This work was supported in part by the Grant-in-Aid for Scientific Research on Innovative Areas (Brain Protein Aging and Dementia Control 26117004; to M.I. and T.M.). This research was partially supported by the Strategic Research Program for Brain Sciences from the Japan Agency for Medical Research and Development (AMED). All experiments were conducted in compliance with the ARRIVE guidelines.

Here, tissue samples were collected at the Tokyo Metropolitan Geriatric Hospital, which provides community-based medical services to the elderly population in Japan. The brain autopsy samples used in the study were registered to the Brain Bank for Aging Research (BBAR) with the informed consent of the deceased&#8217;s relatives. The BBAR is approved by the ethics committee of the Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology. For all brains registered at the brain bank, we obtained written informed consents for their use for medical research from the patients or their families. The patients were placed in a cold (4 &#176;C) room within 2 h after death to reduce postmortem changes in the brain. All methods described here have been approved by Doshisha University and the Brain Bank for Aging Research, Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology.
1. Preparation of Tissue Sections for IMS
<ol>
	<li>Processing tissue specimen of a human autopsied brain 
	Note: Ensure that the sample preparation step for IMS preserves the original state of the tissue. Avoid contamination and post-mortem changes. The following step is crucial.
	<ol>
		<li>Obtain human cortical specimens for IMS from brains that were removed, processed, and stored at -80 &#176;C within 8 h postmortem. Take the brain specimen from the occipital cortex of AD patients and age-matched controls21.</li>
	</ol>
	</li>
	<li>Preparation of frozen tissue sections
	<ol>
		<li>Cut the tissue sections on a cryostat. Place conductive ITO-coated microscope glass slides inside the cryostat21.</li>
		<li>Warm the autopsy brain specimen from -80 &#176;C to -22 &#176;C inside the cryostat.</li>
		<li>Attach a new disposable blade to the cryostat for every experiment. Always try to use a clean part of the blade.</li>
		<li>Put the frozen autopsy brains on the stage along with a small amount of OCT compound (enough to cover the central area of the stage; see Table of Materials).</li>
		<li>Choose the conditions for thin sectioning. For IMS, use a thickness of 10&#8211;12 &#956;m for human brain sections. For IMS and IHC, cut five to six sections from each tissue sample.</li>
		<li>When the blade is just starting to cut the tissue, turn the wheel and &#8220;face&#8221; the block until all of the tissue is exposed. If there is a small streak or tear across the section, wait a little more in the cryostat until the temperature adjustment automatically fixes it. Count a few seconds before opening the anti-roll with a tissue underneath.</li>
		<li>Immediately place the tissue slice on the ITO-coated side of the glass slide. Thaw the tissue slice by putting a finger underneath the slide on the non-ITO-coated side. The tissue will stick to the slide; ensure that the tissue is as flat as possible with no wrinkles. Perform this step at room temperature. 
		Note: Wear gloves, masks, and a laboratory gown because the human samples may contain biological contaminants. When all sections have been made for the day, clean the cryostat, brushes, and chucks with laboratory wipes and 200 mL of 100% ethanol.</li>
	</ol>
	</li>
	<li>Rinsing the tissue sections
	<ol>
		<li>Immerse the samples in 40&#8211;100 mL of 70% ethanol for 30 s to remove endogenous lipids and inorganic salts. Use a glass staining jar.</li>
		<li>Wash the samples with 40&#8211;100 mL of 100% ethanol for 30 s, 40&#8211;100 mL of Carnoy&#8217;s solution for 3 min, 40&#8211;100 mL of 100% ethanol for 30 s, 40&#8211;100 mL of 0.1% trifluoroacetic acid (TFA) for 1 min, and 40&#8211;100 mL of 100% ethanol for 30 s. Use a glass staining jar. 
		Note: Carnoy&#39;s solution is a fixative composed of six parts ethanol, three parts acetic acid, and one part chloroform.</li>
		<li>Dry in a vacuum for 30 min.</li>
	</ol>
	</li>
	<li>Treatment of the tissue sections with a formic acid vapor for a better ionization of the A&#946; proteins from autopsy brain tissues
	<ol>
		<li>Prepare the oven and incubation glass slide to be used for the subsequent vaporization with 5 mL of 100% formic acid. To achieve a satisfactory acid treatment, keep the air humidity in the incubation glass dish at saturation level throughout this step and keep the temperature at 60 &#176;C. Place the tissue slides in the incubation glass dish while avoiding submersion in the formic acid and treat for 6 min.</li>
		<li>Take an optical image of the samples using a film scanner, gel scanner, or a digital microscope, etc. Perform this step at room temperature. The alignment of the optical image of the samples is necessary when the sample target is placed inside the instrument. Usually, it will not be possible to recognize the tissue section underneath the matrix layer. 
		NOTE: The most convenient way to take an optical image is to use an office scanner. It is a good idea to save the image in the imaging data storage folder.</li>
		<li>To correlate the optical images with the samples, make guide marks that are visible both in the optical image and underneath the matrix layer in the camera optic. The easiest way is to spot at least three correction fluid marks around the sample before taking the optical image.</li>
	</ol>
	</li>
	<li>Matrix application
	<ol>
		<li>Preparation of the matrix solution
		<ol>
			<li>In an organic solvent-tolerant microtube, prepare a 10 mg/mL SA solution in 50% acetonitrile (ACN) and 0.1% TFA. Thoroughly dissolve the SA compound by vortexing or brief sonication for 10 min. Store the solution at room temperature until use. 
			NOTE: There are three different options for matrix spraying: using an airbrush, an ultrasonic sprayer, or an automatic sprayer.</li>
		</ol>
		</li>
		<li>Spraying the matrix with an airbrush
		<ol>
			<li>Perform the operation at a constant room temperature (20&#8211;23 &#176;C) and humidity (40%&#8211;60%). The parameters to adjust for optimal spraying include the size of the droplet, the amount of mist, the angle and distance between the spray nozzle and the tissue section, and the laboratory temperature and humidity. Adjust these conditions by checking the microscopy results. 
			Note: As limiting factors include the crystal size and homogeneity of the matrix coverage and the undesirable migration/diffusion of analytes, smaller is better for the drop size. Homogeneity is also the point of inspection.</li>
		</ol>
		</li>
		<li>Spraying the matrix with an ultrasonic sprayer
		<ol>
			<li>Remove the tissue to be sprayed from the desiccator and place it in the chamber. Make sure the tissue is not covering the sensor window.</li>
			<li>Start the preparation by pushing the Start button; usually, prep time is around 90 min. The preparation will be regulated automatically via the monitoring of the matrix layer thickness and wetness. After the preparation is complete, remove the slide and store it in the desiccator for 15 min before reading it in the MALDI instrument.</li>
			<li>Clean the sprayer with 2&#8211;3 mL of 100% MeOH until the spray head appears clean. 
			Note: A fine mist of matrix droplets is allowed to sink into the tissue by a gravitational sheet. An average droplet size of 20 &#956;m is generated; all droplet diameters are less than 50 &#956;m.</li>
		</ol>
		</li>
		<li>Spraying the matrix with an automatic sprayer
		<ol>
			<li>Spray the matrix solution on the tissue surface with an automatic sprayer. A constant &#64258;ow of heated sheath gas (N2, set at 10 psi and 75 &#176;C) will be delivered conjointly with the matrix solution spray. Use a solvent pump system (set at 10 psi and 0.15 mL/min) to deliver the matrix solution. 
			Note: Most importantly, work under a safety cabinet to avoid any inhalation of matrix aerosols. Control the room temperature and humidity to reproduce homogenous matrix crystallization.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. MALDI-IMS
<ol>
	<li>Perform ultra-high-speed mass spectrometry.
	<ol>
		<li>Perform high-throughput and high-spatial-resolution imaging experiments with MALDI-IMS equipped with a 10 kHz Nd:YAG (355 nm) laser.</li>
		<li>For mass spectrometry measurements, define the tissue areas using the MALDI control software and data analysis software.</li>
		<li>Acquire spectra in a positive linear mode with a mass range of m/z 2,000&#8211;20,000 and a spatial resolution of 20 and 100 &#181;m.</li>
		<li>To make the calibration standard, dissolve the peptide calibration standard and the protein calibration standard with a ratio of 1:4 with alpha-Cyano-4-hydroxyl-cinnamic acid (CHCA) in TA30 solution (ACN:0.1% TFA = 30:70) and then dilute it 10x. Place 1 &#956;L of calibration standard on the slide at four different locations.</li>
	</ol>
	</li>
	<li>Using molecular histology software (see Table of Materials), overlay multiple single images to find the spatial correlation of various signals, such as different A&#946; peptides colocalizing in SPs and arterial walls.</li>
</ol>
3. Data Processing
<ol>
	<li>For spectral alignment, align all spectra to a selected m/z list from a previous publication21.</li>
	<li>Import the MALDI-IMS data set into statistical analysis software (see Table of Materials) with baseline subtraction.</li>
	<li>Trace regions of interest (ROIs) based on histological knowledge.
	<ol>
		<li>Perform a univariate analysis for the mean intensities, standard deviations, uncovering of discriminative m/z-markers (ROC analysis), hypothesis tests, and discovery of colocalized m/z values.</li>
	</ol>
	</li>
	<li>Create a segmentation map.
	<ol>
		<li>Perform an unsupervised multivariate analysis for the spatial segmentation of large datasets and a component analysis for the extraction of underlying trends.</li>
		<li>Select anatomical ROIs based on histological characteristics, such as parenchyma and the subarachnoid space.</li>
		<li>Assign structures as individual ROIs and correlate them with the processed MS data in order to identify the associated peptides that allowed the image segmentation of the region.</li>
	</ol>
	</li>
</ol>

