脑缺血动物模型 z-bo 2020-10-13 338

　　Left and right asymmetry in animal models of cerebral ischemia: Adult brain is asymmetry, dominated by one hemisphere. Here, we manually identify the dominant hemisphere, and 90% of people are right-handed. Most right-handed language centers are located in the left hemisphere, which is considered the dominant hemisphere. Compared with the non-dominant hemisphere, the dominant hemisphere has more complex functions. In addition, it has been reported that the pathological symptoms of ischemia are different in the primary and non-primary hemispheres. Studies have shown that brain asymmetry is not a characteristic of humans and is common in vertebrates. Major hemispheric ischemia can cause more severe neurological dysfunction in rats, while animals with non-major hemispheric ischemia recover faster. Other studies have reported significant differences in the infarct volume between the left and right hemispheres in the early MCAO model. Left hemisphere ischemia leads to more severe sensorimotor deficits, and right hemisphere ischemia leads to more severe cognitive deficits. Similarly, there are differences in the biochemical levels between left and right ischemia. A study found differences in the expression of genes encoding growth factors in the left and right cerebral ischemic striatum. The changes of norepinephrine and dopamine in the cerebral cortex and blue dot nuclei are also inconsistent, and the dynorfin level of right amidala reaches its peak 3-5 days after ischemia. These results indicate that the rat brain hemisphere is asymmetric, and the behavior and biochemical changes of the left and right hemispheres are also different after ischemia.

　　Left and right asymmetry in cerebral ischemia animal model: Adult brain is asymmetry, dominated by hemisphere. Here, we manually identify the dominant hemisphere. 90% (90%) of people are right-handed people. The language center of most right-handed people is in the left hemisphere, which is considered to be the dominant hemisphere. Compared with the non-dominant hemisphere, the dominant hemisphere has more complex functions. In addition, it has been reported that the pathological symptoms of ischemia are different in the primary and non-primary hemispheres. Studies have shown that brain asymmetry is not a characteristic of humans and is common in vertebrates. Major hemispheric ischemia can cause more severe neurological dysfunction in rats, but animals with non-major hemispheric ischemia recover faster. Other studies have reported significant differences in the infarct volume between the left and right hemispheres in the early MCAO model. Left hemisphere ischemia leads to more severe sensorimotor deficits, and right hemisphere ischemia leads to more severe cognitive deficits. Similarly, there are differences in the biochemical levels between left and right ischemia. A study found differences in the expression of genes encoding growth factors in the left and right cerebral ischemic striatum. The changes of norepinephrine and dopamine in the cerebral cortex and blue dot nuclei are also inconsistent, and the dynorfin level of right amidala reaches its peak 3-5 days after ischemia. These results indicate that the rat brain hemisphere is asymmetric, and the behavior and biochemical changes of the left and right hemispheres are also different after ischemia. Therefore, brain asymmetry should not be ignored in cerebral ischemia experiments. The difference in hemodynamics in patients with hypertension is more obvious. According to reports, the IMT of the left CCA of untreated hypertensive patients is greater than the IMT of the right CCA. Similarly, in patients under the age of 60, this difference increases with age and gradually decreases after the age of 60. Therefore, the choice of left and right ischemia cannot be ignored when establishing animal models. The heterogeneous distribution of patients with ischemic stroke in the left and right hemispheres indicates that preclinical research can prioritize the treatment of animal models of ischemic stroke in the left hemisphere to promote the transformation of clinical results. However, this does not mean that you are not recommended to study the correct ischemia model. Due to insufficient diagnosis of patients with right brain ischemia, researchers need to pay more attention to the right brain ischemia model. The biochemical and behavioral differences observed in cerebral ischemia in the left and right hemispheres indicate that the ischemic side can be selected according to the purpose of the experiment (left: motor dysfunction; right: memory impairment). However, there are few studies on the difference between left and right cerebral ischemia.

