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. 2026 Jun 15;11(25):36801–36812. doi: 10.1021/acsomega.5c13674

Cutaneous TRPV4 Potentiates the Afferent Transmission of Electroacupuncture Stimulation

Hongyi Cheng †, Xia Li †,‡, Xinyan Gao †, Jing Feng §, Longhua Du †, Bing Zhu †, Kun Liu †,*
PMCID: PMC13325124  PMID: 42395984

Abstract

Electroacupuncture stimulation (ES) exerts therapeutic effects across various conditions, primarily by activating peripheral sensory nerves. The mechanosensitive ion channel transient receptor potential vanilloid 4 (TRPV4), expressed in peripheral non-neuronal cells, represents a potential molecular sensor for ES, yet its specific contribution to ES signal transmission remains unclear. In this study, we combined in vivo calcium imaging in transgenic mice with pharmacological interventions to investigate how cutaneous TRPV4 modulates afferent signaling at the lumbar 4 dorsal root ganglion (DRG) level. An ES intensity of 2 mA at the hindpaw activated more primary sensory and TRPV1-Cre-positive nociceptive neurons and evoked stronger calcium responses compared to 0.5 mA stimulation. Immunofluorescence analysis revealed TRPV4 expression in skin macrophages, but not in primary sensory neurons. To investigate the mechanistic link between ES and TRPV4, the response of primary sensory neurons to ES was assessed following the local activation or antagonism of cutaneous TRPV4. Intradermal injection of the TRPV4 agonist GSK1016790A increased the number of L4 DRG neurons exhibiting spontaneous calcium transients compared with vehicle controlswithout altering mean fluorescence intensityand produced a trend toward a greater proportion of neurons responding to ES alongside a significant enhancement in calcium transient intensity. Conversely, the TRPV4 antagonist GSK2193874 reduced the number of neurons showing basal calcium activity relative to vehicle and significantly attenuated ES-evoked calcium responses compared with the agonist group. These results demonstrate that non-neuronal TRPV4 in the skin potentiates afferent signaling during ES, underscoring a previously overlooked role for neuroimmune interaction in acupuncture signal initiation. These findings improve our understanding of how acupuncture initiates afferent signals at the cellular level and highlight cutaneous TRPV4 as a potential target for modulating ES-evoked afferent signaling.

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Introduction

Acupuncture achieves therapeutic effects by delivering mechanical or thermal stimulation to the body surface. Previous research indicates that these stimuli first activate local Aδ and C-fibers in the skin and subcutaneous tissues, conveying sensory input to neurons within the dorsal root ganglion (DRG). , Subsequent action potentials are then propagated to the central nervous system for integration before being relayed to target organs to mediate regulatory effects. Studies show that low-intensity 0.5 mA electroacupuncture stimulation (ES) at Zusanli (ST36) in mice elicits a systemic anti-inflammatory response via the vagus–adrenal axis. In contrast, a 0.8–1 mA ES excites C-fibers, with the acupuncture effects being mediated through the transient receptor potential vanilloid 1 (TRPV1) channel. , Ablation of TRPV1 receptorswhich are predominantly located on C-fiberswith resiniferatoxin (RTX) impairs acupuncture-mediated regulation of visceral functions such as bladder and gastrointestinal activity, underscoring the crucial role of C-type afferents. − Recent work using ChAT-Cre-GCaMP6s mice demonstrated that a 2 mA ES at Zusanli (ST36) activates TRPV1 nociceptors to drive a somato–vagal–gastric reflex that modulates gastric motility, whereas 0.5 mA and 1 mA intensities failed to evoke this afferent signal. These collective findings demonstrate that ES intensity critically shapes sensory afferent engagement and therapeutic outcomes.

Investigations have progressively extended to non-neuronal cells residing in the local acupoint microenvironment. Acupuncture, as a form of mechanical stimulation, induces relative motion between the needle and the tissue fibers. This mechanical interaction leads to microscopic structural changes in the local tissue, such as the roughening, displacement, winding, tightening, deformation, and rupture of collagen fibers, which can be observed as microtrauma under electron microscopy. − These microstructural changes prompt infiltration and activation of immune cells such as mast cells and macrophages, which release histamine, substance P, and adenosine. , These substances can directly stimulate or sensitize neighboring sensory nerve endings and also alter local microcirculation, suggesting that cutaneous immune cells are pivotal in acupuncture signal transduction.

In this context, the transient receptor potential vanilloid 4 (TRPV4) channel emerges as a molecule of particular interest. TRPV4 is a cation channel sensitive to mechanical force, hypotonic stress, and moderate heat (>34 °C). − It is involved in various neurosensory and inflammatory pathways and is implicated in a broad range of mechanosensory processes. , Although its direct mechanical gating was once debated, , emerging evidence suggests that it functions as an intrinsic mechanosensitive channel in various cell types (e.g., chondrocytes, , endothelial cells).

