Effects of Xingnao Kaiqiao Acupuncture on Brain Functional Connectivity in Patients with Prolonged Disorders of Consciousness

Effects of Xingnao Kaiqiao Acupuncture on Brain Functional Connectivity in Patients with Prolonged Disorders of Consciousness | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of Xingnao Kaiqiao Acupuncture on Brain Functional Connectivity in Patients with Prolonged Disorders of Consciousness Zhijie Cao, Hongzhen Du, Yuxuan Zhang, Ziyuan Cao, Haibo Guo, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7689244/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective To investigate dynamic changes in brain functional connectivity (FC) in patients with prolonged disorders of consciousness (pDOC) during and after Xingnao Kaiqiao(XNKQ) acupuncture therapy using functional near-infrared spectroscopy (fNIRS). Methods 36 patients with pDOC were enrolled. After one patient was excluded because of data quality issues, the final analysis included 35 patients who completed the acupuncture intervention with fNIRS monitoring. fNIRS assessments of changes in oxygenated hemoglobin (HbO) concentration were conducted before, during, and after XNKQ acupuncture sessions. Intra- and interregional FC analyses were performed across eight predefined regions. FC analysis was performed by extracting HbO time series before, during, and after acupuncture. Results Compared with the baseline FC, FC was significantly greater during acupuncture. Subgroup analysis revealed increased FC during acupuncture in patients with unresponsive wakefulness syndrome (UWS). In contrast, no such increase was detected in individuals in the minimally conscious state (MCS) or in the sham acupuncture group. The intraregional FC in the left frontal lobe (LFL), right frontal lobe (RFL), left temporal lobe (LTL), and left dorsolateral prefrontal cortex (LDLPFC) markedly increased during acupuncture. FC remained elevated in the LFL, RFL, LTL, right temporal lobe (RTL), and right primary motor cortex (RPMC) after acupuncture. The interregional FC between the LFL-LTL, LFL-RFL, LFL-RTL, RFL-LTL, RFL-RTL, and LTL-RTL was significantly greater during acupuncture. These enhancements persisted after acupuncture for LFL-LTL, LFL-RFL, RFL-LTL, RFL-RTL, and LTL-RTL FC. In the UWS subgroup, the intraregional FC in the LFL, RFL, LTL, RTL, and LDLPFC, as well as the interregional FC between the LFL and LTL, increased during acupuncture. After acupuncture, FC was increased in the LFL, LTL, and RPMC. In the MCS subgroup, FC in the RFL was elevated after acupuncture. Additionally, interregional FC between the LFL and RFL increased both during and after acupuncture, and connectivity between the RFL and LFL also increased following acupuncture. Conclusion XNKQ acupuncture selectively increases FC in consciousness-related networks, particularly within frontal and temporal cortices, with sustained effects postintervention. These findings support the therapeutic potential of XNKQ for pDOC patients through targeted reorganization of higher-order cognitive networks, providing neurophysiological evidence for its clinical application. XNKQ acupuncture prolonged disorders of consciousness functional near-infrared spectroscopy functional connectivity cerebral oxygenation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Prolonged disorders of consciousness (pDOC) are defined as unconscious states lasting > 28 days after severe brain injury( 1 ). The incidence of VS/UWS is 0.1–0.2 per 100,000 people( 2 ). The diagnosis and treatment of pDOC remain challenging, with patients suffering from persistent loss of cognitive function and self-care capacity while imposing substantial health care resource burdens( 3 , 4 ). Recent advances in emergency medicine and intensive care have increased the incidence of pDOC. Common etiologies include traumatic brain injury, hemorrhagic stroke, and hypoxic-ischemic encephalopathy. pDOC patients are classified as being in either a vegetative state (VS), having unresponsive wakefulness syndrome (UWS), or being in a minimally conscious state (MCS). Patients with VS/UWS exhibit wakefulness without conscious awareness, whereas MCS patients demonstrate reproducible nonreflexive behaviors (e.g., visual tracking)( 5 ). Time-based classifications include acute ( 3 months) phases. Current assessment and treatment options for pDOC remain limited, with suboptimal efficacy in promoting consciousness recovery. Exploring novel clinical strategies and evaluation methods may improve patients' consciousness restoration. Xingnao Kaiqiao (XNKQ) acupuncture, a therapeutic protocol rooted in traditional Chinese medicine, aims to increase consciousness levels and cognitive function in patients. In XNKQ acupuncture, which is based on the principle of "mind regulation", needling techniques are utilized to reactivate brain regions and associated tissues, thereby restoring the governing, conducting, connecting, and controlling functions of the brain. In this therapeutic approach, Neiguan (PC6), Renzhong (DU26), and Sanyinjiao (SP6) are employed as primary acupoints for consciousness restoration, complemented by Baihui (GV20) to increase cognitive modulation, along with Jiquan (HT1), Chize (LU5), and Weizhong (BL40) to treat physical dysfunction and regulate mental function( 6 , 7 ). Jiquan (HT1), located in the axillary region, serves as a convergence point where the tendinomuscular meridians of the hand-taiyin (lung), hand-shaoyin (heart), hand-taiyang (small intestine), and hand-jueyin (pericardium) either "enter the axilla" or "connect at the axilla". Chize (LU5), situated at the cubital crease, is where the tendinomuscular meridians of the hand-taiyin (lung), hand-taiyang (small intestine), and hand-shaoyang (triple burner) "converge at the elbow" or "connect at the elbow center. Weizhong (BL40), positioned at the midpoint of the popliteal crease, functions as a junction for the tendinomuscular meridians of the foot-yangming (stomach), foot-taiyin (spleen), and foot-taiyang (bladder), all of which "converge at the knee" or "connect at the popliteal fossa". Needling these three acupoints, which functionally synchronize the Twelve Sinew Meridians, effectively treats sinew-channel disorders while alleviating limb flaccidity with motor impairment, thus establishing corporeal-spiritual synergy with the primary acupoints' brain-targeted mind regulation. Moreover, since the twelve tendinomuscular meridians are intrinsically affiliated with the twelve primary meridians, which serve as conduits for qi-blood circulation, stimulating these auxiliary points channels qi to activate spiritual governance and dredge spirit pathways, thereby reinstating the spirit's regulatory command over the body. This synchronous modulation of form and spirit clears and refreshes the brain, resulting in the amelioration of consciousness disorders( 8 ). Clinical studies have demonstrated that XNKQ acupuncture can improve consciousness in comatose patients, shorten awakening time, and increase Mini-Mental State Examination and Montreal Cognitive Assessment scores of patients with cognitive impairment( 9 , 10 ). Furthermore, XNKQ acupuncture can modulate functional connectivity (FC) within subcortical networks, thereby promoting motor-related cognitive recovery in patients( 11 , 12 ). Previous studies confirmed that single-acupoint therapy could improve brain FC in patients with pDOC, but the specific mechanisms of multiacupoint combination therapy remain unexplored( 13 ). Although preliminary evidence suggests that XNKQ acupuncture may increase cerebral blood flow and cerebral oxygen metabolism to remodel functional brain networks, the precise neuromodulatory mechanisms underlying its consciousness-promoting effects in patients with pDOC remain unclear, particularly regarding its effects on key brain regions such as the frontal lobe, which are critically associated with cognitive function and consciousness levels. FC is a core metric of brain network functional integration, quantifying the statistical dependence of neural activity between distinct brain regions across time series, such as signal fluctuation correlations. Patients with pDOC exhibit abnormalities in the FC of brain networks( 14 ). FC can be measured using functional near-infrared spectroscopy (fNIRS), which assesses brain functional activity by noninvasively detecting changes in the concentrations of oxygenated hemoglobin (HbO) and deoxygenated hemoglobin (HbR) in the cerebral cortex. Compared with electroencephalography and functional magnetic resonance imaging technologies, it offers superior temporal resolution, lower cost, and better portability, making it a critical tool for evaluating consciousness states in patients with pDOC( 15 ). To estimate FC, resting-state hemoglobin concentration time series data are acquired from the prefrontal, motor, and occipital cortices; Pearson correlation coefficients between interregional time series are calculated to construct FC matrices; and graph-theoretical metrics, including global efficiency and characteristic path length, which reflect neural synchronization and information integration efficiency across brain regions, are evaluated( 16 ). Research has indicated that patients with pDOC exhibit significantly reduced FC in prefrontal brain networks, with patients with MCS demonstrating stronger connectivity than patients with UWS. Further studies confirmed that this trend was more pronounced in the DLPFC( 17 ). FC in brain networks can serve as an objective measurement to reliably distinguish between MCS and UWS( 18 ), as well as a potential preclinical biomarker for predicting the awakening potential of patients with pDOC( 19 ). These findings established the pivotal role of FC in elucidating the dynamic reorganization of brain network topology during consciousness recovery, providing a crucial means to clarify the associations between the therapeutic effects of XNKQ acupuncture and its underlying neural mechanisms. In this study, FC data from the brain network before, during, and after XNKQ acupuncture treatment were collected and analyzed to verify the changing trends, explore the specific mechanisms underlying the effects of XNKQ acupuncture on brain networks, and provide theoretical support for its further clinical application. 