The Investing Fascia–Carotid Sheath–Vagus Nerve Mechanical Coupling Hypothesis: PIEZO Mechanotransduction and Directions for Validation

1. Introduction

The vagus nerve is the tenth cranial nerve and the most widely distributed cranial nerve in the human body. Its cervical course is anatomically well defined. After exiting the skull through the jugular foramen, the vagus nerve descends within the carotid sheath, posteriorly between the common carotid artery and the internal jugular vein. Traditional anatomy and neurophysiology have attributed the cervical functions of the vagus nerve primarily to regulation by its central nuclei and feedback from target organs. The mechanical influence of the surrounding fascial environment has received little attention.

This perspective is now changing. Multiple independent studies have shown that the carotid sheath is not an isolated neurovascular channel. Rather, it has extensive fibrous connections with the deep cervical fascial network. The position of the vagus nerve within the sheath varies markedly among individuals and exhibits left–right asymmetry [1,2,3]. Routine movements of the head and neck alter the spatial relationship between the sternocleidomastoid muscle and the carotid sheath [4]. The vagus nerve and its adjacent structures express mechanosensitive ion channels, providing a molecular basis for converting mechanical signals into neural electrical signals [5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20]. Collectively, these findings point to a hypothesis that has not yet been systematically integrated: the mechanical state of the cervical fascial system may be an important upstream regulator of vagal function.

This article synthesizes these scattered lines of evidence into a systematic theoretical framework. We propose the “Investing Fascia–Carotid Sheath–Vagus Nerve Mechanical Coupling Hypothesis.” Its core proposition is that fibrotic changes in the investing fascia or in the deep cervical fascial layers of the anterior triangle may transmit abnormal mechanical loads through the three-layer continuous or semi-continuous network of the deep cervical fascia to the carotid sheath. This transmission may alter the mechanical microenvironment of the vagus nerve within the sheath and thereby modulate its afferent and efferent functions. We also strictly define the terminological boundaries, levels of evidence, and applicable populations. In the anterior triangle, the mechanical origin should not be broadly termed the investing fascia; it should be described as the deep cervical fascial layers. In the general population with chronic neck pain, the proposition that fibrosis of the superficial deep fascia leads to increased elastic modulus may not hold and should be validated in specific phenotypes.

2. Anatomical Basis: Fascial Continuity and Composition of the Carotid Sheath

2.1. Three-Layer Structure and Operational Definition of the Deep Cervical Fascia

The deep cervical fascia comprises three layers. The superficial layer encircles the neck in a sleeve-like manner, envelops the sternocleidomastoid and trapezius muscles, and forms their muscle sheaths. It attaches superiorly to the skull and continues inferiorly to the thoracodorsal region. In this article, the investing fascia refers to the superficial layer of the deep cervical fascia. In the anterior triangle, its existence as a single continuous layer has been questioned. We therefore use the term “deep cervical fascial layers,” which includes the strap muscle fascia, submandibular gland fascia, and visceral fascia, among others. The middle layer is the pretracheal fascia, which covers the infrahyoid muscles and forms the thyroid sheath. The deep layer is the prevertebral fascia, which covers the prevertebral and scalene muscles. The key feature of the investing fascia is its functional continuity. It is not an isolated local structure. Instead, it forms extensive fibrous connections with the sternocleidomastoid fascia, the trapezius fascia, and deeper layers. Its tension state may be transmitted to surrounding tissues through the fascial network. In an anatomical review, Zhang et al. [21] systematically described the interfascial spaces of the posterior neck and clarified the spatial relationships of the three deep cervical fascial layers with the carotid sheath, sympathetic trunk, and other structures. This work provides supplementary evidence for the anatomical continuity of the deep cervical fascial network.

The anatomical existence of the superficial layer of the deep cervical fascia in the anterior triangle has itself been questioned. Using E12 thin-sheet plastination and confocal microscopy, Nash et al. [22] examined the anterior triangle of the adult neck. They proposed that the so-called investing fascial layer does not exist in the anterior triangle and that the deep potential space of the neck is directly continuous with the subcutaneous tissue. In the upper neck, the strap muscle fascia and submandibular gland fascia form a “dumbbell-shaped” fascial sheet with a free lateral border that is not continuous with the sternocleidomastoid fascia. In the lower neck, no single connective tissue layer extends directly between the two sternocleidomastoid muscles. This finding fundamentally challenges the existence of a single continuous superficial deep fascia in the anterior triangle.

We therefore adopt the following operational definitions. In the posterior triangle and between the sternocleidomastoid and trapezius muscles, the superficial deep fascia may be thin or absent. In the anterior triangle, the starting point of mechanical transmission may more likely involve the combined participation of the deep cervical fascial layers. Zhang and Lee [23] found that the superficial deep fascia is absent between the sternocleidomastoid and trapezius muscles and that this region is entirely filled with adipose tissue. As a result, the carotid sheath communicates directly with the subcutaneous tissue through this channel. This anatomical feature suggests that mechanical tension in the subcutaneous tissue may act directly on the carotid sheath through this channel. Refining these operational definitions will help future studies accurately select measurement sites in ultrasound elastography and avoid conflating the anterior triangle with the posterior triangle as a single mechanical starting point.

2.2. Fascial Origin and Multilayer Composition of the Carotid Sheath

The carotid sheath is classically described as a “fibrous tunnel of the deep cervical fascia,” but its fascial origin has long been debated. A dedicated review systematically searched 22 studies focusing on the fascial wall of the sheath. It stated that the carotid sheath lies between the three layers of the deep cervical fascia—the superficial layer, the pretracheal fascia, and the prevertebral fascia—forming a tubular deep fascial column. However, “the authors’ descriptions of the sheath wall fascia are complex, varied, and inconsistent,” and its specific fascial origin and attachment points still lack consensus [24]. This controversy implies that the fascial composition of the carotid sheath may have marked regional heterogeneity and interindividual variability.

In the skull base region, a cadaveric and radiological correlation study showed that the anterior wall of the carotid sheath comprises multiple layers. These include the tensor–vascular–styloid fascia, stylopharyngeus fascia, pharyngobuccal fascia, and longus capitis fascia. The anterior wall appears as a recognizable hypointense line on magnetic resonance imaging (MRI) [25]. In the suprahyoid region, a fetal histological study confirmed that the fascia of the styloid muscle group attaches to and converges on the anterosuperior aspect of the carotid sheath. The sheath wall forms multipoint, multi-site connections with the pharynx through the visceral fascia, constituting a “mesentery-like fascial bundle” that may be influenced by the mechanical state of the longus capitis [26]. These findings indicate that the carotid sheath is not a single-layer structure. It may be a region-specific multilayer complex intrinsically connected to the deep cervical fascial network.

Liu et al. [27] conducted anatomical studies of 10 cadaveric head specimens and 12 clinical surgical cases. They confirmed from a surgical anatomy perspective that the cervical fascia comprises a superficial layer (the investing fascia), a middle layer (the pretracheal fascia), and a deep layer (the prevertebral fascia). The prevertebral fascia is a key anatomical landmark for protecting the carotid sheath and the parotid gland, and it forms a complete barrier between the carotid sheath and the suboccipital muscles. Surgical approaches using the prevertebral fascia as an anatomical landmark were safe and effective in 12 far-lateral or extreme lateral approach procedures, with no carotid sheath– or parotid gland–related complications. This finding provides direct surgical anatomical evidence for the relationship between the three deep cervical fascial layers and the carotid sheath.

Histological stratification of the adult carotid sheath further supports this understanding. A semi-serial histological study of 8 sides from 5 cadavers showed that the carotid sheath comprises an adventitial layer surrounding each vessel and a common sheath layer. The thickness of the adventitia and common sheath exhibits significant interindividual and site-dependent variation. The adventitial layer is thickest around the common carotid artery and relatively thin around the internal jugular vein. The thickness of the common sheath varies severalfold. The authors explicitly stated that “the carotid sheath is not an anatomical artifact but a distinct histological structure” [28]. Notably, the common sheath does not directly fuse with the investing fascia or the pretracheal layer. Instead, it often fuses with the visceral fascia to form a thick plate. The fusion pattern shows marked interindividual variation: in some specimens, the common sheath is extensively and continuously fused with the visceral fascia; in others, only punctate attachments are present. Mechanical transmission from the investing fascia to the carotid sheath therefore may not occur through direct fascial fusion. It may instead occur indirectly via the deep cervical fascial network or through the visceral fascia and prevertebral fascia pathways. Interindividual differences in fusion patterns may constitute the histological basis for individual differences in mechanical transmission efficiency.

The fibroelastic nature and mechanical heterogeneity of the carotid sheath were confirmed in early anatomical studies. A combined mesoscopic and microscopic study of 40 human specimens showed that the carotid sheath is always present and forms a complete covering around the neurovascular complex. The sheath wall has a fibroelastic structure, and wall thickness varies. Mesoscopic examination revealed that connective tissue fiber bundles connect the wall of the first segment of the internal jugular vein to the inner surface of the carotid sheath. These fiber bundles run radially or obliquely, with densities varying across regions of the sheath wall and being particularly dense around the internal jugular vein. Fibrous continuations fix the sheath anterosuperiorly to the margins of the jugular foramen and the carotid canal [29]. This fiber bundle orientation and density distribution suggests that mechanical deformation of the sheath wall may be preferentially transmitted to the intra-sheath venous wall through these fiber bundles, potentially altering venous return resistance and interstitial pressure. This provides a specific anatomical pathway for sheath wall mechanical deformation, followed by altered intra-sheath venous return and altered intra-sheath mechanical microenvironment [29].

Developmental evidence also supports the multilayer composition of the carotid sheath. A histological study of 15 mid-term human fetuses (9–25 weeks of gestation) showed that the carotid sheath appears only at or after 20 weeks of gestation, clearly separated from the prevertebral layer of the deep cervical fascia but fused with the pretracheal layer covering the strap muscles. The fusion between the sheath wall and the pretracheal layer gradually strengthens from 20 to 25 weeks of gestation, whereas a separable interface is always maintained with the prevertebral layer. The positional relationships of the sheath and its internal structures are established much later than those of the prevertebral and pretracheal layers [30]. This developmental pattern suggests that the fusion relationship between the sheath wall and the pretracheal layer has a developmental basis and may constitute a transmission pathway of pretracheal fascial tension, followed by sheath wall mechanical deformation. In contrast, the separable interface between the sheath wall and the prevertebral layer suggests that the mechanical influence of the prevertebral fascia on the sheath wall may be transmitted through interlayer shear rather than direct fusion. The vagus nerve migrates from a ventral to a dorsal course between 9 and 20 weeks of gestation; its final intra-sheath positional configuration is therefore established before formation of the sheath wall. Subsequent formation of the sheath wall may therefore have “wrapped” rather than “shaped” the intra-sheath position of the vagus nerve. This provides a developmental constraint for the inference that the effect of sheath wall mechanical deformation on the nerve may depend on the connective tissue between the sheath wall and the nerve.

