内毒素诱导的葡萄膜炎中虹膜固有组织细胞上MyD88与TLR-4的共表达

目的观察小鼠内毒素诱导的葡萄膜炎(endotoxin-induced uveitis,EIU)模型中Toll样受体-4(Toll-like receptor-4,TLR-4)、髓样分化因子88(myeloid differentiation factor 88,MyD88)、CD163的共表达。方法 6～8周龄雄性C3H/HeN(野生型)小鼠25只,C3H/HeJ(TLR-4基因缺陷型)小鼠5只,分别腹腔注射霍乱弧菌内毒素细胞壁脂多糖(lipopolysaccharide,LPS)诱导急性前葡萄膜炎动物模型,处死小鼠后虹膜、睫状体铺片,用免疫荧光三标记的方法检测虹膜、睫状体组织内不同时间CD163、TLR-4、MyD88的表达,并对虹膜内CD163、TLR-4和MyD88阳性细胞进行计数分析。结果 C3H/HeN小鼠LPS注射后12h结膜囊内出现炎症反应,24～48h达到高峰,72h炎症逐渐消退,而C3H/HeJ小鼠LPS注射后没有发现炎症反应。在内毒素诱导的C3H/HeJ小鼠虹膜中未检测到CD163、TLR-4和MyD88阳性细胞。C3H/HeN小鼠腹腔注射LPS后在虹膜铺片内0h未见CD163、TLR-4与MyD88阳性表达,12h后可见阳性细胞(40.3±8.9、45.2±6.3、42.5±4.1),24h阳性细胞数(121.0±39.5、138.6±28.3、125.5±36.1)较12h明显增多,差异均有统计学意义(均为P<0.05);48h阳性细胞数(132.3±54.5、129.9±36.2、122.1±29.3)与24h相比,差异均无统计学意义(均为P>0.05);72h阳性细胞数(12.8±3.2、10.4±5.6、9.3±5.2)较48h减少,差异均有显著统计学意义(均为P<0.01)。结论小鼠EIU模型中,LPS激活了CD163标记的巨噬细胞膜表面的TLR-4,TLR-4与MyD88途径可能是EIU主要的信号传导途径。     

TLR-4和CD14在急性前葡萄膜炎小鼠虹膜中的表达

目的 观察内毒素诱导的小鼠急性前葡萄膜炎模型中虹膜Toll样受体-4(Toll-like receptor-4,TLR-4)和CD14的表达.方法 C3H/HeN(野生型)和C3H/HeJ(TLR-4基因缺陷型)小鼠足底注射霍乱弧菌内毒素,建立急性前葡萄膜炎动物模型,注射后每隔2h用裂隙灯进行眼前节观察,并做组织病理学检查.用Western blotting及免疫组织化学法检测小鼠虹膜中TLR-4及CD14蛋白的表达.结果 C3H/HeN小鼠在内毒素注射后4～6 h,小鼠结膜囊内出现大量白色分泌物,12～18 h瞳孔区偶见白色渗出,22～24 h可见虹膜后粘连及渗出物,36～48 h前房内未见炎症消退;C3H/HeJ小鼠未见眼前节炎症反应.通过Western boltting发现正常C3H/HeN小鼠虹膜内有TLR-4蛋白的表达,注射内毒素24 h后TLR-4的表达量显著增高,与12 h相比差异有显著统计学意义(P=0.009).免疫组织化学显示在内毒素诱导后24 h,TLR-4与CD14在急性前葡萄膜炎模型虹膜组织中大量表达,而在C3H/HeJ小鼠中未见TLR-4蛋白的表达.结论 TLR-4在内毒素诱导的C3H/HeJ(TLR-4基因缺陷)小鼠中未见表达,而在C3H/HeN(野生型)急性前葡萄膜炎动物模型虹膜组织中大量表达.TLR-4可能在急性前葡萄膜炎发病环节中发挥着重要作用.     

