RNA sequencing of whole Dorsal Root Ganglia (DRG) and laser captured large diameter DRG neurons upon Environmental Enrichment (EE) compared to Standard Housing (SH)

Purpose: A) compare the gene expression in whole DRG and in large diameter DRG neurons of mice exposed to EE compared to SH Results: we found that EE induces transcriptional changes promoting axon regeneration Method: RNA-seq from sciatic L4-L6 DRG extracted from mice housed for 10 days in EE vs SH. For whole DRG, pool of sciatic DRGs from 2 mice were used for each sample. DRG were collected in RNase later reagent, and crushed with RNase free micropestle for RNA extraction. For laser captured DRG, sciatic large diameter DRG were extracted from 3 mice for each sample. Using RNase-free conditions, DRGs were embedded with Tissue Freezing Medium (Triangle Biomedical Sciences, Inc) and frozen in dry ice-cold 2-methylbutane. 16 µm thick sections were cut on the cryostat and collected on Leica PEN-membrane slides (Leica). From each animal, 4-6 slides were obtained. Toluidine blue (Sigma) staining was used to identify the DRG neurons. Slides containing sections were dehydrated and stained with ethanol series, as follow: 3 times of 30 seconds in 70% ethanol, 60 seconds in 0.5% Toluidine blue dissolved in 70% ethanol, 2 times for 30 seconds each in 70% ethanol, 1 time for 30 seconds in 90% ethanol, 2 times for 30 seconds in 100% ethanol. Once air-dried the slides underwent laser capture using a Leica LMD 6000 microscope. From each animal, large diameter neurons with a diameter of ≥ 30 microns were obtained