| Background and objective:The vascular network is an important component of nutrient transport,signaling,tissue support,and structural maintenance of the central nervous system,and is closely linked to the central nervous system not only anatomically but also functionally.Ischemic stroke is a common and frequent acute vascular disease of the nervous system,and an important vascular change in the process of its occurrence is the opening of side branches,which has the function of regulating tissue perfusion,prolonging the survival of neuronal cells,buffering and reducing reperfusion injury,etc.Some studies have shown that the state of side circulation is positively correlated with the rate of revascularization and clinical outcome,etc.Some studies have shown that tertiary microvascular opening not only provides nutrition,but also plays a neuroprotective role in the formation process.Therefore,the morphological characterization of microvascular changes after cerebral ischemia will provide new possibilities for future therapeutic studies on microvascular neovascularization in ischemic stroke.However,due to the small diameter of microvessels and complex structural variation,it is difficult to assess the morphology of post-ischemic cerebral microvessels,which also limits the possibility of therapeutic studies on microvascular de novo opening.For microvascular collateral assessment,traditional imaging methods are cumbersome,with heavy sample loss,low spatial and temporal resolution,and difficult to reconstruct in 3D.Even the "gold standard" DSA technique for collateral assessment can only achieve millimeter-level resolution due to its contrast-dependent absorption contrast method and is limited by the flow of the contrast agent and its tendency to cause allergy.It is difficult to deeply characterize the post-ischemic microvascular network due to the limitations of contrast agent flow and allergy.The synchrotron radiation(SR)has the characteristics of high collimation,high energy and high coherence,and this imaging technique can achieve the spatial and temporal resolution that cannot be achieved by conventional X-ray,combining with CT technology which brings new hope for the characterization of cerebral microvascular network.At the same time,the phase contrast method based on SR technology can realize soft tissue high-resolution imaging without contrast agent,which provides new opportunities for the characterization of cerebral microvascular networks.Therefore,in this study,we firstly compared the characterization features and advantages of the traditional ACI and the new PCI methods in the murine cerebral microvascular network based on the SRCT technique;secondly,the PCI method based on the SRCT technique deeply explored the multidimensional characterization possibilities of the murine cerebral microvascular network;finally,we used the PCI method based on the SRCT technique to investigate the morphological changes of the microvascular network at different ischemic time points in rats after MCAO.Finally,the morphological changes and characteristics of the microvascular network in rats at different ischemic time points after MCAO were characterized using the SR-based PCI method.This study hopes to provide a new reliable method for brain microvascular network characterization based on SRCT technology,to provide new ideas for characterizing microvascular network changes after the occurrence of ischemic stroke,and to provide new directions for new microvascular neovascularization-based therapies for ischemic stroke.Methods:(1)Male SD rats weighing 280-350 g were selected and randomly divided into two groups,namely phase contrast imaging group and absorption contrast imaging group,5 in each group.The rats in the PCI group were perfused with paraformaldehyde and then executed to remove the brain and tissue gradient dehydration,while the rats in the ACI group were perfused with paraformaldehyde and Microfil contrast agent sequentially,and then executed to remove the brain and tissue gradient dehydration;the brain tissues of both groups were photographed in the SSRF BL13W1 line station for PCI and ACI,and the images were post-processed to obtain two-dimensional and three-dimensional images of the two methods,respectively.The two groups of brain tissues were photographed at the SSRF BL13W1 line station PCI and ACI,and post-processed to obtain two-dimensional and three-dimensional images and data analysis of the two methods.(2)A group of male SD rats weighing 280-350 g were selected,and the brains of the rats were removed after perfusion and tissue gradient dehydration,and the obtained brain tissues were photographed at the SSRF BL13W1 line station for PCI and reconstructed information.(3)Male SD rats weighing 280-350 g were randomly divided into MCAO surgery group and sham surgery group,in which there were 2h,4h,4.5h,6h,1d,7d and 14 d subgroups in the MCAO surgery group,with5 rats in each subgroup and a total 5 of one rat in the sham surgery group as control.The brains were removed,and the tissues were dehydrated in a gradient.In the sham-operated group,the brains were removed,and the tissues were dehydrated in a gradient after perfusion.The obtained brain tissues were photographed by phase contrast imaging at SSRF BL13W1 line station,followed by image reconstruction analysis and post-processing of vascular measurements,and the reconstructed image data were obtained to compare the changes of microvascular opening at different time points after MCAO;the concept of 3D distortion of blood vessels was defined,and statistical analysis was performed to compare the relevant trends at different time points after surgery.Results:(1)Compared with ACI,(1)the PCI method based on SRCT has a clear demarcation of different brain regions,a clear visualization of vessels,and the minimum resolution diameter of vessels was about 10μm;(2)a multidimensional 2D slices and virtual sections of the whole brain in combination with post-processing;(3)a large number of microvascular structures are clearly visible in 3D reconstruction;(4)and a smaller diameter,a larger number of nodes,and more branches in the fourth average characterization of vessels,with statistical difference,P<0.0001.(2)SR-based PCI can,(1)obtain 3D rendering reconstruction of blood vessels in different ROI regions;(2)measure and assign values to the vascular grading and branching analysis maps,resolve depth up to 5grade vessels;(3)extract and observe the 3D morphology of a single vessel from multiple angles,as well as simulate the morphology of the inner surface of the vessel.(3)The microvascular morphology characterization after different ischemic time points was observed by SR-based PCI.(1)One was soft meningeal collateral opening and microvascular opening 2 h after ischemia;(2)two was microvascular opening that persisted until 1d after ischemia;(3)the number of vessel branches were higher than the control group from 2h to 1d after ischemia,with statistical differences,P<0.05,and lower than the control group from 7d to 14 d,with statistical differences,P<0.05;(4)three was microvascular 3D distortion higher than the control group from 2h to 1d after ischemia,with statistical differences,P<0.05,and lower than the control group from 7d to 14 d after ischemia,without statistical differences.Conclusion:(1)This study concluded that SRCT PCI technique is superior to ACI in the complex system of cerebral microvasculature characterization.(2)This study concluded that SRCT PCI technology can realize the multidimensional comprehensive characterization of the complex system of cerebral microvasculature and establish a whole set of analysis methods from whole to local.(3)This study concluded that the SRCT PCI method can detect the microvessels opening,changes and structural characteristics at different time points after cerebral ischemia and confirmed the feasibility of SRCT PCI technique to characterize the steady-state changes of cerebral microvascular system after ischemic stroke. |
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