Deferoxamine deconditioning increases neuronal vulnerability to hemoglobin

Abstract

Concomitant treatment with deferoxamine (DFO) protects neural cells from iron and heme-mediated oxidative injury, but also disrupts cell responses to iron loading that may be protective. We hypothesized that DFO treatment and withdrawal would subsequently increase neuronal vulnerability to hemoglobin. Pretreatment with DFO followed by its washout increased neuronal loss after subsequent hemoglobin exposure by 3-4-fold compared with control vehicle-pretreated cultures. This was associated with reduced ferritin induction by hemoglobin; expression of heme oxygenase-1, which catalyzes iron release from heme, was not altered. Increased neuronal loss was prevented by exogenous apoferritin or by continuing DFO or antioxidants throughout the experimental course. Cell nonheme iron levels after hemoglobin treatment were similar in DFO-pretreated and control cultures. These results indicate that DFO deconditions neurons and subsequently increases their vulnerability to heme-mediated injury. Its net effect after CNS hemorrhage may be highly dependent on the timing and duration of its administration. Withdrawal of DFO while heme or iron levels remain elevated may be deleterious, and may negate any benefit of prior concomitant therapy.

Keywords: Deferoxamine; Intracerebral hemorrhage; Iron chelator; Neuronal death; Stroke; Subarachnoid hemorrhage.

Fig. 1.

Timeline of culture treatment with deferoxamine (DFO), hemoglobin (Hb) and other agents and of outcome assays. All experiments were conducted at ≥ 12 days in vitro (DIV).

Fig. 2.

Deferoxamine pretreatment increases the neurotoxicity of hemoglobin. A) Cultures were pretreated for 24 h with deferoxamine 100 μM (DFO Pre, n = 18 cultures), DFO 100 μM plus hemoglobin 1 μM (Hb + DFO Pre, n = 8 cultures) or DMEM vehicle (DMEM Pre, n = 20 cultures). After medium exchange, cultures were then treated for 24 h with Hb 3 μM alone without DFO. Bars represent mean percentage LDH release (+S.E.M.), normalized to the mean value in sister cultures treated with N-methyl-D-aspartate 300 μM for the duration of the experiment, which releases all neuronal LDH without injuring glial cells. The LDH release in control cultures subjected to washes and medium exchange only were subtracted from all values in this and subsequent experiments to obtain the signal due to neurotoxicity. ***p < 0.001 versus value in DMEM-Pre Hb condition. B) Cultures (10/condition) were pretreated with DFO 100 μM alone as in A; after DFO washout they were then treated with Hb 3 μM alone or with 100 μM each of ascorbate (Asc), Trolox, or DFO. C) Cultures (16/ condition) were pretreated with DFO as in B; after DFO washout they were then treated with Hb 3 μM alone or with horse spleen apoferritin 2 mg/ml ***p < 0.001 versus DFO Pre-Hb condition for B and C.

Fig. 3.

Deferoxamine pretreatment increases neuronal vulnerability to hemoglobin. Photomicrographs of neuron-glia cultures under phase-contrast optics treated as follows: A) Control culture subjected to washes and medium exchanges only. Neuron soma are phase-bright and form clusters (3 clusters marked with arrows) on a monolayer of glial cells. Some clusters are spherical, so individual soma are then slightly out of focus. Multiple intact processes are apparent. B) Culture treated for 24 h with deferoxamine only; neuronal cell bodies and processes are intact. C) Culture pretreated with DMEM vehicle only for 24 h, followed by wash and then treatment with hemoglobin 3 μM alone for 24 h; processes are intact, but some soma appear swollen with poorly defined borders, consistent with a mildmoderate degree of injury. D) Culture pretreated with DFO 100 μM for 24 h, followed by washout and treatment with Hb 3 μM alone; neuronal soma and processes have degenerated to debris; glial monolayer remains intact. Scale bar = 100 μm.

Fig. 4.

Deferoxamine decreases ferritin expression. A) Bars represent mean band density in immunoblots (9 cultures/condition) treated for 24 h with sham media exchange, hemoglobin (Hb) 1 μM alone, deferoxamine (DFO) 100 μM alone or Hb 1 μM with DFO 100 μM, immunostained with antibody to heme oxygenase-1 (HO-1) or actin loading control. B) Cultures (11/condition) were treated as in A; immunoblots were stained with anti-ferritin. C) Mean band density of anti-ferritin stained immunoblots (15 cultures/condition) pretreated for 24 h with deferoxamine (DFO) 100 μM or vehicle (DMEM), then after washout treated with Hb 3 μM or vehicle (Sham) alone for 6 h. D) Cultures (7/condition) treated as in C, stained with anti-glutathione peroxidase-4. Bar order correlates with lane order of representative

Fig. 5.

Deferoxamine pretreatment has no effect on culture iron levels. Cultures (8/condition) were pretreated for 24 h with 100 μM deferoxamine (DFO) or vehicle (DMEM), then after washout treated with hemoglobin (Hb) 3 μM or vehicle (Sham). **p < 0.01, ***p < 0.001 versus corresponding sham condition.

Fig. 6.

Deferoxamine pretreatment protects neurons from hydrogen peroxide. Mean LDH release in cultures (6/condition) pretreated with deferoxamine 100 μM (DFO) or vehicle control (DMEM), and then after washout treated with indicated concentrations of hydrogen peroxide alone. **p < 0.01, ***p < 0.001 versus corresponding DMEM vehicle-pretreated condition.