绿色荧光蛋白标记的恶性组织细胞增生症瘤细胞眼前房移植模型

目的建立绿色荧光蛋白(GFP)标记的恶性组织细胞增生症瘤细胞cy15在眼前房生长及浸润的模型,探讨cy15细胞在前房生长的规律及GFP标记活细胞的优势。方法将带有GFP的逆转录病毒MP71-GFP-PRE转入cy15瘤细胞,获得稳定表达GFP的cy15GFP细胞系。20只BALB/C小鼠(20只眼)每只眼前房注入2μl细胞密度为2×106个/ml的cy15GFP瘤细胞悬液,术后分别在裂隙灯和荧光显微镜下观察瘤细胞在前房的生长情况,连续观察30d,分别在瘤细胞植入前房后第15d、20d、30d时处死小鼠做眼球HE病理切片。结果成功地建立了稳定表达GFP的cy15GFP细胞系。接种的20只BALB/c鼠眼前房均可见肿瘤细胞生长,借助于荧光显微镜能动态地观察到肿瘤细胞在活体小鼠眼前房的生长浸润过程。结论建立的GFP标记的肿瘤细胞眼前房接种模型为研究眼内肿瘤细胞生长与浸润的规律以及抗肿瘤药物的筛选提供了一种新的手段。     

绿色荧光蛋白标记的视网膜母细胞瘤原位移植瘤模型

目的　应用绿色荧光蛋白 (GFP)基因标记人类视网膜母细胞瘤 (RB)细胞 ,建立新型裸鼠原位移植瘤模型 ,探讨RB的生长和转移特征。方法　利用脂质体将GFP真核表达质粒pEGFP N1转入人类RB细胞 (HXO RB44) ,新霉素、荧光显微镜及流式细胞仪筛选稳定表达GFP的细胞克隆。 30(6 0只眼 )裸鼠每只眼视网膜下腔均注入 2 μl细胞密度为 (4 5～ 5 5 )× 10 8个细胞 /ml的RB细胞悬液。术后利用带荧光的体视镜连续观察肿瘤生长情况 ,于不同时间点处死动物 ,荧光显微镜观察视神经、颅底、肺、肝、肾的肿瘤转移情况。结果　术后 34～ 37d可观察到肿瘤生长至眼外 ,并可观察到肿瘤沿视神经向颅内转移 ,肿瘤细胞沿视神经鞘、睫状后长动脉浸润生长 ;移植瘤病理组织学表现类似于人类RB肿瘤 ;免疫组化染色肿瘤细胞GFP呈阳性表达。结论　经GFP基因标记的人类RB细胞裸鼠视网膜下腔移植建立的RB移植瘤模型 ,为研究自然状态下RB肿瘤的生长和转移提供了新的工具。     

绿色荧光蛋白标记的脉络膜恶性黑色素瘤原位移植瘤模型

目的 建立新型裸鼠脉络膜恶性黑色素 瘤原位移植瘤模型。 方法 利用脂质体将绿色荧光蛋白（GFP）真核表达 质粒pEGFP-N₁转入人脉络膜恶性黑色素瘤细胞（OC-1），新霉素、荧光显微镜及流式细胞仪筛选稳定表达GFP的细胞克隆。将2 μl细胞浓度为4.5×10⁷~5.5×10⁷个/ml的细胞悬液注射到麻醉后的40只裸鼠右眼视网膜下间隙，左眼不注射作为对照眼。手术后利用带荧光的体视显微镜连续观察肿瘤生长情况，分别于肿瘤生长的眼内期、眼外期及衰竭期处死动物，荧光显微镜观察裸鼠视神经、颅底、肺、肝、肾的肿瘤转移情况，并行肿瘤组织的GFP免疫组织化学染色。 结果 手术后10~12 d肿瘤开始生长，血管扩张、扭曲，新生血管形成；手术后20~22 d肿瘤占满玻璃体腔；手术后24~26 d肿瘤生长至眼外；眼外期后，迅速进入衰竭期，衰竭期嗅球、肾、肺、肝脏有转移灶。移植瘤病理组织学表现类似于人脉络膜恶性黑色素瘤；免疫组织化学染色肿瘤细胞GFP染色阳性。 结论 以GFP基因标记的OCM-1经裸鼠视网膜下间隙移植建立的脉络膜恶性黑色素瘤原位移植瘤模型，为研究自然状态下肿瘤的生长和转移提供了一个新的方法。 （中华眼底病杂志，2004，20：245-248）     

