实验性大鼠视网膜光损伤的发生机制

目的：研究视网膜光损伤与视细胞凋亡的相互关系，以探讨视网膜光损伤的发生发展机制。方法：所有SD大鼠经循环光环境适应7d，实验前暗适应36h，分别于光照3，6，9，12，15和18h，灌流固定，摘除眼球。光镜标本在常规脱水、透明、石蜡包埋切片后，行苏木精-伊红(HE)、TUNEL法染色，光镜观察；电镜标本在树脂包埋、超薄切片、醋酸-柠檬酸铅双重染色后，透射电镜观察；应用CIAS-1000图像分析系统定量检测外核层面积和视细胞凋亡指数，所得数据作统计学分析。结果：可见视网膜出现光损伤和视细胞凋亡现象，随着光照时间的延长，视网膜光损伤逐渐加重，视细胞凋亡逐渐增多。在外核层，透射电镜观察见核染色质浓集，而无炎性反应。外核层面积和视细胞凋亡指数作相关性分析有显著意义。结论：视细胞凋亡是视网膜光损伤的重要机制；光损伤启动了视细胞凋亡的发生，外核层细胞核的丢失是视细胞凋亡的结果；视网膜光损伤与视细胞凋亡有着密不可分的联系。     

蓝光诱导视网膜光感受器变性大鼠模型的实验研究

目的：建立蓝光诱导视网膜光感受器变性大鼠模型,评价其稳定性及可重复性。方法：选用成年雄性SD大鼠,予缝线开睑,1%阿托品扩瞳,经24h暗适应后,放入光照箱中接受24h强度为2 500Lux的宽谱蓝光照射。在光照后的1d、3d、7d、14d应用光镜、TUNEL染色、透射电镜观察实验大鼠视网膜组织结构的改变及细胞凋亡情况;应用闪光ERG评价视网膜功能。结果：光照后视网膜光感受器的内外节破坏,外核层排列紊乱,核质固缩,核层逐渐变薄,14d时减少达60.09%;光照后1d,外核层出现大量TUNEL（＋）细胞,经透射电镜证实光感受器的死亡方式为凋亡;光照后14d,视网膜功能显著下降,0dB光照强度不能诱导出Photic-ERG波形。结论：2 500Lux蓝光照射能够诱导视网膜光感受器发生典型的以凋亡为特征的变性过程,具有较好的稳定性和可重复性。     

Lactacystin对体外C6胶质瘤细胞超微结构影响的研究

目的 探讨蛋白酶体抑制剂Lactacystin 对体外C6胶质瘤细胞超微结构的影响.方法 Lactacystin处理体外培养的G6胶质瘤细胞,HE染色后光镜观察细胞的死亡情况,锇铀铅染色后透射电镜观察细胞超微结构变化.结果 Lactacystin处理体外C6胶质瘤细胞24 h后,光镜下见胞浆浓缩深染,胞核染色质致密浓缩,透射电镜下见细胞表面微绒毛消失,核皱缩,染色质致密浓缩、边集.结论 蛋白酶体抑制剂Lactacystin可以诱导体外G6胶质瘤细胞凋亡.     

骨髓间充质干细胞在大鼠视网膜光感受器细胞损伤中的应用研究

目的探讨骨髓间充质干细胞(BMSCs)在大鼠视网膜光感受器细胞损伤中的作用机制。方法选取24只SD大鼠,分为正常组(n=6)、光损伤组(n=6,视网膜下注射无菌生理盐水4μl)、磷酸盐(PBS)视网膜下注射组(n=6,视网膜下注射PBS 4μl,其中溶质为BMSCs)和BMSCs视网膜下移植组(n=6,视网膜下移植2×10~5个BMSCs),除正常组外,其余各组光照射24 h,光照后10 d进行手术。术后14 d将眼球摘除,培养大鼠BMSCs原代及传代,制作冰冻切片,行HE染色,计算视网膜外核层层数。采取免疫荧光法,检测大鼠BMSCs表面标志物表达情况。采取TUNEL法检测细胞凋亡情况。结果与正常组比较其视网膜外核层层数明显减少,细胞凋亡百分比有所升高;与光损伤组、PBS组相比,BMSCs组视网膜外核层层数明显增加,细胞凋亡百分比有所降低;其中BMSCs细胞在视网膜下腔不表达MAP2、CD11b、Rh0dopsin、Calretintin,部分表达Nestin阳性;同时4',6-二脒基-2-苯基吲哚二盐酸盐(DAPI)阳性的BMSCs细胞视网膜下腔表达b FGF,不表达BDNF和CNIF。结论大鼠实验结果表明,BMSCs视网膜下移植,可减少视网膜光感受器细胞凋亡,作用原理是可能通过视网膜下腔分泌的b FGF发挥作用。     

