Real-Time Monitoring of Acupuncture-Induced Dynamics in Subcutaneous Connective Tissue via In Vivo Laser Confocal Imaging

Summary

This study establishes in vivo laser confocal imaging as a real-time method for tracking acupuncture-induced morphological dynamics, such as vasodilation and collagen remodeling, in live rats. Using fluorescein sodium labeling, the approach captures both immediate and delayed tissue responses, offering a new tool for quantitatively evaluating physical therapies, including acupuncture and moxibustion.

Abstract

Real-time dynamic monitoring of acupuncture effects is crucial for elucidating its biological mechanisms, yet traditional histological methods fail to capture transient responses in the live microenvironment. Here, we established a methodological framework based on in vivo laser confocal imaging, enabling high-resolution, real-time observation of morphological changes in local tissues following acupuncture intervention. After intravenous injection of sodium fluorescein in rats, dynamic alterations in microcirculation and extracellular matrix (e.g., collagen fiber arrangement) were tracked during acupuncture. The results demonstrated that this method clearly distinguished immediate (e.g., vasodilation) and delayed (e.g., inflammatory response) tissue reactions induced by acupuncture, with a spatial resolution of 0.5 µm and a temporal resolution of 3.5 fps. Subsequent quantitative analysis enabled objective measurement of morphological parameters. This protocol, described in this study, provides a visualization tool for in vivo studies of acupuncture mechanisms and serves as a technical reference for evaluating the effects of other physical stimuli (e.g., moxibustion).

Introduction

Acupuncture, as a cornerstone of traditional medicine, exerts therapeutic effects through complex tissue-level responses^1,2,3,4. However, mechanistic insights remain limited by conventional histology, which captures static snapshots but fails to resolve real-time dynamics of morphological adaptations (e.g., vasomodulation, extracellular matrix remodeling) in live tissue⁵. While intravital microscopy offers dynamic imaging potential, its application in acupuncture research is scarce, particularly for quantifying spatio....

Protocol

All procedures were approved by the Animal Ethics Committee of the Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences (No. D2023-01-29-01). Eight Male Sprague Dawley (SD) rats (8 weeks old, 200-220 g) were obtained from Biotechnology Co., Ltd. (License No. SCXK (Jing) 2020-0001). Animals were housed in sterilized isolation enclosures under controlled conditions: 12-h light-dark cycle, 21 ±± 2 °C temperature, with ad libitum access to standard chow pellets and water. All experiments complied with institutional guidelines and the Guide for the Care and Use of Laboratory Animals.​

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Results

This protocol demonstrates the utility of pCLE for real-time dynamic assessment of acupuncture's effects on microcirculation and extracellular matrix architecture. After intravenous administration of sodium fluorescein via the tail vein, changes were evaluated by observing alterations in erythrocyte velocity within local capillaries and reorganization of collagen fiber arrangement.

Representative results at 5 min post-acupunctur.......

Discussion

As demonstrated in the results, the in vivo confocal imaging protocol established in this study enables simultaneous quantitative analysis of microcirculatory and extracellular matrix dynamics during acupuncture intervention using standard commercially available equipment and routine fluorescent labeling. The success of this protocol relies on two critical steps: precise timing of sodium fluorescein injection, ensuring sustained microvascular contrast¹⁴, and rigorous animal immobilization.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 cun acupuncture needle	Hwato, Suzhou, China	N/A	13 mm/0.25 mm
10% fluorescein sodium	Guangxi Wuzhou Pharmaceutical Co Ltd, China	H45021477
Animal anesthesia machine	RWD, Shenzhen, China	R620IE
Body temperature monitor	RWD, Shenzhen, China	69026
Electric acupuncture apparatus	Hwato, Suzhou, China	SDZ-III
Hair removal cream	Veet, France
ImageJ	National Institutes of Health, USA	https://imagej.nih.gov/ij/
In vivo probe confocal laser endomicroscopy	Optiscan, Australia	ViewnVivoB30	Diameter: 4 mm handheld rigid probe, resistant to bending and maintains structural integrity over time. Length: ≥46 mm. Scanning Mode: Zoom scanning capability.
Isoflurane	RWD, Shenzhen, China	R510-22-16
Urethane	Sigma-Aldrich, USA	94300