经绿色荧光蛋白基因修饰的人脉络膜黑色素瘤细胞株OCM-1-gfp的生物学行为与基因表达

目的 研究经绿色荧光蛋白（GFP）基因修饰的人脉络膜黑色素瘤细胞株OCM-1-gfp在小鼠体内的生长过程、转移规律，以及可能影响肿瘤生长和转移的因素。 方法 用脂质体将GFP基因导入人脉络膜黑色素瘤细胞OCM-1，建立稳定、高水平表达GFP的克隆;分别接种到Balb/c裸鼠视网膜下和后大腿皮下，建立原位和异位肿瘤模型。眼内肿瘤生长情况用荧光显微镜直接观察，皮下肿瘤大小用游标卡尺测量;采用免疫组织化学方法对肿瘤内13种基因的表达进行检测。 结果 稳定表达GFP的脉络膜黑色素瘤细胞OCM-1-gfp基本保持了亲代细胞的特征;能在裸鼠体内形成肿瘤并继续生长和转移;在基因表达方面，肿瘤抑制基因p16染色呈阴性，p53染色呈强阳性。其他的肿瘤抑制基因:视网膜母细胞瘤易感基因（Rb）、p21，转录调控因子（E-2F）、核因子κB（NFκB），细胞增生相关基因细胞周期素D1（cyclin D1）、增生细胞核抗原（PCNA），细胞凋亡相关基因bcl-2、bcl-XL/S和bax，以及表皮生长因子（EGF）及其受体（EGFR）呈现不同程度的阳性表达。 结论 GFP为直接观察脉络膜黑色素瘤在体内的生长和转移提供了标记;脉络膜黑色素瘤原位与异位移植瘤模型在成瘤率和生长情况方面无明显差异;由OCM-1-gfp生长形成的肿瘤p16、p53及NFκB、cyclin D1、PCNA及EGF和EGFR等多种基因表达异常。 (中华眼底病杂志, 2006, 22: 170-173)     

大鼠骨髓间充质干细胞视网膜下移植观察

目的：鉴定骨髓间充质干细胞（mesenchymal stem cells,MSCs)在体外不诱导的条件下视网膜下移植后的定位。方法：体外培养雄性大鼠MSCs，直接作为细胞供体视网膜下移植于成年雄性大鼠视网膜下，4周后将动物处死，取眼球做石蜡切片，用Y染色体鉴定，为做进一步验证，将MSCs用重组腺相关病毒AAV－gfp感染后移植，分别于4、8周取动物眼球作冰冻切片，于荧光显微镜下做绿色荧光蛋白（green fluorescence protein，GFP）表达观察。结果：培养的MSCs集落生长迅速，均一性好；Y染色体原位杂交鉴定表明来源于MSCs的阳性细胞融合入了原来的视网膜结构，在光学显微镜下可分布于视锥，视杆细胞层，双极细胞层及节细胞层；在荧光显微镜下可见GFP标记的阳性细胞存在，分布于视网膜色素上皮层、视锥，视杆细胞层，双极细胞层及节细胞层，细胞形态与结构同周围的视网膜相似；2种标记方法检测到的视网膜结构完整，未见到玫瑰花结样结构。结论：MSCs可在视网膜下移植后4周与原视网膜结构相融合，2种方法检测到的阳性细胞分布于视网膜色素上皮层，视细胞层，双极细胞层及节细胞层。     

建立人眼葡萄膜黑色素瘤裸鼠移植瘤模型的初步探讨

目的研究建立人眼葡萄膜恶性黑色素瘤动物模型的方法,对比不同的移植方法对成瘤率的影响,为葡萄膜恶性黑色素瘤的实验研究提供体内实验平台。方法分别采用原位移植及异位移植的方法,选取BALB／L-nu裸鼠35只,随机分为3组,将原代培养的第三代人眼恶性黑色素瘤细胞制成细胞悬液,分别接种于裸鼠前房(前房组——原位移植)及皮下(皮下A组——异位移植),另以手术切除的新鲜人眼葡萄膜恶性黑色素瘤制作成的完整瘤组织块移植到皮下(皮下B组),在同样条件下饲养,观察其成瘤率和大体及组织病理学形态。结果裂隙灯显微镜下观察可见前房组成瘤率46．6％,14例在接种后第3天即可见前房内黑色片状小肿物,紧贴在角膜内皮及虹膜表面。2个月时肿瘤基本充满前房。皮下A组成瘤率20％,2例于接种1周后可见局部出现黑色斑块,呈椭圆形,轻度隆起。1个月后肿物逐渐增大,接种后60 d长至1．0 cm×1．0 cm,色黑,呈圆形。病理切片检查证实为黑色素瘤。皮下B组接种肿物后,10例中有8例未见肿瘤长大,3个月后自行消退。另2例肿物先扁平,后弥散,不符合肿瘤不断生长的特性,不计人成瘤率。结论将原代培养的人眼葡萄膜黑色素瘤细胞混悬液分别注入裸鼠眼前房及皮下,能建立裸鼠的原位及异位移植瘤模型,成瘤率明显高于组织块皮下移植。其中原位移植瘤模型成瘤率远高于异位移植,且更符合生理特点,有利于眼科操作和裂隙灯显微镜观察,是建立人葡萄膜黑色素瘤裸鼠模型的良好选择。     

