光刺激对新生小鼠视网膜发育的影响及基质糖蛋白Lumican异位表达的意义
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摘要
小鼠出生时睑裂是闭合的,这为其相对幼稚的视网膜出生后的继续发育成熟提供了生理性的黑暗环境。我们通过人工开启单侧眼睑的方法,利用C57BL/6J(黑色近交系)小鼠成功建立了过早接受形觉刺激的动物模型,为研究过早光照对视网膜发育的影响提供了较理想的动物模型。前期工作表明,过早光刺激可引发小鼠可逆性的非轴性近视,并伴随视网膜组织中Lumican基因转录活性的显著性激活。本研究在CD1(白色远交系)小鼠中重建并优化了这一模型,详细量化过早光照引发的视网膜结构的发育异常,描述了Lumican异位表达的时间空间分布情况以及相应区域细胞生长与EMT状态的变化。实验从细胞与分子水平为探讨提前光照影响视网膜发育的病理机制、解析过早光刺激与MOP发生的关联提供了佐证。
课题主要的研究方法和内容如下:
(1)优化过早光刺激小鼠模型,选择CD1新生小鼠替代原有的C57BL/6J作为实验动物,一方面提高了新生小鼠出身率与健康存活比率,更为重要的是,为基于CD1遗传背景的Lumican基因敲除小鼠的引进和深入的机理研究提供可能,实验要求严格杜绝对照眼球的意外感光。我们选择P4作为开睑时间,钝性开启右侧眼睑(记为实验眼,R),以左侧眼球为自身对照(L)。采集P6、P9、P12时间点的眼球制作冰冻切片或于对应时间点抽提视网膜组织总蛋白、总RNA,观察、量化不同时间点新生小鼠视网膜各个核层的细胞增殖与凋亡水平,分析提前光照对视网膜发育影响的时间空间特性。
(2)观察上述三个时间点视网膜中Lumican在mRNA与蛋白水平上的异位表达和分布情况;比较在该糖蛋白异常积蓄的时期,相关视网膜细胞增殖、凋亡和EMT的变化,为全面而深入分析该基因的蛋白产物影响神经发育的潜在机制提供第一手实验数据。
主要结果与结论如下:
(1)过早光刺激对视网膜细胞生长状态的影响
H-E染色显示,光刺激对内、外网层及视杆视锥的发育进程并不产生显著性影响,却能明显的改变视网膜细胞的数量:P6-P9H寸期可促进内、外核层与节细胞数量的维持,P10-P12则转而加剧细胞数量的减少。据此,我们将光刺激影响视网膜发育的时相分作早期的生长促进期(P6-P9)与晚期的生长抑制期(P10-P12)。
(2)过早光刺激对视网膜细胞增殖水平的抑制
以PCNA为细胞增殖指标,量化视网膜细胞的增殖状态。感光细胞增殖活力受过早光刺激的抑制最为敏感,在P9时,ONL中零散分布于边缘区域的PCNA核内荧光信号完全消失,但该异常刺激却并不能改变生长促进期(P6-P9)视网膜细胞的总体增殖状态。进入生长抑制期(P10-P12)之后,光刺激对细胞增殖水平的抑制效应进一步扩展到INL中间神经元,节细胞在P12时生理性的PCNA再度上扬现象在实验眼视网膜中被彻底抑制,视网膜细胞总体增殖水平呈显著性的低下,仅极少数细胞核内呈现出微弱的PCNA阳性信号。
(3)过早光刺激对视网膜细胞凋亡水平的影响
TUNEL染色分析视网膜细胞的凋亡状态。生理状态下,内外核层细胞凋亡峰值形成时间不同:ONL细胞凋亡高峰出现在P12(自然睁眼前),而INL则发生在P6前后。光照可全程抑制感光细胞的凋亡;对视网膜INL细胞凋亡的影响相对复杂:于生长促进期(P6-P9)抑制中间神经元凋亡,于生长抑制期(P10-P12)则明显延滞INL细胞凋亡水平的迅速下滑。
综上所述,光刺激在生长促进期(P6-P9),主要通过抑制神经视网膜(INL+ONL)细胞早期(P6时)凋亡峰的形成、导致大量的细胞存活,从而引起这一时期细胞数量的增多。P12时,尽管光照对感光细胞与中间神经元的凋亡施加了截然相反的影响,但在此时刻,实验眼INL细胞的增殖活力却极度减弱,加之节细胞的PCNA峰未能出现,故生长抑制期(P10-P12)期间,光刺激主要通过对细胞增殖的全面抑制效应来实现细胞数量的回落。
(4)过早光刺激诱导视网膜中Lumiean的表达
