Whole Central and Peripheral Nervous System Mice Dissection

Abstract

Animal models represent the workhorse of the neuroscience field. Despite this, today, there is still no step-by-step protocol to dissect a complete rodent nervous system, nor is there a complete schematic representing it that is freely available. Only methods to harvest the brain, the spinal cord, a specific dorsal root ganglion, and the sciatic nerve (separately) are available. Here, we provide detailed pictures and a schematic of the central and peripheral murine nervous system. More importantly, we outline a robust procedure to perform its dissection. The 30 min pre-dissection step allows isolating the intact nervous system within the vertebra with muscles free of viscera and skin. A 2-4 h dissection follows it under a micro-dissection microscope to expose the spinal cord and the thoracic nerves, and finally peel the whole central and peripheral nervous system off the carcass. This protocol represents a significant step forward in studying the anatomy and pathophysiology of the nervous system globally. For example, the dissected dorsal root ganglions from a neurofibromatosis type I mice model can be further processed for histology to unravel changes in tumor progression.

Introduction

The overall goal of this method is to isolate a mouse's central and peripheral nervous system in one piece. There is currently no protocol to dissect the whole nervous system of a rodent to study it on a global level. Neuroscientists typically use the sciatic nerve as a surrogate for any peripheral nerve¹, and the L3 to L5 ganglions² as a surrogate for any ganglions. Using these methods, it is impossible to conclude if the results are specific to the particular nerve/ganglion. As it is known that at least some nerve pathologies do not affect all nerves and ganglions equally^3,4,5, one must develop a technique to allow the isolation of the complete rodent nervous system to study it globally.

Over the years, we have developed and refined a method to dissect the complete central and peripheral nervous systems of mice. The first step is essentially a gross dissection of the mice in preparation for the micro-dissection steps under the dissection microscope. In steps 2 to 4, the spinal cord and the thoracic nerves are exposed, the brain is dissected, and the whole spinal cord and peripheral nerves are peeled off the carcass.

This method is powerful when coupled to imaging or histology to document any macroscopic or microscopic change^6,7,8,9. Neuroscientists interested in surveying global change or conducting non-hypothesis-driven experiments should use this method to survey the global nervous system.

Protocol

The protocols used in this study have been approved by the Comité Institutionnel des Animaux de l'Université de Sherbrooke, a Canadian Council on animal care certified institution.

1. Preparation for micro-dissection (pre-dissection)

1. Perform anesthesia with 5% isoflurane, followed by euthanasia with 2% isoflurane and 10 psi of CO₂ until there are no vital signs.
   NOTE: Do not perform cervical dislocation, as this damages the spinal cord and cervical ganglions.
2. Place the euthanized mouse facing up (anterior view) on a dissection pad, and spray the fur with 70% ethanol.
3. Then, pin the upper and lower limbs, using four small-diameter pins to hold the mouse in position during the dissection.
4. Using surgical scissors, cut the skin open from the lower abdomen to the throat.
5. Peel off the skin to expose the internal organs, and use additional pins to maintain the skin on each side while exposing the abdominal cavity.
6. Cut the thoracic cage open to expose the heart and lungs.
7. Using a pair of standard anatomical forceps, grip the esophageal and trachea, and cut just above the forceps.
8. Then, begin to peel off all the internal organs in a cranial to caudal approach. To do so, cut the diaphragm along the vertebral column to remove all the internal organs in one piece.
9. Unpin the mouse and rinse off extra mouse blood from the abdominal cavity in a sink.
10. Place the mouse face down (posterior view). Pin the upper and lower limbs, using four pins to hold the mouse in position during the dissection.
11. Using surgical scissors, peel off the skin from the head to the hind limbs.
12. Expose the left sciatic nerve (lower limb).
    1. Cut the muscles open in the lower part of the left lower limb.
    2. Locate and expose the sciatic nerve by removing the muscle around it.
    3. Carefully cut the sciatic nerve at the sural, tibial, and peroneal nerve ramification, and spare the blood vessels.
    4. Continue to isolate the sciatic nerve using standard anatomical forceps and extra fine Bonn scissors, until it becomes parallel to the vertebral column, leaving a free nerve of about 2 cm.
    5. Insert the surgical scissors parallel to the spinal cord, and cut the hip.
    6. Dislocate the hip by pulling apart the sacrum and the femur using fingers.
       NOTE: During this process, it is required to stop frequently to gently pull the sciatic nerve using forceps to avoid disruption.
    7. At the end, delicately tear the hind limb off the mouse carcass, leaving a sciatic nerve of about 4 cm in length.
       NOTE: During this process, locate the L2 nerve, and tease it out of the hind limb to avoid damage to the L2. Repeat step 1.12 for the right side.
13. Expose the brachial nerves (upper limbs).
    1. Manipulate the mouse carcass in hands, rather than on a flat surface (dissection pad).
    2. Locate the left brachial plexus by teasing apart the fat and muscles in the left armpit.
    3. Once located, cut the main brachial plexus ramifications (radially, axillary, suprascapular) and their sub-ramifications around the ulna with extra fine Bonn scissors, leaving free nerves of around 1.5 cm. Gently peel off the plexus out of the upper left limb.
    4. Dislocate the upper left limb; ensure it is nerve-free. Repeat step 1.13 for the right side.
14. Expose the brain.
    1. Now, place the mouse face up (anterior view)
    2. Insert one blade of the extra fine Bonn scissors in the mouth, and cut the mandible through the throat.
    3. Remove the mandible by further cutting from the cheeks to the throat.
    4. Then, cut the skull bone passing from one ear to the other.
       NOTE: Be careful to avoid going too deep and damaging the brain.
    5. Cut and remove the palate and nasal bones to open the skull.
    6. Cut the C1 vertebra at the base of the skull, releasing the cerebellum and the beginning of the spinal cord.
    7. Finally, cut the skull transversely up to the eye, and remove the pieces of the skull to expose the brain.
       NOTE: Keep the brain in its cavity until the nerves are peeled off.
15. Remove any extra fat or muscles from the carcass. Place the carcass in 10% formalin for 15 min at room temperature (RT), followed by brief 1x phosphate buffered saline (PBS) washes until there is no more fixative odor, and proceed to the next step.
    ​NOTE: If the nervous system is dissected in the next 2 weeks, store it at 4 °C in PBS. Otherwise, store it in 10% formalin indefinitely. Always use a chemical hood when manipulating formalin.