视紫红质基因启动子的分离及视网膜特异表达载体的构建

目的 分离视紫红质基因启动子，构建以绿色荧光蛋白(GFP)为报告基因的视网膜特异表达载体，为今后视网膜细胞特异的靶向基因转移，特别是为视网膜疾病的基因治疗提供T具。方法 根据小鼠视紫红质基因5’端DNA顺序合成引物，通过PCR技术从小鼠基因组DNA中扩增524bp DNA片段，然后插入质粒pEGFP-1GFP编码基因上游的多克隆位点处，构建表达载体pmRho-EGFP。采用脂质体包裹pmRho-EGFP，转染体外培养的人视网膜色素上皮(RPE)细胞及其他来源的细胞；注射到SD大鼠视网膜下或玻璃体腔内；或通过电穿孔直接转移到RCS大鼠腓肠肌内。GFP表达采用荧光显微镜观察。结果 pmRho-EGFP在体外培养的人RPE细胞中的表达水平明显高于其他组织来源的细胞；体内转染实验证明pmRho-EGFP可在大鼠视网膜神经细胞中表达，但在大鼠腓肠肌中不表达。结论 小鼠视紫红质基因5’端524hp片段具有基本的启动子活性，能够调控基因在视网膜神经细胞及色素上皮细胞中表达，并具有一定的组织和细胞特异性。     

应用GFP基因转染技术示踪组织工程技术构建角膜基质

目的 应用基因转染技术将绿色荧光蛋白(GFP)标记角膜基质绌胞，以此为种子细胞构建角膜基质并对构建过程进行示踪。方法 构建携带EGFP基因的重组逆转录病毒载体PLNCX2-EGFP，转染兔角膜基质细胞，然后将该细胞接种于PGA，形成细胞-生物材料复合物，移植于母兔角膜基质板层内。术后8周，组织学切片，HE染色，荧光显微镜下对绿色荧光蛋白(GFP)进行示踪观察。结果 8周后，组织工程化角膜组织形成，组织学切片显示PGA完全降解，新生组织形成，组织排列规整，荧光显微镜下见新生组织呈绿色，提示EGFP表达。结论 组织工程角膜基质的组织学结构与角膜基质相类似。新生组织表达GFP，证实组织工程角膜基质组织的构成源于供体细胞。     

视网膜母细胞瘤的组织培养

将临床诊断为RB 病人的眼球标本,取玻璃体肿瘤细胞和肿瘤组织块分别置199—1640混合培养液,37℃恒温培养。玻璃体肿瘤细胞呈单个悬浮生长,细胞单一,不易结成团块。肿瘤组织块培养的肿瘤细胞从组织块边上移行出来,脱落到培养液内呈团块状悬浮生长。二者的肿瘤细胞增殖均按延迟期、生长期和静止期这样一个周期生长,大约需3—4周。肿瘤组织块培养的瘤细胞增殖情况和细胞形态,较玻璃体肿瘤细胞为好。     

大鼠房水外引流手术后前房抗原引流变化的实验研究

目的建立大鼠房水外引流手术模型,通过前房注射荧光标记抗原,观察房水外引流手术对前房内抗原引流的影响。方法 SD大鼠右眼行房水外引流手术后前房内注入荧光标记抗原FITC-dextran,对照组大鼠不行房水外引流手术,仅前房注射FITC-dex-tran。于注射后24h取出颈部淋巴结,一半组织制作冰冻切片荧光显微镜下观察,另一半组织制备单细胞悬液,流式细胞仪检测各组织FITC阳性细胞百分数。结果大鼠前房注射后24h,手术及非手术大鼠在颈部淋巴结均可见到荧光标记抗原分布。手术组大鼠颈部淋巴结FITC阳性细胞百分数为(2.96±0.67)%,明显多于正常大鼠的FITC阳性细胞百分数(1.57±0.48)%,二者比较差异有显著统计学意义(P=0.001)。结论房水外引流手术影响了前房内抗原的引流,使其与局部淋巴结接触增多,可能对眼部的结构和功能产生一定的影响。     