Developmental histological studies of the middle layer of the deep cervical fascia further supplement this evidence. Using histological sections from human embryos (6–8 weeks) and fetuses (9–14 weeks), Martínez-Sanz et al. [31] identified three stages in the development of the middle layer of the deep cervical fascia and found that its development parallels mechanical activity in the neck. The middle layer of the deep cervical fascia is associated with lymphatic and venous structures. In specimens at 14 weeks of gestation, fibrous connections are visible between this layer and the wall of the internal jugular vein. These connections are synchronized with development of the muscular layer and may promote venous and lymphatic circulation. This finding suggests that the mechanical association between the middle layer of the deep cervical fascia and the intra-sheath venous system has a developmental basis, providing developmental evidence for the transmission pathway of deep cervical fascial network, followed by intra-sheath venous return and intra-sheath microenvironment.

The three-layer fascial architecture of the carotid sheath and its mechanical response have also gained developmental histological support. A histological study of 20 New Zealand white rabbits at four developmental stages (early fetus, late fetus, neonate, and adult) showed that three layers of fascial tissue—an inner fascial layer, an intermediate zone, and an outer fascial layer—were consistently present around the common carotid artery, internal jugular vein, and vagus nerve. The arterial inner fascial layer appeared earliest and thickened most markedly, forming a dense multilayered fibrous structure. The venous and neural inner fascial layers were relatively thin. The arterial inner fascial layer was approximately 2–3 times thicker than the venous inner fascial layer, and its collagen fiber orientation formed a specific angle with the long axis of the vessel. This differential thickening pattern is consistent with the intrinsic pulsatile hemodynamic stress levels borne by the three structures. The outer fascial layers of adjacent structures showed potential fusion, most pronounced between the artery and vein and relatively weaker between the vein and nerve, reflecting the extrinsic mechanical interactions of the surrounding tissues [32]. This differential fusion pattern suggests that the strength of mechanical coupling among structures within the sheath may not be uniform. Arterial pulsation may be preferentially transmitted to the venous wall through outer fascial layer fusion, potentially affecting intra-sheath venous return and interstitial pressure. Because this evidence comes from a rabbit model whose cervical anatomy differs from that of humans, extrapolation to humans requires caution.

2.3. Histological Composition of the Carotid Sheath

The carotid sheath is not a homogeneous fibrous membrane. A histopathological study of the carotid sheath in patients with oral squamous cell carcinoma showed that the sheath wall comprises fibrofatty tissue and scattered nerve bundles, with an active lymphatic network and immune cell infiltration [33]. Combined with the findings of Hayashi [28] and Piffer [29], the histological heterogeneity of the sheath wall is reflected not only in cellular composition but also in interindividual differences in fascial layer thickness, fibroelastic composition, and fusion relationships. This histological heterogeneity suggests that the mechanical properties of the sheath wall, such as elastic modulus, may differ across regions. The efficiency of tension transmission from the investing fascia to the sheath wall may therefore vary with the histological composition of the sheath wall. The presence of nerve bundles within the sheath wall further suggests that the sheath wall itself may be innervated and that its mechanical deformation may directly activate local nerve endings. Struthoff et al. [34] performed immunofluorescence analysis of 17 human carotid arteries. They confirmed dense tyrosine hydroxylase (TH)–positive sympathetic nerve fibers at the carotid bifurcation, ultimately located approximately 2.5 mm from the intimal surface. This provides direct human histological evidence for rich innervation in the region adjacent to the carotid sheath and suggests that changes in the peri-sheath mechanical environment may directly affect local neural structures.

2.4. Positional Variation and Left–Right Asymmetry of the Vagus Nerve Within the Sheath

The position of the vagus nerve within the carotid sheath is not fixed. A systematic review of 971 specimens showed that the textbook-described “posterolateral to the common carotid artery” position was present in only 73.5% of individuals. Another 26.5% had significant positional variation, including anterior (3.1%), medial (0.7%), and posteromedial (0.2%) configurations [1]. More notably, an anatomical study of 50 cadavers revealed marked left–right asymmetry. The right vagus nerve was located posterior to the common carotid artery in 64% of cases; the left vagus nerve was located anterior to the common carotid artery in 68%. Fewer than one-third of cases showed symmetric positioning [2]. Randhawa et al. [3] further found in an in vivo ultrasound study of 347 cases that 38% of subjects had anatomical variations of the vagus nerve. Left-sided variations accounted for 67.1%, and right-sided variations for only 6%. The anteromedian type was most common, suggesting that the mechanical exposure configuration of the left vagus nerve within the sheath may be more heterogeneous.

This anatomical heterogeneity implies that the degree of mechanical exposure of the vagus nerve within the sheath may differ systematically among individuals. Nerve configurations located anteriorly and medially within the sheath wall may be more sensitive to tension changes in the investing fascia and pretracheal fascia. In contrast, the rare configuration adjacent to the posterior sheath wall may produce the most direct mechanical response to tension changes in the prevertebral fascia. Left–right asymmetry further suggests that the mechanical effects of investing fascia–sheath coupling may differ by side. The right nerve may be more susceptible to tension changes in the posterior sheath wall (prevertebral fascial layer); the left nerve may be more susceptible to tension changes in the anterior sheath wall (pretracheal fascial layer) [2,3].

This left–right asymmetry is not limited to vagal anatomy. In humans, Williamson et al. [35] applied unilateral carotid sinus compression and directly recorded muscle sympathetic nerve activity (MSNA). Left carotid sinus afferent input had a significantly greater effect on efferent muscle sympathetic output than right-sided input. During left-sided compression, MSNA burst amplitude was 4.07 ± 0.67 μV, compared with 2.48 ± 0.46 μV on the right (*p* < 0.05). This study provides direct human electrophysiological evidence for side-specific effects of mechanical manipulation in the carotid region on autonomic neural output and forms a cross-system contrast with anatomical findings of left–right asymmetry in the vagus nerve. The intervention consisted of external collar compression of the carotid sinus, and the output measured was sympathetic rather than vagal. Its mechanical pathway therefore does not fully correspond to the core hypothesis of fascial tension, followed by sheath wall deformation and vagal function. Nevertheless, it indicates that mechanical input in the carotid region may have side-dependent effects on autonomic neural output.

3. Core Hypothesis of Mechanical Coupling

3.1. From the Investing Fascia and Deep Cervical Fascial Network to the Carotid Sheath: The Mechanical Transmission Chain

3.1.1. Overall Framework of the Transmission Chain

The core of the hypothesis is a mechanical transmission chain. It begins with fibrosis or mechanical remodeling of the investing fascia or of the deep cervical fascial layers in the anterior triangle. This is followed by tension redistribution, strain of the deep cervical fascial network, and deformation of the carotid sheath, including changes in sheath wall tension and sheath lumen geometry. The next step is change in the intra-sheath mechanical microenvironment, including sheath wall tension, sheath lumen geometry, venous return, interstitial pressure, and nerve epineurial strain. The final step is change in vagal mechanical exposure and afferent and efferent activity.

3.1.2. Evidence Gap in the Middle Segment

The middle segment of this chain currently lacks direct evidence from synchronized measurements in the same participant. This segment includes fibrosis or mechanical remodeling of the investing fascia or the deep cervical fascial layers in the anterior triangle, deformation of the carotid sheath, changes in the intra-sheath mechanical microenvironment, and changes in vagal mechanical exposure. The missing measurements include investing fascial stiffness, carotid sheath geometric deformation, surrogate indicators of intra-sheath mechanics, and vagal function. Available support comes mainly from three sources: anatomical continuity, intraoperative ultrasound evidence of changes in the spatial relationship between the sternocleidomastoid muscle and the sheath, and the necessity of PIEZO channels in vagal mechanotransduction. This segment should therefore be explicitly labeled as theoretical inference or pending direct validation rather than an established mechanism.

3.1.3. Mechanical Consequences of Investing Fascial Fibrosis

The starting point of this chain is fibrosis of the investing fascia or of the deep cervical fascial layers in the anterior triangle. Chronic forward head posture, long-term stress, aging, and chronic low-grade inflammation may synergistically drive these fibrotic changes. The essence of fibrosis is increased collagen cross-linking and decreased elastic fibers, causing the fascia to lose its elastic buffering capacity. In this state, shear stress generated by routine micro-movements of the head and neck is no longer absorbed by elastic deformation. It may be transmitted as “rigid conduction” through the three-layer network of the deep cervical fascia to the carotid sheath.

3.1.4. PIEZO1-Mediated Mechanical–Fibrotic Conversion

Fibrosis of the investing fascia itself may be regulated by mechanotransduction pathways. Research in the cardiovascular field has demonstrated that mechanical stretch activates Piezo1 in vascular smooth muscle, upregulates miRNA-582-5p, and promotes collagen deposition through the PTEN/PI3K/Akt pathway, leading to carotid fibrosis and stiffness. Specific knockout of Piezo1 significantly attenuates these changes [36]. This mechanism suggests that fibrotic changes in the investing fascia under chronic mechanical loading may follow a similar Piezo1-mediated pathway, but direct validation in fascial tissue is required.

Xu et al. [37] demonstrated in a pulmonary fibrosis model that PIEZO1 is highly expressed in periostin-positive myofibroblasts and that conditional knockout of Piezo1 significantly attenuates pulmonary fibrosis. Mechanistically, Piezo1 deficiency disrupts actin organization and impairs nuclear localization of YAP/TAZ, shifting myofibroblasts from a proliferative state to a stressed and apoptotic state. This provides a causal template for fibrotic transformation of investing fascial fibroblasts under chronic mechanical loading via the PIEZO1–YAP/TAZ pathway. Because its tissue source is lung rather than fascia, extrapolation requires independent validation.

Wang et al. [38] confirmed in POSTN-enriched lung fibroblasts that PIEZO1 drives fibroblast activation through YAP/TEAD-dependent profibrotic signaling, providing independent replication for the PIEZO1–YAP/TAZ pathway and identifying its downstream transcription factor as YAP/TEAD. Huang et al. [39] revealed in a spinal epidural fibrosis model a second independent pathway of PIEZO1-mediated fibrosis. After collagen fibers envelop fibroblasts, Piezo1-mediated calcium influx stabilizes endothelin-1 (ET-1), which activates HIF-1α and the TGF-β1/Smad3 pathway, forming a positive feedback loop of collagen accumulation and fibroblast activation. Blockade of Piezo1 attenuates fibrotic progression in a mouse laminectomy model. This finding provides an alternative signaling pathway for PIEZO1-mediated mechanical–fibrotic conversion beyond the YAP/TAZ pathway. Its tissue source—spinal epidural—is closer to the cervical fascia than pulmonary or skin fibrosis models. The model nonetheless involves post-laminectomy fibrosis, and its pathological driver differs from the chronic mechanical loading of the superficial deep cervical fascia. Extrapolation to the investing fascia therefore requires independent validation.

