Analysis Of Relevant Factors That Affects The Quantity And Status Of Circulating Neutrophils In Patients With Acute Ischemic Stroke

Object:The study was aimed at exploring changes in the absolute number of circulating neutrophils(NE#),the neutrophil to lymphocyte ratio(NLR)as well as the expression of their specific markers(including CD16,CD66 b and Annexin V)in patients with acute ischemic stroke(AIS)over time,and discussing the correlation of above indicators with pro-resolving factors(Annexin A1)and various clinical parameters of patients.Methods:We enrolled 31 patients with AIS hospitalized in the unit of Neurology of the First Hospital of Jilin University from July to December 2020.Patients were included if they had a stroke less than 72 hours before admission and showed no signs of infection or heart and lung diseases at the admission.The 21 age-and gender-matched healthy controls(HCs)were recruited during the same period.We prospectively collected the following data for each: general clinical data,NIHSS score at admission(day 1),time to onset from admission,cerebrovascular risk factors(including past medical history,such as hypertension,diabetes,heart disease,smoke,previous stroke history and treatment;clinical parameters,such as serum low-density lipoprotein,cholesterol,glycosylated hemoglobin,uric acid,homocysteine,C-reactive protein,folic acid and vitamin B12 tested after admission),blood routine examination at 1 and 8 days after admission,and imaging data related to cerebral infarction.Moreover,plasma and neutrophils were collected from HCs and patients in the early morning of 1 day,2 days and 8 days after admission.Then utilizing flow cytometry to analyze the expression of CD16,CD66 b and Annexin V in circulating neutrophils.Moreover,enzyme-linked immunosorbent assay(ELISA) was used to detect the plasmic concentration of Annexin A1.Subsequently,we analyzed changes of both the quantity(NE#,NLR)and status(the expression of CD16,CD66 b and Annexin V)of circulating neutrophils in AIS patients over time,followed by the discussion about their correlation with the concentration of Annexin A1 in plasma and various clinical parameters of patients.Flowjo V10 software was used for flow cytometry analysis,SPSS Statistics 22.0 software was used for statistical analysis and Graphpad Prism 7 as well as Inkscape 0.92.3 software were used for chart making.Results:(1)Compared with the HCs,the NE# was slightly higher in the AIS group,and NLR of AIS patients increased significantly(P<0.05)on 1d after admission.Then at8 days after admission,both NE# and NLR had fallen back to the control level(P>0.05).Statistical analysis further indicated that there were no significant relevant in NE# and NLR with patients’ clinical parameters such as gender,age,past history,location of onset,NHISS score at admission,time to onset at admission,serum low-density lipoprotein,cholesterol,glycosylated hemoglobin,uric acid,and homocysteamine,C-reactive protein,folic acid and vitamin B12(P>0.05).(2)The CD16 MFI of circulating neutrophils in AIS patients increased significantly on 1d and 2d(P<0.05)compared with the HCs,then fell back to the control level on 8d after admission(P>0.05).Although there was no significant correlation between the CD16 MFI at each time point and clinical parameters regarded in the study(P>0.05).The MFI value of CD16 in patients with previous diabetes was slightly higher than that in patients without diabetes at 1d after admission(P=0.084).The CD16 MFI showed a negative correlation trend with serum uric acid(R=-0.324,P=0.086),and a positive correlation trend with serum glycosylated hemoglobin(R=0.323,P=0.088)at 2d.Moreover CD16 MFI was slightly higher in patients with previous hypertension at 8d after admission than patients without hypertension(P=0.084). (3)The CD66 b MFI of circulating neutrophils in AIS patients on 1d,2d and8 d was significantly higher than that in the HCs(P<0.05),and there was no significant difference between different times(P>0.05).Although,there was no significant correlation between CD66 b MFI at each time point and clinical parameters regarded in the study(P>0.05).CD66 b MFI and serum uric acid levels showed a negative correlation trend(R=-0.331,P=0.097)at 2d.(1)The Annexin V MFI of circulating neutrophils in AIS patients on 1d,2d and 8d was significantly higher than that in the HCs(P<0.05).Among them,Annexin V MFI was higher at 1d and 2d after admission,and Annexin V MFI at 8d was significantly lower than the above two groups(P<0.05).Furthermore Annexin V MFI showed a negative correlation with serum uric acid at 2d(R=-0.443,P=0.016).Although there was no significant correlation with other clinical parameters(P>0.05),it showed a positive correlation trend between glycosylated hemoglobin at 2d(R=0.325,P=0.086).(2)The plasmic level of Annexin A1 in AIS patients was significantly higher than that of the HCs at 1d and 2d after admission(P<0.05),among them the level at1 d was slightly higher than at 2d(no statistical difference,P>0.05).Then,it fell back to the baseline level at 8d after admission(P>0.05).The level of Annexin A1 on the 1d after admission showed a positive correlation with the Annexin V MFI on the 2d(R=0.47,P=0.05).In addition,there was no significant correlation between Annexin A1 levels at each time point and other clinical parameters(P>0.05).At 8days after admission,Annexin A1 showed a negative correlation with CD16 and Annexin V MFI at 1d(no statistical significant,RCD16 =-0.42,PCD16 = 0.10,RAnnexin V=-0.45,PAnnexin V = 0.08).Conclutions:(1)In the early stage of AIS,the quantity of circulating neutrophils were increased slightly with their activation increased rapidly,then both quantity and activation gradually decreased.The adhesion and apoptosis of circulating neutrophils also increased significantly in the early stage of AIS and then gradually decreased,but the decline rate was slightly slower.(2)The level of plasma Annexin A1 in the early stage of AIS could promote the initiation of neutrophil apoptosis which prompt the pro-inflammatory response move to pro-resolving response.(3)Hypertention may prolong the activation of circulating neutrophils,leading to the failure of the inflammatory resolution.(4)The higher levels of serum uric acid at the onset of AIS lead the lower extend of apoptosis in circulating neutrophils.