Dissection and Isolation of Mouse Dorsal Root Ganglia

Summary

Dorsal root ganglia (DRG) contain primary sensory neuron cell bodies and relay peripheral sensory signals to the spinal cord. This protocol describes a method for mouse DRG dissection that supports standardized isolation of structurally intact ganglia, anatomical segmental identification, intuitive guidance for novice users, and reduced procedural tissue injury.

Abstract

Dorsal root ganglia (DRG), which contain the cell bodies of primary sensory neurons, serve as critical relay structures for the transmission of sensory signals from peripheral tissues to the spinal cord. They play essential roles in studies of pain, nerve injury, and neurodegenerative diseases. The acquisition of intact DRG tissue is crucial for investigating the functional properties, molecular expression, and pathological alterations of sensory neurons. However, due to their small size and deep location within the vertebral canal, accurate identification and isolation of DRG present significant technical challenges. In this study, we developed a standardized protocol for the isolation of mouse DRG. This method provides a detailed description of the anatomical localization of DRG, exposure of the vertebral canal, and the key steps involved in ganglion isolation. The entire procedure is illustrated with representative images and video recordings. This approach supports the acquisition of structurally intact DRG with anatomical guidance for segmental identification and is suitable for H&E staining and immunofluorescence analysis. Compared with previous DRG dissection methods, this method allows standardized recovery of segmentally identified ganglia, provides intuitive anatomical guidance for novice users, and reduces procedural tissue injury.

Introduction

Dorsal root ganglia (DRG), which contain the cell bodies of primary sensory neurons, are essential structures for transmitting peripheral sensory signals—including mechanical, thermal, and nociceptive stimuli—to the spinal cord^1,2,3 . Although high-quality resources describing DRG are currently available, detailed procedural guidance is still required to support accurate identification and isolation of DRG from thoracic to sacral segments within a single specimen, while minimizing the risk of DRG damage during tissue collection.

DRG neurons are key components of the peripheral sensory pathway. Their axons bifurcate, with one branch extending to peripheral tissues and the other projecting into the dorsal horn of the spinal cord, thereby playing critical roles in pain transmission, nerve injury, inflammatory responses, and the pathogenesis of various neurological disorders⁴. In addition, DRG have attracted considerable attention in research areas such as nerve injury repair, mechanisms of chronic pain, sensory neuron plasticity, and neuroimmune regulation^5,6. A wide range of experimental studies require intact DRG tissue for applications including immunohistochemical staining, RNA sequencing analysis, primary neuronal culture, and electrophysiological studies⁷. Therefore, the acquisition of structurally intact and accurately segment-identified DRG tissue is fundamental for studies of sensory neural circuits, neuronal molecular characteristics, and pathological alterations.

DRG are located near the intervertebral foramina and are connected to the spinal cord via the dorsal roots. Due to their small size, enclosure within bony structures, and the complexity of surrounding anatomy, intact isolation presents considerable technical challenges. In addition, the lack of clear anatomical landmarks and standardized procedures increases the risk of structural damage or segmental misidentification during tissue collection.

Compared with existing mouse DRG dissection methods, the method established in this study has several advantages. First, previously reported mouse DRG isolation methods generally require removal of the spinal column from the animal, followed by segmentation and midline opening of the isolated spine, after which the DRG are dissected from the intervertebral foramina. During this process, transection of the spinal column and repeated cutting can easily cause mechanical damage to the DRG and may also result in omission of certain segments or unclear segmental identification⁸. In contrast, our method is performed directly in situ in the mouse, without prior isolation or transection of the vertebral column, thereby reducing the risk of DRG loss during tissue collection. Throughout the procedure, the instruments are applied primarily to the bony structures and connective tissues surrounding the DRG rather than directly grasping or pulling the ganglion itself, thereby preserving its integrity.

Second, existing methods often lack clear, stable anatomical landmarks for identifying different DRG segments. Because the nerves connected to the DRG are frequently transected during spinal column removal, it becomes difficult to accurately determine the segmental origin of individual DRG based on nerve course and branching patterns. In contrast, our method is performed directly in situ in the mouse and therefore preserves the natural anatomical relationships among the DRG, the connected nerves, and the surrounding structures, allowing identification of different DRG segments according to nerve connections and local anatomical landmarks. We propose the use of the morphological characteristics of L2 and L3 as key anatomical reference points, together with the anatomical relationship between L3–L5 and the sciatic nerve, followed by sequential identification of adjacent segments in both the rostral and caudal directions.

Furthermore, this method provides a relatively systematic description of the key steps, including exposure of the vertebral column and vertebral canal, opening of the vertebral canal with subsequent exposure of the spinal cord, exposure of the DRG, and stereomicroscope-guided localization and dissection. The workflow is clear and the anatomical landmarks are well defined, which may improve procedural standardization and reproducibility, particularly for beginners. The DRG obtained with this method retain good tissue integrity and are therefore well suited for downstream analyses, including immunofluorescence and H&E staining.

