兔视网膜脉络膜经瞳孔温热疗法生物学效应初探

目的：探讨经瞳孔温热疗法（TTT）对灰兔视网膜脉络膜的生物学效应,为临床合理应用提供参考依据.方法：使用半导体激光器对8只灰兔视网膜进行照射,光斑直径1.2mm,曝光时间1min,激光功率分别为150,200,250,300mW,分别于激光后1h及1mo行光、电镜观察.结果：150mW激光照射时,仅使视网膜色素上皮（RPE）轻度变化;200mW时除RPE变化外,脉络膜细胞变性,毛细血管内红细胞淤滞、变形;随能量加大（250mW）,RPE受损加重,外层视网膜也同时受累,晚期视细胞层及外颗粒层消失;当激光能量达到300mW时,全层视网膜均受到损伤,并可引起脉络膜出血,甚至出现新生血管内皮细胞.结论：TTT对正常灰兔视网膜脉络膜的效应与激光能量成正比,RPE及邻近脉络膜是TTT的主要作用部位.     

兔眼经瞳孔温热疗法阈值能量治疗的组织病理反应和细胞凋亡

目的:观察兔眼视网膜经瞳孔温热疗法(transpupillary thermotherapy,TTT)阈值能量照射后组织病理反应和细胞凋亡情况.方法:健康青紫兰兔20只,采用阈值能量对实验眼采用1.2mm光斑的810nm激光照射60s.采用眼底镜和眼底照相对光斑进行形态学研究,采用光镜和电镜的方法研究光斑的组织病理和超微结构的改变,采用TUNEL法、荧光素标记Annexin Ⅴ-FITC/PI双染色流式细胞测定法观察视网膜细胞的凋亡.结果:TTT后1d可见视网膜轻度灰白色水肿,后视网膜逐渐出现色素沉着.组织病理学切片显示神经节细胞无显著性破坏.TUNEL染色可见视网膜全层均有细胞凋亡的发生,以内颗粒层为主,流式细胞双染测定显示以凋亡为主.结论:阈能级TTT照射未引起神经节细胞严重损伤,较安全,其作用机制以细胞凋亡为主.     

氩绿激光致兔视网膜感光细胞凋亡的实验研究

目的:观察氩绿激光对兔视网膜损伤及修复的组织学改变及对兔视网膜感光细胞凋亡的影响。方法:将8只有色兔(16眼)中每只眼的上、下方视网膜随机分配为激光光凝区及空白对照区,光凝后24h、4wk通过光镜和电镜及TUNEL技术观察视网膜改变及感光细胞凋亡。结果:(1)光镜观察:光凝处视网膜损伤不明显但脉络膜小静脉充血,4wk后消退。(2)电镜观察:光凝处外节膜盘排列稀疏,神经节细胞轻微肿胀,外核层细胞异染色质增多,4wk后色素增殖,胶原增生。(3)TUNEL染色观察:光凝后24h,光凝处外颗粒层可见较多阳性细胞,内颗粒层偶见,4wk后接近正常。(4)感光细胞凋亡率:光凝后24h,感光细胞凋亡率较正常对照组增加,差异有统计学意义(P<0.01)。光凝后4wk较24h凋亡率显著降低,差异有统计学意义(P<0.01)。结论:轻度氩绿激光光凝视网膜损伤轻微。     

