| Part OneIsolated, cultivated, identified and compared of brain and spinal cord microvascular pericytes from Wistar ratsObjective:To compare the migrating rate, tube formation, proliferation and BBB and BSCB associated proteins of brain and spinal cord microvacular pericytes, they were isolated, cultured and identified.Methods:Pericytes were isolated from brain and spinal cord from3-week-old male rat. High-speed centrifugal method was used in this experiment and the capillaries and other impurities were separated into different layers. Then the microvessels were cultivated in the pericyte-specific medium. Pericyte was isolated from the microvessels. The cultured pericyte was performed in the following experiments: identification using NG2and PDGFRβ, migration ability using scratched test assay, proliferation using BrdU incorporation assay and the cell cycle assay, tubular formation ability using matrigel tubular assay and detected the associated proteins of BBB and BSCB using western blot assay. At last, these results were used to compare BMP to SCMP in their characteristic.Results:It was observed and compared the different morphology of BMPs and SCMPs. The results indicated that BMPs possessed bigger cell nucleus and more unfolded cytoplasm than SCMPs. BMPs and SCMPs were identified by using two markers NG2and platelet-derived growth factor receptorβ (PDGFRβ). Meanwhile, BMP and SCMP had the negative results with von Willebrand Factor (vWF) and Glial fibrillary acidic protein (GFAP) markers. Thus, it was indicated that the isolated BMPs and SCMPs did not contaminate with endothelial cells and astrocytes. The result showed that BMPs expressed much more F-actin than SCMPs (P<0.01). The scratch test was used to determine the migration of BMPs and SCMPs. The number of BMPs that migrated into the sound area was79±5, while that of SCMPs was140±4, P<0.01. There was a significant difference between the two types of pericytes. It was found that both the two types of pericytes could form vascular tube on the matrigel. However, we observed the tube length of BMPs (4.66±0.25mm) was larger than SCMPs (2.56±0.49mm). There was a significant difference between them (P<0.01). Using BrdU incorporation assay, the result indicated that there was no significant difference between BMPs and SCMPs. The cell cycle of BMPs and SCMPs was detected by Flow Cytometer. The result listed as follows: BMP:Gi=71.0±2.5%; G2=12.0±1.2%; S=17.1±1.7%; SCMP:Gi=74.9±2.8%; G2=10.4±1.3%; S=14.7±1.1%. There was no significant difference between the two types of pericytes in cell cycle. The results of BrdU incorporation and cell cycle indicated the proliferation of BMPs was no significant difference compared to that of SCMPs. Both BMPs and SCMPs expressed a-SMA, connexin43, NG2, VEGF, desmin, N-cadherin, PDGFRβ and TLR4. BMPs expressed more a-SMA, connexin43, NG2and VEGF. However, SCMPs expressed more N-cadherin, PDGFRβ and desmin. There were significant differences in the above results of WB. Meanwhile, both the two types of pericytes expressed TLR4, and there was no significant difference.Conclusion:This report showed that there were some different characteristics between BMPs and SCMPs when they were cultured alone. It was observed at the first time:a. Pericyte was successfully isolated from spinal cord microvessel. b. Both BMPs and SCMPs expressed N-cadherin, connexin43, desmin and TLR4. c. Monoculture BMPs and SCMPs formed vascular-tube. d. BMPs had stronger tube-formation ability. e. SCMPs possessed stronger ability of migration. These results strongly supported that the BBB integrity and stability could be perfective than BSCB from the angle of pericyte. The different characteristics in pericytes will help us to understand the defense mechanisms and inflammatory response mediated by pericytes in brain and spinal cord. These distinguishing features may reflect the more widespread differences between the BBB and BSCB which directly impact pathophysiological processes in various major diseases. Part TwoStudy on the mechanism of melatonin treated spinal cord injury in C57BL/6miceObjective:To investigate the mechanism of melatonin treated spinal cord injury in mice, the research studied the affection of melatonin on blood spinal cord barrier.Methods:Mice were randomized into the following three groups:(a) Sham+saline containing5%ethanol group (n=30), only removing vertebral plate but no impacting;(b) SCI+saline containing5%ethanol group (n=30), removing vertebral plate and impacting;(c) melatonin group (n=54), removing vertebral plate, impacting and administrating melatonin. The animals were subjected to an impact of50g/mm (5g weight from10mm height) to the dorsal surface of the spinal cord. Melatonin at the doses of5,10,25,50and100mg/kg was administered within30min,24h and48h after spinal cord injury. The permeability of different groups was determined using two tracers (Evans blue and NaFlu). The perfused micro vessels were labeled by LEA. Labeling CD31was used to analyze the endothelial cells surrounding the perfused microvessels. Double-labeling PDGFRβ and CD31immunofluorescence was used to analyze endothelial cells and pericytes on the microvessels. Double-labeling claudin5and CD31immunofluorescence was used to analyze the tight junction protein claudin5mainly secreted by endothelial cells. TUNEL assay was used to determine the apoptotic cells. Immunohistochemistry assay was used to analyze the tight junction proteins ZO-1, claudin5and occludin. Western blot assay was used to analyze the permeability and angiogenesis associated proteins, which included MMP3, AQP4, HIF-la, VEGF and VEGFR2. Results:The permeability of spinal cord injury was determined by two tracers Evans blue (EB) and NaFlu. The results listed as follows: the amount of EB0.4997±0.0729μg/mg and NaFlu822.1±153.7ng/mg in the injured groups. EB0.2643±0.0386μg/mg, NaFlu447.9±102.3ng/mg in the melatonin treated group. There were significant differences between the injured groups and the melatonin-treated groups (P<0.05). The apoptotic cell was detected by TUNEL assay. The result showed that the number of apoptotic cells in melatonin treated group (151.0±9.7) decreased more than those in the injured group (82.3±6.9)(P<0.05). LEA/CD31and CD31/claudin5were co-labeled perfused microvessels and endothelial cells and the tight junction protein claudin5that mainly secreted by endothelial cells using immunofluorescence assay. The results showed that the microvessels and endothelial cells were disrupted and claudin5was destroyed after impacted. After melatonin treatment, the amounts of perfused microvessels, endothelial cells and the expression of claudin5were markly increased. The change of tight junction proteins ZO-1, occludin and claudin5was tested using immunohistochemistry. The expression of tight junction proteins was increased after melatonin treated. The BSCB associated proteins were determined by western blot assay. It was found that melatonin treatment could decrease the expression of MMP3, AQP4, VEGF, VEGFR2and HIF-la (P<0.05).Conclusion:After SCI, the permeability of BSCB increased. Edema formation was found in spinal cord tissue. Tight junction proteins ZO-1, occludin and claudin-5were disrupted. Microvessels were destroyed. The amount of perfused microvessels was decreased. A large number of apoptosis cells appeared, which partly contained endothelial cells and pericytes. The expression of AQP4was increased, which indicated that the edema generated in spinal cord. The up-regulated expression of VEGF suggested that angiogenesis was in progress. All of the above pathological changes indicated that BSCB was disrupted after SCI. While, the permeability of BSCB was decreased after melatonin treated. This therapeutic measure could increase the expression of tight junction proteins, protect the microvessels from disruption, increase the perfusing microvessels, decrease the number of apoptosis cells, inhibit angiogenesis and decrease the edema in the spinal cord. All of the above results in the melatonin treatment indicated that melatonin could protect the disrupted microvessels and BSCB after SCI. |
PDF Full Text Request