We would like to acknowledge Elizabeth Sumner for her help editing this manuscript. Funding for this project was provided in part by The Moody Project for Translational Traumatic Brain Injury Research and RO1NS052532 to HLH.

NOTE: All animal experiments are approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch, Galveston, Texas as directed by the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8th Edition, National Research Council).
1. Tissue Collection, Region Identification, and Sectioning
<ol>
	<li>Subject adult, male, Sprague-Dawley Rats, approximately six weeks old and 250 - 300 g, to experimental fluid percussion injury, as described in Shimamura et al.4.</li>
	<li>Twenty-four hours after experimental TBI, humanely euthanize the animals by placing them in a chamber with an isoflurane concentration of 4% until deeply anesthetized. Ensure death by decapitation, excise the brains, and immediately freeze in powdered dry-ice. Store frozen brains in a -80 &#176;C freezer until sectioning.</li>
	<li>Prior to any LCM procedure that includes transcriptional analysis, clean all surfaces with RNase eliminating detergent to mitigate the risk for contamination and RNA degradation. 
	NOTE: This cleaning includes the area used for staining, the cryostat where tissue is sectioned, and the area around the LCM device.</li>
	<li>Retrieve the brain tissue from storage and place into a cryostat cooled to -20 &#176;C. Allow the brain to equilibrate to the temperature of the cryostat chamber for ~10 min.</li>
	<li>Place the brain ventral side up on a gauze sheet on top of the cryostat stage. With a clean, RNase-free razor blade, cut off the posterior portion just rostral to the cerebellum (can be saved or discarded) and the portion just anterior to the optic chiasm (och).</li>
	<li>Place a small amount of optimal cutting temperature (OCT) medium into two separate cryomolds. Place the brain (containing the frontal association cortex (FrA) and nucleus accumbens core (AcbC)) into the cryomolds anterior side down. Add sufficient OCT medium to cover the tissue.
	<ol>
		<li>Repeat this step with the brain tissue containing the hippocampus (Hp) and suprachiasmatic nucleus (SCN).</li>
		<li>Allow the OCT medium to freeze completely for ~20 min in the cryostat.</li>
	</ol>
	</li>
	<li>Once the tissue and OCT medium are completely frozen, squeeze a small amount of OCT onto a mounting head and press the frozen cryomolds containing the tissue onto the mounting head. Allow the OCT to freeze completely so that the mold containing the tissue is securely attached to the head.</li>
	<li>Secure the head attachment to the sectioning mount and tighten the screw. Adjust the cutting angle in relation to the cryostat blade with the adjustment levers to ensure sections are cut evenly.</li>
	<li>Set the section thickness to 30 &#181;m. When sectioning the tissue block containing the FrA and AcbC, first begin sectioning until cerebral cortex is apparent and then start collecting.</li>
	<li>Referring to the Rat Brain Atlas12, section and collect brain sections by gently placing room-temperature polyethylene napthalate (PEN) membrane slides on top of the sectioned tissue for the FrA until the secondary motor cortex (M2) is reached at Bregma 5.16 mm.
	<ol>
		<li>Visually ensure adherence to slide by inspecting if tissue and OCT are completely melted onto slide. Store all slides with tissue sections in the cryostat until staining.</li>
	</ol>
	</li>
	<li>Continue sectioning until the anterior commissure (aca) becomes visible and unites into one complete commissure underneath the now apparent lateral ventricles (LV) at Bregma 1.80 mm.</li>
	<li>Collect sections until the LVs appear to connect with the aca at Bregma 0.84 mm.</li>
	<li>Once sectioning for the FrA and AcbC has been completed, remove the mounted tissue block, and replace with the block containing HP and SCN.</li>
	<li>Section until the och is flattened at Bregma -0.48 mm, when the third ventricle (3V) becomes apparent.</li>
	<li>Collect sections for the SCN from this point until Bregma -0.72 mm, when the supraoptic decussation (sox) begins. 
	NOTE: It may be necessary to collect more tissue sections throughout the och as the SCN is easily passed over.</li>
	<li>For HP collection, section until the horns of granule cell layer dentate gyrus (GrDG) are visibly apparent at Bregma -3.00 mm.</li>
	<li>Collect sections until the hippocampal CA subfields are fused in the coronal sections at Bregma -4.78 mm. This detail allows for complete collection of the hippocampus under the craniotomy and injury site. 
	NOTE: As demonstrated in previous publications from this lab, most injured cells will be apparent within this range and it is appropriate for gene or microRNA expression analysis.</li>
</ol>
2. Staining Protocol
<ol>
	<li>Prior to staining, wash all dishware and the staining area in a chemical fume hood with RNase-eliminating detergent. This preparation mitigates the risk of RNA degradation due to contamination.</li>
	<li>Prepare all staining reagents and solutions in nuclease free water.</li>
	<li>Take a rack of slides stored in the cryostat and place in the fume hood. Allow slides to warm for 30 s. Place them into staining solutions (prepared with nuclease free water) as follows: 75% EtOH for 1 min, nuclease free water for 1 min, 1% cresyl violet for 1 min, nuclease free water for 30 s, nuclease free water for 30 s, 95% EtOH for 30 s, 100% EtOH for 30 s, xylene for 3 min, and xylene for 3 min.</li>
