Sex differences in the mouse photothrombotic stroke model investigated with X-ray fluorescence microscopy and Fourier transform infrared spectroscopic imaging.

Stroke is a leading cause of death and disability around the world. To date, the majority of pre-clinical research has been performed using male lab animals and results are commonly generalized to both sexes. In clinical stoke cases females have a higher incidence of ischemic stroke and poorer outcomes, compared to males. Best practices for improving translatability of findings for stroke, encourage the use of both sexes in studies. Since estrogen and progesterone have recognized neuroprotective effects, it is important to compare the size, severity and biochemical composition of the brain tissue following stroke in female and male animal models. In this study a photothrombotic focal stroke was induced in male and female mice. Vaginal secretions were collected twice daily to track the stage of estrous. Mice were euthanized at 24 h post-stroke. Histological staining, Fourier transform infrared imaging and X-ray fluorescence imaging were performed to better define the size and metabolic markers in the infarct core and surrounding penumbra. Our results show while the female mice had a significantly lower body mass than males, the cross-sectional area of the brain and the size of infarct and penumbra were not significantly different between the groups. In addition to the general expected sex-linked differences of altered NADH levels between males and females, estrus females had significantly elevated glycogen in the penumbra compared with males and total phosphorus levels were noted to be higher in the penumbra of estrus females. Elevated glycogen reserves in the tissue bordering the infarct core in females may present alternatives for improved functional recovery in females in the early post-stroke phase.

Is Mini Beam Ready for Human Trials? Results of Randomized Study of Treating De-Novo Brain Tumors in Canines Using Linear Accelerator Generated Mini Beams.

The main challenge in treating malignant brain neoplasms lies in eradicating the tumor while minimizing treatment-related damage. Conventional radiation treatments are associated with considerable side effects. Synchrotron generated micro-beam radiation (SMBRT) has shown to preserve brain architecture while killing tumor cells, however physical characteristics and limited facility access restrict its use. We have created a new clinical device which produces mini beams on a linear accelerator, to provide a new type of treatment called mini-beam radiation therapy (MBRT). The objective of this study is to compare the treatment outcomes of linear accelerator based MBRT versus standard radiation treatment (SRT), to evaluate the tumor response and the treatment-related changes in the normal brain with respect to each treatment type. Pet dogs with de-novo brain tumors were accrued for treatment. Dogs were randomized between standard fractionated stereotactic (9 Gy in 3 fractions) radiation treatment vs. a single fraction of MBRT (26 Gy mean dose). Dogs were monitored after treatment for clinical assessment and imaging. When the dogs were euthanized, a veterinary pathologist assessed the radiation changes and tumor response. We accrued 16 dogs, 8 dogs in each treatment arm. In the MBRT arm, 71% dogs achieved complete pathological remission. The radiation-related changes were all confined to the target region. Structural damage was not observed in the beam path outside of the target region. In contrast, none of the dogs in control group achieved remission and the treatment related damage was more extensive. Therapeutic superiority was observed with MBRT, including both tumor control and the normal structural preservation. The MBRT findings are suggestive of an immune related mechanism which is absent in standard treatment. These findings together with the widespread availability of clinical linear accelerators make MBRT a promising research topic to explore further treatment and clinical trial opportunities.

Multimodal imaging of hemorrhagic transformation biomarkers in an ischemic stroke model.

Hemorrhagic transformation of ischemic stroke has devastating consequences, with high mortality and poor functional outcomes. Animal models of ischemic stroke also demonstrate the potential for hemorrhagic transformation, which complicates biochemical characterization, treatment studies, and hinders poststroke functional outcomes in affected subjects. The incidence of hemorrhagic transformation of ischemic stroke in animal model research is not commonly reported. The postmortem brain of such cases presents a complex milieu of biomarkers due to the presence of healthy cells, regions of varying degrees of ischemia, dead and dying cells, dysregulated metabolites, and blood components (especially reactive Fe species released from lysed erythrocytes). To improve the characterization of hemorrhage biomarkers on an ischemic stroke background, we have employed a combination of histology, X-ray fluorescence imaging (XFI), and Fourier transform infrared (FTIR) spectroscopic imaging to assess 122 photothrombotic (ischemic) stroke brains. Rapid freezing preserves brain biomarkers in situ and minimizes metabolic artifacts due to postmortem ischemia. Analysis revealed that 25% of the photothrombotic models had clear signs of hemorrhagic transformation. The XFI and FTIR metabolites provided a quantitative method to differentiate key metabolic regions in these models. Across all hemorrhage cases, it was possible to consistently differentiate otherwise healthy tissue from other metabolically distinct regions, including the ischemic infarct, the ischemic penumbra, blood vessels, sites of hemorrhage, and a region surrounding the hemorrhage core that contained elevated lipid oxidation. Chemical speciation of deposited Fe demonstrates the presence of heme-Fe and accumulation of ferritin.

Tracking elemental changes in an ischemic stroke model with X-ray fluorescence imaging.

