Near-Infrared Fluorescent Probe for the In Situ Visualization of Oxidative Stress in the Brains of Neuroinflammatory and Schizophrenic Mice

Schizophrenia is a common mental disorder with unclear mechanisms. Oxidative stress and neuroinflammation play important roles in the pathological process of schizophrenia. Superoxide anion (O2•–) is an important oxidative stress biomarker in vivo. However, due to the existence of the blood–brain barrier (BBB), few near-infrared (NIR) fluorescent probes have been used for the sensing and detection of O2•– in the brain. With this research, we developed the first near-infrared fluorescent probe (named CT–CF3) for noninvasive detection of endogenous O2•– in the brain of mice. Enabling fluorescence monitoring of the dynamic changes in O2•– flux due to the prolonged activation of microglia in neuroinflamed and schizophrenic (SZ) mice brains, thereby providing direct evidence for the relationship between oxidative stress, neuroinflammation, and schizophrenia. Furthermore, we confirmed the O2•– burst in the brains of first-episode schizophrenic mice and assessed the effect of two atypical antipsychotic drugs (risperidone and olanzapine) on redox homeostasis.


Materials and instruments
All raw materials are analytical grade and are used directly unless otherwise stated.
All ethanol in the synthesis step are further dried. Schizophrenia stimulating drugs MK-801(+) was purchased from Bide Pharmatech Ltd (China). O2 •− was produced from KO2 in dry DMSO by an ultrasonic method. The concentration of O2 •− was determined from the absorption at 250 nm (ε = 2682 M -1 cm -1 ). The preparation of reactive oxygen species and reactive nitrogen species refers to the methods reported previously. 1 UV-vis absorption and fluorescence spectra were obtained on HITACHI U-3900H and HITACHI F-4600 spectrophotometers, respectively. The cells used were purchased from China Cell Bank. The MTT cytotoxicity test kit was purchased from Solarbio. In vitro imaging was carried out using a laser scanning confocal microscope (Leica SP5). In vivo imaging was conducted using an IVIS Spectrum Imaging System.

General Procedure for Fluorescence Detection
We prepared different concentrations of O2 •and CT-CF3 in DMSO solutions (300 μL).

In vitro cytotoxicity assay
The cytotoxicity of the CT-CF3 was tested by the MTT (methyl thiazolyl tetrazolium) method. In a humid environment of 37°C and 5% CO2, PC-12 cells were incubated in high sugar Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum. Subsequently, the cells were collected and planted into 98-well plates.
After 24 h of incubation, the original medium was removed, and the cells containing CT-CF3 (0, 2, 5, 10, 15 and 20 μM) serum-free DMEM was added to the plate. After 24 h of co-incubation, 50 μL of MTT was added to each well to interact with live cells to produce metsan. After another 4 h incubation, the old medium was removed and 150 μL of dimethyl sulfoxide (DMSO) was added to each well and placed on a S3 shaking incubator for 10 min. Finally, the cell viability was determined by measuring the ultraviolet absorption value in different well plates by a microplate reader.

Hemolysis experiment
Take 1mL of mouse blood, add 1mL PBS and dilute to 2 mL. The sample was then centrifuged at 8000 rpm for 10 min. After removing the supernatant, add PBS again to 2 mL for 10 min (8000 rpm). Repeat this process 5 times to obtain blood cells from the serum. Subsequently, the blood cells were diluted into 10 mL PBS, and each time 0.2 mL of blood cell solution was taken out and mixed with 0.8 mL distilled water, PBS and 0.8 mL CT-CF3 gradient solutions (10, 20, 50, 100, 150, 200 μM) let stand for 3 h at room temperature. Finally, centrifuge (12000 rpm, 5 min) and aspirate the supernatant, and tested its ultraviolet absorption (Abs) by a microplate reader. Use the following formula to calculate the hemolysis rate (HR) of red blood cells:
We determined the BBB penetration rate of CT-CF3 by High-performance liquid chromatography (HPLC). The instrument for HPLC is Agilent.1260 infinity ll, and Poroshell 120 column (4 μm C18, 4.6 mm × 150). A soulution of CT-CF3 (0.5 mg/kg, in 3:7 DMSO/PBS) was inject into C57BL/6J mice (n=3, 15-18 g, 7 weeks, female) through intravenous injection. The mice were sacrificed and dissected at 5 min. Brain samples (The weight of the brain = 0.42 g  0.2 g) were removed, weighted and homogenized with 1.0 mL acetonitrile and then the leftover homogenate was extracted with 1.0 mL acetonitrile for twice, the total volume of acetonitrile was 3.0 mL. The brain tissues of all three mice were extracted in the same way. The extracted acetonitrile was filtered by flashing nylon membrane (0.22 μm) to analyze by HPLC.
We mapped the standard curve of CT-CF3 by HPLC, and determined the Cprobe by the standard curve. Quantitative analysis was derived from peak area and the brain uptake was presented by % injected dose per gram (% ID/g). The calculation formula was shown below.

