| BackgroundRetinal ganglion cells(RGCs)is the third level neuron of the retina.Its axons form the optic nerve to transmit the visual information to the lateral geniculate body.Glaucoma,optic nerve trauma,diabetic retinopathy and so on can all cause RGCs axonal damage,vision loss and even blindness.Promoting the regeneration and functional recovery of RGCs axons is the key to the repair of visual impairment.Previous experiments have shown that phosphodiesterase inhibitor papaverine(PAP)can reduce the damage of RGCs and promote RGCs axon regeneration.This study:by improving the separation method of primary RGCs,primary RGCs of SD rats with high yield and high purity were isolated and obtained;To study the promoting effect of papaverine on axonal regeneration of primary RGCs and its molecular regulation mechanism in vitro;The SD rat optic nerve injury model was used to observe the effect of papaverine on RGCs axon regeneration under in vivo conditions,and to explore the mechanism of its regeneration,so as to further verify the effect of papaverine on axon regeneration.Part 1:Isolation,identification and culture of primary retinal ganglion cellsObjectiveTo study the separation and purification of primary RGCs by two-step immunopanning method in order to obtain high-purity and high-yield cells,so as to provide cytological basis for the study of RGCs axon regeneration.MethodsRetinal tissue was removed from 25 SD rats after birth 72h,and the primary RGCs of SD rats were isolated by two-step immunopanning method.Firstly,the retinal tissue was digested with papain to produce a single cell suspension,which was combined with rabbit anti-rat macrophage polyclonal antibody.Secondly,non-RGCs were removed by negative screening petri dishes pre-coated with goat anti-rabbit IgG(H+L)secondary antibody,and RGCs were tightly adsorbed by positive screening petri dishes pre-coated with goat anti-mouse IgG+IgM(H+L)and CD90 antibody,some residual non-RGCs were rinsed by D-PBS for several times.Finally,the RGCs adsorbed at the bottom of the petri dish was digested and separated by trypsin,and the digestion was terminated after the addition of fetal bovine serum.After centrifugation,3mL of DMEM-SATO medium(added with Forskolin,BDNF and CTNF)was used to suspend RGCs.After counting by Count Star cell counter,dilution to 6 mL,the cells were planted into a 24 well plate preset with glass coverslips(0.01%poly-lysine and 10μg/mL laminin were pre-coated glass coverslips).After 24 hours of culture,the cells were fixed and immunofluorescence staining was performed with BRN3A antibody(1:500)to identify the obtained RGCs and calculate the cell purity.Results1.The purity of primary RGCs obtained by two-step immunodishing method was 72.57±0.6191%.2.The average yield of primary RGCs obtained by two-step immunodiscination method was 18889±484.4 cells/retina.Conclusion1.The purity of primary RGCs isolated and purified by two-step immunopanning method was not high,which was not conducive to the identification of physiological characteristics of primary RGCs.2.In addition,the cell yield and total protein content were extremely low,which could not meet the requirements of subsequent Western Blot experiments,hindering further studies on the axon regeneration mechanism of primary RGCs.Part 2:The improvement of the separation and purification technology of primary retinal ganglion cellsObjectiveIn the first part,the purity of RGCs obtained by two-step immunopanning method is not high and the cell yield is very low.This study explored and improved the original two-step immunopanning method to obtain higher purity and higher yield of primary RGCs.MethodsRetinal tissue was removed from 25 SD rats after birth 72h,the primary RGCs were isolated and purified by improved two-step immunopanning method.The retinal tissue was digested with papain to produce a single cell suspension,which was combined with rabbit anti-rat macrophage polyclonal antibody.Secondly,the negative screening dish pre-coated with Goat anti-rabbit IgG(H+L)secondary