Standardized Preparation of Isolated Rabbit Duodenal Smooth Muscle For In Vitro Pharmacological Assessment of Contractile Activity

Summary

Here, we present a protocol to standardize the preparation of isolated rabbit duodenal smooth muscle for in vitro pharmacological assessment, utilizing a Horizontal Constant-Temperature Smooth Muscle Experiment System for real-time data recording of contractile activity.

Abstract

The coordinated contractile activity of gastrointestinal smooth muscle forms the physiological basis for maintaining normal digestion, absorption, and transport functions, with its dysfunction closely associated with motility disorders. The isolated organ perfusion technique eliminates complex in vivo interferences—such as neural, endocrine, and hemodynamic factors—serving as a classic model for pharmacological studies. To this end, this study details a stepwise protocol for the standardized preparation of rabbit isolated duodenal smooth muscle. The procedural workflow highlights the critical steps of preparing solutions, euthanizing the rabbit, performing a precise aseptic mid-abdominal laparotomy for rapid tissue excision, suspending the smooth muscle strips, and calibrating transducers for real-time tension recording. Based on this technical platform, we further validated the method by evaluating the effects of Huoxiang Zhengqi Oral Liquid (HXZQ-OL) on spontaneous contractions and acetylcholine/barium chloride-induced tetanic contractions. In conclusion, this protocol yields a robust and highly reproducible methodological framework, providing a broadly applicable in vitro screening platform for the pharmacological assessment of therapeutic agents targeting gastrointestinal motility.

Introduction

The coordinated contractile activity of gastrointestinal smooth muscle serves as the physiological foundation for maintaining normal digestive, absorptive, and transport functions¹. Its dysfunction is closely associated with various common clinical disorders, including irritable bowel syndrome², functional dyspepsia³, and postoperative gastrointestinal motility disorders⁴. Elucidating the molecular and pharmacological mechanisms regulating smooth muscle activity is crucial for developing novel prokinetic or antispasmodic therapies. In this research field, the isolated organ perfusion technique serves as a classic model. It offers practical value by eliminating complex in vivo interferences such as neural, endocrine, and hemodynamic factors, thereby enabling direct and precise assessment of the effects of drugs or active substances on smooth muscle tone^5,6.

Establishing stable and reliable protocols for in vitro tissue preparation and data recording is essential for obtaining reproducible, high-quality results. Existing in vitro intestinal muscle experimental methods require further optimization and standardization in several areas: standardized management of experimental animals, meticulous surgical techniques for tissue harvesting, consistent environmental control of perfusion systems, and automated data acquisition coupled with standardized analysis. For instance, perfusion fluid temperature and pH levels, mechanical damage during tissue harvesting, and tissue preload settings can all significantly impact tissue viability and introduce experimental errors. These factors compromise the reliability of results and hinder comparability across different studies^7,8.

Therefore, this study aims to establish and elaborate a standardized protocol for preparing isolated duodenal smooth muscle specimens from rabbits and performing real-time tension recording. This protocol systematically describes the entire process, from experimental animal ethics and welfare, aseptic laparotomy, and precise intestinal segment isolation, to achieving environmental control, standardized tension preload settings, and high-fidelity data acquisition using an integrated horizontal constant-temperature perfusion system and a multi-channel physiological signal acquisition system. Based on this standardized technical platform, we further applied cumulative dosing methods to quantitatively evaluate the effects of Huoxiang Zhengqi Oral Liquid (HXZQ-OL), meeting the quality standards of the Chinese Pharmacopoeia (2025 edition)⁹, on spontaneous contractions and acetylcholine- and barium chloride-induced tetanic contractions in isolated duodenal smooth muscle. Huoxiang Zhengqi Oral Liquid (HXZQ-OL), an oral dosage form of the classic Huoxiang Zhengqi traditional Chinese medicine formula, is commonly used in China for gastrointestinal disorders, providing the rationale for evaluating its direct effects on isolated intestinal smooth muscle. This study not only provides direct experimental pharmacological evidence elucidating the gastrointestinal smooth muscle regulatory effects of HXZQ-OL, but the experimental protocol also serves as a standardized, reproducible methodological model for pharmacological screening and in vitro tissue studies of gastrointestinal smooth muscle.

Protocol

Healthy adult New Zealand White rabbits of either sex (weighing 2.0–2.5 kg) were used in this work. All animal experimental procedures were conducted in accordance with the guidelines for the care and use of laboratory animals and were approved by the Laboratory Animal Ethics Committee of Chengdu University of Traditional Chinese Medicine (Approval No.: [2026070]). All efforts were made to minimize animal suffering and to reduce the number of animals used. The workflow diagram is illustrated in Figure 1.

