The overall goal of this procedure is to successfully induce Experimental Autoimmune Neuritis or EAN in a mouse model for the functional and pathological analysis of the disease process. The EAN model can be used to address key questions relating to peripheral neuropathy. It's also a wonderful model to potentially investigate the efficacy of novel therapeutic agents.
The main advantage of this approach is that it provides a simple and objective functional testing paradigm that can be applied when using the EAN model in the mouse. Demonstrating the procedure with me today is Sang Won Yoo, who is an undergraduate student in the neurotrophin and myelin biology lab at the University of Melbourne. Three days before the disease induction, turn on the motor function assessment apparatus and switch on the light button.
Pick up the mouse and lower it onto a container of red food dye. Place the mouse into the walking compartment and set the treadmill speed to 15 centimeters per second. Turn on the treadmill and click record to use the gait function imaging system to capture the running motion of the mouse for 36 seconds.
After 36 seconds, stop the recording and the treadmill and save the practice run video file of the practice run in the designated folder. One day before the first immunization, use a 0.5 milliliter syringe equipped with a 30-1/2 gauge needle to administer 1.6 micrograms per milliliter of Pertussis toxin in 250 microliters of mouse isotonic PBS IP to anesthetized six to eight-week-old male C57BL/6 mice. Then, score the mice for clinical signs of EAN on a scale from zero to four according to the table and perform a motor function assessment as just demonstrated.
The next day, combine equal volumes of freshly prepared solutions of peptide in 0.9%saline and 20 milligrams per milliliter of Mycobacterium tuberculosis in complete Freund's adjuvant in a bead beader and mix the solutions at the maximum speed for one minute at room temperature. Load a syringe equipped with a 23 gauge needle with the inoculum and invert and shake the syringe before evacuating any air within the syringe shaft. Before delivering the inoculum, remove the hair just lateral to the midline between the scapulae and disinfect the exposed skin with 70%ethanol.
Then, use a pair of Adson forceps to grasp the skin and deliver 50 microliters of the solution to the Pertussis toxin injected mouse via subcutaneous injection. The next morning, deliver 1.2 micrograms per milliliter of Pertussis toxin in 250 microliters of PBS IP as just demonstrated followed by another 1.2 micrograms per milliliter injection of Pertussis toxin IP 48 hours later. Three days later after the third Pertussis injection, administer another 50 microliters of freshly prepared inoculum at the same injection site as before and monitor the clinical score and motor function of the animals twice a week until the end of the experiment.
P0 180-199 peptide-induced EAN in C57BL/6 mice leads to a monophasic disease with a clinical score onset from six days post first immunization and a maximal score severity at 25 days post first immunization, followed by some clinical score improvement at 40 days post first immunization. The mice begin to fail at a simple running task as early as six days post first immunization with little capacity to complete a simple treadmill running task by 35 days post immunization that does not improve in running ability at 55 days post first immunization. The examination of semi-thin sections of the sciatic nerves of peptide-induced EAN mice reveals areas of demyelination compared to sciatic nerves from age-matched healthy controls.
High power transmission electron microscopy images allow the identification of specific neuropathological features such as demyelination, damage to the myelin lamella, and signs of axon stress such as mitochondrial swelling. These animals also display acute axonal damage as evidenced by traumatically elevated beta-amyloid precursor protein expression in the sciatic nerves that is not observed in age-matched healthy control tissues. A widening of the nodes and a disruption to the paranodes are also observed.
Once mastered, the subcutaneous injections can be performed within about five to 10 minutes per mouse with minimum chance of any injection error. While attempting this procedure, it's really important that you observe the injection site to ensure that all of the inoculum remains within the injection site and that none of it leaks out. Video capture of treadmill gait is great because it enables other parameters to be computed such as swing phase or stride length and these measures may become important when testing the efficacy of novel therapeutics.
After watching this video, you should have a really good understanding of how to induce EAN in C57BL/6 male mice. In addition, you should have a really good understanding of how to place subcutaneous injections safely and really accurately ensuring that there is no injecting error. Don't forget that working with needles and inoculum, there is the chance of injury especially when using sharps.
So, to avoid any chance of injury, it's a really good idea to always use the forceps and never grasp skin or tissue by hand and also to use single-use syringes, never recapping in the process.
