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.
In vitro 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. 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 root 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.
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 pre-mixed 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 un-reactive or residual photo initiator in the media. After imaging the dorsal root ganglion cultured in a multi compartment device, launch ImageJ 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 plain gels.
