Tianding Wu

The Angiogenic Effect of microRNA-21 Targeting TIMP3 through the Regulation of MMP2 and MMP9.

microRNAs are a novel set of small, non-protein-coding nucleotide RNAs that negatively regulate the expression of target mRNAs. miRNA-21 is a microRNA that is highly enriched in endothelial cells. miRNA-21 has been shown to be a potential pro-angiogenic factor in some biological systems. Our previous study showed that the expression of miRNA-21 was up-regulated after spinal cord injury. However, the effect of miRNA-21 on angiogenesis in the spinal cord was unclear. In this study, to understand the role of miRNA-21 on injured endothelial cells exclusively, an oxygen and glucose deprivation model of endothelial cells was constructed, and the up-regulation of miRNA-21 was discovered in this model. An increased level of miRNA-21 by mimics promoted the survival, migration and tube formation of endothelial cells, which simultaneously inhibited tissue inhibitor of metalloproteinase-3 (TIMP3) expression and promoted matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9) expression and secretion. A decreased level of miRNA-21 by antagomir exerted an opposite effect. As is well known, survival, migration and tube formation of endothelial cells are necessary prerequisites for angiogenesis after injury. TIMP3 was validated as a direct target of miRNA-21 by dual-luciferase reporter assay. Silencing with small interfering RNA against TIMP3 promoted tube formation and increased MMP2 and MMP9 expression at the protein level. In vivo, we found that decreased levels of miRNA-21 inhibited angiogenesis after spinal cord injury in rats using synchrotron radiation micro-computed tomography. In summary, these findings suggest that miRNA-21 has a protective effect on angiogenesis by reducing cell death and promoting cell survival, migration and tube formation via partially targeting the TIMP3 by potentially regulating MMP2 and MMP9. TIMP3 is a functional target gene. Identifying the role of miRNA-21 in the protection of angiogenesis might offer a novel therapeutic target for secondary spinal cord injury, in which angiogenesis is indispensable.

Visualization of microvasculature by x-ray in-line phase contrast imaging in rat spinal cord

Computed tomography combined with angiography has recently been developed to visualize three-dimensional (3D) vascular structure in experimental and clinical studies. However, there remain difficulties in using conventional x-ray angiography to detect small vessels with a diameter less than 200 μm. This study attempted to develop a novel method for visualizing the micro-angioarchitecture of rat spinal cord. Herein, synchrotron radiation-based x-ray in-line phase contrast computed tomography (IL-XPCT) was used to obtain 3D micro-vessel structure without angiography. The digital phase contrast images were compared with conventional histological sections. Our results clearly demonstrated that the resolution limit of the spatial blood supply network in the normal rat thoracic cord appeared to be as small as ~10 μm. The rendered images were consistent with that obtained from histomorphology sections. In summary, IL-XPCT is a potential tool to investigate the 3D neurovascular morphology of the rat spinal cord without the use of contrast agents, and it could help to evaluate the validity of the pro- or anti-angiogenesis therapeutic strategies on microvasculature repair or regeneration.

Three-dimensional alteration of microvasculature in a rat model of traumatic spinal cord injury

Acute spinal cord injury (SCI) always leads to severe destruction of the microvascular networks. To investigate the three-dimensional (3D) alterations of microvasculature following SCI, we utilized an established rat SCI model. Based on the hypothesis that the spinal cord would undergo reorganization and postinjury modification of the vascular networks after SCI, we reconstructed the normal and injured angioarchitecture using micro-CT images of silicone rubber microsphere-perfused specimens. Several morphometric parameters were used to study the 3D vascular alterations in the SCI rat model, including the casting-based vessel volume fraction, connectivity density, separation, thickness and thickness distribution. Our results indicated that the microvascular spatial conformations were significantly different between the normal and injured spinal cord segments. The morphometric changes showed an increase of the vessel volume fraction and separation and a decrease of vessel connectivity density during the vascular healing process after SCI. Our results may contribute to elucidation of the mechanisms of compensatory vascular reconstitution in traumatized spinal cord. The method used here has the potential to improve our understanding of changes in the spatial architecture of vascular networks after SCI compared to the conventional histomorphology techniques. In summary, we developed a new methodology to analyze neurovascular pathology based on 3D vascular network patterns and features in an experimental rat SCI model. This technique could be used as a complementary tool to investigate the efficacy and side effects of therapeutic drugs or rehabilitation regimens.

