General
The mouse imaged in Figs. 2, 3, 4 was a 3 month old (P86) wild-type C57BL/6 male. Mouse sensory neurons can be dissected and cultured as soon as they are formed in the embryo [about embryonic day 13 (E13)] [29]; however, this particular dissection protocol can only be used on mice approximately P5 and above (the older and larger the animal, the easier the dissection). Protocols to dissect embryonic rodent DRG are available [30, 31]. Images of in vitro and ex vivo DRG neurons were taken using an EVOS™ XL Core Cell Imaging System (Thermo Fisher, Fig. 1c) and a Zeiss LSM 780 laser scanning microscope (Fig. 1d, e), respectively, while dissection images (Figs. 2, 3, 4) were taken with an eFlex™ digital microscope (Carson Optical, MM-840). Scale bars in images taken using the EVOS™ system were incorporated by imaging a ruler in parallel images, and are thus approximate. To decrease blood contamination, animals can be transcardially perfused with saline or phosphate buffered saline (PBS) prior to dissection. This is not necessary for generating healthy, contamination-free primary cultures or immunohistochemical tissue sections of high clarity; however, perfusion is likely to improve specificity of proteomic or RNA analyses. Dissections to produce primary cultures should be performed in a laminar flow hood under sterile conditions to reduce the chances of contamination; nevertheless, in our experience, dissection under the hood is not strictly required. To preserve neuronal health, DRG should be kept as cold as possible throughout the dissection. A modified version of this protocol is available for the isolation of adult rat DRG [32].
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.
Dissection protocol
Animals
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 [17]. 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].