Study On The Effects And Mechanism Of C5a After Spinal Cord Injury In Mice

The mechanism of spinal cord injury (SCI) includes primary and secondary injury. Inflammation plays an important role in the secondary injury. Previous research found complement is closely related to inflammation. The complement fragment C5a was thought as the most powerful complement component in mediating inflammation. The blood-spinal cord barrier was broken-down after SCI, much more complement enter the injuried spinal cord, The complement of blood-derived and synthesized by the local injuried spinal cord, which was activated and produced a lot of functional complement fragment such as C5a and C3a. So C5a may affect the glial scar formation and hindlimb locomotion recovery by mediating the inflammatory reaction after SCI. In this study, female C57BL/6J mice were used to establish the SCI model. C5a receptor antagonist (C5aRA),AcF-[OP(D-Cha)WR](PMX53) was used to block the effects of C5a by antagonizing its receptor. Controls were established as normal saline injection group, methylprednisolone (MP) injection group and sham operation group. The effects of C5a in SCI were evaluated by compared the character of pro-inflammatory cytokines (tumor necrosis factor alpha (TNF-α) and interleukin-1β(IL-1β)) expression, microglia/macrophage reaction, glial scar formation and behavior score in each group. Those items were further evaluated in the SCI models developed by C5aR-/- and C5aR+/+ mice to identify if the C5a gene deletion method can lead to less secondary injury and better function recovery after SCI.Materials and Methods:Part I: Adult female C57BL/6J mice were used as experimental animals. C5a receptor antagonist injection group, normal saline injection group, MP injection group as well as the sham operation group were established respectively. In C5aRA group, mice were injected i.p with C5a receptor antagonist peptides (1.0 mg/kg) 45min before and 24h after SCI. In MP injection group, mice were injected i.p with MP immediately after the spinal cord injury and at another 4 times with a 6h interval from the first injection (30mg/kg for each time). In control group, saline was injected i.p into the mice after SCI (10 ml/kg). For shame operation mice, only the vertebral lamina was opened, the dura mater and spinal cord were not damaged. The IL-1βand TNF-αconcentration in spinal cord were detected by ELISA method at normal mice and at 1h, 12h, 24h, 72h after SCI in each experimental group. The expression of IL-1βand TNF-αmRNA in spinal cord were also detected by RT-PCR at normal mice and at 1h, 12h, 24h, 72h after SCI in each experimental group. Flow cytometry detection was used to detect the percentage of microglia/macrophage in spinal cord at normal mice and at 1d, 3d, 7d, 14d after SCI in each experimental group. HE staining method was used to observe the pathological character of the spinal cord 4 weeks after injury in each group. Immunochemistry and Western blot detection of GFAP were used to evaluate the reaction character and degeree of astrocytes in the spinal cord 4 weeks after injury in each group. The mice hindlimb locomotion function recovery was evaluated by BMS score at 12h, 1d, 3d, 7d, 14d, 28d after SCI for each group.Part II: With the use of the SCI model developed with the C5aR-/- and C5aR+/+ mice, we compared the miroglia/ macrophage percentage, pathological character, astrocyte reaction in spinal cord, as well as BMS score between those two groups. All the experimental methods and detection time points were adopted the same as part I.Results:Part IWe successfully developed a vessel clamp crush SCI model with mice. The spinal cord of T11 segment was crushed to 0.2mm for 15 seconds. The spinal meninges were intact after SCI. Mice with a BMS score of 0 after SCI were thought to be handled successfully. C5aRA injection group, normal saline injection group, MP injection group and sham operation group were successfully established as we designed.1.Mice in each SCI group gained a BMS score of 0 at 12h after operation. Mice in the sham operation group didn't show the hindlimb dysfunction after SCI. At 7d after SCI, C5aRA injected mice showed better recovery than those injected with saline, but showed poor recovery in contrast to those injected with MP. The BMS score varied obviously in the three SCI groups at 28d after injury. The BMS score was significantly higher in the C5aRA injection group than in the saline injection group. The MP injected mice still showed better recovery than those in the other two groups.2.TNF-αand IL-1βconcentration in the spinal cord in each injury group increased greatly at 1h following SCI. The peak of those two pro-inflammation cytokines appeared at 1h and 12h respectively, and nearly returned to the normal level at 72 h. Mice in the shame operation control also showed mild increase of these two pro-inflammation cytokines. Compared with the saline injection group, MP injection group showed significantly lower level of TNF-αand IL-1βconcentration at each detection point. TNF-αconcentration was significantly suppressed in the C5aRA injection group in contrast to the saline injection group at each point, but the suppression degree was milder than the MP injection group. Significant IL-1βconcentration suppression was only detected at 12h after SCI in the C5aRA injection group when