恒河猴经瞳孔温热疗法与热休克蛋白相关研究

目的探讨恒河猴经瞳孔温热疗法（transpupillary thermotherapy，TTT）治疗后视网膜和脉络膜组织热休克蛋白70（heat shock protein 70，HSPT0）表达。方法利用恒河猴做动物模型。于1TIrr治疗后不同时间点（1h、1d、1周、2周、1月、4月）摘除双眼固定。应用免疫组化技术，分析TTT治疗对猴眼视网膜和脉络膜组织HSP70的影响。结果TTT治疗后，于1d起，在强反应光斑边缘视网膜和脉络膜有HSP70的表达，4个月时仍有弱表达。弱反应光斑1d时，在视网膜全层及脉络膜均有HSP70表达，4个月时消失。同时,TTT治疗可以引起视网膜不同程度组织病理学的损害。结论TTT可以引起视网膜、脉络膜组织病理学损害。局部温度的升高会诱导视网膜、脉络膜内源性HSP70的产生，并且这种表达在激光治疗后1～4个月的猴眼中仍可以存在。     

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。     

TTT治疗脉络膜黑色素瘤实验研究

目的:探讨人脉络膜黑色素瘤经瞳孔温热疗法(transpupil-lary thermotherapy,TTT)治疗后的组织病理学变化和细胞凋亡相关研究。方法:对经临床TTT治疗不能控制的巨大脉络膜黑色素瘤患者摘除眼球,石蜡包埋切片,行组织病理学检查,VIII因子免疫组织化学研究,并应用TUNEL方法对TTT治疗区域行细胞凋亡检测。结果:TTT治疗后,在激光斑照射的肿瘤部位,最外层为湿性坏死区,其细胞结构消失。中间为干性坏死区,细胞核固缩,在干性坏死区及其下方的肿瘤组织中,可见大量凋亡染色阳性细胞及巨噬细胞,在激光斑基底部可见肿瘤毛细血管呈扩张状态。结论:TTT治疗脉络膜黑色素瘤可以直接摧毁肿瘤组织导致坏死,同时也会引起肿瘤细胞凋亡。但如果瘤体组织太厚,不能完全破坏,则肿瘤下方组织激光后会引起局部毛细血管扩张,是否会加重转移,尚需进一步探讨。     

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.     

不同激光强度经瞳孔温热疗法对色素兔视网膜细胞凋亡的影响

目的 观察不同激光能量的经瞳孔温热疗法（TTT）对色素兔视网膜的病理损伤及对细胞凋亡的影响。 方法 将14只有色家兔随机按不同激光能量分为空白组、50、70、90、110、130、150 mW组，每组2只兔4只眼行TTT治疗照射后,分别于24、48 h后取视网膜组织进行光学显微镜检查及用原位末端转移酶标记（TUNEL）技术和流式细胞仪检测细胞凋亡。 结果 直接检眼镜下激光反应斑颜色随能量增加由灰变白逐渐变浅，即由灰白——白——浓白，直径逐渐增大。苏木素 伊红（HE）染色：50~70 mW组光学显微镜下视网膜各层结构无明显改变；90~130 mW组视网膜结构完整,视锥、视杆细胞肿胀，内颗粒层可见少量的固缩核和细胞浆空泡化。以上能量组激光照射后24、48 h，光学显微镜下视网膜结构与空白对照组无明显差别。150 mW组激光照射后24 h视网膜组织各层肿胀、变性，感光细胞内外节部分缺失；而激光照射后48 h视网膜组织可见明显坏死，全层均可见细胞缺失。TUNEL检测结果显示，各能量组外颗粒层均可见阳性细胞，随激光能量的增加而增加，并逐渐累及内颗粒层和神经节细胞层。流式细胞仪检测结果显示，激光照射后24 h，各能量组均可见细胞凋亡峰。 结论 兔视网膜在能量为50~70 mW的TTT照射后，视网膜组织未见明显改变，但感光细胞凋亡显著增加。随着TTT激光能量增加，视网膜组织出现肿胀、变性甚至坏死。细胞凋亡逐渐累及内颗粒层和神经节细胞层。 (中华眼底病杂志, 2006, 22: 249-252)     

脉络膜黑色素瘤盯TTT治疗后细胞凋亡的初步研究

对5例经瞳孔温热疗法（transpupillary thermotherapy.TTT）治疗挫败的脉络膜黑色素瘤患者手术摘除眼进行分子生物学检测,研究TTT对人脉络膜黑色素养瘤的组织病理学影响及结细胞凋亡的作用,以进一步了TTT的治疗机制.     

