动物造模,脑缺血动物模型 z-bio 2021-02-23 294

　　Left and right asymmetry in the animal model of cerebral ischemia: There is asymmetry in the adult brain, and one hemisphere is dominant. Here to identify the dominant hemisphere by hand. Ninety percent (90%) of people are right-handed. The language center of most right-handed people is 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 is reported that the pathological manifestations of ischemia in the dominant and non-dominant hemispheres are different. Studies have shown that brain asymmetry is not a unique feature of humans and is common in vertebrates. Dominant hemisphere ischemia leads to more severe neurological dysfunction in rats, while animals with non-dominant hemisphere ischemia recover faster. Other studies have reported significant differences in infarct volume between the left and right hemispheres in the early MCAO model. Left hemisphere ischemia leads to more severe sensorimotor impairment, while right hemisphere ischemia leads to more severe cognitive impairment. 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 striatum of the left and right cerebral ischemia. The changes of norepinephrine and dopamine in the cerebral cortex and blue dot nucleus were also inconsistent, and the concentration of dynorphin in the right amygdala reached a peak 3-5 days after ischemia. These results indicate that there is a left-right asymmetry in the rat cerebral hemisphere, and the behavior and biochemical changes of the left and right hemispheres are also different after ischemia. Therefore, in cerebral ischemia experiments, the asymmetry of the brain should not be ignored.

　　The incidence of cerebral ischemia in the left hemisphere is higher than in the right hemisphere: Arterial structure and blood flow: Other researchers explained this difference from the perspective of arterial structure, because there is a significant difference between carotid artery intima-media thickness (IMT) and stroke Correlation, especially non-lacunar strokes (atherosclerotic stroke and cardiogenic stroke), the incidence is higher in the left hemisphere than in the right hemisphere. The results show that this is significantly related to the difference in IMT between the left and right arteries. This difference is mainly due to the different anatomical structures of the left and right arteries. As shown in Figure 2, the middle cerebral artery originates from the common carotid artery (CCA). The left CCA originated directly from the aortic arch, parallel to the ascending aorta, while the right CCA originated from the main trunk of the right arm, causing the hemodynamic difference between the left and right CCA. In this case, the flow rate of the left CCA is faster, and its shear force is higher than that of the right CCA, resulting in the adaptive thickening of the IMT of the left CCA. It is believed that high shear force or oscillating shear force is a powerful determinant of IMT adaptive thickening. Although IMT under high shear stress itself does not cause atherosclerosis, the significant oscillation in the wall shear direction may promote the occurrence of atherosclerosis at a distance. In addition, the blood flow of the aortic arch may also cause left ulcer plaque, which may lead to cerebral embolic stroke.

　　The hemodynamic difference of hypertension patients is more obvious. According to reports, the left CCA of untreated hypertensive patients has a greater IMT than the right CCA. And in patients under 60 years of age, this difference increases with age, and gradually decreases after 60 years of age. Therefore, in the establishment of animal models, the choice between left and right ischemia cannot be ignored. The uneven distribution of patients with ischemic stroke in the left and right hemispheres suggests that preclinical studies can give priority to animal models of ischemic stroke in the left hemisphere to promote the transformation of clinical results. However, this does not mean that it is not recommended to study the right ischemia model. The insufficient diagnosis of patients with right cerebral ischemia reminds researchers to pay more attention to the right cerebral ischemia model. The observed biochemical and behavioral differences 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: permanent cerebral ischemia models can simulate patients without recanalization, while cerebral ischemia reperfusion models can simulate patients with vascular occlusion and timely recanalization. The pathological mechanisms of the two models are obviously different. Whether to make a permanent cerebral ischemia model or an ischemia-reperfusion model remains a question. The MCAO model is widely used in the research of ischemic stroke. Therefore, temporary MCAO (tMCAO) and permanent MCAO (pMCAO) are also used in research. Searched CNKI using the following search terms: "permanent cerebral ischemia" or "cerebral ischemia-reperfusion", "tMCAO" or "pMCAO", the results showed that from 1952 to 2020, about cerebral ischemia-reperfusion The number of publications is much higher than studies on permanent cerebral ischemia. Due to the limitations of the CNKI database, a search was conducted on the PubMed database and similar results were obtained.

　　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 used cerebral ischemia-reperfusion models to develop neuroprotective agents. Moreover, compared with permanent ischemia, blood flow reperfusion can allow the drug to reach the infarct area and fully exert its 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 reasons are as follows: 1) Rats with 0.5-1h reperfusion after occlusion have mild infarction, large variability, and no cognitive dysfunction. In addition, the neurobehavior of these rats recovered quickly after surgery, which did not meet the requirements of long-term testing. 2) When blood vessels are blocked for 3 or 4 hours, reperfusion can lead to an increase in infarct volume, which is commensurate with permanent ischemia; however, mortality increases. 3) MCAO reached by intracavitary suture and lasted for ≥2h caused spontaneous hyperthermia, which is related to hypothalamic injury in rats; this has not been observed in human clinical stroke. Clinical results indicate that the best results will be observed if thrombolytic therapy is started within the first 90 minutes after the onset of symptoms. Reperfusion after 90-120 minutes of vascular occlusion can induce stable neurological diseases and reperfusion injury, with a higher success rate, lower mortality and longer survival time, which can meet the experimental requirements. It is worth noting that compared with rats, mice are more susceptible to occlusion time.

