C57小鼠急性脊髓损伤微循环功能障碍机理及治疗机制研究
|
摘要
第一部分
Wistar大鼠脑和脊髓微血管周细胞的分离、培养、鉴定及其功能差异比较
目的：
分离培养并鉴定Wistar大鼠脑微血管周细胞(brain microvascular pericyte, BMP)和脊髓微血管周细胞(spinal cord microvascular pericyte, SCMP),系统比较两种周细胞的异同点。
方法：
3周龄雄性Wistar大鼠,取其大脑和脊髓组织,运用超高速离心方法使得微血管和其他杂质分成不同的层次,吸出微血管片段层,然后用含10%FBS的DMEM培养基培养,观察周细胞从微血管中爬出。用NG2和PDGFRβ鉴定周细胞、用划痕实验测定细胞迁移能力、用BrdU和细胞周期测定细胞增殖、用基质胶成管实验测定成管能力和血管新生能力和用免疫印记分析实验测定屏障相关蛋白。
结果：
本部分实验观察并比较了BMP和SCMP形态差异,发现BMP较SCMP连接更为紧密,并且BMP较SCMP拥有更大的细胞核,展开面积更大的胞浆；运用周细胞非特异性标记物NG2和PDGFRβ鉴定分离得到的细胞为周细胞,并用vWF和GFAP排除了内皮细胞和星形胶质细胞的污染；发现相同细胞数目的BMP表达F-actin显著多于SCMP (P<0.01);用划痕实验测定了两种周细胞的迁移能力,BMP的迁移数目为79±5,显著低于SCMP的140±4(P<0.01)；将周细胞种在基质胶上,发现两种周细胞均能够成管,且BMPs成管长度4.66±0.25mm显著大于SCMPs的2.56±0.49mm(P<0.05):用BrdU结合新生的周细胞,和所有的周细胞merge以后发现两种周细胞和BrdU的结合率并无显著性差异(P>0.05),说明两种周细胞的增殖能力无显著性区别；归类统计处于不同分裂时期的细胞,结果发现BMP:G1=71.0±2.5%；G2=12.0±1.2%；S=17.1±1.7%；SCMP: G1=74.9±2.8%；G2=10.4±1.3%;S=14.7±1.1％.两种周细胞在上述细胞周期的3个分裂时期无显著性差异,故认为它们的细胞增殖能力没有显著性区别；BMP和SCMP均表达a-SMA.connexin43.NG2.N-cadherin、PDGFRβ、VEGF、desmin和TLR4.BMP表达的α-SMA.connexin43、NG2、PDGFRβ和VEGF显著高于SCMP(P<0.05),而SCMP表达的N-cadherin和desmin显著高于BMP(P<0.05)。同时,两种周细胞均表达TLR4,表达量没有显著性差异(P>0.05)。
结论：
通过提取、分离、鉴定Wistar大鼠脑和脊髓微血管周细胞及比较两种周细胞之间的不同,我们首次从脊髓微血管中分离得到周细胞；发现BMPs和SCMPs均表达N-cadherin、connexin43、desmin、VEGF和TLR4；发现BMPs和SCMPs分别在单独培养状态下均能成管；发现BMPs相比SCMPs有更强的成管能力；发现SCMPs相比BMPs有更强的迁移能力。BMPs和SCMPs间的不同特征有助于我们认识由周细胞调节的防御机制和炎症反应,更广泛地反映BBB和BSCB的不同之处,这些不同可能与许多重大疾病的病理生理进程相关。
第二部分褪黑素治疗C57BL/6小鼠急性脊髓损伤机制研究
目的：
通过研究褪黑素对血脊髓屏障的保护作用,探讨其保护C57小鼠脊髓损伤的相关机制。
方法：
C57BL/6小鼠被随机分成三组：假手术组(n=36),仅摘除椎板不打击,每只小鼠腹腔注射0.4mL生理盐水(内含5%乙醇)；SCI组(n=36),既摘除椎板又打击,每只小鼠腹腔注射0.4mL生理盐水(内含5%乙醇)；褪黑素组(n=60),既摘除椎板又打击后腹腔给药(剂量为5、10、25、50和100mg/kg)。模型组和给药组是用Aliens打击装置对小鼠脊髓造成50g/mm的中度打击(重量为5g的打击棒从10mm高处坠落)。给褪黑素或给生理盐水时间三组一致,为打击后或摘除椎板后10min、24h和48h三个时间点。使用通透性示踪剂Evans blue (EB)和荧光素钠(NaFlu)检测脊髓损伤及给药以后损伤部位的EB和NaFlu渗透量变化,对通透性进行定量分析；取注射EB小鼠的脊髓做冰冻切片,于荧光显微镜下观察,比较渗出EB的荧光面积,对损伤进行定量分析。用LEA标注有灌注功能的微血管,检测微血管在损伤前后血流灌注情况。用CD31标注内皮细胞,检测微血管的变化。用CD31和PDGFRP共同标记内皮细胞/周细胞,分析微血管上内皮细胞和周细胞的变化。用CD31和claudin-5共标,以检测主要由内皮细胞分泌的紧密连接蛋白claudin-5变化。用TUNEL测定损伤前后细胞凋亡情况,分析给药对细胞的保护作用。用免疫组化方法(IHC)检测紧密连接蛋白ZO-1,occludin和claudin-5,观察损伤前后蛋白的变化。应用免疫印记分析方法(Western blot),检测通透性相关蛋白：MMP3, AQP4, HIF-1a, VEGF和VEGFR2等。
结果：
本实验通过运用Evans Blue (EB)和NaFlu两种荧光示踪剂,定量测定SCI给药前后通透性的变化,发现给药后测得EB渗透量0.26±0.04μg/mg显著低于损伤组的0.50±0.08μg/mg(P<0.05)；给药后测得NaFlu渗透量822.1±153.7ng/mg显著低于损伤组的447.9±102.3ng/mg(P<0.05)；通过TUNEL实验,给药组后角部位细胞凋亡数目为82.3±6.9显著低于损伤组的151.0±9.7(P<0.05)；通过IF实验,发现有灌注功能的微血管和总微血管,在给药后均得到保护；微血管上最重要的两种细胞周细胞和内皮细胞,在给药后也得到保护；通过IHC实验,定性观测SCI给药前后连接蛋白的变化,发现给药后连接蛋白的表达量较损伤组均增加；用Western blot来进行BSCB相关蛋白检测,发现褪黑素均能够显著减少脊髓损伤后MMP3、AQP4、VEGF、VEGFR2和HIF-1α的表达水平(P<0.05)。
结论：
脊髓损伤后脊髓屏障的通透性升高,组织水肿,屏障中紧密连接蛋白ZO-1,occludin和claudin-5被破坏,微血管被破坏,其灌注水平下降,细胞凋亡,周细胞和内皮细胞大量死亡,AQP4表达增多,VEGF表达量增多,以上这些病理现象均表明脊髓损伤后血脊髓屏障被破坏。用褪黑素治疗后可以降低脊髓损伤时血脊髓屏障的通透性,提高屏障中紧密连接蛋白的含量,保护原有的微血管不被破坏,提高微血管灌注水平,减少组织内细胞凋亡,抑制血管新生,减少组织水肿。这些结果均提示褪黑素可以通过保护脊髓损伤后被破坏的微血管和血脊髓屏障,改善微循环来达到治疗脊髓损伤的目的。
Part One
Isolated, cultivated, identified and compared of brain and spinal cord microvascular pericytes from Wistar rats
Objective:
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 Two
Study on the mechanism of melatonin treated spinal cord injury in C57BL/6mice
Objective:
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.
Wistar大鼠脑和脊髓微血管周细胞的分离、培养、鉴定及其功能差异比较
目的：
分离培养并鉴定Wistar大鼠脑微血管周细胞(brain microvascular pericyte, BMP)和脊髓微血管周细胞(spinal cord microvascular pericyte, SCMP),系统比较两种周细胞的异同点。
方法：
3周龄雄性Wistar大鼠,取其大脑和脊髓组织,运用超高速离心方法使得微血管和其他杂质分成不同的层次,吸出微血管片段层,然后用含10%FBS的DMEM培养基培养,观察周细胞从微血管中爬出。用NG2和PDGFRβ鉴定周细胞、用划痕实验测定细胞迁移能力、用BrdU和细胞周期测定细胞增殖、用基质胶成管实验测定成管能力和血管新生能力和用免疫印记分析实验测定屏障相关蛋白。
结果：
本部分实验观察并比较了BMP和SCMP形态差异,发现BMP较SCMP连接更为紧密,并且BMP较SCMP拥有更大的细胞核,展开面积更大的胞浆；运用周细胞非特异性标记物NG2和PDGFRβ鉴定分离得到的细胞为周细胞,并用vWF和GFAP排除了内皮细胞和星形胶质细胞的污染；发现相同细胞数目的BMP表达F-actin显著多于SCMP (P<0.01);用划痕实验测定了两种周细胞的迁移能力,BMP的迁移数目为79±5,显著低于SCMP的140±4(P<0.01)；将周细胞种在基质胶上,发现两种周细胞均能够成管,且BMPs成管长度4.66±0.25mm显著大于SCMPs的2.56±0.49mm(P<0.05):用BrdU结合新生的周细胞,和所有的周细胞merge以后发现两种周细胞和BrdU的结合率并无显著性差异(P>0.05),说明两种周细胞的增殖能力无显著性区别；归类统计处于不同分裂时期的细胞,结果发现BMP:G1=71.0±2.5%；G2=12.0±1.2%；S=17.1±1.7%；SCMP: G1=74.9±2.8%；G2=10.4±1.3%;S=14.7±1.1％.两种周细胞在上述细胞周期的3个分裂时期无显著性差异,故认为它们的细胞增殖能力没有显著性区别；BMP和SCMP均表达a-SMA.connexin43.NG2.N-cadherin、PDGFRβ、VEGF、desmin和TLR4.BMP表达的α-SMA.connexin43、NG2、PDGFRβ和VEGF显著高于SCMP(P<0.05),而SCMP表达的N-cadherin和desmin显著高于BMP(P<0.05)。同时,两种周细胞均表达TLR4,表达量没有显著性差异(P>0.05)。
结论：
通过提取、分离、鉴定Wistar大鼠脑和脊髓微血管周细胞及比较两种周细胞之间的不同,我们首次从脊髓微血管中分离得到周细胞；发现BMPs和SCMPs均表达N-cadherin、connexin43、desmin、VEGF和TLR4；发现BMPs和SCMPs分别在单独培养状态下均能成管；发现BMPs相比SCMPs有更强的成管能力；发现SCMPs相比BMPs有更强的迁移能力。BMPs和SCMPs间的不同特征有助于我们认识由周细胞调节的防御机制和炎症反应,更广泛地反映BBB和BSCB的不同之处,这些不同可能与许多重大疾病的病理生理进程相关。
第二部分褪黑素治疗C57BL/6小鼠急性脊髓损伤机制研究
目的：
通过研究褪黑素对血脊髓屏障的保护作用,探讨其保护C57小鼠脊髓损伤的相关机制。
方法：
C57BL/6小鼠被随机分成三组：假手术组(n=36),仅摘除椎板不打击,每只小鼠腹腔注射0.4mL生理盐水(内含5%乙醇)；SCI组(n=36),既摘除椎板又打击,每只小鼠腹腔注射0.4mL生理盐水(内含5%乙醇)；褪黑素组(n=60),既摘除椎板又打击后腹腔给药(剂量为5、10、25、50和100mg/kg)。模型组和给药组是用Aliens打击装置对小鼠脊髓造成50g/mm的中度打击(重量为5g的打击棒从10mm高处坠落)。给褪黑素或给生理盐水时间三组一致,为打击后或摘除椎板后10min、24h和48h三个时间点。使用通透性示踪剂Evans blue (EB)和荧光素钠(NaFlu)检测脊髓损伤及给药以后损伤部位的EB和NaFlu渗透量变化,对通透性进行定量分析；取注射EB小鼠的脊髓做冰冻切片,于荧光显微镜下观察,比较渗出EB的荧光面积,对损伤进行定量分析。用LEA标注有灌注功能的微血管,检测微血管在损伤前后血流灌注情况。用CD31标注内皮细胞,检测微血管的变化。用CD31和PDGFRP共同标记内皮细胞/周细胞,分析微血管上内皮细胞和周细胞的变化。用CD31和claudin-5共标,以检测主要由内皮细胞分泌的紧密连接蛋白claudin-5变化。用TUNEL测定损伤前后细胞凋亡情况,分析给药对细胞的保护作用。用免疫组化方法(IHC)检测紧密连接蛋白ZO-1,occludin和claudin-5,观察损伤前后蛋白的变化。应用免疫印记分析方法(Western blot),检测通透性相关蛋白：MMP3, AQP4, HIF-1a, VEGF和VEGFR2等。
结果：
本实验通过运用Evans Blue (EB)和NaFlu两种荧光示踪剂,定量测定SCI给药前后通透性的变化,发现给药后测得EB渗透量0.26±0.04μg/mg显著低于损伤组的0.50±0.08μg/mg(P<0.05)；给药后测得NaFlu渗透量822.1±153.7ng/mg显著低于损伤组的447.9±102.3ng/mg(P<0.05)；通过TUNEL实验,给药组后角部位细胞凋亡数目为82.3±6.9显著低于损伤组的151.0±9.7(P<0.05)；通过IF实验,发现有灌注功能的微血管和总微血管,在给药后均得到保护；微血管上最重要的两种细胞周细胞和内皮细胞,在给药后也得到保护；通过IHC实验,定性观测SCI给药前后连接蛋白的变化,发现给药后连接蛋白的表达量较损伤组均增加；用Western blot来进行BSCB相关蛋白检测,发现褪黑素均能够显著减少脊髓损伤后MMP3、AQP4、VEGF、VEGFR2和HIF-1α的表达水平(P<0.05)。
结论：
脊髓损伤后脊髓屏障的通透性升高,组织水肿,屏障中紧密连接蛋白ZO-1,occludin和claudin-5被破坏,微血管被破坏,其灌注水平下降,细胞凋亡,周细胞和内皮细胞大量死亡,AQP4表达增多,VEGF表达量增多,以上这些病理现象均表明脊髓损伤后血脊髓屏障被破坏。用褪黑素治疗后可以降低脊髓损伤时血脊髓屏障的通透性,提高屏障中紧密连接蛋白的含量,保护原有的微血管不被破坏,提高微血管灌注水平,减少组织内细胞凋亡,抑制血管新生,减少组织水肿。这些结果均提示褪黑素可以通过保护脊髓损伤后被破坏的微血管和血脊髓屏障,改善微循环来达到治疗脊髓损伤的目的。
Part One
Isolated, cultivated, identified and compared of brain and spinal cord microvascular pericytes from Wistar rats
Objective:
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 Two
Study on the mechanism of melatonin treated spinal cord injury in C57BL/6mice
Objective:
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.
