中洪博元,动物造模,脑缺血 z-bo 2020-07-21 774

　　In the experimental research of cerebral ischemia-reperfusion injury, it is very important to establish an appropriate animal model, which directly affects the reliability and stability of the experimental results. For the replication of cerebral ischemia-reperfusion animal models, the method of blocking blood flow for a certain period of time and then restoring hemoperfusion is generally adopted. Commonly used experimental animals are gerbils, rats, rabbits, mice, dogs, cats and so on. So far, there are more than a dozen methods for replicating animal models of cerebral ischemia-reperfusion injury. This article summarizes several of the commonly used modeling methods, and analyzes and compares their pros and cons and applicability.

　　1 The establishment of an animal model of global cerebral ischemia reperfusion injury

　　1.1 Bilateral common carotid artery occlusion method

　　 This method blocks the bilateral common carotid arteries for a certain period of time and then restores blood flow, causing global cerebral ischemia and reperfusion injury. The advantage is that it is very simple and easy to implement. However, because common experimental animals have relatively complete cerebral artery rings (Willis rings) except for gerbils, and gerbils have only about 20%-30% of animals with cerebral Willis ring hypoplasia. Therefore, simply blocking the bilateral common carotid arteries is difficult to cause complete cerebral ischemia. Therefore, the success rate of cerebral ischemia-reperfusion injury caused by this method is low, and the scope of application is limited, and it is generally only used for the preparation of gerbil cerebral ischemia-reperfusion injury models.

　　1.2 Bilateral common carotid artery occlusion combined with circulatory blood pressure reduction method

　　 Use to clamp the bilateral common carotid arteries and femoral arteries of rabbits (rabbits, cats, dogs and other larger experimental animals) or the retro-orbital venous plexus [2] (mice) to quickly bleed, resulting in an acute cerebral ischemia model. EEG, biochemical indicators and morphological tests show that this model has obvious effects on cerebral ischemia. However, this model is not easy to observe reperfusion. Some authors used bilateral common carotid artery blockade combined with control drugs (sodium nitroprusside, etc.) [3] blood pressure reduction method to create an acute cerebral ischemia model. Hemoperfusion can be restored after ischemia for a certain period of time for observation of cerebral ischemia reperfusion injury. As systemic hypotension can cause damage to the heart, kidneys and other important organs, the model is complicated.

　　1.3体循环控制性降压复合颅内注液增压法

　　通过给狗快速滴注0.02%硝普钠溶液，使其平均动脉压(MAP)于2～3min内降至6.65kPa；之后向小脑延髓池快速注入脑脊液近似溶液，使颅内压(ICP)于5s内升高至13.3kPa。这样由于降低MAP而使脑灌注压下降，同时升高颅内压使脑灌流阻力增大，导致脑血流阻断，造成脑缺血。维持MAP及ICP于上述水平一定时间后，颅内减压使ICP降至正常水平，即可恢复血液灌流。经组织学、生理学、生物化学及放射性同位素等多项技术检测，证明应用体循环降压结合颅内增压技术制造的脑缺血再灌注损伤模型，具有缺血效果可靠、并发症少等优点，可用作脑复苏的实验研究。但该模型不适用于大鼠等小动物，并且也具有因全身性低血压导致其它脏器损伤，而使模型复杂化的缺点。

　　1.4 Four-vascular block method

Pulsinelli et al. first reported in 1979 that bilateral vertebral arteries were combined with ligation of bilateral common carotid arteries in electrocoagulated rats, and after a certain period of time, both common carotid arteries were removed for reperfusion, resulting in severe cerebral ischemia-reperfusion injury. A highly reproducible method for global cerebral ischemia modeling. Li Linxian and others improved on this basis, using direct vision to separate the common carotid and vertebral arteries on both sides of the rabbit, and clip the four arteries to cause cerebral ischemia. This method is simple and easy to implement, and is a commonly used animal model for studying cerebral ischemia reperfusion injury. However, because there are communicating branches between the vertebral artery and the anterior spinal artery, and the differences between different species and individuals of animals are large, the success rate of the model is low, and the stability is also poor.