<ol><li>Kovacs, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+Pathological+Classification+of+Neurodegenerative+Diseases%3A+Turning+towards+Precision+Medicine%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Sciences%22%5BJournal%5D)">Molecular Pathological Classification of Neurodegenerative Diseases: Turning towards Precision Medicine.</a> International Journal of Molecular Sciences. 17, E189(2016).</li>
<li>Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alzheimer%27s+disease%3A+genes%2C+proteins%2C+and+therapy%5BTitle%5D)+AND+%22Physiological+Reviews%22%5BJournal%5D)">Alzheimer's disease: genes, proteins, and therapy.</a> Physiological Reviews. 81, 741-766 (2001).</li>
<li>Selkoe, D. J., Hardy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+amyloid+hypothesis+of+Alzheimer%27s+disease+at+25+years%5BTitle%5D)+AND+%22EMBO+Molecular+Medicine%22%5BJournal%5D)">The amyloid hypothesis of Alzheimer's disease at 25 years.</a> EMBO Molecular Medicine. 8, 595-608 (2016).</li>
<li>Braak, H., Braak, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuropathological+stageing+of+Alzheimer-related+changes%5BTitle%5D)+AND+%22Acta+Neuropathologica%22%5BJournal%5D)">Neuropathological stageing of Alzheimer-related changes.</a> Acta Neuropathologica. 82, 239-259 (1991).</li>
<li>Braak, H., Alafuzoff, I., Arzberger, T., Kretzschmar, H., Del Tredici, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Staging+of+Alzheimer+disease-associated+neurofibrillary+pathology+using+paraffin+sections+and+immunocytochemistry%5BTitle%5D)+AND+%22Acta+Neuropathologica%22%5BJournal%5D)">Staging of Alzheimer disease-associated neurofibrillary pathology using paraffin sections and immunocytochemistry.</a> Acta Neuropathologica. 112, 389-404 (2006).</li>
<li>Bateman, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dominantly+Inherited+Alzheimer+Network.+Clinical+and+biomarker+changes+in+dominantly+inherited+Alzheimer%27s+disease%5BTitle%5D)+AND+%22The+New+England+Journal+of+Medicine%22%5BJournal%5D)">Dominantly Inherited Alzheimer Network. Clinical and biomarker changes in dominantly inherited Alzheimer's disease.</a> The New England Journal of Medicine. 367, 795-804 (2012).</li>
<li>Iwatsubo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualization+of+A+beta+42%2843%29+and+A+beta+40+in+senile+plaques+with+end-specific+A+beta+monoclonals%3A+evidence+that+an+initially+deposited+species+is+Abeta+42%2843%29%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Visualization of A beta 42(43) and A beta 40 in senile plaques with end-specific A beta monoclonals: evidence that an initially deposited species is Abeta 42(43).</a> Neuron. 13, 45-53 (1994).</li>
<li>Reinert, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deposition+of+C-terminally+truncated+A%CE%B2+species+A%CE%B237+and+A%CE%B239+in+Alzheimer%27s+disease+and+transgenic+mouse+models%5BTitle%5D)+AND+%22Acta+Neuropathologica+Communications%22%5BJournal%5D)">Deposition of C-terminally truncated Aβ species Aβ37 and Aβ39 in Alzheimer's disease and transgenic mouse models.</a> Acta Neuropathologica Communications. 4, 24(2016).</li>
<li>Saido, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dominant+and+differential+deposition+of+distinct+beta-amyloid+peptide+species%2C+A+beta+N3%28pE%29%2C+in+senile+plaques%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Dominant and differential deposition of distinct beta-amyloid peptide species, A beta N3(pE), in senile plaques.</a> Neuron. 14, 457-466 (1995).</li>
<li>Harigaya, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amyloid+beta+protein+starting+pyroglutamate+at+position+3+is+a+major+component+of+the+amyloid+deposits+in+the+Alzheimer%27s+disease+brain%5BTitle%5D)+AND+%22Biochemical+and+Biophysical+Research+Communications%22%5BJournal%5D)">Amyloid beta protein starting pyroglutamate at position 3 is a major component of the amyloid deposits in the Alzheimer's disease brain.</a> Biochemical and Biophysical Research Communications. 276, 422-427 (2000).</li>
<li>Vinters, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cerebral+amyloid+angiopathy.+A+critical+review%5BTitle%5D)+AND+%22Stroke%22%5BJournal%5D)">Cerebral amyloid angiopathy. A critical review.</a> Stroke. 18, 311-324 (1987).</li>
<li>Saito, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Potent+amyloidogenicity+and+pathogenicity+of+A%CE%B243%5BTitle%5D)+AND+%22Nature+Neuroscience%22%5BJournal%5D)">Potent amyloidogenicity and pathogenicity of Aβ43.</a> Nature Neuroscience. 14, 1023-1032 (2011).</li>
<li>Sergeant, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Truncated+beta-amyloid+peptide+species+in+pre-clinical+Alzheimer%27s+disease+as+new+targets+for+the+vaccination+approach%5BTitle%5D)+AND+%22Journal+of+Neurochemistry%22%5BJournal%5D)">Truncated beta-amyloid peptide species in pre-clinical Alzheimer's disease as new targets for the vaccination approach.</a> Journal of Neurochemistry. 85, 1581-1591 (2003).</li>
<li>Suzuki, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+tissue+content+of+soluble+beta+1-40+is+linked+to+cerebral+amyloid+angiopathy%5BTitle%5D)+AND+%22The+American+Journal+of+Pathology%22%5BJournal%5D)">High tissue content of soluble beta 1-40 is linked to cerebral amyloid angiopathy.</a> The American Journal of Pathology. 145 (2), 452-460 (1994).</li>
<li>Miyasaka, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualization+of+newly+deposited+tau+in+neurofibrillary+tangles+and+neuropil+threads%5BTitle%5D)+AND+%22Journal+of+Neuropathology+%26+Experimental+Neurology%22%5BJournal%5D)">Visualization of newly deposited tau in neurofibrillary tangles and neuropil threads.</a> Journal of Neuropathology & Experimental Neurology. 64, 665-674 (2005).</li>
<li>Portelius, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mass+spectrometric+characterization+of+brain+amyloid+beta+isoform+signatures+in+familial+and+sporadic+Alzheimer%27s+disease%5BTitle%5D)+AND+%22Acta+Neuropathologica%22%5BJournal%5D)">Mass spectrometric characterization of brain amyloid beta isoform signatures in familial and sporadic Alzheimer's disease.</a> Acta Neuropathologica. 120, 185-193 (2010).</li>