大黄多糖对内毒素诱导急性前葡萄膜炎TLR4/NF-κB传导通路干预作用的实验研究

目的 通过观察大黄多糖对内毒素（LPS）诱导的急性前葡萄膜炎TLR4通路的影响,揭示大黄多糖对急性前葡萄膜炎TLR4通路的分子干预机制,为TLR4 通路介导的炎症治疗提供新的思路和途径。设计 实验研究。研究对象 野生型Wistar大鼠、RAW264.7巨噬细胞系。方法 野生型Wistar大鼠15只,随机分为3组,正常对照组、模型组（LPS组）、大黄多糖处理组,每组5只,大黄多糖处理组腹腔注射400 mg/kg大黄多糖,对照组和模型组注射等量PBS。各组腹腔注射2小时后模型组和大黄多糖处理组每只大鼠足底注射0.1 ml霍乱弧菌内毒素LPS,对照组注射等体积的PBS,24小时后裂隙灯下观察大鼠眼前节炎症反应并按炎症分级评分,RT-PCR观察大鼠虹膜TLR4表达的变化。RAW264.7巨噬细胞系进行体外培养,用大黄多糖、LPS分别刺激,通过Western blot、RT-PCR、ELISA、免疫荧光技术观察两者对巨噬细胞TLR4及相关分子的影响。主要指标 大鼠眼前节炎症反应程度、巨噬细胞TLR4表达变化。结果 裂隙灯下观察,LPS组相比于正常对照组虹膜充血严重,前房闪辉及瞳孔区纤维素渗出均较多;大黄多糖干预组大鼠仅轻度虹膜充血,瞳孔缘无明显渗出膜,瞳孔无缩小。RT-PCR示大黄多糖干预组TLR4 mRNA 表达较LPS组明显降低。Western blot和RT-PCR结果显示大黄多糖在体外分别能引起巨噬细胞TLR4、髓样分化蛋白-88（MyD88）、核因子-κB（NF-κB） p65蛋白和mRNA表达量的增加;ELISA显示大黄多糖100 ?滋g/ml作用于Raw264.7细胞24小时后能引起上清液炎症因子IL-17,IL-10,TNF-α,INF-γ,IL-1β的表达增高;免疫荧光显示大黄多糖能引起培养细胞的激活、TLR4复合物及NF-κB p65表达。结论 大黄多糖对内毒素诱导的大鼠急性前葡萄膜炎模型具有明显的抑制作用。体外实验显示大黄多糖同LPS一样,在体外具有激活巨噬细胞TLR4及其下游MyD88和NF-κB、调节细胞因子表达的作用。（眼科, 2013, 22: 134-140）     

TLR4-MyD88在大鼠急性前葡萄膜炎虹膜中的表达

目的观察内毒素诱导的大鼠急性前葡萄膜炎（EIU）虹膜组织内Toll样受体4（TLR4）、髓样分化因子88（MyD88）及核因子-κBp65（NF-κB p65）的表达。方法Wistar大鼠50只,随机分为5组,0、12、24、48、72h,每组10只。0h组为正常对照组,其余4组均足垫部注射霍乱弧菌内毒素脂多糖（LPS）200μg,建立EIU动物模型,每隔2h用裂隙灯观察大鼠眼前节炎症反应。通过铺片免疫组织化学染色,检测虹膜睫状体组织内TLR4、MyD88和NF—κB p65的表达,并对虹膜内TLR4^＋和MyD88^＋及NF—κB p65^＋细胞进行计数。结果注射后24～48h大鼠眼前段的炎症反应达到高峰,72h炎症反应逐渐缓解。组织病理学检查表明,虹膜睫状体组织的炎性细胞浸润在24～48h达到高峰,与临床反应结果相符。TLR4在模型鼠虹膜睫状体炎复合体中表达的免疫组织化学检测结果表明,0h组虹膜铺片内无阳性细胞,12h后可见细胞形态大多为类圆形的阳性细胞,48h达高峰,72h阳性细胞数开始减少,各组阳性细胞数总体差异有统计学意义（F=46．79,P〈0．05）。MyD88和NF—κB p65的表达与TLR4的改变趋势相一致（F=54．37,P〈0．05;F=85．32,P〈0．05）。结论内毒素诱导的EIU虹膜内,TLR4及其下游信号传导分子的表达量发生改变,提示TLR4-MyD88依赖传导途径可能参与了EIU的发病.     

