Name: Auteur in de schijnwerpers: in vitro en in vivo modellen om de doeltreffendheid van de behandeling van chronische pijn te verbeteren
Uploaded: 2024-06-28T14:10:00
Description: The most common peripheral neuronal feature of pain is a lowered stimulation threshold or hypersensitivity of terminal nerves from the dorsal root ganglia ...

Dit werk werd ondersteund door een NSF Grant (2152065) en een NSF CAREER Award (1846857). De auteurs willen alle huidige en voormalige leden van het Wachs Lab bedanken voor hun bijdrage aan dit protocol. De diagrammen in figuur 1 zijn gemaakt in Biorender.

De DRG-oogst werd uitgevoerd in overeenstemming met het Institutional Animal Care and Use Committee (IACUC) van de Universiteit van Nebraska-Lincoln. Vrouwelijke Sprague Dawley-ratten van 12 weken (~250 g) werden gebruikt voor het onderzoek. De details van de dieren, reagentia en apparatuur die in het onderzoek zijn gebruikt, staan vermeld in de materiaaltabel.
1. Fabricage en assemblage van apparaten met meerdere compartimenten
<ol><li>Computerondersteund o...

Geavanceerd in vitro model voor het bestuderen van neuronale overgevoeligheid in DRG-culturen

<ol>
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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+hyaluronic+acid+hydrogels+with+tunable+mechanical+properties+on+neural+progenitor+cell+differentiation.">The effects of hyaluronic acid hydrogels with tunable mechanical properties on neural progenitor cell differentiation.</a> Biomaterials. 31 (14), 3930-3940 (2010).</li><li>Tomal, W., Ortyl, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water-soluble+photoinitiators+in+biomedical+applications.">Water-soluble photoinitiators in biomedical applications.</a> Polymers. 12 (5), 1073 (2020).</li><li>Leary, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Avma guidelines for the euthanasia of animals: 2013 edition. , (2013).</li><li>Sleigh, J. 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Dit protocol schetst een methode om volwassen Sprague Dawley DRG's te oogsten en ze te kweken in natuurlijke 3D-hydrogels. In tegenstelling tot deze methode omvatten andere benaderingen voor het oogsten van DRG's van muizen en ratten het isoleren van de wervelkolom. De uitgesneden wervelkolom wordt gehalveerd en het ruggenmerg wordt verwijderd om DRG's 23,24,25 bloot te leggen. Schade aan het ruggenmerg beperkt de bloedtoevoer, ...

