Name: collection of frozen rodent brain regions for downstream analyses
Uploaded: 2020-04-23T13:00:00
Description: Watch this Scientific Journal Video about Collection of Frozen Rodent Brain Regions for Downstream Analyses at JoVE.com

This work was supported by the NIH, DA043982 and DA046196.

Animals used in this study were treated in an ethical and humane manner as set forth by Indiana University&#8217;s Institutional Animal Care and Use Committee (IACUC) and National Institutes of Health (NIH) guidelines.
NOTE: All tools and surfaces should be washed with an appropriate solvent to remove nucleases18 before starting any work.
1. Storing brains
<ol>
	<li>Quickly remove the brains from euthanized adult CD1 wildtype mice weighing ...

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+Rodent+Brain+Regions.">Dissection of Rodent Brain Regions.</a> Neuroproteomics, Neuromethods. 57, 13-26 (2011).</li><li>Palkovits, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+removal+of+hypothalamic+or+other+brain+nuclei+of+the+rat.">Isolated removal of hypothalamic or other brain nuclei of the rat.</a> Brain Research. 59, 449-450 (1973).</li><li>Palkovits, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Enzymology. 103, 368-376 (1983).</li><li>Paxinos, G., Franklin, K. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. 2 edn. , (2001).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Rat Brain in Stereotaxic Coordinates. 4 edn. , (1998).</li><li>RNaseZap. Ambion Available from: <a target="_blank" href="https://assets.thermofisher.com/TFS-Assets/LSG/">https://assets.thermofisher.com/TFS-Assets/LSG/</a> (2008)</li><li>. RNA Integrity Number (RIN)- Standardization of RNA Quality Control Available from: <a target="_blank" href="https://www.agilent.com/cs/library/">https://www.agilent.com/cs/library/</a> (2016)</li><li>Scheyer, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabinoid+Exposure+via+Lactation+in+Rats+Disrupts+Perinatal+Programming+of+the+Gamma-Aminobutyric+Acid+Trajectory+and+Select+Early-Life+Behaviors.">Cannabinoid Exposure via Lactation in Rats Disrupts Perinatal Programming of the Gamma-Aminobutyric Acid Trajectory and Select Early-Life Behaviors.</a> Biological Psychiatry. , (2019).</li><li>Choi, D. W., Rothman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+glutamate+neurotoxicity+in+hypoxic-ischemic+neuronal+death.">The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death.</a> Annual Review of Neuroscience. 13 (1), 171-182 (1990).</li><li>Guhaniyogi, J., Brewer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+mRNA+stability+in+mammalian+cells.">Regulation of mRNA stability in mammalian cells.</a> Gene. 265 (1-2), 11-23 (2001).</li><li>Sampaio-Silva, F., Magalh&#227;es, T., Carvalho, F., Dinis-Oliveira, R. 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This work describes a technique to isolate small, specific regions of brain while limiting degradation of nucleic acid and protein. Damage to brain tissues happens quickly once an organism dies. This is partially due to a rapid buildup of extracellular glutamate and the resultant excitotoxicity that occurs21. Messenger RNA is particularly vulnerable to degradation22,23. Breakdown of protein and nucleic acid is greatly reduced at low temper...