Toll样受体-4在葡萄膜炎中的表达和作用

Toll样受体-4(TLR4)作为内毒素的识别受体在固有免疫与适应性免疫之间的桥梁作用越来越受到重视.葡萄膜炎发病中TLR4的作用不断被证实:TLR4基因突变的C3H/HeJ小鼠不能诱导出葡萄膜炎;正常人眼葡萄膜中有TLR4表达而且是有功能的;葡萄膜炎患者外周血中单核细胞TLR4功能下降.本文对TLR4在葡萄膜炎发病中的信号传导通路的进展及其在抗感染中的作用进行了综述.     

枸杞多糖对LPS诱导的人视网膜色素上皮细胞炎性反应的影响及机制

目的:探索枸杞多糖(LBP)对细菌脂多糖(LPS)诱导的人视网膜色素上皮(ARPE-19)细胞的炎性反应的影响及其可能介导的信号通路。方法:通过LPS刺激体外培养的ARPE-19细胞构建炎性反应模型。将细胞随机分为5组,空白组使用完全培养基培养,LPS组给予含10μg/mL LPS的完全培养基刺激24h,低、中、高浓度LBP组分别给予0.1、0.5、1mg/mL LBP完全培养基孵育24h后给予含10μg/mL LPS的完全培养基刺激24h。应用CCK-8观察细胞存活率、实时荧光定量PCR检测相关炎性因子的mRNA表达量及Western-blot检测NF-κB/MAPK信号通路相关磷酸化蛋白表达量的变化。结果:与正常细胞相比,LPS刺激后,ARPE-19的存活率下降。同时,随着外源性LBP浓度增加,ARPE-19的细胞存活率升高,且细胞内炎性因子IL-1β、IL-6和MCP-1的表达量降低,并伴随NF-κB/MAPK通路的相关磷酸化蛋白(p-p65、p-IκBα、p-JNK、p-ERK及p-p38)的表达下降。结论:枸杞多糖通过抑制胞内炎性因子及NF-κB/MAPK通路相关因子的磷酸化,从而阻止LPS诱导人视网膜色素上皮细胞产生的炎性反应。     

针对不同靶位点的siRNA对人晶状体上皮细胞NF-κB P65表达的抑制作用

目的 探讨针对核因子-κB(NF-κB)P65亚基的小干扰RNA(siRNA)对晶状体上皮细胞(LECs)NF-κB表达的影响.方法 设计并合成3条NF-κB siRNA,1条阴性对照siRNA.实验分为6组,A组为正常对照组,常规体外培养HLE-B3细胞;B组为肿瘤坏死因子-α(TNF-α)刺激组;C、D、E、F组为转染组,分别转染4条siRNA,24h后加入含TNF-α的培养基培养.免疫印迹法、实时荧光定量PCR法分别检测NF-κB P65蛋白与mRNA的表达.结果 B组与A组比较,NF-κB P65表达明显增加,差异有统计学意义(P＜0.01).C组、D组与B组比较,NF-κB表达明显减少,差异有统计学意义(P＜0.01).结论 特异性siRNA可以明显降低HLE-B3细胞株NF-κB P65蛋白及mRNA的表达,为RNA干扰治疗外伤性白内障奠定了基础.     

低氧预适应对小鼠光损伤视网膜HIF-1α和NF-κB表达的影响

目的 研究低氧诱导因子(HIF-1α)和核因子(NF-κB)在小鼠视网膜光损伤后的表达及低氧预适应对其表达的影响,探讨低氧预适应对视网膜光损伤的保护机制.方法 将54只BALB/C小鼠随机分成单纯光照组、低氧预适应组和正常组,前2组又按光照后处死时间的不同分为6、12、36 h和7 d组.应用免疫组织化学技术检测小鼠视网膜组织HIF-1α和NF-κB蛋白的表达,并分析低氧预适应组与单纯光照组HIF-1α和NF-κB蛋白的表达差异.结果 正常小鼠视网膜组织HIF-1α几乎不表达,NF-κB有微量表达.低氧预适应组与单纯光照组比较,HIF-1α的表达强度明显增强,而NF-κB的表达强度明显减弱,两组比较差异有统计学意义(P＜0.01).结论 低氧预适应可能通过提高HIF-1α的表达有效抑制NF-κB信号通路,从而通过降低凋亡的发生而起到保护作用.     

