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. 2015 May 20;6(5):761-70.
doi: 10.1021/acschemneuro.5b00037. Epub 2015 Mar 2.

A new method to image heme-Fe, total Fe, and aggregated protein levels after intracerebral hemorrhage

Mark J Hackett  1 , Mauren DeSouza  2   3 , Sally Caine  4 , Brian Bewer  5 , Helen Nichol  4 , Phyllis G Paterson  6 , Frederick Colbourne  2
Affiliations

A new method to image heme-Fe, total Fe, and aggregated protein levels after intracerebral hemorrhage

Mark J Hackett et al. ACS Chem Neurosci. .

Abstract

An intracerebral hemorrhage (ICH) is a devastating stroke that results in high mortality and significant disability in survivors. Unfortunately, the underlying mechanisms of this injury are not yet fully understood. After the primary (mechanical) trauma, secondary degenerative events contribute to ongoing cell death in the peri-hematoma region. Oxidative stress is thought to be a key reason for this delayed injury, which is likely due to free-Fe-catalyzed free radical reactions. Unfortunately, this is difficult to prove with conventional biochemical assays that fail to differentiate between alterations that occur within the hematoma and peri-hematoma zone. This is a critical limitation, as the hematoma contains tissue severely damaged by the initial hemorrhage and is unsalvageable, whereas the peri-hematoma region is less damaged but at risk from secondary degenerative events. Such events include oxidative stress mediated by free Fe presumed to originate from hemoglobin breakdown. Therefore, minimizing the damage caused by oxidative stress following hemoglobin breakdown and Fe release is a major therapeutic target. However, the extent to which free Fe contributes to the pathogenesis of ICH remains unknown. This investigation used a novel imaging approach that employed resonance Raman spectroscopic mapping of hemoglobin, X-ray fluorescence microscopic mapping of total Fe, and Fourier transform infrared spectroscopic imaging of aggregated protein following ICH in rats. This multimodal spectroscopic approach was used to accurately define the hematoma/peri-hematoma boundary and quantify the Fe concentration and the relative aggregated protein content, as a marker of oxidative stress, within each region. The results revealed total Fe is substantially increased in the hematoma (0.90 μg cm(-2)), and a subtle but significant increase in Fe that is not in the chemical form of hemoglobin is present within the peri-hematoma zone (0.32 μg cm(-2)) within 1 day of ICH, relative to sham animals (0.22 μg cm(-2)). Levels of aggregated protein were significantly increased within both the hematoma (integrated band area 0.10 AU) and peri-hematoma zone (integrated band area 0.10 AU) relative to sham animals (integrated band area 0.056 AU), but no significant difference in aggregated protein content was observed between the hematoma and peri-hematoma zone. This result suggests that the chemical form of Fe and its ability to generate free radicals is likely to be a more critical predictor of tissue damage than the total Fe content of the tissue. Furthermore, this article describes a novel approach to colocalize nonheme Fe and aggregated protein in the peri-hematoma zone following ICH, a significant methodological advancement for the field.

Keywords: Spectroscopic imaging; hemoglobin; intracerebral hemorrhage; iron; oxidative stress; stroke.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Resonance Raman and XFI analysis of fresh and aged blood from the proof-of-principle experiment. (A, B) Bright-field visible light images of dried drops of blood prepared from (A) fresh blood and (B) dried blood. (C, D) XFI images of total Fe in dried drops of blood prepared from (C) fresh blood and (D) aged blood. (E, F) Resonance Raman images of hemoglobin distribution in dried drops of blood prepared from (E) fresh blood and (F) aged blood. (G) Representative resonance Raman spectra (514 nm excitation) collected from fresh and aged blood. The intensity scale for panels C and D is in μg cm−2. The intensity scale for panels E and F is integrated Raman counts (AU). Vertical arrows indicate characteristic resonance Raman heme bands. Scale bar = 500 μm.
Figure 2
Figure 2
Visible light microscopy and histological characterization of the hematoma boundary following ICH. Visible light microscopy of (A, D, G) unstained tissue sections, (B, E, H) cresyl violet stained tissue sections, and (C, F, I) H&E stained tissue sections. (A–C) Sham tissue. (D–F) Tissue 1 day after ICH. (G–I) Tissue 1 day after ICH with annotations to demarcate hematoma boundary (H) and peri-hematoma boundary (PHZ) (based on spread of edema into the tissue). Scale bar = 500 μm.
Figure 3
Figure 3
Resonance Raman, XFI and FTIRI analysis of the distribution of hemoglobin, total Fe, and aggregated protein in the rat striatum 1 day after ICH. (A–D) Representative images from a sham animal. (E–H) Representative images from an animal 1 day after ICH. (A, E) Visible light microscopy images of the unstained tissue. (B, F) Resonance Raman images of hemoglobin distribution. (C, G) XFI images of total Fe distribution. (D, H) FTIRI images of the aggregated protein distribution. Regions of interest that correspond to the hematoma (H) and peri-hematoma zone (PHZ) and a similar-sized area in sham animals have been annotated in white. The intensity scale for panels B and F is integrated Raman counts (AU). The intensity scale for panels C and G is in μg cm−2. The intensity scale bar for panels D and H is second-derivative intensity (AU). Scale bar = 500 μm. Asterisks indicate the location of blood vessels, which were excluded from analysis.
Figure 4
Figure 4
Representative resonance Raman spectra. (A) Cresyl violet histology showing the hematoma (H) and peri-hematoma zone (PHZ). (B) Enlarged resonance Raman map from Figure 3F. (C) Resonance Raman spectra extracted from positions that correspond to the hematoma (P1), hematoma/peri-hematoma boundary (P2), and peri-hematoma (P3). Vertical arrows indicate characteristic resonance Raman heme bands. Horizontal arrows indicate bands from the plastic thermanox substrate. Scale bar = 500 μm.
Figure 5
Figure 5
Quantification of total Fe from sham animals and the hematoma and peri-hematoma zone from ICH rats 1 day after ICH. Data are shown as the mean ± SD. Dagger (†) indicates a significant difference relative to sham animals. Asterisk (*) indicates a significant difference between the hematoma and peri-hematoma zone. Each group contained six animals. A significant difference was determined using a one-way ANOVA and two-tailed posthoc test (as described in Methods) and 95% confidence limit (p = 0.05).
Figure 6
Figure 6
FTIRI analysis of the relative aggregated protein content. (A) Normalized nonderivatized FTIR spectra from a representative sham animal and the hematoma and peri-hematoma zone in an animal 1 day after ICH. Spectra have been offset vertically for clarity. (B) Second-derivative FTIR spectra from a representative sham animal and the hematoma and peri-hematoma zone in an animal 1 day after ICH. The difference in spectra intensity at 1625 cm−1 between the blue and red spectra is not significant. Arrow indicates location (1625 cm−1) of the characteristic second-derivative minima of aggregated proteins. (C) Average integrated band area for the band centered at 1625 cm−1 (aggregated protein) from the results of curve fitting spectra from sham animals and the hematoma and peri-hematoma zone 1 day after ICH. (D) Average second-derivative intensity at 1625 cm−1 from sham animals and the hematoma and peri-hematoma zone 1 day after ICH. Dagger (†) indicates a significant difference relative to sham animals. Asterisk (*) indicates a significant difference between the hematoma and peri-hematoma zone. Each group contained six animals. Data in panels C and D are shown as the mean ± SD. A significant difference was determined using a one-way ANOVA and two-tailed posthoc test (as described in Methods) and 95% confidence limit (p = 0.05). Note that in second-derivative spectra increased concentration results in lower (more negative intensity) values.
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