antibody was used to remove non RGCs as much as possible;Different from the two-step immunopanning method,RGCs were isolated by the difference of deposition rate between RGCs and non-RGCs caused by goat anti-mouse IgG+IgM(H+L)antibody and CD90 antibody on the bottom of positive petri dish.At the same time,when separating RGCs adsorbed by positive screening,taking advantage of the fact that RGCs are not closely adsorbed with the Petri dish,D-PBS was used to gently blown for separation.After centrifugation,3mL of DMEM-SATO basal medium(added with Forskolin,BDNF and CTNF)was used to suspend RGCs.After counting by Count Star cell counter,dilution to 6 mL,the cells were planted into a 24 well plate preset with glass coverslips(0.01%poly-lysine and 10μg/mL laminin were pre-coated glass coverslips).After 24 hours of culture,the cells were fixed and immunofluorescence staining was performed with BRN3A antibody(1:500)to identify the obtained RGCs and calculate the cell purity.Results1.The purity of primary RGCs obtained by improved two-step immunopanning method was 88.50±0.4113%.2.The average yield of primary RGCs obtained by improved two-step immunopanning method was 186211±5263 cells/retina.Conclusion1.The purity of primary RGCs isolated and purified by the improved two-step immunopanning method is significantly higher than that by the two-step immunopanning method.2.The yield of primary RGCs isolated and purified by the improved two-step immunopanning method is much higher than that by the original two-step immunopanning method,which can meet the cytological research of RGCs.Part 3:Effect of papaverine on axon growth of primary retinal ganglion cells in SD ratsObjectivecAMP is an important second messenger in cells,which plays an important role in the survival of nerve cells and axon regeneration,and papaverine can increase the level of cAMP.This study intends to research the effect of papaverine on axon growth of retinal ganglion cells in SD rats.MethodsThe primary RGCs of SD rats were isolated and purified by the improved two-step immunopanning method,and were planted in the 24-well plate with a density of 1×105/ml.After adaptive culture for 24 hours on DMEM-SATO base medium(added with Forskolin,BDNF and CTNF),the following treatments were performed respectively:1.Effect of PAP on axon regeneration of primary RGCs and optimum concentration screening.Grouping:control group,0.08 μg/ml PAP group,0.4 μg/ml PAP group,2 μg/ml PAP group and 10 μg/ml PAP group(the control group used equal volume PBS instead of PAP);After adding different concentrations of PAP,the cultured RGCs were fixed for use after intervention for 24 hours,48 hours and 72 hours with β-Tubulin Ⅲ antibody was used for immunofluorescence staining of axons.After taking photos,ImageJ software(simple neurite tracer)was used to measure the axon length,analyze the axon length of different groups and different time points,and screen out the best PAP concentration to promote axon regeneration for subsequent experiments.2.Papaverine promotes the axon growth of primary RGCs to overcome the influence of glial scar(chondroitin sulfate proteoglycan,CSPGs).After 24 hours of adaptive culture,RGCs were divided into control group,CSPGs group,and CSPGs+PAP group(the control group used equal volume PBS instead of PAP;CSPGs concentration 100μg/mL;PAP concentration 2 μg/mL);After 24 h,48 h and 72 h treatment,the axon length was stained by immunofluorescence,and the axon length was measured by ImageJ software(Simple Neurite Tracer)to analyze the effect of PAP on axons overcoming CSPGs to growth.3.cAMP blockers and activators were added to further observe the effect of PAP on axonal regeneration of primary RGCs.After 24 hours of adaptation,they were divided into Control group,PAP group,PAP+rp-cAMP group and rp-cAMP group(the Control group used equal volume PBS instead of PAP,rp-cAMP 50μM,PAP 2μg/mL).Control group,PAP group,PAP+db-cAMP group,db-cAMP group(Control group used equal volume PBS instead of PAP,db-cAMP 500μM,PAP 2 μg/mL).After 24 h,48 h and 