引文
[1]. Xiu RJ IM. Computer analysis of the microvascular vasomotion [J]. Chin Med J (Engl) 1986; 99:351-360.
[2]. Xiu RJ IM. [Microvascular vasomotion: I. Long-term observation and computer analysis of the vasomotion] [J]. Zhonghua Yi Xue Za Zhi 1985; 65:129-135.
[3].修瑞娟,B.W.Zweifach.微血管自律运动研究[J].中华医学杂志1985；65：6.
[4]. Xiu RJ e. The correlation of microcirculatory dynamics and clinical symptoms of diseases. [J]. Basel: Karger 1984.
[5]. Xiu RJ HD, Coppo PA, et al. Anisodamine inhibits thromboxane synthesis, granulocyte aggregation, and platelet aggregation. A possible mechanism for its efficacy in bacteremic shock [J]. JAMA 1982; 247:1458-1460.
[6].史晓瑞,修瑞娟.微血管周细胞的研究进展[J].中国微循环杂志2009；13(6)：582-584.
[7].鹿文葆,秦伟伟,张秋菊,李宏伟,刘淑英,修瑞娟.脑微血管内皮细胞和周细胞共培养构建体外血脑屏障模型[J].国际脑血管病杂志2012；20(5)：338-342.
[8]. Etchevers HC, Vincent C, Le Douarin NM, Couly GF. The cephalic neural crest provides pericytes and smooth muscle cells to all blood vessels of the face and forebrain [J]. Development 2001; 128(7):1059-1068.
[9]. Korn J, Christ B, Kurz H. Neuroectodermal origin of brain pericytes and vascular smooth muscle cells [J]. J Comp Neurol 2002; 442(1):78-88.
[10]. Kurz H. Cell lineages and early patterns of embryonic CNS vascularization [J]. Cell Adh Migr 2009; 3(2):205-210.
[11]. Winkler EA, Bell RD, Zlokovic BV. Central nervous system pericytes in health and disease [J]. Nat Neurosci 2011; 14(11):1398-1405.
[12]. Gaengel K, Genove G, Armulik A, Betsholtz C. Endothelial-mural cell signaling in vascular development and angiogenesis [J]. Arterioscler Thromb Vase Biol 2009; 29(5):630-638.
[13]. Lindahl P, Johansson BR, Leveen P, Betsholtz C. Pericyte loss and microaneurysm formation in PDGF-B-deficient mice [J]. Science 1997; 277(5323):242-245.
[14]. Tallquist MD, French WJ, Soriano P. Additive effects of PDGF receptor beta signaling pathways in vascular smooth muscle cell development [J]. PLoS Biol 2003; 1(2):E52.
[15]. Hellstrom M, Kalen M, Lindahl P, Abramsson A, Betsholtz C. Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse [J]. Development 1999; 126(14):3047-3055.
[16]. Leveen P, Pekny M, Gebre-Medhin S, Swolin B, Larsson E, Betsholtz C. Mice deficient for PDGF B show renal, cardiovascular, and hematological abnormalities [J]. Genes Dev 1994; 8(16):1875-1887.
[17]. Soriano P. Abnormal kidney development and hematological disorders in PDGF beta-receptor mutant mice [J]. Genes Dev 1994; 8(16):1888-1896.
[18]. Lindblom P, Gerhardt H, Liebner S, Abramsson A, Enge M, Hellstrom M, et al. Endothelial PDGF-B retention is required for proper investment of pericytes in the microvessel wall [J]. Genes Dev 2003; 17(15):1835-1840.
[19]. Bell RD, Winkler EA, Sagare AP, Singh I, LaRue B, Deane R, et al. Pericytes control key neurovascular functions and neuronal phenotype in the adult brain and during brain aging [J]. Neuron 2010; 68(3):409-427.
[20]. Winkler EA, Bell RD, Zlokovic BV. Pericyte-specific expression of PDGF beta receptor in mouse models with normal and deficient PDGF beta receptor signaling [J]. Mol Neurodegener 2010; 5:32.
[21]. Geraldes P, Hiraoka-Yamamoto J, Matsumoto M, Clermont A, Leitges M, Marette A, et al. Activation of PKC-delta and SHP-1 by hyperglycemia causes vascular cell apoptosis and diabetic retinopathy [J]. Nat Med 2009; 15(11): 1298-1306.
[22]. Li F, Lan Y, Wang Y, Wang J, Yang G, Meng F, et al. Endothelial Smad4 maintains cerebrovascular integrity by activating N-cadherin through cooperation with Notch [J]. Dev Cell 2011; 20(3):291-302.
[23]. Sieczkiewicz GJ, Herman IM. TGF-beta 1 signaling controls retinal pericyte contractile protein expression [J]. Micro vasc Res 2003; 66(3):190-196.
[24]. Lebrin F, Deckers M, Bertolino P, Ten Dijke P. TGF-beta receptor function in the endothelium [J]. Cardiovasc Res 2005; 65(3):599-608.
[25]. Walshe TE, Saint-Geniez M, Maharaj AS, Sekiyama E, Maldonado AE, D'Amore PA. TGF-beta is required for vascular barrier function, endothelial survival and homeostasis of the adult microvasculature [J]. PLoS One 2009; 4(4): e5149.
[26]. Braun A, Xu H, Hu F, Kocherlakota P, Siegel D, Chander P, et al. Paucity of pericytes in germinal matrix vasculature of premature infants [J]. J Neurosci 2007; 27(44):12012-12024.
[27]. Vinukonda G, Dummula K, Malik S, Hu F, Thompson CI, Csiszar A, et al. Effect of prenatal glucocorticoids on cerebral vasculature of the developing brain [J]. Stroke 2010; 41(8):1766-1773.
[28]. Van Geest RJ, Klaassen I, Vogels IM, Van Noorden CJ, Schlingemann RO. Differential TGF-{beta} signaling in retinal vascular cells: a role in diabetic retinopathy? [J]. Invest Ophthalmol Vis Sci 2010; 51(4):1857-1865.
[29]. Gerhardt H, Wolburg H, Redies C. N-cadherin mediates pericytic-endothelial interaction during brain angiogenesis in the chicken [J]. Dev Dyn 2000; 218(3): 472-479.
[30]. Winkler EA, Bell RD, Zlokovic BV. Lack of Smad or Notch leads to a fatal game of brain pericyte hopscotch [J]. Dev Cell 2011; 20(3):279-280.
[31]. Saharinen P, Eklund L, Miettinen J, Wirkkala R, Anisimov A, Winderlich M, et al. Angiopoietins assemble distinct Tie2 signalling complexes in endothelial cell-cell and cell-matrix contacts [J]. Nat Cell Biol 2008; 10(5):527-537.
[32]. Wakui S, Yokoo K, Muto T, Suzuki Y, Takahashi H, Furusato M, et al. Localization of Ang-1,-2, Tie-2, and VEGF expression at endothelial-pericyte interdigitation in rat angiogenesis [J]. Lab Invest 2006; 86(11):1172-1184.
[33]. Hofmann JJ, Iruela-Arispe ML. Notch signaling in blood vessels: who is talking to whom about what? [J]. Circ Res 2007; 100(11):1556-1568.
[34]. Liu H, Kennard S, Lilly B. NOTCH3 expression is induced in mural cells through an autoregulatory loop that requires endothelial-expressed JAGGED 1 [J]. Circ Res 2009; 104(4):466-475.
[35]. Liu H, Zhang W, Kennard S, Caldwell RB, Lilly B. Notch3 is critical for proper angiogenesis and mural cell investment [J]. Circ Res 2010; 107(7):860-870.
[36]. Regan JN, Majesky MW. Building a vessel wall with notch signaling [J]. Circ Res 2009; 104(4):419-421.
[37]. Stewart KS, Zhou Z, Zweidler-McKay P, Kleinerman ES. Delta-like ligand 4-Notch signaling regulates bone marrow-derived pericyte/vascular smooth muscle cell formation [J]. Blood 2011; 117(2):719-726.
[38]. Walshe TE, Connell P, Cryan L, Ferguson G, Gardiner T, Morrow D, et al. Microvascular retinal endothelial and pericyte cell apoptosis in vitro:role of hedgehog and Notch signaling [J]. Invest Ophthalmol Vis Sci 2011; 52(7): 4472-4483.
[39]. Jin S, Hansson EM, Tikka S, Lanner F, Sahlgren C, Farnebo F, et al. Notch signaling regulates platelet-derived growth factor receptor-beta expression in vascular smooth muscle cells [J]. Circ Res 2008; 102(12):1483-1491.
[40]. Winkler EA, Sengillo JD, Bell RD, Wang J, Zlokovic BV. Blood-spinal cord barrier pericyte reductions contribute to increased capillary permeability [J]. J Cereb Blood Flow Metab 2012; 32(10):1841-1852.
[41]. Shi X HW, Yamamoto H, Tang W, Lin X, Xiu R, Trune DR, Nuttall AL. The Cochlear Pericytes [J]. Microcirculation 2008; 15(6):515-529.
[42]. Sacharidou A, Stratman AN, Davis GE. Molecular mechanisms controlling vascular lumen formation in three-dimensional extracellular matrices [J]. Cells Tissues Organs 2012; 195(1-2):122-143.
[43]. Oudega M. Molecular and cellular mechanisms underlying the role of blood vessels in spinal cord injury and repair [J]. Cell Tissue Res 2012; 349(1): 269-288.
[44]. Sharma HS. Pathophysiology of blood-spinal cord barrier in traumatic injury and repair [J]. Curr Pharm Des 2005; 11(11):1353-1389.
[45]. Wiranowska M, Gonzalvo AA, Saporta S, Gonzalez OR, Prockop LD. Evaluation of blood-brain barrier permeability and the effect of interferon in mouse glioma model [J]. J Neurooncol 1992; 14(3):225-236.
[46]. Ge S, Pachter JS. Isolation and culture of microvascular endothelial cells from murine spinal cord [J]. J Neuroimmunol 2006; 177(1-2):209-214.
[47]. Abbott NJ. Evidence for bulk flow of brain interstitial fluid: significance for physiology and pathology [J]. Neurochem Int 2004; 45(4):545-552.
