| Part I: Construction of mouse brain injury model and evaluation of mirna-210 expression Objective: to establish and verify the model of brain injury in mice.Methods: 1)Male C57BL/6 mice were used for MCAO modeling;2)Longa and Bederson’s 5-point preparation method was used for neurological impairment score after mouse modeling;3)Morris water maze test was used to test the behavioral ability of mice;4)TTC staining and infarct size measurement;5)immunohistochemistry was used to analyze infarction;Results: 1)After awakening,the mice were subjected to cranial nerve injury and water maze to detect the brain injury status of the mice,and the results of neurological impairment score and Morris water maze test showed that the MCAO model of the mice in this study was successfully modeled,and the injury of the mice belonged to mild-moderate injury;2)The results of TCC staining showed that the mice in the model group had different degrees of cerebral infarction,with different infarct sizes and the boundary between the non-injured area was not obvious,but the injured tissue could be significantly distinguished,the infarction mainly existed in the cortical part,and the necrotic area of the severely infarcted brain extended to most of the neocortex,amygdala and hypothalamus adjacent to the striatum;3)The degree of neuronal injury was determined by the loss and attenuation of MAP2 immunoreactivity,and the decreased expression of MAP2 could be detected in the striatum and neocortex as early as after reperfusion,in addition,the expression of HSP72 and HSP27 could also be observed;the damage surrounding areas included the hippocampus,thalamus and hypothalamus.The ipsilateral and posterior cingulate and splenic cortices as well as the hippocampus and the septum were identified as distant affected areas.HSP72 expression was barely detectable in the neocortex and was restricted to very few neurons near the infarct core.HSP72 expression in the hippocampus of the pyramidal cell layer in some mice;4),the gene quantification of miRNA-210 was detected in the bilateral brain tissues of mice at 1 day,3 days and 7 days after hand surgery,and its mean value was taken as the standard for statistics,and the results revealed that the expression level of miRNA-210 in the model group was significantly increased compared with the sham mice in the control group,reaching the highest level on the third day,and then showing a decreasing trend,with a significant difference between the two groups,P <0.05;5)the mice were euthanized at 24 hours,3 days and 7 days after modeling,and perfusion fixation was performed,and the sham mice were used as controls.The brains were collected for cryosectioning and immunofluorescence staining.After MCAO,CD31-positive vascular endothelial cells were found in the infarct border zone.The number of vascular endothelial cells increased over time,which indicated that new blood vessel formation gradually increased with the extension of MCAO time;Conclusion: the model was successfully constructed.Part II:Correlation between miRNA-210 expression and brain injury in mouse model of brain injuryObjective:To analyze The correlation between mirna-210 expression and brain injury.Methods:1)lentiviral transfection was used to knock down the expression of miRNA-210;2)real-time PCR was used to analyze the expression levels of chemokines,cytokines and proinflammatory factors in mice before and after modeling;3)ELISA was used to analyze the expression of chemokines;4)Bruker11.7-TBio Spec was used to analyze the brain after modeling by MRI;Results: 1)The results of miRNA quantitative analysis showed that the expression level of miRNA-210 in the hemispheres of mice in the miRNA-210-silenced expression group was significantly reduced after modeling,and cerebral infarction and related edema were evaluated using MRI T2-weighted images 48 hours after modeling the mice.Compared with the negative control group,the infarct volume was significantly reduced in the miRNA-210-LAN group pretreatment group(32.1% ± 0.9% vs 50.3% ± 3.3%,P < 0.01),and miRNA-210-LAN significantly reduced MCAO induced brain edema(12.5% ± 1.3% vs.3.5% ± 2.5%,P < 0.05);2)the neurological function score was significantly reduced in the miRNA-210-LAN pretreatment group by 13.5 ± 0.7 vs.8.5 ± 0.9,P < 0.01,2A),demonstrating that,miRNA-210 expression can promote the degree of cranial nerve injury in mice.The behavioral ability was tested by water maze.The results showed that the average swimming speed,platform finding time and platform quadrant retention time of mice in miRNA-210-LAN group were significantly lower than those of mice in control group;3)the expression of inflammatory cytokines TNF-α,IL-1β and IL-6 in mice after MCAO modeling showed a tendency of up-regulation,which was significantly higher than that of mice in control group;4)there was no significant difference in the levels of cytokines TGF-β and IL-10 between the two groups,P > 0.05;5)MCAO significantly increased the m RNA abundance of chemokines CCL2 and CCL3 in the brain tissue