72 h intervention,the axon length was stained by immunofluorescence,and the axon length was measured by ImageJ software(Simple Neurite Tracer)to analyze the effect of rp-cAMP and db-cAMP on the promotion of axon regeneration by PAP.Results1.The optimal concentration of PAP to promote axonal regeneration of primary RGCs:At 24 hours,the axon regeneration length of RGCs in 2 μg/mL group was significantly longer than that in the control group,0.08 μg/mL group and 0.4 μg/mL group.However,there was no difference in length compared with 10 μg/ml.At 48 hours and 72 hours,the axon regeneration length of 2μg/mL PAP group was the largest,and this concentration was selected as the optimal concentration 2μg/mL PAP was used for subsequent intervention experiments.2.Effect of papaverine on overcoming glial scar during promoting axonal growth of primary RGCs:At 24 hours,axons length in the control group were significantly longer than those in the CSPGs and CSPGs+PAP groups.However,there was no significant difference between the CSPGs and CSPGs+PAP groups.At 48 hours,the axon length of the control group was significantly greater than that of the CSPGs group,but the difference was not statistically significant compared with that of the CSPGs+ PAP group.The axon length in CSPGs+PAP group was larger than that in CSPGs group.At 72 hours,there was no significant difference in axon length between the Control group and the CSPG+PAP group.However,the axon length of the CSPG+PAP group was much longer than that of the CSPG group.3.cAMP blockers and activators were added to observe the effect of PAP on axon regeneration of primary RGCs:The length of axons in PAP group increased significantly within 24,48 and 72 hours.However,the axon growth of PAP+rp-cAMP group was significantly inhibited in the three time periods,and there was no significant difference in axon length between PAP+rp-cAMP group and Control group.In addition,there were significant differences in axon length among Control group,PAP group,PAP +db-cAMP group and db-cAMP group,and the axon length of PAP+db-cAMP group was the largest.ConclusionPrimary RGCs culture results showed that:1.The effect of 2 μg/mL papaverine on axon regeneration of primary RGCs in SD rats was most obvious.2.2μg/ml papaverine can promote the axons of primary RGCs to overcome the regrowth of CSPGs.3.rp-cAMP can block the axonal growth of primary RGCs.4.db-cAMP promoted the axonal growth of primary RGCs.Part 4:Mechanism of papaverine promoting axon regeneration of primary SD rat retinal ganglion cellsObjectivePrevious studies showed that papaverine can significantly promote axon regeneration of primary RGCs.This part of the study explored the possible signaling pathways and molecular regulatory mechanisms of papaverine in the process of promoting axon regeneration.MethodsThe primary RGCs of SD rats were obtained by improved two-step immunopanning method,and RGCs were planted into 24-well plates and 6-well plates,respectively.After 24 hours of adaptive culture,cAMP/PKA pathway inhibitor H89(5μM),PI3K/AKT pathway inhibitor LY294002(25μM)and MAPK/ERK pathway inhibitor U0126(5μM)were added according to the groups.After 48 h intervention,RGCs in different groups were stained by immunofluorescence and RGCs protein were extracted,respectively,to further study the mechanism of PAP promoting primary RGCs axon regeneration in SD rats.1.cAMP/PKA pathway was divided into Control group,2μg/mL PAP group,H89 group and 2μg/mL PAP+H89 group.2.PI3K/AKT pathway was divided into Control group,2μg/mL PAP group,LY294002 group and 2μg/mL PAP+LY294002 group.3.MEK1/2/ERK1/2 pathway was divided into Control group,2μg/mL PAP group,U0126 group and 2μg/mL PAP+U0126 group.Results1.cAMP/PKA pathway were blockade to observation:The length of regenerated axons in H89 group and 2μg/mL PAP+H89 group was significantly shorter than that in 2μg/mL PAP group.There was no significant difference between 2μg/mL PAP+H89 group and H89 group.The expression level of RAC1 in 2μg/mL PAP group was significantly higher than that