S4
Where mprobe_inject means the quality of injection. mbrain means the wet weight of brain.
mprobe_brain means the quality of probe in brain, which was calculated according to the fomula below.
Where Cprobe was calculated by the peak area according to the standard curve, V means volume of extract (3 mL).

Prediction of the molecular lipid water partition coefficient (c Log P)
The c Log P values were calculated using the online ALOGPS 2.1 program.

Preparation of LPS-Induced neuroinflammation mouse model and fluorescence imaging
Mouse models of neuroinflammation were prepared according to previously reported methods. 2 7-weeks-old male C57BL/6J mice (n =12) that were supplied by Beijing HFK BIOSCIENCE Co., Ltd. On the eighth day, one mouse was randomly selected from each of the three groups, and were simultaneously injected with CT-CF3 (0.5 mg/kg, in 3:7 DMSO/PBS, v/v) through the tail vein, and then immediately placed in the IVIS Spectrum imaging system with an excitation filter of 500 nm and the collection wavelength range is from 640-720 nm.
After the imaging experiments were completed, the mice were sacrificed by cervical dislocation. Immediately dissect out the brain, heart, liver, spleen, lung, kidney and other tissues for fluorescence imaging. Subsequently, they were placed in a 10% formalin solution for paraffin embedding and tissue sectioning.

Detection of pro-inflammatory cytokines
Brain tissue sampling was performed one day after the end of administration. Three mice from different groups were randomly selected and sacrificed with CO2, and the brain tissue was removed on an ice bath. Subsequently, the blood was quickly washed with PBS (4°C, pH = 7.4) solution and the surface water of the brain tissue was blotted dry and placed in a sterile 1.5 mL EP tube. Immediately, perform liquid

Paraffin Section Immunofluorescence
The paraffin section immunofluorescence experimental procedure is divided into 9

Preparation of schizophrenia-first-episode mouse model and fluorescence imaging
Mouse models of schizophrenia were prepared according to previously reported methods. The nonecompetitive NMDA receptor antagonist dizocilpine hydrogen maleate (MK-801) was used to make a mouse model of schizophrenia, which was a widely used schizophrenia stimulating drug. 3 Low-dose and long-term repeated administration of MK-801 can induce long-lasting cognitive deficits and changes in brain nerve structure in adolescent or adult rats. 4 6-weeks-old male C57BL/6J mice (n =12) that were supplied by Beijing HFK BIOSCIENCE Co., Ltd. On the fifteenth day, one mouse was randomly selected from each of the three groups, and were simultaneously injected with CT-CF3 (0.5 mg/kg, in 3:7 DMSO/PBS, v/v) through the tail vein, and then immediately placed in the IVIS Spectrum imaging system with an excitation filter of 500 nm and the collection wavelength range is from 640-720 nm.
After the imaging experiments were completed, the mice were sacrificed by cervical dislocation. Immediately, brain, heart, liver, spleen, lung, kidney and other tissues were dissected out for fluorescence imaging. Various types of tissues were placed in 10% formalin solution and further used for paraffin embedding and tissue sectioning.
We prepared mice of schizophrenia-first-episode with a single injection of MK-801 into healthy adolescent mice. 6-weeks-old male C57BL/6J mice (n =12) that were supplied by Beijing HFK BIOSCIENCE Co., Ltd., were randomly divided into four   Absorption and fluorescence can be used for the study of the pKa of small molecule compounds ( Figure S3). The pKa values were studied by testing the fluorescence intensity of three fluorophores (CN-OH, CF-OH and CT-OH) at 665 nm in different pH solutions, which were 9.07, 7.41 and 5.61 respectively. There was no significant difference in the pKa obtained using absorption or fluorescence.    Figure S14 H&E staining of major organs including heart, liver, spleen, lung, and kidney from three mice. The liver, spleen and kidney of the experimental group (LPS) were damaged.
Compared to the control group, there were obvious liver damages in the experimental group (LPS): More congestion and hemorrhage spots appear in the liver tissue in the yellow box. Figure S15 (A) NIR Fluorescence images of isolated organs of the three mice. (B) Quantification of fluorescence intensity in isolated organs of three mice. Figure S16 The representative immunofluorescence photographs of microglia in the cortex region.

S17
Iba-1-positive microglia (green) and DAPI-granule cells (blue). Figure S18 H&E staining of major organs including heart, liver, spleen, lung, and kidney from three mice. Compared to the control group, there were obvious liver and kidney damages in the treatment group: significant congestion and hemorrhage appear in the liver and kidney (black arrow).    Figure S24 1 H NMR spectrum of CT-CF3 in CDCl3. Figure S25 13 C NMR spectrum of CT-CF3 in CDCl3. Figure S26 MS spectrum of CN-OH. Figure S27 MS spectrum of CF-OH. Figure S28 HRMS spectrum of CT-OH. Figure S29 HRMS spectrum of CT-CF3.