【摘要】： 隨著神經(jīng)生物學(xué)和分子生物學(xué)研究的進(jìn)展，視神經(jīng)損傷和再生的研究成為眼科學(xué)和神經(jīng)科學(xué)共同關(guān)注的課題。 為了從不同的角度對(duì)視神經(jīng)損傷進(jìn)行研究，學(xué)者們建立了多種視神經(jīng)損傷模型。間接性損傷模擬臨床上閉合性顱腦損傷，由于人和動(dòng)物解剖結(jié)構(gòu)的差異，并且間接性視神經(jīng)損傷機(jī)制復(fù)雜，不能完全代替臨床間接性視神經(jīng)損傷。直接損傷可控性強(qiáng)，損傷可以具體化，易于統(tǒng)一損傷標(biāo)準(zhǔn)。目前直接視神經(jīng)損傷模型多為完全損傷，視神經(jīng)橫斷傷是視神經(jīng)損傷動(dòng)物模型中最易建立、最易于統(tǒng)一損傷標(biāo)準(zhǔn)的動(dòng)物模型，致傷量一致，便于對(duì)照研究。國(guó)外對(duì)視神經(jīng)部分損傷定量分析主要有兩種模型方法：牛頓測(cè)力計(jì)模型和螺旋測(cè)微計(jì)標(biāo)定反向鑷損傷模型，但兩者也只是一種半定量損傷，雖然這兩種模型均可造成不同程度的視神經(jīng)損傷，但不夠簡(jiǎn)便，而且對(duì)致傷強(qiáng)度和損傷程度的量化探討不夠深入。 為了深入研究視神經(jīng)損傷，要建立一種易于量化、標(biāo)準(zhǔn)化、可重復(fù)的不同程度視神經(jīng)損傷的動(dòng)物模型，對(duì)致傷強(qiáng)度、損傷程度進(jìn)行量化，對(duì)視神經(jīng)損傷程度在結(jié)構(gòu)上和功能上進(jìn)行定量分析；明確致傷強(qiáng)度和損傷程度之間的聯(lián)系，即多大的致傷強(qiáng)度能造成多大程度的損傷以及損傷后結(jié)構(gòu)和功能的恢復(fù)，同時(shí)要使這種模型影響因素單一。這對(duì)視神經(jīng)損傷后再生研究有重要價(jià)值。 我們對(duì)顱內(nèi)段視神經(jīng)給予不同強(qiáng)度的電刺激，，用電刺激視神經(jīng)所用電功量化損傷強(qiáng)度，用視網(wǎng)膜節(jié)細(xì)胞計(jì)數(shù)量化損傷程度，以此來標(biāo)準(zhǔn)化、量化視神經(jīng)損傷，探討致傷強(qiáng)度和視神經(jīng)損傷程度之間的關(guān)系。 材料和方法： 選用健康成年雄性wister大鼠180只，體重260-300g，雙眼屈光間質(zhì)清澈及眼底檢查正常。 160只動(dòng)物分成5個(gè)刺激電流強(qiáng)度組，0.1mA，0.25mA，0.5 mA，0.75 mA，1.0 mA；每組根據(jù)刺激時(shí)間再次分為8個(gè)亞組(每個(gè)亞組4只)，5secs，10 secs，20 secs，30 secs，45 secs，60 secs，75 secs，90 secs，動(dòng)物隨機(jī)分配到各組中。對(duì)照組：20只未處理的大鼠作為對(duì)照，隨機(jī)分配到5個(gè)不同刺激強(qiáng)度組。 統(tǒng)一選取右眼作為試驗(yàn)對(duì)象。在腦立體定位儀下，根據(jù)大鼠腦立體定位圖譜確定右側(cè)視神經(jīng)電損傷鉆顱位置(Bregma點(diǎn)前移0.2mm，中線旁開3mm)，微電極毀損顱內(nèi)段視神經(jīng)，毀損電壓為5 V，頻率為60 KHz，通過改變電流強(qiáng)度和刺激時(shí)間，造成不同程度視神經(jīng)損傷。2周后再次麻醉大鼠，4％多聚甲醛磷酸緩沖液心臟灌注后取標(biāo)本，去除中央?yún)^(qū)角膜和晶狀體，盡可能完全去除玻璃體，沿矢狀位方向行視網(wǎng)膜全層切片，片厚4μm，HE染色后計(jì)數(shù)視神經(jīng)節(jié)細(xì)胞層的視神經(jīng)節(jié)細(xì)胞。 刺激電流強(qiáng)度及刺激時(shí)間對(duì)視神經(jīng)節(jié)細(xì)胞的影響采用析因設(shè)計(jì)的方差分析；視神經(jīng)節(jié)細(xì)胞計(jì)數(shù)的均數(shù)在刺激電流及刺激時(shí)間間差異采用LSD法做多重比較，P＜0.05為顯著性標(biāo)準(zhǔn)；刺激強(qiáng)度和刺激時(shí)間單獨(dú)效應(yīng)采用one-wayANOVA分析。電功(電功=電壓×電流×?xí)r間)和視神經(jīng)節(jié)細(xì)胞計(jì)數(shù)之間的關(guān)系采用多個(gè)獨(dú)立樣本非參數(shù)檢驗(yàn)Kruskal-Wallis Test；檢驗(yàn)電功和視神經(jīng)節(jié)細(xì)胞計(jì)數(shù)是否為正態(tài)分布采用One-Sample Kolmogorov-Smirnov Test；雙變量相關(guān)分析采用等級(jí)相關(guān)系數(shù)(Spearman相關(guān)系數(shù))非參數(shù)檢驗(yàn)；畫出散點(diǎn)圖，電功(致傷量)進(jìn)行變換后進(jìn)行曲線擬合。 