甘油醛后巩膜交联治疗豚鼠形觉剥夺性近视的实验研究

发布时间：2018-04-01 20:08

本文选题：形觉剥夺性近视　切入点：豚鼠　出处：《天津医科大学》2012年博士论文

【摘要】：研究目的病理性近视(Pathologic myopia, PM)是成年人重要的致盲性眼病之一,目前尚无良好治疗对策。近视发病机制的研究提示：巩膜是各种致病因素作用的主要靶器官,巩膜病理性的薄弱是PM作用的结果,也是导致其它相关病变的诱因,而针对巩膜生物力学强度变化的干预有可能是阻断PM进展的一条可行途径。本研究通过建立豚鼠形觉剥夺性近视(form deprivation myopia, FDM)模型,应用甘油醛促进巩膜交联,探讨甘油醛对巩膜生物力学特征的影响及其对实验性近视的抑制作用,从控制巩膜重塑并增加其强度的角度,为防治PM提供新思路。研究内容和方法 1.甘油醛对离体巩膜条带的交联作用：选用15只三周龄三色豚鼠,按随机数字表法分成3组,每组5只。均取右眼为实验眼。矢状位取2mm×6mm的巩膜条带,给予0.005mol/L甘油醛组,0.05mol/L甘油醛组和空白对照液室温下浸泡4天。应用微材料力学性能测试系统,对各组巩膜条带的极限应力(σmax (MPa)、极限应变εmax (%)、6%弹性模量E(MPs)进行统计分析。 2.豚鼠FDM模型的建立：用6号乳白色半透明乳胶气球,依照豚鼠头部形状剪制成头套,暴露出动物的左眼,双耳及口鼻。制作动物右眼形觉剥夺模型。室温下饲养,正常昼夜节律。分别于实验开始前、形觉剥夺7、14、21天时测量眼轴长度、玻璃体腔长度及屈光度,进行统计学分析。 3.甘油醛后巩膜交联：选用35只三周龄三色豚鼠,将动物按随机数字表法分成5组,每组7只,右眼为实验眼,左眼为对照眼。A组：右眼遮盖7天；B组：右眼遮盖21天,0.9%生理盐水第1、8、15天Tenon's囊下球后注射；C组：右眼遮盖21天,0.05mol/L甘油醛第1、8、15天Tenon's囊下球后注射；D组：右眼遮盖21天,0.5mol/L甘油醛第1、8、15天Tenon's囊下球后注射；E组：正常对照组。并于实验开始前、形觉剥夺7、14、21天时测量眼轴长度、玻璃体腔长度及屈光度。在实验结束时,应用微材料力学性能测试系统测量5个组巩膜的极限应力σmax (MPa)、极限应变εmax (%)、6%弹性模量E(MPa),并与对侧眼比较,进行统计学分析。绘制5个组的应力-应变曲线,比较甘油醛交联对豚鼠巩膜生物力学强度的影响。 4.后巩膜交联术对豚鼠FDM眼超微结构影响的观察：选用18只三周龄三色豚鼠,随机抽取其中的15只豚鼠,按随机数字表法分成5组。右眼为实验眼,左眼为对照眼。实验方法同3。实验结束后处死豚鼠,在视神经颞侧连续切取5片组织,厚度0.5μm,2片行苏木精-伊红染色,3片行免疫组化染色,检测后极部巩膜、脉络膜、视网膜MMP-2表达,光学显微镜下观察并照相。余下的3只豚鼠给予0.5mol/L的甘油醛Tenon's囊下球后注射,分别在第3、7、14天处死。取注射相邻部位的角膜缘、眼外肌和视神经筛板做切片,行苏木精-伊红染色。判断甘油醛对视网膜、视神经是否有毒性。结果 1.离体巩膜实验：不同浓度甘油醛组与对照组实验眼巩膜的应力-应变曲线有明显的梯度变化。空白对照组巩膜的极限应力σmaax、极限应变smax、6%弹性模量E分别为(7.198±0.991)MPa、(21.480±0.853)%和(22.808±2.159)MPa。甘油醛交联组中,6%弹性模量E0.005mol/L甘油醛组、0.05mo/L甘油醛组分别为(25.903±0.892) MPa (P=0.019)和(36.156±2.026) MPa (P=0.000),较空白对照组分别增加了13.57%和58.52%。0.005mol/L甘油醛组、0.05mo/L甘油醛极限应力σmax分别为(10.386±1.023)MPa(P=0.0012)和(14.851±1.602)MPa (P=0.000),较空白对照组分别增加了44.30%和106.32%；0.005mol/L甘油醛组、0.05mo/L甘油醛组极限应变smax分别为(19.320±±0.672)%(P=0.002)和(14.140±±1.001)%(P=0.000),较空白对照组分别降低10.06%和34.17%。 2.