2. Spinal cord exposure

1. To expose the spinal cord, use a cranial-to-caudal approach. To remove each vertebra and their muscle layer above, cut at the ten o'clock and two o'clock positions on the ventral side, using Vannas spring scissors. Chip away the vertebra using Dumont mini-forceps. Continue this process through the cervical and thoracic vertebrae.
2. For the lumbar section, cut the transverse process on each side of the vertebra. Then, cut at the two o'clock and ten o'clock positions by inserting the blade of a pair of Vannas spring scissors into the vertebral canal. Remove the tissues, paying attention to the nerves which are sometimes stuck to the bones.
3. Proceed similarly for the caudal part.

3. Thoracic nerve exposure

1. Put the carcass under the dissection microscope to visualize the anterior side.
2. Using Vannas spring scissors, cut along each rib (from the sternum to the lower extremities) to expose the peripheral nerves.
3. Next, cut the vertebra on both sides (laterally) of the ganglion to expose the ganglion.

4. Peripheral nerve peel off

1. To peel off the spinal cord and further dislodge the peripheral nerves, use the Dumont mini-forceps to gently roll the spinal cord and pull out the nerves one by one, starting with the caudal part of the spinal cord.
   NOTE: Before peeling off, an additional fixation can be performed in 10% formalin for 15 min at RT, followed by brief 1x PBS washes until there is no more fixative odor, as performed in step 1.15.
2. For the thoracic part, pull the nerve at a 90° angle (perpendicular to the spinal cord).
3. For the brachial plexus, cut the vertebra next to the nerve, to make room for the nerve.
   NOTE: The brachial plexus cannot be passed under the vertebra, as in the thoracic region.
4. Remove the excess muscles to clear the nerves.
5. Store in PBS at 4 °C for up to a total of 2 weeks, or indefinitely in 10% formalin at RT.

Representative Results

Rodents have been instrumental to our understanding of nervous system biology and pathophysiology¹⁰. Intriguingly, no methods present the complete dissection of a rodent's central and peripheral nervous system to asses the anatomy and pathological variation in a real-time model^1,2,11. Figure 1 presents an overview of step 1 to step 4 (the preparation for the micro-dissection, spinal cord exposure, thoracic nerve exposure, and peripheral nerve peel off). In step 1, all the viscera are removed, the skin is peeled off, and the main nerves' plexuses (brachial and lumbosacral) are teased out of their limbs. In steps 2 and 3, the vertebras are partly removed (ventral), exposing the spinal cord. In step 4, the whole nervous system is isolated from the mice carcass. Overall, it takes 2.5 to 5 h to complete the procedure.

The spinal nerves are annotated based on their corresponding vertebras. Cervical spinal nerves are labeled according to the vertebra above which they arise. In contrast, the sacral, lumbar, and thoracic spinal nerves are named according to the vertebra below which they come out. As shown in Figure 2, mice harbor two coccygeal, four sacral, six lumbar, 13 thoracic, and eight cervical nerves (although the exact number can slightly differ from one strain to another¹²). The brachial (from cervical 5 to thoracic 2) and lumbosacral (lumbar 3 to sacral 3) nerve plexuses are the easiest to distinguish. The spinal cord is larger in diameter in the cervical and lumbar region than the thoracic region, making it easier to spot the cervical to thoracic and thoracic to lumbar boundaries. The sacral nerves do not have an apparent ganglion, distinguishing them from the lumbar nerves. The two coccygeal nerves do not have formal nerve extensions.