1. Solution preparation

NOTE: All working solutions, including Tyrode's solution, acetylcholine (ACh), barium chloride, atropine, and verapamil, must be prepared fresh immediately before the experiment.

1. Prepare 1 L of Tyrode's solution by dissolving 137 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.4 mM NaH₂PO₄, 1.0 mM MgCl₂, 12 mM NaHCO₃, and 5.6 mM D-glucose. Before use, saturate with a 95% O₂ / 5% CO₂ mixture for 15–30 min, adjusting the pH to 7.3–7.4 (see the Table of Materials).
2. Prepare a 1 mM ACh stock solution. Take 20 µL and add to 20 mL of the Tyrode's solution. Mix thoroughly to obtain a 1 µM ACh solution.
3. Prepare a 1 M barium chloride (BaCl₂) stock solution. Take 20 µL and add to 20 mL of Tyrode's solution. Mix thoroughly to obtain a 1 mM BaCl₂ solution.
   NOTE: BaCl₂ is highly toxic and irritating. When handling, wear personal protective equipment in a fume hood and dispose of waste liquid in accordance with regulations.
4. Prepare a 10 mM atropine sulfate stock solution. Take 20 µL and add to 20 mL of Tyrode's solution. Mix thoroughly to obtain a 10 µM atropine sulfate solution.
5. Prepare a 1 mM verapamil hydrochloride stock solution. Take 2 µL and add to 20 mL of Tyrode's solution. Mix thoroughly to obtain a 0.1 µM verapamil hydrochloride solution.
   NOTE: The concentrations of the prepared solutions were determined by referencing previous studies^6,10,11 and were further optimized through our preliminary experiments to ensure optimal experimental conditions.

2. Preparation of in vitro rabbit duodenal smooth muscle specimens

1. Rabbit euthanasia
   1. Secure the rabbit in the rabbit restraint device (Figure 2A,B and Figure 3A). Perform euthanasia by administering an overdose of sodium pentobarbital (120 mg/kg) via the auricular vein in rabbits (Figure 2C and Figure 3B).
      ​NOTE: Complete death must be confirmed by the following criteria: dilated pupils, absence of pupillary light reflex, cessation of respiration and heartbeat, and loss of plantar reflex.
2. Preparation and disinfection of the surgical site
   1. Secure the rabbit in the supine position on the operating table (Figure 2D and Figure 3C). Using surgical scissors (Figure 2E) or an electric shaver, thoroughly remove all abdominal hair to expose the skin in the designated surgical area (Figure 3D).
   2. Using sponge forceps (Figure 2E), grasp an iodine-soaked cotton ball (Figure 2F,G). Starting at the center of the planned surgical incision, apply the antiseptic solution in a concentric, circular pattern from the inside out (Figure 3E).
      NOTE: The disinfection area should extend beyond the incision edges to ensure complete coverage of the entire surgical field skin. After disinfection, wait at least 3 min to allow the antiseptic to dry naturally on the skin surface.
   3. Place a sterile drape (Figure 2H) over the disinfected abdominal area to expose the surgical field (Figure 3F).
3. Positioning and incision of the abdominal incision
   1. Using a scalpel (blade #23) (Figure 2E), make an 8 cm longitudinal incision along the midline of the abdomen from below the xiphoid process to above the symphysis pubis.
   2. Dissect the skin and subcutaneous tissue layer by layer. Following the linea alba, dissect the anterior sheath of the rectus abdominis muscle, muscle fibers, and peritoneum layer by layer to enter the abdominal cavity (Figure 3G).
   3. NOTE: During the procedure, avoid damaging abdominal organs while ensuring hemostasis to prevent blood contamination of the surgical field and abdominal organs.
4. Duodenal separation
   1. Open the abdominal cavity along the incision (Figure 3H). Gently retract the hepatic lobes cephalad using moist saline gauze (Figure 2I) to fully expose the stomach and duodenum (Figure 3I).
   2. Select the initial segment of the duodenum (bulbar or upper descending portion) located 2–3 cm from the pylorus as the tissue harvesting site.
   3. NOTE: Avoid direct traction or clamping of the target intestinal segment whenever possible to minimize mechanical injury. Instead, use blunt forceps to gently grasp the adjacent mesentery, or use saline-moistened cotton swabs to delicately manipulate and position the tissue.
   4. Using sterile ophthalmic scissors (Figure 2E), quickly and accurately resect a 1–2 cm (Figure 2J) segment of the duodenal tube (Figure 3J,K).
   5. Immediately transfer the excised intestinal segment to a culture dish (Figure 2K) containing prechilled (4 °C) Tyrode's solution continuously perfused with a mixed gas (95% O₂ / 5% CO₂).
5. Intestinal segment trimming, cleaning, and temporary storage
   1. Using ophthalmic forceps (Figure 2E), gently dissect the mesentery and surrounding adipose tissue adhering to the intestinal wall.
   2. Using a syringe filled with cold Tyrode's solution (equipped with a blunt-tip needle) (Figure 2L), gently inject the enema solution into the intestinal cavity. Slowly flush the interior of the intestinal cavity 2–3x to thoroughly clear its contents.
   3. Place the cleaned intestinal segments in fresh oxygenated cold Tyrode's solution for temporary storage until subsequent experiments.

