Fidgetin Like 2-siRNA Regulation Of Retinal Ganglion Cell Survival And Axon Regeneration After Optic Nerve Injury

Background and purpose:Vision provides humans with more than 85%of total information about the external world.Traumatic optic neuropathy involves damage to the optic nerve,which is composed of the axons of retinal ganglion cells(RGCs)—a group of neurons that have cell bodies located in the retina to convey visual signals to the brain.Damage to RGCs or their axons is a common cause of irreversible visual impairment in daily life.Like other neurons in the mammalian central nervous system(CNS),the axons of RGCs display an extremely limited capacity to regenerate after injury,which can,in severe cases,result in irreversible blindness.This failure of axonal regeneration in mature RGCs can be broadly attributed to:an intrinsic inability of axons to regrow after injury;injury-induced formation of a glial scar,which contains inhibitory factors such as chondroitin sulfate proteoglycans that actively inhibit axonal growth.Scientific studies have confirmed that microtubule skeleton splicase Fidgetin Like 2(FL2)can fully affect the regeneration of axons of central neurons,and inhibition of the activity of FL2 can significantly promote the regeneration of axons of dorsal spinal cord neurons,showing a negative correlation between them.Retinal ganglion cells and neurons in the spinal cord dorsal body are similar,all belong to the central neurons system,the recent circumstantial evidence shows FL2 is widely expressed in the cytoplasm of retinal ganglion cells,therefore whether fully inhibit the expression of retinal ganglion cells FL2 can promote axonal regeneration of retinal ganglion cells after optic nerve injury can effectively restore vision is still not get system research.On the basis of the above research background,this paper studied in vitro and in vivo,and fully discussed the systematic study of the survival of retinal ganglion cells and the regapses with different expression levels of FL2-siRNA,so as to give some clinical enlightenment.Part ?:Fidgetin Like 2-siRNA regulating the survival of Retinal ganglion cells and axon regeneration in vitroObjective:Loss of optic nerve fibers and progressive apoptosis of retinal ganglion cells(RGCs)after optic nerve injury lead to irreversible decline of visual function.On the basis of vitro culture,the effects of FL2-siRNA on the optic nerve axon growth and cell survival of retinal ganglion cells were investigated.Materials and Methods:We culture the retinal ganglion cell-5(RGC-5)in primitive culture environment,select the third generation of cells as the research object,then culture in glucose solution for 24h.it is divided into three groups:Control group(cells without any processing),U-FIGN group(FL2 expression plasmid transfection in retinal ganglion cells),D-FIGN group(design FL2 siRNA sequences silence FL2 expression in retinal ganglion cells);The test items are as follows:RT-PCR detect the concentration expression of FL2 mRNA in each group;Immunohistochemical staining was performed to observe the expression of FL2 protein in cells of each group.The expression of FL2,GFAP,GAP-43 and SYN in each group was observed by immunofluorescence.Mitochondrial membrane potential was measured in each group.ATP synthesis ability test.TUNEL was used to detect apoptosis in all groups.CCK-8 was used to detect cell proliferation in each group.The changes of organelles in each group were detected by transmission electron microscopy.Scratch test and Transwell test were used to detect the migration ability of cells in each group.The expression of FL2,GFAP,GAP-43 and SYN in each group were determined by western-blot.Results:RT-PCR detection:the concentration expression of FL2 mRNA in each group was D-FIGN<Control<U-FIGN.Immunohistochemical staining:the expression trend of FL2?IL-6 and IL-1? were D-FIGN<Control<U-FIGN.Immunofluorescence detection:the expression trend of FL2 and GFAP trend were D-FIGN<Control<U-FIGN.The GAP-43 and SYN trend were U-FIGN<Control<D-FIGN.Mitochondrial membrane potential analysis:the mitochondrial membrane potential of the cells with low expression of FL2 presented red fluorescence.With the increase of the content of FL2,the red fluorescence of the cells gradually changed to green fluorescence,suggesting that the mitochondrial energy gradually changed from high energy state to low energy state.ATP synthesis analysis:the data trend among three groups was U-FIGN<Control<D-FIGN.The overexpression of FL2 can reduce the ATP synthesis ability of mitochondria,and then the insufficiency of intracellular energy supply will lead to apoptosis,which is consistent with the above conclusions.Apoptosis and proliferation:the trend of apoptosis proportion of cells in each group was D-FIGN(0.78%)<Control(1.68%)<U-FIGN(2.06%).Transmission electron microscopy:control group,and group D-FIGN organelles form more normal,mitochondria and apparatus,the overall form around the lysosome and lipid deposition distribution is less,and after the FL2 overexpression,cell obviously with lysosomes and lipid deposition,cell inflammatory effect is obvious,at the same time,mitochondrial swelling,prompt poisoning occurs.Cell migration:wound healing test and Trans well test the expression trend between groups was U-FIGN<Control<D-FIGN,P<0.05.WB results:FL2 group presented the same conclusion as above;The expression trend of GFAP in each group was D-FIGN<Control<U-FIGN group.The expression of GAP-43 and SYN showed that the expression trend of each group was U-FIGN<Control<D-FIGN.Conclusions:(1)FL2 has been successfully overexpressed or silenced in RGC-5;FL2 has a certain inflammatory effect,and the overexpression of FL2 can fully aggravate the inflammatory infiltration degree of ganglion retinal cells,which has certain clinical significance.