<li>Kelley, A. R., Perry, G., Castellani, R. J., Bach, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Laser-Induced+In-Source+Decay+Applied+to+the+Determination+of+Amyloid-Beta+in+Alzheimer%27s+Brains%5BTitle%5D)+AND+%22ACS+Chemical+Neuroscience%22%5BJournal%5D)">Laser-Induced In-Source Decay Applied to the Determination of Amyloid-Beta in Alzheimer's Brains.</a> ACS Chemical Neuroscience. 7, 261-268 (2016).</li>
<li>Stoeckli, M., Staab, D., Staufenbiel, M., Wiederhold, K. H., Signor, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+imaging+of+amyloid+beta+peptides+in+mouse+brain+sections+using+mass+spectrometry%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">Molecular imaging of amyloid beta peptides in mouse brain sections using mass spectrometry.</a> Analytical Biochemistry. , 33-39 (2002).</li>
<li>Stoeckli, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MALDI+MS+imaging+of+amyloid%5BTitle%5D)+AND+%22Methods+in+Enzymology%22%5BJournal%5D)">MALDI MS imaging of amyloid.</a> Methods in Enzymology. 412, 94-106 (2006).</li>
<li>Seeley, E. H., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+imaging+of+proteins+in+tissues+by+mass+spectrometry%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Molecular imaging of proteins in tissues by mass spectrometry.</a> Proceedings of the National Academy of Sciences of the United States of America. 105, 18126-18131 (2008).</li>
<li>Kakuda, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distinct+deposition+of+amyloid-%CE%B2+species+in+brains+with+Alzheimer%E2%80%99s+disease+pathology+visualized+with+MALDI+imaging+mass+spectrometry%5BTitle%5D)+AND+%22Acta+Neuropathologica+Communications%22%5BJournal%5D)">Distinct deposition of amyloid-β species in brains with Alzheimer’s disease pathology visualized with MALDI imaging mass spectrometry.</a> Acta Neuropathologica Communications. 5, 73(2017).</li>
<li>Weller, R. O., Djuanda, E., Yow, H. Y., Carare, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lymphatic+drainage+of+the+brain+and+the+pathophysiology+of+neurological+disease%5BTitle%5D)+AND+%22Acta+Neuropathologica%22%5BJournal%5D)">Lymphatic drainage of the brain and the pathophysiology of neurological disease.</a> Acta Neuropathologica. 117, 1-14 (2009).</li>
<li>Weller, R. O., Boche, D., Nicoll, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microvasculature+changes+and+cerebral+amyloid+angiopathy+in+Alzheimer%27s+disease+and+their+potential+impact+on+therapy%5BTitle%5D)+AND+%22Acta+Neuropathologica%22%5BJournal%5D)">Microvasculature changes and cerebral amyloid angiopathy in Alzheimer's disease and their potential impact on therapy.</a> Acta Neuropathologica. 118, 87-102 (2009).</li>
<li>Weller, R. O., Subash, M., Preston, S. D., Mazanti, I., Carare, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perivascular+drainage+of+amyloid-beta+peptides+from+the+brain+and+its+failure+in+cerebral+amyloid+angiopathy+and+Alzheimer%27s+disease%5BTitle%5D)+AND+%22Brain+Pathology%22%5BJournal%5D)">Perivascular drainage of amyloid-beta peptides from the brain and its failure in cerebral amyloid angiopathy and Alzheimer's disease.</a> Brain Pathology. 18, 253-266 (2008).</li>
<li>Morris, A. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vascular+basement+membranes+as+pathways+for+the+passage+of+fluid+into+and+out+of+the+brain%5BTitle%5D)+AND+%22Acta+Neuropathologica%22%5BJournal%5D)">Vascular basement membranes as pathways for the passage of fluid into and out of the brain.</a> Acta Neuropathologica. 131, 725-736 (2016).</li>
<li>Hawkes, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perivascular+drainage+of+solutes+is+impaired+in+the+ageing+mouse+brain+and+in+the+presence+of+cerebral+amyloid+angiopathy%5BTitle%5D)+AND+%22Acta+Neuropathologica%22%5BJournal%5D)">Perivascular drainage of solutes is impaired in the ageing mouse brain and in the presence of cerebral amyloid angiopathy.</a> Acta Neuropathologica. 121, 431-443 (2011).</li>
<li>Kakuda, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Equimolar+production+of+amyloid+beta-protein+and+amyloid+precursor+protein+intracellular+domain+from+beta-carboxyl-terminal+fragment+by+gamma-secretase%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Equimolar production of amyloid beta-protein and amyloid precursor protein intracellular domain from beta-carboxyl-terminal fragment by gamma-secretase.</a> Journal of Biological Chemistry. 281, 14776-14786 (2006).</li>
<li>Matsumura, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%CE%B3-Secretase+associated+with+lipid+rafts%3A+multiple+interactive+pathways+in+the+stepwise+processing+of+%CE%B2-carboxyl-terminal+fragment%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">γ-Secretase associated with lipid rafts: multiple interactive pathways in the stepwise processing of β-carboxyl-terminal fragment.</a> Journal of Biological Chemistry. 289, 5109-5121 (2014).</li>
<li>Morishima-Kawashima, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+apolipoprotein+E+allele+epsilon4+on+the+initial+phase+of+amyloid+beta-protein+accumulation+in+the+human+brain%5BTitle%5D)+AND+%22The+American+Journal+of+Pathology%22%5BJournal%5D)">Effect of apolipoprotein E allele epsilon4 on the initial phase of amyloid beta-protein accumulation in the human brain.</a> The American Journal of Pathology. 157, 2093-2099 (2000).</li>
<li>Takami, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%CE%B3-Secretase%3A+Successive+tripeptide+and+tetrapeptide+release+from+the+transmembrane+domain+of+%CE%B2-carboxyl+terminal+fragment%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%22%5BJournal%5D)">γ-Secretase: Successive tripeptide and tetrapeptide release from the transmembrane domain of β-carboxyl terminal fragment.</a> The Journal of Neuroscience. 29, 13042-13052 (2009).</li>
<li>Kakuda, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Altered+%CE%B3-secretase+activity+in+mild+cognitive+impairment+and+Alzheimer%27s+disease%5BTitle%5D)+AND+%22EMBO+Molecular+Medicine%22%5BJournal%5D)">Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.</a> EMBO Molecular Medicine. 4, 344-352 (2012).</li>
<li>Carlred, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Probing+amyloid-b+pathology+in+transgenic+Alzheimer%E2%80%99s+disease+%28tgArcSwe%29+mice+using+MALDI+imaging+mass+spectrometry%5BTitle%5D)+AND+%22Journal+of+Neurochemistry%22%5BJournal%5D)">Probing amyloid-b pathology in transgenic Alzheimer’s disease (tgArcSwe) mice using MALDI imaging mass spectrometry.</a> Journal of Neurochemistry. 138, 469-478 (2016).</li>
</ol>

