大鼠视神经电刺激损伤定量模型的建立和评价

发布时间：2018-08-26 10:04

【摘要】： 随着神经生物学和分子生物学研究的进展，视神经损伤和再生的研究成为眼科学和神经科学共同关注的课题。为了从不同的角度对视神经损伤进行研究，学者们建立了多种视神经损伤模型。间接性损伤模拟临床上闭合性颅脑损伤，由于人和动物解剖结构的差异，并且间接性视神经损伤机制复杂，不能完全代替临床间接性视神经损伤。直接损伤可控性强，损伤可以具体化，易于统一损伤标准。目前直接视神经损伤模型多为完全损伤，视神经横断伤是视神经损伤动物模型中最易建立、最易于统一损伤标准的动物模型，致伤量一致，便于对照研究。国外对视神经部分损伤定量分析主要有两种模型方法：牛顿测力计模型和螺旋测微计标定反向镊损伤模型，但两者也只是一种半定量损伤，虽然这两种模型均可造成不同程度的视神经损伤，但不够简便，而且对致伤强度和损伤程度的量化探讨不够深入。为了深入研究视神经损伤，要建立一种易于量化、标准化、可重复的不同程度视神经损伤的动物模型，对致伤强度、损伤程度进行量化，对视神经损伤程度在结构上和功能上进行定量分析；明确致伤强度和损伤程度之间的联系，即多大的致伤强度能造成多大程度的损伤以及损伤后结构和功能的恢复，同时要使这种模型影响因素单一。这对视神经损伤后再生研究有重要价值。我们对颅内段视神经给予不同强度的电刺激，，用电刺激视神经所用电功量化损伤强度，用视网膜节细胞计数量化损伤程度，以此来标准化、量化视神经损伤，探讨致伤强度和视神经损伤程度之间的关系。材料和方法：选用健康成年雄性wister大鼠180只，体重260-300g，双眼屈光间质清澈及眼底检查正常。 160只动物分成5个刺激电流强度组，0.1mA，0.25mA，0.5 mA，0.75 mA，1.0 mA；每组根据刺激时间再次分为8个亚组(每个亚组4只)，5secs，10 secs，20 secs，30 secs，45 secs，60 secs，75 secs，90 secs，动物随机分配到各组中。对照组：20只未处理的大鼠作为对照，随机分配到5个不同刺激强度组。统一选取右眼作为试验对象。在脑立体定位仪下，根据大鼠脑立体定位图谱确定右侧视神经电损伤钻颅位置(Bregma点前移0.2mm，中线旁开3mm)，微电极毁损颅内段视神经，毁损电压为5 V，频率为60 KHz，通过改变电流强度和刺激时间，造成不同程度视神经损伤。2周后再次麻醉大鼠，4％多聚甲醛磷酸缓冲液心脏灌注后取标本，去除中央区角膜和晶状体，尽可能完全去除玻璃体，沿矢状位方向行视网膜全层切片，片厚4μm，HE染色后计数视神经节细胞层的视神经节细胞。刺激电流强度及刺激时间对视神经节细胞的影响采用析因设计的方差分析；视神经节细胞计数的均数在刺激电流及刺激时间间差异采用LSD法做多重比较，P＜0.05为显著性标准；刺激强度和刺激时间单独效应采用one-wayANOVA分析。电功(电功=电压×电流×时间)和视神经节细胞计数之间的关系采用多个独立样本非参数检验Kruskal-Wallis Test；检验电功和视神经节细胞计数是否为正态分布采用One-Sample Kolmogorov-Smirnov Test；双变量相关分析采用等级相关系数(Spearman相关系数)非参数检验；画出散点图，电功(致伤量)进行变换后进行曲线拟合。结果：正常对照组视网膜层次清晰，各层排列整齐而致密，视网膜视神经节细胞层的细胞单层排列，大小不一，轮廓不规则，胞核大小不一，染色质分布均匀。从核形态学上可明确分为两类：一类为大而浅染的细胞核，胞核有时可见核仁；另一类为小而深染的细胞核。另外可见少量的呈新月形的血管内皮细胞分布于毛细血管内表面。不同强度视神经损伤后同侧视网膜出现不同程度的以下改变：散在空泡化视神经节细胞、节细胞排列紊乱、节细胞数目减少。刺激电流和刺激时间对大鼠视神经节细胞的影响：析因方差分析结果提示，不同刺激时间之间差异有统计学意义(F=3472.142，P＜0.001)，视神经节细胞计数由高到低依次为对照组(45.55±1.19)，5sec(36.65±2.92)，10sec(32.25±4.58)，20sec(25.40±7.07)，30sec(19.35±5.78)，45sec(13.75±3.80)，60sec(10.35±1.87)，75sec(8.20±1.11)，90sec(7.25±0.85)；电流强度组间差异也有统计学意义(F=335.83，P=0.000)，以0.1mA水平测得的视神经节细胞计数最多(26.67±13.99)，其余各组依次为23.64±13.34，21.64±13.26，19.98±12.96，18.69±13.17；选LSD法做多重比较，视神经节细胞计数均数在刺激电流之间及刺激时间之间差异在α=0.05水平均有统计学意义(P＜0.001)；刺激强度和刺激时间的单独效应，除对照组和刺激时间点为90sec外(F=0.792，P=0.548；F=1.538，P=0.242)，同一刺激时间随着电流强度增加，细胞计数呈下降趋势；同一电流组随着刺激时间的增加，细胞计数也呈下降趋势，刺激电流强度和刺激时间之间交互效应显著(F=27.298，P＜0.001)。致伤强度(电功)对视神经节细胞计数的影响行多个独立样本非参数检验Kruskal-Wallis Test。不同电功组间差异有统计学意义(X~2=168.083，P=0.000)，不同电刺激强度对视神经节细胞计数的影响不同。经One-Sample Kolmo-gorov-Smirnov Test，视神经节细胞计数为非正态分布，因此选择等级相关系数(Spearman相关系数)非参数检验，经相关分析，Spearman相关系数r_s=-0.953，P=0.000(双侧)，故认为致伤量和视神经节细胞计数之间存在负相关关系。将致伤量(电功)做变量变换后进行曲线拟合，致伤量和视神经节细胞计数之间有幂函数关系。结论： 1．电毁损颅内段视神经大鼠动物模型致伤因素单一，对致伤强度、损伤程度易于控制和量化。 2．该模型在致伤强度和损伤程度间建立了对应关系：致伤强度和损伤程度间成幂函数曲线关系。因此，电毁损大鼠颅内段视神经动物模型是一种标准化的大鼠视神经损伤模型，该模型致伤因素单一、操作简便、易于重复，而且能够进行定量分析。