Chronische pijn is wereldwijd de belangrijkste oorzaak van arbeidsongeschiktheid en verlies van werk1. Chronische pijn treft ongeveer 20% van de volwassenen wereldwijd en legt een aanzienlijke maatschappelijke en economische lastop 2, met totale kosten die in de Verenigde Staten elk jaar tussen $ 560 en $ 635 miljard worden geschat3.
Het belangrijkste perifere kenmerk van chronische pijnpatiënten is een verlaagde stimulatiedrempel van zenuwen, wat ertoe leidt dat het zenuwstelsel beter reageert op stimuli 4,5. De verlaagde stimulatiedrempel kan resulteren in een pijnlijke reactie op een voorheen niet-pijnlijke stimulus (allodynie) of een verhoogde respons op een pijnlijke stimulus (hyperalgesie)6. De huidige behandelingen voor chronische pijn hebben een beperkte werkzaamheid, en behandelingen die slagen in diermodellen falen vaak in proeven bij mensen vanwege mechanistische verschillen in pijnmanifestatie7. In vitro modellen die perifere sensibilisatiemechanismen nauwkeuriger kunnen nabootsen, hebben het potentieel om de vertaling van nieuwe therapieën te verhogen 8,9. Verder, door het modelleren van belangrijke aspecten van gesensibiliseerde zenuwen in een kweeksysteem, zouden onderzoekers een dieper begrip kunnen ontwikkelen van de mechanismen die verlaagde drempels veroorzaken en nieuwe therapeutische doelen kunnen identificeren die deze omkeren10.
De ideale in vitro platforms of microfysiologische systemen zouden de fysieke scheiding van distale neurieten en dorsale wortelganglia (DRG) cellichaam, een driedimensionale (3D) cellulaire omgeving en de aanwezigheid van native ondersteunende cellen omvatten om in vivo omstandigheden nauwkeurig na te bootsen. Een recent artikel van Caparaso et al.11 toont echter aan dat de huidige DRG-cultuurplatforms een of meer van deze belangrijke kenmerken missen, waardoor ze onvoldoende zijn om in vivo omstandigheden te repliceren. Hoewel deze platforms eenvoudig in te stellen zijn, bootsen ze de biologische basis van perifere sensibilisatie niet na en vertalen ze zich dus mogelijk niet naar in vivo werkzaamheid. Om deze beperking aan te pakken, is een fysiologisch relevant in vitro model ontwikkeld voor de kweek van dorsale wortelganglia (DRG) binnen een hydrogelmatrix met drie geïsoleerde compartimenten om temporele vloeistofisolatie van neurieten en DRG-cellichamen mogelijk te maken11. Dit model biedt zowel fysiologische als anatomische relevantie, wat het potentieel heeft om perifere sensibilisatie van neuronen in vitro te bestuderen.
De groeiende belangstelling voor het gebruik van DRG-explantaten in een 3D-cultuur is te danken aan hun vermogen om robuuste neurietgroei te bevorderen, wat dient als een indirecte indicator van de levensvatbaarheid van DRG12. Hoewel primaire neonatale of embryonale DRG-explantaten voornamelijk worden gebruikt in de huidige in-vitrokweekplatforms 13,14, biedt het gebruik van explantaten van volwassen knaagdieren een beter model van volwassen neuronale fysiologie, die de menselijke DRG-fysiologie nauw nabootst in vergelijking met explantaten van neonatale of embryonale knaagdieren15. Explantatie DRG's verwijzen naar het behoud van het cellulaire en moleculaire weefsel van native DRG-weefsel, voornamelijk door het behoud van native niet-neuronale steuncellen. Hierin beschrijft dit protocol de methodologie om DRG-explantaten van volwassen Sprague Dawley-ratten te oogsten en te kweken in een apparaat met meerdere compartimenten (MC) (Figuur 1).
Werkzaamheid is aangetoond bij het kweken van DRG's uit de cervicale, thoracale en lumbale wervelkolom zonder waarneembare verschillen in neurietgroei. Voor deze toepassing was het doel om neurietgroei op te wekken in de buitenste compartimenten van het apparaat; daarom werd in dit artikel geen onderscheid gemaakt tussen DRG-niveaus. Indien nodig voor een specifiek experiment kan het DRG-niveau echter worden aangepast aan de behoeften van de onderzoekers. Er zijn momenteel andere gecompartimenteerde kweekmodellen voor de 3D-cultuur van DRG's16, maar deze apparaten bevatten geen geconserveerde native niet-neuronale steuncellen, wat de translatie kan beperken. Het behoud van de oorspronkelijke structuur van geoogste DRG's is belangrijk omdat het zorgt voor het behoud van niet-neuronale steuncellen, waarvan de interacties met DRG-neuronen essentieel zijn voor het behoud van de functionele eigenschappen van deze neuronen. Verschillende studies hebben gedissocieerde DRG's samen gekweekt met niet-inheemse neuronale cellen zoals Schwann-cellen om myelinisatie van neuronen te bevorderen 17,18,19.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#5 forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
			<td>For trimming and cutting DRG</td>
		</tr>
		<tr>
			<td>10x DMEM</td>
			<td>MilliporeSigma</td>
			<td>D2429</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS (autoclaved)</td>
			<td>Prepared in lab</td>
			<td></td>
			<td>7.3 - 7.5 pH</td>