The authors have nothing to disclose.

Here we demonstrated a detailed protocol and results of the visualization of A&#946; and its isoforms from several autopsied brains with AD and CAA with MALDI-IMS. The deposition profile of A&#946;s was drastically changed from A&#946;1-42 to its N- and C-terminal variations. A&#946;1-41 was first identified and visualized in human brains with the current protocol and was further validated with IHC21. Considering that the morphology of deposited A&#946; by IMS with a high-resolution analysis (20 &#956;m) must be in good agreement with IHC, IMS and IHC equally contribute to distinguishing A&#946; deposits by their location and protein contents, as well as by their morphology. As the entire experiment described here was conducted in the occipital cortex, studying the localization of different beta-amyloid species across all brain regions will be generalized with future experiments using the current protocol.
Critical steps within the protocol are tissue preparation steps to obtain an effective ionization of aggregated proteins in human brain tissues. A matrix layer is required to absorb the laser energy and induce desorption and ionization of analytes. In this process, an entire tissue section is homogeneously coated with small crystals. Homogenous cocrystallization of the analyte with the matrix is crucial for high-sensitivity and artifact-free imaging. Each of the three spray methods has its own advantages. Manual spray coating is one of the most frequently used methods. An airbrush is convenient; however, it requires skillful operation. As precise and reproducible experimental technique is essential, using an ultrasonic sprayer and/or an automatic sprayer as described in the current protocols is recommended. With respect to an ultrasonic sprayer, it will not be influenced by the humidity and temperature of the room because it is sprayed into a chamber. Meanwhile, with an automatic sprayer, relatively preserved spatial resolution with good reproducibility is obtained. Generally, the spatial resolution obtained with the three methods increase in the order of 1) airbrush, 2) automatic device, and 3) ultrasonic sprayer.
Most importantly, this protocol was originally generated to detect and visualize A&#946;s in human autopsied brain samples. The visualization of A&#946;s with MALDI-IMS for APP23 mice, which is generated as an animal model of AD based on the Swedish-type mutation of human APP, has been reported earlier by others with an existing method18,19. However, the former protocol applied to APP23 was not sufficient to visualize A&#946; in its lateral resolution and sensitivity. Earlier works discussed that high A&#946; concentrations outside the tissue boundary are clearly artifacts in APP23 imaging with their protocol18,19. That means the so-called &#39;blur&#39; between real SPs and IMS images due to the matrix extraction step was inevitable with MALDI-type imaging. However, in the current protocol, the blur disappeared and each spectrum represented every single SP in the brain parenchyma.
As shown here, we can trace A&#946;1-41 for the first time in AD and CAA brains with MALDI-IMS, as well as with IHC, with a specific antibody by our own generation21. According to the A&#946; processing model, A&#946;1-38 derives from A&#946;1-45 via A&#946;1-42, while A&#946;1-41 derives from A&#946;1-45 by &#947;-secretase stepwise cleavage27,28,29,30,31. This means that the current protocol supports this model. As for the limitations of this technique, we must consider the heterogeneity of the samples from human autopsy brain. The most critical step, in a sense, is to assess qualified autopsy brain tissue with ethical proof. With the current protocol on those qualified autopsy brain tissues, MALDI-IMS can individually track the whole distribution of the complex molecules having multiple modifications, as well as unknown factor(s) regulating the pathogenesis of AD, that are yet to be defined. Furthermore, in understanding the overall pathogenesis of various neuropathology in aged human brains, it must be feasible to adopt MALDI-IMS as a standard approach, in combination with clinical, genetic, and pathological observations in neurological diseases.
Another critical step of MALDI-IMS is the data mining process from the obtained data set, which is always time-consuming. Manual data mining of each peak distribution requires the users to click through every image and look for distributions that may correlate to the morphology of the analyzed sample. Automatic spatial segmentation can be used as the first step of data mining, providing an overview of the data set and allowing the quick detection of prominent features. In this approach, similarities between spectra of a given region are statistically determined, and similar spectra are grouped into one cluster. All pixels are color-coded according to their cluster assignment (Figure 5). In the present AD/CAA study, the area of interest is the space of A&#946; depositions in parenchymal plaque and the subarachnoid and parenchymal vascular structures. The two distinctive peaks which were further validated with IHC were the m/z values from A&#946;1-40 and A&#946;1-4221. Thus, it is easy to find colocalized m/z with A&#946;1-40 and A&#946;1-42 which was already annotated to N- and C-terminal truncated A&#946;, as well as unknown peptides, for further analysis.
Weller and colleagues have reported that A&#946; accumulates in vessel walls, more around arteries than around veins21. Moreover, it has been proposed that the interstitial fluid (ISF) includes A&#946;s excreted from the cerebral parenchyma to the lymph node via a perivascular drainage pathway22,23,24,25,26. The current protocol to generate a segmentation map based on a MALDI-IMS data set at a 20 &#181;m resolution supports the possible existence of perivascular drainage pathways of the brain (Figure 5), which contribute significantly to CAA in AD21,32. Furthermore, we can discover marker proteins colocalized with plaque and subarachnoid vasculature by calculating the correlation of each m/z values. In understanding the overall pathogenesis of various neuropathology in aged human brains, it must be feasible to adopt MALDI-IMS as a powerful approach in combination with established IHC data of clinical, genetic, and pathological observations in neurological diseases.

In order to make the diagnosis and to understand the pathogenesis of neurodegenerative disorders, precise molecular identification of pathological deposits is essential1. In the course of AD, A&#946; is produced to make SPs in the cerebral parenchyma and deposited in vessels long before disease onset2,3,4,5,6. While A&#946;1-42 is the predominant peptide in the SP of AD brains, other A&#946; variants, such as N-terminal or C-terminal truncated or modified A&#946;s, are also identified in affected AD brains7,8,9,10. A full picture of the broad range of A&#946; species in human brains, especially with AD and cerebral amyloid angiopathy (CAA)11, will help scientists understand A&#946; production, metabolism, and deposition.
As the classical approach of neuropathology, immunohistochemistry (IHC) has been the most conclusive method to determine the location of A&#946;s in the brain tissues12,13,14,15. In general, IHC cannot distinguish molecules when several epitopes coexist simultaneously. By contrast, an emerging mass spectrometry-based proteomic analysis is a valuable approach especially for analyzing a variety of A&#946; species in brain tissues, which cannot be differentiated with antibodies16,17. The conventional mass spectrometry-based analysis of brain lysates and immuno-precipitated samples fails to detect minor A&#946; peptides and loses distribution information of A&#946; in the brain tissue.
In earlier works, visualizing A&#946; deposits in mouse brains has been successful using a transgenic animal model of AD, such as APP23. However, this process still needs technical advancements to compare IMS and IHC with respect to resolution and sensitivity18,19,20. AD neuropathology should be studied on human brains, and we used MALDI-IMS technology on human autopsy brain tissues to obtain comprehensive protein mapping21. For this purpose, we developed a protocol for an advanced type of mass spectrometry that has advantages in its rapidity, sensitivity, and reproducibility.