Notably, TRPV4 is primarily expressed in non-neuronal cell typesincluding endothelial cells, keratinocytes, and macrophagesbut not in sensory neurons or their terminals. , Research indicates that TRPV4 is essential for chondrocyte responses to physiological mechanical stretch. In pulmonary ventilation disorders and aseptic inflammation, TRPV4 mediates macrophage polarization toward a pro-inflammatory phenotype that promotes tissue damage, whereas in infectious settings it facilitates bacterial clearance. , This mechanosensitivity suggests TRPV4 could act as a molecular sensor for needle-prick mechanical stimuli within local acupoint tissues. However, whether and how TRPV4 channels on local non-neuronal skin cells, particularly immune cells, participate in transmitting ES signals to DRG sensory neurons remains an open question.

To elucidate the mechanisms underlying differential ES signaling between stimulation intensities and define the role of non-neuronal TRPV4, we combined in vivo calcium imaging in transgenic mice with pharmacological interventions. We hypothesized that higher-intensity ES preferentially recruits specific DRG neuron subpopulations and that cutaneous TRPV4 modulates neuronal excitability via neuroimmune interactions. Our specific aim was to determine whether TRPV4 activation affects both basal calcium activity and evoked responses in sensory neurons, including TRPV1-positive nociceptors. These findings advance our understanding of electroacupuncture’s peripheral mechanisms and establish a basis for developing TRPV4-targeted therapies.

Results and Discussion

DRG neurons are the primary afferent neurons of the somatosensory system. As pseudounipolar cells, their somata extend axonal branches that project to peripheral and central targets with distinct functional properties. DRG neuronal somata exhibit a broad diameter range of approximately 20–150 μm and are typically classified as small (<25 μm), medium (25–35 μm), or large (>35 μm). This size gradient broadly correlates with axonal myelination and conduction velocity: small neurons give rise to unmyelinated C-fibers, medium-sized neurons to thinly myelinated Aδ-fibers, and large neurons to heavily myelinated Aβ-fibers. These fibers relay peripheral sensory information to their corresponding somata within the DRG. Functionally, they mediate distinct sensory modalities: Aβ-fibers, with their low mechanical threshold, transmit non-noxious tactile and proprioceptive signals, while Aδ-fibers mediate sharp, fast pain, and C-fibers convey slow nociceptive pain, thermal sensations, and itch. Consequently, the DRG’s highly heterogeneous neuronal population integrates multiple sensory modalitiesincluding proprioception, touch, temperature, pain, and itchestablishing it as the pivotal initial site for signal transmission and modulation elicited by physical interventions such as acupuncture. Neurotransmitter release underlies the propagation of these neural impulses. Arriving action potentials activate voltage-gated calcium channels at axon terminals, prompting rapid calcium influx that induces vesicular neurotransmitter release and produces a cascading amplification. Calcium ions can surge from a resting concentration of 50–100 nM to 1–10 μM during neuronal firinga shift far exceeding that of sodium or potassium ions (≈0.001%). Their subsequent slow clearance via endoplasmic reticulum and mitochondrial buffering makes them a reliable proxy for neuronal excitability. The maturation of genetically encoded calcium indicators has enabled the use of in vivo calcium imaging to study acupoint sensitization and acupuncture analgesia at the level of DRG neurons. These approaches confirm that peripheral neuronal excitation is a fundamental component of acupuncture’s mechanism. −

Different ES Intensities Elicit Distinct Activation Patterns in Primary Sensory Neurons

ES primarily exerts its therapeutic effects by activating local Aδ and C-fibers in the somatic area, with the clinically optimal ES intensity slightly exceeding the Aδ fiber activation threshold (0.5–2 mA). To characterize how primary sensory neurons in the DRG respond to different stimulation intensities, we applied 0.5 mA or 2 mA ES to the medial dorsum of the right hindpaw of Pirt-GCaMP6s mice and recorded calcium activity in the ipsilateral L4 DRG (Figure A). The 2 mA stimulation evoked stronger calcium transients synchronously with ES onset and activated more primary sensory neurons than 0.5 mA (Figure C,E). Analysis of neuronal size further indicated that medium- and large-diameter neurons exhibited particularly enhanced responses (Figure D,F), consistent with the known activation threshold of A-fibers and suggesting that 2 mA ES more effectively recruits neuronal populations putatively corresponding to mechanosensitive Aδ and Aβ afferents.

