We thank Hanna W&auml;rner for contributing the data for Figure 3 and Prof. Jonas Bergquist for valuable input. The Swedish Research Council (Grant 522-2006-6416 (MA), 521-2007-5407 (MA); The &Aring;ke Wiberg's Foundation (MA, JH), The Royal Swedish Academy of Sciences (MA, JH), and the Swedish Chemical Society (JH) are gratefully acknowledged for financial support.

The protocol is adjusted for the purpose of statistical analysis of MALDI IMS data from multiple rat brain sections, typically 20-30 sections, and consists of five different steps comprising tissue preparation, matrix application, MALDI-TOF MS analysis, data evaluation, and neuropeptide identification. The procedures are outlined and described in more detailed below:
1. Tissue preparation
This procedure includes the collection of the respective tissue samples as well as tissue sectioning for IMS analysis. A particular objective in protein and peptide analysis is to avoid proteolytic degradation. Therefore it is essential to work fast and diligent during tissue dissection.
<ol>
	<li>Sacrifice rats (typically 250-300 g) by decapitation, remove rat brain within a maximum post-mortem time of &lt;30s and freeze on powdered dry ice before transferring to -80&deg;C freezer. Faster freezing using liquid nitrogen increase the risk of microtears in brain tissue, which will negatively affect matrix crystallization and thereby reduce MS quality (Fig. 2D). Whole brains can be stored for several years before sectioning without loss of MS signal quality.</li>
	<li>Cut frozen tissue on a cryostat microtome to 12 &mu;m slices and thaw-mount tissue sections on conductive MALDI glass slides (indium tin oxide coated slides, Bruker Daltonics) or MALDI target (Fig. 2A-C).</li>
	<li>Dry sections for 15 min under vacuum and store slides at -80&deg;C until further use. Tissue sections should be analyzed within the shortest possible time after sectioning, even if stored at -80&deg;C. We find that MS signal quality will be noticeably reduced after a year in storage. In order to reduce oxidation of proteins and peptides, the air in storage container can be replaced with an inert gas (e.g. argon or nitrogen).</li>
</ol>
2. Matrix application
The matrix application step has a significant impact on spectrum quality and requires optimization of multiple parameters depending on the kind of tissue as well as the analyte of interest. These factors include chemical parameters such as the kind of matrix, matrix concentration, pH, tissue washing and organic modifiers as well as instrumental settings including the deposit volume, lateral resolution and number of depositions10 (Fig. 2D). For large scale experiments, it is of great importance to reduce variance, for example by applying the matrix to all sections within one day and by the same operator. Although there are many strategies to apply matrix solution such as by sublimation or by spray, the automated deposition of arrays of small matrix droplets, about 100-150 picoliter in size, has been used successfully for the analysis of small proteins and neuropeptides in various tissues, including brain sections9, 10,11, 12, 13.
<ol>
	<li>Defrost the sections in a dessicator for 1 hour.</li>
	<li>Make sure that the experiment is blinded by a person other than the operator. Re-label all samples.</li>
	<li>Wash sections 1x in 70% ethanol (EtOH, at room temperature, RT) for 10 sec and twice in 95% EtOH (RT) for 10 sec. For large experiments, perform the washing for all glass slides together using a cuvette in order to minimize variation.</li>
	<li>Dry the sections in a dessicator for 10 min.</li>
	<li>Evaluate tissue sections under a microscope and check for tissue distortion, microtears and small cracks that will impair MALDI MS quality (Fig. 2D).</li>
	<li>Prepare fresh matrix solution consisting of 50 mg/mL DHB in 50% methanol, 10% 150mM ammonium acetate (AmAc) and 0.3% trifluoroacetic acid (TFA) in water.</li>
	<li>Matrix application is performed by discrete droplet deposition in a rectangular pattern using a chemical inkjet printer (CHIP, Shimadzu). The first step is to optimize the experimental parameters of matrix application for neuropeptide analysis including the number of droplets per pass, number of passes. This experiment is performed by applying multiple matrix arrays with different application parameters on the same tissue section, while making sure that each array is covering similar brain regions such as corpus callosum, cortex, and striatum. The same experiment has to be performed each time parameters are changed, including different brain structures, different matrices targeting specific analytes, and if different matrix solvents are needed for extraction of specific analytes.</li>
	<li>Scan the glass slide holder with the tissue section and align the holder. Define your array for matrix application on the tissue section and specify the spatial resolution i.e. spot to spot distance. Apply matrix using the optimized protocol on the chemical inkjet printer. For this experiment we used an optimized protocol for peptide imaging with the following printing parameters: 10 drops (100 pL/drop), 10 application passes and a spot to spot distance of 300 &mu;m.</li>
	<li>Scan final matrix spotted sections and save the picture for registration prior to MALDI data acquisition (step 3.4).</li>
	<li>Store the sections until further use in a dessicator under vacuum.</li>
</ol>
3. MALDI MS data acquisition and processing
MS analysis of neuropeptides is performed on a MALDI time of flight instrument (Ultraflex II, Bruker Daltonics, Germany) operating in reflector mode, using software assisted data acquisition from every single matrix spot14. Therefore accurate spatial teaching is imperative. It is essential that the MALDI optimization, acquisition and especially the target registration experiments are performed by the same operator that should preferably be blinded to the experimental groups. In a large scale experiment with multiple glass slides, the MALDI experiments can be performed by one operator while another person is operating the chemical inkjet printer.