微脉冲半导体激光照射对正常大鼠视网膜损伤的观察

目的观察810 nm微脉冲半导体激光照射对正常棕色挪威大鼠（BN 大鼠）视网膜的损伤。方法使用不同能量及负载系数（duty cycle, D C）的810 nm微脉冲半导体激光对130只BN大鼠眼进行照射。分别于激光照射后第1、3、7、1 4、28 d进行彩色眼底照相、荧光素眼底血管造影及组织病理学观察，并检测热休克蛋白(HSP-70)在视网膜的表达情况，用TdT介导dUTP缺口末端标记法(TUNEL)检查细胞凋亡 。结果阈值及阈上能量条件下，低DC时激光照射部位无光学显微镜下的组织病理学改变，高DC时出现可累及视网膜内核层组织的严重损伤；微脉冲半导体激光照 射后1 d大鼠视网膜内核层细胞HSP-70阳性表达细胞即较正常视网膜明显增加，3 d时达到高峰，以后逐渐下降，14 d时恢复近正常水平。HSP-70阳性细胞数量 随激光能量提高而增加。TUNEL染色可见激光照射部位凋亡细胞主要存在于视网膜色素上皮（RPE）层、外核层、内核层，甚至脉络膜层，其数量随激光能量增高而增多。在激光照射后第3 d，凋亡细胞数量最多。结论810 nm微脉冲半导体激光照射后，视网膜损伤程度与激光能量及DC呈正相关。低能量高负载系数（50 mW，50％）或高能量低负载系数（100 mW，5％～15％）时，损伤限于RPE层，避免了神经上皮层的损伤。激光照射后HSP-70高表达及细胞凋亡可能在组织损伤修复过程发挥重要作用。 （中华眼底病杂志，2008，24：122-126）     

阈下能量经瞳孔热疗对正常大鼠视网膜的影响

目的 研究正常棕色挪威大鼠(BN大鼠)视网膜在810 nm激光阈下能量经瞳孔热疗(TTT)后的改变。方法 雄性BN大鼠36只，使用810 nm激光，采用不同阈下能量对BN大鼠进行TTT。分别于TTT后第1、3、7、14 d进行彩色眼底照相和眼底荧光造影(FFA)。TTT后6、12h，1、3、7、14d各处死6只大鼠，进行组织病理学观察。6、12h和1 d的组织用TdT介导dUTP缺口末端标记法(TUNEL)检查细胞凋亡。结果 TTT后第1 d可见大部分光斑处视网膜灰白色水肿。第3 d视网膜水肿减轻，RPE脱色素。其后视网膜逐渐出现色素沉着。FFA中可见到不同程度的高荧光。组织病理学切片上可见所有在眼底照相上曾出现灰白水肿的病灶，视网膜结构均有显著破坏。TUNEL染色可见视网膜全层均有细胞凋亡。TTT能量最低组6 h时凋亡细胞最多；激光斑旁视网膜较激光斑中央视网膜凋亡细胞多。结论 TTT阈下能量可引起不可逆的视网膜损伤，即使无病理改变也能引起视网膜细胞的凋亡。     

模拟强日光照射导致兔眼视网膜光损伤的实验研究

目的:探讨短时间模拟强日光照射对兔视网膜组织结构的影响及光损伤的致伤机制。方法:健康新西兰白兔25只随机分为5组,30000lux模拟日光照射兔眼15min和30min。照射后不同时间取视网膜进行光镜、透射电镜观察并对视网膜感光细胞凋亡率进行检测。结果:15min光照组2d时观察;光感受器细胞外节盘膜结构紊乱,板层结构离散,内节线粒体肿胀,部分嵴断裂;外颗粒层细胞胞浆有空泡样改变;感光细胞凋亡率为3.26%±0.98%。30min光照组2d时观察,外节盘膜结构紊乱,板层结构重度离散,内节线粒体肿胀,嵴断裂;外颗粒层细胞胞浆有空泡样改变;感光细胞凋亡率为3.63%±1.25%。30min光照组7d时观察,外节盘膜组织结构紊乱,板层结构消失,空泡化,内节线粒体肿胀,嵴断裂;外颗粒层细胞胞浆肿胀,大量空泡样改变,排列紊乱,部分胞膜破坏;感光细胞凋亡率为19.63%±1.32%。30min光照组14d时观察,外节盘膜组织结构恢复较规则,板层结构较致密,内节线粒体肿胀,嵴断裂;外颗粒层细胞胞浆肿胀减轻,排列较整齐;感光细胞凋亡率为18.98%±1.13%。结论:30000lux模拟强日光照射15min可导致兔视网膜急性光损伤,模拟强日光照射可导致视网膜感光细胞发生退行性变性,感光细胞凋亡是损伤发生的重要机制。     