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Changes in calcium activity in dorsal root ganglion sensory neurons during ES stimulation at varying intensities. (A) Top panel: breeding strategy for Pirt-GCaMP6s transgenic mice; bottom panel: stimulation sites for ES in mice. (B) Representative in vivo calcium imaging of the L4 DRG, with the white dashed line indicating the DRG border. Scale bar = 100 μm. (C) Percentage of activated DRG sensory neurons across different ES intensities. (D) Changes in calcium fluorescence intensity of DRG sensory neurons across different ES intensities. (E) Percentage of activated DRG neurons, categorized by soma diameter, under different ES intensities. (F) Changes in calcium fluorescence intensity of sensory neurons, grouped by soma diameter, under different ES intensities. Statistical significance was assessed using Student’s t-test (C), the nonparametric Mann–Whitney U test (D and F), and two-way ANOVA followed by Bonferroni’s post hoc test (E). Comparisons with 0.5 mA are indicated as *P < 0.05, **P < 0.01, ***P < 0.001. n = 5.

The pronounced activation of large-diameter neurons may correspond to the more evident local muscle contractions observed under higher-intensity stimulation. Stronger muscle contractions induced by 2 mA ES may additionally activate proprioceptive Aβ fibers embedded in skeletal muscle, indirectly contributing to the pronounced activation of large-diameter neurons observed at higher stimulation intensity. It is important to note that neuronal diameter classification in this in vivo study relied on fluorescence imaging with semimanual tracing rather than the electrophysiological gold standard, where C-fiber identification requires a conduction velocity below 2 m/s. This methodological distinction is relevant given the observed overlap between medium and small neuron classifications. Nevertheless, the significantly stronger activation of medium-diameter neuronsputatively associated with Aδ fibersby 2 mA ES suggests preferential recruitment of this neuronal population at higher stimulation intensity. Thus, although the data robustly show greater overall neuronal activation with 2 mA ES, subclassification into specific diameter-based categories should be interpreted cautiously. The absence of significant differences in small-diameter neuronal (C-fiber) responses across intensities may result from both methodological limitations and the inherent biological characteristics of these neuronal populations.

TRPV4 Expression Was Detected in the Skin and DRG

As a mechanosensitive ion channel, TRPV4 mediates calcium homeostasis and cellular responses across diverse cell types, facilitating mechanical transduction in chondrocytes, inflammatory signal amplification in colonic epithelium and macrophages, barrier regulation in endothelial cells, itch mediator release in keratinocytes, and microglia-mediated neuropathic pain. − Previous studies have established that TRPV4 is predominantly expressed in non-neuronal cellsparticularly keratinocytes, endothelial cells, and immune cellsbut is absent from sensory neurons. ,,

This distinct expression profile implies a potential role in mediating tissue-level responses to physical stimuli such as acupuncture, possibly via neuroimmune interactions. To determine the cellular localization of TRPV4 in neurons and immune cells, TRPV4-GCaMP6s transgenic mice were euthanized via anesthetic overdose (Figure A). For precise spatiotemporal control over TRPV4 lineage labeling and to circumvent potential developmental artifacts, we employed a tamoxifen-inducible Cre-LoxP system (TRPV4-CreER) with induction at 8 weeks of age. The TRPV4 gene is expressed in multiple cell types in mouse skin, including cutaneous endothelial cells, fibroblasts, macrophages, and neutrophils, with relatively higher expression levels observed in macrophages (Figure B). Immunofluorescence analysis of tissue sections from the L4 DRG and its cutaneous receptive field confirmed definitive TRPV4 expression in CD68-positive macrophages within the skin, while verifying its absence from DRG neurons (Figure C,D). This expression pattern, which aligns with prior reports of TRPV4’s mainly non-neuronal distribution, offers an anatomical foundation for its potential involvement in ES signal transduction. The specific localization of TRPV4 to cutaneous macrophagesresident immune cells strategically situated in the skin microenvironmentsupports the view of this mechanosensitive channel as a compelling non-neuronal molecular sensor in acupoints. These findings suggest a plausible mechanism in which needle insertion and electrical stimulation may activate TRPV4 on immune cells, initiating signaling cascades that modulate sensory neuron excitability and contribute to acupuncture effects.

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Immunofluorescence analysis of TRPV4 in the DRG and skin. (A) The breeding strategy for generating TRPV4-GCaMP6s transgenic mice is shown. (B) UMAP plot of trpv4 gene expressed on immune cells sorted from mouse skin epidermis. (C) Representative immunofluorescence images depict GFP (green) and CD68 (red) in the skin of TRPV4-GCaMP6s. Enlarged views of the boxed regions are presented, with white arrows highlighting double-labeled cells. (D) An immunofluorescence image illustrates GFP (green) and Nissl (red) staining in the L4 DRG of a TRPV4-GCaMP6s mouse. The boxed area is shown at higher magnification. Scale bar = 100 μm.

