Correction: The efficacy and safety of low-intensity focused ultrasound pulses for prolonged disorders of consciousness: a study protocol for a randomized controlled trial

dorsolateral prefrontal cortex in 16 patients with DoC (5 MCS and 11 VS/UWS) resulted in increased (the Coma Recovery Scale-Revised ) CRS-R scores in 36% of VS/UWS patients and all MCS patients. Moreover, a 2022 study applying 20Hz rTMS 16 demonstrated significant improvements in consciousness in the real stimulation group compared to the sham group. However, in-depth analysis revealed that only a subset of patients experienced clinically meaningful gains. Third, for transcutaneous auricular vagus nerve stimulation (taVNS), a 2023 randomized double-blind controlled trial 17 involving 57 DoC patients found that while the intervention group showed higher CRS-R scores post-treatment compared to the control group, the difference did not reach statistical significance. Finally, in the realm of Low-intensity focused ultrasound pulses (LIFUP), Monti and colleagues 18 pioneered the use of non-invasive ultrasound to stimulate the thalamus (100Hz TUS) in a patient with MCS following traumatic brain injury in 2016. The patient exhibited significant clinical improvement, marking a pivotal moment in the application of LIFUP for DoC. Subsequent studies have reported increases in CRS-R scores in both acute 19 and chronic 20 DoC cases. However, The absence of rigorous comparison with a sham stimulation group limits these findings, highlighting the need for controlled investigations.LIFUP holds substantial promise as a noninvasive modality for modulating human neural circuits 21 . Compared to conventional non-invasive brain stimulation techniques, ultrasound offers unparalleled spatial precision, capable of targeting both cortical and deep subcortical brain structures with a resolution on the order of just a few cubic millimeters. This high degree of focus allows for the precise stimulation of small subcortical regions, such as the thalamus, which previously required invasive deep brain stimulation (DBS) techniques 21 . Moreover, LIFUP is regarded as a highly safe method of neuromodulation 22,23 . Given the heterogeneity among patients with DoC, LIFUP presents a promising therapeutic avenue, though its clinical efficacy remains to be confirmed through well-designed randomized controlled trials. The underlying mechanisms of LIFUP are not yet fully elucidated. A prevailing hypothesis suggests that the acoustic pressure delivered by LIFUP induces nanoscale deformation of neuronal membranes, which may modulate the activity of ion channels and mechanosensitive receptors embedded in the membrane 22,23 . In particular, LIFUP may alter the function of mechanosensitive ion channels, influencing the excitability and spontaneous firing rates of neurons, thereby inducing both short-term and long-term neuroplastic changes 21,22,23 . These physiological effects may further result in modulations of cerebral blood flow, the release and uptake of neurotransmitters, and ultimately, alterations in neural circuit function 24,25,26 . As such, LIFUP represents a compelling tool for investigating and potentially restoring consciousness in patients with DoC.In 2021, Monti and colleagues 20 conducted a pilot intervention using thalamic LIFUP on three patients with pDoC, of whom two demonstrated significant improvements in behavioral responsiveness compared to baseline. In 2022, a similar intervention 19 was applied to 11 patients with acute DoC, with nine exhibiting behavioral improvements. Both studies reported favorable safety profiles and a high response rate. However, these proof-of-concept case series lacked a sham-controlled condition, limiting the strength of causal inferences. In a separate 2022 study 27 , an 80-second train of theta-burst patterned transcranial ultrasound (tbTUS) was applied to the motor cortex of 15 healthy participants. The results revealed elevated corticospinal excitability sustained for up to 30 minutes, alongside reduced short-interval intracortical inhibition and enhanced intracortical facilitation, suggesting that tbTUS may promote long-term cortical plasticity. Given the operational characteristics of LIFUP, thalamic stimulation using specific parameter sets may likewise elicit excitatory effects and induce plastic changes within thalamic circuits. However, no studies to date have directly compared the effects of different LIFUP parameter configurations, specifically, 100Hz TUS and tbTUS in patients with pDoC. This represents a critical gap in current knowledge. Investigating whether these parameters produce differential outcomes and how they modulate neural functional connectivity, neurotransmitter dynamics, and EEG signal features is essential. Such insights could optimize treatment protocols and advance our understanding of the neurobiological mechanisms underlying recovery in pDoC.Based on the above, this study aims to evaluate the efficacy and safety of LIFUP as a potential neuromodulatory treatment. Another major focus of our investigation is to explore the relationship between multimodal neurofunctional assessments and behavioral outcomes in patients with pDoC. We hypothesize that participants receiving LIFUP delivered via either 100 Hz TUS or tbTUS will exhibit significantly improved behavioral recovery metrics compared to sham-controlled participants. Furthermore, we predict that 100 Hz TUS 19,20 will induce superior neuroplastic effects relative to tbTUS 27 , as quantified through multimodal functional connectivity analyses. These differential effects are anticipated to manifest as: (1) increased phase-locking value (PLV) in whole-scalp or region-specific high-density electroencephalography (HD-EEG), and (2) enhanced effective connectivity (EC) in task-based functional magnetic resonance imaging (fMRI), particularly within targeted neuromodulation networks.This is