Figure 8.

Co-inhibition of LAR and PTPσ enhances neuronal differentiation, morphologic complexity, and arborization of drNPC-O2-derived neurons in the injured spinal cord. A–D, ILP/ISP co-administration promoted neuronal differentiation of drNPC-O2 while decreased their astrocyte differentiation. A–C, Representative images of co-immunostaining of engrafted drNPC-O2 using HNA or Ku80 human nuclei antigen and NeuN (mature neuron marker), GFAP (astrocyte marker), and Olig2 (oligodendrocyte marker) are depicted. D, Quantitative analysis showed ILP/ISP treatment significantly promoted neuronal differentiation of transplanted drNPC-O2 compared with vehicle treated rats. This was associated with a decrease in astrocyte differentiation under ILP/ISP co-treatment while oligodendrogenesis remained unchanged. E–H, Immunohistochemical analysis indicated that ILP/ISP co-treatment significantly increased the number of ChAT-expressing motoneurons, EN.1 V1, and SIM.1 V3 spinal interneurons compared with vehicle treatment. Values represent mean ± SEM, *p < 0.05; multiple t test, N = 5 rats/group. I, Representative reconstructed images generated from z-stack confocal imaging of drNPC-O2-derived neurons (co-immunostained with STEM121/NeuN) is depicted using Blender software. J–R, Quantification of macromorphological values extracted from NeuroM morphology analysis software showed that ILP/ISP treatment significantly enhanced total neurite length, neurite volume, occupied surface area by neurons, number of primary neurites, number of bifurcations, secondary and tertiary sections and branch order indicating that co-inhibition of LAR and PTPσ improved neuronal complexity and arborization. Q–R, Inhibiting LAR and PTPσ receptors also significantly increased number of terminal distal segments in neurons as another evidence for advanced geomorphological parameter of neurons. R, ILP/ISP treatment did not affect global bifurcation angel of neurons. Values represent mean ± SEM, *p < 0.05; nonparametric Student's t test, N = 8–10 neurons per animal and 3 animals per group. ns: non-significant.