The Role And Mechanism Study Of The Activation Of Microglia Mediated By P2Y12 Receptor In Delayed Encephalopathy After Acute Carbon Monoxide Poisoning

CO poisoning is the highest incidence and death rate of poisoning,especially in the northern part of China.It can cause poisoning when the concentration in air is more than 30 mg/m3.The common source is steelmaking in industry.Coking and the chemical industry,which uses CO as raw materials,car exhaust gas and trains passing through tunnels,mine explosions and fire fighting,winter hearth heating doors and windows,etc.The Inner Mongolia Autonomous region is located in the northern highlands.The population in the farming and pastoral areas is large and the heating period is longer,CO poisoning and delayed encephalopathy are more common.CO poisoning can cause multiple organ lesions,especially central nervous system dysfunction.Delayed encephalopathy(DEACMP)is the most common and severe complication of CO poisoning,with an incidence of about 13-50% and a mortality rate of 31%.There is a period of similar recovery from CO poisoning to the onset of DEACMP.it is easy to be mistaken for cure by the patients and their families.Patients often miss the best treatment time.Most of the patients with DEACMP have acute andsevere clinical symptoms,mainly dementia symptoms,there is a high disability rate and fatality rate,which seriously affect the quality of life of patients.Once the patient occurs,there are no effective treatment methods and strategies,which will bring pain to the patient for life,and bring great burden to the family and society.It is urgent to clarify the pathogenesis of deacmp in clinic.It has important clinical significance for early intervention and reducing the occurrence of deacmp events.The pathogenesis of DEACMP has not been fully clarified.Hypoxia alone cannot fully explain its pathogenesis.Many studies have shown that brain tissue injury involves early and secondary injury.A series of changes have taken place in the number and morphology of microglia in the brain after injury.The process of CO poisoning and DEACMP is very similar to that of brain injury and secondary injury.However,recent research progress shows that Immune mechanism is more likely to explain the characteristics of the DEACMP from the point of view of the pathogenesis of "pseudo healing" and the pathological damage in the early stage of the disease.Microglia are recognized as important immune effectors in the central nervous system.At present,there are few reports about immune mechanism of DEACMP.We're going to investigate immune mechanism of DEACMP,and that might help to clarify the pathogenesis of DEACMP and provide evidence for clinical detection,treatment and prevention.Many studies showed that resting microglia become activated bymorphological changes after CO poisoning.Our previous studies have found that myelin basic protein was attacked by releasing inflammatory cytokines after poisoning,then causing demyelination.The release of inflammatory cytokines and the demyelination of myelin basic proteins were significantly inhibited after treatment of dexamethasone.However,whether the activated microglia after CO poisoning can induce chronic inflammatory reaction and demyelination by releasing a large number of inflammatory cytokines,and the pathway by which CO mediates the activation of microglia deserves further study.Adenosine triphosphate(ATP)and adenosine diphosphate(ADP)are necessary energy carriers to maintain normal metabolism of cells,but they are also important neurotransmitters.P2 purine receptors are physiological receptors of purines such as ATP,including 7 ion channel P2 X receptors and 13 gprotein-coupled P2 Y receptors,among which P2Y12 receptor is an important member of the P2 Y receptor family and is widely expressed in the central nervous system.The positive cells were mainly microglia.It has been reported that ATP and ADP was released by many nociceptive stimuli,then could activated microglia.However,Haynes found that P2Y12 receptor was activated under the stimulation of ATP in the rat model of cerebral cortex injury,then activated the microglia.The morphology of the microglia did not change significantly in knockouting the P2Y12 receptor gene model.The results suggested that the activation of P2Y12 receptorplayed an important role in the activation of microglia.Many mechanisms have some limitations to explain the pathogenesis of DEACMP.Combining with the literature reports of previous scholars and our previous research results,In the study,firstly,DEACMP model was established after CO poisoning.Then,injected the P2Y12 receptor agonist.At different time point,water maze was used to evaluate cognitive function,high performance liquid chromatography