Figure 1.

Extracellular calcium and proteasomes participated in neurofilament degradation in sciatic nerve explant cultures.

(A) Immunofluorescence staining against high molecular weight neurofilament (NF). Immunofluorescence microscopic images of cross-sections of sciatic nerve explants cultured for 3DIV were analyzed under a laser confocal microscope. Green fluorescence dots indicate neurofilament-positive axons. DIV: day in vitro. Scale bar: 100 μm. (B) Quantitative analysis of the number of high molecular weight NF in the sciatic nerve explant cultures. aP < 0.05, vs. vehicle-treated nerve controls. (n = 3; mean ± SD). 1: Vehicle; 2: nicotinamide adenine dinucleotide (NAD; 5 mmol/L); 3: nicotinamide (NAM; 20 mmol/L); 4: methyl pyruvate (20 mmol/L); 5: NAD (5 mmol/L) + methyl pyruvate (20 mmol/L); 6: ethylene glycol tetraacetic acid (EGTA, an extracellular calcium chelator; 5 mmol/L); 7: calpeptin (50 μmol/L; a calpain inhibitor); 8: MG132 (20 μmol/L; a proteasome inhibitor). (C) Western blot analysis showing the degradation of medium chain neurofilament (NF-M) in sciatic nerve explants cultured for 3DIV. MG5: 5 μmol/L of MG132; MG20: 20 μmol/L of MG132, EGTA (5 mmol/L). (n = 3; mean ± SD). (D) Quantitative analysis of NF-M immunoreactive bands. The intensity of bands was displayed as relative intensity to uncut nerve control. At least three independent experiments were performed for each condition. -: No treatment. aP < 0.05. Differences in the means between groups were statistically assessed using one-way analysis of variance followed by Bonferroni post hoc test.