| Background:Acute cerebral infarction is one of the common diseases of the nervous system,with high morbidity and mortality.A series of cascade reactions occur after cerebral ischemia,leading to aggravation of ischemic brain damage.Among them,inflammatory response plays an important role in cerebral ischemia and reperfusion injury.Both central and peripheral immune cells are involved in the inflammatory response after cerebral ischemia.Neutrophils usually participate in the inflammatory response at an early stage,and their numbers increase rapidly after acute cerebral infarction and infiltrate into the brain tissue.In the past,neutrophils were considered to be a group of homogeneous cells that mainly play a role in phagocytosis.In recent years,more and more studies have shown that neutrophils contain at least two functionally opposed subgroups.So far,there is no perfect and unified classification system for neutrophils because researchers have not yet formed a consensus on neutrophil clustering markers.Previous studies on the role of neutrophils in acute cerebral infarction mostly believed that the role of neutrophils was negative.However,the discovery of neutrophil heterogeneity has a great impact on the previous understanding.The roles of two groups of neutrophils with opposite functions in cerebral infarction are completely opposite.The changes of each subset of neutrophils after cerebral infarction and their roles and mechanisms in cerebral ischemic injury are still unclear.Therefore,this research aims to investigate the changes of neutrophils in peripheral blood subsets after acute cerebral infarction,and to explore the effect of remote ischemic conditioning on neutrophils subsets.Purpose:Analyzing the expression changes of peripheral neutrophil subsets and signaling pathways after cerebral ischemia and reperfusion;Analyzing the effects of remote ischemic conditioning on neutrophil subsets and clinical outcomes.Purpose:Method:1.Using the method of middle cerebral artery occlusion(MCAO)to construct a mouse model of cerebral ischemia and reperfusion.Before the establishment of the ischemia-reperfusion model for 24 hours,the mice in the remote ischemic preconditioning group were subjected to ischemic preconditioning of both hindlimbs for 7 days(complete ischemia of both hindlimbs for 5 minutes followed by remission for 5 minutes,repeated for 3 cycles,once a day).2.The expression changes of neutrophil subsets and signaling pathways in peripheral blood of mice after 45 min cerebral ischemia and 24 h and 72 h reperfusion were analyzed by single-cell transcriptomic sequencing.3.The changes of neutrophil ratios in peripheral blood and spleen of mice in 45 min cerebral ischemia and 24 h and 72 h reperfusion as well as ischemic preconditioning group were detected by flow cytometry.4.Thirty-eight patients with acute cerebral infarction within 72 hours of onset were recruited and divided into groups according to their wishes,including 24 in the routine treatment group and 14 in the remote ischemic postconditioning group.On the basis of conventional treatment,the patients in the remote ischemic postconditioning group underwent 7-day ischemic postconditioning for both upper extremities using an ischemic preconditioning apparatus(complete ischemia of both upper extremities for 5 minutes followed by remission for 5 minutes,repeated 5 cycles,twice a day).Another 15 volunteers with no history of cerebral infarction and other inflammation-related diseases matched with those in the conventional treatment group were recruited as a control group.5.Evaluate the NIHSS on admission and discharge to evaluate the neurological impairment of the patients.Patients were assessed for m RS at admission and 3 months after discharge to assess patients’ ability to live.6.Use flow cytometry to detect the proportion of neutrophils in the peripheral blood of each group of patients or volunteers.Results:1.The results of single-cell sequencing and cluster analysis of the peripheral blood leukocytes of each group showed that the peripheral blood leukocytes of mice can be divided into 21 subgroups,of which 5 subgroups belong to neutrophils,including: C4,C6,C14,C16,C18.Compared with other subgroups,C6 subgroup expressed lower pro-inflammatory factors such as IL1 b,IL15,IL16 and TNFα(P < 0.05)and chemokines such as CCL6,CCR1,CCRL2,CXCL2,CXCR2,CXCR4(P < 0.05),and higher