阳离子聚合体介导MYOC基因真核表达载体体内转染研究

目的探讨大鼠前房内注射阳离子聚合体／MYOC基因复合物转染小梁组织的有效性和安全性。设计实验研究。研究对象SD大鼠。方法用微量进样器将表达myocilin（MYOC）基因的重组质粒PCDNA3-MYOC与阳离子聚合体（PEI）的混合溶液20μl注射于SD大鼠前房内，于注射后不同时间点（1、3、7、14d）摘除眼球，进行病理切片HE染色、荧光免疫组织化学染色以及透射电镜观察。主要指标病理切片、荧光免疫组织化学染色以及透射电镜观察小梁组织的变化。结果病理切片HE染色未见小粱组织明显炎症细胞浸润以及结构改变；荧光免疫组织化学染色显示注射后1d可见到眼组织荧光着色；于转染后3d小梁网荧光强度明显增强，至14d荧光渐消失；透射电镜证实小梁细胞内可见PEUDNA颗粒，溶酶体增多。结论前房内注射阳离子复合体介导的DNA质粒可将外源性基因安全有效地转染人小梁网组织。     

Centripetal movement of corneal epithelial cells in the normal adult mouse

PURPOSE: To study the natural movement of corneal epithelial cells in the normal adult mouse with histology and in vivo microscopy. METHODS: A transgenic mouse line that was engineered to ubiquitously express green fluorescent protein (GFP) was used to visualize corneal epithelial cells. For histology, epithelial GFP was imaged in a wholemounted cornea en face, and also in frozen cross-sections, under a fluorescence microscope. For in vivo studies, the anesthetized mouse was placed on a custom-made observation platform under a fluorescence microscope. Epithelial fluorescence was digitally recorded two to three times a week, and a rate of cell movement was determined from the time-lapse sequences. RESULTS: The GFP expression in the corneal epithelium was nearly ubiquitous up to about 1 week after birth, and thereafter it gradually became sporadic, resulting in a mosaic pattern of GFP positive cells, with the brightest cells present in the basal and suprabasal layer of the epithelium. Both high- and low GFP-cells formed radial streaks toward the central cornea, frequently displaying vortex patterns at the center. Clusters of several high-GFP cells were tracked in living mice for up to 7 weeks, and an analysis of time-lapse sequences revealed that they moved centripetally at an average rate of 26 micro m/d. CONCLUSIONS: Corneal epithelium of adult GFP mice exhibits a pattern of GFP expression that is suitable for studying cell movement in the normal cornea. Epithelial cells at the basal or suprabasal layers move centripetally in these mice at an average rate of 26 micro m/d.     

基质金属蛋白酶抑制剂抑制兔晶状体后囊膜混浊的研究

目的 观察基质金属蛋白酶(MMP)抑制剂对兔晶状体后囊膜混浊的抑制作用和对眼内组织的毒性.方法 实验研究.选用新西兰白兔行超声乳化晶状体吸除术,用不同浓度的MMP抑制剂GM6001溶液(实验1组:100 μmol/L,实验2组:200 μmol/L,实验3组:500 μmol/L)和GM6001阴性对照液(500 μmol/L)进行术毕及术后隔日晶状体囊袋内灌注3次,观察术后第12周内后囊膜混浊情况,同时观察药物对眼前房反应、眼压、角膜内皮、虹膜、睫状体和视网膜的影响和毒性.采用四格表确切概率法分析使用GM6001后前房反应和对后囊膜混浊的抑制作用;采用单因素方差分析法分析GM6001对眼压的影响.结果 裂隙灯显微镜下观察,可见术后12周对照组后囊膜混浊明显,实验1组后囊膜混浊较对照组轻,实验2组和3组均无后囊膜混浊(P=0.007);病理学结果显示:对照组和实验1组后囊膜表面有多层排列紊乱的上皮细胞和成纤维细胞,而实验2组和3组后囊膜表面几乎无细胞生长;前房反应轻:用药2 d实验组和对照组兔眼前房闪光情况比较,差异无统计学意义(P=0.380);对眼压的影响:实验组与对照组用药2 d(F=0.642,P=0.597)、7 d(F=0.179,P=0.909)眼压比较,差异均无统计学意义;用药7 d,实验3组角膜内皮细胞呈规则的六边形,无变形脱落,与对照组眼角膜内皮细胞形态无明显差别;光镜观察发现对虹膜、睫状体和视网膜均无明显毒性.结论 MMP抑制剂可明显抑制兔眼超声乳化晶状体吸除术后晶状体后囊膜混浊的发生,对眼内组织无明显毒性,安全有效.

