Near Infrared Quadruple Wavelength Spectrophotometry of the Rat Head

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Abstract
Cytochrome oxidase is the terminal enzyme in the respiratory chain, which ultimately transfers electrons to molecular oxygen. Its redox state can be used as a direct indicator of the sufficiency of oxygen supply to the site of cellular energy production, the mitochondria. The enzyme when oxidized has a broad absorption band in near infrared region and this absorption disappears when its copper component is reduced. Because the extinction coefficients for most biological materials are low in near infrared region, it is not difficult to detect near infrared light that penetrated intact tissues. Thus near infrared spectroscopy is expected to be a new tool for noninvasively monitoring tissue hypoxic injury. The technique was first introduced by Jöbsis(1977), and has been applied in several clinical and physiological situations (e.g. Wiernsperger et al., 1981; Cairns et al., 1985; Ferrari et al., 1985; Wyatt et al., 1986). The main obstacle to quantitation of the cytochrome oxidase redox state in intact organ has been the overlap of the spectral changes due to variation in the amount and oxygenation state of hemoglobin. Algorithms previously reported include dual and triple wavelength analysis of difference spectrum. Wray et al. (1988) assumed the additive absorption of three major components, namely oxygenated and deoxygenated hemoglobin and the redox state of cytochrome oxidase, and calculated the change of each component by solving simultaneous equations at three separate wavelengths. The absorptivity constant of the cytochrome change was determined in fluorocarbon exchange transfused rat brain, while those of hemoglobin were measured in a clear solution. Hazeki et al. (1987) proposed a similar method where the standard spectra of hemoglobin were obtained directly in perfused rat head preparations.