	<li>Allow the rack to air-dry for no more than 10 min at room temperature in the fume hood. Alternatively, place racks into an Rnase-free vacuum desiccator for quicker drying. Once dried, proceed immediately to LCM.</li>
</ol>
3. Stereotactic Atlas Guided Laser Capture Microdissection with the LCM System
<ol>
	<li>Prior to beginning any LCM procedure for RNA analysis, wipe down the collection area and the area around the device with RNase-eliminating detergent and 100% EtOH.</li>
	<li>Flip the power switch on the IR laser generating unit before turning on microscope base, and allow the system to initialize before software is started from the desktop. 
	NOTE: If not completed in this order, the software will not recognize the microscope.</li>
	<li>Once the software has booted and run through its initializing steps, press the &#34;Present Stage&#34; button in the software &#34;Setup Panel.&#34; The modular stage will move into position where LCM Macro Caps and slides can be loaded and off-loaded.</li>
	<li>Place the PEN membrane slides into the holders (up to three at a time) with the frosted edge facing rightward. 
	NOTE: If placed in the holder in the wrong orientation, the software may not be able to properly recognize the desired IR or UV cutting area.</li>
	<li>Click the &#34;Mem&#34; checkbox in the &#34;Cap and Slide Handling Area&#34; next to the respective slide (i.e., A, B, or C). This step denotes if the slide is a membrane glass slide. Then click on the desired slide to be captured first.</li>
	<li>To make the camera take a tiled image for tissue location and orientation for cap placements, select &#34;Image&#34; on the Toolbar and select &#34;Acquire Overview.&#34; 
	NOTE: If the user is familiar with the tissue being collected, this step can be omitted by using the manual scroll ball to position the &#34;Region Center&#34; for collection.</li>
	<li>Place the cursor over the area on the tiled image (or relative location without tiled image), right click and select &#34;Place Cap at Region Center.&#34; The software will automatically place a cap over the selected position.</li>
	<li>Select a location. 
	NOTE: To ensure that the IR laser is properly aligned and will pick up only areas where spots are laid by the software it must be located before proceeding.
	<ol>
		<li>Move to an area within the cap where no tissue is present. Click &#34;i&#34; in the &#34;Microdissect Tools Pane&#34; and select the &#34;IR Locate&#34; tab. From the Pane, fire an IR test shot to assess where the IR laser is firing and the size of the spot.</li>
		<li>With the cursor, right click in the middle of IR spot and select &#34;Located IR Spot.&#34; 
		NOTE: Size and intensity of the IR beam can be changed by manually altering the &#34;Power, Diameter, or Duration&#34; under each spot size designator or by moving the sliding scale at the bottom of the dialog box. Several test shots may need to be fired to ensure proper spot size. The IR laser must be re-located each time the position of the cap changes or slides are switched.</li>
		<li>For UV cutting, locate the UV laser by selecting the &#34;UV Locate&#34; tab in the dialog box. 
		NOTE: Because the UV laser and the IR laser move in unison, there is no need to continually re-locate the UV laser each time the position is changed.</li>
	</ol>
	</li>
	<li>Choose the &#34;Freehand Drawing&#34; option in the &#34;Select Tools Pane&#34;. Using a touchscreen stylus, draw around the perimeter of the region of interest while making sure not to lose contact between the touchscreen and the stylus head. Make sure to connect the beginning and end-points together. 
	NOTE: IR spots will be laid down automatically through the software but the user may choose to lay down additional spots to ensure the tissue is adhered to the cap after UV cutting has been performed.</li>
	<li>For FrA collection, perform a gross laser capture of cortical tissue up to when the M2 cortex appears at Bregma 5.16 mm. 
	NOTE: This detail can be amended such that only specific portions of the FrA or specific cell types are collected.</li>
	<li>For AcbC collection, laser capture an area close in proximity to the aca. 
	The core and shell of the AcbC perform different functions and are physiologically unique. Thus it is important to follow the brain atlas as close as possible. It is recommended to collect from no further past bregma 0.84 mm, as the two subregions become too closely associated with each other.</li>
	<li>For Hp collection, laser capture the CA 1, 2, and 3 hippocampal subfields starting at Bregma 3.00 mm and using the completely formed GrDG as a physical landmark for morphological identification. 
	NOTE: For the purposes of this study, do not collect past Bregma -4.78 mm, which can be visually demarcated as the point when the Hp subfields are fused and point ventrally. This area was chosen as most injured neurons can be discovered directly under the injury site6.</li>
	<li>For SCN collection, first visually identify the densely packed nuclei that sit dorsal to the och and then collect.</li>
	<li>After collection of regions (listed above), move LCM Macro Caps to the &#34;QC position&#34; and inspect for complete tissue adherence by visually ensuring UV cut tissue is attached to the membrane. Quickly place the cap ontoan RNase-free 0.5 mL thin walled reaction tube containing 100 &#181;L of cell lysis buffer.</li>
	<li>Vortex samples briefly to ensure complete lysis and store at -80 &#176;C. At this point, LCM can be paused and samples stored until used for downstream genomic analysis6.</li>
</ol>

