Harvesting, Embedding, and Culturing Dorsal Root Ganglia in Multi-compartment Devices to Study Peripheral Neuronal Features
Summary
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.
Abstract
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.
Introduction
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.
Protocol
Representative Results
Discussion
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</s…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| #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) |
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