RCS大鼠与SD大鼠的视网膜结构和光感受器形态观察

目的：比较遗传性视网膜色素变性动物模型RCS大鼠与SD大鼠视网膜及光感受器细胞形态学差异。方法：取变性中期（日龄30 d）RCS（RCS-p＋rats）大鼠及SD大鼠各8只,断颈处死后取新鲜眼球,于80%乙醇、40%甲醛和95%冰醋酸混合固定液（85∶10∶5）中固定24 h,垂直切开眼球,常规乙醇脱水,石蜡包埋,作经线方向4μm的垂直切片,苏木素-伊红染色,光镜下观察两组大鼠视网膜形态和结构,显微镜测微器测定两组光感受器细胞外核层厚度,记录外核层细胞层数,并进行比较。结果：RCS大鼠和SD大鼠视网膜形态学有明显差异,主要表现为光感受器细胞层菲薄（P〈0.05）,细胞核数目减少。结论：视网膜色素变性动物模型RCS大鼠视网膜厚度、外核层变薄,光感受器细胞减少,这可能与视网膜色素变性光感受器细胞的凋亡有着直接的因果关系。     

牛磺酸对大鼠视网膜光损伤导致的细胞凋亡及bcl-2表达量的影响

目的 :探讨牛磺酸与视网膜光损伤诱发的细胞凋亡及与凋亡相关基因bcl- 2蛋白表达量的关系。方法 :建立大鼠视网膜光损伤模型 ,30只wistar大鼠被随机分为 :正常对照组、阳性对照组、牛磺酸用药组。绿色荧光持续照射 2 4h形成视网膜光损伤。用流式细胞术检测视细胞的凋亡率及bcl- 2基因蛋白表达量的变化。结果 :在牛磺酸用药组 ,视细胞的凋亡率明显低于阳性对照组 (P <0 0 1) ,而bcl- 2蛋白表达量却显著高于阳性对照组 (P <0 0 5 )。结论 :牛磺酸通过上调bcl- 2基因蛋白的表达量 ,抑制了视网膜光损伤诱发的细胞凋亡 ,避免视网膜的光损伤。     

脊髓缺血再灌注损伤后神经细胞凋亡的实验研究

目的：研究细胞凋亡在脊髓缺血再灌注损伤中的作用。方法：建立兔脊髓缺血再灌注损伤模型，再灌注后24h和48h取脊髓标本，应用HE染色、透射电镜、TUNEL和免疫组化染色方法观察脊髓标本中运动神经元的细胞凋亡情况。结果：兔脊髓缺血再灌注后，HE染色可见运动神经元呈凋亡形态改变，TUNEL染色阳性，免疫组化染色示Bax蛋白表达明显升高，Bcl-2蛋白表达轻度升高，Bax／Bcl-2比值升高，电镜观察可见典型凋亡小体出现。再灌注24h时凋亡细胞较48h时为多。结论：细胞凋亡是兔脊髓缺血再灌注损伤后运动神经元的主要死亡方式。     

异搏定对大鼠视网膜缺血再灌注损伤的保护作用

目的：在大鼠视网膜缺血再灌注损伤模型上，研究异搏定对视网膜缺血再灌注损伤后视网膜内细胞凋亡的影响。方法：60只大鼠随机分为对照组及异搏定处理组，每组30只。通过结扎大鼠左颈总动脉1h，然后再灌注。异搏定处理组腹腔注射异搏定1mg／kg，对照组腹腔注射生理盐水1mL／kg，分别检测1、6、12、24、48、72h各时段大鼠视网膜内的细胞凋亡，每时段大鼠各5只。结果：再灌注后，细胞凋亡主要出现于视网膜的神经节细胞层和内核层，大鼠视网膜在再灌注6h逐渐出现细胞凋亡，12h逐渐增加，24h细胞凋亡达到高峰，偶尔在外核层可见凋亡细胞。然后细胞凋亡逐渐减少，但是，到了术后72h，仍然可见视网膜内有细胞凋亡，而异搏定可以减少视网膜细胞凋亡的发生。结论：异搏定可以抑制再灌注后细胞凋亡的发生。对大鼠视网膜具有保护作用。     

