沉默信号调控因子1在1型糖尿病模型小鼠角膜和三叉神经节中的表达及意义

背景 糖尿病是引发角膜神经病变的高危因素之一.沉默信号调控因子1(Sirt1)在糖代谢、脂代谢、胰岛素分泌调节中发挥着重要的作用,并与神经系统性疾病密切相关.目前,关于Sirt1与糖尿病性角膜神经病变的关系尚不完全清楚. 目的 探讨Sirt1在1型糖尿病C57BL/6-Ins2Akita/J小鼠角膜及三叉神经节中的表达及其意义,探讨Sirt1与糖尿病性角膜神经病变的关系. 方法 取C57BL/6-Ins2Akita/J雄性小鼠与同窝出生的野生型C57BL/6小鼠各8只,分别作为1型糖尿病模型组和正常对照组.两组小鼠均在12月龄时过量麻醉处死,处死前检测空腹血糖、测体质量.取小鼠三叉神经节和角膜组织,采用苏木精-伊红染色法检测两组小鼠三叉神经节和角膜组织的组织病理学变化,用免疫组织化学法检测三叉神经节和角膜组织中Sirt1蛋白的表达和定位;用荧光定量PCR法检测Sirt1 mRNA在三叉神经节和角膜组织中的表达;采用Western blot检测两组小鼠三叉神经节和角膜组织中Sirt1蛋白的相对表达量,并进行比较.结果 C57BL/6-Ins2Akita/J 小鼠三叉神经节细胞大小不均,细胞排列较为疏松,神经纤维排列紊乱,角膜上皮细胞层数减少,角膜变薄;野生型C57BL/6小鼠神经节细胞排列紧密,细胞形态均一,神经纤维排列整齐.免疫组织化学法检测结果显示,C57 BL/6-Ins2Akita/J小鼠角膜中Sirt1蛋白的表达强度低于野生型C57BL/6小鼠.荧光定量PCR结果显示,Sirt1 mRNA在C57B L/ 6-Ins2Akita/J小鼠角膜表达的灰度值显著低于野生型C57BL/6小鼠(0.56±0.03 vs.0.98±0.13),差异有统计学意义(t=5.853,P=0.010);C57BL/6-Ins2Akita/J小鼠三叉神经节中Sirt1 mRNA表达的灰度值为2.45±0.18,低于野生型C57 BL/6小鼠的2.51±0.22,但差异无统计学意义(t=0.587,P=0.599).Western blot检测结果显示,C57 BL/6-Ins2Akita/J小鼠角膜中Sirt1蛋白的表达显著低于野生型C57BL/6小鼠(0.780±0.017 vs.1.300±0.012),差异有统计学意义(t=33.140,P=0.001);两组小鼠间三叉神经节中Sirt1蛋白的表达差异无统计学意义(1.100±0.015 vs.1.110±0.017) (t=0.430,P=0.709).结论 12月龄C57B L/ 6-Ins2Akita/J小鼠角膜的神经和结构发育异常,Sirt1参与糖尿病角膜病变的发病,有可能是潜在的靶点分子.     

基于流式细胞术快速定量分析小鼠角膜组织中嗜中性粒细胞方法的建立

目的:建立一种基于流式细胞术快速定量分析小鼠角膜组织中嗜中性粒细胞的技术和方法。方法:选取6～8周龄SPF级雌性C57BL/6小鼠15只,使用高尔夫样刀机械性刮除小鼠角膜上皮细胞层,生成直径2 mm的创面,在创伤后18 h切除带有完整角膜缘的小鼠角膜,采用胶原酶I和DNA酶联合消化法获得单细胞悬液,采用FACSCant...     