The Response of Primary Sensory Neurons to ES Was Assessed Following the Local Activation or Antagonism of TRPV4 in the Skin

To investigate the functional role of cutaneous TRPV4 in ES signaling, we performed pharmacological interventions by injecting TRPV4 modulators locally into the right dorsal hindpaw and systematically recorded changes in neuronal calcium activity following the sequential protocol detailed in Figure (Figure A,C). A subset of primary sensory neurons activated by locally injected GSK101 also responded to ES (Figure A,B). Local application of the TRPV4 agonist GSK101 markedly increased basal calcium activity in L4 DRG neurons, with more neurons exhibiting spontaneous calcium transients compared to the vehicle group, although the mean fluorescence intensity of this basal activity did not change (Figure D,E). During 2 mA ES, GSK101 administration produced a strong trend toward a higher proportion of responsive neurons (P = 0.0692) and significantly enhanced their calcium transient intensity (Figure D,F). This facilitatory effect was neuron-subtype-specific, appearing most prominent in large-diameter neurons (Figure H).

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Experimental operation mode and flow diagram. (A) shows the exposed mouse L4 dorsal root ganglion (DRG) and the experimental configuration. (B) Illustrates the sequential protocols for in vivo calcium imaging. Frames 1 and 2 show the baseline calcium activity in L4 DRG neurons and its modulation following the local activation or blockade of TRPV4 channels within the receptive field. The upper series, frames 3–20, depicts the altered neuronal calcium activity in TRPV1-GCaMP6s mice after injecting a TRPV1 agonist into the L4 DRG receptive field. The lower series, frames 3–6, records the calcium responses of L4 DRG neurons to graded intensities of electrical stimulation applied to the receptive field. These lower frames also capture the pharmacologically modulated responses after the local injection of TRPV4 agonist, TRPV4 antagonist, or vehicle. (C) Outlines the pharmacological experiment workflow, in which calcium imaging was performed 5 min after each drug intervention, with the specific calcium imaging sequence detailed in Figure B. 1% CAP: 1% capsaicin, TRPV1 agonist; vehicle: 1% DMSO in saline; GSK101: TRPV4 agonist; GSK219: TRPV4 antagonist.

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Role of TRPV4 in modulating basal and ES-evoked calcium activity in DRG sensory neurons. (A) In vivo calcium image in the L4 DRG of Pirt-GCaMP6s mice. Representative pseudocolor image shows neuronal responses to electrical stimulation (ES- 2 mA, green) and the TRPV4 agonist GSK101 (red). White outlines denote ganglion boundaries. Neurons activated by both stimuli are indicated by solid white arrows. Scale bar = 100 μm. (B) Venn diagram showing the absolute numbers of L4 DRG neurons activated by ES-2 mA alone (gray), GSK101 alone (green), or both stimuli (overlap), as a percentage of total neurons recorded (n = 1533). (C) Representative in vivo calcium image from Pirt-GCaMP6s mice after intradermal injection of TRPV4 agonist, TRPV4 antagonist, or vehicle into the L4 hindpaw sensory field (dashed white line outlines the DRG). Activated neurons are marked with colored circles and arrows. Scale bar = 100 μm. (D) Percentage of activated DRG neurons before and after 2 mA ES following injection of TRPV4 agonist, TRPV4 antagonist, or vehicle. (E and F) Calcium fluorescence intensity before stimulation and after 2 mA electro-stimulation in mice treated with TRPV4 agonist, TRPV4 antagonist, or vehicle. (G) Percentage of activated sensory neurons across different diameters before stimulation following TRPV4 agonist or vehicle injection. (H) Calcium fluorescence intensity in sensory neurons of varying diameters responding to 2 mA ES after injection of TRPV4 agonist, TRPV4 antagonist, or vehicle. Statistical comparisons used Student’s t-test (D), the nonparametric Mann–Whitney U test (E, F and H), or two-way ANOVA followed by Bonferroni’s post hoc test (G). Compared with vehicle and GSK219, *P < 0.05, ***P < 0.001. n = 6. Vehicle: 1% DMSO in saline; GSK101: TRPV4 agonist; GSK219: TRPV4 antagonist.

In contrast, treatment with the TRPV4 antagonist GSK219 suppressed basal neuronal excitability, significantly reducing the number of neurons with spontaneous calcium activity relative to both the GSK101 and vehicle groups (Figure D). This suppression particularly affected small-diameter neurons, showing a strong trend toward reduction compared with the agonist group (P = 0.0540) (Figure G). During 2 mA ES, GSK219 significantly decreased both the number of responsive neurons and their calcium fluorescence intensity compared to GSK101, with large-diameter neurons exhibiting notably diminished calcium transients (Figure D,F,H). No significant differences were observed between the GSK219 and vehicle groups in ES-evoked responses.

These complementary pharmacological manipulations demonstrate that TRPV4 activation in non-neuronal skin cells tonically modulates sensory neuron excitability and selectively facilitates ES-evoked afferent signaling. The differential effects on neuronal subpopulations indicate that TRPV4-mediated signaling preferentially enhances specific afferent pathways, likely via immune-cell-derived mediators that sensitize adjacent nerve endings.