糖尿病大鼠神经干细胞视网膜下移植后TLR4、NF-κB的表达

目的 观察糖尿病大鼠视网膜下移植大鼠胚胎神经干细胞(NSCs)后转录因子(NF-KB)、Toll样受体4(TLR4)的表达.方法 大鼠糖尿病成模后,A组视网膜下移植大鼠NSCs,B组视网膜下填充DMEM,C组不做手术处理.NSCs移植组术后1、2、4周分别取双眼颞上视网膜做对照检测.Western blot法检测TLR4和NF-κB的蛋白表达.结果 A组糖尿病大鼠TLR4、NF-κB持续表达阳性,B组仅术后3 d、1周TLR4表达阳性,术后1周NF.KB表达阳性,C组TLR4和NF-KB表达为阴性.各组TLR4、NF-KB表达差异有统计学意义(P<0.05).结论 糖尿病大鼠NSCs视网膜下移植后TLR4、NF-KB可持续表达.     

TLR2/MyD88信号系统在大鼠角膜移植术后排斥反应中的作用

目的 探讨Toll样受体2(Toll like receptor,TLR2)/MyD88信号系统在大鼠角膜移植术后排斥反应中的作用.方法 取14只SD大鼠为供体,28只Wistar大鼠为受体,建立28个同种异体穿透性角膜移植模型.建模后分为同种异体角膜移植组14只(6只用于术后观察排斥情况),同种异体角膜移植TLR2单克隆抗体组14只(6只用于术后观察排斥情况).另选16只Wistar大鼠,分为同种自体角膜移植组8只,正常对照组8只.3个手术组大鼠分别行穿透性角膜移植术.术后TLR2单克隆抗体组给予0.5 g·L-1TLR2单克隆抗体,分别于术后0d、2d、4d、6d、8d分5次经球结膜下注射给药.正常对照组、同种自体角膜移植组及同种异体角膜移植组给等量生理盐水.术后每天于裂隙灯下观察角膜透明度、新生血管,并用排斥反应指数评分.第9天取材,行HE、免疫组织化学染色和实时荧光定量PCR检测.结果 术后随时间变化各手术组角膜植片均出现不同程度的水肿、混浊和新生血管生长,以同种异体角膜移植组最为明显.HE染色结果显示在同种异体角膜移植组,角膜基质层出现水肿、大量炎症细胞浸润和新生血管,而在同种自体角膜移植组和TLR2单克隆抗体组中炎症反应较轻.免疫组织化学检测结果:TLR2和MyD88分子在正常对照组、同种自体角膜移植组及TLR2单克隆抗体组的角膜上皮中均有微量表达,同种异体角膜移植组角膜上皮、基质细胞中TLR2和MyD88分子的表达明显增多,尤以基质层明显.实时荧光定量PCR结果显示同种异体角膜移植组角膜组织TLR2 mRNA、MyD88 mRNA的表达较同种自体角膜移植组(P =0.000、0.004)和正常对照组(P=0.000、0.000)显著增加,两者的表达亦较TLR2单克隆抗体组显著增加(P =0.000、0.003).结论 TLR2单克隆抗体抑制TLR2及其下游信号分子MyD88的表达;TLR2/MyD88信号系统参与了角膜移植术后排斥反应,影响了角膜植片的转归.     

Endotoxin-induced uveitis in mice. 1. Induction of uveitis and role of T lymphocytes.

