We present a hands-on, step-by-step, rapid protocol for mouse brain removal and dissection of discrete regions from fresh brain tissue. Obtaining brain regions for molecular analysis has become routine in many neuroscience labs. These brain regions are immediately frozen to obtain high quality transcriptomic data for system level analysis.
The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain consists of an outer covering of grey matter over the hemispheres known as the cerebral cortex. Underneath this layer reside many other specialized structures that are essential for multiple phenomenon important for existence. Acquiring samples of specific gross brain regions requires quick and precise dissection steps. It is understood that at the microscopic level, many sub-regions exist and likely cross the arbitrary regional boundaries that we impose for the purpose of this dissection.
Mouse models are routinely used to study human brain functions and diseases. Changes in gene expression patterns may be confined to specific brain areas targeting a particular phenotype depending on the diseased state. Thus, it is of great importance to study regulation of transcription with respect to its well-defined structural organization. A complete understanding of the brain requires studying distinct brain regions, defining connections, and identifying key differences in the activities of each of these brain regions. A more comprehensive understanding of each of these distinct regions may pave the way for new and improved treatments in the field of neuroscience. Herein, we discuss a step-by-step methodology for dissecting the mouse brain into sixteen distinct regions. In this procedure, we have focused on male mouse C57Bl/6J (6-8 week old) brain removal and dissection into multiple regions using neuroanatomical landmarks to identify and sample discrete functionally-relevant and behaviorally-relevant brain regions. This work will help lay a strong foundation in the field of neuroscience, leading to more focused approaches in the deeper understanding of brain function.
The brain, along with the spinal cord and retina, comprise the central nervous system that executes complex behaviors, controlled by specialized, precisely positioned, and interacting cell types throughout the entire body1. The brain is a complex organ with billions of interconnected neurons and glia with precise circuitry performing numerous functions. It is a bilateral structure with two distinct lobes and diverse cellular components2. The spinal cord connects the brain to the outside world and is protected by bone, meninges, and cerebrospinal fluid and routes messages to and from the brain2,3,4. The surface of the brain, the cerebral cortex, is uneven and has distinct folds, called gyri, and grooves, called sulci, that separate the brain into functional centers5. The cortex is smooth in mammals with a small brain6,7. It is important to characterize and study the architecture of the human brain in order to understand the disorders related to the different brain regions, as well as its functional circuits. Neuroscience research has expanded in recent years and a variety of experimental methods are being used to study the structure and function of the brain. Developments in the fields of molecular and systems-level biology have ushered in a new era of exploring the complex relationship between brain structures and the functioning of molecules. Additionally, molecular biology, genetics, and epigenetics are rapidly expanding, enabling us to advance our knowledge of the underlying mechanisms involved in how systems function. These analyses can be carried out on a much more localized basis, to help target the investigation and development of more effective therapies.
The mammalian brain is structurally defined into clearly identifiable discrete regions; however, the functional and molecular complexities of these discrete structures are not yet clearly understood. The multi-dimensional and multi-layered nature of the brain tissue makes this landscape difficult to study at the functional level. In addition, the fact that multiple functions are performed by the same structure and vice versa further complicates the understanding of the brain8. It is vital that the experimental approach executed for the structural and functional characterization of brain regions uses precise research methodologies to achieve consistency in sampling for correlating neuroanatomical architecture with function. The complexity of brain has been recently explained using single cell sequencing9,10 such as the temporal gyrus of the human brain which is composed of 75 distinct cell types11. By comparing this data to those from an analogous region of the mouse brain, the study not only reveals similarities in their architecture and cell types but also presents the differences. To unravel the complex mechanisms, it is therefore important to study diverse regions of the brain with full precision. Conserved structures and function between a human and mouse brain enable the use of a mouse as a preliminary surrogate for elucidating human brain function and behavioral outcomes.
With the advancement of systems biology approaches, obtaining information from discrete brain regions in rodents has become a key procedure in neuroscience research.While some protocols such as laser capture microdissection12 can be expensive, mechanical protocols are inexpensive and performed using commonly available tools13,14. We have used multiple brain regions for transcriptomic assays15 and have developed a hands-on and rapid procedure to dissect mouse brain regions of interest in a step-by-step manner in a short time. Once dissected, these samples can be stored immediately in cold conditions to preserve the nucleic acids and proteins of these tissues. Our approach can be performed faster leading to high efficiency and permitting less chances for tissue deterioration. This ultimately, increases the chances of generating high quality, reproducible experiments using brain tissues.
