复合性眼球破裂视网膜病变的实验研究
摘要
目的 眼球破裂伤常造成严重视力下降或视力丧失，损伤机制尚不十分清楚。本研究利用汽枪枪弹制做复合性兔眼球破裂伤，观察兔眼破裂伤后视网膜早期临床表现、病理变化以及光感受细胞损伤机制。
方法 健康无眼疾新西兰白兔48只，眼前段及视网膜检查正常，体重为2.5～3.0kg。随机分为正常对照、伤后1h、3h、6h、12h、24h共6组，每组8只。家兔麻醉后，用自制汽枪自兔耳根后缘与眼球正中连线的角膜缘内1mm沿角膜平面射击，汽枪子弹从面鼻根部射出，形成眼球破裂伤模型。于伤后1h、3h、6h、12h、24h时行眼电生理检查并处死实验兔，立即摘除眼球。切除角膜后以4％多聚甲醛磷酸缓冲液固定24h，逐级酒精脱水，二甲苯透明，石蜡包埋备用。将包埋好的眼球标本，平行于视神经矢状轴且以其为平面的视网膜进行连续切片8张，切片厚度为4μm，每2张分别行HE染色、p53、bcl-2、细胞原位凋亡检测。HE染色组织切片行光镜及超高倍显微镜观察(ACT-2000型超高倍显微诊断系统)。光镜下观察：TUNEL法细胞凋亡、p53和bcl-2蛋白阳性表达图像经过高清晰彩色病理图像分析系统(HPISA-1000)处理(每张切片取4个视野，每个视野面积为0.2mm×0.2mm)，计算视野中呈棕黄色阳性染色的细胞数，定量分析视网膜组织中p53、bcl-2的表达及细胞凋亡情况。应用SPSS10.0统计软件，进行变量转换，单因素方差分析和相关分析等统计学方法处理
结果
1．临床表现 汽枪弹击伤兔眼后见伤眼结膜立即充血、水肿、角膜伤口可见房水溢出，虹膜、晶体脱出，随后15秒至1分钟发生眼球内出血自伤口处溢出。
2．眼电生理结果 正常8只兔眼视网膜电图b波振幅为103.5±16.841my，实验组1h、3h、6h、12h、24h所有伤眼ERG检查b波振幅消失，呈熄灭型波型。
3．病理检查 HE染色光镜及超高倍显微镜观察：所有伤眼可见脉络膜上腔出
郑州衅2004年蟀士毕业论文一一退丝垄塑里鲤鳖燮业些燮叁一
血，玻璃体内可见大量红细胞，视网膜脱离，偶见浅层巩膜出血、睫状体与巩膜分离。
1h一3h时视网膜感光细胞的锥杆层有碎裂改变，其它结构无明显改变。6h一12h时视
网膜内、外核层明显空泡变性，神经节细胞核少量固缩等明显改变。24h时视网膜
变薄、结构紊乱不清。
4.免疫组织化学及细胞凋亡对照组及各实验组bCI一2均无明显表达。p53在
3h开始在内、外核层及神经节细胞层有少量阳性表达，6h一12h明显增多，24h略
有下降。细胞凋亡6h在神经节细胞和内、外核层有少量表达，12h一24h染色阳性的
细胞明显增多。本试验中:Bd一2对照组与实验各组经方差检验，差异均无显著性
(尸>0.05);正常视网膜组织细胞未见p53表达，经单因素多重比较方差分析，对
照组与伤后lh组差别无显著性片0.053，与伤后3h、6h、12h、24h之间的比较差
异极显著口KO.01);伤后6h和24h p53蛋白的表达无显著性(p=0，807);视细胞
凋亡在伤后6h出现，方差分析，对照组与伤后1h组差别无显著性(户0.062)，对
照组与伤后3h差异具有显著性(户0.005)、与6、12、24h之间的比较有极显著差异
性(尸<0.01)。;伤后12h和24h细胞凋亡的阳性表达无显著性(产0.628)。凋亡
指数统计结果对照组与伤后1h组差别无显著性(产0.019)，伤后lh与伤后3h差异
无显著性(产0.022)，伤后12h、24h之间的比较无显著差异性(片0.392);伤后3h、
6h和12h细胞凋亡指数有有极显著差异性护<0.01)。相关分析显Bcl一2与p53及
细胞凋亡无明显关系，而p53与细胞凋亡存在显著相关(只0.05)。
结论本研究运用汽枪制做眼球破裂伤模型，伤情基本一致，制做简单、重复
性好，便于对比研究，是眼球破裂伤理想的动物模型。眼球破裂伤伤情非常严重，
光镜下视网膜在6h出现明显病理损害，12一24h即出现不可逆改变。眼球破裂伤视
细胞的损伤是多因素共同作用的，其中视细胞凋亡是伤后视网膜损伤的重要机制，
p53蛋白的过表达与视细胞凋亡关系密切，bcl一2蛋白在正常及伤后视网膜中几乎无
表达，与视细胞凋亡无关。
Objective: The globe rupture is especially complicated, which is very bad after treatment. This research use air gun bullet to make wound on habbit eyeball, then observe the early clinical manifestation , the pathology changes, and the injury mechanism of photoreceptors cell of the eyeball , supply experimental basis for clinical treatment.