to determine the ATP content in hippocampucs,flow cytometer to examine the activated microglia cells.The study not only would invstigate the pathogenesis mechanism of DEACMP,but also provide new information to understand the treatment of DEACMP in future clinic practice.PART ? Establishment of delayed encephalopathy rat model of carbon monoxide poisoningAbstractObjective To establish the delayed encephalopathy rat model of carbon monoxide(CO)poisoning and investigate the therapeutic mechanism.Methods 204 intelligence qualified rats of Morris water maze training were randomly divided into experimental group and control group.the experimental group using acute static inhalation of CO,according to CO poisoning different concentrations were randomly divided into 1000 ppm group,3000 ppm group,4000 ppm group and 5000 ppm group.each group inhaled CO 40 min,the control group filled with the same amount of air.Dynamic monitoring of carboxyhemoglobin(C0hb)in rats by spectrophotometer.The Morris water maze test was used to evaluate the cognitive function.The pathological sections were made to observe the damage of hippocampal cells by HE staining.Results1.The typical CO poisoning occurred in rats after poisoning.2.There was a significant positive correlation between C0 Hb content and inhaled CO concentration(r=0.748,P<0.05).3.The mortality of the experimental group was 0%,40.74% and58.97% respectively.4.Compared with before poisoning,the escape latency in control group and 1000 ppm group was not significant difference after poisoning,the escape latency in 3000 ppm group and 4000 ppm group was not significant difference at 7th and 14 th days after poisoning,but there was significant difference at 21 th and 28 th days after poisoning.Compared with the control group,the escape latency in 1000 ppm group was no significant difference at each time point,the escape latency in 3000 ppm group and4000 ppm group was not significant difference at 7th and 14 th days after poisoning,but there was significant difference at 21 th and 28 th days after poisoning.Compared with the 1000 ppm group,the escape latency in3000 ppm group and 4000 ppm group was not significant difference at 7th and 14 th days after poisoning,but there was significant difference at 21 th and 28 th days after poisoning.Compared with the 3000 ppm group,the escape latency in 4000 ppm group was no significant difference at each time point.5.Hippocampal pyramidal cells in the control group and 1000 ppm group were arranged in order,with a complete cell structure.Hippocampal pyramidal cells 21 days after poisoning in the 3000 ppm group and4000 ppm group were deranged with an irregular cell shape,a shrunk cell body,nuclear pyknosis,anachromasis and an unclear cell structure.Conclusion The CO poisoning concentration of 3000 ppm and the time of 21 d were the best methods to establish DEACMP rat model.PART ? Correlation between microglia and Delayed encephalopathy after acute carbon monoxide poisoningAbstractObjective To investigate the correlation between microglia and delayed encephalopathy after acute carbon monoxide poisoning(DEACMP).Methods 244 intelligence qualified rats of Morris water maze training were randomly divided into CO poisoning group and DEACMP group.CO poisoning rat model was established in CO poisoning group according to static inhalation concentration of 1000 ppm carbon monoxide for 40 minutes.DEACMP rat model was established in DEACMP group according to static inhalation concentration of 3000 ppm carbon monoxide for 40 minutes.Dynamic monitoring of carboxyhemoglobin(COHb)in rats by spectrophotometer.The Morris water maze test was used to evaluate the cognitive function.The pathological sections were made to observe the damage of hippocampal cells by HE staining.Flow cytometry was adopted to examine the microglial activation in hippocampus area.Results1.The typical CO poisoning occurred in rats after poisoning.2.Compared with before poisoning,the escape latency in controlgroup and CO poisoning group was not significant difference after poisoning,the escape latency in DEACMP group was not significant difference at 7th and 14 th days after poisoning,but there was significant difference at 21 th and 28 th days after poisoning.Compared with the control group,the escape latency in CO poisoning group was no significant difference at each time point,the escape latency in DEACMP group was not significant difference at 7th and 14 th days after poisoning,but there was significant difference at 21 th and 28 th days after poisoning.Compared with the CO poisoning group,the escape latency in DEACMP group was not significant difference at 7th and 14 th days after poisoning,but there was significant difference at 21 th and 28 th days after poisoning.3.Hippocampal