Name: laser capture microdissection of enriched populations of neurons or single neurons for gene expression analysis after traumatic brain injury
Uploaded: 2013-04-10T12:00:00
Duration: 13 min 32 s
Description: Watch this Scientific Journal Video about Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury at JoVE.com

This work is funded by R01 NS052532 (to HLH), the Moody Foundation, and the Department of Anesthesiology. We thank Laurie Bolding, Christine Courteau Butler and Christy Perry for their editorial assistance, and Christy Perry for layout and excellent production of all figures, tables and artwork.

1. Surgical Procedures and Fluid Percussion TBI
<ol>
 <li>All animal experiments are first approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch, Galveston, Texas and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8th Edition, National Research Council).</li>
 <li>Rats (adult male Sprague-Dawley rats, 400-500 g obtained from vendor Charles Rivers, Portland, Maine) are housed two per cage and provided food and water ad libitum in a vivarium with these constant conditions: light cycle (600 hr to 1,800 hr), temperature (21 &deg;C to 23 &deg;C), and humidity (40% to 50%) one week prior to use.</li>
 <li>Anesthetize rats with 4% isoflurane, intubate, and mechanically ventilate (NEMI Scientific; New England Medical Instruments, Medway, MA) the rats with 1.5-2.0% isoflurane in oxygen: air (70:30) and prepare them for parasagittal fluid-percussion injury as previously described.8,10</li>
 <li>Sacrifice rats at the appropriate time point after injury depending on the experimental design. Quickly remove brains, freeze immediately on dry ice, and store at -80 &deg;C in a 50 ml tube or proceed immediately to embed in OCT for frozen sectioning.</li>
</ol>
2. Sectioning and Staining of Rat Brain
<ol>
 <li>Brain tissue that is not used immediately can be kept at -80 &deg;C for up to one month if kept at a constant temperature. Brains that are frozen at -80 &deg;C, then thawed to -20 &deg;C for sectioning, and then re-frozen do not yield quality RNA after the second thawing. Once the brain is thawed, mounted in OCT and sectioned, slides should be stained within 24 hr and used for LCM within 30 min to one hour of staining. This will ensure good quality RNA. Following LCM, captured cells on LCM caps can be stored in lysis buffer at -80 &deg;C for up to one week, but RNA should be isolated in a timely manner to ensure the highest quality.</li>
 <li>Prior to sectioning the brain, wipe the cryostat with RNase-Zap and clean the brushes with ETOH (replace the disposable blade between each brain).</li>
 <li>Retrieve the brain in the 50 ml tube from the -80 &deg;C freezer; place it into the cryostat at a temperature of -22 &deg;C and thaw in tube for approximately 10 min. Remove the brain from the tube and place onto the stage on gauze, ventral side up.</li>
 <li>Using a razor blade, slice the brain to remove the posterior portion of the brain just rostral to the cerebellum and the anterior portion at the optic chiasm. Fill a cryomold with OCT mounting medium (Tissue Tek), and place the brain into the mold with the anterior side down. Allow the brain tissue to freeze in the mounting medium until it turns white (approximately 10 min).</li>
 <li>Freeze the specimen disc (Tissue Tek) onto the brain with OCT. Remove the brain from the mold. Insert the brain into the specimen head and tighten the screws.</li>
 <li>Insert a disposable, low profile blade (Fisher Scientific) into the knife holder and tighten the lever down.</li>
 <li>Begin slicing the brain at 20 &mu;m to remove the outer layer of OCT. Once the hippocampal region is reached, set the micron dial to 10. Collect coronal serial sections by placing a glass slide or plus-glass slide onto the tissue section (Fisher Scientific). Slides are preserved at -20 &deg;C in an RNase-free staining rack until sectioning is complete.</li>
 <li>To remove all RNases from glassware where tissue sections are processed, wipe down all staining containers and graduated cylinders with Eliminase (Fisher Scientific) and rinse in Milli Q water. Prepare all solutions with RNase-free water and filter the cresyl violet (Sigma-Aldrich) and Fluoro-Jade (Histo-chem) stain with a 0.2 &mu;m filter prior to use.</li>
