Figure 4.

Fate-mapping monocytes in LPS/HI-injured CCR2-CreER; R26R-GFP mice. (A) Scheme of monocytic fate-mapping via CCR2-CreER; R26R-GFP mice. Tamoxifen-dosing labels CCR2⁺ monocytes and their derivatives with constitutive GFP fluorescence regardless of the CCR2 promoter activity. (B) GFP⁺ monocytic infiltrates exhibited stronger anti-Ki67 immuno-signals at 4 d than 30 d post-LPS/HI. (C-F) GFP⁺ monocytic infiltrates were found in ipsilateral hemisphere at 4 d post-LPS/HI (D), including the hippocampus (E) and cerebral cortex (F), but not in contralateral hemisphere (C). Magnified images of the ipsilateral cortex and hippocampus showed both amoeboid (arrowhead) and ramified morphology (arrow) of GFP⁺ cells. (E, F) are magnified images of the squares shown in (D). (G-I) Immunostaining showed that many GFP⁺ monocytic infiltrates expressed Iba1 (G), CD68 (H) and TNFα (I) at 4 d post-LPS/HI. (J-P) At 30 d post-LPS/HI, GFP⁺ monocytic infiltrates were still absent in contralateral hemisphere (J), but found in the damaged ipsilateral hemisphere (K), including the hippocampus (L) and cerebral cortex (M). More GFP⁺ monocytic derivatives displayed a ramified microglia-like morphology (arrows in M). (L, M) are magnified images of squares shown in (K). (N-P) Immunostaining shows that ramified GFP⁺ monocytic derivatives expressed Iba1 (N) and TNFα (P), but only amoeboid GFP⁺ derivatives expressed CD68 (O) at 30 d post-LPS/HI. (Q-T) At 5 months post-LPS/HI, GFP⁺ monocytic derivatives were still restricted in the ipsilateral hemisphere (Q-R), exhibiting both ramified (arrows in S, T) and fewer amoeboid morphology (arrowhead in S). (S) is magnified image of square shown in (R). N = 3 mice for each time point in A-B, n = 6 in C-F, and n = 3 in Q-T. Scale bar: 50 μm (B-P), 100 μm (Q-T).