810nm半导体激光致色素兔虹膜组织损伤的研究

目的观察810nm半导体激光不同能量参数对色素兔虹膜组织的损伤。方法将12只色素兔按不同激光能量随机分为空白组和100、150、200、250、300mW6组，每组2只兔4只眼，予以810nm半导体激光照射。激光光斑直径1．2mm，曝光时间60s，分别于24h、48h行眼压和眼前段观察，摘除眼球，取虹膜组织行光镜和电镜检查。结果裂隙灯下眼前段变化：100、150mW组未见明显反应；200、250、300mW组随照射能量的增加，虹膜皱缩速度加快，激光反应斑时间缩短，颜色加深，直径增大。苏木精-伊红染色：100mW组无明显改变；150mW组为可复性损伤改变；200、250、300mW组虹膜血管闭锁，色素颗粒弥散脱失，随能量增加而加重。扫描电镜：100mW组无明显改变；150mW组虹膜前表面连接疏松，未见热凝固征象；200、250、300mW组虹膜表面收缩形成凹陷，呈凝固性坏死，并随能量增加而加重。同一能量组激光术后24h、48h行苏木精一伊红染色及扫描电镜比较无明显差别。结论直径为1．2mm的810nm半导体激光光斑照射色素兔虹膜组织60s致虹膜组织损伤的阈值为200mW。     

视网膜静脉阻塞的高压氧治疗电镜观察

目的 探讨高压氧治疗对视网膜静脉阻塞(RVO)后视网膜感光细胞层超微结构的影响。方法 通过氩激光光凝法制作兔眼RVO模型，对比观察高压氧治疗前后正常兔眼以及高压氧治疗(HBOT)1、3、5、10d与非HBOT RVO眼视网膜感光细胞层超微结构的改变。结果 非高压氧治疗(NHBOT)组RVO细胞线粒体外形膨大，内嵴肿胀、溶解，内质网肿胀、核糖体脱落、核膜溶解等损伤明显较HBOT组RVO眼为重；但也发现高压氧对正常兔眼视细胞线粒体造成一定损伤。结论 HBO对视网膜静脉栓塞后感光细胞层治疗有效，但应注意其毒副作用。     

经瞳孔温热疗法对急性高眼压大鼠视网膜神经节细胞的保护作用

目的研究低阈值经瞳孔温热疗法（TTT）对急性高眼压大鼠视网膜神经节细胞（RGC）是否具有保护作用。设计实验研究。研究对象BN大鼠。方法采用810nm二极管激光机对10只大鼠视网膜进行热刺激，照射光斑1．2mm，能量50mW，照射时间20s，干预后3d光镜下观察视网膜形态结构的改变，免疫组化方法检测HSP70、HSP27在视网膜组织表达。采用上述激光参数，照射视网膜后3d，制作急性高眼压模型（TTT＋I/R组，n=10），采用TUNEL法检测RGC层细胞凋亡数量，及计数高倍镜下RGC层细胞数，与未干预的急性高眼压模型组（I/R组，n=10）、单纯TTT干预组（TTT组）及正常对照组（n=6）进行比较。主要指标免疫组化染色RGC细胞数及RGC层细胞凋亡数。结果采用低阈值TTT可诱导BN大鼠视网膜神经节细胞HSP70及HSP27表达，且光镜下未出现明显视网膜脉络膜形态的改变。TTT＋I／R组RGC层细胞凋亡数量明显少于I／R组（P=0．048），且前者RGC层细胞数量明显多于后者（辟0．016）；TTT组与正常对照组比较RGC层细胞凋亡数量无显著性差异（P=0．882），但RGC层细胞数明显少于正常对照组（P=0．001）。结论低阈值TTT可诱导BN大鼠视网膜HSP70、HSP27表达，并在急性高眼压损伤下对大鼠RGC凋亡具有抑制作用。（眼科，2007,16：48—51）     

兔眼球破裂伤视网膜细胞内RNA表达与凋亡

目的 观察兔眼球破裂伤后视网膜细胞内RNA的表达和凋亡情况.方法 健康新西兰白兔制作眼球破裂伤模型,用甲基绿-派罗宁-马休黄（MG-P-MY）染色法行细胞RNA检测,TUNEL法细胞原位凋亡检测.结果 伤后3 h组在内核层及神经节细胞层有少量RNA阳性表达;6 h内核层阳性表达增多;12 h阳性表达明显;24 h可见大量RNA阳性细胞.伤后3 h组视网膜细胞中偶见凋亡细胞;6 h组凋亡细胞显著增加;12 h～24 h组见大量凋亡细胞.结论 眼球破裂伤后视细胞内早期即有RNA表达,TUNEL法检测存在凋亡现象.     