2. Results 2.1 Whole-Channel FC Changes Before, During and After XNKQ Acupuncture Whole-channel FC analyses of the 23 patients across the resting state, acupuncture state, and postacupuncture state were performed. Compared with that in the resting state, the FC strength significantly increased during acupuncture ( p = 0.003). However, no statistically significant differences in FC were detected between the resting and postacupuncture states ( p > 0.05). In the UWS subgroup, FC strength also increased significantly during acupuncture ( p = 0.002), whereas no significant differences were detected between the resting and postacupuncture states ( p > 0.05). For both the MCS subgroup and the sham acupuncture group, no statistically significant differences in FC were detected during acupuncture or postacupuncture compared with the resting state ( p > 0.05) (Fig. 1 ). 2.2 FC Changes Within Specific Brain Regions Across Pre, During-, and Post-XNKQ Acupuncture Significant differences in FC were observed in all patients across the resting, acupuncture, and postacupuncture states within distinct brain regions. Compared with those in the resting state, FC in the LFL ( p = 0.011), RFL ( p = 0.005), LTL (p = 0.008), and LDLPFC ( p = 0.010) significantly increased during acupuncture. After acupuncture, FC was further increased in the LFL ( p = 0.032), RFL ( p = 0.003), LTL ( p = 0.020), RTL ( p = 0.040), and RPMC ( p = 0.047). In contrast, no statistically significant differences in FC were detected in the LPMC or RDLPFC across the three states ( p > 0.05). In the UWS subgroup, compared with that in the resting state, the FC strength in the LFL ( p = 0.012), RFL ( p = 0.010), LTL ( p = 0.017), RTL ( p = 0.048), and LDLPFC ( p = 0.038) significantly increased during acupuncture. After acupuncture, significant increases in FC were observed in the LFL ( p = 0.031), RFL ( p = 0.043), and LTL ( p = 0.043) compared with those in the resting state. In the MCS subgroup, only the FC in the RFL ( p = 0.045) significantly increased after acupuncture compared with that in the resting state. No statistically significant differences in FC were detected between the acupuncture or postacupuncture states and the resting state in the sham acupuncture group ( p > 0.05) (Fig. 2 ). 2.3 FC Changes between Brain Regions Before, During, and After XNKQ Acupuncture Significant differences in FC between brain regions were observed in all patients across the resting, acupuncture, and postacupuncture states. Compared with that in the resting state, FC across multiple interregional pairs, including the LFL-LTL ( p = 0.008), LFL-RFL ( p = 0.014), LFL-RTL ( p = 0.030), RFL-LTL ( p = 0.025), and RFL-RTL ( p = 0.005), significantly increased during acupuncture. After acupuncture, FC between the LFL-RFL ( p = 0.006) and RFL-RTL ( p = 0.013) remained elevated. Conversely, no statistically significant differences in FC were observed in the LTL-RTL across all three states ( p > 0.05). In the UWS subgroup, only the FC between the LFL-LTL regions ( p = 0.004) significantly increased during acupuncture compared with that during rest. In the MCS subgroup, compared with that in the resting state, the FC between the LFL-RFL regions ( p = 0.045) was significantly increased during acupuncture. After acupuncture, significant increases in FC were observed in the LFL-RFL ( p = 0.004) and RFL-LTL ( p = 0.033) compared with the resting state. No statistically significant differences in FC were detected between the acupuncture or postacupuncture states and the resting state in the sham acupuncture group ( p > 0.05) (Fig. 3 ). 3. Discussion In this study, an fNIRS system was used to analyze dynamic FC changes in the brain networks of patients with pDOC before treatment, during treatment, and after treatment. The results demonstrated that whole-brain FC during acupuncture was significantly greater than that in the resting state, whereas FC remained elevated following needle withdrawal relative to that in the resting state, although the difference was not significant. No significant changes in FC were observed in the sham acupuncture group throughout the intervention.​ These findings align with those of previous studies documenting the beneficial effects of acupuncture on functional brain networks in patients with pDOC( 29 , 33 ). Reduced cortical synchronization and diminished activity in brain regions associated with cognitive capacity and consciousness have been reported in patients with pDOC, particularly in the prefrontal cortex. The observed FC alterations during XNKQ acupuncture treatment suggest that this intervention can transiently increase interregional synchronization, improve cortical hemodynamics, and modulate functional activation in the cerebral cortex of these patients. Previous studies have focused predominantly on cerebral functional changes during acupuncture while neglecting its sustained posttreatment effects. Research on Hegu (LI4) acupuncture revealed the strongest cortical hemodynamic responses after needle withdrawal, a phenomenon similar to that observed in our study( 34 ). Furthermore, similar increases in FC were observed in the UWS subgroup. While the changes in the MCS subgroup did not reach statistical significance, a discernible increasing trend was observed. At the regional level, changes in FC in patients before, during, and after XNKQ acupuncture generally followed the whole-brain trend. However, after needle removal, the FC in the LFL, RFL, LTL, RTL, and RPMC exhibited more pronounced and sustained enhancement than the whole-brain average did. The frontal lobe governs primarily higher executive functions, including decision-making, behavioral planning, working memory, and emotional regulation, while also controlling voluntary motor functions through the motor cortex. The temporal lobe specializes in auditory processing, language comprehension, memory encoding and retrieval, and higher-order visual integration. Increased persistent FC in the frontal and temporal lobes may indirectly regulate thalamic-brainstem-related arousal networks through neural projections to the thalamus, thereby facilitating the awakening process in patients with pDOC( 35 , 36 ). The thalamus‒primary motor cortex (M1) pathway is a critical circuit for consciousness recovery ( 37 ). The arousal network, which is mediated via thalamus–M1 connectivity, may explain the delayed FC enhancement in the RPMC relative to other regions( 38 ). Both the LPMC and RDLPFC showed significant differences, with the RDLPFC exhibiting FC suppression and connectivity disruption in MCS( 39 ). The aberrant inhibitory interaction between the LPMC and RDLPFC has been reported to contribute to motor inhibition in Parkinson's disease patients( 40 ), which may also contribute to motor suppression in patients with pDOC and account for the asymmetric FC changes between the LPMC and RPMC. FC between different brain regions reflects the degree of synchronization and information exchange. In patients with pDOC, long-range FC between distinct brain regions and interhemispheric FC is significantly reduced ( 17 ), and the integrity of brain network connectivity encompassing both low-order and high-order networks is compromised ( 41 ). During XNKQ acupuncture treatment, FC between different brain regions markedly increased, with a more pronounced trend than changes in local intraregional connectivity. Notably, the increase in connectivity between the left and right frontal lobes as well as between the frontal and temporal lobes is particularly significant. The FL serves as the primary hub for information processing, and consciousness relies on the integration of information across hemispheres and networks. The disruption of LFL-RFL connectivity leads to fragmented information processing ( 42 ), which is reversed by XNKQ acupuncture. However, XNKQ acupuncture demonstrates limited efficacy in enhancing FC within the RDLPFC. Repetitive transcranial magnetic stimulation has been shown to effectively strengthen DLPFC connectivity ( 43 ), suggesting that a combined therapeutic approach may yield superior outcomes for patients with pDOC. 4. Limitations This study had several limitations, including its small sample size. The current findings are primarily based on observations from a single treatment session. We plan to verify the results through larger-scale, multicenter trials in the future and track long-term treatment to validate the sustained effects of XNKQ acupuncture therapy. 