Animal handling and experimental procedures were conducted in accordance with European, national and institutional guidelines for animal care. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at the US Army Center for Environmental Health Research now Walter Reed Army Institute of Research (WRAIR) and performed in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC).
NOTE: The procedure will be performed on six to eight week old male mice of the C57BL/6j strain euthanized by cervical dislocation16. No perfusions are performed in our lab but this protocol could be modified whereby perfusions to clear blood from the vasculature could be performed. All supplies required for the dissection are listed in the Table of Materials. The dissection is subdivided into three components, including removal of the brain, the removal of the pituitary gland and the brain dissection. The intent of brain tissue collection is to process them for transcriptomic assays following RNA extractions. As soon as the brain region is dissected, we immediately transfer each of the brain regions into an already labeled freezer vial and then store the vial in liquid nitrogen or -80 °C.
NOTE: Thorough knowledge of neuroanatomy is essential to perform this procedure. Horizontal and longitudinal sections, as well as the usual transverse sections, should be studied and learned. Since brain tissue degrades very quickly, there is no time to consult an atlas while performing this procedure.
1. Mouse Brain Removal
2. Dissection of the Anterior and Posterior Pituitaries
NOTE: The pituitary glands are covered by a very tough tent-like membrane, with a ridge that runs laterally between the left and right trigeminal nerves. These structures are extremely soft and delicate and, as such, it is recommended the posterior and anterior lobes of pituitary glands be dissected separately in stages, in situ, directly from the skull. Immediately after dissection, transfer the respective pituitary gland to a pre-labeled vial and store the vial in liquid nitrogen preferably otherwise -80 °C. The pituitary gland rests exactly over the junction of the occipital and basisphenoid bones; if they flex, the pituitary architecture is disrupted.
3. Mouse Brain Dissection
NOTE: Immediately after brain and pituitary removal, further dissection is performed on a pre-chilled stainless-steel block (Figure 3). Post dissection, transfer the brain regions to pre-labeled vials and transfer the vials preferably to liquid nitrogen otherwise -80 °C. Structures produced by the top-down method (in chronological order) potentially include the following: cerebellum (CB), brain stem/hind brain (pons and medulla oblongata) (HB), olfactory bulbs (OB) as accessory olfactory bulbs, medial prefrontal cortex (MPFC), lateral prefrontal cortex (FCX), anterior and posterior corpus striatum (ST), ventral striatum (VS) comprised of the nucleus accumbens (NAC) and olfactory tubercle (OT), septum (SE), preoptic area, piriform cortex (PFM), hypothalamus (HY), amygdala (AY), hippocampus (HC), posterior cingulate cortex (CNG), entorhinal cortex (ERC), midbrain (MB) with thalamus and rest of the cerebral cortex (ROC) (Table 1). Specific regions will be discussed in order of isolation, working with a single hemisphere.
Our understanding of the complex brain structure and function is rapidly evolving and improving. The brain contains multiple distinct regions and building a molecular map can help us better understand how the brain works. In this method paper, we have discussed the dissection of the mouse brain into multiple distinct regions (Table 1). In this protocol, the structures are identified based on the critical landmarks and is achieved by keeping the tissue moist with saline solution by retaining its sturdiness for immediate dissection. The method covers more regions than other reports21 and is complementary to dissection methods using frozen brains22,23. These dissected tissues can be preserved and processed later depending on the requirements of the study. The method discussed here is immediate removal of the brain from the skull followed by dissection which provides enough tissue in a quick way for downstream assays given the size of the mouse brain. Our team has extracted RNA from multiple dissected tissues and assayed for gene expression profiling using microarrays for brain tissues; AY, HC, MPFC, septal region, corpus striatum and VS and the results have been published15. These harvested brains were stored at -80°C for several months before RNA extraction. This method can be adopted in combination of other methods to diversify downstream utility of brain regions. This dissection method is focused on following landmarks among anatomically distinct adjoining regions instead of being strictly coronal or sagittal dissections. The distinct brain regions along with weight data was collected under the study aggressor exposed social stress model of PTSD16 and RNA concentrations along with spectrophotometric readings is shown as Figure 5.
Figure 1: Representation of Mouse Brain with Distinct Tissue Types Collected. This figure is a cosmetic representation of the structures and is not scaled to any mapped database. Please click here to view a larger version of this figure.