The Activation of Nociceptive Neurons Was Compared across Different ES Intensities

To characterize nociceptive pathway activation under different ES intensities, we used TRPV1-GCaMP6s transgenic mice. In this model, TRPV1-positive nociceptors are specifically labeled with the calcium indicator GCaMP6s (Figure A), enabling real-time recording of their activity. TRPV1 is a suitable marker for this purpose as it is predominantly expressed on C-fibers and some Aδ-fibers. It is found on nociceptive neurons, including small-diameter neurons of the DRG and trigeminal ganglion (TG), , as well as on certain immune cells. − Its neuronal role is primarily for sensing noxious stimuli like heat and capsaicin, and mediating pain signaling. To confirm the functionality and specificity of our model, we first applied the classic TRPV1 agonist, 1% capsaicin. As expected, capsaicin evoked robust calcium responses in these neurons (Figure B–D), validating our experimental system for subsequent ES. Shun Dong et al. proved that a 2 mA ES at the Zusanli (ST36) point activates TRPV1 nociceptors. , When ES of two intensities (0.5 mA vs 2 mA) was applied to the hindpaw of TRPV1-GCaMP6s mice (Figure B), distinct differential activation patterns emerged. The 2 mA stimulation produced significantly greater activation of TRPV1-positive nociceptive neurons than 0.5 mA, as shown by both increased numbers of activated neurons and elevated calcium fluorescence intensity (Figure E,F). These results align with observations in Pirt-GCaMP6s mice and together demonstrate that nociceptive afferent transmission differs between 0.5 mA and 2 mA ES, with higher thresholds required for optimal TRPV1-mediated signaling.

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Changes in calcium imaging within dorsal root ganglion nociceptive neurons under ES at varying intensities. (A) Breeding strategy for TRPV1-GCaMP6s transgenic mice. (B) Representative in vivo calcium imaging of the L4 dorsal root ganglion in TRPV1-GCaMP6s mice (white dashed line indicates DRG outline). Scale bar = 100 μm. (C) The percentage of nociceptive neurons in the dorsal root ganglion responding to 1% capsaicin. (D) Changes in calcium fluorescence intensity among dorsal root ganglion nociceptive neurons responding to 1% capsaicin. (E) The percentage of nociceptive neurons in the dorsal root ganglion responding to 0.5 mA and 2 mA ES. (F) Changes in calcium fluorescence intensity among dorsal root ganglion nociceptive neurons responding to 0.5 mA and 2 mA ES. Statistical comparisons employed Student’s t-test (E) and the nonparametric Mann–Whitney U test (F). Compared with 0.5 mA, *P < 0.05, ***P < 0.001. n = 8. 1% CAP: 1% capsaicin.

The enhanced TRPV1 activation at 2 mA offers a mechanistic explanation for the previously reported intensity-dependent acupuncture effects. Interestingly, although earlier work indicates that 2 Hz 1 mA ES activates C-fibers, the absence of significant differences in small-diameter neuron responses in Pirt-GCaMP6s mice suggests possible methodological factors. The partial overlap of calcium signals between medium- and small-diameter neurons in vivo, together with the likelihood that different frequencies such as 2 Hz may better target specific C-fiber populations, requires further study to clarify the interplay of intensity and frequency in ES-mediated nociceptor recruitment.

Baseline Calcium Activity in Nociceptive Receptors Was Recorded after the Local Activation of TRPV4 Channels in the Skin, and Their Subsequent Response to ES Was Then Assessed

To investigate the specific role of non-neuronal TRPV4 in nociceptive transmission, we locally activated TRPV4 in TRPV1-GCaMP6s mice and recorded neuronal responses to 2 mA ES (Figure A,C). Vehicle was first injected intradermally, followed by GSK101 at the same site, with baseline calcium activity and ES-evoked responses (2 mA) recorded 5 min after each injection. A subset of nociceptive sensory neurons activated by locally injected GSK101 also responded to ES (Figure A,B). Compared to vehicle controls, TRPV4 activation significantly increased baseline calcium activity in dorsal root ganglion neurons, elevated the proportion of neurons responding to stimulation, and enhanced calcium transient intensity (Figure D–F). These facilitatory effects mirror observations in primary sensory neurons and provide direct functional evidence that TRPV4 activation in skin cells sensitizes nociceptive pathways.

