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Mass Spectrometric Imaging of Ceramides Tracks Therapeutic Response in Traumatic Brain Injury Damon Christopher Barbacci, Aurélie Roux, Ludovic Muller, Shelley N. Jackson, Jeremy Post, Kathrine Baldwin, Barry Hoffer, Carey Balaban, Shawn Gouty, Brian M. Cox, and Amina S. Woods ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00189 • Publication Date (Web): 26 Jul 2017 Downloaded from http://pubs.acs.org on July 27, 2017
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ACS Chemical Neuroscience
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Mass Spectrometric Imaging of Ceramide Biomarkers Tracks Therapeutic Response in
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Traumatic Brain Injury
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Damon Barbacci3; Aurelie Roux1; Ludovic Muller1; Shelley N Jackson1; Jeremy Post1; Kathrine
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Baldwin1; Barry Hoffer5; Carey D Balaban4; J. Albert Schultz3; Shawn Gouty2; Brian M Cox2;
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Amina S. Woods1*
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Abstract: Traumatic brain injury (TBI) is a serious public health problem and the leading cause
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of death in children and young adults. It also contributes to a substantial number of cases of
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permanent disability. As lipids make up over 50% of the brain mass and play a key role in both
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membrane structure and cell signaling, their profile is of particular interest. In this study, we
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show that advanced mass spectrometry imaging (MSI) has sufficient technical accuracy and
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reproducibility to demonstrate the anatomical distribution of 50 micron diameter microdomains
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that show changes in brain ceramide levels in a rat model of controlled cortical impact (CCI) 3
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days post injury with and without treatment. Adult male Sprague-Dawley rats received one
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strike and were euthanized 3 days post trauma. Brain MS images showed increase in ceramides
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in CCI animals compared to control as well as significant reduction in ceramides in CCI treated
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animals demonstrating therapeutic effect of a peptide agonist. The data also suggests the
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presence of diffuse changes outside of the injured area. These results shed light on the extent of
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biochemical and structural changes in the brain after traumatic brain injury and could help to
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evaluate the efficacy of treatments.
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Key Words: Traumatic brain injury, controlled cortical impact, lipids, sphingomyelin, ceramides,
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mass spectrometry imaging, silver nanoparticles implantation, diffuse axonal injury.
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Introduction
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Traumatic brain injury (TBI) research has identified cellular mechanisms involved in
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response to focal or primary, to secondary diffuse injury, and repair. The focal injury causes
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immediate disruption in cell membranes upsetting the normal balance of extra-cellular
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neurotransmitters, homeostasis and medium 1. Intact cells within the focal injury are
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immediately exposed to the stressed environment. Pathways involved in ion transport (Na, K,
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Ca), excitatory amino acids such as glutamate and neurotransmitters are overwhelmed 2–8.
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Cerebral blood flow is also impaired which seemingly has two effects: the focal site retains an
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imbalanced ionic state while the proximal cellular environment is protected from the same fate
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2,5,6,9–12
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cytotoxicity 2,5,6,13–18. Opioid and non-opioid receptors 2,19, cyclooxygenase 4,16, inflammatory
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cytokines 2,4,7,15,20–23 have been investigated.
. Secondary injury involves multiple cellular processes including inflammation and
One non-opioid pathway is NMDA- mediated cell cytotoxicity and NMDA mediated
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dynorphin neurotoxicity. While extra-cellular glutamate and aspartate concentrations are
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elevated in TBI and affect cell potentials, activation of the NMDA receptor by dynorphin is an
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additional pathway to disrupting cell functions 4,8,11,16,24–37. In the central nervous system (CNS),
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dynorphin normally circulates at nanomolar concentrations 2,25,38. However, CNS injury results
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in release of micromolar concentrations of dynorphin resulting in saturation of opioid receptors
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2,38
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resulting in reversal of flow at the membrane causing cell death 2,38. Dynorphin-NR1 subunit
. The excess dynorphin interacts with an epitope of the NR1 subunit of the NMDA receptor
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interaction is driven by strong non-covalent ionic attraction between an acidic epitope of NR1
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and the basic residues of dynorphin. We previously demonstrated that a peptide with the same
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sequence as the NR1 epitope of NMDA receptor, that we termed “decoy peptide” interacts with
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excess dynorphin, thus preventing NMDA mediated dynorphin neurotoxicity 39. It was surmised
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that if dynorphin is involved in TBI, treatment with the decoy peptide could improve outcome in
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injured animals.
