Figure 3｜Number of myelinated nerve fibers in injured and non-injured motor nerves.
Numbers of axons and degree of myelination in non-injured and regenerated nerves were estimated in toluidine blue-stained semithin sections. The total numbers of myelinated axons in the motor branch of the non-injured femoral nerve were similar in both genotypes, whereas at 2 months after injury the total numbers of myelinated axons were reduced in both genotypes (Figure 3A–C). Notably, there was a tendency towards higher numbers of myelinated axons in Pfp–/– mice, but without statistically significant difference (Figure 3C).
Figure 4｜Myelinated nerve fibers in motor nerve branches.
Analysis of myelination, measured by g-ratio, revealed differences between the genotypes on the injured side, in regenerated nerves, indicating improved myelination in Pfp–/– mice, in comparison with wild-type mice (Figure 4A–F). On the non-injured contralateral side, both genotypes showed a similar degree of myelination (Figure 4F).
Figure 5｜Soma size of motoneurons and nerve terminals around retrogradely labeled motoneurons.
The number of synaptic terminals was determined from the immunostainings around motoneurons which were retrogradely labeled in the lumbar spinal cord, following procedures described (Guseva et al., 2018). Spinal cord sections of Pfp–/– and control mice were stained with anti-ChAT (Figure 5A and B) and anti-VGAT (Figure 5C and D) antibodies to visualize perisomatic cholinergic and inhibitory synapses, respectively. The size of motoneuron cell bodies in Pfp–/– and wild-type mice with or without injury was evaluated for motoneurons innervating the muscle branch, as detected by blue dye labeling. There was a tendency for larger cell bodies to be seen in the injured versus non-injured animals of both genotypes, but no significant differences were found between genotypes, neither for injured nor for non-injured animals (Figure 5E). Analysis of cholinergic (ChAT+) terminals at motoneuronal cell bodies showed reduced numbers after injury in both genotypes when compared to the corresponding non-injured groups, but this reduction was less prominent in Pfp–/– than in wild-type mice (Figure 5A, B, and F). The density of inhibitory (VGAT+) terminals did not differ between genotypes without injury and showed a tendency to lower values after injury, but with no significant differences between genotypes (Figure 5C, D, and G).
Figure 6｜Analysis of immune cells in regenerating femoral nerve tissue.
As perforin was previously implicated in opening the blood-brain and blood-spinal cord barriers (Huggins et al., 2017; Liu et al., 2019), we tried to estimate the infiltration of immune cells at the site of nerve injury, thus assessing the function of the blood-nerve barrier. Numbers of immune cells in regenerating femoral nerves were determined 10 days after transection with toluidine blue-stained semithin sections of the nerve within the polyethylene tube. At this stage after injury, cell debris, regenerating axons and newly formed blood vessels were seen (Figure 6). Lymphocytes were found in blood vessels and in damaged tissue (Figure 6). In injured Pfp–/– nerves, only a few lymphocytes were seen outside of blood vessels (Figure 6B and D), whereas in wild-type nerves lymphocytes were more abundant (Figure 6A and C).