视网膜色素变性Rd1小鼠眼球中增殖细胞核抗原（PCNA）的表达

目的　探讨增殖细胞核抗原（proliferating cell nuclear antigen,PCNA）在Rd1小鼠视网膜上的表达情况。方法　取出生后14 d、21 d、28 d的Rd1小鼠和C57BL/6J小鼠各5只,摘出眼球进行HE染色,观察视网膜形态学结构变化;免疫组织化学染色与实时荧光定量PCR检测PCNA蛋白和基因在不同鼠龄小鼠中的表达。结果　HE染色结果显示:PCNA蛋白均在两种小鼠视网膜神经节细胞中表达;C57BL/6J小鼠视网膜结构完整;与C57BL/6J小鼠相比,Rd1小鼠视网膜发育随着鼠龄的增加,发生进行性退化。免疫组织化学染色结果显示:PCNA蛋白表达仅局限于视网膜神经节细胞,免疫阳性信号出现在第14天和第21天,而在第28天未检测到。PCR 实验结果显示:与C57BL/6J小鼠相比较,鼠龄21 d时Rd1小鼠PCNA基因表达水平增高,是C57BL/6J小鼠的2.23倍;鼠龄14 d和28 d时 Rd1小鼠PCNA基因表达均低于C57BL/6J小鼠（均为P<0.05）。结论　PCNA基因参与了Rd1小鼠视网膜发育退化过程。     

C57BL／6小鼠葡萄球菌性角膜感染模型的建立及相关研究

目的以乙基亚硝基脲（ENU）诱变技术建立小鼠葡萄球菌性角膜感染模型,探讨其主要条件致病菌的生物学特性。方法以ENU（150mg／kg）腹腔注射10周龄雄性C57BL／6小鼠,60d后与其同品系雌性小鼠配种,F1代小鼠中发现角膜混浊小鼠,以具有角膜混浊表型小鼠与C57BL／6小鼠回交的方式繁育。对角膜混浊小鼠角膜感染菌进行分离、培养、纯化、鉴定,并使用不同抗生素进行药物敏感试验。结果建立了稳定的小鼠葡萄球菌性角膜感染模型。从自发性角膜混浊（B6一Co）小鼠眼部成功分离纯化了松鼠葡萄球菌,筛选出了对该菌敏感及耐药的抗生素。对该菌敏感的抗生素有阿奇霉素、克林霉素、氯霉素、庆大霉素、利福平、四环素、阿米卡星、复方新诺明、米诺环素、左氟沙星、头孢噻吩、头孢噻肟、呋喃唑酮;对该菌耐药的抗生素有头孢西丁、青霉素、氨苄西林、新生霉素;属于中间态的抗生素为呋喃妥因。结论C57BL／6小鼠葡萄球菌性角膜感染模型为自发性动物模型,系凝固酶阴性葡萄球菌感染所致,以松鼠葡萄球菌为主。     

小分子化合物J2在抑制小鼠角膜移植排斥反应中作用的初步研究

目的 探讨小分子化合物J2在抑制小鼠角膜移植排斥反应中的作用。方法以23只C57BL／6小鼠作为供体,76只BALB／c小鼠作为受体建立角膜移植实验模型,随机数字法分为A、B、C及D组,A组为BALB／c小鼠自体原位角膜移植,B、C及D组为C57BL／6-BALB／c小鼠间同种异体角膜移植。术后灌胃给药,A组和B组给予不含药物的空白液,C组和D组分别给予环孢素A（CsA）和小分子化合物J2,连续灌胃12d,比较各组小鼠角膜植片的存活时间和存活率,术后21d对各组小鼠行外周血单核细胞行流式细胞学检查,并做角膜植片的组织学检查。结果A组观察期内角膜植片未发生排斥,B组角膜植片平均存活时间为（17．8±2．1）d,C组为（38．1±9．9）d,D组角膜植片存活时间为（40．6±8．3）d,D组与A组（P=0．04）及B组（P=0．00）比较存活时间差异均有统计学意义,与C组比较差异无统计学意义（P：0．99）。流式细胞学检查显示J2给药小鼠外周血CD^＋细胞、CD8^＋细胞未发生增殖,组织学检查证实术后21dD组角膜植片未见明显的淋巴细胞浸润。结论小分子化合物J2能够抑制排斥的发生,延长小鼠角膜植片存活时间。（中华胺群条峦,2007,43：608-612）     