5. Conclusion In this study, we collected and analyzed brain FC data before, during, and after XNKQ acupuncture in patients with pDOC and demonstrated that XNKQ acupuncture effectively enhances whole-brain, intraregional, and interregional FC, particularly in the frontal and temporal lobes, with sustained effects after needle withdrawal. Notably, this increase in FC was also observed in both the UWS and MCS subgroups but not the sham acupuncture group. These findings provide a theoretical foundation for elucidating the neuromodulatory effects of XNKQ acupuncture on brain networks and improving awakening efficacy in patients with pDOC. 6. Materials and Methods 6.1 Study Population With the approval of the Ethics Committee of the Binzhou Medical University Hospital (under the Ethical Approval Number KYLL-186), 36 patients with pDOC were recruited to receive the XNKQ acupuncture intervention. All patients were enrolled from the Department of Rehabilitation Medicine at Binzhou Medical University Hospital between January 2024 and May 2025. This trial was registered in the Chinese Clinical Trial Registry (Identifier: ChiCTR2400090915) on 15 October 2024.​ The inclusion criteria were as follows: ( 1 ) aged > 18 years( 13 ); ( 2 ) had a DOC lasting more than 28 days( 20 ); and ( 3 ) were diagnosed with VS/UWS or MCS according to the CRS-R scale. All patients met the diagnostic criteria for pDOC, according to the European Academy of Neurology guidelines for the diagnosis of coma and other disorders of consciousness in 2020( 21 ). The exclusion criteria were as follows: ( 1 ) unstable clinical conditions or vital signs; ( 2 ) scalp lesions or cranial defects and concurrent intracranial tumors, infections, or space-occupying lesions; ( 3 ) severe endocrine metabolic disorders or persistent status epilepticus( 22 ); and ( 4 ) a history of neurological disorders or the use of anesthetic agents. Legally authorized representatives of all the subjects signed written informed consent forms. All enrolled patients received standard clinical care and conventional rehabilitation therapy as part of the baseline treatment protocol. Thirty-six participants completed the study, including 16 patients with MCS and 20 patients with UWS. However, among the 36 participants, one MCS patient was excluded because of poor data quality. Prior to the experiment, the level of consciousness in patients with pDOC was evaluated using the CRS-R scale. Each patient underwent five assessments on nonconsecutive days within a 10-day period, and the highest score obtained was used to classify the patient as UWS or MCS( 20 ). Table 1 summarizes the demographic and clinical assessment scores of all the participants. No significant differences in age distribution were observed between the two groups. However, significant disparities emerged in consciousness assessment scores, including Coma Recovery Scale-Revised (CRS-R) scores( 23 ). The MCS group presented higher CRS-R scores (7.27 ± 2.31), whereas the UWS group presented lower CRS-R scores (3.50 ± 1.32). Table 1 Basic characteristics of the patients. Variable Total (n = 35) MCS (n = 15) UWS (n = 20) Age, years, mean ± SD 59.69 ± 13.79 59.20 ± 14.36 60.05 ± 14.36 Female, n (%) 7(20%) 4 (27%) 3 (15%) CRS-R, mean ± SD 5.11 ± 2.60 7.27 ± 2.31 3.50 ± 1.32 Time from onset to enrollment, mean ± SD(days) 42.46 ± 1.42 42.53 ± 1.19 42.40 ± 1.60. Stroke, n (%) 27(77%) 13(87%) 14(70%) Traumatic brain injury, n (%) 1(3%) 0(0%) 1(5%) Hypoxic-ischemic encephalopathy, n (%) 7(20%) 2(13%) 5(25%) CRS-R, Coma Recovery Scale-Revised; UWS, unresponsive wakefulness syndrome; MCS, minimally conscious state; SD, standard deviation. 6.2 Acupuncture and Point Selection Among the 35 patients, 23 received acupuncture stimulation, while 12 received sham acupuncture stimulation. The acupuncture points applied in this study included the main acupoints Neiguan (PC6, bilateral), ​Renzhong (DU26, unilateral), ​Sanyinjiao (SP6, bilateral) and the secondary acupoints Jiquan (HT1, bilateral), Chize (Lu5, bilateral) and Weizhong (BL40, bilateral)( 24 ). The anatomical location of these points was defined as follows: Renzhong (DU26) is situated at the junction between the upper one-third and middle one-third of the philtrum groove. Neiguan (PC6) is located 2 cun proximal to the transverse wrist crease, between the tendons of the flexor carpi radialis and palmaris longus muscles. Sanyinjiao (SP6) is positioned 3 cun above the medial malleolus, along the posterior border of the tibia. Jiquan (HT1) is situated at the apex of the axilla, where the axillary artery beats. Chize (Lu5) is located in the transverse stripes of the elbow, the radial depression of the bicipital brachii tendon. Weizhong (BL40) is located in the posterior region of the knee at the midpoint of the popliteal transverse stripes (Fig. 4 ). For the sham acupuncture control group, non-acupoints near the main acupoints were selected for minimal, superficial stimulation to control for nonspecific effects. These control points were chosen on the basis of a validated protocol from a previous study and through consultation with acupuncture experts. They are not situated on any recognized meridian or over major nerves but are all within a 2.5 cm radius of the corresponding true acupoint. The specific locations were defined as follows:​ The sham point for PC6 was located lateral to PC6, in the intermeridian space between the Hand-Taiyin Lung Meridian and the Hand-Jueyin Pericardium Meridian. The sham point for DU26 was situated on the vertical line of the mouth, lateral (left) to DU26. The sham point for SP6 was located 6 cun proximal to the tip of the medial malleolus, in the intermeridian space between the Foot-Taiyin Spleen Meridian and the Foot-Jueyin Liver Meridian. This point is 3 cun proximal to the SP6 and 1.25 cun anterior to it on the medial side of the tibia. The sham point for HT1 was located 1 cun inferior and 1 cun anterior to the axillary apex, avoiding axillary hair, on the muscle belly of the bicipital brachii. The sham point for Lu5 was located at the midpoint of the line connecting Lu5 and LI11 (Quchi) when the elbow was flexed at 120 degrees on the radial side of the tendon of the radial wrist extensor longus muscle( 11 , 25 ). The sham point for BL40 was located inferior and lateral to the midpoint of the popliteal transverse crease on the muscle belly of the peroneus longus muscle. The acupuncture procedures were conducted by a licensed acupuncturist with a decade of clinical experience from the Binzhou Medical University Hospital. All the subjects remained in a supine position on a scanning bed throughout the intervention. Sterile, single-use titanium needles (0.20×40 mm; Wujiang CloudDragon Medical Apparatus Co., Ltd.,Wujiang District, Suzhou, China) were utilized. For the XNKQ acupuncture protocol, the needling techniques were executed as described below. At Neiguan (PC6), bilateral insertion was performed to a depth of 0.5–1.0 cun. A reducing stimulation method was applied for 1 minute, involving lifting-thrusting and rotating manipulations, with the left hand rotating the needle counterclockwise and the right hand rotating it clockwise. Subsequently, the needle at Renzhong (DU26) was obliquely inserted toward the nasal septum to a depth of 0.3–0.5 cun, followed by the sparrow-pecking technique until ocular moistening or lacrimation was observed. Finally, at Sanyinjiao (SP6), bilateral oblique insertion (0.5–1.0 cun depth) along the medial tibial border was performed by reinforcing manipulation via the lifting-thrusting method, characterized by heavy thrusting and light lifting, which was maintained for 1 minute. After the main points were needled, the patient’s secondary points, bilateral Jiquan (HT1), bilateral Chize (Lu5), and bilateral Weizhong (BL40), were needled using the lifting-thrusting method( 24 ). The acupuncture intervention, encompassing both needle manipulation and retention time, lasted for 30 minutes in both the verum and sham acupuncture groups( 26 ). 6.3 Data Acquisition fNIRS data were acquired using a 48-channel NirSmart system (Danyang Huichuang, China). The fNIRS cap, designed according to the international 10–20 system, comprised 15 light sources and 16 detectors to obtain resting-state brain network data. The source‒detector distance was maintained within 3 cm (2.9–3.1 cm). Each channel primarily detected hemodynamic changes in the cortical region beneath the midpoint of the source‒detector pair, with channel localization mapped to Brodmann areas. The signals were recorded at wavelengths of 760 nm and 850 nm, with a sampling frequency of 11 Hz. The 48 channels were assigned to eight key regions of interest (ROIs) in the cerebral cortex: the left frontal lobe (LFL; channels 9, 10, 11, 27, 29, 30, 31, 32, 43, 44, 45, 46, and 47), right frontal lobe (RFL; channels 6, 7, 8, 21, 22, 23, 24, 26, 37, 39, 40, 41, and 42), left temporal lobe (LTL; channels 12, 13, 14, 15, 16, 33, and 35), right temporal lobe (RTL; channels 1, 2, 3, 4, 5, 17, and 18), left primary motor cortex (LPMC; channels 34 and 48), right primary motor cortex (RPMC; channels 19 and 38), left dorsolateral prefrontal cortex (LDLPFC; channels 10, 29, 30, 43, 44, and 45), and right dorsolateral prefrontal cortex (RDLPFC; channels 21, 23, 40, and 41) ( 27 )(Fig. 5 ). During fNIRS data acquisition, the participants initially rested in a comfortable supine position for 5 minutes to stabilize their physiological parameters before donning the fNIRS cap. The data acquisition protocol consisted of three phases: the preacupuncture resting-state phase, during which baseline brain activity was recorded for 8 minutes; the acupuncture intervention phase, during which real-time fNIRS signals were acquired throughout the acupuncture procedure (duration adjusted according to the actual manipulation time, typically 20 minutes); and the postacupuncture resting-state phase, during which postintervention resting-state signals were recorded for 8 minutes following needle removal. The final analyses were based on three 8 minute resting-state fNIRS epochs: pretreatment, middle segment of the during-treatment period, and posttreatment. Adopting uniform 8-minute windows aligns with established methodological standards in the fNIRS literature and ensures comparable signal quality across phases( 13 , 16 , 28 , 29 ). 