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>

<ol><li>Bota, M., Dong, H. W., Swanson, L. W. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/12886225">From gene networks to brain networks.</a> Nat. Neurosci. 6 (8), 795-799 (2003).</li>
<li>Mills, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unique+transcriptome+patterns+of+the+white+and+grey+matter+corroborate+structural+and+functional+heterogeneity+in+the+human+frontal+lobe%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Unique transcriptome patterns of the white and grey matter corroborate structural and functional heterogeneity in the human frontal lobe.</a> PLoS One. 8 (10), e78480(2013).</li>
<li>Doppenberg, E. M., Choi, S. C., Bullock, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+trials+in+traumatic+brain+injury%3A+lessons+for+the+future%5BTitle%5D)+AND+%22J+Neurosurg+Anesthesiol%22%5BJournal%5D)">Clinical trials in traumatic brain injury: lessons for the future.</a> J Neurosurg Anesthesiol. 16 (1), 87-94 (2004).</li>
<li>Shimamura, M., Garcia, J. M., Prough, D. S., Hellmich, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Laser+capture+microdissection+and+analysis+of+amplified+antisense+RNA+from+distinct+cell+populations+of+the+young+and+aged+rat+brain%3A+effect+of+traumatic+brain+injury+on+hippocampal+gene+expression%5BTitle%5D)+AND+%22Mol+Brain+Res%22%5BJournal%5D)">Laser capture microdissection and analysis of amplified antisense RNA from distinct cell populations of the young and aged rat brain: effect of traumatic brain injury on hippocampal gene expression.</a> Mol Brain Res. 122 (1), 47-61 (2004).</li>
<li>Bast, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Toward+an+integrative+perspective+on+hippocampal+function%3A+from+the+rapid+encoding+of+experience+to+adaptive+behavior%5BTitle%5D)+AND+%22Rev+Neurosci%22%5BJournal%5D)">Toward an integrative perspective on hippocampal function: from the rapid encoding of experience to adaptive behavior.</a> Rev Neurosci. 18 (3-4), 253-281 (2007).</li>
<li>Rojo, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+stochastic+gene+expression+on+the+cell+survival+rheostat+after+traumatic+brain+injury%5BTitle%5D)+AND+%22PLoS.One%22%5BJournal%5D)">Influence of stochastic gene expression on the cell survival rheostat after traumatic brain injury.</a> PLoS.One. 6 (8), e23111(2011).</li>
<li>Shah, S. A., Prough, D. S., Garcia, J. M., DeWitt, D. S., Hellmich, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+correlates+of+age-specific+responses+to+traumatic+brain+injury+in+mice%5BTitle%5D)+AND+%22Exp+Gerontol%22%5BJournal%5D)">Molecular correlates of age-specific responses to traumatic brain injury in mice.</a> Exp Gerontol. 41 (11), 1201-1205 (2006).</li>
<li>Shearer, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stochastic+fluctuations+in+gene+expression+in+aging+hippocampal+neurons+could+be+exacerbated+by+traumatic+brain+injury%5BTitle%5D)+AND+%22Aging+Clin.Exp.Res%22%5BJournal%5D)">Stochastic fluctuations in gene expression in aging hippocampal neurons could be exacerbated by traumatic brain injury.</a> Aging Clin.Exp.Res. 28 (2), 363-367 (2015).</li>
<li>Caeyenberghs, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Altered+structural+networks+and+executive+deficits+in+traumatic+brain+injury+patients%5BTitle%5D)+AND+%22Brain+Struct.Funct%22%5BJournal%5D)">Altered structural networks and executive deficits in traumatic brain injury patients.</a> Brain Struct.Funct. 219 (1), 193-209 (2014).</li>
<li>Bewernick, B. H., Kayser, S., Sturm, V., Schlaepfer, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+effects+of+nucleus+accumbens+deep+brain+stimulation+in+treatment-resistant+depression%3A+evidence+for+sustained+efficacy%5BTitle%5D)+AND+%22Neuropsychopharmacology%22%5BJournal%5D)">Long-term effects of nucleus accumbens deep brain stimulation in treatment-resistant depression: evidence for sustained efficacy.</a> Neuropsychopharmacology. 37 (9), 1975-1985 (2012).</li>
<li>Karatsoreos, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+Circadian+Disruption+on+Mental+and+Physical+Health%5BTitle%5D)+AND+%22Curr.Neurol.Neurosci+Rep%22%5BJournal%5D)">Effects of Circadian Disruption on Mental and Physical Health.</a> Curr.Neurol.Neurosci Rep. 12 (2), 218-225 (2012).</li>
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<li>Huusko, N., Pitkanen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Parvalbumin+immunoreactivity+and+expression+of+GABAA+receptor+subunits+in+the+thalamus+after+experimental+TBI%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">Parvalbumin immunoreactivity and expression of GABAA receptor subunits in the thalamus after experimental TBI.</a> Neuroscience. 267, 30-45 (2014).</li>
<li>Paxinos, G., Watson, C. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPaxinos%2C+G+%22The+rat+brain+in+stereotaxic+coordinates%22">The rat brain in stereotaxic coordinates</a>. , 7th ed, Academic Press. (2013).</li>
<li>Boone, D. R., Sell, S. L., Hellmich, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Laser+capture+microdissection+of+enriched+populations+of+neurons+or+single+neurons+for+gene+expression+analysis+after+traumatic+brain+injury%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Laser capture microdissection of enriched populations of neurons or single neurons for gene expression analysis after traumatic brain injury.</a> Journal of Visualized Experiments. (74), 1-7 (2012).</li>
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<li>Boone, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pathway-focused+PCR+array+profiling+of+enriched+populations+of+laser+capture+microdissected+hippocampal+cells+after+traumatic+brain+injury%5BTitle%5D)+AND+%22PLoS.One%22%5BJournal%5D)">Pathway-focused PCR array profiling of enriched populations of laser capture microdissected hippocampal cells after traumatic brain injury.</a> PLoS.One. 10 (5), e0127287(2015).</li>
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</ol>

