Impacts Of Light Stimuli On The Maturation In Neonatal Mice And Effects Of The Ectopic Expression Of Stromal Glycoprotein Lumican

Mouse is born with eyelid closed,which creates a considerable dark environment for the further retinal development after birth.Lighting ahead animal model has been established in C57BL/6J mouse(black strain) to identify its potential impacts on retinal maturation via surgically opening the unilateral eyelid.The light-affected eye developed significantly reversible refractive error in our previous research with ectopically transcriptional activation of lumican gene in retina.In our present study we reconstructed the lighting ahead model in CD1 mouse(white strain) with a slight modification,described the retinal developmental abnormalities induced by this stress,and corroborated the temporal and spatial profile of the ectopic Lumican expression in the light-affected retina with an attention to the neuronal growth and EMT in the Lumican accumulated region.And the results may be contributed to the exploration of the pathological mechanisms involving retinal developmental abnormalities induced by early lighting and the analysis of the potential relation between this stimuli and MOEThe methods in our study are as following:(1) The lighting ahead mouse model has been optimized.CD1 neonatal mouse was selected instead of the C57BL/6J counterpart to further study the mechanisms via the introduction of lumican gene knock-out mice based upon the CD1 heredity in addition to the higher birthrate and better health.And the opposite eyelid was strictly blacked to avert unexpected photoreception in retina.Right eyes(test eyes,R) were imposed the eyelids-separating operation at P4,while the left eyes(L) served as the self-control.Total RNA and protein were harvested at P6,P9 and P12,and eye sections were prepared to access the retinal neuronal proliferation and apoptosis at the identified time points,and the influence of lighting ahead on retinal development has been investigated temporally and spatially. (2) We semi-quantified Lumican mRNA and protein level in the light administrated retinae at the individual time points mentioned above and compared the alterations of cell proliferation,apoptosis and EMT in the Lumican deposited retinal region with its self negative control.These results provide first-hand materials to discover the potential functions of this gene product in neuronal development.Results and conclusions:(1) The effect of light stimuli on retinal cell growthH-E staining showed that early lighting did alter the retinal cell growth with no significant effects either on the formation of inner and outer plexiform layer or the development of cones and rods,that is,the stress maintains the cell number in INL, ONL and GCL during P6-P9,while accelerates the loss of neurons within P10-P12. The effects of early lighting on retinal maturation could thus be divided into two phases:earlier cellular growth promoting phase(P6-P9) and later cellular growth inhibiting phase(P10-P12).(2) Early-lighting inhibits retinal cell proliferationRetinal cell proliferation was measured via the cell proliferating marker PCNA.At P9 light stimuli almost diminished the PCNA nuclear immunoactivity dispersed in the marginal region of ONL due to the most susceptibility of the photoreceptor cells, while no significant alterations of general retinal cell proliferation were observed within P6-P9,the cellular growth promoting phase.However,during P10-P12,the cellular growth inhibiting phase,the negative effects of light stimuli on cell growth further extended to the intermediate neurons in INL.Merely faint PCNA signals could be detectable in INL and its physical augment within GCL was inhibited absolutely,resulting in acceleration of cell proliferation decline in neural retinae.(3) Early lighting influences retinal cell apoptosisTUNEL staining was applied to estimate cell apoptosis.Neural cells within INL and ONL possess quite different apoptosis peak:for ONL the apoptosis peaked at P12, the native eyelid separation;while for INL apoptosis reached the summit at P6. Lighting ahead alleviated cell apoptosis of photoreceptors and exhibited more complicated effects on INL cells:for cellular growth promoting phase(P6-P9) this stress inhibited cell apoptosis of intermediate neurons,while for cellular growth inhibiting phase(P10-P12),on the contrary,retarded the rapid apoptotic decline of INL cells.In a word,light stimuli increase retinal cell number during cellular growth promoting phase(P6-P9) mostly due to its inhibitive effects on cell apoptosis both in INL and ONL at P6.Despite of the complicated actions of early lighting on intermediate neurons,it is noticeable that at P12 the cell proliferation activity was sharply dropped in INL and the proliferation peak vanished in GCL in the light affected retina.Therefore,light stimuli aggravated cell loss in cellular growth inhibiting phase(P10-P12) primarily due to its comprehensive inhibition in cell proliferation.