Effect Of C3 Deficiency On Inflammation And Regeneration Following Spinal Cord Injury Of Mouse

Spinal cord injury (SCI) has being paid much attention for its high deformity and mortality rates. The ultimate impairment of SCI is caused by two mechanisms, that is, initial injury and secondary injury. The former includes mechanical pressure, hemorrhage, electrolyte overflow and etc. The latter includes the impairment from edema, inflammation, local ischema, growth factors, cytokines, Ca2+ overflow and the abnormal change of peroxide radicles. These years the research about SCI has been focused on the tissue regeneration during the late stage of injury, for example, the transplantation of neural stem cells or olfactory ensheath cells, with not many effects. The main reason is that the secondary injury is neglected and the survived neurons during early stage of injury are not protected in time. These lead to the lack of innate condition for regeneration. As for SCI, the initial injury is a process that cannot be reversed, while the secondary injury is a process that can be reversed and controled. So more and more attention has been paid to alleviate the secondary injury through kinds of methods such as reducing inflammation.There is an important inflammation process during the early phase of secondary injury which can lead to a series of reactions being harmful to the cells at the injury site. Besides, inflammation can result in the formation of glial scar which inhibits regeneration. Reducing inflammation can improve regeneration and functional recovery of central nervous system (CNS). It is worthy of notice that inflammation can activate complement system, and the activated complement segments can induce inflammation and aggravate the secondary injury to self tissues. Can we reduce inflammation through inhibiting the activation of complement system? How can we do it? We choose to knock out the C3 gene. Because C3 is a pivotal factor in complement activate pathways and is necessary for activation of complement system. In this study, our objective is to knock out the C3 gene to reduce inflammation and secondary injury to improve regeneration and functional recovery after SCI. Animals were divided into WT group (wide type) and KO group (knock out type). All experiments were done at the same time between these two groups. Allen`s weight-drop method was adopted to establish the C3 deficient mouse spinal cord contusion model. By Basso, Beattie and Bresnahan (BBB) score method we observed the functional recovery of SCI mouse. By immunohistochemstry staining method, western blot, RT-PCR, we observed the expression of TNF-α, the activation of astrocytes (AST), the expression of regeneration inhibitory factors NgR and RhoA and the regeneration of nerve fibers. To exclude the individual differences of animals and interferential factors in vivo, we established AST and neuron mechanical injury models and detected their secretion of TNF-αat different time points. After that dosal root ganglions (DRG) were cocultured with AST mechanical injury model and the growth of DRG neurites were detected on different culture days.Main results:1. A mouse spinal cord weight-drop contusion model(5g×6cm) was established. The contused site was at T12. Typical paraplegia symptoms were occurred;2. Both WT and KO mice showed similar pathological results after SCI: the spinal cord fabric at injury site was damaged, lots of hemorrhagic foci could be detected mainly in grey matter, and cavums appeared at injury centre with extendting areas;3. The BBB score of KO mice after SCI was higher than WT mice obviously. The score of KO mice on 21d was 17±0.8, which was close to normal mice, while the WT mice just get 11±1.2 at the same time;4. Western blot and immunohistochemistry showed that the number of TNF-αpositive cells and immunohistohemical intensity of KO mice after SCI were stable and higher than KO mice with sham operation but lower than WT mice at 6h;5. The activated AST number and its activating duration of KO mice decreased obviously after SCI when compared to WT mice;6. The length and density of regenerating neural fibers of KO mice after SCI were higher than WT mice obviously;7. The immunohistochemistry staining method showed that the expression of NgR and RhoA were enhanced after SCI in KO mice, with no obvious difference to WT mice however. Western blot showed that the expression of RhoA mRNA in WT mice increased after SCI, but not in KO mice; 8. TNF-αsecreted by AST or neurons was detected by ELISA after mechanical injury. Its secretion in WT mice, whether from AST or neurons, increased during 1h to 48h, with peak at 6h. However, the secretion in KO mice did not increase;9. DRG was cocultured with AST mechanical injury model. The length and density of neurites growth from DRG were measured. DRG+AST(WT , uninjured)and DRG+AST(KO,uninjured)groups had the longest and densest neurites. DRG+AST(WT, injured) group had the shortest and sparsest neurites. As for DRG+AST(KO, injured), its length and density of neurites were between DRG+AST(WT/KO, uninjured) and DRG+AST(WT, injured).Main conclusions:1. C3 knock-out can inhibit the expression of TNF-αafter SCI and reduce inflammation;2. C3 knock-out can reduce the activated AST number and its activating duration, which is favorable to reduce inflammation, alleviate secondary injury and improve the regeneration environment;3. C3 knock-out can improve the neurite regeneration and functional recovery after SCI;4. C3 knock-out can inhibit the TNF-αsecreted by AST and neurons in vitro and promote neurite outgrowth.In conclusion, our study showed that C3 gene knock-out can inhibit inflammation to some degree, reduce the secondary injury and improve the neurite regeneration and functional recovery after SCI. It is a new feasible way to promote the regeneraton of CNS through inhibiting the activation of complement system.