This protocol provides a technique to harvest and culture explanted dorsal root ganglion (DRG) from adult Sprague Dawley rats in a multi-compartment (MC) device.
The most common peripheral neuronal feature of pain is a lowered stimulation threshold or hypersensitivity of terminal nerves from the dorsal root ganglia (DRG). One proposed cause of this hypersensitivity is associated with the interaction between immune cells in the peripheral tissue and neurons. In vitro models have provided foundational knowledge in understanding how these mechanisms result in nociceptor hypersensitivity. However, in vitro models face the challenge of translating efficacy to humans. To address this challenge, a physiologically and anatomically relevant in vitro model has been developed for the culture of intact dorsal root ganglia (DRGs) in three isolated compartments in a 48-well plate. Primary DRGs are harvested from adult Sprague Dawley rats after humane euthanasia. Excess nerve roots are trimmed, and the DRG is cut into appropriate sizes for culture. DRGs are then grown in natural hydrogels, enabling robust growth in all compartments. This multi-compartment system offers anatomically relevant isolation of the DRG cell bodies from neurites, physiologically relevant cell types, and mechanical properties to study the interactions between neural and immune cells. Thus, this culture platform provides a valuable tool for investigating treatment isolation strategies, ultimately leading to an improved screening approach for predicting pain.
Chronic pain is the leading cause of disability and loss of work globally1. Chronic pain affects about 20% of adults globally and imposes a significant societal and economic burden2, with total costs estimated between $560 and $635 billion every year in the United States3.
The main peripheral feature exhibited by chronic pain patients is a lowered stimulation threshold of nerves, which leads to the nervous system being more responsive to stimuli4,5. The lowered stimulation threshold can result in a painful response to a previously non-painful stimulus (allodynia) or a heightened response to a painful stimulus (hyperalgesia)6. Current chronic pain treatments have limited efficacy, and treatments that succeed in animal models often fail in human trials due to mechanistic differences in pain manifestation7. In vitro models that can more accurately mimic peripheral sensitization mechanisms have the potential to increase the translation of new therapeutics8,9. Further, by modeling key aspects of sensitized nerves in a culture system, researchers could develop a deeper understanding of the mechanisms that drive lowered thresholds and identify novel therapeutic targets that reverse them10.
The ideal in vitro platforms or microphysiological systems would incorporate the physical separation of distal neurites and dorsal root ganglia (DRG) cell body, a three-dimensional (3D) cellular environment, and the presence of native support cells to closely mimic in vivo conditions. However, a recent paper by Caparaso et al.11 shows that current DRG culture platforms lack one or more of these key features, making them insufficient in replicating in vivo conditions. Even though these platforms are easy to set up, they do not mimic the biological basis of peripheral sensitization and thus may not translate to in vivo efficacy. To address this limitation, a physiologically relevant in vitro model has been developed for the culture of dorsal root ganglia (DRG) within a hydrogel matrix with three isolated compartments to allow temporal fluidic isolation of neurites and DRG cell bodies11. This model offers both physiological and anatomical relevance, which has the potential to study peripheral sensitization of neurons in vitro.
The growing interest in the use of DRG explants in a 3D culture is due to their ability to facilitate robust neurite growth, which serves as an indirect indicator of DRG viability12. While primary neonatal or embryonic DRG explants are predominantly used in current in vitro culture platforms13,14, using explants from adult rodents provides a better model of mature neuronal physiology, which closely mimics human DRG physiology compared to explants from neonatal or embryonic rodents15. Explant DRGs refer to the preservation of the cellular and molecular tissue of native DRG tissue, primarily by maintaining native non-neuronal support cells.Herein, this protocol describes the methodology to harvest and culture DRG explants from adult Sprague Dawley rats in a multi-compartment (MC) device (Figure 1).
