Study On The Inflammatory Effects Of Serum Albumin And Complement In Central Nervous System Injury

Aim:Constant blood-brain barrier dysfunction can occur in a lot of neurological diseases, which may cause the exposing of central nervous system (CNS) cells to serum factors with the potential to trigger neurological inflammation signalling cascades. Previous studies demonstrated that inflammation was constant activated at the sites of BBB injury. Since constant and over-activated inflammation may be harmful to the CNS, intervention on the BBB dysfunction related inflammation may decrease the injury to CNS and promote healing. So it's valuable to elucidate the mechanism of BBB injury related inflammation.Albumin is approximately absent from the adult brain in physiological situations whilst it's greatly abundant in the blood system. Increase of intracerebral albumin levels has been reported in a lot of neurologic diseases with BBB impairment. But little has been known about the effects of serum albumin on CNS in those conditions. For this purpose, we focused on the inflammatory effects of serum albumin on microglia, the CNS major inflammatory cells, to identify the potential mechanism of serum albumin in CNS injury.It has been reported that activation of complement, another serum factor, can mediate and aggratate the CNS inflammation. Inflammation can cause secondary injury and glial scar formation in CNS injury. So the second objective of our study was to investigate the possible neuroprotective effects of knocking out the C3 (a key component of complement activation) gene in a mouse spinal cord injury (SCI) model.Materials and Methods:Part I: Microglia, residential macrophages in the central nervous system, can be activated and release a variety of factors including cytokines, chemokines, etc. to mediate the inflammation reaction when the microenvironment was changed in CNS. As our study object, microglial cells were treated with 0.5 mg/ml serum albumin, and untreated PBS controls were set up as well. Total RNAs were extracted at different time points of treatment (3, 6, 9, and 12 h). RT-PCR and Real Time RT-PCR were used to detect the expression of IL-1βand TNF-αmRNA. Serum albumin (0.5 mg/ml) was used to examine the time course of IL-1βand TNF-αproduction. The culture supernatants were collected at 3, 6, 12, 24 h of treatment. IL-1βand TNF-αconcentration were detected by ELISA method. 0.1-2 mg/ml serum albumin were used to investigate the relationship of IL-1βand TNF-αproduction with the stimulator concentration. Further more, 0.5μl serum albumin solution with a concentration of 100 mg/ml was injected into the cortex of C57BL/6J mouse. We checked the microglia activation in vivo with the method of immunochemistry.Part II: To develop and characterize a clinically relevant, graded model of clip compressive SCI in the mouse. C57BL/6J mice were subjected to laminectomy of T11. With the use of a modified vessel clamp, the spinal cords at this level were laterally compressed to 0.1, 0.25, and 0.5 mm, respectively. Functional and pathological changes of spinal cord were evulated by BMS scoring and HE staining. In the 0.18-mm injured mice, the spinal cord time-course changes of histological character, GFAP expression and microglia activation were studied with HE staining, immunohistochemistry and flow cytometry. Nerve regeneration was evaluated by anterograde tract tracing technique.Part III: With the use of the model developed in part II, we compared the BMS score, histopathologic change, microglia activation and GFAP expression in the C3-/- and C3+/+ mice after SCI.Results:Part I:1. Our results showed significant increases in the mRNA levels of IL-1βand TNF-αat 3, 6, 9, and 12 h of treatment in serum albumin-treated microglial cells when compared with untreated controls.2. Serum albumin (0.1-2 mg/ml) enhanced the secretion of IL-1βand TNF-αby microglia in a dose-dependent manner after 24 h of treatment. In addition, LPS (2μg/ml) significantly induced the increases in IL-1βand TNF-αrelease.3. In 0.5 mg/ml serum albumin-treated cultures, the IL-1βlevel of the culture supernatants began to increase significantly from 6 h of serum albumin treatment and continued to increase by 24 h of serum albumin treatment. The TNF-αcontent in the supernatants increased significantly early at 3 h of serum albumin treatment and the maximum level was detected at 24 h of serum albumin treatment. 4. Activated microglia with larger cell bodies and short, thick, or no processes were observed only along the needle tract because of mechanical damage in PBS-injected mice. However, serum albumin triggered more profound activation of microglia in the mice cortex.Part II:1. We developed a vessel clamp crush SCI model that was readily available, easy to reproduce, affordable and clinically relevant. Mice exhibited varying degrees of functional loss and spontaneous recovery depending on injury severity. There were significant differences among the three laterally compressed injury groups in behavioral measures. The more severe injury caused poorer recovery.2. Histological observation showed disrupted structure, inflammatory cells infiltration and microcystic cavitations at the injury epicenter of spinal cord at 8w post-injury. In accordance with the behavior result, the more severe injury caused less spared normal tissues and a wider scar.3. A serial histological observation of spinal cord was taken at 3d, 1w, 2w and 4w after injury. The injury epicenter showed degeneration and necrosis at 3d post-injury. A large number of inflammatory cells could be observed at the epicenter and a secondary injury zone could be identified at the first week. Then the range and degree of inflammatory cells infiltration and secondary injury decreased as time goes by.4. GFAP staining showed that astrocytes at the epicenter died at 3d after injury while the astrocytes around had no obvious change. Activated astrocytes with larger cell bodies and long, thick processes were found at the rostral and caudal sites around the epicenter and there were no GFAP-ip cells in the epicenter. The number of activated astrocytes increased at 2w after injury while the degree of activation decreased. At 4w and 8w post-injury, the range of activated astrocytes significantly attenuated. But there were still a number of activated astrocytes around the epicenter which formed the glial scar.5. In 0.25 mm injury group, immunohistochemistry and BDA anterograde tracing showed a dense glial scar formation at each side of injury epicenter which hindered most regenerative corticospinal tract fibers from reaching the caudal side of injury. But there is still a small number of BDA staining nerve fibers passed through the lesion epicenter and reached the caudal spinal cord via a path located at the dorsal site above corticospinal tract.6. Flow cytometry detection showed the CD68 positive cells (activated microglia/ macrophages) appeared at 3d post injury with a high activation degree, reached the peak at 7d and lasted for at least 8 weeks.Part III:1. BMS score indicated that C3-/- mice gained a better recovery than C3+/+ mice.2. HE staining suggestd that there were less inflammatory cells infiltration and more spared tissues in the spinal cord injury epicenter in C3-/- mice than in C3+/+ mice at 4w post injury.3. The number of activated microglia/macrophages at injury epicenter was less in C3-/- mice than in C3+/+ mice at 3d and 7d post injury.4. Immunochemistry and Western blot results both suggestd that GFAP expression at injury epicenter was more intensive in the C3+/+ mice than in C3-/- mice.Conclusion:In this study, we observed that serum albumin not only promoted the activation and proliferation of microglia, but also induced significant upregulation of mRNA expression and release of pro-inflammatory cytokines, IL-1βand TNF-α, by microglial cells. Secondly, we developed a vessel clamp crush SCI model in mice which was readily available, easy to reproduce, affordable and clinically relevant. Our model can simulate the pathological changes of tissue injury, inflammatory cells infiltration, microglia and astrocytes activation after spinal cord injury in human beings and is suitable for related researches. Thirdly, complement system may be harmfult to the repair of spinal cord injury by enhancing the inflammatory reaction and glial scar formation.In summary, our results will help further understand the inflammatory effects of serum factors on CNS injury. It will provide more evidence for the means of curing CNS disease by mediating the inflammation reaction in CNS.