6.4 Data Processing The fNIRS data were processed and analyzed using NirSpark software. Raw light intensity signals were first converted into optical density (OD) curves, and oxygenated hemoglobin (HbO) and deoxygenated hemoglobin (HbR) concentrations were derived using the modified Beer‒Lambert law. During preprocessing, bandpass filtering (0.01–0.2 Hz) was applied to eliminate motion artifacts and baseline drift caused by physiological fluctuations (e.g., cardiac and respiratory cycles)( 30 ). Subsequent analyses focused exclusively on HbO signals because their signal-to-noise ratio is superior to that of HbR. FC analysis was performed by extracting HbO time series before, during, and after acupuncture. For each ROI, HbO time series were aggregated across corresponding channels. Pairwise Pearson correlation coefficients were computed between the HbO time series of all channel pairs, with these coefficients defined as the functional connectivity strength between associated brain regions( 31 ). For further network-level analysis, time-averaged HbO signals from all 48 channels were subjected to correlation analysis. Pearson correlation coefficients underwent Fisher-z transformation to normalize their distributions, generating standardized metrics for quantifying dynamic changes in FC strength across the three experimental phases( 32 ). 6.5 Statistical Analysis Statistical analyses were performed using SPSS 27.0 software. Between-group comparisons were conducted using two-tailed Student's t tests, whereas multigroup comparisons were evaluated with one-way analysis of variance (ANOVA). A threshold of P < 0.05 was applied to define statistical significance. Declarations Ethics approval and consent to participate This study was reviewed and approved by the Ethics Committee of Binzhou Medical University Hospital (Approval Number: KYLL-186). All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from the legal guardians of all individual participants included in the study. Consent for publication Not applicable Competing interests The authors declare that they have no competing interests. Funding This research did not receive any specific grant. Author Contribution Z.C. and H.D. wrote the main manuscript text; Y.Z. and Z.C. performed data analysis; H.G. and Z.X. reviewed and revised the manuscript. All authors reviewed the manuscript. 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Disturbed functional connectivity and topological properties of the frontal lobe in minimally conscious state based on resting-state fNIRS. Frontiers in neuroscience. 2023;17:1118395. Wang Z, Wei J, Song Y, Li Y, Wu Y, Chen R, et al. Role of right dorsolateral prefrontal cortex-left primary motor cortex interaction in motor inhibition in Parkinson's disease. Frontiers in aging neuroscience. 2025;17:1524755. Medina Carrion JP, Stanziano M, D'Incerti L, Sattin D, Palermo S, Ferraro S, et al. Disorder of consciousness: Structural integrity of brain networks for the clinical assessment. Annals of clinical and translational neurology. 2023;10(3):384–96. Amico E, Marinazzo D, Di Perri C, Heine L, Annen J, Martial C, et al. Mapping the functional connectome traits of levels of consciousness. NeuroImage. 2017;148:201–11. Wan X, Zhang Y, Li Y, Song W. Effects of parietal repetitive transcranial magnetic stimulation in prolonged disorders of consciousness: A pilot study. Heliyon. 2024;10(9):e30192. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7689244","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":540406009,"identity":"0576d1a3-365b-47ca-8533-ea1043a25124","order_by":0,"name":"Zhijie Cao","email":"","orcid":"","institution":"Shandong University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhijie","middleName":"","lastName":"Cao","suffix":""},{"id":540406010,"identity":"c1d2d644-42d6-49dc-92f1-6ce5cf84f167","order_by":1,"name":"Hongzhen Du","email":"","orcid":"","institution":"Binzhou Medical University 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10:02:27","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":131071,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7689244/v1/858bc51e168bcbf56e5ff478.html"},{"id":95321124,"identity":"2feecd0c-c53c-4e48-aea9-ac4e90edf53b","added_by":"auto","created_at":"2025-11-06 16:47:22","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":119376,"visible":true,"origin":"","legend":"\u003cp\u003eStatistical comparisons of whole-channel FC across pre-, during-, and post-XNKQ acupuncture states. (a) All patients; (b) UWS subgroup; (c) MCS subgroup; (d) nearby non-acupuncture control points. **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01. Functional connectivity (FC), unresponsive wakefulness syndrome (UWS), minimally conscious state (MCS).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7689244/v1/18f40d2167c49026f028b966.jpeg"},{"id":95321128,"identity":"c4b00c8b-cc5a-46c8-aaac-ed74e32d38a6","added_by":"auto","created_at":"2025-11-06 16:47:23","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":153498,"visible":true,"origin":"","legend":"\u003cp\u003eStatistical comparisons of FC within specific brain regions across pre-, during-, and post-XNKQ acupuncture states. (a) All patients; (b) UWS subgroup; (c) MCS subgroup; (d) nearby non-acupuncture control points. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. Functional connectivity (FC), unresponsive wakefulness syndrome (UWS), minimally conscious state (MCS).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7689244/v1/72ff7aa4a7b207b7f761000e.jpeg"},{"id":95524142,"identity":"131325ca-9451-48e4-b9c1-00bf3c91b406","added_by":"auto","created_at":"2025-11-10 10:02:21","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":128589,"visible":true,"origin":"","legend":"\u003cp\u003eStatistical comparisons of FC between brain regions across pre-, during-, and post-XNKQ acupuncture states. (a) All patients; (b) UWS subgroup; (c) MCS subgroup; (d) nearby non-acupuncture control points. *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. Functional connectivity (FC), unresponsive wakefulness syndrome (UWS), minimally conscious state (MCS).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7689244/v1/142fc853ece35ce58b1aad0d.jpeg"},{"id":95523669,"identity":"7a02ce5e-a3d7-4410-b774-cbdb0fb06e3e","added_by":"auto","created_at":"2025-11-10 09:59:53","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":85975,"visible":true,"origin":"","legend":"\u003cp\u003eXNKQ Acupuncture Location Map. Top row (left to right): Neiguan, Renzhong, and Sanyinjiao; bottom row (left to right): Jiquan, Chize, and Weizhong.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7689244/v1/fcf3b0f20757788aa5b6463f.jpeg"},{"id":95321135,"identity":"a91c4150-342c-4459-b859-221d84ea8bca","added_by":"auto","created_at":"2025-11-06 16:47:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":412566,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of channels in the prefrontal lobe of the brain.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7689244/v1/8f62072ae39e5bef43acc63c.png"},{"id":101224022,"identity":"e67e537f-aaea-4fdd-8b9b-b0724060da41","added_by":"auto","created_at":"2026-01-27 12:12:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1904184,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7689244/v1/ec54987f-fe00-4b18-9f9d-92bc34d89691.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of Xingnao Kaiqiao Acupuncture on Brain Functional Connectivity in Patients with Prolonged Disorders of Consciousness","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eProlonged disorders of consciousness (pDOC) are defined as unconscious states lasting\u0026thinsp;\u0026gt;\u0026thinsp;28 days after severe brain injury(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). The incidence of VS/UWS is 0.1\u0026ndash;0.2 per 100,000 people(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The diagnosis and treatment of pDOC remain challenging, with patients suffering from persistent loss of cognitive function and self-care capacity while imposing substantial health care resource burdens(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Recent advances in emergency medicine and intensive care have increased the incidence of pDOC. Common etiologies include traumatic brain injury, hemorrhagic stroke, and hypoxic-ischemic encephalopathy. pDOC patients are classified as being in either a vegetative state (VS), having unresponsive wakefulness syndrome (UWS), or being in a minimally conscious state (MCS). Patients with VS/UWS exhibit wakefulness without conscious awareness, whereas MCS patients demonstrate reproducible nonreflexive behaviors (e.g., visual tracking)(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Time-based classifications include acute (\u0026lt;\u0026thinsp;28 days), subacute (28 days\u0026ndash;3 months), and chronic (\u0026gt;\u0026thinsp;3 months) phases. Current assessment and treatment options for pDOC remain limited, with suboptimal efficacy in promoting consciousness recovery. Exploring novel clinical strategies and evaluation methods may improve patients' consciousness restoration.