None of the methods currently available in the literature describe the dissection of all the peripheral nerves. That being said, and despite optimization and practice, it is expected to retrieve less than 100% of the nerves and ganglions at all times. For example, the first two cervical ganglions and nerves are extremely difficult to dissect due to their deep location. It is also not uncommon to miss some thoracic nerves (compare the final dissection in Figure 1 and Figure 2).

Dissecting the whole nervous system allows monitoring the impact of mutations, drugs, development/aging, etc. on specific nervous system parts, rather than using one ganglion or nerve as a surrogate. For example, this methodology was applied to study neurofibroma development in Hoxb7-cre Nf1^f/f mice⁷. Neurofibroma originates from Schwann cells, and mainly develops in dorsal root ganglions in mice. As shown in Figure 3, histological evaluation of a Hoxb7-cre Nf1^f/f mouse's brachial plexus confirms the presence of a neurofibroma in C7. In Figure 3A,B, the spinal cord appears in the center, whereas the pairs of ganglions and peripheral nerves show on the sides from C4 (up) to T1 (bottom). Right C7 is enlarged compared to the other ganglions, indicating a tumor. Histologically, a neurofibroma presents all the cellular components of a ganglion, but in a disorganized way¹³. The immunohistochemistry for S100 confirms the presence of Schwann cells in ganglions and peripheral nerves, and helps to visualize the disorganized nature of the enlarged right C7 ganglion compared to others (Figure 3C-F).

Figure 1: Overview of the key steps of whole mouse central and peripheral nervous system dissection. White arrows point to the brachial plexuses, whereas white arrowheads point to the sciatic nerves. Please click here to view a larger version of this figure.

Figure 2: Murine central and peripheral nervous system. Picture (left) and schematic (right) of the numeration and naming of the murine central and peripheral nervous system. Please click here to view a larger version of this figure.

Figure 3: Histological evaluation of a dissected brachial plexus from a transgenic mouse. Neurofibroma from a Hoxb7-cre Nf1^f/f mouse was evaluated by (A,B) hematoxylin and eosin and (C,D) immunohistochemistry using anti-S100 antibodies. (E,F) Negative immunohistochemistry control without anti-S100 antibody. The bottom panels represent zooms on a neurofibroma in C7. The scale bar represents 2.5 mm (A,C,E) and 250 µm (B,D,F). Please click here to view a larger version of this figure.

Discussion

To prevent the muscles and nerves from drying out, the carcass should be soaked in PBS every 10 min. When dislocating the lower limbs (step 1.12.6), it is important to always have the sciatic nerve plexus and L2 in sight to avoid damaging/tearing it. When dissecting the brain (step 1.14.4), it is critical to avoid going too deep so as not to damage the brain. When dissecting the dorsal root ganglions and peripheral nerves in general, it is critical to use high-quality (not damaged) Dumont mini-forceps to avoid damaging the nerve structures.

Instead of a 15 min 10% formalin fixation step, a 4% paraformaldehyde perfusion, as in Chen et al.⁵, is a viable alternative. Suppose one is interested only in a specific part of the mouse's nervous system (i.e., the brachial plexus). In that case, it is possible to adapt the protocol by omitting the dissection in other parts (i.e., thoracic, lumbar, and sacral). Although much more tedious, performing this dissection protocol without fixation is possible, allowing one to perform primary cell isolation and culture. Similarly, the protocol can be adapted to minimize time if one is interested in cells from a specific part of the mouse's nervous system. It is also possible to perform retrospective dissection from the whole archived mice in formalin buffer, the main difference being that tissues need to be cut rather than mostly torn. This entire protocol could be adapted to any rodent or small mammal.

The main drawback of this method is that it requires highly qualified personnel, specifically trained to perform dissection. A person with zero experience in rodent dissection may require extensive training to reach a satisfactory and consistent level of dissection. Also, it takes 2.5 to 5 h to complete the whole protocol. Magnetic resonance imaging has been used to study the mice's nervous system in vivo¹⁴; although this technique is noninvasive, it is limited to a fraction of the nervous system simultaneously.

This protocol allows for assessing the nervous system's overall status instead of using one/few ganglions or nerves as surrogates^1,2. It can be used to perform a thorough histological evaluation^7,9 or visual inspection⁸ of the nervous system in transgenic mice to identify tumor location. It could also be used to assess drug efficacy, the impact of genetic insult, etc. Figure 3 presents an example where the histological analysis of the brachial plexus of a Hoxb7-cre Nf1^f/f mouse confirms the presence of a para-spinal plexiform neurofibroma in C7.

Materials

Name	Company	Catalog Number	Comments
Dumont mini-forceps	Fine Science Tools	#11200-10
Extra Bonn scissors	Fine Science Tools	#14084-08
Formalin 10%	Fischer Scientific	#22-046-361
PBS 1x	BioShopCanada	#PBS404.500
Standard anatomical forceps	Fine Science Tools	#91100-12
Surgical scissors	Fine Science Tools	#140001-12
Vannas spring scissors	Kent Scientific	#INS600124