Sickle Cell Mice with Hypoxia-Reoxygenation Have Oxidative and Inflammatory Liver Injury - toward a New Experimental Model for Vaso- Occlusive Injury
Authors: Hsu Lewis L. , Shiva Sruti , Mendelsohn Caroline , Hsu Michael R , Noguchi Audrey C , Mendelsohn Laurel , Gladwin Mark 
Published: April 2020

Abstract
INTRODUCTION: Vaso-occlusion causes tissue ischemia and severe pain in patients with sickle cell disease (SCD). The liver is frequently involved in human SCD complications and elevated serum levels of hepatic transaminases often accompany vasoocclusive pain. It is unknown whether in sickle cell disease, the liver sustains oxidative damage after hypoxia-reoxygenation and if so, how long does it take to recover. Both mitochondrial and cytosolic aconitases are potential targets of oxidants in cells due to the oxidant-mediated loss of iron from the [4Fe-4S] cluster. Oxidant damage can occur from the reactive oxygen species generated by dysfunctional mitochondria dysfunction following ischemia-reperfusion and also from peroxidases later released during the inflammatory reaction to tissue injury. A mouse model of SCD can be used to investigate the cellular mechanisms and temporal sequence of vaso-occlusive tissue damage.
OBJECTIVES: After experimental vaso-occlusion by exposing sickle cell mice to hypoxia-reoxygenation, we will determine whether hepatocellular injury shows temporal correlation with a marker of oxidative damage (diminished aconitase activity) and a marker of inflammation (increased myeloperoxidase concentration). No changes are predicted to occur in littermate control mice whose erythrocytes do not sickle with hypoxic challenge.
METHODS: Mice expressing exclusively human sickle cell hemoglobin (“Berkeley sickle mice”) and non-sickling littermate controls (“hemizygotes”) were exposed to 10% oxygen for 2 hours, and then restored to normoxia. At 6, 18, 48, or 72 hrs after hypoxia, animals were euthanized to harvest blood and liver tissue. Normoxic control mice of both types had blood and liver harvested without hypoxic exposure. Additional sickle cell mice received intraperitoneal injection of nitrite (2.4 millimole/g body weight) or saline control at the end of 2 hours of hypoxia, then had blood and liver harvested 18 hours after they were restored to normoxia. Serum alanine aminotransferase (ALT) level was assayed as a quantitative measure of hepatocellular injury. Aconitase and myeloperoxidase (MPO) results were compared by t-test.
RESULTS: Serum ALT level rose 3-fold in sickle mice by 18 hr after hypoxia, and then declined by 48 and 72 hrs after hypoxia. Hemizygotes showed no change in ALT after hypoxia. Liver homogenate aconitase activity was significantly lower in sickle mice than in hemizygotes at the first time point measured, 6 hrs after hypoxia (p=0.003, n=4 per group), suggesting that oxidative damage to the enzyme had occurred early after hypoxia. MPO concentration in livers harvested 48 hr after hypoxia-reoxygenation was 3-fold elevated in sickle mice vs. hemizygotes (p=0.006, n=4 per group), but not at baseline or 72 hr after hypoxia-reoxygenation. Nitrite injection was associated with complete abrogation of rise in ALT in sickle mice challenged with hypoxia-reoxygenation, but was not associated with any differences in aconitase activity or MPO concentration.
CONCLUSION: Serum ALT, liver aconitase activity, and liver MPO concentration had different temporal patterns of changes in sickle mice after hypoxia-reoxygenation challenge as an experimental model of vaso-occlusive tissue injury. Oxidative stress appears to be present within hours after hypoxia, followed by tissue injury (serum ALT rise), which is then followed by inflammation may take 48 hr after experimental vasoocclusion. Although the nitrite is not sufficient as antioxidant to protect against reactive oxygen species and inflammatory leukocytes, nitrite may be protective against tissue injury at the mitochondrial level. This experimental model system may be well-suited for pre-clinical testing of therapy for sickle cell vaso-occlusion.