脉络膜黑色素瘤TTT治疗后细胞凋亡的初步研究

对5例经瞳孔温热疗法(transpupillary thermotherapy,TTT)治疗失败的脉络膜黑色素瘤患者手术摘除眼进行分子生物学检测,研究TTT对人脉络膜黑色素瘤的组织病理学影响及对细胞凋亡的作用,以进一步了解TTT的治疗机制。1材料与方法1.1实验对象选取5例TTT治疗失败的脉络膜黑色素瘤患眼。所有患者均在同仁眼科中心门诊行TTT治疗。激光的光斑直径为3mm,能量800mW,时间1min×2次。患者治疗后1周肿瘤不能控制,无萎缩迹象。为了防止全身转移,在患者知情同意的情况下,门诊摘除患眼,送同仁医院眼科病理取材并制作蜡块,进行组织病理学、免疫组织化…     

810nm激光治疗视网膜母细胞瘤后脉络膜视网膜瘢痕扩大

经瞳孔温热疗法(TTT)可有效治疗视网膜母细胞瘤,但产生的脉络膜视网膜瘢痕可损害患者视力。为探讨TTT治疗视网膜母细胞瘤后眼底瘢痕随时间推移的变化,作者回顾性分析比较了9例(10眼,14个瘤体)视网膜母细胞瘤患者810nm激光TTT照射后即刻及随访期间的眼底情况。患者均     

810nm激光治疗视网膜母细胞瘤后脉络膜视网膜瘢痕扩大[英]／Lee TC…∥Ophthalmology．

经瞳孔温热疗法(TTT)可有效治疗视网膜母细胞瘤，但产生的脉络膜视网膜瘢痕可损害患者视力。为探讨TTT治疗视网膜母细胞瘤后眼底瘢痕随时间推移的变化，作者回顾性分析比较了9例(10眼，14个瘤体)视网膜母细胞瘤患者810nm激光TTT照射后即刻及随访期间的眼底情况。患者均为一次照射后治愈，随访时间16～31个月。瘢痕直径及面积的测量由两人分别独立完成，     

经瞳孔温热疗法新进展

经瞳孔温热疗法（transpupillary thermotheraphy，TTT）采用810nm激光，治疗后极部多种体积较小的脉络膜视网膜肿瘤。近年来，人们摒弃了治疗时视网膜上需产生不同程度可见光斑的传统光凝治疗观念，TTT试图以不产生可见反应或仅见轻微颜色反应来治疗脉络膜新生血管膜。本文综述了此方法的研究进展。     

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）     

Heat shock protein hyperexpression on chorioretinal layers after transpupillary thermotherapy.

PURPOSE: To assess a biological effect induced by temperature elevation during transpupillary thermotherapy (TTT). METHODS: Six pigmented rabbits were anesthetized, and TTT was performed on the right eye using an 810-nm diode laser installed on a slit lamp (spot size, 1.3 mm; duration, 60 seconds; power, 92-150 mW). A series of laser pulses were aimed at the posterior pole of the retina. The left eyes were used as the control. Twenty-four hours after laser irradiation, a histologic study was performed on the chorioretinal layers. Tissue samples were fixed in formalin and embedded in paraffin. A monoclonal antibody was used to detect heat shock protein (Hsp)70 immunoreactivity, followed by a biotinylated goat anti-mouse antibody, revealed by the avidin-biotin complex and the 3-amino-9-ethyl-carbazole (AEC) chromogen. Retinal structures were further identified by hematoxylin erythrosin saffron (HES) coloration. RESULTS: The photocoagulation threshold was found to be at the 150-mW laser power. Under this threshold, Hsp70 immunostaining was the strongest at the 127-mW power, with staining of some choroidal cells, including capillary endothelial cells. No Hsp70 immunoreactivity was observed on the retina. At the 107-mW power, Hsp70 reactivity was observed only in occasional choroidal cells. At the 98-mW power, only mild, diffuse Hsp70 immunoreactivity was observed in the choroid. At the 92-mW power, as in nonirradiated eyes, no Hsp70 immunoreactivity was detected. CONCLUSIONS: Subthreshold transpupillary 810-nm laser irradiation induces choroidal Hsp hyperexpression. This confirms that choroidal Hsp hyperexpression can be induced during TTT, as has been recently hypothesized by several investigators.     