Methods: 48 white New Zealand rabbites were Selected ,of which the anterior sequecment eyes and fundus checks are nomal .The weight is 2.5kg to 3. Okg . Then with random figure form method , the rabbits were randomly divided into 6 groups , 1h,3h,6h, 12h, 24h, wound groups and control groups , each had 8 rabbits respectively. Anaesthetize the rabbits, the eye were shoot from the back costa of the rabbit's ear root and the middle line 1mm inside corneoscleral limbus with an air gun , the bullet projected out from the nose root. 1h, 3h, 6h, 12h, 24h. After the shooting electroretinogram was taken. Then execute the experimental rabbits , and enucleate the eyeball immediately . Cut off the cornea , fix the eyeball for 24 hours with 4% polyformaldehyde phosphate buffer ,then dehydrate it with alcohol gradually .Make sure the xylene is transparent , and embed it with paraffin for backup .Slice the well-embed eyeball specimen into 8 pieces, 4 m thickness in succession , and paralleled with arrowy axes of optic nerv
e and retina with it as plane. Carry through HE stain , the P53, bcl-2, cell original
location apoptosis examines respectively to each two pieces . the HE dyed tissue slices were observed under super times microscopes (AUT-2000 TYPE super times microscopes diagnosis system ).
Methods of light microscopes observation: Disposal TUNEL method cell apoptpsis ,P53 and bcl-2 albumen masculine expressing image by high-definition color pathology image analyse system (HPIAS-1000) (take four visual fileds , each visual field area is 0.2mm*0.2mm). And count the brown-yellow masculine cells in the visual area .Analyse the P53, the expression of bcl-2 and the apoptosis of cell quantitatively , all the original data were analysed with SPSS10. 0 statistics software ,proceeding variable convert and related analysis .
Results
1, clinical manifestation
After being wounded ,the rabbit' s wound eye conjunctiva immediately manifested congestion, edema, and aqueous overflowed from the cornea, then the iris and lens emerged, 15s after injury to 1 miniute , intraocular hemorrhage eyeball internal bleeding overflowed the wound .
2 electrooculography results
The normal electroretinogram ERG- b wave flaps as 103.5+16841 mv, the experiment group 1 h, 3 hs, 6 hs, 12 hs, 24 hs, the ERG- b wave of all disappeared, and manifested as a put-out type.
3 pathology
the HE stain Observed by light microscopes and extremely high times microscopes : epichoroidal space bleed could be seen in all injured eyes, numerous red blood cells were observed in vitreous ,the retina detached , episclera the sclera bleeding was occassionally seen in thin layers, the sclera seperated . ectoretina the layers of the photoreceptor cells of the retina chip between 1 and 3 hours , the other tissue had no change, the inner nuclear layer and outer nuclear layers of the retina
changed obviously, ganglion cell nuclear shrunk alittle ,other changes like these obviously appeared between 6-12 hours, and while 24 hours the retina became thin and its structure is disordered .