(2)The FL2 expression ability can reduce mitochondrial ATP synthesis and membrane potential changes,and energy supply in cells occur,mitochondrial state disorders,respiratory chain uncoupling phenomena,such as cell cannot get enough ability to supply,will happen apoptosis or necrosis,reduce the process of cell proliferation,cell inflammatory effect increase at the same time,around with lysosomes and lipid into electricity,to a certain extent poisoning occur mitochondria.(3)In retinal ganglion cells,FL2 expression can promote GFAP protein expression,effectively activated astrocytes,the optic nerve injury,FL2 silence,nerve growth factor protein expression GAP-43 content rise,promote the growth of retinal ganglion cells and synaptic growth process,providing the basis for the research of below in the body.FL2-siRNA can promote the expression of GAP-43 and play a protective role in RGC-5 cells.Part ?:Fidgetin Like 2-siRNA regulating the survival of ganglion cells and axon regeneration after optic nerve injury in vivoObjective:Optic nerve damage can lead to the loss and apoptosis of retinal ganglion cells(RGCs),resulting in different degrees of visual impairment.On the basis of mice traumatic optic neuropathy model,the effect of FL2-siRNA on the synaptic growth and cell survival of retinal ganglion cells was fully discussed.Materials and Methods:In this study,45 YFP mice were selected as the study subjects to perform surgery for optic nerve splinting in the eyes to establish the model of optic nerve injury.Mice were divided into three groups:control group(normal saline injection),I-FIGN group(vitreous injection of FL2 inhibitor,1×10-3g/L,5?L),and P-FIGN(vitreous injection of FL2 promoter,1×10-3g/L,5?L).The specific investigation items were as follows:HE staining was used to observe the cell morphology of mice retinal tissue in each group;Retinal ganglion cell viability test;The protein expressions of FL2,GFAP,GAP-43 and SYN in each group were analyzed by immunofluorescence assay.Observation of degenerative changes of optic chiasm and contralateral axon;CTB staining was used to observe the degeneration of optic nerve.The protein concentrations of FL2,GFAP,GAP-43 and SYN were analyzed by western-blot.Results:HE staining:the inflammatory response of cells in the control group was severe,and the hierarchy between cells was poor,and the overall structure was gradually confused,and the connectivity between cells was poor,while the dispersion increased.After the addition of FL2 inhibitor,the cells gradually became more distinct,with obvious contour and regular overall structure.After the addition of the promoter,the messy cells were significantly increased,and the connectivity was worse than that of the control group.Cell survival rate:the survival rate of cells in each group was P-FIGN<Control<I-FIGN.Immunofluorescence detection:The expression trend of GFAP groups:I-FIGN<Control<P-FIGN;GAP-43 and SYN:P-FIGN<Control<I-FIGN.Neurodegenerative changes:some axons of the optic nerve in the control group gradually became partial moniliform,and turbidity appeared around the optic nerve fibers.When the expression concentration of FL2 was reduced,the nodular shape and inflammatory reaction of the axon were reduced.After the overexpression of FL2,the nodular shape of the axon continued to extend and gradually deteriorated,the axon changed to fragmental shape,and the degeneration of the optic nerve was more serious.CTB staining:the axons of the cells in the control group were dark blue,and myelin sheaths were mostly round and oval.The cells were moderate in size and evenly distributed.However,after the overexpression of FL2,the glial cell's axis protruded and became swollen and variable,gathering into a mass,and the arrangement of nerve fibers was also chaotic.WB results:The expression trend of FL2 and GFAP in each group was I-FIGN<Control<P-FIGN;The expression of GAP-43 and SYN showed that the expression trend of each group was P-FIGN<Control<I-FIGN.Conclusions:(1)The mice traumatic optic neuropathy model was successfully prepared,and FL2 was successfully overexpressed or inhibited in mouse retinal ganglion cells;FL2 can promote the inflammatory response of mice,effectively activate astrocytes,damage the optic nerve,reduce the expression of nerve growth factor protein,and inhibit the growth process of retinal ganglion cells and the development process of synapses.(2)FL2 can reduce the survival rate of retinal ganglion cells,accelerate the apoptosis process of optic nerve cells,and promote the degenerative changes of optic nerve synapses,resulting in the swelling of synaptic clusters and the appearance of rosary or even fragment,which is consistent with the conclusion of the above in vitro study and worthy of clinical investigation.