Simultaneous monitoring of nitric oxide, oxyhemoglobin and deoxyhemoglobin from small areas of the rat brain by in vivo visible spectroscopy and a least-square approach

Nitric oxide (NO) is a versatile molecule involved in a wide range of biological processes. Under physiological conditions, NO reacts with oxyhemoglobin (OxyHb) to form methemoglobin (MetHb) at a very high rate. Previous works have shown that MetHb is proportional to NO and that MetHb absorption contributes to the in vivo absorption spectrum recorded with visible spectroscopy using optical fibers. However, in vivo spectrophotometric monitoring of a single molecule has long been a problem because the overlapping of OxyHb, deoxyhemoglobin (DeoxyHb), MetHb, cytochromes and other chromophores absorption spectra make in vivo quantitative spectroscopy difficult. The aim of this work was to evaluate the possibility of obtaining the contribution of each main endogenous chromophore, especially OxyHb, DeoxyHb and MetHb, to the in vivo visible absorption spectrum recorded from rat cortex, hippocampus and striatum. A least-square approach with a fitting equation containing the in vitro spectrum of the main endogenenous chromophores was used. The validity of this approach was studied by increasing the endogenous MetHb level with NO infusion and by biliverdin perfusion. Data indicate that in vivo visible spectroscopy in combination with the least-square fitting method may be a useful tool for gaining insight into the roles of NO, hemoglobins and the interaction between them even from small cerebral areas.