形觉剥夺后,实验眼均出现了玻璃体腔长度和眼轴长度的增加以及相对近视度数的增加,在A组、B组和C组中,玻璃体腔长度、眼轴长度和屈光度与对侧眼相比,差异有统计学意义(P玻璃体腔=0.018,0.002,0.001；P眼轴=0.019,0.002,0.000；P屈光=0.000,0.005,0.001)。D组、E组玻璃体腔长度、眼轴长度与对侧眼相比,差异无统计学意义(P玻璃体腔=0.607,0.539；P眼轴=0.607,0.539)D组实验眼与对侧眼屈光度相比差异有统计学意义(P屈光=0.020)。 3.在实验第21天结束时,B、C、D、E组实验眼的屈光度变化值分别为(8.800±0.616)D,(7.236±2.198)D,(6.271±1.112)D和(0.934±0.158)D,总体差异有统计学意义(F=61.249,P=0.000),B、C、D组的屈光度变化值较正常对照组大,差异有统计学意义(PB=0.000,PC=0.000,PD=0.000)；B、C、D组的眼轴变化值较正常对照组大,但差异无统计学意义(PB=0.430,PC=0.840,PD=0.386) 4.在实验第21天结束时,B组和E组对侧眼比较,B组玻璃体腔长度的增加值为(0.198±0.038)mm,E组为(0.139±0.026)mm,差异有统计学意义(t=-3.689,P=0.002)；B组眼轴长度的增加值为(0.481±0.062)mm,E组为(0.438±0.068)mm,差异无统计学意义(t=-1.295,P=0.216) 5.B组实验眼眼轴长度与屈光呈高度负相关(r=-0.832,P=0.000)；B组实验眼玻璃体腔长度与屈光度呈高度负相关(r=-0.804,P=0.000)；E组右眼的眼轴长度和屈光度呈中度负相关(r=-0.604,P=0.000)；E组双眼的眼轴长度呈高度相关(r=0.940,P=0.000)。 6.试验结束时,B组实验眼的极限应力和6%弹性模量分别为(7.988±3.677)MPa(P=0.002)和(19.938±4.871)MPa(P=0.001),较对侧眼分别降低了34.21%和34.38%,极限应变实验眼为(28.6±3.6)%(P=0.034),较对侧眼增加了19.17%。甘油醛交联后,C组实验眼的极限应力和6%弹性模量分别为(9.244±0.806)MPa(P=0.001)和(26.180±4.388)MPa(P=0.031),较对侧眼分别降低了23.13%和13.34%,极限应变实验眼为(26.2±1.0)%(P=0.016),较对侧眼增加了12.93%；D组实验眼的极限应力为(12.476-2.507)MPa(P=0.580),较对侧眼降低了5.50%,6%弹性模量为(30.446±3.410)MPa(P=0.314),较对侧眼增加了6.53%,极限应变为(23.8±1.8)%(P=0.253),较对侧眼降低了4.42%。在D组(0.5mol/L甘油醛)攻膜交联21天后与B组的实验眼相比,巩膜的极限应力增加了35.85%,6%弹性模量增加了52.70%,极限应变降低了16.78%。不同浓度甘油醛交联与对照组实验眼的应力-应变曲线有明显的梯度变化,D组的应力-应变曲线接近正常对照组,高于其它3组。 7.组织学检查：随着豚鼠FDM的出现,表现为眼外组织疏松,巩膜厚度明显降低,纤维排列整齐,脉络膜厚度降低,可见血管腔,但未见大的窦腔,视网膜层次清晰。MMP-2在眼球表层结缔组织、巩膜表层结缔组织、浅层巩膜、脉络膜实质层以及视网膜外丛状层阳性表达比正常对照眼明显。甘油醛交联后出现了眼球表面结缔组织结构变得相对致密,排列有序,与巩膜连接紧密。巩膜组织的厚度变薄,细胞密度增加。脉络膜管腔扩大,厚度变化不明显。视网膜光感受器细胞核清晰,排列整齐,无炎性细胞浸润。MMP-2在除眼表结缔组织外的巩膜表面结缔组织,浅层巩膜、脉络膜实质层以及视网膜外丛状层的阳性表达明显降低。结论： 1.甘油醛是一种安全有效的交联剂,能够显著增强巩膜的生物力学强度。 2.头套法形觉剥夺成功诱导出以豚鼠为对象的动物近视模型,而且这种近视是以眼轴延长特别是玻璃体腔长度增加为主的轴性近视。 3.随着眼轴的延长及近视度数的加深,巩膜的生物力学强度明显降低。 4.本实验进一步证明了MMP-2与近视形成的相关性,而且发现甘油醛能够降低MMP-2的阳性表达。 5.采用甘油醛交联的方法能够有效控制FDM动物模型眼近视的发展。为防治PM提供新思路。
[Abstract]:Purpose of study