Figure 2: Brain Removal from the Cranial Cavity Followed by Pituitary Removal. Stepwise procedure for brain removal (i) clamping and holding after hair removal (ii) securing the clamp with a Kimwipe to keep hair away from the tissue (iii) before removing of muscles (iv) after removal of muscles (v) separation of méninges (vi) removal of globe/eye (vii) cut on orbital ridge (viii) removal of parietal and frontal bones (ix) brain display and removal (x) brain detachment (xi) pituitary view (xii) pituitary removal. Please click here to view a larger version of this figure.
Figure 3: Dissection Setup Station. This image shows the set up for brain dissection post-brain and pituitary tissues removal. Please click here to view a larger version of this figure.
Figure 4: Brain Dissection. Stepwise procedure for brain removal (i) top view of brain (ii) dorsal view of brain (iii) cerebellum removal (iv) cerebral separation (v) hemispheres dissection (vi) olfactory bulb removal (vii) MPFC, FCX and accessory olfactory dissection (viii) SE, VS and ST dissection (ix) PFM removal (x) Limbic system dissection Please click here to view a larger version of this figure.
Figure 5: Data Generated after Sample Collection (A) Brain tissue weight and (B) RNA concentration and (C) OD 260/280 from each of the brain tissue is shown. Here the data is gathered from control groups (n= 3-6) from a study group as reported earlier15,16. Please click here to view a larger version of this figure.
# | Abbreviations | Description of brain region |
1 | CB | Cerebellum |
2 | HB | Brain stem/hind brain (pons and medulla oblongata) |
Separate into the two hemispheres | ||
3 | OB | Olfactory bulbs and accessory olfactory bulbs |
4 | MPFC | Medial prefrontal cortex |
5 | FCX | Lateral prefrontal cortex |
6 | SE | Septum or septal region |
7 | VS | Ventral striatum includes the nucleus accumbens (NAC) and olfactory tubercle (OT) |
8 | ST | Anterior and posterior corpus striatum |
9 | HY | Hypothalamus |
10 | PFM | Piriform cortex |
11 | HC | Hippocampus |
12 | ERC | Entorhinal cortex |
13 | CNG | Posterior cingulate cortex |
14 | AY | Amygdala |
15 | MB | Midbrain with thalamus |
16 | ROC | Remainder of the cerebral cortex |
Table 1: Description of Distinct Regions from Mouse Brain. This table contains list of all brain regions collected with its abbreviation.
The mammalian brain is a complex organ composed of an array of morphologically distinct and functionally unique cells with diverse molecular signatures and multiple regions that perform specialized and discrete functions. The dissection procedure reported here can have multiple goals depending on the requirements of the lab. In our lab we assessed transcription in multiple brain regions collected from mice exposed to PTSD like stress16 . We would like to study further the impact of strain genetic background24 on expression levels in multiple brain regions. This protocol has multiple critical steps that needs to be considered for successful reproducibility of the experiments. Each of the localized regions of brain play a distinct role in neuropathological condition and detailed knowledge of the appropriate brain region to study is lacking. Therefore it is important to generate the dataset pertaining to brain region. Thus, the data can not only be queried by selecting brain region but also by data category (e.g., Transcriptome, protein, cell (cytoarchitectural), or other) leading to more precise information. Previously, the tissues such as olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissues have been shown to be dissected using a microscope25. Alternately sections can be dissected while the brain is frozen14 followed by RNA and protein extractions but this method is limited to brain regions that can be identified by clear landmarks. Total RNA extractions have been carried post microdissection25 from major brain regions as well as using non-laser capture microscopy approach for gene expression studies13. Here we focus on the dissection of fresh mouse brain to separate out the specific brain structures that have dedicated control over physiological and behavioral functions. Our method explains the dissection of more regions than already published reports however it is complementary to other dissection methods available. This approach can help provide a comprehensive assessment of the tissues and its association with debilitating conditions. This dissection strategy provides a viable option to existing sample collection strategies opening possibilities for new discoveries.
With the method described herein, brain tissues are snap frozen in liquid nitrogen before transferring to -80 °C for long term storage. It is important that the tools and tissues are kept cold during the entire procedure for preservation of nucleic acids or proteins. These frozen tissues are homogenized later using lab standard operating procedures. Some of these brain tissues are very small and care should be taken during the homogenization and extraction process protecting target molecules from degradation at higher temperatures.
