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).

This protocol establishes a comprehensive experimental framework for investigating cortical circuit dynamics during EA (electroacupuncture) stimulation. The methodology integrates three key components: chronic cranial window implantation in Thy1-GCaMP6f transgenic mice, standardized EA stimulation at the ST36 acupoint using 2/100 Hz biphasic pulses, and high-resolution two-photon calcium imaging. This integrated approach enables real-time visualization of neural ensemble activity in the primary somatosensory (S1) cortex with millisecond temporal precision, capturing previously inaccessible details of network-level responses to neuromodulation. The technique successfully reveals distinct layer-specific activation patterns and longitudinal plasticity changes, providing critical insights into the cortical mechanisms underlying the effects of EA. By overcoming the fundamental spatiotemporal limitations inherent in conventional fMRI and electrophysiological techniques, this high-resolution platform offers unprecedented analytical capabilities for mapping dynamic neural circuits. The protocol's robust design and reproducible outcomes make it particularly valuable for optimizing targeted neuromodulation therapies and advancing our understanding of circuit-level plasticity in response to peripheral stimulation.

This protocol establishes a comprehensive experimental framework for investigating cortical circuit dynamics during EA (electroacupuncture) stimulation. The methodology integrates three key components: chronic cranial window implantation in Thy1-GCaMP6f transgenic mice, standardized EA stimulation at the ST36 acupoint using 2/100 Hz biphasic pulses, and high-resolution two-photon calcium imaging. This integrated approach enables real-time visualization of neural ensemble activity in the primary somatosensory (S1) cortex with millisecond temporal precision, capturing previously inaccessible details of network-level responses to neuromodulation. The technique successfully reveals distinct layer-specific activation patterns and longitudinal plasticity changes, providing critical insights into the cortical mechanisms underlying the effects of EA. By overcoming the fundamental spatiotemporal limitations inherent in conventional fMRI and electrophysiological techniques, this high-resolution platform offers unprecedented analytical capabilities for mapping dynamic neural circuits. The protocol's robust design and reproducible outcomes make it particularly valuable for optimizing targeted neuromodulation therapies and advancing our understanding of circuit-level plasticity in response to peripheral stimulation.

Objective: This study explores the material basis and biological functions of meridian interstitial channels in mini-pigs proximal to the stomach meridian by analyzing differential proteomics between interstitial channels and adjacent non-interstitial channel tissues. Methods: Liquid chromatography–mass spectrometry (LC-MS) under data-dependent acquisition mode was employed to analyze and identify the proteome of subcutaneous connective tissues along the stomach meridian and adjacent tissues. SWATH MS^ALL method and omicsbean online analysis platforms were used for protein quantification and differential proteomic analysis. Differential proteins were subjected to Gene Ontology annotation and KEGG pathway analysis to understand their functions and biological processes. Combining traditional Chinese meridian theory with modern meridian research, proteins most relevant to meridian functions were selected, and their expression levels were assessed using Western blotting. Results: GO annotation and KEGG pathway analysis revealed differences in molecular functions, biological processes, and metabolic pathways among differential proteins. Most downregulated proteins were enzyme functional proteins involved in amino acid metabolism (GOT1), adenosine nucleotide balance conversion (AK1), and calcium ion-binding processes (ANXA6). Most upregulated proteins were structural proteins in the extracellular matrix—collagen proteins (COL3A1, COL6A1, COL6A3, COL6A6, COL12A1, COL14A1) and proteoglycans (DCN, BGN, FMOD)—involved in influencing and regulating collagen fiber generation and arrangement. Intriguingly, almost all differential proteins were associated with gastrointestinal diseases, implying a pathological correlation of differential proteins in the stomach meridian interstitial channel. Conclusions: The stomach meridian interstitial channels in mini-pigs show 72 differentially expressed proteins compared to adjacent tissues. These differences include the upregulation of structural proteins and downregulation of functional proteins, potentially forming the molecular biological basis for the structural and functional specificity of meridians.

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).