Endotoxin-induced uveitis (EIU) with a high frequency of posterior iris synechiae was induced by the systemic injection of 200 micrograms of endotoxin into C3H/HeN mice, an endotoxin-responsive strain. The cell number and the protein concentration within the aqueous humor began to increase 6 hours after the injection, achieving a peak at 24 hours, and decreased gradually thereafter. Inflammatory cells were observed in the anterior chamber, the vitreous body and near the iris-ciliary body histologically. Most of the inflammatory cells were polymorphonuclear cells. On the other hand, C3H/HeJ mice, an endotoxin-unresponsive strain, showed no increase in either cell number or protein concentration in the aqueous humor after endotoxin administration. Pretreatment of C3H/HeN mice with anti-Thy-1.2 antibody significantly decreased both the cell number and the protein concentration in the aqueous humor and the incidence of the posterior synechiae, as compared with the control group. Anti-CD4 antibody also significantly reduced the severity of EIU, while anti-CD8 antibody had no influence on the disease. Anti-IFN-gamma antibody increased the cell number in the aqueous humor. These observations indicate that T lymphocytes, especially CD4+ T lymphocytes, have an extremely important role in the development of EIU in mice.     

Regulation of endotoxin-induced keratitis by PECAM-1, MIP-2, and toll-like receptor 4

PURPOSE: Bacterial lipopolysaccharide (LPS, endotoxin) is a potent stimulator of inflammatory responses and is likely to contribute to microbial keratitis and to the pathogenesis of sterile corneal ulcers. The purpose of the present study was to identify specific mediators of endotoxin-induced keratitis. METHODS: The corneal epithelium of BALB/c, C3H/HeJ, and C3H/HeN mice was abraded, and Pseudomonas aeruginosa endotoxin (10 microg in 1 microL) was added. Stromal thickness and haze were measured by in vivo scanning confocal microscopy, and neutrophil recruitment determined by immunohistochemistry. RESULTS: Pseudomonas endotoxin induced a significant increase in stromal thickness and haze compared with untreated control corneas at each time point examined, and the severity coincided with neutrophil infiltration into the corneal stroma. Furthermore, systemic depletion of neutrophils completely abrogated endotoxin-induced increases in stromal thickness and haze, indicating an essential role for these cells. Expression of platelet endothelial cell adhesion molecule (PECAM)-1 on vascular endothelium and production of macrophage inflammatory protein (MIP)-2 in the corneal stroma were also significantly elevated after exposure to endotoxin, and antibody blockade inhibited neutrophil recruitment to the cornea and abrogated endotoxin-induced increases in stromal thickness and haze. In LPS-hyporesponsive C3H/HeJ mice, PECAM-1 and MIP-2 were not upregulated after exposure to endotoxin, and endotoxin-induced keratitis did not develop in these mice. CONCLUSIONS: These findings demonstrate that endotoxin-induced keratitis is regulated by toll-like receptor-4 (TLR4)-dependent expression of PECAM-1 and MIP-2, which are essential for recruitment of neutrophils to this site and for development of endotoxin-induced stromal disease.     

Recurrent intraocular inflammation in endotoxin-induced uveitis

PURPOSE: Endotoxin-induced uveitis (EIU) in rats and mice peaks 24 hours after endotoxin injection and is commonly assumed to be a monophasic disease. This study examined intraocular inflammation at later time points to determine whether endotoxin injection can induce recurrent intraocular inflammation in strains of mice with high or moderate levels of susceptibility to EIU. METHODS: EIU was elicited in two mouse strains with high (C3H/HeN) and moderate (FVB/N) susceptibility, by means of intraperitoneal injections of Salmonella typhimurium endotoxin. Inflammatory cells in the anterior and posterior segments of the eye were counted by a masked observer on histologic sections of eyes from 1 to 17 days after endotoxin injection. RESULTS: A bimodal distribution of inflammatory cell infiltration was noted in eyes from C3H/HeN mice. As previously reported, inflammation peaked at 24 hours after endotoxin injection. However, a second, more pronounced peak of intraocular inflammation occurred approximately 5 days after endotoxin injection. FVB/N mice had a single peak of intraocular inflammation 4 days after injection. CONCLUSIONS: Endotoxin injection in C3H/HeN elicits recurrent intraocular inflammation. The previously unrecognized second peak of inflammation is more severe than the initial inflammatory disease. Studies on this second inflammatory peak may be useful in determining the pathogenesis of recurrent uveitis in humans.     