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The authors have nothing to disclose.

For molecular studies of the mammalian brain, LCM has become an essential technique. This article demonstrates that using the combination of the IR and UV cutting lasers in the LCM system can capture genomic changes in any region of the mammalian brain. These regions include those identifiable with conventional LCM-compatible stains, such as cresyl violet or hematoxylin and eosin. The speed of the laser-capture process and ability to perform LCM on thicker 30 &#181;m sections on PEN membrane slides allows to not only obtain sufficient quantities of cell samples, but also to isolate RNA from LCM samples of a suitable quality for all types of downstream genomic analysis; these analyses include microarrays16, PCR arrays17, and quantitative real-time PCR18.
Our data provide a rationale for studies which utilize LCM tissue. We find that miR-15b is upregulated in the hippocampus and cortex (Figure 4) but downregulated in the nucleus accumbens and may be biologically relevant to the understanding of differential effects of TBI in the brain. A previous study suggested that increases in cortical neuronal vulnerability to injury result from overexpression of several miRNAs that negatively regulate pro-survival genes, such as Bcl-219. Target scan analysis shows Bcl-2 is also regulated by miR-15b; thus, our data suggest a mechanistic explanation for why specific regions of the brain (i.e., FCx) may be selectively vulnerable to TBI. It is important to remember most genes are regulated by multiple miRNAs and correlating changes in any one miRNA to a specific target gene is difficult. Moreover, these data indicate that certain changes in gene and microRNA expression may be used as biomarkers of region-specific brain damage. Indeed, we are currently using these data in studies of how new neuroprotective drug compounds with antidepressant properties can differentially affect gene and microRNA expression in brain regions linked to neuropsychiatric disorders. One limitation of our study is that during the multiple steps of slide preparation processes for LCM, RNA integrity may become compromised. This protocol describes the necessary steps to mitigate the risk of RNA degradation. Another limitation is the relatively small sample size used for statistical calculation. In future studies, increasing sample size should lessen the effects of gene and miRNA expression variations among individual animals.
The benefit of LCM is realized in translational genomic studies using animal models of human disease and diseased tissues20,21,22,23. Without the ability to capture specific cell populations, the transcriptional profiles of different brain regions would be an unknowable and undecipherable mix of many cell types. Using LCM methods in brain injury studies has led to current efforts to delineate brain region specific biomarkers and to understand how they correlate with circulating biomarkers of TBI.

