Single Cell Transcriptome Profiling Of Corneal Wound Healing After Infrared Laser Irradiation

Objective:Single-cell RNA sequencing（sc RNA-seq）was used to identity cells involved in corneal wound healing process in mice after injury induced by infrared laser at the wavelength of 3.74μm,explored the repair mechanism of corneal injuryed by laser.Methods:The cornea of C57BL/6J mice was damaged by infrared laser at the wavelength of 3.74μm for exposure duration of 0.8s and the beam diameter of 2mm with corneal radiant exposures of 23.2J/cm2.Histological evaluation of the cornea were preformed pre-exposure and 3h,6h,12h,1d,3d,7d,14d,21d after injury.The sc RNA-seq was conducted on corneal tissue before and 3d,21d after laser injury,using a 10X Genomics pipeline.t-distributed stochastic neighbor embedding（t-SNE）was used for visualizing sc RNA-seq data.Cell types were identified according to marker genes expression and compared between normal and injured corneal tissue.Immunohistochemistry of the cornea were performed to detect the expression of cell-specific proteins involving in corneal wound healing,and compared with sequencing results.Results:By gross observation,it was showed that the corneal lesion was grey-white and could be seen immediately after laser exposure.Histological section showed that at 3h,the epithelial nuclei were hyperchromatic with pyknosis,most chromatin in the stroma cells lost and the endothelial layer disappeared completely.At 6h,the number of epithelial chromatin staining faded and the chromatin of stromal nuclei disappeared.At 12h,the chromatin of epithelial disappeared,infiltrating cells appeared in the stroma layer,and the anterior and posterior surfaces of cornea wrinkled.At 1d,the necrotic epithelium exfoliated,and 1-2 layers of newborn epithelial cells covered the injury area completely.Infiltrating cells increased in the stroma and a few endothelial cells appeared in the endothelium.At 3d-7d,3-4 layers of epithelial cells could be observed,the infiltrating cells in the stroma migrated from the deep layer to the shallow layer,the endothelial cells increased and flattened,and the corneal anterior and posterior surfaces became smooth.From 14d to 21d,the epithelium and endothelium returned to normal and the infiltrating cells in the stroma decreased,but the stromal fibers were still irregularly arranged.scRNA-seq of normal corneal revealed 28 cell clusters.Based on marker genes,five cell types were identified.The major cell types were epithelial cells,stromal cells,endothelial cells,immune cells and Schwann cells.Three corneal epithelial cell subpopulations were identified and designated as epithelial stem cells,proliferating epithelial cells and differentiated epithelial cells.Three stromal cell subpopulations were identified to be stromal stem cells,myofibroblasts and normal stromal cells.The identified mmune cells included neutrophils,CD68 macrophages,dendritic cells,and a few CD4 T cells.The results of sc RNA-seq of the cornea injured by infrared laser radiation showed that at 3d,new differentiated epithelial cells could be found,and immune cells increased significantly,mainly in neutrophils,and small amounts of CD163 macrophages were identified.The proportion of epithelial cells decreased,mainly in differentiated epithelial cells,while epithelial stem cells and proliferating epithelial cells kept almost unchanged.The proportion of myofibroblasts and stromal stem cells increased significantly,while the normal stromal cells decreased obviously.Endothelial cells and Schwann cells decreased.At 21d,the proportion of newly differentiated epithelial cells remained unchanged,while immune cells decreased significantly,mainly in neutrophils.CD163 macrophages could not be identified.The epithelial cells increased,mainly in differentiated epithelial cells,while epithelial stem cells and proliferating epithelial cells kept unchanged.The proportion of normal stromal cells and stromal stem cells increased,and myofibroblasts decreased.Endothelial cells and Schwann cells increased.Immunohistochemical staining showed that the neutrophils infiltrated the injured area at 12h,the number of neutrophils peaked at 1d,then decreased gradually,and only a few positive staining cells were found at 21d.The myofibroblasts was detected at 1d,then decreased gradually,and no positive cells could be seen at 21d.The CD68 and CD163 macrophages infiltrated the injured area at 7d,then decreased,only a few positive staining cells were found at 21d.Neovascularization was found at 14d.Conclusion:The cornea lesion induced by 3.74μm laser at the irradiation dose of23.2J/cm~2in C57BL/6J mice model involves the whole cornea layer.The wound healing process includes the inflammatory phase and the late recovery phase.The cells involved in corneal wound healing after laser injury include epithelial stem cells,proliferating epithelial cells,differentiated epithelial cells,normal stromal cells,myofibroblasts,stromal stem cells,endothelial cells,immune cells and Schwann cells.At 3d after laser injury,new subpopulations of differentiated epithelial cells can be found,immune cells increase and differentiated epithelium decrease significantly.At 21d,new differentiated epithelial cells keep unchanged,immune cells decrease and differentiated epithelial cells increase.Immunohistochemistry shows that neutrophils,CD68 macrophages and myofibroblasts involve in corneal wound healing at 3d after laser injury,which agrees well with sc RNA-seq data,while a small number of CD163 macrophages can be identified by sc RNA-seq.At 21d,immunohistochemistry shows that neutrophils,CD68and CD163 macrophages and myofibroblasts involve in corneal wound healing process.However,CD163 macrophages can’t be identified by sc RNA-seq.In conclusion,the study was the first to perform single-cell transcriptome sequencing to identify cell of mouse corneal tissue.Meanwhile,we conducted Single-cell transcriptome sequencing of corneal damaged induced by infrared laser,which reveals cells involved in corneal wound healing,which provides an experimental basis for the laser corneal treatment.