Abnormal guanosine 3', 5'-monophosphate during photoreceptor degeneration in the inherited retinal disorder of C3H/HeJ mice.

Elevated levels of guanosine 3'-5'-monophosphate (cyclic GMP) are associated with photoreceptor degeneration in the retina of C3H/HeJ mice. This abonormality has been shown to result from a deficiency in the activity of a receptor-specific cyclic GMP phosphodiesterase. The apparent susceptibility of photoreceptor cells to an abnormality in cyclic GMP metabolism suggests that cyclic GMP may play a role in photoreceptor function which is essential to the viability of these specialized cells.     

Epithelium-deficient corneal allografts display immune privilege beneath the kidney capsule

PURPOSE: To determine whether corneal tissue as an allograft displays immune privilege in a nonprivileged site and, if so, whether CD95 ligand expression contributes to the privileged status. METHODS:Syngenic and allogeneic corneal tissues deprived of epithelium were transplanted beneath the kidney capsule of normal mice. Syngeneic BALB/c, allogeneic C57BL/6, and allogeneic B6Smn.C3H-gld (CD95 ligand-deficient) mice were used as donors for BALB/c recipients, and syngeneic C3H/HeJ-gld (CD95 ligand-deficient) mice were used for normal C3H/HeJ recipients. Allogeneic conjunctival segments served as positive grafting controls. Graft fate was assessed by visual inspection at 4, 7, 14, and 21 days and was confirmed by histologic study. Viability of graft endothelium was assessed by immunocytochemical analysis. RESULTS. Syngeneic corneas and C57BL/6 corneas survived almost indefinitely beneath the kidney capsule. Both the stroma and the endothelial layers remained healthy and intact. Allogeneic conjunctiva evoked a strong inflammatory response attended by neovascularization. Similarly, B6-gld corneas deficient in CD95 ligand expression showed acute destruction beneath the kidney capsule. Circumstantial evidence implicates alloimmune rejection as the mechanism. CONCLUSIONS. Epithelium-deprived corneas from normal mice possess inherent immune privilege that protects them from alloimmune rejection even at nonprivileged sites. Constitutive expression of CD95 ligand contributes to the privileged status. It is inferred that the extraordinary success of orthotopic corneal allografts owes as much to the qualities of the graft as an immune-privileged tissue as to the qualities of the eye as an immune-privileged site.     

Toll样受体4对2型糖尿病小鼠视网膜病变的影响

目的　探讨Ｔｏｌｌ样受体４（ｔｏｌｌ-ｌｉｋｅｒｅｃｅｐｔｏｒ４,ＴＬＲ４）对２型糖尿病小鼠模型糖尿病视网膜病变（ｄｉａｂｅｔｉｃｒｅｔｉｎｏｐａｔｈｙ,ＤＲ）的影响。方法　取１６周龄的健康雄性ＴＬＲ４基因缺陷小鼠Ｃ３Ｈ／ＨｅＪ（ｍｕｔａｔｉｏｎ,Ｍｕｔ）及相应野生型小鼠Ｃ３Ｈ／ＨｅＮ（ｗｉｌｄｔｙｐｅ,ＷＴ）各１２只,用链脲佐菌素（ＳＴＺ）腹腔注射建立２型糖尿病动物模型,另取４只未处理小鼠做对照,分为Ｍｕｔ组、ＷＴ组与对照组。于模型建成后１个月、２个月、４个月分别取材,每个时间点模型组分别取材４只,对照组分别取１６周龄的两种小鼠各２只眼球。通过电镜观察小鼠视网膜组织超微结构的变化,并应用ＲＴ-ｑＰＣＲ检测视网膜ＴＬＲ４及巨噬细胞炎性蛋白２（ｍａｃｒｏｐｈａｇｅｉｎｆｌａｍｍａｔｏｒｙｐｒｏｔｅｉｎ-２,ＭＩＰ-２）的ｍＲＮＡ表达。结果　糖尿病模型造模后１个月的超微电镜显示,小鼠视网膜组织即有神经节细胞及神经纤维水肿,核周间隙增宽,核固缩,胞浆颜色变淡;模型造模后２个月时毛细血管内皮细胞胞浆线粒体水肿,嵴变短,周细胞水肿;模型造模后４个月时基底膜节段性增厚,微绒毛突起,管腔狭窄,各时间点的病变ＷＴ组均重于Ｍｕｔ组。视网膜组织ＲＴ-ｑＰＣＲ结果显示,ＷＴ组和Ｍｕｔ组ＴＬＲ４及ＭＩＰ-２的ｍＲＮＡ的表达在糖尿病模型造模后１个月、２个月、４个月时依次上调（均为Ｐ＜０．００１）,各时间点ＴＬＲ４及ＭＩＰ-２的ｍＲＮＡ的表达以ＷＴ组强于Ｍｕｔ组（均为Ｐ＜０．０５）。结论　２型糖尿病小鼠模型中,随病程发展出现视网膜病变并不断恶化,此过程中视网膜中ＴＬＲ４与ＭＩＰ-２的ｍＲＮＡ表达呈显著性上调趋势;ＴＬＲ４基因突变小鼠的视网膜ＴＬＲ４及ＭＩＰ-２的表达比野生型小鼠减弱,同时视网膜病变减轻。     

