The Effects Of Optic Nerve Crush On The Dendrites Of Retinal Ganglion Cells And Amacrine Cells

Section one:Changes in dendrites of RGC subtype BD cells after ONCObjective:To observe the death of retinal ganglion cells and the dendritic changes of remnant BD cells after ONC,so as to provide evidence for understanding the process of ganglion cell death and functional changes after ONC.Methods:10 BD transgenic mice(FSTL4-CreER:Thyl-Stop-GFP)were randomly divided into day 4 and day 7 after ONC.The right eye was used as normal control group,all of them were reared in the same environment.For the expression of GFP specific in BD cells of BD transgenic mice,postnatal day 14 mice were intraperitoneal injection of tamoxifen 150mg/kg.The establishment of ONC model:BD transgenic mice(8-12 weeks)after isoflurane inhalation anesthesia and ocular surface anesthesia,under the operating microscope,the lateral skin was exposed,and the lateral eyeball was exposed.Obtuse separation and exposure of the optic nerve,the optic nerve was clamped with the reverse forceps for 10 seconds at 1.5-2mm after the ball,and the ocular vessels under ON were avoided.The ocular fundus examination confirmed the blood flow state of the retinal vessels.No suturing conjunctival incision after operation,with Ofloxacin Eye Ointment.The left eye was selected as the model eye and the right eye was the normal control.At day 4 and day 7 after ONC,counted the number of retinal GCL DAPI staining cells by the whole mount retina immunofluorescence staining.Choosing BD cells that express GFP but not affected by other single cells to analyze the dendritic morphological by using Neurolucida software.Results:The numer of GCL cells of retina decreased significantly at day 7 after ONC(P<0.01),and the difference was more obvious with the time prolonging.There's no significantly difference between non-polar BD cells and polar BD cells.At day 4 and day 7,the number of non-polar BD cell dendritic spines were decreased by 53%(P<0.001)and 86%(P<0.0001),respectively,and the dendritic area,total length and number of dendritic branch and dendritic arborizations,no obvious change.At day 4 after ONC,although there were change of dendritic morphology of polar BD cells,but the difference was not statistically significant,.At day 7 after ONC,dendritic dendritic area,total length,the number dendritic branches and dendritic arborizations,respectively decreased by 31%(P<0.01,34%(P<0.01)),45%(P<0.05)and 45%(P<0.05)compared with normal control group,the number of dendritic spines at day 4 and day 7 after ONC decreased(44%P<0.01)and 88%(P<0.001),respectively.Conclusion:After ONC,the number of cells in the retinal ganglion cell layer is significantly reduced,and polar BD cells are more sensitive to ONC damage than non-polar BD cells.There will be a significant decrease in synaptic connections between neurons and a decrease in the receptive field after ONC,indicating a gradual decrease in cell function.And The damage caused by ONC increase with time.Section two:Changes in dendrites of amacrine cell subtype ChAT cells after ONCObjective:To investigate the effects on the dendrite structure of two ChAT cells(SACs/DSACs)after the death of RGC caused by the ONCMethods:10 ChAT transgenic mice(ChAT-CreER:Thyl-Stop-GFP)were randomly divided into day 7 and day 10 after ONC.The right eye was used as normal control group,all of them were reared in the same environment.For the expression of GFP specific in ChAT cells of ChAT transgenic mice,postnatal day 14 mice were intraperitoneal injection of tamoxifen 150mg/kg.The establishment of ONC model:ChAT transgenic mice(8-12 weeks)after isoflurane inhalation anesthesia and ocular surface anesthesia,under the operating microscope,the lateral skin was exposed,and the lateral eyeball was exposed.Obtuse separation and exposure of the optic nerve,the optic nerve was clamped with the reverse forceps for 10 seconds at 1.5-2mm after the ball,and the ocular vessels under ON were avoided.The ocular fundus examination confirmed the blood flow state of the retinal vessels.No suturing conjunctival incision after operation,with Ofloxacin Eye Ointment.The left eye was selected as the model eye and the right eye