RMT1-10体外诱导耐受性树突状细胞对小鼠高危角膜移植排斥反应的抑制作用及其机制

目的探讨RMT1-10体外诱导耐受性树突状细胞(Tol-DCs)对小鼠高危角膜移植排斥反应的抑制作用及其机制。方法选取SPF级雄性BALB/c小鼠100只和C57BL/6小鼠50只, 获取C57BL/6小鼠骨髓来源的未成熟树突状细胞(imDCs), 按照诱导干预不同分为imDCs组(不干预)、成熟树突状细胞(mDCs)组(加入脂多糖)、RMT1-10组(加入RMT1-10和脂多糖)和IgG同型对照组(加入IgG同型抗体和脂多糖), 培养7 d后采用流式细胞术检测各组DCs表型CD11c、CD80、CD86、主要组织相容性复合物(MHC)-Ⅱ、T细胞免疫球蛋白和黏蛋白结构域分子(Tim)-4和CD103表达水平;采用酶联免疫吸附法测定细胞培养液上清液中白细胞介素10(IL-10)和转化生长因子β(TGF-β)质量浓度。建立混合淋巴细胞培养体系, 采用细胞计数试剂盒8法检测各组DCs刺激CD4+ T细胞增生的刺激指数(SI)。以角膜基质缝线法诱导BALB/c小鼠角膜新生血管, 以4个象限新生血管均匀长入角膜中周区的小鼠作为受体。将80只受体小鼠采用随机数字表法随机分为imDCs组、mDCs...     

肠道真菌菌群失调对小鼠角膜创伤修复的影响

目的：观察抗真菌药物诱导的肠道真菌菌群失调对小鼠角膜创伤修复的影响。

方法：选用健康无眼疾的C57BL/6J雄性小鼠,将实验小鼠随机分为两组：对照组和两性霉素B组。对照组给予正常饮食,两性霉素B组给予添加两性霉素B的饮食,以诱导小鼠肠道真菌菌群失调,4wk后对两组小鼠角膜进行上皮创伤,使用荧光素钠染色角膜创伤区域,动态观察上皮修复情况,并利用免疫荧光染色观察角膜上皮细胞、炎症细胞变化,通过HE染色观察角膜厚度的变化。

结果：两性霉素B组与对照组相比,再上皮化速度和创伤修复延迟,炎症细胞明显减少,角膜上皮厚度变薄。

结论：肠道真菌菌群失调延迟角膜创伤的修复,降低角膜创伤后的炎症反应。     

鼠角膜TRAIL mRNA表达与角膜移植排斥反应

目的观察正常鼠眼角膜组织中“肿瘤坏死因子相关凋亡诱导配体（TRAIL）的表达分布情况。方法采用RT-PCR技术和免疫组化技术检测TRAIL，定性、定位分析TRAIL在正常BALB／c鼠角膜组织中的表达。结果TRAIL mRNA在正常BALB／c鼠角膜组织中表达，免疫组织化学检测显示：角膜上皮层、内皮层强表达，闻质层弱表达。结论TRAIL广泛存在于角膜组织中，有抑制或减轻角膜移植免疫排斥反应的作用，但其机制有待进一步深入研究。     

视网膜新生血管形成中Cathepsin B的变化

目的通过观察Cathepsin B的变化,探讨视网膜新生血管的发病机制。方法取C57BL/6J7d龄小鼠30只,随机分为对照组和实验组,实验组在缺氧环境中建立视网膜新生血管动物模型。采用苏木精-伊红染色、视网膜ADP酶染色铺片及免疫组织化学法进行检测,同时测定眼组织Cathepsin B活性。结果组织病理学见突破内膜新生血管内皮细胞核数,无血管区面积及无血管区面积/视网膜面积,实验组与对照组比较差异均有统计学意义(P<0.01);对照组与实验组Cathepsin B平均光密度、Cathepsin B活性比较差异有统计学意义(P<0.01)。结论C57BL/6J构建小鼠视网膜新生血管动物模型后,小鼠视网膜组织Cathepsin B表达增加、活性增强,CathepsinB可能是视网膜新生血管产生的一个重要因素。     

17β-雌二醇抗BALB/c与C57BL/6小鼠视网膜光照损伤的比较

目的：通过建立BALB/c与C57BL/6小鼠视网膜光损伤模型，研究17β-雌二醇(17β-estradiol， E2)的视网膜神经保护作用，为成功构建E2抗视网膜光损伤模型提供实验数据。

方法：成年雌性BALB/c、C57BL/6小鼠各40～45只，实验分组如下：正常对照组，去势手术对照组，去势光照组(小鼠去势手术14d后进行持续10 000lx白光光照刺激4、8、12、16、24h组)，玻璃体腔注射假手术组，生理盐水组和E2预处理组(去势手术14d后暗适应24h后分别行玻璃体腔注射2μL生理盐水或10^-5mol/L E2)，每组各6只。通过石蜡切片HE染色、TUNEL染色、视网膜电图检测视网膜形态及功能变化。

