The Effect And Pathogenesis Of Sleep Deficiency On Murine Corneal Epithelial Stem Cells And Retina

Part I:The effects and mechanism of short-term sleep deficiency on corneal epithelial stem cellsPURPOSE:To investigate the effects of short-term sleep deficiency(SD)on corneal epithelial stem cells in mice.METHODS:SD was induced in eight-week-old male C57BL/6J mice by using sticks over water.Mice in short-term SD model were deprived of sleep for 5 days(SD 5D)and 10 days(SD 10D).Normal control(NC)consisted of the same age and sex reared in normal environment.The mouse,TKE2 cell and human limbal stem cells(hLSCs)oxidative stress model were established by topical application of hydrogen peroxide(H2O2).The expressions of Ki67,Cdk1,P63,Krt14,3-NT,glutathione peroxidase(Gpx)2,Gpx3 and AKT in cornea and/or lacrimal glands were detected by immunofluorescence,quantitative real-time polymerase chain reaction(qRT-PCR)and/or Western Blot.EdU and TUNEL staining were used to detect the changes of corneal proliferation and apoptosis.Total antioxidant capacity,reactive oxygen species(ROS)and glutathione(GSH)were detected in murine tears and corneal epithelial cells.Gpx content was detected in murine lacrimal glands.Murine corneal epithelium was detected by fluorescein sodium staining.The PI3K inhibitor LY294002 was used to treat the oxidative stress model of TKE2 cells and hLSCs to determine the effect of PI3K/AKT signaling pathway on the proliferation of ROS-mediated corneal epithelial stem cells.RESULTS:Compared with NC group,short-term SD murine corneal epithelium have the increased expression levels of Ki67,Cdk1,P63,Krt14,Nrf2,p-AKT,EdU staining positive cells,and the decreased expression levels of 3-NT and Nox4.In addition,tears of short-term SD mice had higher ROS levels and lower antioxidant capacity and Gpx,but no significant changes in GSH levels,while corneal epithelial cells had no significant changes in ROS levels,but decreased antioxidant capacity and GSH levels.The gene expression of Gpx2 and Gpx3 were decreased in short-term SD murine lacrimal gland.Murine corneal epithelium treated with 250μm and 500μm H2O2 have increased levels of Ki67 expression and EdU staining positive cells and decreased levels of 3-NT expression.TUNEL staining and corneal sodium fluorescein staining were negative in 250μm and 500μm H2O2 treated mice.LY294002 down-regulated the proliferation of H2O2-induced TKE2 cell and hLSCs,while inhibited the phosphorylation of AKT.CONCLUSION:Upregulated ROS levels accompanied with reduced antioxidant capacity in the tear film could directly affect the corneal epithelial stem cell proliferation by activating the PI3K/AKT signaling pathway in short-term SD mice.Part II:The effects of long-term sleep deficiency on corneal epithelial stem cells PURPOSE:To investigate the effects of long-term sleep deficiency(SD)on corneal epithelial stem cells in mice.METHODS:SD was induced in eight-week-old male C57BL/6J mice by using sticks over water.Mice in long-term model were deprived of sleep for 1 months(SD 1M)and 2 months(SD 2M).Normal control(NC)consisted of the same age and sex reared in normal environment.Immunofluorescence staining,quantitative real-time polymerase chain reaction(qRT-PCR)and Western Blot were used to detect the expression of P63 and Krt14 in murine cornea.QRT-PCR was used to detect the expression of Muc5ac in murine corneal epithelial cells.The fluorescein sodium staining and H&E staining were used to detect the defect and structure of murine corneal epithelium,respectively.Clonal culture of mouse corneal epithelial primary cells and crystal violet staining were used to detect the colony formation rate of murine corneal epithelial cells.The expressions of Pax6,Krt12 and P63 in corneal epithelial primary cells were detected by immunofluorescence staining and immunohistochemical staining.RESULTS:Compared with the NC group,the surface of corneal epithelium was rough,the cell arrangement was irregular,and the corneal epithelium thickness was thinned in the SD 1M and SD 2M groups.And the gene expression level of Muc5ac was higher in cornea of the SD 1M and SD 2M groups than that in the NC group.The results of Wersten Blot and qRT-PCR showed that the expression levels of P63 and Krt14 were lower in cornea of SD 1M and SD 2M groups than those in NC group.The corneal epithelial cells of primary sleep-deprived mice expressed Pax6,Krt12 and P63.The crystal violet staining showed that the colony formation rate of corneal epithelial cells was decreased in SD 1M and SD 2M groups.CONCLUSION:Long-term SD could lead to the limbal stem cells deficiency in mice.Part III:The effects and mechanism of sleep deficiency on murine retinaPURPOSE:To investigate the pathological changes and related mechanisms of sleep deficiency(SD)on murine retina.METHODS:SD was induced in eight-week-old male C57BL/6J mice by using sticks over water.Mice in long-term model were deprived of sleep for 2 months(SD 2M)and 4 months(SD 4M).Normal control(NC)consisted of the same age and sex reared in normal environment.The murine retinal changes were detected by fundus color photography,fluorescein fundus angiography(FFA),spectral-domain optical coherence tomography(SD-OCT)and electroretinogram(ERG).The morphological changes of retina were detected by H&E staining,and the ultrastructure of retina was detected by transmission electron microscopy(TEM).Quantitative real-time polymerase chain reaction(qRT-PCR)and/or immunofluorescence staining and Western Blot were used to detect levels of VE-cadherin,Claudin5,Occludin,Mfn1,Opa1 and Tim23.The level of ROS was detected by DHE staining.Immunohistochemical staining(IHC)and/or qRT-PCR were used to detect the expression changes of oxidative stress related indexes 3-NT,Nox4,Nrf2,Nqo1,SOD2 and Catalase.The changes of the above indexes were observed after a half month of sleep recovery.RESULTS:Compared with NC group,the results of H&E staining,FFA and SD-OCT showed no significant changes in the retinal structure,but there were scattered abnormal bright spots in the murine retina of SD 4M group.The amplitudes of a and b waves of dark adaptive and bright adaptive ERG were significantly reduced.Western Blot and qRT-PCR showed that the expressions of VE-cadherin,Claudin5,Occludin,Mfnl,Opal and Tim23 were significantly decreased in murine retina of SD 4M group.TEM showed that the morphology of retina mitochondria was abnormal and the bilayer structure disappeared in SD 4M group.DHE staining showed that the level of ROS was significantly increased in retina of SD 4M group.IHC and/or qRT-PCR results showed that the expressions of 3-NT,Nox4,Nrf2,Nqo1,SOD2 and Catalase were up-regulated in murine retina of SD 4M group.After 0.5 months of sleep recovery,the above indexes of retina in SD mice were improved.CONCLUSION:Long-term SD causes retinal oxidative stress and mitochondrial dysfunction resulting in blood-retinal barrier dysfunction and eventually retinal dysfunction in mice.