| Cerebrovascular disease is characterized as high rate of incidence, mortality, handicap, and relapse. Therein, over 80% are anemic CVD (cerebrovascular disease). With the development of people's living level and medical level, old-aged people take a much larger proportion than ever before. Carotid stenosis is one of the most common geriatric diseases. It will lead to cerebral ischemia. There are some clinical symptoms for TIA, including sleepiness, temporary syncope and so on. Aggravation of the disease or repeatedly attacks of TIA, and even blockness is the main pathogeny of anemic cerebral infarction. Carotid stenosis in various degrees will cause different clinical symptoms resulted in different degrees of cerebral ischemia. Attention starts to be paid to different outcomes and even different malfunctions of recognition. The system of cerebral ischemia is complex, and there are a lot of risky factors. But the fundamental mechanism and ultimate outcome of anemic cerebrovascular accident is cerebral ischemia and hypoxia for cerebral tissue. In the studies of the pathological mechanism, neural protection, therapeutic drug and other therapeutic methods in terms of anemic cerebral accidents, experiments on animals play a particular role that could not be replaced. However, there is lots of limits on such animal models with cerebral ischemia. For example, the cerebral cells will die or the animals will die in a quite short time; the experiment is difficult to control; the survivability of cerebral tissue on ischemia can not be directly observed, the pathological and physical process of ischemia caused by TIA could not be veritably simulated, etc. Therefore, it is quite necessary to work out an ideal controllable model of carotid stenosis, and it will be helpful to further study the pathological change caused by carotid stenosis in various degrees as well as the intervention.In consequence, the animal model established by placing a hollow tube to make carotid stenosis which could lead to cerebral ischemia provides an authentic and effective way to study the cerebral ischemia mechanism of TIA patients. This experimental model could be used to observe the symptoms of cerebral ischemia. The survivability of cerebral tissue on ischemia can be directly observed as well. This experimental is completely creative in terms of methodology. Since there has been no record of similar models so far, the establishment of this new model will bring an optimistic and promising future to further studies on cerebral ischemia caused by carotid stenosis, especially on morphology of TIA, mechanic metabolism, pharmacology, clinical therapeutics, neural intervention radiology and neurosurgery. This study is to establish an animal model by making controllable carotid stenosis of rabbits which could lead to cerebral ischemia. And it involves intervention through antihypertensive therapy. The detaild of this study are as follows:Part 1 Establishment of the model to create controllable rabbit's carotid derived by controllable rabbit's carotid stenosis through intervention Purpose: to establish an animal model concerning cerebral ischemia led by carotid stenosis in different degrees through intervention.Method: New Zealand Rabbit is used as the experimental animal,which is divided into four groups(A: normal;B:50%stenosis on the right side; C:75%stenosis on the right side,D: obstruction on the right side). Each group has 6 animals.The cylindrical hollow tube is immersed in heparin water for later use before the experiment. The external diameter of the cylindrical hollow tube is equal to that of the carotid artery, While the inner diameter equals that of half and 25% of the external diameter of the external diameter. Before embolization, dispersed and common scanning T1WI and T2WI of nuclear magnetic resonance will be respectively conducted at the time when the cylindrical hollow tube has been stayed in the artery for 15 minutes, 30 minutes, 45 minutes, 1 hours, 2 hours and 4 hours, and then statistical analysis will be worked out based on dispersing value that has been recorded.Result: all operations concerning separation of femoral artery, puncture and intubation have been successfully done. This experiment takes example of 75% carotid stenosis, which is controlled by placing the cylindrical hollow tube after successful operation to be re-imaged. It is observed that stenosis appears in some parts of the general carotid artery. Accordingly, the blood flow becomes slow, and it is narrower compared with the opposite side. Making a comparison between that before embolization and that after embolization, the alternation of inner diameter of the general carotid artery in terms of embolization achieves anticipated effect (p <0.05), with an error of -5%～+5%. The compensation of lateral artery after embolization becomes thicker. There is no significant difference concerning the inner diameter of general carotid artery (p>0.05). it is proved by statistical analysis that when there is 75% carotid stenosis, the difference for the embolized side seems not significant between the ADC value after embolization for 30 minutes, 60 minutes, 90 minutes and 120 minutes and the ADC value