Abstract

Abstract 2569

Poster Board II-546

Sickle cell vaso-occlusion causes tissue ischemia, injury, and severe pain in patients with sickle cell disease (SCD). Investigations of the pathophysiology of vaso-occlusion have largely focused on the mechanisms for microcirculatory obstruction, rather than the tissue damage. The liver is frequently involved in human SCD complications and elevated serum levels of hepatic transaminases often accompany vaso-occlusive pain. Animal models can help to study mechanisms of how vaso-occlusive microcirculatory abnormalities cause tissue damage. Sickle cell mice originally developed by Pastzy (“Berkeley sickle mice”) expressing exclusively human sickle hemoglobin have severe hemolytic anemia and physiologic fragility, and are well-suited for examining an acute vaso-occlusive challenge.

HYPOTHESIS:

Moderate hypoxic exposure will cause liver hypoxia and tissue injury only in homozygote sickle mice, as signs of vaso-occlusive injury.

METHODS:

We examined vaso-occlusion by challenging male sickle mice with moderate normobaric hypoxia (10% oxygen) for up to 2 hours then restoring them to normoxia and assaying plasma alanine aminotransferase (ALT) as a quantitative measure of hepatic injury. Pimonidazole (HypoxyProbe 60mg/kg, NPI, Inc) was injected intraperitoneally before hypoxia, in order to provide irreversible labeling of cells with pO2 less than 10 torr. Controls included sickle mice without hypoxia, and non-sickle mice with the same hypoxic exposure. Mice were euthanized for blood and tissue harvest. Liver, kidney, and lungs were fixed in formalin, and processed for immunohistochemical staining (HP-100 kit, NPI). A veterinary pathologist unaware of the treatment group scored histologic sections under light microscopy for the extent of staining on a scale of 1 to 4, and intensity of staining on a scale of 1 to 3.

RESULTS:

Sickle cell mice have significantly greater extent of Hypoxyprobe labeling of liver than wild-type mice with the same hypoxia-reoxygenation challenge (2.6 + 0.5 vs 1.3 + 0.5, p=0.027 by t-test). At normoxic baseline, liver HypoxyProbe staining from sickle mice and wild-type controls had the same extent (1 vs 1) and intensity of liver staining. Kidney had a higher baseline of Hypoxyprobe staining in all normoxic mice, and this did not increase with hypoxia in sickle mice. Lungs had very little Hypoxyprobe staining at baseline, and a few bronchial cell stained under hypoxia in sickle mice, but the change was not statistically significant. ALT was mildly elevated in sickle mice at normoxic baseline, and rose 3-fold to a peak by 18 hours after hypoxia, then declined back to baseline by 48 – 72 hours.

CONCLUSION:

The extent of HypoxyProbe staining shows that sickle mice suffer significantly greater liver tissue hypoxia with hypoxic exposure than controls with the same mild hypoxia. Sickle mice in normoxia have no greater extent of HypoxyProbe staining than control mice. These results are consistent with hypoxia-triggered vaso-occlusion as the cause of ALT elevation in the sickle mice. The two outcome measures after hypoxic challenge of sickle mice, HypoxyProbe staining for tissue hypoxia and plasma ALT for tissue injury, provide a bioassay system for pre-clinical screening of antisickling therapy.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.