 <li>Thaw brain sections at RT for 30 sec and fix in 75% ETOH (1 min).</li>
 <li>For LCM of single injured neurons after fixation, rinse slides in RNase-free water (1 min), counterstain with 1% cresyl violet (15-20 sec), rinse in RNase-free water (2 &times; 30 sec), stain with Fluoro-Jade (4 min), rinse in RNase-free water (3 &times; 1 min), dehydrate with 95% ETOH made from RNase-free water (30 sec), 100% ETOH (30 sec), and xylene ( 2 &times; 3 min).</li>
 <li>For LCM of swaths of neurons after fixation, rinse slides in RNase-free water (1 min), stain with 1% cresyl violet (1 min), rinse in RNase-free water for (3 &times; 1 min), dehydrate with 95% ETOH (2 &times; 30 sec), 100% ETOH (2 &times; 30 sec), and xylene (2 &times; 3 min).</li>
 <li>Air dry all sections in a fume hood for 15 min prior to LCM. Slides can be stored, section sides up, in a slide box lined with dessicant, if they need to be transferred from room to room. However, optimal results are obtained if LCM is performed immediately after sections are dry. LCM should be limited from 30 min to 1 hr. In the next section, we describe first how to capture continuous swaths of cells, which is easiest to master for those who are learning this technique. Then we describe how to precisely capture single neurons. In our procedure, we identify dying neurons by their affinity for Fluoro-Jade-a marker of degenerating neurons. Fluoro-Jade-negative cells are presumed to be surviving neurons.</li>
</ol>
3. Laser Capture Microdissection (LCM)
LCM of swaths of enriched populations of hippocampal neurons
<ol>
 <li>LCM is performed using a PixCell IIe microscope with an infrared diode laser (Life Technologies).</li>
 <li>Adjust the center joystick to the vertical position. Rotate and lock the 4X objective into position.</li>
 <li>Place a slide at the center of the microscope stage and manually adjust until the region of the hippocampus is in view. Use coarse and fine focus adjustments to bring the image into focus.</li>
 <li>Activate the vacuum chuck by pressing the vacuum button on the controller. Rotate and lock the 10X objective into place. Focus again with coarse and fine adjustments.</li>
 <li>Load the CapSure Macro LCM caps (Life Technologies) into the CapSure cassette module and position a cap at the load line position. Rotate the Cap Placement arm and position around the cap. Raise the Cap Placement arm to remove the CapSure cap from the cassette module.</li>
 <li>Rotate the Cap Placement arm over the slide and lower the arm down over the slide; this will place the cap onto the slide. Adjust the fine focus and move the joystick to check if cells are inside the black circle on the cap. All cell captures should be inside this black circle.</li>
 <li>Set the laser parameters. First, press the laser-enable button on the controller. The laser target spot will be visible as a pink dot in the field of view on the computer monitor.</li>
 <li>Set the laser spot size to small (7.5 &mu;m) to focus the laser and adjust the power and duration to 65 - 75 mW, for 1.0-2.0 msec as needed for optimal cell capture.</li>
 <li>To test the laser, use the joystick to move the laser spot to an area where there are no cells to pick up. Fire the laser using the thumb switch. Use the joystick to move the laser spot away from the melted polymer spot and check to see if a visible dark ring surrounds a clear area where the laser was fired.</li>
 <li>To capture cells, use the joystick to move the laser spot over the area of cells to capture. Fire the laser using the thumb switch. Move the joystick while firing the laser to capture a large area of cells. When the laser is fired it melts the thermoplastic polymer film on the cap to the surface of the target cells. The polymer absorbs the laser radiation, thus not altering the RNA, DNA or protein ensuring the integrity of the sample for future molecular applications.</li>
 <li>After firing all cells of interest, lift the Cap Placement arm. The captured cells will separate from the tissue section and adhere to the CapSure cap. The remaining tissue and missing cells will be visible in the field of view. The cells on the cap can also be viewed by placing the cap on an empty portion of the slide.</li>
