The Changes Of Plasminogen Activators And Other Molecules In Optic Nerve Regeneration And Functional Recovery After Injury

After nerve in peripheral nervous system (PNS) injury, axons can successfully regenerate. Proximal axons can regenerate and remyelination and re-innervate their targets, allowing recovery of function. This contrasts with what is observed in the adult central nervous system (CNS), where axonal regeneration and functional restoration is impossible. About over twenty years ago, the researchers have demonstrated that adult CNS neurons show a similar intrinsic capacity for axon outgrowth following injury as in the PNS,and the ultimate permissive microenvironment in the CNS,which inhibit axonal regeneration and remylination, responsible for abortive regeneration.These studies demonstrated that the environment is a critical determinant of axon regeneration.Damage to the adult CNS results in the formation of glia scar consists of damaged myelin debris,cellular debris, deposited fibrin and collagenous fiber in the microenvironment of injured area.Sprouting of uninjured axons is restrained from crossing the glia scar.For the purpose of crossing the glia scar and synaptic connections building-up,several different proteolytic enzymes might counteract the inhibitory factors present in CNS.Plasminogen activators (PAs) is a serine protease that converts the inactive precursor plasminogen into its active counterpart, plasmin. Plasmin is a broad-spectrum protease with a long list of extracellular targets including cellular receptors, growth factors, and other ECM components.Matrix metalloproteases are a family of zinc-dependent endopeptidases that produced by normal tissue cells and inflammatory cells and tumour cells as nactive zymogens.MMPs can degrade a variety of protein constituents in the ECM and modulate numerous pathological and tissue repair processes in the nervous system.In order to test the changes of ECM proteinases after optic nerve injury as well as to find the relationship between the changes of proteinase activities and nerve injury and nerve regeneration, We built a model of optic nerve crush injury on adult C57BL/6J male mice. We use morphological and biological methods to observe the expression of IgG, fibrin(ogen), PA and MMP-9 extravasating from blood vessel at crush area at different time point after optic nerve crush injury,in order to identify the role of many factors in the environment in the degeneration and regeneration of CNS.MethodsAdult male C57BL/6J mice after deep anesthesia with chloral hydrate intraperitoneal injection later canthotomy was performed and the right optic nerve was crushed for 45 s at 2mm behind the globe,suture skin incision.Left eyes underwent sham-surgery and served as the self-control group. 1,2,4,8,16 hours,1,2,7, and 28 days later,the mice were transcardially perfused with ice-cold PBS under deep anesthesia and dissected optic nerve.Then longitudinal sections (5μm) of nerve segments with the crush sites were prepared and the protein of nerve segments with the crush sites were extracted.The morphology of the optic nerve degeneration were observed in model group by hematoxylin and eosin dyeing.The expression of neurofilaments,fibrin(ogen), IgG at different time point after optic nerve crush injury were observed by Western blot. The changes of enzymatic activities of plasminogen activators at different time point after optic nerve crush injury were observed by in situ zymography.ResultsThe optic nerve degeneration is gradually severe over time after optic nerve crush injury showing that nerve fiber circuity, astrocyte hypertrophy,vacuolar degeneration by hematoxylin and eosin dyeing.Western blot showing:In crushed optic nerves,the expression of neurofilaments proteins did not change much until day 7 comparing to the control, but by day 28 little NF can be detected. IgG was little in normal optic nerve parenchyma and can be found in a lot the nerve parenchyma after injury,by day 28,there was still a lot IgG in the crush sites.Little fibrin(ogen) deposition can be found in normal optic nerve parenchyma; crush injury resulted in BNB disruption; thus, fibrin(ogen) extravasates into the insulted nerve.Within the injured optic nerves, fibrin(ogen) deposition appeared one hour after crush, by day 7,it was completely cleaned from the crush sites.MMP-9 was barely detectable in naive optic nerves.In the injured optic nerves MMP-9 began to accumulate and significantly elevated up to two days after crush,lasted up to day 28 on a considerable level.The expression of MMP-9 is as the inactive zymogen with 92KDa molecular weight.In situ zymography showing:The black dissolvable zone can be found at the circumference of the nerve 1st day after crush, by day 7, the zone extended to the maximum, while black dissolvable zone disappeared with amiloride added into reaction system. PAs enzymatic activity was significantly increased at the 7th day after crush and lasted up to the 28th day on a considerable level and uPA was the critical enzyme.ConclusionsThe animal model of optic nerve injury is established successfully by crushing optic nerve of adult C57BL/6J mice, which offers a way to study the extracellular environments for axonal regeneration and remyelination after the nerve degeneration in CNS.The clearance of fibrin(ogen) and IgG deposition in the injured optic nerves are differentially regulated. The clearance of fibrin(ogen) deposition are independent of the complete repair of BNB.This may indicate that PA/Plg/Pln cascade quickly activate after nerve injury in CNS.The expression of MMP-9 as an inactive precursor in the CNS following injury may be activated to be inhibitory for nerve regeneration. uPA can clear fibrin(ogen) deposition in the crush sites and repair the adverse microenvironments, even activate Plg/Pln cascade to degrade ECM.