in the other groups,and the relative expression level of P-MLC2/MLC2 in 2μg/mL PAP group was lower than that in the other groups.2.PI3K/AKT pathway were blockade to observation:The axon regeneration length in 2μg/mL PAP+LY294002 group was significantly shorter than that in 2μg/mL PAP group.There was no significant difference between LY294002 group and 2μg/mL PAP+LY294002 group.The expression levels of RhoA and P-GSK3β/GSK3βin 2μg/mL PAP group were significantly lower than those in Control group.However,the protein levels of RhoA and P-GSK3β/GSK3β in 2μg/mL PAP+LY294002 group were higher than those in 2μg/mL PAP group.The expression level of Neuropilin-1 in 2μg/mL PAP group was significantly higher than that in Control group,and the expression level in 2μg/mL PAP+LY294002 group was lower than that in 2μg/mL PAP group.The expression levels of PI3K and P-AKT/AKT in 2μg/mL PAP group were significantly higher than those in Control group.3.MEK1/2/ERK1/2 pathway were blockade to observation:Axon length of RGCs in 2μg/mL PAP+U0126 group was significantly shorter than that in 2μg/mL PAP group.However,there was no statistically significant difference between U0126 group and 2μg/mL PAP+U0126 group.The expression level of BDNF in 2μg/mL PAP group was significantly higher than that in other groups.The relative expression levels of P-MEK1/2/MEK1/2 and P-ERK1/2/ERK1/2 in 2μg/mL PAP group were also significantly higher than those in Control group.ConclusionStudies on primary RGCs signaling pathways show that:1.Papaverine(2μg/mL)up-regulated RAC1 expression and down-regulated p-MLC2/MLC2 relative expression level through cAMP/PKA pathway to promote axonal regeneration of primary SD rats RGCs.2.Papaverine(2μg/mL)inhibited the expression of RhoA and P-GSK3β/GSK3βand increased Neuropilin-1 expression level through PI3K/AKT pathway to promote axonal regeneration of primary SD rats RGCs.3.Papaverine(2μg/ml)promoted BDNF expression and accelerated axonal regeneration of primary SD rats RGCs through the MEK1/2/ERK1/2 pathway.Part 5:The effect and mechanism of papaverine on optic nerve axons regeneration in SD rats with optic nerve injuryObjectivePrevious studies have found that pappapine can promote axon regeneration of primary RGCs in SD rats in vitro,and promote axon regeneration by activating cAMP/PKA,PI3K/AKT and MAPK1/2/ERK1/2 pathways.Whether papaverine has the same effect in vivo,this study used SD rat nerve injury model to further verify the role and mechanism of papaverine in promoting axonal regeneration.MethodsMale SD rats of 4-6 weeks old(body weight:170-180 grams)were fed adaptively for one week and randomly divided into groups(10 rats/group)to construct the optic nerve injury model,the axonal regeneration was quantified by labeling with nerve growth protein GAP43.1.Grouping:Sham group,Crush group,Crush+50μg/mL PAP group,Crush+200μg/mL PAP group,Crush+500μg/mL PAP group(PBS was used instead of PAP for Sham group and Crush group),After successful modeling,different concentrations of PAP were injected into the vitreous cavity.The rats were sacrificed 7 and 14 days later,the optic nerve tissue was obtained,section after paraffin embedding,immunofluorescence staining was performed on GFAP and GAP43 and the fluorescence intensity of GAP43 was measured by ImageJ software to reflect the effect of PAP on promoting axon regeneration.At the same time,the protein of optic nerve tissue was extracted and the protein of optic nerve tissue was extracted and the expression level of protein was detected to screen out the optimal concentration of PAP to promote optic nerve regeneration.2.The optimal concentration of PAP(500μg/mL)was used to explore the mechanism of optic nerve regeneration.After the construction of optic nerve injury model,500μg/mL of PAP,cAMP/PKA pathway inhibitor H89(20μM),PI3K/AKT pathway inhibitor LY294002(100μM)and MEK1/2/ERK1/2 pathway inhibitor U0126(20μM)were intravitreal injected immediately.Rats were sacrificed 14 days after the intervention.Optic nerve was obtained,embedded in paraffin