4 the immnohistochemistry and cell apoptosis
The bcl-2protein had no obvious expression in the control group and each experimental group , 3 hours latter , a lower positive expression of p53 were observed in the inner and outer layer of the nuclear and ganglion cells , it increased obviously between 6-12 hours, and decreased slightly after 24 hours .The apoptosis had lower expression in ganglion cells and the inner and outer nuclear layer 6 hours latter , an obvious increasing of dye positive cells was obsered between 12 and 24 hours. In the experiment :when checked by One-way ANOVA, there was no significant difference between bcl-2 group and each experimenta
方法 健康无眼疾新西兰白兔48只，眼前段及视网膜检查正常，体重为2.5～3.0kg。随机分为正常对照、伤后1h、3h、6h、12h、24h共6组，每组8只。家兔麻醉后，用自制汽枪自兔耳根后缘与眼球正中连线的角膜缘内1mm沿角膜平面射击，汽枪子弹从面鼻根部射出，形成眼球破裂伤模型。于伤后1h、3h、6h、12h、24h时行眼电生理检查并处死实验兔，立即摘除眼球。切除角膜后以4％多聚甲醛磷酸缓冲液固定24h，逐级酒精脱水，二甲苯透明，石蜡包埋备用。将包埋好的眼球标本，平行于视神经矢状轴且以其为平面的视网膜进行连续切片8张，切片厚度为4μm，每2张分别行HE染色、p53、bcl-2、细胞原位凋亡检测。HE染色组织切片行光镜及超高倍显微镜观察(ACT-2000型超高倍显微诊断系统)。光镜下观察：TUNEL法细胞凋亡、p53和bcl-2蛋白阳性表达图像经过高清晰彩色病理图像分析系统(HPISA-1000)处理(每张切片取4个视野，每个视野面积为0.2mm×0.2mm)，计算视野中呈棕黄色阳性染色的细胞数，定量分析视网膜组织中p53、bcl-2的表达及细胞凋亡情况。应用SPSS10.0统计软件，进行变量转换，单因素方差分析和相关分析等统计学方法处理
结果
1．临床表现 汽枪弹击伤兔眼后见伤眼结膜立即充血、水肿、角膜伤口可见房水溢出，虹膜、晶体脱出，随后15秒至1分钟发生眼球内出血自伤口处溢出。
2．眼电生理结果 正常8只兔眼视网膜电图b波振幅为103.5±16.841my，实验组1h、3h、6h、12h、24h所有伤眼ERG检查b波振幅消失，呈熄灭型波型。
3．病理检查 HE染色光镜及超高倍显微镜观察：所有伤眼可见脉络膜上腔出
郑州衅2004年蟀士毕业论文一一退丝垄塑里鲤鳖燮业些燮叁一
血，玻璃体内可见大量红细胞，视网膜脱离，偶见浅层巩膜出血、睫状体与巩膜分离。
1h一3h时视网膜感光细胞的锥杆层有碎裂改变，其它结构无明显改变。6h一12h时视
网膜内、外核层明显空泡变性，神经节细胞核少量固缩等明显改变。24h时视网膜
变薄、结构紊乱不清。
4.免疫组织化学及细胞凋亡对照组及各实验组bCI一2均无明显表达。p53在
3h开始在内、外核层及神经节细胞层有少量阳性表达，6h一12h明显增多，24h略
有下降。细胞凋亡6h在神经节细胞和内、外核层有少量表达，12h一24h染色阳性的
细胞明显增多。本试验中:Bd一2对照组与实验各组经方差检验，差异均无显著性
(尸>0.05);正常视网膜组织细胞未见p53表达，经单因素多重比较方差分析，对
照组与伤后lh组差别无显著性片0.053，与伤后3h、6h、12h、24h之间的比较差
异极显著口KO.01);伤后6h和24h p53蛋白的表达无显著性(p=0，807);视细胞
凋亡在伤后6h出现，方差分析，对照组与伤后1h组差别无显著性(户0.062)，对
照组与伤后3h差异具有显著性(户0.005)、与6、12、24h之间的比较有极显著差异
性(尸<0.01)。;伤后12h和24h细胞凋亡的阳性表达无显著性(产0.628)。凋亡
指数统计结果对照组与伤后1h组差别无显著性(产0.019)，伤后lh与伤后3h差异
无显著性(产0.022)，伤后12h、24h之间的比较无显著差异性(片0.392);伤后3h、
6h和12h细胞凋亡指数有有极显著差异性护<0.01)。相关分析显Bcl一2与p53及
细胞凋亡无明显关系，而p53与细胞凋亡存在显著相关(只0.05)。
结论本研究运用汽枪制做眼球破裂伤模型，伤情基本一致，制做简单、重复
性好，便于对比研究，是眼球破裂伤理想的动物模型。眼球破裂伤伤情非常严重，
光镜下视网膜在6h出现明显病理损害，12一24h即出现不可逆改变。眼球破裂伤视
细胞的损伤是多因素共同作用的，其中视细胞凋亡是伤后视网膜损伤的重要机制，
p53蛋白的过表达与视细胞凋亡关系密切，bcl一2蛋白在正常及伤后视网膜中几乎无
表达，与视细胞凋亡无关。
Objective: The globe rupture is especially complicated, which is very bad after treatment. This research use air gun bullet to make wound on habbit eyeball, then observe the early clinical manifestation , the pathology changes, and the injury mechanism of photoreceptors cell of the eyeball , supply experimental basis for clinical treatment.
