Aminoguanidine and alagebrium are the most widely used MG scavenger and AGEs breaker, respectively

for induction and 2% for maintenance. Animals were kept in environmentally controlled animal facilities at the University of Birmingham and every effort was made to minimise animal suffering. Preparation of adult DRG cultures MedChemExpress Piclidenoson primary DRG cultures were prepared using 68 week-old female Sprague-Dawley rats killed by CO2 exposure, as described by us previously. Briefly, L4L7 DRG pairs were dissected, dissociated into single cells in a solution of Neurobasal-A containing 0.1% collagenase and 200 U/ml DNaseI. Dissociated DRGN were pelleted and re-suspended in Dulbecco’s Modified Eagle Medium containing 5-Flouro-2-deoxyuridine at a final concentration of 30 mM and 1% Penicillin-Streptomycin, with and without 10% fetal bovine serum and plated at a cell density of 300 cells/well on sterile glass chamber slides pre-coated with 100 mg/ml poly-D-lysine followed by 20 mg/ml Laminin-I and cultured for 72 h at 37uC in a humidified atmosphere containing 5% CO2. In vivo DRGN injury models To investigate the role of CASP2 in DRGN apoptosis, SN injury models were established as described previously. SN were transected unilaterally in adult female Sprague-Dawley rats at the level of the sacrotuberous ligament. Spontaneous regeneration of SN was inhibited by excising a 3 mm segment of distal nerve stump, ligating both nerve stumps and burying the proximal stump deep into gluteus maximus muscle. Animals were allowed to survive for 1 month, intracardially perfused with 4% formaldehyde, the L4/ L5 DRG harvested, cryoprotected and blocked up in OCT mounting compound and stored at 220uC until required. For all experiments, we used L5 DRG sections for immunohistochemistry and analysis. Immunocytochemistry To detect DRGN survival and expression of CASP2, CASP3, C-CASP2 and C-CASP3, cultures were fixed in 4% formaldehyde for 10 min, washed 63 in PBS, blocked in PBS-T-BSA for 10 min at 7925608 room temperature and incubated with polyclonal anti-rabbit cleaved CASP2 or rabbit polyclonal C-CASP3 and monoclonal anti-mouse bIII-tubulin, all diluted at 1:200 in PBS containing BSA. Cells were then washed 63 in PBS and incubated with anti-mouse Alexa Fluor 488 and anti-rabbit Alex Flour 594 diluted 1:400 in PBS-BSA for 1 h at room temperature. After the final 3 washes with PBS, cover slips were mounted in Vectamount with or without DAPI and viewed under a fluorescent microscope. To assess antibody specificities of caspases, negative controls were included in each run for each antibody tested including omission of the primary antibody. A blinded observer divided each 8-well chamber into 9 quadrants and randomly took photomicrographs from each quadrant using a fluorescent microscope attached to a computer running the Axiovision software. The observer then counted the number of bIII-tubulin+ DRGN with DAPI stained nuclei to assess the degree of survival in each relevant experiment. Experiments were performed in duplicate and repeated on at least 3 independent occasions. Immunohistochemistry Longitudinal 15 mm thick sections of L5 DRG were cut using a Bright cryostat, and adhered onto charged glass slides and immunohistochemistry performed as described by us previously. Briefly, sections were fixed in 100% ethanol, washed 63 in phosphate buffered saline, permeabilised 2837278 in PBS containing 1% Triton X-100 and blocked in PBS containing 0.5% bovine serum albumin and 0.05% Tween 20. Sections were then incubated overnight at 4uC in a humidified chamber with the relevant primary antibody dil