The systematic review by Lin et al. [40] integrated the core role of Piezo1 in fibrosis across multiple tissues, including skin, lung, cardiovascular tissue, liver, and kidney, and discussed the therapeutic potential of Piezo1 as an antifibrotic target. This provides review-level evidence across organ systems for PIEZO1-mediated mechanical–fibrotic conversion. The review did not directly address the cervical fascia or the vagus nerve, and extrapolation to the investing fascia requires independent validation.

Dong et al. [41] systematically reviewed the mechanisms of Piezo-mediated mechanically activated currents in cardiac, pulmonary, hepatic, renal, and skin fibrosis. Wang et al. [42] integrated the roles of Piezo1 in heart failure in regulating calcium homeostasis, reactive oxygen species (ROS) production, fibrosis, and immune activation through multiple pathways. Together, these two reviews provide supplementary review-level evidence across organ systems for PIEZO1-mediated mechanical–fibrotic conversion.

The systematic review by Chen et al. [43] pointed out that the roles of Piezo1 and Piezo2 in fibrosis are cell type-specific and organ-specific and may even include both pro-fibrotic and anti-fibrotic effects. The specific role of PIEZO1 in the investing fascia therefore requires evaluation in the context of its cellular microenvironment. Tu et al. [20] demonstrated in fibroblasts that mechanical stretch mediates fibroblast activation through Piezo1 channels, providing more direct evidence for this cell type-matched causal chain. Its tissue source is still not the cervical fascia, and extrapolation requires caution.

Chen et al. [18] revealed an upstream mechanism of PIEZO1 upregulation. GPR146, a mechanosensitive G protein-coupled receptor (GPCR), directly upregulates PIEZO1 expression through the cAMP-CREB1 pathway. Either GPR146 deficiency or PIEZO1 deficiency abolishes the hypertension and vascular remodeling phenotypes. PIEZO1 upregulation in the investing fascia under chronic mechanical loading may therefore not occur independently but may be mediated by upstream mechanosensitive GPCRs. The expression and function of GPR146 in fascial tissue nonetheless require validation. In contrast to GPR146-mediated PIEZO1 upregulation, Wu et al. [44] found that ZBED6 ameliorates cardiac fibrosis by inhibiting Piezo1 transcription and YAP nuclear translocation, indicating that PIEZO1 expression is also regulated by transcriptional repression mechanisms. This further enriches the regulatory network of PIEZO1 expression. Wang et al. [45] systematically reviewed the structural pharmacology and mechanotransduction mechanisms of Piezo1 and noted that its dysfunction is associated with multiple neurological diseases, providing structural pharmacological support for PIEZO1 as a core molecule in investing fascia–sheath coupling.

3.1.5. Mechanical Interaction Between Immune Cells and Fibroblasts

Yang et al. [46] integrated a mechanical–immune–fibroblast interaction framework. They proposed that extracellular matrix (ECM) stiffening drives a self-reinforcing loop between SPP1+ macrophages and POSTN+ fibroblasts through a core mechanosensing machinery composed of integrin–focal adhesion kinase (FAK), Piezo1, and YAP/TAZ, jointly maintaining a pro-fibrotic microenvironment. This framework suggests that fibrosis of the investing fascia may involve not only PIEZO1–YAP/TAZ activation in fibroblasts themselves but also mechanosensing and pro-fibrotic polarization of resident macrophages within the fascia.

Ezzo et al. [19] provided direct causal evidence for this framework. Direct contact between pro-fibrotic macrophages and fibroblasts recruits and activates Piezo1 on fibroblasts via αvβ3 integrin, triggering fibroblast contraction within seconds and driving YAP/NFAT1 nuclear translocation and pro-fibrotic phenotypic transformation within hours, independent of soluble factors. This provides a transferable molecular template for the possibility that investing fascial fibrosis involves macrophage–fibroblast mechanical interaction. Because the evidence comes from pulmonary and skin fibrosis models, extrapolation to the cervical fascia requires independent validation.

Su et al. [47] demonstrated using myeloid-specific Piezo1 knockout mice that macrophage Piezo1 deficiency attenuates radiation-induced pulmonary fibrosis. The mechanism involves Ca²⁺-dependent calpain signaling that upregulates MCP-1/CCR2/NF-κB and the endoplasmic reticulum (ER) stress ATF6/CHOP axis. This provides direct genetic evidence for the causal necessity of macrophage PIEZO1 in fibrosis. Cao et al. [48] demonstrated that PIEZO1 drives neutrophil NETosis through calcium overload, exacerbating pulmonary fibrosis, and that PIEZO1 inhibitors suppress this process, adding the neutrophil lineage to the immune cell spectrum.

Zeng et al. [49] revealed in a pressure overload model that Piezo1 mediates “mechanical dialogue” between epicardial cells and fibroblasts through Chemerin-Cmklr1 paracrine signaling, activating the PI3K-Akt1-Pou3f1 pathway to drive fibrosis. This provides a second independent molecular bridge for PIEZO1-mediated intercellular mechanical coupling. Zhang et al. [50] used single-cell multiomics to discover that endothelial cell PIEZO1 drives pulmonary fibrosis through the CAPN2-STAT3-IL-33 axis, providing a third independent molecular bridge. Yang et al. [51] demonstrated that intestinal epithelial cell PIEZO1 regulates the macrophage-to-myofibroblast transition via TGF-α signaling, supplementing the epithelial–macrophage mechanical dialogue pathway. Lim et al. [52] found that PIEZO1 selectively enhances IL-13 protein translation through the Ca²⁺-mTOR axis in group 2 innate lymphoid cells (ILC2), promoting type 2 inflammation and pulmonary fibrosis. This reveals a causal chain of mechanical signal, translational control, and cytokine output.

The systematic review by Lei et al. [53] integrated the dual regulatory mechanisms of the mechanotransduction–immune axis in organ fibrosis, covering the coordinated regulation of inflammatory signaling and ECM remodeling by integrin–FAK, Piezo1, and TRPV4, as well as how macrophages, T cells, and neutrophils sense mechanical input and amplify pro-fibrotic responses. Zou et al. [54] demonstrated that mechanical stretch directly activates fibroblast PIEZO1, which through Ca²⁺ influx activates the AP-1 transcription factor, driving PTGS2/PGE2 upregulation, promoting inflammation, and impairing fibroblast repair function. The PIEZO1 inhibitor GsMTx4 significantly reduces the inflammatory response, providing direct evidence for the PIEZO1–Ca²⁺–AP-1–PTGS2/PGE2 mechano-inflammatory pathway. Collectively, these findings indicate that PIEZO1 within the investing fascia may drive fibrosis through mechanical interactions among multiple immune cell and stromal cell populations, but extrapolation to the cervical fascia requires independent validation.

3.1.6. Molecular Framework of Fascial Mechanotransduction

The recently proposed myofascial rheostat model further provides a molecular framework for this step. The fascia, as a hyaluronan-rich adaptive interface, senses mechanical and inflammatory signals through the calcium–hyaluronan axis. Hyaluronan molecular weight dynamics and purinergic signaling constitute a feedback system that may determine the switch of the fascia from elastic buffering to rigid conduction [55]. Kirkness and Scarlata [56] elaborated the Ca²⁺–hyaluronan axis at the molecular level of the fascia, integrating Ca²⁺ channels such as Piezo1, TRPV4, and P2Y2 with hyaluronan synthesis and CD44/RHAMM receptor signaling into a unified fascial mechanotransduction framework. They explicitly noted that Ca²⁺-dependent regulatory mechanisms still require experimental validation in fascial cells, providing a direct theoretical basis for research on PIEZO expression in the investing fascia.

Fan et al. [57] proposed a multiscale framework for mechanosensing and remodeling of soft tissue, emphasizing that Piezo1 and Piezo2 and TRPV4 channels convert mechanical signals into intracellular signals such as Ca²⁺, activating the YAP/TAZ, NF-κB, and TGF-β pathways. The healthy ECM acts as a “spatial filter” that buffers mechanical loads, whereas fibrotic cross-linking erodes this protection, exposing fibroblasts and sensory nerve endings to elevated mechanical and inflammatory signals and forming a “stiffness–inflammation–fibrosis/pain” cycle. This framework provides multiscale theoretical support for investing fascial fibrosis, followed by rigid conduction and altered neural mechanical exposure. It also supports the statement that PIEZO channels are necessary but not necessarily the only molecular components.

The mechanical consequences of investing fascial fibrosis are reflected not only at the level of molecular pathways but also in quantitative changes in tissue stiffness. A shear-wave elastography (SWE) study of radiation-induced cervical fibrosis showed that cervical fascial stiffness increased from 22.7 kPa to 46.4 kPa and muscle stiffness from 21.5 kPa to 47.0 kPa, confirming that fibrosis can cause an almost-fold change in the mechanical properties of the cervical fascia [58]. These quantitative data from a human clinical model are complementary to the mechanical pressure required to reach the deep cervical fascia—approximately 6.06 N/cm²—reported by Pires et al. [59]. The former describes the rigid state of the fascia after fibrosis; the latter describes the feasibility of mechanical loads reaching the deep fascia. Together, they provide quantitative anchors for investing fascial fibrosis and rigid conduction. Because this evidence comes from a radiation-induced fibrosis model whose pathological driver differs from chronic mechanical loading, extrapolation to the investing fascia requires caution. Although the measurement data of Pires et al. [59] were obtained mainly from the trapezius region, their study design covered both the cervical and thoracolumbar regions. If the original article reported cervical-specific pressure thresholds, the parameters of investing fascial mechanical transmission should be calibrated in a region-specific manner.

The superficial layer of the prevertebral fascia, one of the main constituent layers of the carotid sheath, plays a key role in this transmission. Because the prevertebral fascia lies anterior to the cervical spine, its tension state is directly influenced by cervical curvature and the prevertebral muscle group. Changes in cervical curvature or tension in the prevertebral muscles may directly alter the morphology and internal diameter of the carotid sheath through changes in prevertebral fascial tension. The multilayer fascial composition of the anterior sheath wall in the skull base region [25] and the “mesentery-like fascial bundle” in the suprahyoid region [26] provide more superior anatomical anchors for this mechanical transmission.

The systematic review of carotid sheath anatomy by Bond et al. [24] confirmed that the sheath lies between the three layers of the deep cervical fascia. The complexity of its fascial composition and the variability of its attachment points constitute the structural basis for mechanical transmission. The histological evidence of Hayashi [28] suggests that, because the common sheath does not fuse directly with the investing fascia, the mechanical load of the investing fascia may act indirectly on the sheath wall through interlayer shear transmission within the deep cervical fascia or through the visceral fascial pathway. The mechanical efficiency of this pathway may be influenced by interindividual variation in fascial layer fusion.

The findings of Piffer [29] indicate that direct fibrous bundle connections exist between the sheath wall and the internal jugular vein. Mechanical deformation of the sheath wall may therefore be transmitted directly to the intra-sheath venous wall through these fibrous bundles, indirectly altering the balance of the intra-sheath mechanical microenvironment, including venous return and interstitial pressure. The developmental evidence of Miyake [30] indicates that the fusion relationship between the carotid sheath and the pretracheal fascia has a developmental basis, whereas the vagus nerve has already established its intra-sheath positional configuration before formation of the sheath wall. This provides a developmental constraint for the transmission pathway of sheath wall mechanical deformation, followed by connective tissue transmission and altered neural mechanical exposure.