\u003c/p\u003e\u003cp\u003eXingnao Kaiqiao (XNKQ) acupuncture, a therapeutic protocol rooted in traditional Chinese medicine, aims to increase consciousness levels and cognitive function in patients. In XNKQ acupuncture, which is based on the principle of \"mind regulation\", needling techniques are utilized to reactivate brain regions and associated tissues, thereby restoring the governing, conducting, connecting, and controlling functions of the brain. In this therapeutic approach, Neiguan (PC6), Renzhong (DU26), and Sanyinjiao (SP6) are employed as primary acupoints for consciousness restoration, complemented by Baihui (GV20) to increase cognitive modulation, along with Jiquan (HT1), Chize (LU5), and Weizhong (BL40) to treat physical dysfunction and regulate mental function(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Jiquan (HT1), located in the axillary region, serves as a convergence point where the tendinomuscular meridians of the hand-taiyin (lung), hand-shaoyin (heart), hand-taiyang (small intestine), and hand-jueyin (pericardium) either \"enter the axilla\" or \"connect at the axilla\". Chize (LU5), situated at the cubital crease, is where the tendinomuscular meridians of the hand-taiyin (lung), hand-taiyang (small intestine), and hand-shaoyang (triple burner) \"converge at the elbow\" or \"connect at the elbow center. Weizhong (BL40), positioned at the midpoint of the popliteal crease, functions as a junction for the tendinomuscular meridians of the foot-yangming (stomach), foot-taiyin (spleen), and foot-taiyang (bladder), all of which \"converge at the knee\" or \"connect at the popliteal fossa\". Needling these three acupoints, which functionally synchronize the Twelve Sinew Meridians, effectively treats sinew-channel disorders while alleviating limb flaccidity with motor impairment, thus establishing corporeal-spiritual synergy with the primary acupoints' brain-targeted mind regulation. Moreover, since the twelve tendinomuscular meridians are intrinsically affiliated with the twelve primary meridians, which serve as conduits for qi-blood circulation, stimulating these auxiliary points channels qi to activate spiritual governance and dredge spirit pathways, thereby reinstating the spirit's regulatory command over the body. This synchronous modulation of form and spirit clears and refreshes the brain, resulting in the amelioration of consciousness disorders(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Clinical studies have demonstrated that XNKQ acupuncture can improve consciousness in comatose patients, shorten awakening time, and increase Mini-Mental State Examination and Montreal Cognitive Assessment scores of patients with cognitive impairment(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Furthermore, XNKQ acupuncture can modulate functional connectivity (FC) within subcortical networks, thereby promoting motor-related cognitive recovery in patients(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Previous studies confirmed that single-acupoint therapy could improve brain FC in patients with pDOC, but the specific mechanisms of multiacupoint combination therapy remain unexplored(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Although preliminary evidence suggests that XNKQ acupuncture may increase cerebral blood flow and cerebral oxygen metabolism to remodel functional brain networks, the precise neuromodulatory mechanisms underlying its consciousness-promoting effects in patients with pDOC remain unclear, particularly regarding its effects on key brain regions such as the frontal lobe, which are critically associated with cognitive function and consciousness levels.\u003c/p\u003e\u003cp\u003eFC is a core metric of brain network functional integration, quantifying the statistical dependence of neural activity between distinct brain regions across time series, such as signal fluctuation correlations. Patients with pDOC exhibit abnormalities in the FC of brain networks(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). FC can be measured using functional near-infrared spectroscopy (fNIRS), which assesses brain functional activity by noninvasively detecting changes in the concentrations of oxygenated hemoglobin (HbO) and deoxygenated hemoglobin (HbR) in the cerebral cortex. Compared with electroencephalography and functional magnetic resonance imaging technologies, it offers superior temporal resolution, lower cost, and better portability, making it a critical tool for evaluating consciousness states in patients with pDOC(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). To estimate FC, resting-state hemoglobin concentration time series data are acquired from the prefrontal, motor, and occipital cortices; Pearson correlation coefficients between interregional time series are calculated to construct FC matrices; and graph-theoretical metrics, including global efficiency and characteristic path length, which reflect neural synchronization and information integration efficiency across brain regions, are evaluated(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Research has indicated that patients with pDOC exhibit significantly reduced FC in prefrontal brain networks, with patients with MCS demonstrating stronger connectivity than patients with UWS. Further studies confirmed that this trend was more pronounced in the DLPFC(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). FC in brain networks can serve as an objective measurement to reliably distinguish between MCS and UWS(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), as well as a potential preclinical biomarker for predicting the awakening potential of patients with pDOC(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). These findings established the pivotal role of FC in elucidating the dynamic reorganization of brain network topology during consciousness recovery, providing a crucial means to clarify the associations between the therapeutic effects of XNKQ acupuncture and its underlying neural mechanisms.\u003c/p\u003e\u003cp\u003eIn this study, FC data from the brain network before, during, and after XNKQ acupuncture treatment were collected and analyzed to verify the changing trends, explore the specific mechanisms underlying the effects of XNKQ acupuncture on brain networks, and provide theoretical support for its further clinical application.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Whole-Channel FC Changes Before, During and After XNKQ Acupuncture\u003c/h2\u003e\u003cp\u003eWhole-channel FC analyses of the 23 patients across the resting state, acupuncture state, and postacupuncture state were performed. Compared with that in the resting state, the FC strength significantly increased during acupuncture (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003). However, no statistically significant differences in FC were detected between the resting and postacupuncture states (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In the UWS subgroup, FC strength also increased significantly during acupuncture (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002), whereas no significant differences were detected between the resting and postacupuncture states (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). For both the MCS subgroup and the sham acupuncture group, no statistically significant differences in FC were detected during acupuncture or postacupuncture compared with the resting state (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 FC Changes Within Specific Brain Regions Across Pre, During-, and Post-XNKQ Acupuncture\u003c/h2\u003e\u003cp\u003eSignificant differences in FC were observed in all patients across the resting, acupuncture, and postacupuncture states within distinct brain regions. Compared with those in the resting state, FC in the LFL (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.011), RFL (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005), LTL (p\u0026thinsp;=\u0026thinsp;0.008), and LDLPFC (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.010) significantly increased during acupuncture. After acupuncture, FC was further increased in the LFL (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.032), RFL (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003), LTL (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.020), RTL (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.040), and RPMC (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.047). In contrast, no statistically significant differences in FC were detected in the LPMC or RDLPFC across the three states (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In the UWS subgroup, compared with that in the resting state, the FC strength in the LFL (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.012), RFL (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.010), LTL (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.017), RTL (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.048), and LDLPFC (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.038) significantly increased during acupuncture. After acupuncture, significant increases in FC were observed in the LFL (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.031), RFL (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.043), and LTL (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.043) compared with those in the resting state. In the MCS subgroup, only the FC in the RFL (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.045) significantly increased after