The overall goal of this sensory specific, neurotoxin delivery procedure for mice is to establish a small animal sensory neuropathy model. This technique helps expose the laws of injuries small, sensory fibers in sensory neuropathy. Its main advantage is that it is fast and easy.
These mice can help answer key questions in the sensory neuropathic field such as what pathway is crucial contribution for large sensory fibers. To begin, put on personal protective equipment and take proper precautions while making the Resiniferatoxin, or RTX, stock solution. Dilute one aliquot of 600 microliters in saline for the experiment and divide the rest into 12 microliter aliquots for storage.
For test mice, use eight-week old, adult male ICR mice, weighing between 35 and 40 grams. Administer a single dose of RTX solution intraperitoneally with a micro-injection syringe. Next, inject a second group of mice with an equal volume of vehicle to serve as a control.
After making all of the injections, house the mice in their home cages, and be certain they have ad lib access to food and water. After seven days, start conducting behavioral tests once per week. Perform both tests on the same day in a quiet testing room, with a stable environment.
Begin with measuring the animals'thermal latencies with the hot plate test. Set a metal hot plate to 52 degrees Celsius. Then, gently place an animal into a transparent, plexiglass cage on the hot plate.
Start a timer as soon as the animal's hind paws touch the hot cage. If the animal starts shaking, licking its hind paws, or jumping, remove it and record how long it withstood the hotplate to the nearest tenth of a second. If the animal shows no response on the hotplate, stop the session after 25 seconds to avoid potential tissue damage.
Perform three trials per animal, with a 30 minute rest time between trials. Later, use the average time of the three tests as the animal's final score. Next, measure the animal's mechanical sensation threshold.
Put the animal into a transparent, round cage, with a metal mesh for a floor. After the acclimation, apply the different calibers of von Frey hair filaments to the plantar region of the hind paw, using the up and down method. For the first stimulation, apply a middle force filament for 5 to 8 seconds.
After each stimulation, wait two minutes, then apply the next force based on the animal's previous response. If the hind paw withdrew, use the next lower caliber filament. And if the hind paw did not withdraw, use the next larger filament.
Continue this process until the mouse has been poked six times. Then, calculate the mechanical threshold using an established formula. On each testing day, perform three trials on each hind paw.
Express the average of these six mechanical thresholds as the mean mechanical threshold of each animal. For euthanization, use 5 percent isoflurane, followed by an intracardiac perfusion. Then, remove the first foot pads of the two hind paws, and post fix them in 4 percent PFA for six hours.
After the fixation, transfer them to 0.1 Molar phosphate buffer at 4 degrees Celsius. This serves as a long-term storage solution. Prior to cryosectioning, soak the foot pads in 30%sucrose in PBS overnight.
The next day, use a cryostat to make 30 micron tissue slices cutting vertical to the plantar surface. Label the foot pad sections sequentially and then store them in an anti-feeze solution at negative 20 degrees Celsius. When sampling the sections, select every third section and transfer it to a coated glass slide to air dry.
Next, cover each side with a cover plate and process them with a standard immunostaining procedure. First, quench the foot pad sections with 1%hydrogen peroxide in methanol for 30 minutes. Then, wash the tissues three times using room temperature, 0.5 Molar Tris buffer.
Next, incubate the tissues in block solution for one hour. Then, incubate the sections with an anti-sera against pan-axonal marker overnight at 4 degrees Celsius. The next day, wash the sections with Tris buffer three times and then incubate the sections in biotinylated anti-rabbit IgG at room temperature for an hour.
Later, use three washes with Tris buffer to remove the unbound antibodies. Then, incubate the tissues with the avidin biotin complex at room temperature for 45 minutes. To stain the reaction product, briefly apply DAB solution for 45 seconds, then wash the foot pad sections with distilled water and air dry them before mounting.
After removing the foot pads from the euthanized animal, dissect the fourth and fifth lumbar DRG. Then, post fix the dissected DRG tissues for two hours in four percent PFA at room temperature. Next, cryoprotect the DRG tissues with 30%sucrose in PBS overnight.
The following day, cut them into eight micron sections. Place them on slides and label them. Store these sections at negative 80 degrees Celsius.
Use the same method as used for the foot pad sections, except when labeling, include ATF-3, an injury marker, and peripherin, a small diameter neuronal marker in the primary anti-sera, and run that reaction overnight. After washing the sections, incubate them with the appropriate secondary anti-sera. Then after another series of washes, mount the sections for quantification.
This protocol, describes a novel mouse model of Resiniferatoxin neuropathy, which specifically affects small diameter neurons including IENF degeneration associated with sensory disorders. The 50 microgram per kilogram dose was deemed critical and a higher dose was frequently lethal. Seven days post RTX injection, the animals exhibited thermal hypoalgesia, and mechanical allodynia.
At the critical dose, there was IENF degeneration and marked ATF-3 induction. The injured neurons were peripherin positive, small diameter neurons. In contrast, the low dose of RTX did not establish small fiber neuropathy and showed no changes in innervation.
Nor did the low dose show any ATF-3 induction, a marker of neuronal injury. Thus, the critical dose of 50 micrograms of RTX per kilogram is suggested for application of the described method. After watching this video, you should have a good understanding of how to establish a pure sensory neuropathy model.
After it's development, this technique paved the way for the research in the field of small sensory neuropathy, to its product contribution of injury small sensory nerves, in development neuropathic pain. Don't forget that RTX is neurotoxic and hazardous and can act as an irritant to eyes, mucous membrane, upper respiratory tract. Avoid inhalation and wear lab eyeglasses and coat while handling RTX.
Rinse with plenty of water in case of skin contact and after handling.