The developmental histological evidence of Martínez-Sanz et al. [31] indicates that the middle layer of the deep cervical fascia forms an anatomical connection with the carotid sheath early in development and that its development parallels mechanical activity in the neck. This provides supplementary evidence that the mechanical continuity between the deep cervical fascial network and the carotid sheath has a developmental basis. The developmental histological evidence of Zhao [32] indicates that the differential thickening pattern of the three-layer fascial architecture of the sheath wall is consistent with the hemodynamic stress levels borne by each structure and that fusion of the outer fascial layers of adjacent structures reflects extrinsic mechanical interactions. This provides a histological basis for mechanical transmission pathways among the multilayer structures of the sheath wall.

Liu et al. [27] confirmed from a surgical anatomy perspective that the prevertebral fascia is a key anatomical landmark for protecting the carotid sheath and that its anatomical principles can be used to optimize exposure and protection of the carotid sheath. This provides surgical anatomical evidence for the prevertebral fascia playing a key role in mechanical transmission to the carotid sheath.

Mechanical transmission from the investing fascia to the carotid sheath not only has an anatomical basis but also a certain degree of quantitative mechanical feasibility. A study published in Sensors measured the mechanical pressure required to reach the deep cervical fascia and found it to be approximately 6.06 ± 0.186 N/cm². Mechanosensitivity and subcutaneous fat thickness did not affect this pressure value [59]. These data provide quantifiable mechanical parameter support for the investing fascia transmitting mechanical loads to the carotid sheath through the deep cervical fascial network under abnormal tension. The measurement site in this study was the trapezius region (posterior neck), not the investing fascia of core interest in the hypothesis (anterolateral neck), and extrapolation of its values to the investing fascia requires caution. Combined with Nash et al.’s [22] questioning of the existence of the investing fascia in the anterior triangle, future studies need to distinguish the mechanical roles of fascial structures in the anterior and posterior triangles.

The sensitivity of nerves to mechanical compression provides a quantitative reference for the pathophysiological consequences of increased intra-sheath pressure. Dahlin et al. [60] demonstrated in an experimental nerve compression model that continuous compression as low as approximately 30 mmHg causes changes in fast axonal transport and that continuous compression at 50 mmHg for 2 hours causes reversible conduction block. After release of compression, the recovery time of axonal transport is positively correlated with the intensity and duration of compression. Recovery occurs within hours after 2 hours of compression at 30 mmHg; several days are required for complete recovery after 2 hours of compression at 50 mmHg. This threshold provides a referable pathophysiological anchor for increased intra-sheath pressure and impaired vagal function. Its recovery time data suggest that the pathological consequences of acute compression and chronic fascial mechanical loading may differ fundamentally. Acute compression mainly causes reversible functional changes; chronic sustained loading may lead to cumulative, irreversible structural changes. Because this study is an acute compression model and its nerve type, species, and chronic fascial mechanical loading differ, extrapolation requires caution.

Shahabinejad et al. [61] systematically reviewed the rare clinical entity of vagal neuralgia. They found that vascular compression of the vagal nerve root causes VANCOUVER syndrome (chronic cough) and HeLPS (hemilaryngopharyngeal spasm) and that microvascular decompression relieves symptoms. This provides clinical causal evidence for mechanical compression of the vagus nerve, followed by dysfunction, relief of compression, and functional recovery. The compression site in this literature is the vagal nerve root near the skull base or brainstem, not the cervical carotid sheath segment, and its mechanical pathway does not fully correspond to the core hypothesis of cervical fascia, followed by sheath wall and vagal function. Extrapolation therefore requires caution.

Price et al. [62] reported the occurrence of internal jugular phlebectasia in Menkes disease. They speculated that decreased activity of the copper-dependent enzyme lysyl oxidase reduces the tensile strength of the deep cervical fascia constituting the carotid sheath, causing intra-sheath venous dilation. Lysyl oxidase is a key enzyme in collagen cross-linking; its reduced activity leads to insufficient collagen cross-linking and decreased fascial tensile strength. This mechanism contrasts with the increased collagen cross-linking in fibrosis. This clinical genetic disease model demonstrates from another perspective that the mechanical properties of the deep cervical fascia may affect the carotid sheath and its internal structures. Although the direction of its mechanical change is decreased tensile strength rather than fibrotic stiffening, it supports the transmission direction of sheath wall mechanical state and altered intra-sheath microenvironment. More importantly, this model suggests that regardless of whether the direction of change in fascial mechanical properties is stiffening or softening, significant changes in mechanical properties may affect intra-sheath structures through transmission via the sheath wall.

3.2. In Vivo Biomechanical Evidence: Dynamic Coupling Between the Sternocleidomastoid Muscle and the Carotid Sheath

The first half of the aforementioned mechanical transmission chain has in vivo experimental support. A clinical study using intraoperative ultrasound showed that 15° contralateral head rotation displaces the sternocleidomastoid muscle—the main muscular carrier of the investing fascia—from lateral to the carotid sheath to above or medial to the sheath, with high statistical significance (*p* < 0.001). In contrast, the positional change of the vagus nerve within the sheath was not significant (*p* = 0.198) [4]. The authors explicitly stated that this is the first study to discuss the relationship between head rotation and displacement of the sternocleidomastoid muscle relative to the carotid sheath.

This finding has multiple implications. First, it demonstrates that a direct mechanical contact relationship exists between the sternocleidomastoid muscle and the carotid sheath. The configuration in which the sternocleidomastoid muscle lies above the sheath implies direct physical contact between the two. During head rotation, the mechanical movement of the sternocleidomastoid muscle may be transmitted to the sheath wall through fascial connections. Second, a 15° rotation is a routine movement, meaning that the mechanical transmission described in the hypothesis may occur during normal daily life, not only under pathological conditions. Third, the relative stability of the vagus nerve within the sheath (*p* = 0.198) indicates that the effect of mechanical deformation of the sheath wall on the nerve may depend mainly on the connective tissue connections between the sheath wall and the nerve, not primarily on displacement of the nerve itself. This indirectly supports the transmission pathway of sheath wall deformation, followed by connective tissue transmission and altered neural mechanical exposure.

3.3. Mechanical Signal Conversion into Neural Signals: Molecular Mechanisms of PIEZO Channels

3.3.1. Existence and Necessity of PIEZO Channels

How are mechanical signals converted into neural signals? The current hypothetical framework points to the involvement of mechanosensitive ion channels. PIEZO1 and PIEZO2, well-characterized mechanosensitive cation channels, convert membrane stretch or compression into ion influx signals [15]. The systematic review by Prescott and Liberles [63] integrated the cellular and molecular basis of vagal interoceptive function and confirmed that PIEZO channels are core molecular components of vagal mechanotransduction. Pirri [64] elaborated the core role of PIEZO channels in mechano-inflammation, emphasizing their role as “gatekeepers” of neuroimmune crosstalk and suggesting that PIEZO channels may influence neural function by regulating the local inflammatory microenvironment. This provides a supplementary mechanism for investing fascial mechanical loading, followed by local inflammation and altered neural function.

The systematic review by In et al. [65] positioned the vagus nerve as a neurovisceral interface. Vagal sensory receptors detect mechanical stimuli through multiple channels, including PIEZO1, PIEZO2, TRPC5, ASIC2, and Tentonin3. This provides systematic review–level support for the statement that PIEZO channels are necessary but not necessarily the only molecular components. The available evidence can be organized according to the logic of existence, necessity, regulation, and cross-organ universality.

A foundational study published in Science demonstrated that PIEZO1 and PIEZO2 are expressed in the vagal ganglia and nodose ganglia and that double knockout of PIEZO1 and PIEZO2 completely abolishes the baroreceptor reflex. PIEZO channels are therefore necessary molecular components for the vagus nerve to convert mechanical signals into neural electrical signals [5]. Min et al. [16] demonstrated through single-gene knockout of PIEZO2 that loss of PIEZO2 is sufficient to abolish the baroreceptor reflex. PIEZO2 neurons also form a unique “decorated aortic claw” structure in the aortic arch. This advances the necessity of PIEZO channels from dual-gene redundancy to the irreplaceable role of PIEZO2 as a single gene and provides neural-side molecular mechanistic evidence for mechanical deformation of the carotid sheath wall and altered vagal afferent activity.

3.3.2. Single-Gene Necessity of PIEZO2

The single-gene necessity of PIEZO2 in vagal mechanosensation. This principle was subsequently extended to cardiac mechanoreceptors. A study published in Nature demonstrated that vagal PIEZO2 neurons form characteristic “end-net endings” in the heart and that their blood volume–dependent responses are time-locked to atrial and ventricular systole. Single knockout of PIEZO2 abolishes heartbeat-coupled neural activity, causing orthostatic hypotension and impairing cardiovascular stability during traumatic hemorrhage [8]. This finding advances the principle that PIEZO channels are core molecules of vagal mechanotransduction from baroreceptors to blood volume receptors and demonstrates that PIEZO2 has single-gene necessity in vagal mechanosensation.

3.3.3. Cross-Organ Universality of PIEZO2

PIEZO2 in the cross-organ universality of vagal mechanotransduction. Evidence for PIEZO2-dependent mechanotransduction in the vagus nerve has been extended from the cardiovascular system to the respiratory system. A study published in Nature demonstrated that vagal parvalbumin (PVALB) neurons innervate pulmonary neuroepithelial bodies and that pulmonary neuroepithelial bodies uniformly express PIEZO2. Targeted knockout of Piezo2 in pulmonary neuroepithelial bodies abolishes the airway closure response; stimulation of pulmonary neuroepithelial bodies or PVALB neurons induces gasping in the absence of an airway threat [10]. This finding, together with Liu [8], indicates that PIEZO2 is a universal molecular basis for the vagus nerve to sense mechanical stimuli across multiple organs.

3.3.4. PIEZO Expression on the Fascial Side and the Receptor Side

On the fascial side, a human histological study confirmed PIEZO2 protein expression in the human trapezius fascia and detected the distribution of calcitonin gene-related peptide (CGRP)–positive nociceptive nerve fibers and TH-positive sympathetic nerve fibers [6]. The trapezius fascia is continuous with the investing fascia in the superficial layer of the deep cervical fascia. Its PIEZO2 expression provides human histological evidence that the investing fascia has the capacity to convert mechanical signals into cellular signals. This evidence comes from the trapezius fascia rather than the investing fascia itself. García-Martínez et al. [17] confirmed PIEZO2 protein expression in the human palmar aponeurosis, suggesting that PIEZO2 expression in human fascial tissue may be conserved across anatomical sites. This evidence comes from the palmar aponeurosis rather than the cervical investing fascia, and extrapolation to the cervical fascia requires independent validation.