[48]. Abbott NJ, Patabendige AA, Dolman DE, Yusof SR, Begley DJ. Structure and function of the blood-brain barrier [J]. Neurobiol Dis 2010; 37(1):13-25.
[49]. Abbott NJ. Blood-brain barrier structure and function and the challenges for CNS drug delivery [J]. J Inherit Metab Dis 2013; 36(3):437-449.
[50]. Abbott NJ, Ronnback L, Hansson E. Astrocyte-endothelial interactions at the blood-brain barrier [J]. Nat Rev Neurosci 2006; 7(1):41-53.
[51]. Reese TS, Karnovsky MJ. Fine structural localization of a blood-brain barrier to exogenous peroxidase [J]. J Cell Biol 1967; 34(1):207-217.
[52]. Bernacki J, Dobrowolska A, Nierwinska K, Malecki A. Physiology and pharmacological role of the blood-brain barrier [J]. Pharmacol Rep 2008; 60(5): 600-622.
[53]. Armulik A, Genove G, Mae M, Nisancioglu MH, Wallgard E, Niaudet C, et al. Pericytes regulate the blood-brain barrier [J]. Nature 2010; 468(7323):557-561.
[54]. Daneman R, Zhou L, Kebede AA, Barres BA. Pericytes are required for blood-brain barrier integrity during embryo genesis [J]. Nature 2010; 468(7323): 562-566.
[55]. Peppiatt CM, Howarth C, Mobbs P, Attwell D. Bidirectional control of CNS capillary diameter by pericytes [J]. Nature 2006; 443(7112):700-704.
[56]. Zlokovic BV. The blood-brain barrier in health and chronic neurodegenerative disorders [J]. Neuron 2008; 57(2):178-201.
[57]. Diaz-Flores L, Gutierrez R, Madrid JF, Varela H, Valladares F, Acosta E, et al. Pericytes. Morphofunction, interactions and pathology in a quiescent and activated mesenchymal cell niche [J]. Histol Histopathol 2009; 24(7):909-969.
[58]. Hamilton NB, Attwell D, Hall CN. Pericyte-mediated regulation of capillary diameter: a component of neurovascular coupling in health and disease [J]. Front Neuroenergetics 2010; 2.
[59]. Chen CJ, Ou YC, Li JR, Chang CY, Pan HC, Lai CY, et al. Infection of pericytes in vitro by Japanese encephalitis virus disrupts the integrity of endothelial barrier [J]. J Virol 2013.
[60]. Hirooka T, Yamamoto C, Yasutake A, Eto K, Kaji T. Expression of VEGF-related proteins in cultured human brain microvascular endothelial cells and pericytes after exposure to methylmercury [J]. J Toxicol Sci 2013; 38(6): 837-845.
[61]. Liu S, Agalliu D, Yu C, Fisher M. The role of pericytes in blood-brain barrier function and stroke [J]. Curr Pharm Des 2012; 18(25):3653-3662.
[62]. Sagare AP, Bell RD, Zhao Z, Ma Q, Winkler EA, Ramanathan A, et al. Pericyte loss influences Alzheimer-like neurodegeneration in mice [J]. Nat Commun 2013; 4:2932.
[63]. Bartanusz V, Jezova D, Alajajian B, Digicaylioglu M. The blood-spinal cord barrier: morphology and clinical implications [J]. Ann Neurol 2011; 70(2): 194-206.
[64]. Hammes HP LJ, Renner O, Shani M, Lundqvist A, Betsholtz C, Brownlee M, Deutsch U. Pericytes and the pathogenesis of diabetic retinopathy. [J]. Diabetes 2002; 51:3107-3112.
[65]. Ciulla TA, Amador AG, Zinman B. Diabetic retinopathy and diabetic macular edema: pathophysiology, screening, and novel therapies [J]. Diabetes Care 2003; 26(9):2653-2664.
[66]. Crisan M, Yap S, Casteilla L, Chen CW, Corselli M, Park TS, et al. A perivascular origin for mesenchymal stem cells in multiple human organs [J]. Cell Stem Cell 2008; 3(3):301-313.
[67]. Kang SG, Shinojima N, Hossain A, Gumin J, Yong RL, Colman H, et al. Isolation and Perivascular Localization of Mesenchymal Stem Cells From Mouse Brain [J]. Neurosurgery 2010; 67(3):711-720.
[68]. Farrington-Rock C, Crofts NJ, Doherty MJ, Ashton BA, Griffin-Jones C, Canfield AE. Chondrogenic and adipogenic potential of microvascular pericytes [J]. Circulation 2004; 110(15):2226-2232.
[69]. Lamagna C, Bergers G The bone marrow constitutes a reservoir of pericyte progenitors [J]. J Leukocyte Biol 2006; 80(4):677-681.
[70]. Caplan AI. All MSCs are pericytes? [J]. Cell Stem Cell 2008; 3(3):229-230.
[71]. Renner O, Tsimpas A, Kostin S, Valable S, Petit E, Schaper W, et al. Time- and cell type-specific induction of platelet-derived growth factor receptor-beta during cerebral ischemia [J]. Mol Brain Res 2003; 113(1-2):44-51.
[72]. Lange S, Trost A, Tempfer H, Bauer HC, Bauer H, Rohde E, et al. Brain pericyte plasticity as a potential drug target in CNS repair [J]. Drug Discov Today 2013; 18(9-10):456-463.
[73]. Bergers G, Song S. The role of pericytes in blood-vessel formation and maintenance [J]. Neuro Oncol 2005; 7(4):452-464.
[74]. Stallcup WB. The NG2 proteoglycan: past insights and future prospects [J]. J Neurocytol2002; 31(6-7):423-435.
[75]. Kucharova K, Chang Y, Boor A, Yong VW, Stallcup WB. Reduced inflammation accompanies diminished myelin damage and repair in the NG2 null mouse spinal cord [J]. J Neuroinflammation 2011; 8:158.
[76]. Goritz C, Dias DO, Tomilin N, Barbacid M, Shupliakov O, Frisen J. A pericyte origin of spinal cord scar tissue [J]. Science 2011; 333(6039):238-242.
[77]. Liao Y, Day KH, Damon DN, Duling BR. Endothelial cell-specific knockout of connexin 43 causes hypotension and bradycardia in mice [J]. Proc Natl Acad Sci U S A 2001; 98(17):9989-9994.
[78]. Li AF, Sato T, Haimovici R, Okamoto T, Roy S. High glucose alters connexin 43 expression and gap junction intercellular communication activity in retinal pericytes [J]. Invest Ophthalmol Vis Sci 2003; 44(12):5376-5382.
[79]. Mogensen C, Bergner B, Wallner S, Ritter A, d'Avis S, Ninichuk V, et al. Isolation and functional characterization of pericytes derived from hamster skeletal muscle [J]. Acta Physiol (Oxf) 2011; 201(4):413-426.
[80]. Liu X, Sun L, Torii M, Rakic P. Connexin 43 controls the multipolar phase of neuronal migration to the cerebral cortex [J]. Proc Natl Acad Sci U S A 2012; 109(21):8280-8285.
[81]. Bandopadhyay R, Orte C, Lawrenson JG, Reid AR, De Silva S, Allt G Contractile proteins in pericytes at the blood-brain and blood-retinal barriers [J]. J Neurocytol 2001; 30(1):35-44.
[82]. Goldfarb LG, Vicart P, Goebel HH, Dalakas MC. Desmin myopathy [J]. Brain 2004; 127(Pt 4):723-734.
[83]. Gumbiner BM. Regulation of cadherin-mediated adhesion in morphogenesis [J]. Nat Rev Mol Cell Biol 2005; 6(8):622-634.
[84]. Tepass U, Truong K, Godt D, Ikura M, Peifer M. Cadherins in embryonic and neural morphogenesis [J]. Nat Rev Mol Cell Biol 2000; 1(2):91-100.
[85]. Scheef EA, Sorenson CM, Sheibani N. Attenuation of proliferation and migration of retinal pericytes in the absence of thrombospondin-1 [J]. Am J Physiol Cell Physiol 2009; 296(4):C724-734.
[86]. Ezan P, Andre P, Cisternino S, Saubamea B, Boulay AC, Doutremer S, et al. Deletion of astroglial connexins weakens the blood-brain barrier [J]. J Cereb Blood Flow Metab 2012; 32(8):1457-1467.
[87]. Karen J, Rodriguez A, Friman T, Dencker L, Sundberg C, Scholz B. Effects of the histone deacetylase inhibitor valproic acid on human pericytes in vitro [J]. PLoS One 2011; 6(9):e24954.
[88]. Thanabalasundaram G, Schneidewind J, Pieper C, Galla HJ. The impact of pericytes on the blood-brain barrier integrity depends critically on the pericyte differentiation stage [J]. Int J Biochem Cell Biol 2011; 43(9):1284-1293.
[89]. Li BW, Yuan, X.C., Li, H.W.&Xiu, R.J.15d-PGJ2 attenuates CSE-induced inhibition of HUVECs migration [J]. Basic & Clinical Medicine 2011; 31: 394-399.
[90]. Stratman AN, Malotte KM, Mahan RD, Davis MJ, Davis GE. Pericyte recruitment during vasculogenic tube assembly stimulates endothelial basement membrane matrix formation [J]. Blood 2009; 114(24):5091-5101.
[91]. Belanger M, Allaman I, Magistretti PJ. Brain energy metabolism: focus on astrocyte-neuron metabolic cooperation [J]. Cell Metab 2011; 14(6):724-738.
[92]. Quaegebeur A, Lange C, Carmeliet P. The neurovascular link in health and disease: molecular mechanisms and therapeutic implications [J]. Neuron 2011; 71(3):406-424.
[93]. Fonta C, Imbert M. Vascularization in the primate visual cortex during development [J]. Cereb Cortex 2002; 12(2):199-211.
[94]. Liang M, Ekblad E, Hellstrand P, Nilsson BO. Polyamine synthesis inhibition attenuates vascular smooth muscle cell migration [J]. J Vasc Res 2004; 41(2): 141-147.
[95]. Bedekovics J, Szeghalmy S, Beke L, Fazekas A, Mehes G Image analysis of platelet derived growth factor receptor-beta (PDGFRbeta) expression to determine the grade and dynamics of myelofibrosis in bone marrow biopsy samples [J]. Cytometry B Clin Cytom 2014.
[96]. Wallow IH, Burnside B. Actin filaments in retinal pericytes and endothelial cells [J]. Invest Ophthalmol Vis Sci 1980; 19(12):1433-1441.
[97]. Sims JR, Karp S, Ingber DE. Altering the cellular mechanical force balance results in integrated changes in cell, cytoskeletal and nuclear shape [J]. J Cell Sci 1992; 103 (Pt 4):1215-1222.
[98]. Meazzini MC, Toma CD, Schaffer JL, Gray ML, Gerstenfeld LC. Osteoblast cytoskeletal modulation in response to mechanical strain in vitro [J]. J Orthop Res 1998; 16(2):170-180.
[99]. Kristensson K, Olsson Y. Accumulation of protein tracers in pericytes of the central nervous system following systemic injection in immature mice [J]. Acta Neurol Scand 1973; 49(2):189-194.
[100]. Balabanov R, Dore-Duffy P. Role of the CNS micro vascular pericyte in the blood-brain barrier [J]. J Neurosci Res 1998; 53(6):637-644.
[101]. Allt G, Lawrenson JG Pericytes: cell biology and pathology [J]. Cells Tissues Organs 2001; 169(1):1-11.
[102]. Fisher M, French S, Ji P, Kim RC. Cerebral microbleeds in the elderly: a pathological analysis [J]. Stroke 2010; 41(12):2782-2785.
[103]. Fang H, Wang PF, Zhou Y, Wang YC, Yang QW. Toll-like receptor 4 signaling in intracerebral hemorrhage-induced inflammation and injury [J]. J Neuroinflammation 2013; 10:27.
[104]. Figueiredo RT, Fernandez PL, Mourao-Sa DS, Porto BN, Dutra FF, Alves LS, et al. Characterization of heme as activator of Toll-like receptor 4 [J]. J Biol Chem 2007; 282(28):20221-20229.
[105]. Smiley ST, King JA, Hancock WW. Fibrinogen stimulates macrophage chemokine secretion through toll-like receptor 4 [J]. J Immunol 2001; 167(5): 2887-2894.
[106]. Edelman DA, Jiang Y, Tyburski JG, Wilson RF, Steffes CP. Lipopolysaccharide activation of pericyte's Toll-like receptor-4 regulates co-culture permeability [J]. Am J Surg 2007; 193(6):730-735.
