Neuroendocrine Cells Exist In The Corneal Stroma

Background:The cornea is a transparent film located on the anterior wall of the eyeball.It is divided into three layers including the epithelial cell layer,stromal layer and endothelial cell layer from the outside to the inside.The cornea stromal layer is located in the middle,accounting for about 90%of the full thickness of the cornea.The structure of the corneal stroma is closely related to the maintenance of corneal transparency according to the traditional view.The stroma consists of a collagen-rich extracellular matrix and stromal cells scattered in it.Among them,according to the previous literature,the corneal nerves originate from the trigeminal ophthalmic branch,and their distribution in the corneal stroma is from the outside to the inside of the dendritic arrangement,and it emits countless tiny branches(neuro terminals)to the corneal epithelium.In addition,in patients with diabetes,corneal complications are less common during disease development.After corneal alkali burn,there is inflammatory cell infiltration and neovascularization.Neuroendocrine cells(NECs)is a kind of cells who produce neurotransmitters,neuromodulators,or neuropeptide hormones and are believed to be closely related to the development of tumors,which was discovered in the gastrointestinal tract and pancreas previously.Objective:After using GFP rat corneas which were removed the corneal epithelium,visual observation of the morphological structure of the corneal stromal cells,followed by immunofluorescence to explore its type and source,and establishing an Alloxan anterior chamber injection animal model to study its function,and finally using Tomato/GFP-Camk?-cre Knockout(CaMK2?-cremTmG(F/-))mouse model to investigate the distribution of such cells in corneal stroma and corneal epithelium under normal conditions.Methods:Methods:The experiment was divided into four parts.The first part was to study the morphology and structure of the cells in corneal stroma.By tearing off the corneal epithelium of GFP rats after enzymolysis,we could observe the corneal stroma by fluorescence microscopy directly.The second part was exploring the type of this kind of cells in corneal stroma.Using serial immunofluorescent staining,we had made corneal whole mount and trigeminal frozen sections of GFP rats,whose forms of antibodies included neuro-related markers and endocrine-related markers.The third section was to explore the origin of such cells in corneal stroma,and the method was comparing the results of immunofluorescence staining of corneal whole mount and trigeminal frozen sections of GFP rats.The CaMK2a-cre mTmG(F/-)mouse model was used to observe the distribution of this type of cells in the corneal stromal layer and corneal epithelial layer and the connection between them in the same section.In the fourth part,the repair after damage and functional characteristics of the cells were explored by injecting Alloxan to the GFP rats' anterior chamber through a tunnel.The experiment had 2 groups:Normal group and Alloxan group.There were 6 rats in each group.The Alloxan group contained P1,P7 and P60 on the basis of the completion time of anterior chamber injection;It had two conditions including normal one and after alkaline burn.Results:In the first part,after observing cells in corneal stroma of GFP rats,we found a group of cells that had different morphology from the stromal cell but resembled the nerve structure.However,its unique nucleus structure proved it was not the nerve terminal described in previous literature but a new type of cell.They had the aggregation among the cell bodies,and had processes connected each other to form a mesh in the entire corneal stroma.In the second part,the epithelial was removed from the GFP rat cornea,the rest of the cornea was used as a whole mount with immunofluorescence staining.It was found that it expressed neural markers including type ? beta-tubulin(TUBB3)and neurofilament light chain protein(NEFL),synaptophysin(SYP),ubiquitin carboxyl terminal hydrolase L1(UCHL1),glutamate transporter 1(Vglutl)and calcium/calmodulin dependent protein kinase 2(CaMK2),and endocrine hormone markers including insulin 2(Insulin2),neuropeptide Y(NPY),somatostatin(SST),etc.These discoveries have given a new name to such cells-neuroendocrine cells.In the third part:Immunofluorescence staining of frozen sections of trigeminal nerve of GFP rats,and comparison of corneal slices,the results showed that trigeminal neurons do not express GFP protein,but their nerve-related markers and endocrine-related markers were positive.The whole mount of CaMK2?-cre/mTmG(F/-)mice corneas showed that the protrusions possibly coming out from trigeminal will give out finer branches to the corneal epithelium,and they may have connection with the neuroendocrine cells in corneal stroma.In the fourth part:Comparing with the normal group,this cell disappeared after anterior chamber injection with Alloxan.And as the time lasped,the previous cell structure did not recover,accompanied by corneal neovascularization growing.Conclusion:A kind of neuroendocrine cell existing in the stromal layer of the cornea'which is not originated from trigeminal and secretes the specific protein expressed by the nerve structure and endocrine-related proteins such as insulin.This is different from the nerve terminal described in previous viewpoints.Such cells whose nucleis have direct contact with each other send out protrusions connection to each other or entangled with each other to form a network structure may have connection with free terminals in corneal epithelium.This type of cell does not change its structure during the pathogenesis of diabetes,but it cannot regenerate after specific damage caused by Alloxan,which promotes permanent corneal neovascularization.This neuroendocrine cell may be involved in comeal metabolism and neovascularization.