This work illustrates the dissection of frozen brain regions for extraction of high-quality nucleic acid or protein using a reference, such as the Allen Mouse Brain Atlas1, as a guide. In this technique, brains are flash-frozen and stored at -80 &#176;C for later sectioning and dissection while being maintained in a frozen condition. This process allows the researcher to harvest a large number of brains in one session and later dissect them for an accurate collection of multiple brain regions.
The accurate collection of brain regions of interest (ROIs) is often required when answering questions related to gene and protein expression. While pharmacology, electrophysiology and optogenetics can be used on wildtype or genetically modified rodents to help elucidate molecular changes underpinning observed behaviors2,3,4, the measurement of induced changes in transcriptomes and proteomes is often used to support these findings. Techniques such as quantitative reverse transcription polymerase chain reaction (RT-qPCR), western blotting, RNAseq5, MAPSeq6 and HPLC7 are robust and relatively low in cost, allowing many labs to study induced molecular changes within small brain regions2,4,5,6.
There are several ways to extract and purify nucleic acid or protein from brain regions8,9,10,11,12. Many labs harvest brain regions by chilling and cutting brains on ice at the time of harvest9,13. While this approach can result in high quality nucleic acid and protein, it is somewhat time-limited as degradation within the microenvironment of the tissue may take place at these temperatures. This may be particularly true when attempting to dissect a large number of animals or ROIs in one sitting. Keeping samples frozen helps maintain labile target molecules while providing the researcher time to carefully compare landmarks on both sides of each section in the effort to collect relatively pure samples. Laser capture is another way to collect tissue for RNA or protein analysis from brain areas10. This procedure is superior to mechanical dissection in that very small and irregularly shaped ROIs can be identified and isolated. However, laser capture is limited by the use of expensive equipment and reagents, is time consuming and may also be more susceptible to sample degradation.
Micropunch dissection on frozen tissues is not new. Early papers by Miklos Palkovits and others describe the basic techniques in detail14,15. This presentation largely follows the original work, with some improvements to facilitate efficiency and decrease the expense of the equipment needed. For instance, brain sections are made in a frozen brain block rather than on a cryostat. This produces thicker sections which reduces the number of sections needed to collect ROI samples. This method also dissects samples on a frozen glass plate that sits on dry ice within an insulated box. This produces a sub-freezing stage at the bench on which to work. Sections dissected in this way are easily manipulatable, allowing the researcher to compare both sides of each section with a reference in order to limit contamination from regions outside the desired ROI.
Advantages of this protocol are that 1) the brain is kept in a frozen condition throughout the process, which helps preserve protein and nucleic acid and gives the researcher time to carefully harvest ROIs, and 2) the reagents required are inexpensive and are found in most molecular biology labs. In this process, whole brains are sectioned to 0.5&#8211;1.0 mm in a brain matrix and placed on a frozen glass plate that is continuously chilled with dry ice. Landmarks found in the Allen Brain Atlas1 or other brain atlases16,17 are used to identify regions of interest, which are then dissected using either a cold punch or scalpel. Because the tissue is never thawed, regions harvested in this manner provide high quality RNA and protein for downstream analyses.

