Figure 2
Changes in microglial dynamics and interaction between microglia and the pyramidal tract axons after spinal cord injury (SCI). (a) Experimental scheme. Each mouse underwent in vivo imaging once at POD2 (green arrow), POD4 (blue arrow) or POD7 (purple arrow) after SCI or Sham operation. (b) tdTomato expression under the CAG promoter-coded adeno-associated virus (AAV) was injected into the primary motor cortex (M1) of Cx3cr1eGFP/+ mice to visualize the microglia and pyramidal tract axons in the spinal cord using in vivo two-photon microscopy. (c) Whole mounts of the brain, spinal cord, and coronal sections. Pyramidal tract axons from M1 to the spinal cord in (i) primary motor cortex, (ii) internal capsule, (iii) cerebral peduncle, (iv) cervical pyramidal tract axons, and (v) thoracic pyramidal tract axons are labeled with tdTomato. (d) Typical images of microglia–pyramidal tract axon interactions in the spinal cord at post-operation day (POD) 2, POD4, and POD7. Scale bar: 50 µm. (e) Density of Cx3cr1+ cells in the Sham tDCS − PLX− and SCI + tDCS − PLX− groups at POD2, POD4, and POD7. (f) Cell body area of Cx3cr1+ cells in the Sham tDCS − PLX− and SCI + tDCS − PLX− groups at POD2, POD4, and POD7. (g) Aspect ratio, (h) circularity, and (i) roundness of Cx3cr1+ cells in the sham-operated and SCI groups at POD2, POD4, and POD7. (j) Colocalization ratio of axonal and microglial fluorescent intensities in the Sham tDCS − PLX− and SCI + tDCS − PLX− groups at POD2, POD4, and POD7. (k) Proportion of Cx3cr1+ cell process angles (p = 2.2E − 16, Sham vs. SCI group at POD2; p = 2.99E − 6, at POD4; p = 0.00835, at POD7). (l) Proportion of angles between the Cx3cr1+ cell processes and pyramidal tract axons (p = 0.0293, Sham vs. SCI group at POD2; p = 3.41E − 16, at POD4; p = 2.36E − 6, at POD7). n = 5 mice per group (e–l). Each black circle and black triangle represent the average data for an individual Sham tDCS − PLX− and SCI + tDCS − PLX− mouse, respectively (e–l). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; Sham tDCS − PLX− and SCI + tDCS − PLX− groups were compared for each day. Statistical analysis was performed using two-way analysis of variance followed by Bonferroni post-hoc tests (e–j) and Mardia–Watson–Wheeler test (k, l).
