Study On The Effect Of The Formation And NETs On The Progression And Prognosis In Pseudomonas Aeruginosa Keratitis

Background:Bacterial keratitis is one of the most common blinding ketatopathy.If not treated in time,it often leads to corneal scar;corneal perforation and even enucleation of eyeball.Pseudomonas aeruginosa is the main pathogen of bacterial keratitis.Because of its high bacterial virulence,Pseudomonas aeruginosa keratitis is characterized by acute onset,rapid progress and severe injury.Therefore,early intervention and effective treatment are very important for the prognosis of Pseudomonas aeruginosa keratitis.In order to improve the prognosis of Pseudomonas aeruginosa keratitis,the basic research on anti-inflammatory and bactericidal effect is particularly important.Neutrophils are key inflammatory cells in infectious keratitis.It can engulf and destroy bacteria with reactive oxygen species(ROS)and pro-inflammatory cytokines.The chemokines released by neutrophils induce many inflammatory cells to enter the injury which amplifies the inflammatory reactions.Neutrophil extracellular traps(NETs)is a newly discovered bactericidal mechanism of neutrophils.Neutrophils are activated by the pathogen to mix and extrude their DNA,NE and MPO creating NET-like structures called NETs,and then NETs capture and destroy the pathogen.This process is called NETosis.NETs have been proved to play a role in fungal and Stahylocous aureus infection.However,there are only a few reports about the effect of NETs in Pseudomonas aeruginosa infection.In addition,the persistent presence of NETs can damage the tissue,and the elimination of NETs can reduce tissue damage.In this paper,we mainly study the bactericidal effect of NETs and explore its influence on the progression and prognosis of keratitis by regulating the formation of NETs in Pseudomonas aeruginosa keratitis.Objective:To study the bactericidal effect of neutrophil extracellular traps(NETs),and explore its influence on progression and prognosis of keratitis by regulating the formation of NETs in Pseudomonas aeruginosa keratitis.Materials and methods:C57BL/6 mice of SPF were used in the experiment.All were 6-8 weeks of age.They were randomly divided into and experimental group,and experimental group included tobramycin and dexamethasone(TOB and DXM)treated group,tobramycin(TOB)treated group,saline treated group.The model of bacterial keratitis was established by a modified corneal surface lens method.The injury control group was inoculated with sterilized saline,while experimental groups were inoculated with Pseudomonas aeruginosa(106 CFU).After the cornea was infected,injury control group was treated without any eye drops,and the experimental groups were respectively treated with TOB and DXM eye drops,TOB eye drops,and saline,three times a day.Keratitis was observed until the recovery of keratitis,slit lamp with photography,clinical score and the number of corneal perforation were recorded on the 1st,3rd,5th,7th,10th day after infection.During the course of different period,immunofluorescence staining,hematoxylin-eosin(HE)staining,scanning electron microscope(SEM)and pathogen burden assay were used to assess inflammatory reaction,the formation of NETs and antibacterial efficiency.All data were processed by SPSS 24.0 and converted into graphics by GraphPad Prism 6.Results:1.During the natural course of Pseudomonas aeruginosa keratitis in mice model,numerous neutrophils swam to the cornea rapidly,and NETs were observed on 1d after infection.Inflammatory reaction reached peak,and there were more neutrophils and NETs on 3d.Keratitis relieved along with a reduction of neutrophils and NETs at 7d,and completely recovered at 30d.2.The injury control group showed corneal epithelial defect on the 1st day,and the corneal epithelial healing on the 3rd day.In the experimental groups,they showed corneal opacity,corneal edema,epithelial defect,and inflammatory cell infiltration on the 1st day.There were significantly differences among the three groups after medical treatments.TOB and DXM treated group showed mildest corneal damage,fastest recovery and best prognosis.On the 3rd day,structure of cornea was in mild disorder,the inflammatory cells infiltrated the cornea.SEM revealed that NETs was less and sparse.No significant changes were observed on the 5th day.On the 7th day,the inflammation began to lessen,and NETs was still visible.On the 10th day,it showed corneal epithelial healing,and structure of the cornea was regular,without NETs.On the 20th day,keratitis completely recovered.Saline group showed heaviest coneal damage,lowest recovery and worst prognosis.On the 3rd day,it showed dense corneal opacity,corneal edema and corneal melting.HE staining showed that the structure was in severe disorder,a large number of inflammatory cells infiltrated into the whole corneal layers.Dense NETs were seen by SEM.There was no significant change between the 3 rd and 5th day.On the 7th day,the inflammation began to lessen,and neutrophils and NETs were visible.On the 10th day,NETs were still observed.On the 30th day,keratitis completely recovered.The condition of TOB treated group was in the middle.During the observation period,the clinical scores of and TOB and DXM treated group were lower than the saline group(FD3 4.35,PD3=0.04,FD5=6.81,PD5=0.02,FD7=6.17,PD7=0.02,FD10=17.60,PD10=0.002).The difference was significant between TOB treated group and the saline group on the 3rd and 7th days(FD3=4.95,PD3=0.03,FD7=5.07,PD7=0.04).However,there was no significant difference between TOB and DXM treated group and TOB treated group(p>0.05).3.The results of pathogen burden assay showed there was no significant difference among keratitis groups on the 1st and 3rd day after medical treatment.On the 7th day,bacteria amount of TOB and DXM treated group and TOB group was much less.On the 10th day,no bacteria grew in these two groups,while culture of saline group was positive(F2'D7=39.16,P2'D7=0.003,F3'D7=87.63,P3'D7=0.001,FD10=21.00,PD10=0.002).And there was no significant difference between TOB and DXM treated group and TOB group,which indicated that glucocorticoid did not reduce antibacterial efficiency of antibiotics.4.There were 2 cases of corneal perforation in TOB and DXM treated group,3 cases in TOB group,12 cases in the saline group.The difference was significant between TOB and DXM treated group,TOB treated group and the saline group(F4=8.88,P4=0.004;F5=6.65,P5=0.01).However,there was no significant difference between TOB and DXM treated group and TOB treated group(p>0.05).Conclusions:1.NETs participates in the process of Pseudomonas aeruginosa keratitis.The formation of NETs is related to progression and prognosis of keratitis.2.In Pseudomonas aeruginosa keratitis,glucocorticoid inhibits the formation of NETs,leading to less corneal damage and better prognosis.3.In Pseudomonas aeruginosa keratitis,glucocorticoid does not reduce the antibacterial efficiency of antibiotics.