经瞳孔温热疗法后视网膜和脉络膜的细胞凋亡研究

目的探讨经瞳孔温热疗法（TTT）对视网膜、脉络膜层细胞凋亡表达的影响。方法利用恒河猴做动物模型，于治疗后不同时间点摘除双眼。应用组织学及TUNNEL检测技术，研究TTT治疗与细胞凋亡的相互关系，及对视网膜、脉络膜的组织病理学影响。结果正常猴眼视网膜及脉络膜均未见细胞凋亡发生。TTT治疗后，可见光斑中央细胞坏死、周边细胞凋亡发生。1h至4周，均可见凋亡阳性细胞，4个月时，仍可见少量凋亡细胞存在。同时，TTT治疗可引起视网膜不同程度的组织病理学损害。结论TTT可引起视网膜组织病理学损害。局部温度的升高会诱导视网膜脉络膜细胞凋亡的产生，减少坏死和炎症的程度。     

577nm阈下微脉冲激光与577nm激光光凝对兔视网膜组织影响的比较

背景 近年来研究表明,577 nm阈下微脉冲激光治疗视网膜疾病可达到传统577 nm激光光凝的治疗作用且对视网膜组织损伤小,但其具体作用机制和敏感的靶细胞尚未完全阐明. 目的 探讨和比较577 nm阈下微脉冲激光与577 nm激光光凝视网膜后成年中华黑兔视网膜组织形态学变化,为577 nm阈下微脉冲激光光凝在临床上的应用提供依据.方法 采用抽签法按照视网膜光凝条件不同将26只中华黑兔分为正常对照组(2只)、577 nm激光组(6只)和阈下微脉冲激光组(18只),其中阈下微脉冲激光组按照激光工作负载率的不同亚分为9％、12％和15％阈下微脉冲激光组,每组各6只,正常对照组不做任何处理.光凝后行彩色眼底照相及OCT检查,摘取兔眼球壁行苏木精-伊红染色,光学显微镜下观察兔脉络膜和视网膜组织结构的变化.结果 彩色眼底照相和OCT显示正常对照组兔眼视网膜组织结构清晰.9％阈下微脉冲激光组OCT扫描可见视网膜神经上皮层稍模糊;12％阈下微脉冲激光组光凝斑处视网膜神经上皮层轻度水肿,视网膜色素上皮(RPE)层稍模糊;15％阈下微脉冲激光组可见光凝斑处视网膜神经上皮层明显水肿,RPE层局限性隆起;各阈下微脉冲激光组彩色眼底照相均未见光凝斑.577 nm激光组兔眼彩色眼底照相可见灰白色光凝斑,OCT扫描层面可见视网膜呈多灶性隆起,视网膜各层组织结构模糊,伴浆液性神经上皮层脱离.视网膜组织病理学检查可见,与正常对照组兔眼相比,9％和12％阈下微脉冲激光组兔眼脉络膜血管变形或出血,但视细胞形态结构、双极细胞层和视网膜神经节细胞(RGC)层未见明显改变;15％阈下微脉冲激光组视细胞扁平状膜盘肿胀,双极细胞层和RGC层未见明显改变;577 nm激光组兔眼视细胞层、双极细胞层和RGC层结构紊乱,RPE层变薄.结论 577 nm阈下微脉冲激光对脉络膜层和RPE层具有高度选择性,视网膜光凝后对视网膜神经上皮层的损伤程度轻微,既可发挥治疗作用,又不损伤视网膜神经上皮;577 nm激光视网膜光凝可对视网膜全层造成损伤.     