		</tr>
		<tr>
			<td>24 well plates</td>
			<td>VWR</td>
			<td>82050-892</td>
			<td>To temporarily store harvested and cut DRGs</td>
		</tr>
		<tr>
			<td>3 mL Syringe sterile, single use</td>
			<td>BD</td>
			<td>309657</td>
			<td></td>
		</tr>
		<tr>
			<td>48 well plates</td>
			<td>Greiner Bio-One</td>
			<td>677180</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Petri dish</td>
			<td>Fisher Scientific</td>
			<td>FB0875713A</td>
			<td>To hold media for trimming and cutting</td>
		</tr>
		<tr>
			<td>Aluminium foil</td>
			<td>Fisherbrand</td>
			<td>01-213-104</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Plus 50x</td>
			<td>ThermoFisher</td>
			<td>17504044</td>
			<td>For DRG media</td>
		</tr>
		<tr>
			<td>Collagen type I</td>
			<td>Ibidi</td>
			<td>50205</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved cup Friedman Pearson Rongeur</td>
			<td>Fine Science Tools</td>
			<td>16221-14</td>
			<td>For dissection</td>
		</tr>
		<tr>
			<td>Dumont #3 forceps</td>
			<td>Fine Science Tools</td>
			<td>11293-00</td>
			<td>For dissection</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>ThermoFisher</td>
			<td>16000044</td>
			<td>For DRG media</td>
		</tr>
		<tr>
			<td>Form cure</td>
			<td>Form Labs</td>
			<td></td>
			<td>curing agent</td>
		</tr>
		<tr>
			<td>Form wash</td>
			<td>Form Labs</td>
			<td></td>
			<td>To wash excess resins off MC</td>
		</tr>
		<tr>
			<td>Glass bead sterilizer</td>
			<td>Fisher Scientific</td>
			<td>NC9531961</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass vials (8 mL)</td>
			<td>DWK Life Sciences (Wheaton)</td>
			<td>224724</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>ThermoFisher</td>
			<td>35050-061</td>
			<td>For DRG media</td>
		</tr>
		<tr>
			<td>HEPES (1M)</td>
			<td>Millipore Sigma</td>
			<td>H0887</td>
			<td></td>
		</tr>
		<tr>
			<td>High temp V2 resin</td>
			<td>FormLabs</td>
			<td>FLHTAM02</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronic Acid Sodium Salt</td>
			<td>MilliporeSigma</td>
			<td>53747</td>
			<td>Used to make MAHA</td>
		</tr>
		<tr>
			<td>Irgacure</td>
			<td>MilliporeSigma</td>
			<td>410896</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>R&#38;D Systems</td>
			<td>344600501</td>
			<td></td>
		</tr>
		<tr>
			<td>Large blunt-nose scissors</td>
			<td>Militex</td>
			<td>EG5-26</td>
			<td>For dissection</td>
		</tr>
		<tr>
			<td>Large forceps (serrated tips)</td>
			<td>Militex</td>
			<td>9538797</td>
			<td>For dissection</td>
		</tr>
		<tr>
			<td>Large sharp-nosed scissors</td>
			<td>Fine Science Tools</td>
			<td>14010-15</td>
			<td>For dissection</td>
		</tr>
		<tr>
			<td>Low Retention pipette tips</td>
			<td>Fisher Scientific</td>
			<td>02-707-017</td>
			<td>For pipetting collagen and MAHA</td>
		</tr>
		<tr>
			<td>Methacrylated hyaluronic acid (MAHA)</td>
			<td>Prepared in lab</td>
			<td>N/A</td>
			<td>85 - 115 % methacrylation</td>
		</tr>
		<tr>
			<td>Nerve Growth Factor (NGF)</td>
			<td>R&#38;D Systems</td>
			<td>556-NG-100</td>
			<td>For DRG media</td>
		</tr>
		<tr>
			<td>Neurobasal A Media</td>
			<td>ThermoFisher</td>
			<td>10888022</td>
			<td>For DRG media</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (PS)</td>
			<td>EMD Millipore</td>
			<td>516106</td>
			<td>For DRG media</td>
		</tr>
		<tr>
			<td>pH test strips</td>
			<td>VWR International</td>
			<td>BDH35309.606</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips (1000 &#181;L)</td>
			<td>USA Scientific</td>
			<td>1111-2021</td>
			<td></td>
		</tr>
		<tr>
			<td>Preform 3.23.1 software</td>
			<td>Formslab</td>
			<td></td>
			<td>To upload STL file</td>
		</tr>
		<tr>
			<td>Rat</td>
			<td>Charles River</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Resin 3D printer</td>
			<td>Form Labs</td>
			<td>Form 3L</td>
			<td>3D printing MC device</td>
		</tr>
		<tr>
			<td>Small sharp-nosed scissors</td>
			<td>Fine Science Tools</td>
			<td>14094-11</td>
			<td>For dissection</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>MilliporeSigma</td>
			<td>S6014</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight cup rongeur</td>
			<td>Fine Science Tools</td>
			<td>16004-16</td>
			<td>For dissection</td>
		</tr>
		<tr>
			<td>Straight edge spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15024-10</td>
			<td>For dissection</td>
		</tr>
		<tr>
			<td>Surgical Scaplel blade (No. 10)</td>
			<td>Fisher Scientific</td>
			<td>22-079-690</td>
			<td></td>
		</tr>
		<tr>
			<td>Syring filters, PES (0.22 &#181;m)</td>
			<td>Celltreat</td>
			<td>229747</td>
			<td></td>
		</tr>
		<tr>
			<td>Tiny spring scissors</td>
			<td>World Precision Instruments</td>
			<td>14003</td>
			<td>For trimming and cutting DRG</td>
		</tr>
		<tr>
			<td>UV lamp</td>
			<td>Analytik Jena US</td>
			<td></td>
			<td>To photocrosslink hydrogel (15 - 18 mW/cm2)</td>
		</tr>
	</tbody>
</table>

