pressure-Volume Conductance Technique

Real-Time Detection of Ferulic Acid Effects on Rat Left Ventricle Using Pressure-Volume Conductivity Catheter

Decreased cardiac function can have a negative impact on other organs. The left ventricular pressure-volume relationship is considered to be a valid method for evaluating cardiac function. Real-time monitoring of cardiac function is important for drug evaluation. Under closed-chest conditions, the miniature transducer, which is an important component of the pressure-volume catheter, enters the left ventricle of the rat through the right carotid artery. The device visualizes the changes in cardiac function during the experiment in the form of a pressure-volume loop. The actual volume of the ventricle is calculated by altering the conductivity of the blood by injecting 50 &#181;L of a 20% sodium chloride solution into the rat's left jugular vein. The actual volume of the rat's ventricular cavity is calculated by measuring the conductivity of the blood in a known volume using a pressure-volume conductance catheter. This protocol allows for continuous observation of the effects of drugs on the heart and will promote the rationale for the use of specialty ethnic drugs in cardiovascular disease.

Author Spotlight: Advantages of Pressure-Volume Loop Measurement Method in Cardiovascular Research

This protocol describes a method for measuring left ventricular pressure and volume using the pressure-volume conductance technique. This method enables continuous real-time monitoring of the effects of drugs on the heart.

Decreased cardiac function can have a negative impact on other organs. The left ventricular pressure-volume relationship is considered to be a valid ...

Enhancing Pulmonary Infection Treatment Using M-ROSE

Microbiological Rapid On-Site Evaluation for Pulmonary Infectious Diseases

The prompt initiation of empirical anti-infective therapy is crucial in patients presenting with unexplained pulmonary infection. Although imaging acquisition is relatively straightforward in clinical practice, its lack of specificity often necessitates additional time-consuming tests such as sputum culture, bronchoalveolar-lavage fluid culture, or genetic sequencing to identify the underlying etiology of the disease accurately. Moreover, the limited efficacy of empirical anti-infective treatment may contribute to antibiotic misuse. Recent advancements in interpreting microbial background on rapid on-site evaluation (ROSE) slides have enabled clinicians to promptly obtain samples through bronchoscopy (e.g., alveolar lavage, mucosal brushing, tissue clamp), facilitating bedside staining and interpretation that provides essential microbial background information. Consequently, this establishes a foundation for developing targeted anti-infection treatment and individualized drug therapy plans. With a better understanding of which pathogens are causing infections in real-time, physicians can avoid unnecessary broad-spectrum antibiotics contributing to antibiotic resistance. Establishing a rapid and standardized M-ROSE workflow within respiratory medicine departments or intensive care units will greatly assist physicians in formulating accurate treatment strategies for patients, which holds significant clinical implications.

Author Spotlight: Advancing Pathogen Detection and Disease Assessment in Real-Time Using M-ROSE

The protocol here demonstrates a fast and standardized microbiological rapid on-site evaluation (M-ROSE) workflow, including three steps: slide making, staining, and interpretation. This protocol will help physicians make rapid clinical decisions.