Unraveling Spatiotemporal Metabolic Perturbation of Amino Acids Associated with Ischemia-Reperfusion Injury by MALDI...

Various complex metabolic perturbations are involved in cerebral ischemia-reperfusion (I/R) injury. However, limited data have been reported on dynamic spatiotemporal metabolic perturbations of amino acids (AAs) in I/R injured rat brains. In this work, a combination of laser-assisted chemical transfer (LACT) and hexafluoroisopropanol (HFIP) was applied to the enhancement of on-tissue derivatization of AAs for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) of their dynamic spatiotemporal changes during I/R injury. The spatiotemporal distribution patterns of 15 derivatized AAs in the rat subjected to middle cerebral artery occlusion (MCAO) followed by different periods of reperfusion were unraveled by using MALDI MSI, including excitatory amino acids (e.g., Glu, Asp), inhibitory amino acids (e.g., GABA, Gly) and the other amino acids (e.g., Val, Pro, Gln, Phe). The metabolic perturbations of AAs showed similarities and differences in cerebral cortex (CTX) and striatum (STR) of MCAO rat brains at different periods of I/R injury. Generally, in 0.5-1.5 h of ischemia followed by 1-6 h of reperfusion, I/R triggered a dramatic increase in the levels of some AAs such as Glu, Asp, Gly, GABA, Gln, and Phe in both CTX and STR. After 6 h of reperfusion, AA levels gradually decreased. Immunohistochemistry (IHC) assay further showed that the immunoreactivities of selected enzymes related to Glu and GABA metabolism increased at the later stage of reperfusion. This present work provided spatiotemporal metabolic perturbations of AAs, which will further help us understand the mechanisms of I/R injury and also have great significance in developing region-specific therapeutic drugs and diagnostic markers.