Lumican主要由间充质来源的细胞(如成纤维细胞、角膜细胞)合成,在视网膜、脑等外胚层起源的神经器官组织中表达低下。光照引发视网膜中Lumican转录水平的激活始发自P9,于P12达高峰,睁眼后迅速回落,至P21重新恢复沉寂。P9时,极其微量的Lumican蛋白产物可在视网膜组织总蛋白提取液中检出,以后其含量急剧增多,P12时可见65kDa的宽幅蛋白条带——视网膜中表达的Lumican是糖蛋白而非KSPG。P12时,Lumican糖蛋白主要分布在外网层以内的神经组织中,这包括:内外网层、INL与GCL,主要影响中间神经元与节细胞的生长、转化行为。
(5) Lumican异位表达与相关细胞增殖水平的抑制
以PCNA为细胞增殖指标,对P9和P12时的INL与GCL的细胞增殖状态进行了进一步的量化。随着Lumican在神经视网膜INL与GCL中的蓄积,相应区域新生细胞数量锐减明显,这一方面体现在P12时(Lumican异常蓄积高峰时刻)INL细胞PCNA核内信号显著性减少,一方面表现为GCL中PCNA上扬现象的完全消失。
(6) Lumican异位表达与相关细胞凋亡水平的维持
为了准确评价视网膜INL与GCL细胞的凋亡状态,我们选择了Caspase-939kDa活性肽段——p39作为细胞凋亡量化指标。免疫荧光检测证实,Caspase-9标记的细胞分布在视网膜(INL+GCL)核层中,而ONL中未见荧光信号;Western-blot确证该分子在这两个核层中的存在形式主要是p39活性肽段,未见全长(49kDa)条带出现,说明此抗体可以特异性标记INL与GCL中的凋亡细胞。随着Lumican在视网膜中的聚集,P12时相对于对照组细胞凋亡水平的进行性锐减,实验眼视网膜Caspase-9 p39依旧维持在相对较高的水平,说明光刺激可于生长抑制期(P10-P12)引发这两个核层细胞较为明显的凋亡。
综合上述结果我们发现,随着Lumican糖蛋白在神经视网膜内侧区域的积蓄,相应分布区域的视网膜细胞表现出增殖活性的锐减与凋亡水平的相对维持,其综合效应将导致(INL+GCL)细胞生长活性的减低、细胞数量的减少。这一发现揭示了,Lumican可能参与了光刺激对生长抑制期间(P10-P12)相关核层细胞生长的调节。参考Lumican对间充质来源细胞(如角膜细胞)具有抑增殖促凋亡的调控作用,我们据此推测,该分子对来源于神经上皮的视网膜细胞也有相似的作用。正是由于光刺激引发Lumican转录水平的激活以及其糖蛋白产物在P9-P12的迅速蓄积,导致相同的刺激对不同时期的视网膜(尤其是INL与GCL)发育产生截然相反的影响——由前期的促进生长转入后期持续的抑制。
(7) Lumican异位表达与EMT
值得我们注意的是,光照并非立即诱发Lumican在视网膜中的异常表达。而上皮细胞尽管在特殊应激诱发(如损伤)下,可以重新开启Lumican的合成潜力,但通常需经历EMT,且Lumican异位表达后还可以进一步维持这种异常转化状态。以α-SMA作为EMT量化指标、分析P6、P9、P12时间点视网膜EMT状态,发现光刺激可引发P6时α-SMA在视网膜中的广泛提升以及P9时(INL+GCL)中α-SMA的富集效应,提示Lumican的表达继发于光刺激诱发的P6-P9时期EMT的上扬。P12时,视网膜EMT还出现了一次较高水平的维持,这一反弹现象伴随着Lumican表达高峰的出现,提示二者之间潜在的关联性。
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 MOE
The 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 growth
H-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 proliferation
Retinal 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 apoptosis
TUNEL 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 retinae
Lumican,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 inhibition
Using 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 EMT
It 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.
