Ferumoxytol administration does not alter infarct volume or the inflammatory response to stroke in mice

Abstract

Ferumoxytol is an ultrasmall superparamagnetic iron oxide (USPIO) nanoparticle that is FDA-approved as an intravenous iron replacement therapy for the treatment of iron deficiency anemia in patients with chronic kidney disease. Ferumoxytol has also been used as a contrast agent for cerebral blood volume mapping by magnetic resonance imaging (MRI), which suggests it could be used for imaging hemodynamic abnormalities after stroke. However, circulating macrophages can internalize USPIOs, and recent data indicate that the accumulation of iron in macrophages can lead them to adopt the M1 pro-inflammatory phenotype. Therefore, the uptake of intravenously administered iron particles by circulating macrophages that home to the stroke core could potentially alter the inflammatory response to stroke. To test this possibility in vivo we administered a dose of ferumoxytol previously used to obtain cerebral blood volume maps in healthy humans by steady-state susceptibility contrast (SSC) MRI to BALB/cJ mice 48h after stroke and examined cytokine levels, microglial/macrophage activation, and lesion volume in the brain 5 days later. Treatment with ferumoxytol did not lead to any differences in these parameters. These data indicate that the use of ferumoxytol as a contrast agent for brain imaging after stroke does not alter the inflammatory response to stroke in mice, and is therefore unlikely to do so in human subjects.

Keywords: Ferumoxytol; Inflammation; Magnetic resonance imaging; Stroke.

Figure 1

Administration of ferumoxytol does not alter cytokine or chemokine expression 7 days after stroke. Graphs showing cytokine and chemokine levels in the stroke lesion 5 days post ferumoxytol or saline treatment. There were no significant (P<0.05) differences between treatment groups (n=11 for saline group, n=12 for ferumoxytol group).

Figure 2

Administration of ferumoxytol does not alter CD68 immunoreactivity 7 days after stroke. A.) Whole section images showing CD68 immunoreactivity in the lesion and in areas of axonal degeneration 5 days after saline and ferumoxytol administration and 7 days after DMCAO. B.) There is no difference in % area covered by CD68 immunoreactivity in mice that received ferumoxytol (n=9 per group). C.) There is also no overt difference in CD68⁺ cell morphology in mice that received ferumoxytol. Error bars are SD, Scale bar: 50µm.

Figure 3

Ferumoxytol administration leads to increased Perls' iron staining in the corpus callosum but not in peri-infarct regions. A.) Representative image of iron staining (blue) among macrophages (brown) in the stroke core (asterisk) and border. Scale bar: 50µm. B.) Low magnification image of iron staining in the corpus callosum underlying the stroke (asterisk). Scale bar: 50µm. C.) Higher magnification image of co-localization (marked by arrowheads) of Perls’ iron staining in CD68-expressing activated microglia or macrophages in the corpus callosum adjacent to the stroke. Scale bar: 10µm. D–G.) Quantification of the percentage of sections per mouse with iron staining in the cortex (p = 0.76), stroke border (p = 0.49), stroke core (p = 0.58), and corpus callosum (*p = 0.03).

Figure 4

Lesion size is not altered 5 days after administration of ferumoxytol compared to after administration of saline. A.) Representative whole section image showing cresyl violet staining and NeuN immunoreactivity 7 days after DMCAO. These sections were used to calculate infarct volume. The area of the ipsilateral hemisphere excluding the stroke core (outlined in red) was subtracted from the area of the contralateral hemisphere (outlined in blue) and then divided by the area of the contralateral hemisphere to calculate infarct volume as a percentage of the contralateral hemisphere. B.) There is no difference in % infarct volume between saline- and ferumoxytol-treated mice (n=9 per group).