of MCAO mice.CCL2 increased at 6 hours after MCAO and remained elevated at 12 and 24 hours,while the expression level of CCL2 did not change significantly in mice in the miRNA-210 silenced expression group;the chemokine CCL3 showed a rapid increase in control MCAO mice,2.3-fold increase within 6 hours after modeling,4.9-fold increase within 12 hours after modeling,and a significant decrease from 12 to 24 hours after modeling,but its expression level was still significantly higher than that of mice in the miRNA-210-LAN group.Our findings suggest that pretreatment with Mi RNA-210-LAN prior to MCAO significantly inhibited MCAO induced CCL2 and CCL3 expression;6)mice after 6 hours of brain injury modeling were subjected to intraventricular injection of miRNA-210 inhibitor to inhibit miRNA-210 expression in mouse brain tissue.The results of m RNA detection suggested that the expression levels of proinflammatory cytokines TNF-α,IL-1β,IL-6,and proinflammatory cytokines CCL2 and CCL3 in mice injected with miRNA-210-LAN showed a significant decreasing trend,and the levels were not significantly different from those in mice pretreated with mi R-RNA-LAN before modeling at 2 days after injection;7)After MCAO modeling by MRI,the brains of mice injected with miRNA-210-LAN into the ventricles were examined,and the results showed that after modeling in mice,the mice showed significant cerebral infarction,but after intracerebroventricular injection of miRNA-210-LAN,the cerebral infarction of mice was repaired to a certain extent,and their cerebral infarction area was significantly reduced,which was calculated to be reduced to 1/6 of that before miRNA-210-LAN injection.The results of neurological function score showed that after MCAO modeling,the neurological function score of mice suggested that the mice had moderate-severe brain injury,and the neurological function score results of mice injected with miRNA-210-LAN 6 hours after brain injury modeling suggested that the brain injury of mice showed a significant improvement;Conclusion:miRNA-210 can promote the development of brain injury,and is expected to be a new target for the prediction and treatment of ischemic brain injury.Reducing the expression of mirna-210 can significantly reduce ischemic brain injury.Part III:Correlation between miRNA-210 expression and brain injury in mouse model of brain injuryObjective:To explore the predictive value of miRNA-210 in patients with brain injury.Methods: 1)this study included 50 neonates with MCAO and 50 healthy neonates who visited the Department of Pediatrics of Union Hospital from January 2017 to December 2020,and the newborns were ensured to be within 48 hours after birth.2)Test Within 6 hours after birth of the newborn,blood samples are collected through the femoral vein,and a total of 2 m L of blood samples are collected,and all subjects should be in a fasting state.The levels of serum NSE and S100 B proteins were measured with ELISA kits,and NSE and S100 B proteins were detected with a model 550 microplate reader;3)Real-time PCR was used to detect the expression of miRNA-210;4)trained doctors assessed their behavioral neurology on the 7th day of life in term infants,while neonatal behavioral neurological assessment of preterm infants was evaluated at term;5)newborns were divided into:(1)high NBNA group with NBNA score higher than 37;(2)low NBNA group with NBNA score < 35;and(3)medium NBNA group with NBNA score between 35 and 37.Results:1)The results showed that there was no significant difference in gender,weight,gestational age and mode of birth between MCAO newborns and normal newborns,P > 0.05;newborns were divided into:(1)high NBNA group with NBNA score higher than 37;(2)low NBNA group with NBNA score < 35;and(3)medium NBNA group with NBNA score between 35 and 37.2)The expression levels of NSE,S100 B and miRNA-210 were up-regulated in neonates with MCAO,and the expression levels increased correspondingly with the deepening of the degree of MCAO;3)The miRNA levels in MCAO patients,and the cutoff values of three relative expression levels were selected as the criteria for diagnosis(0.5,0.6,0.7).Based on the analysis of the average detection levels in MCAO patients and the levels in healthy subjects in the control group,it was found that when the relative expression level of miRNA-210 was set at 0.5,the maximum number of confirmed patients was 42;4)The diagnostic performance of miRNA-210 was studied when the miRNA-210 cutoff value was 0.5.After calculation,the sensitivity,specificity,positive predictive value(PPV),negative predictive value(NPV)and diagnostic efficiency(Diagnostic Efficiency)of miRNA-210 diagnosis were 84%,90%,89.4%,84.9% and 87%,respectively.Conclusion: miRNA-210 can inhibit hypoxic-ischemic brain damage and is expected to be a novel treatment for ischemic brain damage.In addition,miRNA-210 has a good diagnostic performance in neonates with brain injury and is expected to play a role as a neonatal diagnostic biomarker. |
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