(4) Early lighting activates Lumican expression ectopically in retinaeLumican,usually synthesized by cells of mesenchymal origin(such at fibroblasts, keratocytes),possesses the lowest quantification within retina,brain and other nervous organs/tissues coming from embryonic ectoderm.Early lighting reopened the silenced Lumican expression in retinae on P9 partially at the transcriptional level, and the ectopic glycoprotein accumulated evidently at P12,then reversed to silence till P21.A specific 65kDa board band was evident at P12,implies that Lumican deposited in retina is glycoprotein instead of the KSPG form isolated in the corneal stroma,and distributed dominantly in the inner portion inside the outer plexiforrn layer,including INL,GCL,outer and inner plexiform layer as well.Its localization suggests that Lumican aberrantly appearing in the light affected retinae mainly possesses potential functions on the cell behaviors of intermediate neurons and RGCs.(5) Lumican ectopic expression and relative neuronal proliferative inhibitionUsing the PCNA cell proliferating marker,the INL and GCL cell proliferation was valued generally and respectively at both P9 and P12.With the Lumican accumulation,the renewal cells in INL and GCL sharply decreased.When Lumican was mostly accumulated at P12,the nuclear PCNA signals dropped moderately,and PCNA boost observed in normal developing retina vanished completely in GCL. (6) Lumican ectopic expression and relative neuronal apoptosis maintenance Caspase-9 activate peptide,p39(Mr 39kDa),was selected to measure the special cellular apoptosis in INL and GCL.Immunofluorencent assay showed that caspase-9 could specially stain the cells localized in INL and GCL,with no unexpected label of ONL cells.Western-blot analyses demonstrated that the isoform distributed in these two layers is prominently p39 and no 49kDa band(whole length) was observed, proving it is a perfect antibody marking the apoptotic cells specially in INL and GCL. Compared with the progressive drop of cell apoptosis within the INL and GCL, caspase-9 p39 isoform in the light affected retinae still kept at a considerable higher level,suggesting during cellular growth inhibiting phase(P10-P12) lighting ahead induced obviously higher cell apoptosis in these two layers.In conclusion,with the deposition of Lumican glycoprotein in the inner portion of neural retinae,the relative retinal neurons manifested a sharp decline of cell proliferation and a considerable maintenance of cell apoptosis,and the combinative effects is to give rise to a profound falling phenomenon of cell growth and an aggravating loss of cells.This finding implies that Lumican might participate in the influence of early lighting on the cell growth in relative retinae during the cellular growth inhibiting phase(P10-P12).Considering its matrikine effects on mesenchyme-derived cells,we deduced that Lumican also inflicts similar functions on the retinal cells of neural epithelial origin,that is,inhibits cell proliferation and promots cell apoptosis.It is lighting administration induced the activation of Lumican transcription and its glycoprotein swift accumulation during P9-P12 which results in diverse effects of the same stress on retina in different development status (especially for INL and GCL):from earlier growth promoting to later inhibiting.(7) Lumican ectopie accumulation and EMTIt should be noticeable that light induced Lumican expression in immature retinae is not an immediate event.Epithelial cells regaining the capacity of Lumican synthesis in answer to some stimuli(such as injury) usually undergo previous EMT,and Lumican depositing as a result of EMT is reversely contributed to the establishment of the pathological transition.α-SMA was applied as the EMT marker and the EMT status in retina at P6,P9 and P12 was measured,respectively.Light stimuli elicited the generalα-SMA upregulation in P6 retinae and the followedα-SMA abundance in INL and GCL at P9 give us a clue that the Lumican expression is a secondary effect evoked by the EMT activation in P6-P9 in light-affected retinae.At P12,EMT in light-affected retinae restored to a higher lever again,which is accompanied by Lumican accumulation up to its maximum,indicating a potential correlation between the two incidents.