Subthreshold transpupillary thermotherapy reduces experimental choroidal neovascularization in the mouse without collateral damage to the neural retina

PURPOSE: Transpupillary thermotherapy (TTT) is currently being evaluated for treatment of choroidal neovascularization (CNV) in age-related macular degeneration. To optimize TTT for CNV, the effect was analyzed of invisible (subthreshold) or visible (threshold) doses of TTT on the normal mouse retina and on experimental CNV. METHODS: TTT was delivered to the normal retina of 42 mice with a diode laser at increasing power settings (50, 60, 70, or 80 mW), to obtain thermal lesions ranging from invisible (subthreshold) to visible (threshold) burns. CNV was induced in 53 mice by krypton laser photocoagulation of the fundus, after which the CNV lesions were treated with TTT (50, 60, or 80 mW). Eyes were enucleated 7 days after TTT and prepared for histology, and the CNV complex was evaluated on hematoxylin-eosin stained serial sections by measuring the maximum height of the CNV lesions. Ultrastructural changes were examined by transmission electron microscopy. RESULTS: Increasing the TTT laser power yielded gradually more visible effects. At 50 mW, which induced subthreshold burns, no damage was seen in the neural retina, retinal pigment epithelium (RPE), or choroid at any time point. By contrast, eyes treated with higher power exhibited progressively more damage to the neural retina, including a complete disruption of the outer nuclear layer. When TTT was applied to the laser-induced CNV lesions, the height of lesions was significantly reduced (P < 0.001) in response to all three power settings at 7 days after treatment. The mean relative thickness of the CNV lesion was 3.29 +/- 0.89 in untreated mice, whereas in TTT-treated mice it was 1.69 +/- 0.35, 1.69 +/- 0.41 and 1.70 +/- 0.17 at power settings of 50, 60, and 80 mW, respectively. The overlying neural retina showed no apparent damage with the 50- or 60-mW settings, whereas outer nuclear layer disruption occurred with a power of 80 mW. Electron microscopy confirmed the presence of vascular occlusion at 1 day and a fibrotic scar at 7 days after TTT. CONCLUSIONS: Subthreshold TTT can effectively occlude newly formed vessels and cause regression of the experimental CNV complex without damaging the neural retina. The results demonstrate the importance of using subthreshold laser power in experimental and clinical evaluation of TTT.     

Krypton laser photocoagulation induces retinal vascular remodeling rather than choroidal neovascularization

The purpose of this study is to analyze the retina and choroid response following krypton laser photocoagulation. Ninety-two C57BL6/Sev129 and 32 C57BL/6J, 5-6-week-old mice received one single krypton (630 nm) laser lesion: 50 microm, 0.05 s, 400 mW. On the following day, every day thereafter for 1 week and every 2-3 days for the following 3 weeks, serial sections throughout the lesion were systematically collected and studied. Immunohistology using specific markers or antibodies for glial fibrillary acidic protein (GFAP) (astrocytes, glia and Muller's cells), von Willebrand (vW) (vascular endothelial cells), TUNEL (cells undergoing caspase dependent apoptosis), PCNA (proliferating cell nuclear antigen) p36, CD4 and F4/80 (infiltrating inflammatory and T cells), DAPI (cell nuclei) and routine histology were carried out. Laser confocal microscopy was also performed on flat mounts. Temporal and spatial observations of the created photocoagulation lesions demonstrate that, after a few hours, activated glial cells within the retinal path of the laser beam express GFAP. After 48 h, GFAP-positive staining was also detected within the choroid lesion center. "Movement" of this GFAP-positive expression towards the lasered choroid was preceded by a well-demarcated and localized apoptosis of the retina outer nuclear layer cells within the laser beam path. Later, death of retinal outer nuclear cells and layer thinning at this site was followed by evagination of the inner nuclear retinal layer. Funneling of the entire inner nuclear and the thinned outer nuclear layers into the choroid lesion center was accompanied by "dragging" of the retinal capillaries. Thus, from days 10 to 14 after krypton laser photocoagulation onward, well-formed blood capillaries (of retinal origin) were observed within the lesion. Only a few of the vW-positive capillary endothelial cells stained also for PCNA p36. In the choroid, dilatation of the vascular bed occurred at the vicinity of the photocoagulation site and around it. Confocal microscopy demonstrates that the vessels throughout the path lesion are located within the neuroretina while in the choroid (after separation of the neural retina) only GFAP-positive but no lectin-positive cells can be seen. The involvement of infiltrating inflammatory cells in these remodeling and healing processes remained minimal throughout the study period. During the 4 weeks following krypton laser photocoagulation in the mouse eye, processes of wound healing and remodeling appear to be driven by cells (and vessels) originating from the retina.     

