Expression Changes And Effection Of Neuroglobin In Mechanical Injury Of Nerve

Oxygen is the essential element of life.The metabolism and utilization of oxygen are two important propositions in life science.It is well known that brain is one of major oxygen-consurning organ and sensitive to hypoxia.But if there exists a special globin for oxgen's metalolism and transport in brain is not known. Till 2000,Burmester reported a newly identified protein-neuroglobin(NGB), mainly expressed in nervous systems and retina of vertebrate,which is a monomer with high oxygen affinit5.Studies revealed NGB binded oxygen reversibly and played an important role in oxygen homeostasis of neural tissues. Other studies demonstrated that expression of NGB could be up-regulated under hypoxic conditions and had neuro-protective function both in vitro and in vivo. The discovery of NGB and the results of recent studies provide new opportunities to further understanding of neuroprotection and may lead to a novel therapeutic and diagnostic and therapeutic strategy for cerebral stroke and other neurodegenerated diseases.However.relatively little is known about the expression changes and effect of neuroglobin in mechanical injury of nerve.We designed experiments in vitro and in vivo to research the subject as follows:1,We cultured primary neuron in vitro after obtaining cerebral cortex from fetus rats brain and purifying it.Then the cultured neurons were injured mechanically through the model designed by ourselves,and the result of injury was proved by observing the changes under light microscope and dyeing immunohistochemically.2,We choosed two methods to observe the expression changes of neurons NGB at different time points after mechanical injury:One was immunohistochemical method with image analysis technology using NIH software-Image J,the other was Western Blot technology.The two both demonstrated that the expression of NGB was up-regulated afer mechanical injury and reached the peak after 16 hours since injury.3,Because of the low transfection efficiency of neuron,we choosed the neuroblastoma cell-B104 cell to undertake the experiments.Firstly,the B104 cell was proved to appear similar time course of NGB's expression change after mechanical injury using Western Blot technology.Then we used the pCMV-Myc-NGB plasmid to transfect the B104 cell and proved that the expression of NGB was more than the cell untrasfected or transfected by pure plasmid(pCMV-Myc plasmid).The viability of B104 cell transfected by pCMV-Myc-NGB plasmid was better than that of the cell untransfected or transtfected by pure plasmid through measuring LDH activity or MTT absorbance.4,We set up acute local traumatic brain injury(TBI)graded model in rats using improved device of Feeney's weight-dropping model.The three groups of brain trauma were made by different impact(200g×cm,600g×cm,1000g×cm). Then the injured brain was cut sheets and dyeing with TTC.The volume of necrotic brain tissue were calculated by NIH software-Image J and proved to be different statistically among different-impact groups.5,We measured the NGB's levels in the rat serum at the same time point of the mild,moderate and severe TBI using the home-made ELISA kit.The serum levels of NGB were different statistically among different grade TBI.Also we measured the serum NGB's levels at different time points after TBI of the same grade to explore the time-course of NGB's serum concentration.In summary,the NGB's expression was up-regulated by the mechanical injury in neuron and NGB had neuroprotection effection.And NGB's serum level was relative to the severity of TBI.it may be a new diagnostic tool to judge the TBI's severity.