课题主要的研究方法和内容如下:
(1)优化过早光刺激小鼠模型,选择CD1新生小鼠替代原有的C57BL/6J作为实验动物,一方面提高了新生小鼠出身率与健康存活比率,更为重要的是,为基于CD1遗传背景的Lumican基因敲除小鼠的引进和深入的机理研究提供可能,实验要求严格杜绝对照眼球的意外感光。我们选择P4作为开睑时间,钝性开启右侧眼睑(记为实验眼,R),以左侧眼球为自身对照(L)。采集P6、P9、P12时间点的眼球制作冰冻切片或于对应时间点抽提视网膜组织总蛋白、总RNA,观察、量化不同时间点新生小鼠视网膜各个核层的细胞增殖与凋亡水平,分析提前光照对视网膜发育影响的时间空间特性。
(2)观察上述三个时间点视网膜中Lumican在mRNA与蛋白水平上的异位表达和分布情况;比较在该糖蛋白异常积蓄的时期,相关视网膜细胞增殖、凋亡和EMT的变化,为全面而深入分析该基因的蛋白产物影响神经发育的潜在机制提供第一手实验数据。
主要结果与结论如下:
(1)过早光刺激对视网膜细胞生长状态的影响
H-E染色显示,光刺激对内、外网层及视杆视锥的发育进程并不产生显著性影响,却能明显的改变视网膜细胞的数量:P6-P9H寸期可促进内、外核层与节细胞数量的维持,P10-P12则转而加剧细胞数量的减少。据此,我们将光刺激影响视网膜发育的时相分作早期的生长促进期(P6-P9)与晚期的生长抑制期(P10-P12)。
(2)过早光刺激对视网膜细胞增殖水平的抑制
以PCNA为细胞增殖指标,量化视网膜细胞的增殖状态。感光细胞增殖活力受过早光刺激的抑制最为敏感,在P9时,ONL中零散分布于边缘区域的PCNA核内荧光信号完全消失,但该异常刺激却并不能改变生长促进期(P6-P9)视网膜细胞的总体增殖状态。进入生长抑制期(P10-P12)之后,光刺激对细胞增殖水平的抑制效应进一步扩展到INL中间神经元,节细胞在P12时生理性的PCNA再度上扬现象在实验眼视网膜中被彻底抑制,视网膜细胞总体增殖水平呈显著性的低下,仅极少数细胞核内呈现出微弱的PCNA阳性信号。
(3)过早光刺激对视网膜细胞凋亡水平的影响
TUNEL染色分析视网膜细胞的凋亡状态。生理状态下,内外核层细胞凋亡峰值形成时间不同:ONL细胞凋亡高峰出现在P12(自然睁眼前),而INL则发生在P6前后。光照可全程抑制感光细胞的凋亡;对视网膜INL细胞凋亡的影响相对复杂:于生长促进期(P6-P9)抑制中间神经元凋亡,于生长抑制期(P10-P12)则明显延滞INL细胞凋亡水平的迅速下滑。
综上所述,光刺激在生长促进期(P6-P9),主要通过抑制神经视网膜(INL+ONL)细胞早期(P6时)凋亡峰的形成、导致大量的细胞存活,从而引起这一时期细胞数量的增多。P12时,尽管光照对感光细胞与中间神经元的凋亡施加了截然相反的影响,但在此时刻,实验眼INL细胞的增殖活力却极度减弱,加之节细胞的PCNA峰未能出现,故生长抑制期(P10-P12)期间,光刺激主要通过对细胞增殖的全面抑制效应来实现细胞数量的回落。
(4)过早光刺激诱导视网膜中Lumiean的表达
Lumican主要由间充质来源的细胞(如成纤维细胞、角膜细胞)合成,在视网膜、脑等外胚层起源的神经器官组织中表达低下。光照引发视网膜中Lumican转录水平的激活始发自P9,于P12达高峰,睁眼后迅速回落,至P21重新恢复沉寂。P9时,极其微量的Lumican蛋白产物可在视网膜组织总蛋白提取液中检出,以后其含量急剧增多,P12时可见65kDa的宽幅蛋白条带——视网膜中表达的Lumican是糖蛋白而非KSPG。P12时,Lumican糖蛋白主要分布在外网层以内的神经组织中,这包括:内外网层、INL与GCL,主要影响中间神经元与节细胞的生长、转化行为。
(5) Lumican异位表达与相关细胞增殖水平的抑制