On the receptor side, another study confirmed PIEZO1 and PIEZO2 protein expression in the sensory nerve structures of the human carotid sinus wall, including mechanoreceptor morphologies such as Ruffini-like corpuscles and dendritic nerve structures [7]. The carotid sinus is adjacent to the carotid sheath and is a direct downstream sensory structure for changes in the mechanical environment of the sheath wall. Alba et al. [7] also detected PIEZO1 and PIEZO2 expression in the human carotid body, suggesting that vagal mechanical sensing of cervical vascular structures may not be limited to baroreceptors but may also involve mechanical modulation of chemoreceptors. This finding, together with PIEZO expression in the carotid sinus, indicates that vagal mechanotransduction in the cervical vascular region is characterized by multi-receptor cooperation.

3.3.5. PIEZO Expression in Peripheral Mechanosensory Structures

Human evidence for PIEZO expression in peripheral mechanosensory structures has been extended from the carotid sinus to the aortic arch. A study integrating histology, proteomics, and transcriptomics identified for the first time candidate baroreceptor channels PIEZO1, TRPV2, and TRPM4 in the human aortic arch and confirmed that these ion channels do not originate from aortic wall cells but may originate from vagal neuronal cell bodies [11]. Yundung et al. [11] not only identified PIEZO1 in the human aortic arch but also found that TRPV2 and TRPM4 are co-expressed as candidate baroreceptor channels. Vagal mechanotransduction therefore may not rely solely on the single molecule PIEZO1 but may involve a multi-channel cooperative system. Extrapolating this principle to the cervical fascia–sheath continuum, activation of the vagus nerve by mechanical deformation of the sheath wall may likewise involve the cooperative participation of PIEZO1 and PIEZO2 and other mechanosensitive channels.

Linz et al. [66] identified two mechanosensitive potassium channels in cardiac-projecting vagal afferent neurons of the nodose ganglion. Both were predominantly K⁺-selective and sufficient to generate depolarization and trigger action potentials. The study explicitly stated that nonselective cation channels such as Piezo1 and Piezo2 mediate baroreceptor function but are not directly responsible for repolarization, and that mechanosensitive potassium channels shape vagal afferent signals by terminating action potentials. This provides direct electrophysiological evidence for the statement that PIEZO channels are necessary but not necessarily the only molecular components. Xie et al. [67] demonstrated that PIEZO1 is functionally expressed in enteric neurons, suggesting that PIEZO1 may also play a mechanosensory role in nonsensory neurons and expanding the understanding of mechanical responses of autonomic nerve endings within the investing fascia.

Tereshenko et al. [6] confirmed PIEZO2 protein expression in the human trapezius fascia and detected the distribution of TH-positive sympathetic nerve fibers and CGRP-positive nociceptive nerve fibers. The trapezius fascia is continuous with the investing fascia in the superficial layer of the deep cervical fascia. The presence of TH-positive sympathetic fibers suggests that fascial tissue itself may be a target region for sympathetic nerve endings and that changes in the mechanical state of the fascia may directly affect local sympathetic tone. Alba et al. [7] detected high expression of PIEZO1 and PIEZO2 (71% and 86%, respectively) in the human superior cervical ganglion. Combined with these findings, a more complete “fascia–sympathetic–vagus” interaction hypothesis can be formulated. The mechanical load of the investing fascia may be transmitted not only to the carotid sheath through the deep cervical fascial network but may also regulate sympathetic and vagal tone through sympathetic nerve endings within the fascia and PIEZO channels in the superior cervical ganglion. This bidirectional regulatory mechanism may explain why upper versus lower cervical spinal manipulation produced opposite directions of heart rate variability (HRV) change in Section 4.2 [72]. Mechanical interventions at different segments may produce differences in net effects through different weightings of sympathetic and vagal components.

The mechanistic implications of this finding warrant further elaboration. The superior cervical ganglion is the final common pathway for sympathetic output in the head and neck, and its postganglionic fibers innervate the carotid artery, internal jugular vein, thyroid gland, salivary glands, and facial blood vessels. If mechanical deformation of the sheath wall is transmitted to the superior cervical ganglion through the prevertebral fascia and activates its PIEZO1 and PIEZO2, the following cascade effects may occur. Excitability of sympathetic postganglionic neurons may be altered. Tension of the carotid artery and internal jugular vein may change. The intra-sheath hemodynamic microenvironment may be altered. Vagal mechanical exposure may be further changed. This indirect mechanics–sympathetic–vessels–vagus pathway forms a parallel relationship with the direct mechanics–sheath wall–vagus pathway in the original hypothesis.

The net autonomic effect of investing fascia–sheath coupling may therefore be the algebraic sum of bidirectional vagal and sympathetic regulation rather than a change in vagal tone alone. This provides a new perspective for explaining the inconsistency of clinical study results in Section 4.2. The direction of HRV changes observed in different studies may depend on the relative weighting of sympathetic and vagal components. This weighting may be influenced by individual differences in PIEZO expression levels in the superior cervical ganglion, the mechanical state of the prevertebral fascia, and cervical segmental differences. This mechanism also provides a more specific theoretical basis for the testable prediction in Section 5.1 of the hypothesis, namely immunohistochemical detection of PIEZO1 and PIEZO2 in the superior cervical ganglion. Direct validation of this inference nonetheless requires selective activation or inhibition of PIEZO1 and PIEZO2 in the superior cervical ganglion in animal models, and no such functional study is currently available.

3.3.6. Mechanosensitive Cell Populations in the Fascia

Identification of mechanosensitive cell populations in the fascia. A study published in Scientific Reports used single-cell RNA sequencing to identify a previously unrecognized CD34+ membranous cell population in rat subcutaneous fascia. This population accounted for 20.0% of total fascial cells and significantly upregulated mechanosensitive genes such as Piezo2, elastin, and integrin β3. The cells were laminar and arranged alternately with collagen bundles. They were stretched by extracellular fibers into a “hammock-like” state of tension and extensively surrounded blood vessels, nerves, and collagen bundles. They formed a “distributed sensory network” through Cx43+ gap junctions and tunneling nanotubes [12]. This finding provides direct cellular-level evidence that the investing fascia itself has the capacity to convert mechanical signals into cellular signals and provides an anatomical anchor at the cell–nerve contact interface for the transmission of fascial mechanical signals to neural signals. Because this study was based on rat abdominal subcutaneous fascia, extrapolation of its conclusions to the human cervical investing fascia requires independent validation.

3.3.7. PIEZO Channel Regulatory Mechanisms

The mechanotransduction efficiency of PIEZO channels is not constant; it is regulated by auxiliary subunits. A study published in PNAS identified a previously completely uncharacterized member of the MyoD family of inhibitory proteins, MDFIC2. MDFIC2 is selectively and abundantly expressed in dorsal root ganglia, trigeminal ganglia, and vagal sensory neurons. It physically binds to PIEZO1 and PIEZO2, alters their mechanosensitivity and inactivation kinetics, and converts them into high-threshold, slow-inactivating mechanoreceptors. Cryo-electron microscopy revealed a conserved binding pocket for the auxiliary subunit in the pore module of PIEZO1 and PIEZO2 [9].

The regulation of PIEZO channels by MDFIC2 has been characterized at the molecular structural level, and its functional consequences have been validated in vivo. A study published in PNAS demonstrated that in a mouse model of neuropathic pain, Mdfic2 expression is downregulated and that adeno-associated virus (AAV) delivery of MDFIC2 cDNA alleviates mechanical allodynia [13]. This established a causal chain. First, MDFIC2 expression level changes. Second, PIEZO channel mechanosensitivity changes. Third, response intensity to mechanical stimuli changes. Extrapolating this to the present hypothesis, differences in MDFIC2 expression levels among individual vagal sensory neurons may partially explain differences in conversion efficiency of investing fascial mechanical loading and altered vagal afferent activity. This inference still requires validation in local cervical tissues.

In addition to MDFIC2, PIEZO2 protein expression levels are regulated by ubiquitination. Huo et al. [68] demonstrated that the E3 ubiquitin ligase Nedd4-2 directly binds to the PPSY motif in the intracellular segment of PIEZO2 and downregulates PIEZO2 expression and current through ubiquitination. In a hypertensive rat model, Nedd4-2 was highly expressed in baroreceptor neurons of the nodose ganglion and significantly downregulated Piezo2, leading to impaired baroreceptor reflex sensitivity and increased sympathetic tone. This provides a second independent mechanism for PIEZO functional variation. MDFIC2 regulates the mechanosensitivity and inactivation kinetics of PIEZO; Nedd4-2 regulates the protein expression level of PIEZO2. Together, the two constitute an upstream mechanism of PIEZO functional variation. Differences among individuals in the efficiency of converting investing fascial mechanical loading into vagal afferent activity may be jointly influenced by MDFIC2 expression levels and Nedd4-2–mediated PIEZO2 ubiquitination levels.

Bai et al. [69] found that proinflammatory cytokines released by microglia upregulate astrocytic Piezo1 expression and thereby regulate neuroinflammation, suggesting that PIEZO1 expression may also be regulated by the neuroimmune microenvironment. This adds a new dimension to the regulation of PIEZO1 expression.

3.3.8. PIEZO1-Mediated Mechanical–Fibrotic Conversion

Piezo1 in vascular smooth muscle mediates mechanical stretch–induced collagen deposition and fibrosis [36], suggesting that Piezo1 may have broader biological functions in the conversion of mechanical loading into tissue remodeling. Konishi et al. [14] found in patients with symptomatic carotid atherosclerotic plaques that Piezo1 expression was significantly increased in plaque myofibroblasts, providing direct human histological evidence for the association between Piezo1 expression in mesenchymal cells adjacent to the carotid sheath and mechanical load–driven tissue remodeling. This evidence comes from carotid plaques rather than the sheath wall or the investing fascia itself, and extrapolation requires caution. Nevertheless, it indicates that mesenchymal cells in the carotid region possess the molecular basis for Piezo1 mechanotransduction.

Taken together, the above evidence forms a complete hypothetical chain of macroscopic mechanical input, PIEZO molecular conversion, and neural electrical signal output. This chain extends from peripheral mechanosensory structures (carotid sinus, aortic arch, heart, and airway) to fascial tissues (trapezius fascia, investing fascia, subcutaneous fascia, and palmar aponeurosis) and then to ganglia (vagal ganglia). PIEZO1 and PIEZO2 and their auxiliary subunit MDFIC2 constitute a multilevel, regulatable mechanotransduction system [15,16]. All PIEZO-related evidence nonetheless comes from tissues such as the vagal ganglia, heart, lung, trapezius fascia, carotid sinus, aortic arch, subcutaneous fascia, and palmar aponeurosis. No study has yet directly validated the expression and function of PIEZO1 and PIEZO2 in the investing fascia or the carotid sheath wall. The hypothesis extrapolates the necessity of PIEZO channels in vagal mechanotransduction to PIEZO activation in the cervical fascia–sheath complex. This extrapolation requires immunohistochemical and functional validation in local cervical tissues.