Objective : To study the effect of glyceraldehyde on the biomechanical properties of sclera and to investigate the effect of glyceraldehyde on the biomechanical properties of sclera and to investigate the inhibitory effect of glyceraldehyde on the biomechanical properties of sclera .

Research content and methods

1 . The cross - linking effect of glyceraldehyde on scleral strap in vitro : 15 three - week - old three - color guinea pigs were selected and divided into 3 groups according to the random number table method . The scleral bands of 2 mm 脳 6 mm were taken for the right eye . The ultimate stress ( 蟽max ( MPa ) , ultimate strain 蔚max ( % ) and 6 % elastic modulus E ( MPs ) of scleral strips were analyzed statistically .

2 . Establishment of a guinea pig FDM model : Using a 6 # milky white translucent latex balloon , the head cover is cut according to the shape of the head of the guinea pig to expose the left eye , the double ears and the mouth nose of the animal . The right eye shape deprivation model of the animal is made . The eye axis length , the length of the vitreous cavity and the power of the glass body are measured before the experiment is started , and the length and the power of the vitreous cavity are analyzed statistically .

3 . Posterior scleral cross - linking of glyceraldehyde : 35 three - week - old three - color guinea pigs were selected , the animals were divided into 5 groups according to the random number table method , 7 in each group , the right eye was the experimental eye , the left eye was the control eye . Group A : the right eye covered 7 days ;
Group B : The right eye was covered in 21 days , 0.9 % saline was injected at 1 , 8 , 15 days Tenon ' s capsule .
Group C : The right eye covered 21 days , 0.05 mol / L glyceraldehyde group 1 , 8 , 15 days Tenon ' s capsule was injected after balloon ;
Group D : The right eye was covered in 21 days , 0.5 mol / L glyceraldehyde group 1 , 8 , 15 days Tenon ' s capsule was injected after balloon ;
At the end of the experiment , the ultimate stress 蟽max ( MPa ) , the ultimate strain 蔚max ( % ) , the 6 % elastic modulus E ( MPa ) of the sclera in the five groups were measured and compared with the contralateral eye , and the effects of glyceraldehyde crosslinking on the biomechanical strength of the sclera of guinea pig were compared .

4 . The effects of posterior scleral cross - linking on the ultrastructure of FDM eyes in guinea pigs were observed : 15 guinea pigs were randomly selected from 18 guinea pigs with three - week - old three - color guinea pigs .

Results

1 . In vitro scleral experiment : The stress - strain curve of sclera was significantly changed in different concentration glyceraldehyde group and control group . The limit stress 蟽maax , ultimate strain smax , and 6 % elastic modulus E of the scleral were ( 7.198 卤 0.981 ) MPa , ( 21.480 卤 0.853 ) % and ( 22.808 卤 2.159 ) MPa respectively . The limit stress 蟽max of the 0 . 05 mol / L glyceraldehyde group was ( 10.386 卤 1.023 ) MPa ( P = 0.0012 ) and ( 14.851 卤 1.602 ) MPa ( P = 0.000 ) , respectively , and the blank control group increased 44.30 % and 106.32 % respectively .
The limit strain smax of the 0.05 mol / L glyceraldehyde group was ( 19.320 卤 0.672 ) % ( P = 0.002 ) and ( 14.140 卤 1.001 ) % ( P = 0.000 ) , respectively , which were 10.06 % and 34.17 % respectively .

