Ferric iron chelation lowers brain iron levels after intracerebral hemorrhage in rats but does not improve outcome

Abstract

Iron-mediated free radical damage contributes to secondary damage after intracerebral hemorrhage (ICH). Iron is released from heme after hemoglobin breakdown and accumulates in the parenchyma over days and then persists in the brain for months (e.g., hemosiderin). This non-heme iron has been linked to cerebral edema and cell death. Deferoxamine, a ferric iron chelator, has been shown to mitigate iron-mediated damage, but results vary with less protection in the collagenase model of ICH. This study used rapid-scanning X-ray fluorescence (RS-XRF), a synchrotron-based imaging technique, to spatially map total iron and other elements (zinc, calcium and sulfur) at three survival times after collagenase-induced ICH in rats. Total iron was compared to levels of non-heme iron determined by a Ferrozine-based spectrophotometry assay in separate animals. Finally, using RS-XRF we measured iron levels in ICH rats treated with deferoxamine versus saline. The non-heme iron assay showed elevations in injured striatum at 3 days and 4 weeks post-ICH, but not at 1 day. RS-XRF also detected significantly increased iron levels at comparable times, especially notable in the peri-hematoma zone. Changes in other elements were observed in some animals, but these were inconsistent among animals. Deferoxamine diminished total parenchymal iron levels but did not attenuate neurological deficits or lesion volume at 7 days. In summary, ICH significantly increased non-heme and total iron levels. We evaluated the latter and found it to be significantly lowered by deferoxamine, but its failure to attenuate injury or functional impairment in this model raises concern about successful translation to patients.

Fig. 1

Non-heme iron levels (mean±SEM) measured with a Ferrozine-based spectrophotometry assay in naïve rats and at 1, 3 and 28 days after ICH. Samples were taken contra-lateral to ICH (A), on the side of the ICH (B), in the liver (C) and cerebellum (D). Non-heme iron was significantly elevated in the damaged hemisphere at 3 and 28 days compared to the naïve and day 1 ICH group. Day 3 was also significantly higher than naïve rats in the contralateral hemisphere, but not from other times after ICH. No significant differences were observed in either the cerebellum or liver samples. An * denotes significance from naive whereas # denotes a significant difference from day 3 (p<0.05).

Fig. 2

Collagenase-induced ICH primarily damaged the striatum, but other structures were sometimes directly injured, such as the corpus callosum. A) Representative cresyl violet stained lesions illustrate swelling of the damaged hemisphere on day 1, which therefore precluded an accurate determination of tissue loss. B) Tissue loss (mean±SEM) significantly increased between 7 and 21 days post stroke (* denotes p<0.05), which likely stems from continuing cell death, atrophy and more rarely resolution of any residual edema at day 7.

Fig. 3

The total concentration (mean±SEM) of iron in contralateral (A) and ipsilateral (B) hemispheres of ICH rats were measured at 1, 7 and 21 following collagenase injection. Contralateral levels of iron did not differ from naïve levels at any time. Iron levels in the ipsilateral hemisphere were elevated from naïve levels at 7 and 21 days following ICH (* p<0.05 vs. naïve rats) and were further elevated at day 21 as compared to day 1 (# p<0.05). Representative images display the distribution and concentration of iron after ICH (C), the color legend represents μg/cm² of iron.

Fig. 4

Part A indicates the regions of interest where measurements were taken to determine spread of iron from the hematoma. At 21 days post stroke the level of iron in I1 was significantly higher than a corresponding region in naïve rats. The remaining images (B) demonstrate the injury as identified with cresyl violet staining, the location and concentration of iron mapped with XRF and the merger of these images. High iron regions overlie the border zone of the injury.

Fig. 5

Several elements were mapped simultaneously with RS-XRF. The top row of data comes from a single rat brain 7 days after ICH. In this case there was elevated calcium in and around the hematoma. The bi-color map indicates the spatial relationship of these two elements. The second group of images is from a rat at 21 days post-ICH. Elevations of calcium, zinc and sulfur co-localize to a region outside of the iron rich area. All XRF images are scaled independently with the respective legends representing concentrations in μg/cm².

Fig. 6

Neurological function (NDS scores) was assessed prior to (baseline — BL) and on days 1, 3 and 7 following ICH. Each symbol represents the NDS score for one animal. Also shown is this median and inter-quartile range. There were no significant group differences at any time indicating that DFX did not improve outcome.

Fig. 7

Total iron levels (mean±SEM) were quantified in the ipsilateral and contralateral hemispheres with RS-XRF. Giving DFX significantly lessened iron levels in both hemispheres (A; * p<0.05). Representative iron images are shown in B. The color scale is in μg/cm².