アダルト マウス後根神経節外植体し、解離細胞モデル神経可塑性とウイルス感染を含む環境の侮辱への応答を調査するため

The overall goal of this procedure is to create an ex vivo model of dorsal root ganglia explant and/or DRG-derived primary dissociated cell model to investigate neuronal responses to environmental cues. This method can help answer key questions in the field of neuroplasticity, or neuro inflammation in response to pathogens exposure. The main advantage of this technique is that the dorsal root ganglia explants maintains the organ-like complexity useful to start cell cycle into action and changes to intercellular microenvironment due to specific treatments.

Watchful demonstration of this method is very critical as in the identification of DRGs as well as harvesting them can be quite difficult due to their size and location. To begin this procedure, expose the vertebral column by cutting down the skin layer of the animal, dorsally using fine scissors. Next, isolate the vertebral column by cutting through the ribs on both sides of the column and through the sacrum, separating the vertebral column from the rest of the animal.

Afterward, mount the vertebral column with the ventral side up onto a surgical mat using needles and pins. Then make a double cut on both sides of the vertebral bodies to expose the ventral side of the vertebral canal. Under a surgical microscope gently move the spinal chord on the side to expose the contralateral dorsal spinal roots and to locate the DRG along the dorsal roots of the peripheral nerves.

Each ganglion is partially hidden inside the intervertebral foramina. To harvest DRG, pinch the dorsal root with one forceps and gently pull out the DRG from the intervertebral foramen. Then place the second forceps on the spinal nerve peripheral to the DRG and pull the DRG together with the spinal nerve and spinal root.

Subsequently, place the collected DRG in a 35 millimeter Petri dish containing three milliliters of ice cold, serum-free media. Transfer each DRG to a dry glass Petri dish. Under a surgical microscope, clean and trim off excess fibers and connective tissue still attached to the DRG using a blade.

The DRG is easily identifiable as a bulgy transparent structure along the white spinal nerve, and blood vessels are often found surrounding the DRG. After that place the cleaned DRG in a new Petri dish containing ice cold, serum-free media. Dilute the gelatinous protein mixture in ice cold serum-free media in a 1:1 ratio.

Then plate the DRG ex vivo in the 12 well plates pre-coated with 10 or 20 microliters of gelatinous protein mixture. And keep them at 37 degrees Celsius for 30 to 60 minutes. Now gently add 1.5 to two milliliters of serum-free media to the culture system to cover the entire explant and maintain the explants at culturing conditions.

Change the DRG growth medium every 72 hours and let the DRG grow for as long as needed. This is a critical step since DRG is anchored to the glass plate using the gelatinous protein mix. Therefore, time oporization and pipetting skills are critical to avoid floating.

In this procedure, place all the DRG collected in a 1.5 milliliter sterile tube with F12 media containing Collagenase 4 and incubate it at 37 degrees Celsius for 45 minutes. Afterward, replace the fresh media containing Collagenase 4 and incubate the sample for another 45 minutes. Then treat the explants with two milliliters of F12 media containing 0.025%trypsin at 37 degrees Celsius for 30 minutes immediately after the Collagenase 4 treatment.

Incubate them with two milliliters of F12 media containing fetal bovine serum at 37 degrees Celsius for 15 minutes. Afterward, wash the explants three times with two milliliters of F12 media and proceed to mechanically disassociate them with a glass pipette until the media turns cloudy. Following that, filter the dissociated cell culture through a 0.22 micrometer filter to remove any impurities and excess connective tissue.

Then centrifuge the filtered cell lysate for two minutes. Remove the supernatant and re-suspend the cell pellet in 500 micro liters of Nurobasal Media. Place the dissociated cells onto laminate-coated cover slides at a preferred cell density.

In this step prepare the virus in the correct dilutions in serum-free media to infect the model by using one unit of MOI when working with DRG derived dissociated cells. Place the explants to be infected with virus in a sterile cell plate containing a mixture of serum-free media and virus. Then place the cell plate or tube at 37 degrees Celsius for infection to take place.

Now fix the sample in 4%formalin. Wash it three times for 10 minutes each in PBS. Then incubate the sample with the desired primary antibody diluted in PBST and 10%normal goat serum.

Store the sample at four degrees Celsius overnight. The next day wash the sample three times for 10 minutes each with PBS. Incubate the sample at room temperature for one hour in the appropriate secondary antibody diluted in PBS.

Then wash it three times for 10 minutes each with PBS. Incubate the sample with Hoechst dye in PBS for 20 minutes prior to mounting step. Subsequently mount the sample on glass slides using a fluorescence mounting medium and cover slip.

In this image HSV-1 infection of dissociated satellite cells depicted along bata tubulin positive neurites in green is revealed using an antiviral GD antibody in red. Satellite glial cells nuclei are stained blue with Hoechst. The same antibody reveals viral entry in dissociated DRG neurons.

Cells are co-labeled with an antibody against Heparan sulfate expressed on the cell membrane. Heparan sulfate is also a component of the extracellular matrix and plays an important role for axonal growth. Neurons are labeled with Pariferin in red while Hoechst blue is used to identify satellite cells nuclei.

Once mastered, the ex vivo DRG extractinate can completed in two to two and a half hours if performed properly. While attempting this procedure it is important to remember to respect timing. And not to let the specimens dry out in order to keep the explants alive.

Following the harvesting of the DRG, other technique like the dissociation of single cells can be performed in order to answer additional questions such as the effect of environmental ques for the intercellular metabolism. After it's development, this technique paved the way for researchers in the field of neural neurology to explore the neuronal immune response to pathogens. After watching this video, you should have a good understanding on how to generate dorsal root ganglion explants and how to further generate dorsal root ganglion derived primary dissociated cell cultures.

Don't forget that working with viruses or any other pathogens can be extremely hazardous and precautions such as via gloves and lab coats, properly dispose your wastes should always be taken while performing this procedure.