Oxyhemoglobin produces necrosis, not apoptosis, in astrocytes

Objective: Subarachnoid blood, resulting from traumatic brain injury or subarachnoid hemorrhage, has been linked with cell injury and stress gene induction. We investigated whether oxyhemoglobin (OxyHb), a major component in blood clots, exerts a cytotoxic effect on cultured astrocyte cells, and the pattern of cell death. Methods: A murine astrocyte cell line was used (passages 28-35). Cell growth studies were performed 24, 48, and 72 h after exposure to OxyHb (1, 10, and 30 μM). Western blot analysis of poly adenosine diphosphate [ADP]-ribose polymerase (PARP) cleavage and TUNEL stain analysis were performed to determine the presence of apoptosis. Cells treated with OxyHb were also evaluated with transmission electron microscopy to determine changes that may have occurred at the ultra-structural level. Results: OxyHb (10-30 μM), after 72-h incubation, inhibited cell growth. Western blot analysis of PARP and TUNEL staining for the presence of apoptosis were essentially negative in all groups. Ultrastructural analysis revealed an abundance of necrosis and random occurrences of apoptosis in a few cells. Conclusion: Cultured astrocytes exposed to OxyHb causing cell growth inhibition could possibly be a result of cellular cytotoxicity and necrosis.