Despite major advances in the field of psychopharmacology in recent years, the majority of treated schizophrenia patients retain disabling symptoms, most commonly a variety of negative symptoms. Currently, clinical treatment of schizophrenia remains dominated by pharmacological control. The current use of antipsychotic medications is effective in controlling the positive symptoms of schizophrenia, but has little effect on the negative symptoms. Neuroimaging and neurophysiological studies have shown that negative symptoms are associated with abnormal brain activity in the combined right and left dorsolateral prefrontal and temporoparietal joint regions, and that physical therapy techniques can modulate cortical activity. Therefore, this study aims to investigate the efficacy of transcranial direct current stimulation(tDCS) combined with repetitive transcranial magnetic stimulation(rTMS) on negative symptoms in patients with schizophrenia and to explore possible mechanisms.

The double-blind randomized placebo-controlled study comparing active tDCS stimulation combined with active rTMS stimulation, active rTMS stimulation combined with sham tDCS stimulation, and active tDCS stimulation combined with sham rTMS stimulation to sham tDCS stimulation combined with sham rTMS stimulation at 4 weeks of treatment and 2 weeks of follow-up in patients with predominantly negative symptoms with schizophrenia was studied for efficacy. In addition to the primary observation of changes in the Negative Symptom Assessment Scale (SANS), secondary outcomes include changes in Positive and Negative symptom scale (PANSS) total and negative total scores, changes in the MATRICS Consensus Cognitive Battery (MCCB), changes in local brain activity (functional magnetic resonance imaging, fMRI), white matter integrity (diffusion tensor imaging, DTI), changes in laboratory examination indices changes and changes in psycho-behavioral and EEG index.

This is the first clinical trial combining tDCS with rTMS for the treatment of schizophrenia patients with predominantly negative symptoms. This study will provide solid evidence for the combination of tDCS with rTMS for the treatment of negative symptoms in schizophrenia. This study will also help to further explore the mechanisms of tDCS combined with rTMS for the treatment of negative symptoms in schizophrenia in terms of imaging and behavior.

This study was designed as an interventional, double-blind, sham-controlled, randomized open-label trial. The main objective is to investigate whether tDCS combined with rTMS is effective in improving negative symptoms in patients with schizophrenia and whether it is superior to single physical therapy. We will also study the effects of tDCS combined with rTMS on blood biomarkers, brain imaging, EEG and psycho-behavioral aspects in patients with schizophrenia.

Schizophrenia patients with prominent negative symptoms who met the inclusion criteria were randomly assigned in a 1:1:1:1 ratio to the active tDCS stimulation combined with active rTMS stimulation, active rTMS stimulation combined with sham tDCS stimulation, and active tDCS stimulation combined with sham rTMS stimulation to sham tDCS stimulation combined with sham rTMS stimulation. General information such as age, gender and marital status were matched between the different groups.

Firstly, a well-trained team is involved in the enrollment process, including a senior psychiatrist, two research nurses, two master students working full-time, and 20 psychiatrists who were actively referring patients for scientific research. Secondly, by giving a more elaborate explanation of the study goals, including the pros and cons, to both patients and their guardians they now better understand the background and possible relevance of the study.

The study population will include 120 patients with schizophrenia mainly with negative symptoms recruited from the inpatient departments of Tianjin Anding Hospital. Schizophrenia is diagnosed according to the Diagnostic and Statistical Manual of Mental Disorder, 5th Edition (DSM-5). All patients are stabilized with no changes in their treatment over the past 1 month prior to their inclusion.

For all patients, clinicians assess the subject's eligibility and provide to each subject and to the patient's legal representative comprehensive verbal and written information regarding the objectives and procedures of the study as well as the possible risks. A signed informed consent is obtained from each participant and/or legal representative for schizophrenia prior to undertaking any study-related procedure. Schizophrenia who do not wish to take part in the study will continue to undergo treatment as usual.

Examinations and assessments during treatment include physical examination, scale assessments, medication use inventory, and laboratory examinations. Subjects were not intervened to take medication during this period. Only detailed recordings were performed.

EEG recording and testing procedures were performed using a 64-conductor Ag/AgC1 electrode cap (Easycap, Berlin, Germany) based on the International 10/20 system extension, and EEG signals were recorded using the BrainAmp DC (Brain Products, Germany) EEG system. There are 4 paradigms in EEG recordings, including P50, P300, MMN and Gamma (20/30/40 Hz). Signal processing was performed offline using BrainVision Analyzer 2.2 (Brain Products, Germany) software.

Four paradigms are used in the experiment to explore the responses related to emotions and cognition, namely task-switching, the color Stroop task, the n-back task, and the Taylor aggression paradigm, at baseline and after interventions. The paradigm order is determined by the Latin square design. The presentation of stimuli and recording of responses are controlled by E-Prime 2 software.

Patients undergo two MRI scan sessions, both including T1, BOLD and DTI. The first session takes place before the treatment starts while the second session takes place in week 4. During this procedure, participants will be exposed to a 3 Tesla magnetic field strength with switching gradient fields, radio waves, and scanner noise. An anatomical scan will be made to obtain the structural image necessary for the localization of functional activation. Patients will only be included in the study upon agreeing to be informed of any coincidental pathological findings. A statement to that effect must be signed before entering into the study. A functional MRI scan (either ASL or EPI) is recorded during the resting state, i.e. the subjects do not do any tasks during this scan. Breathing and heart rate are measured in order to remove variance that correlates with these rhythms.

The primary endpoints of all participants were assessed at the beginning (baseline), at the end of 2 weeks (midpoint) and at the end of 4 weeks (endpoint), then at the 2 weeks after intervention (follow-up point). Other visits were conducted between baseline and 6-week to assess secondary endpoints. The first assessment occurred at baseline prior to randomization. The final assessment occurred after the completion of the 2-week follow-up.

Subjects received care as usual while participating in this study and receiving conventional antipsychotic medication. No adjustment of the type or dose of atypical antipsychotic medication during the previous 1 month and the next 1 month. No interference with the drug regimen, just detailed records. Subjects receive the assessments of clinical symptoms and cognitive function during the week before and after treatment, while blood samples and brain imaging data and behavioral indicators data are collected.

Inclusion Criteria:

1. Meets Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (DSM-5) diagnostic classification criteria for schizophrenia.
2. Age 18-60 years old, gender is not limited.
3. Predominantly negative symptoms (PANSS-negative symptom score≥20; or PANSS negative scores are higher than positive scores).
4. Stable psychiatric symptoms (negative symptoms, positive symptoms) for more than 4 weeks or more.
5. No adjustment in the type or dose of antipsychotics taken in the past 1 months and in the next 1 months
6. Patients and Guardians agree to participate in this study and sign an informed consent form.

Exclusion Criteria:

1. Patients with severe physical illness, infectious diseases and immune system diseases, severe neurological diseases, mental retardation or organic brain diseases.
2. Pregnant or lactating women.
3. Other brain stimulation treatment (ECT, MECT, etc) within past 3 months.
4. History of previous seizures.
5. Those evaluated as unsuitable for tDCS and rTMS and those who do not cooperate with treatment.

Additional exclusion criteria for patients participating in the MRI scanning Participants have to fill out a detailed questionnaire covering safety aspects in relation to research in a 3 Tesla magnetic field and MRI environment. These criteria are:

1. MRI incompatible implants in the body (such as cochlear implant, insulin pump, pace maker or other metal implants).

2. Any risk of having metal particles in the eye, due to manual work without proper eye protections.

3. Tattoos containing red pigments.

4. Claustrophobia.

5. The refusal to be informed of structural brain abnormalities that could be detected during the experiment.

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