 <li>Lift the Cap Placement arm and rotate it to the Cap Unload Station. Lower the cap into the station and rotate the Cap Placement arm back into its previous position.</li>
 <li>Slide the CapSure insertion tool along the platform and onto the cap. Lift the tool from the platform. The cap will remain attached. Lightly touch the cap to the CapSure clean pad to clean away unwanted tissue.</li>
 <li>Place the cap into a 0.5 ml RNase-free tube filled with 100 &mu;l of lysis buffer obtained from the RNAqueous Micro-Isolation Kit. Immediately vortex the cap for 30 sec to lyse the cells. Incubate the samples at 42 &deg;C for 30 min and freeze at -80 &deg;C until RNA isolation.</li>
</ol>
LCM of single FJ+ neurons in the hippocampus
<ol start="15">
 <li>To capture single cells, load CapSure HS LCM caps into the CapSure cassette module and position a cap at the load line position. Rotate the Cap Placement arm and position it around the cap. Raise the Cap Placement arm to remove the CapSure cap from the cassette module.</li>
 <li>Set the laser spot size to small (7.5 &mu;m) to focus the laser and adjust the laser power and duration to 65 -75 mW for 0.45 to 0.50 msec for optimal cell capture of single cells.</li>
 <li>Rotate the Cap Placement arm over the slide and lower the arm down toward the slide. This will place the cap onto the slide. The Capsure HS cap surface sits elevated from the sample during LCM.</li>
 <li>Use the joystick to move the laser over single FJ+ cells. All cells being captured must be inside the small black ring on the cap. Fire the laser using the thumb switch.</li>
 <li>When all cells within the black circle area have been fired with the laser, lift the Cap Placement arm. The captured cells will separate from the tissue section and adhere to the CapSure HS cap.</li>
 <li>Place another slide on the microscope stage and collect FJ+ cells until the HS cap is full. Place the cap into a 0.5 ml RNase-free tube filled with 40-50 &mu;l of lysis buffer. Immediately vortex the cap for 30 sec to lyse the cells. Incubate the samples at 42 &deg;C for 30 min and freeze at -80 &deg;C until RNA isolation.</li>
</ol>
4. Total RNA Isolation From LCM Samples (This section will just be described in the video presentation)
<ol>
 <li>Total RNA from cells is isolated using the RNAqueous-Micro Kit (Ambion) following the manufacturer's protocols. All reagents and wash solutions are provided in this kit. Pre-wet the micro filter cartridge assembly by adding 30 &mu;l of lysis solution buffer to the filter. After 5 min, centrifuge the filter for 30 sec at 10,000 &times; g.</li>
 <li>Add 3 &mu;l of LCM additive to the sample lysate, pipette to mix, and centrifuge.</li>
 <li>Add 52 &mu;l of 100% ETOH (1/2 volume) to the sample lysate; pipette to mix, and transfer the lysate to the filter column.</li>
 <li>Centrifuge the filter column at 10,000 &times; g for 1 min to bind the RNA to the column. If there are multiple caps for one sample, spin each lysate through the same column separately.</li>
 <li>Complete the RNA isolation procedure as described in the RNAqueous Micro kit.</li>
 <li>Perform DNase treatment and DNase inactivation of LCM RNA samples as described in the kit, transfer the RNA to a RNase-free tube and store at -80 &deg;C.</li>
</ol>
5. RNA Quantification and Downstream Applications
Assessment of RNA quality and quantification is performed with an Agilent Bioanalyzer using reagents from the Pico Kit (Agilent Technologies, Santa Clara, CA) following the manufacturer's protocol.
<ol>
 <li>Total RNA is analyzed on an Agilent Bioanalyzer 2100 (Agilent Technologies). The software evaluates the concentration and the integrity of the RNA sample by assigning an RNA Integrity Number (RIN) to each sample ranging from 1 to 10. 1-5 indicates degraded RNA and 7-10 indicates good quality RNA.</li>
 <li>Non-degraded intact RNA will be used for Real-Time PCR; individual Taqman assays or RT2 profiler arrays using Sybr-green chemistry. The RNA can also be used for mRNA microarray analysis and microRNA microarray analysis.</li>
</ol>