[107]. Edelman DA, Jiang Y, Tyburski J, Wilson RF, Steffes C. Toll-like receptor-4 message is up-regulated in lipopolysaccharide-exposed rat lung pericytes [J]. J Surg Res 2006; 134(1):22-27.
[108]. Choi JJ, Choi YJ, Chen L, Zhang B, Eum SY, Abreu MT, et al. Lipopolysaccharide potentiates polychlorinated biphenyl-induced disruption of the blood-brain barrier via TLR4/IRF-3 signaling [J]. Toxicology 2012; 302(2-3): 212-220.
[1]. Qiu J. China Spinal Cord Injury Network: changes from within [J]. Lancet Neurol 2009; 8(7):606-607.
[2]. Cripps RA, Lee BB, Wing P, Weerts E, Mackay J, Brown D. A global map for traumatic spinal cord injury epidemiology: towards a living data repository for injury prevention [J]. Spinal Cord 2011; 49(4):493-501.
[3]. Ray SK, Dixon CE, Banik NL. Molecular mechanisms in the pathogenesis of traumatic brain injury [J]. Histol Histopathol 2002; 17(4):1137-1152.
[4]. Rossignol S, Schwab M, Schwartz M, Fehlings MG Spinal cord injury: time to move? [J]. J Neurosci 2007; 27(44):11782-11792.
[5]. Tator CH. Biology of neurological recovery and functional restoration after spinal cord injury [J]. Neurosurgery 1998; 42(4):696-707; discussion 707-698.
[6]. Hulsebosch CE. Recent advances in pathophysiology and treatment of spinal cord injury [J]. Adv Physiol Educ 2002; 26(1-4):238-255.
[7]. Oudega M. Molecular and cellular mechanisms underlying the role of blood vessels in spinal cord injury and repair [J]. Cell Tissue Res 2012; 349(1): 269-288.
[8]. Griffiths IR, Burns N, Crawford AR. Early vascular changes in the spinal grey matter following impact injury [J]. Acta Neuropathol 1978; 41(1):33-39.
[9]. Noble LJ, Wrathall JR. Distribution and time course of protein extravasation in the rat spinal cord after contusive injury [J]. Brain Res 1989; 482(1):57-66.
[10]. Casella GT, Bunge MB, Wood PM. Endothelial cell loss is not a major cause of neuronal and glial cell death following contusion injury of the spinal cord [J]. Exp Neurol 2006; 202(1):8-20.
[11]. Sadrzadeh SM, Eaton JW. Hemoglobin-mediated oxidant damage to the central nervous system requires endogenous ascorbate [J]. J Clin Invest 1988; 82(5): 1510-1515.
[12]. Mautes AE, Weinzierl MR, Donovan F, Noble LJ. Vascular events after spinal cord injury: contribution to secondary pathogenesis [J]. Phys Ther 2000; 80(7): 673-687.
[13]. Makinde T, Agrawal DK. Intra and extravascular transmembrane signalling of angiopoietin-l-Tie2 receptor in health and disease [J]. J Cell Mol Med 2008; 12(3):810-828.
[14]. Wolburg H, Lippoldt A. Tight junctions of the blood-brain barrier: development, composition and regulation [J]. Vascul Pharmacol 2002; 38(6):323-337.
[15]. Armulik A, Genove G, Betsholtz C. Pericytes: developmental, physiological, and pathological perspectives, problems, and promises [J]. Dev Cell 2011; 21(2): 193-215.
[16]. Hardebo JE, Kahrstrom J. Endothelial negative surface charge areas and blood-brain barrier function [J]. Acta Physiol Scand 1985; 125(3):495-499.
[17]. Lossinsky AS, Shivers RR. Structural pathways for macromolecular and cellular transport across the blood-brain barrier during inflammatory conditions. Review [J]. Histol Histopathol 2004; 19(2):535-564.
[18]. Nag S, Manias JL, Stewart DJ. Expression of endothelial phosphorylated caveolin-1 is increased in brain injury [J]. Neuropathol Appl Neurobiol 2009; 35(4):417-426.
[19]. Hawkins BT, Lundeen TF, Norwood KM, Brooks HL, Egleton RD. Increased blood-brain barrier permeability and altered tight junctions in experimental diabetes in the rat: contribution of hyperglycaemia and matrix metalloproteinases [J]. Diabetologia 2007; 50(1):202-211.
[20]. Brightman MW, Zis K, Anders J. Morphology of cerebral endothelium and astrocytes as determinants of the neuronal microenvironment [J]. Acta Neuropathol Suppl 1983; 8:21-33.
[21]. Bracken MB, Shepard MJ, Holford TR, Leo-Summers L, Aldrich EF, Fazl M, et al. Administration of methylprednisolone for 24 or 48 hours or tirilazad mesylate for 48 hours in the treatment of acute spinal cord injury. Results of the Third National Acute Spinal Cord Injury Randomized Controlled Trial. National Acute Spinal Cord Injury Study [J]. JAMA 1997; 277(20):1597-1604.
[22]. Eck JC, Nachtigall D, Humphreys SC, Hodges SD. Questionnaire survey of spine surgeons on the use of methylprednisolone for acute spinal cord injury [J]. Spine (Phila Pa 1976) 2006; 31(9):E250-253.
[23]. Geisler FH, Dorsey FC, Coleman WP. Past and Current Clinical-Studies with Gm-1 Ganglioside in Acute Spinal-Cord Injury [J]. Ann Emerg Med 1993; 22(6): 1041-1047.
[24]. Chinnock P, Roberts I. Gangliosides for acute spinal cord injury [J]. Cochrane Db Syst Rev 2005; (2).
[25]. Courtine G, Roy RR, Raven J, Hodgson J, Mckay H, Yang H, et al. Performance of locomotion and foot grasping following a unilateral thoracic corticospinal tract lesion in monkeys (Macaca mulatta) [J]. Brain 2005; 128:2338-2358.
[26]. Tobias CA, Han SSW, Shumsky JS, Kim D, Tumolo M, Dhoot NO, et al. Alginate encapsulated BDNF-producing fibroblast grafts permit recovery of function after spinal cord injury in the absence of immune suppression [J]. J Neurotraum 2005; 22(1):138-156.
[27]. Bracken MB, Holford TR. Effects of timing of methylprednisolone or naloxone administration on recovery of segmental and long-tract neurological function in NASCIS 2 [J]. J Neurosurg 1993; 79(4):500-507.
[28]. Kong LL, Yu LC. It is AMPA receptor, not kainate receptor, that contributes to the NBQX-induced antinociception in the spinal cord of rats [J]. Brain Res 2006; 1100(1):73-77.
[29]. Bozbuga M, Izgi N, Canbolat A. The effects of chronic alpha-tocopherol administration on lipid peroxidation in an experimental model of acute spinal cord injury [J]. Neurosurg Rev 1998; 21(1):36-42.
[30]. Faden AI, Halt P. Platelet-activating factor reduces spinal cord blood flow and causes behavioral deficits after intrathecal administration in rats through a specific receptor mechanism [J]. J Pharmacol Exp Ther 1992; 261(3):1064-1070.
[31]. Kubeck JP, Merola A, Mathur S, Brkaric M, Majid K, Shanti N, et al. End organ effects of high-dose human equivalent methylprednisolone in a spinal cord injury rat model [J]. Spine (Phila Pa 1976) 2006; 31(3):257-261.
[32]. Zawilska JB, Nowak JZ. Melatonin: from biochemistry to therapeutic applications [J]. Pol J Pharmacol 1999; 51(1):3-23.
[33]. Kaptanoglu E, Tuncel M, Palaoglu S, Konan A, Demirpence E, Kilinc K. Comparison of the effects of melatonin and methylprednisolone in experimental spinal cord injury [J]. J Neurosurg 2000; 93(1 Suppl):77-84.
[34]. Pyter LM, Adelson JD, Nelson RJ. Short days increase hypothalamic-pituitary-adrenal axis responsiveness [J]. Endocrinology 2007; 148(7):3402-3409.
[35]. Genovese T, Mazzon E, Crisafulli C, Esposito E, Di Paola R, Muia C, et al Effects of combination of melatonin and dexamethasone on secondary injury in an experimental mice model of spinal cord trauma [J]. J Pineal Res 2007; 43(2): 140-153.
[36]. Topsakal C, Kilic N, Ozveren F, Akdemir I, Kaplan M, Tiftikci M, et al. Effects of prostaglandin E1, melatonin, and oxytetracycline on lipid peroxidation, antioxidant defense system, paraoxonase (PON1) activities, and homocysteine levels in an animal model of spinal cord injury [J]. Spine (Phila Pa 1976) 2003; 28(15):1643-1652.
[37]. Yuan X, Li B, Li H, Xiu R. Melatonin inhibits IL-lbeta-induced monolayer permeability of human umbilical vein endothelial cells via Rac activation [J]. J Pineal Res 2011; 51(2):220-225.
[38]. Reiter RJ, Tan DX, Manchester LC, Tamura H. Melatonin defeats neurally-derived free radicals and reduces the associated neuromorphological and neurobehavioral damage [J]. J Physiol Pharmacol 2007; 58 Suppl 6:5-22.
[39]. Akakin D, Kiran D, Ozkan N, Ersahin M, Ozdemir-Kumral ZN, Yegen B, et al. Protective effects of melatonin against spinal cord injury induced oxidative damage in rat kidney: A morphological and biochemical study [J]. Acta Histochem 2013; 115(8):827-834.
[40]. Ersahin M, Ozdemir Z, Ozsavci D, Akakin D, Yegen BC, Reiter RJ, et al. Melatonin treatment protects against spinal cord injury induced functional and biochemical changes in rat urinary bladder [J]. J Pineal Res 2012; 52(3):340-348.
[41]. Park S, Lee SK, Park K, Lee Y, Hong Y, Lee S, et al. Beneficial effects of endogenous and exogenous melatonin on neural reconstruction and functional recovery in an animal model of spinal cord injury [J]. J Pineal Res 2012; 52(1): 107-119.
[42]. Ray SK, Samantaray S, Smith JA, Matzelle DD, Das A, Banik NL. Inhibition of cysteine proteases in acute and chronic spinal cord injury [J]. Neurotherapeutics 2011; 8(2):180-186.
[43]. Chen HY, Chen TY, Lee MY, Chen ST, Hsu YS, Kuo YL, et al. Melatonin decreases neurovascular oxidative/nitrosative damage and protects against early increases in the blood-brain barrier permeability after transient focal cerebral ischemia in mice [J]. J Pineal Res 2006; 41(2):175-182.
[44]. Sommansson A, Nylander O, Sjoblom M. Melatonin decreases duodenal epithelial paracellular permeability via a nicotinic receptor-dependent pathway in rats in vivo [J]. J Pineal Res 2013; 54(3):282-291.
[45]. ALLEN A. Surgery of experimental lesion of spinal cord equivalent to crush injury of fracture dislocation of spinal column. A preliminary report [J]. JAMA 1911; 57:878-880.
[46]. Echeverry S, Shi XQ, Rivest S, Zhang J. Peripheral nerve injury alters blood-spinal cord barrier functional and molecular integrity through a selective inflammatory pathway [J]. J Neurosci 2011; 31(30):10819-10828.
[47]. Basso DM, Fisher LC, Anderson AJ, Jakeman LB, McTigue DM, Popovich PG Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains [J]. J Neurotrauma 2006; 23(5): 635-659.
[48].孙源君,马坚妹.小鼠脊髓损伤模型的行为学评估方法[J].解剖科学进展2012；18(2)：173-176.
[49]. Myers SA, DeVries WH, Gruenthal MJ, Andres KR, Hagg T, Whittemore SR. Sildenafil improves epicenter vascular perfusion but not hindlimb functional recovery after contusive spinal cord injury in mice [J]. J Neurotrauma 2012; 29(3): 528-538.
[50]. Han S, Arnold SA, Sithu SD, Mahoney ET, Geralds JT, Tran P, et al. Rescuing vasculature with intravenous angiopoietin-1 and alpha v beta 3 integrin peptide is protective after spinal cord injury [J]. Brain 2010; 133(Pt 4):1026-1042.
[51]. Morita K, Sasaki H, Fujimoto K, Furuse M, Tsukita S. Claudin-11/OSP-based tight junctions of myelin sheaths in brain and Sertoli cells in testis [J]. J Cell Biol 1999; 145(3):579-588.
[52]. Forster C. Tight junctions and the modulation of barrier function in disease [J]. Histochem Cell Biol 2008; 130(1):55-70.
[53]. Gul S, Celik SE, Kalayci M, Tasyurekli M, Cokar N, Bilge T. Dose-dependent neuroprotective effects of melatonin on experimental spinal cord injury in rats [J]. Surg Neurol 2005; 64(4):355-361.
