Purpose: Our previous studies showed that tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 are induced after hemorrhagic shock and that their induction is attenuated by hyperbaric oxygen treatment. In this study, we tried to identify the cell types in the liver that are responsible for the increase in TNF-alpha mRNA and IL-6 mRNA after hemorrhage using in situ reverse transcription-polymerase chain reaction (in situ RT-PCR).
Methods: Chronically cannulated rats were subjected to hemorrhage, maintaining a mean arterial blood pressure of 40 mmHg for 1 h. They were resuscitated with the collected blood and saline, and the livers were removed at designated times, then fixed and frozen.
Results: Standard in situ hybridization could not detect the mRNAs of TNF-alpha and IL-6 in the livers; however, in situ RT-PCR detected TNF-alpha mRNA and IL-6 mRNA mainly in the vascular endothelial cells and perivascular nonparenchymal cells of the bled rats. Sinusoidal cells were positive for TNF-alpha mRNA, but negative for IL-6 mRNA. No signal was found in normal rats, or when the experimental protocol was modified to: omit the RT step; precede the RT step with RNA digestion; or use an irrelevant probe.
Conclusion: These results show that vascular endothelial cells and perivascular nonparenchymal cells are activated after massive hemorrhage to produce inflammatory cytokines.