　　Comparison of permanent cerebral ischemia and cerebral ischemia reperfusion models:

　　Preclinical studies mainly use cerebral ischemia-reperfusion models: patients with permanent cerebral ischemia models do not need to be reventilated. However, cerebral ischemia-reperfusion models can simulate patients with vascular obstruction and recanalization in time. The pathological mechanisms of the two models are obviously different. Whether to create a permanent cerebral ischemia model or an ischemia-reperfusion model is still a question. Since the MCAO model is widely used in the study of ischemic stroke, transient MCAO (tMCAO) and permanent MCAO (pMCAO) are also used in research. Searching CNKI using the search terms "permanent cerebral ischemia" or "cerebral ischemia reperfusion", "tMCAO" or "pMCAO" led to the publication of many publications on cerebral ischemia reperfusion from 1952 to 2020. Far beyond the research on persistent cerebral ischemia. Due to the limitations of the CNKI database, a search was performed on the PubMed database and the results were similar.

　　Timely recanalization of blood vessels after cerebral ischemia is the most direct and effective treatment. However, reperfusion will cause new damage. Therefore, many studies have developed neuroprotective agents using the cerebral ischemia-reperfusion model. In addition, compared with permanent ischemia, reperfusion of blood flow allows the drug to reach the infarct area and exert its full effect. This may be one of the reasons why most researchers use cerebral ischemia-reperfusion models. In addition, in most studies, reperfusion was performed 90-120 minutes after occlusion. The reason is as follows. 1) Rats reperfused 0.5 to 1 hour after occlusion have no mild infarction, large variability and cognitive impairment. In addition, the neurobehavior of these rats recovered quickly after surgery and did not meet the requirements of long-term research. 2) If the blood vessel is occluded for 3 or 4 hours, reperfusion will increase the infarct volume, which is equivalent to permanent ischemia, but will increase mortality. 3) The intracavitary suture reaches MCAO and lasts for more than 2 hours, causing spontaneous hyperthermia in rats with hypothalamic injury. It has not been observed in human clinical stroke. Clinical results show that the best effect can be observed if thrombolytic therapy is started within the first 90 minutes after the onset of symptoms. Reperfusion 90-120 minutes after vascular occlusion can induce stable neurological diseases and reperfusion injury, with high success rate, low mortality, long survival period and meeting experimental requirements. It is worth noting that mice are more susceptible to occlusion time than mice.

　　The difference between permanent cerebral ischemia and cerebral ischemia-reperfusion: The pathological mechanisms of permanent cerebral ischemia and cerebral ischemia-reperfusion are obviously different. Permanent cerebral ischemia is mainly hypoxic-ischemic primary injury. A slight decrease in blood flow will not cause obvious dysfunction or metabolic disorders. With the extension of the ischemic time, the ischemic core gradually expands to the ischemic penumbra, and the infarct volume reaches the maximum. If blood flow is restored in time, the damage to the infarct core can be eliminated. Otherwise, interventional therapy can only prevent the expansion of the infarct core. In addition, if reperfusion is delayed (more than 3 hours), its reversal effect will become very weak, leading to more severe ischemia-reperfusion injury. Cerebral ischemia-reperfusion mainly causes secondary (non-ischemic) cell death. Reperfusion will change the physiological and pathological processes after ischemia, resulting in significant differences in acute, subacute (3d) and chronic recovery (7d) strokes. Genetic map analysis also showed that cerebral ischemia-reperfusion injury is related to inflammation and apoptosis pathways, while permanent cerebral ischemia diseases are related to neurotransmitter receptors, ion channels, growth factors and other pathways.

　　At present, the difference between the mechanisms of these two modes needs further clarification. Accurately explaining the temporal and spatial changes of injury in the two models will help to achieve accurate treatment of the two models from the acute phase to the recovery phase. In addition, the preclinical choice of this model depends on the type of drug being studied and its perceived mechanism of action, which is also the basis for this choice. For example, the cerebral ischemia reperfusion model should be the first choice for testing free radical ablation and anti-inflammatory drugs, while the permanent cerebral ischemia model should be the first choice for glutamate antagonist testing. .. However, in preclinical studies, researchers focused on the cerebral ischemia-reperfusion model, and the gradual enrichment of its mechanism provides more references for the choice of this model. On the contrary, there are few studies on permanent cerebral ischemia. Clinical benefits of patients with persistent cerebral ischemia: At present, preclinical trials mainly use cerebral ischemia-reperfusion models, but most patients with ischemic stroke have successfully recanalized. Not... t-PA is currently the only solubilizer approved by the US FDA for stroke treatment, but its narrow time limit and high bleeding risk limit its clinical application. In recent years, the utilization rate of rt-PA has continued to increase from 9.9% in 2006 to 21.8% in 2018. However, the recanalization rate needs to be improved, and only a few patients who cannot dissolve rt-PA thrombosis can successfully undergo mechanical thrombectomy. Usually, most patients with permanent cerebral ischemia who have not reopened still exist. The contradiction between research hotspots and clinical needs may partly explain the failure of clinical drug conversion. In addition to established experimental goals or drug mechanisms, it is recommended to give priority to animal models of permanent cerebral ischemia in preclinical research. However, considering the heterogeneity of human stroke, the drug takes into account the preclinical effects, short-term and long-term histology and function in various models and species and before switching to clinical trials. It is necessary to consider improving the target parameters. In short, the support for permanent cerebral ischemia models cannot be ignored. The accurate model reflects most clinical stroke cases and supports clinical translation. In addition, in the process of clinical transformation, please do not confuse the corresponding pathological mechanisms of the two models. However, this does not mean that the cerebral ischemia reperfusion model should not be used. Advances in medical technology will allow more patients to reopen quickly and successfully. The combination of thrombolytics and neuroprotective agents has gradually matured.