[54]. Taskiran D, Tanyalcin T, Sozmen EY, Peker GO, Gulmen V, Cagli S, et al. The effects of melatonin on the antioxidant systems in experimental spinal injury [J]. Int J Neurosci 2000; 104(1-4):63-73.
[55]. Esposito E, Genovese T, Caminiti R, Bramanti P, Meli R, Cuzzocrea S. Melatonin reduces stress-activated/mitogen-activated protein kinases in spinal cord injury [J]. J Pineal Res 2009; 46(1):79-86.
[56]. Park K, Lee Y, Park S, Lee S, Hong Y, Kil Lee S. Synergistic effect of melatonin on exercise-induced neuronal reconstruction and functional recovery in a spinal cord injury animal model [J]. J Pineal Res 2010; 48(3):270-281.
[57]. Tavukcu HH, Sener TE, Tinay I, Akbal C, Ersahin M, Cevik O, et al. Melatonin and tadalafil treatment improves erectile dysfunction after spinal cord injury in rats [J]. Clin Exp Pharmacol Physiol 2014.
[58]. Lee Y, Lee S, Lee SR, Park K, Hong Y, Lee M, et al. Beneficial effects of melatonin combined with exercise on endogenous neural stem/progenitor cells proliferation after spinal cord injury [J]. Int J Mol Sci 2014; 15(2):2207-2222.
[59]. Schiaveto-de-Souza A, da-Silva CA, Defino HL, Del Bel EA. Effect of melatonin on the functional recovery from experimental traumatic compression of the spinal cord [J]. Braz J Med Biol Res 2013; 46(4):348-358.
[60]. Diehl KH, Hull R, Morton D, Pfister R, Rabemampianina Y, Smith D, et al. A good practice guide to the administration of substances and removal of blood, including routes and volumes [J]. J Appl Toxicol 2001; 21(1):15-23.
[61]. Fan ZK, Lv G, Wang YF, Li G, Yu DS, Wang YS, et al. The protective effect of salvianolic acid B on blood-spinal cord barrier after compression spinal cord injury in rats [J]. J Mol Neurosci 2013; 51(3):986-993.
[62]. Sharma HS, Ali S, Tian ZR, Patnaik R, Patnaik S, Lek P, et al. Nano-drug delivery and neuroprotection in spinal cord injury [J]. J Nanosci Nanotechnol 2009; 9(8):5014-5037.
[63]. Morita K, Furuse M, Fujimoto K, Tsukita S. Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands [J]. Proc Natl Acad Sci U S A 1999; 96(2):511-516.
[64]. Furuse M, Tsukita S. Claudins in occluding junctions of humans and flies [J]. Trends Cell Biol 2006; 16(4):181-188.
[65]. Liebner S, Czupalla CJ, Wolburg H. Current concepts of blood-brain barrier development [J]. Int J Dev Biol 2011; 55(4-5):467-476.
[66]. Strazielle N, Ghersi-Egea JF. Physiology of blood-brain interfaces in relation to brain disposition of small compounds and macromolecules [J]. Mol Pharm 2013; 10(5):1473-1491.
[67]. Daneman R, Zhou L, Kebede AA, Barres BA. Pericytes are required for blood-brain barrier integrity during embryogenesis [J]. Nature 2010; 468(7323): 562-566.
[68]. Sharma HS. Pathophysiology of blood-spinal cord barrier in traumatic injury and repair [J]. Curr Pharm Des 2005; 11(11):1353-1389.
[69]. Oyinbo CA. Secondary injury mechanisms in traumatic spinal cord injury: a nugget of this multiply cascade [J]. Acta Neurobiol Exp (Wars) 2011; 71(2): 281-299.
[70]. Happel RD, Smith KP, Banik NL, Powers JM, Hogan EL, Balentine JD. Ca2+-accumulation in experimental spinal cord trauma [J]. Brain Res 1981; 211(2):476-479.
[71]. Imaizumi T, Kocsis JD, Waxman SG. Anoxic injury in the rat spinal cord: pharmacological evidence for multiple steps in Ca(2+)-dependent injury of the dorsal columns [J]. J Neurotrauma 1997; 14(5):299-311.
[72]. Xiong Y, Rabchevsky AG, Hall ED. Role of peroxynitrite in secondary oxidative damage after spinal cord injury [J]. J Neurochem 2007; 100(3):639-649.
[73]. Ray SK, Hogan EL, Banik NL. Calpain in the pathophysiology of spinal cord injury: neuroprotection with calpain inhibitors [J]. Brain Res Brain Res Rev 2003; 42(2):169-185.
[74]. Espino J, Bejarano I, Ortiz A, Lozano GM, Garcia JF, Pariente JA, et al. Melatonin as a potential tool against oxidative damage and apoptosis in ejaculated human spermatozoa [J]. Fertil Steril 2010; 94(5):1915-1917.
[75]. Esposito E, Genovese T, Caminiti R, Bramanti P, Meli R, Cuzzocrea S. Melatonin regulates matrix metalloproteinases after traumatic experimental spinal cord injury [J]. J Pineal Res 2008; 45(2):149-156.
[76]. Goldberg AL, Kershah SM. Advances in imaging of vertebral and spinal cord injury [J]. J Spinal Cord Med 2010; 33(2):105-116.
[77]. Lee JY, Kim HS, Choi HY, Oh TH, Ju BG, Yune TY. Valproic acid attenuates blood-spinal cord barrier disruption by inhibiting matrix metalloprotease-9 activity and improves functional recovery after spinal cord injury [J]. J Neurochem 2012; 121(5):818-829.
[78]. Xu WB, Gu YT, Wang YF, Lu XH, Jia LS, Lv G. Bradykinin preconditioning modulates aquaporin-4 expression after spinal cord ischemic injury in rats [J]. Brain Res 2008; 1246:11-18.
[79]. Saadoun S BB, Verkman AS, Papadopoulos MC. Greatly improved neurological outcome after spinal cord compression injury in AQP4-deficient mice. [J]. Brain 2008; 131:1087-1098.
[80]. Semenza GL. Hydroxylation of HIF-1:oxygen sensing at the molecular level [J]. Physiology (Bethesda) 2004; 19:176-182.
[81]. Josko J, Mazurek M. Transcription factors having impact on vascular endothelial growth factor (VEGF) gene expression in angiogenesis [J]. Med Sci Monit 2004; 10(4):RA89-98.
[82]. Kaur C, Ling EA. Blood brain barrier in hypoxic-ischemic conditions [J]. Curr Neurovasc Res 2008; 5(1):71-81.
[83]. Shibuya M. Vascular endothelial growth factor receptor-1 (VEGFR-1/Flt-1):a dual regulator for angiogenesis [J]. Angiogenesis 2006; 9(4):225-230; discussion 231.
[84]. Haddadi G, Shirazi A, Sepehrizadeh Z, Mahdavi SR, Haddadi M. Radioprotective effect of melatonin on the cervical spinal cord in irradiated rats [J]. Cell J 2013; 14(4):246-253.
[85]. Fraga CA, Oliveira MV, Alves LR, Viana AQ Sousa AA, Carvalho SF, et al. Immunohistochemical profile of HIF-lalpha, VEGF-A, VEGFR2 and MMP9 proteins in tegumentary Ieishmaniasis [J]. An Bras Dermatol 2012; 87(5): 709-713.
[86]. Shibuya M. Differential roles of vascular endothelial growth factor receptor-1 and receptor-2 in angiogenesis [J]. J Biochem Mol Biol 2006; 39(5):469-478.
[2]. Xiu RJ IM. [Microvascular vasomotion: I. Long-term observation and computer analysis of the vasomotion] [J]. Zhonghua Yi Xue Za Zhi 1985; 65:129-135.
[3].修瑞娟,B.W.Zweifach.微血管自律运动研究[J].中华医学杂志1985；65：6.
[4]. Xiu RJ e. The correlation of microcirculatory dynamics and clinical symptoms of diseases. [J]. Basel: Karger 1984.
[5]. Xiu RJ HD, Coppo PA, et al. Anisodamine inhibits thromboxane synthesis, granulocyte aggregation, and platelet aggregation. A possible mechanism for its efficacy in bacteremic shock [J]. JAMA 1982; 247:1458-1460.
[6].史晓瑞,修瑞娟.微血管周细胞的研究进展[J].中国微循环杂志2009；13(6)：582-584.
[7].鹿文葆,秦伟伟,张秋菊,李宏伟,刘淑英,修瑞娟.脑微血管内皮细胞和周细胞共培养构建体外血脑屏障模型[J].国际脑血管病杂志2012；20(5)：338-342.
[8]. Etchevers HC, Vincent C, Le Douarin NM, Couly GF. The cephalic neural crest provides pericytes and smooth muscle cells to all blood vessels of the face and forebrain [J]. Development 2001; 128(7):1059-1068.
[9]. Korn J, Christ B, Kurz H. Neuroectodermal origin of brain pericytes and vascular smooth muscle cells [J]. J Comp Neurol 2002; 442(1):78-88.
[10]. Kurz H. Cell lineages and early patterns of embryonic CNS vascularization [J]. Cell Adh Migr 2009; 3(2):205-210.
[11]. Winkler EA, Bell RD, Zlokovic BV. Central nervous system pericytes in health and disease [J]. Nat Neurosci 2011; 14(11):1398-1405.
[12]. Gaengel K, Genove G, Armulik A, Betsholtz C. Endothelial-mural cell signaling in vascular development and angiogenesis [J]. Arterioscler Thromb Vase Biol 2009; 29(5):630-638.
[13]. Lindahl P, Johansson BR, Leveen P, Betsholtz C. Pericyte loss and microaneurysm formation in PDGF-B-deficient mice [J]. Science 1997; 277(5323):242-245.
[14]. Tallquist MD, French WJ, Soriano P. Additive effects of PDGF receptor beta signaling pathways in vascular smooth muscle cell development [J]. PLoS Biol 2003; 1(2):E52.
[15]. Hellstrom M, Kalen M, Lindahl P, Abramsson A, Betsholtz C. Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse [J]. Development 1999; 126(14):3047-3055.
[16]. Leveen P, Pekny M, Gebre-Medhin S, Swolin B, Larsson E, Betsholtz C. Mice deficient for PDGF B show renal, cardiovascular, and hematological abnormalities [J]. Genes Dev 1994; 8(16):1875-1887.
[17]. Soriano P. Abnormal kidney development and hematological disorders in PDGF beta-receptor mutant mice [J]. Genes Dev 1994; 8(16):1888-1896.
[18]. Lindblom P, Gerhardt H, Liebner S, Abramsson A, Enge M, Hellstrom M, et al. Endothelial PDGF-B retention is required for proper investment of pericytes in the microvessel wall [J]. Genes Dev 2003; 17(15):1835-1840.
[19]. Bell RD, Winkler EA, Sagare AP, Singh I, LaRue B, Deane R, et al. Pericytes control key neurovascular functions and neuronal phenotype in the adult brain and during brain aging [J]. Neuron 2010; 68(3):409-427.
[20]. Winkler EA, Bell RD, Zlokovic BV. Pericyte-specific expression of PDGF beta receptor in mouse models with normal and deficient PDGF beta receptor signaling [J]. Mol Neurodegener 2010; 5:32.
[21]. Geraldes P, Hiraoka-Yamamoto J, Matsumoto M, Clermont A, Leitges M, Marette A, et al. Activation of PKC-delta and SHP-1 by hyperglycemia causes vascular cell apoptosis and diabetic retinopathy [J]. Nat Med 2009; 15(11): 1298-1306.
[22]. Li F, Lan Y, Wang Y, Wang J, Yang G, Meng F, et al. Endothelial Smad4 maintains cerebrovascular integrity by activating N-cadherin through cooperation with Notch [J]. Dev Cell 2011; 20(3):291-302.
[23]. Sieczkiewicz GJ, Herman IM. TGF-beta 1 signaling controls retinal pericyte contractile protein expression [J]. Micro vasc Res 2003; 66(3):190-196.
[24]. Lebrin F, Deckers M, Bertolino P, Ten Dijke P. TGF-beta receptor function in the endothelium [J]. Cardiovasc Res 2005; 65(3):599-608.
[25]. Walshe TE, Saint-Geniez M, Maharaj AS, Sekiyama E, Maldonado AE, D'Amore PA. TGF-beta is required for vascular barrier function, endothelial survival and homeostasis of the adult microvasculature [J]. PLoS One 2009; 4(4): e5149.
