Imaging mass spectrometry to visualize biomolecule distributions in mouse brain tissue following hemispheric cortical spreading depression☆
Graphical abstract
Highlights
► Imaging MS analysis of metabolites, neuropeptides and proteins following cortical spreading depression. ► Imaging MS of biomolecular changes following a transient neurological disorder that does not cause morphological changes. ► 3D imaging MS of peptides and proteins. ► Multivariate analysis of 3D imaging MS datasets.
Introduction
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) can generate biomolecular profiles directly from tissue that contain hundreds of distinct biomolecular ions [1]. Spatially-correlated analysis, imaging MS, can simultaneously record the distribution of each of these ions in heterogeneous tissue samples [2], [3]. There is growing evidence that imaging is having an impact in disease detection and investigation [4], [5], [6]. By combining imaging MS with histology the differential MS profiles found in specific histopathological entities can be used to identify candidate biomarkers [7].
A major advantage of imaging MS is that it can annotate tissues based on their MS profiles and thereby distinguish biomolecularly distinct regions even if they are not distinct using established histological and histochemical methods [8]. Imaging MS-based molecular histology has been used to differentiate histologically overlapping/identical tumors, identify patient subgroups [9], reveal intratumor heterogeneity that may indicate clonal development [9], [10], [11] and uncovered evidence of early infiltration at tumor interface zones [12], [13]. These capabilities offer enormous potential to investigate the biomolecular changes that take place prior to, or without, morphological change, or for which a molecular specific stain is unavailable. This may be especially true for metabolic and neuropeptide changes following transient events, for instance for neurological diseases such as migraine or epilepsy that are defined by their episodic nature, and which may lack histopathological features and stains for specific metabolites/neuropeptides.
In many neurological diseases the pathophysiology is not entirely known and would benefit from systematic investigations of the biomolecular differences between diseased and healthy tissue. Information of such biomolecular differences can guide the search for reliable biomarkers in neurological diseases that are currently lacking.
Cortical Spreading Depression (CSD) is a self-propagating wave of intense neuronal and glial cell depolarization that occurs in one hemisphere of the cerebral cortex which is followed by a marked neuronal silencing [14], [15], which in humans is associated with for instance the aura phase during migraine attacks [16], [17]. Apart from migraine, spreading depression can occur in brain tissue in relation to ischemic insults or seizures [15], [18], [19]. CSD can be easily induced experimentally in animals by local stimulation of the cortex by current injection or by topical application of a high concentration of K+ on the brain surface [15], [17]. When induced in one hemisphere, CSD does not cross to the other hemisphere [15], [20] Previous studies have described a number of neuropeptides and metabolites that are transiently released after CSD such as changes in lactate [21] and glutamate [22]. However the spatial distributions of these peptides and metabolites, and the evolution of their spatiotemporal signatures, have not been established.
Here we report the results of a proof-of-principle imaging MS study of the biomolecular changes following CSD, including metabolites, neuropeptides and proteins. CSD was unilaterally induced, enabling the contralateral hemisphere to be used as an internal control (as introduced by Andrén and workers for Parkinson's disease research [23], [24] and recently used by Hanreider et al. [25]). A SHAM-operated mouse, in which Na+ instead of K+ is applied to the brain surface and which does not result in CSD events, is included to control for possible changes related to the surgical procedures.
Section snippets
CSD experiments
Two male C57Bl/6 J mice of 3 months of age were used. In one animal 7 CSDs were evoked by repeated application of 1 M KCl onto the right visual cortex with a 5-min interval. The other animal received SHAM treatment by repeated application of 1 M NaCl which does not induce CSDs. CSD induction and monitoring was carried out by topical KCl application as described previously [26] with some modifications. In short, surgery was carried out using 1.5% isoflurane anesthesia in surgical air. After
Results and discussion
The non-targeted nature of imaging MS led us to investigate whether it could be used to investigate the chemical and spatial extent of the disturbances that follow CSD, evoked in one hemisphere leaving the other contralateral hemisphere as an internal control. Seven CSDs, spaced 5 min apart, were evoked in one hemisphere of C57Bl/6 J mice, after which the animals were immediately sacrificed and the brains removed and flash frozen (< 1 min postmortem time).
MALDI Imaging MS is able to analyze
Conclusion
The results reported here represent the first 3D MALDI imaging MS that explicitly targets biomolecular changes that are not associated with distinct morphological features, and which includes the first multivariate analysis of 3D MALDI imaging MS datasets. The 2D and 3D imaging MS investigations revealed metabolite and peptide disturbances immediately following CSD in wild-type mice, but the analogous protein datasets did not show similar profound changes. The short time between CSD and animal
Acknowledgements
This work is financially supported by NWO Horizon project 93511027, the ICT consortium COMMIT project ‘e-biobanking with Imaging’, the Cyttron II project ‘Imaging Mass Spectrometry.’, the Vici award 918.56.602, and the Center of Medical System Biology (CMSB) established by the Netherlands Genomics Initiative/Netherlands Organisation for Scientific Research (NGI / NWO) and Community.
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This article is part of a Special Issue entitled: Imaging Mass Spectrometry: A User’s Guide to a New Technique for Biological and Biomedical Research.
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Equal contributions.