Altered excitatory-inhibitory balance within somatosensory cortex is associated with enhanced plasticity and pain sensitivity in a mouse model of multiple sclerosis

S1 IHC in pre-symptomatic and clinical-onset EAE: PV+ cell counts, PNN counts, and Iba-1+ microglia counts. A) Representative fluorescence photomicrographs of PV+ staining (low-mag) in S1 from control (CFA) and EAE animals at the pre-symptomatic stage (7–9 dpi PRE) or clinical onset (ONS). B) Group mean (±S.E.) total PV+ cell counts from S1HL of CFA (n = 8), PRE (n = 4), and ONS (n = 4) EAE animals. No significant differences were observed between groups (one-way ANOVA N.S.). C) Representative fluorescence photomicrographs of WFA+ staining (PNNs) in S1 from control (CFA) and EAE animals at the pre-symptomatic stage (7–9 dpi PRE) or clinical onset (ONS). D) Group mean (±S.E.) total PNN counts from S1HL of CFA (n = 11), PRE (n = 4), and ONS (n = 8) EAE animals. EAE animals exhibited significantly reduced PNN-counts vs. CFA-controls at clinical onset (one-way ANOVA, p = 0.007, post hoc comparisons vs. CFA-controls by Dunnett’s method). E) Representative fluorescence photomicrographs of Iba-1+ staining (PNNs) in S1 from control (CFA) and EAE animals at the pre-symptomatic stage (7–9 dpi PRE) or clinical onset (ONS). F) Group mean (±S.E.) total Iba-1+ counts from S1HL of CFA (n = 13), PRE (n = 4), and ONS (n = 8) EAE animals. EAE animals exhibited significantly increased numbers of Iba-1+ cells (microglial activation) in S1HL vs. CFA-controls at all time points (one-way ANOVA, p = 0.012, post hoc comparisons vs. CFA-controls by Dunnett’s method). (PDF 6418 kb

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

© 2015 American Chemical Society.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<sup>-2</sup>), 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<sup>-2</sup>) within 1 day of ICH, relative to sham animals (0.22 µg cm<sup>-2</sup>). 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