Simultaneous monitoring of intracellular pH changes and hemodynamic response during cortical spreading depression by fluorescence-corrected multimodal optical imaging

Cortical spreading depression (CSD) plays an important role in trauma, migraine and ischemia. CSD could induce pronounced hemodynamic changes and the disturbance of pH homeostasis which has been postulated to contribute to cell death following ischemia. In this study, we described a fluorescence-corrected multimodal optical imaging system to simultaneously monitor CSD associated intracellular pH (pHi) changes and hemodynamic response including hemoglobin concentrations and cerebral blood flow (CBF). CSD was elicited by application of KCl on rat cortex and direct current (DC) potential was recorded as a typical characteristic of CSD. The pHi shift was mapped by neutral red (NR) fluorescence which was excited at 516-556 nm and emitted at 625 nm. The changes in hemoglobin concentrations were determined by dual-wavelength optical intrinsic signal imaging (OISI) at 550 nm and 625 nm. Integration of fluorescence imaging and dual-wavelength OISI was achieved by a time-sharing camera equipped with a liquid crystal tunable filter (LCTF). CBF was visualized by laser speckle contrast imaging (LSCI) through a separate camera. Besides, based on the dual-wavelength optical intrinsic signals (OISs) obtained from our system, NR fluorescence was corrected according to our method of fluorescence correction. We found that a transient intracellular acidification followed by a small alkalization occurred during CSD. After CSD, there was a prolonged intracellular acidification and the recovery of pHi from CSD took much longer time than those of hemodynamic response. Our results suggested that the new multimodal optical imaging system had the potential to advance our knowledge of CSD and might work as a useful tool to exploit neurovascular coupling under physiological and pathological conditions.

Research highlights► Simultaneous monitoring of intracellular pH and hemodynamic changes during CSD. ► Multimodal imaging of fluorescence, dual-wavelength reflectance and laser speckle. ► Spatiotemporal changes in CBF, Hb, HbO2, CMRO2 and pH during spreading depression. ► In vivo fluorescence correction by using multimodal optical imaging.