4. Clinical Inferences and Functional Validation

4.1. In Vivo Evidence at the Functional Output End

The clinical evidence cited in this section does not directly test the middle segment of the chain—fascial mechanical changes, carotid sheath deformation, changes in the intra-sheath microenvironment, and changes in vagal mechanical exposure—and serves only as indirect support.

The validation step from mechanical manipulation to neural functional output has been supported by multiple independent studies, but the available evidence is not consistent. Most studies have limited levels of evidence and cannot serve as a basis for clinical recommendations.

A repeated-measures self-controlled study showed that after cervical myofascial release under 50° head-up tilt (maximal sympathetic activation), 17 healthy subjects produced a vagal response sufficient to overcome sympathetic tone, reflected by a significant increase in the high-frequency (HF) component of HRV. Touch placebo and no-touch control conditions produced no such effect [70]. This study demonstrates that mechanical release targeting the cervical fascia may directly alter the net output of the autonomic nervous system. The study nonetheless had a small sample size (*n* = 17) and did not control for the influence of respiratory rhythm on HRV, limiting its level of evidence.

Another double-blind, sex-balanced validation study showed that a standardized vagus nerve neurodynamic test—applying stretch to the cervical vagus nerve segment through a series of neck movements—elicited a significant decrease in heart rate in healthy subjects (approximately 12 bpm in men and 8 bpm in women, *p* < 0.017). The magnitude of heart rate reduction was significantly greater in men than in women (*p* < 0.05), suggesting that sex may influence the modulation of vagal function by cervical mechanical manipulation. No adverse events were reported [71]. This sex difference indicates that future studies should include sex as an important covariate when designing intervention protocols and assessing autonomic effects. The study remains a small-sample physiological study, and its clinical significance is uncertain.

A randomized controlled crossover preliminary study provided an additional dimension of segmental specificity. Upper cervical (C1–C2) spinal manipulative therapy enhanced parasympathetic dominance (increased normalized high-frequency power [nuHF], *p* = 0.03), whereas lower cervical (C6–C7) manipulative therapy enhanced sympathetic dominance (increased low-frequency/high-frequency ratio [LF/HF], *p* = 0.02) [72]. After upper cervical manipulation, nuHF increased by approximately 15%–20% from baseline; after lower cervical manipulation, LF/HF increased by approximately 20%–25% from baseline. Mechanical interventions at different segments may therefore modulate autonomic neural output in opposite directions. The influence of the mechanical state of the cervical fascia on the autonomic nervous system is thus not uniform but may depend on the specific anatomical segment subjected to mechanical manipulation. This study nonetheless had a very small sample size (*n* = 20) and was designated by its authors as a “preliminary study.” Its segmental specificity conclusions have not been consistently confirmed by subsequent systematic reviews and cannot be regarded as definitive.

A randomized controlled trial in female workers in the garment industry also showed that suboccipital fascial release may affect HRV parameters, with an effect superior to rest, suggesting that mechanical intervention targeting the suboccipital region may help improve occupational stress–related autonomic dysfunction [73]. This study supports the inference that mechanical manipulation targeting specific cervical regions, including the suboccipital region, may produce autonomic effects. Its intervention target was nonetheless the suboccipital region, not the main muscular attachment area of the investing fascia of core interest in the hypothesis, and its sample size was limited.

Kazantseva et al. [74] published a randomized controlled trial (*n* = 128) evaluating the effects of myofascial release on HRV in patients with autonomic dysfunction. The subjects were patients with autonomic dysfunction in whom sympathetic activity was dominant. They were randomly divided into a myofascial release group (*n* = 64) and a control group (*n* = 64). Both groups received a physical rehabilitation program comprising aerobic exercise, flexibility training, and breathing training; the myofascial release group additionally received self-massage for 12 weeks. The intervention protocol consisted of self-myofascial release three times per week for approximately 20 minutes per session, mainly targeting the neck, shoulder, and upper back muscle groups.

The myofascial release group showed a significant increase in high-frequency power (*p* = 0.008) and a significant decrease in the LF/HF ratio (*p* = 0.013). HF increased by approximately 25% from baseline; LF/HF decreased by approximately 20% from baseline. The control group showed smaller improvements in the corresponding parameters. These findings suggest that myofascial release may reduce sympathetic activity and increase parasympathetic activity. The 12-week intervention period and specific protocol parameters of this study provide a direct reference for the design of future randomized controlled trials (RCTs) targeting fascial nodes. The limitations are nonetheless evident. The article was published in Russian, and the journal’s level of evidence is relatively low. Both groups received exercise and breathing training, so the independent effect of myofascial release cannot be fully isolated. The intervention consisted of self-massage rather than clinical manual therapy, and its mechanical load intensity and precision are limited. The abstract reported only *p* values and did not provide effect sizes or confidence intervals. This study therefore serves as a supplementary example of the positive effects of soft tissue techniques but not as core evidence.

Overmann et al. [75] reported in patients with chronic neck pain and depression that a single session of myofascial release along the trapezius muscle significantly improved HRV, pain perception, and cervical range of motion. This study simultaneously measured muscle tone, elasticity, and stiffness. The myofascial release group showed a significant reduction in stiffness (*p* < 0.05), whereas elasticity and muscle tone showed no significant changes. The mechanical effects of myofascial release may therefore act mainly on the viscoelastic or stiffness properties of the fascia rather than on the elastic modulus itself. This differential result contrasts with the inference in Section 3.1 of the hypothesis that the essence of fibrosis is increased collagen cross-linking and decreased elastic fibers. Myofascial release may improve the mechanical state of the fascia by reducing stiffness rather than restoring elasticity, and this mechanistic hypothesis can be tested through future SWE measurements. The intervention target of this study (the trapezius muscle) is consistent with the key fascial node recommended by the hypothesis, and the study population partially overlaps with the specific phenotype characterized primarily by cervical mechanical loading and accompanied by autonomic symptoms, as defined in Section 5.6 of the hypothesis. Its HRV was nonetheless a secondary outcome, the intervention duration was short, effect sizes were not reported in detail, and the journal’s level of evidence is moderate. It therefore serves as supplementary positive evidence rather than core evidence.

The randomized controlled trial by Navarro-Rico et al. [76] showed that visceral manual therapy improves HRV parameters in patients with bruxism (SDNN time × group interaction effect, *p* = 0.04), providing another independent RCT showing that soft tissue techniques may affect autonomic nervous function. The study population consisted of patients with bruxism, which differs from the specific phenotype defined by the hypothesis. The sample size was small, and the target was the orofacial or visceral fascia rather than the investing fascia, so extrapolation requires caution.

4.2. Negative Evidence and Controversies

The clinical evidence cited in this section likewise does not directly test the middle segment of the chain and serves only as indirect support.

Regarding the effects of cervical mechanical manipulation on autonomic nervous function, the available evidence is not consistent. The results of the above positive studies must be examined together with negative or conditional evidence. For clarity, the available evidence can be divided into three tiers.

The first tier comprises systematic reviews and meta-analyses. Amoroso Borges [77] included 9 studies but did not perform a meta-analysis, and its methodological quality was limited. Kovanur Sampath [78] included 14 trials with 618 subjects; GRADE assessment indicated low-quality evidence, with the conclusion that spinal manipulation as a whole did not affect autonomic nervous measures but that the cervical subgroup may affect HRV-HF. Hansen [79] specifically evaluated thoracic mobilization or manipulation and concluded that it had no significant effect on autonomic nervous system markers, contrasting with previous directional conclusions that thoracic stimulation produces sympathetic enhancement. Villani [80] included 50 studies with 2147 subjects and found that joint mobilization increased skin conductance (a sympathetic marker), whereas soft tissue techniques and non–high-velocity low-amplitude (HVLA) techniques were associated with reduced heart rate and increased root mean square of successive differences (RMSSD). Manual therapy may therefore interact with regulatory systems in a technique-dependent manner. Gordon [81] suggested that osteopathic manipulative treatment may produce some effects on psychophysiological indicators, especially in pain populations, but the primary outcomes were psychometric measures and HRV was a secondary outcome. Harper [82] specifically evaluated the effects of manual therapy on HRV in patients with long-standing neck pain, included only 3 RCTs, and was unable to perform a meta-analysis, concluding that no single technique has been proven superior. The scoping review by Monteiro [83] suggested that cervical manipulation may reduce systolic blood pressure and improve sympatho-vagal balance, but the level of evidence is limited.

The second tier comprises negative RCTs. Budgell [84] (*n* = 95, sham manipulation control, standardized respiratory rhythm) was unable to replicate short-term autonomic nervous function changes after cervical manipulation in healthy young adults. Monteiro [85] showed in female subjects that upper cervical manipulation and massage failed to modulate sympatho-vagal balance or blood pressure. These inconsistent results suggest that the effects of cervical mechanical manipulation on vagal function may be regulated by multiple factors, including manipulation site, manipulation type, subject state, and respiratory rhythm control.

The third tier is target selection bias. The HRV changes observed in previous positive studies, such as Henley [70] and Win [72], may partly arise from confounding due to uncontrolled respiratory rhythm. The technique-specific analysis by Villani et al. [80] indicates that HVLA manipulation and soft tissue techniques may have different directions of effect on the autonomic nervous system. The stimulation targets of existing negative RCTs were all cervical joints or nonspecific cervical massage; they did not target the main muscular attachment area of the investing fascia, the suboccipital margin, or the peri-auricular vagal afferent region. Negative results therefore cannot exclude the possibility that mechanical intervention targeting key nodes may produce autonomic effects. They instead suggest that future studies require more precise target selection and intervention protocol design.

4.3. Tools for Validating Morphology–Function Associations

The imaging validation pathway of the hypothesis must answer the following question: if mechanical changes in the carotid sheath environment lead to morphological changes in the intra-sheath vagus nerve, are these morphological changes correlated with functional changes?

A cross-sectional study of 88 healthy subjects was the first to establish a negative correlation between cervical vagus nerve CSA and HRV parasympathetic parameters on the left side [86]. Several reference sources can be combined to construct a toolchain of normal reference benchmarks, morphology–function association, and pathological deviation assessment. These sources include CSA reference values from Drakonaki et al. [87] (330 cases/657 nerves), sheath-specific reference values from Bedewi et al. [88], pooled reference values from the systematic review by Fisse et al. [89], and the standardized protocol and side-to-side differences from Pelz et al. [90]. Sijben et al. [91] confirmed CSA reduction in Parkinson disease. Abičić et al. [92] confirmed that CSA is associated with cardiac vagal function in multiple sclerosis. Lee et al. [93] confirmed that right-sided CSA is associated with an abnormal heart rate response to deep breathing in Parkinson disease. Laucius et al. [94] confirmed that CSA is associated with autonomic symptom scores in Parkinson disease. Additional studies have expanded the evidence on the disease spectrum, developmental independence, functional associations, and sex or neck circumference associations of CSA changes, including Laucius et al. [95] in ALS, Miyamoto et al. [96] in longitudinal follow-up of isolated rapid eye movement sleep behavior disorder and Parkinson disease [iRBD/PD], Nezu et al. [97] in patients with vascular risk factors, Wang et al. [98] in healthy children, Onodera et al. [99] in community-dwelling older adults, and Hegde et al. [100] in healthy adults. Di Giovanni et al. [101] confirmed in patients with genetically confirmed CANVAS that bilateral vagus nerve CSA was significantly reduced, extending the disease spectrum of CSA reduction to hereditary neurodegenerative diseases. The systematic review and meta-analysis by Kheradpishe et al. [102] suggested that vagus nerve CSA may be increased in diabetic neuropathy, in contrast to CSA reduction in neurodegenerative diseases. This further supports the notion that the direction of CSA change may be disease-specific and emphasizes the necessity of standardized ultrasound protocols.

