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C57B L/6小鼠分为出生后1 d(P1 d)、P7d、P13 d(睁眼前1d)、P14 d(半睁眼)、P17 d(完全睁眼后1d)和P23 d(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、腹鼻侧和腹颢侧的神经纤维图像,根据体视学测量线性结构的原理,运用Image-Pro Plus 5.1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53 μm)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、P23 d的角膜面积分别为(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,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)、(16.821±2.102)、(17.660±1.216)mm/mm2,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；背颢侧区: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P13 d到P14 d,背鼻侧区的神经纤维密度减少了6.0％,差异无统计学意义(t=0.589,P=0.572)；而且从P14 d到P17 d,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P17 d开始,小鼠角膜神经纤维呈追赶性增长,恢复增长趋势.
揹景 目前已有許多關于哺乳動物髮育階段角膜神經分佈的研究,但是尚無對睜眼前後這幾箇時間段角膜神經分佈的研究.目的 瞭解小鼠睜眼前後角膜神經纖維長度與密度隨著年齡增長的變化規律,為進一步的實驗和臨床研究提供依據.方法 SPF級C57B L/6小鼠分為齣生後1 d(P1 d)、P7d、P13 d(睜眼前1d)、P14 d(半睜眼)、P17 d(完全睜眼後1d)和P23 d(完全睜眼後7d),各組均5隻鼠10隻眼.採用β-微管蛋白Ⅲ抗體進行整張角膜鋪片免疫熒光染色,使用Delta Vision Core拍攝角膜揹鼻側、揹顳側、腹鼻側和腹顥側的神經纖維圖像,根據體視學測量線性結構的原理,運用Image-Pro Plus 5.1軟件中的“手動測量-建立多邊形特徵”計算角膜麵積,併且使用LnsgRand.grd麯線(間距為53 μm)分彆計算各組小鼠的角膜麵積、神經纖維總長度以及各分區的神經纖維密度.結果 C57/BL6小鼠P1、P7、P13、P14、P17、P23 d的角膜麵積分彆為(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,神經纖維總長度值分彆為(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,神經纖維密度分彆為(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/mm2,均隨鼠齡的增長而增加,差異均有統計學意義(F=22.906、0.424、2.375,P=0.000),角膜麵積和神經纖維長度之間呈正相關(r=0.983,P＜0.01).角膜4箇分區的神經纖維密度隨年齡的增長而增加(揹鼻側區:F=0.159,P=0.000；揹顥側區:F=2.172,P=0.001；腹鼻側區:F=1.998,P=0.000；腹顳側區:F=2.352,P=0.000).其中從P13 d到P14 d,揹鼻側區的神經纖維密度減少瞭6.0％,差異無統計學意義(t=0.589,P=0.572)；而且從P14 d到P17 d,揹顳側區、腹鼻側區和腹顳側區的神經纖維密度分彆減少瞭4.6％、5.5％和0.1％,差異均無統計學意義(t=0.549,P=0.596:t=0.701,P=0.501；t=-0.100,P=0.919).結論 小鼠睜眼過程對整箇角膜或角膜各分區神經纖維的髮育都有不同程度的影響.從P17 d開始,小鼠角膜神經纖維呈追趕性增長,恢複增長趨勢.
배경 목전이유허다관우포유동물발육계단각막신경분포적연구,단시상무대정안전후저궤개시간단각막신경분포적연구.목적 료해소서정안전후각막신경섬유장도여밀도수착년령증장적변화규률,위진일보적실험화림상연구제공의거.방법 SPF급C57B L/6소서분위출생후1 d(P1 d)、P7d、P13 d(정안전1d)、P14 d(반정안)、P17 d(완전정안후1d)화P23 d(완전정안후7d),각조균5지서10지안.채용β-미관단백Ⅲ항체진행정장각막포편면역형광염색,사용Delta Vision Core박섭각막배비측、배섭측、복비측화복호측적신경섬유도상,근거체시학측량선성결구적원리,운용Image-Pro Plus 5.1연건중적“수동측량-건립다변형특정”계산각막면적,병차사용LnsgRand.grd곡선(간거위53 μm)분별계산각조소서적각막면적、신경섬유총장도이급각분구적신경섬유밀도.결과 C57/BL6소서P1、P7、P13、P14、P17、P23 d적각막면적분별위(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,신경섬유총장도치분별위(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,신경섬유밀도분별위(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/mm2,균수서령적증장이증가,차이균유통계학의의(F=22.906、0.424、2.375,P=0.000),각막면적화신경섬유장도지간정정상관(r=0.983,P＜0.01).각막4개분구적신경섬유밀도수년령적증장이증가(배비측구:F=0.159,P=0.000；배호측구:F=2.172,P=0.001；복비측구:F=1.998,P=0.000；복섭측구:F=2.352,P=0.000).기중종P13 d도P14 d,배비측구적신경섬유밀도감소료6.0％,차이무통계학의의(t=0.589,P=0.572)；이차종P14 d도P17 d,배섭측구、복비측구화복섭측구적신경섬유밀도분별감소료4.6％、5.5％화0.1％,차이균무통계학의의(t=0.549,P=0.596:t=0.701,P=0.501；t=-0.100,P=0.919).결론 소서정안과정대정개각막혹각막각분구신경섬유적발육도유불동정도적영향.종P17 d개시,소서각막신경섬유정추간성증장,회복증장추세.
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 human.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 from 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,P7d,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±1.044),(19.065±3.350),(23.687±0.907),(27.309±2.477),(31.989±3.976),(41.214±1.573)mm,respectively ; the densities of 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/mm2,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 =2.1 72,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 P17 d(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.
