Effects of hepatic zonal oxygen levels on hepatocyte stress responses

Hepatocytes spend their lifetimes in a gradient of oxygen, hormones, and enzymes. We used a three-dimensional Matrigel model to determine whether hepatocytes cultured at perivenous (zone 3) oxygen levels differed in susceptibility to anoxia-induced cell injury compared with hepatocytes cultured at periportal (zone 1) oxygen levels.

Hepatocytes were harvested from Sprague Dawley rats and cultured at 9% oxygen (hepatic zone 1) or 5% oxygen (hepatic zone 3) and stressed at 0% oxygen. Microscopy, real-time reverse transcriptase-polymerase chain reaction, and enzyme-linked immunosorbent assay were used to assess cell viability, mitochondrial potential, acute phase responses, and membrane blebbing.

Hepatocytes cultured in Matrigel with HepatoZyme medium at zone 1 and zone 3 oxygen conditions were viable for 1 wk and showed acute phase responses as measured by interleukin-6-induced fibrinogen production. In response to 3 h anoxia, cells maintained at the perivenous oxygen level showed increased membrane blebbing and increased loss of mitochondrial membrane potential in comparison to the periportal oxygen cultured cells. Cells at perivenous oxygen also showed a reduced ability to recover following reoxygenation.

Hepatocytes can remain viable and functional for extended periods in culture at low oxygen levels that mimic the hepatic perivenous environment, yet these cells are more susceptible to anoxia-induced damage than hepatocytes cultured at the periportal oxygen level. The small population of perivenous hepatocytes may be critical in determining the fate of the liver during ischemia/reperfusion since hepatocytes cultured at that concentration appear to be more labile in response to anoxia.