Studies Of The Function And Mechanism Of TPA On Sciatic Nerve Regeneration And Functional Recovery After Injury

Tissue plasminogen activator (tPA) is a member of serine protease family. The major function of tPA is to cleave plasminogen (Plg) into biologically active protease plasmin, which degrades fibrin of thrombus. Recently, many studies have revealed that tPA/plasmin proteolytic system was involved in both physiological and pathological processes in the nerve system. It took part in the development of seizure and Alzheimer's disease, and also played a role in the process of learning and memory. In peripheral nerve system, tPA activity was increased after nerve crush. Mice lacking tPA gene showed delayed functional recovery after sciatic nerve crush. These findings suggested that tPA might play a very important role in peripheral nerve regeneration and functional recovery after injury.In order to study the mechanism of tPA in nerve regeneration and recovery after injury, we analyzed the change of tPA and other molecules involved in nerve regeneration at different time point after nerve injury and tried to find the relationship between tPA activities and nerve regeneration. We made a model of sciatic nerve crush injury on adult C57BL/6J male mice. We emploied gel zymography to analyze tPA enzymatic activity at every stage after sciatic nerve injury. The data showed that nerve function began to recover 8 days after injury. 8 days after injury, the numbers of total axons and myelinated axons were increased, and the deposited fibrin and myelin debris were cleared. TPA enzymatic activity was significantly increased from 2 to 7 days after crush as compared with uncrushed nerves. These results revealed that tPA was involved in the process of clearing fibrin and myelin debris. And it offered a positive environment for nerve regeneration.Peripheral nerve injury is a regular disease and there has no effective therapy method. Here we showed that exogenous tPA or tPA/Plg promoted axonal regeneration, remyelination and functional recovery after sciatic nerve injury in adult C57BL/6 mouse. Local application of tPA or tPA /Plg on the 7th days after sciatic nerve crush significantly increased the total number of axons and myelinated axons as compared to controls. The increased axonal regeneration was accompanied by enhanced expression of neurofilament (NF). Treatment of tPA or tPA/Plg reduced thedeposition of fibrin(ogen) after nerve injury. Moreover, tPA or tPA/Plg enhanced the number of MMP-9 positive macrophages, reduced the collagen scar formation and accelerated clearance of myelin and lipid debris during demyelination. Consequently, tPA or tPA/Plg treatment protected muscular atrophy after nerve injury, and it was indicated that functional recovery of the nerve. These results suggested that administration of exogenous tPA or tPA/Plg promoted axonal regeneration and re-myelination through removal of fibrin deposition and activation of MMP-9 positive macrophages which may be responsible for myelin debris clearance and prevention of collagen scar formation. Therefore tPA may be useful for treatment of peripheral nerve injury.Regeneration of peripheral nerves is dependent on the ability of regenerating neurites to penetrate through structurally altered extracellular matrix (ECM), surrounding tissue, and of infiltrating cells at the injury site to reach their synaptic targets. These conditions seem to impede axonal growth and migration of growth cones. Thus, it appears advantageous for regenerating neurites to use proteases, such as the tPA and MMP-9, that are capable of degrading ECM and dissolving adhesive contacts to promote growth cone migration and axonal outgrowth. Many previous data showed that tPA could regulate the expression and activition of MMP-9. Mice lacking tPA gene showed attenuated MMP-9 activity after sciatic nerve crush. A marked decrease in MMP-9 was found in the obstructed kidneys of tPA knockout mice. In vitro, tPA induced MMP-9 gene expression and protein secretion in renal interstitial fibroblasts. In order to investigate the effect of tPA on MMP-9 expression in sciatic nerve after injury, we subjected tPA knockout and matching wild-type mice to sciatic nerve crush. The data showed that disruption of tPA gene in mice reduced MMP-9 protein level in sciatic nerve after injury. After 2 days injury, MMP-9 positive cells appeared in sciatic nerve. To determine what kind of the cells express MMP-9, we performed MMP-9 and F4/80 (a marker antibody for macrophage) double immunostaining. The immunofluorescence staining showed that most of the MMP-9-expressed cells were also F4/80 positive stainning, it was indicated that most of the MMP-9-expressed cells were macrophages. Loss of tPA gene reduced MMP-9 protein level by attenuating macrophages migrated into sciatic nerve after injury.