Choroidal heat shock overexpression in transpupillary thermotherapy: preliminary results

PURPOSE: To assess retinochoroidal overexpression of heat shock proteins (HSP-70) induced by a transpupillary laser irradiation below the photocoagulation threshold. METHODS: Four pigmented rabbits were anesthetized and TTT was performed on the right eye using a 810nm diode laser (Iridis, Quantel-Medical, France) adapted on slit lamp (spot size: 1.3 mm, duration: 60 seconds; power 92-150 mW). Series of laser impacts were aimed at the posterior pole of the retina. Left eyes were used as control. Twenty-four hours after laser irradiation, a histological study was done on chorioretinal layers. Tissue samples were fixed in formalin and embedded in paraffin. A monoclonal antibody was used to detect HSP-70 immunoreactivity (mouse IgGl, SPA-810, Stress Gen, Canada), followed by a biotinylated goat antimouse antibody (Dako, Denmark), revealed by the avidin-biotin complex (Vectastain kit, Vector, USA) and the AEC chromogen. Retinal structures were further identified by HES coloration. RESULTS: The photocoagulation threshold was obtained for laser power at 150 mW. Under this threshold, HSP-70 immunostaining was the strongest for power 127 mW with a staining of some choroidal cells, including capillary endothelial cells. No HSP-70 immunoreactivity was observed on the retina. For the laser power 107 mW, HSP-70 reactivity was observed only in occasional choroidal cells. For the laser power 92 mW, as for nonirradiated eyes, no HSP-70 immunoreactivity was detected. CONCLUSIONS: Transpupillary 810 nm laser irradiation under the photocoagulation threshold induces choroidal HSP overexpression. This study concludes that choroidal HSP overexpression can be induced during TTT.     

微脉冲半导体激光与氩激光对兔视网膜损伤及修复的组织学研究

目的观察微脉冲半导体激光与氩激光对兔视网膜损伤和修复的组织学反应。方法在黄斑水肿治疗参数下分别对兔眼行微脉冲阈下光凝和氩激光光凝,观察视网膜组织学改变。结果微脉冲阈下光斑不可见而氩激光可见;激光后两者光镜下视网膜感光细胞损伤不明显但脉络膜小静脉轻度充血,4周后消退;电镜下氩激光光斑处外节排列稀疏,色素上皮细胞（RPE）和节细胞肿胀,外核层细胞异染色质增多,4周后RPE和胶原增生明显,脉络膜内色素颗粒增生;而微脉冲光斑变化不明显。结论微脉冲激光光凝视网膜较氩激光损伤更轻微。     

The levels of cytokines and nitric oxide in rabbit vitreous humor after retinal laser photocoagulation

OBJECTIVE: To determine the levels of nitric oxide (NO) and cytokines such as interleukin 1-beta (IL-1beta), interleukin 6 (IL-6), interleukin 8 (IL-8), and tumor necrosis factor-alpha (TNF-alpha) on vitreous humor following retinal laser photocoagulation. MATERIALS AND METHODS: The rabbits were divided into 3 groups of 4 animals (8 eyes) each. Twelve pigmented rabbit eyes underwent modified grid pattern photocoagulation with a power of 240 mW (group I); 300 mW (group II); and 360 mW (group III). The eyes received 200 burns using a spot size of 200 micro, and duration of 0.2 s. Vitreous humor samples were collected from each eye preoperatively and at 24 and 72 hours after the laser. RESULTS: When compared to preoperative levels, IL-6 levels were increased in all groups; IL-1beta levels were increased significantly only in group III. IL-8 levels were high in groups II and III only at 72 hours (P <0.05). TNFalpha levels were elevated significantly in group II and III only at 24 hours (P <0.05). NO levels were significantly higher than preoperative values in all groups at all times. CONCLUSION: Our results support that especially IL-6, IL-8, and NO levels increase significantly following laser photocoagulation. This preliminary study suggests that IL-6, IL-8, and NO might be dominant contributing factors in the occurrence of the inflammation postoperatively.