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Roux et al 40 first reported that ceramide (CER), diacylglycerol (DAG), cholesterol ester
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(CE), sphingomyelin (SM), and phosphatidylcholine (PC) species abundance changed in the
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region of controlled cortical impact (CCI) and trended with the severity of the injury with respect
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to controls across multiple regions at seven days. In particular, they showed that MSI with
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implanted AgNPs has accuracy and reproducibility to use the 0.5th percentile outliers to the
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control group distribution of m/z peaks as metrics for tracking biochemical sequelae of CCI.
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Ceramides were of particular interest because they are known to be biomarkers of cell death41–44
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and may have the potential for monitoring loci therapeutic treatment if the decoy peptide in fact
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improves outcome in injured animals. This manuscript will illustrate the potential utility of four
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Ceramides m/z 644.4171 CER 34:1, m/z 670.4328 CER 36:2, m/z 674.4481 CER 36:1, and m/z
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700.4797 CER 38:1 as biomarkers for monitoring effects of brain injury and drug treatment in an
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animal model of TBI.
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Results and Discussion Technical improvements in laser desorption MSI with silver nanoparticles permit the
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effects of CCI and treatment to be examined meaningfully by graphical visualization of
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suprathreshold pixels in affected regions and by direct statistical comparisons of m/z peaks
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within brain regions of interest.
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areas above threshold (1220000 counts) are white and peak areas less than or equal to threshold
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are black of CER 36:1 (m/z 674.4481) is presented in Figure 1. Each section image was then
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symmetrically divided into four areas (quadrants) as defined in Figure 1b to enable intra- and
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inter-section statistical evaluation of regional effects of CCI and decoy peptide treatment.
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Quadrant 1 was the dorsal aspect of the section on the side contralateral to the craniotomy in
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control and CCI rats. Quadrant 2 was the ventral half of the section on the side contralateral to
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the craniotomy. On the side ipsilateral to the craniotomy, Quadrant 3 occupied the ventral half
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and Quadrant 4 was the dorsal half of the section, which contained the direct CCI site in the
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impacted animals.
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An illustration of binary image representation where peak
The supra-threshold data for each m/z value serve as an objective, reproducible and
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quantitative guide for counts of spatial features such as the number of supra-threshold pixels,
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total peak area of supra-threshold pixels, and the average peak area per pixel for pixels exceeding
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threshold, per section. A spatial overview of the effects of CCI and decoy peptide treatment on
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regional up regulation of CER species are documented by maps of samples that exceed a
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threshold at the upper 0.5% of the respective control-vehicle group cumulative distribution
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functions (see Figure 1b for example).
