| Purpose:To investigate the effects of sub-Tenon injection of genipin on scleral biomechanics in guinea pig model of high myopia and chronic glaucoma,to explore the effects of genipin scleral cross-linking on the progression of myopia and glaucoma and its mechanism,and to compare the similarities and differences of the effects of genipin on the two models.Methods:(1)selecting 90 guinea pigs which meet the conditions,and randomly dividing into blank group(group A)(n=18),high myopia group(n=36),glaucoma group(n=36).The right eye was used as the experimental eye,and the left eye was not processed.Building high myopia,chronic high intraocular pressure models by latex facemask and anterior chamber injection of 2% methyl cellulose.(2)One month after successful modeling of high myopia model and chronic glaucoma model,guinea pigs in high myopia group were randomly divided into group B(n=18)and group C(n=18).The guinea pigs in groups B and C were respectively injected with normal saline 0.25 ml and genipin 0.25ml(5mmol/L)sub-Tenon of the right eye,once every 4 days,for a total of 7times.At the same time continue to induce form deprivation of high myopia.The guinea pigs in the glaucoma group were randomly divided into group D(n=18)and group E(n=18).The guinea pigs in groups D and E were respectively injected with normal saline 0.25 ml and genipin0.25ml(5mmol/L)sub-Tenon of the right eye,once every 4 days,for 7times,and the IOP was maintained at more than 25 mm Hg.One day after the last injection(group A1,B1,C1,D1,E1),one week(group A2,B2,C2,D2,E2),and one month(group A3,B3,C3,D3,E3),the axial length,diopter,IOP and other parameters of guinea pigs were measured.(3)Micron IV Small Animal retinal imaging system was used to scan the fundus of guinea pigs,and the thickness of RNFL was measured at the temporal,upper,nasal,and lower directions on a ring with a diameter of500μm centered on the optic disc.(4)Sacrificing guinea pigs,dissecting the optic nerve of the right eye to observe the changes of optic nerve axons by transmission electron microscopy.Three eyeballs in each group were randomly selected to observe the damage degree of ocular wall tissue by HE and TUNEL staining.(5)Takeing temporal sclera from the other 3 eyes,and cutting sclera 1mm away from the optic disc to make a10*4mm band,measuring thickness under a light microscope.Using MTS Insight electronic universal tension test machine to peform stress relaxation,stress stretching,and creep tests.(6)Detecteig the expression of tissue inhibitor of matrix metalloproteinase-1(TIMP-1)m RNA and matrix metalloproteinase-2(MMP-2)m RNA in sclera was by quantitative real-time polymerase chain reaction.Detecting the expression of TIMP1 and MMP2 related proteins in sclera by Western blotting.Observing the morphological changes of scleral collagen around optic disc by scanning electron microscope.Results:Axial length and diopter changes of guinea pigs1.1 Results of guinea pig model of high myopia and chronic glaucomaAfter one month of intervention,the average diopter of guinea pigs in high myopia group was-6.58±1.66 D,showing significant myopic drift.The mean IOP of glaucoma group was 29.55±2.93 mm Hg,which was consistently higher than 25 mm Hg for 1 month.The modeling success rates of high myopia and chronic glaucoma model were 45% and 87.8%,respectively.1.2 Changes of biological parameters of the guinea pigs eyeThe axial length of high myopia group: the axial length of group B1(8.62±0.16mm)was significantly longer than that of group A1 and C1(group A1:8.29±0.16mm;Group C1:8.43±0.11 mm).The axial axis of group B2 and C2(group B2:8.67±0.19 mm;Group C2:8.57±0.15 mm)was significantly longer than group A2(8.17±0.15 mm).The axial axis of group B3(8.70±0.31 mm)was significantly longer than that of group A3(8.34±0.17 mm)(all P < 0.05).The diopter of group C(C group-2.54±0.87 D,C2 group-3.00±0.44 D,C3 group 4.58±0.70D)was significantly lower than that of group B(B1 group:-5.04±0.87 D,B2 group:-5.62±0.75 D,B3 group:-6.54±0.60D)(all P < 0.05).The changes of axial length in glaucoma group: there was no significant difference in axial length between groups A1,D1 and E1.