A simple, step-by-step dissection protocol for the rapid isolation of mouse dorsal root ganglia
© Sleigh et al. 2016
Received: 8 December 2015
Accepted: 3 February 2016
Published: 11 February 2016
The cell bodies of sensory neurons, which transmit information from the external environment to the spinal cord, can be found at all levels of the spinal column in paired structures called dorsal root ganglia (DRG). Rodent DRG neurons have long been studied in the laboratory to improve understanding of sensory nerve development and function, and have been instrumental in determining mechanisms underlying pain and neurodegeneration in disorders of the peripheral nervous system. Here, we describe a simple, step-by-step protocol for the swift isolation of mouse DRG, which can be enzymatically dissociated to produce fully differentiated primary neuronal cultures, or processed for downstream analyses, such as immunohistochemistry or RNA profiling.
After dissecting out the spinal column, from the base of the skull to the level of the femurs, it can be cut down the mid-line and the spinal cord and meninges removed, before extracting the DRG and detaching unwanted axons. This protocol allows the easy and rapid isolation of DRG with minimal practice and dissection experience. The process is both faster and less technically challenging than extracting the ganglia from the in situ column after performing a dorsal laminectomy.
This approach reduces the time required to collect DRG, thereby improving efficiency, permitting less opportunity for tissue deterioration, and, ultimately, increasing the chances of generating healthy primary DRG cultures or high quality, reproducible experiments using DRG tissue.
Rodent DRG neurons have been used to study sensory nerve development, function, and regeneration [9–11], to aid in drug discovery , and to elucidate the mechanisms underlying peripheral nerve disorders, such as diabetic neuropathy [13, 14] and Charcot-Marie-Tooth disease [15, 16]. Dependent on genetic background, mice possess 30 or 31 pairs of DRG—8 cervical, 13 thoracic, 5 or 6 lumbar and 4 sacral (Fig. 1b) [17–19]. By axotomy of associated axon bundles, DRG can be carefully removed from healthy or disease model mice and then be enzymatically disrupted to produce live sensory neuron cultures (Fig. 1c, d), or fixed, sectioned, and immunohistochemically analysed (Fig. 1e). Importantly, the cultured sensory neurons faithfully retain features of the in vivo neurons and reflect the diverse population of DRG cells at the time of dissection . DRG can alternatively be processed for other downstream experiments, such as protein or RNA analyses [21, 22].
Here, we describe a simple and rapid technique for dissecting DRG from the spinal column of postnatal day 5 (P5) mice or older. This protocol can be combined with previously published culturing methods [17, 23–25], immunohistochemistry techniques [26, 27], or RNA (e.g. reverse transcriptase PCR) and protein (e.g. western blotting) evaluation , for in depth sensory nerve analyses.
Reagents, equipment and set up
The following materials and reagents (Sigma, unless otherwise stated), or similar alternatives, are required for the dissection: bone scissors (Fine Science Tools [FST], 14110-15), fine curved and straight scissors (FST, 14095-11 and 14094-11), small spring scissors (FST, 15000-08), thick and fine forceps (FST, 11000-25 and 11251-10), 70 % (v/v) ethanol in distilled water, fine marker pen, 12 × 0.2 mm minutiens insect pins (Austerlitz, 0.20), PBS (137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 2 mM KH2PO4), Hank’s balanced salt solution (HBSS, Thermo Fisher, 14170-112), 60 × 15 mm petri dishes (BD Biosciences, 351007) lined with Sylguard 184 silicone elastomer (Dow Corning, 01015311), disposable surgical scalpel blade (Swann-Morton, 0208), and SZB 250 dissection microscope (VWR, 630-1577). Sylguard 184 silicone elastomer was prepared by combining the elastomer base with the curing agent (10:1), using the mix to line petri dishes, and allowing to set for at least 48 h. To remove bubbles from the Sylguard, a vacuum desiccator can be used before setting.
All animal handling and experiments conformed to the Home Office Animals (Scientific Procedures) Act (1986) and were approved by the University College London—Institute of Neurology Ethics Committee. Animals were sacrificed using carbon dioxide before confirmation of death, rather than cervical dislocation, as the latter may damage cervical DRG.
Isolation of the spinal column
To restrict fur contamination, the deceased animal should be doused with 70 % ethanol. The torso may also be shaved (prior to this); however, this is not necessary. Forceps are used to pinch the external layer of fur and skin, while a small, dorsal incision is made using fine scissors in the region of the pelvis (Fig. 2a). The pelt is then removed from the head to the base of the tail (Fig. 2b), by either cutting or careful tearing of the skin in the transverse plane, followed by pulling the pelt up and over the head. The arms and head are then removed by cutting with scissors beneath the shoulder blades and at the C1–2 region of the column, found adjacent to the base of the skull (Fig. 2c). The abdominal wall musculature is cut on the ventral side (Fig. 2d) and continued laterally, one direction at a time, until the spinal column is reached (Fig. 2e). The scissors are then turned at right angles to face the rostral direction, and all ribs are detached close to the spinal column on both sides (Fig. 2f). At this point, a fine marker pen can be used to highlight the most caudal ribs (Fig. 3c), which act as a landmark for the T13 ganglia found just caudal to the ribs . The diaphragm, viscera, and rib cage are removed from the anterior side of the column. The femurs are then cut using bone scissors close to the column (Fig. 2g), and the whole spinal column removed (Fig. 2h) by making a transverse cut at the level of the femurs.
