A novel multi-modal platform to image molecular and elemental alterations in ischemic stroke

Abstract

Stroke is a major global health problem, with the prevalence and economic burden predicted to increase due to aging populations in western society. Following stroke, numerous biochemical alterations occur and damage can spread to nearby tissue. This zone of "at risk" tissue is termed the peri-infarct zone (PIZ). As the PIZ contains tissue not initially damaged by the stroke, it is considered by many as salvageable tissue. For this reason, much research effort has been undertaken to improve the identification of the PIZ and to elucidate the biochemical mechanisms that drive tissue damage in the PIZ in the hope of identify new therapeutic targets. Despite this effort, few therapies have evolved, attributed in part, to an incomplete understanding of the biochemical mechanisms driving tissue damage in the PIZ. Magnetic resonance imaging (MRI) has long been the gold standard to study alterations in gross brain structure, and is frequently used to study the PIZ following stroke. Unfortunately, MRI does not have sufficient spatial resolution to study individual cells within the brain, and reveals little information on the biochemical mechanisms driving tissue damage. MRI results may be complemented with histology or immuno-histochemistry to provide information at the cellular or sub-cellular level, but are limited to studying biochemical markers that can be successfully "tagged" with a stain or antigen. However, many important biochemical markers cannot be studied with traditional MRI or histology/histochemical methods. Therefore, we have developed and applied a multi-modal imaging platform to reveal elemental and molecular alterations that could not previously be imaged by other traditional methods. Our imaging platform incorporates a suite of spectroscopic imaging techniques; Fourier transform infrared imaging, Raman spectroscopic imaging, Coherent anti-stoke Raman spectroscopic imaging and X-ray fluorescence imaging. This approach does not preclude the use of traditional imaging techniques, and rather it should be use to complement traditional methods such as MRI or histology and immunohistochemistry, to gain a greater insight into disease mechanisms. We demonstrate the potential of this approach by characterizing biochemical alterations within the PIZ 24h after the induction of photothrombotic stroke in mice. Substantial molecular and elemental alterations were identified in the PIZ 24h after stroke that are consistent with tissue swelling and edema, but not oxidative stress. This reveals important mechanistic information, that could not previously be obtained, which should be considered in future studies aimed at developing therapeutic intervention from this model.

Elemental characterisation of the pyramidal neuron layer within the rat and mouse hippocampus

“…A major reason for this is that chemical stains and chemical fixation procedures often remove, re-distribute or even add chemical content to biological samples. [5][6][7][8][9][10] Consequently, it has only been through the recent advances in direct, label free elemental mapping techniques, which have provided the opportunity to examine the elemental composition of neurons, or other brain cells such as glia, [11][12][13][14][15][16][17][18] with respect of their anatomical location and brain function.…”

“…14,[52][53][54][55][56][57] For these reasons, XFI has found increasing use within the field of neuroscience, to study elemental alterations that correlate with anatomical structure, 58,59 or occur as a consequence of diseases such as Alzheimer's disease, [60][61][62][63][64] Parkinson's disease, 65,66 damaging events that occur during epilepsy, [67][68][69] traumatic brain injury, 70 and stroke. 17,71,72 XFI has also been used to study the elemental phenotype of individual cells and neurons within in vitro cultures, 64,[73][74][75][76] and to study the elemental distribution of the rat hippocampus, 12,[67][68][69]77 but detailed characterisation of elemental signatures of cells within CA1 medial, CA1 lateral, CA2 or CA3 sub-regions has not been previously undertaken.…”

A Review of ex vivo Elemental Mapping Methods to Directly Image Changes in the Homeostasis of Diffusible Ions (Na+, K+, Mg2 +, Ca2 +, Cl–) Within Brain Tissue

“…Prior to imaging analyses, cryo-sections are typically cut at a temperature of −15 to −25 • C, and typical section thickness is 1-50 µm. To achieve sub-cellular resolution imaging section thickness of <5 µm is often necessary (Hackett et al, 2016), with cellular detail well preserved in sections 5-20 µm thick (Chwiej et al, 2008(Chwiej et al, , 2011(Chwiej et al, , 2012aHackett et al, 2015b;Caine et al, 2016;Lins et al, 2016;Williamson et al, 2016). Sections 50 µm or thicker are more suitable for regional or sub-regional analyses (Popescu et al, 2009;Pushie et al, 2011).…”

“…Sections 50 µm or thicker are more suitable for regional or sub-regional analyses (Popescu et al, 2009;Pushie et al, 2011). Tissue sections are often analyzed dehydrated (air-dried or freeze-dried) (Chwiej et al, 2008(Chwiej et al, , 2011(Chwiej et al, , 2012aHackett et al, 2015b;Caine et al, 2016;Lins et al, 2016;Williamson et al, 2016), however it has been shown that analyses of frozen samples that do not thaw at any point during sample preparation is required to best preserve sub-cellular elemental distribution on the sub-micron scale (Matsuyama et al, 2010;Roudeau et al, 2014).…”

“…Direct elemental mapping techniques include X-ray Fluorescence Microscopy (XFM), Proton Induced X-ray Emission (PIXE), Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS), and Secondary Ion Mass Spectrometry (SIMS). These techniques (shown schematically in Figure 1), have enabled neuroscientists to directly study the distribution of Na + , K + , Mg 2+ , Ca 2+ , and Cl − , in situ within ex vivo tissue sections at cellular and sub-cellular spatial resolution, in conditions such as traumatic brain injury (Chwiej et al, 2011;Lozić et al, 2014), ischemic stroke (Caine et al, 2016;Pushie et al, 2018), hemorrhagic stroke (Hackett et al, 2015b;Williamson et al, 2016), schizophrenia (Lins et al, 2016), and epilepsy (Inamura et al, 1990;Ren et al, 1999;Chwiej et al, 2008;Chwiej et al, 2012a), to name a few (Figure 2). This mini-review highlights the recent applications of XFM, PIXE, LA-ICP-MS, and SIMS, to study the role of ions in healthy brain function and during disease or injury.…”