2 . After deprivation , the vitreous cavity length and the axial length of vitreous cavity and relative myopic degree increased in the experimental eyes . In group A , group B and C , the length of vitreous cavity , the length of the eye axis and the refractive power were significantly different from that of the contralateral eye ( P glass body cavity = 0 . 018 , 0.002 , 0.001 ;
P - eye axis = 0.019 , 0.002 , 0.000 ;
P refractive index = 0.000 , 0.005 , 0.001 ) . Compared with the contralateral eye , there was no significant difference in the length of vitreous cavity , the length of the vitreous cavity and the length of the vitreous cavity in group D and E ( P glass body cavity = 0.607 , 0.539 ;
P - eye axis = 0.607 , 0.539 , the difference was statistically significant ( P refractive index = 0.020 ) .

3 . At the end of the 21st day of the experiment , the refractive changes of the experimental eyes of B , C , D and E groups were ( 8.800 卤 0.616 ) D , ( 7.236 卤 2.198 ) D , ( 6.271 卤 1.112 ) D and ( 0.934 卤 0.158 ) D , respectively . The total difference was statistically significant ( F = 61.249 , P = 0.000 ) , and the difference was statistically significant ( PB = 0.000 , PC = 0.000 , PD = 0.000 ) ;
The changes of ocular axis in group B , C and D were higher than those in the control group , but the difference was not statistically significant ( PB = 0.430 , PC = 0.840 , PD = 0.386 ) .

4 . At the end of the 21st day of experiment , the increase of vitreous cavity length in group B and group E were ( 0.198 卤 0.038 ) mm and ( 0.139 卤 0.026 ) mm , respectively , and the difference was statistically significant ( t = - 3.689 , P = 0.002 ) .
The increase of ocular axial length in group B was ( 0.481 卤 0.062 ) mm , E group was ( 0.438 卤 0.068 ) mm , there was no significant difference ( t = - 1.295 , P = 0.216 ) .

In group B , the ocular axial length was negatively correlated with the refractive index ( r = - 0.832 , P = 0.000 ) .
In group B , the vitreous cavity length was negatively correlated with the refractive power ( r = - 0.804 , P = 0.000 ) .
There was a moderate negative correlation between the ocular axis length and the refractive power of the right eye ( r = - 0.604 , P = 0.000 ) .
The ocular axial length of both eyes was highly correlated ( r = 0.940 , P = 0.000 ) .

6 . At the end of experiment , the ultimate stress and 6 % elastic modulus of the experimental eyes of group B were ( 7.988 卤 3.677 ) MPa ( P = 0.002 ) and ( 19.938 卤 4.871 ) MPa ( P = 0.001 ) , respectively . The ultimate stress and 6 % elastic modulus of experimental eyes were ( 9.244 卤 0.806 ) MPa ( P = 0.031 ) and ( 26.180 卤 4.388 ) MPa ( P = 0.031 ) , respectively .
In group D , the ultimate stress was ( 12.476 - 2.507 ) MPa ( P = 0.580 ) , the side - eye was reduced by 5.50 % , the elastic modulus of 6 % was ( 30.446 卤 3.410 ) MPa ( P = 0.314 ) , the limit strain was ( 23.8 卤 1.8 ) % ( P = 0.253 ) .

7 . Histological examination : With the appearance of FDM in guinea pigs , the thickness of sclera decreased significantly , the fibrous arrangement was orderly , the thickness of the sclera was decreased , and the retinal layer was clear . The thickness of the scleral tissues became thinner and the density of the sclera was increased . The expression of MMP - 2 in the scleral surface connective tissue , scleral surface connective tissue , the superficial sclera , the choroidoidal substance and the outer retina of the sclera decreased significantly .

Conclusion :

1 . glyceraldehyde is a safe and effective crosslinking agent , which can significantly enhance the biomechanical strength of sclera .

2 . The myopia model of the guinea pig is induced successfully by the method of the method of headgear , and the myopia is the axial myopia which mainly increases the length of the vitreous cavity , especially the length of the vitreous cavity .

3 . With the extension of the ocular axis and the degree of myopia , the biomechanical strength of sclera decreased significantly .

4 . This experiment further proved the correlation between MMP - 2 and myopia , and found that glyceraldehyde could reduce the expression of MMP - 2 .

5 . The method of crosslinking glyceraldehyde can effectively control the development of ocular myopia in FDM animal model .

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R778.11

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