In this study, we established a standardized dissection and isolation method for mouse DRG and provided schematic illustrations for anatomical localization, together with representative images and video documentation of the sampling procedure. This method supports isolation of DRG from the thoracic to sacral levels in a single specimen while reducing the risk of DRG damage during tissue collection.

Protocol

All animal experiments were conducted in strict accordance with the protocol approved by the Institutional Animal Care and Use Committee of Shanghai East Hospital. The dissection procedures described in this study were performed using adult C57BL/6 mice (male or female, 12 weeks of age, 25–30 g). Figure 1 shows the dissection instruments used in this protocol.

1. Preparation

NOTE: For perfusion-fixed specimens, adopt appropriate protective measures to avoid exposure to fixative. Perform the dissection in a fume hood or on a well-ventilated bench, and wear appropriate personal protective equipment throughout the procedure, including gloves, protective clothing, and safety goggles.

1. Maintain the mice under a 12 h light/12 h dark cycle at an ambient temperature of approximately 24 °C and a relative humidity of 55%, with ad libitum access to food and water.
2. Before beginning the dissection, euthanize the mouse in a carbon dioxide chamber until respiration has completely ceased. Confirm death, then place the animal in the supine position on a dissection board and secure the limbs with pins to maintain a stable posture.
3. Spray the fur over the thoracoabdominal region with 70% ethanol to reduce hair contamination and maintain a clean operative field.
4. Use scissors to open the skin along the midline of the thorax and abdomen. Extend the incision cranially along the ventral midline to fully expose the thoracic cage. Elevate the inferior end of the sternum and cut the thoracic wall along both sides of the costal arch to open the thoracic cavity and expose the heart. Remove part of the sternum and ribs if necessary to obtain a clear view of the heart.
5. Make a small incision in the right atrium with scissors to establish an outlet for blood and perfusate. Fill a 20 mL syringe with precooled PBS (4 °C), and slowly insert the needle into the left ventricle near the apex of the heart. Stabilize the needle and avoid puncturing through the ventricular wall.
6. Perfuse precooled PBS slowly and evenly until the liver becomes pale, the outflow runs clear and most of the blood has been washed out. Then, replace the PBS with 20 mL of precooled 4% paraformaldehyde (4 °C) and continue perfusion at a slow, steady rate until the limbs become rigid, the tail curls, and the tissues change from red to pale, indicating that fixation is largely complete.
7. After perfusion, withdraw the needle, remove any residual fluid from the thoracic cavity, and proceed immediately to tissue collection. If unfixed tissue is required for downstream analyses, perfuse only with precooled PBS and omit the 4% paraformaldehyde fixation step.
   NOTE: Maintain a slow, steady, and uniform perfusion rate throughout the procedure to avoid cardiac rupture or tissue swelling caused by excessive pressure. Perform all of the above procedures at room temperature.