a single-site, controlled randomized trial conducted at the Intensive Care Rehabilitation Ward of Shanghai Yangzhi Rehabilitation Hospital, also known as the Shanghai Sunshine Rehabilitation Center. In this parallel-group study, 78 patients with pDoC will be prospectively randomized in a 1:1:1 ratio to one of three arms: Group A (100Hz TUS), Group B (tbTUS), or Group C (sham LIFUP stimulation).All enrolled patients will receive two sessions of TUS treatment whether it is real or sham stimulation. In addition to CRS-R assessments, comprehensive neurophysiological and neuroimaging assessments, including HD-EEG, Somatosensory Evoked Potentials (SSEPs), fMRI, proton magnetic resonance spectroscopy (MRS) and positron emission tomography-computed tomography (PET-CT), will be performed at baseline (pre-treatment) and follow-up (post-treatment) time points. Longitudinal outcome measures will be assessed at three timepoints: 1 month (30±7 days), 3 months (90±14 days), and 6 months (180±30 days) following the final intervention.During each treatment session, vital signs including body temperature, heart rate, blood pressure, and respiratory rate will be monitored to ensure safety. The vital signs of the patients will be continuously monitored 24 hours after the intervention. The trial will be supervised by a team comprising at least one neurologist, one neurorehabilitation specialist, and associated research staff. All procedures and evaluations will adhere strictly to the study protocol. A schematic flow chart of the trial design is provided in Figure 1.1. Participants must have sustained their injury or onset more than 28 days prior to enrollment; 2. Participants must be over 18 years of age; 3. Participants must exhibit a behavioral profile consistent with either a VS/UWS or a MCS, as assessed by the CRS -R; 4. Participants must not have experienced any significant fluctuations in their medical condition during the past week. Common causes of instability include fever exceeding 37.3°C within the past seven days, epileptic seizures, paroxysmal sympathetic hyperactivity, and inability to maintain adequate oxygenation for 40 minutes. The above conditions will be assessed by two ICU physicians based on the patient's status over the recent week.2.2.2.Exclusion Criteria: 1. Participants with significant/severe cardiac disease; 2. Participants with a history of neurological illness prior to the current injury; 3. Participants who are unable to safely undergo MRI scanning due to a severe infection; 4. Participants with intracranial metal implants. All patients will be recruited from the intensive care and rehabilitation wards. The study will be conducted from March 2025 to December 2025. Potential participants will be identified by reviewing electronic medical records within the hospital information system (HIS). Researchers will explain the details of the study to the patients' legal guardians. Upon obtaining consent, a neurologist will evaluate each candidate based on the inclusion and exclusion criteria. Those who meet the eligibility requirements will be formally invited to participate in the study and will undergo a comprehensive neurological examination, followed by a rehabilitation assessment, prior to the intervention.For the behavioral assessment using the CRS-R, this study adopts a randomized controlled intervention design. Repeated measures will be analyzed using ANOVA to account for potential confounding variables. Based on a power analysis conducted using G*Power, with an effect size of 0.40, a significance level (α) of 0.05, statistical power (1-β) of 0.80, a two-tailed test (α=0.05), and an equal allocation ratio among the three groups (N1: N2: N3 = 1:1:1), the required each group sample size is calculated to be 18. After applying Bonferroni correction, the significance threshold for individual comparisons should be adjusted to α=0.0167. To maintain this corrected alpha level, each group would require a sample size of 22. Considering a potential 15% dropout or exclusion rate, a minimum of 76 participants is required. Therefore, the study will enroll a total of 78 participants: 26 in Group A, 26 in Group B, and 26 in Group C. In the event of patient withdrawal or death during the study, we will implement standardized procedures. All withdrawal cases will be documented systematically, including: 1) voluntary withdrawal (participant-initiated consent revocation), 2) loss to follow-up (≥3 consecutive contact attempts failed), 3) clinical deterioration (≥2-point GCS decrease sustained for 48 hours), and 4) investigator-determined withdrawal (with protocol-specified justification). For data management, all collected pre-withdrawal data will be retained per intention-to-treat principles, with final evaluable assessments carried forward for primary analysis. Mortality cases will trigger immediate reporting to the Ethics Committee of Shanghai Yangzhi Rehabilitation Hospital (Shanghai Sunshine Rehabilitation Center), followed by survival analysis using Kaplan-Meier methodology and independent adjudication of causes by a clinical endpoint committee. Statistical approaches will include sensitivity analyses (completers vs. non-completers) and multiple imputation for missing data.Randomization will be performed using the random number generation function in the R programming language (set.seed). All eligible participants will be randomly assigned to one of three groups in a 1:1:1 ratio: Group A (100Hz TUS), Group B (tbTUS), and Group C (sham stimulation). To ensure allocation concealment and fairness, a randomization sequence will be generated by an independent researcher who is not involved in the trial's implementation or outcome evaluation. The randomization sequence will be kept confidential and will not be disclosed to outcome assessors or statisticians. Baseline assessments will be completed after group assignment. Healthcare providers and