重组人促红细胞生成素对小鼠视网膜光损伤的防护作用

目的　探讨重组人促红细胞生成素(rHEPO)通过小鼠血视网膜屏障的情况及其对视网膜光损伤的保护作用。方法　24只BALB/c小鼠腹腔注射rHEPO,采用酶联免疫吸附测定(ELISA)法检测小鼠视网膜中促红细胞生成素(EPO)的含量; 24只BALB/c小鼠建立光损伤动物模型,通过光镜和核苷酸末端转移酶介导的dUTP缺口标记(TUNEL)法观察实验组小鼠(12只,腹腔注射rHEPO)和对照组小鼠( 12只,腹腔注射生理盐水)视网膜细胞的变化情况。结果　腹腔注射rHEPO后2、4、6及8h小鼠100μg视网膜蛋白中EPO的含量分别为(0 .68±0 .24)、(1 .87±0 .37)、(0. 96±0 .24)及(0. 47±0. 13)mU,差异有统计学意义(F=2 .113,P<0 .05),在腹腔注射药物后4h视网膜中EPO的浓度达到高峰。随光照时间延长,光镜下可见对照组视网膜杆细胞的内、外节破坏明显加重,外核层逐渐变薄,出现核固缩,甚至核碎裂;实验组各观察时间点损伤变化均较轻,仅见感光细胞内、外节排列紊乱,形成空泡,外核层细胞排列稍紊乱,厚度无明显变化。荧光显微镜下观察,对照组视网膜外核层的凋亡细胞随光照时间延长不断增多,至光照后7d凋亡细胞数量减少;实验组视网膜外核层仅见少量凋亡细胞,光照后72h凋亡细胞的数量明显减少,至光照后7d几乎无凋亡细胞。实验组视网膜外核层凋亡细胞的数量     

Histopathological study on the monkey retina and choroid by diode laser endophotocoagulation

The fundi of three cynomolgus monkeys were coagulated using a diode laser endophotocoagulator. The histopathological changes of the retina and the choroid were investigated periodically up to 3 months. The damage of the retina extended into the outer plexiform layer following mild burn, into the inner nuclear layer with moderate burn and into the nerve fiber layer on severe burn. The pigment epithelium proliferated with multiple layer cells 1-3 months after the irradiation. The choriocapillaries were obstructed, regardless of the degree of burns, immediately after the irradiation, but recanaliculation was observed at the mild and moderate burns 1-3 months after the irradiation. The effects on the choroid were localized to the inner layer following mild burn, but the damage extended into the outer layer with moderate burns. Following severe burns obstruction of the medium size vessels was observed. The fibrous changes of the choroid increased in accordance with the degree of the burn. The damaged melanophore in the choroid recovered considerably 1 month after the mild burn. On the other hand, many macrophages still remained in moderate and severe burn lesions. These results suggested that diode laser photocoagulation might be effective for the treatment of the choroidal diseases rather than retinal diseases, because diode laser photocoagulation produced the considerable change in the choroid as well as in the retina.     

Retinal break and rhegmatogenous retinal detachment after transpupillary thermotherapy as primary or adjunct treatment of choroidal melanoma