Efficacy has been shown in culturing DRGs from the cervical, thoracic, and lumbar spine with no observable differences in neurite growth. For this application, the objective was to elicit neurite growth into the outer compartments of the device; therefore, this article did not discriminate among DRG levels. However, if needed for a specific experiment, the DRG level can be tailored to meet experimenters' needs. There are currently other compartmentalized culture models for the 3D culture of DRGs16, however, these devices do not contain preserved native non-neuronal support cells, which can limit translation. Preserving the native structure of harvested DRGs is important because it ensures the retention of non-neuronal support cells, whose interactions with DRG neurons are essential for maintaining the functional properties of these neurons. Several studies co-cultured dissociated DRGs with non-native neuronal cells such as Schwann cells to promote myelination of neurons17,18,19.
DRG harvest was performed in compliance with the Institutional Animal Care and Use Committee (IACUC) at the University of Nebraska-Lincoln. Female Sprague Dawley rats aged 12 weeks (~250 g) were used for the study. The details of the animals, reagents, and equipment used in the study are listed in the Table of Materials.
1. Multi-compartment device fabrication and assembly
2. Hydrogel preparation
3. Photoinitiator solution preparation
NOTE: A photoinitiator is necessary to crosslink the MAHA under UV light. It is commonly used at percentages from 0.3% to 0.6%.
4. Methacrylated hyaluronic acid dissolution
5. Device assembly
6. Animal preparation
7. Dorsal root ganglia (DRG) harvest
8. DRG trimming and cutting
9. Collagen neutralization
10. Hydrogel fabrication
11. DRG embedding
12. Control DRG embedding
13. DRG imaging
14. DRG neurite quantification
The present protocol described a technique to harvest and culture DRG from adult Sprague Dawley rats in a multi-compartment (MC) device. As shown in Figure 1, DRG harvested from adult rats was trimmed and cut into ~0.5 mm. The trimmed and cut DRGs were then embedded in a hydrogel in the soma region of the MC device (Figure 2) and cultured for 27 days before neurite quantification. DRG was cultured in plain gel to serve as the control. The concentration of hydrogel formulation used for this experiment was 4.5/1.25 mg/mL collagen: MAHA. On days 27 and 21 for multi-compartment and plain gels, respectively, there was a robust growth of neurites (Figure 3). The average length of neurites in MC (894.22 µm ± 308.75 µm) was comparable to neurite length in control plain gels (864.26 µm ± 362.84 µm) (Figure 4). This demonstrates the ability of the MC device to support DRG culture and neurite growth. Neurite lengths were quantified using ImageJ software.
Figure 1: Schematic diagram showing the experimental procedure. (A) Dorsal root ganglia (DRG) harvest from adult Sprague Dawley rats. (B) Trimming and cutting of harvested DRG. (C) Hydrogel formulation, DRG embedding, and culture in hydrogel within MC fitted into a 48-well plate. (D) Growth of DRG neurites after 21-30 days of culture. Please click here to view a larger version of this figure.
Figure 2: Printed multi-compartment with three isolated compartments and can be used in a 48-well plate. (A) A representative image showing the top view of the printed MC device shows the DRG and neurite compartments (red lines) and the DRG embedding area (green). (B) Side view of MC. (C) An image of MC fitted into a 48-well plate. Scale bar = 10 mm. Please click here to view a larger version of this figure.
Figure 3: Neurite growth in multi-compartment (MC) device, and control plain gels. (A) A representative image showing traced neurite growth in a multi-compartment (MC) device in brightfield. (Below) Neurites that grew through the tunnels of MC are indicated with arrows. (B) Image of neurite growth in control plain gels (without MC). (C) A representative image showing neurite tracing of twelve long neurites in control plain gels (purple lines). Six long neurites at both sides of the DRG were quantified to give the average neurite length. Images were captured with a fluorescent plate imager at 4x magnification, and neurites were quantified using FIJI (ImageJ). Scale bars = 1000 µm. Please click here to view a larger version of this figure.
Figure 4: Neurite length in multi-compartment (MC) compared to that of plain gel (PG). Scatter plot showing individual neurite lengths with means and standard deviations represented by error bars. The average neurite length in MC was 1204.40 µm ± 690.43 µm (mean ± SD) compared to 864.26 µm ± 362.84 µm (mean ± SD) in the plain gel, indicating MC support neurite growth. Please click here to view a larger version of this figure.
Supplementary Coding File 1: STL file of multi-compartment (MC) device generated using computer-aided design software. Please click here to download this File.
