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 th e intravenous administration of lipopolysaccharide (LPS) in rabbits. Immuno -histochemical analysis (IA), in situ hybridization and in situ reverse tra nscription-polymerase chain reaction (in situ RT-PCR) were also performed i n lung sections. Results iNOS mRNA expression was found using Northern blotting in lung 5 ho urs after LPS injection, while it was not found in control. The positive st ain was found only in macrophages in lung 5 hours after LPS injection by st andard hybridization and IA; while by in situ RT - PCR, the amplification p roducts were found in macrophages, airway epithelial cells, vascular endoth elial cells, smooth muscle cells and leukocytes, in addition to macrophages distributed abundantly throughout the lung. The signal was absent in contr ol 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 duri ng endotoxemia in rabbits. This suggests that in situ RT-PCR is much more s ensitive than in situ hybridization, and can be used to examine genes with low expression.