米诺环素对视网膜色素变性小鼠光感受器细胞的保护作用

目的 观察米诺环素对视网膜色素变性（RP）的rd小鼠［C3H/HeN (Pde6brd-/rd-)］RP过程的影响。方法 40只新生rd小鼠随机分为10组，5组为实验组，5组为对照组，每组4只小鼠。实验组，出生后每日腹腔注射米诺环素22.5 mg/kg；对照组，出生后每日腹腔注射生理盐水10 ml/kg。在出生后1、7、14、21、28 d各处死一组实验组和对照组小鼠，取眼球做组织学观察并行凋亡细胞检测，并对两组视网膜光感受器细胞数、外核层厚度以及凋亡细胞数目进行统计分析。结果 （1）rd小鼠出生后7 d光感受器细胞开始凋亡，14 d达高峰，28 d光感受器细胞完全消失；（2）出生后7 d实验组光感受器细胞数目和外核层厚度与对照组比较差异无统计学意义；（3）出生后14、21 d实验组光感受器细胞数目和外核层厚度多于对照组相应时间点，差异有统计学意义（14 d：t=-3.03、P=0.016，t=-4.469，P=0.004；21d: t=-8.782、P＜0.001，t=-3.497、P=0.004）；（4）出生后7、14 d实验组外核层凋亡细胞数目少于对照组相应时间点，差异有统计学意义（t=-3.497、P=0.004，t=-8.782、P＜0.0001）。结论 米诺环素在rd小鼠RP早期可以延缓光感受器细胞丢失，但不能完全阻止RP的发生。     

Oxidative stress retards vascular development before neural degeneration occurs in retinal degeneration rd1 mice

Purpose

To investigate the role of reactive oxygen species (ROS) in retinal development during the early postnatal stage of rd1 mice.

Methods

Development of the three retinal vascular layers of C57BL/6 J (WT) and C3H/HeN (rd1) mice was evaluated from 9th postnatal day (P9) to P21. Retinal ROS production was semi-quantitatively measured using dihydroethidium fluorescence. Mice were treated with intraperitoneal injections of 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) at a dose of 275 mg/kg body weight, and PBS as the control from P3 to P8.

Results

Rd1 mice showed retardation of retinal vascular development in the deep layer at P9. No significant difference was observed in the outer nuclear layer thickness of rd1 and WT mice. ROS production in the outer nuclear layer of rd1 mice was significantly higher than that in the outer nuclear layer of WT mice at P9, P13, and P17 (P?<?.05). TEMPOL facilitated the development of the deep vascular layer when compared with injection of PBS.

Conclusions

Retardation of retinal vascular development is observed in rd1 mice; ROS is partially responsible for this finding. When using rd1 mice, we should be aware of this difference in comparison to other retinal degeneration animal models and human pathophysiological changes.