3. Horizontal constant-temperature smooth muscle experimental system startup and solution infusion

1. Add Tyrode's solution to the preheating dish of the DSQG-1 Horizontal Constant-Temperature Smooth Muscle Experiment System (Figure 4B; see the Table of Materials).
2. Turn on the system power, touch the operation panel to set the bath temperature to 37 °C, and begin preheating (Figure 4C).
3. Press the Infusion button, and the system will automatically dispense 20 mL of Tyrode's solution into the bath tank.
4. Press the Drainage button to drain the liquid into the waste collection container completely.
5. Press the Rinse button, and the system will automatically complete the process of first draining the contents and then refilling with liquid.
6. Press the Stop button to immediately terminate all ongoing infusion, drainage, and rinse processes.
7. Press the Inflation button to continuously introduce the mixed gas (95% O₂ / 5% CO₂) into the bath. Adjust the gas flow rate to scale 5 to ensure subsequent experimental tissue oxygenation.
   NOTE: Scale 5 indicates a gas flow rate of 100 mL/min. Excessively high gas flow rates may cause increased solution turbulence, affecting the stability of tension recordings.

4. Connecting multi-channel physiological recording and processing system with software operation

1. Connect the data cable of the Horizontal Constant-Temperature Smooth Muscle Experiment System transducer to Channel 1 of the Multi-Channel Physiological Recording and Processing System (Figure 4D).
2. Turn on the external power supply for the Multi-Channel Physiological Recording and Processing System and start the computer. Double-click the RM6240XC Multi-Channel Physiological Recording and Processing System icon on the desktop to launch the experimental software system (see the Table of Materials).
3. In the menu bar, select Experiment | Digestion | Physiological Characteristics of Gut Smooth Muscle to load the corresponding experimental module.
4. Set the following parameters: Channel mode to Tension, sampling frequency to 400 Hz, sensitivity to 1.5 g, time constant to DC, and filter frequency to 30 Hz.
5. Click the Start button in the upper-right corner of the software interface to begin real-time signal acquisition and display. Click the Record button to simultaneously display the oscilloscope and save the acquired signal to the hard drive in real time.
6. In the menu bar, select Experiment | Rapid Zeroing to adjust the tension signal to the baseline level.
   NOTE: If the signal deviates from the baseline by more than one vertical coordinate unit, manually adjust the zero point of the tension transducer using a screwdriver.
7. In the menu bar, select Experiment | Real time data display method | floating plate. In the dialog box that appears, choose Normal Realtime Measurement-Fast to display real-time tension data of the intestinal muscle in the floating window.

5. Suspension and tension preload setting of duodenal muscle strip

1. Adjust the front-to-back spacing of the tissue hooks by rotating the horizontal screw knob according to the length of the duodenal muscle strip, ensuring it matches the strip's length.
2. Maintain the intestinal segment in its natural cylindrical configuration, and align the line of sight with the circular cross-sectional opening of the intestinal lumen.
3. Using ophthalmic forceps, gently retract the intestinal opening to maintain patency.
4. Gently insert the tip of the tissue hook into the intestinal lumen.
5. At a distance of 2–3 mm from the intestinal margin, pierce the upper single-layer intestinal wall from the inside out using the tip of the tissue hook.
   NOTE: The tissue hook must penetrate the muscle layer to ensure secure anchoring, avoiding superficial attachment only to the serosal layer, so as to prevent mechanical cutting and tearing of the intestinal wall by the hook when preload is applied.
6. The other side of the intestinal wall is identical to the first side. After identifying the parallel corresponding point, penetrate only the single-layer intestinal wall at that point (Figure 3L).
7. Rotate the horizontal screw knob again to adjust the hook spacing, ensuring the duodenal muscle strip remains naturally relaxed.
8. Gradually apply tension by adjusting the one-dimensional fine adjuster until the average tension of the duodenal muscle strip stabilizes at 0.8 ± 0.1 g (Figure 4A).
   NOTE: Visually inspect the entire length of the intestine to ensure it remains straight and cylindrical, free of twisting, rotation, or spiral folds. If this is not the case, immediately reduce the tension and readjust the mounting point at one end.
9. Before commencing the experiment, equilibrate the duodenal muscle strip in the perfusion chamber for 60 min. During this equilibration period, replace the Tyrode's solution with fresh buffer every 15–20 min until its spontaneous contractile rhythm stabilizes.
   NOTE: The 60 min equilibration period was considered complete only when the isolated intestinal segments exhibited stable spontaneous contractile activity for at least 15 consecutive min. The objective criteria for stabilization were defined quantitatively as follows: (1) baseline resting tension drift of < ±5%, (2) variance in mean contractile amplitude of < ±10% across consecutive 5 min intervals, and (3) frequency fluctuation of < ± 1 cycle/min.
10. Record the spontaneous contraction activity curve of duodenal muscle strip as baseline reference data for subsequent experiments.