结果：去势光损组视网膜内核层/外核层厚度从白光10 000lx光照4h组开始显著减小； 玻璃体腔注射E2预处理可显著抑制两种品系小鼠视网膜各层细胞的凋亡(P<0.01)以及C57BL/6小鼠视网膜电图检测中最大混合反应a波和b波波幅的下降(P<0.05)。

结论：相同光照条件对两种品系小鼠光损敏感性存在差异； E2对BALB/c小鼠无论是视网膜形态学及功能学上都产生了保护作用，而对C57BL/6小鼠功能学保护作用显著。     

Effect of alloantibodies on corneal allograft survival

PURPOSE: The precise role of antibodies in corneal transplantation is ambiguous, with evidence to support as well as repudiate their involvement in graft rejection. Accordingly, this study was undertaken to investigate the direct contribution of donor-specific antibodies to corneal graft rejection. METHODS: Serum samples from CB6F1 rejecters of orthotopically grafted C3H/Hej corneas were tested by ELISA for elevated levels of donor-specific alloantibody. Orthotopic corneal allograft rejection was also examined in B-cell-deficient mice. In a prospective study, na?ve BALB/c T-cell-deficient nude mice and complement-depleted nude mice were passively infused with immune donor-specific serum and grafted with fully allogeneic C57BL/6J corneas. The incidence and speed of graft rejection were observed in each case. The susceptibility of corneal cells to antibody-mediated lysis was tested in vitro. RESULTS: Seventy percent of the CB6F1 hosts that rejected the C3H/Hej corneal allografts possessed significantly elevated levels of alloantibody in serum. Although BALB/c corneal allografts were rejected by B-cell-deficient mice at the same incidence as wild-type control mice, their mean survival time (MST) was significantly longer than that of their wild-type counterparts. Serum of BALB/c mice immunized against C57BL/6J alloantigens produced complement-dependent cytolytic activity against C57BL/6J corneal cells in vitro. Passive transfer of this alloantiserum to T-cell-deficient BALB/c nude mice produced complement-dependent corneal lesions, resulting in significantly increased opacity of C57BL/6J corneal grafts, compared with the relatively clear grafts in control hosts. CONCLUSIONS: Alloantibody, although not necessary for corneal graft rejection, can produce extensive injury to corneal allografts in a complement-dependent manner.     

小鼠角膜移植排斥反应中植片内细胞因子表达水平的变化

背景研究表明细胞因子在角膜移植免疫调节中起重要的作用,但关于角膜移植术后角膜植片内细胞因子表达的变化报道较少。目的探讨角膜移植术后不同时间点细胞因子的表达变化。方法采用雌性6～8周龄BALB／c小鼠及C57BL／6小鼠分别建立小鼠自体角膜移植和同种异体角膜移植模型,每组各10只,自体角膜移植术组供体和受体同为BALB／c小鼠,同种异体角膜移植术组供体为C57BL／6小鼠,受体为BALB／c小鼠。于术后1d开始临床观察角膜植片并对炎症程度进行评分,当植片评分总和≥5分或植片混浊≥2分时为发生植片排斥反应。将BALB／c小鼠随机分为正常对照组3只和同种异体角膜移植术组15只,后者分别于术后6h及1、3、7、14d对角膜植片行逆转录PCR（RT—PCR）检测植片中细胞因子白细胞介素4（IL-4）、γ-干扰素（IFN-γ）、IL-10、肿瘤坏死因子α（TNF—α）表达的变化。结果在60d的观察期中,自体角膜移植术后小鼠角膜植片混浊评分均〈2分,植片的炎症评分之和均〈5分,植片存活率为100％,但同种异体角膜移植术后角膜植片水肿、混浊,伴有角膜新生血管形成,至术后24d角膜植片排斥反应评分之和均≥5分,植片混浊评分≥2分,全部发生排斥反应,角膜植片平均存活时间为（17．80±4．66）d。RT—PCR检测结果表明,正常角膜中IL-4和IFN-γ呈阳性表达,IL-10和TNF-α表达阴性。同种异体角膜移植术后6h可检测到IL-4呈阳性表达,IFN-γ和IL-10表达呈强阳性,而未检测到TNF—α的表达。术后1～3d角膜植片中IL-4呈强阳性表达,IFN-γ阳性表达,TNF—α表达增强,IL-10表达逐渐消失。角膜移植术后7d,可见IL-4表达阴性,而IFN-γ、IL-10和TNF—α仍呈强阳性表达。至术后14d,角膜植片中IL-4、IFN-γ和TNF—α．均未见表达,仅检测到IL-10呈阳性表达。结论TNF-α是角膜移植术后局部参与调控的主要因子。     