[Abstract]:With the development of neurobiology and molecular biology, the study of optic nerve injury and regeneration has become a common concern in ophthalmology and neuroscience.
In order to study the optic nerve injury from different angles, many kinds of optic nerve injury models have been established. Indirect injury can not completely replace clinical indirect optic nerve injury because of the difference of anatomical structure between human and animal and the complicated mechanism of indirect optic nerve injury. At present, the direct optic nerve injury model is mostly complete injury, and the optic nerve transection injury is the easiest animal model to establish and unify the optic nerve injury standard. The injury amount is the same, which is convenient for comparative study. There are two kinds of model methods: Newton dynamometer model and spiral micrometer calibration reverse tweezers injury model, but both of them are only a semi-quantitative injury. Although both models can cause different degrees of optic nerve injury, they are not simple enough, and the quantitative study of injury intensity and degree is not deep enough.
In order to study optic nerve injury deeply, it is necessary to establish an animal model of optic nerve injury which is easy to quantify, standardize and repeatable, quantify the intensity and degree of injury, quantify the degree of injury, quantify the structure and function of optic nerve injury, and clarify the relationship between injury intensity and degree of injury. It is important to study the regeneration of optic nerve after injury.
In order to standardize and quantify the optic nerve injury, we gave different intensity electrical stimulation to the intracranial optic nerve, quantified the damage intensity by electrical stimulation of the optic nerve and quantified the damage degree by retinal ganglion cell count.
Materials and methods:
180 healthy adult male Wister rats weighing 260-300 g were selected. The refractive matrix was clear and the fundus was normal.
160 animals were divided into 5 stimulation current intensity groups, 0.1 mA, 0.25 mA, 0.5 mA, 0.75 mA, 1.0 mA; each group was further divided into 8 subgroups according to stimulation time (4 rats in each subgroup), 5 secs, 10 secs, 20 secs, 30 secs, 45 secs, 60 secs, 75 secs, 90 secs. The animals were randomly allocated to each group. To 5 different stimulation intensity groups.