<ol>
	<li>Load glass slides into the mass spectrometer.</li>
	<li>Check the calibration of the MALDI acquisition method using a low molecular weight standard calibration mix (Bruker Daltonics).</li>
	<li>Optimize acquisition parameters.
	<ol>
		<li>In order to optimize MS signal and to avoid ablating matrix from neighboring matrix deposits, the size of the laser and the optimum focus on tissue should be determined.</li>
		<li>The laser energy is set to ensure the maximum MS quality from as many matrix deposits as possible without elevating the baseline, reducing peak resolution or saturating the detector.</li>
		<li>Assess the maximum number of shots per matrix spot until only noise is detected, often 1000-2000 shots. Estimate the number of shots that should be accumulated and the number of shots acquired before the laser position within a spot should change. In order to sample each matrix spot evenly, we accumulate 600 shots in 25 shot steps, for a total number of 24 steps using a random pattern of movement, from each matrix deposition.</li>
	</ol>
	</li>
	<li>Register the scan of all spotted sections to motor coordinates of the MALDI stage using the FlexImaging (v.2.0)10 and perform data acquisition in batch mode by AutoXexuteBatchRunner.exe software.</li>
	<li>Process each single spectra by means of baseline subtraction (Convex hull V3), smoothing and external calibration (optional), followed by export as an ascii file (*.dat, *.txt or *.csv format).15</li>
</ol>
4. Data evaluation
Final data evaluation comprises data post processing and data reduction by focus only on peak information, followed by statistical analysis.
<ol>
	<li>As an initial step, the MALDI IMS sections were evaluated for overnormalization effects. This can be easily achieved by employing data visualization tools such as FlexImaging (Bruker Daltonics) or BioMap (Novartis). As an initial step the total ion images are evaluated before total ion current (TIC) normalization, followed by manual inspection of single ion distribution images of various prominent peptide peaks. Look for characteristic peak intensity distributions and if they are related to tissue features (damages), spotting quality or normalization effects (Fig. 3).</li>
	<li>Delineate regions of interests (e.g. the striatum) according to histological features and export the corresponding spectra in ascii file format. Preferably, normalization of spectra to the total ion current (TIC) can be performed at this stage.</li>
	<li>Import ascii files in a data handling software such as Origin (v.8.1, Originlab), MATLAB (MathWorks, Natick, MA, USA) or R16. Peak detection can be performed using peak finding tools included in the software, for example "peak analysis" in Origin or "mspeaks" in Matlab. Export the peaklists from all spectra as one single tab delimited text file.</li>
	<li>In order to determine bin borders for detected peptide peaks, binning analysis is performed using appropriate software tools (e.g. pbin17) or in-house written scripts for MATLAB or R. Here the single text file containing all peak picked data is loaded into the software and the parameters for peak border determination are specified such as how often a peak should be present in spectra in order to be relevant for the experiment. For example, the experiment contains 2 groups of animals, 5 animals in each group, and 100 spectra are collected from each animal and region of interest. Assume a peak is potentially interesting if it is present in at least the majority of the animals in one group (3/5) and in at least half of the spectra of those animals (3x50=150 spectra), this will give a total percentage of 15% for the 150 positive spectra out of the total 1000 (2x5x100) spectra. Using the pbin tool, this step yields a single binrange file containing all bin widths determined from the acquired data. In order to verify that bin borders are appropriate, it is easy to visualize the bins in Origin together with the original spectra traces.</li>
	<li>Peak-area integration can reduce variance that is important for statistical analysis. We use an in-house written script for R to calculate the area under the curve between peak borders determined in step 4. Integrated peak areas are imported into MS excel (v.2007) and statistical analysis by means of non-parametric unpaired testing using the SAM tool is performed18.</li>
</ol>
5. Peptide identification
Sequence verification of the observed peptide identities is essential in order to conclude biological relevance. The most accurate approach include true top-down determination directly off tissue using peptide fragmentation by means of tandem mass spectrometry (MS/MS), although high peptide concentrations are required for this kind of analysis12,13. For low abundant peptides or multiple peptides with close m/z values (&plusmn;0.5%), on-tissue analysis is impaired and off tissue analysis using a peptidomic strategy is utilized that includes extraction, separation and MS based identification of endogenous neuropeptides. For the experiment presented here, the central focus was on opioid peptide detection, which is a particular challenge since these peptides are quite low abundant compared with other neuropeptides in the spectra. Furthermore, these peptides are rather polar which makes them relatively hydrophilic and difficult to retain with common peptide extraction and separation techniques.. Therefore we applied a previously reported protocol for tissue extraction and opioid peptide prefractionation in combination with standard LC-MS/MS based peptide identification9,19.
<ol>
	<li>Collect coronal sections of target structures of interest (nucleus accumbens, NAc; caudate putamen, CPu). Mount frozen rat brain in a cryostat microtome and remove surrounding brain material (cortex, septum, corpus callosum) with a scalpel. Collect sections (30 &mu;m; n=50) of the dissected NAc and CPu and thaw mount the NAc and CPu parts of the sections on different glass slides.</li>
