Experimental Study Of The Role Of Nerve Growth Factor And Its Receptor In Form Deprivation Myopia

Myopia is commonly and frequently encountered disease in ophthalmology,especially high myopia, which is also called pathological myopia or degenerationmyopia. High myopia is often associated with excessive and progressive elongation ofthe eye globe, resulting in varying degrees of visual deterioration. It can give rise tomany complications leading to blindness, such as degenerative retinopathy, posteriorscleral staphyloma, choroidal neovascularization, macular degeneration andrhegmatogenous retinal detachment, etc. The foundation of form deprivationmyopia(FDM)model is helpful for us to further understand how high myopia occursand develops. Form deprivation modulates the development of local sclera by changing the level of various neurotransmiter and growth factors in the retina. Theseneurotransmiter and growth factors work by means of cooperate parallel and across,and lead to the ultrastructural and biomechanical alteration of sclera through a seriesof signal transduction. Sclera was remodeled and eye axial was lengthened andinduced to axis myopia. With the development of the axial length of eye and funduschanges, retinal photoreceptor cells decrease continuously. Apoptosis is one of thepathways of photoreceptor cell death in retinal degenerated disorders. At present, it islack of investigating the mechanisms of retinal apoptosis and its anti-apoptosistreatment in retinal degenerative disease including high myopia.Nerve growth factor is a member of family of neurotrophins. It exerts effects bysignaling through surface membrane receptors, trkA and p75NTR in the neurotissueand unneurotissue. NGF expresses and influences the shape and function of the threekinds neuron of in the retina. Nastri had discovered that Anti-NGF serum eye dropswere able to elicit a slight contraction on ocular overgrowth in chicks raised incontinuous light and suggested the NGF was possible to take part in the developmentof FDM. It is well known that NGF receptor p75NTR involved in programmed celldeath (PCD) and exogenous NGF may inhibit the apoptosis of retinal cell inexperiment retinal detachment.In this study, form deprivation myopia model is established to investigateexpression of NGF,TrkA,p75NTR and Caspase-3 protein in retina byimmunohistochemistry, and to detect the expression of NGF mRNA,TrkA mRNAand p75NTR mRNA in retina by reverse transcription-polymerase chainreaction(RT-PCR). We would investigate whether there are obvious retinal apoptosisin FDM in guinea pigs and observe the retinal rescue of NGF in order to provide anew additional strategy for the treatment of human high myopia. We also observedthe effect of NGF on human sclera fibroblast in vitro.Part one: Expression of nerve growth factor (NGF) and high affinityNGF receptor (TrkA) in retinal tissue of guinea pigs with form deprivation myopiaMethods1. Establishment of a form deprivation myopia model: Eighty guinea pigs(seven-day-old) were divided into 4 groups randomly, i.e, group A (2 weeks'occluded), group B (4 weeks' occluded), group C (8 weeks' occluded), group D(7weeks' occluded and removed for 1 week). There were 20 guinea pigs in each group.Ten guinea pigs were used for immunohistochemistry, the other ten guinea pigs wereused for RT-PCR. All the right eyes underwent form deprivation with translucentgoggles and the left eyes were used as control.2. Measure of the refractive state and the axial length: Before occluding andafter occluding for A,B,C,D group, the refractive state and axial length weremeasured with retinoscopy and A-scan ultrasonography respectively.3. Hematoxylin-eosin staining and morphology: After the refractive state andaxial length were measured,the eyes were extracted,routine hematoxylin-eosinstaining were done, and morphological changes in form deprived guinea pigs retinaand sclera were observed under light microscope.4. The expression of NGF protein and TrkA protein in retina were detected byimmunohistochemistry.5. The expression of NGF mRNA and TrkA mRNA in retina