The mammalian brain is remarkably complex and heterogeneous with hundreds to thousands of cell types1. Indeed, human studies have shown that in regions such as the frontal cortex, structural and functional differences in white and grey matter are reflected in distinct and divergent transcriptional profiles2. Brain heterogeneity has been a major obstacle to interpret gene expression data and in the field of brain injury. This ambiguity in preclinical studies has subsequently led to hundreds of failed clinical trials of treatments for brain injury3.
We use laser capture microdissection (LCM) methods to study traumatic brain injury (TBI)-induced gene dysregulation in the rat brain4, focusing on the hippocampus, a brain region that is essential for learning and memory5. The ability to laser capture and analyze gene expression in both dying and surviving neurons gives us a greater understanding of the role of stochasticity in gene expression in determining the outcome (neuronal survival) after TBI6. LCM techniques have also proven useful for exploring the effects of TBI on hippocampal neurons when comparing young and aging mice7 or rats8.
In recent studies, we examined other regions of the rat brain adversely impacted by TBI, with a focus on areas in rats and in human TBI patients that are associated with executive function (i.e. frontal cortex9) and TBI comorbidities; these comorbidities include depression (i.e. nucleus accumbens10) and circadian rhythm disorders (suprachiasmatic nucleus11). In previous studies, Huusko and Pitkanen12 and Drexel et al.13 used LCM to examine gene expression in the thalamus and hypothalamus. Our study builds upon these prior observations and includes four other brain regions. To study the region-specific molecular changes induced after TBI, it was necessary to gain expertise in identifying and obtaining cell types in these regions using an LCM system. The UV-cutting and infrared (IR) lasers allow for precise microdissection of desired brain regions. Here, we describe how we use this LCM system, guided by stereotaxic coordinates in the rat brain atlas14, to identify and laser capture four rat brain regions that are differentially affected by the experimental fluid-percussion brain injury method4.

