Establishment Of A New Animal Model Of Dry Eye And Its Pathogenesis Induced By Atomization And Resuspension Of Atmospheric Particulate Matter

Objective:To establish a new dry eye animal model by atomization and resuspension of atmospheric particulate matter(PM)to study the pathogenesis of dry eye caused by environmental pollution.Methods:To simulate the atmospheric pollution environment,a device for atomization and resuspension of atmospheric particulate matter was established.Thirty SD rats were randomly divided into 3 groups:control group,low concentration(300μg/m3)PM treatment group and high concentration(500 μg/m3)PM treatment group.The experimental period was 14 days.Clinical indicators included tear break-up time(TBUT),corneal fluorescein staining,tear secretion test,inflammation index score and corneal irregularity index score.The number of corneal epithelial cells,the number of stromal inflammatory cells,the length of corneal nerve fibers,and the number of corneal endothelial cells were evaluated using intravital confocal microscopy in the three groups.The morphology of tissue cells was observed by HE staining on paraffin sections,the number of goblet cells was observed by PAS staining,and the apoptosis of keratoconjunctival cells was detected by TUNEL staining.Ki67 immunofluorescence staining showed pathological proliferation,and CD45 immunofluorescence staining was used to evaluate inflammatory cell infiltration.The changes of corneal ultrastructure were observed by electron microscope.Then,the expressions of inflammatory factors IL-1β,TNF-α,IFN-γ and the activation of inflammation-related NF-κB signaling pathway were detected by Western Blot.Results:The results of clinical indicators showed that compared with the control group,the PM-treated rats had significantly decreased tear film break-up time,increased corneal fluorescein staining score,decreased tear secretion,increased inflammatory index score,and increased corneal irregularity index score,consistent with the clinical manifestations of dry eye.The results of intravital confocal microscopy showed that compared with the control group,the PM-treated rats increased the volume and number of corneal epithelial cells,increased the number of stromal inflammatory cells infiltrated,and decreased the length of corneal nerve fibers,but the number of corneal endothelial cells did not change significantly.HE staining of histopathological sections further observed that the corneal epithelial cell layer increased in the PM-treated group,the number of goblet cells was decreased by PAS staining in the conjunctiva,the number of apoptotic cells positive for TUNEL staining in the conjunctiva was significantly increased,and the number of proliferating cells positive for Ki67 staining increased,the infiltration of CD45-positive inflammatory cells was also increased in the PM-treated group.Transmission electron microscopy revealed ultrastructural changes in the PM-treated group with reduced number of microvilli and tight junctions in the corneal epithelium.Western Blot results showed that the expressions of inflammatory factors IL-1β,TNF-α and IFN-γ in PM treatment group were all up-regulated,and phosphorylated NF-κB protein was increased,suggesting the activation of inflammation-related NF-κB signaling pathway.All morphological and molecular biological changes correlated with PM concentration.Conclusion:Atomization and resuspension of atmospheric particulate matter can significantly reduce the tear film stability of experimental rats,and induce the establishment of a new dry eye model.The ocular surface toxicity of atmospheric pollution should be paid great attention by clinicians.