Abstract：

Objective To determine whether matrix metalloproteinase (MMP) inhibitor can provide therapeutic effects for rabbits posterior capsule opacification in vivo and to observe the side effects of this drug on surrounding intraocular structures. Methods Experimental research. New Zealand white rabbits were undertaken phacoemulsification operation. GM6001 at different concentrations ( 100,200 and 500 μmol/L)and GM6001 negative control liqueur were infused into the capsule bags of the rabbits at the end of operation and two days after the operation. The incidence of posterior capsule opacification was assessed and the histological sections of posterior capsules were observed under microscope 12 weeks after the surgery. The anterior chamber response was observed on day 2 post-operatively. The changes of intraocular pressure were measured by day 2 and day 7. Corneal endothelial cells were observed under scanning electron microscopeand iris, ciliary body and retina were observed under microscope on day 7. Results GM6001 significantly prevented posterior capsule opacification (P=0. 007 ). No opacification occurred on the rabbit posterior capsule in eyes with 200 and 500μmol/L GM6001 on week 12 post-operatively in vivo. No cells were found on posterior capsule in 500 μmol/L group, whereas lens epithelial cells and fibroblasts were found in the controls under microscope. No difference of anterior chamber flare between the eyes with GM6001 at different concentrations and the control group (P=0. 380) by day 2 after the operation. The intraocular pressure in eyes with GM6001 was the same as that in the control 2-days ( F = 0. 642, P = 0. 597 ) and 7-days ( F =0. 179 ,P =0. 909) post-operation. The corneal endothelial cells in eyes with 500 μ mol/L GM6001 arranged regularly and did not show any difference from that in the control eyes under scanning electron microscope 7-day after the operation. The iris, ciliary body and retina in eyes with 500 μmol/L GM6001 were normal in appearance 7-day after the operation. Conclusions MMP inhibitor can prevent posterior capsule opacification effectively in rabbits in vivo and does not cause damage to surrounding intraocular structures,suggesting that MMP inhibitor may become a medication used for the prevention of lens posterior capsule opacification.     

应用转基因技术体外培养表达人碱性成纤维细胞生长因子的视网膜色素上皮细胞

目的 探讨应用转基因技术体外培养稳定表达人碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)的视网膜色素上皮(retinal pigment epithelium,RPE)细胞的可行性。方法 构建以绿色荧光蛋白(green fluorescent protein,GFP)作为标记基因的人bFGF真核表达载体pcFG。应用脂质体介导法转染人RPE细胞,以G418筛选出表达bFGF的RPE细胞,并进行细胞克隆,传代培养4周。于荧光显微镜下观察GFP的表达情况,用原位杂交和免疫组织化学染色法检测bFGF在RPE细胞的表达情况。结果 酶切结果证实含有GFP的真核表达载体pcFG构建正确。在荧光显微镜下可见RPE细胞表达绿色荧光蛋白。经原位杂交和免疫组织化学染色证实,转染pcFG后的RPE细胞内有大量bFGF—mRNA的转录蛋白表达。结论 应用转基因技术可体外培养稳定表达bFGF的RPE细胞。     

Induction of gene into the rabbit eye by iontophoresis: preliminary report

PURPOSE: To assess the transfer of 6-carboxyfluorescein (6-FAM)-labeled phosphorothioate oligonucleotides(S-ODNs) into the ocular tissues, their stability, and possibility of injury to the ocular tissues. METHODS: The S-ODNs(2 mL/eye)were transduced noninvasively into albino rabbit eyes.The iontophoresis group consisted of 6 rabbits (12 eyes); the control group consisted of 2 rabbits (4 eyes) given eye drops containing S-ODNs. Aqueous humor and vitreous humor were collected after iontophoresis, subjected to electrophoresis with a fluorescence DNA sequencer and analyzed by the Gene Scan program. Frozen sections, 10-microm thick, were prepared for observation under a fluorescence microscope. A plasmid 4.7 kbp in size that expresses green fluorescent protein (GFP) was induced into the 18 eyes of 9 rabbits by the same procedure. RESULTS: In the iontophoresis group, S-ODNs were detected in the anterior chamber 5 minutes after electrophoresis began and in the vitreous after 10 minutes. These S-ODNs maintained the same length as at the initial synthesis. The S-ODNs could also be detected in the posterior retina 20 minutes after electrophoresis. No evidence of degeneration or inflammation due to the above procedure was found in the ocular tissues. Fluorescence showing GFP gene expression was found in the cornea, the anterior chamber angle, and the ciliary subepithelial tissues. CONCLUSIONS: These findings show that iontophoresis is an effective method to induce genes into the rabbit eye.     

Requirement of CD80+ costimulation for rejection of ocular tumors and termination of immune privilege

Immune privilege in the anterior chamber of the eye is very effective at preventing elimination of ocular tumors that express weak tumor antigens. We previously demonstrated that DBA/2 mice immunized in the flank with a P815 tumor cell vaccine that expressed CD80/IL-12 was unable to terminate immune privilege and prevent tumor growth in the anterior chamber. In the present study, we determined whether expression of costimulatory signals on tumor cells injected via an ocular route would terminate immune privilege. We observed that CD80-positive tumors were always rejected from the anterior chamber. However, the pattern of tumor rejection was dramatically different depending upon whether the mice were either na?ve, or immunized in flank against the tumor cells. Na?ve mice that received an anterior chamber injection of CD80-positive tumors did not develop immune privilege and displayed strong delayed hypersensitivity (DH) against the tumor cells. Moreover, the ocular tumors were rejected by ischemic necrosis that induced atrophy of the eye and phthisis. When immunized mice received an identical injection of tumor cells in the anterior chamber, they also did not develop immune privilege and displayed strong DH. However, tumor rejection was considerably different and occurred without destruction of normal ocular tissue, or phthisis. To determine if immune privilege was restored in these eyes, mice received a second injection of P815 cells (CD80-negative) into the anterior chamber 2weeks after the first CD80-positive tumors were eliminated. Surprisingly, immune privilege was not restored and the P815 tumors were eliminated completely, again without phthisis. We conclude that specific rejection of ocular tumors with minimal destruction of the eye requires systemic immunization and expression of CD80 costimulatory signals on tumors within the eye.     

Intraocular in vivo imaging of activated T-lymphocytes expressing green-fluorescent protein after stimulation with endotoxin

BACKGROUND: Intravital microscopy allows imaging of specific cell populations in vivo. The value of this technique is well established, but would be enhanced if one could distinguish functional states of cells in vivo. Interleukin-2 (IL-2) is expressed upon stimulation of T-cells and is a commonly used marker for T-cell activation. This study tests the use of enhanced green fluorescent protein (GFP) as a reporter gene for interleukin-2 (IL-2) expression in vivo. METHODS: Characterization of mice that have the GFP gene under the control of IL-2 regulatory sequences has previously been published. Uveitis was induced by injection of E. coli endotoxin into the vitreous of these IL-2/GFPki transgenic mice. Four hours later, 3 microg of recombinant mouse IL-2 was injected into the anterior chambers of one group of mice. In vivo imaging of infiltrating cells in the iris stroma was performed with fluorescence microscopy at 6, 24, 48, and 72 h after endotoxin injection. The absolute number of fluorescent cells per mm2 was evaluated. RESULTS: Eyes with endotoxin-induced uveitis had cells that expressed GFP and were identifiable by intravital microscopy. The fluorescent cells were exclusively seen in the subset of cells that had infiltrated the iris stroma or arrested along the vascular endothelium. The number of GFP-positive infiltrating cells in the iris increased from undetectable at baseline to 0.5 cells/mm2 at 6 h and 1.3 cells/mm2 at 72 h. The animals that received endotoxin as well as IL-2 tended to have more GFP-positive cells at the 48-h and 72-h time points, but these differences were not statistically significant CONCLUSIONS: GFP is commonly used as a reporter gene for in vitro expression assays. The results presented here document that transgenic mice with GFP under the control of IL-2 regulatory elements can be used with intravital microscopy for in vivo expression assays that allow detection of activated T-cells at multiple time points within the same animal. This provides a novel method for temporal and spatial studies on the state of cell activation in inflammatory responses.