title: A new method to image heme-Fe, total Fe, and aggregated protein levels after intracerebral hemorrhage.
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.
authors.affiliation: †Molecular and Environmental Sciences Group, Department of Geological Sciences, University of Saskatchewan, 114 Science Place, Saskatoon, Saskatchewan S7N 5E2, Canada.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Hackett
authors.affiliation: ‡Department of Psychology and Neuroscience and Mental Health Institute, University of Alberta, Edmonton, Alberta T6G 2E9, Canada.|§Stress, Memory and Behaviour Lab, Graduate Program in Biochemistry, Federal University of Pampa, Uruguaiana, Rio Grande do Sul 97500-970, Brazil.
authors.forename: Mauren
authors.initials: M
authors.lastname: DeSouza
authors.affiliation: ∥Department of Anatomy and Cell Biology, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan S7N 5E5, Canada.
authors.forename: Sally
authors.initials: S
authors.lastname: Caine
authors.affiliation: ⊥Canadian Light Source, Saskatoon, Saskatchewan S7N 2V3, Canada.
authors.forename: Brian
authors.initials: B
authors.lastname: Bewer
authors.affiliation: ∥Department of Anatomy and Cell Biology, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan S7N 5E5, Canada.
authors.forename: Helen
authors.initials: H
authors.lastname: Nichol
authors.affiliation: #College of Pharmacy and Nutrition, University of Saskatchewan, D Wing Health Sciences, 107 Wiggins Road, Saskatoon, Saskatchewan S7N 5E5, Canada.
authors.forename: Phyllis G
authors.initials: PG
authors.lastname: Paterson
authors.affiliation: ‡Department of Psychology and Neuroscience and Mental Health Institute, University of Alberta, Edmonton, Alberta T6G 2E9, Canada.
authors.forename: Frederick
authors.initials: F
authors.lastname: Colbourne
mesh_terms: D000818:Animals; D002543:Cerebral Hemorrhage / Q000473:pathology*; D004195:Disease Models, Animal; D006418:Heme / Q000032:analysis*; D007501:Iron / Q000032:analysis*; D008297:Male; D018384:Oxidative Stress / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013052:Spectrometry, X-Ray Emission; D017550:Spectroscopy, Fourier Transform Infrared; D013057:Spectrum Analysis / Q000379:methods*; D013059:Spectrum Analysis, Raman
chemical_list: D006418:Heme; D007501:Iron
keywords: Spectroscopic imaging; hemoglobin; intracerebral hemorrhage; iron; oxidative stress; stroke