gma; freshly added from 50 g/ml stock solution)]. Neurobasal medium was replaced every three days. Cells have been fixed just after 1, five or eight days in culture, stained with anti-neurofilament (1:2000; Novus Biologicals, Littleton, CO) and neurite lengths had been measured applying the Easy Neurite Tracer plug-in in ImageJ (v1.48). Per time point per genotype five wells were analyzed.
DN-NFIL3 [11] was subcloned in to the AAV-IRES-EGFP construct kindly supplied by Dr. Dietmar Fischer (University of Ulm, MEDChem Express Artemotil Germany). As a handle, an empty AAV-IRES-EGFP construct was employed. AAV serotype five (AAV5; plasmid generously supplied by Dr. Jurgen Kleinschmidt, University of Heidelberg, Germany) particles have been developed and purified working with an iodixanol cushion as previously described [168]. Viral vector stocks were stored at -80 in Dulbecco’s phosphate buffered saline with MgCl2 and CaCl2 (Invitrogen) supplemented with 5% sucrose. Virus titers had been determined by quantitative PCR and had been commonly 1-3×1012 genomic copies/ml.
Adult male Wistar rats (18020 g) have been anesthetized with Hypnorm/Dormicum (0.08/0.02 ml/100 g I.M.). L4 and L5 DRGs were exposed and injected with 1 l (1-3×109 genomic copies)virus per DRG making use of a glass capillary pulled to a fine point and attached to a Hamilton syringe, at a speed of 0.2 l/min [18]. Just after the injection the muscle layers have been sealed with dissolvable sutures as well as the skin was closed with Michell-clips. Two weeks later the animals received a sciatic nerve crush. Animals have been sedated with 1.8% isoflurane in 0.9 L/min medical compressed air. The sciatic nerve was exposed in the left hind leg and crushed by closing locking forceps with ribbed jaws for 30 sec. The crush site was marked by 10/0 
ethilon surgical sutures within the epineurium 15723094 and afterwards the skin was closed with Michell-clips. A single week later animals had been sedated with 1.8% isoflurane as well as the sciatic nerve was exposed once more. At 1 cm distal towards the crush web site the sciatic nerve was transsected along with the proximal finish was submerged for 30 min inside a tiny cup containing the retrograde tracer FastBlue. Also, a part of the distal finish on the sciatic nerve was removed for immunohistochemical analysis. Afterwards the nerve was washed to take away excess tracer as well as the muscle and skin were closed as before. A single week later animals had been anesthetized with Avertin (250 mg/kg I.P.) and perfused with saline followed by 4% paraformaldehyde (PFA) in saline. Injected DRGs have been dissected, post-fixed overnight and transferred to a 25% sucrose resolution overnight at 4. Tissue was embedded in Tissue-Tek, snap frozen in dry ice-cooled isopentane and stored at -80.
DRGs have been sectioned at 20 m, post-fixed in 4% PFA, blocked in blocking buffer (PBS containing 1% BSA, 5% FCS and 0.3% Triton X-100) and stained with anti-III-tubulin (1:500; clone Tuj1; Covance, Berkeley, CA) and anti-GFP (1:4000; Abcam, Cambridge, UK), followed by donkey anti-mouse Alexa594 (1:400), biotinylated goat anti-rabbit (1:300) and streptavidinAlexa488 (1:400). All sections have been captured at fixed exposure settings employing an Axioplan two fluorescence microscope (Zeiss, Sliedrecht, The Netherlands) as well as a 10x objective. Pictures have been analyzed using a custom algorithm in Image-Pro Plus software program (MediaCybernetics, Rockville, MD) as previously described [18]. The algorithm automatically identifies all DRG nuclei determined by the Tuj1 staining, which specifically stains neuronal cytoplasm and leaves the nucleus unstained and visible as a dark round object. GFP and FastBlu