以PCNA为细胞增殖指标,对P9和P12时的INL与GCL的细胞增殖状态进行了进一步的量化。随着Lumican在神经视网膜INL与GCL中的蓄积,相应区域新生细胞数量锐减明显,这一方面体现在P12时(Lumican异常蓄积高峰时刻)INL细胞PCNA核内信号显著性减少,一方面表现为GCL中PCNA上扬现象的完全消失。
(6) Lumican异位表达与相关细胞凋亡水平的维持
为了准确评价视网膜INL与GCL细胞的凋亡状态,我们选择了Caspase-939kDa活性肽段——p39作为细胞凋亡量化指标。免疫荧光检测证实,Caspase-9标记的细胞分布在视网膜(INL+GCL)核层中,而ONL中未见荧光信号;Western-blot确证该分子在这两个核层中的存在形式主要是p39活性肽段,未见全长(49kDa)条带出现,说明此抗体可以特异性标记INL与GCL中的凋亡细胞。随着Lumican在视网膜中的聚集,P12时相对于对照组细胞凋亡水平的进行性锐减,实验眼视网膜Caspase-9 p39依旧维持在相对较高的水平,说明光刺激可于生长抑制期(P10-P12)引发这两个核层细胞较为明显的凋亡。
综合上述结果我们发现,随着Lumican糖蛋白在神经视网膜内侧区域的积蓄,相应分布区域的视网膜细胞表现出增殖活性的锐减与凋亡水平的相对维持,其综合效应将导致(INL+GCL)细胞生长活性的减低、细胞数量的减少。这一发现揭示了,Lumican可能参与了光刺激对生长抑制期间(P10-P12)相关核层细胞生长的调节。参考Lumican对间充质来源细胞(如角膜细胞)具有抑增殖促凋亡的调控作用,我们据此推测,该分子对来源于神经上皮的视网膜细胞也有相似的作用。正是由于光刺激引发Lumican转录水平的激活以及其糖蛋白产物在P9-P12的迅速蓄积,导致相同的刺激对不同时期的视网膜(尤其是INL与GCL)发育产生截然相反的影响——由前期的促进生长转入后期持续的抑制。
(7) Lumican异位表达与EMT
值得我们注意的是,光照并非立即诱发Lumican在视网膜中的异常表达。而上皮细胞尽管在特殊应激诱发(如损伤)下,可以重新开启Lumican的合成潜力,但通常需经历EMT,且Lumican异位表达后还可以进一步维持这种异常转化状态。以α-SMA作为EMT量化指标、分析P6、P9、P12时间点视网膜EMT状态,发现光刺激可引发P6时α-SMA在视网膜中的广泛提升以及P9时(INL+GCL)中α-SMA的富集效应,提示Lumican的表达继发于光刺激诱发的P6-P9时期EMT的上扬。P12时,视网膜EMT还出现了一次较高水平的维持,这一反弹现象伴随着Lumican表达高峰的出现,提示二者之间潜在的关联性。
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 MOE
The 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 growth
H-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 proliferation
Retinal 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 apoptosis
TUNEL 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 retinae
Lumican,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 inhibition
Using 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 EMT
It 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.
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