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Effect of a TRPV4 agonist on basal and ES-evoked calcium activity in TRPV1-expressing nociceptive neurons. (A) In vivo calcium image in the L4 DRG of TRPV1-GCaMP6s mice. Representative pseudocolor image shows neuronal responses to electrical stimulation (ES-2 mA, green) and the TRPV4 agonist GSK101 (red). White outlines denote ganglion boundaries. Neurons activated by both stimuli are indicated by solid white arrows. Scale bar = 100 μm. (B) Venn diagram showing the absolute numbers of L4 DRG neurons activated by ES-2 mA alone (gray), GSK101 alone (green), or both stimuli (overlap), as a percentage of total neurons recorded (n = 1152). (C) Representative in vivo calcium image of TRPV1-expressing nociceptive neurons within the L4 dermatome’s dorsal footpad following intradermal injection of either the TRPV4 agonist GSK101 or vehicle (dashed white line indicates DRG boundary). Activated neurons are highlighted with colored circles and arrows (scale bar = 100 μm). (D) Percentage of responsive neurons under Baseline and ES-2 mA conditions following vehicle or GSK101 injection. (E and F) Calcium fluorescence intensity at baseline and in response to 2 mA ES following vehicle or GSK101 injection. Statistical comparisons used two-way ANOVA followed by Bonferroni’s post hoc test (D) and the nonparametric Mann–Whitney U test (E and F). Significant differences relative to the vehicle control are indicated as *P < 0.05, **P < 0.01. n = 6. Vehicle: 1% DMSO in saline; GSK101: TRPV4 agonist.

The modulation of TRPV1-positive nociceptors by non-neuronal TRPV4 suggests an indirect signaling mechanism, likely mediated by macrophage-derived inflammatory factors such as IL-1β. This neuroimmune interaction paradigm is further supported by documented increases in neuropeptide release (CGRP and substance P) and mast cell degranulation following acupuncture stimulation. , Importantly, while TRPV4 is absent from mast cells, as confirmed by Luo et al., our immunofluorescence results clearly localize TRPV4 expression in cutaneous non-neuronal cells, including macrophages (Figure ), which are identified as the primary cellular mediators in this pathway.

The functional relevance of non-neuronal TRPV4 was further demonstrated using pharmacological approaches in Pirt-GCaMP6S and TRPV1-GCaMP6S mice. TRPV4 activation significantly enhanced both basal and stimulation-evoked neuronal responses, whereas its inhibition produced calcium activity indistinguishable from vehicle controls and substantially lower than that of agonist-treated groups. This bidirectional modulation indicates that cutaneous TRPV4 channels play an essential role in needle-induced afferent transmission via immune–neural cross-talk. The attenuated response following TRPV4 antagonism suggests that ES may initially activate nerve endings directly, with subsequent engagement of neuropeptide–immune cell positive feedback mechanisms that require longer temporal resolution for full characterization.

The anatomical basis for this signaling pathway is well established. Skin-resident macrophages, strategically positioned as primary immune sentinels, play vital roles in pathogen clearance and skin barrier repair. − Previous studies have shown that TRPV4 is essential for crystal-induced macrophage release of IL-1βin acute gout, which activates TRPV1 nociceptors, ,, providing a mechanistic precedent for our findings. On the other hand, the epidermal layer of the skin forms a critical barrier and protective structure, playing a key role in normal skin function. Within this layer, the synapse-like connection model between keratinocytes and intraepidermal free nerve endings (IEFNs) is important for sensory transmission. , Studies have shown that TRPV4 can promote the release of inflammatory factors from skin keratinocytes by modulating ATP release, suggesting that TRPV4 may also contribute to the afferent signaling of electrical stimulation via keratinocytesa direction that warrants further investigation.

The tibial nerve innervating the mouse paw and the peroneal nerve innervating the knee joint represent the primary distal branches of the sciatic nerve, which originates from the L4–L6 dorsal root ganglia. Therefore, both the dorsal paw and lateral lower leg fall within the receptive field of the L4 dorsal root ganglion. The increased excitability of L4 DRG neurons following local TRPV4 agonist application within this receptive field strongly supports non-neuronal cells’ involvement in TRPV4–sensory neuron communication. Future studies could assess inflammatory factor levels, keratinocytes, and macrophage polarization status after local injection of TRPV4 agonists or antagonists, to further examine whether TRPV4 mediates acupuncture-induced nerve fiber activation via macrophage/keratinocytes-derived inflammatory mediators such as IL-1β, IL-6.

This study introduces in vivo, real-time dorsal root ganglion calcium imaging to clarify how cutaneous non-neuronal TRPV4 channels regulate ES afferent signaling through neuroimmune interactions. Our results establish that non-neuronal TRPV4 acts as a key mediator in sensory signal modulation, dynamically regulating neuronal excitability during acupuncture stimulation. It should be acknowledged that the present study employed pharmacological tools (TRPV4 agonist and antagonist) to manipulate TRPV4 activity under physiological baseline conditions, and the translational relevance of these findings to native pathological states remains to be established. Under physiological conditions, needle-induced mechanical deformation of the skin represents a direct and plausible activating stimulus for cutaneous TRPV4, whereas under pathological conditionssuch as local inflammation or tissue edemaadditional biophysical stimuli, including sustained hypo-osmotic stress and elevated mechanical loading, may further augment TRPV4 activation, potentially amplifying neuroimmune signaling and ES-evoked afferent responses. Defining the threshold and dynamic range of TRPV4 activation across these conditions would clarify its contribution to acupuncture efficacy in both healthy subjects and diseased states. These findings provide a mechanistic framework for understanding acupuncture’s peripheral actions and offer new insights for developing TRPV4-targeted therapies, advancing our comprehension of neuroimmune interactions in ES afferent transmission.