　　The difference between permanent cerebral ischemia and cerebral ischemia-reperfusion: The pathological mechanism of permanent cerebral ischemia and cerebral ischemia-reperfusion is obviously different. Permanent cerebral ischemia is mainly a hypoxic-ischemic primary injury. A slight decrease in blood flow will not cause obvious functional or metabolic disorders. With the extension of 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 infarct core injury can be reversed. Otherwise, interventional therapy can only inhibit the expansion of the infarct core. In addition, if reperfusion is delayed (≥3 hours), its reversal effect will be very weak and cause more serious ischemia-reperfusion injury. Cerebral ischemia-reperfusion mainly leads to secondary (non-ischemic) cell death. Reperfusion changes the physiological and pathological processes after ischemia, resulting in significant differences in the acute phase (24h), subacute phase (3d), and chronic recovery phase (7d) of stroke. Gene profile analysis also showed that cerebral ischemia-reperfusion injury is related to inflammation and apoptosis pathways, while permanent cerebral ischemic injury is related to neurotransmitter receptors, ion channels, growth factors and other pathways. In addition, the effect of the drug on the two models is different.

　　At present, the difference in mechanism between these two modes deserves further clarification. Accurately describing the temporal and spatial changes of the damage development of the two models will help to achieve precise treatment of the two models from the acute phase of ischemia to the recovery phase. In addition, the preclinical choice of the model depends on the type of drug under study and its perceived mechanism of action, which also provides a reference for this choice. For example, the cerebral ischemia-reperfusion model should be preferred to test free radical scavenging and anti-inflammatory drugs, while the permanent cerebral ischemia model should be preferred for glutamate antagonist testing. However, in preclinical studies, researchers focused on the cerebral ischemia-reperfusion model, and the gradual enrichment of its mechanism provided more reference for the choice of this model. On the contrary, there are few studies on permanent cerebral ischemia.

　　Clinical advantages of patients with permanent cerebral ischemia: At present, preclinical studies mostly use the cerebral ischemia-reperfusion model. However, most patients with ischemic stroke have not successfully recanalized. rt-PA is currently the only dissolving drug approved by the FDA for the treatment of stroke, but its narrow time window 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 still needs to be improved, and only a small percentage of patients who fail rt-PA thrombolysis can successfully undergo mechanical thrombectomy. In general, patients with permanent cerebral ischemia that have not been recanalized still account for the majority. The contradiction between research hotspots and clinical needs may partly explain the failure of clinical drug conversion. It is recommended that in addition to the established experimental purpose or drug mechanism, animal models of permanent cerebral ischemia should be given priority in preclinical research.

　　However, considering the heterogeneity of human stroke, drugs should consider preclinical efficacy in a variety of different models and species, and consider improving short-term and long-term histological and functional parameters before being translated into clinical trials. In short, the advocacy of permanent cerebral ischemia model cannot be ignored. The accurate model reflects most clinical stroke cases and helps clinical transformation. In addition, during clinical transformation, the corresponding pathological mechanisms of the two models should not be confused. However, this does not mean that cerebral ischemia-reperfusion models should not be used. With the advancement of medical technology, more patients will be able to recanalize quickly and successfully. The combined application of thrombolysis and neuroprotective agents will gradually mature.