外周血Tfh细胞比例及其胞内IL-21含量与蓝光视网膜损伤的相关性

目的 探讨外周血滤泡辅助性T细胞（Tfh细胞）比例及其胞内白介素-21(IL-21)含量与蓝光视网膜损伤的相关性。方法 随机将Brown Norway(BN)大鼠均分为4组，采用蓝光全身每天照射3小时建立大鼠视网膜光损伤模型。按照射时间分为光照0天组即对照组（0 d组）、3天组（3 d组）、7天组（7 d组）和14天组（14 d组）。光照后采用流式细胞术和ELISA法分别检测大鼠外周血中Tfh细胞的比例及其胞内白介素21(IL-21)含量；视网膜电流图（ERG）检查评价视网膜功能；眼底照相和HE染色分别检查眼底改变和视网膜外核层厚度变化。结果 与对照组相比，视网膜蓝光损伤后外周血Tfh细胞比例和胞内IL-21含量均随光照时间延长显著增加（均P <0.05）;ERG显示随光照时间延长视网膜功能损害加重，a、b波潜伏期和振幅分别随光照时间延长而延长和降低（均P <0.01）；大鼠眼底在光照3 d出现色素脱失样改变，随光照时间延长出现视网膜血管变细及渗出样改变；HE染色显示随光照时间延长ONL厚度显著变薄（均P <0.05）。相关性分析提示外周血Tfh细胞比例及其胞内IL-...     

局灶脑缺血边缘区星形胶质细胞的超微结构变化

目的:观察缺血再灌边缘区星形胶质细胞的形态学和超微结构变化,了解其坏死过程中形态学变化特点。方法:SD大鼠随机分为假手术组、单纯缺血组、缺血再灌组。标本冠状面按A,B,C,D4等分切脑。采用线栓并环扎的方法建立大鼠局灶脑缺血模型,单片脑组织TTC染色定位边缘区。取缺血周围区和中心区脑组织,经固定包埋,半薄切片(1μm),超薄切片。JEOL100透射电镜下观察。结果:缺血3h既可见星形胶质细胞明显变化,以细胞和核肿胀为主。再灌3h胞浆内可见肿大的线粒体和空泡变。缺血6h:细胞水肿加重。缺血6h再灌3h可见细胞肿胀破溃,染色质漏出核周,核周明显水肿。缺血12h,水肿进一步加重。缺血24h及再灌3h星形胶质细胞核破溃变形,核周明显水肿。结论:星形胶质细胞在缺血3h即可发生明显的结构变化,缺血中心区的星形细胞随着缺血时间的延长变化逐渐加重,而边缘区细胞的损伤与缺血时间呈不一致性。星形胶质细胞对缺血呈高度的敏感性,可以作为判断早期缺血的一个指标。     

