Intradermal Thumbtack Needle Alleviates Depressive Symptoms and Resets Executive Control System Function in Young Patients with Subthreshold Depression: A study protocol of a randomized, controlled, and single-blind trial {1}

Background Subthreshold depression is the prodromal stage of a depressive episode, which is characterized by depressive symptoms but fails to meet the diagnostic criteria for depression. At present, there is no recognized therapy for subthreshold depression worldwide. Studies have also shown that acupoint stimulation can significantly decrease depressive symptoms by modulating different executive control systems, such as reward circuits and motivational circuits. In this proposed study, our objective is to evaluate the efficacy of intradermal thumbtack needle therapy, a continuous and effective acupoint stimulation therapy, on subthreshold depression and generate hypotheses on the mechanism by which this therapy resets executive control system function in young patients with subthreshold depression. Method In this randomized controlled trial, all eligible participants are diagnosed with subthreshold depression. Participants are randomly assigned to two groups at a ratio of 1:1 and receive either intradermal thumbtack needle or sham intradermal thumbtack needle therapy. The primary outcome is self-reported depression severity on the 9-item Patient Health Questionnaire (PHQ-9) from baseline to 6 weeks. The secondary outcomes include the Short Form Survey (SF-12) and the Generalized Anxiety Disorder Assessment (GAD-7) complementarity. Two psychological paradigms, the Attention Network Test (ANT) and the Psychomotor Vigilance Task (PVT), and functional magnetic resonance imaging (fMRI) will be used to evaluate the mechanism. These two psychological paradigms will explain the mechanism from the perspective of executive control in psychology, and fMRI will reflect the change in functional reset of the executive control system from an imaging point of view. The results are obtained at the start of treatment and and protocol optimizing the laboratory protocols. LDY: Protocol development, optimizing laboratory protocols. Optimizing the laboratory protocols, acquiring of data. ZJW: Trial coordination, acquiring of data. JTL: Proposal and protocol development. Design of study. YBW: Proposal and protocol acquiring of JZ: and

the end of treatment. The entire duration of the study will be approximately 12 months.
Discussion This study is designed to evaluate the efficacy of intradermal thumbtack needle on StD patients. Additionally, mechanisms by which this therapy resets executive control system function will be studied

BACKGROUND {6a}
Depression is one of the most common mental disorders; it is caused by various reasons and characterized by noticeable and persistent depression, loss of interest and lack of energy. [1] According to the latest statistics from the World Health Organization (2017), depression is growing at a rapid rate every year, increasing by 18.4% from 2005 to 2015. [2] It is estimated that by 2030, depression will become the first disease burden in the world. [3] Subthreshold depression refers to the state of having depression symptoms but falling to meet the clinical diagnostic criteria for depression. From the perspective of the continuity of depression development, subthreshold depression is a transitional stage between the absence of depressive symptoms and depressive episodes. [4] Studies have shown that compared with patients who were not depressed, those who had subthreshold depression, a precursor to a depressive episode, exhibited a 5.5-fold risk for major depression 1 year later. [5] Domestic studies show that the incidence of subthreshold depression among college students is as high as 36.56%. [6] Therefore, subthreshold depression interventions should receive attention.
Given that subthreshold depression does not meet the diagnostic criteria for depression, medication is not recommended. At present, there is no recognized therapy for subthreshold depression worldwide.
As a traditional Chinese medicine therapy, acupuncture has been used in the clinical treatment of depression for a long time. Acupoint stimulation could significantly alleviate the symptoms of depression. [7,8] Our team has been focusing on depression for more than 20 years. We have also found that acupoint stimulation exhibits good clinical effect in treating depression. The neurobiological mechanism of the treatment of acupuncture and moxibustion for depression through neuronal protection and brain plasticity was clarified.
In terms of treatment, greater than 70% effectiveness has been reported. In addition, our previous studies have shown that intradermal thumbtack needle therapy for subthreshold depression exhibits the characteristics of good efficacy and easy promotion. However, high-quality clinical evidence for the treatment of subthreshold depression is lacking, and the treatment mechanism has not been elucidated to date.
In recent years, psychological researchers have tried to study depression from the perspective of how cognitive change affects mood, [9] while the core of cognitive function is executive control. Executive control (also called executive functions (EFs)) refers to a family of top-down mental processes needed when one needs to concentrate and pay attention and when relying on automatic responses, instinct or intuition would be ill advised, insufficient, or impossible. [10] The lack of psychological resources will lead to the decline of executive function [11] and subsequently change executive control capacity, attention, memory and other advanced cognitive activities. In addition, cognitive dysfunction of depression may exist beyond the depressive symptoms, [12,13] which is the main reason that leads to the failure of patients with depression to return to normal social functions. The cognitive impairment of subthreshold depression is manifested as time perception, obviously impaired cognitive control ability, and attention bias and memory bias to negative events. [14] Our previous studies have also shown that acupoint stimulation can significantly decrease depressive symptoms by modulating different executive control systems, [15,16] such as reward circuits and motivational circuits. These findings provide an operational approach for us to fully explore the relationship between executive control systems and depression.
Therefore, based on previous research, this project will use cognitive function of young people with subthreshold depression as the entry point and executive function control system as perspective. Then, psychological paradigms and functional magnetic resonance imaging technology will be used to compare the brain executive control system of the subthreshold depression group and normal group and to observe the specificity of brain structure, network and function of the subthreshold depression group. Finally, comparison and analysis between the effects of intradermal thumbtack needle and sham intradermal thumbtack needle treatment in the brain executive control system will be performed, and the neural mechanism promoting the functional reset of the brain executive control system will be hypothesized based on the evaluation of the efficacy of the intradermal thumbtack needle therapy.