Stroke is a leading cause of long-term disability in adults and a leading cause of death in developed nations. The cascade of cellular events and signalling that occur after cerebral ischemia are complex, however, analyzing global element markers of metabolic state affords the means to monitor stroke severity, status of injury, and recovery. These markers provide a multi-parameter method for assessing changes through the post-stroke time course. We employ synchrotron-based elemental mapping to follow elemental changes in the brain at 1 h, 1-, 2-, and 3-days, and at 1-, 2-, 3-, and 4-weeks post-stroke in a photothrombotic stroke model in mice. Our analysis reveals a highly consistent metabolic penumbra that can be readily identified based on the level of dysregulated potassium and other key elements. Maps of elemental distributions are also useful to demarcate events in the cellular response to the inflammatory cascade, including ion dysregulation, recruitment of cells to the lesion, and glial scar formation.

Sample preparation with sucrose cryoprotection dramatically alters Zn distribution in the rodent hippocampus, as revealed by elemental mapping.

Transition metal ions (Fe, Mn, Cu, Zn) are essential for healthy brain function, but altered concentration, distribution, or chemical form of the metal ions has been implicated in numerous brain pathologies. Currently, it is not possible to image the cellular or sub-cellular distribution of metal ions in vivo and therefore, studying brain-metal homeostasis largely relies on ex vivo in situ elemental mapping. Sample preparation methods that accurately preserve the in vivo elemental distribution are essential if one wishes to translate the knowledge of elemental distributions measured ex vivo toward increased understanding of chemical and physiological pathways of brain disease. The choice of sample preparation is particularly important for metal ions that exist in a labile or mobile form, for which the in vivo distribution could be easily distorted by inappropriate sample preparation. One of the most widely studied brain structures, the hippocampus, contains a large pool of labile and mobile Zn. Herein, we describe how sucrose cryoprotection, the gold standard method of preparing tissues for immuno-histochemistry or immuno-fluorescence, which is also often used as a sample preparation method for elemental mapping studies, drastically alters hippocampal Zn distribution. Based on the results of this study, in combination with a comparison against the strong body of published literature that has used either rapid plunge freezing of brain tissue, or sucrose cryo-protection, we strongly urge investigators in the future to cease using sucrose cryoprotection as a method of sample preparation for elemental mapping, especially if Zn is an analyte of interest.

Direct label-free imaging of brain tissue using synchrotron light: a review of new spectroscopic tools for the modern neuroscientist.

The incidence of brain disease and brain disorders is increasing on a global scale. Unfortunately, development of new therapeutic strategies has not increased at the same rate, and brain diseases and brain disorders now inflict substantial health and economic impacts. A greater understanding of the fundamental neurochemistry that underlies healthy brain function, and the chemical pathways that manifest in brain damage or malfunction, are required to enable and accelerate therapeutic development. A previous limitation to the study of brain function and malfunction has been the limited number of techniques that provide both a wealth of biochemical information, and spatially resolved information (i.e., there was a previous lack of techniques that provided direct biochemical or elemental imaging at the cellular level). In recent times, a suite of direct spectroscopic imaging techniques, such as Fourier transform infrared spectroscopy (FTIR), X-ray fluorescence microscopy (XFM), and X-ray absorption spectroscopy (XAS) have been adapted, optimized and integrated into the field of neuroscience, to fill the above mentioned capability-gap. Advancements at synchrotron light sources, such as improved light intensity/flux, increased detector sensitivities and new capabilities of imaging/optics, has pushed the above suite of techniques beyond "proof-of-concept" studies, to routine application to study complex research problems in the field of neuroscience (and other scientific disciplines). This review examines several of the major advancements that have occurred over the last several years, with respect to FTIR, XFM and XAS capabilities at synchrotron facilities, and how the increases in technical capabilities have being integrated and used in the field of neuroscience.

Structural Characterization of Sm(III)(EDTMP).

Samarium-153 ethylenediamine-N,N,N',N'-tetrakis(methylenephosphonic acid) ((153)Sm-EDTMP, or samarium lexidronam), also known by its registered trademark name Quadramet, is an approved therapeutic radiopharmaceutical used in the palliative treatment of painful bone metastases. Typically, patients with prostate, breast, or lung cancer are most likely to go on to require bone pain palliation treatment due to bone metastases. Sm(EDTMP) is a bone-seeking drug which accumulates on rapidly growing bone, thereby delivering a highly region-specific dose of radiation, chiefly through ß particle emission. Even with its widespread clinical use, the structure of Sm(EDTMP) has not yet been characterized at atomic resolution, despite attempts to crystallize the complex. Herein, we prepared a 1:1 complex of the cold (stable isotope) of Sm(EDTMP) under alkaline conditions and then isolated and characterized the complex using conventional spectroscopic techniques, as well as with extended X-ray absorption fine structure (EXAFS) spectroscopy and density functional structure calculations, using natural abundance Sm. We present the atomic resolution structure of [Sm(III)(EDTMP)-8H](5-) for the first time, supported by the EXAFS data and complementary spectroscopic techniques, which demonstrate that the samarium coordination environment in solution is in agreement with the structure that has long been conjectured.