6. Experimental data acquisition

1. For ongoing experiments, in the menu bar, select Tools | Split view to perform real-time statistical analysis on captured waveform data.
2. For stored data files, click the Open button in the toolbar, select a saved data file with the .lsd extension, and load historical experiment records.
3. Click the Measure icon in the toolbar, select Region mode. Move the mouse cursor over the waveform area, hold down the left mouse button to select any two points, and the system will display a data panel showing the average tension value within that segment.
4. Click the Measure icon in the toolbar, select Period mode. Using the left mouse button, click on five consecutive distinct wave peaks (peak points) along the waveform. Right-click the mouse, and the data panel will calculate and display the contraction frequency (in bpm) (Supplemental Figure S1).

Results

This experimental protocol describes the standardized preparation method for isolated rabbit duodenal muscle strips and the process of recording their dynamic tension and frequency changes in real time using an isolated organ perfusion system. Following this protocol, HXZQ-OL was sequentially added via cumulative dosing into a 20 mL Tyrode's solution bath, achieving final volume concentrations of 10, 20, 30, 40, and 50 µL/mL in the bath. Referencing the standards of the Chinese Pharmacopoeia (2025 edition), these concent...

Discussion

Isolated organ perfusion and tension recording techniques represent classic methods for studying smooth muscle physiology and pharmacology. Their findings hold significant importance for elucidating the pathophysiological mechanisms underlying gastrointestinal motility disorders, vascular diseases, and uterine dysfunction^12,13,14,15. Using a standardized rabbit ex vivo duodenal model, ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.5-10 µL Pipette	Dalong Laboratory Instruments Co., Ltd.	7010101004
10 µL Pipette tips	LABSELECT	T-001-10
10 mL Volumetric flask	Sichuan Shuniu Glass Instrument Co., Ltd.	B-043804
100 mL Volumetric flask	Sichuan Shuniu Glass Instrument Co., Ltd.	B-043807
100-1000 µL Pipette	Dalong Laboratory Instruments Co., Ltd.	7010101014
1250 µL Pipette tips	LABSELECT	T-001-1250
20 mL Volumetric flask	Sichuan Shuniu Glass Instrument Co., Ltd.	B-010105
200 µL Pipette tips	LABSELECT	T-001-200
50 mL Conical centrifuge tube	LABSELECT	CT-002-50A
5-50 µL Pipette	Dalong Laboratory Instruments Co., Ltd.	7010101006
Acetylcholine chloride	Supelco	PHR1546
All-in-One Lab Computer	Lenovo	R5-3500U
Atropine sulfate	Macklin	A822893
Barium chloride	Sigma-Aldrich	342920
Calcium chloride	Sigma-Aldrich	499609
D-Glucose	Sigma-Aldrich	G5767
Freezing point osmometer	Shanghai Precision Instruments Co., Ltd.	BS100
Horizontal Constant-Temperature Smooth Muscle Experiment System	Chengdu Instrument Factory	DSQG-1
Huoxiang Zhengqi Oral Liquid	Taiji Group Co., Ltd.	Z50020409
Magnesium chloride	Sigma-Aldrich	M8266
Multi-Channel Physiological Recording and Processing System	Chengdu Instrument Factory	RM6240XC
New Zealand White rabbits	Pizhou Dongfang Breeding Co., Ltd. (License No.: SCXK[Su]2022-0004)	either sex (weighing 2.0–2.5 kg)
pH meter	Shanghai Leici	PHS-25
Potassium chloride	Sigma-Aldrich	P5405
Povidone-iodine	H&H Medical Co., Ltd.	3180132
Precision balance (0.1 mg)	Sartorius	BCE64i
Saline solution	H&H Medical Co., Ltd.	100014438029
Sodium bicarbonate	Sigma-Aldrich	S6014
Sodium chloride	Sigma-Aldrich	S9888
Sodium dihydrogen phosphate	Supelco	1063700250
Sodium Pentobarbital	Sigma-Aldrich	P3761
Verapamil Hydrochloride	Macklin	V820460
Vortex mixer	Kylin-Bell	XW-80A