Fig 2: Traumatic brain injury (TBI) in wildtype (wt) and chemokine-deficient (Ccl3-/- and Ccr2-/-) mice.(a) In situ hybridization revealed intense Ccl3 expression in the perilesional zone four hours after trauma, in a pattern resembling distribution of activated microglial cells. (b) Temporal patterns of Ccl3 and Ccr2 transcript levels in the injured wt neocortex revealed by qRT-PCR. (c) The Ccr2 transcript was further upregulated in Ccl3-/- mice as compared to wt mice three days postinjury. Gfap expression was also strongly upregulated but with no differences between the strains. (d) The Ccl2 transcript, encoding a Ccr2 ligand, was upregulated after injury with no differences between wt and Ccl3-/- mice as shown by microarray analysis. In contrast, Selp transcript expressed by endothelial cells was more upregulated in Ccl3-/- compared to wt brains. (e) Lyz2 expression and the microglial surface marker isolectin B4 (IB4) showed only partial overlap three days post-injury in wt brains (in situ hybridization and histochemistry). The Bst2, characterizing pDCs, exhibited a different expression pattern (insert). (f) At three days postinjury, the Lyz2 increase shifted in opposite directions in the Ccl3-/- and Ccr2-/- compared to wt brains. (g) Cavity volume was reduced in the Ccr2-/- compared to wt mice seven days after injury in accordance with downregulation of inflammatory Lyz2-response.

Fig 3: Inflammatory-related transcripts measured in Ccl3-/- and Ccr2-/- mice with F2 hybrid background three days postinjury. Lyz2 expression was upregulated in homozygous Ccl3 knockouts but downregulated in Ccr2-/- as in the parental strains, as was Bst2. Both Itgax and Cxcl10 levels were less upregulated in the injured F2 Ccr2-/- brains. Gfap expression increased to the same extent in injured wt and F2 hybrid brains.

Fig 4: Inflammatory cells in the injured wt and Ccr2-/- cortex examined by flow cytometry and qRT-PCR.(a) Cells detected with FITC-CD45 in combination with either APC-PDCA-1 (Bst2) or APC-Cd11c (Itgax) in neocortex of wt or Ccr2-/- mice three days after injury. Upper panel left shows gating for Cd11c. The middle and right panels show wt and Ccr2-/- results, respectively, demonstrating reduced number of Cd11c-positive cells in the Ccr2-/- mice. Lower panel shows that PDCA-1 positive cells were reduced in Ccr2-/- brains. (b) Quantitative flow cytometry data from wt and Ccr2-/- mice three days postinjury. Isotype controls showed only trace signals. (c) Temporal expression patterns of six inflammatory-related transcripts (Itgax, H2-Aa, Bst2, Cxcl10, Lyz2 and Hmox1) after injury in wt and Ccr2-/- brains.