Microdissection of Mouse Brain into Functionally and Anatomically Different Regions

요약

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.

초록

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.

서문

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 brain^2,3,4. 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 brain^6,7. 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 sequencing^9,10 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 tools^13,14. 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.

프로토콜

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

1. Clamp the maxilla of the decapitated head with a hemostat (Figure 2i) and use a gauze pad to reflect the scalp rostrally further creating a dry field on the dorsum of skull (Figure 2ii).
2. Insert the fine curved scissors into the foramen magnum to separate the adhering meninges. Here, insert the scissors at the opening on the base of skull is the foramen magnum, where the spinal cord passes with the blade pointing vertically (12 o'clock position), but parallel to and pressing against the interior surface of the basal plate bone (i.e., occipital squama) and rotating the blade forty-five° to the left side and then to the right side.
3. Continue to rotate the wrist to pry off the basal plate bone that will snip up the middle of the remaining bone continuing into the occipital bone and intraparietal bones prying the bones left and right until they are removed. In a similar fashion, remove the occipital bone and the intraparietal bone to expose the cerebellum.
   NOTE: At this point, the hollow bony structure called the tympanic bullae, on the ventral posterior portion of the skull that encloses parts of the middle and inner ear, can be removed unless one is dissecting the posterior and anterior lobes of pituitaries (Figure 2iii).
4. Remove the muscle attachments to the temporal ridge using a curved sharp scissor blade. Place one limb of the curved sharp scissors under the lambdoid suture penetrating into the junction of the transverse and sagittal sinuses (Figure 2iv).
5. Advance the scissors rostrally along the midsagittal suture up to the bregma and cut it very gently while carefully lifting upward to avoid laceration of cerebral cortices. This is a critical step in the procedure (Figure 2v).
6. At this step, parietal bones are lifted and rotated thus scraping the inner surface of the bone to identify and cut remaining meningeal attachments. Grasp the temporal bone prying outwardly, away from brain. Remove the frontal bone from each side using curved scissors or a rongeur into the orbit and cutting coronally at a right angle to the orbital ridge, but no further than the midline (Figure 2vii).
7. Make two cuts parallel and about 4 mm apart in the sagittal plane (Figure 2viii).
   NOTE: Do not cut both sides with one single stroke.
8. Remove the fragments of the frontal bone avoiding lacerating the brain surface (Figure 2ix). At this point, use a fine scissors to cut the dura mater, which should be accessible between the olfactory bulbs.
9. Gently invert the skull to allow gravity to assist in removal of the brain, while continuing to identify and cut remaining meningeal attachments and cranial nerves. The brain will be released by cutting the largest trigeminal nerve attached to the brain and is clearly visible extending from the base of the calvarium (Figure 2x).
   ​NOTE: Transfer the brain to a cold saline solution (ice cold RNase-free sodium citrate (0.9%) or physiological (0.9%) saline) for further dissection.

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.

1. Keep the auditory bullae intact to make sure that the pituitary anatomy is intact and easily identifiable (Figure 2ix).
2. Dissect the posterior lobe of pituitary followed by the anterior lobe of pituitary, from the remainder of the skull.
3. Make an extremely small parasagittal cut on both sides in the ridge of the membranous tent and lift the posterior lobe of pituitary with ultra-fine forceps, taking care not to disrupt the anterior pituitary tissue (Figure 2x).
4. Make a sagittal cut between the lateral margins of the anterior lobe of pituitary and the nearest trigeminal nerve and lift out the anterior lobe of pituitary thereafter (Figure 2x).

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.