蓝色发光二极管光源照射对大鼠视网膜的安全性试验

背景:国外研究证实,波长为470 nm的蓝光对抑制褪黑素分泌,调整生物节律效果最明显.目前国内尚无发光二极管光源用于调节生物节律的实验报道.目的:观察一定强度蓝色发光二极管光源对大鼠视网膜是否存在损伤.设计、时间及地点:随机分组对照动物实验,组织学分析,于2007-05/2008-04在北京大学第三医院动物实验室完成.材料:SD大鼠32只,BN大鼠16只由北京大学第三医院动物部提供.方法:取SD大鼠16只及BN大鼠16只分别以抽签法随机分为实验组与对照组.将实验组大鼠放入光箱中,光源工作参数为:蓝光波长470 nm,光强控制在300～350 μW/cm~2,光照射4 h/d,连续照射3 d.另取SD大鼠16只以抽签法随机实验组(n=8)与对照组(n=8),将实验组大鼠放入光箱中,光源工作参数为:蓝光波长470 nm,光强控制在120~150μW/cm~2,4 h/d,连续照射3 d.对照组均不进行特殊干预.主要观察指标:照射结束后第2天,取大鼠双侧眼球,冰冻切片后苏木精-伊红染色观察各组大鼠视网膜变化.结果:48只大鼠均进入结果分析.强度为300~350μW/cm~2的蓝色发光二极管光源照射时,SD大鼠视网膜变薄,层次不清,细胞排列不整齐;而BN大鼠视网膜结构层次清晰,细胞排列整齐,与对照组比较无明显差异:强度为120~150μW/cm~2 蓝色发光二极管光源照射时,SD大鼠视网膜结构层次清晰,细胞排列整齐,与对照组比较无明显差异.结论;300~350 μW/cm~2蓝光发光二极管光源照射强度对有色素保护的视网膜是安全的,照射强度为120~150 μW/cm~2蓝色发光二极管光源对不同种属大鼠的视网膜都不会造成光损伤.     

POD Nanoparticles Expressing GDNF Provide Structural and Functional Rescue of Light-induced Retinal Degeneration in an Adult Mouse

Peptide for ocular delivery (POD) is a novel cationic cell-penetrating peptide (CPP) which, when conjugated with polyethylene glycol (PEG-POD), can deliver plasmid DNA to the retinal pigment epithelium (RPE) of adult murine retina. PEG-POD nanoparticles containing an expression cassette for glial cell line–derived neurotrophic factor (PEG–POD~GDNF) were investigated for their ability to inhibit light-induced photoreceptor apoptosis. PEG-POD~GDNF, control nanoparticles, or buffer were injected into the subretinal space of adult murine retina and retinal degeneration induced by blue light. Animals injected with PEG-POD~GDNF showed a significant reduction (3.9–7.7 fold) in apoptosis relative to control-injected animals. The thickness of the outer nuclear layer (ONL) of the superior retina of PEG-POD~GDNF-injected eyes was significantly greater (23.6–39.3%) than control-injected retina 14 days post-light treatment. PEG-POD~GDNF-injected eyes showed a 27–39% greater functional response relative to controls, as measured by electroretinogram (ERG) 7 days post-light treatment. This is one of only two studies demonstrating histological and functional rescue of a mouse model of retinal degeneration following nonviral administration of a transgene into adult retina. Although rescue is short lived for clinical application, this study represents an important step in the development of nonviral gene therapy for retinal diseases.     

大鼠肠缺血—再灌注模型中多个器官细胞凋亡的观察

目的：探讨多脏器功能失常综合征发病中细胞凋亡的作用。方法：建立大鼠肠系膜上动脉夹闭模型，常规组织切片，ＨＥ染色，普通光学显微镜检查及吖啶橙溴化乙锭双重染色的荧光显微镜观察肠、肝、肺、肾４个器官组织的细胞死亡现象，并在石蜡切片上计数凋亡细胞的百分比；荧光显微镜下计数缺血再灌注不同时间分离的肝细胞中活细胞、凋亡细胞和坏死细胞数。结果：①肠缺血再灌注后各时间点均可观察到细胞凋亡及细胞坏死现象；②局灶性的肾小球和肾小管坏死主要分布在肾皮质与髓质交界处；③肝细胞凋亡和细胞坏死的百分比均随肠缺血再灌注时间延长而升高，且与假手术组比较均有显著性差异（Ｐ均＜０．０５）；缺血１２０分钟、再灌注６０分钟组与缺血１８０分钟组比较，细胞凋亡与细胞坏死的百分比均较高（Ｐ均＜０．０５）。结论：肠缺血再灌注后多个器官组织在短时间内即可发生大量细胞凋亡，这在多器官功能障碍发病中可能具有重要意义     

