Browsing by Author "Hunt, NH"

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    Chemical alterations to murine brain tissue induced by formation fixation: implications for biospectroscopic imaging and mapping studies of disease pathogenesis
    (Royal Society of Chemistry, 2011-07-21) Hackett, MJ; McQuillan, JA; El-Assaad, F; Aitken, JB; Levina, A; Cohen, DD; Siegele, R; Carter, EA; Grau, GE; Hunt, NH; Lay, PA
    Understanding biochemical mechanisms and changes associated with disease conditions and, therefore, development of improved clinical treatments, is relying increasingly on various biochemical mapping and imaging techniques on tissue sections. However, it is essential to be able to ascertain whether the sampling used provides the full biochemical information relevant to the disease and is free from artefacts. A multi-modal micro-spectroscopic approach, including FTIR imaging and PIXE elemental mapping, has been used to study the molecular and elemental profile within cryofixed and formalin-fixed murine brain tissue sections. The results provide strong evidence that amino acids, carbohydrates, lipids, phosphates, proteins and ions, such as Cl(-) and K(+), leach from tissue sections into the aqueous fixative medium during formalin fixation of the sections. Large changes in the concentrations and distributions of most of these components are also observed by washing in PBS even for short periods. The most likely source of the chemical species lost during fixation is the extra-cellular and intra-cellular fluid of tissues. The results highlight that, at best, analysis of formalin-fixed tissues gives only part of the complete biochemical "picture'' of a tissue sample. Further, this investigation has highlighted that significant lipid peroxidation/oxidation may occur during formalin fixation and that the use of standard histological fixation reagents can result in significant and differential metal contamination of different regions of tissue sections. While a consistent and reproducible fixation method may be suitable for diagnostic purposes, the findings of this study strongly question the use of formalin fixation prior to spectroscopic studies of the molecular and elemental composition of biological samples, if the primary purpose is mechanistic studies of disease pathogenesis.© 2011, Royal Society of Chemistry
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    Investigation of the mouse cerebellum using STIM and mu-PIXE spectrometric and FTIR spectroscopic mapping and imaging
    (Elsevier, 2011-10-15) Hackett, MJ; Siegele, R; El-Assaad, F; McQuillan, JA; Aitken, JB; Carter, EA; Grau, GE; Hunt, NH; Cohen, DD; Lay, PA
    The cerebral biochemistry associated with the development of many neurological diseases remains poorly understood. In particular, incomplete understanding of the mechanisms through which vascular inflammation manifests in tissue damage and altered brain function is a significant hindrance to the development of improved patient therapies. To this extent, a combination of spectrometric/spectroscopic mapping/imaging methods with an inherent ability to provide a wealth of biochemical and physical information have been investigated to understand further the pathogenesis of brain disease. In this study, proton-induced X-ray emission (PIXE) mapping was combined with scanning transmission ion microscopy (STIM) mapping and Fourier-transform infrared (FTIR) imaging of the same tissue sample to study directly the composition of the murine (mouse) cerebellum. The combination of the elemental, density and molecular information provided by these techniques enabled differentiation between four specific tissue types within the murine cerebellum (grey matter, white matter, molecular layer and micro blood vessels). The results presented are complementary, multi-technique measurements of the same tissue sample. They show elemental, density and molecular differences among the different tissue types. (C) 2011 Elsevier B.V.
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    Light and heavy ion beam analysis of thin biological sections
    (Elsevier, 2013-07-01) Lee, J; Siegele, R; Pastuovic, Z; Hackett, MJ; Hunt, NH; Grau, GE; Cohen, DD; Lay, PA
    The application of ion beam analysis (IBA) techniques to thin biological sections (ThBS) presents unique challenges in sample preparation, data acquisition and analysis. These samples are often the end product of expensive, time-consuming experiments, which involve many steps that require careful attention. Analysis via several techniques can maximise the information that is collected from these samples. Particle-induced X-ray emission (PIXE) and Rutherford backscattering (RBS) spectroscopy are two generally non-destructive IBA techniques that use the same MeV ions and can be performed simultaneously. The use of heavy ion PIXE applied to thick samples has, in the past, resulted in X-ray spectra of a poorer quality when compared to those obtained with proton beams. One of the reasons for this is the shorter probing depth of the heavy ions, which does not affect thin sample analysis. Therefore, we have investigated and compared 3-MeV proton and 36-MeV carbon ion beams on 7-μm thick mouse brain sections at the ANSTO Heavy ion microprobe (HIMP). The application of a 36-MeV C4+ ion beam for PIXE mapping of ThBS on thin Si3N4 substrate windows produced spectra of high quality that displayed close to a nine-times gain in signal yield (Z2/q) when compared to those obtained for 3-MeV protons for P, S, Cl and K but not for Fe, Cu and Zn. Image quality was overall similar; however, some elements showed better contrast and features with protons whilst others showed improved contrast with a carbon ion beam. RBS spectra with high enough counting statistics were easily obtained with 3-MeV proton beams resulting in high resolution carbon maps, however, the count rate for nitrogen and oxygen was too low. The results demonstrate that on thin samples, 36-MeV C4+ will produce good quality PIXE spectra in less time; therefore, carbon ions may be advantageous depending on which element is being studied. However, these advantages may be outweighed by the inherent disadvantages including increased ion beam damage, the necessity of very high ion energies resulting in higher neutron fields. © 2013, Elsevier B.V.
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    Mechanisms of murine cerebral malaria: multimodal imaging of altered cerebral metabolism and protein oxidation at hemorrhage sites
    (American Association for the Advancement of Science, 2015-12-18) Hackett, MJ; Aitken, JB; El-Assaad, F; McQuillan, JA; Carter, EA; Ball, HJ; Tobin, MJ; Paterson, DJ; de Jonge, MD; Siegele, R; Cohen, DD; Vogt, S; Grau, GE; Hunt, NH; Lay, PA
    Using a multimodal biospectroscopic approach, we settle several long-standing controversies over the molecular mechanisms that lead to brain damage in cerebral malaria, which is a major health concern in developing countries because of high levels of mortality and permanent brain damage. Our results provide the first conclusive evidence that important components of the pathology of cerebral malaria include peroxidative stress and protein oxidation within cerebellar gray matter, which are colocalized with elevated nonheme iron at the site of microhemorrhage. Such information could not be obtained previously from routine imaging methods, such as electron microscopy, fluorescence, and optical microscopy in combination with immunocytochemistry, or from bulk assays, where the level of spatial information is restricted to the minimum size of tissue that can be dissected. We describe the novel combination of chemical probe–free, multimodal imaging to quantify molecular markers of disturbed energy metabolism and peroxidative stress, which were used to provide new insights into understanding the pathogenesis of cerebral malaria. In addition to these mechanistic insights, the approach described acts as a template for the future use of multimodal biospectroscopy for understanding the molecular processes involved in a range of clinically important acute and chronic (neurodegenerative) brain diseases to improve treatment strategies. 2015 © The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. Distributed under a Creative Commons Attribution Non Commercial Licence 4.0 (CC BY-NC).