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&amp;nbsp;</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/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/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/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/files/ftp_upload/56134/56134fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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

Research

JoVE Journal

Neuroscience

9.1K Views.  University of Texas Medical Branch. We beschrijven het gebruik van laser vangt microdissection te verkrijgen van monsters van afzonderlijke cel populaties van verschillende hersengebieden gen en microRNA p.a.. Deze techniek maakt het mogelijk de studie van Differentiele effecten van traumatische hersenenverwonding in bepaalde gebieden van de hersenen van de rat.

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

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 ...

Industrialized, Artificial Intelligence-guided Laser Microdissection for Microscaled Proteomic Analysis of the Tumor Microenvironment

The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell populations. To characterize proteome-level variation and tumor heterogeneity at scale, high-throughput methods are needed to selectively isolate discrete cellular populations in solid tumor malignancies. This protocol describes a high-throughput workflow, enabled by artificial intelligence (AI), that segments images of hematoxylin and eosin (H&#38;E)-stained, thin tissue sections into pathology-confirmed regions of interest for selective harvest of histology-resolved cell populations using laser microdissection (LMD). This strategy includes a novel algorithm enabling the transfer of regions denoting cell populations of interest, annotated using digital image software, directly to laser microscopes, thus enabling more facile collections. Successful implementation of this workflow was performed, demonstrating the utility of this harmonized method to selectively harvest tumor cell populations from the TME for quantitative, multiplexed proteomic analysis by high-resolution mass spectrometry. This strategy fully integrates with routine histopathology review, leveraging digital image analysis to support enrichment of cellular populations of interest and is fully generalizable, enabling harmonized harvests of cell populations from the TME for multiomic analyses.

This protocol describes a high-throughput workflow for artificial intelligence-driven segmentation of pathology-confirmed regions of interest from stained, thin tissue section images for enrichment of histology-resolved cell populations using laser microdissection. This strategy includes a novel algorithm enabling the transfer of demarcations denoting cell populations of interest directly to laser microscopes.

The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell ...

Cancer Research

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

Medicine

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

A Micro-CT-based Method for Characterizing Lesions and Locating Electrodes in Small Animal Brains

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that requires manual estimation. Here, we describe a simple, straightforward method for quantifying lesions and locating electrodes in the brain that is less laborious and yields more detailed results. Whole brains are stained with osmium tetroxide, embedded in resin, and imaged with a micro-CT scanner. The scans result in 3D digital volumes of the brains with resolutions and virtual section thicknesses dependent on the sample size (12&#8211;15 and 5&#8211;6 &#181;m&#160;per voxel for rat and zebra finch brains, respectively). Surface and deep lesions can be characterized, and single tetrodes, tetrode arrays, electrolytic lesions, and silicon probes can also be localized. Free and proprietary software allows experimenters to examine the sample volume from any plane and segment the volume manually or automatically. Because this method generates whole brain volume, lesions and electrodes can be quantified to a much higher degree than in current methods, which will help standardize comparisons within and across studies.

This article describes a straightforward method to prepare small animal brains for micro-CT imaging, in which lesions can be quantified and electrodes located with high precision in the context of the whole brain.

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that ...