High-resolution three-dimensional visualization of the rat spinal cord microvasculature by synchrotron radiation micro-CT

PURPOSE Understanding the three-dimensional (3D) morphology of the spinal cord microvasculature has been limited by the lack of an effective high-resolution imaging technique. In this study, synchrotron radiation microcomputed tomography (SRµCT), a novel imaging technique based on absorption imaging, was evaluated with regard to the detection of the 3D morphology of the rat spinal cord microvasculature. METHODS Ten Sprague-Dawley rats were used in this ex vivo study. After contrast agent perfusion, their spinal cords were isolated and scanned using conventional x-rays, conventional micro-CT (CµCT), and SRµCT. RESULTS Based on contrast agent perfusion, the microvasculature of the rat spinal cord was clearly visualized for the first time ex vivo in 3D by means of SRµCT scanning. Compared to conventional imaging techniques, SRµCT achieved higher resolution 3D vascular imaging, with the smallest vessel that could be distinguished approximately 7.4 μm in diameter. Additionally, a 3D pseudocolored image of the spinal cord microvasculature was generated in a single session of SRµCT imaging, which was conducive to detailed observation of the vessel morphology. CONCLUSIONS The results of this study indicated that SRµCT scanning could provide higher resolution images of the vascular network of the spinal cord. This modality also has the potential to serve as a powerful imaging tool for the investigation of morphology changes in the 3D angioarchitecture of the neurovasculature in preclinical research.

The effect of estrogen-related receptor α on the regulation of angiogenesis after spinal cord injury

Estrogen receptor-related receptor-α (ERRα) is an orphan member of the nuclear receptor superfamily that interacts with peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) to stimulate vascular endothelial growth factor (VEGF) expression and angiogenesis in a hypoxia-inducible factor-1α-independent pathway. Although it is not regulated by any natural ligand, the action of ERRα can be blocked by the synthetic molecule XCT790. In the present study, Sprague-Dawley rats were randomly allocated to a sham group, injury-saline group or injury-XCT90 group. A modified Allens weight-drop method was applied to induce the acute traumatic spinal cord injury (SCI) model in these rats, and an injection of XCT790 was administered every 24h, starting half an hour after the SCI contusion. Histological analyses revealed that XCT790 significantly aggravated tissue damage and decreased the number of ERRα-positive cells at 1, 3 and 7 days after SCI. Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) analyses also indicated that XCT790 dramatically repressed the expression of ERRα, thus reducing the expression of VEGF and angiopoietin-2 (Ang-2) throughout the duration of the experiment, but the expression of PGC-1α was not affected. Immunofluorescence analyses indicated that vascular density and endothelial cell proliferation were decreased in the injury-XCT90 group compared with the injury-saline group. These results suggest that ERRα is involved in mediating angiogenesis after SCI in the rat traumatic SCI model.

Three Dimensional Quantification of Microarchitecture and Vessel Regeneration by Synchrotron Radiation Microcomputed Tomography in a Rat Model of Spinal Cord Injury.

A full understanding of the mechanisms behind spinal cord injury (SCI) processes requires reliable three-dimensional (3D) imaging tools for a thorough analysis of changes in angiospatial architecture. We aimed to use synchrotron radiation μCT (SRμCT) to characterize 3D temporal-spatial changes in microvasculature post-SCI. Morphometrical measurements revealed a significant decrease in vascular volume fraction, vascular bifurcation density, vascular segment density, and vascular connectivity density 1 day post-injury, followed by a gradual increase at 3, 7, and 14 days. At 1 day post-injury, SRμCT revealed an increase in vascular tortuosity (VT), which reached a plateau after 7 days and decreased slightly during the healing process. In addition, SRμCT images showed that vessels were largely concentrated in the gray matter 1 day post-injury. The maximal endothelial cell proliferation rate was detected at 7 days post-injury. The 3D morphology of the cavity appears in the spinal cord at 28 days post-injury. We describe a methodology for 3D analysis of vascular repair in SCI and reveal that endogenous revascularization occurs during the healing process. The spinal cord microvasculature configuration undergoes 3D remodeling and modification during the post-injury repair process. Examination of these processes might contribute to a full understanding of the compensatory vascular mechanisms after injury and aid in the development of novel and effective treatment for SCI.

Nondestructive imaging of the internal micro­structure of vessels and nerve fibers in rat spinal cord using phase-contrast synchrotron radiation microtomography

The spinal cord is the primary neurological link between the brain and other parts of the body, but unlike those of the brain, advances in spinal cord imaging have been challenged by the more complicated and inhomogeneous anatomy of the spine. Fortunately with the advancement of high technology, phase-contrast synchrotron radiation microtomography has become widespread in scientific research because of its ability to generate high-quality and high-resolution images. In this study, this method has been employed for nondestructive imaging of the internal microstructure of rat spinal cord. Furthermore, digital virtual slices based on phase-contrast synchrotron radiation were compared with conventional histological sections. The three-dimensional internal microstructure of the intramedullary arteries and nerve fibers was vividly detected within the same spinal cord specimen without the application of a stain or contrast agent or sectioning. With the aid of image post-processing, an optimization of vessel and nerve fiber images was obtained. The findings indicated that phase-contrast synchrotron radiation microtomography is unique in the field of three-dimensional imaging and sets novel standards for pathophysiological investigations in various neurovascular diseases.

Tetramethylpyrazine Facilitates Functional Recovery after Spinal Cord Injury by Inhibiting MMP2, MMP9, and Vascular Endothelial Cell Apoptosis.

