| BackgroundAcute spinal cord injury (SCI) is a devastatingly paralytic event accompanied with a series of vulnerable outcomes. Promoting the recovery of nerve function after acute spinal cord injury has been the research focus in neurotrauma field. The severe destruction of local microcirculation is an important cause of acute exacerbation of spinal cord injury. The vascular remodeling is required for the improvement of local microcirculation of injured spinal cord. TMP, an active biomonomer extracted from traditional Chinese Herbal Medicine has been demonstrated a positive anti-ischemia effect in a variety of disease ischemia models including SCI. Our previous studies proved that TMP can increase the number of blood vessels around damage zone in a rat model with traumatic spinal cord injury, but the exact mechanism needs further study. Therefore, In the current study, the synchrotron radiation micro-CT and immunofluorescence techniques were used to investigate the structural and volumetric alterations of both the2D and3D angioarchitecture to ensure the function of TMP in microvascular network repairing using the same rat acute SCI model, in order to provide a theoretical foundation for TMP for the clinical treatment of acute spinal cord injury. Chapter one:The effects of TMP intervention on the2D morphology of the microvasculature in rat traumatic spinal cord injury model.Purpose:To investigate effects of TMP intervention on the angiogenesis and2D morphology of the microvasculature in rat traumatic spinal cord injury model.Method:52rats were randomly divided into three groups:sham group (4), NS group(4X6time point), TMP group(4X6time point).Rats in sham group were subjected to laminectomy only. The total of remaining48rats were subjected a modified Allen’s weight drop impacting to induce traumatic spinal cord injury. Rats in TMP group were treated by TMP30min after surgery (200mg/kg Qd ip), and continuous treatment for five days. T10spinal cord specimens were harvest at1,3,714,21and28day post-injury, followed by serial of frozen sections. Dual-labeled immunofluorescence was used to detect the rat anti-mouse RECA-1and rabbit anti-PCNA. Rats in NS group were receives the same therapeutic intervention except given the equivalent dose of sterile saline. Statistical analysis was performed using SPSS19.0software package. The values were compared by ANOVA followed by SNK method between different time-points, and T test for the comparison between control and experimental groups. P<0.05was interpreted as significant.Results:(1) The effects of TMP on2D morphology of spinal microvasculature after SCI:(1.1)Normal spinal cord microvascular had a regular structure.(1.2)The vascular area fraction (VAF) of spinal cord decreased significantly (P<0.01) at1day post injury. The arrangement of residual blood vessels was disordered. The VAF increased significantly at3day and7day after SCI. Until28day after injury, the VAF could not be restored to normal level still, but the vessels become more organized.(1.3)The vascular area fraction of TMP group at3,7,14,21days after SCI were higher than the NS group (P<0.05).(2) The effects of TMP on angiogenesis after SCI.(2.1)Only few angiogenesis was in normal spinal cord.(2.2)Angiogenesis started from1day post-injury, gradually increased overtime and peaked on day7(P<0.01). Subsequently, the number of neovascularization was significantly decreased. It was almost no neovascularization after14day post-injury.(2.3)The amount of neovascularization in TMP group was higher than that in NS group (P<0.05) at3day,7day post-injury while no significant differences in the rest of the time pointsConclusion:(1) Acute spinal cord injury causes to severely structural and volumetric damage of the microvasculature, and the limited post traumatic angiogenesis is served as an inherent mechanism to improve the dysfunction of microcirculation.(2) TMP can directly promote angiogenesis after acute spinal cord injury.Chapter two:The effects of TMP intervention on the3D morphology of the microvasculature in rat traumatic spinal cord injury model.Objective:To investigate effects of TMP intervention on the3D morphology of the microvasculature in rat traumatic spinal cord injury model.Methods:52rats were randomly divided into three groups:sham group (4), NS group(4×6time point), TMP group(4×6time point).Rats in sham group were subjected to laminectomy only. The total of remaining48rats were subjected a modified Allen’s weight drop impacting to induce traumatic spinal cord injury. Rats in TMP group were treated by TMP30min after surgery (200mg/kg Qd ip), and continuous treatment for five days. Angiography was carried out at1,3,7,14,21and28day post-injury. T10spinal cord specimens were harvest, followed graded alcohol dehydration and methyl salicylate soak. SR-μCT examination was performed in Shanghai Synchrotron Radiation Facility.3D images of vasculature were reconstructed by VG Studio software. Quantization parameter was acquired by USIS software package. Rats in NS group were receives the same therapeutic intervention except given the equivalent dose of sterile saline. Statistical analysis was performed using SPSS19.0software package. The values were compared by ANOVA followed by SNK method between different time-points, and T test for the comparison between control and experimental groups. P<0.05was interpreted as significant.Results:(1) A high-resolution three-dimensional images of rat spinal vascular network was acquired by SR-μCT and could be in-depth analysis.(2)3D pathomorphological of microvasculature in SCI model.(2.1)Total length of vascular decreased (P<0.01) post injury and recovered over time, but could not return to normal levels until28days (P <0.05);(2.2)Contrast agent leakage could be observed on the3D image.(2.3)Blood vessel around damage region changed its orientation.(2.4)Vascular tortuosity increased significantly post injury (P<0.01).(2.5)Vascular continuity decreased post injury:the number of vascular segments increased significantly (P<0.01), while bifurcation point number significantly decreased (P<0.01), the continuity of the blood vessel would partly restore over time.(2.6)28days after SCI, syringomyelia was surrounded by blood vessels. Those vascular were consecutive and branched into the hole.(3)The effects of TMP on3D morphology of spinal microvasculature after SCI3D features of spinal vascular changes mainly reflected in vessels of which diameter were less than40μm. Compared with NS group, the vascular length in TMP group was significantly increased at day1,3and7post-SCI (P<0.05) while no difference was found at14,21and28post-SCI (P>0.05); vascular tortuosity was decreased at day1post-SCI (P<0.05) while no difference was found at other time point (P>0.05); vascular segments density was increased at day1,3,7post-SCI (P<0.05) while bifurcation point number was decreased at day1,3,7,14,21,28post-SCI (P<0.05).Conclusion:(1) Acute spinal cord injury causes to severely three-dimensional structural damage of the microvasculature. Self-repair of Vascular post-SCI included angiogenesis and vascular network structure remodeling(2) After spinal cord injury, TMP could improve the dysfunction of local microcirculation, accelerated vascular network structure remodeling through a variety of ways including decreasing vascular tortuosity, increasing blood volume and vascular continuity. |
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