and then cut into sections for GFAP and GAP43 immunofluorescence staining.The fluorescence intensity of GAP43 was measured by ImageJ software to reflect the effect of PAP on promoting axon regeneration.Optic nerve proteins were obtained to detect the expression of pathway-related proteins.2.1 cAMP/PKA pathway grouping:Sham group,Crush group,Crush+500μg/mL PAP group,Crush+H89 group,Crush+500μg/ml PAP+H89 group.2.2 PI3K/AKT pathway grouping:Sham group,Crush group,Crush+500μg/mL PAP group,Crush+LY294002 group,Crush+500μg/mL PAP+LY294002 group.2.3 MEK1/2/ERK1/2 pathway grouping:Sham group,Crush group,Crush+500μg/mL PAP group,Crush+U0126 group,Crush+500μg/mL PAP+U0126 group.Results1.In 7-day and 14-day groups,the length of optic nerve axon regeneration and the expression level of GAP43 protein in Crush+500μg/m PAP group were significantly higher than those in other groups.The expression level of GFAP was the highest in Crush group,and decreased gradually with the increase of papaverine concentration.In addition,the fluorescence intensity of GAP43 in Crush+500μg/mL PAP group at 14 days was significantly higher than that in Crush+500μg/mL PAP group at 7 days,the repair and regeneration of axons were more significant.2.The addition of pathway blockers inhibited the repair and regeneration of optic nerve axon.2.1 The RAC1 protein expression level in Crush+500μg/mL PAP group was significantly higher than that in Crush group and Sham group.After adding H89,the expression of RAC1 protein was inhibited in Crush+500μg/mLPAP+H89 group,which was significantly lower than Crush+500μg/mL PAP group.However,the relative expression level of P-MLC2/MLC2 was opposite to RAC1.GAP43 immunofluorescence staining showed that the fluorescence intensity of GAP43 in Crush+500μg/mL PAP group was significantly increased,axonal regeneration more obvious,while that in Crush+500μg/mL PAP+H89 group was significantly decreased,axonal regeneration was inhibited.2.2 RhoA and P-GSK3β/GSK3β protein expression levels in Crush+500μg/mL PAP group were significantly lower than those in Crush group.However,the expression levels of RhoA and P-GSK3β/GSK3β in Crush+500μg/mLPAP+LY294002 group were significantly higher than those in Crush+500μg/mL PAP group.Neuropilin-1 protein level was the highest in the Crush+500μg/mL PAP group,but in the Crush+500μg/mL PAP+LY294002 group was significantly lower than that in the Crush+500μg/mL PAP group.GAP43 staining showed that after the addition of blocking agent LY294002,the fluorescence expression intensity of GAP43 was significantly reduced,and axon repair and regeneration were weakened.Meanwhile,the expression levels of PI3K and P-AKT/AKT in Crush+500μg/mL PAP group were significantly higher than those in Crush group.2.3 The expression level of BDNF in Crush+500μg/mL PAP group was the highest and significantly higher than that in Crush+500μg/mLPAP+U0126 group.Fluorescence staining showed that the blocking agent U0126 could significantly inhibit the expression level of GAP43 and weaken axon repair and regeneration.In addition,the results showed that the relative expression levels of P-MEK1/2/MEK1/2 and P-ERK1/2/ERK1/2 in Crush+500μg/mL PAP group were significantly higher than those in Crush group.ConclusionExperimental confirmation of rat optic nerve injury animal model in vivo:1.Papaverine can promote the repair and regeneration of optic nerve axon in SD rats after optic nerve injury in a dose-dependent and time-dependent manner.2.Papaverine up-regulated RAC1 expression and inhibited the relative expression of P-MLC2/MLC2 through cAMP/PKA pathway to promote the repair and regeneration of optic nerve axon injury.3.Papaverine inhibited the expression of RhoA and P-GSK3β/GSK3β and increased the expression of Neuropilin-1 through PI3K/AKT pathway to promote axon regeneration of injured optic nerve.4.Papaverine up-regulated the expression of BDNF through the MEK1/2/ERK1/2 pathway to accelerated the regeneration of injured optic nerve axon. |
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