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 spatiotemporal responses to physical stimuli^6,7. In contrast, this confocal imaging approach enables quantitative extraction of structural and dynamic parameters-such as the rate of change in red blood cell movement velocity and collagen arrangement-in deep tissues (e.g., muscle layers and acupoints ST 36/GB 34), overcoming the limitations of conventional intravital microscopy in spatial resolution, volumetric imaging, and long-term tracking⁸.

Laser confocal imaging has emerged as a powerful tool for in vivo visualization at cellular resolution^{9,10,11,12,13}. Yet, existing studies primarily focus on superficial tissues (e.g., skin or cornea), with few addressing deep acupuncture targets or integrating quantitative morphological analysis. Crucially, to our knowledge, no standardized methodology exists to correlate acupuncture manipulation with live-tissue kinetic changes.

Here, we bridge this gap by establishing a confocal imaging-based methodological platform for real-time tracking of acupuncture-induced morphological events. Using sodium fluorescein (administered intravenously at 1% concentration, 0.1 mL/100g body weight) as a contrast agent, we achieve high-resolution monitoring (0.5 µm spatial/3.5 fps temporal resolution) of microcirculatory and matrix alterations across a 450 µm × 450 µm field of view at up to 400 µm depth-from immediate vasodilation to delayed inflammatory cascades-during acupuncture stimulation. This approach further enables 3D reconstruction and parameter quantification of collagen fiber realignment, overcoming the limitations of endpoint histology.

The practical considerations for implementation of this protocol are (i) recommended hardware includes a confocal system with ≥0.5 µm resolution and ≥3 fps acquisition rate, (ii) typical limitations include photobleaching beyond 90 s of continuous imaging and sensitivity to respiratory or tissue movement, and (iii) depth penetration may be reduced in highly dense or scattering tissues. To our knowledge, this work not only provides the first dynamic atlas of acupuncture-evoked tissue reorganization but also sets a precedent for evaluating physical therapies (e.g., moxibustion) in live systems. By converting transient physiological responses into quantifiable metrics, we offer a new paradigm for mechanistic studies of neuromodulatory therapies.

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.​

NOTE: A flow chart highlighting the main procedures is shown in Figure 1.

1. Preparation of animal and tracer: Fluorescein Sodium (FS)

1. To prepare a 1% fluorescein sodium (FS) staining solution, dissolve 0.1 g of FS in 10 mL of sterile saline (0.9% NaCl). Store the solution at room temperature (RT) for subsequent use.
2. Anesthetize SD rats via isoflurane induction (induction: 5% isoflurane, maintenance: 2.5% isoflurane). Remove hair from the cranial region (excluding the head) using electric clippers, then apply depilatory cream for 5 min. Eliminate residual cream and hair by rinsing with warm water.
   ​NOTE: Maintain the clipper blade parallel to the skin surface during operation to prevent dermal injury that may compromise quantitative imaging analysis. Warm water must be strictly used for rinsing to avoid animal hypothermia affecting experimental outcomes.

2. Injection of FS

1. Secure the rat.
   1. Anesthetize hair-removed rats via intraperitoneal injection of 10% urethane (1 g/10 mL, in saline) at a dosage of 1.0 g/kg.
   2. Upon achieving full anesthesia, secure animals in a supine position on the operating platform. Apply a gentle toe pinch to the hind paw using forceps and observe the absence of withdrawal reflex. Verify the loss of corneal reflex by lightly touching the cornea with a moist cotton tip.
   3. Confirm sustained unresponsiveness and stable respiratory rate before proceeding with the procedure. During anesthesia, maintain the animal body temperature with a 37 °C heating pad.
      NOTE: Full anesthesia must be confirmed to prevent involuntary movements that would compromise relocation of the imaging window.
2. Intravenously administer 1% FS solution via the tail vein at a dosage of 0.1 mL per 100 g body weight.
   1. Perform tail vein injection using a 1 mL syringe fitted with a 27 G needle. Wipe the tail gently with warm water to promote vasodilation, followed by disinfection with 70% ethanol.
   2. Insert the needle bevel-up at a shallow angle (≤15°) along the longitudinal axis of the vein. Inject the solution slowly at a rate of 0.1-0.2 mL/min.
   3. Ensure technical resistance-free flow, absence of perivascular edema, and confirm successful injection by yellow discoloration of the pupils, pinnae, and paws.
      ​NOTE: Accidental intra-arterial injection must be strictly avoided.