harvesting, embedding, and culturing dorsal root ganglia in multi-compartment devices to study peripheral neuronal features

Het meest voorkomende perifere neuronale kenmerk van pijn is een verlaagde stimulatiedrempel of overgevoeligheid van terminale zenuwen van de dorsale wortelganglia (DRG). Een mogelijke oorzaak van deze overgevoeligheid houdt verband met de interactie tussen immuuncellen in het perifere weefsel en neuronen. In vitro modellen hebben fundamentele kennis opgeleverd om te begrijpen hoe deze mechanismen leiden tot overgevoeligheid van de nociceptoren. In vitro modellen staan echter voor de uitdaging om de werkzaamheid te vertalen naar mensen. Om deze uitdaging aan te gaan, is een fysiologisch en anatomisch relevant in vitro model ontwikkeld voor de kweek van intacte dorsale wortelganglia (DRG's) in drie geïsoleerde compartimenten in een plaat met 48 putjes. Primaire DRG's worden geoogst van volwassen Sprague Dawley-ratten na humane euthanasie. Overtollige zenuwwortels worden bijgesneden en de DRG wordt in de juiste maten gesneden voor kweek. DRG's worden vervolgens gekweekt in natuurlijke hydrogels, waardoor een robuuste groei in alle compartimenten mogelijk is. Dit systeem met meerdere compartimenten biedt anatomisch relevante isolatie van de DRG-cellichamen uit neurieten, fysiologisch relevante celtypen en mechanische eigenschappen om de interacties tussen neurale en immuuncellen te bestuderen. Dit cultuurplatform biedt dus een waardevol hulpmiddel voor het onderzoeken van isolatiestrategieën voor behandelingen, wat uiteindelijk leidt tot een verbeterde screeningsaanpak voor het voorspellen van pijn.

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&#39; 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.

Auteur in de schijnwerpers: in vitro en in vivo modellen om de doeltreffendheid van de behandeling van chronische pijn te verbeteren

Het oogsten, inbedden en cultiveren van dorsale wortelganglia in apparaten met meerdere compartimenten om perifere neuronale kenmerken te bestuderen

Our research seeks to understand some of the underlying mechanisms that contribute to chronic musculoskeletal pain, so we can develop novel targeted treatments. Thus, we have developed in vitro and in vivo models of chronic low back pain and neuronal sensitization. Current therapeutics that work in vivo in animal models often fail clinical trials due to mechanistic differences between animal models and human subject chronic pain phenotype.Individual test by that model, nociceptor hypersensitivity present in chronic pain have the potential to identify more promising treatments for chronic pain in humans. Our protocol clearly demonstrates how to isolate primary dorsal root ganglia, and culture them in a multi compartment device. By keeping intact primary dorsal root ganglia, we maintain native support cells and culture them in a multi compartment device, allowing anatomically relevant isolation between neuronal somas and peripheral neurites.This tool will help to culture primary dorsal root ganglia in a more physiologically and anatomically relevant way, thereby increasing translational efficacy of findings. Specifically, we are excited to use this system to identify novel treatment for nociceptor hypersensitivity present in chronic pain.

Het huidige protocol beschreef een techniek om DRG te oogsten en te kweken van volwassen Sprague Dawley-ratten in een apparaat met meerdere compartimenten (MC). Zoals te zien is in figuur 1, werd DRG geoogst van volwassen ratten bijgesneden en gesneden in ~0,5 mm. De bijgesneden en gesneden DRG's werden vervolgens ingebed in een hydrogel in het soma-gebied van het MC-apparaat (Figuur 2) en gedurende 27 dagen gekweekt voordat neurietkwantificerin...

Dit protocol biedt een techniek om geëxplanteerd dorsaal wortelganglion (DRG) van volwassen Sprague Dawley-ratten te oogsten en te kweken in een apparaat met meerdere compartimenten (MC).

The most common peripheral neuronal feature of pain is a lowered stimulation threshold or hypersensitivity of terminal nerves from the dorsal root ganglia ...

harvesting-embedding-culturing-dorsal-root-ganglia-multi-compartment

Research

JoVE Journal

Bioengineering

Harvesting, Embedding, and Culturing Dorsal Root Ganglia in Multi-compartment Devices to Study Peripheral Neuronal Features

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.