Laser-Induced Brain Injury in the Motor Cortex of Rats

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) occlusion of the middle cerebral artery (MCA) using a catheter. This generally accepted technique, however, has some limitations, thereby limiting its extensive use. Stroke induction by this method is often characterized by high variability in the localization and size of the ischemic area, periodical occurrences of hemorrhage, and high death rates. Also, the successful completion of any of the transient or permanent procedures requires expertise and often lasts for about 30 minutes. In this protocol, a laser irradiation technique is presented that can serve as an alternative method for inducing and studying brain injury in rodent models.
When compared to rats in the control and MCAO groups, the brain injury by laser induction showed reduced variability in body temperature, infarct volume, brain edema, intracranial hemorrhage, and mortality. Furthermore, the use of a laser-induced injury caused damage to the brain tissues only in the motor cortex unlike in the MCAO experiments where destruction of both the motor cortex and striatal tissues is observed.
Findings from this investigation suggest that laser irradiation could serve as an alternative and effective technique for inducing brain injury in the motor cortex. The method also shortens the time for completing the procedure and does not require expert handlers.

The protocol presented here shows a technique to create a rodent model of brain injury. The method described here uses laser irradiation and targets motor cortex.

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) ...

Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, microvascular proliferation, mesenchymal transformation and necrosis characterize the histological heterogeneity of high-grade gliomas. Our laboratory has demonstrated that the presence of high densities of mesenchymal cells, named oncostreams, correlate with tumor malignancy. We have developed a unique approach to understand the mechanisms that underlie glioma&#39;s growth and invasion. Here, we describe a comprehensive protocol that utilizes laser capture microdissection (LMD) and RNA sequencing to analyze differential mRNA expression of intra-tumoral heterogeneous multicellular structures (i.e., mesenchymal areas or areas of tumor invasion). This method maintains good tissue histology and RNA integrity. Perfusion, freezing, embedding, sectioning, and staining were optimized to preserve morphology and obtain high-quality laser microdissection samples. The results indicate that perfusion of glioma bearing mice using 30% sucrose provides good morphology and RNA quality. In addition, staining tumor sections with 4% Cresyl violet and 0.5% eosin results in good nuclear and cellular staining, while preserving RNA integrity. The method described is sensitive and highly reproducible and it can be utilized to study tumor morphology in various tumor models. In summary, we describe a complete method to perform LMD that preserves morphology and RNA quality for sequencing to study the molecular features of heterogeneous multicellular structures within solid tumors.

Laser microdissection (LMD) is a sensitive and highly reproducible technique that can be used to uncover pathways that mediate glioma heterogeneity and invasion. Here, we describe an optimized protocol to isolate discrete areas from glioma tissue using laser LMD followed by transcriptomic analysis.

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, ...

Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke

Stroke is the leading cause of disability and the fifth-leading cause of death in the United States. Approximately 87% of all strokes are ischemic strokes and are defined as the sudden blockage of a vessel supplying blood to the brain. Within minutes of the blockage, cells begin to die and result in irreparable tissue damage. Current therapeutic treatments focus on clot removal or lysis to allow for the reperfusion and prevent more severe brain damage. Although transient brain plasticity may salvage some of the damaged tissue over time, significant fractions of patients are left with neurological deficits that will never resolve. There is a lack of therapeutic options to treat neurological deficits caused by stroke, emphasizing the need to develop new strategies to treat this growing patient population. Injectable biomaterials are currently being designed to enhance brain plasticity and improve endogenous repair through the delivery of active agents or stem cells. One method to test these approaches is to utilize a rodent stroke model, inject the biomaterial into the stroke core, and assess repair. Knowing the precise location of the stroke core is imperative for the accurate treatment after stroke, therefore, a stroke model that results in a predictable stroke location is preferable to avoid the need for imaging prior to injection. The following protocol will cover how to induce a photothrombotic stroke, how to inject a hydrogel in a controlled and precise manner, and how to extract and cryosection the brain while keeping the biomaterial intact. In addition, we will highlight how these same hydrogel materials can be used for the co-delivery of stem cells. This protocol can be generalized to the use of other injectable biomaterials into the stroke core.

Stroke is a global issue with minimal treatment options and no current clinical therapy for regenerating the lost brain tissue. Here we describe methods for creating precise photothrombotic stroke in the motor cortex of rodents and subsequent injection of hydrogel biomaterials to study their effects on tissue regeneration after stroke.

Stroke is the leading cause of disability and the fifth-leading cause of death in the United States. Approximately 87% of all strokes are ischemic strokes ...

Biology

Cryopreserved Tissue Section Fixation and Staining: A Procedure to Process Frozen Brain Tissue Sections for Laser Microdissection

Source: Comba, A. et al. <a href="https://www.jove.com/v/60939/laser-capture-microdissection-glioma-subregions-for-spatial-molecular">Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion.</a>&#160;J. Vis. Exp.&#160;(2020)
This video presents the fixation and staining method to process frozen mouse brain tumor tissue. The processed tissue helps differentiate and obtain a specific population of cells during subsequent laser capture microdissection.

Source: Comba, A. et al. Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, ...

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

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