<table><tbody><tr><td>Cryostat</td><td>Leica Microsystems, Wetzlar, Germany</td><td>CM1950</td><td></td></tr><tr><td>Indium tin oxide (ITO)-coated microscope glass slide</td><td>Bruker Daltonics</td><td>#237001</td><td></td></tr><tr><td>Blade (disposable)</td><td>Leica Microsystems, Wetzlar, Germany</td><td>#117394</td><td></td></tr><tr><td>O.C.T. Compound</td><td>Leica Microsystems, Wetzlar, Germany</td><td>FSC 22 Blue</td><td></td></tr><tr><td>Scanner</td><td>EPSON</td><td>GT-980</td><td></td></tr><tr><td>Air-Brush</td><td>GSI Creos</td><td>PS274</td><td></td></tr><tr><td>Compressor</td><td>MRHOBBY</td><td>Mr.Linear compressor L5</td><td></td></tr><tr><td>Ultrasonic sprayer</td><td>Bruker Daltonics</td><td>ImagePrep</td><td></td></tr><tr><td>Automatic sprayer</td><td>HTX Technologies</td><td>TM Sprayer</td><td></td></tr><tr><td>Confocal-laser-scanning-microscope</td><td>Carl Zeiss Inc.</td><td>LSM 700</td><td></td></tr><tr><td>Ultra high speed MALDI instrument</td><td>Bruker Daltonics</td><td>rapifleX MALDI Tissuetyper</td><td></td></tr><tr><td>MALDI control software</td><td>Bruker Daltonics</td><td>FlexControl 3.8</td><td></td></tr><tr><td>Data analysis software</td><td>Bruker Daltonics</td><td>FlexImaging 5.0</td><td></td></tr><tr><td>Molecular histology software</td><td>SCiLS, Bremen, Germany</td><td>SciLS Lab 2016a</td><td></td></tr><tr><td>Statistical software</td><td>SCiLS, Bremen, Germany</td><td>SciLS Lab 2016a</td><td></td></tr><tr><td>Sinapinic acid (SA)</td><td>Nacalai tesque</td><td>30494-91</td><td></td></tr><tr><td>Alpha-Cyano-4-hydroxyl-cinnamic acid (CHCA)</td><td>Wako</td><td>037-19261</td><td></td></tr><tr><td>Calibration standard</td><td>Bruker Daltonics</td><td></td><td></td></tr><tr><td>Biotinylated anti-mouse IgG antibodies</td><td>Vector Laboratories, Inc., Burlingame, CA</td><td></td><td></td></tr><tr><td>Biotinylated anti-rabbit IgG antibodies</td><td>Vector Laboratories, Inc., Burlingame, CA</td><td></td><td></td></tr><tr><td>Avidin and biotinylated HRP complex.</td><td>Vector Laboratories, Inc., Burlingame, CA</td><td>Vectastain Elite ABC kit</td><td></td></tr><tr><td>3,3-diaminobenzidine (DAB)</td><td>Vector Laboratories, Inc., Burlingame, CA</td><td>Vectastain Elite ABC kit</td><td></td></tr></tbody></table>

visualization of amyloid &#946; deposits in the human brain with matrix-assisted laser desorption/ionization imaging mass spectrometry

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into extracellular plaques of the brain. The A&#946; peptides, composed of 40 amino acids, are generated from amyloid precursor proteins (APP) by &#946;- and &#947;-secretases. A&#946; is deposited not only in cerebral parenchyma but also in leptomeningeal and cerebral vessel walls, known as cerebral amyloid angiopathy (CAA). While a variety of A&#946; peptides were identified, the detailed production and distribution of individual A&#946; peptides in pathological tissues of AD and CAA have not been fully addressed. Here, we develop a protocol of matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) on human autopsy brain tissues to obtain comprehensive protein mapping. For this purpose, human cortical specimens were obtained from the Brain Bank at the Tokyo Metropolitan Institute of Gerontology. Frozen cryosections are cut and transferred to indium-tin-oxide (ITO)-coated glass slides. Spectra are acquired using the MALDI system with a spatial resolution up to 20 &#181;m. Sinapinic acid (SA) is uniformly deposited on the slide using either an automatic or a manual sprayer. With the current technical advantages of MALDI-IMS, a typical data set of various A&#946; species within the same sections of human autopsied brains can be obtained without specific probes. Furthermore, high-resolution (20 &#181;m) imaging of an AD brain and severe CAA sample clearly shows that A&#946;1-36 to A&#946;1-41 were deposited into leptomeningeal vessels, while A&#946;1-42 and A&#946;1-43 were deposited in cerebral parenchyma as senile plaque (SP). It is feasible to adopt MALDI-IMS as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD, CAA, and other neurological diseases based on the current strategy.

