Figure 2.
Effects of CBX and ATP on microglia migration and accumulation. (A) Schematic diagram of the leech nerve cord and, for the region depicted in the rectangle, micrographs of crushed connectives to show how cell accumulations are measured. Tissue was stained with Hoechst 33258 dye to show nuclei. The top and bottom micrographs are representative collapsed images (10 each through 18 µm) with and without drug treatment. The crushes are indicated, with squares (100 × 100 µm) outlining regions of measurement. Nuclei are mostly microglia but include some sheath cells. “Treatment” tissue in the micrograph was incubated in 10 µM CBX. (B) Cell accumulation at the site of a crush injury in CBX. Tissues were treated with CBX at 1-, 10-, and 100-µM concentrations at the time of crush, and the microglia were allowed to accumulate for 4 h. Tissues were then fixed and stained. Bars represent mean number of cells at the site of injury in a 100 × 100 × 18–µm3 volume. “Distal” is a measure of the number of cells in an uncrushed region, ∼1 mm from any injury, and is used as a baseline for the number of cells distributed randomly throughout the tissue. The difference between the “control” or other conditions and the distal is the true accumulation. Mean ± SEM are represented (P < 0.001; n = 6). (C) Microglia moving in 30 µM CBX and 100 µM ATP. Microglia nuclei were tracked using time-lapse recording, and those moving >30 µm were counted. CBX did not inhibit movement in ATP, although it did inhibit movement induced by crushing the connectives. Bars represent average number of microglia moving in 30 min ± SEM (P < 0.01; n = 5). (D) Microglia accumulation in CBX and ATP. 10 µM CBX significantly reduced the accumulation of cells at the crush. In the CBX washout condition, the connectives were incubated in CBX for 4 h and washed for 1 h before crushing. “Distal” is a measure of cell nuclei in an uncrushed region. The reduction seen in CBX did not occur after CBX was washed from the tissue, showing that the effect of CBX was reversible. It was also eliminated in the simultaneous presence of 100 µM ATP, consistent with an effect of CBX to block release of ATP, but not the direction of movement (n = 3; *, P < 0.5; **, P < 0.01). (E) Microglial migration toward a crush and the effects of 10 µM CBX and 100 µM ATP. 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. Drugs were added 5 min before crushing. The CBX with ATP condition was similar to the control condition in both total movement and directional movement, but CBX alone decreased the movement significantly (P < 0.05; n = 3).