Migration, chemotaxis and invasion depend on the microglial activation state Based on the observed differences nearly in morphology and MTOC polarization, we hypothesized that the activation state will alter directional microglial migration. First, a scratch wound assay was used to analyze migration in 2 D while viewing the cell morphology. Both untreated and IL4 treated microglia migrated into the cell free area but the response of IL4 treated cells was nearly 2 fold higher. Very few LPS treated microglia mi grated into the scratch wound. Next, migration in 3 D was quantified using the Transwell chambers. Significantly more IL4 treated microglia transmigrated than control cells, whereas, LPS treated cells migrated very little. In all cases, transmigration was increased by a gradient of the chemoattractant, ATP, that is, by 5.

9 fold, 4. 4 fold, and 7. 3 fold. Nevertheless, chemo taxis of IL4 treated cells remained Inhibitors,Modulators,Libraries the highest, 74% higher than control cells, 7 fold higher than LPS treated cells. We recently showed that unstimulated microglia can degrade fibronectin. In the absence of microglia, the substrate fluorescence was uniform. Re gardless of treatment, microglial cells degraded fibronec tin, Inhibitors,Modulators,Libraries leaving cell sized patches of reduced fluorescence. The invasion capacity of microglia was then analyzed using an assay in which migration to the underside of each filter requires degradation of Matrigel. IL4 treated microglia invaded 1. 7 fold more than control cells, whereas, LPS treated cells invaded 66% less. Adding ATP to the lower well increased the invasiveness of unstimulated cells by 2.

6 fold, and IL4 treated cells by 3. 2 fold. IL4 Inhibitors,Modulators,Libraries treated microglia had a 2. 2 fold greater invasion capacity than unstimulated cells. LPS treated cells were not Inhibitors,Modulators,Libraries analyzed because they migrated and invaded very poorly. IL4 treated microglia use a wide range of degradative enzymes for invasion Degradation of ECM can involve any or all of three broad classes of degradative enzymes, MMPs, cathep sins, and heparanase. To analyze their contributions to microglia transmigration and invasion, we first used three class specific but broad spectrum inhibi tors, GM6001, E 64, OGT2115. Then, based on the results, we tested selective inhibi tors of Cat S or Cat K 2 propanone. For each inhibitor, we used a single concentration.

Because the invasion assay was for 24 hr, during which the inhibitor efficacy might decrease, for the broad Inhibitors,Modulators,Libraries spectrum inhibitors, we chose a high concentration in an attempt to inhibit all the subtypes within the relevant enzyme class. Then, for the selective Cat S and Cat K inhibitors we used a concentration that was 10 to 20 times the IC50, which is expected to inhibit 90% of the enzyme activity. Importantly, inhibitor Rapamycin none of the inhibitors was toxic at con centrations and times used.