	<li>Extract peptides off tissue by adding 100 &mu;L 5%ACN/0.1% TFA, incubate for two minutes and collect in eppendorf low protein binding tubes. Repeat this step twice.</li>
	<li>Perform peptide prefractionation by means of strong cation exchange chromatography using stepwise (n=4) elution at increased ionic strength19. Dry down samples under vacuum using a speedvac concentrator.</li>
	<li>Analyze the peptide fractions by means of nanoflow C18 reversed phase liquid chromatography (1100, Agilent Technologies, Santa Clara, CA) interfaced to electrospray tandem mass spectrometry (LC-MS/MS). The MS experiments were performed on a hybrid linear iontrap/Fourier transform ion cyclotron resonance (FTICR) instrument (LTQ FT 7T, Thermo Scientific, Waltham, MA,) Peptide fulscan spectra (m/z 150-2000) were acquired with the FTICR analyzer at high mass resolution (100000) followed by subsequent fragmentation of the 5 most intense peptide peaks in the iontrap by means of collision induced dissociation (CID)9.</li>
	<li>Peptide sequence identification is performed by database matching and can be complemented by de-novo sequencing analysis. For database search, commercially available search engines (Mascot, XTandem or Protein prospector) are employed20. The searches are typically performed against databases containing sequences of known or predicted neuropeptides and sequences of neuropeptide precursor proteins21.</li>
</ol>
6. Representative Results
MALDI imaging mass spectrometry of striatal tissue sections as prepared according to the protocol described here resulted in the detection of more than 1000 peaks corresponding to approximately 300 monoisotopic molecular species (average spectra shown in Fig. 1). Data visualization for prominent molecular ion peaks was achieved using the Flex Imaging software and showed characteristic peak intensity distributions that are well in line with anatomical features (Fig.3). A further feature of MALDI IMS is its relative good reproducibility. In this experiment, the overall coefficient of variance for the peak intensities of all detected molecular species was 30%, but many peaks displayed very low variation and high reproducibility within treatment groups (Fig. 4). The relative peak intensity data of four different regions of interest, including the dorsolateral and dorsomedial part of both lesioned and intact striata were subjected to statistical analysis. In order to adjust for multiple comparisons simultaneously, the statistical analysis was performed by means of nonparametric testing using the SAM tool18. The most prominent changes were found in the dorsolateral part of the dopamine-denervated, parkinsonian striatum. Here significant changes in between the different treatment groups were observed for two dynorphin peptides; dynorphin B and alpha-neoendorphin (Fig.5). In detail, a relative increase of both dynorphin peak intensities by 50-60% was observed in high dyskinetic animals compared to low dyskinetic animals and lesion controls (p&lt;0.05, F(2, 15) = 12.8 DynB, F=5.7 aNeo; Fig. 5).
<img alt="Figure 1" src="/files/ftp_upload/3445/3445fig1.jpg" /> 
Figure 1. Average MS traces obtained from two closely related regions of the striatum; caudate putamen (CPu) and nucleus accumbens (NAc). The two regions display different MS profiles with some molecular species uniquely expressed in one region, or at different peak intensity levels (insert, m/z 2028). The spatial distribution pattern of each peak can be visualized using specialized imaging software (bottom panel).
<img alt="Figure 2" src="/files/ftp_upload/3445/3445fig2.jpg" /> 
Figure 2. (A) The brain is mounted on a cryostat chuck using an embedding media (OTC; arrow), care is taken that the OTC does not contaminate the area of brain to be sectioned since the OTC cause ion suppression of peptides. (B,C) Thin sections (&asymp;12 &mu;m thickness) are thaw-mounted onto MALDI compatible glass slides and dried for a few seconds to avoid freeze damage as seen in C. (D) Microtears may be difficult to detect to the naked eye, but impair MALDI matrix crystallization and obliterate MALDI MS signal. The same section stained with cresyl violet reveals microtears and cracks (bottom right photomicrograph).
<img alt="Figure 3" src="/files/ftp_upload/3445/3445fig3.jpg" /> 
Figure 3. The first step in data evaluation is to visualize several different peaks across the mass range analyzed (A-I). Here, striatal sections from 9 mice were imaged with MALDI MS. Visualization of the average total ion current will reveal areas of conspicuous high or low ion intensities (arrows). These areas can be affected by over- or under-normalization effects and distort data analysis compromising the results. Poor anatomical definition of peak distributions reveal sections with generally low peak signal-to-noise, for example sections 3 and 9, peaks F through I.
<img alt="Figure 4" src="/files/ftp_upload/3445/3445fig4.jpg" /> 
Figure 4. MS reproducibility between treatment groups can be assessed by calculating the average MS trace and the standard error for each m/z value (inserts, m/z 722 and 1749). Good reproducibility ensures valid statistical analysis.
<img alt="Figure 5" src="/files/ftp_upload/3445/3445fig5.jpg" /> 
Figure 5. Dynorphin B and alpha-neoendorphin peak intensities are significantly increased in the 6-OHDA-lesioned, parkinsonian, striatum of high-dyskinetic animals (HD; arrows) compared to the low-dyskinetic (LD) and lesion control group (LC). Peptide peak intensities expressed as average fold-change of intact side &plusmn; SEM (lesion/intact side). * p&lt;0.05; cx cortex; cc corpus callosum. Scale bar 5 mm. Please <a href="https://www.jove.com/files/ftp_upload/3445/3445fig5_lg.jpg">click here</a> to see a larger version of this figure.