Dörschner et al. [103] nonetheless confirmed that ultrasound measurements are significantly smaller than true anatomical dimensions and that differences across settings cannot be eliminated by a single correction factor. The absolute measurement validity of CSA must therefore be interpreted with caution, and the CSA < 4.2 mm² threshold [104] has not been independently validated.

4.4. Clinical Disease Spectrum and Pathological Nomenclature

Hauser et al. [104] proposed the “cervicovagopathy” framework, integrating cervical ligamentous instability, carotid sheath compression, and vagus nerve dysfunction into a nameable entity, and proposed multi-indicator ultrasound validation criteria (CSA < 4.2 mm², C1–C2 instability > 4.0 mm, and internal jugular vein [IJV] CSA < 180 mm²). This study is a retrospective hypothetical review (Level 5) without a control group. Its framework should be positioned as a hypothetical framework rather than clinical evidence.

4.5. Therapeutic Implications: Fascial Release as a Neuromodulatory Strategy

The intervention evidence cited in this section does not directly test the middle segment of the chain and serves only as indirect support. The therapeutic implications remain hypothesis-driven and cannot serve as clinical recommendations.

If the hypothesis holds, release-based interventions targeting the investing fascia may indirectly modulate vagal function through a mechanical unloading effect. Existing clinical observations include improvement of autonomic symptoms by cervical fascial manipulation [70], relief of vagus-related symptoms by cervical soft tissue release [71], segmental-specific effects of cervical manipulation on HRV [72], and potential effects of suboccipital fascial release on HRV [73]. Upper trapezius SWE showed a significant decrease in shear wave velocity after osteopathic manipulative treatment [105]. RCTs by Kazantseva et al. [74], Overmann et al. [75], and Navarro-Rico et al. [76] supplemented the positive HRV effects of soft tissue techniques, but methodological limitations warrant caution.

The negative RCTs by Budgell [84] and Monteiro [85] and the systematic review by Amoroso Borges [77] suggest that the existing supportive evidence comes mainly from small-scale clinical studies and that the results are inconsistent. Although the latest meta-analysis [78] suggests that cervical manipulation may affect HRV-HF, the quality of evidence is low. The technique-specific analysis by Villani et al. [80] indicates that soft tissue techniques may produce parasympathetic effects, whereas HVLA manipulation may not. Gordon et al. [81] suggest that osteopathic manipulative treatment may affect psychophysiological indicators, but HRV was a secondary outcome. The target selection of existing negative RCTs may have an anatomical blind spot: the stimulation targeted cervical joints rather than key fascial nodes related to vagal function. The above interventions are not simple neck massage; they are targeted mechanical interventions aimed at the main muscular attachment area of the investing fascia, the suboccipital region, and the peri-auricular vagal afferent region. These therapeutic implications are nonetheless still hypothesis-driven and cannot serve as clinical recommendations.

At the clinical operational level, Lam et al. [106] reported an ultrasound-guided hydrodissection technique of the cervical sympathetic chain and vagus nerve, providing a referable operational template for targeted intervention. This study is a technical report without a control group and cannot yet demonstrate efficacy.

5. Validation Directions and Key Questions

5.1. Evidence Gaps and Testable Predictions

5.1.1. Established or Relatively Well-Supported Steps

The carotid sheath lies between the three layers of the deep cervical fascia, and its fascial composition exhibits marked regional heterogeneity and interindividual variability [24].

The investing fascia has an anatomical weak point between the sternocleidomastoid and trapezius muscles. This region is filled with adipose tissue, and the carotid sheath communicates directly with the subcutaneous tissue through this channel [23]. The overall existence of the investing fascia in the anterior triangle has nonetheless been questioned by Nash et al. [22].

The carotid sheath has a fibroelastic structure, and its wall thickness varies. Connective tissue fiber bundles connect the internal jugular vein wall to the inner surface of the sheath wall [29].

The position of the vagus nerve within the sheath exhibits marked individual variation and left–right asymmetry [1,2]. Randhawa et al. [3] found a 38% variation rate and a predominance of left-sided variation in 347 ultrasound cases. Williamson et al. [35] found that unilateral carotid sinus compression increased MSNA and that the left-sided effect was greater than the right-sided effect, providing electrophysiological evidence for side specificity.

Contralateral head rotation of 15° significantly alters the spatial relationship between the sternocleidomastoid muscle and the carotid sheath (*p* < 0.001), whereas the position of the vagus nerve within the sheath remains stable (*p* = 0.198) (4).

The mechanical pressure required to reach the deep cervical fascia is approximately 6.06 N/cm² [59]. Nerves are sensitive to mechanical compression. Dahlin et al. [60] demonstrated that continuous compression at 30 mmHg causes changes in axonal transport and that 50 mmHg causes reversible conduction block. Shahabinejad et al. [61] provided clinical causal evidence for compression, symptoms, decompression, and relief. Price et al. [62] indirectly supported, using a Menkes disease model, that the mechanical properties of the deep cervical fascia may affect intra-sheath venous structures.

The reliability of cervical fascial SWE is acceptable [105,107,108]. A radiation-induced cervical fibrosis model showed that fascial stiffness increased from 22.7 kPa to 46.4 kPa [58]. Capron et al. [109] demonstrated that the tubular geometry of blood vessels interferes with SWE estimation, and a geometric correction protocol must be established for SWE of the sheath wall.

Cervical vagus nerve CSA already has normal reference values [87], sheath-specific reference values [88], pooled reference values [89], and a standardized protocol [90]. Pathological changes in CSA have been validated in Parkinson disease [91], multiple sclerosis [92], ALS [95], longitudinal follow-up of iRBD/PD [96], vascular risk factors [97], healthy children [98], community-dwelling older adults [99], healthy adults [100], Parkinson disease [94], CANVAS [101], and diabetic neuropathy [102]. Ultrasound measurements nonetheless differ from true anatomical dimensions [103].

PIEZO1 and PIEZO2 are expressed in the vagal ganglia [5], heart [8], lung [10], trapezius fascia [6], palmar aponeurosis [17], carotid sinus [7], aortic arch [11], and subcutaneous fascia [12] and have mechanotransduction functions [15]. Prescott and Liberles [63] confirmed that PIEZO is a core component of vagal mechanotransduction. Pirri [64] elaborated the neuroimmune crosstalk role of PIEZO in mechano-inflammation. In et al. [65] positioned the vagus nerve as a neurovisceral interface and noted that multiple channels participate in mechanotransduction. Linz et al. [66] demonstrated that mechanosensitive potassium channels participate in the termination of vagal afferent signals. Xie et al. [67] demonstrated that PIEZO1 is also functionally expressed in nonsensory neurons. Min et al. [16] confirmed that PIEZO2 is irreplaceable as a single gene. Alba et al. [7] detected PIEZO1 and PIEZO2 in the superior cervical ganglion and carotid body. Yundung et al. [11] found TRPV2 and TRPM4 co-expressed with PIEZO1 in the aortic arch. Konishi et al. [14] confirmed increased Piezo1 expression in carotid plaques. Struthoff et al. [34] confirmed the presence of dense sympathetic nerve fibers at the carotid bifurcation, providing human histological evidence for innervation of the carotid sheath region.

PIEZO channels are regulated by the auxiliary subunit MDFIC2 [9,13]. Huo et al. [68] demonstrated that Nedd4-2 downregulates PIEZO2 through ubiquitination. Chen et al. [18] demonstrated that GPR146 upregulates PIEZO1. Wu et al. [44] found that ZBED6 inhibits Piezo1 transcription and YAP nuclear translocation. Bai et al. [69] found that a microglia–astrocyte signaling axis regulates astrocytic Piezo1 expression. Wang et al. [45] systematically reviewed the structural pharmacology and disease associations of Piezo1. Ezzo et al. [19], Su et al. [47], Cao et al. [48], Zeng et al. [49], Zhang et al. [50], Yang et al. [51], Lim et al. [52], Lei et al. [53], and Zou et al. [54] supplemented evidence on PIEZO1-mediated immune–mesenchymal cell mechanical interactions and mechano-inflammation at the levels of macrophages, neutrophils, epicardial cells, endothelial cells, epithelial cells, ILC2 cells, systematic reviews, and corneal fibroblasts, respectively. Tu et al. [20], Wang et al. [38], Huang et al. [39], Lin et al. [40], Dong et al. [41], Wang et al. [42], Chen et al. [43], and Fan et al. [57] further enriched the PIEZO1–fibrosis pathway. Most of the above evidence comes from noncervical tissues, and extrapolation to the investing fascia requires independent validation.

Cervical myofascial release produces an HRV vagal response in healthy subjects (*n* = 17) [70]. A standardized cervical movement sequence elicits a decrease in heart rate (*n* = 30) [71]. Upper and lower cervical manipulation produce segmental-specific HRV changes (*n* = 20) [72]. Suboccipital fascial release may affect HRV [73]. Kazantseva et al. [74] (*n* = 128), Overmann et al. [75], and Navarro-Rico et al. [76] reported positive HRV effects, but methodological limitations warrant caution. A 2024 meta-analysis (*n* = 618) suggested that cervical manipulation may affect HRV-HF but that the quality of evidence is low [78]. A technique-specific meta-analysis (50 studies, *n* = 2147) suggested that soft tissue techniques may produce parasympathetic effects [80]. Gordon et al. [81] suggested that osteopathic manipulative treatment may affect psychophysiological indicators. The autonomic effects of thoracic segments remain unclear under higher-level evidence [79]. Harper et al. [82] suggested that manual therapy may improve HRV in long-standing neck pain, but only 3 RCTs were included. The scoping review by Monteiro et al. [83] suggested that cervical manipulation may reduce systolic blood pressure and improve sympatho-vagal balance, but the level of evidence is limited. Lam et al. [106] provided feasibility evidence for the hydrodissection technique but cannot yet demonstrate efficacy.

Farmer et al. [110] established a method for single-axon recording of the human cervical vagus nerve. Park [111] confirmed its safety and noted that approximately 80% of vagal fibers are afferent. This provides a technical pathway for directly testing the effects of fascial mechanical manipulation on vagal afferent and efferent activity.