　　Animal surgery: Many reports on animal strains, gender, age and risk factor models of ischemic stroke are available. Epidemiological surveys show that the incidence of stroke depends largely on age. Human strokes mostly occur in middle-aged and elderly people. Therefore, it is recommended to use middle-aged animals as research objects, rather than old-age animals. The incidence of stroke in women is not lower than in men, and it depends on age. In addition, clinical stroke patients often show risk factors for stroke (hypertension, hyperglycemia, and atherosclerosis). Unfortunately, most preclinical studies use young and healthy male animals as research subjects, which may partly explain the failure of clinical transformation of drugs. The neuroimmune microenvironment of old animals turns into a pro-inflammatory state. In addition, the antioxidant capacity of the brain tissue of elderly animals is reduced, and the early stage of cerebral ischemia and reperfusion shows a strong inflammatory response, accompanied by enhanced activation of microglia and macrophages; in addition, it is more prone to apoptosis, which leads to more Large infarct size and more serious neurological deficits. In addition, changes in the brain environment of old animals may not be conducive to 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 drugs on elderly animals is weakened. Other studies have shown that long-term exposure to low temperatures can reduce large-scale damage in elderly animals by improving metabolism. Granulocyte colony stimulating factor (G-CSF) can promote neurogenesis and is beneficial to the recovery of neurological function in aged rats after stroke. Combined application 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, increasing mortality and disability. Among them, hypertension is the most important variable risk factor. Angiotensin-converting enzyme inhibitors can also reduce post-stroke aspiration pneumonia. Preclinical studies have proven that hypertension can reduce the integrity of the blood-brain barrier, increase white matter damage and edema, and aggravate cerebral ischemic damage. Therefore, spontaneously hypertensive rats (SHRs) show larger infarctions. According to reports, the NMDA receptor antagonists dizocilpine (MK-801) and normal pressure hyperoxia (NBO) can significantly reduce the infarction in normotensive rats, but cannot reduce the infarct size of SHR. Epidemiological studies have shown that more than 50% of stroke patients have high blood sugar, and stroke patients with diabetes tend to be younger. Hyperglycemia can induce stroke by aggravating vascular endothelial dysfunction and promoting early arteriosclerosis, systemic inflammation and thickening of capillary basement membrane or increasing lactic acid production. This can lead to more severe metabolic disturbances and calcium homeostasis, oxidative stress and inflammation, thereby exacerbating ischemic injury, especially cortical infarction, and leading to a poor prognosis. According to reports, hypoglycemic drugs, such as pioglitazone, can also reduce the recurrence rate of stroke in diabetic patients. Studies have shown that in the mouse model of stroke, high blood sugar can cause more severe infarction and edema, aggravate sensorimotor and cognitive impairment, 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 pathogenesis and prognosis of stroke are related to age and comorbidities. In these animal models, the drug may not activate the relevant pathways in young male animals and exert neuroprotective effects.

　　Occluder: The physical characteristics of the tip of the occluder (tip diameter, tip length, tip shape and flexibility) play a crucial role in the occurrence of cerebral infarction variation and subarachnoid hemorrhage (SAH). In theory, matching the size of the occluder to the size of the animal can improve the consistency of the model. For different rodent weights, it is recommended to use commercially available occluders, which can increase the success rate of the operation, increase the consistency of the experiment, and reduce the incidence of SAH. In addition, this also increases the comparability of the results of different laboratories, which is more conducive to systematic reviews and provides more scientific and accurate data for clinical translation.

　　Fasting: In many research reports in this field, animals are fasted for 12-24 hours before surgery. But in fact, rodents lack the vomiting reflex and have a higher metabolic rate. Fasting for 6 hours may cause weight loss and liver glycogen depletion; therefore, fasting overnight may affect the animal's physiological state and surgical tolerance, as well as the neuroprotective effect of the candidate drug. Therefore, it is not recommended to fast before surgery, unless appetite behavior test, blood sugar test or other experiments are required.

　　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 time of anesthesia should be shortened as much as possible to reduce the impact of anesthesia on animals. MCAO can be performed through a CCA or external carotid artery (ECA) incision, and the occluder is introduced into the internal carotid artery (ICA). For pMCAO, the CCA approach may be a simpler surgical procedure, while the ECA approach is a better choice for temporary MCAO because it can maintain the anatomical integrity required for reperfusion. The sham operation group helps to eliminate the influence of the procedure itself on the animal model of stroke. In some articles, the operation of the sham operation group only included 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 did not cause ischemia and neurological dysfunction in rats. This kind of sham operation eliminates the impact of surgery and occluder implantation on rats to the greatest extent.

　　Ischemia assessment: In preclinical studies, neurological score and infarct volume are the first choice indicators for evaluating the efficacy of drug candidates. Of course

　　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 has no effect on infarct volume. In addition, neurological function assessment is usually only performed in the acute or subacute phase of preclinical research, while clinical assessment time is usually extended to 3 months or more. Prolonging the evaluation of preclinical neurological function may be more conducive to the observation of drug efficacy and clinical transformation.

　　Post-surgery care: 1) Live in groups after stroke: According to animal life habits, post-stroke animals should be kept in the same group as pre-stroke animals, and each cage should contain sham and stroke animals, which can reduce post-stroke mortality. 2) Antibiotics: The sterility of surgery is very important. Antibiotics may interfere with the intestinal flora and affect experimental results. Therefore, if a sterile environment is provided for surgery, antibiotics are not required. 3) Analgesia: From an ethical perspective, all animals should be analgesic after surgery, because pain may affect the physiological state of the animals and the neuroprotective effect of the candidate drug; however, the neuroprotective effect of analgesic and the drug candidate must be avoided. Possible response. 4) Animal monitoring: Surgery will cause animal fluid loss and eating disorders. Therefore, the vital signs of animals should be monitored, and body fluids and soft food should be supplemented in time, especially for model animals with risk factors for stroke.

　　Conclusion: For the first time, the left and right hemisphere ischemia of clinical stroke patients were compared with preclinical studies. This article reviews for the first time the mechanism differences between the permanent cerebral ischemia model and the ischemia-reperfusion model at different 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 the mechanism of action of the candidate drug are determined, the permanent cerebral ischemia model should be given priority. This article provides relevant details of animal preoperative treatment, surgical procedures and postoperative care to establish accurate, effective and reproducible cerebral ischemia models, and provide references for researchers in this field.