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+and+origin+of+motor+complications+in+Parkinson's+disease.">The evolution and origin of motor complications in Parkinson's disease.</a> Neurology. 55, S13-S20 (2000).</li><li>O, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noncommunicable+Diseases+and+Mental+Health+Cluster,+Noncommunicable+Disease+Prevention+and+Health+Promotion+Department,+Ageing+and+Life+Course.">Noncommunicable Diseases and Mental Health Cluster, Noncommunicable Disease Prevention and Health Promotion Department, Ageing and Life Course.</a> Active Ageing: A Policy framework. , (2002).</li><li>Schapira, A. 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L., Chaurand, P., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI+imaging+mass+spectrometry:+molecular+snapshots+of+biochemical+systems.">MALDI imaging mass spectrometry: molecular snapshots of biochemical systems.</a> Nat. Methods. 4, 828-833 (2007).</li><li>Ljungdahl, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Mass+Spectrometry+Reveals+Elevated+Nigral+Levels+of+Dynorphin+Neuropeptides+in+L-DOPA-Induced+Dyskinesia+in+Rat+Model+of+Parkinson's+Disease.">Imaging Mass Spectrometry Reveals Elevated Nigral Levels of Dynorphin Neuropeptides in L-DOPA-Induced Dyskinesia in Rat Model of Parkinson's Disease.</a> PLoS ONE. 6, e25653 (2011).</li><li>Groseclose, M. R., Andersson, M., Hardesty, W. M., Caprioli, R. 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The authors have nothing to disclose.

