Alcohol-induced oxidative stress in the liver: in vivo measurements

Abstract

Oxidative stress is increasingly suspected to contribute to the initiation and progression of many disease, including those caused by alcohol exposure. Two major products of reactive oxygen and nitrogen species formation are 4OH-nonenal and 3-nitrotyrosine protein adducts, both of which can be detected by immunohistochemistry. In the past, immunohistochemical techniques have served largely as qualitative measures of changes. However, coupled with digital capture and analysis of photomicrographs, one can now quantitate treatment-related changes with immunohistochemistry. This chapter summarizes techniques for immunohistochemical detection of these products of reactive oxygen and nitrogen species and subsequent image-analysis. Although the methods described herein are based on liver, these techniques have been employed successfully in most tissue types with minor modifications and are therefore broadly applicable.

Fig. 1

Effect of LPS on 4OH-nonenal adduct accumulation in mouse liver as determined by quantitative immunohistochemistry. Representative photomicrographs are shown depicting 4OH-nonenal immunostaining of a liver section (100×) of a mouse 24 h after injection with LPS (10 mg/kg intraperitoneally). The same microscope region is shown before (A) and after (B) white balancing of the image-analysis software. Note that the acellular space in (A) is light yellow instead of white. (C) is the same as (B), but after the blue exposure is increased 10%. Note that the contrast between the DAB chromogen (brown) and the hematoxylin counterstain (blue) is enhanced by digitally increasing the blue exposure. (D) shows the microscope region from (C), determined by the computer to be within the threshold range for positive staining