acupuncture compared with that in the resting state. No statistically significant differences in FC were detected between the acupuncture or postacupuncture states and the resting state in the sham acupuncture group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 FC Changes between Brain Regions Before, During, and After XNKQ Acupuncture\u003c/h2\u003e\u003cp\u003eSignificant differences in FC between brain regions were observed in all patients across the resting, acupuncture, and postacupuncture states. Compared with that in the resting state, FC across multiple interregional pairs, including the LFL-LTL (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008), LFL-RFL (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.014), LFL-RTL (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.030), RFL-LTL (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.025), and RFL-RTL (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005), significantly increased during acupuncture. After acupuncture, FC between the LFL-RFL (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.006) and RFL-RTL (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.013) remained elevated. Conversely, no statistically significant differences in FC were observed in the LTL-RTL across all three states (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In the UWS subgroup, only the FC between the LFL-LTL regions (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004) significantly increased during acupuncture compared with that during rest. In the MCS subgroup, compared with that in the resting state, the FC between the LFL-RFL regions (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.045) was significantly increased during acupuncture. After acupuncture, significant increases in FC were observed in the LFL-RFL (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004) and RFL-LTL (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.033) compared with the resting state. No statistically significant differences in FC were detected between the acupuncture or postacupuncture states and the resting state in the sham acupuncture group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eIn this study, an fNIRS system was used to analyze dynamic FC changes in the brain networks of patients with pDOC before treatment, during treatment, and after treatment. The results demonstrated that whole-brain FC during acupuncture was significantly greater than that in the resting state, whereas FC remained elevated following needle withdrawal relative to that in the resting state, although the difference was not significant. No significant changes in FC were observed in the sham acupuncture group throughout the intervention.​ These findings align with those of previous studies documenting the beneficial effects of acupuncture on functional brain networks in patients with pDOC(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Reduced cortical synchronization and diminished activity in brain regions associated with cognitive capacity and consciousness have been reported in patients with pDOC, particularly in the prefrontal cortex. The observed FC alterations during XNKQ acupuncture treatment suggest that this intervention can transiently increase interregional synchronization, improve cortical hemodynamics, and modulate functional activation in the cerebral cortex of these patients. Previous studies have focused predominantly on cerebral functional changes during acupuncture while neglecting its sustained posttreatment effects. Research on Hegu (LI4) acupuncture revealed the strongest cortical hemodynamic responses after needle withdrawal, a phenomenon similar to that observed in our study(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Furthermore, similar increases in FC were observed in the UWS subgroup. While the changes in the MCS subgroup did not reach statistical significance, a discernible increasing trend was observed.\u003c/p\u003e\u003cp\u003eAt the regional level, changes in FC in patients before, during, and after XNKQ acupuncture generally followed the whole-brain trend. However, after needle removal, the FC in the LFL, RFL, LTL, RTL, and RPMC exhibited more pronounced and sustained enhancement than the whole-brain average did. The frontal lobe governs primarily higher executive functions, including decision-making, behavioral planning, working memory, and emotional regulation, while also controlling voluntary motor functions through the motor cortex. The temporal lobe specializes in auditory processing, language comprehension, memory encoding and retrieval, and higher-order visual integration. Increased persistent FC in the frontal and temporal lobes may indirectly regulate thalamic-brainstem-related arousal networks through neural projections to the thalamus, thereby facilitating the awakening process in patients with pDOC(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). The thalamus‒primary motor cortex (M1) pathway is a critical circuit for consciousness recovery (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). The arousal network, which is mediated via thalamus\u0026ndash;M1 connectivity, may explain the delayed FC enhancement in the RPMC relative to other regions(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Both the LPMC and RDLPFC showed significant differences, with the RDLPFC exhibiting FC suppression and connectivity disruption in MCS(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). The aberrant inhibitory interaction between the LPMC and RDLPFC has been reported to contribute to motor inhibition in Parkinson's disease patients(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e), which may also contribute to motor suppression in patients with pDOC and account for the asymmetric FC changes between the LPMC and RPMC.\u003c/p\u003e\u003cp\u003eFC between different brain regions reflects the degree of synchronization and information exchange. In patients with pDOC, long-range FC between distinct brain regions and interhemispheric FC is significantly reduced (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), and the integrity of brain network connectivity encompassing both low-order and high-order networks is compromised (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). During XNKQ acupuncture treatment, FC between different brain regions markedly increased, with a more pronounced trend than changes in local intraregional connectivity. Notably, the increase in connectivity between the left and right frontal lobes as well as between the frontal and temporal lobes is particularly significant. The FL serves as the primary hub for information processing, and consciousness relies on the integration of information across hemispheres and networks. The disruption of LFL-RFL connectivity leads to fragmented information processing (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e), which is reversed by XNKQ acupuncture. However, XNKQ acupuncture demonstrates limited efficacy in enhancing FC within the RDLPFC. Repetitive transcranial magnetic stimulation has been shown to effectively strengthen DLPFC connectivity (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e), suggesting that a combined therapeutic approach may yield superior outcomes for patients with pDOC.\u003c/p\u003e"},{"header":"4. Limitations","content":"\u003cp\u003eThis study had several limitations, including its small sample size. The current findings are primarily based on observations from a single treatment session. We plan to verify the results through larger-scale, multicenter trials in the future and track long-term treatment to validate the sustained effects of XNKQ acupuncture therapy.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn this study, we collected and analyzed brain FC data before, during, and after XNKQ acupuncture in patients with pDOC and demonstrated that XNKQ acupuncture effectively enhances whole-brain, intraregional, and interregional FC, particularly in the frontal and temporal lobes, with sustained effects after needle withdrawal. Notably, this increase in FC was also observed in both the UWS and MCS subgroups but not the sham acupuncture group. These findings provide a theoretical foundation for elucidating the neuromodulatory effects of XNKQ acupuncture on brain networks and improving awakening efficacy in patients with pDOC.\u003c/p\u003e"},{"header":"6. Materials and Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e6.1 Study Population\u003c/h2\u003e\u003cp\u003e With the approval of the Ethics Committee of the Binzhou Medical University Hospital (under the Ethical Approval Number KYLL-186), 36 patients with pDOC were recruited to receive the XNKQ acupuncture intervention. All patients were enrolled from the Department of Rehabilitation Medicine at Binzhou Medical University Hospital between January 2024 and May 2025. This trial was registered in the Chinese Clinical Trial Registry (Identifier: ChiCTR2400090915) on 15 October 2024.