This protocol outlines a method to harvest adult Sprague Dawley DRGs and culture them in 3D natural hydrogels. In contrast to this method, other approaches to harvesting DRGs from mice and rats involve isolating the spinal column. The excised spinal column is halved, and the spinal cord is removed to expose DRGs23,24,25. Damage to the spinal cord limits blood supply, which affects DRGs and internal neurons26. This reduces the activity of DRGs, making the surgical approaches more appropriate. Further, a method to 3D print a robust MC device that allows for a physiologically relevant culture of DRGs has been outlined.
This protocol outlines a method to culture DRG in vitro, emphasizing studying peripheral sensitization of neurons in an anatomically and physiologically relevant model. This culture method can establish a physical separation of distal neurites and the DRG cell body in a 3D environment. The MC device mimics the in vivo environment since DRG explants with cells are preserved and may be co-cultured with native glial cells. DRG explant contains non-native neuronal cells, which could be co-cultured with other cells to mimic physiological conditions. This physiological relevance promotes studies into how non-neuronal cells regulate neurite activity and helps in the understanding of basic mechanisms involved in nociception. Incorporating these non-neuronal interactions should yield more accurate predictions of cell signaling outcomes compared to simplified culture models with dissociated DRGs. The presence of two neurite compartments maximizes physiological relevance because single DRGs innervate different tissues in vivo. This design permits the treatment of one neurite compartment while the other serves as the control. Control and treatment compartments connected to a single soma could be used to study cross-compartment effects, mimicking in vivo conditions where neurites extend from a single soma and experience different stimuli as they innervate different tissues, providing insight into cross-talk between neurites. If cross-talk occurs between neurites in adjacent compartments, untreated DRGs can serve as a true control.
This device could be used for treatment isolation or to study the interaction between neurons and DRG in the context of pain. Since distal neurites can be selectively treated separately from the DRG soma and proximal neurites, this system could be used for peripheral treatments such as targeted joint therapeutics, where the terminal end of peripheral sensory neurons is directly treated.
A key advantage of the MC device is the high-throughput experiments across multiple wells of the same plate, as the MC device is designed to fit into a 48-well plate. An additional benefit of the in vitro platform is the capacity to alter hydrogel stiffness within the MC by tuning collagen and MAHA concentrations to mimic the properties of various tissue microenvironments. One potential limitation of this protocol is that fluidic isolation between the neurite and DRG soma compartments is not ideal for cultures beyond 72 h without a media change. A previous study confirmed that fluidic isolation is maintained in the compartments for up to 72 h without media changes11. Prior studies have shown that increasing hyaluronic acid concentration significantly enhances collagen crosslinking density, thus restricting the diffusivity of macromolecules across the hydrogel matrix27. Therefore, to improve fluidic isolation for cultures past 72 h, the concentration of MAHA can be increased, as demonstrated in a study by Caparaso et al.7, where an increase in MAHA resulted in improved fluidic isolation. Control embedding is done to ensure comparable growth of DRG neurites in MC and plain gel. This is to ensure MC has no impact on DRG growth. It is not recommended to repeat control embedding with every experiment once DRG growth in MC is validated.
The authors acknowledge that human DRGs are larger, contain more cells, and have varying proportions of neuron subtype compared to rat DRGs28. The use of rodent DRGs is a limitation of this study in terms of translation to the clinic; however, this system could be valuable as a screening platform to identify compounds that impact neuronal excitability. Although there are compositional differences between rodent and human DRGs, there is also a high overlap within the proteome between rat and human DRGs29. This overlap in proteome – and the presence of the same neuronal subtypes – suggests that the rat DRG can be used to screen therapeutics at a high level, and further testing can be conducted to probe if changes in the rat DRG mechanistically translate to the human DRG.
This platform allows for targeted pharmacological stimulation and the study of neurons or cell bodies with temporal control. Changes in neuronal excitability and signal transmission may be easily measured by manipulating neurites or the DRG. The ability to assess neuronal excitability using calcium imaging in plain control hydrogels with explant DRGs has been demonstrated7. Similar methods can be employed in the MC device to assess neuronal excitability.