were detected byRT-PCR.6. Statistical analysis SPSS12.0 software was used to perform t-test andanalysis of variance, less than 0.05 was considered statistical significance.Results1. Comparison of refraction state and axial length between the occluded eyesand control eyes: Monocular occluding for 8 weeks could lead to myopia, therefraction showed significant difference between the occluded eyes (-4.38±1.11)D andthe control ones((3.75±0.71)D, the change of refractive degree was 8.13D(P＜0.01).The longer the eyes were occluded, the axial length was longer, there was significant differences between the occluded eyes(8.18+0.16)mm and the controlones(7.26±0.20)mm, the change of axial length was 0.92mm(P＜0.01).The longerthe eyes were occluded, the more severe the myopia was. The removal of occludingfor I week, the refraction state and axial length was recovered 27ï¼…,43ï¼…respectively.2. Morphology change in retina and sclera: hematoxylin-eosin staining showedthe posterior retina of the occluded eyes were thinner than that of the control eyes,The posterior sclera of the occluded eyes were thin too.3. Immunohistochemistry: NGF and TrkA positive cells were mainlydistributed in the inner nuclear layer and ganglion cell layer of the retina, displayingas pale brown pellet. Through the image analysis, the expression of NGF protein andTrkA protein in retina of the occluded eyes was decreased, there were significantdifference compared with that in the control eyes(P＜0.01)4. RT-PCR: The expression of NGF mRNA and TrkA mRNA in retina of theoccluded eyes decrease, there were significant difference compared with that in thecontrol eyes(P＜0.01).Part two: Experimental study of retinal apoptosis and NGFrescue in form deprivation myopia in guinea pigsResearch one: Expression of retinal apoptosis and NGF receptionp75NTR in form deprivation myopia in guinea pigsMethods:1. Sixty guinea pigs (seven-day-old)were divided into three groups, this is, groupA (4 weeks' occluded), group B (8 weeks' occluded), group C (12weeks' occluded).There were 20 guinea pigs in each group. All the right eyes underwent formdeprivation with translucent goggles and the left eyes were used as control.Establishment of a form deprivation myopia model and measure of the refractive stateand the axial length: refers to part one.2. Histopathological changes in retina were observed by light microscopy. Retinal apoptotic cells were determined by TdT medicated biotin-dUTP nick-endlabeling(TUNEL) staining.3. The expression of p75NTR protein and Caspase-3 protein in retina wasdetected by immunohistochemistry.4. The expression of p75NTRmRNA in retina was detected by RT-PCR.5. Statistical analysis refers to part one.Results1. Comparison of refraction state and axial length between the occluded eyes andcontrol eyes: The refractive degrees and axial length of occluded eyes weresignificantly deeper and longer than control eyes. After occluding for 12 weeks, thechange was -11.47D in refraction degree and 1.06mm in axial length.2. The thickness of choroid and retina occluded 12 weeks became thinneraccompanying with the outer Segment of photoreceptor elongated and RPE arrangeddisorderly. Apoptotie cells were only found in retinal outer and inner nuclear layer ofthe occluded eyes in group C by TUNEL staining.3. Immunohistochemistry:p75NTR and Caspase-3 positive cells were mainlydistributed in the outer segments of photoreceptor cells of the retina,immunohistochemistry staining shows that p75NTR and Caspase-3 positiveexpression display as pale brown. Through the image analysis, the expression ofp75NTR protein and Caspase-3 protein in retina of the occluded eyes increase. Therewere significant difference compared with that in the control eyes (P＜0.01).4. RT-PCR: The expression of p75NTR mRNA and Caspase-3 mRNA in retinaof the occluded eyes increase, there were significant difference compared with that inthe control eyes (P＜0.01).Research two: NGF protective effect on retinal apoptosis in formdeprivation myopia in guinea pigsMethods1. One hundred twenty guinea pigs (seven-day-old)were divided into