Wound healing studies

Experiment:    Wound Healing Experiment

Method:

Day 0:

1. Conduct wound healing studies at 6-10 weeks post-transplantation.
2. Add 15.5 mL tert-amyl alcohol to 25 grams of 2,2,2 tribromoethanol and dissolve overnight in a dark bottle to generate a stock solution. The subsequent solution should be diluted with PBS, dissolved overnight, and filtered through a 0.2 µm filter to generate a working solution (20 mg/ml).
3. Anesthetize animal with 0.4-0.75 mg/g intra-peritoneally.
4. Shave dorsal skin.
5. Make 2 punch biopsies (4 wounds) in dorsal skin with a 3 mm punch biopsy.
6. Save skin samples for 1) paraffin 2) frozen (OCT embedding), and 3) RNA (stored in RNA later).
7. Photograph mice systematically from a fixed distance daily and analyze size of the wound by counting pixels in Adobe Photoshop (This is the wound image on Day 0).
8. Each mouse should be housed singularly during the study.

Day 1, 2, 4, 6, 8, 14:

1. Image wounds.
2. Sacrifice 2 mice per group and harvest skin for 1) paraffin 2) frozen section (OCT embedding), and 3) RNA (stored in RNA later).
3. Harvest bone marrow for flow cytometry and RNA analysis (to check for Cre knock-out efficiency).

Day 3, 5, etc.:

1. Image wounds.
2. Change cages every 2 days.

For analysis:

1. Blind genotypes to analyzer and analyze the size of the wound with Adobe Photoshop and normalize number of pixels per wound to Day 0.
2. Assess the contribution of Rev-erb to wound healing by combining data from independent experiments and analyze using a linear mixed effects model. This was done using the R package ‘nlme’ (R script: wound model <- lme('wound size' ~ 'genotype' * 'time point', random=~1 | 'independent experiment' /'independent mouse' /'nested observation', data=data.file, na.action='na.exclude').