There are several advantages of employing MALDI imaging mass spectrometry in the study of neuropeptides. An unbiased analysis of MS data can reveal that only specific brain nuclei, or as in the results presented here where only the dorsolateral part of the striatum is associated with a certain pathophysiological condition. By retaining the spatial information it is then possible to redefine regions of interest to perform statistical analysis with higher sensitivity and lower variability compared with analysis of whole brain sections or using traditional peptidomics studies on peptide extracts. In addition, it is important to realize MALDI IMS readily can detect previously unknown post-translational modifications, but structural analyses must follow to determine the exact amino acid positions that are modified.
Common pitfalls in visualizing the MALDI IMS data include mapping the maximum peak intensity to a linear optical scale from black (0%) to color (100%) for each and every section in the experimental series (Figure 3), instead of mapping all sections to a common absolute scale where 100% is the maximum peak intensity of all sections (Figure 5). The latter method allows comparison of group data and visualization of differences between treatment groups. 
A major obstacle in MALDI IMS analysis is the assignment of peptides to specific mass peaks. On-tissue tandem-mass spectrometry is sometimes possible, but often proves quite difficult13,14. We find that a more traditional approach including a preparative fractionation on strong cation exchange chromatography, followed by reversed phase LC-MS/MS can be used to successfully sequence many neuropeptides and especially opioid peptides. It is still not uncommon to obtain good quality MS/MS spectra that do not match any database entries using common search engines such as MASCOT. In these cases de novo-sequencing by hand is the only option. The ultimate proof of peak identity can be obtained by MALDI IMS of tissue sections from the appropriate knockout mouse; but this is not always available or possible. An alternative is to validate the results by a diametrically different method, for example by western immunoblotting or immunohistochemistry. This can often include raising antibodies and a significant amount of work validating the new antibodies.
The general strategy presented in this protocol is optimized for large scale neuropeptide MALDI IMS experiments including several sections and experimental conditions. The protocol has been optimized especially for opioid peptides and will have great impact in future studies, as employed in diverse fields of research including the mechanisms underlying pain and the endogenous response to drugs of addiction.

