不同波长单色光对豚鼠视网膜血管活性肠肽动态表达的影响

Effects of different monochromatic lights on the dynamic expression of retinal vasoactive intestinal polypeptide in guinea pigs

目的观察不同波长单色光对豚鼠眼球生长发育及视网膜血管活性肠肽(VIP)动态表达的影响,探讨可能的调节机制。设计实验性研究。研究对象1周龄英国种短毛三色雄性豚鼠36只。方法 36只豚鼠随机分为A、B、C三组,每组12只,A组饲养在红光环境中(610 nm),B组饲养在蓝光环境中(430 nm),C组为对照组饲养在混合白光中(光谱),光照度均为200 lux。分别在实验前和实验后3、6周测量豚鼠眼球屈光度(RX)和眼轴(AL)。并于各时点每组随机抽取4只摘取双侧眼球,获得巩膜干重,并制作视网膜连续切片10张,2张常规HE染色,2张用于对照,其余6张采用SP三步法进行免疫组织化学染色,在光学显微镜下摄像分析(高倍视野×400),计算每张切片高倍镜下阳性细胞数目,并采用北京航空航天大学的Cmias4.0病理图像分析系统软件对切片VIP阳性信号进行图像分析,表达强度用积分光密度(IOD)值表示。PBS缓冲液作为阴性对照,阳性对照取豚鼠小肠石蜡切片。主要指标屈光度、眼轴、巩膜干重、视网膜VIP阳性细胞计数以及VIP积分光密度值(IOD)。结果试验后6周,红光组、蓝光组及对照组豚鼠眼球屈光度分别为(2.696±0.171)D、(5.139±0.151)D和(3.161±0.122)D,(F=605.169,P=0.000);各组眼轴分别为(8.273±0.165)mm、(8.019±0.151)mm和(8.161±0.120)mm,(F=6.009,P=0.009);各组巩膜干重分别为(0.609±0.088)mg、(0.716±0.101)mg和(0.680±0.041)mg,(F=2.292,P=0.126),但红光组与蓝光组相比,差异有统计学意义(F=1.256,P=0.048)。VIP在豚鼠视网膜内丛状层、神经节细胞层及神经纤维层强表达,在内核层、光感受器细胞层散在表达。与对照组相比,红光组视网膜VIP表达最高,阳性细胞计数43.250±9.939,IOD值1.622±0.119;蓝光组视网膜VIP表达最低,阳性细胞计数27.500±4.928,IOD值1.273±0.127,三组差异有显著统计学意义(F=8.478,15.082;P=0.002,0.000)。结论长波长红光诱导近视形成,短波长蓝光起抑制作用。VIP可能通过影响巩膜形态功能参与单色光对眼球生长发育的调节,是长波长红光近视诱导过程中的调控分子,但具体的信号通路有待于进一步探讨。

Objective To observe the effects of monochromatic lights with similar luminance on the preditable changes in eye growth and expression of vasoactive intestinal polypeptide （VIP） in guinea pigs and to discuss the relative mechanism. Design Experimental study. Participants Thirty-six 1 week old British species of tri-color male guinea pigs. Method 36 guinea pigs were randomly divided into A, B and C groups, each with 12, which were respectively raised in red light （610 nm, long wave-length）, bule light （430 nm, short wave-length） and white light （broad spectrum） with same luminance （200 lux）. At the time of 0 w, 3 w and 6 w of experiment, refraction and axial length were measured. Besides, 4 guinea pigs of each groups were randomly selected to be removed eyes at the 6th. The eyes removed were fixed 48 h in 4% Paraformaldehyde phosphate buffer solution, and then separated selera from retina and choroids. Calculated dry weight of sclera obtained. While the left retina tissues were made into paraffin sections to HE staining and detect expression of VIP with immunohistochemistry （SP three steps）. Main Outcome Measures Refraction, axial length, dry weight of selera, counts of retina VIP-immunoreaetive cells and the value of retina VIP integrated optical density （IOD）. Results At the end of study, refraction of red, blue, and white light groups was respectively 2.696±0.171 D, 5.139±0.151 D, and 3.161±0.122 D （F=605.169, P=0.000）, whereas the corresponding axial length of three groups was repeetively 8.273±0.165 mm, 8.019±0.151 mm, and 8.161±0.120 mm （F=6.009,P=0.009）. Average dry weight of sclera in each group was 0.609±0.088 mg,0.716±0.101 mg, and 0.680±0.041 mg with no statistic difference （F=2.292,P=0.126） yet, there was a significant variation between that of Group A and Group B （F=l.256,P=0.048）.VIP was most expressed in inner plexiform layer , ganglion cell layer and nerve fiber layer of retina, with scattered expressed in inner nuclear layer and photoreceptor cells layer. In addition, the expression of VIP in retina was most in Group A, the number of retina VIP-immunoreactive cells anti the value of retina VIP IOD were respectively 43.250±9.939 and 1.622±0.119; while that was least in Group B, corresponding results of the two measurements mentioned above were 27.500±4.928 and 1.273±0.127 respectively. There existed great difference among three groups （F=8.478, 5.082： P=0.002, 0.000）. Conclusions Red light（long wave-length） could promote eye growth and myopia occurrence, while blue light （short wave-length） was on the opponent. VIP, a potential signal molecular, may be involved in the regulation of monochromatic lights on eye growth, by affecting scleral metabolism and morphological structure.