| BackgroundAcute spinal cord injury (SCI) is one of the most common and devastating diseases caused by external force impacting on spinal cord, and is always accompanied with complete or incomplete dysfunction of motor, sensory and sphincter motility. As there is still no effective treatment strategies for SCI, to explore an effective drug is always one of important research topics. Many studies demonstrated that the destruction of micro vessels would aggravate the secondary injury after SCI, and the improvement of local microcirculation is important for the repair process. Tetramethylpyrazine (TMP) is an active biomonomer extracted from traditional Chinese Herbal medicine with demonstrated positive anti-ischemia effect. Our research group had demonstrated the pro-angiogenesis and promoting functional rehabilitation effect by administrating TMP in a rat model with traumatic spinal cord injury. In the current study, using the same rat acute SCI model, we investigated the vascular protection mechanism of TMP by evaluating the effects of TMP on apoptosis of endothelia cells, micro vessel density, collagen Ⅳ, laminin, collagen Ⅰ, collagen ⅩⅧ, matrix metalloproteinase (MMP)-2and MMP-9.Chapter One:The Effects of Tetramethylpyrazine on Apoptosis of Vascular Endothelial Cells and Micro vessel density in a Rat Model of Spinal Cord InjuryObjectiveTo investigate the the effects of TMP on apoptosis of vascular endothelial cells (ECs) and micro vessel density (MVD) in rats after spinal cord injury. Materials and methods130male SD rats (200-250g) were used in this study.10rats were randomly assigned for sham group (n=5) which only received laminectomy. The rest120rats were used to obtain SCI model by a modified Allen’s weight drop apparatus, and randomly divided into NS group (n=60) and TMP group (n=60). NS group were treated intraperitoneally with saline (200mg/kg) while TMP group with TMP (200mg/kg), every24h for5days, starting half an hour after SCI. The NS group and TMP group were divided into6time-period sub-group:1,3,7,14,21and28days (n=20each sub-group). Each group of SCI rats (n=60) were used for immunofluorescence detection of apoptosis vascular ECs (IF, n=30), HE staining and immunohistochemical detection of MVD (IHC, n=30). All data was analyzed with SPSS17.0software. The values were analyzed for the comparison between the two groups by Mann-Whitney U test. P<0.05was interpreted as significant.Results(1) HE staining showed damage in the spinal cord undergo extensive bleeding, hematoma formation, hematoma absorption, collagen proliferation, scar formation and other pathological process.(2) Apoptosis vascular ECs appeared at day1in both groups. The number of apoptosis vascular ECs reached the peak at day3and then decreased in NS group. There was no apoptosis cell after14days in NS group. In TMP group, the number of apoptosis vascular ECs decreased after day1and there was no apoptosis cell after7days. Compared with NS group, the number of apoptosis vascular ECs in TMP group was significantly down-regulated at day3, day7and day14(P<0.05).(3) MVD decreased sharply at day1after SCI and then rose gradually in both groups. After14days, MVD had no obvious growth. Compared with NS group, MVD of TMP group was significantly up-regulated at day3, day7and day14(P<0.05).Conclusions(1) TMP could reduce the apoptosis of vascular ECs after spinal cord injury.(2) TMP could up-regulate MVD after spinal cord injury. Chapter Two:The Effects of Tetramethylpyrazine on Collagen and Laminin in a Rat Model of Spinal Cord InjuryObjectiveTo investigate the the effects of TMP on type Ⅳ, laminin, type Ⅰ and type ⅩⅧ collagen in rats after spinal cord injury.Materials and methods65male SD rats (200-250g) were used in this study.5rats were randomly assigned for sham group (n=5) which only received laminectomy. The rest60rats were used to obtain SCI model by a modified Allen’s weight drop apparatus, and randomly divided into NS group (n=30) and TMP group (n=30). NS group were treated intraperitoneally with saline (200mg/kg) while TMP group with TMP (200mg/kg), every24h for5days, starting half an hour after SCI. The NS group and TMP group were divided into6time-period sub-group:1,3,7,14,21and28days (n=5each sub-group). The samples were obtained and proceeded to immunochemistry detection for the expression of type IV, laminin, type Ⅰ and type ⅩⅧ collagen. All data was analyzed with SPSS17.0software. The values were analyzed for the comparison between control and experimental groups by Mann-Whitney U test. P<0.05was interpreted as significant.Results(1) The expression of Type Ⅳ collagen was enhanced1day after SCI. Type Ⅳ collagen proteins were expressed in vascular ECs, neurons, oligodendrocytes and astrocytes. The number of positive cells rose gradually and reached the peak at day7. After that the number decreased. Compared with NS group, the type Ⅳ collagen positive expression in TMP group was significantly up-regulated at day1, day3, day7and day14(P<0.05).