1) Pre-clinical studies
Subjects and Drugs
Subjects
All experimental procedures were performed in accordance with the guidelines of the Animal Experiment Ethics Committees of …………………….. Timed-pregnant Balb/c mice were purchased from ……………………... They were housed in a temperature and humidity-controlled room (23 ± 1℃, 45–55%) of 12-hour light/dark cycle (lights on at 08:00 a.m.) with free access to water and food. The day of birth was designated as the postnatal day 0 (P0). On the P7, they (weighing 4–6 g) were randomly allocated into three groups: control group (Ctrl group), MK801 group, and MK801 + Sevoflurane group (MK801 + Sev group). At the time of weaning (P21), mice were separated from their mothers and housed in group of four to six per cage.
Drugs administration
MK801 (Ref. M107, St. Louis, MO, USA) was used to induce negative symptoms of schizophrenia as reported previously 43. It was dissolved in 0.9% sterile saline. At the P7, pups received intraperitoneal (i.p.) injection of 0.5 mg/kg MK801 in the MK801group and MK801 + Sev group or an equal volume of saline (Ctrol group) twice a day (at 10:00 am and 15:00 pm) for five consecutive days. Their body weights were measured every day during the treatment period and once/week since cessation of the treatments till the P31. During the P31-P35, the MK801 + Sev group mice were placed in a 25 cm x 20 cm x 15 cm plexiglass chamber, and 1% sevoflurane was delivered via inlet of the chamber via a sevoflurane vaporizer (Easy fill / Cagemount, R58S, ……………………..) at 1 L/min in 30% oxygen balanced with nitrogen for 1hr (10:00 am to 11 am) for five consecutive days. The concentration of sevoflurane was monitored with a gas monitor (BeneView T8, Mindray Bio-Medical Electronics Co.Ltd., ……………………..) via the outlet of the chamber. The MK801 and control groups received identical gases without sevoflurane under the identical setting for 1hr for 5 days. Open field test was performed at the P43-P45, and then three-chamber social test was conducted after two non-stimuli days at the P47. After behavioral tests (see below), they were sacrificed under terminated anesthesia and their brain samples were harvested for electrophysiological recordings, western blot and immunohistochemical analysis, respectively. The experimental timeline is presented in Appendix: Supplementary Fig. 1.
Behavioral test
In order to avoid possible behavioral testing biases, mice were tested in a random order and all trails were carried out in the day at the same time period (9:00–16:00). In addition, the apparatus was cleaned with 70% ethanol after each trail to eliminate any effects from olfactory perception.
Open field test (OFT)
Open field test was adopted for assessing spontaneous locomotor activity. Mice were gently placed in the center of an open-top apparatus with 25 equal squares (50 cm × 50 cm × 40 cm) and allowed to move freely for 15 min. Distances and traces of 15 min movements were recorded with a video camera and analyzed using EthoVision XT 8.0 (Noldus, Wageningen, Netherlands), the total distance travelled and the number of squares crossed during recording period were calculated for further analysis.
Three-chamber sociability test
The three-chamber test was conducted to assess social interaction with time spent in a side chamber with a novel mouse in a wire cup versus time spent in a side chamber with an empty identical wire cup46. A rectangular plexiglass box (60 cm X 40 cm X 20 cm) without a top cover was divided into three chambers (20 cm X 40 cm X 20 cm) with two partitions. Between each chamber, there is a 5-cm opening hole which can be closed or opened with a lever operated door. The middle chamber was empty, while the side chamber contained an empty wire cup or an identical wire cup with a novel sex- and weight-matched mouse in. The test was divided into two phases. In brief, after a ten-minute habituation period (phase 1), the test mouse was placed in the middle chamber and allowed free access to visit each chamber. For the social approach phase (phase 2), the mouse was placed in the central compartment and left to explore with either an empty black wire cup (in left side chamber) or a similar wire cup with a novel mouse inside (the right side chamber). The apparatus and wire cups were thoroughly cleaned with 70% ethanol between phases and after each trial. Behaviors were videotaped by an automated tracking software (TopScan/ObjectScan, Cleversystems, Leesburg, VA, USA). The time spent in each compartment, and the time explored (sniffing) within a 2 cm vicinity of the cup with a novel mouse in was calculated to assess sociability.
