Motor-evoked potential (MEP)

The study will employ single-pulse transcranial magnetic stimulation (sTMS, Xiang Yu Medical; China) to the primary motor cortex (M1 area) of the hand region. This technique will measure motor-evoked potential (MEP) in the first dorsal interosseous muscle via surface electrodes. MEP amplitude, latency, resting motor threshold (RMT), active motor threshold (AMT), and central motor conduction time (CMCT) will be evaluated as indicators of corticospinal tract function. Subjects will be seated comfortably with their head and arm relaxed. The TMS coil will be placed 5 cm lateral to the vertex, aligning at a 45° angle from the brain midline with the handle pointing backwards. The location yielding consistently maximal MEP responses will be identified as the “motor hotspot” [27]. The magnetic stimulus intensity will be set at 20% above the MEP threshold. The TMS operator will adjust the magnetic cortical stimulus intensity to elicit MEPs of peak-to-peak amplitude 1 mV on average [28]. Five sequential stimulations will be administered at each intensity, and the average of the resulting five MEP traces will be used for data analysis. Latency will be reported as mean ± SD, while amplitude will be represented by the median due to data distribution skewness.

CMCT indicates the time it takes for a signal to travel from the motor cortex to spinal motor neurons. To measure CMCT, we will use spinal magnetic stimulation. After cortical stimulation, each participant will undergo cervical stimulation. The coil will be placed above the C7 spinous process, 2 cm lateral to the midline, aiming to activate cervical nerve roots at the intervertebral foramina. Participants will experience an MEP with a latency time from cortical magnetic stimulation of the first dorsal interosseous muscle. They will also experience an MEP with a different latency time due to cervical magnetic stimulation of the same muscle (peripheral motor conduction time). The difference between these two latency times provides the CMCT value.

CT perfusion (CTP)

Cerebral hemodynamic changes will be assessed using CTP in cerebral blood flow before and after the intervention. The imaging examination utilized a Siemens SOMATOM Force 960 + model with a 320-row spiral scanner. Patients underwent CT plain scans initially, followed by cerebral perfusion imaging using dynamic perfusion mode. The scan parameters were set as follows: collimation width of 40 mm, tube voltage of 120 kV, tube current–time product of 320 m As, gantry rotation speed of 0.35 s per rotation, pitch of 1.2375, with 20 samples (each machine movement corresponds to two samples), a sampling interval of 1.5 s, and a scan length of 105 mm. After selecting the appropriate range, the MALLINCKRODT high-pressure injector was used for intravenous injection through the antecubital median vein. Lohexol injection (370 mg I/ml) at a rate of 5 ml/s, 50 ml in total, was administered, and the scan continued for 60 s, encompassing 20 cycles and yielding 640 slices. Upon completion of the scan, the images were automatically transferred to the United Imaging AI post-processing workstation (uai_pacs). All scan data were transferred to the uai_pacs post-processing workstation, and the Stroke Protocol within the CT Brain Perfusion Analysis software was employed for processing and analysis. The software automatically generated pseudo-color maps of cerebral perfusion based on the maximum lesion level for assessment. The regions of interest included the core infarction zone and the ischemic penumbra. The software automatically matched the mirrored zones on the healthy side, obtaining related data and pseudo-color maps, encompassing cerebral blood flow (CBF), cerebral blood volume (CBV), mean transit time (MTT), time to peak (TTP), ischemic penumbra (IP) volume, and other parameters related to perfusion, thereby providing perfusion color maps.

Functional connectivity by magnetic resonance imaging (MRI)

We will use a 3-T MRI scanner (Siemens Skyra, Erlangen, Germany), along with a circular surface coil to investigate changes in the brain. This will include evaluations of gray matter density, cortical thickness, subcortical nuclei volumes, and functional connectivity. Each participant will undergo an MRI scan lasting approximately 25 min. During the scan, they will have their eyes closed and extra padding will be placed around their ears to reduce noise interference. We will analyze changes in neural network connectivity using human connectomics. By integrating intelligent imaging technology with multimodal imaging data, including T1, DTI, and BOLD functional MRI, we can compare alterations in brain fiber connectivity patterns and changes in brain functional regions before and after treatment. This approach allows us to visualize and quantify functional connections between brain regions, alterations in the number of nerve fiber bundles, changes in the direction of nerve fiber bundle pathways, and changes in the volume of functionally abnormal regions.

Gamma oscillation entrainment via electroencephalogram (EEG)

In this study, a 32-channel scalp EEG signal recording system (Brain Amp MR PLUS, Brain Products, Germany) with a sampling rate of 500 Hz will be utilized. The electrode positions will be based on the international 10/20 system, with CPz as the reference electrode and AFz as the ground electrode. Prior to the experiments, the impedance of all electrode channels will be adjusted to below 20 kΩ. Preprocessing of data from all subjects will be done using the Makoto pipeline and EEG Lab toolbox in MATLAB. We will assess gamma oscillation entrainment by calculating changes in power spectral density before, during, and after the intervention using custom MATLAB functions.

RNA sequencing of the blood sample

Every participant will have a 1-mL blood sample taken from a vein by a trained nurse both at the start of the study and 2 weeks after the intervention period. This blood collection will allow us to assess changes in neuroinflammatory factors, nerve growth factors, and genes related to synaptic plasticity in the blood. Prior to the blood draw, participants will be advised to refrain from eating after 8 pm the previous day, as well as drinking and engaging in intense exercise. Breakfast will also be prohibited on the day of the blood draw. Once they have emptied their bladder, they will proceed to a laboratory room where a 1-mL venous blood sample will be collected into an EDTA anticoagulant tube. To this, 3 mL of TRIzol Reagent (LMAl Bio, China) will be added, ensuring thorough mixing. The mixture will then be incubated at 25 °C for 5 min and stored at − 80 °C. This rigorous process maintains the integrity and stability of the blood samples for subsequent biochemical analysis.

Participant timeline

Time schedule of enrolment, interventions, assessments, and visits for participants is shown in Table 1.

Sample size

The sample size calculation was conducted using G*power software (v3.1.9.2). The effect size of this study was estimated from a study conducted by Kaux et al. [29] who investigated the effects of transcranial direct current stimulation associated with physical therapy in acute stroke patients evaluated by the upper extremity section of the Fugl-Meyer Test, which was determined to be 0.995. The experiment employs a 1:1 simple randomization, n1 = n2. According to a prior difference between two independent means analysis t test, with a power of 0.95 and an error probability of 0.05, we applied these values to compute a sample size of n = 28. Accounting for a 10% attrition rate, we determined that each group should comprise 31 participants, yielding a total of 62 stroke patients included in the study. It is important to note that all enrolled patients received clinical medications and conventional rehabilitation treatments that were generally uniform.

Recruitment

The project is based at the Department of Neurology and Neurorehabilitation of the Seventh People’s Hospital affiliated with Shanghai University of Traditional Chinese Medicine. This hospital is a national comprehensive stroke and a member of the national neurology clinical research centre. It possesses outstanding capabilities in acute stroke management and post-stroke functional rehabilitation. Over the past 3 years, it has treated more than 1000 cases of stroke, guaranteeing the enrolment of participants.