(group D1:8.52±0.24mm;E1 group: 8.50±0.10mm).The axial axis of D2 and E2(D2:8.65±0.21 mm;Group E2:8.69±0.31mm)was significantly longer than group A2.The axis of the eye in group D3 and E3(group D3:8.68±0.25mm;Group E3:8.69±0.31mm)was significantly longer than group A3(P < 0.05).There was no significant difference in diopter between groups A,D and E(P > 0.05).1.3 Change of RNFL thickness in guinea pigsRNFL thickness changes in high myopia group: RNFL thickness in all quadrants of group C was greater than that of group B,but no statistical difference(P > 0.05).Changes of RNFL thickness in glaucoma group: RNFL thickness in all quadrants in group E was greater than that in group D.The superior and temporal side RNFL thickness of E1 group(21.67±1.97μm,23.67±1.97μm)was significantly higher than that of D1 group(18.00±1.67μm,19.33±2.58μm).The RNFL thickness of the superior,inferior and temporal side in E2 group(21.33±2.25μm,21.17±1.83μm,23.33±1.86μm)was significantly higher than that in D2 group(17.83±1.83μm,17.50±2.26μm,19.00±2.19μm).The superior side RNFL thickness in E3 group(19.33±1.86μm)was significantly higher than that in D3 group(16.17±1.94μm)(P < 0.05).1.4 Changes in scleral thickness in guinea pigsThe scleral thickness of group C(group C1:230.00±15.17μm,group C2:176.66±22.06μm,group C3:227.50±17.46μm)was significantly higher than that of group B(group B1:199.17±17.15μm,group B2:230.00±15.17μm,group C2:227.50±17.46μm).202.50±6.89μm,B3group: 126.25±19.48μm)(P < 0.05).The scleral thickness of group E(group E1:264.58±32.03μm,group E2:250.00±29.15μm,group E3:224.17±16.25μm)was significantly higher than that of group D(group D1:212.50±10.37μm,group D2:205.42±7.14μm,group D3:264.58±32.03μm,group E2:250.00±29.15μm,group E3:224.17±16.25μm).181.67±22.51μm)(all P < 0.05).1.5 Histological changes of guinea pig eyeballHE staining showed no significant pathological changes in the thickness of retina and choroid in groups A,B,C,D and E.The sclera of group A showed no significant pathological changes.In group B1,the diameter of scleral collagen decreased,the arrangement of collagen was loose,and the space between collagens increased.In groups B2 and B3,the scleral lesions gradually worsened and collagen fibers broke.The scleral lesions in group C were slighter than those in group B.Compared with group C1,the scleral lesions in groups C2 and C3 were gradually aggravated.In group D1,the diameter of scleral collagen decreased,but the diameter of collagen fibers was uniform,arranged neatly,and the structure was dense.The lesions were aggravated in groups D2 and D3.Compared with group D,the diameter of collagen in group E was thickened and the arrangement of collagen was dense.Compared with group E1,the diameter of scleral collagen in groups E2 and E3 decreased.TUNEL staining showed no positive signal in retina and sclera of group A.There were a few scattered positive signals in the retina and sclera of group B1,and the number of positive signals in groups B2 and B3 was higher than that in group B1.The number of retinal and sclera positive signals in group C was less than that in group B,but compared with group C1,the number of positive signals in group C2 and C3 gradually increased.In group D1,there were continuous positive signals in the retina,and a small number of scattered positive signals in the whole sclera.In groups D2 and D3,the number of positive signals in the retina and sclera gradually increased.The positive signals of retina and sclera in group E were less than those in group D.Compared with group E1,the positive signals of retina in groups E2 and E3 gradually increased,and the positive signals of sclera gradually decreased.Transmission electron microscopy showed no significant pathological changes in the optic nerve axons of guinea pigs in group A.In group B,the optic nerve axis protrusion showed uneven size,irregular shape,atrophy and thinning of myelin sheath,and the degree of lesion was aggravated with time.In group D,the optic nerve axon protrusion of guinea pigs showed similar pathological changes to group B,but were more severe,including giant axons,atrophy and disappearance of axon tissue,and myelin fracture,and the degree of lesion increased with the increase of modeling time.The degree of lesions in groups C and E was significantly lower than that in groups B and D,and a certain number of normal axons remained.Scleral biomechanical changesRelaxation modulus: the average instantaneous relaxation modulus(0.71±0.05)and equilibrium relaxation modulus(0.51±0.070)in group C were significantly higher than those in group A(0.31±0.16,0.11±0.013)and group B(0.23±0.07,0.14±0.068).The average instantaneous relaxation modulus(1.21±0.12)and equilibrium relaxation modulus(0.83±0.13)in group E were significantly higher than those in groups A and D(0.55±0.19,0.32±0.10).