was the normal control.At day 4 and day 7 after ONC,observed the number of retinal GCL DAPI staining cells by the whole mount retina immunofluorescence staining and frozensection immunofluorescence staining.Choosing BD cells that express GFP but not affected by other single cells to analyze the dendritic morphological by using Neurolucida software.Results:Two kinds of ChAT cells(SACs/DSACs)were distributed in the GCL and INL of the retina respectively.After ONC damage,the density of SACs/DSACs cells did not change significantly.The dendrite area of SACs is larger than that of DSACs,and the dendrite is longer.After ONC injury,the dendrite area of SACs changed,but the difference was not statistically significant.The length of dendrite of SACs decreased by 21%and 24%on day 7 and day 10 after ONC,respectively,and the difference was statistically significant(P<0.0001).For DSACs,the area of dendrites changed after ONC,but the difference was not statistically significant and the length of dendrites decreased by 17%and 20%on day 7 and day 10 after ONC,respectively,and the difference was statistically significant(P<0.01).Conalusion:1.The optic nerve damage caused by ONC does not cause a decline in the number of SACs/DSACs.2.The decrease of dendrite density in SACs/DSACs after ONC may be the result of synaptic reverse degeneration after RGCs death.Section three:Effect of CD3 gene knockout on BD cells after ONCObjective:To investigate whether CD3 knockout affects the development of BD cells and whether CD3 knockout will affect the sensitivity of BD cells to ONC damage.Methods:The CD3-/-BD transgenic mice(FSTL4-CreER:Thy1-Stop-GFP:CD3-/-)were generated by breeding FSTL4-CreER:Thyl-Stop-GFP mice with CD3-/-mice.The CD3-/-BD mice were randomly divided into 4 days after ONC and 7 days after ONC.5 mice in each group were kept in the same environment.The right eye was used as a normal control.For the expression of GFP specific in BD cells of BD mice,postnatal day 14 mice were intraperitoneal injection of tamoxifen 150mg/kg.The establishment of ONC model:CD3-/-BD mice(8-12 weeks)after isoflurane inhalation anesthesia and ocular surface anesthesia,under the operating microscope,the lateral skin of the lateral eye was exposed,exposing lateral eyeballs.Obtuse separation and exposure of the optic nerve,the optic nerve was clamped with the reverse forceps for 10 seconds at 1.5-2mm after the ball,and the ocular vessels under ON were avoided.The ocular fundus examination confirmed the blood flow state of the retinal vessels.No suturing conjunctival incision after operation,with Ofloxacin Eye Ointment.The left eye was selected as the model eye and the right eye was the normal control.After ONC,counted the number of retinal GCL DAPI staining cells by the whole mount retina immunofluorescence staining.Choosing BD cells that express GFP but not affected by other single cells to analyze the dendritic morphological by using Neurolucida software.Results:After knockout of CD3 gene,there was no significant change in cell density of GCL in mice.After 7 days of ONC,the number of GCL cells in CD3-/-mice decreased by 20%(p<0.001),the difference was statistically significant.The dendritic area of CD3-/-Non-polar BD cells decreased by 23%,the total length of dendrites decreased by 8%,the number of dendritic spines was increased by 38%,but the difference was not statistically significant,but dendritic branches and dendritic arborization were increased by 25%(P<0.05),the difference was statistically significant.The polar BD cells dendrites changed after CD3 knockout,but the difference was not statistically significant.At day 7 after ONC,CD3-/-non-polar BD cells' dendritic area decreased by 11%,but not statistically significant;the total dendrite length and number of dendritic spines and dendrites,dendritic branch number were decreased to different extent,and were associated with the time after injury,the difference was statistically significant.CD3-/-polar BD cells' dendritic area respectively decreased by 10%and 18%at day 4 and day 7 after injury,but the difference was not statistically significant,the total length of dendrites decreased by 30%(P<0.05)significantly after injury at day 7.The