A simple, step-by-step dissection protocol for the rapid isolation of mouse dorsal root ganglia

Abstract

Background: 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.

Findings: 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.

Conclusions: 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.

Fig. 1

Dissected DRG can be enzymatically dissociated and grown in culture or sectioned for immunohistochemical analyses. a DRG are clusters of sensory neuron cell bodies located in the dorsal roots of the spinal column. The schematic portrays a transverse section of the spinal column. b Mice possess 8 cervical, 13 thoracic, 5 or 6 lumbar, and 4 sacral DRG pairs totalling 60 or 62 individual ganglia depending on genetic background. The picture depicts the dorsal aspect of the spinal cord and the spinal nerve roots without bilateral DRG for simplicity. c, d Representative images of primary DRG sensory neuron cultures 24 h post-plating. Live cultures imaged by phase contrast microscopy c or fixed neurons imaged by confocal microscopy d can be used to assess various cellular phenotypes including morphology (e.g. cell soma area and axon length), electrophysiology, and protein localisation. Note how quickly DRG neurons extend axonal processes. e Intact DRG can be fixed, embedded in freezing medium, sectioned on a cryostat (thickness of this example 10 μm), and analysed immunohistochemically. βIII-tubulin (a.k.a. Tuj1) is a pan-neuronal marker, while DAPI identifies nuclei. Note that not all DAPI⁺ cells are Tuj1⁺, indicating that non-neuronal cells are present in vitro and in vivo. DRG were dissected from one month old wild-type animals (c–e). Scale bars 200 μm

Fig. 2

Spinal column isolation. a, b After dousing the fur with 70 % ethanol, a small incision is made in the dorsal skin at the level of the hips (a), and the pelt (arrow) removed from the head to hind limbs (b). c The head is removed by cutting at the base of the skull (C1–2 level) and the arms are cut beneath the shoulders to aid removal of the skin. d, e An incision is made through the abdominal wall muscles (d) and continued laterally to the spinal column in both directions (e). f The ribs are then cut parallel with and close to the spinal column on both sides, before detaching the viscera connected to the anterior side of the spinal column. g, h The femurs are cut (g), and the spinal column removed (h) by making a transverse cut at the level of the femurs. In all panels, the head is to the left and tail to the right. The dorsal aspect is imaged in all panels except for d (ventral aspect) and h (C caudal, D dorsal, R rostral, V ventral). Scale bars 2 cm

Fig. 3

Spinal cord exposure. a, b Muscle, fat, and other soft tissues are cut from the spinal column using curved scissors. c The T13 level DRG pair is found caudal to the most caudal ribs, which are used as landmarks (identified by forceps). d Spinal nerves (arrows) project from the column and can be removed. e, f Once cleared of soft tissues (e), the column can be cut (arrows) into three pieces (f), with one cut at the level of the last rib in order to orientate the dissection. g, h The column segments must be placed dorsal (g), not ventral (h), side facing up. i, j, k Thick forceps are then used to secure the spinal column dorsal side up (i), before cutting it into two equal halves along the midline (j, k). l The spinal column hemi-segments can be pinned out, medial side up, using two insect pins through intervertebral discs in Sylguard-lined petri dishes, before flooding with ice cold PBS. In all panels, the rostral end is to the left and the caudal end to the right. Scale bars 0.5 cm

Fig. 4

DRG extraction and cleaning. a, b The spinal cord is peeled from the pinned column in a rostral to caudal direction. c–e The meninges are identified as translucent sheets of tissue covering the DRG (c), and carefully removed (d), making the DRG easier to see (e). f Individual ganglia are extricated by clasping and lifting with forceps the distally projecting axon bundles found on the lateral side of the DRG. Care must be taken not to damage the DRG with the forceps. g, h DRG are then pinned out via their axons, and any residual meninges removed (g), before cutting the axons close to the DRG (h). In all panels, the rostral end is to the left and the caudal end to the right, and black arrows and arrowheads highlight DRG and axon bundles, respectively, while white arrows identify the meninges. In a–e, the medial aspect is facing up. Scale bars 0.5 cm