(all P < 0.05).Creep rate: the average equilibrium creep rate of group B(0.24±0.0038)was significantly higher than that of group A and group C(group A: 0.0076±0.00064;Group C: 0.012±0.0020);The equilibrium creep rate of group D(0.0050±0.00090)was significantly lower than that of group A,and the average equilibrium creep rate of group E(0.0029±0.00026)was significantly lower than that of groups A and D(all P < 0.05).Maximum tension and maximum stress: the average maximum tension and maximum stress of scleral bands in group B(1.51±0.35N)and 2.18±0.41 were significantly lower than those in group A(3.31±0.28 N,3.51±0.12)and group C(3.50±0.96 N,3.91±0.85).The average maximum tensile force(5.39±0.79N)and maximum stress(6.15±0.89)of the sclera band in group E were significantly higher than those in groups A and D(2.39±0.59 N,3.00±0.060)(all P < 0.05).Young’s modulus: the average Young’s modulus of group C(21.44±2.49 Mpa)was significantly higher than that of group A(15.31±1.18Mpa)and group B(11.56±2.90 Mpa).The average Young’s modulus of scleral bands in group E(40.51±13.04 Mpa)was significantly higher than that in groups A and D(21.98±4.21 Mpa)(all P < 0.05).Expression of MMP-2,TIMP-1 and scleral microstructure changes3.1 PCR: The expression of TIMP1-m RNA in the sclera of guinea pigs in group B was lower than that in group A,and the expression of TIMP1-m RNA in group C was significantly higher than that in group B;The expression of TIMP1-m RNA in group E was significantly higher than that in groups A and D.The expression of MMP2 in group B was significantly higher than that in group A,and the expression of MMP2-m RNA in group C was significantly lower than that in group B.The expression of MMP2-m RNA in the sclera of guinea pigs in group D was higher than that in group A,and the expression of MMP2-m RNA in group E was significantly lower than that in groups A and D(all P <0.05).3.2 WB: The expression of TIMP1 protein in the sclera of guinea pigs in group B was significantly lower than that in group A,and the expression of TIMP1 in group C was significantly higher than that in group B.The expression of TIMP1 in group D was lower than that in group A but higher than that in group B,and the expression of TIMP1 in group E was significantly higher than that in groups A and D.The expression of MMP2 protein in group B was higher than that in group A,and the expression of MMP2 in group C was significantly lower than that in group B.The expression of MMP2 in group D was higher than that in group A,and the expression of MMP2 in group E was significantly lower than that in group D(all P < 0.05).3.3 Scanning electron Microscope:In group A,the diameter of scleral fibers was uniform,the tissue arrangement was regular,and the space between fibers was narrow.In group B,collagen fibers and fiber diameters were uneven and loosely arranged.The scleral lesions in group D were similar to those in group B,but milder than those in group B,degree of pathological changes of two groups were aggravated with time.In groups C and E,the diameter of collagen in the sclera increased,the arrangement of fiber tissues became regular and compact,and the space between fibers decreased,but the enhancement effect weakened with time.Conclusion:1.The guinea pig model of high myopia showed axial growth and diopter increase;the chronic glaucoma model showed increased IOP,and both models resulted in retinal,sclera,and optic nerve axonal damage.Genipin scleral cross-linking can inhibit the development of axial length and diopter in high myopia model,and reduce the damage of retina,sclera and optic nerve axon in myopia and glaucoma model.Genipin scleral crosslinking had good safety,not causing significant damage to the eyeball tissue.2.The creep rate and Young’s modulus of the sclera of high myopic and glaucoma guinea pigs showed opposite changes,but the tensile strength of both decreased.Genipin cross-linking can effectively improve the biomechanical properties of the sclera in the two models.After cross-linking,the Young’s modulus of the sclera is increased,the tensile strength is significantly enhanced,and the expansion of the sclera in high myopia and glaucoma is inhibited.3.Abnormally increased expression of TIMP1 and decreased expression of MMP2 appeared in the eyes of both high myopia and glaucoma guinea pigs,but the degree of related changes in high myopia guinea pigs was higher than that in glaucoma group.Genipin can reverse the imbalance of TIMP1 and MMP2 expression in guinea pig models of high myopia and glaucoma.The scleral collagen fiber lesion of glaucoma guinea pigs was weaker than that of high myopic guinea pigs.Genipin can strengthen scleral collagen fibers,increase the diameter and density of collagen fibers. |
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