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The level of CER 34:1 in control-vehicle and control-treated animals was below the limit
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of detection for all but 0.67% of the pixels (0.62% in the Control-Vehicle and 0.73% in the
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Control-Treated groups). The supra-threshold (upper 0.5%, pixel peak area greater than 0
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counts) distribution was restricted to the choroid plexus and ependymal lining associated with
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the cerebral ventricles in these control-operated animals (Figure 2). CER 34:1 was highly up-
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regulated around the impact site in CCI groups (Figure 2). The average number of supra-
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threshold pixels in the damaged quadrant 4 was elevated significantly in CCI-Vehicle group
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relative to both Control groups (Tukey HSD tests, p=2E-07 and p=6E-07, respectively). The
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CCI-treated animals showed an intermediate number of supra-threshold pixels, indicating
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attenuation of CER 34:1 up-regulation by the decoy peptide. Analyses of the total CER 34:1
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peak areas of the supra-threshold samples (Figure 3) showed regional effects of CCI and
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treatment. In the impacted quadrant of the section (Q4), the total lipid peak was augmented
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significantly in the CCI Vehicle group relative to the Control Treated (Tukey HSD tests, p=1E-
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06) group and the Control Vehicle (Tukey HSD tests, p=6E-07) group. Decoy peptide reduced
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the CCI-induced increase in the total CER 34:1 peak (i.e., relative to the CCI vehicle group,
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Tukey HSD tests, p=4E-04), but it remained greater than the total peak values for the Control
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Treated Q4 and Control Vehicle Q4. Effects of CCI and treatment were not significant in other
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quadrants.
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For CER 38:1 in the Control groups, the supra-threshold (pixel peak area greater than
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306000 counts) sample sites were distributed evenly and diffusely in the neocortex (including
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cingulate cortex) and dorsal thalamus, with a lesser density in the hippocampal formation. The
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CCI sites showed a higher concentration of supra-threshold pixels. The average number of
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supra-threshold pixels in the damaged quadrant Q4 was elevated significantly in CCI-Vehicle
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group relative to the other groups (Tukey HSD tests, p=7E-06, p=0, p=3E-07, respectively).
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Decoy peptide treatment produced a reduction in the distribution and total supra-threshold peak
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magnitude of CER 38:1 in the quadrant that received CCI (Figures 4 and 5). The number of
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supra-threshold pixels in CCI-treated animals did not differ significantly from either Control
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group, indicating that the decoy peptide eliminated CCI-related CER 38:1 up regulation. The
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total peak area data for pixels greater than threshold (Figure 4) was significantly greater in the
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CCI Vehicle group than the two Control injury groups (Tukey HSD tests, p=0 for each
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comparison). The CCI-peptide treated group showed a significant reduction in the CER 38:1
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total peak area in Q4 relative to the CCI-Vehicle group (p=1E-07), and did not differ
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significantly from either Control Treated Q4 or Control Vehicle Q4 group data. Effects of CCI
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and treatment were not significant in other quadrants.
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In the control animals, the samples with supra-threshold peaks for CER 36:1 were most
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concentrated in the caudate-putamen (Figure 6) and the piriform lobe (including amygdala), with
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a scattered distribution in the CA fields of the hippocampal formation. The CCI site showed a
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prominent increase in supra-threshold (pixel peak area greater than 1220000 counts) CER 36:1
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sample sites relative to the Control-Vehicle, Control-Treated and CCI-Treated groups (Tukey
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HSD tests, p=2E-06 and p=7E-06, respectively). The effect was partially attenuated by decoy
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peptide treatment; the CCI-treated group had a greater number of supra-threshold pixels in
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quadrant 4 than either of the Control groups (Tukey HSD tests, p=0.02 and p=0.06, respectively).
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The same pattern of effects was observed for the total peak area data for pixels greater than
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threshold near the CCI site (quadrant 4). The summary in Figure 7 shows the significantly
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higher values in the CCI Vehicle group relative to the CCI Treated, Control Treated, and
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Control Vehicle groups (Tukey HSD tests, p=0.003, p=0, and p=0, respectively). CCI Treated
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Q4 data were intermediate, and were significantly greater than Q4 supra-threshold total peak
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values from either the Control Treated Q4 or the Control Vehicle groups (Tukey HSD tests,
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p=0.013 and p=0.006, respectively). A similar pattern was measured across groups in Q1
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(contralateral to the CCI site). Tukey HSD tests showed that the number of supra-threshold
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pixels was elevated in the CCI-Vehicle group relative to the Control-Vehicle and CCI-Treated
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groups (Tukey HSD, p