视网膜挫伤模型兔视神经细胞的凋亡

背景：视网膜挫伤是眼外伤后导致视力损害的主要原因之一,临床治疗比较棘手。虽然对视网膜挫伤后眼底改变在临床上已有较充分的认识,但在其发病机制、药物治疗上意见仍不一致。 目的：在兔视网膜挫伤模型上,观察视神经细胞的凋亡并探讨其发生机制。 方法：健康成年无眼疾青紫蓝兔48只,随机分为8组,挫伤后1,3 h组、挫伤后1,3,7,14,28 d组及正常对照组,每组6只。右眼为致伤眼,以改良Al en’s重击法制备兔单眼挫伤性视网膜病变模型。分别于建立模型后在相应时间点获取兔眼标本,对筛板后5 mm视神经行病理切片,苏木精-伊红染色,观察组织形态并行神经胶质细胞计数,以电镜及TUNEL法观察视神经细胞凋亡情况。 结果与结论：正常对照组兔视神经纤维排列整齐,胶质细胞与神经纤维走向一致,呈极性排列。挫伤后1,3 h、挫伤后1,3,7 d兔视神经纤维排列紊乱,胶质细胞杂乱无序。挫伤后1,3 h、挫伤后1,3,7,14,28 d胶质细胞计数减少,与正常对照组相比,差异有非常显著性意义（P＜0.01）。视网膜挫伤后存在神经胶质细胞凋亡现象,正常对照组、挫伤后1 h、挫伤后28 d组几乎不见凋亡细胞;挫伤后3 h组、挫伤后1,3,7,14 d组均可见TUNEL染色阳性的凋亡细胞,其中在3 d组数量较多,与正常对照组之间比较,差异有非常显著性意义（P＜0.01）。提示视网膜挫伤后,视神经胶质细胞凋亡可能是视功能恢复不良的原因之一。     

急性肺损伤炎性细胞凋亡异常及粒细胞集落刺激因子调控的研究

目的 探讨急性肺损伤时支气管肺泡灌洗液(BALF)中的中性粒细胞(PMN)凋亡发生规律及其与粒细胞集落刺激因子调控关系.方法 豚鼠30只,分为3组:组1为生理盐水正常对照组,组2为油酸致病组,组3为油酸+粒细胞集落刺激因子组.组2、组3分别由尾静脉注射油酸(0.12 ml/kg)造成豚鼠急性肺损伤模型.组1则注入生理盐水.组3在实验造模前2 d由皮下注射粒细胞集落刺激因子1.0μg/kg,1次/d.组1、组2、组3分别于注射后2 h用生理盐水进行全肺支气管肺灌洗,收集BALF.用梯度密度法离心收集PMN.用原位末端标记法检测BALF中PMN凋亡.结果 组2、组3和组1BALF中PMN凋亡百分比分别为(2.500±1.080)%、(3.500±0.850)%、(6.400±1.505)%.组2、组3较组1 BALF中PMN凋亡均显著降低(均P＜0.01).结论 急性肺损伤炎性细胞PMN凋亡延迟,PMN持续激活和释放毒性内容物与肺损伤有密切关系.粒细胞集落刺激因子能调控干预急性肺损伤时PMN凋亡延迟.     

不同时间给予猪肺表面活性物质对大鼠油酸型急性肺损伤疗效的影响

目的 探讨不同时间给予猪肺表面活性物质(PPS)混悬液对油酸致大鼠急性肺损伤(ALI)的治疗作用.方法 将48只SD大鼠随机分为假手术组、模型组、0.5 h PPS治疗组和2 h PPS治疗组.假手术组仅行气管和颈动脉插管手术操作,其余各组大鼠静脉注入油酸诱发ALI;0.5 h PPS治疗组和2 h PPS治疗组分别于油酸注入后0.5 h和2 h经气道均滴入100 mg/kg和150 mg/kg两个剂量的PPS,实验过程中计数大鼠呼吸频率,测定动脉血气指标;于实验4 h计算大鼠存活率后处死动物,观察肺组织病理学改变,并检测肺系数及支气管肺泡灌洗液(BALF)中总蛋白(TP)含量和血浆中肿瘤坏死因子-α(TNF-α)浓度.结果 与模型组比较,0.5 h给予PPS 100 mg/kg和150 mg/kg以及2 h给予PPS 150 mg/kg治疗都能显著降低大鼠的呼吸频率,提高动脉血氧分压(PaO2)及大鼠存活率,降低肺毛细血管通透性及肺出血、肺水肿发生率,降低血浆中TNF-α和BALF中TP含量(P均＜0.05).而2 h给予100 mg/kg PPS的治疗作用不明显.结论 早期气道内滴入≥100 mg/kg的PPS,能明显改善油酸型ALI大鼠的呼吸功能、减轻肺损伤;晚期较大剂量的PPS(150 mg/kg)才有治疗作用.     