3. In vivo probe confocal laser endomicroscopy (pCLE) set-up

1. Activate the pCLE system and verify the cleanliness of both the scanning probe connector and the distal tip.
2. Clean the distal tip of the probe (also termed the scanning tip) using a cotton swab moistened with physiological saline, then dry gently with lens tissue.
3. Position the laser probe at the target observation site (e.g., medial thigh region). Secure the confocal imaging probe using an adjustable clamp, ensuring stable contact with the tissue surface without compression artifacts, and fix it by tightening the adjustment knob.
4. Connect the probe to the pCLE system and configure acquisition parameters (e.g., field of view [FOV], storage directory). The specific parameters used in this experiment are as follows:Depth: 18 µm; Laser: 219 mW; Brightness: 76%; Speed: 1920 × 540; Gain: 2400; Zoom: 1× (464 µm); Filter: LP 515; Look Up Table: Grey Scan.
   NOTE: Imaging parameters (scanning depth/laser power) were iteratively optimized until a distinct visualization of tissue architecture was achieved. Consistent intensity is critical for longitudinal image comparability. Adding 37 °C saline droplets at the probe-tissue interface eliminated signal loss observed in initial iterations. The laser power used in this study is below the commonly accepted threshold for phototoxicity in in vivo confocal imaging.

4. Imaging of the organization structure with pCLE and acquisition

1. Position the probe on the skin surface and initiate data acquisition to visualize tissue architecture.
   1. Hold the probe in a pencil-grip manner; maneuver it to target regions (e.g., areas with vasculature or fibrous connective tissue within the field of view), place the distal tip directly on the imaging zone, then stabilize via the probe arm.
2. First, acquire baseline tissue architecture for 1 min under physiological conditions, followed by 5 min recording during the acupuncture intervention. Terminate acquisition post-intervention. Data files automatically save to the directory specified in step 3.3-3.4.
   NOTE: Files are saved as layer stacks (.tiff) and individual frames (.tiff).

5. Acupuncture intervention

NOTE: Acquisition in the imaging step is performed during the intervention.

1. Insert a 1-cun (the Chinese unit of length measurement is approximately 13 mm) disposable stainless steel needle into the acupoint adjacent to the probe-observed tissue. Connect one lead to the stainless steel needle, and secure the other reference electrode on the rat's tail.
2. Apply electroacupuncture (EA) stimulation using the following parameters: 1 mA intensity, dense-disperse wave (2/15 Hz), for 5 min.
   NOTE: Maintain sufficient distance (>3 mm) between the needle insertion site and the probe to prevent EA-induced dermal tremors that compromise image stability.

6. Image analysis

1. Open the acquired image stacks in ImageJ. Convert the spatial calibration to micrometers and transform the image format to 8-bit for plugin compatibility (Image > Type > 8-bit).
   ​NOTE: Default image dimensions are in inches (system-specific setting).
2. Define the region of interest (ROIs) for: microcirculation: single capillary segment, matrix structure: collagen fiber region. Maintain identical ROIs across sequential images for longitudinal consistency.
3. Microcirculation analysis
   1. Select the region containing the red blood cells that needs to be observed, and crop the image to remove other interfering areas (Rectangle > Image > Crop). Use the threshold tool to segment the red blood cells (RBC) exclusively (Image > Adjust > Threshold tool).
   2. Load the MTrack2 plugin. Set appropriate tracking parameters based on RBC size (in this experiment, 10-42 pixels) to exclude interference and ensure accurate RBC tracking (Plugin > Tracking > MTrack2).
4. Matrix structural analysis
   1. Load the Directionality plugin, select Fourier components mode, and perform statistical analysis on the distribution proportion of collagen fibers across 0° to 180° orientations (Analyze > Directionality).

7. Data finalization

1. Export the analyzed data in TIFF/XSL formats using the naming convention: [Group]_[Date].extension. Backup all raw and processed files to a designated secure server. Record quantitative outputs in a summary spreadsheet for statistical analysis.

8. Safety and waste management

1. Personal protective equipment (PPE): Wear nitrile gloves, a lab coat, and safety goggles when handling urethane, isoflurane, or fluorescein sodium.
2. Anesthetic handling: Perform procedures involving urethane or isoflurane within a fume hood to minimize inhalation exposure.
3. Sharps precautions: Dispose of all needles and sharp objects immediately in designated puncture-resistant sharps containers.
4. Electroacupuncture (EA) safety: Verify electrical isolation of EA equipment and secure lead connections to prevent leakage currents or short circuits.
5. Waste disposal: Discard isoflurane bottles as medical waste per institutional guidelines. Dispose of dye-contaminated materials (e.g., swabs, tissues) waste in sealed hazardous waste containers. Collect sharps in certified sharps disposal boxes.
   NOTE: All procedures must comply with institutional safety and environmental health regulations.

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