This protocol allows the study of the initiation and development of neuroinflammatory responses in the mouse peripheral nervous system during a viral infection. With this model, infections travel a greater distance from the periphery to the peripheral and central nervous system, allowing a maximal assessment of the kinetics of the neuroinflammatory response. After placing the five to seven week old anesthetized C57BL/6 mouse in the supine position on a surgical pad, confirm a lack of response to pedal reflex.
And use a 100 to 180 grit emery board, to gently abrade the glabrous skin of one hind footpad between the heel and walking pads with about 20 passes of the board. Then use fine forceps to slowly peel off the stratum corneum detached by the abrasion to expose the stratum basale. After gentle mixing, topically apply 20 microliters of the appropriate titer of virus inoculum onto the abraded footpad.
Carry out mock inoculations with medium only in the abraded footpads of control animals in parallel. After each application, gently rub the footpad 10 times with the shaft of a needle to facilitate adsorption of the virus every 10 minutes for 30 minutes. When the abraded footpad is dried, allow the mice to recover with monitoring until sternal recumbency before placing the animals into individual cages for clinical follow-up and sampling.
At the appropriate experimental time points, collect the virus exposed organs, including the heart, lungs, spleen, pancreas, liver, kidneys, and bladder into individual 1.5 milliliter microtubes on ice. Then harvest the abraded footpads between the heel and walking pads and place the samples into individual 1.5 milliliter microtubes on ice. Next, place the mouse in the prone position and wet the fur with 70%ethanol.
To expose the vertebral column, make a small incision in the region of the pelvis and strip the skin from the hind limbs toward the head. To remove the fore and hind limbs, use scissors to cut parallel with and close to the spinal column on both sides. And remove the head at the base of the skull.
Use the scissors to open the skull from the foramen magnum to the frontal bone. And use forceps to pull open the skull in a lateral direction. Then use the forceps to gently scoop out the brain and place the brain into a 1.5 milliliter microtube on ice.
Use curved scissors to clean the spinal column of muscle, fat, and soft tissue, and remove the tail. Then place the intact spinal column into a 15 milliliter tube of sterile ice-cold PBS on ice for no more than three hours. After the dissection, transfer the spinal cord into a tissue culture dish to allow removal of any remaining soft tissue.
Use a razor blade to make three transverse cuts through the spinal column at the S2, L1, and the T1 vertebrae. Transfer the pieces into individual 15 millimeter dishes containing sterile ice-cold PBS. Keeping track and marking the origins of the segments for later analysis.
Next, use forceps to secure one segment dorsal side up and use a razor blade to make a single, longitudinal cut down through the midline to split the column in two equal halves. Place both halves in a new Petri dish containing fresh, sterile ice-cold PBS in the original orientation to be able to distinguish the left and right sides of the spine. Under a dissecting microscope, use fine forceps to gently peel the spinal cord out of the each half of the vertebral column in a rostral to caudal direction.
Then place both spinal cord halves in individual 1.5 milliliter microtubes containing 500 microliters of sterile ice-cold PBS on ice. To expose the dorsal root ganglion, gently remove the meninges from one side of the spinal column to the other. And pull the exposed white spinal nerve out of the spinal column.
Harvest the round, transparent dorsal root ganglion from the spine segment into a 15 millimeter Petri dish of ice-cold PBS. And collect the ipsilateral and contralateral dorsal root ganglion as just demonstrated. After harvesting the spinal nerve and dorsal root ganglion from the other two spinal column segments as just demonstrated, centrifuge all of the tubes of tissue at high speed.
Then aspirate the supernatants and flash-freeze the samples in liquid nitrogen, and store at minus 80 degree Celsius until homogenization. For tissue homogenization, thaw the tubes of frozen tissue on ice before weighing out 100 milligrams of each sample. Transfer into a two milliliter microcentrifuge tube containing sterile beads and 500 microliters of radioimmunoprecipitation assay buffer.
Next, disrupt the tissues at room temperature on a homogenizer at 20 cycles per second for two minutes, followed by a one minute waiting period, and 20 cycles per second for an additional two minutes. After the second round of homogenization, centrifuge the tissues at high speed before transferring 500 microliters of each supernatant into a new appropriately labeled tube. Store at minus 20 degree Celsius until downstream analysis.
The site of abrasion is visible in the control footpad. And it is typical for a crust to form at the abrasion site as part of the healing process. In contrast, mice inoculated with pseudorabies, demonstrate a severe inflammation of the footpad at the humane endpoint that is characterized by swelling and redness.
Histopathological examination of the inoculated footpad and dorsal root ganglion reveals epidermal necrosis and severe dermal inflammation within the pseudorabies infected foot sections. Infiltration of neutrophils is also observed in pseudorabies infected DRG sections. A significant increase in G-CSF protein expression is measured in the footpad and dorsal root ganglion of pseudorabies infected mice compared to controls at both seven and 82-hours post-infection.
Significant G-CSF levels are also observed at 82-hours post-infection in the spinal cord, brain, heart, and liver tissues of pseudorabies infected mice compared to controls. Moreover, significant levels of IL-6 are detected in all tested tissues of pseudorabies infected mice starting at 24-hours post-infection. In a moribund state, pseudorabies was detected in the footpad, dorsal root ganglion, spinal cord, and brain.
With the high cytoplasmic expression of psuedorabies glycoprotein gB in the neurons of the dorsal root ganglion in pseudorabies infected mice. To ensure a successful infection, it is important that the droplet of virus in a column doesn't fall from the abraded footpad. This animal model may serve platform to test the efficacy of anti-inflammatory and antiviral drugs in order to prevent viral-induced peripheral neuropathies.