Theoretical estimation of retinal oxygenation during retinal artery occlusion

The aim of the present work was to simulate the oxygenation of the whole retina under normal conditions as well as during retinal ischemia. A differential equation describing how oxygen is transported from blood to tissue, diffuses through the tissue and is consumed according to Michaelis-Menten kinetics was constructed. The outer retina was divided into three regions of which one was set to have consumption. The inner retina was considered as one uniform region with respect to maximal rate of oxygen consumption and blood flow. The results suggest that extreme hyperoxia would be needed to make the choroid capable of supplying the whole retina during total retinal artery occlusion and moreover confirm that light might to some extent be beneficial. As supplying 100% oxygen by nose cannula or common oxygen mask can hardly increase the arterial oxygen tension to the levels needed to rescue the whole retina, the effects of oxygen treatment of total retinal artery occlusion are expected to be modest, both in darkness and light, unless a non-rebreather face mask system is used.     

Adeno-associated virus type 5: transduction efficiency and cell-type specificity in the primate retina

Gene transfer using adeno-associated viruses (AAVs) has been effective for treating inherited retinal diseases in animal models. Further evaluation in primates must be performed prior to clinical application, however, because of the difference between the retina of the primate and those of other animals. Prior work has shown that AAV2 can transduce rod-photoreceptor and RPE cells in the non-human primate retina and that AAV5 is more efficient at transducing photoreceptor cells than AAV2 in the rodent retina. In this study, we evaluated the efficiency of AAV5 in the non-human primate retina after subretinal injections of the vector to distinct anatomic retinal regions (superior, inferior, nasal, macula, temporal). rAAV5 led to a rapid onset of transgene expression (within 2 weeks), with expression persisting up to 10 months. Postoperative electrophysiology studies showed that global retinal function was preserved following gene transfer. Quantitative analysis of gene transfer demonstrated a maximum transduction efficiency of 22% in the injected areas. Evaluation of cell types using confocal microscopy and cone-specific antibodies revealed that AAV5, expressing reporter genes from the cytomegalovirus (CMV) promoter/enhancer, preferentially transduced rods. No significant differences were found in the regional tropism of AAV5 among the five areas injected despite variation in retinal topography. Immunohistochemical studies revealed that the AAV5 receptor, PDGFR-A, is localized to the outer segments of rods but not cones providing a basis for the observed tropism. Our results support the utility of AAV5 for rod photoreceptor degeneration therapies.     

Neuroprotective Effects of Nicotinamide after Experimental Spinal Cord Injury

OBJECTIVE: To investigate the ability of nicotinamide to protect against secondary damage in spinal cord tissue after an experimental injury. Trauma to the spinal cord induces a cascade of cellular events that lead to progressive tissue injury over time. Nicotinamide has been shown to affect many elements of this cascade, including excitatory amino acid release, inflammation, apoptosis, and cellular energy balance. METHODS: Male Long-Evans (n = 12) rats received an excitotoxic spinal cord injury by intraspinal injection of quisqualic acid (QUIS), a glutamate receptor agonist. A second set of rats (n = 4) received intraspinal saline as a sham injury. Thirty minutes after injury, animals that had QUIS injections received an intraperitoneal injection of either saline (control, n = 4) or nicotinamide (500 mg/kg, n = 8). Seven days postinjury, the spinal cords were removed, and serial sections were cut, mounted on slides, and stained. By using light microscopy, the extent of tissue damage was assessed at the epicenter of injury as well as sections up to 450- microm rostral and 450- microm caudal to the epicenter. RESULTS: Only those animals receiving QUIS injections showed damaged tissue. There was no significant difference in the amount of damage at the epicenter of injury between the saline- and nicotinamide-treated animals. However, when comparing the total amounts of damage over the 975- microm length of cord examined, the rostro-caudal extent of injury was significantly reduced in the nicotinamide-treated animals compared with the saline-treated animals. CONCLUSIONS: Systemic nicotinamide serves to limit the rostro-caudal extent of cell death after experimental spinal cord injury.