　　Animal surgery: Many reports on animal pedigree, gender, age and risk factor models of ischemic stroke have been provided. According to epidemiological studies, the incidence of stroke is highly dependent on age. Stroke mainly occurs in middle-aged and elderly people. Therefore, we recommend that you study middle-aged animals instead of older animals. The incidence of stroke in women is not lower than in men and depends on age. In addition, clinical stroke patients often show risk factors for stroke (hypertension, hyperglycemia, and atherosclerosis). Unfortunately, most preclinical studies use young, healthy male animals for research. This can partly explain the failure of clinical transformation of drugs. The neuroimmune microenvironment of old animals becomes pro-inflammatory. In addition, the antioxidant capacity of the brain tissue of elderly animals is reduced, and the early stages of cerebral ischemia and reperfusion show a strong inflammatory response, and the activation of microglia and macrophages is enhanced; in addition, it is also prone to apoptosis. , Larger infarct size and more serious neurological defects. In addition, changes in the brain environment of older animals may not promote the survival of new cells or the growth of neurons. These factors inhibited the recovery of nerve function in aged rats. According to reports, the neuroprotective effect of the drug on older animals is weakening. Other studies have shown that long-term exposure to low temperatures can reduce large-scale damage to older animals by improving metabolism. Granulocyte colony stimulator (G-CSF) can promote neurogenesis, which helps the nerve function recovery of aging rats after stroke. Combining with bone marrow mesenchymal stem cells can increase angiogenesis, but it cannot further improve recovery after stroke. The incidence of many diseases such as diabetes, hypertension and hyperlipidemia increases with age, which also increases the risk of stroke and increases mortality and disability. Among them, hypertension is the most important variable risk factor. Angiotensin-converting enzyme inhibitors can also reduce aspiration pneumonia after stroke. Preclinical studies have shown that hypertension can reduce the integrity of the blood-brain barrier, increase white matter damage and edema, and exacerbate cerebral ischemic damage. Therefore, spontaneously hypertensive rats (SHR) show larger infarctions. According to reports, the NMDA receptor antagonist diglycoside (MK-801) and atmospheric hyperoxemia (NBO) can significantly reduce the infarction of normotensive rats, but can reduce the infarct size of SHR. Can't. Epidemiological studies have shown that more than 50% of stroke patients suffer from hyperglycemia, while diabetic stroke patients tend to be younger. Hyperglycemia can induce stroke by exacerbating vascular endothelial dysfunction and promoting early arteriosclerosis, systemic inflammation, thickening of capillary basement membrane or increasing lactic acid production. It may lead to more severe metabolic disorders and calcium homeostasis, oxidative stress and inflammation, aggravate ischemic injury, especially cortical infarction, and lead to poor prognosis. According to reports, hypoglycemic drugs, such as pioglitazone, may also reduce the recurrence rate of stroke in diabetic patients. Studies have shown that in mouse models of stroke, hyperglycemia can cause more severe infarction and edema, aggravate sensorimotor and cognitive deficits, and hinder the recovery of nerve function. Interestingly, the infarct size of diabetic rats treated with insulin was similar to that of normal rats. Obesity and atherosclerosis are important risk factors for stroke. The above research results indicate that the etiology and prognosis of stroke are related to age and comorbidities. In these animal models, the drug does not activate related pathways in young male animals and may not exert neuroprotective effects.

