L Length than the maximum distance along a neurite defined ie the distance from the end Soma tL there singer neurites and the end of the L Ngeren leg at each junction of branched axons. When L Longest neurite / branching means the Selected Selected cell extended over the edge of the image, the captured images were additionally Tzlich to z Select and L Length of the procedure. Measurement of neurite outgrowth AC480 in SGN were transfected as above, au He only GFP expressing SGN were labeled as described above. Calpa Ma took Are separate activity Th ganglion cultures initially Highest in NT 3 have been retained and BDNFsupplemented average. After 48 h, the cells were incubated with the cell permeable fluorogenic Calpa Substratl Solution loaded not followed butoxycarbonyl Leu Metchloromethylaminocoumarin in DMEM/N2 media by incubation for 20 minutes by washing with DMEM.
The cultures were then depolarised with 30K or 80K DMEM/N2 average. Control cultures were treated with medium without levying o DMEM/N2. Some cultures were also treated with BMS-708163 the inhibitor Calpa Calpeptin, it has been up to a final concentration of 15 M 15 min before depolarization added and remained in the culture w During depolarization. t Boc LM CMAC fluorescence imaged with a dichroic filter only. The fluorescence Was t using ImageJ software. A circle is just within the limit set each neuronal soma, and the average pixel density measured within the circle. Background fluorescence than the average density of pixels in a circle with a diameter equal to just au Determined outside the boundary of the neurons, the background from the fluorescence value obtained for the neuron was subtracted.
The scale was used arbitrarily, but consistently. We have at least 15 RND Llig Selected Hlten neurons per condition in each of the three replicates measured using different cultures. Statistical analysis, and graphics processing of images were prepared by Excel. Statistical analysis was performed with SigmaStat. Significance of differences between treatment groups in moderation took Neuritenl Length was by Kruskal-Wallis ANOVA on the R Nts determined, followed by Dunn post the art method. Significance of differences between treatment groups in Ma for the percentage of neurites SGN carrying by analysis of variance of Holm Sidak posteriori method using SigmaStat determined. The images were for the Ver Dissemination of prepared with Adobe Photoshop and Illustrator. III.
RESULTS membrane depolarization inhibits SGN neurite cultures of dissociated cells of initiation of rat spiral ganglion P5 produced NT 3, a neurotrophic factor, survival of the and neurite outgrowth SGN f Promoted and depolarized to 30 or 80 mM were maintained oo mM compared to SGN nondepolarized control medium. Use of NT-3, to maintain the survival of SGN, which could impact on the survival of depolarization effects on neurite outgrowth separate. After 48 h, the cultures were fixed and stained with antique Antineurofilament rpern 200t recogn both phosphorylated and non-phosphorylated neurofilament 200, Alexa 568 through secondary Rantik Rpers followed by visualization of the SGN somata and neurites erm by immunofluorescence Resembled immungef rbt. Five to seven randomlychosen fields in each well were imaged digitally. Use ImageJ was Neuritenl length By measuring the L Longest processes from each NF 200 positive SGN determined. Figure 1 shows the cumulative percent histograms of SGN Neuritenl Length.