<table>
	<tbody>
		<tr>
			<td>Arcturus Laser Capture Microdissection System</td>
			<td>Applied Biosystems (Life Technologies)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 2100 Bioanalyzer</td>
			<td>Agilent Technologies</td>
			<td>G2939AA</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 6000 Pico Kit</td>
			<td>Agilent Technologies</td>
			<td>5067-1513</td>
			<td></td>
		</tr>
		<tr>
			<td>Arcturus PEN Membrane Glass Slides&#160;</td>
			<td>Applied Biosystems (Life Technologies)</td>
			<td>LCM0522</td>
			<td></td>
		</tr>
		<tr>
			<td>Capsure LCM caps</td>
			<td>Applied Biosystems (Life Technologies)</td>
			<td>LCM0211/LCM0214</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>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 proof (Histological grade)</td>
			<td>UTMB Pharmacy</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>Taqman Gene Expression Primer/Probes</td>
			<td>Applied Biosystems (Life Technologies)</td>
			<td>4331182</td>
			<td></td>
		</tr>
		<tr>
			<td>Taqman microRNA Expression Primer/Probes</td>
			<td>Applied Biosystems (Life Technologies)</td>
			<td>A25576</td>
			<td></td>
		</tr>
		<tr>
			<td>Lightcycler 96 System</td>
			<td>Roche</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The schematic presented in Figure 1 illustrates the overall workflow of atlas guided LCM of specific brain regions and potential downstream analysis applications. This study focused on four brain regions relevant to TBI pathophysiology and to the development of comorbidities: FrA, AcbC, SCN, and Hp. A limitation present in LCM of specific brain regions is that anatomical locations are often obscured by a lack of defined boundaries, as can be seen in Figure 2 (A, D, G, J). The use of a brain atlas to guide sectioning and laser capture of specific regions reduces the possibility of sample contamination with brain regions other than the target. When care is taken to follow tissue landmarks, both during sectioning and after Nissl-staining, LCM technology can provide a highly consistent means of acquiring discrete populations of cells, nuclei, or regions15. The long-term goals of these studies are to identify genes and microRNAs that can potentially serve as surrogate, non-invasive biomarkers of brain region specific injury. The first step in this biomarker development pipeline is the characterization of tissue-specific transcriptional changes after experimental TBI. These data can then be correlated with injury-induced changes in circulating biofluids.
The presented procedures were optimized to mitigate risk of RNA degradation in order to allow RNA analysis via reverse transcription of total LCM RNA before quantitative real-time PCR (RT-qPCR). Total RNA (including small and large RNA species) was isolated using a column-based isolation method on tissue that was laser-captured from individual brain regions. To assess RNA integrity, quality, and quantity after isolation procedures, RNA samples were briefly denatured at 70 &#176;C and run on an RNA analyzer. Qualitative measurements included analyzing peak amplitudes of the 18s and 28s rRNA bands (Figure 3), which can be representative of overall degradation, and using the &#34;RNA Integrity Number&#34; (RIN). If using careful RNase-free techniques, RNA isolated from LCM samples typically results in RINs that range from 6-8. A lower RIN can imply poor RNA quality and can potentially decrease the accuracy of gene and microRNA expression analysis. Conversely, a higher RIN can improve confidence in the validity of results generated from RNA analysis.
RT-qPCR was performed using primer/probe sets for individual genes and microRNAs (Figure 4). Approximately 1 ng of total RNA was reverse-transcribed into cDNA and pre-amplified before qPCR was performed according to manufacturer&#39;s protocol. Injury-related genes assessed in this study included BCL2 Associated X, Apoptosis Regulator (Bax), B-Cell CLL/Lymphoma 2 (Bcl-2), Caspase 3, Apoptosis-Related Cysteine Peptidase (Casp3), Brain Derived Neurotrophic Factor (Bdnf), and CAMP Responsive Element Binding Protein 1 (Creb). MiR-15b was selected because it has been shown to be altered after experimental TBI in individual dying neurons. It also has experimentally validated and bioinformatically predicted gene targets with pro-survival functions (unpublished data). Normalized fold-change ratios were calculated by &#916;&#916;Ct method comparing gene and microRNA expression levels in TBI animals and na&#239;ve controls, with normalization to an endogenous gene (Gapdh) or small RNA (U6), respectively. A fold-change above 1 indicates an overall upregulation in that gene or microRNA, and conversely, a fold change lower than 1 indicates a downregulation. Statistical analysis showed significant changes in gene expression between TBI and na&#239;ve control (p&#160;&#8804; 0.05) that were brain region dependent. No significant changes in miR-15b expression were detected between TBI and na&#239;ve control, but there were trends towards higher and lower expression in a brain region dependent fashion. These data suggest that further optimization is necessary to assess changes in microRNA expression. It is also possible the sample size was too small to gain statistical significance, in part due to inherent variability in expression. Future studies will include sham-operated animals to ensure that gene and microRNA expression changes are attributed to TBI and not due to the surgical preparation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56134/56134fig1.jpg" /> 
Figure 1. Workflow of Atlas-guided LCM for Downstream Genomic Analysis. (A-F) Procedures from animal preparation to downstream qPCR analysis: (A) Adult, male Sprague Dawley rats (~6 weeks of age and weighing 300 g) are anesthetized, subjected to fluid percussion TBI, and humanely euthanized 24 h after injury. (B) Serial sections (30 &#181;m) of fresh frozen brains are based on coordinates of specific brain regions (FrA, AcbC, Hp, SCN) from Paxinos and Watson&#39;s The Rat Brain atlas. (C) Sections are fixed, Nissl-stained (1% Cresyl Violet), dehydrated, and air dried. (D). LCM performed on identified brain regions with an LCM System. (E) LCM Macro Caps transferred onto an RNase-free 0.5 mL tube with 100 &#956;L of cell lysis buffer and stored at -80 &#186;C until RNA isolation for downstream genomic analysis. RNA can then be reverse-transcribed for gene or microRNA RT-qPCR analysis to examine differential expression of molecular target(s) after TBI and/or between brain regions of interest (F). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56134/56134fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56134/56134fig2.jpg" /> 
Figure 2. LCM of TBI Affected Brain Regions. Representative images of tissue sections collected from the ipsilateral side of injury site with IR and UV laser functions on the LCM system (A-I). Tissue was sectioned on a cryostat (30 &#181;m) and collected on PEN membrane slides. Sections were then fixed, Nissl-stained with cresyl violet (1%), and dehydrated to identify specific brain regions based on anatomical landmarks referenced in Paxinos and Watson&#39;s The Rat Brain Atlas. (A-C) An area of the frontal association cortex (FrA) (D-F) Components of the CA1, CA2, and CA3 pyramidal layers of the hippocampus (Hp) located next to the fully formed horns of the granule layer of the dentate gyrus (GrDG). (G-I) An area of the nucleus accumbens core (AcbC) proximal and rostral to the anterior commissure (aca). (J-K) Suprachiasmatic nucleus (SCN) rostral to the supraoptic chiasm (och). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56134/56134fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56134/56134fig3.jpg" /> 
Figure 3. Representative Scans of RNA Quality. Representative electropherograms and associated gel images of RNA derived from LCM collected tissue (A-D). Electropherograms and associated gel images show intact RNA based on appearance of 18s and 28s rRNA peaks and gel bands. This RNA is suitable for all downstream applications, including genomic and proteomic analyses. (A) Frontal association cortex (FrA) RNA. RIN 6.1. (B) Hippocampus (Hp) RNA. RIN 4.4. (C) Nucleus accumbens core (AcbC) RNA. RIN 7.3. (D) Suprachiasmatic nucleus (SCN) RNA. RIN 7.6. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56134/56134fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56134/56134fig4.jpg" /> 
Figure 4. Downstream Analysis of Brain Region-specific Gene and MicroRNA Expression via RT-qPCR. Individual RT-qPCR for genes of interest (Bax, Bcl-2, Bdnf, Casp3, Creb) and microRNA of interest (miR-15b) was performed on laser captured brain regions after TBI (Hp and AcbC n= 5, FrA n= 4) and compared to uninjured na&#239;ve animals (n= 4). Analysis of genes related to TBI pathogenesis was performed for Hp, AcbC, FCx. Data is presented as normalized fold change ratios compared to na&#239;ve control brain regions (Unpaired t test with Welch&#39;s Correction &#177; SEM; * p &#8804; 0.05) (A). Differential expression of miR-15b in different brain regions is presented as normalized fold changes compared to na&#239;ve control (&#177; SEM) (B). Data from SCN (n=2) not included. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56134/56134fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury at JoVE.com