Electrophysiological recording
Slice preparation
Slices of prefrontal cortex were prepared as previously described47. In brief, coronal brain slices (250 µm) containing the PFC were cut with a vibratome (VT120S, Leica Microsystems) in ice cold solution containing (in mM): 75 sucrose, 85 NaCl, 2.5 KCl, 1.25 NaH2PO4, 4 MgSO4, 0.5 CaCl2, 24 NaHCO3, 25 D-glucose saturated with 95% O2/5% CO2, and recovered in a chamber filled with artificial cerebrospinal fluid (ACSF) (in mM: 119 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 MgCl2, 2 CaCl2, 26 NaHCO3, 10 D-glucose) saturated with 95% O2 and 5% CO2 at 32 ◦C before recording.
Whole-cell recording
After incubation, slices were transferred to a recording chamber where perfused at 2–3 ml/min with ACSF, saturated with 95% O2 and 5% CO2 and maintained at 32 ± 2 °C. The pyramidal neurons of PFC layer II/III were viewed with an Olympus microscope equipped with infrared DIC optics. The patch recording pipettes (4–6 MΩ) were filled with a solution (in mM: 135 cesium methanesulfonate, 8 CsCl, 0.25 EGTA, 10 HEPES, 7 Na2-phosphocreatine, 0.34 Na3-GTP, 2.168 Mg-ATP, pH = 7.2–7.3, with CsOH) and access resistance of recorded cells was less than 30 MΩ. mEPSCs /mIPSCs were recorded at holding potential of -70 mV/0 mV in ACSF supplemented with 1 µM tetrodotoxin. Only cells with membrane potentials lesser than − 65 mV and series resistance below 25 MΩ were included for further analysis. Cells were excluded if input resistance changed 20% over the entire experiment. Whole cell recordings were conducted by using Multiclamp 700B amplifier (Axon Instruments). All data were collected with 2 kHZ Bessel filter at a 10 kHz sampling frequency (DigiDATA 1550A, Axon Insteuments), and analyzed by Clampfit 10.0 software (Axon Instruments) following low-pass filtering at 1000 Hz. The synaptic response belonging to the inhibitory and excitatory amplitude was used to determine the E/I ratio (mEPSC amplitude/mIPSC amplitude) of the recorded pyramidal neuron.
Western blot
PFC samples (n = 6/group) were harvested for western blot analysis and dissociated in lysis buffer (containing protease inhibitors, 50 mM Tris–HCl, pH 7.6) on ice for 30 min and homogenized via ultrasonification (Ningbo scientz biotechnology CO. LTD, Ningbo, China). After centrifugation at 12,000 g for 10 min at 4 °C, supernatants were collected and protein concentrations were measured with a BCA assay kit (Beyotime Institute of Biotechnology, China). Equivalent amounts of protein samples mixed with gel loading buffer (50 mM Tris-HCl, 10% SDS, 10% glycerol, 10% 2-mercaptoethanol, 2 mg/ml bromophenol blue) were boiled for 5 min and loaded to SDS-PAGE gels. The separated proteins were electrophoretically transferred to polyvinylidene difluoride membranes. Then the membranes were incubated in blocking buffer (5% fat-free milk in Tween20) for 2hrs at room temperature and probed with primary antibodies: Rabbit anti-NR2A(1:1000, Cat.No.MAB1555P, Millipore, USA), Rabbit anti-NR2B (1:1000, Cat.No.MAB1557P, Millipore, USA), Rabbit anti-GABAAα1 (1:1000, Cat.No.224203, Synaptic Systems, Germany), Rabbit anti-GABAAβ2 (1:1000, Cat.No.224803, Synaptic Systems, Germany), Mouse anti-SY38 (1:1000, Cat.No.Ab8049, Abcam, UK), Mouse anti-PSD95 (1:1000, Cat.No.MAB1596, Millipore, USA), Rabbit anti-ErbB4 (1:1000, Cat.No.05-1133, Millipore, USA), Mouse anti-NRG1 (1:1000, Cat.No.sc-393006, Santa Cruz Biotechnology, USA) Rabbit anti-GAPDH (1:5000, Cat.No.5174, Cell Signaling Technology, USA) and Rabbit anti-β-Tubulin (1:5000, Cat.No.2128, Cell Signaling Technology, USA) overnight at 4 °C. After washing in TBST for three times, the membranes were incubated with horseradish peroxidase (HRP) conjugated secondary antibodies diluted 1:5000 (Goat anti-Rabbit (1:5000, Cat.No.Ab6721, Abcam, UK) or Rabbit anti-Mouse(1:5000, Cat.No.Ab6728, Abcam, UK) for 2hrs at room temperature. The antigen-antibody complexes were detected by enhanced chemiluminescence system (Bio-rad) and visualized by a computer image system (GENE GNOME Chemiluminescence apparatus, Quantity one, Bio-Rad Laboratories). Image processing and semiquantification were performed with Image J software. Measurements were repeated independently at least 3 times for each experiment. Density of each band was normalized to the internal controls (β-tubulin or GAPDH).
