| Back groundAlthough the inhibitory environment of the CNS has been recognized one of the most difficulties for axonal regeneration, not until the past several decades have CNS myelin and related molecules become appreciated as primary blockades to CNS regeneration after injury and that's why the clinical symptom occurs.In recent several years, some molecules were discovered one after another such as Nogo-A, Myelin associated glycoprotein, Oligodendrocyte myelin glycoprotein and signal transduction pathway mediated by Nogo receptors which enhanced our understanding of mechanism for axonal regeneration in SCI. The treatments aimed at Nogo and Nogo receptors have became a new hot spot.But until now, the outcomes of such research were not enough to overcome the difficulties. Monoclonal antibody IN-1 can block the axonal inhibitory effect of Nogo, but not for MAG and OMgp. NEPI-40, the antagonist of NgRs can only suppress the adverse effect of Nogo-66. It's important to find another method to remove the myelin inhibitors.Macrophage is one kind of inflammatory cell which plays a pivotal role for repair in trauma. Now, with regard to the role of macrophages in CNS repair there are two points of view. One kind of view thinks that macrophages can promote the axonal regeneration, another is the opposite. It was used to consider aseptic inflammation harmful in CNS, especially in SCI. In fact, the whole process of repair is accompanied with inflammation in human body. In the process, macrophages play major roles which could get rid of the necrotic tissues and secrete nutrition factors.Recent research showed that macrophages could have more influence in phagocytosis of myelin pieces, promoting axonal regeneration and re-myelinization. In 1998, Zeev-Brann reported that bone marrow derived inflammatory cell appeared in repair of CNS injury and it was proved by Buss that delayed infiltration of macrophages in the injured region led to the persistent exist of myelin pieces which could inhibit re-myelinization of axon. In 2007, it was confirmed by Samuel David that whether macrophages expressed with NgRs and its infiltration process had close relationship with axonal re-myelinization, which have deep impact on physiologic function of neural cell. We expect to provide a new method for treatment of SCI and accumulate experimental and theoretical evidences for further animal study and clinical application.ObjectiveTo investigate the isolation, culture methods and evaluation of macrophages expressed with NgRs in vitro in preliminary and reveal the biological characteristics of the cells. To explore the separation, cultivation methods and practical potential of primary neural stem cells and olfactory ensheathing cells in vitro. To delivery the macrophages expressed with NgRs and NSCs to the wounds of SCI rat and compare the survival rate, cell differentiation, synapse formation and recovery of neurological function with other transplantation groups. To investigate the mechanism of the macrophages expressed with NgRs in recovery of SCI rat and to establish a foundation for further research in animal experiments and clinical applications.Methods1. Primary macrophages were harvested from injured spinal cord and digested with trypsin, and the morphological, biological characteristics and the NgRs antigen positive cells with methods of immunochemistry staining were observed.2. Primary OECs were isolated from the olfactory bulb of new born rat and digested with trypsin, and purified with delaying differing rates of various cell types and method of Ara-C inhibition. The percent of P75 positive cells was calculated with methods of immunochemistry staining.3. Embryonic brain of rats were chosen to isolate primary NSC with aseptic technique by trypsin digestion. The culture medium was added with nutrition factors in order to increase the amount of cells and the morphological characteristics, growth curve and cell differentiation were recorded.4. Nine to ten days after models of rat spinal cord injury were established, primary macrophages, OECs and labled NSC were selected to deliver to the four groups of SCI rat with different methods. The data of survival rate, cell differentiation, synapse formation and recovery of neurological function in each group were surveyed.Results1. Most of the primary macrophages presented round and elliptic shape after adherence for one hour. The macrophages displayed with active metabolism could survive for about 3 weeks and be marked by CD68 antibody and NgR antibody for majority of the cells.2. Primary OECs were successfully cultured and the body of OECs in the fifth day presented with two kinds of shapes:fusiform and multipolar. Their quality of profile was real and had good refraction. Eight days later, their body became larger and the increased process wove into net each other. Fourteen days later, majority of OECs showed themselves two morphological types:bipolar and tripolar. The percent of P75 positive cells decreased gradually from the beginning of the first week.3. The suspended primary NSCs developed into mulberry-like cells with little process and expressed with nestin antigen. They could also express phenotypic NF-200 marker of neuron and GFAP marker of astrocyte after being induced to differentiation. Being marked by BrdU was adopted to examine the ability of renewing themselves.4. From 4 to 10 weeks ,the BBB locomotion scores of all transplantion groups, especially the co-transplantation group of macrophages and NSCs, were improved significantly contrasting to that of the control group (P<0.05). Transplanted NSCs can survive for at least ten weeks and differentiated into neurons and astrocytes. Immunohistochemitry staining of NF-200 and synaptophysin showed the co-transplantion group of macrophages and NSCs had a higher percent of positive rate than that of the other three groups.ConclusionThe macrophages presented with NgR antigen were isolated from the traumatic spinal cord tissues successfully and the morphological, biological characteristics were discovered. Enough OECs and NSCs were harvested and proved to be eligible resources for potential experimental application in research. Macrophages presented with NgRs antigen could alter the microenviroment and improve the survival and differentiation rate of NSCs grafts, thus promote the synapse formation and neurological recovery of SCI rat. Signal transduction pathway mediated by by Nogo receptors might be the key, which could conduct the process of phagocytosis in myelin pieces elimination and be beneficial for neural regeneration.
|
PDF Full Text Request