before embolization. There is a significant difference between the ADC value after embolization for 240 minutes and that before embolization (p <0.05); the difference for the opposite side seems not significant between the ADC value after embolization for 30 minutes, 60 minutes, 90 minutes, 120 minutes and 240 minutes and that before embolization.Conclusion: the animal model by making controllable carotid stenosis of rabbits through intervention which could lead to cerebral ischemia is initially established. It is a creative model both domestically and globally. This model is characterized as little injury, considerable repeatability, and high success ratio. The success ratio achieves 100%. Besides, it is quite controllable. The degree of stenosis could be accurate within the scope -5%～+5%, and it is directly proved by image of vessel.Part 2 Carotid stenosis in various degrees will cause the change of pathological form of cerebral ischemiaPurpose: to explore the changing rules of cerebral ischemia cells resulted in carotid stenosis in various degrees at a fixed time.Method: the process of model preparation is the same as part 1. There are①the normal group;②50% stenosis on the right side;③75% stenosis on the right side;④100% stenosis (blockness) on the right side;⑤nornal antihypertensive group;⑥antihypertensive group with 50% stenosis on the right side;⑦antihypertensive group with 75% stenosis on the right side;⑧antihypertensive group with 100% stenosis (blockness) on the right side (the brains of all the animals will be removed 4 hours later since the successful establishment of the model). The brains of animals will be removed after the model is successfully established. The general changes of the cerebral surface are observed and recorded with a camera. In addition, it will be HE dyed commonly.Result: 1. general description of the cerebral tissue: the superficial vesels of normal rabbit's cerebral tissue on both sides are generally symmetrical, filled with blood. There is no obvious change in group A compared with group B, C and E. The superficial vessels on the right side of group G is not filled as well as the opposite side in case that they are moderately changed, resulting in the narrowing twig vessels. The superficial vessels on the right side of group H are narrower than the opposite side with little blood. The twig vessels will disappear. Slight hydrocephalus will be found. 2. The result of HE dying: there are no necrotic cells in group B, C, and E; and necrotic cells in group D, F, G and H can be found by microscope, resulting in swelling cells, slightly-dyed chromatin, obscue contour, blurry karyotheca and nucleolus, intensive basophilia and red color. The stuffing in some cells is dissolved and released, the contour of cells left. Thus, the cells will seem like acuoles. Alternatively, there may be some inflammatory cells or birdeye-like cells. Regarding the stenosis groups, as the degree of stenosis increases, 75% stenosis will lead to no change of the state of cerebral cells, and 100% stenosis will lead to slight change of the state of cerebral cells. Regarding the antihypertensive groups, with the degree of stenosis increasing, 50% stenosis on one side will lead to slight change of the state of cerebral cells, 75% stenosis on one side will lead to moderate change of the state cerebral cells, and 100% stenosis on one side will lead to serious change of the state of cerebral cells. The degree and number of cytoclasis in Group H will greatly increase compared with Group G.Conclusion: there is slight or no change of cerbral superficial vessels in the stenosis groups; regarding the antihypertensive group, as the degree of stenosis increases, the hemisphere of superficial vessels on the stenosis side turn narrower and sparser. Finally twig vessels dissappear, and there is slight hydrocephalus. Regarding the stenosis groups, there is slight or no change of the state of cerebral cells; while regarding the antihypertensive groups, the change of the state of cerebral ischemia cells becomes serious. The degree and number of cytoclasis concering serious change is much more than that concerning moderate change.Part 3 The study of cerebral tissues SOD and MDA injured by cerebral ischemia resulted in carotid stenosis in various degrees.Purpose: to explore the changing rules of SOD and MDA in cerebral tissues in the condition of cerebral ischemia caused by carotid stenosis in various degrees at a fixed timeMethod: T-Sod and MDA is mensurated carefully, in accordance with the standard operation methods in specifications of SOD and MDA produced by the first branch of Nanjing Jiancheng Bio- Engineering Institutes,Result: the changing rules of SOD and MDA: intra-group comparison: there is no significant difference among these groups (P > 0.05).Inter-group comparison: there is no significant difference in Group A in comparison with Group B, C and F; there is extremely significant difference in Group A compared with Group G and H (P < 0.01). To sum up, there is no statistical difference in SOD and MDA within each group; SOD is decreasing gradually in inter-group comparison, while MDA is increasing.Conclusion: concerning the stenosis groups, as