Revelation of the dynamic progression of hypoxia-reoxygenation injury by visualization of the lysosomal hydrogen peroxide.

Hydrogen peroxide (HO) plays an important role in pathological conditions, such as cerebral ischemia-reperfusion (I-R) injury. Fluorescent probes may serve as valuable tools to detect the amount, temporal and spatial distribution of HO in living cells. To investigate the role of lysosomal HO involved in cerebral I-R injury, we designed and synthesized a lysosome-targetable two-photon fluorescent probe ztl-4, through expansion and substitution of the original pyridazinone scaffold, conjugation of electronic-donating aromatic ring and precise terminal modification of the alkyl linker. The probe ztl-4 exhibited fast, sensitive and highly selective response toward HO. ztl-4 could image exogenous HO in SH-SY5Y cells and brain slices. In addition, ztl-4 was located in lysosomes with high colocalization coefficient compared with LysoTracker. ztl-4 was further applied for detecting the endogenous generation of HO in SH-SY5Y cells subjected to oxygen and glucose deprivation (OGD) or OGD/reoxygenation (OGD/R) injury. Both OGD- and OGD/R-induced cell injury caused a time-dependent increase of HO production within lysosomes. Moreover, OGD/R-treated cells showed much more amount of HO than OGD-treated cells, indicating that reoxygenation will promote HO accumulation in lysosomes of post-hypoxia cells. Therefore, the probe is suitable for monitoring the dynamic changes of lysosomal HO in cells.