The effect of apyrase on microglia migration and accumulation. (A) Tissues were treated with apyrase at 5, 10, and 20 U/ml beginning at the time of crush, and after 4 h, the tissue was fixed, stained with Hoechst 33258 dye, and photographed. “Distal” was a measure of microglia in an uncrushed region, ∼1 mm from any injury, and was used as a baseline for the number of cells distributed throughout the tissue before injury. The difference between the “control” and the distal is the measure of microglia accumulation, as sheath cells do not move. Bars represent mean number of cells at the site of injury in a 100 × 100 × 18–µm³ volume (P < 0.05; n = 3). (B) Hydrolysis of ATP and microglial migration toward a crush. In the ∼400-µm-long region adjacent to the crush, the number of microglia nuclei moving >50 µm in 2 h was determined from time-lapse images. The connectives treated with apyrase to hydrolyze ATP had approximately one fourth the number of microglia moving relative to the control; the fraction of those moving that moved toward the crush (i.e., the directional movement) was approximately the same as the control (P < 0.05; n = 3). (C) Diffusion of extracellular ATP released after a crush caused microglia to move in an adjacent tissue. Two sets of connectives were dissected, stained with Hoechst 33258 dye, and incubated in L-15 culture medium overnight. The next day, one set was imaged with time-lapse microscopy (every 2.5 min), whereas the other set was crushed and placed parallel to it, 40–50 µm away. The microglia moved in the intact connectives, which would not have occurred in isolation. This movement was blocked in 20 U/ml apyrase, indicating that extracellular ATP released from the crush was required to cause microglial movement. Movement was measured as translocation of microglial cell nuclei by >30 µm in 1 h (P < 0.05; n = 3).