therapists involved in the participants' care, including acupuncturists and cognitive therapists, will remain blinded to group allocation to minimize bias.After initial screening and evaluations, all patients in the three groups will undergo two sessions of LIFUP. Prior to LIFUP, K-Plan simulation will be performed to determine the optimal stimulation trajectory. Prior to the intervention, baseline assessments will be conducted within a 7-to 10-day window, including five repeated CRS-R evaluations, HD-EEG, SSEPs, fMRI, MRS and PET-CT. The first session of LIFUP will be administered between days 11 and 13, with the second session delivered after a 2-to 3-day interval. Each stimulation session will be conducted according to its corresponding parameter specific duration. CRS-R evaluations will be conducted 1 hour before and after each session of LIFUP. Following the second session of LIFUP, repeat assessments including EEG, SSEPs, fMRI, MRS, and PET-CT will be conducted, with all evaluations completed within 5-7 days post-intervention. The intervention phase will last approximately 23 days in total. The entire process of examination and treatment is shown in Figure 2.For the stimulation protocol in Group A (100Hz TUS), LIFUP will be applied with the following parameters: Fundamental Frequency (FF):500 kHz, Pulse Repetition Frequency (PRF) :100Hz, Pulse Width(PW):0.5ms, Duty Cycle (DC):5%, Sonication Duration (SD):30 seconds, Number of Sonication:20, Spatial-peak Pulse-Average Intensity (ISPPA):14.39W/cm 2 , and Spatial-peak Temporal-Average Intensity (ISPTA):719.73 mW/cm². Peak rarefactional pressure:0.68 MPa. The pulse timing parameters of 100Hz TUS are shown in Figure 3.Under stimulation condition Group B, referred to as tb TUS following protocol 27 , the parameters will be set as follows: FF of 500 kHz, PRF of 5 Hz, PW of 20 of of with a total of is 2 , and the is 2 . Peak rarefactional The pulse timing parameters of tbTUS are shown in Figure 4. Group C will undergo the tbTUS the power will remain In order to minimize during TUS 28 , will be delivered to participants through while is The neural activity and safety of both stimulation protocols (100Hz TUS and have in studies . 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The CRS-R 23 that and in patient status will be assessed by post-treatment scores for each the group level, the will be to evaluate behavioral both intervention and control as it is to a more of functional CRS-R assessments will be conducted at before the intervention, and after each treatment session to the for each For patients who undergo two sessions of LIFUP, scores will be and in the outcome and data baseline and following treatment of LIFUP, patients will undergo a series of MRI using a with a The will the following sequence of fMRI, and for revealed a more in connectivity between the thalamus and the of the brain during The primary of this study is to potential changes in connectivity, particularly between the thalamus and before and after intervention. will be quantified using prior have reported or level changes in cortical regions, these remain to cortical In this study, we to whether different stimulation parameters induce changes in of within the and of the This may further the underlying neural mechanisms of recovery in patients with participants will undergo a PET-CT using a PET-CT Each will receive an of at based on body After participants will in a with for before the changes in may as of treatment efficacy and predict clinical have reported in studies on the information and PET-CT procedures be found in the will be assessed during the baseline phase prior to stimulation of LIFUP. A follow-up will be conducted one post-treatment during the on protocols involving thalamic TUS stimulation in healthy 40 , are as a for treatment efficacy and and EEG will be on the before and on the after intervention of LIFUP. EEG as a critical and tool in patients with pDoC. To no studies have reported changes in EEG functional connectivity, or power before and after treatment of LIFUP in this EEG following intervention of LIFUP have not yet This study aims to this gap by investigating changes in neural and connectivity associated with LIFUP, thereby insights its therapeutic mechanisms in pDoC. information on and EEG protocols is provided in the will a comprehensive analysis behavioral outcomes of neuroimaging data MRS, Statistical analyses will be conducted at both the individual and group outcomes before and after as as between intervention and control The statistical will include ANOVA to for potential confounding variables. will be analyzed using or independent sample while will be assessed using the or will be to evaluate and To explore between neurotransmitter and analyses will be For SSEPs, changes in and before and after treatment will be compared using EEG data will be analyzed using to evaluate both and functional connectivity including (PLV) and MRI assessments will be conducted using to functional and effective connectivity during both and task-based MRS will be to post-treatment changes in and within the brain PET-CT imaging will be to alterations in both in the and the following the intervention. 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As in studies of stimulation of the thalamus could both and decrease functional connectivity, suggesting that the brain may connectivity a during recovery . tbTUS excitatory 27 cortical its deep more of in the during intervention of tbTUS in patients revealed enhanced and though the underlying mechanisms remain to be . This and spatial suggests therapeutic effects may by both and Finally, we that the mechanisms underlying the recovery of DoC require more precise of In the this could potentially be with our this is the first randomized controlled trial to the effects and safety of LIFUP on patients with pDoC. 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