Indication of Dynamic Peroxynitrite Fluctuations in the Rat Epilepsy Model with a Near-Infrared Two-Photon Fluorescent Probe

Epilepsy is a chronic neurodegenerative disease that has seriously threatened human health. Accumulating evidence reveals that the pathological progression of epilepsy is closely related to peroxynitrite (ONOO – ). Unfortunately, understanding the physiological roles of ONOO – in epilepsy is still challenging due to the lack of powerful imaging probes for the determination of the level of fluctuations of ONOO – in the epileptic brain. Herein, a near-infrared (NIR) two-photon (TP) fluorescent probe [dicyanomethylene-4 H -pyran (DCM)–ONOO] is presented to trace ONOO – in living cells and in kainate (KA)-induced rat epilepsy models with satisfactory sensitivity and selectivity. The probe is composed of a NIR TP DCM fluorophore and a recognition moiety diphenylphosphinamide. The phosphoramide bond of the probe is interrupted after reacting with ONOO – for 10 min, and then, the released amino groups emit strong fluorescence due to the restoration of the intramolecular charge transfer process. The probe can effectively detect the changes of endogenous ONOO – with excellent temporal and spatial resolution in living cells and in rat epileptic brain. The imaging results demonstrate that the increasing level of ONOO – is closely associated with epilepsy and severe neuronal damage in the brain under KA stimulation. In addition, the low-dose resveratrol can effectively inhibit ONOO – overexpression and further relieve neuronal damage. With the assistance of TP fluorescence imaging in the epileptic brain tissue, we hypothesize that the abnormal levels of ONOO – may serve as a potential indicator for the diagnosis of epilepsy. The TP fluorescence imaging based on DCM–ONOO provides a great potential approach for understanding the epilepsy pathology and diagnosis.

癫痫是一种严重威胁人类健康的慢性神经退行性疾病。越来越多的证据表明，癫痫的病理进展与过氧亚硝酸盐（ONOO – ）密切相关。不幸的是，由于缺乏强大的成像探针来确定癫痫大脑中 ONOO 的波动水平，了解 ONOO 在癫痫中的生理作用仍然具有挑战性。在此，提出了一种近红外（NIR）双光子（TP）荧光探针[二氰亚甲基-4H-吡喃（DCM）-ONOO]，用于在活细胞和红藻氨酸（KA）诱导的大鼠癫痫模型中追踪ONOO，具有令人满意的灵敏度和选择性。该探针由 NIR TP DCM 荧光团和识别部分二苯基膦酰胺组成。与ONOO反应10分钟后，探针的磷酰胺键被中断，然后由于分子内电荷转移过程的恢复，释放出的氨基发出强烈的荧光。该探针可以有效检测活细胞和大鼠癫痫脑中内源性 ONOO 的变化，具有出色的时间和空间分辨率。成像结果表明，ONOO – 水平的升高与 KA 刺激下大脑中的癫痫和严重神经元损伤密切相关。此外，低剂量白藜芦醇可有效抑制ONOO-过度表达，进一步缓解神经元损伤。借助癫痫脑组织中的TP荧光成像，我们假设ONOO – 的异常水平可能作为癫痫诊断的潜在指标。基于 DCM-ONOO 的 TP 荧光成像为了解癫痫病理和诊断提供了一种巨大的潜在方法。