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The authors declare that they have no conflicts of interest.

This LCM technique has enabled us to make significant advances in understanding the molecular mechanisms of TBI.8,10,11,12,13 We were the first investigators to utilize this technique to demonstrate that distinct subregions of the rat hippocampus have gene expression profiles that correlate with their selective vulnerability to injury. Our studies showed that we could quantify gene expression, by qPCR, from as few as 10 laser captured cells and that we could perform genome-wide microarray analysis from as few as 600 captured cells. LCM would be a valuable tool in similar studies of other brain regions that are known to be implicated in diverse neurological and neuropsychiatric disorders. For example, studies using LCM have shed light into the pathogenesis of Parkinson's disease in dopamine neurons of the substantia nigra,14 and aided genome-wide profiling studies of the nucleus accumbens, which is involved in reward circuits implicated in substance abuse disorders.15
Neuronal heterogeneity is reflected at the genome level16 and may contribute to the lack of success of experimental treatments for TBI in clinical trials. Thus, our goal is to utilize this technique to investigate the critical elements influencing neuronal survival after TBI. Our recent genome-wide profiling study of dying and adjacent surviving hippocampal neurons after TBI suggested that a cellular rheostat that reflects the ratio of the expression levels of cell survival to cell death genes regulates cell fate after TBI. These ongoing studies will contribute to the design and development of pharmacotherapeutic strategies that can positively influence the cell survival rheostat. Furthermore, we currently use LCM to track and monitor the effects of potential therapeutic drug treatments in hippocampal neurons after TBI. Thus, our studies demonstrate that given careful RNase-free handling techniques and some modifications of existing protocols, it is possible to obtain high quality RNA samples from LCM for accurate quantitative gene expression analysis.
However there are a few pitfalls associated with LCM techniques. For example, collecting only neuronal cells without any microglia contamination can be almost impossible. In the pyramidal cell layer of the hippocampus it has been estimated that 95% of the cells are neurons with 90% of this population to be pyramidal cells and 10% interneurons, leaving a small percentage of glial and other cell types.8,17,18 In our studies we use Fluoro-jade a fluorescent dye that specifically labels only injured neurons in brain tissue. A different stain that is a marker for GFAP would be necessary to stain microglia.19 Collecting only the Fluoro-jade stained single neuronal bodies ensures a more homogenous population of neurons. Another common problem that may require troubleshooting is that a circle is not defined when the laser is fired. If this occurs, it is necessary to check the laser settings and adjust the power and duration as necessary. Also it is important to ensure that the cap is placed flat on the tissue and seated correctly in the arm. Referring to the LCM technical manual or calling technical support will sometimes be necessary. Following LCM and RNA isolation, the quality of the RNA should always be assessed using a bioanalyzer prior to any gene expression analysis.
There are several types of laser capture instruments currently on the market including laser cutting (Life Technologies, Leica Microsystems) and laser catapulting (Zeiss) instruments. Our LCM system works well for capturing small numbers of cells. Other high-throughput and more automated systems may be more suitable for obtaining larger numbers of cells for genomic and particularly proteomic analysis. Indeed, LCM using these automated systems has great potential for analysis of protein expression in identified cells or enriched populations of cells.20 Furthermore, LCM can facilitate gene expression analysis of immunolabeled cells,21 allowing us to investigate gene expression in defined cell types regardless of the complexity in the most heterogeneous tissues. Thus, LCM is an excellent tool for cutting-edge molecular studies of single or enriched populations of cells.