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

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.

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

stereotactic-atlas-guided-laser-capture-microdissection-brain-regions

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.1K Views.  University of Texas Medical Branch. 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. 

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

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 ...

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

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 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.

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 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 ...

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 ...

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the detonation of explosive devices, however, the mechanisms that underlie the brain damage resulting from blast overpressure exposure are not entirely understood and are believed to be unique to this type of brain injury. Preclinical models are crucial tools that contribute to better understand blast-induced brain injury. A novel in vitro blast TBI model was developed using an open-ended shock tube to simulate real-life open-field blast waves modelled by the Friedlander waveform. C57BL/6N mouse organotypic hippocampal slice cultures were exposed to single shock waves and the development of injury was characterized up to 72 h using propidium iodide, a well-established fluorescent marker of cell damage that only penetrates cells with compromised cellular membranes. Propidium iodide fluorescence was significantly higher in the slices exposed to a blast wave when compared with sham slices throughout the duration of the protocol. The brain tissue injury is very reproducible and proportional to the peak overpressure of the shock wave applied.

A Novel In Vitro Model of Blast Traumatic Brain Injury

This paper describes a novel model of primary blast traumatic brain injury. A compressed-air driven shock tube is used to expose in vitro mouse hippocampal slice cultures to a single shock wave. This is a simple and rapid protocol generating a reproducible brain tissue injury with a high throughput.

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...