Methods: 48 white New Zealand rabbites were Selected ,of which the anterior sequecment eyes and fundus checks are nomal .The weight is 2.5kg to 3. Okg . Then with random figure form method , the rabbits were randomly divided into 6 groups , 1h,3h,6h, 12h, 24h, wound groups and control groups , each had 8 rabbits respectively. Anaesthetize the rabbits, the eye were shoot from the back costa of the rabbit's ear root and the middle line 1mm inside corneoscleral limbus with an air gun , the bullet projected out from the nose root. 1h, 3h, 6h, 12h, 24h. After the shooting electroretinogram was taken. Then execute the experimental rabbits , and enucleate the eyeball immediately . Cut off the cornea , fix the eyeball for 24 hours with 4% polyformaldehyde phosphate buffer ,then dehydrate it with alcohol gradually .Make sure the xylene is transparent , and embed it with paraffin for backup .Slice the well-embed eyeball specimen into 8 pieces, 4 m thickness in succession , and paralleled with arrowy axes of optic nerv
e and retina with it as plane. Carry through HE stain , the P53, bcl-2, cell original
location apoptosis examines respectively to each two pieces . the HE dyed tissue slices were observed under super times microscopes (AUT-2000 TYPE super times microscopes diagnosis system ).
Methods of light microscopes observation: Disposal TUNEL method cell apoptpsis ,P53 and bcl-2 albumen masculine expressing image by high-definition color pathology image analyse system (HPIAS-1000) (take four visual fileds , each visual field area is 0.2mm*0.2mm). And count the brown-yellow masculine cells in the visual area .Analyse the P53, the expression of bcl-2 and the apoptosis of cell quantitatively , all the original data were analysed with SPSS10. 0 statistics software ,proceeding variable convert and related analysis .
Results
1, clinical manifestation
After being wounded ,the rabbit' s wound eye conjunctiva immediately manifested congestion, edema, and aqueous overflowed from the cornea, then the iris and lens emerged, 15s after injury to 1 miniute , intraocular hemorrhage eyeball internal bleeding overflowed the wound .
2 electrooculography results
The normal electroretinogram ERG- b wave flaps as 103.5+16841 mv, the experiment group 1 h, 3 hs, 6 hs, 12 hs, 24 hs, the ERG- b wave of all disappeared, and manifested as a put-out type.
3 pathology
the HE stain Observed by light microscopes and extremely high times microscopes : epichoroidal space bleed could be seen in all injured eyes, numerous red blood cells were observed in vitreous ,the retina detached , episclera the sclera bleeding was occassionally seen in thin layers, the sclera seperated . ectoretina the layers of the photoreceptor cells of the retina chip between 1 and 3 hours , the other tissue had no change, the inner nuclear layer and outer nuclear layers of the retina
changed obviously, ganglion cell nuclear shrunk alittle ,other changes like these obviously appeared between 6-12 hours, and while 24 hours the retina became thin and its structure is disordered .
4 the immnohistochemistry and cell apoptosis
The bcl-2protein had no obvious expression in the control group and each experimental group , 3 hours latter , a lower positive expression of p53 were observed in the inner and outer layer of the nuclear and ganglion cells , it increased obviously between 6-12 hours, and decreased slightly after 24 hours .The apoptosis had lower expression in ganglion cells and the inner and outer nuclear layer 6 hours latter , an obvious increasing of dye positive cells was obsered between 12 and 24 hours. In the experiment :when checked by One-way ANOVA, there was no significant difference between bcl-2 group and each experimenta
引文
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26 Chang CJ, Lai WW,Edward DP, et al. Apoptotic photoreceptor cell death after traumatic retinal detachment in humans.ArchOphthalmol,1995;113(7):880-886.
27 BuchiE R. Cell death in the rat redna after apressure-induce-dischaemia-reperfusion insult:an electron microscopic study. I.Ganglion cell layer and inner nuclear layer. Exp Eye Res, 1992,55:605-613.
28 Kaneda K,Kashii S,Kurosawa T,etal.Apoptotic DNA fragmentation and up regulation of Bax induced by transient ischemia of the ratretina.BrainRes,1999,815:11-20.
29 安美霞，张效房，张金嵩．挫伤性视网膜病变感光细胞凋亡的实验观察．中华眼科杂志．2004，40(5)，118-121．
30 孙立英，王正国，李晓炎，等．微型生物激波管的研制及其在家兔眼冲击伤实验中的应用．第三军医大学学报，1991，13：469-471．
31 大野敦史．钝眼外伤脉络膜循环研究．第1报．实验的钝的外伤眼脉络膜组织血流量．日眼会志，1984，75：1497-1505．
32 HanD P,Mieler WF,Schwartz DM,Abrams GW. Management of traumatic hemorrhagic
retinaldetachmentwithparsplanavitrectomy[J].Arch Opthalmol,1990;108(9):1281-1286.
33 LewisH,ResnickSC,FlanneryJG,StraatsmaBR.Tissueplasminogenactivatortreatmentofexperimentalsubretinalhemorrhage[J].AmJOphthalmol,1991;111(2):197-204.