Mouse strain-dependent heterogeneity of resting limbal vasculature

PURPOSE: Heterogeneity of the extent of angiogenesis induced by exogenous growth factors may be determined by genetic influences. Because angiogenesis is the formation of new vessels from preexisting ones, strain-related influences on na?ve resting limbal vessel phenotype and gene expression were determined in mice having divergently low and high angiogenic responses. METHODS: Resting limbal vessel surface area and density and extent of bFGF-induced corneal angiogenesis were determined in C57BL/6J, BALB/cJ, F1 intercross identical with C57BL/6J X 129S3/SvIM, and 129S3/SvIM mouse strains by quantitative three-dimensional reconstruction confocal microscopy. Strain-related influences on pro- and antiangiogenic gene expression in na?ve cornea were determined by quantitative real-time RT-PCR. RESULTS: The strain-dependent rank order of resting limbal vessel surface area and resting vessel density paralleled bFGF-induced neovascularization: 129S3/SvIM > BALB/cJ, F1 > C57BL/6J (P < 0.0006). Pigment epithelium-derived factor (PEDF) was increased more than 67-fold compared to Ang-2 in resting cornea of both C57BL/6J and 129S3/SvIM strains (P < 0.0001; P < 0.0001), suggesting a strongly antiangiogenic environment. The corneas of the C57BL/6J mice demonstrated 1.8-, 1.5-, and 1.7-fold increased mRNA levels for Flt-1, VEGF, and bFGF, respectively (P < 0.02; P < 0.04; P < 0.02); however, TSP-1 expression was increased 2.4-fold compared with 129S3/SvIM (P < 0.0004). CONCLUSIONS: Strain-dependent differences in the resting limbal vessel surface area and density correlated with heterogeneity in the extent of bFGF-induced angiogenesis. Differences in pro- and antiangiogenic gene expression levels in resting cornea may influence vascular limbal phenotype during quiescence and may predict susceptibility to angiogenesis-dependent diseases.     

Cytotoxic T cells play no essential role in acute rejection of orthotopic corneal allografts in mice

PURPOSE: To determine whether cytotoxic T cells of the direct alloreactive type are activated and responsible for early, acute failure of orthotopic corneal allografts observed in eyes of C57BL/6 but not of BALB/c mice. METHODS: Corneas from BALB/c and BALB.B mice were placed orthotopically in eyes of C57BL/6 and beta-2 microglobulin knockout mice (deficient in CD8(+) cytotoxic T cells). Graft fates were assessed clinically, and the T lymphocytes of recipients were assayed for the capacity to lyse target cells bearing donor major (MHC) and/or minor histocompatibility (minor H) antigens (direct and indirect pathways, respectively). RESULTS. Similar to BALB/c recipients, C57BL/6 mice with rejected cornea allografts acquired donor minor H-specific T cells. Unlike BALB/c recipients, C57BL/6 mice-both rejectors and acceptors-acquired donor MHC-specific T cells. beta-2 Microglobulin knockout mice showed rejection of corneal allografts in a manner indistinguishable from C57BL/6 mice, including early, acute rejection, yet T cells from beta-2 microglobulin knockout recipients of corneal allografts displayed no cytotoxic T cells specific for either donor MHC or minor H alloantigens. CONCLUSIONS: Although C57BL/6 mice acquired donor MHC-specific cytotoxic T cells (direct alloreactive cells), neither these cells nor donor minor H-specific cytotoxic T cells (indirect alloreactive cells) play any essential role in corneal allograft rejection, including the early acute failure uniquely observed in C57BL/6 eyes.     