1.1 Sample collection and preparation
<ol>
<li>Isoflurane (Apoteksbolaget AB, Sweden) </li>
<li>Surgical tools; two pairs of scissors, a rongeur, spatula </li>
<li>Finely crushed dry ice </li>
<li>Aluminum foil and plastic bags for storage </li>
<li>Cryostat(Microm, Heidelberg, Germany) </li>
<li>Indium&#x2212;tin oxide (ITO)-coated MALDI slides (Bruker Daltonics, Germany) </li>
<li>optimal cutting temperature medium (OCT) (Sakura Finetek Europe, Netherlands) </li>
<li>vacuum dessicator </li>
</ol>
1.2 Matrix application
<ol>
<li>Acetonitrile (ACN), methanol (MeOH), ethanol (EtOH) of pro-analysis grade (Merck, Germany). </li>
<li>Trifluoroacetic acid (TFA) (Sigma Aldrich, St. Louis, MO). </li>
<li>Water was purified with a Milli-Q purification system (Millipore, Bedford, MA). </li>
<li>CHIP-1000 (Shimadzu, Japan) or Portrait 630 spotter (LabCyte, Sunnyvale, CA) </li>
</ol>
1.3 Mass spectrometry
<ol>
<li>Ultraflex II (Bruker Daltonics) </li>
<li>FlexImaging software (v 2.0, Bruker Daltonics) </li>
<li>Peptide or Protein Calibration Standard (Bruker Daltonics) </li>
</ol>
1.4 Data processing
<ol>
<li>Origin v.8.1 (OriginLab Corp, Norhampton, MA)</li>
<li>Matlab version 7.9.0.529 (MathWorks, Natick, MA) </li>
<li>pBin (<a href="http://www.vicc.org/biostatistics/software.php" target="_blank">http://www.vicc.org/biostatistics/software.php</a>) </li>
</ol>
1.5 Neuropeptide identification
<ol>
<li>Ultrasound sonicator </li>
<li>10 kDa molecular weight cut-off filter (Millipore, Bedford, MA) </li>
<li>SP Sephadex C 25 gel (Sigma, Germany)</li>
<li>Hypersil column (ThermoFisher) </li>
<li>Pyridine, formic acid (FA), Acetonitrile (ACN), methanol (MeOH), ethanol (EtOH) of pro-analysis grade (Merck, Germany).</li>
<li>Trifluoroacetic acid (TFA) Sigma Aldrich (St. Louis, MO, USA). </li>
<li>Agilent 1100 nanoflow LC, microWPS, C18 column (0.075x150mm) </li>
<li>Agilent 1100 Micro-fractioncollector, Prespotted anchorchip target plate (Bruker Daltonics) </li>
<li>BioTools software (v 3.1 SR2, Bruker Daltonics) </li>
<li>Bio Works software (v.3.3, Thermo Fisher; <a href="http://www.thermoscientific.com" target="_blank">www.thermoscientific.com</a> ) </li>
<li>Mascot software (v 2.2, MatrixScience; <a href="http://www.matrixscience.com" target="_blank">www.matrixscience.com</a> ) </li>
<li>Swepep database (<a href="http://www.swepep.org" target="_blank">www.swepep.org</a>) </li>
</ol>