[26]. Braun A, Xu H, Hu F, Kocherlakota P, Siegel D, Chander P, et al. Paucity of pericytes in germinal matrix vasculature of premature infants [J]. J Neurosci 2007; 27(44):12012-12024.
[27]. Vinukonda G, Dummula K, Malik S, Hu F, Thompson CI, Csiszar A, et al. Effect of prenatal glucocorticoids on cerebral vasculature of the developing brain [J]. Stroke 2010; 41(8):1766-1773.
[28]. Van Geest RJ, Klaassen I, Vogels IM, Van Noorden CJ, Schlingemann RO. Differential TGF-{beta} signaling in retinal vascular cells: a role in diabetic retinopathy? [J]. Invest Ophthalmol Vis Sci 2010; 51(4):1857-1865.
[29]. Gerhardt H, Wolburg H, Redies C. N-cadherin mediates pericytic-endothelial interaction during brain angiogenesis in the chicken [J]. Dev Dyn 2000; 218(3): 472-479.
[30]. Winkler EA, Bell RD, Zlokovic BV. Lack of Smad or Notch leads to a fatal game of brain pericyte hopscotch [J]. Dev Cell 2011; 20(3):279-280.
[31]. Saharinen P, Eklund L, Miettinen J, Wirkkala R, Anisimov A, Winderlich M, et al. Angiopoietins assemble distinct Tie2 signalling complexes in endothelial cell-cell and cell-matrix contacts [J]. Nat Cell Biol 2008; 10(5):527-537.
[32]. Wakui S, Yokoo K, Muto T, Suzuki Y, Takahashi H, Furusato M, et al. Localization of Ang-1,-2, Tie-2, and VEGF expression at endothelial-pericyte interdigitation in rat angiogenesis [J]. Lab Invest 2006; 86(11):1172-1184.
[33]. Hofmann JJ, Iruela-Arispe ML. Notch signaling in blood vessels: who is talking to whom about what? [J]. Circ Res 2007; 100(11):1556-1568.
[34]. Liu H, Kennard S, Lilly B. NOTCH3 expression is induced in mural cells through an autoregulatory loop that requires endothelial-expressed JAGGED 1 [J]. Circ Res 2009; 104(4):466-475.
[35]. Liu H, Zhang W, Kennard S, Caldwell RB, Lilly B. Notch3 is critical for proper angiogenesis and mural cell investment [J]. Circ Res 2010; 107(7):860-870.
[36]. Regan JN, Majesky MW. Building a vessel wall with notch signaling [J]. Circ Res 2009; 104(4):419-421.
[37]. Stewart KS, Zhou Z, Zweidler-McKay P, Kleinerman ES. Delta-like ligand 4-Notch signaling regulates bone marrow-derived pericyte/vascular smooth muscle cell formation [J]. Blood 2011; 117(2):719-726.
[38]. Walshe TE, Connell P, Cryan L, Ferguson G, Gardiner T, Morrow D, et al. Microvascular retinal endothelial and pericyte cell apoptosis in vitro:role of hedgehog and Notch signaling [J]. Invest Ophthalmol Vis Sci 2011; 52(7): 4472-4483.
[39]. Jin S, Hansson EM, Tikka S, Lanner F, Sahlgren C, Farnebo F, et al. Notch signaling regulates platelet-derived growth factor receptor-beta expression in vascular smooth muscle cells [J]. Circ Res 2008; 102(12):1483-1491.
[40]. Winkler EA, Sengillo JD, Bell RD, Wang J, Zlokovic BV. Blood-spinal cord barrier pericyte reductions contribute to increased capillary permeability [J]. J Cereb Blood Flow Metab 2012; 32(10):1841-1852.
[41]. Shi X HW, Yamamoto H, Tang W, Lin X, Xiu R, Trune DR, Nuttall AL. The Cochlear Pericytes [J]. Microcirculation 2008; 15(6):515-529.
[42]. Sacharidou A, Stratman AN, Davis GE. Molecular mechanisms controlling vascular lumen formation in three-dimensional extracellular matrices [J]. Cells Tissues Organs 2012; 195(1-2):122-143.
[43]. Oudega M. Molecular and cellular mechanisms underlying the role of blood vessels in spinal cord injury and repair [J]. Cell Tissue Res 2012; 349(1): 269-288.
[44]. Sharma HS. Pathophysiology of blood-spinal cord barrier in traumatic injury and repair [J]. Curr Pharm Des 2005; 11(11):1353-1389.
[45]. Wiranowska M, Gonzalvo AA, Saporta S, Gonzalez OR, Prockop LD. Evaluation of blood-brain barrier permeability and the effect of interferon in mouse glioma model [J]. J Neurooncol 1992; 14(3):225-236.
[46]. Ge S, Pachter JS. Isolation and culture of microvascular endothelial cells from murine spinal cord [J]. J Neuroimmunol 2006; 177(1-2):209-214.
[47]. Abbott NJ. Evidence for bulk flow of brain interstitial fluid: significance for physiology and pathology [J]. Neurochem Int 2004; 45(4):545-552.
[48]. Abbott NJ, Patabendige AA, Dolman DE, Yusof SR, Begley DJ. Structure and function of the blood-brain barrier [J]. Neurobiol Dis 2010; 37(1):13-25.
[49]. Abbott NJ. Blood-brain barrier structure and function and the challenges for CNS drug delivery [J]. J Inherit Metab Dis 2013; 36(3):437-449.
[50]. Abbott NJ, Ronnback L, Hansson E. Astrocyte-endothelial interactions at the blood-brain barrier [J]. Nat Rev Neurosci 2006; 7(1):41-53.
[51]. Reese TS, Karnovsky MJ. Fine structural localization of a blood-brain barrier to exogenous peroxidase [J]. J Cell Biol 1967; 34(1):207-217.
[52]. Bernacki J, Dobrowolska A, Nierwinska K, Malecki A. Physiology and pharmacological role of the blood-brain barrier [J]. Pharmacol Rep 2008; 60(5): 600-622.
[53]. Armulik A, Genove G, Mae M, Nisancioglu MH, Wallgard E, Niaudet C, et al. Pericytes regulate the blood-brain barrier [J]. Nature 2010; 468(7323):557-561.
[54]. Daneman R, Zhou L, Kebede AA, Barres BA. Pericytes are required for blood-brain barrier integrity during embryo genesis [J]. Nature 2010; 468(7323): 562-566.
[55]. Peppiatt CM, Howarth C, Mobbs P, Attwell D. Bidirectional control of CNS capillary diameter by pericytes [J]. Nature 2006; 443(7112):700-704.
[56]. Zlokovic BV. The blood-brain barrier in health and chronic neurodegenerative disorders [J]. Neuron 2008; 57(2):178-201.
[57]. Diaz-Flores L, Gutierrez R, Madrid JF, Varela H, Valladares F, Acosta E, et al. Pericytes. Morphofunction, interactions and pathology in a quiescent and activated mesenchymal cell niche [J]. Histol Histopathol 2009; 24(7):909-969.
[58]. Hamilton NB, Attwell D, Hall CN. Pericyte-mediated regulation of capillary diameter: a component of neurovascular coupling in health and disease [J]. Front Neuroenergetics 2010; 2.
[59]. Chen CJ, Ou YC, Li JR, Chang CY, Pan HC, Lai CY, et al. Infection of pericytes in vitro by Japanese encephalitis virus disrupts the integrity of endothelial barrier [J]. J Virol 2013.
[60]. Hirooka T, Yamamoto C, Yasutake A, Eto K, Kaji T. Expression of VEGF-related proteins in cultured human brain microvascular endothelial cells and pericytes after exposure to methylmercury [J]. J Toxicol Sci 2013; 38(6): 837-845.
[61]. Liu S, Agalliu D, Yu C, Fisher M. The role of pericytes in blood-brain barrier function and stroke [J]. Curr Pharm Des 2012; 18(25):3653-3662.
[62]. Sagare AP, Bell RD, Zhao Z, Ma Q, Winkler EA, Ramanathan A, et al. Pericyte loss influences Alzheimer-like neurodegeneration in mice [J]. Nat Commun 2013; 4:2932.
[63]. Bartanusz V, Jezova D, Alajajian B, Digicaylioglu M. The blood-spinal cord barrier: morphology and clinical implications [J]. Ann Neurol 2011; 70(2): 194-206.
[64]. Hammes HP LJ, Renner O, Shani M, Lundqvist A, Betsholtz C, Brownlee M, Deutsch U. Pericytes and the pathogenesis of diabetic retinopathy. [J]. Diabetes 2002; 51:3107-3112.
[65]. Ciulla TA, Amador AG, Zinman B. Diabetic retinopathy and diabetic macular edema: pathophysiology, screening, and novel therapies [J]. Diabetes Care 2003; 26(9):2653-2664.
[66]. Crisan M, Yap S, Casteilla L, Chen CW, Corselli M, Park TS, et al. A perivascular origin for mesenchymal stem cells in multiple human organs [J]. Cell Stem Cell 2008; 3(3):301-313.
[67]. Kang SG, Shinojima N, Hossain A, Gumin J, Yong RL, Colman H, et al. Isolation and Perivascular Localization of Mesenchymal Stem Cells From Mouse Brain [J]. Neurosurgery 2010; 67(3):711-720.
[68]. Farrington-Rock C, Crofts NJ, Doherty MJ, Ashton BA, Griffin-Jones C, Canfield AE. Chondrogenic and adipogenic potential of microvascular pericytes [J]. Circulation 2004; 110(15):2226-2232.
[69]. Lamagna C, Bergers G The bone marrow constitutes a reservoir of pericyte progenitors [J]. J Leukocyte Biol 2006; 80(4):677-681.
[70]. Caplan AI. All MSCs are pericytes? [J]. Cell Stem Cell 2008; 3(3):229-230.
[71]. Renner O, Tsimpas A, Kostin S, Valable S, Petit E, Schaper W, et al. Time- and cell type-specific induction of platelet-derived growth factor receptor-beta during cerebral ischemia [J]. Mol Brain Res 2003; 113(1-2):44-51.
[72]. Lange S, Trost A, Tempfer H, Bauer HC, Bauer H, Rohde E, et al. Brain pericyte plasticity as a potential drug target in CNS repair [J]. Drug Discov Today 2013; 18(9-10):456-463.
[73]. Bergers G, Song S. The role of pericytes in blood-vessel formation and maintenance [J]. Neuro Oncol 2005; 7(4):452-464.
[74]. Stallcup WB. The NG2 proteoglycan: past insights and future prospects [J]. J Neurocytol2002; 31(6-7):423-435.
[75]. Kucharova K, Chang Y, Boor A, Yong VW, Stallcup WB. Reduced inflammation accompanies diminished myelin damage and repair in the NG2 null mouse spinal cord [J]. J Neuroinflammation 2011; 8:158.
[76]. Goritz C, Dias DO, Tomilin N, Barbacid M, Shupliakov O, Frisen J. A pericyte origin of spinal cord scar tissue [J]. Science 2011; 333(6039):238-242.
[77]. Liao Y, Day KH, Damon DN, Duling BR. Endothelial cell-specific knockout of connexin 43 causes hypotension and bradycardia in mice [J]. Proc Natl Acad Sci U S A 2001; 98(17):9989-9994.
[78]. Li AF, Sato T, Haimovici R, Okamoto T, Roy S. High glucose alters connexin 43 expression and gap junction intercellular communication activity in retinal pericytes [J]. Invest Ophthalmol Vis Sci 2003; 44(12):5376-5382.
[79]. Mogensen C, Bergner B, Wallner S, Ritter A, d'Avis S, Ninichuk V, et al. Isolation and functional characterization of pericytes derived from hamster skeletal muscle [J]. Acta Physiol (Oxf) 2011; 201(4):413-426.
[80]. Liu X, Sun L, Torii M, Rakic P. Connexin 43 controls the multipolar phase of neuronal migration to the cerebral cortex [J]. Proc Natl Acad Sci U S A 2012; 109(21):8280-8285.
[81]. Bandopadhyay R, Orte C, Lawrenson JG, Reid AR, De Silva S, Allt G Contractile proteins in pericytes at the blood-brain and blood-retinal barriers [J]. J Neurocytol 2001; 30(1):35-44.
[82]. Goldfarb LG, Vicart P, Goebel HH, Dalakas MC. Desmin myopathy [J]. Brain 2004; 127(Pt 4):723-734.
[83]. Gumbiner BM. Regulation of cadherin-mediated adhesion in morphogenesis [J]. Nat Rev Mol Cell Biol 2005; 6(8):622-634.