six groups, this is, group A(normal control), group B(12 weeks' occluded), group C(12 weeks'occluded +saline),group D(12 weeks' occluded +NGF5μg), group E(12weeks'occluded+NGF10μg), group F(12 weeks 'occluded +NGF 20pg). There were 20guinea pigs in each group. Except group A, all the right eyes underwent formdeprivation with translucent goggles and the left eyes were used as control. AfterNGF or saline had been injected into vitreous of occluded eyes in planning time,myopia was confirmed by retinoscopy and A-scan ultrasonography.2. Retinal apoptotic cells were determined by flow cytometry.3. The expression of TrkA proteins, p75NTR proteins, Caspase-3 proteins inretina were detected by immunohistochemical staining.4. The expression of TrkA mRNA, p75NTR mRNA in retina was detected byRT-PCR.5. Statistical analysis refers to part one. Apoptotic rate by several independentsamples ranks test.Results:1. The refractive degrees and axial length of form deprivation eyes weresignificantly deeper and longer in group B,C,D,E,F comparing with its self-controleyes and group A(P＜0.01).2. Retinal apoptotic rate of group B(4.45±0.48)ï¼…, C(4.56±0.32)ï¼…was morethan group A(0.31±0.22)ï¼…; The rate of group D(3.22±0.34)ï¼…,E(1.94±0.42)ï¼…,F(1.05±0.26)ï¼…was less than group B,C(P＜0.01), but more than group A.3. Retinal p75NTR proteins and Caspase-3 proteins in group D,E,F weresignificantly less than group B,C(P＜0.01), but more than in group A, whereas TrkAproteins is more than in group B and C(P＜0.01).4. Retinal p75NTR mRNA in group D,E,F were significantly less than groupB,C(P＜0.01), but more than in group A, whereas TrkA mRNA is more than in groupB and C.Part three: The effect of NGF on the human scleral fibroblast cultured in vitroMethods1. Primary culture of human scleral fibroblast was performed with sclera tissuefrom human embryonic eyes and identified with vimentin immunohistochemistystaining.2. MTT assayed the cell proliferation of scleral fibroblast at varying NGFconcentration. The cell cycle of HSF with stimulation of NGF is analyzed by the flowcytometry.3. Hydroxyproline contents (collagen synthesis) were measured with thechloramine T method.4. Statistical analysis refers to part one.Results1. The human scleral fibroblasts show good biological characteristics in primaryand secondary culture. Vimentin immunohistochemisty staining of HSF is positive.2. NGF can promote the cell proliferation of scleral fibroblast at theconcentration 25-200μg/L in dose and time dependent manner, when theconcentration of NGF is 100μg/L, the proliferative effect is the most significant(P＜0.01). The result of flow cytometry reveals that with stimulation of NGF thepercentage of the cells in G₁ phase is decreased while the percentage of the cells in Sphase is increased and the Prl(cell proliferation index) is increased as well(P＜0.01).3. NGF can stimulate HSF to increase the collagen protein production. Thiseffect is also in dose-depended manner. When the concentration of NGF is 50μg/L,the effect is the most powerful.Conclusions1. Form deprivation can succeed in setting up myopia model of guinea pigs,which cause the eyes axis to lengthen, induce to axis myopia;2. Investigating the dynamic expression and function of the protein and mRNAof NGF and TrkA on retina in form deprivation of guinea pigs. FDM made the expression of NGF and TrkA of protein and mRNA in retina down-regulating. NGFand TrkA may play an important role on the formation and development of myopia.3. Form deprivation in guinea pigs made apoptosis of retinal outer and innernuclear layer and the expression of p75NTR protein and Caspase-3 protein increased,whereas p75NTR mRNA increased significantly, too. The increased expression ofp75NTR and Caspase-3 in retina was consistent with retinal apoptosis.4. NGF can effectively reduce apoptosis by down-regulating of p75NTR andCaspase-3 and increased expression of TrkA. The rescue of NGF on retina was in adose dependent manner.5. NGF can stimulate the proliferation of HSF significantly and increase theproduction of collagen protein in a dose-dependent manner.