Table 1.
Primary antibodies.
Table 2.
Secondary antibodies.
Fig 1.
Both, the trkB receptor (a) and CNTFα receptor (b) were detected in the cultures of dissociated spiral ganglia. To verify the presence of the receptors (labeled in red) on SGN, the neuronal cells were double stained with neuron-specific 200 kD anti-neurofilament antibody (labeled in green).
Fig 2.
Neuronal survival Effect of 50 ng/ml BDNF, 100 ng/ml CNTF and the combination of both factors (B/C) compared to the negative control (NC) on spiral ganglion neurons (SGN) survival rate after 48 h in vitro incubation.
The survival rate is given as a percentage of the initially seeded cells per experiment which was set as 100%, represented as mean and standard error of mean (SEM). Asterisks above the error bars indicate the significance of the conditions compared to the negative control. Both neurotrophic factors improved the survival rate significantly and the combination of BDNF and CNTF resulted in the significantly highest increase of surviving neurons (*p<0.05; **p<0.01; ***p<0.001; independent experiments: 3, wells per experiment: 3).
Fig 3.
Classification of neuronal morphologies Different morphologies of SGN could be detected in the culture.
1: monopolar neurons; 2: bipolar neurons; 3: neurons with no neurites, 4: pseudomonopolar neurons; 5: multipolar neurons. Neurons were stained with DAB against neurofilament. Magnification: 200x.
Fig 4.
NTF effect on neuronal morphologies Effect of 50 ng/ml BDNF, 100 ng/ml CNTF and the combination of both NTFs (B/C) compared to negative control (NC) on neuronal morphology in terms of polarity and missing neurite outgrowth after 48 h in vitro incubation.
All morphologies and their distribution dependent on the treatment are depicted in an overview graph (mean ± SEM) (A). The analysis of the most frequent neuron types is depicted in B-D. The amount of monopolar neurons was increased when treated with BDNF or both NTFs. Only BDNF (15.04%) but not CNTF (10.21%) increased the amount of bipolar neurons. Both factors added simultaneously resulted in a highly significant increase to 42.84% of bipolar neurons compared to the negative control, showing a synergistic effect. The number of neurons without neurites was significantly decreased when both NTFs were administered together. BDNF or CNTF treatment resulted in a statistically lower percentage of neurons without neurites compared to the negative control as well. Furthermore, BDNF treatment caused a lower rate of neurites without neurons than CNTF. The SGN morphology is given as a percentage of the total number of scored neurons represented as mean and standard error of mean (SEM). Asterisks above the error bars show the significance of the conditions compared to the relevant negative control. (mean ± SEM; *p<0.05; **p<0.01; ***p < 0.001; ns = not significant; independent experiments: 3, wells per experiment: 1).
Fig 5.
Neurite outgrowth Neurite length (μm) was measured on spiral ganglion neurons (SGN) cultured for 48 h with addition of growth factors (50 ng/ml BDNF, 100 ng/ml CNTF and combination of both (B/C)) or without factor application (negative control, NC).
All neurotrophic factor treatments caused significantly longer neurites compared to the neurons of the negative control. The significantly longest neurites were detected when BDNF and CDNF were applied concurrently. Asterisks above the error bars indicate the significance in the experimental conditions compared to negative control (**p<0.01; ***p < 0.001; independent experiments: 3, wells per experiment: 3).
Table 3.
Neurite length.