Gene expression analysis of heterogeneous tissues has always been problematic; this is particularly true in the mammalian brain, which has approximately 5,000 different cell types.4 Prior to the development of the laser capture microdissection (LCM) technique, genomic studies of the effects of TBI in vivo were based on analysis of gene expression in a mixed population of brain cells comprised, not only of different neuronal cell types, but also of supporting glial and immunomodulatory cells. The resulting complex gene expression profiles obtained from these heterogeneous tissues, and the often conflicting patterns of injury-induced cellular signals, may be one explanation for the failure in human clinical trials of therapeutic strategies proven effective in pre-clinical studies of TBI.5
To obtain a clear understanding of injury-induced gene expression in vulnerable populations of neurons from the rat hippocampus, we adopted the technique of LCM, first reported by Emmert-Buck et al.3 Subsequently, we modified and optimized this microdissection technique for efficient capture of enriched populations of neurons and single neurons for mRNA profiling using quantitative real time PCR and microarray analysis. To maintain the integrity of mRNA in frozen sections of brain tissue for genomic analysis, we modified existing protocols for fixing, staining and rapid capture of neurons from frozen rat brain sections. For identification and isolation of injured and dying hippocampal neurons, we also optimized the Fluoro-Jade staining technique for LCM. Fluoro-Jade does not distinguish between apoptotic and necrotic cell death. Thus, all types of degenerating neurons can be detected by this stain.6,7
Here, we describe the protocol used in our laboratory to obtain pools of single dying or surviving neurons as well as swaths of enriched populations of distinct neuronal cell types (i.e. CA1-CA3 neurons for gene expression analysis after TBI). The procedure for fluid percussion brain injury performed in our laboratory is described in detail in Shimamura et al.,8 and is very similar to the lateral fluid-percussion injury protocol for mice published in JoVE by Alder et al.9 Since the LCM technique has been shown to have minimal or no effect on the integrity of DNA, RNA and protein in tissues, this is an excellent tool for molecular and protein analysis of defined cell types.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td></td>
 </tr>
 <tr>
 <td>PixCell IIe Laser Capture Microscope</td>
 <td>Life Technologies (Arcturus)</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Agilent 2100 Bioanalyzer</td>
 <td>Agilent Technologies</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>RNAqueous Micro- Kit</td>
 <td>Life Technologies (Ambion)</td>
 <td>AM1931 </td>
 <td></td>
 </tr>
 <tr>
 <td>Agilent 6000 Pico Kit</td>
 <td>Agilent Technologies</td>
 <td>5067-1513</td>
 <td></td>
 </tr>
 <tr>
 <td>Capsure LCM caps</td>
 <td>Applied Biosystems</td>
 <td>LCM0211/LCM0214</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluoro-Jade</td>
 <td>Histo-chem Inc.</td>
 <td>#1FJ</td>
 <td></td>
 </tr>
 <tr>
 <td>Cresyl Violet Acetate</td>
 <td>Sigma-aldrich</td>
 <td>C5042-10G</td>
 <td></td>
 </tr>
 <tr>
 <td>Xylene (Histological grade)</td>
 <td>Fisher Scientific</td>
 <td>X3P-1GAL</td>
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 <td>Ethanol 200 proof (Histological grade)</td>
 <td>UTMB pharmacy</td>
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 <td>RNase free barrier pipette tips</td>
 <td>Fisher Scientific</td>
 <td>2123635, 212364, 212361, 21-236-2A</td>
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 <td><a href="http://tools.invitrogen.com/content/sfs/brochures/cms_086283.pdf" target="_blank">Arcturus Laser Capture Microdissection system (Life Technologies)</a> 
 <a href="http://microscopy.zeiss.com/microscopy/en_de/downloads/brochure-downloads.html?catalog=laser_microdissection" target="_blank">Zeiss Laser Microdissection The PALM family </a> 
 <a href="http://www.leica-microsystems.com/fileadmin/downloads/Leica DM6000 B/Brochures/Leica_DM4000-6000_B-Brochure_en.pdf" target="_blank">Leica Microsystems</a></td>
 </tr>
 </tbody>
</table>

laser capture microdissection of enriched populations of neurons or single neurons for gene expression analysis after traumatic brain injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