Methods

Mice

Adult Pirt-GCaMP6s, TRPV1-GCaMP6s, and TRPV4-GCaMP6s mice weighing between 22 and 30 g were used in this study. These animals were generated by crossing homozygous Pirt-Cre, TRPV1-Cre, and TRPV4-CreER mice with homozygous Rosa26-LoxP-STOP-LoxP-GCaMP6s mice. The Pirt-Cre mice were a gift from Xinzhong Dong’s laboratory at Johns Hopkins University, USA, while the TRPV4-CreER mice were provided by Professor Feng Jing’s laboratory at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Shanghai Southern Model Organism Technology Co., Ltd. handled animal transportation and breeding. Mice were housed either individually or in groups of up to five per cage with ad libitum access to food and water. Housing facilities maintained constant temperature and humidity under a 12 h artificial light cycle from 7:00 to 19:00. All experimental procedures complied with the 2006 Ministry of Science and Technology Guidelines for the Humane Treatment of Laboratory Animals and were approved by the Animal Ethics Committee of the China Academy of Chinese Medical Sciences. To identify TRPV1-CreER expression, 1% capsaicin (Sigma, M2028) was administered into the right dorsal footpad of mice, and real-time calcium imaging was employed to observe dynamic changes in intracellular calcium activity in related neurons following drug administration. To induce TRPV4-CreER expression, TRPV4-GCaMP6s mice received intraperitoneal injections of tamoxifen (Sigma, #T564) for five consecutive days.

In Vivo Calcium Imaging

DRG Exposure

Specific procedural steps were performed as previously described. Mice were anesthetized via intraperitoneal injection of 1.25% tribromoethanol (20 μL/g) and then subjected to tracheal intubation. A 2–3 cm midline dorsal incision was made, and a partial laminectomy was performed to fully expose the right L4 DRG. After dissecting the epineurium, any bleeding was cleared with cotton swabs. Care was taken throughout the procedure to avoid damaging the spinal cord and DRG. Respiratory status was continuously monitored, and the absence of blood exudation in the L4 DRG was ensured to maintain a clear imaging field.

Calcium Imaging Recordings

Mice were fixed in a prone position on a custom microscope stage. A custom spinal cord clamp (Beijing Zhongshi) was used to securely immobilize the spinal cord and prevent displacement. The clamp angle was adjusted to fully expose the L4 dorsal root ganglion under the objective lens. Mice were also connected via tracheal intubation to a ventilator anesthesia machine (Kent Scientific Corporation, USA) for respiratory monitoring. Imaging was conducted using a Leica 10× air objective (Leica TCS Stellaris 8, Germany) at 1× magnification. After locating the target field, the X, Y, and Z axes were adjusted to encompass the entire DRG, followed by a z-axis scan with a step size of 20 μm. With 488 nm excitation light at 5% laser intensity, images of 512 × 512 pixels were acquired at 10–12 s per frame and a 400 Hz scan rate. Six frames were recorded for real-time imaging: the first two captured baseline neuronal calcium activity before stimulation, and frames three to six recorded calcium responses to ES. Neuronal activation causes GCaMP to bind intracellular Ca2+, emitting green fluorescence that is captured for imaging. Anesthesia was maintained with 0.9–1.4% isoflurane, and body temperature was regulated using a warming blanket system (FHC, USA) (Figure ).

Calcium Imaging Data Analysis

A custom MATLAB program quantified the number of activated neurons and their relative fluorescence intensity. The total number of DRGs was manually counted using ImageJ. Neuronal activation was defined by a fluorescence increase exceeding 30% of the baseline level. This was calculated as ΔF/F0 = (Ft–F0)/F0, where ΔF represents the maximal evoked fluorescence change and F0 is the baseline fluorescence intensity. The percentage of responding neurons was calculated using the formula: (Number of activated neurons/Total number of neurons analyzed) × 100%. Neurons were categorized by size as follows: small-diameter neurons had a cell area <400 μm2, medium-diameter neurons measured 400–700 μm2, and large-diameter neurons exceeded 700 μm2.

