In situ RT-PCR detection of inducible nitric oxide synthetase gene expression in lung during endotoxemia in rabbits

Objective: To detect the location of inducible nitric oxide synthetase (iNOS) protein and mRNA in lung during endotoxemia in rabbits. Methods: Northern blotting was performed before, 1 hour and 5 hours after the intravenous administration of lipopolysaccharide (LPS) in rabbits. Immunohistochemical analysis (IA), in situ hybridization and in situ reverse transcription-polymerase chain reaction (in situ RT-PCR) were also performed in lung sections. Results: iNOS mRNA expression was found using Northern blotting in lung 5 hours after LPS injection, while it was not found in control. The positive stain was found only in macrophages in lung 5 hours after LPS injection by standard hybridization and IA; while by in situ RT-PCR, the amplification products were found in macrophages, airway epithelial cells, vascular endothelial cells, smooth muscle cells and leukocytes, in addition to macrophages distributed abundantly throughout the lung. The signal was absent in control or samples. Conclusions: Using an in situ RT-PCR technique, iNOS expression was not only observed in macrophages but also in many other kinds of cells in lung during endotoxemia in rabbits. This suggests that in situ RT-PCR is much more sensitive than in situ hybridization, and can be used to examine genes with low expression.