2. DRG and adjacent nerves (Figure 2)

1. Exposure of the vertebral column and vertebral canal (3–5 min)
   1. Place the mouse in the prone position and secure it on the dissection board. Make a longitudinal incision along the midline of the dorsal skin with scissors, extending the incision from the tail towards the neck.
   2. Use scissors to separate the dorsal skin from the underlying connective tissue, and reflect the skin laterally on both sides to fully expose the dorsal musculature. Wipe away hair adhering to the surface of the connective tissue with absorbent paper, or rinse the surgical field with saline to keep the operating area clean.
   3. Incise the dorsal muscles and connective tissue longitudinally along both sides of the spinal column to fully expose the vertebral structures.
      ​NOTE: Prevent the scissors from penetrating too deeply into the thoracic or abdominal cavity during incision of the dorsal tissues, as this may affect subsequent dissection procedures.
   4. Carefully clear the connective tissue and residual muscle covering the spinal column with a bone rongeur. If residual tissue interferes with visualization, trim it with scissors as needed to achieve full exposure of the vertebral column. Next, cut through the cervical vertebrae from the dorsal aspect with scissors to detach the cranial end of the spinal column from the head and facilitate the following steps.
2. Open the vertebral canal and expose the spinal cord (Figure 3; 8–10 min).
   1. Insert micro curved scissors into the vertebral canal at the cervical level, and cut along both sides of the laminae to open the canal.
      ​NOTE: Take care to avoid injury to the spinal cord during the procedure.
   2. Remove the dorsal laminae to gradually expose the dorsal aspect of the spinal cord.
   3. Continue cutting away the dorsal laminae along the longitudinal axis of the spine with micro curved scissors, extending progressively from the thoracic to the sacral region to fully expose the dorsal aspect of the spinal cord.
3. Exposure of the DRG (Figure 4; 8–10 min)
   1. After fully exposing the dorsal surface of the spinal cord, use a bone rongeur to gently open the vertebral canal structures laterally on both sides, and alternate the manipulation between the left and right sides.
   2. Once the vertebral canal has been completely separated on both sides, observe the DRG located within the intervertebral foramina under a stereomicroscope.
4. Identification of the DRG (Figure 5; 8–10 min)
   1. Once the vertebral canal has been completely reflected bilaterally and the spinal cord has been fully exposed, examine the dorsal root nerve bundles arising from the dorsal surface of the spinal cord under a stereomicroscope. Follow the dorsal roots laterally and identify the DRG near the intervertebral foramina as oval or ovoid structures.
   2. Confirm during identification that the DRG lies at the interface between the dorsal root of the spinal nerve and the intervertebral foramen, and observe that it is arranged as a bilateral pair at each spinal segment. Identify the DRG as a ganglion formed by the cell bodies of sensory neurons, with one side entering the dorsal horn of the spinal cord and the opposite side extending into peripheral tissues.
   3. Use a stereomicroscope to identify the DRG as a faint yellow or gray-white, mildly swollen structure with a fuller shape and comparatively distinct boundaries.
5. Conduct segmental identification and dissection of the DRG (Figure 6; 8–10 min)
   1. Use microsurgical forceps under a stereomicroscope to gently elevate the dorsal root nerve bundle and thereby achieve full exposure of the attached DRG.
   2. Use the following features to determine the segmental identity of the DRG: designate DRG connected to multiply branched nerve fibers (>2 branches) as L2 and more cranial DRGs, and designate DRG connected to bifurcated nerve fibers (two branches only) as L3 and more caudal DRGs. Next, identify the remaining DRG segments sequentially above and below according to the locations of the intervertebral foramina.
      NOTE: The nerve fibers arising from the L3–L5 DRG merge to form the sciatic nerve.
   3. Use microsurgical curved scissors or microsurgical forceps to carefully separate the connective tissue and nerve fibers surrounding the DRG, and progressively free the ganglion from its attachments to the tissues around the intervertebral foramen.
   4. Transect the dorsal root nerve fibers connecting the DRG to the spinal cord, and simultaneously sever the nerve bundle extending toward the periphery to isolate the ganglion.
   5. Carefully isolate the DRG using microsurgical forceps. If long-term preservation and subsequent immunofluorescence analysis are required, immediately place the isolated DRG into a 0.5 mL centrifuge tube containing 4% paraformaldehyde and fix the tissue for 3 h. After fixation, wash the tissue for 3 × 5 min with 0.1 M PBS (main components: NaCl, KCl, Na2HPO4, and KH2PO4), and transfer the tissue into 30% sucrose solution for dehydration until it sinks to the bottom. Finally, embed the tissue in OCT and store it at −80 °C for long-term preservation.