(2) The expression of laminin was enhanced1day after SCI. Laminin proteins were expressed in vascular ECs, neurons, oligodendrocytes and astrocytes. The number of positive cells rose gradually and reached the peak at day7. After that the number decreased. Compared with NS group, the laminin positive expression in TMP group was significantly up-regulated at day3and day7(P<0.05).(3) Type Ⅰ collagen were expressed1day after SCI. The number of positive cells rose gradually and reached the peak at day7. After that the number decreased. Type Ⅰ collagen proteins were expressed in neurons, oligodendrocytes and astrocytes. Compared with NS group, the type Ⅰ collagen positive expression in TMP group was significantly up-regulated at day3, day7and day14(P<0.05).(4) Type ⅩⅧ collagen were expressed at day7in NS group and the number of positive cells rose gradually and reached the peak at day21. After that the number decreased. Positive cells were firstly observed at day14in TMP group and the number rose gradually to reach the peak at day28. Type ⅩⅧ collagen proteins were expressed in glial cells at the beginning, and appeared in neurons soon. Compared with NS group, the type ⅩⅧ collagen positive expression in TMP group was significantly down-regulated at day7, day14and day21(P<0.05).Conclusions(1) TMP up-regulated the expression of type Ⅳ collagen, laminin and type Ⅰ collagen at early phase after SCI.(2) TMP down-regulated the expression of type ⅩⅧ collagen after SCI. Chapter Three:The Effects of Tetramethylpyrazine on MMP-2and MMP-9in a Rat Model of Spinal Cord InjuryObjectiveTo investigate the the effects of TMP on the expression of MMP-2and MMP-9in rats after spinal cord injury.Materials and methods 130male SD rats (200-250g) were used in the study.10rats were randomly assigned for sham group (n=5) which only received laminectomy. The rest120rats were used to obtain SCI model by a modified Allen’s weight drop apparatus, and randomly divided into NS group (n=60) and TMP group (n=60). NS group were treated intraperitoneally with saline (200mg/kg) while TMP group with TMP (200mg/kg), every24h for5days, starting half an hour after SCI. The control group and TMP group were divided into6time-period sub-group:1,3,7,14,21and28days (n=20each sub-group). Each group of SCI rats (n=60) were used for immunohistochemistry (IHC, n=30) and western blot (WB, n=30) to detect the expression of MMP-2and MMP-9. All data was analyzed with SPSS17.0software. The values were analyzed for the comparison between control and experimental groups by T test. P<0.05was interpreted as significant.Results(1) In immunohistochemistry, MMP-2were expressed1day after SCI. The number of positive cells rose gradually and reached the peak at day3. After that the number decreased. MMP-2proteins were expressed in neurons, oligodendrocytes and astrocytes. Compared with NS group, MMP-2positive expression in TMP group was significantly down-regulated at day1, day3, day7and day14(P<0.05).(2) In western blot, MMP-2proteins up-regulated1day after SCI and reached the peak at day3, and expression decreased after that. Compared with NS group, MMP-2protein expression in TMP group was significantly down-regulated at day1, day3, day7and day14(P<0.05).(3) In immunohistochemistry, MMP-9were expressed1day after SCI and the number of positive cells decreased after that. MMP-9proteins were expressed in neurons, oligodendrocytes and astrocytes. Compared with NS group, MMP-9positive expression in TMP group was significantly down-regulated at day1, day3, day7, day14and day21(P<0.05).(4) In western blot, MMP-9proteins up-regulated1day after SCI and decreased after that. Compared with NS group, MMP-9protein expression in TMP group was significantly down-regulated at day1, day3, day7, day14and day21(P<0.05).ConclusionsTMP could down-regulated the expression of MMP-2and MMP-9after SCI. |
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