Sporadic AD patients with CAA (n = 5; mean age = 83.2 y) and aged subjects with senile plaque free (SP O) and CAA (n = 5; mean age = 77.2 y) were analyzed (Table 1). The CAA phenotypes of patient #3 were most prominent in this study. Distributions of A&#946;1-40 and A&#946;1-42 deposits in the brain tissue from patient #3 were visualized with MALDI-IMS (Figure 1). There were no significant signals in nonpathological control brains (patients #9 and #10), as shown in Figure 1. Here, MALDI-IMS clearly visualized that A&#946;1-42 was preferentially deposited as SPs in the cerebral parenchyma. By contrast, shorter A&#946;s, such as A&#946;1-36 to 1-41, were preferentially deposited on the leptomeningeal vascular areas (Figure 1).
Distributions of A&#946;40 and A&#946;42 were further validated with IHC using adjacent frozen sections of the tissues. The anti-A&#946;40 antibody labeled CAA (arrows in Figure 2B), which is in clear contrast to the distribution of A&#946;42 in the cerebral parenchyma as SPs. For A&#946;1-41, we are the first to detect this fragment in human brains, and we have generated specific antibodies that can differentiate A&#946;41 from A&#946;40 andA&#946;4221.
The spatial resolution of the MALDI-IMS is generally the most important factor to be improved upon. In Figure 1 and Figure 2 show MALDI-IMS with a 100 &#181;m pitch resolution and obtain an overall distribution profile for a relatively wide area21. It is difficult to define amyloid deposition exactly at the subarachnoid space, including vascular structure. To portray fine tissue structures of subarachnoid vessels and the surface of the cortex, high-resolution MALDI imaging (40 &#181;m: Figure 3; 20 &#181;m: Figure 4 and Figure 5) was performed. As a result, MALDI-IMS clearly demonstrated that shorter A&#946;s, such as A&#946;1-36 to 1-41, are distributed in the walls of arteries, which is comparable with IHC.
MALDI-IMS demonstrated the detailed distributions of both A&#946;x-40 and A&#946;x-42 (x = 2, 4, 5, 6, 7, 8, 9, and 11pE) in AD accompanied with moderate CAA brain. For example, while A&#946;x-40 species showed a similar distribution profile to A&#946;1-40, A&#946;x-42 species showed a different distribution pattern with A&#946;1-4221. By the current protocol, single ion images of the individual A&#946; peptides observed with MALDI-IMS are assigned to A&#946; species. By contrast, acquired image data can be investigated using unsupervised multivariate statistics in order to obtain image segmentation of anatomical regions of interest for the further analysis of undefined proteins. Figure 5 shows a segmentation map obtained with a bisecting k-means analysis applied to the same section from patient #321. This clustering method successfully identified plaque-like structures in the parenchyma (blue in Figure 5C) and vascular structures in the subarachnoid space (green in Figure 5C). It is interesting to find a small circular area in the parenchyma, which is detected just around a small arteriole in the parenchyma, as well as in the subarachnoid space (purple in Figure 5C). This is verified by single ion images of these individual A&#946; peptides in Figure 5D-5F.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57645/57645fig1.jpg" /> 
Figure 1: MALDI-IMS of a frozen AD/CAA brain section. Various C-terminal truncated A&#946; peptides in AD accompanying severe CAA (patient #3) are visualized in the left panel and controls (patient #9 on the right and patient #10 on the left in all images) in the right panel. A&#946;1-36 to A&#946;1-41 are preferentially deposited in leptomeningeal blood vessels, while A&#946;1-42 and A&#946;1-43 are deposited in the cerebral parenchyma as senile plaques in case #3, while there was no signal in the control patients&#39; brains (cases #9 and #10). Resolution = 100 &#956;m. Scale bar = 5 mm. This figure has been modified with permission from Kakuda et al.21. <a href="https://www.jove.com/files/ftp_upload/57645/57645fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57645/57645fig2.jpg" /> 
Figure 2: MALDI-IMS of frozen AD/CAA brain sections and adjacent sections of the occipital cortex from AD brains. (A - C) The frozen AD/CAA brain sections and (D and E) adjacent sections of the occipital cortex from AD brains were immune-stained and focused on arteriole and cerebral parenchyma,&#160;using antibodies against A&#946;40 (D: BA27) or A&#946;42 (E: anti-A&#946;42 polyclonal). Both analyses demonstrated that A&#946;40 is preferentially deposited in leptomeningeal blood vessels (arrows in panel D) and arterioles in the subarachnoid space and the cerebral parenchyma forming CAA. In contrast, A&#946;42 is mainly deposited in SPs. For IMS, Resolution = 100 &#956;m. Scale bar = 5 mm (A, B, and C) = 5 mm, 500 (D and E). This figure has been modified with permission from Kakuda et al.21. <a href="https://www.jove.com/files/ftp_upload/57645/57645fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57645/57645fig3.jpg" /> 
Figure 3: MALD-IMS of frozen AD/CAA brain sections (patient #3) at a resolution of 40 &#181;m. Various C-terminal and N-terminal truncated and modified A&#946; peptides in AD accompanying severe CAA (patient #3). A&#946;1-36 to A&#946;1-41 are preferentially deposited in leptomeningeal blood vessels, while A&#946;1-42 and A&#946;1-43 are deposited in the cerebral parenchyma as senile plaques. Scale bars = 1 mm (A-B),&#160;2 mm (upper left). Resolution = 40 &#956;m. <a href="https://www.jove.com/files/ftp_upload/57645/57645fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57645/57645fig4.jpg" /> 
Figure 4: MALDI-IMS of frozen AD/CAA brain sections (patient #3) at a resolution of 20 &#181;m. This panel shows various C-terminal and N-terminal truncated and modified A&#946; peptides in AD accompanying severe CAA (patient #3). A&#946;1-36 to A&#946;1-41 are preferentially deposited in leptomeningeal blood vessels, while A&#946;1-42 and A&#946;1-43 are deposited in the cerebral parenchyma as senile plaques. Scale bars = 200 &#956;m (a, b, c), 1 mm (upper left). Resolution = 20 &#956;m. <a href="https://www.jove.com/files/ftp_upload/57645/57645fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57645/57645fig5.jpg" /> 
Figure 5: Segmentation map, obtained by MALDI-IMS of a frozen AD brain section, reveals putative senile plaque, large subarachnoid vessel structures, and small parenchymal arterioles. (A) Segmentation map obtained from a multivariate image analysis of MALDI-IMS data. (B) Bisecting k-means based clustering analysis identified plaque-like and vessel-like structures in the occipital cortex. The clusters and substructures and their relations are shown as nodes (e.g.,1-0-0). (C) Distinct A&#946; peptide localization patterns resembling plaques (blue), subarachnoid vessels (green), and arteriole (red) structures. Note that a few red clusters are also distributed in the subarachnoid space. This is verified by single ion images of these individual A&#946; peptides at a 20 &#181;m resolution: (D) A&#946; 1-40, (E) A&#946; 1-41, and (F) A&#946; 1-42. <a href="https://www.jove.com/files/ftp_upload/57645/57645fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Case</td>
			<td>Gender</td>
			<td>Age at death</td>
			<td>Braak SP</td>
			<td>CAA</td>
		</tr>
		<tr>
			<td>1</td>
			<td>M</td>
			<td>83</td>
			<td>C</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>2</td>
			<td>M</td>
			<td>88</td>
			<td>C</td>
			<td>1</td>
		</tr>
		<tr>
			<td>3</td>
			<td>M</td>
			<td>84</td>
			<td>C</td>
			<td>2</td>
		</tr>
		<tr>
			<td>4</td>
			<td>M</td>
			<td>78</td>
			<td>C</td>
			<td>1</td>
		</tr>
		<tr>
			<td>5</td>
			<td>M</td>
			<td>83</td>
			<td>C</td>
			<td>1</td>
		</tr>
		<tr>
			<td>6</td>
			<td>M</td>
			<td>84</td>
			<td>O</td>
			<td>0</td>
		</tr>
		<tr>
			<td>7</td>
			<td>M</td>
			<td>78</td>
			<td>O</td>
			<td>0</td>
		</tr>
		<tr>
			<td>8</td>
			<td>M</td>
			<td>70</td>
			<td>O</td>
			<td>0</td>
		</tr>
		<tr>
			<td>9</td>
			<td>M</td>
			<td>73</td>
			<td>O</td>
			<td>0</td>
		</tr>
		<tr>
			<td>10</td>
			<td>M</td>
			<td>81</td>
			<td>O</td>
			<td>0</td>
		</tr>
	</tbody>
</table>
Table 1: Clinical and pathological data of AD with CAA cases and aged SP O subjects. Human cortical specimens for IMS and IHC were obtained from the Brain Bank at Tokyo Metropolitan Institute of Gerontology. Each brain specimen was taken from the occipital cortex of five AD patients and five age-matched controls. The extent of amyloid deposition as shown by an A&#946; monoclonal antibody was defined by Braak SP (amyloid) stages. At stage O, there are almost no senile plaques throughout the isocortex. At stage C, virtually all the isocortical areas are affected. AD brains are invariable at stage C. This table has been modified with permission from Kakuda et al.21.

Watch this Scientific Journal Video about Visualization of Amyloid Î² Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry at JoVE.com

Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid &#946; protein on brain tissues of Alzheimer&#39;s disease and cerebral amyloid angiopathy samples using MALDI-IMS.

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into ...

visualization-amyloid-deposits-human-brain-with-matrix-assisted-laser

Research

JoVE Journal

Neuroscience

10.6K Views.  Doshisha University. De belangrijkste bijdrage van ons papier is dat we een nauwkeurig en fijn landschap van amyloïde bètapathologie van de alzheimerhersenen hebben onderzocht met de beeldvormingstechnologie van de MALDI-beeldvormingsmassaspectrometrie. We bereikten technologische vooruitgang door het genereren van een speciaal protocol met mierenzuur voorbehandeling van de weefselglijbanen. Na MALDI-IMS worden segmentatiekaarten gegenereerd en worden berekeningen uitgevoerd om nieuwe markereiwitten of peptiden...

