method of long term study done on C22 and C3 mice

Prep work: The mice were first genotyped to confirm mice’s genotype. The mice that were used for this experiment were created using cc22 mice in a C57BL/6J x CBA/Ca background, which have 7 copies of the human PMP22 gene. The CC22 mice were then backcrossed with wild type for ten generations. The researchers then isolated mice with milder phenotype and reduced PMP22 compared to the CC22 mice (3 or 4 copies instead of 7), and had a more stable genotype. All 3 types, The mild phenotype, C3-PMP, wt, and C22 were used for the experiment.

Experiment 1: neuromuscular SHIRPA

    In this experiment, the mice were assessed for neuromuscular function using the SHIRPA protocol. SHIRPA protocol contains 19 items to check for, such as body tremors and spontaneous activity as well as tail elevation. Using the items, the mice were each given a score from 0 to 43. 

Experiment 2: Electrophysiology

    In this experiment, mice were anesthetized and the sciatic nerve and caudal nerves were studied on an EMG machine to study motor conductivity. The electrodes were placed at the medial ankle and at the sciatic notch. Wave pulses were delivered and CMAP amplitudes were recorded the MNCVs were calculated. The needle electrode was inserted again to study the caudal nerve and CNAP and NCV were recorded

Experiment 3: Histology and Morphology

.     Mice were deeply anesthetized, blood was flushed shout and the body was preserved. Then the peroneal, femoral, and lumbar nerves were physically removed and fixed in formaldehyde overnight.  The samples were then processed into the resin, and light and electron microscopy were performed on the samples. The samples were stained using thionine and acridine orange for light microscopy and stained with methanolic uranyl acetate and lead citrate for electron microscopy. Morphology was performed by fitting the nerve to the stage and cross-section of the nerve were analyzed. 

Post experiment: Statistics. 

The data was gathered from the experiment were analyzed using descriptive statistics. The differences between the different mice were evaluated using the unpaired t-test.