In Vitro Hypoxia/Reoxygenation Modeling of Ischemia/Reperfusion Injury Using Glucose Oxidase in Human Cells

Abstract

Ischemia/reperfusion injury (I/RI) is associated with trauma and various diseases. This research investigated an in vitro system of hypoxia/reoxygenation that may be used as a screen for drugs designed to reduce I/RI. Subjecting cells to reduced oxygen (O2) by purging the atmosphere with nitrogen is a slow process that does not reproduce the rapid local reduction in O2 that occurs in vivo during ischemia. Glucose oxidase (GOX) rapidly oxidizes glucose and produces a very low oxygen level (< 0.4 µM within 5 min) with an insignificant loss of glucose. Human umbilical vein endothelial cells (HUVEC) and HeLa cells were subjected to hypoxia for 10 min and returned to normoxia for up to 6 hrs in complete medium. Noticeable cell death was detected by one hour and most died by 6 hrs. At 3.5 hrs a set of cells were homogenized and assayed for total antioxidant potential (TAP) and activities of select antioxidant enzymes. In HUVECs, TAP was 73% higher in cells made anoxic (A) compared to controls (C). Catalase and xanthine oxidase activities were 2‐fold and 70% higher, respectively, in A cells than C while superoxide dismutase or glutathione peroxidase activities were not significantly different between A and C cells. Similar responses were observed in HeLa cells. Catalase could prevent cell death implicating hydrogen peroxide as an important contributor to this injury. This in vitro system can be used to understand the response to IRI and allow testing of potential therapeutics. Funded by US Army Medical Research Materiel Command.