　　 1.5 three-vessel blocking method

Kamegama et al. replicated the global cerebral ischemia model by electrocautery cutting off the basilar artery and simultaneously clamping the common carotid arteries on both sides. This method not only blocked the main blood supply to the brain, but also blocked the side from the prespinal artery. Compared with the aforementioned four-vessel occlusion model, branch blood vessels have a high success rate of cerebral ischemia and no need to re-screen is its main advantage. On this basis, Tian He Tunqian and others made improvements, and performed the operation under direct vision without a microscope, and used a special arterial clip to clamp the basilar artery without electrocautery. After ischemia for a certain period of time, the clipping can be removed for complete reperfusion, and the disadvantages of arterial damage and hemorrhage caused by cauterization of the basilar artery are avoided, and the effect of ischemia and reperfusion is rapid and stable. During use, the systemic arterial blood pressure has been stable within the physiological range, thereby avoiding insufficient cerebral blood perfusion pressure during reperfusion caused by the decrease of systemic arterial pressure, which is another prominent advantage.

　　1.6 Six Artery Blocking Method

　　 Based on the four-vessel block method, the traditional bilateral common carotid artery blockade was changed to bilateral internal carotid artery and external carotid artery blockage and bilateral vertebral artery blockage. In this method, because the carotid artery is blocked at the distal end of the carotid sinus, the carotid sinus area can maintain a certain pressure. This avoids the traditional four-vessel method from blocking the bilateral common carotid arteries. Because the pressure in the carotid sinus area on both sides drops at the same time, the systemic blood pressure is reflexively increased, leading to increased cerebral collateral circulation and increased cerebral blood flow. In turn, the degree of cerebral ischemia is reduced. However, the procedure of this method is more complicated and difficult.

　　1.7 carotid shunt method

　　 By ligating the left common carotid artery and bleeding from the distal end of the right common carotid artery at the same time, the blood supply to the brain from the common carotid arteries on both sides is stopped, creating a global cerebral ischemia model. At this time, although the blood flow of the vertebral arteries on both sides can still flow to the circle of Willis through the basilar artery, the bloodletting from the distal end of the right common carotid artery causes significant pressure on the common carotid artery, internal carotid artery and posterior communicating artery reduce. Therefore, when the blood flow of the basilar artery enters the posterior communicating artery, it does not perfuse the brain forward, but flows out through the common carotid artery of the internal carotid artery retrogradely, causing cerebral ischemia. The blood released from the common carotid artery is fed into the body through the ipsilateral femoral vein. The blood flow and speed can be effectively controlled during the whole process. The advantage of this model is that it does not affect the blood flow of the basilar artery and vertebral artery, the brainstem ischemia is not obvious, and it has little effect on basic life activities such as breathing and circulation. During reperfusion, bloodletting of the right common carotid artery is stopped, so that the low pressure of the right common carotid artery, internal carotid artery and posterior communicating artery disappears, and the blood flow from the basilar artery can enter the circle of Willis; at the same time, the left common carotid artery is ligated and recovered Its hemoperfusion. In this way, the brain has sufficient blood perfusion, that is, sufficient reperfusion is another characteristic. However, the scope of cerebral ischemia caused by this modeling method is still controversial. Studies have shown that this ischemia is mainly the ischemia of the brain area supplied by the right middle cerebral artery. The reason for this difference may be related to the bloodletting speed and the degree of decrease in internal carotid artery pressure.

　　1.8 Carotid negative pressure shunt method

"The experimental rats were anesthetized intraperitoneally with sodium pentobarbital (40mg/kg). Inject heparin (180U) from the left femoral vein and slowly inject saline, clamp the common carotid arteries on both sides, and continuously suck blood (0.3ml/min) in the common carotid artery from the right external carotid artery. This is the beginning of cerebral ischemia; At the same time, blood was continuously infused from the left femoral vein. After 20 minutes of global cerebral ischemia, blood was stopped, 1ml of normal saline was injected into the carotid artery, the external carotid artery was ligated, and the common carotid arteries on both sides were loosened. This is the start of reperfusion [4 ].

　　 2 Modeling method of focal cerebral ischemia reperfusion injury

　　 The establishment of an animal model of focal cerebral ischemia and reperfusion injury is one of the important methods for studying the pathophysiological mechanism of ischemic stroke and its prevention. The middle cerebral artery (MCA) is the most frequent site of vascular disease in human cerebral infarction. Because the anatomy of the cerebral blood vessels of rats is close to that of humans, the vascular damage is constant and the reproducibility is good. Therefore, the rat middle cerebral artery occlusion (MCAO) model is a relatively accepted standard animal model for studying cerebral ischemic injury. For the preparation of focal cerebral ischemia models, craniotomy, suture insertion, photochemical induction, and embolization are commonly used to block and recanalize the MCA blood flow in rats, resulting in the localization of the corresponding perfusion area. Focal cerebral ischemia reperfusion injury.