level of anti-inflammatory factor TGFβ(P < 0.05).Further surface marker analysis showed that the vast majority of neutrophils in the C6 subgroup were CD53-cells,while the vast majority of neutrophils in the other four subgroups were CD53+ cells.2.Compared with the sham-operated group,the proportion of CD53+ neutrophils in the peripheral blood of MCAO mice was slightly decreased at 45 min of cerebral ischemia(P < 0.01),and significantly increased at 12 h and 24 h of I/R(P < 0.001).There was no significant change at I/R72h(P > 0.05).The CD53+ neutrophils in the peripheral blood of mice in the ischemic preconditioning group were significantly lower than those in the I/R 24 h group(P < 0.01),but not significantly different from the sham operation group(P > 0.05).The change of CD53-neutrophils was opposite to that of CD53+ neutrophils.3.Compared with the sham-operated group,the proportion of CD53+ neutrophils in the spleen of MCAO mice did not change significantly at 45 min of cerebral ischemia(P > 0.05),but tend to decrease at I/R12h(P = 0.061),and significantly decreased at I/R24 h and 72h(P < 0.001).The CD53+ neutrophils in the spleen of mice in the ischemic preconditioning group were significantly higher than those in the I/R24 h group(P < 0.001),but lower than those in the sham-operated group(P < 0.001).The change of CD53-neutrophils was opposite to that of CD53+ neutrophils.4.Compared with the sham-operated group,in the peripheral blood neutrophils of MCAO mice,inflammatory response-related signaling pathways(such as TNFα-NFκB,IL6-Stat3,IFNα,IFNγ,IL2-Stat5 signalings)were significantly inhibited in 45 min of cerebral ischemia and was significantly activated at I/R24h(P < 0.05),and was significantly inhibited at I/R72h(P < 0.05).5.There was no significant difference in the NIHSS score between the ischemic postconditioning group and the conventional treatment group at admission and discharge(P > 0.05),and there was no significant difference in the m RS score at admission and 3 months after discharge(P > 0.05).However,ΔNIHSS and Δm RS in the ischemic postconditioning group were significantly higher than those in conventional treatment(P < 0.05).6.Compared with the control individuals,the proportion of neutrophils in peripheral blood of patients in the conventional treatment group increased significantly(P < 0.01),and the proportion of CD53+ neutrophils was also significantly increased(P < 0.05)at 2-days after admission.The peripheral blood neutrophils pencentages of the patients in the conventional treatment group at 5 and 9-days after admission have no significant difference from those in the control group(P > 0.05),and were significantly lower than those those at 2-days after admission(P < 0.05),while the CD53+ neutrophils were still significantly higher than those in the control group group(P < 0.05),and with no significant difference from those at 2-days after admission(P > 0.05).Compared with the conventional treatment group,the proportion of CD53+ neutrophils in the ischemic postconditioning group was significantly decreased at 7 days after remote ischemic postconditioning(9 days after admission)(P < 0.05).The change of CD53-neutrophils was opposite to that of CD53+ neutrophils.Conclusions:1.Neutrophils in peripheral blood can be divided into 21 subgroups,of which 5 subgroups(C4,C6,C14,C16,C18)belong to neutrophils.The five subgroups of neutrophils can be divided into two subgroups,CD53+(including C4,C14,C16,and C18 subgroups)and CD53-(C6 subgroup).2.Functionally,the CD53+ subset is a group of pro-inflammatory neutrophils,while the CD53-subset is a group of anti-inflammatory neutrophils.3.In the acute phase of cerebral ischemia,CD53+ neutrophils in the spleen of mice were released into the blood,and the peripheral blood CD53+ neutrophils were significantly increased,and the remote ischemic preconditioning inhibited this change.4.After acute cerebral infarction,the neutrophils in peripheral blood of patients with acute cerebral infarction increased significantly and were polarized to CD53+ subsets.After conventional treatment,the proportion of peripheral blood neutrophils decreased,but they were still polarized to CD53+ neutrophils.Remote ischemic condotiongning may play a protective role by inhibiting the polarization of peripheral blood neutrophils towards CD53+ subsets. |
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