　　Occluder: The physical characteristics of the occluder tip (tip diameter, tip length, tip shape, flexibility) play an important role in the development of cerebral infarction changes and submucosal hemorrhage (SAH). .. Theoretically, matching the size of the occluder to the size of the animal can improve the consistency of the model. If the rodents have different body weights, we recommend using a commercially available occluder. This increases the success rate of surgery, increases the consistency of the experiment, and reduces the occurrence of SAH. In addition, this also improves the comparability of results from different laboratories, facilitates systematic review, and provides more scientific and accurate data for clinical translation.

　　Fasting: In many research reports in this field, animals are fasted for 12 to 24 hours before surgery. However, in reality, rodents lack the vomiting reflex and have a high metabolic rate. Fasting for 6 hours can cause weight loss and liver glycogen depletion. Therefore, the overnight fast will affect the animal's physiological condition and surgical resistance, as well as the neuroprotective effect of candidate drugs. Therefore, unless appetite behavior test, blood sugar test or other experiments are required, it is not recommended to fast before surgery.

　　During surgery: Many commonly used anesthetics have been shown to have neuroprotective effects. Therefore, it is necessary to consider the mechanism of action of anesthetics to avoid interaction with candidate drugs. Physiological parameters should be monitored during anesthesia, and the anesthesia time should be as short as possible to reduce the effect of anesthesia on the animal. MCAO can be performed through a CCA or external carotid artery (ECA) incision, and then the occluder is inserted into the internal carotid artery (ICA). For pMCAO, the CCA method may be a simpler surgical procedure, but the ECA method is suitable for temporary MCAO because it can maintain the anatomical integrity required for reperfusion. The sham operation group helps to eliminate the influence of the process itself on the animal model of stroke. In some articles, the false operation group operation only includes the separation of CCA from ECA. However, mechanical stimulation caused by the insertion of the occluder can cause endothelial cell dysfunction and vasomotor dysfunction. In some studies, in the sham operation group, only the occluder was inserted into the CCA without reaching the ICA, which caused rat ischemia and neurological dysfunction. This type of false operation maximizes the effect of rat surgery and occluder transplantation. Ischemia evaluation: In preclinical studies, neurological score and infarct volume are the first choice indicators for evaluating the efficacy of drug candidates. However, it should be noted that the absence of changes in infarct volume does not prove that the candidate drug has no neuroprotective effect. For example, basic fibroblast growth factor can provide neuroprotection during recovery from ischemia, but does not affect infarct volume. In addition, neurological evaluation is usually only performed in the acute or subacute phase of preclinical research, but the clinical evaluation period is usually extended to 3 months or longer. Prolonging the evaluation of preclinical neurological function may further promote the observation of drug efficacy and clinical changes.

　　Post-operative care: 1) Life of the stroke group: According to animal habits, animals after stroke should be in the same group as those before stroke. Each cage should be equipped with sham surgery and stroke animals to reduce mortality after stroke. 2) Antibiotics: surgical sterility is very important. Antibiotics can interfere with the intestinal flora and affect the results of experiments. Therefore, if a sterile environment for surgery is provided, antibiotics are not needed. 3) Pain relief: From an ethical point of view, pain will affect the physiological condition of animals and the neuroprotective effect of candidate drugs. Therefore, all animals must be analgesics after surgery, but the neuroprotection of drugs and candidate drugs should be avoided for pain relief effect. Possible reaction. 4) Animal monitoring: Surgery may cause fluid loss and eating disorders in animals. Therefore, it is necessary to monitor the important physical signs of animals and add body fluids and soft food in time, especially for model animals at risk of stroke.

　　Conclusion: For the first time, we compared left and right hemisphere ischemia in clinical stroke patients with preclinical studies. In this article, we will see for the first time the difference in mechanism between the permanent cerebral ischemia model and the ischemia-reperfusion model at various stages of the disease. According to the type distribution of clinical stroke patients, it is recommended to give priority to the left cerebral ischemia model or choose the model according to the experimental purpose (left: motor dysfunction, right: memory impairment). In addition, unless the experimental purpose and mechanism of action of the candidate drug are determined, the permanent cerebral ischemia model should be given priority. This article provides detailed information about the preoperative, intraoperative and postoperative care of animals to establish accurate, effective and reproducible cerebral ischemia models for researchers in this field.