　　2.1 Craniotomy

　　 Most studies choose to open the lower temporal craniotomy and ligate the MCA across the outer or inner edge of the olfactory tract by electrocoagulation or silk thread ligation, causing cerebral infarction. Kader et al. improved the method of electrocoagulation occlusion of MCA, occluding all visible branches of MCA, and the infarction effect was better. The MCAO model of craniotomy has reliable ischemia effects and is the most widely used classic focal cerebral ischemia model so far. However, the trauma of the craniotomy is large, the cerebrospinal fluid leaks out, and the brain microenvironment is changed. The reperfusion injury effect cannot be observed after MCA occlusion. Although some authors create a reperfusion model by clamping the MCA with a miniature arterial clip or lifting the MCA by threading, and then recanalize the blood flow to make a reperfusion model, they are limited to the study of reperfusion injury after a short period of MCAO. Need to be further improved.

　　2.2 Plug-in method

　　2.2.1 Kuizumi first reported the reversible MCAO model made by nylon thread insertion method in 1986, which solved the problem of focal cerebral ischemia reperfusion injury. The preparation method of the model is to fix the animal supine on the operating table after anesthesia, and use the median neck incision or choose the right neck incision to separate the common carotid artery, external carotid artery and internal carotid artery. Ligation of the common carotid artery, external carotid artery and its branch arteries. Separate the internal carotid artery to the blister to see its extracranial branch pterygopalatine artery, thread the branch and ligate the branch along the starting point. Cut a small opening in the external carotid artery, insert the prepared nylon thread into the external carotid artery, enter the internal carotid artery through the bifurcation of the common carotid artery, and pass through the start of MCA to the proximal end of the anterior cerebral artery (the average insertion depth is about 18.5±0.5mm) . During reperfusion, pull the nylon thread to return the ball end to the external carotid artery to restore the blood supply of MCA for reperfusion. If the surgical approach is selected from the side of the neck, the surgical field of view can be better exposed and the blood vessels can be separated; and compared with the median neck incision, the trachea is less irritated, and no tracheostomy is required, making the operation relatively simple. The results of pathological examination showed that the modeled animals had infarcts in the caudate putamen and dorsolateral cortex on the ischemic side, consistent lesions, good stability, and reliable ischemic effects. It is especially suitable for the study of the pathophysiological mechanism of basal ganglia ischemia-reperfusion and the observation of drug treatment effects and mechanisms. In addition, there is no need for craniotomy, little trauma, no abnormal changes in blood pressure, blood gas and body temperature after occlusion of MCA by thrombus, and no influence on the natural process of cerebral edema and intracranial pressure pathological changes after ischemia. All advantages are better than craniotomy occluded MCA Method of establishing focal cerebral ischemia model.

　　2.2.2 Zea Longa thread bolt method

The preparation method of this model is to anaesthetize the animal, cut the posterior cervical median, and burn the bilateral vertebral arteries to block the posterior circulation; anterior median cut to expose the left common carotid artery (CCA) and internal carotid artery (ICA) Clamp the CCA with the internal carotid artery (ECA), insert a single-strand nylon thread (4-0) into the distal end of the ICA, and block the blood flow at the beginning of the middle cerebral artery. The average incoming wire length is (2±0.2) cm[5].

