| Back groundAx on regeneration and formation of functional synapses with target cells are the main objectives and key issues in repair of spinal cord injury. Large numbers of basic studies on promotion of axon regeneration have shown that failure of early regeneration of the central nervous system in adult mammalians is due to the presence of myelin inhibitors.We have found in previous experiments that neural stem cells(or) olfactory ensheathing cells could divide into neurons and other glial cells after transplanted into the damaged spinal cord. The nerve synapse formation increased and BBB motor function score improved in all rats with spinal cord injury. Although a lot of damage nerve regeneration and (or) budding (to be published data) was observed in experiments, the distance axonal regeneration or sprouting is extremely limited as the damage to local micro-environment changed.It is difficult to break through the barrier between the myelin-inhibitory factor and glial scar to format function synapses with the distal stump.Some specific components of the myelin debris (eg:Nogo66, myelin-associated glycoprotein MAG, myelin oligodendrocyte glycoprotein OMgp, etc.) act on the axon on the Nogo receptor (NgR) and its acceptor-p75NTR complex to regulate cytoskeletal structure of axons within the axon growth inhibition through the small guanosine triphosphatase (small GTPase) Rho way.Thus,it is difficult to obtain satisfactory results of spinal cord injury repair. Nogo monoclonal antibody IN-1 can antagonize with Nogo protein, but not with the other two inhibitors MAG and Omgp. Grand Pre used the competing peptide NEPI-40 to antagonize the role of NgR and showed that NEP1-40 may be against the Nogo-66-induced axon growth inhibition, but not sensitive to other myelin inhibitors. This peptide can not be closed all inhibitory molecule binding site of NgR and the effects in vivo is not very satisfactory. Therefore, most urgent task is to find an effective and highly targeted method to clear myelin debris.At present, there are two opponent views on the role of macrophages in the central nervous system injury. One is that macrophages can inhibit the growth of axons; other is that of macrophages play a catalytic role to the axon regeneration. The inflammatory response appeared through repair process of the tissue. Macrophages play a major role and can remove necrotic tissue and secrete neurotrophic factor, in all the body plays an important role in tissue repair. Mantovani believed that the macrophagecan can chang to "select" activation-type and the "classic" activation of two kinds impacting by the different signals in micro-environment of the inflammatory response. The "select"-activated macrophages are conversed by IL-4 and glucocorticoid-induced and adjust the immune inflammatory reaction process, the debris removal of necrotic tissue, promoting angiogenesis, tissue repair and reconstruction. The "classic"-activated macrophages may secrete pro-inflammatory cytokines and increase the inflammatory reaction, causing further damage to local cell. In recent years, there are rare reports of macrophages in the injured spinal cord injury.Recent studies have shown that the role of macrophage in phagocytosis of myelin debris and axonal regeneration may be greater than we anticipated.In 1998, Zeev-Brann reported that there were spinal cord-derived inflammatory cells appearing in mature mammalian central nervous system after injury. Buss and Schwab found that the ED-1 immunohistochemistry staining positive cells were microglia cells and macrophages which cleaned up myelin debris in SCI rats. MASAAKI researched macrophages and its role through the comparement of the rat spinal cord contusion model (on behalf of contusion caused by a myelin debris and myelin regeneration was inhibited) and the chemical demyelination models (on behalf of the early removal of myelin debris and myelin regeneration in multiple sclerosis).The arthors transplanted green fluorescent protein transgenic mouse bone marrow cells into rat spinal cord contusion model irradiated by the radiation and chemical demyelination models.They studied the infiltration of activated macrophages and myelin debris residual changes in the different levels by immunohistochemical staining of spinal cord. The result showed activated macrophages expressing green fluorescent protein constituted the major cell groups in the damage location, indicating macrophages in the two models primary derived from bone marrow and rarely from intrinsic microglia. The immunohistochemical staining showed myelin debris existed long-time in the contusion model and the activated macrophage infiltration in the contusion model more slowly than that in the chemical model, the number reduction significantly. RT-PCR detection of trend chemokine (MCP-1, GM-CSF) expressed a low level 2 to 4 days after injury, suggesting that the delay infiltration of activation of macrophages post-traumatic spinal cord related to the persistence of myelin debris and lead to remyelination suppression.Samuel et al [5] studied Nogo receptor (NgRs) in peripheral nerve injury. They induced damage in the sciatic nerve in the thigh of rats and found that macrophages, once arriving and starting to remove myelin debris, they performed for expression of NgR1 activated forms and went into the base of Schwann cells. When the restoration of the nerve start to produce new proteins of myelin (such as NgR ligands), this receptor not only inhibits macrophages with a combination of myelin, but also directly inhibits the formation of myelin. When the researchers damaged the nerve again to inhibit the formation myelin, the macrophages remained in the outer layer of Schwann cells and swallowed myelin debris. However, NgR1 knockout mice or use of Y-27632 to inhibit NgR downstream Rho-associated kinase could increase the combination rate of macrophages and myelin debris, indicating that NgR expression in macrophages participated in the withdrawal process at the end of repair in peripheral nerve injury. But whether macrophages express NgR after CNS injury and what role NgR expression plays in CNS repair remain unclear.After spinal cord injury, activated macrophages infiltrated to injury areas and the delay of myelin debris clearance inhibit of axon