We followed the Standard Protocol Items: Recommendations for
Interventional Trials [17] and Standards for Reporting Interventions in Clinical Trials of Acupuncture [18] guidelines for the reporting of our trial protocol.

Design {8} {9}
A randomized, single-blind trial will be conducted at the Guangdong Provincial Traditional Chinese Medicine Hospital and College of Psychology, South China Normal University. Participants will be recruited from community volunteers or from outpatient clinics in Guangdong Provincial Traditional Chinese Medicine Hospital. Participant enrolment started in May 2020 and is expected to end in September 2021. Eligible participants who provide consent will be randomly assigned in a ratio of 1:1, to receive a 6-week intervention with intradermal thumbtack needle (n=40) or sham intradermal thumbtack needle (n=40). Before and after the intervention and during follow-up, the PHQ-9 questionnaire was used as the primary outcome, and SF-12 and GAD-7 questionnaires served as the secondary outcomes to investigate the clinical efficacy of intradermal thumbtack needle on subthreshold depression and the conversion rate from subthreshold depression to depression. In addition, the psychological paradigm (executive function control system) and imaging evaluation were performed on the subthreshold depressed population before and after treatment, respectively, to explore the neural mechanism by which intradermal thumbtack needle promotes the functional reset of the brain executive control system.

Psychological paradigms:
The Attention Network Test (ANT) will be applied to measure the ability of the executive control system. Combined with spatial cue and flanker tasks, ANT can effectively measure three different attention network effects: alertness effect, orientation effect and executive control effect. [19] The Psychomotor Vigilance Task (PVT) is a sustained-attention, reaction-timed task that measures how quickly subjects respond to a visual stimulus. [20,21] Imaging evaluation: Forty participants will be randomly selected for fMRI before the intervention, and the results will be compared with the normal human brain database. After the intervention, all participants will undergo fMRI, and the treatment and control groups will be compared with each other.
The study diagram is presented in Figure 1.

Participants {7} {10} {26}
Participants aged between 18 and 30 years and diagnosed with subthreshold depression who have had no psychological counseling in the last six months or at present and have no intention to do so in the future are eligible to participate in the study. The following criteria are used for diagnosis of SD according to DSM-V: [22] (1) screened positive for subthreshold depressive symptoms by the Center for Epidemiological Studies Depression Scale specialists in psychiatry to diagnose StD. [24,25] Participants will be excluded if they meet any of following criteria: (1) diagnosed with other psychosis; (2) antidepressants; (3) alcohol dependency; (4) recent suicidal risk; (5) significant cognitive impairment; (6) recent bereavement; (7) terminal illness on clinical grounds.
Eligible participants will be informed of the aims and nature of the research both verbally and via an information sheet. They will be asked to complete and sign a consent form before enrolment. Patient information will remain confidential, and patients will have the right to withdraw without prejudice at any time.

Sample size calculation {14}
At present, there is a lack of research, and no previous literature is available for effective support. Thus, it is impossible to calculate the previous efficacy as the sample size. This study was a prospective exploration, so 80 subjects were selected for inclusion.

Recruitment {15}
The participants will be recruited at the Acupuncture clinic of Guangdong  15 ) and Gan (CO 12 ) of the auricular point [26,27] on the right side.
After skin cleansing with a 75% alcohol swab, an intradermal thumbtack needle will be quickly inserted into the skin and embedded at the acupoints mentioned above. The needles will remain in the acupoints for 3 days and will be removed by the therapists at the next visit. This procedure can also ensure

Control group {6}
The acupoint selection in this group is the same as that in the treatment group except that the sham intradermal thumbtack needles will be used. After skin cleansing with a 75% alcohol swab, the sham intradermal thumbtack needles can only stick on the surface of the skin instead of being inserted into the skin.
The sham needles will be removed by the therapists after 3 days. Participants are required to receive this intervention twice a week. The specification of the sham needle is the same as above. (Figure 3) During the intervention, participants will not be allowed to undergo psychotherapy or other drug therapy. All the participants will be followed up for a year after treatment to determine how long the treatment lasts and whether the participant has converted to depression.