5.1.2. Steps That Remain Inferential or Await Direct Validation

The mechanical pathway by which fibrosis of the investing fascia or of the deep cervical fascial layers is transmitted to the carotid sheath through the deep cervical fascial network lacks direct measurement data.

The intermediate steps of sheath wall mechanical deformation, changes in the intra-sheath mechanical microenvironment, and changes in vagal mechanical exposure have no synchronized in vivo measurement data from the same participant and should be labeled theoretical inference or pending direct validation.

The specific expression levels of PIEZO channels in the investing fascia and the carotid sheath wall have not been verified by immunohistochemistry, and the mechanical sensitivity of PIEZO1 and PIEZO2 in the superior cervical ganglion has not been validated at the functional level.

The conversion efficiency of mechanical signals into vagal afferent and efferent activity and its individual differences are indirectly explained by MDFIC2 [9,13], Nedd4-2 [68], GPR146 [18], ZBED6 [44], and Bai et al. [69].

The causal relationship of cervical mechanical manipulation and vagal functional output remains inconsistent across positive studies [70,71,72,73,74,75,76], negative RCTs [84,85], and systematic reviews [77,78,79,81,82,83]. Technique-specific differences [80] and target selection bias may be key factors.

5.1.3. Testable Predictions

In patients with a specific phenotype characterized primarily by cervical mechanical loading and accompanied by autonomic symptoms, as well as in healthy controls, perform ultrasound elastography of the investing fascia and measurement of cervical vagus nerve CSA. We predict that the elastic modulus of the investing fascia will be significantly higher in the patient group than in the control group and that left-sided vagus nerve CSA will be negatively correlated with the elastic modulus of the investing fascia. The measurement site should be limited to the weak point of the investing fascia between the sternocleidomastoid and trapezius muscles or the posterior triangle, not broadly to the nuchal fascia. Zandi et al. [108] did not find significantly increased cervical fascial stiffness in patients with chronic neck pain; this prediction should therefore be limited to a specific phenotype rather than generalized to all patients with chronic neck pain.

Under ultrasound guidance, apply a controlled mechanical load to the investing fascia while simultaneously recording HRV. We predict that individuals with a higher elastic modulus of the investing fascia will show a smaller change in HRV-HF under the same load, based on the inference that MDFIC2, Nedd4-2, GPR146, ZBED6, and Bai et al. [69] may affect PIEZO conversion efficiency.

Perform immunohistochemistry on the investing fascia and the carotid sheath wall. We predict that PIEZO1 and PIEZO2 will be expressed in both and that expression levels will correlate with the degree of fascial fibrosis. Simultaneously detect PIEZO1 and PIEZO2 in the superior cervical ganglion to test the extended hypothesis that fascia–sheath coupling may involve sympathetic mechanotransduction.

A mechanical intervention protocol targeting key fascial nodes related to the vagus nerve—the trapezius fascia, the origin and insertion of the sternocleidomastoid, the suboccipital margin, and the posterior auricular region and depression of the auricular concha—should preferentially use soft tissue techniques rather than HVLA manipulation, with intervention pressure referenced to 6.06 N/cm², and should simultaneously record HRV, CSA, and the elastic modulus of the investing fascia.

A same-participant synchronized measurement design should be used. Under ultrasound guidance, apply graded pressure to the investing fascia while simultaneously measuring investing fascial SWE stiffness, carotid sheath transverse diameter or area, Doppler flow of the internal jugular vein and carotid artery, vagus nerve CSA, and HRV. Directly measure sheath wall tension, intra-sheath tissue pressure, and nerve epineurial strain in intraoperative or animal models. This design directly tests the middle segment of the chain.

5.2. Anatomical Validation Priorities

It must be clarified whether the manner in which the superficial layer of the prevertebral fascia envelops the vagus nerve exhibits systematic variation. Another key question concerns the mechanical transmission pathway between the investing fascia and the carotid sheath. Hayashi [28] found that the common sheath does not directly fuse with the investing fascia, whereas Miyake [30] found that the sheath wall fuses with the pretracheal layer and remains separable from the prevertebral layer. It therefore remains unclear whether transmission occurs through the visceral fascia, the prevertebral fascia, or interlayer shear. The density and orientation of connective tissue connections between the vagus nerve and the sheath wall within the sheath also require clarification. The questioning by Nash et al. [22] of the existence of the investing fascia in the anterior triangle and the 38% variation rate found by Randhawa et al. [3] require the hypothesis to clarify the starting point of mechanical transmission in the anterior triangle.

5.3. Biomechanical and Imaging Validation

SWE provides a quantitative tool for assessing the mechanical properties of the cervical fascia. The intra-rater intraclass correlation coefficient [ICC] for nuchal fascia SWE is 0.63–0.89 [107], the inter-rater ICC for the upper trapezius is 0.80–0.99 [105], and the test-retest reliability for cervical or lumbar fascia is ICC ≥ 0.90 [108]. Zandi et al. [108] found no significant difference in fascial stiffness between patients with chronic neck pain and healthy controls, and statistical power was only approximately 0.30, suggesting that SWE detection of changes in fascial stiffness may be specific to pathological states. High-resolution ultrasound assesses vagus nerve CSA and its dynamic deformation [86,87,88,89,90,92,93,95,96,97,98,99,100,101,102]. Ultrasound measurements nonetheless differ from true anatomical dimensions [103]. Capron et al. [109] demonstrated that the tubular geometry of blood vessels interferes with SWE estimation, and a geometric correction protocol must first be established for SWE of the sheath wall.

5.4. Neurophysiological Validation

HRV analysis is a mature tool for assessing vagal tone. An operational design would include applying a controlled mechanical load to the investing fascia under ultrasound guidance while simultaneously recording HRV, testing whether changes in fascial tension immediately affect vagal tone. In view of the negative results of Budgell [84] and Monteiro [85] and the findings of Amoroso Borges [77], Kovanur Sampath [78], Villani et al. [80], Harper et al. [82], and Monteiro et al. [83], future studies should control respiratory rhythm, include a sham manipulation control, enroll a sufficient sample size, select key fascial nodes related to vagal function, and preferentially use soft tissue techniques rather than HVLA manipulation. Farmer et al. [110] established a method for single-axon recording of the human cervical vagus nerve, and Park [111] confirmed its safety. This technique provides a pathway for directly testing the immediate effects of fascial mechanical manipulation on vagal afferent and efferent activity, but it has not yet been applied in fascial mechanics research.

5.5. Reverse Causality and Multilevel Regulation

The present hypothesis currently considers only the unidirectional pathway of cervical fascial mechanics and vagal function. Several unresolved questions remain.

Reverse causality asks whether altered vagal function affects fascial tension. Autonomic tone influences vascular tone, interstitial fluid flow, and inflammatory status, potentially altering the mechanical properties of the fascia. No dedicated study has yet addressed this question.

Central regulation asks to what extent the central nervous system overrides peripheral mechanical effects. Vagal function is regulated by the nucleus tractus solitarius, the dorsal motor nucleus of the vagus, and other structures, and central drive may partially offset peripheral mechanical signals.

Multilevel interaction asks whether abnormal cervical fascial mechanics could be the result of central drive. Chronic stress may alter fascial tension through changes in sympathetic tone, forming an indirect central–fascia–vagus pathway.

5.6. Boundaries and Limitations of the Hypothesis

This hypothesis does not claim that abnormal cervical fascial mechanics is the sole or primary cause of vagal dysfunction. Vagal function is jointly influenced by multiple levels of factors, including central regulation, neuroinflammation, and metabolic status. The core contribution of the hypothesis lies in proposing a previously overlooked upstream mechanical dimension. Zandi et al. [108] did not observe significantly increased cervical fascial stiffness in patients with chronic neck pain, suggesting that the proposition in the hypothesis that investing fascial fibrosis leads to increased elastic modulus may not hold in the general chronic neck pain population and should be validated in the specific phenotype of cervicogenic autonomic dysfunction. The measurement site should also be limited to the anterolateral attachment area of the investing fascia rather than the posterior nuchal fascia, and the pathological driver should be predominantly chronic mechanical loading rather than acute inflammation or trauma. Several key steps in the hypothesis remain at the stage of theoretical inference or preliminary validation, including the expression levels of PIEZO channels in the investing fascia and the carotid sheath wall, the quantitative relationship between investing fascial fibrosis and the intra-sheath mechanical microenvironment, and the conversion efficiency of mechanical signals into vagal afferent and efferent activity. In particular, the middle segment of this hypothetical chain lacks synchronized in vivo measurements in the same participant and should be labeled theoretical inference or pending direct validation. The value of the hypothesis lies in identifying what can be tested.

6. Conclusion

The relationship between the fascial system of the neck and the vagus nerve has long occupied an ambiguous zone between anatomical description and clinical intuition. Anatomists have documented connections between the investing fascia and the carotid sheath [21,24,25,26,27,28,29,30,31,32], and clinicians have observed the coexistence of cervical tension and autonomic symptoms [104]. The mechanistic link between the two has nonetheless lacked a coherent theoretical framework.

The Investing Fascia–Carotid Sheath–Vagus Nerve Mechanical Coupling Hypothesis attempts to fill this gap. It reorganizes the investing fascia, the three-layer network of the deep cervical fascia, and the carotid sheath into a functional mechanical unit and proposes that the functional state of the vagus nerve—not merely its anatomical position—may be regulated by this mechanical unit. The existing evidence chain includes four major categories. First, anatomical and imaging evidence supports the multilayer fascial composition of the carotid sheath, its connectivity with the deep cervical fascial network, positional variation and left–right asymmetry of the vagus nerve, and dynamic mechanical coupling between the sternocleidomastoid muscle and the carotid sheath [1,2,3,4,21,24,25,26,27,28,29,30,31,32,35,58,59,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103]. Second, molecular and mechanotransduction evidence supports PIEZO channel–mediated vagal mechanotransduction, PIEZO expression on the fascial and receptor sides, Piezo1-mediated mechanical–fibrotic conversion, PIEZO channel regulation by MDFIC2 and Nedd4-2, immune–fibroblast mechanical interactions, and the calcium–hyaluronan and Piezo/TRPV4 frameworks [5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,63,64,65,66,67,68,69]. Third, clinical and functional evidence suggests that cervical mechanical manipulation may influence vagal functional output, although results are inconsistent and most studies have limited levels of evidence [70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85]. Fourth, controversy and limitation evidence highlights the unresolved anatomical existence of the investing fascia in the anterior triangle, the lack of synchronized in vivo measurements in the middle segment of the chain, the unverified expression of PIEZO channels in the investing fascia and carotid sheath wall, and the need for precise target selection and standardized protocols [22,78,79,84,85,103,108].

If this hypothesis is validated, it will change our understanding of the cervical physiology of the vagus nerve and may open a new path for assessing and intervening in autonomic dysfunction by targeting fascial mechanics. Before that, terminological precision, evidence grading, and specification of applicable boundaries must be maintained to avoid misinterpreting theoretical inference as clinical conclusions.