Histology and immunohistochemistry
After antigen recovery, coronal paraffin sections (5 µm in thickness) were incubated with primary antibodies: mouse anti-parvalbumin (PV, 1:500, Millipore, Cat.No.MAB1572), or anti-GAD67 (1:1000, Millipore, Cat.No.MAB5406) at 4 °C. Signal was detected with Alexafluor 546-labeled fluorescent secondary antibodies (1:1000, Invitrogen, Carlsbad, CA, USA) for GAD67 positive cells, while signal for PV positive cells was detected with a mouse-rabbit ABC kit (PK-6200, Universal, Vector). Images were captured using camera system (SP8 and DMi8 DFC7000J, Leica, Germany). GAD67 and PV positive cells were calculated using Image J (NIH, USA) and expressed as cell numbers/mm2.
Statistical analyses
All data were presented as mean ± SEM, and statistical analyses were done with SPSS (SPSS Inc., Chicago, IL, USA). Treatment effects were statistically analyzed by one-way ANOVA followed by Bonferroni’s post hoc tests for comparison when normality (and homogeneity of variance) assumptions are satisfied, otherwise Kruskal-Wallis test was applied to analyze the differences between groups followed by Dunn’s multiple comparisons tests. Body weight was analyzed using a two-way ANOVA followed by Bonferroni’s post hoc tests. A p value < 0.05 was considered to be of statistical significance.
2) An open-labelled single arm trial
Patients’ recruitment
After the completion of pre-clinical experiments, we went to evaluate whether the therapy is effective in patients. An open-labelled single arm, proof-of-concept clinical trial was conducted (registered in ……………………..) from December 24, 2019, to January 20, 2020. After obtaining approval from Ethic committee of …………………….., and written informed consent from patient or family member, 10 schizophrenia patients (7 men and 3 women; age 18–65 years), who were taking antipsychotics and experiencing an acute exacerbation of psychosis and met all the inclusion criteria (appendix A: Table 1), were recruited (appendix B: Flowchart).
Clinical endpoints and outcome measures
The primary endpoint was the percentage of the early response at week 2, and the clinical response was defined as a minimum 30% reduction in Positive and Negative Syndrome Scale (PANSS) total score. The percent change of PANSS total score was defined as a change /(baseline − 30) x100% 48. The secondary endpoints were the change of Brief Psychiatric Rating Scale (BPRS-18) from the baseline to week 1 and week 2, and the early response rate at week 1. Safety and tolerability assessments included treatment-emergent adverse events (TEAEs), clinical laboratory tests, electrocardiograms and physiologic measures.
Trial protocol
Recruited participants received routine antipsychotic medications (in line with standard clinical guidelines; one or two antipsychotics of risperidone, paliperidone, aripiprazole and olanzapine). After fasting for 8hrs and establishing intravenous line, dexmedetomidine, a sedative, was intravenously administrated via an infusion pump with a loading dose of 0.5 µg/kg for 15 min and then maintained at the rate of 0.1–0.3 µg/kg/h. Sevoflurane was delivered at 2.5 L/min in 100% oxygen for 5hrs using a face mask under spontaneously respiration. Sevoflurane concentration was started with 6.0% for first 5-min duration to induce a quick sedation, and then gradually decreased to 0.5–1.5% and adjusted to maintain the deep sedation. Participants were closely monitored with electrocardiography, pulse oximetry, non-invasive blood pressure, bispectral index (BIS), end-tidal partial pressure of carbon dioxide and temperature throughout. This treatment was repeated for 6 times with intervals of 1–2 days for a total of two weeks (see the detailed protocol in the appendix C).
Statistical Analysis
Simon’s two stage design was used in this proof of concept study. Based on findings of an earlier study, the percentage of patients with at least 30% improvement in total PANSS at 2 weeks was about 30% 49. In this study, a 70% or higher early response rate was set as the target of “good” and 30% was set as “poor”. The optimal two-stage design to test the null hypothesis that P < = 0.300 versus the alternative that P > = 0.700 has an expected sample size of 6.08. After testing the drug on 2 patients in the first stage, the trial was planned to be terminated if 0 respond. If the trial goes on to the second stage, a total of 10 patients was planned to be studied. If the total number of patients responding is less than or equal to 5, the drug was rejected.
The efficacy and safety analysis were conducted in the full analysis set, which included all patients who received at least one treatment of sevoflurane inhalation and had at least one evaluation post-baseline. The early response of BPRS-18 total score at week 1 and week 2 was reported as mean with standard deviation.