The Effects Of Tetramethylpyrazine Intervention On Promoting Angiogenesis In A Rat Model Of Spinal Cord Injury

BackgroundAcute spinal cord injury (SCI) is a devastatingly paralytic event accompanied with a series of vulnerable outcomes, one of which is the severe destruction of local micro-vascular architecture that would aggravate the secondary injury to the spinal cord as well as retarding its repair process. The vascular remodeling is required for the improvement of local microcirculation of injured spinal cord. Tetramethylpyrazine (TMP) is an active biomonomer extracted from traditional Chinese Herbal Medicine with demonstrated positive anti-ischemia effect. Recently, studies had demonstrated that TMP played a protective role in the spinal cord injury repair in animal SCI models. Our research group had demonstrated the pro-angiogenesis effect by administrating TMP in a rat model with traumatic spinal cord injury, which probably improved the local posttraumatic ischemia condition. In the current study, using the same rat acute SCI model, the modern micro-radiography and immunofluorescence techniques were used to investigate the structural and volumetric alterations of both the2D and3D angioarchitecture within the injured spinal cord. The expression level of HIF-1α and VEGF was also examined to explain the probable mechanism of TMP treatment on posttraumatic angiogenesis. Chapter One:Three-dimensional (3D) visualization of microvasculature in the rat spinal cord by modern micro-radiographyObjectiveTo explore the application potential in three-dimensional visualization of angioarchitecture of the rat spinal cord using micro-CT and IL-XPCT techniques.Materials and methodsEight SD rats were subjected to angiography after Microfil injection and the spinal cords were harverted.(1) Four samples were followed by micro-CT scanning,3D spinal vascular rendering and morphometric analysis using the micro-view software.(2) Other four samples were immersed in methyl pacetaminosalicylate and prepared for IL-XPCT detection.Results(1) The spinal vasculature was successfully reconstructed by micro-CT and IL-XPCT. The vascular arrangement and distribution were reconstructed similar to that of the the spinal vascular images obtained by histology method.(2) The intramedullary blood supply was composed of sulcal vessels and penetrating vessels, both of them fed the neural parenchyma in gray matter.(3) The vessel density in gray matter significantly increased when comparing with that of peripheral white matter.(4) The smallest vessel diameter measured in IL-XPCT image was approximately7.4μm. Conclusions(1) The neurons in spinal cord are susceptible to pathological ischemia and hypoxia.(2) Micro-CT is an effective stereological methodology for structural and volumetric evaluation of rat spinal vasculature.(3) IL-XPCT can be used as a novel and potent high-resolution imaging tool to investigate the anatomical neurovascular network in rat spinal cord. Chapter Two:The effects of TMP intervention on the2D and3D morphology of the microvasculature of traumatized spinal cordObjectiveTo investigate the protective effects on local microcirculation in the rat injured spinal cord treated with TMP.Materials and methodsA total of85SD rats were used in this experiment. Five rats were only subjected to laminectomy as the Sham group, and the other80rats were subjected to the modified Allen's weight drop impacting to induce the traumatic spinal cord injury, and these animals were divided into the TMP group (n=40) and the NS group (n=40). Rats in TMP group were treated with TMP through intraperitoneal injection (200mg/kg) once a day for five days. Angiography was carried out at1,3,7and14day post-injury. The samples were dissociated from spinal canal, followed by thick tissue section and micro-CT examination, in which the3D morphometric parameters including VVF, M.Th, M.Sp and Conn.D were evaluated. The samples without angiography proceeded to HE staining and micro-vessel immunofluorescence for vessel counts and diameter measurement. Rats in NS group were treated in the same manner except that only the equivalent amount of normal saline solution was injected. All data was analyzed with SPSS13.0software. 