extension. The formation of scar due to the accumulation of astrocytes led to inhibition of axonal regeneration. We speculate that the adult mammalian spinal cord injury, the rapid removal of myelin debris by activated macrophages on the central nervous system regeneration has the same importance as that in the peripheral nervous system, but the specific role of macrophage cells in spinal cord injury still need study.ObjectiveTo construct the spinal cord injury model and to 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 construct the lentiviral vector of rat NgR gene and to transfect macrophages expressing NgR, to explore the feasibility of immune cells transfecting. To compare the effect of NgR expression on macrophage phagocytosis; To construct neuronal injury model in vitro and culture it with NgR-macrophages group, the mock group and the normal macrophage group together, to compare the effect of each group on the repairment, survival, differentiation of the injuried neuron. To explore the possible mechanisms of NgRs expression in macrophages on the spinal cord injury repairment and to give the experimental and theoretical basis for further animal studies and clinical applications.Methods1. To construct the spinal cord injury model and draw the materials at the day 3 and 7. To observe the infiltration of macrophages and expression of NgRs by immunofluorescence staining. 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. RT-PCR technique was used to obtain gene-coding region of rat NgR fragments, digestion using restriction enzyme. Construction of gene recombination lentiviral vector plasmid and packaging plasmid mediated by the liposome. The envelope plasmid co-transfected 293T cells and the lentivirus producted. Infection the rat macrophages using the lentivirus and the insertion and expression of NgR gene were detected by PCR.3. Myelin basic protein (MBP) was added into macrophages (NgR-macrophages group, the mock group and the normal macrophages group) and cultured, and the phagocytic capacity was measured by Western blot. Student's t-test was used for statistical analysis.4. The neuronal injury model was constructed. Macrophages of different groups were co-cultured with the injured nerve plates. 100μL culture medium in each group was extracted respectively at different time points (30min, 1h,6h,12h,24h,48h,72h after injury), testing LDH content and absorption values of the viable cells using MTT colorimetric. morphological changes of neurons in each group was observed under inverted phase contrast microscope at different times and repeated measures analysis of variance was used for statistical analysis.Results1. No macrophage infiltration was seen in the spinal cord of the sham-operated group. Double immunofluorescence labeling of the injured spine in the SCI group showed that NgR positive cells were also CD68 positive macrophages. In addition, immunofluorescence of the tissue sections showed that NgR was on surfaces of these cells, indicating that NgR was expressed in cell surface. To confirm that macrophages expressed NgR, the injured spinal cord at day 7 was isolated and labeled NgR antibody. The results showed that NgR was expressed on cell surfaces in a spotty manner.2. The result of sequencing showed that the sequence of the cloned NgR gene was consistent with that reported in the Gen Bank. The plasmid that was identified showed the correct sequence. After the 3 plasmids of LV vectors were cotransfected to the 293T cells, considerable green fluorescence in 293T cells was observed under the fluorescent microscope; the supernatant was collected and concentrated using ultercentrifugation, and the titer of the replication-defective LV vector particles measured was found to be 6.7×107 TU/mL. After the constructed LV vectors infected the macrophages, the results obtained using RT-PCR showed that the expression of NgR in the NgR-macrophages group(experimental group)was significantly higher than that in the mock group and the macrophages group(control group)at both mRNA and protein level. LV vectors carrying the NgR gene were constructed successfully. The NgR gene-modified macrophages could express NgR to a higher degree.3. The MBP content in NgR-macrophages group was significantly higher than that in the mock group and the normal macrophages group(P<0.05). There was no significant difference between the mock group and the normal macrophages group(P>0.05).4. There was no significant change in optical density (OD) value at 0.5,1,6 and 12 h after culture in each group, and at 24 h a significant difference of decrease was observed in OD value between the injured nerve+NgR-macrophages group and the other groups(p<0.05). There was no significant difference between the mock group and the normal macrophages group(P>0.05). There was also no significant difference between the NgR-macrophages group and the normal neuron group (P>0.05). With the lapse of time, LDH level decreased markedly in the injured nerve groups, while it did not decrease significantly in the NgR-macrophages group. There was no significant difference of LDH contents decrease in each group after co-culture for 0.5,1,6 and 12h. But 24 h later, LDH contents decreased markedly in the injured nerve+NgR-macrophages group as compared with that of the other groups (p<0.05). There was nos significant difference between the mock group and the normal macrophages group (P>0.05). There was no significant difference between the NgR-macrophages group and the normal nerve group.ConclusionThe spinal cord injury model was constructed successful. The expression of NgRs on macrophages infiltrating in the injury spinal cord was confirmed From the histological and cellular level.The lentiviral vector of rat NgR gene was successfully constructed and transfected macrophages expression NgRs. This lays the foundation for treatment of spinal cord injury by transplantation of genetically modified macrophages.Macrophages expression NgRs in vitro can promote repairment of the injured neurons. The mechanism may be that macrophages regulate phagocytosis by intracellular signaling pathways through expression NgRs, thus creating conditions for nerve regeneration. 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. |
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