Sholl Analysis

We performed a Sholl analysis38 to evaluate the effect of neuropathic pain on the hippocampal dendrite morphology. ImageJ software (NIH, USA) was used for all image processing and morphological analyses. For dendrite tracing, images for each individual neuron were converted to 8-bit color images. Dendrites were traced using the NeuronJ plugin (http://www.imagescience.org/meijering/software/neuronj/) as previously described.5 Sholl analysis was performed on the NeuronJ tracings using the Sholl Analysis plugin (http://fiji.sc/Sholl_Analysis). For the Sholl analysis, the single animals were selected as the unit of analysis (5 animals per group). For each animal, 2–3 well-stained neurons were taken for evaluation (separately for CA1 and DG). When calculating the density of dendritic spines, one neuron (15 per group) was used as the unit of analysis. For each mouse (5 mice per group) the evaluation was performed on 3 neurons. Both hemispheres were used to evaluate the neuronal morphology.