The right eye was selected as the experimental object. The drilling position of the right optic nerve was determined according to the stereotaxic map of the rat brain (Bregma point moved forward 0.2 mm, 3 mm by the middle line), and the optic nerve was damaged by microelectrode. The damage voltage was 5 V and the frequency was 60 KHz. Two weeks later, the rats were anesthetized again. After cardiac perfusion with 4% paraformaldehyde phosphate buffer, the central cornea and lens were removed. The vitreous body was removed as completely as possible. The retina was sliced along the sagittal plane. The thickness of the slice was 4 microns. The optic ganglion cells were counted after HE staining.
The effects of stimulation current intensity and stimulation time on optic ganglion cells were analyzed by variance analysis of factorial design; the mean counts of optic ganglion cells were compared by LSD method, P < 0.05 was the significant standard; the single effects of stimulation intensity and stimulation time were analyzed by one-way ANOVA. Kruskal-Wallis Test was used to test the relationship between electrical power = voltage * current * time and the count of optic ganglion cells. One-Sample Kolmogorov-Smirnov test was used to test the normal distribution of electrical power and the count of optic ganglion cells. Spearman correlation coefficient was used for bivariate correlation analysis. Non parametric test; draw scatter plot and transform the electric work (injury volume) into curve fitting.
Result:
In the normal control group, the retinal layers were clear, arranged neatly and compactly, and the cells in the retinal optic ganglion cell layer were arranged in a single layer, irregular in size, irregular in outline, and uniformly distributed in chromatin. In addition, a small number of crescent-shaped vascular endothelial cells were found on the inner surface of capillaries. After optic nerve injury of different intensities, the ipsilateral retina showed the following changes in varying degrees: scattered vacuolated optic ganglion cells, disordered arrangement of ganglion cells, and reduced number of ganglion cells.
Effects of stimulation current and stimulation time on rat optic ganglion cells: Factorial analysis of variance showed that there were significant differences between different stimulation time (F = 3472.142, P < 0.001). The counts of optic ganglion cells in the control group were 45.55 (+ 1.19), 5 sec (36.65 (+ 2.92), 10 sec (32.25 (+ 4.58), 20 sec (25.40 (+ 7.07), 30 sec (1.19), respectively. 9.35 [5.78], 45 sec (13.75 [3.80], 60 sec (10.35 [1.87]), 75 sec (8.20 [1.11], 90 sec (7.25 [0.85]) and the difference of current intensity between groups was also statistically significant (F = 335.83, P = 0.000). The number of optic ganglion cells measured at the level of 0.1 mA was the highest (26.67 [13.99]), and the others were 23.64 [13.34, 21.64 [13.26, 19.98] 12.96, 18.69 [13.17] respectively. The mean number of optic ganglion cells was significantly different between stimulation current and stimulation time (P Cell counts decreased with the increase of current intensity, and decreased with the increase of stimulation time in the same current group. The interaction between stimulation current intensity and stimulation time was significant (F = 27.298, P < 0.001).
Kruskal-Wallis Test was used to examine the effect of injury intensity (electrical power) on the counts of optic ganglion cells. There was a significant difference between different electrical power groups (X~2=168.083, P=0.000). Different electrical stimulation intensity had different effects on the counts of optic ganglion cells. The counts were non-normal distribution, so the Spearman correlation coefficient was selected for non-parametric test. The correlation analysis showed that the Spearman correlation coefficient r_s=-0.953, P= 0.000 (both sides), so there was a negative correlation between the number of injuries and the number of optic ganglion cells. After variable transformation, the injuries were fitted to the curve, the amount of injuries and the amount of injuries. There is a power function relationship between visual ganglion cell counts.
Conclusion:
1. Electric injury of intracranial optic nerve rat model has a single injury factor, which is easy to control and quantify the injury intensity and degree.
2. The model establishes a corresponding relationship between the injury intensity and the degree of injury: the relationship between the injury intensity and the degree of injury is a power function curve.
Therefore, the animal model of intracranial optic nerve injury in rats is a standardized model of optic nerve injury in rats. The model is simple, easy to operate, repeatable and can be quantitatively analyzed.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R-332;R779.1

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