1. Take great care in removing the brain from the calvarium as the landmarks may be destroyed in case there are any lacerations. At this step, perform all dissections using face protection, specifically, a 7x jewelers visor, and illumination will be provided by surgical lamps positioned over each of the dissector's shoulders. Much of the dissection will be accomplished using blunt mode small curved forceps (i.e., Graefe forceps).
2. Place the brain on a stainless-steel block (Figure 4i and Figure 4ii). Keep the block cold by surrounding it with ice and an ice-cold saline solution. Periodically moist the tissue with ice-cold saline solution to preserve the structures.
3. Position the brain such that the cerebral cortices are facing upwards. Using small curved forceps, gently reflect the CB by exposing the superior, middle and inferior cerebellar peduncles and remove the CB (Figure 4iii and Figure 4iv).
4. Make a midsagittal cut starting from the dorsum and between the OB and the cerebral hemispheres (Figure 4v) and make sure not to extend farther than the anterior commissure. At this point, the vermis can be easily separated from the lateral portions and HB will be obtained by a coronal cut at the anterior margin of the pons.
5. Separate the medulla by a coronal cut at the posterior margin of the pons.
   NOTE: At this point, the diencephalon, the posterior part of the forebrain containing the epithalamus, thalamus, hypothalamus, and ventral thalamus and the third ventricle, will be turned ventral side up, and a midsagittal cut will be made from the optic chiasm rostrally. The dissection of cerebral hemispheres followed by removal of OB from one of the hemisphere will be at this step (Figure 4v and Figure 4vi).
6. Separate the cerebral hemispheres (Figure 4v) before dissecting the diencephalon further (Figure 4v). The dorsal approach will be employed by careful blunt dissection to preserve the critically salient midline landmarks. Visualize every interhemispheric connection before severing them as this will minimize the possibility of deviating from the midline.
   NOTE: At this step one of the cerebral hemisphere (Hemibrain) can be preserved and the second hemisphere can be used for further dissection
7. Slip the closed blades of a small, curved forceps, beneath the corpus callosum and gently spread to retract the neocortex bilaterally. The corpus callosum is a broad band of nerve fibers that joins the two hemispheres which will be bluntly dissected by pinching with the forceps, without disturbing the midline structures lying beneath.
   NOTE: Mesial faces of the hemispheres will have multiple landmarks visible such as myelinated structures like the genu of the corpus callosum, the fornices, and the anterior commissure. Also, though a few millimeters lateral to the midline, the mammillothalamic tract and the fasciculus retroflexus may also be visible.
8. Bisect the multiple structures that cross the midline. This will include the corpus callosum, anterior commissure, ventral fornix commissure, posterior commissure, dorsal fornix commissure, supra mammillary decussation, superior colliculus commissure, ventral fornix commissure, and periventricular thalamic fibers.
9. Take special care at this step in or near the midline that might be partially compromised by the dorsal approach method. All follow-up structures are at risk if not performed carefully.
10. Remove the OB (wedge shaped and lighter color) and accessory olfactory bulbs followed by collection of the MPFC (cingulate cortex area 1, prelimbic, infralimbic, medial orbital, and secondary motor cortices [M2]) and FCX by a coronal cut that is made 1mm anterior to the genu of the corpus callosum (Figure 4vii). Divide the resulting section by a parasagittal cut 1/3 of the distance from the medial to the lateral surface, yielding MPFC medially and remainder of FCX laterally. The cingulate cortex is the mouse analog of MPFC.
    NOTE: This tissue slice will also contain a small amount of the M2 (secondary motor cortex) ¹⁷ and is unavoidable.
11. Make a coronal cut at the level of the anterior commissure leading to visibility of pars anterior limb of the anterior commissure in the cross-section on the rostral face of the resulting coronal section whereas the transverse portion will be revealed in the caudal face of the section This is a confirmatory landmark for NAC¹⁸. The VS is composed of the NAC and OT.
    NOTE: There is an anterior limb in addition to the transverse segment in the anterior commissure and it is called anterior commissure, pars anterior.
12. Partially cut horizontally through the transverse portion of the anterior commissure from the midline beneath the anterior horn of the lateral ventricle, to free the septal nucleus dorsally and the VS ventrally. Remove the small amount of the cortex from the lateral surface where NAC and OT will be removed.
13. Separate the rostral portion of the ST from the overlying cortex via curved scalpel cut just outside the external capsule taking care not to include striatal tissue in the cortical sample. At this point, the septal nuclei /SE will be visible and can be easily taken from this slice (Figure 4viii).