Criteria for discontinuing or modifying allocated interventions {11b}
Patients can leave the study at any time for any reason if they wish to do so without any consequences. The patient's participation in this study can also be ended by the investigator if the patient is uncooperative and/or does not attend study visits. The patient data that have been collected up to that moment will be included in the analysis. In case too many data are missing (e.g., missing baseline or all of the follow-up patient-reported outcome measures , the patient will be replaced by a new patient. This study will be prematurely ended in case of any abundance in adverse events or if the psychiatrist advises this termination. Criteria for study termination include any suspected unexpected serious adverse reaction (SUSAR).

Data collection and outcome assessment {12}{13}{18a}{18b}
Data will be documented in the paper CRF by researchers or evaluators and will be double-checked by quality inspectors. All the detailed original data (paper CRF) will be recorded timely, clearly, and completely. Case reports will be modified only by researchers who are documenting the data. The information obtained will be simultaneously recorded on an electrical database by Excel for subsequent statistical analysis.
The schedule of measurements is present in Table1.

Primary outcome
Primary study outcomes will be the measurement of depressive symptoms by the 9-item Patient Health Questionnaire (PHQ-9). The PHQ-9 is a reliable and valid measure of depression severity that directly reflects the subjective feelings of depressed patients and their changes in treatment. [28,29] For the severity measure, the PHQ-9 score ranges from 0 to 27. Each of the 9 items can be scored from 0 (not at all) to 3 (almost every day). The higher the score, the more severe the depressive symptoms.

Secondary outcomes
The secondary study outcomes are as follows: 1. A questionnaire called the Short Form Survey (SF-12) will be used to measure the health-related quality of life of participants. [30] Its score ranges from 0 (lowest level of health) to 100 (highest level of health).

Mechanism evaluation outcomes
There are two outcomes to measure the mechanism evaluation: 1. Two psychological paradigms, ANT and PVT, will be used as indicators for mechanism evaluation. Corresponding results are obtained by calculating reaction times and correct rates under different tasks. These two paradigms can provide objective data from the perspective of psychology, and they are only tested at baseline and at the end of the interventions.

2.
Functional magnetic resonance imaging (fMRI) scanning will help us more intuitively understand the changes in brain function and brain structural connections. The signal of the function, gray matter fiber and white matter fiber of the dorsolateral prefrontal cortex (DLPFC) will be extracted from resting-state fMRI data. [32] These signals will be calculated to assess the functional network of the brain based on graph theory. Table 1 shows the participant timeline.

Sequence generation {16a}
Using a simple randomization method, the estimated sample size will be entered into the PEMS3.1 statistical package. The sequence number, random seed number, and grouping results were obtained, and random cards were made and sealed in an opaque envelope.

Concealment mechanism {16b}
The order in which eligible cases are included in the trial at the time of clinical implementation corresponded to the sequence of serial numbers on the envelopes, which were opened and grouped according to the prompts on the random card.

Implementation {16c}
Generate the allocation sequence, enrol participants, assign participants to interventions will be done by three groups of non-interfering individuals.

Who will be blinded {17a}
Participants, data recorder, statisticians will be blinded, treatment operators will not be blinded.

Procedure for unblinding if needed {17b}
There is no circumstances that unblinding is permissible. The needles in the two groups were identical in appearance. The treatment operators will intervene and replace the needles, and the participants will be blinded.

Data management and monitoring{5d} {21a} {27}
Data will be collected at the time points mentioned above. A data management and oversight committee will be established to supervise the collection and entry of data. Adverse events or unintended effects of intradermal thumbtack needles will first be recorded in the CRFs by physicians who are in charge. The principal investigator will have access to the data. Clinical information will not be released without the permission of the principal investigator, except in cases of urgency or necessity. Members of the data monitoring Committee will not participate in any part of the experiment. The role of the data monitoring committee (DMC) is to audit the trial conducted every month after the trial starts and write monthly reports on recruitment of participants, compliance of participants to the assigned treatment, and whether the CRFs are correctly filled in.