超抗原活化的耐受性淋巴细胞防治高危角膜移植免疫排斥反应的实验研究

目的检测超抗原金黄色葡萄球菌肠毒素B（SEB）体外活化的小鼠淋巴细胞的免疫耐受功能,探讨该细胞对小鼠高危角膜移植免疫排斥反应的防治作用。设计实验研究。研究对象14只BALB／C小鼠作为供体,28只C57BL/J小鼠作为受体。方法无菌取C57BL/J小鼠脾脏淋巴细胞,制备成5×10^6／ml的混悬液,分别与SEB和刀豆蛋白A（ConA）体外共培养,MTF法测定细胞增殖。用流式细胞仪测定SEB和ConA体外活化的淋巴细胞在第0、6天时的CD4^＋CD25^＋调节性T细胞和CD3＋NK1．1＋NKT细胞百分比。以BALB／c小鼠为供体,C57BL,J小鼠为受体,建立小鼠高危角膜移植动物模型,术后分为实验组、对照组,并分别结膜下注射0．05ml培养6天的SEB活化的淋巴细胞悬液（浓度1×10^＋个细胞／m1）及相同体积的生理盐水,术后观察记录植片的存活状况,并进行组织病理学检查和免疫组织化学检测。主要指标角膜植片平均存活时间,组织病理学及免疫组织化学染色检查淋巴细胞浸润情况。结果SEB、ConA与淋巴细胞共培养前OD570cm值均为0．15±0．01（n=6）,共培养后第3天为0．25±0．07和0．59±0．06,第6天为0．43±0．07和0.35±0．05。SEB组培养后的淋巴细胞中CD3^＋NK1．1＋NKT细胞由0天时的（1．21±0．19）％升高到（5．67±0．25）％,CD4^＋CD25＋调节性T细胞由（0．37±0．06）％升高到（0．98±0．12）％。活化淋巴细胞结膜下注射后小鼠角膜植片平均存活（28．60±3．75）天,而生理盐水组为（22．13±4．91）天,两者比较差异有统计学意义（P=0．006）。HE染色显示对照组植片中度水肿,角膜基质纤维板层结构排列紊乱,有炎性细胞浸润,植片中可见新生血管。而实验组植片仅轻度增厚,基质板层纤维排列规则,未见炎性细胞浸润及新生血管长人。免疫组织化学染色显示治疗组小鼠角膜植片中CD4^＋和C     

BALB/c and C57BL6 mouse strains vary in their ability to heal corneal epithelial debridement wounds

Genetically engineered mice are usually produced on a mixed genetic background and can be derived from several mouse strains including 129SvJ, C57BL6, and BALB/c. To determine whether differences in recurrent corneal epithelial erosions (RCEEs), corneal epithelial stem cell deficiency (CESCD), and cell migration rate vary between two different mouse strains (BALB/c and C57BL6), 8-week mice were subjected to 1.5 (small) or 2.8 mm (large) manual debridement wounds and allowed to heal for 4 weeks. Syndecan-1 (sdc-1) null mice backcrossed seven generations onto a BALB/c genetic background were also included in the RCEE and CESCD studies to permit comparisons between genotypes within a single strain. After sacrifice, corneas were assessed for the presence of recurrent erosions; no fewer than 15 corneas were used for each strain or genotype studied. Data show that the frequency of recurrent erosions after small wounds was 81 +/− 9% in the C57BL6 mice, 73 +/− 2% in the BALB/c mice, and 32 +/− 6% in sdc-1 null mice. Neither strain developed CESCD after small wounds. The frequency of erosions after large wounds was greater (88 +/− 8%) in the C57BL6 mice compared to BALB/c (60 +/− 2%), and sdc-1 null mice (32 +/− 5%). Four weeks after the large wounds, fixed, flat mounted corneas were assessed for evidence of CESCD with antibodies against the conjunctival keratin K8 and the goblet cell marker, the mucin Muc5AC. The frequency of CESCD 4 weeks after the large wounds was significantly greater in the C57BL6 mice than in the BALB/c or sdc-1 null mice. To assess cell migration rates, corneas were subjected to 1.5 mm wounds and allowed to heal for 12, 15, 18, 21, and 24 h. After sacrifice, corneas were stained with Richardson stain (BALB/c) or propidium iodide (C57BL6) to assess reepithelialization rates. While reepithelialization rates were similar for the early times after wounding, by 24 h the C57BL6 corneas had healed faster: 16 of 30 corneas from the C57BL6 mice were closed compared to 9 of 30 of the BALB/c wounds. BALB/c corneas appeared larger overall compared to C57BL6 corneas; measurements of the overall mass of the enucleated eyes and diameters of the flat-mounted corneas confirmed that C57BL6 eyes and corneas were 6.8% and 4.4% smaller respectively than those of BALB/c mice even though the masses of the two mouse strains at 8 weeks of age were identical. Using BrdU to label dividing cells, we found that 18 h after wounding, C57BL6 and BALB/c corneal epithelia showed similar numbers of proliferating cells. To determine if the enhanced corneal epithelial cell migration rate seen in the C57BL6 mice was specific to the cornea, we conducted time-lapse studies to assess random cell migration rates in vitro using primary cultures of mouse epidermal keratinocytes. Consistent with the in vivo data, epidermal keratinocytes derived from BALB/c mice migrated 60% slower than C57BL6 cells. These data prove that strain-specific differences in cell migration rate in vivo are present in the cornea and are accompanied by differences in the frequencies of recurrent erosions and corneal epithelial stem cell deficiency.     