​\u003c/p\u003e\u003cp\u003eThe inclusion criteria were as follows: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) aged\u0026thinsp;\u0026gt;\u0026thinsp;18 years(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e); (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) had a DOC lasting more than 28 days(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e); and (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) were diagnosed with VS/UWS or MCS according to the CRS-R scale. All patients met the diagnostic criteria for pDOC, according to the European Academy of Neurology guidelines for the diagnosis of coma and other disorders of consciousness in 2020(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe exclusion criteria were as follows: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) unstable clinical conditions or vital signs; (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) scalp lesions or cranial defects and concurrent intracranial tumors, infections, or space-occupying lesions; (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) severe endocrine metabolic disorders or persistent status epilepticus(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e); and (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) a history of neurological disorders or the use of anesthetic agents. Legally authorized representatives of all the subjects signed written informed consent forms. All enrolled patients received standard clinical care and conventional rehabilitation therapy as part of the baseline treatment protocol.\u003c/p\u003e\u003cp\u003eThirty-six participants completed the study, including 16 patients with MCS and 20 patients with UWS. However, among the 36 participants, one MCS patient was excluded because of poor data quality. Prior to the experiment, the level of consciousness in patients with pDOC was evaluated using the CRS-R scale. Each patient underwent five assessments on nonconsecutive days within a 10-day period, and the highest score obtained was used to classify the patient as UWS or MCS(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e summarizes the demographic and clinical assessment scores of all the participants. No significant differences in age distribution were observed between the two groups. However, significant disparities emerged in consciousness assessment scores, including Coma Recovery Scale-Revised (CRS-R) scores(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). The MCS group presented higher CRS-R scores (7.27\u0026thinsp;\u0026plusmn;\u0026thinsp;2.31), whereas the UWS group presented lower CRS-R scores (3.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.32).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBasic characteristics of the patients.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVariable\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTotal (n\u0026thinsp;=\u0026thinsp;35)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMCS (n\u0026thinsp;=\u0026thinsp;15)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUWS (n\u0026thinsp;=\u0026thinsp;20)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge, years, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e59.69\u0026thinsp;\u0026plusmn;\u0026thinsp;13.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e59.20\u0026thinsp;\u0026plusmn;\u0026thinsp;14.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e60.05\u0026thinsp;\u0026plusmn;\u0026thinsp;14.36\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFemale, n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7(20%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4 (27%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3 (15%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCRS-R, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.11\u0026thinsp;\u0026plusmn;\u0026thinsp;2.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.27\u0026thinsp;\u0026plusmn;\u0026thinsp;2.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.32\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTime from onset to enrollment, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD(days)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e42.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e42.53\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e42.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.60.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStroke, n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e27(77%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13(87%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e14(70%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTraumatic brain injury, n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1(3%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0(0%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1(5%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHypoxic-ischemic encephalopathy, n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7(20%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2(13%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5(25%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eCRS-R, Coma Recovery Scale-Revised; UWS, unresponsive wakefulness syndrome; MCS, minimally conscious state; SD, standard deviation.\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e6.2 Acupuncture and Point Selection\u003c/h2\u003e\u003cp\u003eAmong the 35 patients, 23 received acupuncture stimulation, while 12 received sham acupuncture stimulation. The acupuncture points applied in this study included the main acupoints Neiguan (PC6, bilateral), ​Renzhong (DU26, unilateral), ​Sanyinjiao (SP6, bilateral) and the secondary acupoints Jiquan (HT1, bilateral), Chize (Lu5, bilateral) and Weizhong (BL40, bilateral)(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). The anatomical location of these points was defined as follows: Renzhong (DU26) is situated at the junction between the upper one-third and middle one-third of the philtrum groove. Neiguan (PC6) is located 2 cun proximal to the transverse wrist crease, between the tendons of the flexor carpi radialis and palmaris longus muscles. Sanyinjiao (SP6) is positioned 3 cun above the medial malleolus, along the posterior border of the tibia. Jiquan (HT1) is situated at the apex of the axilla, where the axillary artery beats. Chize (Lu5) is located in the transverse stripes of the elbow, the radial depression of the bicipital brachii tendon. Weizhong (BL40) is located in the posterior region of the knee at the midpoint of the popliteal transverse stripes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). For the sham acupuncture control group, non-acupoints near the main acupoints were selected for minimal, superficial stimulation to control for nonspecific effects. These control points were chosen on the basis of a validated protocol from a previous study and through consultation with acupuncture experts. They are not situated on any recognized meridian or over major nerves but are all within a 2.5 cm radius of the corresponding true acupoint. The specific locations were defined as follows:​\u003c/p\u003e\u003cp\u003eThe sham point for PC6 was located lateral to PC6, in the intermeridian space between the Hand-Taiyin Lung Meridian and the Hand-Jueyin Pericardium Meridian. The sham point for DU26 was situated on the vertical line of the mouth, lateral (left) to DU26. The sham point for SP6 was located 6 cun proximal to the tip of the medial malleolus, in the intermeridian space between the Foot-Taiyin Spleen Meridian and the Foot-Jueyin Liver Meridian. This point is 3 cun proximal to the SP6 and 1.25 cun anterior to it on the medial side of the tibia. The sham point for HT1 was located 1 cun inferior and 1 cun anterior to the axillary apex, avoiding axillary hair, on the muscle belly of the bicipital brachii. The sham point for Lu5 was located at the midpoint of the line connecting Lu5 and LI11 (Quchi) when the elbow was flexed at 120 degrees on the radial side of the tendon of the radial wrist extensor longus muscle(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe sham point for BL40 was located inferior and lateral to the midpoint of the popliteal transverse crease on the muscle belly of the peroneus longus muscle. The acupuncture procedures were conducted by a licensed acupuncturist with a decade of clinical experience from the Binzhou Medical University Hospital. All the subjects remained in a supine position on a scanning bed throughout the intervention. Sterile, single-use titanium needles (0.20\u0026times;40 mm; Wujiang CloudDragon Medical Apparatus Co., Ltd.,Wujiang District, Suzhou, China) were utilized.