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

Summary

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.

Abstract

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.

Introduction

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 diseases^6,7.

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'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 materialize^9,10. 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' 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'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.

Protocol

This protocol is derived from previously published literature^14,15,16,17. 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

1. 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∙6H₂O, 25 mM NaHCO₃, 11 mM D-glucose, and 5 mM HEPES (see the Table of Materials).
2. 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.
3. Precool 1/3 of the PSS to 4 °C and prewarm the rest to 37 °C for subsequent experiments.
   NOTE: the PSS was originally prepared at room temperature in steps 1.1-1.2

2. Rat intestinal canal dissection

1. Gather Petri dishes filled with 4°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.
2. Quickly place the stomach and intestine tubes in a Petri dish filled with 4 °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's edge with scissors. Subsequently, divide the intestine into 1-2 cm segments; this entire process is illustrated in Figure 1.

3. Suspension and fixation of the intestinal canal (Figure 2)

1. Turn on the in vitro tissue perfusion system and adjust the bath temperature in the instrument to 37 °C. Place the PSS (37 °C) into the bath.
2. Prepare a 15 cm surgical suture (see the Table of Materials) and soak it in 4 °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.
3. 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.
4. Open the data acquisition software (see the Table of Materials) and click on Start to ensure the corresponding path signal is being recorded.

4. Normalization

1. 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.
2. 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 °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.

5. Detection of reactivity

1. Observe the rhythmic spontaneous contraction waves in the software and proceed to the next experiment as this indicates a sufficient response.

6. Experimental observation

1. 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'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.

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.

1. 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.
2. 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.
3. Click on Window-Data Pad to copy data to other statistical analysis software (see the Table of Materials) for statistical analysis.
4. 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.
5. 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.

8. Postsurgical treatment

1. After surgery, euthanize the animals following institutionally approved protocols.
   NOTE: For the present study, the animals were euthanized by inhaling excess isoflurane.

Results

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 µmol/L) and barium chloride (1.2 mmol/L) enhanced tension in these tubes, while atropine (300 µmol/L), epinephrine (1 µmol/L) and nifedipine (10 µmol/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 α and β 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 α and β receptors (Figure 3C). It excites the α receptor and β₂ 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.

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 °Cphysiological salt solution; (C) intestinal tube after removal of surrounding tissue; (D) 2 cm long intestinal tube used in the experiment. Please click here to view a larger version of this figure.

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 °Cphysiological salt solution; (C) fixing the intestinal tube in the one of baths containing 37 °Cphysiological salt solution; (D) giving preload and recording normal waveform. Please click here to view a larger version of this figure.

Figure 3: Effects of drugs on isolated intestinal tubes. Effects of (A) acetylcholine (0.3 µmol/L) and atropine (300 µmol/L), (B) barium chloride (1.2 mmol/L), (C) epinephrine (1 µmol/L), and (D) nifedipine (10 µmol/L) on isolated intestinal tubes. Please click here to view a larger version of this figure.

Discussion

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 contraction^20,21. 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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetylcholine	Sigma, USA	A6625
atropine	Sangon Biotech Co., Ltd., Shanghai, China	IA06501
Barium chloride	Macklin Biochemical Co.,Ltd.,Shanghai, China	B861682
CaCl2	Sangon Biotech Co., Ltd., Shanghai, China	A501330
D-glucose	Sangon Biotech Co., Ltd., Shanghai, China	A610219
drawing software	GraphPad Software, San Diego, California, USA	—
Epinephrine	Sigma, USA	E4642
HEPES	Xiya Reagent Co., Ltd., Shandong, China	S3872
In vitro tissue perfusion system	PowerLab, ADInstruments, Australia	ML0146
KCl	Sangon Biotech Co., Ltd., Shanghai, China	A100395
KH2PO4	Sangon Biotech Co., Ltd., Shanghai, China	A100781
LabChart Professional version 8.3	ADInstruments, Australia	—
MgCl2·6H2O	Sangon Biotech Co., Ltd., Shanghai, China	A100288
NaCl	Sangon Biotech Co., Ltd., Shanghai, China	A100241
NaHCO3	Sangon Biotech Co., Ltd., Shanghai, China	A100865
nifedipine	Macklin Biochemical Co.,Ltd.,Shanghai, China	N5087
statistical analysis software	GraphPad Software, San Diego, California, USA	—
Surgical sutures	Johnson, USA	—