小鼠睁眼前后角膜神经纤维长度与密度的变化及其意义

Changes in corneal nerve fiber length and density before and after eye opening in mouse and its significance

背景目前已有许多关于哺乳动物发育阶段角膜神经分布的研究，但是尚无对睁眼前后这几个时间段角膜神经分布的研究。目的了解小鼠睁眼前后角膜神经纤维长度与密度随着年龄增长的变化规律，为进一步的实验和临床研究提供依据。方法SPF级C57BL／6小鼠分为出生后1d（P1d）、P7d、P13d（睁眼前1d）、P14d（半睁眼）、P17d（完全睁眼后1d）和P23d（完全睁眼后7d），各组均5只鼠10只眼。采用β-微管蛋白Ⅲ抗体进行整张角膜铺片免疫荧光染色，使用Delta Vision Core拍摄角膜背鼻侧、背颞侧、腹鼻侧和腹颞侧的神经纤维图像，根据体视学测量线性结构的原理，运用Image—Pro Plus5．1软件中的“手动测量-建立多边形特征”计算角膜面积，并且使用LnsgRand．grd曲线（间距为53μm）分别计算各组小鼠的角膜面积、神经纤维总长度以及各分区的神经纤维密度。结果C57／BL6小鼠P1、P7、P13、P14、P17、P23d的角膜面积分别为（0．404±O．007）、（1．362±0．154）、（1．573±O．080）、（1．603±0．046）、（1．8474±0．052）、（2．445±O．798）mm^2，神经纤维总长度值分别为（3．718±1．044）、（19．065±3．350）、（23．687±0．907）、（27．309±2．477）、（31．989±3．976）、（41．214±1．573）mm，神经纤维密度分别为（9．592±1．138）、（14．506±1．908）、（15．088±1．241）、（16．772±1．897）、（16．8214±2．102）、（17．660±1．216）mm／mm^2，均随鼠龄的增长而增加，差异均有统计学意义（F=22．906、0．424、2．375，P=0．000），角膜面积和神经纤维长度之间呈正相关（r=0．983，P〈0．01）。角膜4个分区的神经纤维密度随年龄的增长而增加（背鼻侧区：F=0．159，P=0．000；背颞侧区：F=2．172，P=0．001；腹鼻侧区：F=1．998，P=0．000；腹颞侧区：F=2．352，P=0．000）。其中从P13d到P14d，背鼻侧区的神经纤维密度减少了6．O％，差异无统计学意义（t=0．589，P=0．572）；而且从P14d到P17d，背颞侧区、腹鼻侧区和腹颞侧区的神经纤维密度分别减少了4．6％、5．5％和0．1％，差异均无统计学意义（t=0．549，P=0．596；t=0．701，P=0．501；t=-0．100，P=0．919）。结论小鼠睁眼过程对整个角膜或角膜各分区神经纤维的发育都有不同程度的影响。从P17d开始，小鼠角膜神经纤维呈追赶性增长，恢复增长趋势。

Background Recently, there were many studies on corneal innervations during mammalian development. However, there were fewer studies on discussing corneal innervations before and after mouse eye openings. Objective The present study was to investigate the change in the regulation of corneal nerve fiber length and density before and after mouse eye openings to offer a basis for clinical research in hmnan. Methods Thirty SPF C57BL/6 mice were divided into postnatal 1 day （P1 d） , P7 d, P13 d （1 day before eye opening）, P14 d （eye halfopened ） , P17 d （ 1 day after eye opening）and P23 d （7 day after eye opening）groups, with 5 mice and 10 eyes for each group. Entire corneal stretches were prepared and immunostaining with an anti-neuron-specific β-Ⅲ tubulin antibody was performed to label the corneal nerve fibers. Confocal microscopic pictures fi＇om the corneal dorsal-nasal region （ DN ） , dorsal-temporal （DT） , ventral-nasal region （ VN ） and ventral- temporal （VT） were taken using Delta Vision Core. From these pictures, the mouse corneal area, total length and density of nerve fibers in the 4 regions were calculated. The use of the animals complied with Statement of ARVO. Results Corneal areas of P1 d,P7 d, P13 d, P14 d,P17 d and P23 d mice were（0. 404±0. 007） , （1. 362±0. 154） , （1. 573±0. 080） , （ 1. 603±0. 046） , （1. 847± 0. 052）, （ 2. 445 ± 0. 798 ） mm2 , respectively; the total lengths of nerve fibers were （ 3. 718 ± l. 044 ） , （ 19. 065 ± 3. 350 ） , （23. 687 ±0. 907 ） , （27. 309 ± 2. 477 ） , （31. 989 ± 3. 976 ） , （41. 214 ± 1. 573 ） ram, respectively;the densitiesof nerve fibers were （9. 592 ± 1. 138 ） , （ 14. 506 ～ 1. 908 ） , （ 15. 088± 1. 241 ） , （ 16. 772 ～ 1. 897 ） , （ 16. 821 ～2. 102 ） , （ 17. 660～ 1. 216）mm^2mm^2 , respectively, all showing significant increases with age （ F = 22. 906, P = 0. 000; F = 0. 424, P = 0. 000 ; F = 2. 375, P = 0. 000 ）. A positive correlation of the increasing corneal areas and increasing lengths of nerve fibers was found（ r=0. 983 ,P〈0.01 ）. Nerve fiber densities in the four corneal regions significantly increased with age（DN region：F= 0. 159,P = 0. 000; DT region： F = 2. 172,P = 0. 001 ; VN region： F = 1. 998,P = 0. 000; VT region：F=2. 352 ,P=0. 000）. From P13 d to P14 d,the corneal nerve fiber densities in the DN region decreased by 6.0% without significant difference （t = 0. 589,P = 0. 572） ;and the corneal nerve fiber densities in the DT region, VN region and VT region decreased by 4.6% ,5.5% and 0. 1% ,respectively,without significant difference from P14 d to P17d（t=0.549,P=0.596;t=0.701,P=0.501;t=-0.100,P=0.919）. Conclusions The development of nerve fibers in the whole cornea or the four corneal regions is influenced by eye opening in mouse to various extents. From P13 d to P14 d,the corneal nerve fiber densities in the DN region decreased by 6.0% without significant difference. From P14 d to P17 d, the corneal nerve fiber densities in the DT region, VN region and VT region decrease by 4. 6% , 5.5% and 0. 1% ,respectively,without significant difference. Afterwards,the growth of nerve fibers increased in pace and the growth rate is recovered.