Nissl staining

Sections of the spinal cord at the lumbar level (L4-L6) were used for Nissl staining following the protocol previously described²⁸. We used cresyl violet 0.25% with 0.1% of acetic acid. Slices were dehydrated by an increased ethanol concentration series, sealed and cover-slipped with the non-aqueous mounting medium DPX (Sigma-Aldrich, San Luis, MI, USA). A Leica DMRB microscope (Leica, Wetzlar, Germany) and a Leica DFC300FX camera were used for slice observation and for taking photographs of the tissues. The number of total motor neurons in the ventral horn was counted in at least six slices to a minimum of 6 animals per experimental group. To count the number of stained motor neurons (>400 μm²) in the ventral horn, high resolutions photomicrographs were taken with a 10X objective under the same conditions of light, brightness and contrast. The final value for each group is the mean of all animals included in the study.