Study On Dynamic Changes Of Cerebral Microvasculature In Living Rats After Stroke

Purpose and Significance: Stroke is one of the three major causes ofdeath worldwide, which has high morbidity and disability rate. Study onthe mechanisms of stroke incidence, the earlier diagnosis, and explorationof novel stroke therapy is becoming hot topics in neurobiology, neurology,and neuroradiology. There is no effective treatment for the ischemic strokeexcept thrombolysis within4.5hour of stroke attack. Therefore, it isextremely important to study on pre-clinical treatment in experimentalischemic stroke using small animals such as rats. Since the diameter of ratcerebral vasculature is uausally smaller than100μm, it is impossible todirectly observe the dynamic changes of cerebral microvasculature in suchsmall animals via traditional imaging devices. To establish feasible smallanimal model and to achieve real-time in vivo monitoring the changes ofcerebral vascular morphological and hemodynamic changes are nowurgent tasks in experimental stroke research. In current study, we utilizedsynchrotron radiation (SR) X-ray to explore the feasibility of highresolution dynamic imaging in living rats with middle cerebral arteryocclusion (MCAO). We expected to break the bottleneck in experimental stroke research and provide novel concept and unique technique to directlyobserve the changes of cerebral vascular morphology during brainischemia and post-ischemic reperfusion.Methods: After optimizing parameters for SR angiography imaging, weperformed a series of SR angiography by using two different contrastagents (BaSO4and non-ionotropic Iodine). We first established a standardmethod to observe cerebral microvasculature in living rat under normalcondition. We then designed a series of experiments to study the cerebralmicrovasculature in rat MCAO model.1) We optimized the MCAO suturemodel in Sprague-Dawley (SD) rats by in vivo observing the suturelocation with SR angiography and compared the infarct volume with TTCstaining of brain samples. We used the results to determine the optimumsilicone coating length of suture and ensured the reproducibility of themodel.2) We investigated the collateral circulation in the masticatorymuscle area after external carotid artery transection or90-min tMCAO inliving animal using SR angiography; and analyze the result with MR dataand anatomical information.3) We monitored the angiogenesis process atdifferent time points after90-min tMCAO in rat brains using SR liveangiography and micro-CT angiography. We also developed quantitativemethods to evaluate angiogenesis.4) We analyzed the characteristicfeatures of microvasculature in aged rats and the difference between SDand spontaneous hypertension rats (SHR) using SR angiography.Results: We demonstrated that the characteristics of contrast agent playedan important role in the SR angiography. BaSO4contrast agent is adequate for the observation of whole rat brain vascular morphology whilenon-ionotropic Iodine was suitable for the studying the cerebral vascularmorphology and hemodynamic changes in living animals. We optimizedimaging parameter of SR angiography in living animals. Using timesubtraction technique, we reduced the effects of bone and other tissuebackground. As a result, the quality of SR angiography was greatlyimproved. After performing a series of SR angiographic experiments in ratMCAO model, we demonstrated that1) with modified suture siliconecoating length, the outcomes of MCAO in rats was reproducible, whichmakes this model easy to perform and popularize;2) the SR angiographydemonstrated that the collateral between branches of pterygopalatine arteryand external carotid artery in rat masticatory muscle region after MCAO,which could maintain the focal blood supply after external cervical arterytransection;3) After1day of tMCAO, we found that the corticalperforating arteries and cortical branches of MCA dilated in ipsilateralhemisphere. After3days of tMCAO, the number of cortical perforatingarteries increased and the shape of microvessels in corpus striatum areawas stiff and twisted. After7days of tMCAO, the microvessels in corpusstriatum area had twisted and cluster-like changes as well as the number ofcortical perforating arteries decreased. After14days of tMCAO, thedensity of microvessels in corpus striatum area increased and the corticalperforating arteries are still less. After28days of tMCAO, the number anddiameter of cortical perforating arteries recovered to normal, the structureof MCA cortical branches and cortical perforating arteries were detectable,and microvessels in corpus striatum were stiff compare to the normal rats;4) We demonstrated the diameter of distal branches of internal carotid artery became thinner along with aging, and resistant arteries weredetectable with twisted morphology in adult and aged SHR rats.Conclusion: This work proved that non-ionotropic Iodine was suitable forin vivo rat cerebral vasculature imaging. We used SR angiographic data tomodify suture silicone coating length, which yielded rat MCAO suturemodel more reproducible. We established in vivo evidence of collateral inrat masticatory muscle region, which provides valuable information forfurther investigating collateral after ischemia and develop possible therapyregimen via collaterals. Our work revealed focal cerebral vascular changes,indicating endogenous angiogenesis and vascular remodeling. The workon SHR cerebral microvasculature revealed morphological changes alongwith age. Our work suggests that SR angiography is a unique tool for theobservation of microvasculature with high resolution in living animals. SRangiography provides a novo approach and unique technique to betterunderstanding of cerebral vascular changes during stoke.