3. Confirm the ganglion components.

1. H&E staining results (Figure 7)
   1. Fix the isolated DRG immediately in a conventional histological fixative, for example, 4% paraformaldehyde. After fixation, rinse the tissue in 0.1 M phosphate buffer, perform dehydration, clearing, and paraffin embedding according to standard histological procedures, and prepare 4–6 µm tissue sections for downstream staining analysis.
   2. Perform hematoxylin and eosin (H&E) staining on the sections to evaluate the overall structure and cellular morphology of the DRG. Use the stained sections to identify the cellular components and tissue organization of the ganglion and to determine the quality of the harvested DRG.
   3. Examine the H&E-stained sections under a microscope, and confirm that the DRG is composed predominantly of numerous sensory neuronal cell bodies. Identify neurons with large, round to ovoid somata, and observe their clearly visible nuclei as well as the prominent nucleoli present in some neurons. Also confirm the presence of closely apposed satellite glial cells surrounding the neurons in a characteristic ring-like arrangement.
   4. Examine the interior of the ganglion and identify the nerve fiber bundles and connective tissue septa to confirm that the overall tissue architecture is intact and clearly organized. Verify that the cellular boundaries are well defined and that no obvious tissue disruption or structural disorganization is present.
   5. Confirm successful DRG isolation on H&E staining by the presence of preserved overall structure, well-defined cellular morphology, and no evident mechanical injury. Detect sample damage by observing disrupted cellular architecture, indistinct tissue margins or localized tissue destruction arising during dissection or processing. Use these H&E findings as a practical basis for evaluating the quality of DRG collection and the integrity of the tissue.
2. TUBB3 immunofluorescence staining results (Figure 8)
   1. Fix the isolated DRG immediately in 4% paraformaldehyde for 3 h. Once fixation is complete, rinse the tissue three times in 0.1 M PBS, allowing 5 min for each wash. Next, place the tissue in 30% sucrose solution until it settles at the bottom, embed it in OCT, and cut frozen sections at 8–12 µm for downstream immunofluorescence staining.
   2. Warm the sections to room temperature, rinse them 3 × 5 min in PBS, and carry out antigen retrieval with Tris-EDTA antigen retrieval solution. Let the sections cool naturally to room temperature, wash them again 3 × 5 min with PBS, and block with 5% BSA for 30–60 min at room temperature. After the blocking step, apply the fluorophore-labeled TUBB3 primary antibody and incubate the sections overnight at 4 °C. The next day, wash the sections three times with PBS, stain the nuclei with DAPI, mount the sections, and capture images using a fluorescence microscope.
   3. Inspect the DRG sections and verify that well-preserved specimens display a distinct ganglion outline, intact tissue structure, and orderly cellular arrangement. Observe TUBB3 immunofluorescence mainly in sensory neuronal cell bodies and nerve fibers to define the overall neuronal distribution within the ganglion. Use DAPI counterstaining to identify the larger TUBB3-positive cells as sensory neurons, and note that the small DAPI-positive nuclei distributed around these cells show a morphological pattern consistent with satellite glial cell nuclei. Identify dissection- or processing-related damage by disrupted tissue continuity, irregular TUBB3 signal localization, poorly defined neuronal morphology, or focal increases in background signal.
   4. Apply TUBB3 immunofluorescence staining as a supplementary indicator of DRG tissue integrity. Recognize successful DRG isolation by the presence of a preserved ganglion outline, distinct fluorescence signals in sensory neuronal cell bodies and nerve fibers, clear cellular morphology, and minimal background staining.

Results

Successful DRG isolation requires preservation of the ganglion's integrity, with no obvious compression, crushing, or tearing. Gently lift the laterally connected nerve fibers, progressively release the ganglion from the surrounding tissues, and transect the connected nerve fibers only after the ganglion has been fully mobilized, so that it can be removed intact. Avoid direct traction on the ganglion body with instruments during the procedure. After isolation, preserve the native morphology of the DRG within the interver...

Discussion

DRG, as key components of the peripheral sensory nervous system, play a critical role in transmitting mechanical, thermal, and nociceptive sensory information to the central nervous system^9,10,11. DRG neurons exhibit a characteristic pseudounipolar morphology, with one process extending to peripheral tissues and the other projecting into the dorsal horn of the spinal cord, thereby playing critical roles in sensory signal transmi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.5 mL centrifuge tube	Not specified	Not specified	Used for fixed DRG storage
20 mL syringe	Not specified	Not specified	Used for cardiac perfusion
30% sucrose solution	Not specified	Not specified	Used for tissue dehydration before OCT embedding
4% paraformaldehyde	Servicebio	G1101
5% BSA	Solarbio	SW3015
70% ethanol	Not specified	Not specified	Used to reduce hair contamination before dissection
Absorbent paper	Not specified	Not specified	Used to remove hair from connective tissue
Adult C57BL/6 mice	Vital River	Not specified	male or female; 12 weeks of age; 25–30 g
Alexa Fluor 488-conjugated goat anti-rabbit IgG	Servicebio	GB25303
Anti-TUBB3 antibody	Proteintech	CL488-66375	Used for TUBB3 immunofluorescence
Carbon dioxide chamber	Not specified	Not specified	Used for euthanasia
DAPI	Servicebio	G1012
Dissection board	Not specified	Not specified	Used to secure the mouse during dissection
Fluorescence microscope	Not specified	Not specified	Used for imaging immunofluorescence-stained sections
Friedman-Pearson bone rongeurs	RWD	S21022-14	Extracted from manuscript: bone rongeur (RWD, S21022-14)
H&E staining solution kit	Servicebio	G1005
Microsurgical curved scissors	ASSI	SDCT16R8	Extracted from manuscript: micro curved scissors (ASSI, SDCT16R8)
Microsurgical forceps	Fine Science Tools	11254-20	Extracted from manuscript: microsurgical forceps (FST, 11254-20)
OCT compound	Sakura Finetek	No. 4583
PBS	Servicebio	G2156
Pins	Not specified	Not specified	Used to secure limbs on the dissection board
Saline	Not specified	Not specified	Used to rinse the surgical field
Scissors	Fine Science Tools	14060-09	Extracted from manuscript: scissors (FST, 14060-09)
Stereomicroscope	Leica Microsystems, Wetzlar, Germany	10447197	Extracted from manuscript: stereomicroscope (Leica, 10447197)
Tris-EDTA antigen retrieval solution	Solarbio	C1038