| Part One The pathophysiological chang of microcirculation after spinal cord injuryObjective:Spinal cord injury (SCI) is incurable diseases in the world, at present, microvessels has became the hot point of research, and pericytes has been increasingly paid more attention because of its unique location. The pathological response of pericytes after SCI is still not clear. It has been reported that pericytes are closely related with the function of blood-brain barrier and microvessels. The aim of our study was to investigate the role of pericytes on microvessels after SCI.Methods:C57BL/6mice were randomly divided into four groups:sham group,2days (S2),7days (S7) and14days after SCI group (S14)(n=20per group). The injury group received a moderate impacted spinal cord injury. The permeability of blood-spinal cord injury (BSCB) was detected by Evan’s Blue (EB) administered intraperitoneally; perfused blood vessels were detected by FITC-LEA intravenous injection; micro vessel area density (MVA) and microvessel density (MVD) were analyzed by immunofluorescence and immunohistochemistry, respectively; the expressions of VEGF, VEGFR2and Angl were detected by Western blot; pericyte coverage was further calculated by immunofluorescence. Hypoxic condition (95%N2and5%CO2) was applied to mimic the phathological situation of SCI, the secretions of proangiogenic factors (Angl, VEGF and TGF-β) were detected by ELISA; the expressions of HIF-Ia, VEGFR2, PDGFRp, NG2and a-SMA were detected by Western blot; the secretion and expression of MMP2was detected by gelatin zymography analysis and Western blot, respectively. The differentiation of pericytes into endothelial cells and astrocytes were determined by immunofluorescence. The effect of percytes on endothelial tubular network was determined by Matrigel System.Results:1) After spinal cord injury, the results of EB measurement with qualitative and quantitative analysis showed that injury-induced the permeability of BSCB was markedly increased. The amount of EB extravasation was0.06±0.01,0.81±0.16,0.47±0.12and0.15±0.07(μg/mg) in sham group and S2, S7and S14. Compared with sham control, the amount of EB extravasation was markedly increased at2days post injury (p<0.01), subsequently, the amount of EB extravasation was gradually decreased until14days.2) After spinal cord injury, the perfused blood vessels were seriously damaged, especially the gray matter highly vascularized. The areas of perfused microvascular vessels (dimeter<100μm) calculated by Image pro plus7.0were9.46%±0.74%,4.10%±0.44%,4.72%±0.46%,6.07%±0.56%in sham group and S2, S7and S14. There was no significant difference when compared the area of perfused microvessels at S2with that at S7. While, the area of perfused microvessels at S14showed a notable increase compared with that at S2(p<0.05).3) After spinal cord injury, the result of CD31-immunoreactivity showed that significant decrease of blood vessels were observed at S2and arrived by40.24%of sham control; while the blood vessels at S7and S14arrived by48.49%and74.09%of sham group, respectively. The area of microvessels at S7and S14significantly increased compared with that at S2. The result of immunohistochemistry with CD31-positive was consistent with that of immunofluorescence. The decreased microvascular density was gradually upregulated from S7to S14.4) Simultaneously, the expressions of VEGF and VEGFR2began to increase from S2after SCI, which was line with the increase of blood vessels observed at S7and S14. While the expression of Angl was decreased after SCI, the decrease did not be ameliorative at S14.5) In this study, we found that pericyte coverage was largely lost at S2after SCI in mice compared with that at sham group. Consistent with the increase of PDGFR-β or CD13-positive cells was the dencrease of pericyte coverage observed at S7. However, the location of pericyte on microvessels was obscure, thus the quantitative of pericyte coverage was impossible.6) In vitro, we isolate mice microvascular pericytes and endothelial cells, which were further identified by NG2, PDGFRβ, vWF and GFAP. Under hypoxia, the secretions of VEGF, VEGFR-2and TGF-P were upregulated and their mediator HIF-la was also upregulated in pericytes. Simultaneously, the expressions of pericyte markers, PDGFRβ, NG2and a-SMA, were augmented, the expression and secretion of MMP2was increased under hypoxia. However, pericytes did not differentiate into EC and astrocyte under hypoxic condition. Then, we found that addition pericytes to endothelial cells supported endothelial tubular integrity as compared with ECs alone under hypoxia.Conclusions:The alteration of pericyte coverage after SCI might influence the angiogenesis by the secretions of angiogenic factors and the expressions of angiogenic associated proteins, while the decreased secretion of Angl in pericytes might be essencial reason of reduce perfused blood vessels at7days post injury. Part TwoThe protective effect of melatonin on microcirculation after spinal cord injury and mechanism researchObjective:Melatonin treatment is effective for SCI, under hypoxia, melatonin could reduce microvascular injury. The regulatory role of melatonin on microvessels is still unclear after SCI. The aim of this study was to investigate the effect of melatonin exerting on microvascular injury, and primarily elucidate the possible protective mechanism from the perspective of pericytes.MethodsrAdult male C57BL/6(18-22g) mice were randomly divided into three groups of27in each:sham group (Sham), vehicle group (Veh) and melatonin group (Mel). Mice received a moderate impacted SCI (5g weight from10mm height). BSCB permeability, LEA-labeled blood vessels, pericyte coverage and western blot were measured according to the methods introduced in part I at7days post injury. In addition, functional recovery after SCI was assessed using the Basso Mouse Scale (BMS) at2,5,7,10and14days after SCI. Scores were graded in an open-field environment by trained investigators who were blind to the experimental conditions. A aimed spinal cord was removed at indicated time, weigh wet quality, then put it in90℃electric heat drying cabinet for72h dry to permanent quality, then weigh dry quality, calculate the water content of spinal cord according to the formula of Elliot: water content of spinal cord=(wet quality-dry quality)/wet quality×100%. Under Oxygen-glucose deprivation/reperfusion (OGD/R), the secretion of Angl was detected by ELISA and the expression of ICAM-1was detected by immunofluorescence in primary pericyte.Results:At7days following injury, melatonin treatment rescued blood vessels and motor neurons in the injury epicenter and improved locomotor functional outcome; melatonin significantly reduced permeability of BSCB, attenuated the loss of occludin, and inhibited edema formation and upregulation of aquaporin-4(AQP4). Furthermore, our data showed that pericytes covering capillaries were largely lost after SCI and melatonin treatment increased the quantity of pericytes when compared with vehicle controls. Melatonin improved the expression of angiopoietin-1(Angl) following injury and upregulated the secretion of Angl in primary pericytes after OGD/R. Melatonin inhibited the expression of ICAM-1following injury and attenuated the increase of immunofluorescence mediated by ICAM-1in primary pericytes after OGD/R. Melatonin inhibited the expression of Bax, and increased the expression of Bcl-2at7days post injury.Conclusions:Melatonin ameliorates microcirculation (microvessel and BSCB) in mice to exert the protective effect on SCI, which may be partly mediated by increased pericyte coverage. Then the upregulated secretion of Angl mediated by melatonin could inhibit the expression of ICAM-1and inhibit apoptosis to protect the injured spinal cord. |
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