BACKGROUND Spinal cord injury (SCI) is a major public health issue that leads to neurological dysfunctions and morbidities in patients. Tetramethylpyrazine (TMP) plays a neuroprotective role in SCI; however, the underlying mechanism has not been fully elucidated. OBJECTIVE In the present study, we aimed to investigate the mechanisms and therapeutic effects of TMP on SCI. METHODS A contusion SCI model was established that used a modified Allens method. In the TMP group, TMP (200 mg/kg) was injected daily for 5 days post-injury, while in the Negative Control (NC) group, an equal volume of normal saline was injected. Hindlimb motor function was evaluated using the Basso, Beattie, Bresnahan (BBB) scale. The effects of TMP on protein levels of the matrix metalloproteinases 2 (MMP2) and 9 (MMP9), Bax and cleaved caspase-3 were determined by western blotting. Apoptotic changes in vascular endothelial cells were evaluated using immunofluorescence and TUNEL staining. Alterations in 3D vessel morphology after treatment with TMP were assessed by synchrotron radiation micro-CT (SRμCT). RESULTS TMP treatment significantly improved recovery in hindlimb motor function and attenuated vascular endothelial cell apoptosis in rats with SCI. Additionally, TMP treatment markedly decreased the protein levels of MMP2 and MMP9, pro-apoptotic bax and cleaved caspase-3 while promoting angiogenesis, as evidenced by vessel visualization using SRμCT. CONCLUSION These results indicate that TMP attenuated SCI-induced neurological impairments by the down-regulation of the expression of MMP2 and MMP9 proteins, the inhibition of vascular endothelial cell apoptosis, and the promotion of angiogenesis.

Synchrotron radiation micro-CT as a novel tool to evaluate the effect of agomir-210 in a rat spinal cord injury model.

MicroRNA-210 (miR-210) was initially reported to be associated with hypoxia and plays a vital role in modulating angiogenesis. However, the potential effect and underlying mechanisms of miR-210 activity in rat spinal cord injury (SCI) have not yet been fully illuminated. In the present study, differential microRNA expression after SCI was determined by Microarray analysis. To explore the effect of miR-210 after SCI, we intrathecally injected agomir-210 with Alzet Osmotic Pumps to up-regulated the endogenous miR-210 expression. Then, synchrotron radiation micro-CT (SRμCT) imaging was used to investigate the effect of agomir-210 in rat SCI model. We found that the endogenous miR-210 expression could be up-regulated by intrathecal agomir-210 injection. The administration of agomir-210 significantly promoted angiogenesis, as evidenced by increased vessel number and volume detected by SRμCT, attenuated the lesion size and improved functional recovery after SCI. Additionally, agomir-210 attenuated cellular apoptosis and inflammation in the injured rat spinal cord. Expression levels of pro-apoptotic protein (Bax) and pro-inflammatory cytokines (TNF-α and IL-1β) were significantly decreased after agomir-210 treatment, whereas expression levels of anti-apoptotic (Bcl-2) and anti-inflammatory (IL-10) proteins were up-regulated. In conclusion, our results indicated that SRμCT is a powerful imaging tool to evaluate the effects of angiogenesis after agomir-210 administration in rat SCI model. The up-regulation of endogenous miR-210 expression following agomir-210 administration promoted angiogenesis and anti-apoptotic protein expression, and attenuated inflammation. MiR-210 played a positive role in neurological functional recovery and could be a potential new therapeutic target for SCI.

Visualization of mouse spinal cord intramedullary arteries using phase- and attenuation-contrast tomographic imaging

Many spinal cord circulatory disorders present the substantial involvement of small vessel lesions. The central sulcus arteries supply nutrition to a large part of the spinal cord, and, if not detected early, lesions in the spinal cord will cause irreversible damage to the function of this organ. Thus, early detection of these small vessel lesions could potentially facilitate the effective diagnosis and treatment of these diseases. However, the detection of such small vessels is beyond the capability of current imaging techniques. In this study, an imaging method is proposed and the potential of phase-contrast imaging (PCI)- and attenuation-contrast imaging (ACI)-based synchrotron radiation for high-resolution tomography of intramedullary arteries in mouse spinal cord is validated. The three-dimensional vessel morphology, particularly that of the central sulcus arteries (CSA), detected with these two imaging models was quantitatively analyzed and compared. It was determined that both PCI- and ACI-based synchrotron radiation can be used to visualize the physiological arrangement of the entire intramedullary artery network in the mouse spinal cord in both two dimensions and three dimensions at a high-resolution scale. Additionally, the two-dimensional and three-dimensional vessel morphometric parameter measurements obtained with PCI are similar to the ACI data. Furthermore, PCI allows efficient and direct discrimination of the same branch level of the CSA without contrast agent injection and is expected to provide reliable biological information regarding the intramedullary artery. Compared with ACI, PCI might be a novel imaging method that offers a powerful imaging platform for evaluating pathological changes in small vessels and may also allow better clarification of their role in neurovascular disorders.