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) Histological and vascular morphological alterations post-SCI:(1.1) Histology:(a) H&E staining:The injured epicenter and neighboring region were clearly identified at1day post-SCI, in which pathological edema, inflammatory cells infiltration and neural necrosis could be observed, especially in the gray matter. The intramedullary haemorrhage and ischemia regions were expanded into both the cranial and caudal regions. Glial scar and vacuoles were formed with gliacytes activation as healing processed over time.(b) Angiography-based thick tissue section:The microvasculature was profoundly damaged with a large region of haemorrhage as well as ischemia at the acute phase post-SCI. At day14post-SCI, the ischemia area was contracted along with endogenous micro-vessels budding around the injured parenchyma.(1.2)3D/2D morphology:(a)3D vascular morphology:The local vasculature was in disorder with VVF and Conn.D significantly down-regulated (p<0.05), but the M.Th and M.Sp up-regulated when comparing with those in the sham samples (p<0.05).(b)2D vascular morphology:The RECA-1positive micro-vessels were drastically reduced at1day post-SCI (p<0.01) in the anterior horn, then slowly increased with healing time.(2)3D morphology affected by TMP:Compared with NS group, the VVF in TMP group was significantly increased at day3,7and14post-SCI (p<0.05); M.Th was increased at day7post-SCI (p<0.05); no difference was found in M.Sp (p>0.05); Conn.D was significantly decreased at day7post-SCI (p<0.05).(3) Micro-vessel counts and diameter affected by TMP:Compared with NS group, the RECA-1positive micro-vessels in TMP group was significantly increased at day7and14post-SCI (p<0.05), as well as the increase in average micro-vessel diameter at1day (p<0.05).Conclusions(1) The trauma directly causes to structural and volumetric damage of the microvasculature, and the posttraumatic angiogenesis is served as an inherent mechanism to promote the vascular remodeling in the rat SCI model.(2) TMP improves the local spinal microcirculation by increasing of blood flow via the positive effects of vessels dilation and pro-angiogenesis. Chapter Three:The effects of TMP intervention on the expression level of VEGF and HIF-la in traumatized spinal cordObjectiveTo investigate the molecular mechanism of pro-angiogenesis induced by TMP in injured spinal cord. Materials and methodsA total of85SD rats were used in this experiment. Five rats were only subjected to laminectomy as the Sham group, and the other80rats were subjected to the modified Allen's weight drop impacting to induce the contusive spinal cord injury, then divided them into TMP group (n=40) and NS group (n=40). Rats in the TMP group were treated with TMP through intraperitoneal injection (200mg/kg) once a day for five days. The samples were extracted from spinal canal after laminectomy at day1,3,7and14post-SCI, followed by western blot, VEGF immunofluorescence and HIF-la immunochemistry examination. The protein level and position of VEGF and HIF-la were analyzed. Rats in NS group were executed the same way except the use of equivalent amount of normal saline injection. All data was analyzed with SPSS13.0software. The values were analyzed for the comparison between control and experimental groups by T test. P<0.05was interpreted as significant.Results(1) VEGF expression location:The positive VEGF molecules were freely diffuse in both gray matter and white matter at normal and injured spinal cord. VEGF proteins were expressed in neurons, dendrite, axon, glial cells, oligodendrocyte, endothelial cells, neural fibre and ependymocytes.(2) VEGF expression level affected by TMP:Compared with NS group, the VEGF positive expression in TMP group was significantly up-regulated at day7and14post-SCI (p<0.05).(3) HIF-lα expression location:At1and3days post-injury, the positive HIF-1α molecules were mostly located in neurons, dendrite, axon and ependymocytes, as well as partial in glial cells, oligodendrocyte, and endothelial cells.(4) HIF-1α expression level affected by TMP:HIF-1α proteins were significantly up-regulated in the injured spinal cord suffering from acute trauma with the highest level at3days (p<0.01). TMP prevented the HIF-la up-regulation at each time-points post-injury (p<0.05).Conclusions(1) HIF-1a/VEGF plays a role in the process of posttraumatic angiogenesis in the rat spinal cord injury.(2) TMP effectively reduced the local hypoxia in the acutely injured spinal cord.(3) TMP promotes posttraumatic angiogenesis possible with HIF-la-independent up-regulation of VEGF protein in the rat acute SCI model.