結(jié)果： 正常對(duì)照組視網(wǎng)膜層次清晰，各層排列整齊而致密，視網(wǎng)膜視神經(jīng)節(jié)細(xì)胞層的細(xì)胞單層排列，大小不一，輪廓不規(guī)則，胞核大小不一，染色質(zhì)分布均勻。從核形態(tài)學(xué)上可明確分為兩類：一類為大而淺染的細(xì)胞核，胞核有時(shí)可見核仁；另一類為小而深染的細(xì)胞核。另外可見少量的呈新月形的血管內(nèi)皮細(xì)胞分布于毛細(xì)血管內(nèi)表面。不同強(qiáng)度視神經(jīng)損傷后同側(cè)視網(wǎng)膜出現(xiàn)不同程度的以下改變：散在空泡化視神經(jīng)節(jié)細(xì)胞、節(jié)細(xì)胞排列紊亂、節(jié)細(xì)胞數(shù)目減少。 刺激電流和刺激時(shí)間對(duì)大鼠視神經(jīng)節(jié)細(xì)胞的影響：析因方差分析結(jié)果提示，不同刺激時(shí)間之間差異有統(tǒng)計(jì)學(xué)意義(F=3472.142，P＜0.001)，視神經(jīng)節(jié)細(xì)胞計(jì)數(shù)由高到低依次為對(duì)照組(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)；電流強(qiáng)度組間差異也有統(tǒng)計(jì)學(xué)意義(F=335.83，P=0.000)，以0.1mA水平測(cè)得的視神經(jīng)節(jié)細(xì)胞計(jì)數(shù)最多(26.67±13.99)，其余各組依次為23.64±13.34，21.64±13.26，19.98±12.96，18.69±13.17；選LSD法做多重比較，視神經(jīng)節(jié)細(xì)胞計(jì)數(shù)均數(shù)在刺激電流之間及刺激時(shí)間之間差異在α=0.05水平均有統(tǒng)計(jì)學(xué)意義(P＜0.001)；刺激強(qiáng)度和刺激時(shí)間的單獨(dú)效應(yīng)，除對(duì)照組和刺激時(shí)間點(diǎn)為90sec外(F=0.792，P=0.548；F=1.538，P=0.242)，同一刺激時(shí)間隨著電流強(qiáng)度增加，細(xì)胞計(jì)數(shù)呈下降趨勢(shì)；同一電流組隨著刺激時(shí)間的增加，細(xì)胞計(jì)數(shù)也呈下降趨勢(shì)，刺激電流強(qiáng)度和刺激時(shí)間之間交互效應(yīng)顯著(F=27.298，P＜0.001)。 致傷強(qiáng)度(電功)對(duì)視神經(jīng)節(jié)細(xì)胞計(jì)數(shù)的影響行多個(gè)獨(dú)立樣本非參數(shù)檢驗(yàn)Kruskal-Wallis Test。不同電功組間差異有統(tǒng)計(jì)學(xué)意義(X~2=168.083，P=0.000)，不同電刺激強(qiáng)度對(duì)視神經(jīng)節(jié)細(xì)胞計(jì)數(shù)的影響不同。經(jīng)One-Sample Kolmo-gorov-Smirnov Test，視神經(jīng)節(jié)細(xì)胞計(jì)數(shù)為非正態(tài)分布，因此選擇等級(jí)相關(guān)系數(shù)(Spearman相關(guān)系數(shù))非參數(shù)檢驗(yàn)，經(jīng)相關(guān)分析，Spearman相關(guān)系數(shù)r_s=-0.953，P=0.000(雙側(cè))，故認(rèn)為致傷量和視神經(jīng)節(jié)細(xì)胞計(jì)數(shù)之間存在負(fù)相關(guān)關(guān)系。將致傷量(電功)做變量變換后進(jìn)行曲線擬合，致傷量和視神經(jīng)節(jié)細(xì)胞計(jì)數(shù)之間有冪函數(shù)關(guān)系。 結(jié)論： 1．電毀損顱內(nèi)段視神經(jīng)大鼠動(dòng)物模型致傷因素單一，對(duì)致傷強(qiáng)度、損傷程度易于控制和量化。 2．該模型在致傷強(qiáng)度和損傷程度間建立了對(duì)應(yīng)關(guān)系：致傷強(qiáng)度和損傷程度間成冪函數(shù)曲線關(guān)系。 因此，電毀損大鼠顱內(nèi)段視神經(jīng)動(dòng)物模型是一種標(biāo)準(zhǔn)化的大鼠視神經(jīng)損傷模型，該模型致傷因素單一、操作簡(jiǎn)便、易于重復(fù)，而且能夠進(jìn)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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R-332;R779.1

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相關(guān)期刊論文 前2條

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本文編號(hào)：2204521