げっ歯類での後根神経節の脊髄および単離の油圧押出

The overall goal of this method is to isolate the spinal cord by hydraulic extrusion as well as to identify and isolate dorsal root ganglia in mice and rats. This is accomplished by first euthanizing the rodent, followed by decapitation. The skin is cut along the spine and separated from the underlying tissue.

Next, the spinal column is separated from the surrounding tissue by cutting along both sides of the spinal column in distal direction past the pelvic bone. Then the spinal column is cut distal to the pelvic bone. A syringe with an adjusted pipette tip is inserted at the distal-most end of the spinal cord.

By applying steady pressure, the spinal cord is extruded into the Petri dish on ice using ice cold sterile PBS. Using spring scissors, the spinal column is split longitudinally. Next, the attachment sites of the distal-most costa are located.

This allows identification of the distal-most thoracic DRGs on each side denoted T13. Subsequent DRGs can now be identified. The DRGs corresponding to the spinal lumbar enlargement are located distally.

Using spring scissors, the dorsal and ventral roots are cut and the DRGs are gently scooped out of the cavity and transferred to labeled Petri dishes containing sterile PBS on ice. Hydraulic extrusion of the spinal cord is a significantly faster method than traditional laminectomy where the spinal vertebrae are broken one at a time. This method reduces the risk of tissue damage caused by the dissection process and yields tissue applicable to a broad range of analysis.

Dorsal root ganglions may generally be difficult to identify. However, correct identification is highly important for further analysis of the tissue for instance following sciatic nerve injury. So by numbering the dorsal root ganglions according to the localization relative to the costa, dorsal root ganglions can be identified consistently.

Prepare a syringe for spinal cord extrusion by cutting off the large end of an unfiltered pipette tip. The tip will fit tightly when adjusted properly. Aspirate ice cold sterile PBS and leave the syringe on ice until further use.

For adult rodents, euthanize with gas such as CO2 or isoflurane followed by decapitation. Do not perform cervical dislocation as this will disturb the spinal vertebra and render spinal cord extrusion impossible. Pups can be euthanized by direct decapitation.

Sprinkle water onto the fur to control the hair. Next, cut open the fur along the spinal column in distal direction. Isolate the spinal column by cutting on both sides along the spinal column past the pelvic bone and cut the spinal column proximal to the pelvis.

Trim the spinal column to avoid the proximal-most and distal-most areas as these may render the spinal column too S-shaped for successful spinal cord extrusion. Ensure that the spinal cord is visible at both ends. If the spinal cord is not visible, trim the spinal column until the spinal cord becomes visible.

Place a Petri dish containing PBS on ice. Straighten the spinal column by placing the index finger on the bent part at the proximal end of the spinal column. Insert the pipette tip at the distal end of the spinal cord.

Apply steady pressure and extrude the spinal cord into the Petri dish. Cut open the spinal column. Cut a few vertebrae on the ventral side of the column and then shift to cut a few vertebrae on the dorsal side.

Continue to alternate sides all the way along the spinal column. Place the open spinal cord under a microscope. Identify the T13 vertebra segment by locating the attachment site of the distal-most costa in the spinal column.

Be careful not to confuse the distal-most costa with the nearby intercostal nerves. The distal-most costa are attached to the proximal part of the T13 vertebra segment and the T13 DRGs are located distally to vertebra T13 and proximally to vertebra L1.Identify concomitant DRGs in distal direction. Prepare numbered Petri dishes containing ice cold sterile PBS on ice.

To avoid damage to the DRG during removal, grab the dorsal and ventral roots rather than the DRG itself with a pair of 5 tweezers. Using micro scissors, first cut the peripheral nerve, followed by the dorsal and ventral roots. In pups, these nerves are not as clearly visible as in adult rodents.

In order to cut the nerves as close as possible to the DRG, gently let the tip of the scissors glide along the DRG to position the tip of the scissors prior to making the cut. Using the tip of closed tweezers, gently scoop out the DRGs and transfer them to the corresponding Petri dish. In adult rats and adult mice, the DRGs are white with clearly visible dorsal and ventral roots while in pups the DRGs appear as clear spheres.

For western blotting, isolate the tissue of interest and place the tissue in lysis buffer. Next, homogenize the tissue, centrifugate, collect the supernatant and perform western blotting according to standard protocols. For RNA analysis, isolate the tissue of interest and immediately place the tissue in RNA stabilization solution.

Next, perform qPCR according to standard protocols. For immunohistochemistry on snap-frozen tissue, prepare dry ice powder on a piece of silver foil. Place the tissue on dry ice powder to let it snap-freeze.

Then transfer the tissue to a cryostat and embed the tissue for slicing. Post-fixate the tissue in 4%PFA for 10 minutes at room temperature and perform immunohistochemical staining according to standard protocols. For immunohistochemistry on cryosections, fixate the tissue in 4%PFA at four degrees and transfer the tissue to 25%weight per volume sucrose overnight at four degrees.

Place the tissue in embedding material and freeze the tissue using cooled 2-methylbutane. Slice the tissue in a cryostat and perform immunohistochemical staining according to standard protocols. For immunohistochemistry on paraffin sections, fixate the tissue in 4%PFA at four degrees and transfer the tissue to 25%weight per volume sucrose overnight at four degrees.

Embed the tissue in paraffin, slice the tissue on a microtome, and perform immunohistochemical staining according to standard protocols. Depending on the antibodies used for immunohistochemical staining, either of the exemplified tissue treatment methods may be found applicable. After watching this video, you should have a good understanding of how to isolate the spinal cord by hydraulic extrusion and how to identify and isolate the dorsal root ganglia in a consistent manner.

The methods shown here are applicable to mice and rats across all ages and the isolated tissue can be used for a broad range of analysis including methods that require fast isolation of non-fixated tissue.