大鼠视觉发育关键期视网膜相干光断层成像变化与Bcl-2、Bax、Caspase-3mRNA表达的相关性研究

In vivo high-resolution imaging of rat retina with optical coherence tomography and the expression of Bcl-2, Bax, Caspase-3 mRNA during critical period plasticity

目的了解SD大鼠视觉发育关键期内视网膜厚度的变化，探讨细胞凋亡的相关作用。方法实验研究。选择新生SD大鼠50只，其中10只（20只眼）于生后14、18、21、24及42d行双眼眼底相干光断层扫描（OCT），测量视网膜厚度。另10只大鼠（20只眼）分别于生后14、18、21、24及42d各取2只大鼠（4只眼），提取视网膜组织总RNA，实时荧光定量PCR法检测Bcl-2、Bax和Caspase-3mRNA表达水平。另20只大鼠（40只眼）于各对应时间点取4只（8只眼），制作眼球石蜡切片行HE染色并行视网膜厚度测量。同时取10只大鼠（20只眼）于各对应时间点取2只（4只眼）制作冰冻切片行TUNEL染色。对不同时间点的视网膜厚度，Bcl-2、Bax及Caspase-3的mRNA相对表达量进行方差分析，对两种方法所测视网膜厚度进行线性回归分析。结果大鼠生后14、18、21、24及42d视网膜厚度OCT测量值分别为（243．42±13．83）、（218．78±8．21）、（195．42±8．02）、（195．74±14．85）及（190．79±11．70）μm，差异有统计学意义（F=15．425，P=0．000）。在大鼠生后3周内，视网膜厚度逐渐变薄，此后变化不显著。其结果与HE切片视网膜厚度测量呈线性相关（R＝0．794，P=0．000）。比较不同时间点大鼠视网膜组织Bcl-2、Bax和Caspase－3mRNA相对表达量，差异均有统计学意义（F=18．684，F=47．307，F=49．611；P=0．000）。Bax和Caspase－3mRNA表达呈增加趋势，并在生后24d呈现峰值，生后42d表达量明显下降。TUNEL染色结果显示生后18d至42d视网膜神经节细胞层、内核层和外核层均可见较多凋亡细胞，生后42d视网膜细胞凋亡明显减少。结论视觉发育关键期内大鼠视网膜厚度逐渐变薄。CirrusHD—OCT可较准确测量大鼠视网膜厚度，是观察视网膜形态学变化的有效检查方法之一。视觉发育关键期内，细胞凋亡参与大鼠视网膜发育，可能影响视网膜厚度的变化。

Objective To observe the changes of retinal thickness during critical period plasticity in rat, and to investigate whether apoptosis participates in the structural forming of retina. Methods Experimental research. 50 normal newborn pups of SD rat were randomly selected in the experiment. In vivo consecutive scanning of retinal image was taken and the retinal thickness from RPE to ILM was recorded in 10 pups （20 eyes） with spectral domain optical coherence tomography （OCT） at postnatal day 14 （P14）, P18, 1）21, P24 and P42. Bel-2, Bax, Caspase-3 mRNA was assessed with fluorescent quantitative real-time polymerase chain reaction after total RNA extracted from 4 retinas of 2 pups at each time point from P14 to P42. Histological measurement of retinal thickness of sections with HE staining from 4 pups （8 eyes ） at each time point was compared with the results of OCT scanning. TUNEL staining was used to detect the apoptotic cells in retinal cyrosections of 2 pups （4 eyes） at the same time point. The data were analyzed by One-way ANOVA and Linear Regression through SPSS 11.5 software. Results There was significant difference between the retinal thickness measured through OCT in pups from P14 to P42 （F = 15. 425, P = 0. 001）. And the values were （243.42 ± 13.83）μm at P14, （218. 78 ± 8.21 ） μm at P18, （ 195.42 ± 8.02） μm at P21, （ 195.74 ± 14. 85 ） μm at P24, （ 190. 79 ± 11.70） μm at P42. The retinal thickness measured through OCT decreased significantly during the first 3 weeks after birth. The results of OCT measurement had linear correlation with histology measurement （R = 0. 794, P = 0. 000 ）. There was significant difference between mRNA expression of Bcl-2, Bax and Caspase-3 in pups from P14 to P42 （ F = 18. 684, F =47. 307 ,F =49. 611 ;P =0. 000）. The relative expression of Bax and Caspase-3 peaked at P24 while Bcl-2 was much more stable. There were a lot of apoptotic cells in the ganglion ceils layer, the inner nuclear layer and the outer nuclear layer during P18 to P24 by TUNEL staining. And the apoptosis alleviated at P42. Conclusions The retinal thickness decreases when the retina continues to develop during critical period plasticity. Cirrus HD-OCT can be used as an effective instrument to show the layers of retina in rat in vivo. Apoptosis participates in the course of retinal development which possibly leads to the thinning of retina.