Figure 7
Two-photon imaging detection of microglia in CX3CR1-GFP mice and morphometric analysis of cellular morphology in CCI-TBI and anti-ICAM-1/catalase administration. CX3CR1-GFP mice (B6.129P-Cx3cr1 tm1Litt/J, The Jackson Laboratory) were subjected to moderate CCI-TBI with and without anti-ICAM-1/catalase conjugate administration 30 min after impact. No craniectomy (naive) CX3CR1-GFP mice served as control. At 48 hrs following CCI-TBI, subjects were perfusion fixed, brain tissue was collected and subsequently sectioned into 1 mm segments for two-photon imaging. (A) Representative single slice images depict microglial ramification in the area of impact for naive, CCI-TBI, and CCI-TBI+anti-ICAM-1/catalase mice. Arrowheads point to microglial processes, which are finely ramified in the naive condition and thicken and retract following CCI-TBI. Anti-ICAM-1/catalase attenuates changes in microglia cell body enlargement and process retraction. Scale bar 20 μm. Imaris (Bitplane) software was utilized for 3D reconstruction of GFP-expressing microglia from z-stack images obtained by two-photon microscopy. (B) Surface rendering was performed for microglia based on GFP signal threshold to measure cellular surface area (D) (only objects with surface area >1000 μm2 were analyzed) and sphericity (E). (C) Imaris FilamentTracer was employed to map microglial processes to quantify changes in cell ramification in CCI-TBI. Microglial total filament length (F) and number of filament branching points (complexity, (G) and were assessed for naive, CCI-TBI, and CCI-TBI+anti-ICAM-1/catalase groups. Data presented at mean ± SD. (Ordinary one-way ANOVA. Surface area: F = 73.12, P < 0.0001. Sphericity: F = 17.20, P = 0.0013. Sum of filament length: F = 74.57, P < 0.0001. Number of filament branching points: F = 38.50, P < 0.0001).