compared to the saline group.3.The same as TNF-αand IL-1βprotein, TNF-αand IL-1βmRNA in the spinal cord in each injury group increased greatly at 1h following SCI. The peak of those two pro-inflammation cytokines mRNA appeared at 1h and 12h respectively, and nearly returned to the normal level at 72 h. Mice in the shame operation control also showed mild increase of these two pro-inflammation cytokines mRNA. Compared with the saline injection group, MP injection group showed significantly lower level of TNF-αand IL-1βmRNA concentration at each detection point. TNF-αmRNA concentration was significantly suppressed in the C5aRA injection group in contrast to the saline injection group at each point. But the suppression degree was milder than the MP injection group. Significant IL-1βmRNA concentration suppression was only detected at 12h after SCI in the C5aRA injection group when compared to the saline group.4.The percentage of microglia/macrophage in the spinal cord in each injury group begin increaseing at 1d, increased greatly at 3d, the peak of the percentage of microglia/macrophage appeared at 7d, and it already decreased but still higher than normal level at 14d. Mice in the shame operation control also showed mild increase of the percentage of microglia/ macrophage. Compared with the saline injection group, MP injection group showed significantly lower level of the percentage of microglia/macrophage at each detection point. The percentage of microglia/macrophage was significantly suppressed in the C5aRA injection group in contrast to the saline injection group at each point, but the suppression degree was milder than the MP injection group.5.Histological observation of spinal cord was taken at 4w after injury. The injury spinal cord epicenter in each SCI group showed disorganization and central tunnel disappeared. A large number of inflammatory cells could be observed at the epicenter. Compared with the saline injection group, MP injection group showed less inflammatory cells and small cope at detection point. Compared with the saline injection group, C5aRA injection group showed less inflammatory cells and small cope at detection point, but more than the MP injection group. Spinal cord in the sham operation group didn't show the disorganization or inflammatory cells gathered.6 . 4 weeks after injury, Immunochemistry results showed irregularly-shaped cavitations at the injury epicenter. GFAP profusely expressed at cavity wall and near the injury site, GFAP positive astrocytes accumulated and proliferated around the epicenter, with hypertrophied cell bodies, thick prosesses, interwove with each other and constituted the dense glial scar at injury site. Compared with the saline group, the number GFAP positive astrocytes were decreased in C5aRA group, but more intensive than MP group. The GFAP positive astrocytes expression in shame operation control group as in normal spinal cord.PartⅡ1.Mice in the two group gained a BMS score of 0 at 12h after operation. From 7d after SCI, C5aR-/- mice showed better recovery than those C5aR+/+ mice .And at 28d after injury. The BMS score was significantly higher in the C5aR-/- mice than in the C5aR+/+ mice. Hindlimbs of C5aR-/- mice appeared frequent plantar stepping, but paws rotated at initial contact and lift off, gained a BMS score of 5.00±0.61. Hindlimbs of C5aR+/+ group mice showed plantar placing of the paw without weight support, occasional dorsal stepping but no plantar stepping, Gained a BMS score of 3.20±0.76.2.The percentage of microglia/macrophage in the spinal cord in the two group began increaseing at 1d after SCI, Increased greatly at 7d, and it already decreased but still higher than normal level at 14d. Compared with the C5aR+/+ mice group, the percentage of microglia/macrophage at injury epicenter in C5aR-/- mice showed significantly lower level at each detection point post injury. 3.HE staining suggestd that there was less inflammatory cells and more spared tissues in the spinal cord injury epicenter in C5aR-/-mice than in C5aR+/+ mice at 4w post injury.4.Immunochemistry and Western blot results both suggestd that GFAP expression at injury epicenter was more intensive in the C5aR+/+mice than in C5aR-/-mice.Conclusion:1.Used C5aRA to block the effects of C5a after spinal cord injury in mice, we observed that it can inhibit the mRNA expression and release of pro-inflammatory cytokines, IL-1βand TNF-αsignificantly, and decreased the activation and proliferation of microglia/macrophage, it's also can decreased the glial scar formation, at last it can improved hindlimb function. Those indicated that, C5a may be harmful to the repair of the injuried spinal cord by enhancing the inflammatory reaction and glial scar formation.2.Compared with the C5aR-/- mice, The results showed that there was intensive inflammatory reaction and glial scar formation in C5aR+/+mice after spinal cord injury. Once again to indicated that C5a may be harmful to the repair of the injuried spinal cord.In summary, SCI model was established by used the vessel clamp to crush mice spinal cord., the effects of C5a in the secondary injury after SCI was mearned by using C5aRA and C5aR-/- mice. Results suggested that C5a may be harmful to the repair of spinal cord injury by enhancing the inflammatory reaction and glial scar formation , then affect the function of hindlimb in mice.