This method can be used to study various pain conditions where stimulation of peripheral primary sensory neurons is a driving factor, such as disc-associated low back pain. Screening nociceptor hypersensitivity can be a promising tool for discovering novel drugs to reduce hyperexcitability. Reducing hyperexcitability has the potential to translate clinically to reduced pain. This platform can screen high-throughput drugs to identify the most efficacious candidates for in vivo validation using pain-like behavior assays.
The authors have nothing to disclose.
This work was supported by an NSF Grant (2152065) and an NSF CAREER Award (1846857). The authors would like to thank all current and past members of the Wachs Lab for contributing to this protocol. Diagrams in Figure 1 were made in Biorender.
#5 forceps | Fine Science Tools | 11252-00 | For trimming and cutting DRG |
10x DMEM | MilliporeSigma | D2429 | |
1x PBS (autoclaved) | Prepared in lab | 7.3 – 7.5 pH | |
24 well plates | VWR | 82050-892 | To temporarily store harvested and cut DRGs |
3 mL Syringe sterile, single use | BD | 309657 | |
48 well plates | Greiner Bio-One | 677180 | |
60 mm Petri dish | Fisher Scientific | FB0875713A | To hold media for trimming and cutting |
Aluminium foil | Fisherbrand | 01-213-104 | |
B27 Plus 50x | ThermoFisher | 17504044 | For DRG media |
Collagen type I | Ibidi | 50205 | |
Curved cup Friedman Pearson Rongeur | Fine Science Tools | 16221-14 | For dissection |
Dumont #3 forceps | Fine Science Tools | 11293-00 | For dissection |
Fetal Bovine Serum (FBS) | ThermoFisher | 16000044 | For DRG media |
Form cure | Form Labs | curing agent | |
Form wash | Form Labs | To wash excess resins off MC | |
Glass bead sterilizer | Fisher Scientific | NC9531961 | |
Glass vials (8 mL) | DWK Life Sciences (Wheaton) | 224724 | |
GlutaMax | ThermoFisher | 35050-061 | For DRG media |
HEPES (1M) | Millipore Sigma | H0887 | |
High temp V2 resin | FormLabs | FLHTAM02 | |
Hyaluronic Acid Sodium Salt | MilliporeSigma | 53747 | Used to make MAHA |
Irgacure | MilliporeSigma | 410896 | |
Laminin | R&D Systems | 344600501 | |
Large blunt-nose scissors | Militex | EG5-26 | For dissection |
Large forceps (serrated tips) | Militex | 9538797 | For dissection |
Large sharp-nosed scissors | Fine Science Tools | 14010-15 | For dissection |
Low Retention pipette tips | Fisher Scientific | 02-707-017 | For pipetting collagen and MAHA |
Methacrylated hyaluronic acid (MAHA) | Prepared in lab | N/A | 85 – 115 % methacrylation |
Nerve Growth Factor (NGF) | R&D Systems | 556-NG-100 | For DRG media |
Neurobasal A Media | ThermoFisher | 10888022 | For DRG media |
Parafilm | Bemis | PM996 | |
Parafilm | Bemis | PM996 | |
Penicillin/Streptomycin (PS) | EMD Millipore | 516106 | For DRG media |
pH test strips | VWR International | BDH35309.606 | |
Pipette tips (1000 µL) | USA Scientific | 1111-2021 | |
Preform 3.23.1 software | Formslab | To upload STL file | |
Rat | Charles River | ||
Resin 3D printer | Form Labs | Form 3L | 3D printing MC device |
Small sharp-nosed scissors | Fine Science Tools | 14094-11 | For dissection |
Sodium bicarbonate | MilliporeSigma | S6014 | |
Straight cup rongeur | Fine Science Tools | 16004-16 | For dissection |
Straight edge spring scissors | Fine Science Tools | 15024-10 | For dissection |
Surgical Scaplel blade (No. 10) | Fisher Scientific | 22-079-690 | |
Syring filters, PES (0.22 µm) | Celltreat | 229747 | |
Tiny spring scissors | World Precision Instruments | 14003 | For trimming and cutting DRG |
UV lamp | Analytik Jena US | To photocrosslink hydrogel (15 – 18 mW/cm2) |