Accumulation many of microglia was immuno histochemically evaluated by counting Iba 1 positive cells around the injected area. At 48 hours after intras triatal injection of wild type human PAI 1 protein, there were large numbers of Iba 1 positive microglia accumu lated around the PAI 1 injection site. The R346A mutant protein, which is not capable of inhibiting PA, similarly induced microglial accumulation around the injection site. Denatured PAI 1 protein had no effect. Because Inhibitors,Modulators,Libraries the injection alone may cause tissue injuries, a basal level of microglial accumulation was seen after vehicle injection. Because PAI 1 did not in duce microglial activation in vitro, we sug gest that the microglial accumulation seen in this experiment probably results from microglial recruitment rather than activation.

The microglial migration promoting activity of the R346A mutant protein was also seen in an in vitro migration assay, indicating that the PAI 1 effects are independent of the fibrinolysis Inhibitors,Modulators,Libraries system. Additionally, the Q123K mutant of human PAI 1 retained the migration promoting activity in vitro, thereby suggesting that binding of PAI 1 to vitronectin may not be required for the activity. Re combinant human PAI 1 protein has been shown pre viously to be effective in mice. Indeed, human and mouse PAI 1 protein exerted similar effects on the stimulation of microglial migration. To further exclude the possibility that microglial accu mulation around the injection site is not due to cell activation or proliferation, another in vivo migration assay was performed using a stab injury cell injection model, which has been previously used to determine glial cell migration in vivo.

In this method, fluores cently labeled microglial cells were injected into the cortex, and their migration toward the stab injury site monitored. For this, primary microglial cells were treated with 1 ug ml of PAI 1 protein for 12 hours, and the Inhibitors,Modulators,Libraries cells labeled with CMFDA. The CMFDA labeled microglial cells were injected into the mouse brain, and then the stab injury was created. After 72 hours, three dif ferent areas were visible. Iba 1 immunostaining was also performed Inhibitors,Modulators,Libraries to identify microglial cells. Iba 1 CMFDA double labeled cells were accumulated Inhibitors,Modulators,Libraries around the stab injury site in the mouse brains after injection with PAI 1 wild type or R346A mutant protein treated microglia. Denatured PAI 1 protein had no effect.

The results support the notion that PAI 1 promotes microglial migration in vivo. Plasminogen activator inhibitor type 1 derived from astrocytes regulated microglial Ganetespib migration In a series of experiments, we presented evidence that addition of exogenous PAI 1 protein promotes micro glial migration both in vitro and in vivo. We next aimed to determine the role of endogenous PAI 1 protein in the regulation of microglial migration.