Video: Visualization of Amyloid β Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 (Fig.1). A 12 &mu;m thin tissue section is covered with a MALDI matrix, which facilitates desorption and ionization of intact peptides and proteins that can be detected with a mass analyzer, typically using a MALDI TOF/TOF mass spectrometer. Generally hundreds of peaks can be assessed in a single rat brain tissue section. In contrast to commonly used imaging techniques, this approach does not require prior knowledge of the molecules of interest and allows for unsupervised and comprehensive analysis of multiple molecular species while maintaining high molecular specificity and sensitivity2. Here we describe a MALDI IMS based approach for elucidating region-specific distribution profiles of neuropeptides in the rat brain of an animal model Parkinson's disease (PD). 
PD is a common neurodegenerative disease with a prevalence of 1% for people over 65 of age3,4. The most common symptomatic treatment is based on dopamine replacement using L-DOPA5. However this is accompanied by severe side effects including involuntary abnormal movements, termed L-DOPA-induced dyskinesias (LID)1,3,6. One of the most prominent molecular change in LID is an upregulation of the opioid precursor prodynorphin mRNA7. The dynorphin peptides modulate neurotransmission in brain areas that are essentially involved in movement control7,8. However, to date the exact opioid peptides that originate from processing of the neuropeptide precursor have not been characterized. Therefore, we utilized MALDI IMS in an animal model of experimental Parkinson's disease and L-DOPA induced dyskinesia. 
MALDI imaging mass spectrometry proved to be particularly advantageous with respect to neuropeptide characterization, since commonly used antibody based approaches targets known peptide sequences and previously observed post-translational modifications. By contrast MALDI IMS can unravel novel peptide processing products and thus reveal new molecular mechanisms of neuropeptide modulation of neuronal transmission. While the absolute amount of neuropeptides cannot be determined by MALDI IMS, the relative abundance of peptide ions can be delineated from the mass spectra, giving insights about changing levels in health and disease. In the examples presented here, the peak intensities of dynorphin B, alpha-neoendorphin and substance P were found to be significantly increased in the dorsolateral, but not the dorsomedial, striatum of animals with severe dyskinesia involving facial, trunk and orolingual muscles (Fig. 5). Furthermore, MALDI IMS revealed a correlation between dyskinesia severity and levels of des-tyrosine alpha-neoendorphin, representing a previously unknown mechanism of functional inactivation of dynorphins in the striatum as the removal of N-terminal tyrosine reduces the dynorphin's opioid-receptor binding capacity9. This is the first study on neuropeptide characterization in LID using MALDI IMS and the results highlight the potential of the technique for application in all fields of biomedical research.

Dopamine replacement pharmacotherapy using L-DOPA is the most commonly used symptomatic treatment of Parkinson&#x2019;s disease, but is accompanied by side effects including involuntary abnormal movements, termed dyskinesia 1. Here, a protocol for MALDI imaging mass spectrometry is presented that detects changes in rat brain neuropeptide levels related to dyskinesia.

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 ...

Medicine

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization (IR-MALDESI)

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption electrospray ionization (MALDESI) is an example of such methods, where features of matrix-assisted laser desorption/ionization (MALDI) (e.g., pulsed nature of desorption) and electrospray ionization (ESI) (e.g., soft-ionization) are combined. One of the major advantages of MALDESI is its inherent versatility. In MALDESI experiments, an ultraviolet (UV) or infrared (IR) laser can be used to resonantly excite an endogenous or exogenous matrix. The choice of matrix is not analyte dependent, and depends solely on the laser wavelength used for excitation. In IR-MALDESI experiments, a thin layer of ice is deposited on the sample surface as an energy-absorbing matrix. The IR-MALDESI source geometry has been optimized using statistical design of experiments (DOE) for analysis of liquid samples as well as biological tissue specimens. Furthermore, a robust IR-MALDESI imaging source has been developed, where a tunable mid-IR laser is synchronized with a computer controlled XY translational stage and a high resolving power mass spectrometer. A custom graphical user interface (GUI) allows user selection of the repetition rate of the laser, number of shots per voxel, step-size of the sample stage, and the delay between the desorption and scan events for the source. IR-MALDESI has been used in variety of applications such as forensic analysis of fibers and dyes and MSI of biological tissue sections. Distribution of different analytes ranging from endogenous metabolites to exogenous xenobiotics within tissue sections can be measured and quantified using this technique. The protocol presented in this manuscript describes major steps necessary for IR-MALDESI MSI of whole-body tissue sections.

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI

A mass spectrometry imaging (MSI) source operated at atmospheric pressure was developed by coupling mid-infrared laser desorption and electrospray post-ionization. Exogenous ice matrix was used as the energy-absorbing matrix to facilitate resonant desorption of tissue-related material. This manuscript provides a step-by-step protocol for performing IR-MALDESI MSI of whole-body neonatal mouse.

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption ...

Bioengineering

Correlative Light and Electron Microscopy to Study Microglial Interactions with &#946;-Amyloid Plaques

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding immunostaining (specifically for ionized calcium-binding adapter molecule 1 (IBA1), a calcium binding protein expressed by microglia) and tissue processing for electron microscopy (EM). Methoxy-X04 is a fluorescent dye that crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. Injection of the animals with methoxy-X04 prior to sacrifice and brain fixation allows pre-screening and selection of the plaque-containing brain sections for further processing with time-consuming manipulations. This is particularly helpful when studying early AD pathology within specific brain regions or layers that may contain very few plaques, present in only a small fraction of the sections. Post-mortem processing of tissue sections with Congo Red, Thioflavin S, and Thioflavin T (or even with methoxy-X04) can label &#946;-pleated sheets, but requires extensive clearing with ethanol to remove excess dye and these procedures are incompatible with ultrastructural preservation. It would also be inefficient to perform labeling for A&#946; (and other cellular markers such as IBA1) on all brain sections from the regions of interest, only to yield a small fraction containing A&#946; plaques at the right location. Importantly, A&#946; plaques are still visible after tissue processing for EM, allowing for a precise identification of the areas (generally down to a few square millimeters) to examine with the electron microscope.

This article describes a protocol for visualizing amyloid A&#946; plaques in Alzheimer&#39;s disease mouse models using methoxy-X04, which crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. It allows pre-screening of plaque-containing tissue sections prior to immunostaining and processing for electron microscopy.

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding ...

Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS) experiments. Determining the appropriate protocol to follow throughout the sample preparation process can be difficult as each step must be optimized to comply with the unique characteristics of the analytes of interest. This process involves not only finding a compatible matrix that can desorb and ionize the molecules of interest efficiently, but also selecting the appropriate matrix deposition technique. For example, a wet matrix deposition technique, which entails dissolving a matrix in solvent, is superior for desorption of most proteins and peptides, whereas dry matrix deposition techniques are particularly effective for ionization of lipids. Sublimation has been reported as a highly efficient method of dry matrix deposition for the detection of lipids in tissue by MALDI IMS due to the homogeneity of matrix crystal deposition and minimal analyte delocalization as compared to many wet deposition methods 1,2. Broadly, it involves placing a sample and powdered matrix in a vacuum-sealed chamber with the samples pressed against a cold surface. The apparatus is then lowered into a heated bath (sand or oil), resulting in sublimation of the powdered matrix onto the cooled tissue sample surface. Here we describe a sublimation protocol using 1,5-diaminonaphthalene (DAN) matrix for the detection and visualization of gangliosides in the rat brain using MALDI IMS.

A protocol for the sublimation of DAN matrix onto rat brain tissue for the detection of gangliosides using MALDI Imaging Mass Spectrometry is presented.

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass ...

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible for phagocytosis and clearance of debris and detritus. These cells include CNS-resident macrophages known as microglia, and mononuclear phagocytes infiltrating from the periphery. Light microscopy has generally been used to visualize phagocytosis in rodent or human brain specimens. However, qualitative methods have not provided definitive evidence of in vivo phagocytosis. Here, we describe quantitative 3D in silico modeling (q3DISM), a robust method allowing for true 3D quantitation of amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent Alzheimer&#39;s Disease (AD) models. The method involves fluorescently visualizing A&#946; encapsulated within phagolysosomes in rodent brain sections. Large z-dimensional confocal datasets are then 3D reconstructed for quantitation of A&#946; spatially colocalized within the phagolysosome. We demonstrate the successful application of q3DISM to mouse and rat brains, but this methodology can be extended to virtually any phagocytic event in any tissue.

Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer&#39;s disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly using MALDI (matrix&#160;assisted laser desorption/ionization)-based analytical techniques. New matrices for small-molecule MSI, which can improve the analysis of low-molecular weight (MW) compounds, are needed. These matrices should provide increased analyte signals while decreasing MALDI background signals. In addition, the use of ultrahigh-resolution instruments, such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, has&#160;the ability to resolve analyte signals from matrix signals, and this can partially overcome many problems associated with the background originating from the MALDI matrix. The reduction in the intensities of the metastable matrix clusters by FTICR MS can also help to overcome some of the interferences associated with matrix peaks on other instruments. High-resolution instruments such as the FTICR mass spectrometers are advantageous as they can produce distribution patterns of many compounds simultaneously while still providing confidence in chemical identifications. Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging. In this work, a protocol for the use of DT for MALDI imaging of endogenous lipids from the surfaces of mammalian tissue sections, by positive-ion MALDI-MS, on an ultrahigh-resolution hybrid quadrupole FTICR instrument has been provided.

Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging of small molecules; protocols for the use of DT for the MALDI imaging of endogenous lipids on the surface of tissue sections&#160;by positive-ion MALDI-MS on an ultrahigh-resolution quadrupole-FTICR instrument are provided here.

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly ...

Biology

Detecting Amyloid-&#946; Accumulation via Immunofluorescent Staining in a Mouse Model of Alzheimer's Disease

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies on accumulation of amyloid-&#946; (A&#946;) in the brain. A&#946; is produced from the proteolytic cleavage of the beta-amyloid precursor protein (APP) by &#946;-secretase and &#947;-secretase. In pathological circumstances, the increased &#946;-cleavage of APP leads to overproduction of A&#946;, which aggregates into A&#946; plaques. Since A&#946; plaques are a characteristic of AD pathology, detecting the amount of A&#946; is very important in AD research. In this protocol, we introduce the immunofluorescent staining method to visualize A&#946; deposition. The mouse model used in our experiments is 5&#215;FAD, which carries five mutations found in human familial AD. The neuropathological and behavioral deficits of 5xFAD mice are well-documented, which makes it a good animal model to study A&#946; pathology. We will introduce the procedure including transcardial perfusion, cryosectioning, immunofluorescent staining and quantification to detect A&#946; accumulation in 5&#215;FAD mice. With this protocol, researchers can investigate A&#946; pathology in an AD mouse model.

In the neuropathology of Alzheimer's disease, one of the most crucial characteristics is the deposition of amyloid-&#946;. In this protocol, we describe the method of immunofluorescent staining in 5&#215;FAD transgenic mouse to detect amyloid-&#946; accumulation in plaques. The process of perfusion, cryosectioning, staining and quantification will be described in detail.

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies ...

 Advanced MALDI-MSI Mapping of Metabolites in Brain Organoids

Molecular Imaging of Human Brain Organoids Using Mass Spectrometry 

Brain organoid models serve as a powerful tool for studying human brain development and function. Mass spectrometry imaging (MSI), a cutting-edge technology, allows us to map the spatial distribution of diverse molecules such as lipids, sugars, amino acids, drugs, and their metabolites within these organoids, all without the need for specific molecular probes. High-quality MSI data hinge on meticulous sample preparation. Fixatives play a pivotal role, but conventional options such as glutaraldehyde, paraformaldehyde, and cryopreserving such as sucrose may inadvertently impact tissue metabolites. Optimal fixation entails flash freezing in liquid nitrogen. However, for small organoids, a more suitable approach involves transitioning the organoids directly from the incubator into a warmed embedding solution, followed by freezing in dry ice-cooled ethanol. Another critical step is the embedding prior to cryosectioning, which also requires materials compatible with MSI, as traditional options can interfere with matrix deposition and ionization. Here, an optimized protocol for high resolution-MALDI-MSI of human brain organoids is presented, encompassing sample preparation, sectioning, and imaging using mass spectrometry. This method showcases the molecular distribution of small metabolites, such as amino acids, with high mass accuracy and sensitivity. As such, coupled with complementary studies of brain organoids, it can assist in illuminating complex processes governing early brain development, metabolic cell fate trajectories, and distinctive metabolite signatures. Furthermore, it provides insights into the precise locations of molecules within the organoid, enriching our understanding of the spatial organization of 3D brain organoid models. As the field continues to advance, a growing number of studies leveraging MSI to delve into brain organoids and complex biological systems is anticipated, thereby deepening the understanding of the metabolic aspects of human brain function and development.

Molecular Imaging of Human Brain Organoids Using Mass Spectrometry

An advanced method was developed for mass spectrometry imaging (MSI) of brain organoids that allows mapping metabolite distributions within these models. This technology offers insights into brain metabolic pathways and metabolite signatures during early development and in disease, promising a deeper understanding of the human brain function.

Brain organoid models serve as a powerful tool for studying human brain development and function. Mass spectrometry imaging (MSI), a cutting-edge ...

Fluorescence-Guided Matrix-assisted Laser Desorption/Ionization with Laser-Induced Postionization Mass Spectrometry of Individual Rat Neural Cells

Single-cell measurements are critical to understanding the rich spatiochemical heterogeneity of the brain. Matrix-assisted laser/desorption ionization (MALDI) mass spectrometry (MS) is capable of label-free, high-throughput characterization of endogenous molecules in individual cells. The recent advances in the development of MALDI mass spectrometers with laser-induced post-ionization (MALDI-2) provide greatly enhanced sensitivity of detection for a variety of lipids and other small molecules. However, MS imaging of large samples with MALDI-2 at cellular resolution is prohibitively slow for most applications. In this protocol, primary cells are isolated and dispersed onto conductive slides. Relative cell locations are determined by whole-slide fluorescence microscopy, followed by accurate coregistration of the microscopy coordinates to the stage coordinates of the MALDI-2 mass spectrometer. Targeted MS analysis of only cell locations provides high-throughput, single-cell measurements with high analyte coverage and reduced data size as compared to MS imaging of the entire sample. We describe the critical steps necessary for single-cell preparation, whole-slide fluorescence imaging, matrix application, and MALDI-2 mass spectrometry.

This protocol outlines the use of microMS for fluorescence-guided, single-cell MALDI-2 mass spectrometry, enabling enhanced molecular profiling of primary rat neuronal cells.

Single-cell measurements are critical to understanding the rich spatiochemical heterogeneity of the brain. Matrix-assisted laser/desorption ionization ...

Visualization and Mapping of Methoxy-X04-Labeled Amyloid Plaques in an Alzheimer’s Disease Mouse Brain Section

Source:&#160;<a style='text-decoration:none;' href='https://app.jove.com/v/54060/correlative-light-and-electron-microscopy-to-study-microglial-interactions-with-946-amyloid-plaques'><span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>Bisht, K.,&#160;et. al<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>. Correlative Light and Electron Microscopy to Study Microglial Interactions with &#946;-Amyloid Plaques.&#160;J. Vis. Exp.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> (2016)</a> &#160;This video demonstrates the visualization and mapping of Methoxy-X04-labeled amyloid plaques in Alzheimer&#8217;s disease mouse brain sections using fluorescence and bright-field microscopy, enabling precise correlation of plaques with anatomical structures for studying Alzheimer&#8217;s pathology.

Source:&#160;Bisht, K.,&#160;et. al. Correlative Light and Electron Microscopy to Study Microglial Interactions with &#946;-Amyloid Plaques.&#160;J. Vis. ...

Visualization of Amyloid β Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

Samenvatting

Meer video's verkennen