Imaging Dynamic Peroxynitrite Fluxes in Epileptic Brains with a Near‐Infrared Fluorescent Probe

Abstract Epilepsy is a chronic neurodegenerative disease, and accumulating evidence suggests its pathological progression is closely associated with peroxynitrite (ONOO−). However, understanding the function remains challenging due to a lack of in vivo imaging probes for ONOO− determination in epileptic brains. Here, the first near‐infrared imaging probe (named ONP) is presented for tracking endogenous ONOO− in brains of kainate‐induced epileptic seizures with high sensitivity and selectivity. Using this probe, the dynamic changes of endogenous ONOO− fluxes in epileptic brains are effectively monitored with excellent temporal and spatial resolution. In vivo visualization and in situ imaging of hippocampal regions clearly reveal that a higher concentration of ONOO− in the epileptic brains associates with severe neuronal damage and epileptogenesis; curcumin administration can eliminate excessively increased ONOO−, further effectively protecting neuronal cells. Moreover, by combining high‐content analysis and ONP, a high‐throughput screening method for antiepileptic inhibitors is constructed, which provides a rapid imaging/screening approach for understanding epilepsy pathology and accelerating antiseizure therapeutic discovery.


General materials and experimental methods
All commercially available compounds were used as provided without further purifications.
Chemicals and solvents were purchased from the companies Sigma Aldrich, Aladdin, Bepharm, etc. TLC analysis was performed on silica gel plates and chromatographic purification of products was performed on silica gel (300-400 mesh). 1 H NMR, 13 C NMR were recorded on a Bruker DRX400 (400 MHz), using CDCl 3 , and DMSO-d 6  UV absorption spectra were accomplished on a PerkinElmer 650 spectrophotometer (PerkinElmer Ltd., US). All fluorescence measurements were recorded at room temperature with a FLS-980 fluorescence spectrometer (Edinburgh Instruments Ltd., England) or F-4600 spectrophotometer (HITACHI, Japan). HPLC spectra were performed on an AGILENT 1200 system (AGILENT Co., Ltd., US). The imaging experiments were carried out using a confocal fluorescent microscope (Leica TCS SP8 MP, Nanjing University).

Synthesis and characterization of ONP
Scheme S1. The chemical synthesis route of ONP.
A mixture of methylene blue (374 mg, 1 mmol), 10 mL DCM, and 10 mL water were stirred in a 50 mL round-bottom flask under argon atmosphere. Na 2 S 2 O 4 (525 mg, 1.5 mmol) and NaHCO 3 (168 mg, 2 mmol) were slowly added to the stirred solution. The mixture was then stirred for 20 minutes until the aqueous phase had turned to yellow. The organic layer was separated and the aqueous layer was extracted with dichloromethane (2 X 5 mL (σ), we took 20 data points to obtain the average value before treatment with ONOO -.
Where y i is the average value from calculation and y is the measured data point. The σ noise is calculated as Where N is the number of data points used for the average value. The σ noise of ONP is 50.65.
According to the IUPAC definition, when the signal-to-noise ratio equals 3 (S/N=3), the signal is considered to be a true signal. Thus, the limit of detection (LOD) of ONP can be extrapolated from the linear calibration curve when the signal equals three times of the noise.
The linear regression curve was then fitted according to the data in the range of ONOOfrom 0 to 4 μM and obtained the slope of the curve (1.6235*10 9 M -1 ). The detection limit (3σ/slope) was then determined to be 93. 6

The measurement of lipophilicity
Lipophilicity was presented as log P o/w values, which were determined by the flask-shaking method. An aliquot of a stock solution of the sample in aqueous NaCl (0.9% w/v and saturated with octanol) was added to an equal volume of octanol (saturated with 0.9% NaCl, w/v), and the mixture was shaken overnight at 60 rpm to allow partitioning at 298 K. After the sample was centrifuged at 3000 rpm for 10 min, the probe content of the organic and aqueous phases was determined by UV absorbance (254 nm). Log P was defined as the logarithmic ratio of probe concentrations in the organic and aqueous phase.

HPLC analysis
HPLC analysis of ONP after incubation without or with ONOOat 310 K was performed on an AGILENT 1200 system (AGILENT Co. Ltd., US). The reaction mixture was analyzed with the detection wavelength at 665 nm (for the product of MB) and 254 nm (for ONP), respectively. Running conditions were as follows: mobile phase composition was MeCN/H 2 O: 65/35 (0.1% TFA); temperature of 303 K; Agilent RP-C8 column of 4.6*180mm; and flow rate of 1 mL/min.

Cytotoxicity assay
The cytotoxic effect of ONP and MB were evaluated by employing Cell Counting Kit-8 assay

Screening by high-content analysis
Live SH-SY5Y cells were pretreated with various anticancer agents (20 µM) or different antioxidants (20 µM) for 1 h, cells were then incubated with ONP (10 µM) for another 30 min.
High-content analysis (HCA) was performed after washing with PBS three times. Images and quantitative analysis were used an HCA with an excitation filter of 630 nm and the collection wavelength range is from 680-730 nm.

Frozen sectioning
For the preparation of brain sections, mice were deeply anesthetized with isoflurane and sacrificed. The fresh brain was taken out and directly frozen at liquid nitrogen. Optimal

Paraffin section and HE staining imaging
Brain tissues stored at -80 ºC were removed directly and post-fixed in freshly prepared 4%  Table S1. A comparison of ONP with the reported ONOOprobes.            Figure 4C was quantified using the IVIS Spectrum imaging system.