the degree of stenosis increases, there is no obvious variance in cerebral tissues SOD and MDA when the degree of carotid stenosis on the one side reaches 75%; there is slight variance in SOD and MDA when the degree reaches 100%. In antihypertensive groups, as the degree of stenosis increases, SOD gradually decreases while MDA increases. The difference will get the most significant in the antihypertensive group with 100% stenosis. The obvious increase of oxygen derived free radicals, the obvious decrease of anti-oxidation and the obvious strengthening of overoxidation reaction of lipid prove that the degree of the injuries of cerebral tissues increases as that of stenosis increases.Part 4 Study of expression of relevant genes—Bcl-2, Bax, and Caspase-3 protein, concerning apoptosis led by carotid stenosis in various degrees Purpose: to explore the relations among the apoptosis led by carotid stenosis in various degrees at a fixed time, the location of apoptosis, and Bcl-2, Bax, and Caspase-3 gene protein.Method: immunochemistry—SP methodology (the full name is streptomycin anti-biotin protein—correlation of overoxidated mildew) is made use of to study the expression case of these gene proteins in the epencephalon suffering carotid stenosis.Result: Bcl-2 and Bax protein are distributed in the form of granule and strip, coresponding with the location of organelles. Sometimes, karyotheca needs to be colored. There is hardly of Bax in group A's cerebral tissus, and there is expression of Bax which is slightly positive in most parts of B, C, and E's cerebral tissues. Besides, In the II-IV layer of cortex, hippocampal cells, thalamus, and Purkinje cells, Bax immunocyte which is slightly positive could be observed. In the cerebral cortex, corpus striatum, thalamus, border of hippocampal areas of group D and group F, the increasing intensity of immunity could be observed. In the above parts of group G and group H, a more obvious increasing intensity of immunity could be observed, with signicant difference compared with the normal group; on the contrary, some neuron cells of Bcl-2 show positive expression in the cerebral tissue of group A, and the positive granule mainly consists of cytoplasm. The position of expression is mainly focused in the cerebral cortex of frontal brain and hippocampal areas. The number of positive neuron cells will slightly decrease in the frontal cerebral tissue of group B, C, and E, without signicant diference from group A. Hippocampal pyramidal cells show positive expression, and the main component is cytoplasm. At this time, the expression of positive granules in the hippocampal areas could reach the maximum. Positive granules could be observed on most neuron cells of group G's frontal cerebral tissues, whose main componenet is neuron cells. There is no positive granule expression for vacuole-like cells, however, there is granule expression for just a few pyramidal cells. The expression of positive granule in frontal cortex will reach the maximum at that time. There are only a few neuron cells which show positive expression in the frontal cerebral tissues of group H. postive granule expression could be occasionally observed in vacuole-like cells and birdeye-like cells. The structure of hippocampal pyramidal cells is indistinct, changing as vacuole. Positive granule expression is occasionaly observed; Caspase-3 protein in cytoplasm seems strip-shaped, dyed by brown yellow granule. Expression of Caspase-3 protein could hardly be observed concerning each part of normal rabbit's cerebral tissue of group A. While expression of Caspase-3 protein could occasionally be observed concerning each part of cerebral tissue of group B, C, and E, without without signicant diference from group A. In additon, expression of Caspase-3 protein is slightly positive concerning each part of cerebral tissue of group D and F; there are lots of positive immunocyte cells in frontal cerebral tissues, hippocampal areas and thalamus concerning group G, and there are more positive immunocyte cells in such parts concerning group H. Caspase-3 protein is similarly distributed as Bax in apoptosis cells. It is found that expression of BCL-2 is negatively correlative with the number of apoptosis cells while expression of Bax and Caspase-3 is positively correlative with the number of apoptosis cells, through analysis on the correlation betwwen expression of BCL-2, Bax, and Caspase-3 and the number of apoptosis cells.Conclusion: BCL-2 sometimes shows positive expression in the normal rabbit's cerebral tissues. With the increasing degree of carotid stenosis on one side, early inchemia will reach the peak of expression. The peak of expression in the hippocampal areas will be reached earlier than that in cortex areas. BCL-2 is able to restrain apoptosis. Later, the expression dramatically decreases; however, it is higher than the normal cerebral tissue. In the model of cerebral inchemia led by carotid stenosis, with the increasing expression of the degree of carotid stenosis on one side, Bax and Caspase-3 protein is positively correlative with the number of apoptosis and will accelerate the apoptosis.
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