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFor the XNKQ acupuncture protocol, the needling techniques were executed as described below. At Neiguan (PC6), bilateral insertion was performed to a depth of 0.5\u0026ndash;1.0 cun. A reducing stimulation method was applied for 1 minute, involving lifting-thrusting and rotating manipulations, with the left hand rotating the needle counterclockwise and the right hand rotating it clockwise. Subsequently, the needle at Renzhong (DU26) was obliquely inserted toward the nasal septum to a depth of 0.3\u0026ndash;0.5 cun, followed by the sparrow-pecking technique until ocular moistening or lacrimation was observed. Finally, at Sanyinjiao (SP6), bilateral oblique insertion (0.5\u0026ndash;1.0 cun depth) along the medial tibial border was performed by reinforcing manipulation via the lifting-thrusting method, characterized by heavy thrusting and light lifting, which was maintained for 1 minute. After the main points were needled, the patient\u0026rsquo;s secondary points, bilateral Jiquan (HT1), bilateral Chize (Lu5), and bilateral Weizhong (BL40), were needled using the lifting-thrusting method(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). The acupuncture intervention, encompassing both needle manipulation and retention time, lasted for 30 minutes in both the verum and sham acupuncture groups(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e6.3 Data Acquisition\u003c/h2\u003e\u003cp\u003efNIRS data were acquired using a 48-channel NirSmart system (Danyang Huichuang, China). The fNIRS cap, designed according to the international 10\u0026ndash;20 system, comprised 15 light sources and 16 detectors to obtain resting-state brain network data. The source‒detector distance was maintained within 3 cm (2.9\u0026ndash;3.1 cm). Each channel primarily detected hemodynamic changes in the cortical region beneath the midpoint of the source‒detector pair, with channel localization mapped to Brodmann areas. The signals were recorded at wavelengths of 760 nm and 850 nm, with a sampling frequency of 11 Hz. The 48 channels were assigned to eight key regions of interest (ROIs) in the cerebral cortex: the left frontal lobe (LFL; channels 9, 10, 11, 27, 29, 30, 31, 32, 43, 44, 45, 46, and 47), right frontal lobe (RFL; channels 6, 7, 8, 21, 22, 23, 24, 26, 37, 39, 40, 41, and 42), left temporal lobe (LTL; channels 12, 13, 14, 15, 16, 33, and 35), right temporal lobe (RTL; channels 1, 2, 3, 4, 5, 17, and 18), left primary motor cortex (LPMC; channels 34 and 48), right primary motor cortex (RPMC; channels 19 and 38), left dorsolateral prefrontal cortex (LDLPFC; channels 10, 29, 30, 43, 44, and 45), and right dorsolateral prefrontal cortex (RDLPFC; channels 21, 23, 40, and 41) (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e)(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eDuring fNIRS data acquisition, the participants initially rested in a comfortable supine position for 5 minutes to stabilize their physiological parameters before donning the fNIRS cap. The data acquisition protocol consisted of three phases: the preacupuncture resting-state phase, during which baseline brain activity was recorded for 8 minutes; the acupuncture intervention phase, during which real-time fNIRS signals were acquired throughout the acupuncture procedure (duration adjusted according to the actual manipulation time, typically 20 minutes); and the postacupuncture resting-state phase, during which postintervention resting-state signals were recorded for 8 minutes following needle removal. The final analyses were based on three 8 minute resting-state fNIRS epochs: pretreatment, middle segment of the during-treatment period, and posttreatment. Adopting uniform 8-minute windows aligns with established methodological standards in the fNIRS literature and ensures comparable signal quality across phases(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e6.4 Data Processing\u003c/h2\u003e\u003cp\u003eThe fNIRS data were processed and analyzed using NirSpark software. Raw light intensity signals were first converted into optical density (OD) curves, and oxygenated hemoglobin (HbO) and deoxygenated hemoglobin (HbR) concentrations were derived using the modified Beer‒Lambert law. During preprocessing, bandpass filtering (0.01\u0026ndash;0.2 Hz) was applied to eliminate motion artifacts and baseline drift caused by physiological fluctuations (e.g., cardiac and respiratory cycles)(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Subsequent analyses focused exclusively on HbO signals because their signal-to-noise ratio is superior to that of HbR.\u003c/p\u003e\u003cp\u003eFC analysis was performed by extracting HbO time series before, during, and after acupuncture. For each ROI, HbO time series were aggregated across corresponding channels. Pairwise Pearson correlation coefficients were computed between the HbO time series of all channel pairs, with these coefficients defined as the functional connectivity strength between associated brain regions(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). For further network-level analysis, time-averaged HbO signals from all 48 channels were subjected to correlation analysis. Pearson correlation coefficients underwent Fisher-z transformation to normalize their distributions, generating standardized metrics for quantifying dynamic changes in FC strength across the three experimental phases(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e6.5 Statistical Analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using SPSS 27.0 software. Between-group comparisons were conducted using two-tailed Student's \u003cem\u003et\u003c/em\u003e tests, whereas multigroup comparisons were evaluated with one-way analysis of variance (ANOVA). A threshold of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was applied to define statistical significance.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003e This study was reviewed and approved by the Ethics Committee of Binzhou Medical University Hospital (Approval Number: KYLL-186). All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from the legal guardians of all individual participants included in the study.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis research did not receive any specific grant.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eZ.C. and H.D. wrote the main manuscript text; Y.Z. and Z.C. performed data analysis; H.G. and Z.X. reviewed and revised the manuscript. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eEdlow BL, Claassen J, Schiff ND, Greer DM. Recovery from disorders of consciousness: mechanisms, prognosis and emerging therapies. Nature reviews Neurology. 2021;17(3):135\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOverbeek BUH, van Erp WS, Eilander HJ, Koopmans R, Lavrijsen JCM. Prevalence of the Minimally Conscious State Among Institutionalized Patients in the Netherlands: A Nationwide Study. Neurology. 2023;101(20):e2005-e13.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuxtable R. Dying too soon or living too long? Withdrawing treatment from patients with prolonged disorders of consciousness after Re Y. 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Heliyon. 2024;10(9):e30192.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"XNKQ acupuncture, prolonged disorders of consciousness, functional near-infrared spectroscopy, functional connectivity, cerebral oxygenation","lastPublishedDoi":"10.21203/rs.3.rs-7689244/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7689244/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e\u003cp\u003eTo investigate dynamic changes in brain functional connectivity (FC) in patients with prolonged disorders of consciousness (pDOC) during and after Xingnao Kaiqiao(XNKQ) acupuncture therapy using functional near-infrared spectroscopy (fNIRS).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003e36 patients with pDOC were enrolled. After one patient was excluded because of data quality issues, the final analysis included 35 patients who completed the acupuncture intervention with fNIRS monitoring. fNIRS assessments of changes in oxygenated hemoglobin (HbO) concentration were conducted before, during, and after XNKQ acupuncture sessions. Intra- and interregional FC analyses were performed across eight predefined regions. FC analysis was performed by extracting HbO time series before, during, and after acupuncture.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eCompared with the baseline FC, FC was significantly greater during acupuncture. Subgroup analysis revealed increased FC during acupuncture in patients with unresponsive wakefulness syndrome (UWS). In contrast, no such increase was detected in individuals in the minimally conscious state (MCS) or in the sham acupuncture group. The intraregional FC in the left frontal lobe (LFL), right frontal lobe (RFL), left temporal lobe (LTL), and left dorsolateral prefrontal cortex (LDLPFC) markedly increased during acupuncture. FC remained elevated in the LFL, RFL, LTL, right temporal lobe (RTL), and right primary motor cortex (RPMC) after acupuncture. The interregional FC between the LFL-LTL, LFL-RFL, LFL-RTL, RFL-LTL, RFL-RTL, and LTL-RTL was significantly greater during acupuncture. These enhancements persisted after acupuncture for LFL-LTL, LFL-RFL, RFL-LTL, RFL-RTL, and LTL-RTL FC. In the UWS subgroup, the intraregional FC in the LFL, RFL, LTL, RTL, and LDLPFC, as well as the interregional FC between the LFL and LTL, increased during acupuncture. After acupuncture, FC was increased in the LFL, LTL, and RPMC. In the MCS subgroup, FC in the RFL was elevated after acupuncture. Additionally, interregional FC between the LFL and RFL increased both during and after acupuncture, and connectivity between the RFL and LFL also increased following acupuncture.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eXNKQ acupuncture selectively increases FC in consciousness-related networks, particularly within frontal and temporal cortices, with sustained effects postintervention. These findings support the therapeutic potential of XNKQ for pDOC patients through targeted reorganization of higher-order cognitive networks, providing neurophysiological evidence for its clinical application.\u003c/p\u003e","manuscriptTitle":"Effects of Xingnao Kaiqiao Acupuncture on Brain Functional Connectivity in Patients with Prolonged Disorders of Consciousness","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-06 16:47:18","doi":"10.21203/rs.3.rs-7689244/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"004d96ad-a36a-4809-bc1d-ca774b69b11f","owner":[],"postedDate":"November 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-27T12:11:52+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-06 16:47:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7689244","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7689244","identity":"rs-7689244","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}