[84]. Tepass U, Truong K, Godt D, Ikura M, Peifer M. Cadherins in embryonic and neural morphogenesis [J]. Nat Rev Mol Cell Biol 2000; 1(2):91-100.
[85]. Scheef EA, Sorenson CM, Sheibani N. Attenuation of proliferation and migration of retinal pericytes in the absence of thrombospondin-1 [J]. Am J Physiol Cell Physiol 2009; 296(4):C724-734.
[86]. Ezan P, Andre P, Cisternino S, Saubamea B, Boulay AC, Doutremer S, et al. Deletion of astroglial connexins weakens the blood-brain barrier [J]. J Cereb Blood Flow Metab 2012; 32(8):1457-1467.
[87]. Karen J, Rodriguez A, Friman T, Dencker L, Sundberg C, Scholz B. Effects of the histone deacetylase inhibitor valproic acid on human pericytes in vitro [J]. PLoS One 2011; 6(9):e24954.
[88]. Thanabalasundaram G, Schneidewind J, Pieper C, Galla HJ. The impact of pericytes on the blood-brain barrier integrity depends critically on the pericyte differentiation stage [J]. Int J Biochem Cell Biol 2011; 43(9):1284-1293.
[89]. Li BW, Yuan, X.C., Li, H.W.&Xiu, R.J.15d-PGJ2 attenuates CSE-induced inhibition of HUVECs migration [J]. Basic & Clinical Medicine 2011; 31: 394-399.
[90]. Stratman AN, Malotte KM, Mahan RD, Davis MJ, Davis GE. Pericyte recruitment during vasculogenic tube assembly stimulates endothelial basement membrane matrix formation [J]. Blood 2009; 114(24):5091-5101.
[91]. Belanger M, Allaman I, Magistretti PJ. Brain energy metabolism: focus on astrocyte-neuron metabolic cooperation [J]. Cell Metab 2011; 14(6):724-738.
[92]. Quaegebeur A, Lange C, Carmeliet P. The neurovascular link in health and disease: molecular mechanisms and therapeutic implications [J]. Neuron 2011; 71(3):406-424.
[93]. Fonta C, Imbert M. Vascularization in the primate visual cortex during development [J]. Cereb Cortex 2002; 12(2):199-211.
[94]. Liang M, Ekblad E, Hellstrand P, Nilsson BO. Polyamine synthesis inhibition attenuates vascular smooth muscle cell migration [J]. J Vasc Res 2004; 41(2): 141-147.
[95]. Bedekovics J, Szeghalmy S, Beke L, Fazekas A, Mehes G Image analysis of platelet derived growth factor receptor-beta (PDGFRbeta) expression to determine the grade and dynamics of myelofibrosis in bone marrow biopsy samples [J]. Cytometry B Clin Cytom 2014.
[96]. Wallow IH, Burnside B. Actin filaments in retinal pericytes and endothelial cells [J]. Invest Ophthalmol Vis Sci 1980; 19(12):1433-1441.
[97]. Sims JR, Karp S, Ingber DE. Altering the cellular mechanical force balance results in integrated changes in cell, cytoskeletal and nuclear shape [J]. J Cell Sci 1992; 103 (Pt 4):1215-1222.
[98]. Meazzini MC, Toma CD, Schaffer JL, Gray ML, Gerstenfeld LC. Osteoblast cytoskeletal modulation in response to mechanical strain in vitro [J]. J Orthop Res 1998; 16(2):170-180.
[99]. Kristensson K, Olsson Y. Accumulation of protein tracers in pericytes of the central nervous system following systemic injection in immature mice [J]. Acta Neurol Scand 1973; 49(2):189-194.
[100]. Balabanov R, Dore-Duffy P. Role of the CNS micro vascular pericyte in the blood-brain barrier [J]. J Neurosci Res 1998; 53(6):637-644.
[101]. Allt G, Lawrenson JG Pericytes: cell biology and pathology [J]. Cells Tissues Organs 2001; 169(1):1-11.
[102]. Fisher M, French S, Ji P, Kim RC. Cerebral microbleeds in the elderly: a pathological analysis [J]. Stroke 2010; 41(12):2782-2785.
[103]. Fang H, Wang PF, Zhou Y, Wang YC, Yang QW. Toll-like receptor 4 signaling in intracerebral hemorrhage-induced inflammation and injury [J]. J Neuroinflammation 2013; 10:27.
[104]. Figueiredo RT, Fernandez PL, Mourao-Sa DS, Porto BN, Dutra FF, Alves LS, et al. Characterization of heme as activator of Toll-like receptor 4 [J]. J Biol Chem 2007; 282(28):20221-20229.
[105]. Smiley ST, King JA, Hancock WW. Fibrinogen stimulates macrophage chemokine secretion through toll-like receptor 4 [J]. J Immunol 2001; 167(5): 2887-2894.
[106]. Edelman DA, Jiang Y, Tyburski JG, Wilson RF, Steffes CP. Lipopolysaccharide activation of pericyte's Toll-like receptor-4 regulates co-culture permeability [J]. Am J Surg 2007; 193(6):730-735.
[107]. Edelman DA, Jiang Y, Tyburski J, Wilson RF, Steffes C. Toll-like receptor-4 message is up-regulated in lipopolysaccharide-exposed rat lung pericytes [J]. J Surg Res 2006; 134(1):22-27.
[108]. Choi JJ, Choi YJ, Chen L, Zhang B, Eum SY, Abreu MT, et al. Lipopolysaccharide potentiates polychlorinated biphenyl-induced disruption of the blood-brain barrier via TLR4/IRF-3 signaling [J]. Toxicology 2012; 302(2-3): 212-220.
[1]. Qiu J. China Spinal Cord Injury Network: changes from within [J]. Lancet Neurol 2009; 8(7):606-607.
[2]. Cripps RA, Lee BB, Wing P, Weerts E, Mackay J, Brown D. A global map for traumatic spinal cord injury epidemiology: towards a living data repository for injury prevention [J]. Spinal Cord 2011; 49(4):493-501.
[3]. Ray SK, Dixon CE, Banik NL. Molecular mechanisms in the pathogenesis of traumatic brain injury [J]. Histol Histopathol 2002; 17(4):1137-1152.
[4]. Rossignol S, Schwab M, Schwartz M, Fehlings MG Spinal cord injury: time to move? [J]. J Neurosci 2007; 27(44):11782-11792.
[5]. Tator CH. Biology of neurological recovery and functional restoration after spinal cord injury [J]. Neurosurgery 1998; 42(4):696-707; discussion 707-698.
[6]. Hulsebosch CE. Recent advances in pathophysiology and treatment of spinal cord injury [J]. Adv Physiol Educ 2002; 26(1-4):238-255.
[7]. Oudega M. Molecular and cellular mechanisms underlying the role of blood vessels in spinal cord injury and repair [J]. Cell Tissue Res 2012; 349(1): 269-288.
[8]. Griffiths IR, Burns N, Crawford AR. Early vascular changes in the spinal grey matter following impact injury [J]. Acta Neuropathol 1978; 41(1):33-39.
[9]. Noble LJ, Wrathall JR. Distribution and time course of protein extravasation in the rat spinal cord after contusive injury [J]. Brain Res 1989; 482(1):57-66.
[10]. Casella GT, Bunge MB, Wood PM. Endothelial cell loss is not a major cause of neuronal and glial cell death following contusion injury of the spinal cord [J]. Exp Neurol 2006; 202(1):8-20.
[11]. Sadrzadeh SM, Eaton JW. Hemoglobin-mediated oxidant damage to the central nervous system requires endogenous ascorbate [J]. J Clin Invest 1988; 82(5): 1510-1515.
[12]. Mautes AE, Weinzierl MR, Donovan F, Noble LJ. Vascular events after spinal cord injury: contribution to secondary pathogenesis [J]. Phys Ther 2000; 80(7): 673-687.
[13]. Makinde T, Agrawal DK. Intra and extravascular transmembrane signalling of angiopoietin-l-Tie2 receptor in health and disease [J]. J Cell Mol Med 2008; 12(3):810-828.
[14]. Wolburg H, Lippoldt A. Tight junctions of the blood-brain barrier: development, composition and regulation [J]. Vascul Pharmacol 2002; 38(6):323-337.
[15]. Armulik A, Genove G, Betsholtz C. Pericytes: developmental, physiological, and pathological perspectives, problems, and promises [J]. Dev Cell 2011; 21(2): 193-215.
[16]. Hardebo JE, Kahrstrom J. Endothelial negative surface charge areas and blood-brain barrier function [J]. Acta Physiol Scand 1985; 125(3):495-499.
[17]. Lossinsky AS, Shivers RR. Structural pathways for macromolecular and cellular transport across the blood-brain barrier during inflammatory conditions. Review [J]. Histol Histopathol 2004; 19(2):535-564.
[18]. Nag S, Manias JL, Stewart DJ. Expression of endothelial phosphorylated caveolin-1 is increased in brain injury [J]. Neuropathol Appl Neurobiol 2009; 35(4):417-426.
[19]. Hawkins BT, Lundeen TF, Norwood KM, Brooks HL, Egleton RD. Increased blood-brain barrier permeability and altered tight junctions in experimental diabetes in the rat: contribution of hyperglycaemia and matrix metalloproteinases [J]. Diabetologia 2007; 50(1):202-211.
[20]. Brightman MW, Zis K, Anders J. Morphology of cerebral endothelium and astrocytes as determinants of the neuronal microenvironment [J]. Acta Neuropathol Suppl 1983; 8:21-33.
[21]. Bracken MB, Shepard MJ, Holford TR, Leo-Summers L, Aldrich EF, Fazl M, et al. Administration of methylprednisolone for 24 or 48 hours or tirilazad mesylate for 48 hours in the treatment of acute spinal cord injury. Results of the Third National Acute Spinal Cord Injury Randomized Controlled Trial. National Acute Spinal Cord Injury Study [J]. JAMA 1997; 277(20):1597-1604.
[22]. Eck JC, Nachtigall D, Humphreys SC, Hodges SD. Questionnaire survey of spine surgeons on the use of methylprednisolone for acute spinal cord injury [J]. Spine (Phila Pa 1976) 2006; 31(9):E250-253.
[23]. Geisler FH, Dorsey FC, Coleman WP. Past and Current Clinical-Studies with Gm-1 Ganglioside in Acute Spinal-Cord Injury [J]. Ann Emerg Med 1993; 22(6): 1041-1047.
[24]. Chinnock P, Roberts I. Gangliosides for acute spinal cord injury [J]. Cochrane Db Syst Rev 2005; (2).
[25]. Courtine G, Roy RR, Raven J, Hodgson J, Mckay H, Yang H, et al. Performance of locomotion and foot grasping following a unilateral thoracic corticospinal tract lesion in monkeys (Macaca mulatta) [J]. Brain 2005; 128:2338-2358.
[26]. Tobias CA, Han SSW, Shumsky JS, Kim D, Tumolo M, Dhoot NO, et al. Alginate encapsulated BDNF-producing fibroblast grafts permit recovery of function after spinal cord injury in the absence of immune suppression [J]. J Neurotraum 2005; 22(1):138-156.
[27]. Bracken MB, Holford TR. Effects of timing of methylprednisolone or naloxone administration on recovery of segmental and long-tract neurological function in NASCIS 2 [J]. J Neurosurg 1993; 79(4):500-507.
[28]. Kong LL, Yu LC. It is AMPA receptor, not kainate receptor, that contributes to the NBQX-induced antinociception in the spinal cord of rats [J]. Brain Res 2006; 1100(1):73-77.
[29]. Bozbuga M, Izgi N, Canbolat A. The effects of chronic alpha-tocopherol administration on lipid peroxidation in an experimental model of acute spinal cord injury [J]. Neurosurg Rev 1998; 21(1):36-42.
[30]. Faden AI, Halt P. Platelet-activating factor reduces spinal cord blood flow and causes behavioral deficits after intrathecal administration in rats through a specific receptor mechanism [J]. J Pharmacol Exp Ther 1992; 261(3):1064-1070.
[31]. Kubeck JP, Merola A, Mathur S, Brkaric M, Majid K, Shanti N, et al. End organ effects of high-dose human equivalent methylprednisolone in a spinal cord injury rat model [J]. Spine (Phila Pa 1976) 2006; 31(3):257-261.
[32]. Zawilska JB, Nowak JZ. Melatonin: from biochemistry to therapeutic applications [J]. Pol J Pharmacol 1999; 51(1):3-23.
[33]. Kaptanoglu E, Tuncel M, Palaoglu S, Konan A, Demirpence E, Kilinc K. Comparison of the effects of melatonin and methylprednisolone in experimental spinal cord injury [J]. J Neurosurg 2000; 93(1 Suppl):77-84.