dendritic spines and dendrites and dendritic branch were decreased significantly after ONC injury,and along with the time after injury is more obvious,the difference was statistically significant.The normal mice and CD3-/-mice BD cells in different time points after ONC is found that at day 4 after injury,the number of non-polar CD3-/-mice BD cells remaining dendritic spines is 58%more than normal mice(p<0.01),and dendritic area,total length and number of dendrites and dendritic arborization have no significant difference.Conclusion:1.CD3 molecules regulate the dendrite development and cell differentiation of RGCs.After knockout of CD3 gene,the number of non-polar BD cells' dendrite branches and terminals increased.2.The knockout of CD3 gene does not affect the damage of RGCs caused by ONC and the restructure of the dendritic.Section four:Effect of CD3 gene knockout ChAT cells after ONCObjective:To investigate whether CD3 knockout affects the development of ChAT cells and whether CD3 knockout will affect the sensitivity of ChAT cells to ONC damage.Methods:The CD3-/-chat(ChAT-CreER:Thyl-Stop-GFP:CD3-/-)transgenic mice were generated by breeding ChAT-CreER:Thyl-Stop-GFP mice with CD3-/-mice.The CD3-/-ChAT mice were randomly divided into 7 days after ONC and 10 days after ONC.5 mice in each group were kept in the same environment.The right eye was used as a normal control.For the expression of GFP specific in ChAT cells of ChAT mice,postnatal day 14 mice were intraperitoneal injection of tamoxifen 150mg/kg.The establishment of ONC model:CD3-/-ChAT mice(8-12 weeks)after isoflurane inhalation anesthesia and ocular surface anesthesia,under the operating microscope,the lateral skin of the lateral eye was exposed,exposing lateral eyeballs.Obtuse separation and exposure of the optic nerve,the optic nerve was clamped with the reverse forceps for 10 seconds at 1.5-2mm after the ball,and the ocular vessels under ON were avoided.The ocular fundus examination confirmed the blood flow state of the retinal vessels.No suturing conjunctival incision after operation,with Ofloxacin Eye Ointment.The left eye was selected as the model eye and the right eye was the normal control.After ONC,counted the number of ChAT staining cells by the whole mount retina immunofluorescence staining.Choosing ChAT cells that express GFP but not affected by other single cells to analyze the dendritic morphological by using Neurolucida software.Results:After CD3 gene knockout,the average density of SACs cells increased by 16%(P<0.01),while the number of DSACs cells did not change significantly.SACs/DSACs,like normal mice,had no significant change in cell density after ONC.The dendrite area of SACs after CD3 knockout,the dendrite area did not change significantly,but the dendrite length decreased by 9%(P<0.01).DSACs dendrite area increased by 37%(P<0.05)after CD3 gene knockout,and the length of dendrite increased by 6%,but the difference was not statistically significant.At day 7 after ONC,the SACs dendrite area of CD3-/-mice increased by 13%,compared with that in the normal control group.After 10 days of ONC,it increased by 8%compared with the normal control group,but the difference was not statistically significant.For dendritic length,it decreased by 7%and 14%(P<0.01)at day 7 and day 10 after ONC,respectively,and the dendrite area of DSACs decreased by 2%and 19%at day 7 and day 10 after ONC,but the difference was not statistically significant.The length of dendrites decreased by 12%and 21%(P<0.05)at day 7 and day 10 after ONC.The normal mice and CD3-/-mice ChAT cells in different time points of the injury were compared,at the day 7 after ONC injury,dendritic area of CD3-/-mice DSACs was more 8%(P<0.05)than that of normal mice at day 7 after ONC,although the SACs dendritic area also increased,the difference was not statistically significant,the dendritic length it did not change significantly.Conclusion:1.CD3 molecules regulate the dendrite development and cell differentiation of SACs/DSACs.After knockout of CD3 gene,SACs cell density increased,but dendritic length decreased.2.The knockout of CD3 gene does not affect the damage of ChAT cells caused by ONC and the restructure of the dendritic.