　　2.3 Photochemical induction method

Generally, Wistar rats are used. After anesthesia, the left temporal approach is used (the initial segment of the right MCA has a greater variation), exposing the subsquamous bulge above the foramen ovale, at the front end of the junction between the zygomatic arch and the temporal scale A bone window with a diameter of 5 mm was opened at 2 mm, and the dura was incised. The proximal segment of MCA can be seen across the olfactory tract. The starting part of the MCA or the inside of the olfactory tract 2mm to the intersection with the inferior cerebral vein can be selected as the light spot. Use actinic cold light source instrument (metal halide lamp) to carry out step-by-step irradiation: after 3% rose bengal B (or other photosensitive materials) is injected through the femoral vein, immediately continue to irradiate the MCA self-olfactory beam to the inferior cerebral vein. 15min at the vascular site (local light intensity is 0.5W/cm2); after that, move the light source to the start of MCA, inject the same amount of Rose Bengal B and continue to irradiate MCA for 15min to form a simple MCA occlusion model. The principle is that Rose Bengal B produces and releases singlet nitrogen under the action of a green light source, which causes the lipid peroxidation of polyunsaturated fatty acids in vascular endothelial cells to directly damage vascular endothelial cells, leading to thrombosis and local vascular occlusion. After a certain period of time, 20μl of 20μmol/L nimodipine was locally instilled under the light. After 2-10 minutes, the blood clot material disappeared, allowing the blood vessel to recanalize and form reperfusion. Since the blood vessel has no mechanical damage, it is easy to recanalize, reperfusion is sufficient, and the perfusion flow can reach 100%. When modeling, select the starting end of MCA and the inner side of the olfactory tract 2mm to the blood vessel at the intersection with the inferior cerebral vein for step-by-step irradiation, which not only occludes the proximal end of the MCA, but also blocks the lenticular branches emitted by it, which can cause Infarcts of the cerebral cortex and the anterolateral and posterior parts of the basal ganglia. The main advantages of the photochemically induced MCA occlusion and recanalization model are: ①The success rate is high, the resulting infarct is relatively stable and reproducible; ②The onset of cerebral infarction due to cerebral arterial thrombosis in this model and pathological conditions The process is similar, and it is especially suitable for the research of antiplatelet, antithrombotic drugs and endothelial cell protective agents. However, this method requires special equipment, and the formation of ischemic foci are mostly caused by capillary endothelial damage, which is quite different from the clinical ischemic area formed by large vessel embolism [6].

　　2.4 Embolization

　　This kind of modeling method is to inject embolic agents (such as broken blood clots, carbon particles, arachidonic acid, etc.) into the blood vessel to make a cerebral embolism model. The aseptic and dried blood clot is crushed to make a suppository suspension. After the embolus is injected from the external carotid artery, the external carotid artery is ligated and the common artery is released. The embolus rushes into the internal carotid artery and enters the MCA. The model does not require craniotomy, is simple to make, and has reliable ischemic effects. And it is most similar to the pathological process of clinical embolic stroke, which is more suitable for the observation and research of thrombolytic therapy, especially the selection of human blood clot as an embolic agent is more practical. The disadvantage is that due to the large randomness of emboli, it is impossible to accurately predict the location and size of the infarction. There are also researchers using balloon embolization, but it can only be used for large animals such as baboons and rabbits.

　　2.5 Endothelin-1 induced focal cerebral ischemia-reperfusion model in rats

Sharkey et al. established a rat model of focal cerebral ischemia-reperfusion induced by ET-1. Many people at home and abroad have used this method to study ischemic brain injury. Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (3.5ml·kg-1), fixed on a stereotaxic device in the prone position, maintained anal temperature of 37.0～38.0℃ with a heating pad, and exposed the skull along the midline by cutting the front skin. The stereotactic map of the rat brain is marked by fontanelle. The head is 0.9mm, the right side is 5.2mm, and the ET-1 catheter is implanted at 8.7mm under the skull. After 5 minutes, aCSF diluted with different concentrations of ET-1 (4.0μl) 1.0μl·min-1 was injected into the vicinity of MCA; at the same time, tail artery intubation was performed to monitor the blood pressure changes in the tail artery before and after ET-1 administration. Compared with the commonly used thread insertion method, this model only involves simple stereotaxic and craniotomy; it will not cause damage to the larger arteries in the brain and reduce the risk of intracranial hemorrhage; the occurrence of reperfusion is a gradual process and avoids The sharp increase in CBF caused by artificial reperfusion is more in line with the gradual recovery of CBF after thrombolysis in human stroke; the small range of craniotomy (diameter 0.65mm) will not cause major changes in the brain environment; it can be produced and inserted. Similar to a relatively stable infarct range and good reproducibility; in addition, the biggest advantage of this model is that it can study ischemic brain injury in awake animals. Therefore, this model can study the mechanism of cerebral ischemia and reperfusion and the brain It is an ideal model of focal cerebral ischemia and reperfusion for the evaluation of the efficacy of protective new drugs.