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....

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, includ....

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