Statistical methods {20a}
The researcher takes a factual record of the contents of the observation form to ensure that it is truthful and reliable. The observation form may not be altered as originally. If altered, the original record may not be altered; only an additional narrative explaining the reasons and indicating the date of the alteration may be provided. Two investigators will use two input methods to input data and perform proofreading. EpiData software will be used to build the database. All original data will be analyzed with SPSS Statistics version 22.0 software by a statistician. All statistical tests use a two-sided test, and P <0.05 indicates statistical significance. For analysis of the patients between groups that reached the end of the study, t test will be performed to compare the differences between groups. A comparison of the total loss rate and loss rate due to adverse events will use the χ2 test.

Methods for any additional analyses (e.g., subgroup analyses) {20b}
There are no subgroup analyses planned.

Methods in analysis to handle protocol non-adherence and any statistical methods to handle missing data {20c}
Cases with protocol non-adherence or missing data will be taken off the list and not counted.

Interim analyses {21b}
There are no interim analyses planned.

Quality control {23}
All practitioners, assessors and statisticians will receive training uniformly prior to the trial to guarantee the use of consistent practices. The training program includes research implementation, adverse reaction monitoring and treatment, training on operational technical specifications, and the completion of case observation forms and CRFs. Treatment will be performed by acupuncturists with at least 3 years of training in acupuncture. The study will be regularly monitored by clinical monitors every 3 month to ensure the quality of the trial.

Plans for communicating important protocol amendments to relevant
parties (e.g., trial participants, ethical committees) {25} All substantial amendments will be notified to the Guangdong Provincial Traditional Chinese Medicine Hospital's Ethics Committee. Non-substantial amendments will be recorded and filed. In case amendments concern or affect participants in any way, they are informed about the changes. If needed, additional consent will be requested and registered. Also, online trial registries will be updated accordingly.

Provisions for post-trial care {30}
If the condition worsens after the experiment, we will provide professional psychologists and acupuncture treatment to help alleviate the condition.

Dissemination plans {31a}
Results of this research will be disclosed completely in international peer-reviewed journals. Both positive and negative results will be reported.
Patients will receive a laymen summary of the results in case they opted-in to receive outcomes on a study level.
Plans to give access to the full protocol, participant-level data, and statistical code {31c} The datasets used and/or analyzed during the current study can be made available by the corresponding author.

DISCUSSION
In recent years, the issue of cognitive function in patients with depression has attracted the attention of scholars all over the world, and the relationship between depression and cognitive function has been the focus of discussion.
The core of cognitive function is executive control, and executive function consists of three elements: working memory, inhibitory control, and cognitive flexibility [10]. The introduction of this concept to clinical medicine is mainly derived from the study of prefrontal cortex injuries, which cause a range of neuropsychological abnormalities, including difficulties with planning, cognitive flexibility, working memory, inhibitory control, and self-monitoring.
Currently, studies of executive dysfunction in the prefrontal cortex are mainly conducted using psychological or functional imaging methods. The former is used to assess the neuropsychological mechanism of executive dysfunction by evaluating the psychological activity associated with executive function; the latter is used to assess possible functional deficits in these brain regions by observing changes in neurodevelopmental and metabolic activity in the relevant brain regions. The organic combination of the two helps to elucidate the site of executive dysfunction and its functional activity status.
Our team is also committed to the organic integration of advanced brain imaging techniques, cognitive neuroscience research results, and questions about the mechanisms of acupuncture regulation of brain function to reveal the mechanisms of brain cognitive impairment and adaptation, the role of resting-state spontaneous fluctuations in cognitive processing, and the effects of acupuncture regulation. Our preliminary study showed that intradermal thumbtack needle therapy for subthreshold depression exhibits the characteristics of good efficacy and easy dissemination and that needle pricking can improve depressive symptoms by modulating different execution control systems, such as the reward loop and motivation loop.
In view of this information, this project proposes to describe subthreshold depression from a cognitive perspective (e.g., decreased executive function, narrowed attention, decreased working memory, difficult to generate positive images, and constant flashbacks of negative images) and to objectify the clinical manifestations of subthreshold depressed people using the executive control system as an entry point. Two typical psychological paradigms will be used to analyze the changes of executive control system from the perspective of psychology. On the other hand, a comparative analysis of brain executive control systems in subthreshold depressed and normal populations will be performed using functional magnetic resonance imaging to observe the specificity of brain structure, networks and function in subthreshold depressed populations. Finally, a comparative analysis of the brain executive control system in a young population with subthreshold depression treated by intradermal thumbtack needle therapy versus placebo will be used to generate hypotheses on the neural mechanisms that promote functional reset of the brain executive control system based on the assessment of endothelial needle efficacy.

Trial status{3}
Recruiting started in September 2020. The current protocol is version 2 of 21-3-2020. Currently (5th of Dec 2020), we included ten patients. Patient recruitment is estimated to be completed around August 2022.