    NOTE: The anterior and posterior limits of the PFM are defined by imaginary coronal planes that are in line with the anterior commissure and the mammillary bodies respectively. The medial limit is defined by the external capsule¹⁸.
14. On the lateral surface of the remaining hemi-section of the diencephalon, make a partial horizontal cut along the rhinal sulcus extending caudally, but only as far as the imaginary coronal plane level with the mammillary bodies. Make a partial coronal cut, extending medially 1 mm from the lateral cortical surface in the plane of the mammillary bodies of the HY. Here, para-sagittal incision in the plane of the claustrum will free the PFM (Figure 4ix and Figure 4x).
    NOTE: On the medial surface of the remaining hemi-section of the diencephalon, numerous anatomical features will be visible including the mammillary bodies, the fasciculus retroflexus, and the stria medullaris. A semicircular pattern (dorsal concave side) will be visible, which denotes the margin between the thalamus and the HY. The HY will be easily identified on the midsagittal section view per the optic chiasm anteroventrally and anterior commissure anterodorsally and the mammillary bodies along with the fasciculus retroflexus posteriorly. The latter landmarks have been well displayed in the atlas¹⁷ as well as in albino mouse forebrain context¹⁹.
15. Make a partial coronal cut posterior to the mammillary bodies, extending only as far laterally as the hypothalamic sulcus. At this point a parasagittal cut along the length of the hypothalamic sulcus now frees the HY .
16. Entail the eversion of the lateral ventricle to reveal additional intra-limbic connections and the remaining limbic system components. Viewing the medial face of the remaining hemi-section of the diencephalon, curved forceps will be best option as they allow the technician to manipulate around other delicate sections of the surrounding area used to sever the fornix and is inserted into the lateral ventricle and gently expanded, using blunt dissection to open the ventricle and rotate the HC 90° from the vertical to the horizontal plane. It may be necessary to use the #11 blade to section the choroidal artery/choroid plexus as the pointed tip can help with precise cuts.
17. Rotate the CNG 90° (but in the opposite direction from the HC) to be in the horizontal plane. Using forceps, gently rotate the HC another 180° outward and laterally, which will make the inner surface of the ERC visible and facing upwards.
18. A fan-like radiation of myelinated fibers arising from the ERC will be seen converging to form the angular bundle, including the perforant path and its attachment to the HC. Rotate the thalamus and MB 180° dorsally to ventrally reveal the AY.
19. If required, lift the optic tract to reveal the attachment of the stria terminalis as it will contain bands of fibers running along the lateral margin of the ventricular surface of the thalamus to the AY and will make the outlines of the AY clear.
    NOTE: The HC and fornix will be clearly visible in their entirety, nested in the lateral ventricle and, can be easily lifted out. The lateral ventricle will be lined with pia mater and fan-like origins of the perforant path through the very transparent subpial aspect of the ERC will be visible²⁰.
20. The outer surface of medial ERC will have a visible prominent layer of large pyramidal cells. In addition, a dense convergence of Golgi-staining fibers will be a salient feature in the medial ERC. In this dissection procedure, after everting the lateral ventricle, these fibers will be easily visible on the medial surface through the pia mater, which lines the inner surfaces of the ventricles, forming a very prominent fan-like structure.
21. Notice that the fibers are gathering subpially, descending and leaving the ventral ERC, extending posteriorly and then ascending vertically to perforate the HC. This fan-like structure in the ERC will be used to define the margins for the purpose of dissection. Identify and remove in the order of AY, ERC, CNG and HC structures.
    NOTE: Defining a good landmark for AY dissection will be a key for the next step and the junction on posterior margin where stria terminalis meets the AY will be a good point.
22. At this point, the remaining structures include the thalamus and the MB. Confirm the identification of the thalamus by visualization of the stria medullaris on its dorsal surface extending in the midline rostrocaudal plane. Separate the thalamus completely from the midbrain by a coronal cut caudal to the habenula and rostral to the superior colliculi. The ROC is saved at this step.
23. At this point complete collection of all brain regions and, immediately (as each is obtained) store the samples flash frozen till further processing.

결과

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 sturdines...

토론

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 ...

자료

Name	Company	Catalog Number	Comments
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