OBJECTIVE: To report the development of retinal break or rhegmatogenous retinal detachment (RRD) after transpupillary thermotherapy (TTT) as primary or adjunct treatment of choroidal melanoma. METHODS: In this noncomparative, interventional case series, the authors reviewed medical records of 13 patients who developed retinal break or RRD following TTT. The main outcome measures were clinical features and outcome of treatment of retinal break or RRD following TTT. RESULTS: Of 1574 patients managed on the Oncology Service at Wills Eye Institute with TTT as primary or adjunct treatment of choroidal melanoma, 13 (1%) developed retinal break with or without RRD. The mean patient age at diagnosis of choroidal melanoma was 56 years. Treatment for choroidal melanoma included combined plaque radiotherapy and TTT in 10 patients and TTT alone in 3 patients. The median number of TTT sessions before development of retinal break or RRD was 2. Retinal break or RRD developed at a median of 3 months following the last TTT. All the retinal breaks were located in the TTT-treated area. Retinal breaks were atrophic in 11 eyes and horseshoe shaped in 2 cases. The extent of retinal detachment was none in 1 eye, 1 quadrant or less in 5 eyes, 2 or 3 quadrants in 4 eyes, and 4 quadrants in 3 eyes. Seven patients underwent vitrectomy, one received cryotherapy and laser photocoagulation, and five were observed without treatment. In all eight patients who received treatment for RRD, the retina was attached after a mean follow-up period of 54 months with no intraocular or local extraocular tumor dissemination. CONCLUSIONS: Development of retinal break or RRD is a rare complication of TTT for treatment of choroidal melanoma. The majority of these cases develop within 6 months of TTT and most are caused by atrophic retinal holes in the TTT-treated area.     

Early vascular changes induced by transpupillary thermotherapy of choroidal neovascularization

OBJECTIVE: To study early direct effects of transpupillary thermotherapy (TTT) on choroidal neovascularization (CNV) and choroid. DESIGN: Retrospective, noncomparative interventional case series. PARTICIPANTS: Sixty-four eyes with subfoveal CNV. INTERVENTION: TTT was delivered using a diode laser at 810 nm through a contact lens. Exposure time was 60 seconds with a power/diameter ratio of 247 mW/mm. The end point was an invisible treatment with no color change at the retina level. MAIN OUTCOME MEASURES: Fluorescein and indocyanine green angiographic findings within 1 hour, and at 1, 2, and 4 weeks after TTT. RESULTS: Fluorescein angiography (FA) and indocyanine green angiography (ICGA) performed within 1 hour after TTT showed increased leakage of CNV and choroidal vessels. Follow-up at 1 and 2 weeks demonstrated a hypofluorescent area corresponding to the laser spot and absence of angiographic leakage seen on FA and ICGA. At 4 weeks after TTT, FA showed mottled hypofluorescence-hyperfluorescence of the TTT-treated area and absence of angiographic leakage. CONCLUSIONS: TTT induces a characteristic dynamic sequence of vascular changes. Treatment with TTT can lead to absence of angiographic (FA and ICGA) leakage for 4 weeks. Determination of the long-term efficacy and visual implications awaits the results of clinical trials.     

Transpupillary thermotherapy: effect of wavelength on normal primate retina

PURPOSE: To correlate changes in primate fundus after transpupillary thermotherapy (TTT) at two wavelengths. METHODS: Twelve primate eyes were treated with TTT using a wavelength of 635 nm (n=7) or 810 nm (n=5). Laser parameters were as follows: 635 nm (spot size, 1 mm; duration, 30-8 seconds; and fluence [power over time], 20-91.4 J/cm) and 810 nm (spot size, 2 mm; duration, 60 seconds; and fluence, 96-436 J/cm). Fundus photography, fluorescein and indocyanine green angiography, and enucleation were performed at time 0 or 2 weeks after TTT for histologic analysis. RESULTS: Threshold for fundus lesions (91.4 J/cm at 635 nm and 191 J/cm at 810 nm), acute and chronic retinal damage shown by histologic analysis (79.2 J/cm at 635 nm and 96 J/cm at 810 nm), and choroidal vessel occlusion (50 J/cm at 635 nm and 96 J/cm at 810 nm) were lower at 635 nm. Disorganization of the retina and retinal pigment epithelium was seen for both wavelengths at time 0 and 2 weeks after TTT. Occlusion of the choriocapillaris and choroidal stromal vessels was noted only in specimens obtained 2 weeks after TTT. CONCLUSIONS: TTT resulted in acute and delayed damage to the neurosensory retina that persisted at 2 weeks. The 635-nm wavelength demonstrated a lower threshold fluence for visible fundus lesions, retinal damage, and choroidal vascular occlusion than the 810-nm laser.