In our first LCM study, we were able to show that functionally distinct subregions (CA1 and CA3) of the rat hippocampus have gene expression profiles that reflect their vulnerability to TBI.8 Figure 1A-1C shows laser capture of hippocampal pyramidal neurons (CA1-CA3), dentate gyrus neurons and SCN neurons before, and after LCM. Clean captures of pyramidal, granule or SCN neurons, respectively, are shown on the macro caps. Figure 2A-2B illustrates dying, Fluoro-Jade positive pyramidal neurons and surviving, Fluoro-Jade negative pyramidal neurons before and after LCM. The clean capture of the dying or surviving neurons is shown by viewing the captured cells on the LCM caps.
After LCM, the total RNA can be used for a diverse array of molecular studies including quantitative real-time PCR analysis of gene expression using TaqMan or SYBR green chemistries (Figure 3A), small focused PCR arrays with SYBR green probes (Figure 3B) or whole genome microarray studies (Figure 3C).
<img alt="Figure 1" fo:content-width="3in" fo:src="/files/ftp_upload/50308/50308fig1large.jpg" src="/files/ftp_upload/50308/50308fig1.jpg" /> 
Figure 1. Laser capture microdissection of swaths of defined cell populations from the rat brain. Frozen 10 &mu;m coronal sections of the rat brain are fixed in ethanol, stained with a nissl stain (cresyl violet) and prepared for LCM. Shown are pictures of the rat hippocampus and suprachiasmatic nucleus before and after LCM and the captured cells as visualized on the macro caps. A. Pyramidal neurons from the CA1-CA3 subfields of the rat hippocampus. B. Granule cells in the hippocampal dentate gyrus. C. Bilateral suprachiasmatic nuclei located on either side of the third ventricle and located above the optic chiasma. <a href="https://www.jove.com/files/ftp_upload/50308/50308fig1large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 2" fo:content-width="3in" fo:src="/files/ftp_upload/50308/50308fig2large.jpg" src="/files/ftp_upload/50308/50308fig2.jpg" /> 
Figure 2. Laser capture microdissection of single neurons from rat hippocampus. Shown are A. Degenerating, Fluoro-Jade-positive (stained) hippocampal CA3 neurons and B. Surviving, Fluoro-Jade-negative (unstained) CA3 neurons before and after LCM. Captured cells are visualized. <a href="https://www.jove.com/files/ftp_upload/50308/50308fig2large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 3" fo:content-width="6.5in" fo:src="/files/ftp_upload/50308/50308fig3large.jpg" src="/files/ftp_upload/50308/50308fig3.jpg" /> 
Figure 3. Gene expression analysis of total RNA isolated from laser captured neurons. A. Quantitative real time PCR data of circadian clock gene expression in SCN. B. Heatmap of gene expression in dying and surviving hippocampal neurons showing expression of apoptosis-related genes using focused PCR arrays. C. Agilent whole-genome microarray analysis of gene expression in hippocampal CA1-CA3 neurons from rats that received sham injury, TBI or TBI plus a recombinant adeno-associated siRNA virus designed to knockdown expression of genes induced by brain injury (neuronal nitric oxide synthase and glutathione peroxidase-1). <a href="https://www.jove.com/files/ftp_upload/50308/50308fig3large.jpg" target="_blank">Click here to view larger figure</a>.