[34]. Pyter LM, Adelson JD, Nelson RJ. Short days increase hypothalamic-pituitary-adrenal axis responsiveness [J]. Endocrinology 2007; 148(7):3402-3409.
[35]. Genovese T, Mazzon E, Crisafulli C, Esposito E, Di Paola R, Muia C, et al Effects of combination of melatonin and dexamethasone on secondary injury in an experimental mice model of spinal cord trauma [J]. J Pineal Res 2007; 43(2): 140-153.
[36]. Topsakal C, Kilic N, Ozveren F, Akdemir I, Kaplan M, Tiftikci M, et al. Effects of prostaglandin E1, melatonin, and oxytetracycline on lipid peroxidation, antioxidant defense system, paraoxonase (PON1) activities, and homocysteine levels in an animal model of spinal cord injury [J]. Spine (Phila Pa 1976) 2003; 28(15):1643-1652.
[37]. Yuan X, Li B, Li H, Xiu R. Melatonin inhibits IL-lbeta-induced monolayer permeability of human umbilical vein endothelial cells via Rac activation [J]. J Pineal Res 2011; 51(2):220-225.
[38]. Reiter RJ, Tan DX, Manchester LC, Tamura H. Melatonin defeats neurally-derived free radicals and reduces the associated neuromorphological and neurobehavioral damage [J]. J Physiol Pharmacol 2007; 58 Suppl 6:5-22.
[39]. Akakin D, Kiran D, Ozkan N, Ersahin M, Ozdemir-Kumral ZN, Yegen B, et al. Protective effects of melatonin against spinal cord injury induced oxidative damage in rat kidney: A morphological and biochemical study [J]. Acta Histochem 2013; 115(8):827-834.
[40]. Ersahin M, Ozdemir Z, Ozsavci D, Akakin D, Yegen BC, Reiter RJ, et al. Melatonin treatment protects against spinal cord injury induced functional and biochemical changes in rat urinary bladder [J]. J Pineal Res 2012; 52(3):340-348.
[41]. Park S, Lee SK, Park K, Lee Y, Hong Y, Lee S, et al. Beneficial effects of endogenous and exogenous melatonin on neural reconstruction and functional recovery in an animal model of spinal cord injury [J]. J Pineal Res 2012; 52(1): 107-119.
[42]. Ray SK, Samantaray S, Smith JA, Matzelle DD, Das A, Banik NL. Inhibition of cysteine proteases in acute and chronic spinal cord injury [J]. Neurotherapeutics 2011; 8(2):180-186.
[43]. Chen HY, Chen TY, Lee MY, Chen ST, Hsu YS, Kuo YL, et al. Melatonin decreases neurovascular oxidative/nitrosative damage and protects against early increases in the blood-brain barrier permeability after transient focal cerebral ischemia in mice [J]. J Pineal Res 2006; 41(2):175-182.
[44]. Sommansson A, Nylander O, Sjoblom M. Melatonin decreases duodenal epithelial paracellular permeability via a nicotinic receptor-dependent pathway in rats in vivo [J]. J Pineal Res 2013; 54(3):282-291.
[45]. ALLEN A. Surgery of experimental lesion of spinal cord equivalent to crush injury of fracture dislocation of spinal column. A preliminary report [J]. JAMA 1911; 57:878-880.
[46]. Echeverry S, Shi XQ, Rivest S, Zhang J. Peripheral nerve injury alters blood-spinal cord barrier functional and molecular integrity through a selective inflammatory pathway [J]. J Neurosci 2011; 31(30):10819-10828.
[47]. Basso DM, Fisher LC, Anderson AJ, Jakeman LB, McTigue DM, Popovich PG Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains [J]. J Neurotrauma 2006; 23(5): 635-659.
[48].孙源君,马坚妹.小鼠脊髓损伤模型的行为学评估方法[J].解剖科学进展2012；18(2)：173-176.
[49]. Myers SA, DeVries WH, Gruenthal MJ, Andres KR, Hagg T, Whittemore SR. Sildenafil improves epicenter vascular perfusion but not hindlimb functional recovery after contusive spinal cord injury in mice [J]. J Neurotrauma 2012; 29(3): 528-538.
[50]. Han S, Arnold SA, Sithu SD, Mahoney ET, Geralds JT, Tran P, et al. Rescuing vasculature with intravenous angiopoietin-1 and alpha v beta 3 integrin peptide is protective after spinal cord injury [J]. Brain 2010; 133(Pt 4):1026-1042.
[51]. Morita K, Sasaki H, Fujimoto K, Furuse M, Tsukita S. Claudin-11/OSP-based tight junctions of myelin sheaths in brain and Sertoli cells in testis [J]. J Cell Biol 1999; 145(3):579-588.
[52]. Forster C. Tight junctions and the modulation of barrier function in disease [J]. Histochem Cell Biol 2008; 130(1):55-70.
[53]. Gul S, Celik SE, Kalayci M, Tasyurekli M, Cokar N, Bilge T. Dose-dependent neuroprotective effects of melatonin on experimental spinal cord injury in rats [J]. Surg Neurol 2005; 64(4):355-361.
[54]. Taskiran D, Tanyalcin T, Sozmen EY, Peker GO, Gulmen V, Cagli S, et al. The effects of melatonin on the antioxidant systems in experimental spinal injury [J]. Int J Neurosci 2000; 104(1-4):63-73.
[55]. Esposito E, Genovese T, Caminiti R, Bramanti P, Meli R, Cuzzocrea S. Melatonin reduces stress-activated/mitogen-activated protein kinases in spinal cord injury [J]. J Pineal Res 2009; 46(1):79-86.
[56]. Park K, Lee Y, Park S, Lee S, Hong Y, Kil Lee S. Synergistic effect of melatonin on exercise-induced neuronal reconstruction and functional recovery in a spinal cord injury animal model [J]. J Pineal Res 2010; 48(3):270-281.
[57]. Tavukcu HH, Sener TE, Tinay I, Akbal C, Ersahin M, Cevik O, et al. Melatonin and tadalafil treatment improves erectile dysfunction after spinal cord injury in rats [J]. Clin Exp Pharmacol Physiol 2014.
[58]. Lee Y, Lee S, Lee SR, Park K, Hong Y, Lee M, et al. Beneficial effects of melatonin combined with exercise on endogenous neural stem/progenitor cells proliferation after spinal cord injury [J]. Int J Mol Sci 2014; 15(2):2207-2222.
[59]. Schiaveto-de-Souza A, da-Silva CA, Defino HL, Del Bel EA. Effect of melatonin on the functional recovery from experimental traumatic compression of the spinal cord [J]. Braz J Med Biol Res 2013; 46(4):348-358.
[60]. Diehl KH, Hull R, Morton D, Pfister R, Rabemampianina Y, Smith D, et al. A good practice guide to the administration of substances and removal of blood, including routes and volumes [J]. J Appl Toxicol 2001; 21(1):15-23.
[61]. Fan ZK, Lv G, Wang YF, Li G, Yu DS, Wang YS, et al. The protective effect of salvianolic acid B on blood-spinal cord barrier after compression spinal cord injury in rats [J]. J Mol Neurosci 2013; 51(3):986-993.
[62]. Sharma HS, Ali S, Tian ZR, Patnaik R, Patnaik S, Lek P, et al. Nano-drug delivery and neuroprotection in spinal cord injury [J]. J Nanosci Nanotechnol 2009; 9(8):5014-5037.
[63]. Morita K, Furuse M, Fujimoto K, Tsukita S. Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands [J]. Proc Natl Acad Sci U S A 1999; 96(2):511-516.
[64]. Furuse M, Tsukita S. Claudins in occluding junctions of humans and flies [J]. Trends Cell Biol 2006; 16(4):181-188.
[65]. Liebner S, Czupalla CJ, Wolburg H. Current concepts of blood-brain barrier development [J]. Int J Dev Biol 2011; 55(4-5):467-476.
[66]. Strazielle N, Ghersi-Egea JF. Physiology of blood-brain interfaces in relation to brain disposition of small compounds and macromolecules [J]. Mol Pharm 2013; 10(5):1473-1491.
[67]. Daneman R, Zhou L, Kebede AA, Barres BA. Pericytes are required for blood-brain barrier integrity during embryogenesis [J]. Nature 2010; 468(7323): 562-566.
[68]. Sharma HS. Pathophysiology of blood-spinal cord barrier in traumatic injury and repair [J]. Curr Pharm Des 2005; 11(11):1353-1389.
[69]. Oyinbo CA. Secondary injury mechanisms in traumatic spinal cord injury: a nugget of this multiply cascade [J]. Acta Neurobiol Exp (Wars) 2011; 71(2): 281-299.
[70]. Happel RD, Smith KP, Banik NL, Powers JM, Hogan EL, Balentine JD. Ca2+-accumulation in experimental spinal cord trauma [J]. Brain Res 1981; 211(2):476-479.
[71]. Imaizumi T, Kocsis JD, Waxman SG. Anoxic injury in the rat spinal cord: pharmacological evidence for multiple steps in Ca(2+)-dependent injury of the dorsal columns [J]. J Neurotrauma 1997; 14(5):299-311.
[72]. Xiong Y, Rabchevsky AG, Hall ED. Role of peroxynitrite in secondary oxidative damage after spinal cord injury [J]. J Neurochem 2007; 100(3):639-649.
[73]. Ray SK, Hogan EL, Banik NL. Calpain in the pathophysiology of spinal cord injury: neuroprotection with calpain inhibitors [J]. Brain Res Brain Res Rev 2003; 42(2):169-185.
[74]. Espino J, Bejarano I, Ortiz A, Lozano GM, Garcia JF, Pariente JA, et al. Melatonin as a potential tool against oxidative damage and apoptosis in ejaculated human spermatozoa [J]. Fertil Steril 2010; 94(5):1915-1917.
[75]. Esposito E, Genovese T, Caminiti R, Bramanti P, Meli R, Cuzzocrea S. Melatonin regulates matrix metalloproteinases after traumatic experimental spinal cord injury [J]. J Pineal Res 2008; 45(2):149-156.
[76]. Goldberg AL, Kershah SM. Advances in imaging of vertebral and spinal cord injury [J]. J Spinal Cord Med 2010; 33(2):105-116.
[77]. Lee JY, Kim HS, Choi HY, Oh TH, Ju BG, Yune TY. Valproic acid attenuates blood-spinal cord barrier disruption by inhibiting matrix metalloprotease-9 activity and improves functional recovery after spinal cord injury [J]. J Neurochem 2012; 121(5):818-829.
[78]. Xu WB, Gu YT, Wang YF, Lu XH, Jia LS, Lv G. Bradykinin preconditioning modulates aquaporin-4 expression after spinal cord ischemic injury in rats [J]. Brain Res 2008; 1246:11-18.
[79]. Saadoun S BB, Verkman AS, Papadopoulos MC. Greatly improved neurological outcome after spinal cord compression injury in AQP4-deficient mice. [J]. Brain 2008; 131:1087-1098.
[80]. Semenza GL. Hydroxylation of HIF-1:oxygen sensing at the molecular level [J]. Physiology (Bethesda) 2004; 19:176-182.
[81]. Josko J, Mazurek M. Transcription factors having impact on vascular endothelial growth factor (VEGF) gene expression in angiogenesis [J]. Med Sci Monit 2004; 10(4):RA89-98.
[82]. Kaur C, Ling EA. Blood brain barrier in hypoxic-ischemic conditions [J]. Curr Neurovasc Res 2008; 5(1):71-81.
[83]. Shibuya M. Vascular endothelial growth factor receptor-1 (VEGFR-1/Flt-1):a dual regulator for angiogenesis [J]. Angiogenesis 2006; 9(4):225-230; discussion 231.
[84]. Haddadi G, Shirazi A, Sepehrizadeh Z, Mahdavi SR, Haddadi M. Radioprotective effect of melatonin on the cervical spinal cord in irradiated rats [J]. Cell J 2013; 14(4):246-253.
[85]. Fraga CA, Oliveira MV, Alves LR, Viana AQ Sousa AA, Carvalho SF, et al. Immunohistochemical profile of HIF-lalpha, VEGF-A, VEGFR2 and MMP9 proteins in tegumentary Ieishmaniasis [J]. An Bras Dermatol 2012; 87(5): 709-713.
[86]. Shibuya M. Differential roles of vascular endothelial growth factor receptor-1 and receptor-2 in angiogenesis [J]. J Biochem Mol Biol 2006; 39(5):469-478.