Exposure of the spinal cord
Once the spinal column has been excised (Fig. 3a), extraneous muscle, fat, spinal nerves and other soft tissue found on the exterior of the column are removed using fine curved scissors with the blade tips facing up (Fig. 3b–e), reducing the likelihood of accidentally cutting into the column. The column is then cut in the transverse plane into three pieces, with one cut just below the most caudal rib to orientate the spinal cord region (Fig. 3f). To limit the chances of damaging a DRG pair, try to perform transverse cuts through the vertebrae between the discs. These preceding steps (Fig. 3a–f) are performed to facilitate the next step of halving the spinal column. Thick forceps are used to hold the spinal column segments straight and steady, dorsal side facing up, while a sterile surgical scalpel blade is used to divide the spinal column in half down the midline (Fig. 3g–k). It is vital that this cut is done as close to the midline as possible, in order to improve subsequent access to the DRG. A rolling motion along the midline, starting at one end of the segment and finishing at the other, using a curved scalpel blade is perhaps the easiest way to assure accurate cutting. The more soft tissue removed from the exterior of the column, the easier this cutting process will be. Using a dissection scope, the two halves of the spinal column are then pinned out in Sylguard-lined petri dishes, medial side facing up, using insect pins through the exterior intervertebral discs. Sterile, ice cold PBS is then added to the petri dish to aid dissection and keep the sample from desiccation. To keep the dissection cold, PBS should be regularly replaced.
Extraction and cleaning of DRG
The spinal cord can now be slowly peeled in a rostral to caudal direction from the column, revealing the DRG below (Fig. 4a, b). Care should be taken at this point not to remove all ganglia with the spinal cord, as this will complicate DRG identification, dissection, and cleaning. To prevent this, the underlying meninges are held with fine forceps if they begin to be pulled away with the cord. Once the cord has been discarded, the meninges must be identified and detached in order to reduce cellular contamination of cultures and restrict sample rolling when sectioning on the cryostat (Fig. 4c–e). The meninges surround the spinal cord in situ, and cover DRG once the cord is taken out. To extract the meninges, use fine forceps to carefully peel them back from one end of the spinal column segment to the other. If they prove difficult to identify, try grasping at the vertebrae between the DRG using fine forceps. Sometimes the DRG are extracted with the meninges. This is not a problem, just pin out the meninges using insect pins and carefully extricate the individual DRG. If the DRG remain in the column, use fine forceps to grasp the distally projecting axon bundle found on the lateral side of the ganglia, and lift the DRG up and out of the column taking care not to touch and damage the cell ganglion at all times. DRG can then be pinned out using their axons in the Sylguard (Fig. 4f), before removing residual meninges (Fig. 4g), and using fine spring scissors to cut away the axon bundles found on the outside of the DRG (Fig. 4h). This last step will reduce myelin debris and glial cell contamination, which is important for both culturing and protein/RNA profiling. The long and thin white axons are easily distinguished from the round and darker DRG. Once dissected and cleaned, DRG can then be collected in HBSS for subsequent enzymatic digestion of the extracellular matrix and culturing (Fig. 1c, d), fixed for sectioning and immunohistochemistry (Fig. 1e), or processed for assessment of protein/RNA levels [21, 22].
Dissection of up to 40 individual DRG, from the time of animal euthanisation to commencing enzyme treatment or fixation, can be easily completed in 20–30 min per animal using this simple dissection protocol. The method is technically easier to perfect and, in our hands, considerably quicker than performing a spinal cord laminectomy with subsequent DRG removal from the in situ spinal column, which appears to be the most common dissection method described in the literature [17, 24, 32, 33]. A limitation specific to the dissection technique outlined here is that is requires the cutting of the spinal cord down the midline, which may affect its subsequent analysis if required. Nonetheless, the reduced dissection time diminishes the opportunity for cellular damage of DRG neurons, leads to a greater preservation of neuronal viability, and therefore increases the possibility of performing accurate and reproducible downstream experiments. Given its simplicity, the technique can be quickly perfected with little practice and anatomical knowledge by researchers of any dissection experience.
Examination of DRG neurons has provided a wealth of information on the development, regeneration, and function of not only sensory nerves, but the nervous system as a whole. Moreover, in combination with analyses of the neuromuscular system, for example neuromuscular junction phenotyping [34, 35] and primary embryonic motor neuron cultures [36, 37], assessment of in vitro sensory neurons or ex vivo DRG can provide a powerful approach to improving understanding of the pathological cellular events underlying disorders of the peripheral nervous system.
JNS and GS conceived the study, JNS and GAW performed and optimised the dissection protocol, and JNS, GAW, and GS wrote the manuscript. All authors read and approved the final manuscript.
The authors are grateful to members of the David L. Bennett, Linda Greensmith, and Giampietro Schiavo laboratories for helpful discussions, and to Ione Meyer and Bernadett Kalmar for critical reading of the manuscript. This work was supported by a Wellcome Trust Sir Henry Wellcome Postdoctoral Fellowship (103191/Z/13/Z) [JNS], a Wellcome Trust Senior Investigator Award (107116/Z/15/Z) [GS], and UCL [GS]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The authors declare that they have no competing interests.
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