| Objective Stroke is one of the most common cause of death worldwide, and cerebral ischemia disease accounts for about 70%-90% of the stroke. The mortality and morbidity of the stroke are high. Establishing an efficient animal model of cerebral with a good repeatability, a stable infarct location and area which mimic the human’s stroke in the actual situation is important to study the pathophysiology and means of prevention and treatment of stroke. Endothelin-1 (ET-1) has been widely used in research rodent model of cerebral ischemia abroad, but less in primates. ET-1 is used little in domestic research of the model of cerebral ischemia. Loss of neurons and accompanied reactive gliosis and scarring after cerebral ischemia are difficult to reverse with existing treatment approaches. Chen Gong, etc. repoeted that a nerve transcription factor (NeuroD1) could directly reprogram and translate the reactive astrocytes into functional neurons after brain injury in mice in 2014, which providing a new method of thinking for the treatment of brain injury. In this study, for the first time we use ET-1 to induce a focal cerebral ischemia model in macaque, and study the cytological changes around the ischemic injury. It provides a preliminary reference for future study on reactive astrocytes reprogramming into neurons in macaque focal cerebral ischemia mode.Method1. This experiment experienced twice, the first is explore experiment, we take two disability macaques (both male). Three disability macaques were used (2 males,1 female) in the second experiment, disability macaque selection criteria:no visible deformity or only part of the body portion of the limb and joint deformities, but no head deformities. Limited of number of samples, we use the left brain hemispheres contrast with the right. The experimental side was induced focal cerebral ischemia by injection of ET-1 in the ccortex, control side use saline injection by the same way as the experimental group. The experimental group of first test, ET-1 doses were 0.2ug/10ul, lug/lOul,5ug/10ul. The second experiment,2.5ug/10ul,5ug/10ul, lOug/lOul. Solvent of all the experimental group and the control group were lOul normal saline.2. Macaque brain tissue of the first experiment were perfused by paraformaldehyde after injuryed one week, two weeks, three weeks, four weeks. Macaque in s econd test were perfused after injuryed two weeks, four weeks, six weeks, eight weeks later. We use NEUN, GFAP and IBA1 staining, then analysis the variation law of neurons, astrocytes, microglias around the ischemia injure between the experimental group and the control group. We also do Nissle dyeing to analyze the neuronal damage range in second experiment.Results1. After ET-1 injection, it produce a wider range of neuronal injury compared to saline control group. And the position of ischemic injury is fixed, always is the region surrounding the drug injection needle tract, and neurons loss scope of different time periods 5ugET-1 is stable, about the needle tract outside 1500um.2. Cytology variation law around ischemia injury:Timeliness:When dose of ET-1 is 2.5ug, astrocyte activation process rise for at least 28d in time, fall appeared not later than in 56d, the peak occurs between28d and 42d or 42d and 56d, the degree of microglia activation:28d> 42d> 56d, the peak occurs probably before 28d or between 28d and 42d. When dose of ET-1 is 5ug, astrocyte activation from 19d to 33d is a rising process, the peak occurs after 33d, the degree of microglia activation:19d> 33d, the peak occurs probably before 19d or between 19d and 33d. When dose of ET-1 is lOug, astrocyte activation from 15d to 29d is a rising process, the peak occurs probably after 29d, the degree of microglia activation:15d> 29d, the peak occurs probably before 15d or between 15d and 29d. The peak of GFAP +astrocytes contrast with EBA1+ microglia activation, peak of IBA1+microglia appear early in each dose.Spatiality:The degree of GFAP+ astrocytes and IBA1+microglia proliferation and NEUN+neuron damage near the needle tract compare with outside 500um in each dose of ET-1 are decreasing in each time point, until the normal zone.Dose relationship:Suppose three periods (15d-19d,29d-33d,28d-29d), due to the time is close, the degree of activation of astrocytes and microglia changed little. And each time period, under the ET-1 low-dose, the degree of IBA1+ microglia and GFAP+ astrocytes proliferation were higher than high-doses. Under Nigeria’s staining With the increaseing doses of ET-1, the trend of neuronal damage range is on the increase.Conclusion1. Macaque focal cerebral ischemia model induced by Endothelin-1 injected into cortex is a minimal injury, easy to locate reusable, simple operation, ideal focal cerebral ischemia model.2. The proliferation of GFAP+ astrocytes and IBA1+ microglia is temporal and spatial dependent around the ischemis injury in this model.3. We speculate that with the increasing doses of ET-1 may lead to the increase of neuronal damage range, and the decrease of IBAl+microglia and GFAP+ astrocytes proliferation under the same time in the ischemia injury core. |
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