ES in Mice

Localization

The receptive field of the L4 dorsal root ganglion (DRG) in the mouse hindlimb was mapped using brush stimulation. During calcium imaging, a 10 mm wide soft brush was stroked from the knee joint to the dorsal paw at 2 cm/s across the skin innervated by the L4 segment. The dorsal paw area exhibiting the strongest calcium signal in response to brushing was defined as the optimal receptive field for L4 DRG neurons. All subsequent ES and pharmacological stimulations were consistently targeted to the medial right dorsal hindpaw. Electroacupuncture stimulation: Two acupuncture needles (0.25 × 13 mm, Zhongyan Taihe) were inserted approximately 2 mm deep into the dorsal skin of the right foot. A HANS acupoint nerve stimulator (Nanjing Jisheng Medical Technology Co., Ltd.) delivered different stimulation intensities (0.5 or 2 mA) with a pulse width of 0.2–0.6 ms and a frequency of 10 Hz. This stimulation was applied during recording frames 3–6.

Pharmacologic Activation/Inhibition of TRPV4 Function

Intradermal injections of either the TRPV4 agonist GSK1016790A (30 μM, 10 μL; Sigma, #G0798), the TRPV4 antagonist GSK2193874 (30 μM, 10 μL; Sigma, #SML0942), or a vehicle (1% DMSO in saline, 10 μL) were administered into the right dorsal footpad of mice. Electrical stimulation was applied 5 min later.

Immunofluorescence

Tissue processing: mice were anesthetized with an overdose of urethane (0.2 mL/10 g). The right L4 dorsal root ganglion and its innervated skin region from the lateral calf were harvested. Tissues were fixed in 4% paraformaldehyde for approximately 12 h, dehydrated in 30% sucrose, and cryoprotected overnight at 4 °C. After blotting moisture with filter paper, samples were embedded in OCT compound. Cryosections were cut at a thickness of 20 μm. DRG sections were mounted directly onto adhesive slides, while skin sections were stored in 0.1 M PB solution before mounting for subsequent immunofluorescence staining. Immunofluorescence staining: sections were washed three times for 5 min each in 0.1 M PB to remove residual embedding medium. They were then blocked for 1 h at room temperature with 3% goat serum and 0.5% Triton X-100 in 0.1 M PBS. After washing, sections were incubated overnight at 4 °C on a shaker with primary antibodies: chicken polyclonal anti-GFP (Abcam, #AB13970) and rabbit polyclonal anti-CD68 (Abcam, #AB125212), both diluted 1:500. Following primary antibody incubation, sections were washed three times with 0.1 M PB. They were then incubated for 2 h at room temperature with secondary antibodies: goat antichicken Alexa Fluor 488 (Invitrogen, #A-11039), goat antirabbit Alexa Fluor 594 (Invitrogen, #A-11012), and Nissl 594 (Thermofisher, N21479), all at 1:500 dilution. After final washes, sections were coverslipped with an antifade mounting medium containing DAPI and imaged using a confocal fluorescence microscope.

Single-Cell RNA-seq Analysis

Publicly available single-cell RNA sequencing (scRNA-seq) data of mouse epidermal cells were retrieved from the Single Cell Portal (singlecell.broadinstitute.org). Data sets were filtered by species (Mus musculus), organ (skin), and sex (both male and female). TRPV4 gene expression was examined specifically within the immune cell population, and the results are presented in Figure B.

Statistical Analysis

Data analysis was conducted using MATLAB, ImageJ, Excel, SPSS, and GraphPad Prism 8.0. Figures were prepared with BioRender (https://BioRender.com), GraphPad Prism 8.0, Origin 2024, and Adobe Illustrator CC 2020. Normality was assessed for all data sets prior to statistical testing. Non-normally distributed data are reported as median with interquartile range [M (Q1 Q3)] and were evaluated using the Mann–Whitney U test. Normally distributed data are expressed as mean ± standard error of the mean (Mean ± SEM). Within-animal comparisons of pre- and poststimulation conditions employed Student’s t-test. Differences between stimulation diameters in the same animal were assessed using two-way analysis of variance (ANOVA). Statistical significance was defined as P < 0.05.

Acknowledgments

We thank Xinzhong Dong and Professor Feng Jing for supporting the transgenic mice. Special thanks to BioRender.com for providing the platform to create the schematic figures in this study. Figures and were Created in BioRender. Cheng, H. (2026) https://BioRender.com/3bhcwai.

Glossary

Abbreviations

DRG

dorsal root ganglion

ES

electroacupuncture stimulation

TRPV1

transient receptor potential vanilloid 1

TRPV4

transient receptor potential vanilloid 4

H.C. and K.L. conceptualized and designed the research; H.C., X.L., and L.D. performed all in vivo calcium imaging; H.C. performed all pharmacology-based experiments; H.C. analyzed the data, prepared all figures and the Table of Contents (TOC) graphic, and wrote the manuscript; and X.G., J.F., and B.Z. supervised the study. All authors have given approval to the final version of the manuscript.

This study was funded by the National Natural Science Foundation of China (Nos. 82230123, 82174513), Scientific and technological innovation project of China Academy of Chinese Medical Sciences (No. CIZJS2025016), and the Fundamental Research Funds for the Central public welfare research institutes (No. ZZ-JQ2023003).

The authors declare no competing financial interest.

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