<table>
	<tbody>
		<tr>
			<td>0.5 mm Mouse coronal brain matrice</td>
			<td>Braintree Scientific</td>
			<td>BS-SS 505C</td>
			<td>Cutting block</td>
		</tr>
		<tr>
			<td>0.5 mm Rat coronal brain matrice</td>
			<td>Braintree Scientific</td>
			<td>BS-SS 705C</td>
			<td>Cutting block</td>
		</tr>
		<tr>
			<td>1.0 mm Biopsy Punch with plunger</td>
			<td>Electron Microscopy Sciences</td>
			<td>69031-01</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>Dot Scientific</td>
			<td>229443</td>
			<td>For storing frozen ROIs</td>
		</tr>
		<tr>
			<td>1.5 mm Biopsy Punch with plunger</td>
			<td>Electron Microscopy Sciences</td>
			<td>69031-02</td>
			<td></td>
		</tr>
		<tr>
			<td>2.0 mm Biopsy Punch with plunger</td>
			<td>Electron Microscopy Sciences</td>
			<td>69031-03</td>
			<td></td>
		</tr>
		<tr>
			<td>4-12% NuPage gel</td>
			<td>Invitrogen</td>
			<td>NPO323BOX</td>
			<td>protein gradient gel</td>
		</tr>
		<tr>
			<td>Bioanalyzer System</td>
			<td>Agilent</td>
			<td>2100</td>
			<td>RNA analysis system</td>
		</tr>
		<tr>
			<td>Dounce tissue grinder</td>
			<td>Millipore Sigma</td>
			<td> 
			D8938</td>
			<td>Glass tissue homogenizer</td>
		</tr>
		<tr>
			<td>Dry Ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber-Lite</td>
			<td>Dolan-Jenner Industries Inc.</td>
			<td>Model 180</td>
			<td>Cool lamp</td>
		</tr>
		<tr>
			<td>Glass plates</td>
			<td>LabRepCo</td>
			<td>11074010</td>
			<td></td>
		</tr>
		<tr>
			<td>HALT</td>
			<td>ThermoFisher</td>
			<td>78440</td>
			<td>protease inhibitor cocktail</td>
		</tr>
		<tr>
			<td>Low profile blades</td>
			<td>Sakura Finetek USA Inc.</td>
			<td>4689</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse anti-actin antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>JLA20</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Nanodrop</td>
			<td>Thermo Scientific</td>
			<td>2000C</td>
			<td>Used in initial RNA purity analysis</td>
		</tr>
		<tr>
			<td>No. 15 surgical blade</td>
			<td>Surgical Design Inc</td>
			<td>17467673</td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey Blocking buffer</td>
			<td>LiCor Biosciences</td>
			<td>927-40000</td>
			<td>Western blocking reagent</td>
		</tr>
		<tr>
			<td>Omni Tissue Master 125</td>
			<td>VWR</td>
			<td>10046-866</td>
			<td>Tissue homogenizer</td>
		</tr>
		<tr>
			<td>rabbit anti-KCC2 antibody</td>
			<td>Cell Signaling Technology</td>
			<td>94725S</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>RNA Plus Micro Kit</td>
			<td>Qiagen</td>
			<td>73034</td>
			<td>Used to extract RNA from small tissue samples</td>
		</tr>
		<tr>
			<td>RNaseZap</td>
			<td>Life Technologies</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>Excelta Corp.</td>
			<td>16050103</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard razor blades</td>
			<td>American Line</td>
			<td>66-0362</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>15596026</td>
			<td>Used to extract RNA from tissue</td>
		</tr>
	</tbody>
</table>

collection of frozen rodent brain regions for downstream analyses

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex (mPFC) or nucleus accumbens. The challenge in this work is to dissect the correct area while preserving the microenvironment to be examined. In this paper, we describe a simple, low-cost method using resources readily available in most labs. This method preserves nucleic acid and proteins by keeping the tissue frozen throughout the process. Brains are cut into 0.5&#8211;1.0 mm sections using a brain matrix and arranged on a frozen glass plate. Landmarks within each section are compared to a reference, such as the Allen Mouse Brain Atlas, and regions are dissected using a cold scalpel or biopsy punch. Tissue is then stored at -80 &#176;C until use. Through this process rat and mouse mPFC, nucleus accumbens, dorsal and ventral hippocampus and other regions have been successfully analyzed using qRT-PCR and Western assays. This method is limited to brain regions that can be identified by clear landmarks.

In order to validate this method, the medial prefrontal cortex was collected from adult CD1 wildtype male mice and RNA and protein were extracted and characterized. RNA was analyzed by capillary electrophoresis. Degraded RNA displays a loss in the intensity of the 28S and 18S ribosomal bands and also shows degradation products as a smear between 25 and 200 nucleotides (Figure 5A, sample 1). High quality RNA shows distinct ribosomal bands with little to no signal in the lower molecular weight...

Watch this Scientific Journal Video about Collection of Frozen Rodent Brain Regions for Downstream Analyses at JoVE.com

Collection of Frozen Rodent Brain Regions for Downstream Analyses

This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools.

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex ...

This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools. Because brain regions are kept frozen from harvest through dissection, target molecules are preserved and the researcher has time to carefully dissect and store regions of interest. Identifying regions of interest can initially be challenging.Using common brain landmarks as they emerge and recede through the sections in relation to the desired regions will make identification clear. After removing brains from euthanized adult CD-1 wild type mice, flash freeze the tissue for 60 seconds in either liquid nitrogen or isopentane. Pre-chill with dry ice and store it at minus 80 degree Celsius.24 hours before dissecting the tissue, place a clean brain matrix on a stack of thawed freezer packs. And sandwich the sides to the matrix between two freezer packs making sure to leave approximately half a centimeter between the bottom of the razor slots and the top of the packs. Set up a frozen glass plate for the dissection by filling an insulated box with ice up to approximately five centimeters from the top.Then place a 2.5 centimeter layer of dry ice on top. And cover it with black plastic sheeting. Place a glass plate on top of the plastic.And dry ice around the borders of the plate. Remove the frozen brain matrix from the freezer and insert the brain, cortex side up, into the matrix. Allow the tissue to equilibrate to the temperature in the box for 10 minutes.Keeping the lid open during this time. Use cold forceps to adjust the brain's position in the matrix so that the sagittal sinus and transverse sinus line up with the perpendicular grooves of the block. Once the brain is in position, place a chilled razor blade near its center and press it approximately one millimeter into the tissue.Next, place one chilled blade at each end of the brain and press all the way down into the matrix. Then begin adding chilled blades at the rostral end, placing them into the slots one at a time and gently pressing them one millimeter into the tissue. Continue to add blades at one millimeter intervals, working toward the caudal end.When all blades are in place, press down on top with fingers, palm, or a blunt object and rock them slowly from side-to-side to move them through the tissue. Once the blades have reached the bottom of the slots, grasp each side of the group of blades and free them from the matrix by rocking back and forth. After freeing the group of blades place them rostral side up on the glass plate, and put dry ice next to or on top of the stack to further freeze the samples for easier separation.Then place the stack with sharp edges down and separate the blades by shifting the stack between the thumb and fingers. Line up the section on the glass plates from rostral to caudal and separate the tissue from the blades by flexing them between the fingers, or by separating them with a second chilled blade. At times, tissue may stick to both sides of a blade and care must be taken to maintain rostral to caudal orientation.Before starting the dissection, open the Allen Mouse Brain Atlas or another reference and find the landmarks necessary to identify regions of interest. Use chilled forceps or blades to flip the section. And make sure that the region of interest is consistent throughout the section.Cut into the section with a clean scalpel or a punch, pushing the metal gently but firmly into the tissue. Rocking it back and forth to make the cut. After harvesting the region of interest, place it into labeled pre-chilled 1.5 millimeter tubes and store them at minus 80 degree Celsius.To process the tissue, add cold RIPA buffer for protein extraction or a guanidinium containing solvent for RNA extraction and immediately homogenize it with a glass downs or mechanical homogenizer. To validate this method, the medial prefrontal cortex was collected from adult CD-1 wild type male mice, and RNA and protein were characterized. When analyzed with capillary electrophoresis, degraded RNA displays a loss in the intensity of the 28S and 18S ribosomal bands, as well as a smear between 25 and 200 nucleotides.While high-quality RNA shows distinct ribosomal bands little to no signal in the lower molecular weight region. The RNA obtain using the frozen dissection method was compared with RNA prepared from freshly harvested tissue. Both methods produce RNA with high integrity numbers and strong ribosomal banding.To confirm that this method can be used to preserve the microenvironment of dissected tissue for later analysis, RNA was extracted from dissected medial prefrontal cortex that had been stored over several weeks. All samples produced high-quality RNA with distinct ribosomal banding and RNA integrity numbers above eight. To confirm protein integrity of the frozen dissected samples, the protein was collected, transferred to nitrocellulose and probe with antibody against the high molecular weight target KCC2, and the lower molecular weight protein actin.In all cases bands were sharp and distinct with no discernible breakdown products. It is important to keep the tissues frozen throughout the process as this preserves the microenvironment of the sections and maintain section morphology for comparison with brain atlas images. Regions of interest collected in this manner can be stored for several months at negative 80 degree Celsius and can be utilized in many downstream applications, including RT-qPCR, RNA-Seq, Western blot analysis, and HPLC.This method has been used to explore molecular changes within the limbic systems of perinatal rodents exposed to THC and other cannabinoids to better understand how these drugs may affect brain development.

Research

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