In this process, it is important to identify clear landmarks to pinpoint the specific tissue regions. This is achieved by keeping the tissue moist with saline solution to keep it from becoming quickly soft and retaining its sturdiness for a while. Our previous studies compared gene expression changes between control and aggressor exposed mouse tissues15 and did not study any changes caused due to the saline used during dissection. In our experience, this is especially important during the incision starting from the hypothalamus and the 180° flipping as it exposes and makes the regional separation shading of the limbic regions (AY, HC, ERC) obvious and more clear. The limbic system is situated deep within the brain and gets damaged by a variety of stimuli, and hence is important diagnostically and therapeutically. There limbic system consists of brain regions however there is no universal agreement on this list26. Though not studied, we think that there are minimal or no effects of saline use. This is because the entire procedure lasts about 20 minutes following cold conditions during the entire process.
Regions within brain tissue are identified using landmarks mentioned in brain atlases. Using this technique, the landmarks need to be clear and this procedure should be done sequentially. The dissection has to be done while the brain is still fresh and sturdy; (has to be done with the first 15 to 20 minutes) – otherwise the landmarks will not be clear and regions will not be distinct if the brain stays for longer time and became softer.
As described above, within the brain are sub regions, containing multiple functional areas that act independently or in coordination with intrinsic connective networks. It is important to retrieve these regions with great precision in order to study its broad dimensions. This will help to integrate these concepts by combining the specialized regions where each is serving a distinct process with a therapeutic potential.
The authors have nothing to disclose.
We thank Ms. Seshmalini Srinivasan, Mr. Stephen Butler and Ms. Pamela Spellman for experimental assistance and Ms. Dana Youssef for editing the manuscript. The funding support from USAMRDC is gratefully acknowledged. The Geneva Foundation contributed to this work and was supported by funds from the Military and Operational Medicine Research Area Directorate III via the US Army Research Office.
Disclaimer:
Material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the author, and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense. Research was conducted under an approved animal use protocol in an AAALAC accredited facility in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, NRC Publication, 2011 edition.
Brain Removal | |||
Deaver scissors | Roboz Surgical Store | RS-6762 | 5.5" straight sharp/sharp |
Deaver scissors | Roboz Surgical Store | RS-6763 | 5.5" curved sharp/sharp |
Delicate operating scissors | Roboz Surgical Store | RS-6703 | 4.75" curved sharp/sharp |
Delicate operating scissors | Roboz Surgical Store | RS-6702 | 4.75" straight sharp/sharp |
Light operating scissors | Roboz Surgical Store | RS-6753 | 5" curved Sharp/Sharp |
Micro spatula, radius and tapered flat ends | stainless steel mirror finish | ||
Operating scissors 6.5" | Roboz Surgical Store | RS-6846 | curved sharp/sharp |
Tissue forceps | Roboz Surgical Store | RS-8160 | 4.5” 1X2 teeth 2mm tip width |
Rongeur (optional) | Roboz Surgical Store | RS-8321 many styles to choose | Lempert Rongeur 6.5" 2X8mm |
Pituitary Dissection | |||
Scalpel handle | Roboz Surgical Store | RS-9843 | Scalpel Handle #3 Solid 4" |
and blades | Roboz Surgical Store | RS-9801-11 | Sterile Scalpel Blades:#11 Box 100 40mm |
Super fine forceps Inox | Roboz Surgical Store | RS-4955 | tip size 0.025 X 0.005 mm |
Brain Dissection | |||
A magnification visor | Penn Tool Col | 40-178-6 | 2.2x Outer and 3.3x Inner Lens Magnification, Rectangular Magnifier |
Dissection cold plate | Cellpath.com | JRI-0100-00A | Iceberg cold plate & base |
Graefe forceps, full curve extra delicate | Roboz Surgical Store | RS-5138 | 0.5 mm Tip 4” (10 cm) long |
Light operating scissors | Roboz Surgical Store | RS-6753 | 5" curved sharp/sharp |
Scalpel handle | Roboz Surgical Store | RS-9843 (repeated above) | Scalpel Handle #3 Solid 4" |
and blades (especially #11) | Roboz Surgical Store | RS-9801-11 (repeated above) | Sterile Scalpel Blades:#11 Box 100 40mm |
Spatula | Amazon | MS-SQRD9-4 | Double Ended Spatula Square AND Round End |
Tissue forceps | Roboz Surgical Store | RS-8160 (repeated above) | 4.5” 1X2 teeth |