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.

Assembly of Multi-Compartment Device and Harvesting of Dorsal Root Ganglia (DRG) from Adult Sprague Dawley Rats

To begin, use a three milliliter syringe to apply a thin line of silicone gel into the groove beneath the MC device. Using number three forceps gently position the MC device into the well of a 48 well plate. Place the device into a 48 well plate one day before harvesting the dorsal root ganglion.Place the euthanized rat on the dissection platform. Using large blunt nose scissors, make an incision through the skin at the base of the cervical spine and extend it down the length of the spine. To further drain blood from the spinal canal, cut beneath the shoulder blades and through the muscle to sever major blood vessels.Using large sharp nose scissors, cut the muscle away from the spine, then remove the dorsal segments of the vertebrae. Use straight rongeur and sharp nose scissors to remove the spinous and transverse processes of the vertebrae starting at the cervical end and moving towards the lumbar end. Afterward, remove the spinal cord.Using small sharp nose scissors, carefully sever the nerve roots connected to the dorsal root ganglia along both sides of the spinal cord. Now cut the cervical end of the spinal cord and gradually lift it out of the spinal canal, severing any remaining nerve roots connected to the dorsal root ganglia. To expose the dorsal root ganglia, use a curved rongeur to remove additional lateral parts of the vertebrae.Using number three forceps and straight edge spring scissors, extract the dorsal root ganglia between each vertebra level. Grip the spinal nerve roots from the dorsal root ganglia. Gently pull them out of the pocket and cut through the other end of the spinal nerve.Lastly, place the dorsal root ganglia into wells of a 24 well plate on ice.

Montage van een apparaat met meerdere compartimenten en oogsten van dorsale wortelganglia (DRG) van volwassen Sprague Dawley-ratten

Trimming, Cutting, and Embedding the Dorsal Root Ganglion for Culturing in a Multi-Compartment (MC) Device

To begin, take the harvested dorsal root ganglia. On a clean bench, fill a 60-millimeter Petri dish with trimming media. Arrange the dissecting scope and glass bead sterilizer, and place an autoclave toolbox with trimming tools next to the scope.Using the dissecting scope, remove any excess tissue around the dorsal root ganglion, and trim the nerve roots close to the dorsal root ganglion body. Now, fill a second 60-millimeter Petri dish with fresh media. Using the dissecting scope, cut the trimmed dorsal root ganglia to approximately 0.5 millimeters.Transfer the cut dorsal root ganglia into a fresh 24-well plate containing media on ice. Then, pipette 65.1 and 52.8 microliters of the premixed hydrogel into the soma and neurite compartments, respectively. Carefully embed the cut dorsal root ganglia into the soma compartment.Thermally cross-link the hydrogel in the incubator at 37 degrees Celsius for 30 minutes. Then, UV cross-link the hydrogel for 90 seconds. Afterward, add dorsal root ganglia growth media to the hydrogel and dorsal root ganglia.Perform a complete media change after one hour to remove any unreactive or residual photo-initiator in the media.

Het dorsale wortelganglion trimmen, knippen en inbedden voor kweek in een apparaat met meerdere compartimenten (MC)

Quantification of Dorsal Root Ganglion Neurite Growth in a Multi-Compartment (MC) Device Using Fiji

After imaging the dorsal root ganglion cultured in a multi-compartment device, launch Image J and open the image file. To enhance the contrast and make the smallest neurites visible using the shortcuts Control with Shift and C, open the Brightness and Contrast option. Adjust the brightness and contrast sliders to visualize the neurites.Then click on Apply. Now, navigate to Plugins, select Segmentation, and click Simple Neurite Tracer to open the neurite tracer. Then, click on one end of the neurite at the dorsal root ganglion body to initiate a new trace, and continue adding points along the neurite until it is fully traced.Then click on Complete Path. Using the straight line tool in Fiji, draw a line of the same length as the scale bar without releasing the end of the line. And note the length value under the toolbar.Set the scale to convert the neurite length to real units and copy the results to Excel. Then, convert the lengths to real units in Excel. Then, measure the length of six long neurites extending from both sides of the dorsal root ganglion, and calculate the average length and standard deviation of the dorsal root ganglion.On days 27 and 21, there was robust growth of neurites in the multi compartment and plain gels, respectively. The average length of neurites in the multi compartment device was comparable to neurite length in control plane gels.