Watch this Scientific Journal Video about Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury at JoVE.com

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

laser-capture-microdissection-enriched-populations-neurons-or-single

Research

JoVE Journal

Neuroscience

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) to provide an abundant supply of cells. NPCs were differentiated in the presence of a new combination of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), dibutyryl cAMP (DBC) and retinoic acid on dishes coated with poly-L-lysine and mouse laminin to obtain neuron-rich cultures. NPCs were also differentiated in the absence of neurotrophins, DBC and retinoic acid and in the presence of ciliary neurotrophic factor (CNTF) to yield astrocyte-rich cultures. Differentiated NPCs were characterized by immunofluorescence staining for a panel of neuronal markers including NeuN, synapsin, acetylcholinesterase, synaptophysin and GAP43. Glial fibrillary acidic protein (GFAP) and STAT3, astrocyte markers, were detected in 10-15% of differentiated NPCs. To facilitate cell-type specific molecular characterization, laser capture microdissection was performed to isolate neurons cultured on polyethylene naphthalate (PEN) membrane slides. The methods described in this study provide valuable tools to advance our understanding of the molecular mechanism of neurodegeneration.

Human neuroprogenitor cells (NPCs) were expanded under proliferating conditions. NPCs were differentiated into neuron-rich cultures in the presence of a combination of neurotrophins. Neuronal markers were detected by immunofluorescence staining. To isolate a pure population of neurons, NPCs were differentiated on PEN membrane slides and laser capture microdissection was performed.

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium carbonate. Animal studies as well as human genetic studies indicate that lithium affects molecular targets that are involved in neuronal growth, survival and maturation, and notably molecules involved in Wnt signaling. Given the ethical challenge to obtaining brain biopsies for investigating dynamic molecular changes associated with lithium-response in the central nervous system (CNS), one may consider the use of neurons obtained from olfactory tissues to achieve this goal.The olfactory epithelium contains olfactory receptor neurons at different stages of development and glial-like supporting cells. This provides a unique opportunity to study dynamic changes in the CNS of patients with neuropsychiatric diseases, using olfactory tissue safely obtained from nasal biopsies. To overcome the drawback posed by substantial contamination of biopsied olfactory tissue with non-neuronal cells, a novel approach to obtain enriched neuronal cell populations was developed by combining nasal biopsies with laser-capture microdissection. In this study, a system for investigating treatment-associated dynamic molecular changes in neuronal tissue was developed and validated, using a small pilot sample of BD patients recruited for the study of the molecular mechanisms of lithium treatment response.

In this study, a novel platform to investigate intraneuronal molecular signatures of treatment response in bipolar disorder (BD) was developed and validated. Olfactory epithelium from BD patients was obtained through nasal biopsies. Then laser-capture microdissection was combined with Real Time RT PCR to investigate the molecular signature of lithium response in BD.

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific sets of sparsely distributed activated neurons called neuronal ensembles that mediate learned associations. Fluorescence Activated Cell Sorting (FACS) has recently been optimized for adult rat brain tissue and allowed isolation of activated neurons using antibodies against the neuronal marker NeuN and Fos protein, a marker of strongly activated neurons. Until now, Fos-expressing neurons and other cell types were isolated from fresh tissue, which entailed long processing days and allowed very limited numbers of brain samples to be assessed after lengthy and complex behavioral procedures. Here we found that yields of Fos-expressing neurons and Fos mRNA from dorsal striatum were similar between freshly dissected tissue and tissue frozen at -80 &#186;C for 3 - 21 days. In addition, we confirmed the phenotype of the NeuN-positive and NeuN-negative sorted cells by assessing gene expression of neuronal (NeuN), astrocytic (GFAP), oligodendrocytic (Oligo2) and microgial (Iba1) markers, which indicates that frozen tissue can also be used for FACS isolation of glial cell types. Overall, it is possible to collect, dissect and freeze brain tissue for multiple FACS sessions. This maximizes the amount of data obtained from valuable animal subjects that have often undergone long and complex behavioral procedures.

Fluorescence Activated Cell Sorting FACS and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

Here we present a Fluorescence Activated Cell Sorting (FACS) protocol to study molecular alterations in Fos-expressing neuronal ensembles from both fresh and frozen brain tissue. The use of frozen tissue allows FACS isolation of many brain areas over multiple sessions to maximize the use of valuable animal subjects.

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.