Animal Surgery
Twelve healthy BALB/c mice (all males, weight between 20–25 g, Beijing Life River Laboratory Animal Technology Co., Ltd. China) were used in this study. All procedures were performed under institutional review and approved by the Animal Ethics and Management Committee of Yanshan University.
Mice were randomly divided into two groups: Sham group (n = 6) and TUS group (n = 6). After anesthetized with 2% isoflurane, the mice were fixed in a stereotaxic apparatus (68002, 68030, RWD Co., China), then 1.2% isoflurane were given continuous by a face mask. Then, the fur covering the neck was shaved, and the skin was washed with 0.9% saline solution.
The skin was incised along the midline and the anterior fontanelle, posterior fontanelle, herringbone suture and sagittal suture of the skull, the subcutaneous tissue and periosteum was cleaned to facilitate the ultrasound intervention. Homemade microfilament electrodes were inserted into the neck of the mice in a standard way to facilitate electromyogram (EMG) of muscles acquisition, and a cranial drill (78001, Warroad, China) was used to perforate the skull of the mice at the sagittal suture. Three electrodes were applied for local field potential (LFP) acquisition in mice, one microfilament electrode was inserted into the motor cortex of the brain (coordinates: anteroposterior (AP) = 1 mm, mediolateral (ML) = 1.5 mm, dorsoventral (DV) = − 1 mm); the other two electrodes were respectively ground and reference electrodes, located 5 mm above the sagittal suture in mice. After completion, electrodes were fixed using glue and dental cement.
After 24 hours from the surgery, the LFP and EMG signals were recorded before the administration of propofol. Then propofol (Diprivan, Aspen Pharma Trading Limited, Ireland) 70 mg/kg was administered by intraperitoneal injection and, these mice of TUS group were stimulated with ultrasound on the motor cortex after the administration of propofol.
Litus Protocol
LITUS Protocol
The pulsed signal generated by two arbitrarily connected function generators (AFG3022C, Tektronix, USA) was amplified by a linear power amplifier. An unfocused ultrasound transducer (V301-SU, Olympus, USA) was used in our experiment (Fig. 1). An ultrasound transducer with a fundamental frequency of 500 kHz was used in the experiment, in addition, a 3D printed conical collimator filled with US coupling gel was used to connect the mouse head with the transducer in order to reduce the absorption and distortion of the sound waves, at a 45° angle to the electrodes. The stimulation duration, pulse repetition frequency and duty cycle were set as 50ms, 1Hz, 5%, respectively. The ultrasound pressure under the skull measured by a calibrated needle-type hydrophone (HNR500; Onda, USA) in the water tank is 0.51MPa and its corresponding Isppa and Ispta are 8.67 W/cm2 and 0.43 W/cm2. We determined the placement of the ultrasound transducer and coupling cone based on the mouse’s brain atlas and the distribution map of ultrasound field [14]. It can ensure that the ultrasound was targeted to the motor cortex. The total time for each stimulation was 5 minutes.
Data Acquisition
The data was sent to the neural signal processor (Cerebus Data Acquisition System, Blackrock Microsystems, USA), through a pre-amplifier (63386, A-M Systems Inc., USA), which were transmitted to a computer for data storage and processing.
Power Spectrum Analysis And Time-frequency Diagram And Sample Entropy
We divided the data into several groups (Pre-Anes, Post-Anes, tus-5min / 10min / 15min / 20min). The LFP signals with one minute after the starting time of each group were used to calculate the power. The power spectrum of LFPs was analyzed using the Welch algorithm [19]. We performed 1-200hz filtering and power frequency removal (50Hz) processing on the LFP data and 300-1000Hz filtering on the EMG data. The mean absolute power was obtained and analyzed from these different frequency bands of [1–4 Hz], [4–12 Hz], [13–30 Hz], [30–45 Hz], [55-100Hz], [100-140Hz] and [140-200Hz]. The total absolute power of frequency bands was obtained from the band (1-200Hz) by summing the absolute power of the above all frequency bands. The relative power of each frequency band was equal to the corresponding absolute power dividing by the total absolute power. The time-frequency diagram was calculated using short-time Fourier transforms with a hamming window. The Sample entropy of the signals in different frequency bands based on the literature [20]. The Sample entropy can be expressed by the following equation:
$$SampEn(m,r,N)=-\text{ln}\left[{C}^{m+1}\right(r)/{C}^{m}(r\left)\right]$$
1
where N is the length of data, m is the vector dimension, and r is the tolerance.
Statistical Analysis
The SPSS 21.0 statistical software was used for statistical analysis. Continuous variables were presented as the mean ± SEM. The results at the statues of different times were evaluated with one-way ANOVA using multiple comparisons (least significant difference (LSD)). Differences were considered significant when P < 0.05.