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.

Watch this Scientific Journal Video about MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease at JoVE.com

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson&#039;s Disease

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 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 ...

maldi-imaging-mass-spectrometry-neuropeptides-parkinson-s

Research

JoVE Journal

Medicine

25.8K Views.  Uppsala University. Dopamine replacement pharmacotherapy using L-DOPA is the most commonly used symptomatic treatment of Parkinson’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.

Video: MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate &gt;80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes &gt;95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson&#039;s Disease

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing &#62;95% loss of striatal dopamine and 90.63&#177;-4.02 % ipsiversive rotational bias towards the side of the lesion.

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone.&#160;To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder.&#160;Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD.&#160;The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates.&#160;This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson&#039;s Disease

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration.&#160;A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Controlling Parkinson's Disease With Adaptive Deep Brain Stimulation

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time according to fluctuating disease and medication state. In the present realization of adaptive DBS we record and stimulate from the DBS electrodes implanted in the subthalamic nucleus of patients with Parkinson&#39;s disease in the early post-operative period. Local field potentials are analogue filtered between 3 and 47 Hz before being passed to a data acquisition unit where they are digitally filtered again around the patient specific beta peak, rectified and smoothed to give an online reading of the beta amplitude. A threshold for beta amplitude is set heuristically, which, if crossed, passes a trigger signal to the stimulator. The stimulator then ramps up stimulation to a pre-determined clinically effective voltage over 250 msec and continues to stimulate until the beta amplitude again falls down below threshold. Stimulation continues in this manner with brief episodes of ramped DBS during periods of heightened beta power.
Clinical efficacy is assessed after a minimum period of stabilization (5 min) through the unblinded and blinded video assessment of motor function using a selection of scores from the Unified Parkinson&#39;s Rating Scale (UPDRS). Recent work has demonstrated a reduction in power consumption with aDBS as well as an improvement in clinical scores compared to conventional DBS. Chronic aDBS could now be trialed in Parkinsonism.

Controlling Parkinson&#039;s Disease With Adaptive Deep Brain Stimulation

Adaptive deep brain stimulation (aDBS) is effective for Parkinson&#8217;s disease, improving symptoms and reducing power consumption compared to conventional deep brain stimulation (cDBS). In aDBS we track a local field potential biomarker (beta oscillatory amplitude) in real time and use this to control the timing of stimulation.

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

Development of an Alpha-synuclein Based Rat Model for Parkinson&#039;s Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS)

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a specific anatomical region. Within the brain, metals have a highly compartmentalized distribution, depending on the primary function they play within the central nervous system. Imaging the spatial distribution of metals has provided unique insight into the biochemical architecture of the brain, allowing direct correlation between neuroanatomical regions and their known function with regard to metal-dependent processes. In addition, several age-related neurological disorders feature disrupted metal homeostasis, which is often confined to small regions of the brain that are otherwise difficult to analyze. Here, we describe a comprehensive method for quantitatively imaging metals in the mouse brain, using laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) and specially designed image processing software. Focusing on iron, copper and zinc, which are three of the most abundant and disease-relevant metals within the brain, we describe the essential steps in sample preparation, analysis, quantitative measurements and image processing to produce maps of metal distribution within the low micrometer resolution range. This technique, applicable to any cut tissue section, is capable of demonstrating the highly variable distribution of metals within an organ or system, and can be used to identify changes in metal homeostasis and absolute levels within fine anatomical structures.

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry LA-ICP-MS

Quantitatively mapping metals in tissue by laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) is a sensitive analytical technique that can provide new insight into how metals participate in normal function and disease processes. Here, we describe a protocol for quantitatively imaging metals in thin sections of mouse neurological tissue.

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a ...

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI&#8212;Application in Premanifest Huntington's Disease

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy begins many years prior to the motor onset (during the &#34;premanifest&#34; period), but the spatiotemporal pattern of regional atrophy across the brain has not been fully characterized. Here we demonstrate an online cloud-computing platform, &#34;MRICloud&#34;, which provides atlas-based whole-brain segmentation of T1-weighted images at multiple granularity levels, and thereby, enables us to access the regional features of brain anatomy. We then describe a regression model that detects statistically significant inflection points, at which regional brain atrophy starts to be noticeable, i.e. the &#34;change-point&#34;, with respect to a disease progression index. We used the CAG-age product (CAP) score to index the disease progression in HD patients. Change-point analysis of the volumetric measurements from the segmentation pipeline, therefore, provides important information of the order and pattern of structural atrophy across the brain. The paper illustrates the use of these techniques on T1-weighted MRI data of premanifest HD subjects from a large multicenter PREDICT-HD study. This design potentially has wide applications in a range of neurodegenerative diseases to investigate the dynamic changes of brain anatomy.

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI&amp;#8212;Application in Premanifest Huntington&#039;s Disease

This paper describes a statistical model for volumetric MRI data analysis, which identifies the &#34;change-point&#34; when brain atrophy begins in premanifest Huntington&#39;s disease. Whole-brain mapping of the change-points is achieved based on brain volumes obtained using an atlas-based segmentation pipeline of T1-weighted images.

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy ...