Collection of Frozen Rodent Brain Regions for Downstream Analyses

Summary

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

Abstract

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 0.5–1.0 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 °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.

Introduction

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 °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 behaviors^2,3,4, 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 regions^2,4,5,6.

There are several ways to extract and purify nucleic acid or protein from brain regions^8,9,10,11,12. Many labs harvest brain regions by chilling and cutting brains on ice at the time of harvest^9,13. 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 detail^14,15. 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 0.5–1.0 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 atlases^16,17 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.

Protocol

Animals used in this study were treated in an ethical and humane manner as set forth by Indiana University’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

1. Quickly remove the brains from euthanized adult CD1 wildtype mice weighing approximately 30 g using a conventional approach¹³ and flash-freeze for 60 seconds in either liquid nitrogen or isopentane pre-chilled with dry ice.
2. Store frozen brains at -80 °C in aluminum foil or conical tubes until use.

2. Preparing the brain matrix

1. Twenty-four hours before dissecting tissue, place a clean brain matrix (see Table of Materials) on a stack of thawed freezer packs. Sandwich the sides of the matrix between two freezer packs making sure to leave approximately 0.5 cm between the bottom of the razor slots and the top of the gel packs (Figure 1A). Place aluminum foil on the ends to aid in cooling.
   NOTE: When purchasing a brain matrix, buy one that is large enough to encase the entire volume of the brain to be dissected.
2. Place the box containing the brain matrix and freezer packs in a -20 °C freezer with the top ajar overnight.

3. Setting up a frozen glass plate

NOTE: The purpose of this setup is to prepare a frozen surface on which to dissect brain sections.

1. Place ice in an insulated box up to approximately 5 cm from the top. Then place a 2.5 cm layer of dry ice on top of the ice and cover with black plastic sheeting to aid in visualizing the sample (Figure 1B, Figure 1C).
2. Place a glass plate (should just fit in the opening of the box) on top of the plastic and place dry ice on top of the plate in the far corners (Figure 1C).
3. Take the frozen brain matrix from the freezer and insert the brain to be cut cortex-side up. Allow it to equilibrate to the temperature in the box for 10 min. Keep the lid on the box during this time.
4. Adjust the brain’s position in the matrix with cold forceps so that the sagittal sinus and transverse sinus line up with the perpendicular grooves of the block (Figure 1D). This will help ensure symmetric sections for easier dissection. Touch tips of forceps to dry ice briefly to chill before adjusting the brain.
5. Once the brain is in position, place a chilled razor blade near its center and press the blade approximately 1 mm into the tissue. Add chilled blades to the rostral and caudal ends to help hold the brain in place (Figure 2A and Figure 2B).
6. Starting on the rostral end, add blades one at a time, placing them into the slots and pressing them gently down into the tissue approximately 1 mm (Figure 2B). Continue to add blades at 1 mm intervals working towards the caudal end.
7. Make sure the brain does not shift during this time. Line up blades horizontally and vertically (Figure 2B). In order to cut sections into 0.5 mm widths, low profile microtome blades (see Table of Materials) may be used. Place these between the larger razor blades (Figure 2C).
8. Once blades are in place, press down on top of the group with fingers, palm or some other flat surface (Figure 2C, Figure 2D). Rock the group of blades slowly from side to side to move them through the tissue.
   NOTE: This may take some time (1–2 min) and requires patience. There should be resistance to the blades moving through the tissue. Easy entry means the brain is thawing and the block should be cooled with dry ice.
9. When all blades have reached the bottom of the slots, grasp each side of the group of blades and work free of the matrix by rocking back and forth.
   NOTE: Exercise caution to avoid cutting oneself on the sharp edges of the blades.
10. Once the group is free, place the stack, rostral side up, on the glass plate (Figure 2E). Place dry ice next to and/or on top of the stack to further freeze the samples for easier separation.
11. Place the stack with sharp edges down on the glass plate and separate blades by shifting the stack between thumbs and fingers. The blades should separate from each other with sections attached.
12. Line up sections on the glass plate from rostral to caudal (Figure 2F).
13. Separate tissue from the blades by flexing blades between fingers, or by separating with a second cold razor (Figures 2G,2H,2I).

4. Dissecting sections

1. Open the Allen Mouse Brain Atlas or another reference and find landmarks necessary to identify regions of interest. Some obvious landmarks include the anterior commissure, the corpus callosum, the lateral ventricles, and the hippocampus (Figure 3).
2. Flip the section to be cut with chilled forceps and make sure the region about to be collected is consistent throughout the section. During harvesting, consult the reference atlas often to make sure the correct ROI is obtained.
   NOTE: A magnifying glass or dissecting microscope is often helpful in this process. Good lighting is also essential. Low wattage LED lamps or a cool lamp (see Table of Materials) can be used for this purpose.
3. With a clean scalpel or punch, cut into the section (Figure 4). Prechill each tool before cutting by touching it briefly to dry ice. Push the blade gently but firmly into the tissue, rocking it back and forth to make the cut. Do not push too hard or the tissue will fracture.
   NOTE: Tools will warm over time. Slightly warmed blades can be helpful in making clean cuts and can limit fracturing, but be careful to avoid thawing of tissue. Periodically chill tools on dry ice.
4. Once an ROI is harvested, place it into labeled, prechilled 1.5 mL tubes. Store harvested tissues at -80 °C until needed.
5. Process frozen tissue collected in this manner by adding cold RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% CHAPS and protein inhibitor cocktail (see Table of Materials)) for protein extraction, or a guanidinium-containing solvent (see Table of Materials) for RNA extraction to the frozen sample and immediately homogenize using either a glass Dounce or mechanical homogenizer (see Table of Materials). In this way, protease and nuclease inhibitors are in place as the sample warms to protect protein and nucleic acids from degradation.

Results

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...

Discussion

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 degradation^22,23. Breakdown of protein and nucleic acid is greatly reduced at low temper...

Materials

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