pyramidal cells in the control group and CO poisoning group were arranged in order,with a complete cell structure.Hippocampal pyramidal cells 21 days after poisoning in DEACMP group were deranged with an irregular cell shape,a shrunk cell body,nuclear pyknosis,anachromasis and an unclear cell structure.4.Compared with the control group,the activation level of microglia in CO poisoning group was not significant difference at 7th,21 th and 28 th days after poisoning,but there was significant difference at 14 th days after poisoning,the activation level of microglia in DEACMP group was not significant difference at 7th days after poisoning,but there was significant difference at 14 th,21th and 28 th days after poisoning.Compared with theCO poisoning group,the activation level of microglia in DEACMP group was not significant difference at 7th days after poisoning,but there was significant difference at 14 th,21th and 28 th days after poisoning.Conclusion1.The activation of microglia is related to the occurrence of DEACMP.2.The activation state of microglia is different with the concentration of CO poisoning,with the increase of toxic concentration and the prolongation of poisoning time,the activation of microglia was mainly characterized by classical activation.PART ? The role and mechanism study of the activation of microglia mediated by P2Y12 receptor in delayed encephalopathy after acute carbon monoxide poisoningAbstractObjective To investigate the role and mechanism of the activation of microglia mediated by P2Y12 receptor in delayed encephalopathy after acute carbon monoxide poisoning.Methods DEACMP rat model was established according to static inhalation concentration of 3000 ppm carbon monoxide for 40 minutes.192 surviving rats of DEACMP after modeling were randomly divided into intervention group and DEACMP group by random number table method.The control group was placed in the toxic box filled with air.The intervention group was given the intraperitoneal injection of Sura 100 u M,the DEACMP group and the control group were given equal dose of normal saline.Three groups of rats were in 7,14 21 and 28 d after poisoning with Morris water maze test to evaluate the change of cognitive function;to observe the damage of hippocampus by HE staining;the application of high performance liquid chromatography,flow cytometry analysis,to change of ATP content in hippocampus of rat and the situation of microglial cells activation.Results1.The typical CO poisoning occurred in rats after poisoning.2.Compared with before poisoning,the escape latency in control group was not significant difference after poisoning,The escape latency in DEACMP group and intervention group was not significant difference at7 th and 14 th days after poisoning,but there was significant difference at21 th and 28 th days after poisoning.Compared with the control group,the escape latency in DEACMP group and intervention group was not significant difference at 7th and 14 th days after poisoning,but there was significant difference at 21 th and 28 th days after poisoning.Compared with DEACMP group,the escape latency intervention group was not significant difference at 7th and 14 th days after poisoning,but there was significant difference at 21 th and 28 th days after poisoning.3.Hippocampal pyramidal cells in the control group was arranged in order,with a complete cell structure.Hippocampal pyramidal cells 21 days after poisoning in DEACMP group were deranged with an irregular cell shape,a shrunk cell body,nuclear pyknosis,anachromasis and an unclear cell structure.These pathological changes were also found in the intervention group,but were lighter than those in DEACMP group.4.Compared with the control group,the activation level of microglia in DEACMP group and intervention group were not significant difference at 7th days after poisoning,but there were significant difference at 14 th,21th and 28 th days after poisoning.Compared with the DEACMP group,the activation level of microglia in intervention group was not significantdifference at 7th days after poisoning,but there was significant difference at 14 th,21th and 28 th days after poisoning.5.Compared with the control group,the ATP content in DEACMP group and intervention group were not significant difference at 7th days after poisoning,but there were significant difference at 14 th,21th and 28 th days after poisoning.Compared with the DEACMP group,the ATP content in intervention group was not significant difference at each time point.Conclusion ATP was able to mediate the activation of microglia through p2y12 receptor pathway.The activated microglia have changed their function and secreted a variety of inflammatory stimulators,thus participating in the development of DEACMP.