Recovery of herpes simplex virus from ocular tissues of latently infected inbred mice

Evidence for latent infection of ocular tissues following topical corneal inoculation with herpes simplex virus type 1 (HSV) was sought in three strains of inbred mice that differ in susceptibility to HSV stromal keratitis. Corneas of BALB/c, C57BL/6, and DBA/2 mice were inoculated topically with HSV. At 6-8 weeks after inoculation, when no active ocular infection was present, minced whole eyes and trigeminal ganglia were assayed for latent virus. Virus was recovered by explantation from minced eyes of all three strains (DBA/2 = 20%; BALB/c = 17%; C57BL/6 = 7%). In order to determine which ocular structures harbored virus, corneas, retinas and choroid-sclera were cultivated separately. Virus was activated from corneas of DBA/2 and BALB/c mice, but not from corneas of C57BL/6 mice. These findings suggest that HSV is capable of establishing latent infection in ocular tissue of inbred mice and that the rate of establishment of latency is under host genetic control. Since neural cell bodies are not present in the cornea, the data suggest that latency is established in cells other than neurons.     

ACAID induced by allogeneic corneal tissue promotes subsequent survival of orthotopic corneal grafts

PURPOSE: To determine whether immune deviation is induced by allogeneic corneal tissue implanted in the anterior chamber and whether survival of subsequent orthotopic corneal allografts is thereby enhanced. METHODS: Corneal tissue from C57BL/6 mice was implanted in the anterior chamber of eyes of BALB/c mice. The fate of these implants was assessed histologically, and the donor-specific immune response of recipient mice was tested for donor-specific delayed hypersensitivity and the capacity to accept or reject C57BL/6 corneas grafted orthotopically into the fellow eye. RESULTS: C57BL/6 cornea implants in the anterior chamber failed to induce donor-specific delayed hypersensitivity but impaired donor-specific delayed hypersensitivity in a proportion of recipients with implants in place for 2 weeks. Mice with donor-specific delayed hypersensitivity rejected the intraocular implants. Mice bearing C57BL/6 cornea implants in the anterior chamber for 2 (but not 4) weeks accepted the C57BL/6 corneas grafted orthotopically into the fellow eye at a high rate and with few rejection reactions. CONCLUSIONS: Implantation of allogeneic corneal tissue in the anterior chamber of the eye has the transient capacity to alter the recipient alloimmune response in a manner that promotes survival of subsequent orthotopic corneal allografts.     

Herpetic keratitis in inbred mice

The corneas of four inbred strains of mice (BALB/c, DBA/2, C3H and C57BL/6) were inoculated with the RE strain of herpes simplex virus, type 1. The corneas were examined at frequent intervals and graded on a scale of 0 (clear cornea) to +5 (severe necrotizing stromal keratitis). At 3 weeks postinfection, the mean corneal scores were: DBA/2, 4.0; BALB/c, 2.2; C3H, 0.7; and C57BL/6, 0.15. The differences between the scores are statistically significant (P less than 0.05), except for the C3H and C57BL/6 strains. The order of severity of corneal disease in these mice corresponds to the order of susceptibility to systemic infection found in these same inbred strains. Additional studies of herpetic keratitis in inbred mice should prove helpful in understanding the genetic and immunologic basis of herpetic stromal keratitis.