| ObjectivesMicrovasculature plays an important role in the maintenance of primary physiological brain function. After ischemic stroke, cascade reactions initiate to destruct the homeostasis of microvascular network, thus leading to remodeling of neurovascular niche. Cerebral ischemia induces endogenous angiogenesis. However, up to now, it still remains unclear about the mechanism, development course and detailed morphology of angiogenesis. Visualization of microvessels using histological methods is confined to two-dimensional field, which is unable to reveal the spatial and temporal distribution of vascular network. Conventional imaging techniques such as MRA, CTA, DSA are incapable of detecting small vessels <50μm in diameter without angiography. Advanced synchrotron radiation technique has broken through the diffraction limitation of traditional optical imaging methods, which achieves nondestructive, three-dimensional imaging of microstructure on a micron or sub-micron scale. In the present study, synchrotron radiation (SR) X-ray in-line phase contrast imaging (ILPCI) was used to acquire high resolution and high contrast images of rat brain tissues in both normal and ischemic states. We attempted to explore the dynamic development course of cerebral microvascular plasticity and provide detailed three-dimensional imaging information and morphometry evidence.Methods ILPCI was performed at the Shanghai Synchrotron Radiation Facility without the use of contrast agents. Computed tomography (CT) slices obtained were reformatted using filtered back projection and then converted into three-dimensional reconstruction images to analyze subtle details of the cerebral microvascular network. Subsequently, quantitative analysis of angioarchitecture was performed based on bioinformatics. Meanwhile stereomicroscope and HE staining of brain tissues were compared with ILPCI results.Then we observed dynamic process of angiogenesis after permanent focal ischemia in rat brain and attempted to establish quantitative analysis of neovasculature. Combining with immunohistochemistry, comprehensive evaluation was carried out in terms of angioarchitectural remodeling process after ischemic stroke.Results:1. By using ILPCI, we established a hierarchical visualization system of cerebral microvasculature in the sequence of projection data,2D slice and integral3D images. By using CCD with per pixel of5.9μm, brain vessels up to9.4μm in diameter were resolved. In contrast with HE staining, we achieved clear visualization of microvessels in hippocampus without any contrast agents and complex section procedure.3D rendered images of vascular trees on the surface of the brain were highly congruent with the casting corrosion Microfil perfusion results. Multi-level anastomosis of pia vessels could be clearly discriminated.2. We established horizontal, coronal, sagittal virtual slices and digitized images of brain vasculature3D based on conversion of spatial dimensions. Blood supply of different functional areas were revealed from3D perspective.3. We extracted skeleton of vascular network and demonstrated that microvessels <20μm are the major component of the whole brain angioarchitecture. We further clarified a series of vessel parameters, including vascular volume, vascular density, number of junctions, the number of nodes, adjacent vessel space, diameter distribution.4. In terms of middle cerebral artery occlusion (MCAO) model by SR imaging, we found that of microvessels were densely distributed in the periphery of the ischemic area and gradually extended from the ischemic penumbra to the infarct zone at2hours,4hours and6hours after MCAO. And these microvessels exhibited a sustained increasing tendency in quantity. However, with prolonged ischemic time, both the distribution and density of neovasculature showed a decreasing tendency at1day,3days after MCAO. Local microvessels were tortuous, forming irregular vascular loop. Malacia was formed and glial scar began to appear at7days after ischemia. Brain volume on the lesion side significantly decreased and intraventricle was compressed at18days after MCAO. The organization of infarct tissues progressively developed and glial scars were shown in the slice. Stroke cysts without parenchymal tissue were also identified.5. With the increase in ischemic time, the brain volume on the lesion side gradually decreased from3days after MCAO. Moreover, brain volume was significantly reduced compared with the control group (p<0.05). Compared with the control group, vascular density was significantly increased at2hours,4hours,6hours after MCAO (p<0.05). However, vascular density was significantly decreased at7days and18days after MCAO (p<0.05). Number of vessel junctions and nodes were significantly increased at2hours,4hours and6hours after ischemia in comparison with the control group (p<0.05). While these two indexes were gradually reduced at3days,7days and18days (p<0.05). Microvessls with diameter of10-20μm showed a significant increase in quantity at2hours,4hours and6hours after ischemia compared with the control group (p<0.05). Significant decrease in microvessel quantity was revealed at7days and18days (p<0.05). The tortuosity showed a significant increase at2hours,4hours,6hours,1day and3days after ischemia (p<0.05).6. CD31positive microvessels maintained a sustained increase in quantity at2hours,4hours and6hours in striatum. They were densely distributed in the periphery of the ischemic area. These microvessels exhibited various shapes, for instance, spotty, short-curved or tubular. Newly generated microvessels gradually decreased at1day,3days and7days after ischemia. The aforementioned trend of microvasculature was consistent with SR imaging results.Conclusions1.SR-based ILPCI-CT can serve as a powerful tool to accurately visualize brain microvasculature. The morphological parameters of blood vessels in both CT slices and three-dimensional reconstructions were determined.2. Ischemic injury leads to the structural and functional plasticity of microvasculature. The process of angiogenesis initiates in an early stage and quickly reached the culmination. Newly generated microvessels showed an increase in tortuosity and branching, thus intertwining into a complicated network. It remains as an important compensatory approach to local blood supply.3. SR based ILPCI is able to provide detailed imaging evidence of dynamic angiogenesis course from both2D and3D perspective. |
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