Biochemical and Biophysical Research Communications
Protective effect of vitreous against hemoglobin neurotoxicity
Introduction
Hemoglobin (Hb) is a pro-oxidant protein that is released into CNS tissue in millimolar concentrations after spontaneous or traumatic hemorrhage. Its location within erythrocytes provides an effective barrier that prevents its toxicity within the circulation, and also in the initial hours after parenchymal or subarachnoid hemorrhage. However, subsequent erythrophagocytosis by microglia and infiltrating macrophages is apparently insufficient to prevent significant local Hb release and breakdown. At one week after experimental subarachnoid hemorrhage, heme concentrations within the hematoma are two orders of magnitude above those required to kill cultured neurons [1], the cell population most vulnerable to Hb and iron [2,3]. In vivo, parenchymal injection of autologous blood or Hb produces a delayed iron-dependent injury that is attenuated in rodent and pig models by the ferric chelator deferoxamine [[4], [5], [6], [7]].
In contrast to its toxicity in the brain parenchyma and subarachnoid space, the deleterious effects of extravascular blood appear to be mitigated in the eye. Retinal neurons and photoreceptors sustain relatively little injury after hemorrhage localized to the retina or extending into the vitreous [8], the hyaluronan-based gel in the posterior segment of the eye. Accordingly, management is primarily conservative, limited to postural changes to promote erythrocyte settling or observation alone, and usually results in a satisfactory outcome [9,10]. While this phenomenon may merely indicate that retinal cells are selectively resistant to heme or iron-mediated injury, two observations suggest otherwise. First, photoreceptor degeneration is observed when hemorrhage is localized to the subretinal space rather than the retina or vitreous, and can be reduced by deferoxamine [[11], [12], [13]]. Second, the vulnerability of cultured retinal neurons to iron resembles that of other central neurons [3,14,15].
An alternative hypothesis is that vitreous is inherently protective against Hb, and reduces the vulnerability of adjacent cells to its oxidative toxicity. If that is so, then elucidation of its protective mechanisms may have implications beyond ocular hemorrhage, and may provide information relevant to the design of safe and effective therapies for hemorrhagic stroke and trauma. As an initial step towards this end, we investigated the effect of bovine vitreous in a characterized model of Hb neurotoxicity.
Section snippets
Materials
Bovine vitreous was purchased from InVision Bioresources, Seattle, Washington, USA. It was frozen after harvesting and shipped on dry ice. For use in experiments, it was quickly thawed, homogenized while ice-cold, and sterile-filtered. Aliquots were then stored at −80 °C until used.
Human Hb A was obtained as a gift from Hemosol, Inc, Etobicoke, Ontario, Canada.
Apotransferrin was purchased from Millipore-Sigma, Burlington, MA, USA and apoferritin was purchased from Sigma-Aldrich, St Louis, MO,
Results
Vitreous protects against Hb toxicity. Consistent with prior observations [17], treatment with 10 μM Hb for 24 h resulted in degeneration of most phase-bright cells with the typical appearance of neurons in this model, without injury to the background glial monolayer (Fig. 1A–D). This morphological change was associated with increased LDH activity in the culture medium and increased culture fluorescence after PI staining (Fig. 1 E, F). Both cell injury markers were significantly attenuated in a
Discussion
This study provides novel evidence that vitreous robustly protects neurons from Hb neurotoxicity. At a concentration 3% of that in vivo, bovine vitreous completely prevented the widespread neuronal death produced by sustained exposure to 10 μM Hb. At this vitreous dilution, a primary protectant was its ascorbate, and the result was replicated by treating cells with the same concentrations of ascorbate in culture medium alone. However, at concentrations found in undiluted vitreous, other
Acknowledgements
This study was supported by NIH grants R21NS088986 and RO1NS095205.
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