Histology
At the 16-week of age mice sacrificed with an overdose of pentobarbital
and underwent transcardial perfusion with a 4% paraformaldehyde
solution. Following perfusion, the lumbar segment of the spinal cord was
carefully extracted, post-fixed for 2 hours, and subsequently
cryopreserved in a solution of 30% sucrose in phosphate buffer (PB) at
4ºC. Then, transverse 20-µm thick sections of the spinal cord were
serially cut with a cryostat. For assessing the number of MNs, slices of
L4-L5 spinal cord segments separated 100 μm were stained with a solution
of 3.1mM cresyl violet for 3 h. MNs were identified by their anatomical
localization in the ventral horn of the spinal cord and counted
following strict criteria of their size (diameters lager than 20µm) and
morphological characteristics (polygonal shape and prominent nucleoli).